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·RENAL FUNCTION
Transactions of the Second Conference
October 19 20, 1950, Neu I ork, NY
Edited by
STANLEY E BRADLEY
DEPARTMENT OF MEDICINE
( OF LFGF OF PHYSICIAS & TR FONS
COLUMBIA IVERSITY
Sponsored by the
JOSIAH MACY, JR FOUNDATION
565 Park Avenue New York 21 NY

·Copyright 1951, by the
JOSIAH MACY JR FOUNDATION
Prne $300
Printed m the United States of America
By Corlirs, MACY & COMPANY, INC, New York NY

·PARTICIPANTS Second Conference on Renal function MEMBERS ROBERT I PITTS Cbirmai Depa tment of Phys ology Co nell Un esty Med cal College New York NY STANLEY E BRADLEY Secretary Der rtment fMdn College of Phys cans & Surgeons Columbu Un ers ty New York, NY PHYLLIS A BOTT Depa tm t f Phys olog cal and Cincal Chem stry Wom As Med cal College of Penns tvan a P! ladelph a P DANIEL C DARROW Departm nt of Ped at s Yale Un versty School of Medcne Mew Ha en con WILLIAM DOCK Depatment of Medcne College of Mel ne State in vers ty of New York Med cal Con er at New Yok City Brooklyn NY ARTHUR M VISHBERG Depatment of Medcne College of Ved ne New Yok in ersty New Yok NY ROY P FORSTER D partn nt { Zoologs Dartmouth College Han er H ALLAN L GRAITLIN Department of An tomy Shool of Medicne Johns Hopka in vers ty Bait mo e Md CHARLES A JANEWAY Deparment of Ped at cs Harvard Mel cal School Bo on, M255 JOHN A LUETSCHER JR Depin ent of MJ Re Stanfo Jln versty Shool of Met ine San Francs Calf JEAN OLIVER Departm nt of Pa hol gy Gallese of Med ine Saclo ers y flew lok Mlej cal Cen er at New York Cry Brooklyn, A Y EW ALD E SELLURT Deriment of Pology School of Medco s tem Reserve in ersty Cleveland Oho Absent ·JAMIES A SHANNON Nitsonal Heart Institute, National Institutes of Health, U S Public Health Service Bethesda, MD NATHAN W. SHOCK Section on Gerontology, National Heart Institute National Institutes of Health and Baltimore City Hospitals Baltimore, MJ JOHN V TAGGART Department of Medicine College of Physicians & Surgeons, Columbs University New York, NY GEORGE W THORN Department of Medicine, Harvard Medical School Boston, Mass GUESTS FRANCIS BINKLEY Department of Pathology (Biochemistry), University of Utah School of Medicine Salt Lake City Utah CHARLES H BURNETT Department of Medicine Southa estern Medical School, University of Texas Dallas, Texas ROBERT GAUNT Department of Zoology, College of Medicine State Unnersity of New York Medical Center at Syracuse Syracuse, NY ALFRED GILMAN Department of Pharmacology College of Physicians & Surgeons Columbus University New York NY HENRY LAUSON Department of Pediatrics Cornell University Medical College New York NY ELAINE P RALLI Department of Medicine College of Medicine, New York University New York N) EPHRAIM SHORR Department of Medicine Cornell University Medical College Nen York NY HARRY B VAN DYKE Department of Pluarmacology College of Physicians and Surgenns Columbu UOL Nen York NY HARVEY L. WHITE Department of Physiology Washington University School of Medicine St Louis, MO JOSIAH MACY, JR FOUNDATION Frank Fremont Smith, Medical Director Janet Freed Arwant for Conference Program ·TABLE OF CONTENTS Second Conference on Renal Function 9 30 Josiah Macy }r Foundation Conference Progrim Franh Fromont Smith Introductory Remarks Robert I Patts Adrenal Cortical Hormone and Related Hormones in Water Vetabohsm Robert Count Discussion References Antiduretic Factors Harry 8 ian Dyke Discussion References Some P.tuttary and Adrenul Influences on Renal Function Harvey L White Discussion References The Actions of ACTH and Cortisone on Renal Function in lian Charles II Burncit Discussion Icferences 18 57 74 79 100 104 106 115 125 227 129 141 The Excretion of Sodum in iclation to Glomerular Tiltration Markey L White Discussion Refcrences Endocrine Factors in the Utilization of Glutathione Francis Bakley Discussion References Bioissa of Sodium Retuning Corticoids and Some Changes in Crcretion of these Substances in Disease John A Luetscher Jr and Quentin B Doming Discussion References 142 19 15 155 170 198 ·Renal Tunction 6 In contradistinction to the usual scient fic meetmg we like to hue the presentations relatively brief ind to bring out the difficulties encountered in problems rather than the neat solutions of those problems in other words we feel that the heart of these meetings is in the discussion and that only in 1 really inform dI and friend y atmosphere is it possible for people to discuss freely all of their Ideas Therefore we hope that you wont hesitate to sperk spon taneously and informally What you say may not be too wise but how cın you be sure that it wont evoke wisdom in someone elsel One point which should be stressed is that between the disciplines there are real difficulties in communication - prtly emohonal and partly semanhc Emotionally some im estigators accept only data derived from methods or disciplines with which they are fanular On the semintic level the physical and biological sciences can understand each other without difficulty ns can the medical psy chitric and social sciences However to bridge the gap betweets the physical and biological sciences on the one hand and the psychological and social sciences on the other is very difficult Through the Conference Progrim and in the published trinsictions we hope to give a clearer reproduction of shat takes place in the laboratory and what goes on in the minds of scientists thun nou appears in the scientific hiterature Unfortunately somewhere along the line scientists themselves hare been responsible for hiding the artistic creative aspects of scientific endervor bclund the cold, white light of logic the essential testing method wluch miles possible the practical apphcation and use of the crertive idea This program is an experiment and you ne part of that experi ment The success of the undertaking is measured entirely by what each participant gains from this expenence We hope that here you w?}} feel the freedom inherent m the scientific method ind that you will help us to improve our conference procedure ·INTRODUCTORY REMARKS ROBERT F PITTS, Chairman IT is w pleasure to welcome you as members and guests of this Scrond Conference on Renal Function sponsored by the ſosiah Macy Foundation, and to wish you a very pleasant and profitable series of discussion sessions today and tomorrow The chuironment which has been prepared by Dr Fremont- Smith, Viss Freed and the Foundation staff is a most favorable one furthermore, this assembled company has as one of its many assets congeniality I am, therefore, sure that we will have a pleasant time I would also like to emphasize the statement of Dr Fremont-Smith that it will be a profitable time only if we freely and openly discuss our problems, contnbute our own experience and seek the council of the assembled group The topic chosen by you at the last session, namely, hormonal control of renal function, has been narrowed by me to include only pituitary and adrenal cortical influences The latter, especially, is a timely topic for discussion, since the adrenals are now so much in the public eye However, we all realize that, important as they me, the adrenal cortical hormones operate through a background of other hormonal activities, perhaps through a background of nervous vasomotor regulation, and certainly through an mherent or putonomous capacity of the kidney to work independentis Nevertheless, some have questioned the wisdom of diffusing our cfforts by considering two endocrine systems rather than concentrating on one, obviously the adrenals, in an effort to exhaust them in five straight sessions My answer is that our adrenal resen e is very great and that we cannot exhaust it in one conference Further. more I feel that maintaining a proper perspectre is more in portant than exhausting a single phase of hormonal regulation That perhips is adequate justification of the adnuxture of anterior pitan tir, posterior pituitary, and adrenal cortical influences which you ahserie in the conferener program At least it is my justification ile shall start our program with 3 consideration of the action of the adrenal cortex and related hormones on water ercretion De ashed Dr Gaunt, Professor of Zoology at the State University of New York Medical Center nt Syracuse to open this discussion ·ADRENAL CORTICAL HORMONE AND RELATED HORMONES IN WATER METABOLISM* ROBERT GAUNT Department of Zoology, College of Medicine State Untersity of New York Medical Center at Syracuse I want to begin with a little matter of personal orientation that to me at least is pertinent I was very pleased when De Pitts invited me a year ago to attend the first of these conferences on renal function but also embarrassed because I felt I had nothing to contribute to the subject Although we have studied vanous aspects of water metabolism for some time, anv precise work on kidney function itself has been of a most elementary sort. There is however, one personally reassuring feature today we talk about the adrenal and the kidney and when it comes to the adrenal I have no such inhibitions as those mentioned for the kidney Indeed if adrenal workers had kept still when they did not know what they we talking about the history of physiology during the last quarter tury would have been very drab -- a fact that encourages me proceed When we discuss the effects of cortical hormones on water excre ion we are faced immediately with a dilemma, for these hormones may either inhibit or they may enhınce the excretion of water by the kidney I have been interested to note that among my friends only casually interested in this subject, some regard the cortical hormones as diuretic agents some as water retaining agents The same is true of sodium excretion Although the adrenal hormones are best known as agents which cause sodium retention, there are numerous circumstances and conditions in wluch they enhance sodium excretion Dr Pitts and his colleagues(1) have shown that the renal tubule of the adrenalectomized animal is not always deficient in its ability to reabsorb sodium it is only deficient when the plasma sodium level is low At high plasma sodrum levels the renal tubule of the adrenalectomized dog reabsorbs more socluum thin normal from tubular urine • The work seferred to here from our laboratory was conducted primarily lwy Drs TH Burnie W R Boss W I Eversole and C N Osborn It was aided by grants from the US Publc Ilealth Service and Cibi Pharmacentical Products Inc 10 ·Ilarmoncs in Water Metabolism 11 So in the midst of this rather bewildering variability of response to cortical hormones or to their absence, about the only generalization that can be made is that no generalization is possible We are dealing with hormones of adaptation and they do different things under different circumstances We could bring to bear on this problem a great mass of facts and figures from many sources which would take a long time to present * I would prefer to let them come out in the discussion, and in order to get the discussion going limit myself to some broad statements and suggestions as to the pattern of endocrine interrelationships and interactions which impinge upon the kidney and affect water metabolism I will take advantage of the fact that m these sessions we have been invited to indulge in speculatae con siderations and in discussion of work in progress Now one of the clearest and best agreed upon facts with which no might begin is that an adrenalectomized animal cannot escrete a water lord rapidly Why is thus the case? There are a combination of actual or possible reasons A) These are extra renal factors - one being a delayed mtestin absorption of flud(8) Although with large water loads that facto may be of considerable magnitude it is of relatnes little importance to the question here, because it is easy enough to show that if the water is absorbed or of one by-passes the gut in administering it, it still cannot be ercreted at a normal rate B) After adrenalectomy, there my le althongh not in arial's - a reduced renal plasma flow and glomenılır filtration rate (CFA) (4,9) The filtration rate in certain circumstances may be ses erely depressed - in which case presum ahh it could contribute mirhedh a delay in water excretion It is easy enough to muntain the Crn in adrenalectomyzed animals as many workers have done but water still cannot be excreted normally C) There is an enhanced tubular reabsorption of water(1,4,9, 10,11,12) I believe that has been seen by all workers who have koled specifically for it and it is probably the major cause of the inzbults to excrete utter after adren lectors Thuis abnormality en difficult to repur Con ersek it is easy to show that the rate of water druiresis in normil ammals and men can be greath • The sul wet matter of this discussion is Eren estraneh covered in terrat resa W34587) The teletences citrd here therefore will br brgely to these 13 ani to very recent parts ·12 Renal Function exceeded when water loaded animals are given extra imounts of cortical hormone(4) This effect occurs without essential elevation in the filtration rate and hence is due to an inhibited tubular reabsorption of water While these effects of cortical hormones on water excretion are presumably not entirely independent of sodium excretion they cannot be in many instances correlated with sodium excrehon in any way to suggest casual relationships (4 13 14 15) What then is the cruse of this enhanced tubular rcabsorption of unter after adrenalectomy-n defect of such magnitude that an animal may be thrown into a state of lethal water intoxication at relatively small water londs? For various reasons we inve been favorably disposed to the idea that it is due to a hypersensitivity to or an accumulation of antidiuretic humorni agents perhaps of posterior pituutary origin This brings us to the concept of a functional antagonism between the hormones of the posterior pituitary and adrenal corter -- a con cept that was first elaborated by Silvette Britton and Corey (16) some ten years ago This hypothesis in its simplest form states that the cortical hormones enhance water excretion and inlubit sodum excretion and that antidiuretic hormone of the posterior pituitary (ADH) has precisely opposite actions There are many of us who would now_accept this is a general rule with certain definite exceptions It might be mentioned that both cortical and posterior pituitary hormones enhance the excretion of potassium probably acting in a supplementary rather than an antagonistic fashion (17) I think there is little doubt that a hypersensitivity to ADHI can be demonstrated in the adrenalectomized animal(18 19) but such animals are probably hypersensitive to any antidiuretic influence Various workers including our group have found evidence of the accumulation of some antiduretic substance in the body fluds of adrenalectomized animals(20 1921) Pitts und lus co workers(1) huse elaborated the ider that the increased sodium excretion of adrenal insufficiency is due to in action of ADil uinuntigonized by cortic al hormones Therefore it may be suggested that the un antagonized action of ADII or some similar antidiuretic agent is at least one cause of the frilure to excrete water and to retain sodium in adrenal insufficiency If thus be acceptable at least as 7 s orling hypothesis the ques tion arises 1s to the identity of the antidiuretic subst unce thrt accumulates or is hyperactive after adrenilectomy ·Hormones in Watır Metabolism 13 In the first place none of us have conclusive proof that the substance which accumulates in the body fluids of adrenalectomized animals or Addisonian patents is actually ADH It could be ADH, it could be a substance which mimics ADII, it could be a substance which causes the release of ADH Nevertheless, there is something there which disappears from the blood of hypophysectomized rats and which has the attributes of ADHI(19) I will not elaborate upon these points because they will probably be discussed by Dr ian Dyhe There are reasons why it might be expected that ADHI would accumulate in the absence of the adrenals It has been shown that various tissues, particularly hepatic tissue inactivate pitressin (22, 23) Bimie's worl(21) in our laboratory has indicated that this hepatic agent is an enzyme (probably not a specific one) and that its ability to inactuate pitressin is reduced after adrenalectomy can Dyke May I ask whether the differences shown in your hepatic inactivation experiments with pstressin have been examined for their statistical significance? Gaunt Yes Dr Bimie did The significance of that kind of observation will not be fully apparent until it is related quantita tively to wliat goes on in the whole animal Nevertheless it obrtously provides a basis for the suggestion that ADII may accumulate after adrenalectomy because it is not destroyed at a normal rate On theoretical grounds one would not expect hypersecretion of ADII after adrenalectomy I think work of this sort may have widespread usefulness m related studies Dr C H Lloyd at Syracuse has some evidence that the liver of cirrhotic patients cannot inactuate pitressin norm illy either in tro or in vitro That obviously would correlate with the work of Rulli Hoagland and their associates ( 25 26) concerning the genesis of the abnormal fud metabolism in laver disease Dr E Moller Christensen of Copenhagen told me in a recent Ictter that he has shown that in water intoxication in rabbits the inner loses its abihty to inactivate pitressin Such an observation might be related to the fact that as ansials go into water mtonic? ton there is at a certain point a sharp break in the diuresis Ad mitted the posterior pituitani should not be actise at all under these conditions fiowever the conditions are extreme ones charac terized by profound neurological and metabolic abnormalities I sul rules may not be followed Input 1 shell data ·Renal l'unction Mly colleague Dr ) H Birnie who is working in Dr Hans Heller s laboratory in England this ycar tells me they have found that the livers of protein deficient animals inactivate pitressin less effectively than normal Obviously agun this may be associated with the water retention seen in such mimals As far as what happens in adrenal insufficiency is concerned I want to emphasize the point that the general interpretation which has been offered to cxplun the aberrations of sodium and water metabolism in adrenal insufficiency is not dependent upon an accumulation of ADH It would buttress the case if this were true but if there is some ADH present in effective amounts after adrena lectomy a hypersensitivity to it might explain the tendency to cxcrete water slowly and to lose sodium and chloride lou ma recall in this connection that I inter Ingrım and Cross(27) showca that the fall of plasma sodum in the adrenalectomized cnt 125 dependent upon 7 functional posterior pitutury The most common problem in the clinical use of DCA or cortisone is a retention of water that may lend to frank cdemi And this brings us hich to our original st itement that the cortical steroids may be either diuretic or water retuning agents We have tried to reduce to digrammatic terms the work and thinking( 4 28 29 30 31) of many people relating to this apparent paradox (See Figure 1) As indicated we think that the major diuretic action of steroids such as DCA is brought about by their inhibition of the tubular reabsorption of water Vost people would grec that the major water retuning action of DCA results from its stimulation of sali reabsorption Under some conditions as with large water loud the diuretic fnctors seem to be dominant Under other conditios which are yet to be precisely defined the water retaining factors ure dominant If how ever one gives the hormone for some time the resulting retention of salt may lead to thirst polydipsia and polyuria In the end 1 combination of factors would contribute both to ? large water exchange and an expanded extracellular flund volume ic to retention of fluid Such a combination of events is 2 probable explanation of the DCA induced dubetes insipidus like syndrome observed by the group at Columbia(29) To sec the operation of this syndrome in eraggerated form it is only necessary to combine DCA administration with a high salt intake ·We he obtuned suggestive but not conclusive evidence that 1 huge crcess of cortical hormone increases the antidiuretic iclish of blood serum(19) Si ahen and Creen(33) found that Di Chical ments were all designed for other purposes and month the cycrction of an antıdaturetic substance in urine Thiest ten needed particularl with the use of low des In their plicability is clear to the question at issue I ere Rall. Are you speaking of DC I specifcalls or of therlar Hormones in Water Metabolisin DCA Overdosage 15 Enhanced tubular reabsorpt on of salt Thrst Inh 6 ted tubular reabsorpt on of water Polyd ps a Increased Extracellular Volume Edema FICURE 1 Polyuria Finally I would like to explore a little the nature of thus sug gested antagonistic interrelationship between the adrenal cortex and posterior pituitary Certainly the hormones of these two glands do huse some antagonistic properties The queshon ts are those hormones secreted and do they act in a closely integrated check ind balance system or does each follow its own more or less inde pendent way with the antagonism being incidental? D: Shannon raised that question several years ago (32) If they operate in a check and balance manner it might be t. pected that the admnistration of a slight excess of one of thes hormones would call forth a compensatory hypersetetion of thu other ·Renal Function 16 Gaunt Shahen and Green (33) used DCA We used both extract and DCA and found increased antidiuretic activity of serum but at the time we were looking for sometłung else and we do not have enough data to establish the significance of the results More precise quantitative information has been obtained by the opposite approach to the problem in work conducted by Dr Nagareda (34) in our laboratory She tried to determine whether an excess of pıtressin would cause a compensatory activation of the adrenal cortex She had the advantage of a sensitive method, namely, the Sayers ascorbic acid depletion test for adrenal stimulation She found, in brief, that large doses of pitressin (100 or 400 mU) would cause marked adrenal stimulation as judged by the ascorbic acid depletion test This may hue been due to a nonspecific stress like reaction In any case small but physiologically effective doses (5 mV) of pitressin produced no evidence of adrenal stimulation This leaves is with no convincing reason for saying that slight variations in the circulating levels of either ADH or cortical hormone per sc causes compensatory hypersecretion of the other There is however, another approach to the problem It is in accord with all current theory that excesses of water inhibit the secretion of ADH and thus allow such excess water to be excreted rapidly Rapid water excrction cannot occur, hou ever, in the ab sence of some effective amount of cortical hormone When excess water is present and ADHI secretion is more or less Inalted, is an increased amount of cortical hormone relensed to help as it were, push the ercess water out? Dr Nagared, has investigated thus by finding what water load would cause a measurable depletion of adrenal ascorbic acid and therefore, presumably, adrenal stimula tion She found that whereas a small water load (5 ml in ? 220 gm rat) would not cause adrenal stimulation a lırger amount (10 ml in 1 220 gm rat) would do so Thirty milliliters of water, given in three doses, was still more effective We concluded from this that whereas small water loads presumably are handled simply by reducing the output of ADH, heavier lords elicit adrenal stimulation which contributes to the duretic process When excess water is present, it is generally behei cd that the stimulus which inlbits the posterior pituitary is a fall in plasma osmotic pressure What is the stimulus to which the adrenal cortex responds when it is stimulated to activity by a water lond? (What ever it is, it might be expected to be one that acts on the anterior pituitary and thus indirectly on the adrenal corter Whether that ·Hormones in Waler Vetabolism 17 is actually the case is being currently investigated ) Me do not know what the stimulus is but are designing further experiments on the basis of a hint emerging from this data that the adrenal might be responding in a converse manner to the same stimulus that is acting on the posterior pituitary All of these water doses would be expected to reduce the plasma sodium level and we have verified that they do so If one gave normal salıne instead of water there was no fall in adrenal ascorbic acid even when the doses of saline were huge (30 ml) Can it be then that the basis for such integration as exists betueen the adrenal cortex and postenor prłustary lies in the fact that their hormones are turned on" or "turned off m a conuerse manner by the same stimulus namely the osmotic pressure of plasma? Ile have not answered the question there are other alternatives - but it will be an interesting one to investigate further Or can it be hit one reason 7 load of normal salme is excreted less rapidl than lord of pure water is that the water may evoke an adrenal par spation of a quintitatac or qualitatne sort which sahne docs +? I raise that question also without attempting an answer but too is one susceptible to direct erperimental inqurs by mcthods now available In summary there are several Inown means by which the hor mones of the adrenal cortex can affect water metabolism a) by indusbiting the tubular rerbsorption of water b) by stimulating or inhibiting the tubular reabsorption of sodium and other electro lytes c) by maintaining the filtration rate d) by muntaining an enzyme system in hepatic and perlaps other tissues wuch machiatos intidurretic hormone e) by maintaining 7 normal rate of flud absorption from the gut ind I) by presenting an accumul tion of antidrore tic sulistance (ADU?) in the blood The sum toti of these effects is generally to produce an enhanced water druresis In certain conditions however corticni hormone induced salt reten tron mat lend to 1 fland retention and edem or indirectly to a dorbetes musipdns lite syndrome The actions of cortical hormones on water excretion and to a certain extent those on electrolyte cxcretion are antagonized h the hormones of the posterior pittutin DISCUSSIO! I runont Smuth Dr Caunt you said 1 little curler til under stool correcth that an animal en en water load with adrena!

·16 Renal function Gaunt Shalien and Green(33) used DCA We used both extract and DCA and found increased antidurctic activity of serum but at the time we were looking for something else and we do not have enough data to establish the significance of the results More precise quantitative information has been obtained by the opposite approach to the problem in work conducted by Dr Nagued (31) in our lalyoratory She tried to determine whether an excess of pıtressin would cause a compensatory activation of the adrenal cortex She had the advantage of a sensitive method, namely, the Sayers ascorbic acid depletion test for adrenal stimulation She found, in brief, that furge doses of pitressin (100 or 400 mv ) would cause marked adrenal stimulation as zudged by the ascorbic acid depletion test Tlus may be heen due to a nonspecific stress like reaction in any case small but physiologically effective doses (5 ml) of pitressin produced no evidence of adrenal stimulation This leaves us with no convincing reason for saying that slight variations in the circulating levels of either ADII or cortical hormone per sc causes compensatory lypersecretion of the other There is however, another approach to the problem It is in accord with all current theory that excesses of water inhibit the secretion of ADH and thus allow such excess water to be excreted rapidly Rapid water excretion cannot occur, however, in the ab sence of some effective amount of cortical hormone When cxcess water is present and ADHI secretion is more or less Inited, is an increased amount of cortical hormone released to help, as it were, push the excess water out? Dr Nagareda hus investigated this by finding what water Jond would cause a measurable depletion of adrenal ascorbic acid and therefore, presumably, adrenal stimula tion She found that whereas a small water load (5 mi in a 220 gm rat) would n : - - amount (10 ml in a 220 gm water, given in three doses, * from this that whercas small water loads presumably are handled simply lảy reducing the output of ADHI, heavier loads elicit adrenal stimulation which contributes to the durctic process When excess water is present, it is generally believed that the stimulus which inhibits the posterior pitutary is a fall in plasma osmatic pressure What is the stimulus to which the adrenal corter responds when it is stimulated to actuity by a water loud? (What ever it is, it might be expected to be one that acts on the anterior pituitar, and thus indirectly on the adrenal cortex Whether thit ·Hormoncs in Water Ictabolism 17 is actually the case is being currently mestigated ) We do not know what the stimulus is but are designing further experiments on the basis of a hunt emerging from this data that the adrenal might be responding in a converse manner to the same stimulus that is acting on the posterior pituitary All of these water doses would be expected to reduce the plasma sodium level and we have senfied that they do so If one gase norinal saline instead of water there was no fall in adrenal ascorbic acid eten when the doses of salıne were huge (30 ml) Can at he then that the basis for such integration as exists between the adrenal corter and posterior pituitary hes in the fact that their hormones are turned on" or "turned off" in a converse manner by the same stimulus namely the osmotic pressure of plasma? We have not answered the question - there are other altematies -- but it will be an interesting one to mestigate further Or can it be that one reason a load of normal saline is etcreted less rapidly than a load of pure water is that the water may evoke an adrenal par ticipation of a quantitative or qualitative sort which sahne does not I raise that question also without attempting an answer but it, too is one susceptible to direct expenmental inquiry by methods nos available In summary there ire ses eral known means by which the hor nones of the adrenal corter can affect water metabolsm a) bir nhibiting the tubular reabsorption of water b) by stimulating or inhibiting the tubular reabsorption of sodium and other electro lytes c) bs maintaining the filtration rate d) by maintaining an enzyme system in hepatic and perhaps other tissues which inactuates antidiuretic hormone e) by maintaining a nonnal rate of fund absorption from the gut and f) by preventing an accumula tion of antidiuretic substance (ADH?) in the blood The sum total of these effects is generally to produce an enhanced water druresis In certain conditions hor eser cortical hormone induced salt reten ton mai lead to a fwd retention and edema or indirectly to a diabetes insipidus like syndrome The actions of cortical hormones on water excretion and to a certain extent those on electrolyte excretion are antagonized by the hormones of the posterior pituutar DISCUSSION Freriont Smith Dr Gaunt you said a little carlier if I under stood correctly that an animal gren a water load with adrenal ·16 Renal I'tinction Gaunt. Shahen and Green(33) used DCA We used both extract and DCA and found increased antidiuretic activity of serlim but at the time we were looking for something else and we do not have enough data to establish the significance of the results Moie precise quantitative information has been obtained by the opposite approach to the problem in work conducted by Dr Nagareda (34) in our laboratory She tried to determine whether an excess of pitressin would cause a compensatory activation of the adrenal cortex She had the advantage of a sensitive method, namely, the Sayers ascorbic acid depletion test for adrenal stimulation She found, in brief, that large doses of pitressin (100 or 400 mU) would cause marked adienal stimulation as judged by the ascorbic acid depletion test This may have been due to a nonspecific stress like reaction In any case, small but physiologically effective doses (5 mU ) of pitressin produced no evidence of adrenal stimulation This leaves us with no convincing reason for saying that slight variations in the circulating levels of either ADH or cortical hormone per se causes compensatory hypersecretion of the other There is, however, another approach to the problem It is in accord with all current theory that excesses of water inhibit the secretion of ADH and thus allow such excess water to be excreted rapidly Rapid water excretion cannot occur, however, in the ab sence of some effective amount of cortical hormone When excess water is present and ADH secretion is more or less halted, is an increased amount of cortical hormone released to help, as it were, push the excess water out? Dr Nagareda has investigated this by finding what water load would cause a measurable depletion of adrenal ascorbic acid and therefore, presumably, adrenal stimulation She found that whereas a small water load (5 ml in a 220 gm rat) would not cause adrenal stimulation a larger amount (10 ml in a 220 gm rat) would do so Thirty milliliters of water, given m three doses, was still more effective We concluded from this that whereas small water loads presumably are handled simply by reducing the output of ADH, heavier loads elicit adrenal stimulation which contributes to the diuretic process When excess water is present, it is generally believed that the stimulus which inhibits the posterior pituitary is a fall in plasma osmotic pressure What is the stimulus to which the adrenal cortex responds when it is stimulated to activity by a water lond? (Whatever it is, it might be expected to be one that acts on the anterior pituitary and thus indirectly on the adrenal cortex Whether that ·Hormones en later Mctabolism 19 ments one must always ei aluate whether in attempting to obt un a definite response one has gone beyond the range of physiological dosage into what might more properly be termed a toxic dose The fourth point is that in considering the relationshups of adrenal steroids to antidmresis it is well to recull that in min deso y corts costerone acetate n quantities sufficient to establish normal elec trols te balance does not correct the abnormal water tolerince test (Robinson kepler Power) wherers Il 17 oxy steroids do in a high proportion of patients Gaunt 1 dont the to see you rule adrenal cortical extinct out of the picture TI orn I do not feel that adrenrl cortical extract studies should be crcluded from consideration I do feel however that it is extremely difficult to interpret 1 negative or n positive findmg when an adrenal citrict of unknown composition is employed Gaunt As yet we have not been able to stimulate diuresis markedly with cortisone in normal animals lle cun with whole extract There may be something qualitatively different or it may be due to a slow absorption of cortisone in our short term experi ments Cortisone will work in adrenalectomized rits Thorn In man ke have been able to get a muhed effect In patients with Addisons disense we have definitely shown that cortisone inhibits the increased antidiuretic activity present in these patients A demonstrable effect may be observed with 100 mg o cortisone given for a few days However in these patents it is necessary to treat them for 1 month or two with 25 mg daily to obtain maximum response The difference between the effects of desorycorticosterone-hic steroids and cortisone lhe steroids empla sizes again the difficulty of interpreting positise or negatise results with extracts of unknown composition Gaunt That is true II hite Did I understand correctly that the ceretic response to 110 mi dose of water is greater with adrenal cortical hormone administration than without? Or is it merely that these hormones protect against the water intoxication produced by excessive do es of water? Gaunt With cortical extracts you can get it with small water loads W7ite In acute erperiments?

·18 Renal Function cortical hormone would produce a greater diuresis thm with the water alone Gaunt Much greater Fremont-Smith What, roughly speaking, is the maxımum amount that you can get with water alone? A 100 percent incre se? Gaunt It is closer to 50 or 75 percent Ticmont-Smith That is without an increase in glomerular sate? Gaunt in water intoncation it has not been metsured There is no rise when diuresis is increased following smaller water louds(9) It is almost impossible to produce water intoxication when cortical extract is given with the water We have given up to twenty of our standard doses at half-hour intervals at the end of much time the investigator is worn out and the rat is still going strong Iremont-Smith Was there any major difference in the compose tion of that urine than if they were getting straight wates? Gaunt As far as sodium chloride is concerned it was much the same but the experiments were complicated by the fact that the animal going into water intoxication develops severe diarrhez le had no means of separating completely the sotestinal and renal electrolytes The total overal loss was about the same in treated and control animals Salt itself, of course, will prevent water intoxta catron T}le cortical hormones do not give protection as a result of salt conservation Thorn There are foun general comments which might be appropriate at this time First, it is important to bear in mind the fact that the zona glomerulosa of the rat has been shown to persist after hypophysectomy and that in rats, therefore this arcs of the adrenas corter may not be under control of ACTH It is assumed generally that the zona glomerulosa secretes the desory corticosterone like steroids In contrast, in minst appears that all known frictions of the adresal cortex are stimulated by log beef, and sheep ACTII preparations The second pomt which must be bome in mind in attempting to interpret experiments carried out with whole extract is that sh most instances the exact content of steroids in such extracts is not knoun The third point to consider is the general principle that where is one obtains à certain effect with small doses of steroid hormones, there may be a complete reversal of this effect with or large quanhties of hormones Thercfore, in evaluating animal exper- ·Hormones in Water Vetabolism 21 Shorr I wonder whether that is necessarily the case with respect to adrenal cortical extract? For example to maintain rats we have found it necessary to give as much as 15 ml a day of adrenal cortical extract which would seem a disproportionately large dose on any human weight basis In other words it is very hard to transfer to the rat the dosage requirements that we would assume for other species Thorn With adrenal extracts one must consider the rapid absorp tion as a factor in diminishing the efhcacy of a given dosc It has been shown that rapid small injechons of extract are the most efficient means of administering hormone of this type The main tenance dose for patients with Addisons disease under normal circumstances is small shereas very large quantities of aqueous extract are necessary to reverse the water test or the abnormal electroencephalograpluc changes Ralli Dr Patts mentioned in his opening remarks that the adrenal hormones are operating on a background of other cellular activities I believe this to be an important consideration The nutritional situation within the cell may affect the reaction to 1 given dose of a given hormone There are substances particularly some of the fractions of the vitamin B complex which are involved in many of the coenzyme systems within the cells It may well be that the state of these enzyme systems will determine the activity of a hormone We have observed that the amount of pintothenic acid in the diet will affect the response of the wlute blood cells in intact and adrenalectomized rats to stress and to ACTH(35) Thorn May I ash Dr Pitts if Dr Grunt quoted him correctly when he said that he beheves that the serum sodium level is the governing factor relating to sodum excretion in the absence of the adrenal? Gaunt What I meant to say was that at low plasma levels the adrenalectomized animal reabsorbed less sodam than normal and that at high plasmı levels it reabsorbed more than normal Petts That is the concept we had Burnett However is I will bring out tomorrow the abihty m umans with Addison s disease to excrete in excess load of hyper mic saline is not precisely related to the serum sodium level bulvr reabsorption of sodium to be sure may be us demonstrated Dr Pitts increased only at a high serum sodium level(1) but ·20 Renal Tunction Gaunt les White Administration of the cortici! hormone simultaneofil with the water causes the water to be crcreted faster? Gaunt Yes With small water loads one sometimes sees it uitli DCA and sometimes not we have never failed to see it with extract Whute Are lurge doses of DCA required to incrc ise rate of * iter excretion? Gaunt That is a relative matter ig in Winte Docs it take a milligram or less thin a milligram? Gaunt The dosages we hrve given me been generally more than that We dont know the minimal effechac dose White More than a milligram is a rather large dose for a rat Gaunt I used to think so In terms of shit the adrenal corter itself can do I dont know what a big dose is any more White Compared to what is necessary? Gaunt You cannot go by that Certunly what it takes just to maintain lufe say that slight residurl amouint wluch the adrenal of the hypophysectomized animal can put out beurs no quantitat tive relation to what the normal adrenal dous anyway I think the work with whole extract is more meaningful Here 2 ml or so of the Upjohn product will give a good response and that is not an erces sive amount by any criterion Thorn It is important to consider the dosage employed in these experiments in an adrenalectomized animal ench increment of adrenal hormone administered may be considered to be an actin increase in effective hormonal dose ulerers cu adren il prepr1 trons are administered to animals with an intact adrenil it is well known that an inlubation of pituitary ACTHI production by the administered hormone will result in subsequent ?rophy of the adrenal corter Under these circnmstances the net cfect of an guen quantity of adrenal hormone administered will have to be calculated as the difference betucen the anal secretory activity of the grand and the total dosage guven It is obu sous that nt ? cert un dosage level the administered hormone will do no more than replice endogenous hormone This is most likely to be a scrime frctor in considering the results of the experiments when the adresy is already at a high level of actiuity before substitution of hormone ·Hormones in IVater Metabolism 23 Luetscher Cortisone or ACTII In Figure 2, let us confine our attention to the bottom two cures, representing unine volume and sodium and to the weight curve, which is the continuous line at the top The patient, a kve year old girl with nephrosis, was being treated with cortisone During the initial stage, there was a sum in weight which was apparently related to a diminution both in trine flow and in the excretion of sodium In contrast to that, Figure 3 shous the course of a patient whose Airine sodum was virtually zero right from the start and in whom the action of cortisone apparently was not mediated through the retention of sodium At the same time, houever, the urine volume fell and a gam in seight occurred The serum sodium concentration fell during this stage Then after the fifth day there was a tendency but WEPOSE CORY SONE BODY WEIGHT AK PROTEINURIA 12 DOC ASSAY Y 847 127 100 PLASMA TOTAL PROTEINS Oce ALRA. URINE VOLUME SODIUM ESCALES tes Or t. .... er.I...NAIM FIGURF 3 Prolonged crical remission folowing administration of cortisose Note temporary rise in proteinuria aud weight during therapy with some im provement after the fourth day of therapy followed after end of treatment by e) mination of Osez 10 kg of edema reduction of proteisuria to less than 05 m per day, and an increase in serum protein and albumst concentration approaching por dere's Assay of sodium reddising corticoids fell to normal levels after treat ment Chocal improvement has bres masztuned for four months ·29 Renal Function the ability to excrete a lord of hypertonic salıne was impaired in our human experiments at both low and high serum sodium les cls Luctscher Mav I ask Dr Gaunt one question about interpreta tion? We have all observed patients under treatment with cortisone OF ACTH whose body weight his first increased for a few days and then subsequently has decreased agın Most of us have inter preted this diphasic response as Dr Grunt's scheme indicated that the initial retention of water was related to the retention of sodum and that subsequent loss of water might be due to some opposite phase of action However, in the nephrotics we have run into a situation where the patients show a similır retention of water and a gun of weight during the first few days of treatment without an change in the excretion of sodium Thorn This is cortisone, not ACTH? 24 SNEROSIS DOC ASSAYY CORTISONE DO ANONY GMC 177 207 PROTEINURIA 19Y BODY WEIGHT PLASMA TOTAL PROTEINS O GM Mock AL MUMIN • URINE VOLUME - ANO SODIUM - . 10 . COS NOPHILES DAYS 2 3 4 5 6 711 +3.9.1 34 9.750# FIGURE 2 Temporary remission following administrat on of cortisone Note the increased proteinuna d minished sodium ercretson and rise in weight during administration followed after therapy by cessation of proteinuria diuress and return of serum protein and albumin concentration to virtually normal levels Edema recurred two weeks Liter • Figures 2 and 3 are reprinted from a piper by J A Luetscher Jr and B Deming Treatment of nephrosis with cortisone I Clin Investigation (in press) ·Hormoncs m Water Metabolism 23 Luetscher Cortisone or ACTH In Figure 2, let us confine our attention to the bottom tuo curies representing urine volume and sodium and to the weight cure uhich is the continuous line at the top The patient a fileser old girl with nephrosis was being treated with cortisone During the initial stage there was a gun in weight włuch was apparently related to a dimmutton both in unne flow and in the excretion of sodium In contrast to that Figure 3 shows the conrse of a patient whose urine sodium wis virtually zero night from the start and in whom the action of cortisone apparently was not mediated through the retention of sodium At the same time hou ever the une volume fell and a gain in neight occurred The serum sodium concentration fell during this stage Then after the fifth day there was a tendency DNTNEM VOSS CDA SONE BOOY WEIGHT G PROTEINURIA DOC ASSAY Y 843 27 nyo PLASMA to PROTESSO 100 MA A. URINE VOLUWE ++ Jorg SODIUM EOSINOPALES Ders FICLRE 3 along din cal rem ss on following adm ustration of cuttuone R . a ·Renal Fruction for the body weight to decrease again, as the urine volume 1 creased, again with very small chinges in sodum excretion During this stage, the serum sodium concentration increased. In answer to Dr Thorn's question about ACTH, Figure 4 shows the course of a patient treated first with cortisone, and later with ACTH Both with cortisone and ACTH, we see a tendency for the Weight to increase during the inual stage of treatment and to fill during the later stage of treatment with the expected changes in urine volume Yet the urine sodium at all these stages us insignificant unt) the onset of the final durresis at the end of ACTH administra tron The fall in sertis sodum concentration during the muta water retention, and the rise during the phase of diurests are evident both with cortisone and wsth ACTH These findings suggest that cortisone and ACTH may have some form of antıduretic activity not mediated through the retention of sodium alone Thor Would you say that there is a rough clinical parallelts between the degree of edenia and the effectu eness of a guen GORYESONE 100 ALHUNIN 107 mu Hoga JD 65 40- WEIGHT Win 110 303 100 CREATININE CLEARANCE - L 2014 do VH 70% URINE PADEN VOLUNE SODIUM ft sag 200 - sed . DO LaL 6 MO SEAUW SODIUN PROTEIN . ALAUNIN • 44t 1201 341 QAYS 12:438|1234961. goal: 3454133412 343 2:438763123456 FIGURE 4 Some messurements on a pilient treia successssely with cortisone albuma and ACTI ·Hormones in 11 ater Betabolism 25 quantity of these agents? The greater the edem: the more likely one is to obtain good diuresis whereas the closer the patients weight approaches normal the more difficult it is to induce a good dunesis with either cortisone or ACTH? Luetscher Thit seems to be true Thorn It is not alu zys? Luetscher I was speaking pumarily of the changes which were seen during the early days of treatment and shich seemed to indı cate some water ictuning icinuity of cortisone and ACTII It is interesting to observe at the same time that in the patient described the creatinine was incre used during this phase when the water was being retuned and it seems very difficult to interpret except 1s ? primary tubular action The changes which occur during the final duesis are complex involving filtrition rite tubular function and extra renal factors Thorn Smce patients on a very low sodrum intake may retun only 5 or 6 mEq of sodium 1 day and show no additional excretion of potassium before druresis occurs it is difficult to assign in im portant role to sodrum retention and potassium loss as factors con ditioning the response to ACTH and cortisone The diuresis which occurs on cortisone or ACTH with evidence of drenal coitical actition would appear to be on a different basis than a diuresis initiated after ACTII or cortisone has been discontinued when temporary adrenal cortical insufficienci mu ber facilitating factor in the sodium loss In 1938 we reported that 11 17 oxy steroids induced increased sodium excretion when administered to normal dogs and to adrenal ectomized dogs maintained on desoxycorticosterone (36) When adequate quantities of cortisone first became a uzlable for clinical trial se obsered that 100 mg of cortisone duly induced sodum chloride retention These patients were not recerung desory corti costerone acetite The pardor apperred to be resolved when patients with Addisons dise ise were gnien desori corticosterone acetate in 5 mg doses per day followed by the addition of 100 mg of cortisone acetate Under these circumstances we did not obserie an additae sodium retaining effect but rather an increase in sodım excretion abose the base line excietion observed on desorycorti costerone Our impression at present is that all three varieties of adrenal steroids il desory 11 orv and 11 17 hvdroxy steroids pre silt returning compounds the last being relatively inactic in ·26 Renal Function this respect There does appear to be the possibility that under certain conditions 11 17 hydroxysteroid compounds compete in action with II desory compounds under wluch circumstances less sodium retention will occur in our experience it is not possible to maintain patients with Addisons disease on 25 mg of cortisone daily in a normal salt balance without the addition of large quantı ties of sodium chloride or a small muntenance dose of desoxy corti costerone acetate whereas this quantity of cortisone will usually result in restoration of the electroencephalogram toward normal and reversal of the abnormal water test It is true that adequate salt retention may be obtained in patients with Addison s disease given 100 mg of cortisone daily but this dose is excessive and results in hypercorticism The majority of our patients have done extremely well over long periods of time on a dose of cortisone of 125 to 25 mg duly with 1 to 2 mg of desoxycorticosterone duly During periods of stress much larger quantities of cortisone are needed ie 100 mg duly In patients whom we have studied with complete bilateral adremlectomy for hypertension it is noteworthy that a normal sodium chloride balance cannot be maintuned with 25 to 50 mg of cortisone daily and that ultimately supplementury sodun chloride or sınall doses of desoxycorticosterone ure required These studies ruse three interesting points First at would appear that 11 17 hydroxysteroids are not the only hormonal secretions of the adrenal cortex since they are incapable of inducing sufficient sodium retention in most patients with Addison s disease and in bilaterally adrenalectomized hypertensive subjects when given in a quantity adequate to reverse the other hnown distu, bances This fact suggests that other sub stances secreted by the adrenal with greater salt retaining capacity are essential for normal function These could be either 11 desory or 11 ory compounds such 25 compounds A and B Second in the treatment of patients with adrenal insufficiency there ippears to be an optimum ratio between the quantities of cortisone and desoxycorticosterone which are employed requiring approximately 5 to 10 mg of cortisone to 1 mg of desory corticosterone Third in relation to Dr Grunts experiments it may interest you to know that in comparison to cortisone the adrenal extracts such as Eschatı of Parke Davis and Co and Upjohns Adrenal Cortex Fxtract are salt retaining mdicating the presence of substances other than compounds E or F ·Hormoncs in Water Metabolisni 27 Burnett As I understand it your finding is that nerge doses of DCA plus small doses of cortisone cruse salt loss What about the average dose of DCA plus large doses of cortisone? He found no such salt loss with 100 to 200 mg of cortisone a day in Addi sonans on muntenance quifitics of DCA Thorn I believe that the sodnim chlonde response of a patient with Addison s disease will be modified significantly by the dosages of desory corticosterone and cortisone employed if the dosage of desory corticosterone is small and the closige of cortisone 1 cry large one may obtain in overall change in the degree of salt retention Another important point is that with cortisone acetate yien intra ruscularly all these effects observed in man or anımıls are slow fects If you want an immediate renal effect cortisone is best en intravenously Dr Gaunts point regarding the aqueous er tracts is ? very good one because these are very rapidly absorbed ACTH effect is observed within three to four hours after administra tron whereas an intramuscular injection of cortisone acetate does not att un maximum action for three or four days on a constant dose level Recently we have shown that very mpid absorption and action as far as the eosinopenja is concerned may be obtuned with corti sone administered orally Gilman How do you give adrenocortical steroids intravenously? Thorn We have guen cortisone intravenously slowly as a sus pension in sahne As with DCA we have used the glucoside in travenously Luetscher When you speak of additive or competitive effects against the salt retaining hormone I wonder whether we ought to try to distinguish betuten the additive and competitive effects of cortisone versus desorycorticosterone as the crystalline compound and versus the normal secretion of the adrenal Have you seen any evidence of sodium loss during cortisone treatment in normal patients aside from the secondary release of sodium and water retained during the mtnl days of treatment? Thorn I would think Dr Luietscher is right Of course in all of pur normal studies we have gone to high dosage levels to be sure that He are grung an excess However I would say that in the majority of experiments 100 mg of cortisone almost antarably cilises sorne salt retention initially in 1 normal induu dnal ·30 Renal Fuchon ing severe hypoglycemic manifestations, b) abnormal election cephrlogram(36,38) and electrocardiogram, and, c) excessive antidiuretic activity with a positive Robinson-Kepler-Power water test Cortisone, in adequate doses, will in almost all instances correct these metabolic defects The effect of cortisone may be limited in the presence of liypothyroidism and obviously cortisone therapy in muntenance doses will not restore insulin sensitivity since in a large measure this is dependent upon the intact adrenal medulli as well as upon intact liver glycogen stores ilhute That is what I really wanted to ask How does the adlı tion of cortisone to DCA regimen change the salt balance? If the patient was in salt balance on DCA alone does adding 5 mg of cortisone per gram of DCA change that salt balance? Thorn Defimtely, and we have seen prtients admitted in neurcrisis when they weie gnen 125 mg of cortisone, although on the sume dose of DCA which had been adequnte in the preceding yeu Burnett If the cortisone is increased is the process rciersed? Thorn If you go to the larger dose With 100 mg of cortisone, I would assume that such a patient mght remun in adequate salt balance but would show signs of hypercorticism We liave no patients carried for a long period Gilman Do you have any idea what metabolic defect you are correcting with cortisone when you are changing the abnormil electroencephalogram to normal? Is it related to water metabolism or carbohydrate metabolism? Thorn We have no idea as to the mechanism of cortisone in corecting the abnormal electroencephalogram Lan Dyke What are the electroencephalographic changes Dr Thorn? Thorn We are using the changes in the electroencephalogram as evidence for a metabolic disturbance in brain tissue The Addi sonian presents a pattern which is duplicated in our experience onlv in certain lypothyroid prtients This is characterized by a) the presence of oscillations which are slower than the normal alpha rhythm, have a predilection for the frontal area of the cerehral cortex, and are relatnely refractory to the usunl effect of opening the eves b) an unusual sensituusty of the electroencephalogram to voluntary hyperventilation, and s) a reduction in meidence of low-

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·32 Renal Function Gaut I dont know van Dijke It would seem that the continued liberation of inte duretic principle despite a fill in the osmotically actre constituents of plasm? imples ? derangement of the neurohypophysıl system Gaunt It would seem to me that the point we mentioned rould not explain all that occurs I dont know Lan Dujhe Certuinly important information concerning the source of the antidiuretic substance of adrenalectomized animals could be obtuncd niter removal of the adrenals from animals with diabetes insipidus Gaunt le borrowed a few diabetes insipidus animals from Di Pitts while he was in Syracuse to start a collaboratre project on that Dr Birmie und Dr Capps did this work and what little data they ohtumed indicited that the scrum of dubetes insipidus dogs contuned less antıduretic activity than normal Shorr Would you refresh me Dr Grunt about the specificity of the inactivation mechanism as far as general tissues in the body tre concerned? Is it confined to the liver? Gaunt No It is also present in whole blood and skeletal muscle for instance It may well be present in all tissues Shorr in the kidney? Gaunt les but the laser is most cffectue Thorn We have carried on one experiment which might be of interest in relation to the possible site of action of the antagonism between cortisone and posterior pituitary antidiuretic activity in 1 patient with dnbetes insipidus mu en pıtressin cortisone failed to inlubit the antidiuretic actity of the latter Although on jously one should not attach significince to a single experiment it does suggest the fact that the most likely site of action of cortisone in inhibiting antıdiuretic activity is at the hypothalamic or posterior pituutan, levei Ralli lehre an unusual case 1 patient with unbetes insipidus duc to 1 chromophobic tumor of the pituitary - 1ho after eighteen months of observation developed adrenal cortical insufficiency and Kent into Addisom in crisis Pror to the des elopment of the adrenal insufficiency he studied the effect of injecting pitressin in this patent on the excretion of antıdutetic substince A normal subject 495 ·Hormones in II ater Metabolism . 33 studied at the same time The antidiuretic assays were done by the method of Burn(39) in rits hydrated to 5 percent of their body weight by stomach tube One millibter of the urine to be tested was injected intraperitoneally The rats in groups of four were placed in metal cages over a glass funnel and urine was collected directly into gridunted cylinders The volume of urine excreted was noted every thirty minutes The results are shown in the two figures Figure 5 gres the results in the normal subject the clear circles show the effect of the subjects urine collected prior to the injection of pitressin and there was no antidiuretic effect The sold circles show the antidiuretic effect of the urine two hours after the injection of pıtressin This was equivalent to the effect of 5 mU of pitressin (crosses) Concentrating the urine to one half of its original volume did not increase the intidiuretic effect (triangles) but did increase the chloruretic effect the figures for chloride ercre tion by the rats is given in the figure Figure 6 shows the results of injecting the urine from the patient with diabetes insipidus Three urine samples were assayed The clear circles are the assay of the urine sample vorded at 7 o clock in the morning the patient GO RAY ASSA OF URNE ROW MOANAL SV ECT STORE. ATTER WAT ON ORCH RESOR wom) URNE METEO 45 ADEO COLLECTED IN AFTER WJEC 08 NEFORE TRESS NEWCOO VOL ITS & .APTER TRESS NESAC OR 1 Z WAS VO 345 V>• PITRE UN # AT URNE CONCENTRACOTO VOL 30 PERCENT OF NGESTED WATER EXCRETED CHLORIOL EXCRETION OPREME REPRESEN MC 25% DERE NON My x megn 1 na 19 60 Y ME 90 IN ca MINUTES 120 FICURE 5 20 ·84 Renal Function ANTIDIURETIC ASSAYS OF URINE FROM PATIENT WITH DIABETES POUR FOLLOWING INJECTIONS OF PITRESSIN 60 0 10 UN S MIC 0 2 E UR NE VDOED 3410E SRC SR ez 20 UN 13 PI METEO URNE COL EGTES AFTEA MOUR MOC OS VANE COL ECTED SSD NOVA) 41 tc ott BO PERGEHT OF INBESTED WATER EXCRETED CH QR DE EL METOWN VWINE RC RCSONT NG 6 tretton o 30 33 1 9 GO 190 DO 20 90 120 MINUTES FICURE B haung taken 20 pressor units of pitressin at midnight The sold circles represent the assay of the urine voided 1 hour after the injection of n second dose of 20 pressor units of pitressin The tringles are the assay of the urine voided from the first to the thurd hour after thus dose of pitressin It is interesting that the unne sample that remained in the bladder overnight had no antıd uretic effect nor did the simple voded in the second and third hours after another dose of pitressin The urine sample voided in the first hour after pitressin was injected had a murhed antidmretic effect One may infer that whatever pstressin is excreted in the urine is excreted principally wi fun the first hour after the injection and that of the urine remuns in the bladder the antiduretic principle ex creted is destroyed This patient with diabetes insipidus has now also adrenal cortical insufficiency and has been in Addisonian crisis three times The serum sodium has been at very low levels and he has required very large doses of whole adrenal cortical extract and salt to return him to sodium balance Ile is now controlled on lipordrennl extract DCA silt about 18 gm dully and pitressin ·र Hormoncs in Water Metabolism 35 every other night He always excretes water - even during periods of crisis Shannon I am a httle surprised to see a group of people interested in renal physiology sit around a table as they have this morning without protesting results presented which represent overall effects rasolving the animal as a whole particularly when the experimental preparations are difficult of not impossible to control and when the animal utilized in some experimental preparations has not been studied in order to determine the extent to which mechanisms utilized in the formation of urme are under specific nervous and/or humoral control All of us who hase worked in the field of renal physiology appreciate that one can get the same overall net effect by a variety of combinations of mechantsms Consequently at is disturbing to me to see net effects utilized in an attempt to interpret the underlying mechanisms involved in the net effect without measuring specific viables Such experiments apper to me to have the nature of pilot experiments which can be used in the design of a more definitise tupe experimental Thorn Dc Shannons criticism is a good one On the other hand one must remember the very abnormal situations which surround an attempt to set up definitive studies in whuch only one vanable is accepted We all realize that the overall retention of 50 mEq of sodium per das in a careful balance study may represent 1 very significant change in the adı durls response to a hormone prepara tron although the sensitivity of the clearance technique is not great enough to detect this change Shannon It nught be helpful in order to clarify my point of view to descnbe very briefly two sets of experiments performed upon the dog In each case there was a dramatic increase in the renal excretion of water and in each the urine was characterized by sodrum concentrations which were low as compared to plasma The first observations were obtained in a study of a limited num ber of dogs with diabetes insipidus as the result of the more or less complete ablation of posterior pituitary tisstie These animals weighing from 7 to 12 Irlos were charicterized by urine flows in the order of 3 to 4 liters per day with free access to water glo merular 6ltration rates and renal plasma fons were in low normal ranges When dilute salıne (05 percent NaCl) was the only fuid available for drinking 2 sequence of events occurred wherein there occurred 7 dramatie increase in urine flou to range of 12 to 16 approach ·36 Kenal Function uters per day Sodium concentration of the urine in the latter condition was essentially that of the ingested fud(40,41) An eximination of the experimental data collected in these experiments suggests that following the beginning ingestion of the saline there were progressive increases in the amount of sodium localized in the body, progressive increases in the volume of extracellular fud, accompamed by a progressive expansion of plasma yolume Presumably as a result of the latter there were increases in glomeru lar filtration rate of roughly 100 percent as comprred to the control levels The physiological defect which permitted this sequence of events was possibly due in part to an enhanced ability on the part of the renal tubules to reabsorb sodium and certainly to a dımınıshed ability to reabsorb water against a concentration gradient The second set of observations were those reported by Ragan Ferrebee Phyfe Atchley, and Loeb(42) wherein a syndrome of polydipsia and polyuna was produced by the administration of DCA and the polyuria enhanced by the further addition of sodium chloride in the diet The detailed data presented in relation to the latter studies give httle indication of any marhed retention of sodium and a concurrent expansion of extracellular fluid and plasma solume, although it is possible that these things did occur to a minor degree in any case, the initial polvuria in the diabetes insipidus ammal was due to a specıhe absence of posterior pituitary hormones, and the enhancement of the polyuria was due to the administration of sodium and the physiological consequences of the primary deficiency Polyuria in the latter series of observations was presumably due to the specific administration of DCA and its effect in cnhancing sodum reten tion the latter having more dramatic effects when the sodrum intake was high than when the sodium intake was lou Quiltatu ely then one must conclude that the same net effect is obtainable from the administration of sodium in the diabetes insipidus animal, on the one hand, and the normal anımal recensing DCA, on the other There is no necessary relationship hetween the two experimental preparations other than the limited number of mechanisms available to the kidnet with which to accomplish a physiological task Consequently, obtaining the same net effect under two different circumstances can by no means be taken as an indication that the same pnman controlling factors are inoked in producing a giien experimental effect ·Hormones in later Metabolism 37 There is little information available on the rat which will permit the definition of how this species accomplishes overall salt and water adjustment in terms of the discrete renal mechanisms which are involved We do have a fair amount of information about the human The human has a rather ngid rate of glomerular filtration When isotonic salt solution is administered there is little change in glomerular filtration rate and little immediate change in the amount of sodium excreted It would appear that the human organism does not have the capacity to increase salt and water excretion acutely as a result of a simple expansion of extracellular flud it could be that this results from an inability on the part of the human to expand glomerular filtration rate Janeuay I think some experiments that Dr Gamble and Dr Wallace(43) did in children during the last two or three years showed that it takes about four days to reach equilibrium as you described for sodium salts On the fourth day they begin to excrete the excess sodrum Shannon We have comparable studies in the Dock The dog will come into equlibrium very quickly Shannon Yes qmte quickly Thorn Why do you state that in the intact animal one can dis regard the possibility of reciprocal changes in posterior pituut rry secretion in response to salt loading? Shannon I do not mean to say that the two preparations increase unne flow by two completely different mechanisms Rather as the result of two distinct physiological vanables the same net renal effect is obtained Large doses of DCA to a dog do not consistently produce a diabetes insipidus like state Nonetheless one must suppose that there is an interplay of various hormones as the result of DCA administration The diabetes insipidus only appears consistently when one increases the salt intake above normal One could argue young adult f 1 j tonicity of the fluud reaching the gland From another point of view it would be unlikely to suppose that the simple absence of posterior pituitary as in the diabetes insipidus animal would produce 1 marked increase in salt retaine hormones under constant of inte ·38 Renal Function loading since the physiological defect in diabetes insipidus animals is apparent with very moderate salt loads Dock The salt load is always given with a lot of water Shannon The experiments I have described were all with hypo tonic saline The animal comes quickly into equilibrium in such a situation If the normal dog is loaded with isotonic siline and not too rapidly he also will come into equilibrium rather rapidly whereas the normal human does not come into equilibrium in any mersurable period of time during an acute experiment Gaunt There is no question about the limitations of the rat for detuled considerations of pure renal physiology I tried to make it clear in my introductory remarks that we at least and not at tempted any detailed analyses of renal function in these various endocrine imbalances I think it could be donc if that were ones primary interest For other aspects of the problem the rat can supply useful information with great ease and economy Bott I should like to know how much water balance varies from animal to animal and from species to species I received the impres sion that adrenalectomized rats reabsorb more water from the tubules than do normal ones Is this due to difference in the state of hydration? The blood volume is low in the adrenalectomized rat isnt it Dr Grunt? Gaunt Yes If one follows daily water intake in the adrenalectomized animal during the time when the animal is relatively heilthy there is a lugher than normal urine volume In some cases 1 nega tue water balance Adrenalectomized animals can excrete water in higher than normal amounts Whnt they cannot do is to increase their urme volume i response to a water lord anything like normal animals They will get rid of it in twenty four hours not in two As to the state of dehydration that exists in the animal as a whole Dr Darrow has done work on that(44) I behere he found that there was no difference from normal in the total body water of drenalectomized rats calculated on a fat free basis Such tissues ?s skin and muscle show a higher than normal water content There is extracellular dehydration and intracellular hydration Thorn There is one important point sluch Dr Shannon has brought up in relation to the great load of sodium chloride which the dog can tolerate It might be that the large intake of dietary potassilim ingested by dogs and their large potassilim reserves ·Hormoncs in Water Metabolism 39 might be important factors in enhancing this apparent species difference Pitts I think there is real species difference here at least in a quantitative sense Ladd and Raisz(45) demonstrated most con vincingly that the normal dog can tolerate a phenomenal amount of sodium chloride in the diet without appreciable gain in body weight As salt load increases, glomerular filtration increases and the animal attains salt balance by ercreting a fair proportion of the extra filtered load The situation in man is at least quantitatively different We studied some ten convalescent patients loaded with 27 gm of sodium chloride intranvenously each day for three days The salt was administered as 3 liters of isotonic saline Before dur ing and after this loading procedure we studied filtration rate and renal blood flow These patients came in to cquilibrium with the elevated salt intake after three or four days with a weight gain of 5 to 18 lbs These patients did not show any uniform and cer tainly no impressive increase in filtration rate or renal blood flow I would infer that the greater capacity of the dog to handle sodium loads with minimal weight gain is in some way related to the capacity of this animal to increase the filtered sodium load Burnett Yet in some humans the filtration rate is raised by acute loading Pitts We gave five patients 1500 ml of 0 9 percent saline in thirty minutes at 1 rate of 50 ml per minute Clearances were measured before during and after this acute loading procedure Not infrequently did we find a fall in filtration rate during the infusion no rise afterwards yet a fairly significant increase in sodium excretion Burnett Hypertonic? Pitts Isotonic Shannon We have done much the same experiment only giving 3 liters in the course of a comparable period of time That is about twice the rate of Dr Pitts experiments Towards the end of salıne administration one observes a slight disturbance in renal plasma flow and glomerular filtration rate but this is transient Shortly thereafter these functions have returned to the control level Burnett Administration of hypertonic saline solution has incon stantly caused increases in glomerulus filtration rates in our hands yet Dr Leiter and Dr VicCance have been able to increase filtra tion rate with hypertonic stline solutions ( 46 47) ·Renal Tunction 40 Pitts I have taken hypertonic sodium bicarbonate intravenously ind my filtration was rused roughly 20 percent There is I believe 1 considerable variability in man in the extent that filtration rate is raised by the infusion of isotonic and hypertonic solutions A modernte increase in sodium excretion results whether or not an increase in filtration rite occurs It is true that excretion is greater if filtration rate increases Shannon The dog also has the capacity to retain both witer und salt beyond that of 2 hunn He has 7 much more labile system Some of the observations on children by Farr seem to indicate certun cases of nephrosis are characterized by a much greater labılıty of the glomerular apparatus than there is commonly found in the normal adult To my know ledge i comparable study of te normal child has not been made Burnett I dont know of any work on the normal child Janeitay Dr Vletcoff( 48) has contrasted salt loading in acute experiments in nephrotic children and normal children The loading has been incident to the measurement of renal clearances In tie two groups there tre big differences in how the sodum load is handled in the nephrotics 1 great deal of it is du erted cxtra renally the blood sodium rises and there is an actual increase in potassium excretion in the normal child most of the sodium load is excreted without a rise in blood sodium Dr Gladys Fashena* has shown similar responses to large doses of sodium chloride in nephrotics Burnett We have found it true that administration of hypertonic sodium bicarbonate results in a rather striking excretion of potassium in the adult patient with nephrosis(49) Lauson The labılıty of the nephrotics clearances with changes in protein intale is particulurly marked in the super normals thosc whose clearances are relatively super normal are more apt to have marhed depressions of put on very low protein intake Sometimes this is not the case we had one careful experiment in a five year old chuld in which changing his protein intake from 50 to less than 20 gm of protein per day had no effect at all on around the clock creatinine clearances † Furthermore I think it would not be Personal commun cat on + TI serper ment and those referred to below were carred out at the sloptal of the Rockefeller Inst tute for Vled cal Research the Department of Dr DD Van Style by Drs r P Ch nard HA Eder RL Cref A 11 ller GC Cotz s and Il D Lauson ·Hormones in Water Vetabolism difficult to demonstrate this lability in some patients whose filtration rate on an ordinary diet is subnormal The Imman being certainly can change huis glomerular filtration rate or uren clearance at least by means of drastic changes in protein intihe even in the adult One experiment that I have in mind is that of Longley in Cleve land (50) who by greatly resinching prolem minhe reduced lus vrea clearances I believe by 30 percent or more it is necessary to reduce intake drastically to get much of 1 decrease On very high protein intake the urea clearance did not increase very much I would ble to return to Dr Pitts experiments on man in which he failed to find a umform increase in glomerular filtration rate Do you mean De Pitts that after equihbration occurred following the infusion the glomerular filtration rate did not increase even by a httle or were the experiments confined to measurements made eich day during the infusion of the material? Putts There were two different types of experiments in the first type filtration rate and blood flow were measured prior to and after three days of a hydrating procedure consisting of the intra enous administration of 3 liters a day of isotonic salıne Surlyse quently the indırdual was dehydrated by the daily administration of 2 ml of mercuhydon for three days and the measurements were again made in the second type of experiment filtration rate and blood flou were measured before durmg and after the administra tion of 1500 ml af sal na at rate of so mi nar mn of tio ch would conclude that there is a qu intitative species difference in the way the dog and man reret to hydration and dehydration Louson I remember the experiment of Stew art and Rourke(51) which is graphed in Gambles hook on extracellular Aud I think it would be desirable to repeat that same experiment They had a patient presumably norinal who had undergone minor surgery in whom four or five liters of isotonic silme a dis produced a seien Inter gun in extracellular fud and eventual equilinum of output to duly intake of salt I have always felt Dr Shannon that antil proved otherwise this obsersation was at least qualitatively similar to your dog experiment(40) except that unfortunately the filtration rale sas not measured by Stewart and Rourke I wonder if you would expect filtration rate to increase somewhat after saline reten tson had become really substantsal as in their patient? HA Rathmor ·Renal Function 42 Shannon Our experiments simply indicate that the normal human in a chronic balance experiment can be expected to handle different salt loads without 7 measurable difference in glomerular filtration rate as conventionally measured Pitts What prompted us to do our experiments was Ladd and Raisz (45) implication that conclusions reached in studies of electrolyte excretion in the dog could not be applied to man At the outset I disagreed with this view After finishing our studies I have changed my mind somewhat The relatively slight changes in filtra tion rate and blood flow in man in comparison with the large changes in the dog are such that they shake my faith in the safety of trans ferning too completely from dog to man concepts of the relatase importance of one mechanism versus another Fremont Smith Is there any evidence that urine volume is ever directly and primarily related to changes in glomerular filtration? It seems to me from all of the evidence I have seen whatever the glomerular filtration is doing that the change in volume is primarily related to reabsorption phenomenon Shannon It is six of one and half a dozen of the other You have two processes going on all the time Filtration presents a volume of fluid to the tubular system which then reabsorbs the vast majority of solute and solvent in normal circumstances It is only when there is a defect in the system that ? relationship can be demonstrated between the two at normal salt and water intakes The diabetes insipidus animal of about 10 hilos will excrete in the order of 5 liters a day Honeer this level of urinary excretion can be markedly reduced as the result of severe restriction on witer and thus is usually accompanied or made possible by z marked lowering of glomerular filtration rate Fremont Smith But also even greater change in fırs tubular reabsorption? Shannon No sir Fremont Smith Am I wrong. There is no change in the tubular reabsorption? Shannon As far as sodium is concerned it is almost complete in the diabetes insipidus animal in either state Fremont Smith I meant of water Shannon This is apparently dominated by presence or absence of the antidiuretic hormone in the normally hydrated animal ·Hormones in Water Vetabolism 13 Burnett Jou think the cutback in your animals was due to a decrease in filtration rate? Shannon In that particular expenment I would assume that this is most likely One must appreciate that it is very difficult to obtain an animal completely devoid of all tissue capable of elaborating the antidiurehc hormone Lauson In those experiments would you be willing to say fath that the increased percentage reabsorption of that diminished fil trate volume represented an intrinsic tubular adjustment possibly ndependent of hormones? Shannon I would not say that I dont beheve one can be certain in any experimental preparation that one has completely ablated the tissue from which the antidiuretic hormone can arise Lauson I wonder if the comerse experiment that you reported in the second paper on the control of water excretion (41) doesnt also indicate a dependence of urne flow on filtration rate In this experiment you had a diabetes insipidus dog on a constant infusion of 20 mU per hour of pitutrin, wluch was more than adequate to reduce urine flow to a minimum when the filtration rate was about 25 ml per minute Then when vou gave a large dose of water by stomach tube actual duresis occurred in spite of continuation of the infusion of excess pituitrın is this druresis not then altrbutable to the fact that the filtration rate simultaneously rose from 25 to tround 36 to 38 ml a minute? Is this not a clearcut converse of the ther experiment, in which simple dehydration żn the diabetes insipidus dog led to increased percentage reabsorption of water by the tubules in association with a reduced filtration rate Shannon I suspect so, although not published in the series other experiments were performed on the dog with isotonic sodium chloride being administered to produce an expansion of 5 to 10 percent of extracellular body fuid The pattern of response of the dubetes insipidus dog receiving what one might consider normal amounts of antidiuretic hormone was essentially the same as that of the normal ·Renal Function REFERENCES 1 ROEMMELT JC SARTORIUS OW and PITTS RF Excte tion and reabsorption of sodium and water in the adrenalecto mized dog Am | Physiol 159, 124 (1949) 2 HARTMAN F A and BROWNELL KA Tbe Adrenal Gland Philadelphia Lea and Febiger 1949 3 KENDALL E C The influence of the adrenal cortex on the metabolism of water and electrolytes Vitamine und Hormone 6, 277 (1948) 4 GAUNT R BIRNIEJ H and EVERSOLE W J Adrenal cortex and water metabolism Physiol Rer 29, 281 (1949) 5 PITTS RF and SARTORIUS O W Mechanism of action and therapeutic use of diuretics | Pharmacol & Exper Tl erap 98 161 (1950) 6 GAUNT R The adrenal cortex in salt and water metabolism Progress in Hormone Research Proc Laurentian Horm Conf 1951 (in press) 7 SAYERS G The adrenal cortex and homeostasis Physiol Ret 30, 241 (1950) 8 GAUNT R Water d urests and water intoxication in relation to adrenal cortex Endocrinology 34 400 (1944) 9 Boss WR BIRNIE H and GAUNT R Renal factors in the adrenal cortical control of water metabolism Ibid 46 307 (1950) 10 GERSH I (Baltimore) and GROLLMAN A Kidney function in adrenal cortical insufficiency Am J Physiol 125, 66 (1939) 11 LOTSPEICH W D The effect of adrenalectomy on the renal tubular reabsorption of water in the rat Endocrinology 44, 314 (1949) 12 FRIEDMAN SM MACKENZIE KR and FRIEDVAN CL Renal function in the adrenalectomized rat ibid 43 123 (1948) 13 ELMAD JIAN F Dose response data in man following orally administered or injected lipo adrenal cortical extract Federation Proc 9 38 (1950) 14 GREEN D M et al Mechanisms of desoxy cort costerone action effects of the water soluble glycoside on human citculatory and renal functions Endocrinology 47, 102 (1950) 15 GREEN DM FARAH A and KLEMPERER W W Mecha nisms of desoxycorticosterone action Renal effects of the water soluble glycoside Ibid 281 16 COREY E L SILVETTE H and BRITTON S W Hypophyseal and adrenal influence on renal function in sat Am I Pbysiol 125 644 (1939) ·Hormones in Water Metabolism 21 SARTORIUS O W and ROBERTS K The effects of pitressin and desoy corticosterone in low dosage on the excretion of sodrum potassiumn and water by the normal dog Endocrinology 45, 273 (1949) 18 SILVETTE H and BAITTON S W Renal function in normal and adrenalectomized opossums and effects of post prutary and cortico adrenal extracts Am / Physiol 121, 328 (1938) 19 BIRNIEJ Helal An antidiuretic substance in the blood of normal and adrenalectomized rats Endocrinology 47, 1 (1950) 20 MARTIN S HERRLICH H and FAZEKAS JF Relation between electrolyte imbalance and excretion of an antidiuretic substance in adrenalectomized cats Am 1 Physiol 127, 51 (1939) LLOYD CW and LOBOTSKY J Serum antidiuretic substances and waary corticosteroid l of Clin Endocrinol 10, 3 (1950) 22 HELLER H and URBAN F F Fate of antidiuretic principle of postpituitary extracts » 11o and in suro Physiol 85, 502 (1935) 23 EVERSOLE W I BIRNIC J Hand GAUNT R Inactivation of posterior pituxtary antidiuretic hormone by the liver Endocrinologs 45, 378 (1949) 24 BIRNIE H laactivation of posterior pituitary antidiuretic hormone by laser extracts Federation Proc. 12 (1950) 25 LABBY D H and HOAGLAND CL Water storage and move ments of body fluids and chlorides during acute liver disease 1 Clin Intestigation 26, 343 (1947) 26 LESLIE SH and RALLI EP Efect in rats of high fat diets on renal excretion of water and antidiuretic substances Endoc molog) 41, 1 (1947) 27 WINTER CA INGRAM WR and GROSS E G Effect of patressin injections upon serum electrolytes and a ater exchange of Cats with diabetes insspidus and adrenal insufficiency Am] Pbyrol 127 67 (1939) 28 AULINOS M G SPINGARN CL and LOJKIN ME Diabetes insipidus like condition produced by small doses of desoxy corto costerone acetate in dogs Ibid 135, 102 (1941) 29 RACAN Celal A syndrome of polydipsia and polyunsa in duced in normal animals by desoxy corticosterone acetate Ibid 131 73 (1940) 30 RICE and RICHTER CP Increased sodium chloride and water intale of normal rats treated with desoxycorticosterone acetate Endocrinology 33, 106 (1943) 31 PETERS } P Role of sodium in production of edema New England | Med 239, 333 (1948) 32 SHANNON Í A Control of renal excretion of water rate of liberation of posterior pituitary antidruretic hormone in dog 1 Exper Afed 76 387 ( 1942) ·46 Renal Function 33 SLAHIN G, and GREEN, D M Mechanisms of desoxy carti costerone action IV Relationship of fluid intake and pressor responses to output of antidiuretic factor Am | Physiol 155, 290 (1948) 34 NAGARCDA S and GANT, Tunctional relationship between the adrenal cortex and the posterior pituitary Endosninology 1950 (in press) 35 RALLI E P The relation of vitamins to the adrenal cortex Adrenal Cortex Raffi, E P, Bloch K, and Pins G, Editors Trans First Conf New York Josiah Masy, Is Foundation, 1950 (P 159) 36 THORN, G W ENGLE, L I, and EisENNEPG, H Effect of corticosterone and related compounds on renal excretion of elec trolytes | Exper Aled GB, 181 (1938) 37 Cosmos, E DUELL H , and GAUNT, R Some biological proper tres of desoxycosticosterone glucoside Endocrinology 46, 30 (1930) 38 FORSHAM P Hef al Clinical and metabolic changes in Addison s desease following the administration of Compound E acetate Tr A An Physicians 62, 233 (1949) 39 BURN ) H Estimation of the antidiuretic potency of pituitary posterior lobe extract Quart } Pharm 4, 317 (1931) 40 SHANNON JA Control of renal excretion of water, effect of variations in state of hydration on water excretion in dogs with diabetes insipidus / Exper Aled 76, 371 (1912) 41 Control of cenal excretion of water rate of liberation of postersor pituitary antidiuretic hormone in dog Ibra 387 42 RAGAN C e al A syndrome of polydspssa and polyuria a duced in normal ansmals by desoxycorticosterone acetate Am Physol 131, 73 (1940) 43 GAMBLE, J L " al The effect of farge loads of electrolytes Pediatrics (in press) 44 HARRISON, HE and DARROW, D C The distribution of body water and clectrolytes and adrenal insufficiency / Clm freshga rron 17, 77 (1938) 45 LIDD M and Aisz, L. G Response of the normal dog to dietary sodium chloride Am | Physiol 159, 149 (1949) 46 DEAN RFA and MCCANCE, R A The renal response in infants and adults to the administration of hypertonic solutions of sodium chloride and uzca Physiol 109, 81 (1919) 47 MOLOTOFP R G, and LEITER, L Renal plasma flow and sodium reabsorption and excretion is congestive heart failure / Clw Intestigation 27, 1 (1918) 46 METCOFF, ), and WALLACE W M The nephrotic syndrome in children Response to intravenous sodium loads Ibid 29, 835 (1950) ·Hormones in Water Metabolism 49 BURNETT CH, BURROWS, BA, and COMMONS, R R The lack of correlation between glomerular filtration rate, and serum electrolyte concentration changes, urinary electrolyte excretion or edema formation following sodrum loads in subjects with normal kidneys, glomerulonephritis, and the nephrotic syndrome Ibud 28, 773 (1949) 50 LONGLEY, LP, and MILLER, ME The effect of diet and meals on the maximum urea clearance Am, Med Sc 203, 253 (1942) 51 STEWART, J D, and ROURKE, G M Effects of large intravenous infusions on body fluid Clin Intestigation 21, 197 (1942) ·ANTIDIURETIC FACTORS HARRI B VAN DYKE Department of Pl armacolog) College of Physicians and Surgeons Columbia Unnt ersity ANTIDIURETIC FACTORS may be defined as substances which reduce diuresis by water Depending upon their action they may belong to one of three groups of substances First a factor may be identical with neurohypophysial antidiuretic hormone This is probably the factor which is found in the urine of mammals after dehydration or after osmotic stimulation of the cerebrum Secondly, a factor may cause the hberation of antidiuretic hormone as is the case with several drugs such as acetylcholine or vohimbine Thirdly, the antidiuretic effect of a factor may have no relationship to the neurohypophysial secretion Such a factor may reduce urine flow by reducing the rate of renal plasma flow or the glomerular filtra tion rate or both A substance in this category could conceivably have an extra renal action No antidiuretic factor resembling closely the neurohypophysial antidiuretic hormone in its action on the kidney but not originating in the neurohypophysis has been dis covered It is natural that antidiuretic factors especially those found in body fluids or secretions are often thought to be identical with the neurohypophysial secretion Therefore in the discussion which follows I shall speak briefly of the physiology of the neuro hypophysis The assay of an antidiuretic factor is of such great importance for its characterization that I shall deal with this topic in some detul Lastly I shall discuss various antidiuretic factors which have been reported The experiments of Ranson and his colleagues placed great emphasis on the physiological unity of the neurohvpophysis con sisting of the paired hypothalamic nuclei (especially the supraoptic and paraventricular nuclei) the infundibulum or stalk and the infundibular process or neural lobe of the pıtıntary Today most investigators agree that the structures comprising this physiological unit must be intact for the normal secretion of antidiuretic hormone in response to a physiological demand The signal for secretion may be released by a rise in the osmotic pressure of plasma Nearly all the antidiuretic hormone of the neurohypophysis is stored in the infundibular process which in the adult man contains about 48 ·Antidiuretic l'actors 49 15,000 mV The size of this stored secretion in terms of its activity can be appreciated from the human experiments of Barclay and Cooke(1) who demonstrated antidiuretic effects from intravenous doses of 05 to 2 mU A distinction must be made between the antidiuretic principle and the protem of the posterior pituitary which was isolated by van Dyle et al (2) This protein contained both the antidiuretic and the oxytocic principles and had a potency corresponding to 61 micrograms per unt of standard powder Under discussion today is the antidiuretic principle which possibly is a fragment of a large protein molecule The most potent extract reported con tained a umt in about 2 micrograms (0002 mg ) Perhaps its true potency is as great as the pure or nearly pure orvtocic principle of Pierce and du Vigneaud(3) whose product was nearly twice as potent in terms of orz tocic assay The antidiuretic principle is dialyzable and ultrafiltrable it is readily adsorbed in blood and on numerous other substances such as artificial zeolites It resists digestion by pepsin but is readily inactivated by trypsin Heat as well as high or low hydrogen ron concentrations destroy it Apparently the S S-group of cystine must be intact if its activity is to be retained This amino acid is an important constituent both of the antidiuretic principle and of the hvpothetical parent protein In lowering the rate of urine formation by purely renal mechanisms, antıdjuretic hormone increases the renal reabsorption of water fost investigators have concluded that it has no effect on the glomerular filtration rite However Barclar and Cooke(1) in their recently described human experiments, obsen ed that in some of the subjects the anbdruretic effect may be accompanied by reduchon of the glomerular filtration rate, in fact in five of thirteen subjects, there was a reduction of both the glomerular Gltration rate and the rate of renal plasma fow Barclay and Cooke used small doses totaling 05 to 2 mU administered as 2 to 80 microunits per minute during or after such doses they observed antidiuresis in each of thirteen subjects It is not surprising that both glomerular filtration rate and the rate of renal plasma flow hare been reduced in association with antidiuresis after the enonnous doses which have been used in either animals or man As recently as 1947 Little and has collengues(4) injected 10 units of pitressin intramuscularly into the human subjects under study So far as the normal rate of secretion is concerned there is good ·Renal Function 0 agreement at least in the dog that the rate is about 1 to 5 mV per hour per dog(56) Antidiuretic hormone is an example of a stored internal secretion which is liberated according to the body s needs and in this respect resembles the stored secretion of the thyroid in contrast with adrenal cortical secretion which is rapidly but temporarily elabo rated according to the demands of stress The control of the liberation of antidiuretic hormone is principally effected by the osmoreceptors associated with the neurohypophysis The connec tions with other parts of the central nervous system must be numerous although it is not possible to give any accurate picture of the relationships In what form the hormone is liberated is unknown It can be detected in the internal jugular blood after osmotic stimulation An unknown fraction is excreted in the urine Pre sumably in the interval between liberation into the blood streim and excretion in the urine 7 considerable part of the hormone is inactivated by reducing substances and enzymes such as pro teinases and polypeptidases After enormous intravenous doses 10 to 25 percent of the dose may be excreted in the urine The endogenously secreted hormone which appears in the urine under goes sedimentation in the ultracentrifuge like a large pırtıcle This urinary hormone easily undergoes fragmentation with liberation of the nonsedimentable antidiuretic principle(7) Vuch effort has been expended in attempts to demonstrate antı druretic activity in various body fluids The hormone is not present in cerebrospinal fluid despite early suggestions to the contrary The hormone can be detected in the blood of the dog after osmotic stimulation and in the serum of rats especially after adrenulectomy Jwing to its relatively simple composition the urine is perhaps the Jest fluid in which to demonstrate the hormone inequivocally in man the maximum concentration appears to be 1 to 2 mV per millilter In other mammals with maximum osmotic stimulation the greatest concentration is about 6 mu per milliliter (dog and rat) In a desert rodent the hangaroo rat (Dipodomys meram) nearly ten times as high a concentration has been found(8) Any study of antidiuretic factors in bods Auds has to take into account the great difficulties of assay Without accurate and quantı tatrs ely acceptable assays no conclusion can be reached concerning the alleged presence of true antidiuretic hormone ror clample chloruresis is sometimes accepted as a necessary feature of the action of the hormone and may be the basis for denying its ·Antidruretic Factors 51 neurohypophysal origin let a careful survey of the literature does not support this conclusion and it appears certain that depending upon conditions, an increased rate of excretion of chloride or of cations such as sodium or potassium does not necessarily occur after the administration of the principle Anslow and his colleagues (9) concluded that an effect on electrolyte excretion depends upon the water load Their experi ments suggest that without suppression of the endogenously secreted hormone an effect will not be obsers ed Although Shannon (5) believed that the principles administered as pituitrin inhibited the tubular reabsorption of chlonde, he never observed this effect in one of the four dogs with diabetes insipidus which were used for his experiments Other experiments have been performed by Sartorius and Roberts(10) in the dog and they concluded that whether or not hydration was present the intras enous injection of the antidruretze principle increased the rate of excretion of sodium and that this effect was antagomized by desorycorticosterone acetate Human experiments have been reported by Hart and Verney(11), McQuarrie and co workers(12), White and Findley(13), Barclay and Cooke(1) and others From these obsers atons it may be concluded that in man the antidiuretic principle has no consistent effect on the rate of excretion of chloride Some workers have attnbuted a chloruretic effect to the oxytocic principle (Fraser(14), Kuschunsky and Bundschuh{15), Dicker nd Heller (16), Heller and Stephenson(17) and Schaumann(18, 19) Some of the later work led to the conclusion that the primary effect is on cations and that chlonde was the accompanying anion It appears and to conclude that many factors modify electrok te etcretion whether antidiuretic principle or oxytocic principle or both be administered Among these are the electrolyte metabolsm the dietary electrolyte and possibly the rate of secretion of adrenal cortical hormone Chloruresis and an increased excretion of sodium or potassium or both are not necessarily characterishe of the action of the antidiuretic principle The principal factors in the bioassay of antidiuretic hormone are specificity, sensitivity and quantitate precision Pertinent examples are summarized in Table 1 The hydrated dog reseiving a body frud for assay is considered to be particularis appropriate, espea cially the dog with diabetes insipidus Particularly to be as orded is the comparison of an intravenous assay in one species with subcutaneous or intraperitoneal assy in another in all anstances ·52 Renal Function matenal under examination should be assived by the same method, thus avoiding the error into which Heller(20) fell in believing that he had demonstrated separate antidiuretic and pressor hormones Fraser(21) was able to point out that when all of Heller's extracts were assayed intravenously they did not differ in their antidiuretic and pressor effects The rat receiving an extract subcutaneously may absorb hormone slowly owing to contaminating substances so that the apparent titer of potency may be deceptively high Noble Plunkett and Taylor(22) stated that intraperitoneal injection into rats may provoke an antidiuretic effect from toxic materials TABLE I METHODS OF ASSAYING ANTIDIURETIC PRINCIPLE Route Quantitative Animal Specificity Sensitivity* precis on Rat Subcut Imperfect 1 mU/100 gm Poor with extracts Intraper 2 mU/100 gm Retter than Subcut Intraven 002 mV /rat Good Rabbit Intraven Poor? 105m2 (rabbit Fair Dog Intraven Imperfecto 25 mU/dog Fair (diabetes Intraven Best 025 mV /dog Fair insipidus) *Optimum doses for quantitative estimates are higher The following considerations suggest that intravenous assay in dogs heavily hydrated is especially valuable a) the assay animal is so sensitive that no chemical manipulation of the body Aud is necessary before the intravenous injection, b) if there is a gross renal effect, such as an interference with glomerulır filtration rate this can be readily detected by creatinine clearances or, less exactly, by determining the rate of excretion of endogenous creatinine, c) inactivation of the hypothetical hormone can easily be under taken fyrti a reducing agent such as sodiam chog'culate trually there is a lag of a few minutes in the antıdiuretic effect of true hormone If sympathetic vasoconstrictors are stimulated there is a rapid supression of the flow of urine Precautions also have to be taken to avoid an unusual release of neurohumoral transmitting agents affecting neurohypophysial secretion For example O'Connor and Verney(23) found that epinephrine can interfere with the release of antidiuretic hormone Pickford's numerous etperi ments(24) support her contention that acetvicholine acts on the ·Antidiuretic Factors 53 neurons of the supraoptic nuclei thus provoking a liberation of antidiuretic hormone The possible types of antidiuretic factors were discussed in the first paragraph of thus communication and will again be mentioned briefly here The specific factor is derived from the neurohypophysis and acts directly on the kidney usually with little interference with glomerular filtration rate or the rate of plasma flow Some substances are thought to stimulate the neurohypophysis so that the hormone is liberated In this group belong acetylcholine and the derivatives of phenylcınchonınıc acid studied by Marshall and his colleagues (25 26)* No antidiuretic substance physiologically resembling the true hormone but not derived from the neuro hypophysis has been discovered Lastly a toxic effect of an unknown substance may provoke an apparent antidiuresis owing to inter ference with the circulation of the kidney either locally or by general cardiovascular effects In Table II are presented the drugs which have been discovered to have an antidiuretic effect Their mechanism of action is usually to produce a liberation of the neurohvpophysial hormone When antidiuretic hormone has been thought to have been identified in the urine various alternative interpretations are pos suble Usually one of the following hypotheses has been adopted on the assumption that true antıdıuretic hormone has been detected a) the rate of liberation of the hormone is increased b) the rate of destruction of the hormone has been reduced as in hepatic disease c) there is a hormonal imbalance so that the rate of liberation is altered as when there is a deficiency of adrenal cortical secretion (38) d) true antidiuretic hormone is not present but its identification is an assay artifact and e) an abnormality of the kidney permits exceptional amounts of the hormone to appear in the urine It is not possible to speculate on the rate of secretion of true antidiuretic hormone from the amounts detected in the urine All the observations have been made with great quantı tres (eg 2000 to 5000 mU injected intravenously into man by Taylor and Noble(39)) after such enormous doses in both men and animals 5 to 23 percent of the injected dose have been recovered When injections are made subcutaneously or intraperi toneally interference with absorption owing to a contaminating substance may yield an artificially high hiter Hence intravenous +410 by personal commun cat on ·51 TABLE II DRUGS AS ANTIDIURETIC AGENTS Antidiuretic effect in diabetes insipidus Investigators Drug No ? No Acetylcholine Yohimbine Morphine* Phenobarbital Nicotinet Dımercaprol (BAL) 3 hydrory 2 phenyl cinchoninic acid and related compounds Choline Atropine Pickford (24), others Zunz(27), Fugo(28) de Bodo (31) de Bodo(32) Bum et al (31) Earle and Berliner (35) Marshall and Blanchard (25) Molitor and Pick(36) Cow(37), others Renal Function . Probably No No expmts reported *Also reported to reduce rates of GF and RPF (Handley and Keller(29)). (Brown, Ilodges and Bradbury(30)) Antidiuretic effect may be absent in nonsmokers (Eggleton(33)) ·Antıdırctic factors 55 assays in the dog as used by Hare and his co workers and by Ames Moore and van Dyke appear to yield the most reliable information The first unequivocal demonstration of antidiuretic hormone in urine was made by Gilman and Goodman in 1937(40) Other experiments employing the rat have been performed by Robinson and Far (41) as well as by a number of later investigators Usually the urine was prepared for injection by concentrating it and then dialyzing the concentrate To circumvent this procedure by which the final extract may contain toxic material attempts have been made to adsorb the hormone and later to elute it The first of these methods was that of Noble Rinderknecht and Williams(42) who used zinc ferrocyanide as the adsorbent eluted the hormone with ammonia ethanol and then fractionated the eluted material with ethanol Two examples of the latest methods in which urine has been the starting material are those of Stuech Leslie and Rall(43) who adsorbed the hormone from concentrated urine on permutit and accomplished elution by 5 percent sodium chloride in I molar acetic acid In the same year Grollman and Woods(44) described adsorption on charcoal with elution by glacial acetic acid and precipitation of the active material by a mixture of equal parts of absolute ethanol and petroleum ether An increase in antidiuretic factor usually detected in urine and usually considered of neurohypophysıal ongin has been descnbed for a number of clinical conditions The report of Robinson ind Farr(41) correlated a high urinary antidiuretic titer with clinical edema in the premenstruum nephrosis and Cushings disease Toxemias of pregnancy have been thought to be accompanied by an increased secretion of antidiuretic hormone( 45 46 47 48) Stander and his colleagues questioned the neurohu pophysial origin of the urinary principle Ham und Landis believed that it was derived from the placenta According to Ellis and Grollman(49) antidiuretic principle is present in the urine of z large proportion of human subjects with hypertension (eles en of fifteen) but cannot be detected in the urine of normal persons (none of nine) Also these workers believed that hypertensive dogs excreted two to four times as much antidiuretic principle as normal dogs Experimental pro cedures in normal subjects may lead to an increased rate of secretion of antidiuretic principle Among these are dehydration electroconvulsant therap: and fainting The titers described by ·56 Renal Function Taylor and Noble(39) are unusually high and it seems certain that the authors meant to express their results in microunits rather than in millunits Especially favored for study is hepatic disease whether in experi mental animals as in rats on a high fat diet or in man with severe hepatic disease such as cirrhosis with ascites In most instances the rat has been used for assay and there re unexplained incon sistencies in the reports which tend to nullify the conclusions (compare the results of Leslie and Rall(50) with those of Hall Frame and Drill(51)) Human luer disease may be associated with a low rate of urinary excretion of water and with a considerable diuresis as ascites vecedes An attractive hypothesis has been that of Rall and her co workers in 1945(52) and later who postulated a decreased destruction of antidiuretic hormone in such subjects particularly in association with ascites This presumably in part would explain water retention Quantıtatively there is a wide dif ference between their results and those of van Dyke Ames ind Plough(53) who found by assays in dogs a slight but real increase in the titer of antidiuretic principle in the urine of persons with cirrhosis and ascites as compared with cirrhotic patients without ascites They never were able to detect the high titers reported by Ralli and her co workers who performed their assay in rats Sims (51) also believed that infectious hepatitis could lead to a diminished destruction of antidiuretic hormone as revealed by increased urinary titers Sims likewise used rats for his assays An antidiuretic effect due to the hormone relaxın has been described by Zarrow(55) He employed extracts of sow ovaries or pregnant rabbit blood and reported that the volume of urine excreted by rabbits is diminished after the injection of such extracts None of his observations justify his conclusion that such extracts induce water retention since he offers no record of the water intake of his animals The whole effect could be a tonic reduction in food and water consumption Brez Mazur and Shon(56) attributed the olguna of cirrhosis infectious hepatitis nephrosis and congestive heart failure to the indirect effect of a liberation of their vasodepressor material (VD) They considered VDV to be identical with ferntın or apoferritin and believed that either of these proteins acting on the neurohypophysis causes an increased liberation of antidiuretic hormone ·Antidiuretic Factors SUVVARI 57 The possible origin and nature of antidiuretic factors are discussed with special consideration of the neurohy pophy sial antı. diuretic hormone The difficulties of assay and of characterization of an antidiuretic substance are described Examples of antidiuretic factors such as drugs or constituents of tuine are discussed and an evaluation of their significance is attempted DISCUSSION Lauson I think it might be of interest to mention some experiments that were done on me some years ago which were a direct imitation of Shannon's experiments(57) in which we infused pitressin at a constant rate while I was maximally hidrated The results indicated that my kidney sensitivity to pitressin was of the same order of magnitude that he had observed in the dog The creatinine U/P ratios achieved at the end of two hour Infusions in me and after forty five minutes in Shannons Dog D were approximately the same for the same rates of infusion when the Latter nere expressed as millunits of pitressin per hour per kilo gram of body weight I might also point out that the lowest effective dose was as httle as 75 mU per hour This rate reduced the urine flow to something over 1 mi a minule, not a maximal effect but quite substantial Therefore the extraordinary renal sensitivity to these extracts applies to man as well as to the dog White In a sencs of perhaps a dozen normal human subjects the results were about the same In our experience 0 25 to 05 mU will give a good but not maxmal antidiuretic response in an habetic insipidus dog that we have used. The normal human subjects have shown about the same sensitivity on the basis of body weight, it takes about 1 to 3 mU per average size man to give about the same percentage drop in urine flow when he has been hydrated, putting out 5 to 6 ml a minute Lauson As a single wjection? White As a single intravenous injection Normal young subjects were compared with normal old subjects no difference in sensitivity was found So the dose of pitressin for unmistakable threshold response is about 0 25 to 0 50 mV per 15 or 20 kilos of either dog or man Perhaps a thurd of the time you may get a barely perceptible ·58 Renal Function response with 01 mu in the dog with dubetes insipidus So I think there is enough evidence to indicate that the sensitivities of the dog and the man are about the same Dock Your normal subjects here were all well hydrated? van Dyke They were adequately hydrated, the specimens of trine were obtained in the morning before food or water Dock They were not restricted the previous day? Lan Dyhc No Dock In water restriction of man hou high does the urinary concentration of ADH go? van Dyke We have very httle data ourseles The highest we have ever observed is about 2 mU equivalent of pitressin per milli liter of urine Dock As high as any cirrhotics? can Dyke I don't know how high cirrhotics go Rall. We have assayed the urine of normal human subjects who have been dehydrated for forty eight hours and have obtained a very high antidiuretic titer If I may, I should like to discuss some of the points mentioned by Dr van Dyke, but first I would like to explain how I happened into this field As you know it had been thought for many years that the determining factor in the control of ascites in patients with cirrhosis of the liver was the level of serum albumin Mly associates and I had been studying and treating a group of patients with cirrhosis and ascites and had determined the serum constituents. including serum albumin at regular intervals of two to four week for periods of four to tieke months Interestingly enough we found that the serum albumin level did not increase before the ascites was controlled but rather that following diuresis and the associated decrease in ascites, the level of albumin in the serum rose grndunlly(58) In addition we observed as had been reported many times in the literature that the unne output in patients with Iner disease was greatly reduced and that the twenty four hour output was often as low as 350 to 500 ml This suggested to us that the hidney might play an important role in the production of the antidiuretic state encountered in patients with cirrhosis and that this state of antidiuresis was a factor in the production of ascites The report by Robinson and Farr(41) on the presence of an anti- ·Antıdıurctic Factors 59 diuretic substance in the urine of patients with edema stimulated us to do antidiuretic assays on the urines of patients with cirrhosis of the lner We found significant amounts of an antidruretic substance in the urine of these patients, particularly when ascites was present(52) All assays were done by the method of Burn* in rats hydrated to 5 percent of their body weight by gavage We have continued to use this method since 1914 and we believe it to be a reliable method of assay as judged by control assays on known amounts of patressin în continuing these studies we have attempted to determine the nature of the antidiuretic substance present in urine and in the course of this work have done innumerable assass of both unne and pitressin treated in a vanety of ways An interest ing species difference was observed in simultaneous assays done with pitressin in hydrated dogs as compared to rats Water duiresis was established in the dog by administering 75 to 150 ml of water per kilogram of body weight The material to be tested was given intravenously through an indwelling needle and injections of MILLJUNITS / CC GO 30 2 28 10 0-06 Ajo - Ro GO 40 10 PERCENT OF (WESTED WATER EXCRETED 30 MINUTE 20 160 FIGURE 77 Results of assays of diferent doses of pitxessia in hydrated sats .Loc CReference 39 P48 Figures 7 and 9 are reprinted from Am J Phynal 163, No 1 14 11" CO DO ·60 Renal Function EFFECT OF O OIM SODIUM THIOGLYCOLATE ON THE ANTIDIURETIC EFFECT OF PITRESSIN ASSAYED N THE RAT PITRESSIN 1OO MU/CC+ o OIM SOD THIOGLYCOLLATE (36 RATS) PITRESS 100 VUI CC+ OOM SOD ACETATE (36 RATS) 0-0 OM SOD TH OGLYCOLLATE (20 RATS) PERCENT OF INGESTED WATER EXCRETED 30 201 JO 60 120 190 180 210 240 TIME IN MNUTES AFTER HYDRATION FIGURE 8 freshly diluted commercial pitressin* were given during the assa experiments in order to quantitate the antıdıuretic potency of the trented pitressin We assay ed commercial pitressin* as such in varying dilutions dmlyzed pıtressin and pitressin treated with sodium thiogly collate according to the method reported by Dr van Dyke and his associates(7) The results are illustrated in Figures 7 8 and 9 Figure 7 shows the antidiuretic response in hydrated rats to doses of pitressin varying from 2 to 60 mV and also shows the consistency of these assays in rats Figure 8 shows the effect of treating pitressin with sodium thioglycollate and it is quite clear that the treatment sith thioglycollate did not inactuate the antidiuretic effect of the pitressin in rats Figure 9 shows assays of thioglycollate treated pitressin in the dog ar the antı diuretic activity was completely abolished acil to the result reported by Dr van Dyke It the rat and the dog react to thoglycol itressin This and other erper ated to us liuretic activity of commerci n be frs when one substance one of nts are ont active in the dog th rit1 gre Danes & Co ·Antidiuretic Factors 61 -J6z VOLUME OF VRINE ML mit MINUTE Lingo REPLACED TIME IN MINUTES O to 60 100 120 140 160 100 200 220 FIGURE 9 Antidiuretic effect of p tress n treated with 001 11 sodium thioglycollate and pitressin treated with 001 M sodium acetate injected 1 v into a female dog during water diuresis detail in a recent paper(58) As far as the antidiuretic substance in the urine of patients with liver disease is concerned, we have found the dog much less sensitive than the rat Lauson What did the thioglycollate alone do in the rat? Ralli Thioglycollate alone has no appreciable antidiuretic effect when injected intraperitoneally in the rat Whenever hydrated rats are given any solution intraperitoneally the rate of urine excretion is slightly less than if they were hydrated and no injection at all were given Lan Dyke If you performed assays intravenously in the rat what would you expect?

·64 Renal Function ties along these lines Certainly if the dog gets large amounts of antidiuretic hormones he shows no such effect He merely decreases his water intake and gets along very well indeed, and I imagine most of the patients would have the intelligence of the dog insofar as regulating their water intake is concerned Surely they would not have much difficulty in getting rid of their electrolytes Binhley What is the evidence that there is an enzyme in the Iver that destroys the antidiuretic hormone? Rallı I think Dr Gaunt could also discuss that question There were some experiments by Heller (59) in which pitressin was incubated with lever slices and they reported that the antidiuretic effect of the pitressin was destroy ed I do not know if this has been confirmed There is no absolute evidence that the lver of the patient with cirrhosis is incapable of inactivating pitressin Me have suggested this as a possibility but it remains to be proved Gaunt I do not have the evidence at hand These were Dr Birne's(60) experiments with which I had nothing to do He studied the effects of pH, heat, heavy metals, etc on the hepatic material and the evidence convinced him and others who reviewed at that he was dealing with an enzyme that enactnated pitressin I cannot elaborate the point except to say that in all of its chemical properties the material was identical to what has been called insulinase Binhley The order of activity you gave for the concentration of enzyme is exactly the order of actity for concentration of free sulfhydry in the luer suggesting that inactivation occurs as a result of combination with sultay dryl groups rather than by enzymic activity Rath I would like to suggest another possible explanation for the impured or disordered kidney function in the patient with severe liver damage Cirrhosis of the Iner y a disease with widespread metabolic effects which inolve the “nutrbodal" state of the tissue cells The kidney cells undoubtedly are also affected by this situation and the question anses as to whether the condition of the intracellular constituents of the kidney cells may not be affect ing their physiological capacity to function normally Bradley I would like to ask Dr Shannon if he believes antı diuretic hormone acts solely on water reabsorption Shannon The facts are not available I bellele that it is purely a matter of opinion and speculation First, a pure antıdınetic ·Antidiuretic Factors 65 hormone is not available Second the same preparation when used m four dogs may not produce identical results in all animals As Dr van Dyke has pomted out that is quite common in any one series of intestigations If sodium excretion is plotted against dosage of antidiuretic hormone administered continuously in the range of 5 to 20 mV per lour there is no change in sodium excre ton accompanying the change in water excretion At higher dosage some dogs progressively increase sodium excretion but on the basis of our evidence only when the hormone is administered above the physiological range Rallı Is the relationship between hormone administration and sodium excretion roughly a logarithmic one Shannon The data would not permit that type of analysis Dock Did the group in using the artificial hidney on patients with severe jaundice ind anurut observe a marked rise in blood pressure and urine excretion after dialysis of the patients blood? Thorn les(62) Dock This dialyzable shock factor can not be either pitressin or apoferrıtın neither of which is dialyzable? Thorn No these molecules are too large to pass through our cellophane membrane Dock Some substance of that sort may be in Dr Rallis urine extracts or at any rate a substance not destroved by reducing substances which inactis ate pitressin It probably is quite different from the antidiuretic substances apoferntin and pitressin Shorr At the risk of introducing another variable in the problem of diuresis I would like to present some of the results of our experi ments on the antidiuretic effects of ferritin (56) Our early experi ments were run with concentrates of the liver vasodepressor material VDV which though crude were pure enough to be injected intravenously The animals were given a continuous infu $ion of 5 percent dextrose until the urinary output became relatnely constant At this point the addition of VDI to the infusion flud caused a sharp curtailment of urine flow which persisted for some time after the infusion of dextrose alone was resumed After the identification of VDV as ferntın or apoferritin these experiments were repented using both oral and intravenous hydra tion Both ferritin and apoferritin were effective in mounts ranging from about 150 to 300 microgrims ferritin N per kilogram ·68 Renal Function less sensitive We have been able to utilize a combination of the immunochemical and inesoappendix methods to identify very small amounts of ferritin The reaction between antiferritin sera and such minute amounts of ferritin does not produce 1 visible pre cipitate but it does abolish the vasodepressor activity as measured by the meso ippende test(63) The liver possesses a cry active mechanism for removing ferritin from the blood and activating st so that the amount of ferrain injected is much larger than the amount remaining in the blood stream to cause intdruresis The discovery that ferritin cxists in two forms grietis considerable understanding of tus pictation process The actre form which produces 1 visodepressor response in the rat meso ippendix test ontuins sulfhydryl groups When these groups are oxidized to the disulfide form the ferntı is inactivated In the normal liver ferritin is present largely if not entirely in the disulfide form The oudition of sulfhvdni groups appears to the place readily in the normal laver Tlus prompt and efficient inactis non results in a very low concentration of ferrtın in the blood stream Rat bioassays of blood simples taken from a dog during an intidiuretic test revered activities corresponding to approumately 0001 to 0002 microgram of ferritin V per milli liter In other words about 99 9 percent of the injected ferritin is immediately inactivated(61) Janellay When you talk about actie ferrıtın do you mean its vasodepressor activity? Shorr les is measured by the rit mesoappender test Jancu ay It is 1 250depressor actritv? Shorr Yes Jancılay The activity is present only when there are sulfhydryl groups and bears no relation to its ability to bind iron? Shorr That is true but it is also interesting that treatment of disulfide ferritin with reduced glutathione or cysteine con tits it to the sulfhydryl forin and at the sime time labilizes the ferritin iron converting it to the ferrous state(65) Thorn Did you say the disulfide form has no antidiuretic effect? Shorr We do not know lle huent cnongh data so far The normal liver only releases reduced or actie ferritin under anaerobic conditions in nutritional cirrhosis in rits the ner *** ·Antidiuretic Factors 69 metabolism is disturbed so that actue ferritin is released under both aerobic and anaerobic conditions and as a result ferritin is constantly present in the blood stream It was therefore of interest to see whether or not the potential participation of ferritin in antidruresis was supported by its presence in conditions associated with antidiuresis in man(66) We studied a group of eight cirrhotics with edema ascites and antidiuresis as measured by the Fremont Smith water test A}had very considerable amounts of ferritin in the blood unopposed by the oppositely acting factor VEV Two other patients with cirrhosis but without edema and ascites and with normal water tests revealed only traces of ferritin The con centration of ferritin in the serym of patients with antıdıuresis is of about the same order of magnitude 75 we found in the circulation of the dogs during antidiuretic experiments In seien patients with the nephrotic syndrome ferritin was present not only in the plasma but also in the edem find Here too the oppositely acting 1 El was not present We are very much indebted to Dr Lauson for permission to study one of his cases before during and after treatment with intravenous albumin There was a significant fall in ferritin activity in the blood during the albumin druresis with a prompt return to the original high value on the re accumulation of edema following cessation of albumin therapy Finally seien cases of congestre heart failure with edem; and ascites were studied None of these cases had hypertension In all both pirsma and edema fluid contained considerable amounts of ferritin without admixture with VEV The difference between the results in these cases and in hyper tensne patients is that in the latter both ferntın and VEV are present in the plasma in amounts which are approximately equivalent physiologically Rat tests of hypertensive plasmas are neutral or shows 7 slight predominance of ferritin or VCV How ever trentment of the plasma to inactivate either ferntın or VIVI always unmasks 1 considerable concentration of the oppositely act ing factor Thus the possibility exists that these two factors with opposite actions on the blood vessels mu also be oppositely acting is regards their effect on the posterior pituitary houever our purification of VEV is not yet at a stage permit us to test this hypothesis We are still activel intestigating the antidiuretic phenomena in the dog and are seeking more conclusie data to support our ·70 Renal Function hypothesis At this stage in our work it appears at least possible that ferritin may constitute another element participating in antı duresis There are two mechanisms which could operate in these patients In congestive heart failure with edema hypoxia might lead to the continuous release of reduced ferritin from the liver Protein undernutrition which leads to the relense of active ferntı in cirrhotic rats may be responsible for its occurrence in nephrosis and decompensated cirrhosis Thorn You dont think that the response in 1 nephrotic is just an uncovering of ferritin by reduction in the VEM factor? Shorr In nutritional cirrhosis in rats there is a renal defect as a result of which the kidney is not able to form VEM We are guessing that in nephrosis the same defect occurs Thorn Our dialysis experiment would support your hypothesis In the patient in shock we apparently dialyze a factor which does allow the blood pressure to return to normal levels and to be muntained for four to six hours and then return toward a low level If the liver particularly has not improved in this time? second run of five or six hours of dialysis is done and the patient becomes responsive again to vasopressor stimuh The interesting thing along with Dr Shorr s concept is that the hypertensive does exactly the same thing when you dialyze a hypertensive patients blood pressure goes ever so much higher than the patient with normal blood pressure suggesting that possibly a diffuse depressor substance is balanced at a high level and the fact is we dont know whether we are dializing off an actuator of ferritin or an inactivator of renin system but the substance is diffusible and goes through a cellophane membrane Shorr Ferritin is not diffusible Thorn We have not been able to explain the druresis We observed it in patients run without renal shutdown on the basis solely of improvement in blood pressure It has been pretty obvious from the start that something modified the diuresis or accelerated the djuresis in a patient who has been run on the kidney five or six hours of his kidney has capacity to respond Doch The salt output goes up too? Thorn Yes Janeray Ds Shorr can you characterize VEV1 chemically?

·Antidiuretic Factors 71 Short I wish we could We are working on it actively now Jancuay It is not renin? Shorr So far, it doesnt seem to be renin We dont think it will tum out to be hypertensin since it is not musculotropic Lauson I would like to ask Dr Shorr if he has done any experi ments in which homologous ferritin has been continuously admin istered to see if it is chronically antıdiuretic Shorr Homologous ferritins usually continue to be intidiuretic when repeatedly injected in dogs However out of a large series of dogs receiving homologous ferritins two no longer responded with antidiuresis We huve no explanation at present for the failure in these two animals White Is it your concept that the overproduction of posterior lobe hormone is crusally related to any of these diseases? Short If I am allowed to speculate at this stage in our knowledge of these factors I would postulate that in nephrosis cirrhosis and heart failure accompanied by edema the normal Iner metabolism of ferritin is disturbed As a result of this defect actrie ferritin is released into the blood stream and accumulates in amounts which are of the same order of magnitude as those found in the circula tion in dogs in the course of our antidiuretic experiments with mjected ferritin From preliminary animal studies we infer that ferritin acts in some way upon the posterior pituitary causing release of antidiuretic factors from that gland It appeurs that the antidiuretic activity present in the urine in our animal experiments is not ferritin White It might be worth while to mention liere some observ ? ions bearing on the matter that was discussed earlier I know of o human subjects not suffering from diabetes insipidus who for a different reason were kept on large doses of pitressin or pituitrin one of them for over six months and one for about eighteen months with no retention of water or anything else Their intake was just cut down to balance their output and they got along perfectly well These were hypertensases the work was done to see whether they could be helped with more pitressin than they were making for themselves all that I am prepared to say - it was not my work - is that it did not hurt them at all Short There is one other possible effect of the presence of ferritin in the blood stream It may bring about in the peripheral ·72 Renal l'unction vascular bed, a state of vasodepression with reduction of spon taneous vasomotion and relaxation of the precapıllary splunters When this occurs the capılır, bed becomes hyperemic with cou tinuous flow through many capillaries which ordinarily have only intermittent flow These changes would all favor hydrostatic rather than osmotic forces in the exchange of fud between the capillaries and the tissues and might contribute to edema in that way Perhaps both effects on the pituitary and on the periplieral vascular bed are needed to produce edema White That would seem to me more important than overpro duction of posterior hormone Lauson Would you say this circumstance would favor outward filtration rather than absorption? Shorr We do not think of the capillary bed as consisting of inert endothelial tubes There are muscular units, the precapıllary sphincters from which the true capillaries arise These are capable of vasomotron by which the relative relations between osmouc and hydrostatic forces may be altered according to need Fremont Smith That means you have effectie capillary filtra tion increase Shorr Ses That would be in cases of edema Fremont Smith As far as we know the effect of capillan pressure as measured has not been shown to be effective White How often has it been satisfactonly measured? Thorn Does the ferritin level of serum go down after ferntın extraction? Shorr I cannot tell you Janeuay Do all capillaries behare the same in response to this material? What about the capillanes in the kidney itself? Shorr We have made no direct observations on the capillary bed of the kidney but we would expect a sanety of factors to operate Norm)the senin hypertensin system might oppose the action of ferritin or VENI might have a local action Honen er, under certain conditions the kidney cannot form V'El and concert ably the formation of renin may be similarly presented in such instances the vasodepressor action of ferntin could predominate and we suspect this may hue been the case in De Van Sile's studies on ·Antidiurctic Factors 73 hemorrhagic shock He found a striking alteration in the filtration fraction which fell profoundly as irrei ersibility des eloped We have found hyporeactive shock to be associated with predominance of ferritin in the blood and with an inability of the kidney to form VEV The capacity of the kidney to form VEM is destroyed by prolonged hypona I am wondering, Dr Gaunt, whether an observation we have made may have a bearing on your problem When we assayed the blood of a few adrenalectomized animals after withdrawal of DCA we found ferritin predominance If this is confirmed in a larger series it might be at least a contributing factor in the subsequent antidiuretic state Thorn But DCA in man does not correct the excessive antidiuretic effect Shorr That is right I had in mind the measurable antidiuretic factor in such animals Gaunt DCA in doses which do not repair the deficient diuretic response to water will reduce the concentrations of serum antidiuretic substance to normal Short In the adrenalectomized anımal the kidney loses the capacity to form VEM This capacity is restored by DCA or adrenal cortical extracts Shannon Is there any possibility of hyperreactivity in these antidiuretic states? Shorr Dr Lee has observed a curious hvperreactivity to epinephrine in the conjunctival vessels of patients with cirrhosis There is no hyperplasia and the vessels are dilated Shannon Hyperreactivity? Short The eye vessels of cirrhotic patients were hyperreactie Our studies in animals with nutritional currhosis were confined to the mesenteric vessels, these evidenced hyporeactivity which is what one might expect to find in vex of the predominance of VDV in the blood stream ·74 Renal Function REFERENCES 2 1 BARCLAY, ) A and COOK W T Effect of continuous intra venous infusion of pituitrin on renal function in man Proc 1815 Internat Plynol Congr 86 (1950) VAN DYKE HB, et al Isolation of protein from the pars neuralis of ox pituitary with constart oxytocic, pressor and druresis inhibiting activities ✓ Pharmacol & Exper Therap 74, 190 (1942) 3 PIERCE J G and DU VIGNEAUD V Preliminary studies on the anino acid content of a high potency preparation of the oxytocic hormone of the posterior lobe of the pituitary gland | Biol Chem 182 359 (1950) 4 LITTLE J M et al Effect of pitressin on the urmary excretion of chloride and water in the human Amer 1 Plyssol 151, 174 (1947) 5 SHANNON LA Control of renal excretion of water rate of liberation of posterior pituitary antıdıuretic hormone in dog | Exper Med 76, 387 (1942) 6 VERNEY E B Agents determining and influencing the functions of the pars nervosa of the pituitary Brit Aled 2, 119 (1948) 7 AMES RG MOORE DH and VAN DYKE H B The excre tion of posterior pituitary antidiuretic hormone in the urine and its detection in the blood Endocrinology 46, 215 (1950) AMES RG and VAN DYKE HB Antidiuretic hormone in the urine and pituitary of the kangaroo rat Proc Soc Exper Biol & Med 75, 417 (1950) 9 ANSLOW WP JR et al Chloruretic action of the pressor antidiuretic fraction of posterior pituitary extract Federation Proc 7, 3 (1948) 10 SARTORIUS OW and ROBERTS K Effects of pitressin and desoxycorticosterone in low dosage on excretion of sodium potassiumn and water by normal dog Endocrmology 45 273 (1949) 11 HART PD and VERNEY E B Obseryatrons on rate of water loss by man at rest description of constant temperature and humidity room spontaneous druresis during prolonged rest Clix Sc 1, 367 (1934) 12 MCQUARRIE I MANCHESTER RC and HUSTED C Study of water and mineral balances in epileptic children effects of diuresis catharsis phenobubital therapy and water storage Am Dis Cbild 43, i519 (1932) 13 WHITE H L and FINDLEY T JR Responses of normal sub jects and of patients with diabetes insipidus to water and salt ingestion ? Clin Intestigation 18, 377 (1939) 8 ·Antidiuretic Factors 75 14 FRASER, A M Diuretic action of orytocic hormone of pituitary gland and its effect on assay of pituitary extracts Pharmacol & Exper Therap 60, 89 (1937) 15 KUSCHINSKY, G, and BUNDSCHUH, HE Uber eine diuretische und Kochsalz ausschwemmende Substanz in Hypophysenhinter lappen Praparaten Arch f exper Paib u Pharmakol 192, 683 (1939) 16 DICKER, S E, and HELLER, H Renal action of posterior pitui tary extract and its fractions as analyzed by clearance experiments on rats | Physiol 104, 333 (1946) 17 HELLER, H, and STEPHENSON RP Effect of posterior pitur tary extract and its fractions on renal electrolyte excretion Nature 165, 189 (1950) 18 SCHAUMANN, O, and SCHMIDT L Uber die Beeinflussung der Wirkung von Oxytocin und Vasopressin auf die Salzdrurese durch Salyrgan Arcb exper Path u Pharmakol 205, 367 (1948) 19 SCHAUMANN, O Uber den Einfluss von Oxytocin und Vaso pressin auf die Ausscheidung von Kalium und Natrium durch die Niere Experientia 5, 360 (1949) 20 HELLER H Effect of hydrogen son concentration on stability of antidiuretic and vasopressor activities of posterior pituitary ex tracts | Pbysiol 96,337 (1939) 21 FRASER, A M Ratio between antidiuretic and pressor activities of posterior pituitary extract subjected to mild hydrolysis | Physiol 100, 233 (1941) 22 NOBLE, RL, PLUNKETT ER and TAYLOR NBG Factors affecting the control of the pituitary gland Rec Progr Hormone Res 5, 263 (1950) 23 O CONNOR W J and VERNEY, E B The effect of increased activity of the sympathetic system in the inhibition of water druresis by emotional stress Quart Exper Physiol 33, 77 (1914) 24 PICKFORD, M Inhibitory effect of acetylcholine on water drure $15 in dog and its pituitary transmission Physiol 95, 226 (1939) (and later reports) 25 MARSHALL, EK JR, and BLANCHARD, KC The antidruretic effect of 3 hydroxy Cinchonsnic acid derivatives / Pharm 100l & Exper Therap 95, 185 (1949) 26 MARSHALL, EK JR, BLANCHARD K C and DEARBORN EH Further observations on the antidiuretic effect of cinchoninic acid derivatives Bull Jobns Hopkins Hosp 86, 89 (1950) 7 ZUNZ, E, and VESSELOVSKY O Action de la corynanthine et de la yohimbine sur la diurese aqueuse Compi rend for de Biol 131, 135 (1939) ·Renal Function 76 32 28 FUGO, NW Antidiuretic action of yohimbine Endocrinology 34, 143 (1944) 29 HANDLEY, CA, and KELLER, A D Changes in renal function produced by morphine in normal dogs and dogs with diabetes insipidus | Pharmacol & Exper Tberap 99, 33 (1950) 30 BROWN WE HODGES, R E, and BRADBURY, J T A study of the antidiuretic effect of the depressant drugs used in eclampsia Tr Am A Obst Gynec, Abdom Surg 60, 177 (1949) 31 DE BODO, R C Antidiuretic action of morphine and its mechan ism J Pharmacol & Exper Therap 82, 74 (1944) DE BODO, RC, and PRESCOTT, K F Antidiuretic action of barbiturates (phenobarbital, amytal pentobarbital) and mechan ism involved in this action Ibid 85, 222 (1945) 33 EGGLETON, MG The effect of nicotine on the diuresis induced by ethyl alcoholPhysiol 108, 482 (1949) 34 BURN J H, TRUELOVE, LH, and BURN, I Antidiuretic action of nicotine and of smoking Brf m 1, 403 (1915) 35 EARLE DP JR and BERLINER, RW Effect of 2 3 dimer captopropanol on diuresis Am J Physiol 151, 215 (1947) 36 MOLITOR H and PICK E P Uber Druresehemmung durch Histamin und Cholin Arch f exper Path , Pharmakol 101, 198 (1924) 37 COW, D Einige Studien uber Drurese Arch f exper Path u Pharmakol 69, 393 (1912) 38 LLOYD C Wand LOBOTSKY, ) Relationship between serum antidiurectic substances and uninary corticosteroid in the human Am J Med 7, 415 (1949) 39 TAYLOR, N BG and NOBLE, R L Appearance of an antı diuretic substance in the urine of man after various procedures Proc Soc Exper Biol & Aled 73, 207 (1950) 40 GILMAN A and GOODMAN L Secretory response of posterior pituitary to need for water conservation I Physiol 90, 113 (1937) 41 ROBINSON, F H JR and FARR LE Relation between clinical edema and excretion of antidiuretic substance in urine Ann Int Mied 14, 42 (1940) 42 NOBLE, R L, RINDERKNECHT H, and WILLIAMS, PC Ap parent augmentation of pituitary antidiuretic action by various fetarding substances / Physiol 96, 293 (1939) 43 STUECK GH, JR, LESLIE, SH and RALLI, E P Preliminary obsers ations on the antidiuretic substance recovered from the urines of patients with currhosis of the liver Endocrinology 44, 325 (1949) 44 GROLLMAN A and Woods B A ner procedure for the de termination of the antıduretic principle in the unine Ibrid 409 ·Antidiuretic Factors 45 ANSELM VO K HOFFMANN F and KENNEDY V P Relation of hyperfunction of posterior lobe of hypophysis eclamps3 and nephropaths of pregnancy Edmburgl Al ] 39 376 (1932) 46 TIEL HM and Rrib. DE Observations upon occurrence of antidsuretic substance in urine of patients with preeclampsia and eclampsia Endocrinology 24, 297 (1939) 47 SCHAFFER NK CADDEN JF and STANDER HJ Measure ment of antidiuretic actasty as applied to edamptıc urine and properties of antidiuretic substances in rat urine pituitary and beef liver ford 28 702 (1941) 48 HAM G Card LANDIS EN Compactson of pituit in with antidiuretic substance found in human usine and placenta | Cm imestigation 21, 455 (1942) 49 ELLIS ME and GROLLMAN A The antidiuretic hormone in the ar de in experimental and clinical hypertension Endocrinology 44, 415 (1949) 50 LESLIE S H and RALL EP The effect in rats of high fat drets on the excretion of water and antidiuretic substances loid 41 1 (1947) 51 HALL CA FRAME B and DRILL VA Renal excretion of water and antidiuretic substances in patients with hepatic tirchosis and rats with dietary Iner injury this 44, " (1949) 2 RALLI E P et al Factors influencing asc tes in patients with Cirrhosis of the Inet Clm Jmestigation 24 316 (1945) 13 VAN DYKE HB ANTES R G and PLOUGH IC The excre tron of antidiuretic hormone in the urine of patients with cirrhosis of the boxer Ts A Am Physicians 63 35 (1950) 54 SIMSL Antidiuretic activits of urine in acute hepatitis Lab & Clin Med 33, 1476 (1948) 55 ZARROW M X The antidiuretic action of relaxin containing preparations Proc Soc Exper Brol & Med 71, 705 (1949) 56 BALZ S MAZUR A and SHORR E Hepatorsnal factors in circulatory homcostasis XX Antidiuretic act on of hepatit vaso depressor VDM (ferritin) Am J Physiol 162 198 (1950) 57 SHANNOS JA Control of renal excretion of water 11 Rate of liberation of posterior pituitary antidiuretic hormone in dog Exper Aled 16, 387 (1942) 58 RALLI EP et al Evidences for more than one antidiuretic substance on pitressin Am, Physiol 163 141 (1950) 59 HELLER Hand URBAN T r The fate of antid utetic principle of post pituitary extracts Physiol 85 502 (1935) 60 BIRNIE JH Inactivation of posterior pituitary antidiuretic hormone by Iyer extracts Peder iion Proc 9 12 (1950) ·78 Renal Function 61 RALLI, E P, et al The course of arrhosis of the liver in patients treated with large doses of liver extract intravenously Niedıcme 28, 301 (1949) 62 MERRILL, J P, et al Use of an artificial kidney 11 Clinscal experience ] Clin Investigation 29, 425 (1950) 63 MAZUR, A, and SHORR, E A quantitative immunochemical study of ferritin and its relation to the hepatic vasodepressor material Biol Chem 182, 607 (1950) 64 MAZUR, A LITT I, and SHORR, E Oxidation and reduction of ferritin sulfhydryl disulfide groups by liver Ibud 187, 497 (1950) The relation of sulfhydryl groups in ferritin to its vasodepressor activity Ibid 485 66 SHORR, E et al The antidiuretic action of the hepatıc vaso depressor ferritin (VDM) and its occurrence in conditions asso ciated with antıdıuresis in man Tr A Am Physicians 63, 39 (1950) 65 ·SOME PITUITARY AND ADRENAL INFLUENCES ON RENAL FUNCTION HARVEY L WHITE Department of Physiology. Wasbougion Unversity Scbool of Áledicrue MY FIRST remarks will be on work that we have been doing on some endocrine influences on renal circulation (PAH and inulin clearances) and on PAH Tm, and more recently on the ability of the kidney to form ammonia in acidosis We know that hypophysectomy lou ers these clearances and PAH Tm, we believe it does thus through loss of growth hormone Pure growth hormone, as pure as has been prepared so far, will restore the low values in hypophysectomized animals to normal and will raise the values in normal dogs to sell above normal(1) We are convinced that these great falls in clearances and Tm on hypophysectomy are not due to the following factors a) loss of thyrotropic hormone, because the drops are much greater than are seen on thyroidectomy, b) loss of gonadotropic hormone, because no fall occurs on castration, and c) loss of ACTH, because ACTH given to the hypophysectomized animal does not have any effect in restormg the low les els to the normal, which growth hormone does Our best growth hormone preparations (Ammours 22KR2 and a preparation made in the Biochemistry Department at our school by Dr Krahl, Dr Park, and Dr Daughaday) have been almost but not completely homogeneous electrophoretically On bioassay, the best preparations have been almost free from other hormones The enhancing effects on renal functions produced by the growth hormone cannot be ascribed to the thyrotropin content, because if we give a normal dog a dose of "pure" thyrotropin eight to ten times as large as the amount of thyrotropin contained in the doses of grous th hormone that we are giving we get nses of a few percent, whercas the growth preparations cause up to 100 percent increase We have further evidence that the fall in these clearances and Tm on hypophysectomy is not due to lack of ACTH, in that not Only is ACTH ineffectre in restonng these depressed values of the hypophy sectomized animals but neither is DCA nor whole 79 ·80 Renal Function adrenal extract in amounts adequate to keep the adrenalectomized dogs hidney function normal nor a combination of the two We therefore feel that the most reasonable viewpoint at present is that these falls in renal circulation and in Tm are due to lack of growth hormone or to something not yet separted from it The situation became less clear however when we found that the second (3PLS3R) and third (H1902) Armour batches did not work on normal dogs although thev did on hypophysectomized A dose of 05 mg per kg per day of the first batch (3PKR3) was effective but when amounts of the second and third batches equivalent to 05 mg of the first batch were given no effects were obtained on normal dogs although the hypophysectomized dogs still responded(2) Dr I M Bunding of the Armour Laboratories has been very kind in giving us several batches of growth hormone and in supplying data on the bioassays for contaminant hormones Table III shows that when 05 mg per kg per day of Armour s first batch (3PKR3) is given subcutaneously to a normal dog there is no effect on the clearances by the fifth day but a striking effect by the twelfth day Filtration fraction practically always falls The lower part of Table III shows the depressant effect of hypophysectomy on the clearinces and Tm and the restoration with growth hormone The effect comes on sooner than with the normal dog and we will see later that the hypophysectomized dog will respond to relatively inactive preparations which have no effect on normals Table IV shows the effects of growth hormone (3PKR3 active on normal and hypophysectomized dogs) and of growth hormone plus lpoadrenal extract (Upjohn) on the clearances and Tmn of a DCA supported adrenalectomized dog The DCA support was adequate in that PAH Tm was essentially normal and clearances and plasma sodium and potassium levels were normal although plasma NPN was somewhat elevated I may say that we have never been able on DCA alone to keep the NPN of adrenalectomized dogs down completely to normal it may not be much above normal but is always slightly elevated even though the inulin clearance may be quite normal Thorn How much DCA did you gue the DCA supported animal? White Three pellets of 75 mg eich That was on January 31 1948 and the dog lived for eighteen months with no subsequent ·TABLE ET FEFECT OF CROWTII JIORVONE OY RENAL FUNCTIONS IV NOR VAI AND HYPOPISECTOMIZED DOGS VORVAL DOC K43 PANT clearance Inulin clearance PAH Tm ml/min/ Plasma M12 352 ml/mm/ Glucose M? 116 mg / min 20 mg % 81 336 819 GOS 308 117 170 138 91 16 10/28/48 Normal 10/29/48 through 11/10/48-05 mg/g growth hormone (3PKR3) 11/3/19 5 days of growth hormone 11/10/48 12 days of growth hormone 11/12/48 11/16/48 HIYIOPHYSECTOMIZFD 39 6/13/47 Normal 6/16 17 Mornni 8/20/47 Simple hypopity sectomy 7/17/47 9/10/17 12/31/47 2/16/48 11/22/18 11/21/49 through 12/3/18 - 05 mg/g growth hormone (3PAR3) 11/29/18 5 days of growth hormone 12/3/48 9 days of growth hormone 81 91 79 80 264 263 95 96 19 23 Some Patuntary and Adrenal Influences 158 176 151 129 121 63 44 45 52 51 61 88 88 56 08 66 71 65 335 261 82 95 • Talles III & Il reprinteri from im I 1 huol 157 17 (1919) + Crowth fur une bilen daily sulk uztancously 17 18 106 99 18 ·82 TABLE IV EFFECTS OF GROWTH HORMONE ON RENAL FUNCTION IN AN ADRENALECTOMIZED POG* PAH clearance Joulin clearance PAH Tin Plasına NPN Plasma Glucose K41 mg % mg min /112 mga 12 14 37 46 75 85 ml/min / ml/min/ MI? M? 1/21/48 Left adrenalectomy, 3 pellets (75 mg each) desoxycorticosterone acetate implanted 1/28/48 Right adrenalectomy 2/24/48 213 86 10/25/48 276 96 10/27/48 through 11/8/48 -05 mg/kg growth hormone (3PKR3) 11/1/48 5 days of growth hormone 313 110 11/8/48 12 days of growth hormone 265 76 11/12/48 through 11/24/48 - 05 mg/kg growth hormone (PKR3) and 6 2 ml lipo adrenal cortex 11/18/48 5 days of hormones 229 89 11/24/48 12 days of hormones 182 96 • Growth hormone or growth hormone plus lipoadrenal cortex given daily Renal Function 17 16 115 91 48 60 48 74 78 57 ·24 Date Remarks TABLE V EFFFCTS OF RELATIYFLY INACTIVE GROWTII JORNIOVE PREPARATIONS ON REVAL FUYCTION IY NORMAL DOGS PAIT Inulin anl/min/11 CO L/m / 0: ml/min/11 246 91 260 3 85 121 188 91 92 206 83 DOCH MORVAL 4/11 & 4/12 Control 1/29 After 9 days of 1 23 mg/kg/day 3PKS3R 5/5 6 more days of 25 mg/kg/day 3PKS3R 5/16 10 more days of 05 mg/kg/day H1902 Interval of 3 weeks without treatment 6/10 Ifter 7 days of 195 mg Ag/dny 3PKS3R DOG 146 MORTAL 5/19 & 5/20 Control 5/31 Ifter 10 days of 07 mg/kg/day I{1902 6/8 6 more days of 14 mg Ag/day 111902 6/7 1 more day of 14 mg/kg/day H11902 178 Some Pituitary and Adrenal Influences 101 100 222 91 236 3 46 121 192 123 81 116 83 ·84 Renal Function therapy, finally dymg of adrenal insufficiency The growth hormone experiments were nine to ten months after pellet implantation We cannot say that there is no response, but it is not compırıble to that of normal or hy pophysectomized dogs Table V shows that our second and thurd batches in doses con taining amounts of growth hormone equal to or double the amount in 05 mg of 3PAR3 had no effect on clearances of normal dogs except once on the inulin The following table, Table VI, shows that these same preparations were active on hypophysectomized dogs ercept for a failure with inulin in K12 on 4/18 the hypophy sectomized dog is much more sensitive than the normal Bradley Did the body temperature remun constant? White Yes There is an increase in orygen consumption Ile have never seen a renotropically active preparation that did not increase the orygen consumption, this increase is not due to its thyrotropin content An amount of thyrotropin equivalent to the amount contained in these preparations gives no increase in oxygen consumption or clearances in either normal or hypophysectomized dogs While we have not ghen these prepirations to thyroidec tomized dogs we have in the past geen crude interior lobe preparations to thyroidectomized dogs with consistent increases in oxygen consumption This has some direct effect on tassues in general though presumably it is not thy rotropin and does not act through the thyroid gland Dock Are there any effects on the kidney with this? White Withn the time that these were given ten to twelve days there has been no change in the dogs body weight These are all adult dogs We have not killed these dogs at the end of twelve days to see whether or not their kidneys were hyper trophied we would huse to kill many dogs to find out I do not believe that these doses of growth hormone for this short period of time will produce any significant increase in kidney size llc have positive information about that in the rat obtained by Dr Bunding in the Armour Laboratories Rits were liy poplysec tomized at 50 grams Two weeks later they were started on growth hormone one group receiving 125 and the other 50 gamma per rat per day continued for fourteen days The body weight increased between 09 and 1 gm per dat while liver per body weight and kidney per body weight ratios remained the same as in tintreated ·Date Remarks TABLE YI EFFECTS OF RELATIVELY INACTIVE GROWTII HORVOVE PREPARATIONS OY REYAL FUNCTIONS IN IN POPHY SECTIOVIZED DOGS PATI Inulin ml /mn/11 CO 0 L/min/1 ml/mn/1* 126 221 39 287 95 63 DOG A39 HYPOPHY SECTOVIZED 4/7 & 4/8 Control 4/21 After 13 days of 125 mg/kg/day 3PLS3R 4/22 After 14 days of 125 mg Ag/day 3PASUR 1/25 Ifter 17 days of 125 mg Ag/day 3PKS3R Inten al of 6 weeks without treatment 6/13 After 10 days of 1 mg Ag/day 111902 6/14 After 11 days of 1 mg/kg/day 111902 2 86 274 100 101 205 Some Pituitary and Adrenal Influences 187 102 87 1/18 1457 DOG 442 HYPOPIO SFCTOVIZFD 4/5 & 1/8 Control After 11 days of 1 25 mg Ag/day 3PLS3R 4/19 After 12 days of 125 mg/kg/day 3PKSR 140 195 58 54 225 78 191 103 ·86 Renal Function animals ie the percentage of organ weight increase on growth hormone was no greater than that of body weight increase Taggart Vight not the proximal segment have hypertrophied out of proportion to the rest of the kidney as reported with the balance of the adrenal corticoids? White No measurements of nephron segments were made me do not know whether there was a demonstrable increase in proximal tubule size whether the machinery was larger or just tuming over more rapidly I do not believe that growth hormone administration of this duration would produce anything like the percentage increase in size of even one segment comparable to that of increase in PAH Tm which may go up from a third or fourth of normal to normal Thorn I take it the adrenalectomized animals did not show increase in oxygen consumption with this particular preparation White Unfortunately no oxygen determinabons were made on the adrenalectomized dog The renotropically active preparations always produced a rise in oxygen consumption in normal dogs and still more so in those hypophvsectomized Thorn We have shown that cortisone will cause an increase in basal metabolic rate which is not mediated by the thyroid(3) Wlute Some preparations have produced a rise in oxygen con sumption without rises in clearances but we have never seen a rise in clearances not accompanied by a rise in Oxygen consumption Gilman Will you comment on the filtration factors? Wlute We not infrequently see filtration fractions higher than usually found - we had one about 040 Our average is abopt 0 30 or 031 As does every one else we always find it much higher than in mın I find it difficult to believe that a dog ever normally filters 40 percent of the water going through the glomeruli but this is what he sometimes find on PAH and inuln clearances The extraction of diodrast - the work was done before the days of PAH - at low plasma levels is the same in the hypophysectomized as in the normal dog ie we believe that diodrast and presumably PAH clearance is just as good a measure of renal plasma flow in the hypophysectomized dog as it is in the normal We have no observations as to the behavior in the adrenalectomized animal We have also seen filtration fractions of 04 in normal dogs not ·Some Patuitary and Adrenal Influences 87 too infrequently It happens that these are high filtration fractions here but no higher than we sometimes see in the normal dog I am sure everybody has had that experience and probably not been quite sure what it means I admit it seems to me an awfully high percentage of filtrate Latison Have you considered the possibility of measuring actual size change in the kidney by intravenous pyelography? A radiolo gist assured me once that one could obtain at least an indication You could then control it in the same animal Dock With straight films of belles on dogs you can get lovely kidney shadows and if you take them at four or five feet you can make measurements with great accuracy White Do you think you could detect a 20 percent change? Dock A 20 percent change in length would be striking 11 lute We have not done that I dont believe that there is a change in mass comparable with the change in clearances We have been concerned about the ineffectiveness of certain growth hormone preparations in normal dogs One can think of three logical possibilities to explain this if one doesnt stretch the connotation of the word explain too far One possibility is that the material is not growth hormone at all that the actic prepara tions are active because they contain some contaminant which is really the renotropically active substance I use the term "renotropic to save time meining a substance which influences kidney function without any commitment as to how or why it does In other words maybe it was not growth hormone but something that was present in the active preparations and the inactive preparations were inactive not because they did not have enough growth hormone but because they did not have this contaminant The second and just as logical possibility is that the active preparations were actise because they were reasonably pure growth hormone and the inactie were inactn c because they contuned in inhibitory substance which presents exhibition of the renotropic without prei enting the exhibition of growth promoting activity The third possibility is that the mother molecule of the grouth hormone has both growth promoting and renotropic activity but in preparation somc batches suffer some damage in such a way that the renotropic activity is impured without the growth pro moting activity being correspondingly impaired ·88 Ronal Function These are three possibilities one just as good as the other from the standpoint of logic but with no evidence so far to permit a choice among them Thorn Were these different animals in the second assays or the same animals? White Some were the sime and some were different We spent considerable time trying to decide between these three possibili ties we are not sure which it is I think it is the last and will give you boetly some of the evidence One thung that seemed worth doing was to get some intermediate fractions that occur in the preparation of the purification of growth hormone in the hope of finding one that would be renotropic but not growth promoting The other possibility that one of these fractions contains the hypothetical inhibitor could be tested by adding fractions to active growth hormone preparation if inhibition occurred it could be argued that it is the presence of such postulated inhibitor in the inactive preparations that makes them inactive I will make the story brief by saying that neither such activity has been found Dr Wilhelmi was kind enough to send us some of the fractions that he had obtained in various preparations that he made He sent us some of hus fractions CD and E and one which he calls PH5dep those who are acquainted with the process will recognize these All we can say is that none of these substances when given to an animal had much effect on clearances in other words there was no evidence that any one of these frictions investigated contained the postulated renotropic contaminant in any concentrated form Of course if we tried five more fractions we might find one that did the other attempt to see if addmg any of these fractions to an active preparation would make it mactive also has failed to produce evidence of inhibition although we have not tried all of them yet This work is time consuming and I think we have spent about as much time on it as is justified Table III illustrates what I mean It shows a normal dog that we tried out to be sure that she did respond to a known reno tropically active preparation D2XR2 Armours purest growth hormone preparation which is almost homogeneous electro phoretically That produced the expected effects on clearances and as you can see the filtration fraction fell The inimal was permitted to recover and six weeks later was given 05 mg per kilogram per day of Wilhelmi s fraction D This liad perhaps some ·89 Some Pituitary and Adrenal Influences effect though not much on the PAH That is certainly significant on the inulin I wont say that none of these fractions did anything to the clearances but the point I am trying to make is that if they consisted largely of thus hypothetical active contaminant thes should have been even more active than the presumably pure growth hormone preparation was but none of them were any where near as actie The animal was then given thyrotropin in amount eight times as much as is contuned in our dose of growth hormone and its effect on the clearances is not comparable to that of the grou th hormone TABLE VII EFFECTS OF A ACTIVE PREPARATION OF A RELATIVELY INACTIVE FRACTION AND OF THROTROFI Date 143 NORMAL PAN Inulin FF 0 11/21 10/5/49 243 80 032 122 10/18 through 10/31 042 mg ng day CH 22 R2 10/28 482 132 027 168 10/31 451 125 028 156 11/14 31" 104 118 237 85 036 110 12/6 through 12/13 05 mg Ag/day Wilhelmi fraction D 12/15 293 112 03S 114 12/30 229 6 033 112 12/31/49 through 1/9/50 01 mg kg/day thyrotropm PRR3 1/9/50 250 99 035 116 1/19/50 278 83 032 033 Jancu ay It is interesting that it does not increase the orygen consumption White There is no increase in origen consumption in normal dogs with this dose of thyrotropin which is still a small one This is merely to confirm that the thyrotropin content of this preparation 22LR2 is erceedingly low its renotropic action cannot be explained br thyrotropin contamination ·90 Renal Function Table VIII shows results with another fraction Here is another normal dog who responded to 22LR2 in the expected fashion Wilhelmis fraction E had a possibly significant effect on PAHI clearance and it apparently had a considerable effect on inulin clearance here we are suspicious of this inulin value however because it was done at unusually low plasma levels and because the same preparation had no effect on either PAH or unuln clear ance in another dog who responded well to 22R2 In other words the effect if any is much less than that of the pure growth hormone preparation In considering the effects of all these fractions in several dogs we obtained no evidence for either the active con tamınant or the inhibitor theory We have had two or three more extracts that were prepared in the Biochemistry Department at our school and while it does not settle the question all we can say is that no evidence has been obtained in favor of either of these first two possibilities By exclusion this would perhaps favor the view of some change in the protein molecule which influenced one activity more than it did the other TABLE VIII EFFECTS OF AN ACTIVE PREPARATION AND OF FRACTION E 149 NORMAL Date РАН Inul n FF 0 11/8/49 216 97 0 45 11/12 through 12/2/49 0 42 mg/kg/day G H 22R2 11/21 431 163 0 38 181 11/23 507 151 030 192 12/2 381 130 0 34 12/20 236 95 040 118 12/20 through 12/29 05 mg/kg/day Wilhelmi s fraction E 270 052 108 12/29 1 1 142 1/1/50 Sick not eating died on 1/8/50 cause tindetermined van Dyke You did not try to alter the original extract which had this effect?

·91 Some Pituitary and Adrenal Influences Wlute No We have thought about trying either gentle onda tion or reduction to see if we could artificially damage one property without the other van Dyke Could you fractionate it at all? Could you separate it into your active and less actise fractions and test those? W lute In what way? Lan Dyke Perhaps by the alcohol fractionation technique White These fractions have come out of the Wilhelm Fishman Russell procedurel 4) and are less homogeneous electrophoretically than the 224 R2 as well as being much less actre in promoting growth I think it would be quite a job if you really tried to frac tionate then we have not tried to do so can Dyhe I thought that the original Armour preparation with which you had your first difficulty was less potent White The first one was potent Fortunatel we obtained a good one the first time Lan Dyhe But the second one? White That was not Lan Dyke That was inactive in normal dogs White That is right can Dyhe If you fractionated that extract might you not possibh find the source of the discrepancy? White If we knew enough about how to fractionate it possibly we could It was prepared by a procedure identical with that used for the first preparation tan Dyke Perhaps this could be done if an adequate suppk of extract were available 1 late None of it is left That is the trouble lle get the extract in simples of abont 100 to 200 mg at a time Obviously the best way to show whether the renotropic principle is grou th hormone or not is to get a prepustion which you can be sure does not have anything but growth hormone in it and sce if that works I would like to discuss briefly one other point Our 22LR2 which 118 a very actre preparation was sent to us in three hottles The ·92 Renal Function first bottle was used while it was still quite actre The second was quite active, but after having been opened about two months it became less active and at the end of six months it had no activity at all in the normal dog, although in the hypophysectomized dog it still acted In other words, it spontaneoush became the same material as H1902 and 3PKS3R We did nothing to it - it had been in the desiccator all the time -- and I don't know why it went bad We assay ed it for growth promoting activity, as seen in Figure 11 We used eleven rits, hypophysectomized at 115 gm I want to point out that these are not the conventional conditions for growth hormone bioassivs Most people use 60 gm rats and start the hormone administration ten days after operation We happened to have some 115 gm rats and they were not used until six weeks after the operation So these cannot be quantitatively compared with the responses of rats of 60 gm begun ten days after the operation But the magnitude of the weight increase is about what one would expect to find with these sume doses of growth hormone preparations Four rats recewed 5 gumma four recerved 10 and three of them recenved 20 and the average increase was about 09 to 1 gm per rat per day, the 20 gamma rats increasing a little more than the other two This shows that growth promoting 14 12 - 310 013) 014) (4) WEIGHT INCREASE IN GM HYPOPHYSECTOMIZED AT 115 GM GH BEGUN 6 WEEKS POST-OP 0-0 50 GH/RAT/DAY IOT 0-0208 - NO OF RATS USED 6 DAYS OF GROWTH HORMONE 8 10 12 ADMINISTRATION 19 FIGURE 11 Growth promoting activity of a sample of Amours 22KR2 growth horinone originally highly active renotropically which on standing had lost its renotropic activity in normal dogs ·93 Some Pituitary and Adrenal Influences activity had not been greatly impaired after renotropic activity in normal dogs had been lost although it does not prove that there 11d been no impairment of growth promoting activity Dr Bunding took the few remaining millgrams of tlus sample and has sub sequently found that its grou th promoting activity is about 60 percent of the original so the decline in renotropic activity has been much greater than that in growth promoting activity Thorn I want to be clear on one point The material that was active in the hypophy sectomized ammal and not in the normal when given tuo or three times the dose still had no effect? II lute That is right le went up to two and one half times le normal dose and it still did not work in normal dogs The third ittle which was opened after we found the second bottle hnd ne bad was just as active as the first one The only difference handling it and the second bottle was that the top had not been en off Yet it was not hermetically senled So I cannot explain it was the matter it may have been a matter of moisture one animal the response fell off with repeated administrations e same active preparation We knou that it was not becouse preparation * 2s becoming less active becouse at the time of the last administration to this normal dog which failed to respo it was giving good responses in other normal dogs That is t only normal dog who has acquired a tolerance to this material other dogs where repeated courses have been given thes him continued to respond except in the case where the material weni bud Thorn is the antibodi titer to the matenal higher in the refractory dog? W lute We have no obsen ations on it The arterial pressure is not changed in these animals so obviously the renal vascular resistance is decreased by what mechinism we dont know We hue no observations on what would happen if the materal were gren to an animal with denerated Lidncis although I believe that we would still get increases Selhurt The hematocnt stays normal? Does the cell olume go down or anything of that sort? White le did not do careful hematocrit determinations but I am sure they did not change significant Just looking at the tubes ·94 Renal Function as they were centrifuged I know there was no significant rise or fall in hematocrit We had no reason to think there would be any change and did not measure it We leave the matter then by saying that these preparations do raise the clearances and the Tm in normal and in hypophysectomized dogs that these effects are not due to thyrotropin content that the hypophysectomized dog is more sensitive than the normal and that the failure of some preparations to show the effect in normal dogs may be due to the fact that there has been differential damige done to the molecule We offer that simply because we do not have any other explanation No evidence was obtained for the inhibitor theory or the active contaminant theory although these possibilities are not excluded It may be that the active miterial is not really growth hormone but some hitherto undescribed substance which is present in active form in some of these growth hormone preparations and not in others and which has essentially the same electrophoretic mobility as the growth hormone I would like to say just a few words about another type of work in which we have been interested We felt that some other kidney functions which are susceptible to quantitative evaluation in addı tion to the clearances and Tm might be worth investigating as to their being influenced by endocrine factors So we produced acidosis in normal dogs to stimulate ammonia formation What we wanted to do was to establish conditions which would give us a repro ducible ammonia response to acidosis and see whether the hypo physectomized and the adrenalectomized dog responded in the same way We found that when 35 to 5 mEq ammonium chloride per kilogram are given by stomach tube or subcutaneously to normal dogs that the plasma CO falls usually to 16 or 17 mEq within one or two hours TI ammonia production rises at the same rate se rate of ammonia production reaches its maximum and levels off within about two hours with somewhat larger doses 1e 7 to 8 mEq per kilogram the plasma CO drops to 12 to 14 mEg and there is usually a greater ammonia Output than with the lesser dose On two occasions we have given 10 mEq per kilogram and seen no greater ammonia Output than with the 8 mEq which we give as a standard dose In such acidosis ie in response to 7 or 8 mEq ammonium chloride per kilogram with plasma CO dropping from the normal of 23 or 24 down to perhaps 13 14 or 15 the ammonia output rises from a control value of about 05 to 15 mEq per hour to 45 to 55 mEq per hour for 12 to 15 kg dogs That will happen within two hours This happens to be about ·95 Some Patustary and Adrenal Influences the same per unit body weight as man's maximum ammonia output, which has been described as in the neighborhood of 20 mEq per hour(5,6) I believe Di Pitts found about 55 to 6 mEq per hour as the highest outputs in dogs If this is not ammonia Tm, it is close to it Even if this is not Tm, we have established a predictable response of the normal dog to this particular dose of acid, reproducible wathin a reasonably narrow range With the smaller doses, eg, 35 to 5 mEq per kilogram, the plasma CO has already begun to rise and the rate of ammonia excretion begun to fall at the end of three or four hours after the acid administration With the larger doses, up to 7 or 8 mEq per kilogram, the plasma Co. is still down four hours later and the ammonia output has not yet begun to fall By the next morning, however, prouded the doses are not larger than 8 mEq per kilogram - and we have not in estigated larger doses from this standpoint-twenty-four hours after the acid is ghen, the CO, level and ammoma output are back to normal in normal dogs, in that respect they differ quite markedly from the human subject If we now compare a hypophy sectomized dog with the normal we find that he behaves the same as the normal The number of experiments on this is not as large as it should be but so far our finding is that the ability of the kidney to form ammonia is unchanged in the first few wechs after lypophy sectomy Thus finding contrasts with that in a single adrenalectomized dog with inadequate DCA support This dog had been adrenalectomized a year and one-half before these erperiments and up until about a month before the experiment had remained in excellent condition with the 225 mg DCA subcutaneously She began then to show definite signs of adrenal failure, loss of appetite, wealness, and beginning fall in plasma sodum which had been between 140 and 145 all this time but nou gradually went down It fell as low as 130, with some rise in potassium During this time the animal was going down hill, and was in muhed adrenal insuficiency when the ability to form ammonia was tested Of course, the animal had acidosis cien before she was given the ammonium chloride the plasma CO, was running around 19 compared to a normal of 23 or 21 With relatively small acid administration (22 to 33 mEq per kg), pisma CO, fell to 11-13 mq, but there was no increase in ammonia production. This was not because the ammonia production was alreadı high The control outputs, with out giving acid, were 0.2 to 13 mcg per hour, the same as with normal animals on a comparable diet This animal was kept on a low meat and high milk diet So it appears then that the kidney ·96 Renal Function in an inadequately supported drenalectomized dog shows great impairment of ability to form ammonia in response to acute acidosis Most of you have seen in the September 1950 issue of the American Journal of Medicine that Dr Pitts reported somewhat similar findings on adrenaſectomized rats and found at least a partial restoration of ammonia forming ability on DCA Or cortisone(7) In Figure 12 is plotted the highest ammon a excretion in meq per kg per hour against the lowest plasma CO, in a gven experi ment Usually the lowest plasma CO coincided with the period of highest ammonia dever more than an hours difference most of the plotted points being in the third or fourth hour after the acid dose When the normal animal has the plasma co lowered to 14 to 17 mEq the ammoma output is in the neighborhood of 04 mEq per kilogram per hour With still greater acidosis where plasma CO Jei els are belowy 13 zate of ammonia output is usually between 4 and 5 per kilogram per hour The hypophysectomized dog - one animal -- shows a response not significantly different 05- 8 HIGHEST NH, EXCR MEQXKG HOUR *A 0.2 X NORMAL HYPOX ADRENEX X x 12 13 14 15 16 17 LOWEST PLASMA CO2, MEONLITER FIGURE 12 ou put plotted at int lo est plasma co nelsn * mot dog ncy 6 toman cente ·Some Pituitary and Adrenal Influences 97 from the normal, whereas in two observations on the adrenalectomized dog, even though acidosis was more severe than it had been in the normal or the hypophysectomized, there is essentially no rise in ammonia output We have no observations on restoration with adrenal cortical therapy, the only observation being on this dog which is already pretty well down hill We have known for quite a while that the hypophysectomized dog on a normal diet handles electrolytes in a quite normal fashion, mrintains normal plasma sodium and potassium, stays in electrolyte and water balance, and does not develop edema or salt depletion It seems then that the formation of the salt-regulatıng hormone is not under anterior lobe control We also know that the zona glomerulosa of the dog does not atrophy after hypophy sectomy, whatever that may be worth m concluding anything as to the site of formation of the DCA lle hormones as compared with the others That is also true in the rat We also know that ACTII in human subjects usually produces some salt retention, at least as the initial effect This could mean that there is an overlapping of function, that the hormones not primarily concerned with salt retention have some salt-retamıng effect, or it could mean that the zona glomcrulosa, if that is the place where DCA-like substances are formed, is a little bit response to ACTH It seemed to us that the obvious thing to do, in order to see whether the ability of the hypophy sectomized dogs' adrenal corter to form salt regulating hormones was normal or not, was to put it under stress In other words, the fact that he maintains normal plasma sodium and stays normal when he is on a normal salt intake is not a very sciere test So we put such dogs on a very low sodium intale, 1 mEq a day This was clone by giving Lon lac, a sodum free null powder, as the source of protein, plus fat and carbohydrate and added vitamins On such a regimen the hypophy sectomized dog docs go into sodum deficit a very extensive negative sodrum balance The plasma sodnim dropped to 123 where it had been 143, and the dog would have been dead in another day, I am sure, if we had not gui en hier 1 luge dose of sult and resumed a normal diet In one case it took only tuo weeks for thus to come on, in another case it took sir wuchs It appears that the adrenal corter of the lypophy sectomized dog is not completel normal so far as its ability to forin DCA-like hormones is concerned, but it is autonomous enough to get along qute well, so far as salt regulating hormone is concerned as long as the animal is on a normal, not necessirik lugh, sılt intale He cannot stand sesere salt deprna.

·98 Renal Function tion indefinitely If at this point we give these dogs a large dose of salt, they retain it We gave them 130 mEq and they did not put any out in the next twenty four hours, not more than 2 or 3 mEq, they soak it up It seems as though they hold more salt than you would think they had room for as calculated on the basis of our rather inadequate calculations of body fluids Selkurt Does the hypophysectomized dog without preliminary salt deprivation have a tendency to hold on to salt or lose it when loaded? White He has a perfectly normal salt test, 1e, output response to loading Selhurt Only when you initially deprive them does that phenomenon come on? White We give the normal dog a big dose of salt, 60, 70, 80 to 100 mEq in the form of 0 9 percent sodium chloride by stomach tube and follow the rate at which that comes out in the urine The normal dog gets rid of it in about eighteen to twenty four hours while it takes a man about four days It follows a fairly reproducible curve, the maximum output being in either the second or the third three hour period all or most of it is out in eighteen hours and all is out in twenty four The hypophysectomized animal behaves in the same way as the normal, there is no evidence that he loses it any faster or any more slowly than the normal Also, when the normal dog is put on this very restricted salt dret, he gets along perfectly well He just reduces his output to the level of his intake if there is any negative balance it is very small The normal dog we can say can stand at least eight weeks of this very restricted salt intake whereas the hypophysectomized dog stands it from two to six weeks Shorr You might consider another type of experiment, that is, raising the metabolic rate with thyroid Occasionallv, when we attempt to restore the basal rate of a patient with myxedema by giving thyroid, we find that the myxedema is of pituitary origin As a result of thyroid therapy the relative adrenal insufficiency will now become apparent and the patient will go into an adrenal crisis Winte Yes Thorn You cannot raise the BMR of the hypophysectomized animals with thyroid? Shorr No, you cannot ·Somc Pituitary and Adrenal Influences Thorn It is uell known that moderate doses of thyroid ve ineffective in correcting the hypometabolism of pıtıntıy-deficient patients Whte I cannot speak from expenence about the giving of thyroid to the hypophy sectomized dog but they seem to be even more sensitive than the normal dog to thyrotropin Thorn But the metabolic response is very poor to administered thyrox hormone White We have no experience on tlint I do know that they will respond to thyrotropin We have also found that mulin and PAN clearances and PAN Tm are increased by oral thvroid administration in the lypophysectomized dog even more than in the normal It appears then that the adrenil cortex of the hypoplry sectomized! dog is not impaired in its function of maintaining the ability of the kidney to form ammonia but that it is somewhat impaired in its function of maintaining the hidney s ability to conserve silt, although st has to be put to great stress before we can demonstrate such impairment Dr Pitts asked me first if I would say sometlung about endocrine influences on intraccllular tubular processes I told him I did not know enough about it to discuss the matter with any authority or confidence I might however point out one or two speculations It is possible that in the absence of the adrenal cortex the kidneys bulity to hydrolyze glutamine into ammonia and glutamic acid is impared Some of you may remember that Jiminez Diaz(8) reported decreased ammonia formation in ustro by kidney tissue of adrenalec tomized cats in a di alanıne mcclum indicating lowered amino Oxidase actuity or concentration So fu 15 I know that is the only observation on the change in the enzyme content or ictivity of kincs tisstie ys influencrid las some endocrine factor i dont know thit this has ever been confrmed or denied and I do not know of any nhsersations on the glut imın ise content of the kidnes of the nelrendectomized arumul as compared with the normal It may up) by shut ths is the reson u kunt does not work s7 «c!? Igual I know of no obsers abons regard ng an change in the ammonia prestursors in the blood in an endocrine condition I dont knos of ans reason to think that there is an deficit af glutamine sur- ·100 Renal Function plied to the kidney in adrenal insufficiency One might, of course, postulate that if in response to acidosis there is an increased stimulus to adrenal cortical activity, that as the result of the neoglucogenesis, protein breakdown, there might be an increased supply of amino acids available as precursors for ammonia produce tion That seems a little farfetched to me, I just don't know We know that Lotspeich and Pitts(9) showed some time ago that if you do gre the animal more amino acids when he is in acidosis you can increase his ammonia production, and it is conceivable that some such mechanism exists It has also been known since the time of Walter back in 1877 that the rabbit is much less capable of forming ammoma in response to acidosis than is the dog or man(10), and again one might speculate that perhaps the rabbit's kidney does not have enough glutaminase or renal amino oxidase Perhaps someone here Inous some published work which I have missed, or some work in progress and not yet published, on the enzyme content, either glutaminase or amino oxidase, of the kidneys of hypophysectomized or adrenalectonuized animals as compared with the normal We would assume from these findings, if they turn out to be consistently true, that the hypophysectomized animal's kidney enzy me system is all right as far as ammonia production is concerned, but not the adrenulectomized DISCUSSION Gaunt I would like to ask two questions of Dr White One concerns your work on how these hypophy sectomized animals handle salt loads Apparently you do not agree with Dr de Bodo(11) since you found that sensitivity to salt restriction took a long time to develop, did you not? Second, how do the hypophy sectomized ammals handle water? White We have been fortunate in this work in some ways The first time, the very first growth hormone preparation we tried worked well If it had not we probably would have qut then The very first hypophy sectomized dog we put on 1 mEq of sodium a day went into severe salt deficiency in two ucels The next one took six weeks If it had been the first one we probably would have stopped after about four weeks I should have refreshed my memory as to how long de Bodo followed these dogs on restricted salt intake ·Some Patutary ond Adrenal Influences 101 1 might say thus, we had one hypophysectomized dog which neser did go ito salt deficiency, and about the time we were becoming concened about that she came into heat and we found at autopsy that there was some anterior lobe left That is, of course, always a possibility Any conclusion that the anterior lobe was not necessary must be held in abeyance until the autopsy shows com. plete absence of anterior lobe We late repeatedly seen small fragments of anterior lobe regenerate I don't mean we have watched it We have seen females that would come into heat after two scars This may sound fantastic, but one of those dogs was bred and did not become pregnant, six months later she came in again and was bred and did become pregnut but mscarried, si months later she was bred again and carried her pups to term but could not murse them sıx inonths later she had pups at term and nursed them normally We interpret that as a small bit of anterior lobe growing larger just such isolated observations as that show that it can and does happen Shock Do you have any ideas about the mechanism whereby the Tm can be increased in normal animals Winute I don't know except that it must have something to do with enzymatic processes, which is another way of saving i don't know It makes the wheels go around faster I thinh rather than making them Ingger although this is only a guess Taggart We have a limited amount of information wuch may have some bearing on this point Last year I described our techmquc for studymg PAll transport in kidney slices Since then we use treated a number of rabbits with either testosterone or thyroxin for sex days before they were sucrificed for the propır: tion of kidney slices The doses administered were essentially the same as those which have previously been found to produce significant increases in dioxirast Tm in dogs( 11.12) Thyroun administration increased the respiration of Ladiney slices to approva. mitely 30 percent alone the normal controls, whereas testosterone liud no appreciable effect on respiration Neither agent significantly Altered the rate of PAll transport Certainly in the case of thyroxin, the trachuners of oxidative metalxolism was tuming over faster than normally, Inut this was not reflected in the rate at which PAII was trinsported m the slicy White Ilnd those closes increased the Tm in the lang ralınt? Taggart We do not hos ·102 Renal Function White You did not determine that? I dont remember about the rabbit I remember that testosterone is said to raise it in the dog(11) but not in man(13) Taggart The point which I wished to emphasize was simply that a speeding up of the metabolic machinery as observed with thyroxin apparently does not increase the transport capacity White The enzymologists point out instances where the oxygen consumption is not impaired and still the transfer mechanism is impaired Taggart A good example is dinitrophenol which can increase the respiration of kidney slices up to 100 percent above normal but at the same time markedly depresses PAH transport White An instance where an agent is known to produce increased transfer in the living animal but not increased transfer per unit weight of kidney in vitro would favor of course the view which you no doubt hold that maybe this is merely an incre isc in muss of tubular tissue and not a faster turnover Taggart I was wondering whether the effects which you have observed with growth hormone were associated with any morpho logical alteration of the nephron White We have no observations on that It might be possible to detect whether there is a change in kidney size through x ray obserı ations as Dr Dock suggests I did not realize you could do that Taggart An overall measurement of kidney size would not be entirely satisfactory I am more interested in knowing what happens to the various portions of the nephron Grafflın Acromegaly as is well known from the work of Cushing and Davidoff (14) and others is usually characterized by hep? tomegaly and nephromegaly Cushing and Davidoff reported that the enlarged kidneys (and liver) were hustologically well within normal limits and I believe this has been the general experience of interested pathologists I should like to call attention to the work of Turley who around 1916 made an entirely unique war reconstruction of the glomerulus ind proximal convoluted tubule in acromegaly(15) In this case the weight of the kidneys was approximately twice the normal and no abnormal histologic change was observed The essential facts contributed by the model are ·Some Pituitary and Adrenal Influences 103 that the pars concoluta of the proumal tubule of the particular nephron reconstructed had undergone a striking increase in length but not in diameter, and that the wcrease in length was roughly proportional to the enlargement of the ladney as a whole (appron mately twice normal) The size of the glomerulus was within the normal range Taggart Selye has described the effect of testosterone adminis tration in mice(16) The kidneys of treated animals sucreased their Weight by 50 percent or more but essentially all of the increase was nitrbutable to hypertrophy of the proumal and distal con soluted tubules Thorn To check the growth hormone work on the dog you would like to know the Tm is very high in the acromegalic would you not? Ilas not any body ever reported one? Graffin I dont know of one Schurt May I ask a question in regard to the change in plasma flow relative to Tm Does the Tm increase to a greater degree than the effective plasma flow or does it go up proportionately? It might throw some light on whether this is real growth or whether the kinetic mechanism is speeded up lou would expect that the vascular channels would grow in proportion of the effect is due to lıypertrophy White The Tm in the hypophy sectomized dog is lowered more than the plasma flow is and they are bath raised essentially to normal ly growth hormone so that the im is raised more than the PAII clearances in the hypophy sectomized animal in the normal dog the Tm is usually not raised quite as much as the clearance as we frequently double the PAlf clearance in the normal dog and usually somewhat less than double the Tm In the hypo ply sectomized dog we again double the clearance and inple the Tm it has been down to a thud of normal and is used to about normal Shannon One thought occurs to me that might be pertinent to this discussion We are considering whether increased alulity to transport PAll is representatise of hypertrophy or representatic of increased activity l'ossibly Di Taggit might comment on hus experience with the simple administration of acctate Acctate increases transport rapidly and about as stnhingly as Dr Whites growth hormone ·104 Renal Function Taggart Naturally I am interested in the possibility of differen tating the effect of acetate, which occurs within ten to fifteen minutes, from that of the hormones which I believe develops only over the course of several days White In noimals we usually dont see anything in the first five days It will be seen in ten days but usually not in five In the hypophysectomized the increase will be seen in five days Shannon How rapidly does it decline Dr White? White After five days of administration the PAH clevrance was 336 ml per minute with a normal of 352 ml per minute thus no change was observed in twelve days it was doubled or more Two days after we stopped it was well on its way down and six days after we stopped it was below 1 REFERENCES WHITE H L HEINBECKER P and ROLF D Enhancing effects of growth hormone on renal function Am, Physiol 157, 47 (1949) 2 Variability of effects of growth hormone preparations on renal functions lbl 159, 596 (1919) 3 HILL S R JR et al Effect of ACTH and cortisone on thyroid function Clm Endocrinol 10, 1375 (1950) 4 WILHILMI A E FISHMAN J Band RUSSELL JA A new preparation of crystalline anterior pituitary growth hormone Biol Chem 176 735 (1948) 5 TOLLING A On the mechanism of the ammonium chloride acidosis Acta med Scand mat 71, 221 (1929) 6 SALTER W T FARQUHARSON R F and TIBBETTS DM Studies of ion of acid base balanc on of phos phates 7 PITTS R F Acid base regulation by the hidneys An i Med 9 356 (1950) 8 JIMINIZ DIAZ C Death in Addison s disease (functional renal failure) Lancet 2 1135 (1936) 9 LOTSPIICH WD and PITTS RF Role of amino acids in renal tubular secretion of ammonia / Biol Cher 168 611 (1947) 10 WALTER F Untersuchungen uber die Wirkung der Sauren auf den thierischen Organismus Arch f exper Paib u Pharmakol 7, 148 (1877) ·1 Some Pıtutary and Adrenal Influences 105 11 WELSH CA, ed effects of testosterone propionate on renal function in dog as measured by creatinine and diodrast clearance and diodrast Tm Am Pbystol 137, 338 (1912) 12 EILER J ), ALTHAUSEN TL, and STOCKHOLM, M Efect of thyroxin on maximum cate of transfer of glucose and diodrast by the renal tubules Ibıl 140, 699 (1944) 13 KLOPP C YOUNC NT and TAYLOR HC JR Effects of testosterone and of testosterone propionate on renal functions in man | Clm inuestigation 24, 189 (1915) 14 CUSHING Hand DAVIDOFF L M The Patbologueal Imdings 19 Tour Autopsied Cases of duromegdy usb a Ducussion of il - Significance (Monograph 22 ) New York Rockefeller Institute for Medical Research 1927 15 GRAFFLIN AL The normal the acromegalic and the hyper plastic nephritic human nephron A further consideration of the plastic reconstruction of Louis A Turley And Path 27, 691 (1939) 16 SELYI H1 Effect of testosterone on kidney | Urol 42, 637 (1939) ·THE ACTIONS OF ACTH AND CORTISONE ON RENAL FUNCTION IN MAN* CHARLES H BURNETT Department of Internal Medicine Southwestern Medical School University of Texas OUR INQUIRY into the effects of ACTH and cortisone was conducted along two general lines(1,2,3) (A) The effects of large doses of these substances on standard renal clearances and on the simul taneous urinary excretions and plasma levels of a number of the constituents of the urine have been determined in normal human subjects, in patients with Addison's disease, and in isolated patients with glomerulonephntis and with periarteritis nodosa (B) We have attempted to quantitate certain aspects of the abnormalities of water and salt metabolism in Addison s disease before and after cortisone therapy These latter investigations were directed espe cially toward studying the effect of this steroid on the inability of patients with adrenal insufficiency to excrete excessive quantities of sodium and water Special mention should be made of the dosages which were employed in these studies Really very large doses -- up to 400 to 600 mg of ACTH a day and comparable quantities of cortisone - were given to the normal subjects The reason for this was that in these normal individuals it was found that doses of this magnitude were required in order to demonstrate an ACTH or cortisone effect in terms of increased uric acid excretion cosinopenia and weight gain appreciable effects on renal function were obtained also only with the large doses With ACTH, Cin and Cpan were both significantly increased as shown in Figure : - ne instance and was unchang to 200 mg a day and Cras similarly changed in the one instance where measured By increas ing the dose to very large quantities Ciu now was significantly Jses of 100 nor 21 100 ·Actions of ACTII and Cortisone on Renal 107 EFFECT OF ACTH AND CORTISONE ON RENAL HEMODYNAMICS CCM CN NORMALS DC Control Cr* ACTH Crth Cort sone CCM ADDISON'S DISEASE CUA С. MILA PM AC RP CHAM CPAN . 00아 mo 300 700 Cp Control CPanth ACTH Chow wth Cori sone no poo BOO 400 XOO 100 DO noo f coo yo ro yoo OS I AC FICURF 13 increased in both instances and Cral in the one instance where measured The filtration fraction did not change in the group of patients with Addison s disease control filtration rates and from very low to normal ales in each instance a significant increase in Cin was cffected by the aclministration of cortisone Thic doses in these patients were not as large is in the normals - 100 to 200 mg a day - but ns has been pointed out for en Adelsonian this is far above phu siologic requirement for maintenance Cratt also increased slightly but here the change was not significant, therefore among the Axldisonians filtration fraction rose In the entire group inut more particul url in the normal group Although plasma flox is mere used the crkulated lslood flow wis not simbc intls altered the reason for this was thut under the condition of the experiments liem tocnits timſormly fell in lout the proportion that Can Llerriner 175 incre used Tm 15 an affected los {CTII and regularly chimed with cortisone rising In some inst 111ces w fallug in others ·108 Renal Function EFFECT OF ACTH AND CORTISONE ON CREATININE- INULIN RATIO IN 9 PATIENTS Сс % Сп 160 KO 150 30 140 40 130 10 120 MEAM MEAN CONTROL 20 10F . .. 0 00 MEAN DUR NG THERAPY 100 90F 90 80 80 70 70 O. CONTROL . DUR NG ACTH OR CORT SONE 60H 160 50F 150 Por ont EA OS 1 FW PM AP AC CP AD ACH FIGURC 14 Simultaneous crentimne ind inulin clenrances were measured in ninc patients (Figure 14) During the control studies hierige C/Cto exceeded 10 in all groups wernging 12 During ther py there was a significant drop with in werige now for the entire group of 10 Examination of changes in these ratios in individual patients demonst ites two exceptions to these rerige figures in one patient with Addisons disease (CP) and one with glomeru lonephritis (A Ch ) the ratios rose on therapy These decreuses in ratio of Cer/Cia ucre therefore fairly consistent and significant In only one patient (CA) who received ACTH was actual crcrction or clearance of creatinine significmtly changed by either igent In this patient Cc fell from 100 ml per minute during control studies to 67 ml per minute during ACTH administration No adequate explanation for these findings is apparent from the data Increase in extracellul ır sprct is inferred from the decreased hematocht and weight guns occurred regularly Since at least in the noimals blood pressure did not rise there is no evidence that glomerular hydrostatic pressure was increased Hemodilution per sc would be expected to increase plasma flow if all the other relevint ·Actions of ACTH end (ortisone on Renal 109 factors remuned constant and Clatt was indeed metused on each of the normal subjects in approumite proportions to the ed inges in lemntocrit If it is assumed that the amount of filtrate formed is limited ly the increase in collord osmotic pressure of the nnfiltered plasma as it flows through the glomerulus then it would seem reasonable to conclude that with a greater plusmı flou more filtrite would be formed per unit time before the same limuting protein concentration would be reached With mreised plasma Now greater diffusion of furch might also be expected to occur irre spectue of changes in pressure relationships This increased prisma fou might increase filtrition rate withont tht intenention of changes in glomerular pressure or of changes in the size and permeabilits of the filtering bed In the patients with adrenal insufficiency Csall increases were slight while those in Cin were usually large it is more difficult therefore in this group to uttribute increased glomerul ir filtration mata to increase glomerular plin floss in three of these four patients increased glomerular las drostatic pressure can be postulated la cause small increases in arterial blood pressure occurred follow ing cortisone Alterations in glomerular hudrostatic pressure might also lire Ixcus produced changes 1 fferent or afferent arteriolis tone and could have resulted in lirke linges in filtration independent of those conditioned in glomerulur plisnin flow or systemie internal pressure There are no olusers tions in these experiments whicos mould indicite u lucther or not such isomotor changes did m fict Occtir If the improved Con su presented in increase in the total fume tioning rewal miss similar to the renal lupertropluy produced in timuls in luge dones of steroids(45) or if there the result of in icin ation of previoush functionicss nephrons one would «rpect Proportional teises in The Changes 11 Co Tmru Kert pot it all constant loncier no chrnce increases ind decreases were oliserica The rise in Cix without proportionste increase or with rio rise it im Cr can be foush interpreted if creatine is secretex) 116) nun nefully me ores filtration rate the onyt must conclude tut decreased tubulir secretion resulter and indeed in there must lies been actual sorption of creatine Ixo me tre itipu Geirince fell well lielos mulin cle ir wice Alternatuck inulin is Teilysorbed reduces tmbulir perma bult to this subast mee must ·110 Renal Function hue occurred in any event the conclusion seems inescapable that tubular transport of creatinine or inulin or both must have been altered Excretion studies of uric acid and phosphorus further support the premise that adrenal steroids act in part at least by changing renal tubular function In keeping with other reports(67) urine uric acid excretion increased muhedly despite lowered serum con centrations in three of our four patients and further this increased excretion was accomplished in the presence of a diminished filtered lond of uric acid Since it appears that uric acid is normally totally filterable at the glomerulus(89) and enbsorbed by the tubules (10) the data suggest that the uricosuric effects of ACTH and cortisone are the result of altered renal tubul ir transport Adrenal hormones may accelernte tubular secretion of uric acid if such occurs(11) or inhibit tubular reabsorption or both Turtliei evidence for 7 primarily renal mechanism for the liri cosuric effect of ACTH and cortisone is furnished by the observa tion that the administration of drenal cortical extract to nephrec tomized animals does not alter the rate of accumulation of uric acid in the serum(12) Similu conclusions may be drawn from phosphorus ercretion The well established phosphaturia resulting from administration of these hormones was demonstrable despite diminished or constant filtered lords of these anions Therefore it seems possible that alterations in the renal tubulu transport of phosphorus may well account for the losses of phosphnte exceeding those of nitrogen and for those losses occurring in the absence of negative nitrogen and calcnum balance In one patient (E A ) who received ACTII renal tubular secretion of phosphate apparently occurred Phosphate clearance was 150 to 190 percent of the inulin clearance during tuo consecutive periods We hue been unable to repeat this obser sation in subsequent patients esen by combining sodium para aminohippurite and phosphate loads The data are therefore pre sented merely for your consideration but so far as we have been able to determine they are right At any rate it does secm likely that tubului transport of phosphorus has been altered The data on sodi im and potassium excretion in normal subjects can be quickly summarized Day to day excretion in keeping with the finding of many others showed sodium retention wuth both ·Actions of ACTII and Cortisone on Renal 112 EFFECT OF ACTH ON ELECTROLYTE EXCRETION DURING NAPAK LOAD PLEA CONTROL ACTH W/ tor MOHOU 10 08 06 NAPAM LOAD NAPAH LOAD wear PAN vene AU gy 041 02 FICURE 15 2 agents usually more with ACTH thin with cortisone Potassium crcrction was inconstantly increased or not moreased at all At tempts to demonstrate changes in tubular activity relative to chinges in quantities of these cations filtered and reabsorbed during route cxperiments were by and large unsuccessful Under one circumstance hosever a reproducible and interesting phenomenon occurred as shown in Figure 15 Whereas Tmrak determinations during control studies resulted in increased urinary 1933 demon drun by potassium so that the administered solum pari immohippuate was now being crcreted as potassium pari ammohippurnte Al (laughs there tre no good explinations for these plicnomes i one might speculate that the effect of adrenal steroids on potassium excretion mu depend upon an ugmentation of sodium reabsorp con al upon the nature and quantity of the union change scre tion Possibly related to these findings is the fact that in patients with the nephrotic syndrome administration of solum louls results in the crcrction of large amounts of potassium( 1311) Turtlıcanon it has recenth been noted that DCA does not produce increased ·112 Renal Function EFFECT OF HYPERTONIC SALINE ON SODIUM EXCRETORY RATE NO EXCRETION MM/MA 19 ADDISONS DISEASE NORMAL SUBJECTS 18 7 Average Control Maximum Attoined 16 15 23 1.2 10! 9 3 3 Pot on GM AC RP СР AP MM MO AT PS AK FIGURE 18 potassium excretion when given to patients receiving a low sodium dict* and m whom sodium renbsorption wis ulready neur maximal This liypothesis of the mechanism underlying the increased potas sium excietion which sometimes accompanies ACTII or cortisone administration is consistent with a recent suggestion(15) that intra turulur exchinge of sochum and potassium may occur The effects of hypertonic solutions of saline were studied in five patients with Addisons discuse (Figure 16) As expected from previous experimental and clinical observations there was a miled chfference between patients with adrenal insufficiency and normil subjects studied under similar conditions Thus m four of five patients urinuy sodium excietion increased only slightly following administration of npproximately 4 percent salıne intravenously and there was no overlapping between these four and the normals in the fifth patient durug control studies wlule on a previously high salt intake but no DCA the urioary excretion following hypertone salıne equilled or exceeded that of two norint's Tlie sinc test HD Seld n L Welt and Cort prsonal com un et on ·Actions of ACTII and Cortisone on Renal EFFECT OF HYPERTONIC SALINE ON YONG 113 MATU CHUNGE ZOF ADD SONS OISEASE + NORMAL SUBJECTS WAR NUM CHANGE Llu CONTROL 20F TREATMENT PAT KYT GN CP MD AT I IGLRE ! is CR erformed in three patients after cortisone (150 to 200 mg per 1 for four to seven days) slightly improved ercretion in two ilients ind definitch improved crcrction in the third patient I had no effect on the response of one normal and in inother rhaps the response was conditioned ind diminished by thus art rond This in tbility to crcrete the sodium load illows it le ist tuo gruss explanations First urine fu did not increase lurests simpl did not follow idmistration of his pertonie sulme Second the poor sexlum excretion was also explained the relatie in abults of these patients to merene urinir concentration above control kwels (figure 1) Control concentration ind i Pof soclum inicd rather widely in the patients with diren al insufficiency as indicated by the blochs below the line blits to increase I p was sharphs limited before artisane I compared with the normals The one trpirent arception to this imong the norm its (I) should lx mentioned in this unst incr 15 result of 1 hrn previous salt int the control concentration was so lugli tint presumably maximal urin in 41 ng KI Inry I to 8 nba pridrag the contu niinnn wom! (txt trat in clunsre son Toater that li hung softest 1 1994 nrt an ritrat M ·112 Renal Function EFFECT OF HYPERTONIC SALINE ON SODIUM EXCRETORY RATE NO EXCRETION M/A 19 ADDISON'S DISEASE NORMAL SUBJECTS 18 17 Average Control Maximumn Altained 16 13 14 13 11 NaCl dhe & DCA ID OGA Abrant G.H AC RP CP AP MM MD AT fs AK E.RO rigunr 16 potassium excretion when given to patients icceiving a lou sodium dict* and in u hom sodium realisorption was already ncar maximal This hypothesis of the mechanism underlying the increased potissium cruction wluch sometunes accompanies ACTII o cortisone adınınıstration is consistent with a recent suggestion (15) that intratubulai exchange of sodium and potassium may occur The effects of lıypertonic solutions of salınc were studied in five patients with Addison's discase (Figure 16) As expected from previous experimental and clinical olisen ations, there was a marked difference between patients with adrenal insufficiency and normal subjects studied under similar conditions Thuis in four of five patients urmary sochum evciction increased only slightly follow administration of approximately 4 percent salıne intravenously an! there was no overlapping between these four and the normals In the fifth patient during control studies, wlule on a previously lugh salt intaho bạt no DCA, the urinary excretion following hypertonic salme equalled or exceeded that of two normals The sime test D1 Scklin, L G Welt ind J Cort Personal communication ·Actions of ACTI and Cortisone on Renal EFFECT OF HYPERTONIC SALINE ON PINO 113 up WAT WUN CHANGE 205 ADDISONS DISEASE 154 NORMAL SUBJECTS NAY MUM CHANGE TOF $ LI 104 cownino ISH 20F OCA S CORNSONEL OCA TPEATMENT OCA & CONTISOWE IDCA 11 PATIENT 8PCA 19 NOIN NOSEN WOIMI NIG MOCI GN NO CI SI oci ed I & DCA RP AP MM MO AT FICURE 17 FS AK performed in three patients after cortisone (150 to 200 mg per day for four to seven days) sughtly improved excretion in two patients and definitely improved excretion in the third patient DCA had no effect on the response of one normal and in another perhaps the response was conditioned and diminished by this steroid Thuis inability to excrete the sodrum load allons at least two gross explanations first urine flow did not increase druresis simpl did not follos administration of hypertonic saline Second the poor sodium excretion was also explained by the relative inabihty of these patients to increase urinary concentration above control levels (Figure 17) Control concentration and U P of sodium varied mther widel in the patients with adrenal insufficiency as indicated by the blocks below the line Ability to increase U/P* was sharply limited before cortisone is compared with the normals The one apprent exception to this among the normals (AA) should be mentioned in this instance, as a result of a high prestouts salt intake, control concentration was so high that presumably maximal urinary Although serum sexlrum did rise 3 to 8 nEq per L during the experiments urnary sodium concentration changes were so much greater that V/P changes reflect essentially changes in urinary concentration .

·Renal Function concentration of sodium had already been reached prior to the experiment In the three patients who recewed cortisone sught to marked increase in urmary concentrating ability resulted It should be emphasized that prior to cortisone treatment impairment of capacity to increase urinary sodium concentration was present in all patients including the one not on DCA and this defect was not demonstrated in tho normals receiving DCA The administration of sodium para aminohippurate during deter mination of Tmran in two patients resulted in a very surprising finding for which there is no apparent explanation Here sodium excretions following sodium loads given in lower concentribon at slower rates of administration resulted in greater excretory rates of sodium than had previously been observed with the same patients with hypertonic salıne both before and after cortisone In tuo instances this was due to both increased urine flow and increased urmury sodium concentration following administration of sodium par ammohippurate In another it was due to an unexpected durc SiS with little change in concentration Administration of water loads to these same patients before and after cortisone demonstrated in keeping with Dr Thorns findings a reversal of the Robinson Kepler Passer test in three or four patients 2. sodium concentration is relately inilexible and second ability tu increase urine flow is limited The precise renal abnormality in adrenal insufficiency has not been defined but there is good evidence that the characteristic salt loss is due to decreased tubular verbsorption of sodum(16) let in adrenalectomized animals receiving hypertonic snline at 1 rate rapid enough to produce serum sodum concentrations significantly above normal increased tubular reabsorption in the presence of normal glomerular filtration rates were demonstrated(17) In our patients with Addison s disease who excreted lords poorly the per centage of filtered sodium excreted u as defimtely lower than in the normal subjects in whom this measurement was made under similar experimental conditions Hossever the individual mulin clearances in the patients varied from low to normal values In addition it was not considered safe to attempt to achieve as high serum sodium ·Actions of ACTI and Cortisone on Renal 115 levels as were produced in the animal experiments cited above A close comparison of the two sets of data is therefore not possible One thing does seem clear If the inulın clearance measures the glomerular filtration rate, the failure to excrete salt was not due to dımınıshed filtration of sodium The hghest sodium excretion occurred in a patient with the lowest inulin clearance and one of the poorest sodium excretions occurred in a patient with a normal filtration rate The data reported here, and those of others cited, support the hypothesis that the defects of renal salt excrchon in adrenal insuffi ciency are the result of impaired responsiveness of the renal tubules to the homeostatıc requirements of the moment Some conditions demand increased tubular reabsorption of sodium and other condi tions the reverse The renal abnormality in jespect to water is chiefly one of mability to reduce the proportion of filtered water which is neabsorbed, despite the demands for water excretion or the organism The delayed removal of salt following hypertonic saline infusion, the inability to respond to water diuresis and the hyponatremia following salt restriction can all be expluned by these tuo renal defects of salt and water excretion in adreni! insufficiency Cortisone appears usually to correct the defect of meie ised water reabsorption and to improve but not entirely remove the inabilit to change reabsorptive rate of sodium DISCUSSION Shock Were all the reported measurements made during the period of salt and fluid retention or were some determinations made during the period following withdrawal of ACTH? Burnett The measurements were made among the normal group between three and fle days after starting ACTII They were therefore studied during the period when one could expect maximum bud and salt retention, and they did uniformly show salt and water loss following withdrawal of ACTH Thorn To clarify this point, Dr Bumett you maintained your Addisonian patients on DCA is that correct? There was also one patient maintained on salt? Burnett That is right In two of the patients on DCA there was reason to believe that they were just about out of the DCA because ·316 Renal Function pellets had been implanted tuelle und eight months previously The patient who had had pellets implanted eight months preuzously went into crisis on the night following the test so there was fairly good er dence that she was out or almost out of DCA Two others were adequately controlled on DCA at the time of testing, whether in these the presence of the DCA conditioned the responses hus bothered 228 night along From evidence of other observers it seems probable that DCA did not have too much effect on the results reported here, first, because it has been obserted in animals that were not on DCA is that correct, Dr Patts? Pitts They had been on small maintenance doses of DCA Ent were untreated for a period of three to five days prior to an experi Bent Burnett Other expenmental and clinical observations similar to these had been observed in patients with Addison's disease who had not previously received DCA(18,19,20,21) Fonever, it is clear that in observations reported here previously, DCA may have condi tioned the response Thorn But the essential difference in the untrerted Addisonin is a greater reduction in glomerular filtration when he isn't prepared with DCA beforehani? Burncit Yes Thorn Are filtration rates uniformly down Burnett Filtration rate may be someu hat improved by DCA but we were able to find no inst inces where improvement was as strih- 1kg as was obsersed with cortisone Premont Smith I would like to ask a question about this water excretion The Addisoni ms line a delayed response to water excre tion or failure to excrete svater normally As far as I can remember, this is the onls condition in which edema does not occur Why dont patients with Addison's disease des elop edema? Thors They do as soon as you gre sodium chloride or anything that improves their capacity to ietain sodium chlonde Fremont Smith Why don't the desclop cdemı spontaneously the way any other person does sho has a failure to ercrete water normally? Thorn Tossibly patients with other diseases develop edem i in conjunction with salt retention There is a lotit do vater retention, isn't there without accompanying sodium chloride?

·Actions of ACTH and Cortisone on Renal 117 Fremont-Smith Then it must mean that they don't fail to cxcrete their water if they don't become edematous They must either get rd of the water or become edematous If this is not so, then they don't take the water Thom They don't take the water Their mtale of water is very low and they do eliminate it over the tuenty four-hour period Fremont-Smith Thes have a failure of thurst, and if you give an average or a slightly elevated water load to an Addisonian patient, if he does not somit it or does not have diarrhea, he develops edema is that right? Thorn No If you give him thuis test load of water which Dr Burnett describes he eliminates it but his diuresis is delayed and it comes on soughly after the twelve-o clock period The test runs from eight in the morning to twele noon in the normal individual and the normal individual excretes 50 percent or more of his water Soad in the first three to four hours Fremont Smith If he cannot get rid of it or if he does not get d of it before drinking again will he des elop edem za Burnett Actually we see again and agun that the patient who has been treated for Addisonian crisis is edematous the morning following his treatment Thorn That is one of the reasons why DCA has been so difficult to administer in proper dosage because of the inherent underly mg tendency to retain water Thus, if one adds a very potent sodium retaining agent to a patient who has a tendency to retain water, one can predict the development of edema in a relatively short time Fremont Smith Has anybody seen an untreated Addisonian with edema? Thorn Never Fremont Smith They are dehydrated Thorn But their total body water content per gram of tissue is high Their extracellular fluid is dehydrated Fremont Smith The blood olume is down Thorn The blood volume is down The water content of the tissue is not down Fremont Smith It is a striking example of contrast because they have a failure to excrete water and yet they are dehydrated The ·118 Renal Function curious thing is that in a sense they are similar to the diabetes insipidus because the dubetes insipidus if left alone would get a diminished extracellular fluid and diminished blood volume but he saves himself by drinking to keep up with his hidneys The Addi sonian develops the same condition because he does not the water isnt that night? Thorn Yes Tremont Smith It is rather striking because I cannot think of any other clinical condition where there is failure to cxcrete water normally where the person does not become or have a seal tendency to become edematous Thorn These experiments that Dr Burnett carried out are really heroic ind extremely difficult experiments to conduct in these patients regardless of the fact that they are muntuned with DCA since the injection of even small quantities of sodium chloride intra venously will oftentimes precipitate a fever of 105° or 106' that evening Salt solutions may be extremely toxic to Addisonin patients maintained on DCA alone I might add one point of interest for those investigating endoge nous creatinine methods in Addisonıın patients There are striking changes in creatinine and creatine metabolism following cortisone therapy Shorr How long do your experiments last? Burnett The actual infusion of hypertonic salıne usually lasts an hour The actual experiment is usually about three hour pro cedure Short Some time ago we became interested in the response of the urinary uric acid creatinine ratio following administration of a test dose of ACTH(22) We found that without any ACTH normal subjects frequently exhibited 1 rise in the unc acid creatinine ratio which was as large or larger than the total nse achies ed with ACTH In other words something happens to this ratio in the course of just fisting which apparently is not associated with any regular fall in eosinophils but which may gule a pseudo response in the ACTH test Indeed in Addisonrins fasting brings ont the defect in response of the uric acid creatinine ratio almost as regularly as does ACTH We then repeated the studies without ACTH on normal subjects modifying the imtial level of unc acid ercretion bi grung on the ·ors of ACTII and Cortisone on Renal 119 day previous to the test two meals high in purines An individual who responds to fasting alone with an increase in unc acid creatinine ratio of 80 to 210 percent may after a day on high purine diet show practically no increase in the ratio Similarly when ACTH is administered to a normal subject the response may be either in increase in this ratio of 90 percent or following two meals of Meetbreads it may be reduced to an abnormal 2 percent Another thing which became apparent from these studies was lat both with fasting and after ACTH an increase in urinary pH ually accompanied the rise in uric acid creatinine ratio Burnett I cannot answer those questions in reference to uric d and creatinine but I do not remember any difference between Addisonians and the normals In regard to the pll we thought had something early and measured pil in the urine but there not any constant change erther in the normal or the Addisonian ip including the urinan pH after administration of sodium aminohippurate load Ort The time intervals in our studies were the same as those in the Forsham Thorn ACTH test and it may very well be hese are longer than yours We have a two hour control period viure the injection and four hours afterwards That prolongs the experiment beyond your time relationships Shannon What is the time relationship of the whole test as you do it? Short We run the test as follows No food after supper the evening before the test two hours of urine collection from six to eight o clock in the morning ACTH injection at eight o clock urme collection from nine to twelve noon We use the same time relations in the mock test Burnett I doubt whether there is even that great a difference As I think back some of our ACTII experiments ran over to twelve or one o clock because we had trouble getting under wiv There was in these patients therefore a long fast Rall. Did you say that one of the major defects in the Add sonian was salt loss due to a decrease in the reabsorption of sodium? Burnett Under some circumstances Ralli At what level of sodium ingestion would the decreased abihidi to reabsorb sodium take place in the Addisoniin?

·118 Renal Function curious thing is that in a sense they are similar to the diabetes insipidus because the dubetes insipidus if left alone would get a diminished extracellular fud and diminished blood volume but he saves himself by drinkmg to keep up with his kidneys The Add: sonian de clops the same condition because he does not take water isnt that right? Thorn Yes Fremont South It is rather striking because I cannot think of any other clinical condition where there is failure to excrete water normally where the person does not become or have a real tendency to become edematous Thorn These experiments that Dr Burnett carried out ue really heroic and extremely difficult experiments to conduct in these patients regardless of the fact that they are muntained with DCA since the injection of even small quantities of sodium chloride intra enously will oftentimes precipitrte 1 feier of 105° or 106º that evening Salt solutions may be extremely toxic to Addison in patients maintained on DCA alone I might add one point of interest for those ins estigating endoge nous creatınıne methods in Addisonian patients There are striking changes in crentinine and creatine metabolism following cortisone therapy Shorr How long do your experiments last? Burnett_The actual infusion of hypertonic salınc usually lasts an hour The actual experiment is usually about 7 three hour pro cedure Shorr Some time ngo we became interested in the response of the urinary uric acid creatinine ratio following administration of 1 test dose of ACTH(22) We found that without any ACTH normal subjects frequently exhibited a rise in the uric acid creatınınc ratio which was as large or larger than the total rise achieved with ACTII In other words something happens to this ratio in the course of juist fasting which apparently is not associated with any regular fall in eosinophils but wluch may give a pseudo response in the ACTH test Indeed in Addisonians fasting brings out the defect in response of the uric acid creatinine ratio most as regularly as docs ACTH We then repeated the studies without ACTII on normal subjects modifying the initial les el of uric ned excretion bi ghing on the ·Actions of ACTH and Cortisone on Renal 119 day previous to the test two meals high in purines An individual who responds to fasting alone with an increase in uric acid-creatınme ratio of 80 to 240 percent may after a day on high purine diet show practically no increase in the ratio Similarly when ACTH is administered to a normal subject the response may be either an increase in this ratio of 90 percent or following two meals of sweetbreads it may be reduced to an abnormal 2 percent Another thing which became apparent from these studies was that both with fasting and after ACTH an increase in urinary pH usually nccompanied the rise in uric acid creatınıne ratio Burnett I cannot answer those questions in reference to uric acid and creatinine but I do not remember any difference between the Addisonians and the normals In regard to the pH we thought we had something early and measured pH in the urine but there was not any constant change either in the normal or the Addisonian group including the urinary pH after administration of sodium para aminohippurate load Shorr The time intervals in our studies were the same as those used in the Forsham Thorn ACTH test and it may very well be that these are longer than yours We have a two hour control period before the injection and four hours afterwards That prolongs the experiment beyond your time relationships Shannon What is the time relationship of the whole test as you do it? Shorr We run the test as follows No food after supper the evening before the test two hours of urine collection from six to eight o clock in the morning ACTH injection at eight o clock urine collection from nine to twelve noon We use the same time relations in the mock test Burnett I doubt whether there is even that great a difference As I think back some of our ACTH experiments ran over to twelve or one o clock because we had trouble getting under way There was in these patients therefore a long fast Rall: Did you say that one of the major defects in the Addisonian was salt loss due to a decrease in the reabsorption of sodium? Burnett Under some circumstances Rall. At what level of sodium ingestion would the decreased ability to reabsorb sod um take place in the Addisonian ·118 Renal l'inction curious thing is that in a scrise they are similir to the dullis Insipidus Ixodels thic caletes fusipidus, ff left alone, would get a chiminished extracellular flufel une diminished blood volume but he Salves limself luy drinking to keep up with buis kidneye llue Added« sconi in develops the same conditions leo.Los lic cloes not the water, innt that right? I horn Yes I remon-Smith. It is uither striking lecitisse I camot think of iny other clinical condition where there is failure to escrito widler normally where the peason clocs jiot Ixcomc, or linvi at real tundency to become rematous, Thorn 'Ilıc se purposlenits thint IDr. Burnett carried out fie reilly Lerok a strunely difficult experiments to conduct In this patients regardless of the fact that they are maintained with ICA Since this infection of cove's soul quotiile of sodium clioride Intra remonly will oftentimes precipiente a favor of 10174 or 100% tlett evening Silt solutions may be extremely toxic to Ackelsonil in pollende mantined on DCA clone I miglit ndil one point of interest for these luvrstig ting endosco O creatinino ne thels in Adlelisonline patients 'Iliere are seriklis changes in creatinine and creative metabolism following cortisone therapy Shorr. Ilow long do your experiments frust? Burnt illor actorat infusion of liypertonic suline 1180 illy Jouts 1110 Hour 'lle se toll cosperiment lx 11Sally about a thirre Pour pro. (dure Slurr some time ago we las ame loterested in the response of the urinyurle 10 Idee reallloe ratlo following, suministration of it to (les of ACIII(22). We found that without any ACIII, normal subofects (rrepently coxlublied at the fu the urle nella reitinin ratio which was sy large or larger than the total rise aculeased with ACTI In other words, something to uppers to this rullo in the course of 111st finsting, which apparently is not ressouchatel with any regulur Call to castoplille, Tullt which may shoe al puerulo rapuse du te ACTII test Indeed, In Addisonans fasting Torings out the defect In response of the nile sicid.creatinine rallo almost regularly als curs ACTIE Wetlirsimpated thic studies wituut ACU a formal subofects, mwlifying tle Inital Imse of urk add cxcrciten ly gludus, on thic ·Actions of ACTH and Cortisone on Renal 119 day previous to the test, two meals high in purines An individual who responds to fasting alone with an increase in uric acid creatinine ratio of 80 to 240 percent, may, after a day on high purine diet, show practically no increase in the ratio Similarly, when ACTH is administered to a normal subject, the response may be either an increase in this ratio of 90 percent, or following two meals of sweetbreads, it may be reduced to an abnormal 2 percent Another thing which became apparent from these studies was that, both with fasting and after ACTH, an increase in urinary pH usually accompanied the rise in uric acid creatinine ratio Burnett I cannot answer those questions in reference to uric acid and creatinine but I do not remember any difference between the Addisonians and the normals In regard to the pH we thought we had something early and measured płł in the urine but there was not any constant change either in the normal or the Addisonian group, including the urinary pl after administration of sodium para aminohappurate load Shorr The time intervals in our studies were the same as those used in the Forsham Thorn ACTH test, and it may very well be that these are longer than yours We have a two hour control period before the injection and four hours afterwards That prolongs the experiment beyond your time relationships Shannon What is the time relationship of the whole test as you do it? Short We run the test as follows No food after supper the evening before the test, two hours of urine collection from six to eight o clock in the morning ACTII injection at eight o'clock urine collection from nine to twelve noon We use the same time relations in the mock test Burnett I doubt whether there is even that great a difference As I think back some of our ACTII experiments run over to twelve or one o'clock because we had trouble getting under way There was in these patients therefore 1 long fast Rall. Did you say that one of the major defects in the Addisonian was salt loss due to a decrease in the reabsorption of sodium? Burnctt Under some circumstances Rallt At what level of sodium ingestion would the decreased ability to reabsorb sodium take place in the Addisonian?

·118 Renal Function curious thing is that in a sense they are similar to the diabetes insipidus because the diabetes insipidus if left alone would get a diminished extracellular fud and diminished blood solume but he sives himself by drinking to keep up with his kidneys The Addı sonian develops the same condition because he does not the water isnt that right? Thorn les Fremont Smith It is rather strikmg because I crnnot think of any other clinical condition where there us farlure to excrete water normally where the person does not become or have a reni tendency to become edematous Thorn These experiments that Dr Burnett carried out are realls not avtremely difficult experiments to conduct in these not th DCA S enoussy Hd t evening Salt solutions muy he extremely toxic to Auus maintained an DCA alonc I might add one point of interest for those investigating endoge nous creatinine methods in Addisonii patients There we striking changes in creatinine and creatine metabolism following cortisone therapy Shorr How long do your experiments last? Burnett The actual infusion of hypertonic saltne usually lasts in hour T?e actual experiment is usually about three hour pro J na of the test subjects frequently exhibited a 15 ratio which was as large or larger than the total tise tehesed with AUTUI In other words something happens to this rato in the course of just fasting which ypparently is not 1550ciated with any regular fall in ensinophils hut słuck may give a pscudo response in the ACTH test Indeed in Addisonians fasting brings out the defect in response of the tric acid creatinine ratio almost as regularly as does ACTH lle then reported the studies without ACTII on normal subjects modifying the initial level of uric acid excretion by giving on the ·Actions of ACTH and Cortisone on Ronal 119 Hay previous to the test two meals high in purines An individual who responds to fasting alone with an increase in uric acid creatinine atio of 80 to 240 percent may after a day on high purine diet how practically no increase in the ratio Similarly when ACTH s administered to a normal subject the response may be either an increase in this ratio of 90 percent or following two meals of weetbreads it may be reduced to an abnormal 2 percent Another thing which became apparent from these studies was hat both with fasting and after ACTH an increase in urinary pH usually accompanied the nse in uric acid creatinine ratio Burnett I cannot answer those questions in reference to uric cid and creatinine but I do not remember any difference between he Addisonans and the normals In regard to the pll we thought ve had something carly and measured pH in the urine but there was not any constant change either in the normal or the Addisonian group including the urinary pH after administration of sodium para aminohippurate load Shorr The time intervals in our studies were the same as those used in the Forsham Thom ACTH test and it may very well be that these are longer than yours We have a two hour control penod before the injection and four hours afterwards That prolongs the experiment beyond your time relationships Shannon What is the time relationship of the whole test as ou do it? Shorr We run the test as follows No food after supper the evening before the test two hours of urine collection from sıx to eight o clock in the morning ACTII injection at eight o clock urine collection from nine to twelve noon We use the same time relations in the mock test Burnett I doubt whether there is even that great a difference As I think back some of our ACTH experiments run over to twelve or one o clock because we had trouble getting under way There was in these patients therefore a long fast Rall. Did you say that onc of the major defects in the Addison was salt loss duc to a decrease in the reabsorption of sodium? Burnett Under some circumstances Rallı At what level of sodium ingestion would the decreased ability to reabsorb sodium take place in the Addisoni in?

·120 Renal Function Burnett I have not iny idea I simply picture the Addisoni in a narrower range that is he is unable to conserve salt below a certain level of intile and unable to excrete excess salt above another given higher level of intake I dont know where you put those lies Do you D. Thorn? Chorn No Tley depend upon an intact kidney since renal disease complıcating adrenal diseise would alter the threshold I would judge from clinical experience tlint 1 norinal salt intake consisting of 8 to 10 gm would be well within the capacity of the Addison in keeping in mind that the potassium intake is very important in determutng the threshold for sodium tolerance Dock If you keep in Addisonian on 1 high salt intake for a long period of time does he ngain come into balance? Burnett Not according to Nerfensteins study As I remember the patient with Addisons disease stayed in positive balance(20) Thorn He stars in positive brlance but agun thus will depend on the extent of sodium intake Shannon He cannot stay in positive balance indefinitely Burnett As I remember the data as far as they carried the experi ment for four to five days they stayed in positive induce Shannon A normal individual if you increase salt will be in z positive balınce Thorn He comes into balance ultimately with increased total body NICI Shorr Like the hyperparathyroid patient receiving cloum hic is const intly getting more and losing more Bott Dr Burnett would you mind reviewing the conditions under which you obtuned a revers al of sodium and potassuum that is winle giung sodum puri iminoluppurite you found that potis sium para uminohippurite was excreted? Burnett Referring back to Figure 15 the pattern of electrolyte and para iminohippurate excretion in the urine before and nie Tm determmations during the control period and after ACTII rre shown It is apparent that on ndministration of the load isefore ACTII administration 2 Juger ainout of pıra aminohuppitrate anpeared in the urine lint that most of it is in association with sodium with not too great in mercise in potassium ercretion ·Actions of ACTH and Cortisone on Renal 121 although in some inst inces there was increased potassium excretion in the urine Under similar conditions after ACTH was admmistered with the increased para aminohippurate following loading, the quantity of sodium was decreased as compared with the control experiment whereas the quantity of potassium was incrersed This phenomenon was observed in all patients with ACTH and cortisone that we studied We have no explanation for this phenomenon but it has occurred to us that this reflects the so called potassium diuretic effect of sodium solutions In our experience, however, this effect is not constant Hypertonic saline, sodium bicarbonate, and sodum para aminohippurate m some instances may effect increased potassiun excretion but not always Under the stimulus of adrenal steroids apparently, however, it has been brought out Bott But this particular example was in a normal individual? Burnett The patient shown in Figure 15 is a normal individual We did not observe this effect in two patients with Addison s disease in whom we studied it following administration of cortisone Rall What were the serum levels of potassium in the Addisonian at the time that you were doing these tests? Burnett They were normal Darrow That fits in with the findings in dietary potassium de. ficiency If you put rats on dsets low in potassium and also low in sodium chloride, you get practically no changes in the muscles It requires a load of sodium chloride to produce potassium deficiency with desoxy corticosterone also Burnett Dr Donald W Seldın* has shown in animals on a low sodium intake that it is impossible to demonstrate increased potassium excretion on administration of DCA I interpret that this effect In some way is related to the one you have mentioned Darrow That is the general belief Luctscher It would be important to study the sodium potassium exchange after para aminohippurate during the later phase of ACTH or cortisone administration when sodnm is being released Burnett I am sure it would • Personal connunication ·122 Renal ranchon Luctscher That might well show a reversion to normal A similar exchange of potassium for sodium was observed by Dr Elliott Neuman( 23 ) in patients with nephrosis when he measured the clerrance of thiosulfate Sodium thosulfate was given by infusion Patients with edema and with little sodium in the urine excreted potassium with the thuosulfate When sodium appeared in the urine normally thiosulfate was normally excreted with sodium The im prirment of sodium excretion demonstrated under this stress is usually visible in ordinary daily balances Jancuay We hrve seen regularly in nephrosis thnt we get a big potassium diuresis during infusions of sadam salts Burnett lou have observed it with thiosulfate and we have with sodium bicarbonate with patients with nephrosis It seems to be a very common phenomenon regardless of the sodium salt which is employed Gaunt I wonder if anybody has considered the possibility that potassium diuresis when it occurs after a heavy sodium load might be due to action of the posterior pituitary hormones If you give enough sodium to obtain release of antıduretic hormone you might expect to get possibly the excretion of potassum Burnett That may well have been a factor in some of our er periments with ACTH because we know that certain lots were contaminated with posterior pituitary It also appeared however with cortisone and in these instances one would have to assume that cortisone stimulated the release of whidiuretic hormone Caunt Another point I would like to make relates to the question of what would happen in case the silt load followed the water load I saw some figures recently that have been collected by Dr Drill and Dr Hays at Wayne University* They found as everyone else has that if you give adrenalectomized animals 7 water lord they cannot excrete it normally If you give them a load of normal sahne they cannot excrete it normally But they worhed with various con centrations of salt in their witer load and found a point roughly as I remember it about 04 percent sodium chloride at which the adrenalectomized animal would excrete just like the normal unmai This impressed me because we all have had so much difficulty getting that response back to normal They did it just by adjusting the concentraton of salt in the wnter Ung bl bed data ·Actions of ACTII and Cortisone on Renal 123 Rall We have been determining the critical requirement for sodum chloride in adrenalectomized rats on high pantothenic acid intake As you may recall we have observed that if the diet of the rat follow ing adrenalectomy is supplemented with large amounts of calcium pantothenate( 24,25) and a 1 percent solution of NaCl as drinking water surya al is greatly prolonged If the rat is glen no NaCl the pintothenic acid content of the diet has no effect on surnal and if the diet is deficient or normal in its panto thenate content 1 percent NaCl will not prolong surural and the rats die in an average of eleven days after adrenalectomy in the present experiments srity day old rats were adrenalectomized and were placed on mcısured amounts of MC1 and 1 diet contuning 4 mg of calcium pantothenate per 10 gm of diet The first group were given 1 ml of NaCl duls (6 ml of a 0 167 Volar solution) A second group recen ed 3 m mols of Nac1 daily (18 ml of a 0 167 Volar solution) ind third group received 5 mm of NaCl daily (30 ml of 0 167 Volar MCI) Distilled water was provided in 1 separate drinking bottle to assure 1 sufficient ftud intale In the adrenalectomized animals recerung the NnCl solu tions of 1 and 3 ml survival was sharply limited the median survival time being nine and fourteen days respectuek When the NaCl concentration was increased to 5 mV survival was increased but it still did not equal the survival in adrenalectomized rats per mitted a 1 percent solution of NaCl ad lib The experiments are still in progress and when we have deter mined the duly amount of NaCl necessary for prolonged survival in adrenalectomized rats on the high pantothenate intake we plan to study whether the concentration of the NaCl solution conditions the animals requirement under the circumstances of these experi ments Gaunt in the muntenance experiment the animals would get along still better with more than 05 percent sodium chlonde wonki they not? Ralli They would Gaunt What I was speaking about is 311 acute experiment - how to make an adrenalectomized animal e crete simultaneously 1 salt and water load If you get the salt and water in proper proportions according to Drill and Havs the animals ercrete it at normal rate whereas in other proportions they do not Thorn It might be worth while for the group to keep in mind correlated experiments that might be helpful The control of the ·121 Renal Function sodium chloride concentration in the sweat has been shown to be modified significantly by alterations in adrenal hormone les el(26) Frau ley(27) in our group recently has shown that within a certain range of serum sodium level the sodium chloride concentration in saliva provides another means of demonstrating the effect of adrenal steroids In analysing these problems, I wonder whether any evi dence has ever been obtained which might suggest that the kidney also secretes" sodium Shannon I tlunk that is pretty close to the point I was going to male in relation to Dr Burnett's data Om information on the sequence of processes wluch condition the formation of urine i the mammalian nephron is wholly inadequate for one to answer the many questions raised by experiments such as huve been presented Burnett Do you think we can ever do that without getting back into the tubule? Shannon I believe one can obtain a part of the solution there, that is by an extension of the studies by Oliver, Richards and Walker, but under more physiological conditions It is quite essential to Inow whether the absorption of morganic ions in the proximal tubule proceeds at a more rapid rate than the movement of water This must be settled before one can attempt a placement of certain functions in the proximal or distal tubule I don't see any other way of doing it than by tubular punctures in wholli physiological preparations Pitts We tred one possible means of attack on the problem using Inppurate infusions with the view, if we could markedh lower filtration rate without affecting tubular transport, that we might be able to demonstrate societion of sodium We couldn't demonstrite secrction Burnett We have been struck with one fact i all of these experi ments Originally some patients were studied simply on a general ward diet We soon found, however, tinnt we had to have them on at least a week of constant intake before we could get any kind of reproducible result All the patients shown liere had been on a constant intake for a week prior to the control test and during the time of hormone administration If this program is not chered to the results mein absolutely nothing Putts What is the effect of cortisone or ACTH on phosphate excretion without loudig?

·Actions of ACTH and Cortioone on Renal 125 Burnett It incre uses but not is markedly as it did iftei loading There also was an increase in phosphate excietion duing the day to day measurements REFERENCES 1 RELMAN AS el dl Effects of ACTH and cortisone on renal function in normals and patients with Addisons disease (Sub mitted for publication) 2 INCBAR SH 11 e effects of ACTH and cortisone on the renal tubular transport of unic acid phosphorus and elec trolytes in patients with normal renal and adrenal function (Subm tted for publication) 3 BURNETT CHeld Renal excretion of hypertonic sodium solutions by normal human subjects and patients with Addisons disease with observations on the influence of DCA and cortisone (Submitted for publication) SELYE H Effect of hypophysectomy on morpholog cal appear ance of kidney and on renotrophic action of steroid hormone 1 Urol 46 110 (1941) S KOCHAKIAN CD Role of hydrolytic enzymes in some of the metabolic activities of steroid hormones Recent Progr Hormone Research I 177 (1947) 6 SPRAGUE RG et al Observations on the effect of cortisone and ACTH in man Anb Ini Afest 85 199 (1950) 7 FORSHAM P H e al Clinical studies w th pituitary andreno corticotropin Clm Endocrmol 8 15 (1948) BORDLEX J II and RICHARDS AN Quant tative studies of glomerular ursoe concentration of uric acid in glomerular urine of snakes and frogs determined by ultramicroadaptation of Folın s method / Biol Cbem 101 193 (1933) 9 BERGLUND H and FRISK AR Urk acid elimination in man Acto me. Siandmat 86 233 (1935) 10 BERLINER RW et al The renal mechan sin for urate excre tion in man Clm Imestigation 29 396 (1950) PRAETORIUS E and KIRK J E Hypouricemia with evidence for tubular elimination of uric acid Lab & Clm Med 35 865 (1950) 12 FRIEDMAN M BERNSTEIN D and BYERS SO Role of the adrenal cortex in the excretion of purines Pederalion P10 8 52 (1949) 13 BURNETT C H BURROWS BA ind COMAIONS R R The lack of correlation between glomerular filtration rate and serum electrolyte concentration changes urinary electrolyte excretion or edema formation following sodiur lords in subjects with normal kidneys glomerulonephritis and the nephrotic syndrome 1 Chin Imestigation 28 773 (1949) 8 11 ·26 Renal Function 14 METCOFF J and WALLACE V M The nephrotic syndrome in children Response to intravenous sodium loads Ibid 29, 835 (1950) 15 BERLINER RW KENNEDY TJ JR and HILTON J G Renal mechanisms for excretion of potassium An ! Physiol 162, 348 (1950) 16 HARRISON HE and DARROW D C Renal function in experi mental adrenal insufficieny Ihd 125, 631 (1939) 17 ROEMMELT JC SARTORIUS O W and PITTS R F Excre tion and reabsorption of sodium and water in the adrenaincto mized dog lb / 159, 124 (1949) 18 WILLSON DM and SUNDERAAN FW Studies in serum electrolytes effect of water restriction in patient with Addisons disease receiving sodium chlonde | Clm Int estigalion 18 35 (1939) 19 GREINE JA DAVID A JOHNSTON GW Effect of adrenal cortical extract desoxycorticosterone and added potassium upon electrolyte balance in normals and in Addison s disease 1 Clin Endocrinol 2, 49 (1942) 20 Poror A et al Effect of adrenal cortical compounds on elec trolyte metabolism of a patient with Addison s disease during high sodium chloride intake Ibid 8 608 (1948) 21 REFORZO MEMBRIVES ) POWIRS M H and KEPLER EJ Studies on renal excretion of water and electrolytes in cases of Addison s disease Ibid 5 76 (1945) 22 TAUSSKY HA SWAN RC and SHORR E An inquiry into the specificity of the uric acid-creatinine ratio as a measure of adrenal cortical responsiveness Proceedings of the Second Clinical ACTH Conference John R Mote editor Philadelphia The Blaliston Co 1951 (in press) 23 NEWMAN EV GILMAN A and PHILIPS FS Renal clear ance of thiosulfate in man Bull Johns Hopkins Hosp 79, 229 (1946) 24 RALLI E P Factors affecting survival in adrenalectomized rats Er docrmology 39, 225 (1946) 25 DUMIM ME and Ralur EP The critical requirement for pantothenic acid by the adrenalectomized rat Ibid 43 283 (1948) 26 CONN J W Electrolyte composition of sneat clinical implica tions as an index of adrenal cortical function Arch in Bred 83 416 (1949) 27 FRAU LEY T F and THORN GW Relation of salivary sodium potassium ratio and adrenal cortical activity Proceedings of the Second Clinical ACTH Conference John R dote editor Philadelphia The Blakıston Co 1951 (in press) ·THE EXCRETION OF SODIUM IN RELATION TO GLOMERULAR FILTRATION HARVEY L WHITE Department of Physiology, Tasbingian University School of Asedicine I would like to say a few words about the relation of glomerular filtration to sodium and water output If glomerular filtrate is sud dently reduced by 8 or 9 percent, as when one changes from hing down to standing up, the salt and water outputs drop to a third or fourth of what they had been Thus is what one would expect Nothing has happened to the tubules to inform them of any change so they continue to reabsorb, as nearly as they can, what would approach the same amounts of salt and water as were being reabsorbed before, so there is a great deal less remaining to be excreted That is an acute condition if this kept on for very long it would result in an accumulation of surpluses of salt and water, and the tubules would somehow be informed of this and would start reject ing more, so that the output would come back to where it was before In other words, a stort-lasting, small reduction in glomerular filtration would be expected to produce a murly greater acute percestage reduction in sodium and water outputs, which it does, whereas a long-lastrag, even great reduction, in glomerular filtration would not be expected to produce any chronic reduction in salt and water output of the tubules behaved normally The question is how to test this by producing long-lasting reductions in glomerular output while still having normal kidneys and body finds it one seduces filtration by hypophs sectomızıng an ansmal, brings it dow to kalf the normal, there is no retention of entfer salt or water The animal stays in balance because his tubules reject a larger fraction of the filtered salt and water, and he can get nd of suddenly imposed loads of salt just as fast as the normal animal can, but that still isn't quite the answer One of the things we have been doing during the past year is prepare asumals so that we can produce a permanent fall in filtration sate with the kidneys undamaged and without any accumulation of salt or water Thus was done by esplanting the bladder trigone so we could collect from the two kidneys separately These to 127 ·128 Renal Function animals sunne for many weeks or months If then a ligature is put on one renal artery so as to restrict very slightly the blood flow through that kidney there will be a great reduction in the salt and water output of that kidney, with a corresponding increase in the salt and water output of the other kidney The output, with intake constant, is the same as it was before, the animal stays in balance indefinitely The kidney that was constricted does not know that any thing has happened to it in fact, nothing has happened to the composition of the blood, because the other kidney prevents any accumulation from occuring, so the constricted kidney continues to reabsorb at somewhere near the same rate that it would have if there had not been this slight reduction in filtration The fact that the companion kidney increases its sodum output might be regarded as evidence that some accumulation has occurred This cannot be definitely answered at present, the important point is that any presumptive accumulation is negligible compared to what would have occurred if the output of both kidneys had suffered the same percentage decrease as that of the ligated kidney I will cite a few specific cases If one puts on a ligature of just the proper tightness, and a few days Inter determines inulin and PAH clearances and sodium and water outputs of the separate kidneys there will be no measurable fall in the clearances of the ligated kidney, but a fall of 50 percent in sodium and water outputs At the same time the sodium and water outputs of the other kidney have gone up so that the two are putting out the same as they did before These animals are kept on constant intake in other words a unilateral fall in filtration rate too small to be detected by our methods will resnit in a imteral 50 percent fall in sodium output and that will last indefinitely, whereas if both kidneys had been 50 ligated as to reduce their filtration rates by 5 percent, there would have been no change in the salt or water output of either kidner after a short time If we make the construction somewhat greater by progressive tightening so that the PAH and mulin clearances are obviously reduced, say to 80 percent or 70 percent of normal, the sodium and water outputs of the constricted kidney drop to about 3 percent of the companion's outputs The companion kidney's outputs sale increased so that the total outputs remain the same with a still further constriction, but not complete obhteration of the arter, since one cu st}} feel the pulse distal to the constriction, urine output ceases entirely from this kidney, although it sta}} Iras a liload ·Glomerular Filtration 129 flos I don't know what fraction it is because we hne no way of measuring st The other kidney then puts out the same amount originally put by the two kidneys If we take an animal vluch has a constricted kidney with a filtration rate of 70 percent of normal but with only 3 percent of the sodium coming out of that kidnev and nou remove the good lidney, the remaining ligated kidney will of course increase its PAl and inulin clearances but is presented by the ligature from incre ising them as much as happens after a unilateral nephrectomy as ordmarılı done The imłın clearance of the remaining ligated kidney thus rises somewhat but never reiches the level of tuo normal kidneys Houever the salt Output riscs so that within three days after the removal of the good kidncy the salt output of the iemaining kidney is nearlv normal, and eventually becomes normal, ie, it is taking care of all the sait that both kidneys had been doing This emphasizes u liat has perhaps not always been adequately emphasized, the distinction between transitory and long lasting changes in filtration rate as they affect salt and water outputs To me it seems onks to be expected that a sudden small drop in filtration rate would produce a great drop in salt and water outputs but that drop would not last very long In a short time it would be right back to normal, but when the other good kidney prevents a surplus from accumulatmg then this small drop of say 5 percent in filtration rate will permanently reduce the salt output of that kidney But when you take the other kidney out then the salt begins to pile up even though it isn t easily detectable by analy. sis, the tubules promptly responding by rejectmg a bigher fraction of the filtered salt than they did before DISCUSSION Parts When one renal arters is moderately constricted, does the remaining kidney increase its filtration rate apprecubly? 11Inte Yes, if the constricted kidney has been reduced in its filtration enough to be sure about it Of course we cannot be really sure of drops in filtration today as comprred with two weeks ago unless there is at least a 15 percent diop However, the sum of the tuo clearances will be someu hat less than before, the companion hidney does not show as much of an increuse as there is a decrease in the constricted kidney Pitts Would you interpret that as meaning that perhaps the somewhat discredited concept of glomerulo tubular bulance las an element of truth in it?

·130 Renal Function Winte The actual immediate response of tlie tubules to a change in filtration rate without change in composition of blood or filtrite is somewhere between that of continuing to reabsorb, within limits the same amount of salt per unit time as before, and of reabsorbing the same percentage of the filtered amounts as they did before Selkurt Another interesting speculation on your experiment is the fact that when you begin to ligate the renal artery you reduce the pulse pressure without reducing mean pressure In 1913 Gesell (1) came out with a paper that indicated that the pulse pressure might be an effective means of varying the salt and urine output His experiments demonstrated that when the pulse pressure was reduced there was no change in renal blood flow There was a drop in renal output of chloride It may be possible that in your experi ment the first effect might be the reduction of pulse pressure with its concomitant effect on the urine volume and sodium excretion That could go on with very little change in filtration mte und blood flow particularly since the circulatory autonomy of the kidney tends to muntain the flow rather well even as you begin to impair the arterial input It might be a factor to consider in that experiment White I doubt if we had any very significant fall in pulse pres sure distal to these lightest constrictions, but whether we did or not, the fact remains that there was no measurable fall in filtration rate We felt that both ureters should be accessible because we wanted to be sure that we had the same errors of collection in the two These are not twenty four-hour collections, but the animals were on a constant diet We chose arbitrarily a three hour period from ten o'clock in the morning to one o'clock in the afternoon, always the same time of day, always in the postabsorptive state no food since five o'clock the evening before The outputs then are furly reproducible from one day to the next, although there is considerable fluctuation But you see we alu ays hve the other kidney for control It is actuully easier to transplant the two ureters than one, because when the trigone is transplanted the ureter 1) orifices are still in the bladder mucosa they do not slough out or become stenosed, and no trouble is encountered with ascending infections I dont remember now whose operation that is but it is not original with us It makes a very satisfactory prepiration The anımais survive a long time without any ascending infection Shannon How do you make the urine collections in those preparations?

·Glomerular Filtration 131 11 hutc Just put a catheter in them Shannon The animals dont develop infections White We don t put it in very fur We have done it in two ways Sometimes the animal stands in a cinvas sling Then somebody has to hold a little cup up against each ureteral orifice with or without suction but it usually works perfectly well if you just put in a small catheter I dont mean we go up to the kidney pelvis just an inch or so Pitts How do you heep from getting superficial skin erosion skin infection and breakdown? That was our problem in the use of such dogs a number of years ago Il Rute There has been no difficulty with the wound That is qute different from our experience of a number of years ago when we explanted the ureters into the separate flanks but when the bladder mucosa is there it heuls up well 1 dont mean that all of them get along with a perfectly uneventful postoperative course but most of them do I have not done these operations myself they Mere done by a competent surgeon Dr Barber Mueller It is apparently a relatively simple procedure in such hands Darrow What worries me is why the tubules on two different sides should be adjusted to different rates of absorption Do you calculate terms of load from filtrate? Is there anything common? After all they are presented presumably with the sime sort of fil trate arent they? White Yes presumably they are getting the sume kind of filtrate Darrow How is your kidney wise enough to do different things on the two sides of the body? 1 hite Both sides are being perfused with the same perfusate and the tubules on both sides are being exposed to essentially the same funds as before we put on the construction So how do they know that any thing has happened? Why should they change their behavior? Darrow Eventually they do It lite so they do not what I mein is they continue to do as nearly as they can the sime thing as before Darrou At first but not later ·132 Renal Function White They keep on permanently so long as the companion kidney remains If you permitted an accumulation of salt to take place then they would not heep on They would finally realize there is too much salt here and quit taking back so much of it Dont ask me how they recognize that That is 1 question of a different category Lauson Do you believe this is relatively independent of hormonal factors White We know that in this case both kidneys are in the same dog They are subjected to the same hormonal influences unless we want to postulate that one kidney is itself making a hormone that the other one is not making That is the only possible difference Lauson Would this be an intrinsic renal adjustment of the good side to the necessity for excreting extra salt on that side because of the intrinsic inibility of the opposite side to excrete its share? White That is right because if it did not put out more salt then salt would accumulate It probably does accumulate at first It seems to me this is quite analogous with our problems in the regulnt on of respiration Why do you start breathing harder when you make niore CO ? Because if you did not the CO would pile up until you got into trouble One step netrer un explınation is of course that the respiratory center has responded to the increased CO tension While we all hope to know some day more about how the insides of cells work we do not need to know the intimate det uls of how 1 response is effected in order to know that it occurs As soon as salt or CO has accumulated by some threshold increment the behavior of the tubules or of the respiratory center respectively changes Rallı Is this not largely due to the difference between the filtra tion rate in the two kidneys? White The tubule is being advised that the composition of the plasma and filtrate is relatively unchanged so it continues with a relately unchanged reabsorptive behavior Unchanged tubular reabsorptive behavior toward salt is here defined as that which affects the reabsorption of a constant amount of salt per unit time subject only to the limitations of a decrezsed amount available for reabsorption and of the practical impossibility of extracting the last residuum of salt the tubule is set to reabsorb the same amount of salt per unit time is before if it is given the opportunity Such ·Glomerular Filtration 133 tancy of behavior depends on a constancy of environment but not be altered by changes in filtration rate only It does not a reabsorption of a constant percentage of the filtered salt a constant amount per unit volume of filtrate nor a constant of total osmotic work As mentioned before complete con cy so defined is not actually realized somewhat more salt pes on decreased filtration than that predicted on this basis somewhat less on increased filtration all. Would it not be reasonable to say that tlus change in ation rate in the kidney might set about 1 series of reactions ch would have a wider effect than would be inticipated hate That is possible of course I dont visualize what those be I can "explın it to my own satisfaction by saying that if didnt happen salt would pile up I do not mean to endou the ales with the faculty of anticipating changes in salt level I only n that as soon as some slight increase in level has occurred the mules in some was recognize this and depart from their constancy alt reabsorptie behavior as defined here they nos begin to sorb less salt per unit time even if filtration rate has not nged since the plasma and filtrate are now saltier The result at more salt is excreted and accumulation of any great surplus revented The unilateral ligation experiments enable us to see at happens when only one factor filtration rate us changed e the presence of the companion Lednev prevents the surplus ch would otherwise accumulate in this case the state of con it reabsorptive behavior as defined above is approached but no longer holds when conditions are such that the usually duced surplus can develop as when fall in filtration rite is teral or after the companion kidney in these experiments is hoved Darrow Either you have or you do not have evidence that as tubular reabsorption changes on the good side an analogous nge takes place in the other kidney s tubules What I have in d is that one could calculate the tubular load of sodium from glomerular filtration Then f one assumes that 80 percent of filtrate is reabsorbed in the proximal tubules one has a value the reabsorption in the distal tubules on the two sides If the tal reabsorption is at the same rate on the two sides then the nary excretion should be explained by the same rates of distal bsorption but different distal loads on the two sides Otherwise ·134 Renal Function one might have to assume a local adjustment in the rate of distal reabsorption dependent on the different loads and other local factors If one can explun the rates of excretion in terms of glomerular filtrates one has ? generalized adjustment on the two sides that are referable to such general factors as hormones White I think that is right Luctscher Does any accumulation of water and sodium actually occur? White We do not yet hale for compuson an observations made immediately after the ligation Luctscher Two important questions arise First does the filtra tion rate go up on the unimpured side equally with the diminution on the side on which the lighture wis placed? Second does the body weight or extracellular flud volume go up at any time so as to provide a stimulus to such 1 readjustment as Dr Darrow suggests? White The body weight does not go up Bradley Do the two kidney s respond differently to a sodnim Jord? W lute Yes The good kidney still puts out the major fraction of the salt Pitts But both will put out more salt? White Yes If in the unloaded state the ligated kidney is putting out 5 percent of the sodium it will still put out 5 percent when londed ie the two kidneys show the same percentage increase in output on loading Pitts Isnt it merely a quantitative conceptual difficulty? If both kidneys to begin with are reabsorbing 99 percent of the salt and water and you cut filtration rate slightly on one side so that that hidney now reabsorbs 99 8 percent of the salt und unter the other kidney has only to decrease its ibsorption to 98 2 percent foi the system to come into balance ngun Gilman I think Dr Whites experiments serve to remind us of the fact that the kidney is an end organ and an end organ charged with tremendous responsibilities It has a Irrge amount of sork to do All of the reabsorptive mechanisms that we have discussed are actne transport mechanisms and require the expenditure of energy Also we should remind ourselves that the subject of this Conference has to do with the hormonal control of renal function ·Glomerular Filtration 135 Although we can imagine some autonomous function on the part of the kidney, certainly its homeostatic mechanisms must be under the control of a gamut of endocrme and possibly other types of controls It is not surprising therefore that when tuo kıdneys are receiving the same humoral messages their tubular function would tend to be equal insofar as the extent to which transport mechanisms are carried out Under these circumstances when you decrease the load to one and it is still under the same humoral influences as the other, it is going to attempt to do the same reabsorptive job that the unoperated kidney does It is not going to do a complete job of reabsorption of electrolytes because all sorts of things are going on in the tubule which will prevent complete reabsorption of electrolytes despite the dımınıshed amount of filtrate The most obvious of these is the presence in the tubule of substances which are not actively reabsorbed and their presence in the tubule prevents reabsorption of sodium, chloride and other electrolytes Probably urea is the outstanding substance in that category Therefore we can expect these tubules receiving a reduced load to behave in the same way and to have essentially the same reabsorptive capacity as the normal kıdney but we cannot expect complete reabsorption because of the osmotic effects of substances which are not actively transported Shorr I wonder whether we don't have to consider the possibility of an influence by locally produced humoral factors because this is essentially a hypertensive kidney White There is no hypertension in these dogs Shorr Is there not a temporary hypertension? White I can not answer as to a transitory hypertension The measurements were made about once a week and there was no hypertension seen Lauson I would like to raise two or three specific questions If one had a 30 percent reduction in glomerular filtration rate on the affected side and at that time had this modest increase in salt excretion on the good side, one would assume that the kidneys were not under excessive stimulation by endogenous desorycorta costerone like hormones Now what would you anticipate would happen to the affected kidney with excessive DCA adminustration? Could the affected kidney be made to reabsorb more NaCl under these conditions? It seems to me this has a bearing on the question ·136 Renal Function of whether excessive DCA like activity does exist in certun states in which the glomerular filtration rate is low such as congestive failure and the nephrotic syndrome and if it does eust could such adrenal hyperactivity cause more complete NaCl reabsorption than is already taking place due to lowered filtration rate? I wonder if NaCl balance could be restored without edema formation if one had a chronic constriction like this on both sides giving a chronic reduction in glomerular filtration of about 30 percent I inte We didnt carry out bilateral construction but it is well known that Goldblatt dogs do not develop edema We did not want to produce hypertension we thought if we ligated both kidneys we would probably get hypertension which would confuse the issue I dont know whether 30 percent reduction in blood flow will usually produce hypertension or not even if it is acutely put on Does anybody here know? There we only two papers that I have found one by Corcoran and Page(2) and one by Blalock(3) years ago It is hard to cut down renal blood flow it gradually rises again and after a while is almost back to normal but thev still have hypertension The question as to how much you have to reduce the blood flow to the dogs kidney to get hypertension is one to wluch I do not know the answer Pitts It seems to me that Dr White in his chronic experiments has very beautifully shown that a small reduction in renal arterial caliber and filtration rate on one side can disturb balance The work of Blale Wegria et al (4) at the College of Physicians and Surgeons has shown that an increase in venous pressure accom pinied perhaps by a small change in filtration rate can likewise produce this same imbalance 0 W Sartorius* has shown that an increase in ureteral pressure can likewise throw the system o it of balance with only a very small change in filtration rate it is true that all these effects might be mediated through some change in the pulse pressure in the kidney but the common factor to me would appear to be the change in filtration rate The question is how can one reduce pulse pressure without producing some minor but highly significant change in filtration rate Dock In Blakes experiments the filtration rates were unchanged Pitts That is debatable if you examine their data closely Selkurt I would like to think that in similar experiments that * Personal commun cat on ·Glomerular Filtration 187 we did(5) the obstruction to blood flow was small However wc frequently obtained decreases in filtration rate, though slight in some cases I was very interested in the remarks that Dr White made They seemed to indicate that very small changes in filtration common ground on these phenomena where the whole explanation and better tubular reabsorption of the reduced loud White As I remember hatz' group(6) showed that chronic elevation of renal vein pressure does not result in any permanent changes in sodium output Pults He did not do the two separately? White Mo Pitts I think repetition of his experiments using your technique would give an entirely different picture Whute les I would expect to find essentially the same changes with unilateral renal vein construction as with arterial I think that whereas slight bilateral changes in filtration rate will temporarily produce big changes in output, the changes in output wont Inst long In a short time the total output goes back to normal even though the rise of venous pressure persists whether it be on one 1 monal influences unless it is renin doing it and renın apparently would work the other way because presumably there should be more renin in the ligated kidney than in the other - then the effects should be permanent as they turned out to be Gatint Dr Pitts I would like to mention some observations of Dr Boss and Dr Osborn in our laboratory (7) These show an effect of cortical hormones on water reabsorption under conditions of a chronically low filtration rate The work was done on hypophy sectomized rats ·138 Renal Function Several years ago Chen and Geiling(8) showed that the hypo physectomized rat despite its potential diabetes insipidus could not excrete a water load normally In extending the work we found that the urinary loss of fluid after water was given by stomach tube was no greater than the insensible loss(9) We tried to repair the deficiency with various hormones given singly and in combina tion The only thing that helped was adrenal cortical preparations but with them it was difficult if not impossible ever to restore a completely normal diuretic response to water The observations of Boss and Osborn show why A few days after hypophysectomy the glomerular filtration rate fell to about half of normal "This was accompanied by a great increase in the tubular reabsorption of water The conditions of the experiment were such that under a water load 10 percent of the glomerular filtrate was recovered as urine in normal animals Only about 4 percent of the filtrate was recovered in the hypophysectomized animals and as already indı cated the filtrate itself was of low volume When cortical extract was given the hypophysectomized animals in acute experiments 10 percent of the glomerular filtrate could be recovered is urine as in normal anımıls but the urine volume itself was only about half normal The reason apparently was that the cortical hormones did nothing to restore the filtration rate They restored reabsorptive rates to normal but the urme volume was limited even after treat ment by the chronically low filtration rate and in an amount pro portional to the reduction in filtration The failure of cortical hor mones to affect filtration accords with the work Dr White reported ezrlier on the hypophysectomized dog As seen here however these hormones can affect reabsorptive processes in the absence of the pituitary Seihurt Does sodium excretion parallel the water? Gaunt The flame photometer broke down in the middle of the series and I cannot say with certainty We got only some of them and they indicated that there may be a disproportionately high sodium excretion associnted with the increased urine volume induced by cortical extract We will know definitely about that soon Lauson I would like to describe some observations on a series of around the clock mersurements of endogenous creatinme clear ance ind of excretions of sodium and water which strongly suggest that glomerulır filtration rate plays an important role In a five year old girl with nephrotic syndrome the creatinine clearance was rused ·Glomerular Filtration 139 acutely on successive days by the infusion of albumin, which is a relatively simple way of changing the circulation without doing much to the endocrine system in this study there was a striking rise in sodium excretion and diuresis associated with relatively modest mcreases in glomerular filtration rate as estimated from the creatinine cle urance We have been inclined to feel that this effect is more an intrinsic renal affair than an endocrine one, but of course one cannot be entirely certain of this Luetscher The injection of concentrated human albumin in nephrosis leads to a series of changes which are very complex In addition to the increased creatinine clearance, there are increases in plasmı albumin concentration, in plasma volume, in urine flow and in serum sodium concentration No one of these factors alone apperrs to guarantee the normal excretion of sodium in the urine To complicate the matter further, Dr Deming and I have noted a fall in the sodium retaining activity of the urinary corticoid fraction during and after albumin-induced diuresis in a few patients with nephrosis Lauson These are the acute mmifestations within the same day I would be the last to deny that long term adjustments are made These probably include such factors as ferritin which Dr Shor has already discussed Luetscher Another problem in the study of nephrosis is that Various patients respond quite differently to the same stimulus In Figure 18, you can see some variations in the effects of albumin on the mulin clearance In these acute experiments, we were impressed by the large, rapid changes in glomerular filtration which were followed only after some time by the sluggish and reluctant appearance of sodium in the urine We had the feeling that an increased glomerular filtration rate was an important factor, but not the only one concerned There is again a variation in response, which depends on the situation If the serum sodium concentration is very low, there is usually a clear-cut initial diuresis of water, during which very little sodium is excreted The serum sodium concentration increases Such a loss of water without sodium has been observed at the very beginning of spontaneous diuresis and after the administration of albumin A striking example is seen in Figure 4* during treatment * See p 24 ·140 Renal Function CASE AR t2 EN PLASMA VOLUME RELAT VE TO CON ROL مرمر مرممل 3 3 PLASMA PROTEIN vih CONCEN TRATION WULAN CLEARANCE ru 31 URINE FLOW HA top SODIUM OUTPUT www ww www OUS ملنا فسنقنقي مينفقينفقا seo LAWW N *4 HOURS sod um FIGURE 18 Efect of injections of albumin on several factors affect ng the excret on of Each small arro v ind cates the inject on of 25 gm of album n Aote that the scale of unne flor and sod um output are magnified ten times in comparison wth the inulin clearance Reprinted from Can It test gation 29 896 (1950) of nephrosis with cortisone when water was lost without sodium and again during administration of ACTH when the patient lost several hilograms of fud before any sodium appeared in the urine All of these patients had a considerable reduction in serim sodum concentration In this setting a release of water regularly appears as the first event in a diuresis During albumin or cortisone treat ment it is not invariably followed by complete diuresis however and the serum sodnim concentration may rise to normal or higher without the appearince of sodium in the urine On the other hand if the serum sodnim level is nearls normal before the diuresis the initial water anresis is not scen and com plete elimination of edema may occur with little change in serum sodium concentration ·Glomerular Filtration 141 REFERENCES 1 GESELL, RAOn the relation of pulse pressure to renal secre tion Am J Physiol 32, 70 (1913) 2 CORCORAN, AC, and Page, H Renaf blood flow in experi mental renal hypertension Ibr. 135, 361 (1942) 3 LEVY, S E, LIGHT, RA, and BLALOCK, A Blood flow and oxygen consumption of kidney in experimental renal hyperten sion Ibid 122, 38 (1938) 4 BLAKE, W D, et al Effect of increased renal venous pressure on renal function Ibid 157,1 (1949) 5 HALL, P W, and SELKURT E E The effect of partial graded venous obstruction on electrolyte handling by the dogs kidney Ibid (in press) 6 HWANG, W, et al Effects of sustained elevation of renal venous pressure on sodium excretion in unanesthetized dog Ibid 162, 649 (1950) 7. Boss, WR, and OSBORN, C M The effect of adrenal cortical extract on renal function in hypophysectomized rats Anat Rec (abstract, in press) 8 CHEN, G, and GEILING EM K Antidiuretic effect of posterior pituitary extract in completely and partially hypophysectomized fats Proc Sof Exper Biol & Aled 52, 152 (1943) 9 JOSEPH, S, et al The anterior pituitary and its relation to the adrenal cortex in water diuresis Endocrinology 35, 338 (1944) ·ENDOCRINE FACTORS IN THE UTILIZATION OF GLUTATHIONE FRANCIS BINKLEY Department of Pathology (Biochemistry) University of Ulah School of Medicine I WOULD like to report certain of our experiments* which may beai on our ideas of the mechanism of action of the renal tubule Certuin of our experiments hue consisted of moderate saline loading of the dog with physiological sodium chloride so that there was a constant urine flow of about 25 ml and a relatively constant excretion of sodium Then a mixture of glutamine and sodium glutathione (18 and 50 gm respectively per 500 ml of saline) was infused with the salıne Within fifteen minutes there was ? sharp decline in the rate of urine flow and within sretz minutes the urine flow was reduced to about one tenth of its original amount The creatinine clearance either increased or remained con stant The sodium reabsorption was about the same as the water reabsorption We huve not had a dissociation of the two Except for one possible catch our series of experiments do show a very definite relationship between the metabolism of glutathione and the reabsorption of sodium by the kidney The catch is that glutathione contains a small amount of pyrogen when solutions of glutathione are injected into a rabbit there is a small temperature rise I should mention as a part of the further evidence for the relationship of glutathione and sodrum transfer that Grunert and Phillips(1) in experiments with alloran diabetic rats on low sodium diets have found that those rats develop low sodium levels ind glutathione lev such that there is a correlation between the two levels In Figure 19 is an illustration of what happens as measured by our methods of analysis(2) following the treatment of patients with ACTH This patient recened only 40 mg of ACTH daily There was a gradual decrease of the levels of glutathione over a period of several days and at the sime time there was an increase of * Ti ese stud es were supported by grants from the United States rubl e Ilealth Service F H Tyler Hi P Plenk I R himmel, C i Olson SF FC 110 and GM Chr stensen were collaborators in the work reported here r 142

·Utilization of Glutathione
143
ACTH
Patient GC
CYSTEINYLGLYCINE
سم
GLUTAMYLCYSTEINE
البلبلا
الليلة
البيليبرا
ار
19 26
33 40 4850
DAYS
FIGURE 19
Ermit montent (leukemia) on levels of gluta
The levels are expressed as ng
to a hematoent of 50 rrent

·144
Renal Function
Patient M
20-
CYSTEINYLGLYCINECO
10-
40-
GLUTAMYLCYSTEINE
10
بيل
20
10-
IB 25
DAYS
FIGURE 20
tah.
y glutamylcysteine As we have reported(1), thus fraction is charac
teristic of diabetic bloods The increase of this fraction also corre
sponded to the period when excess creatine was found in the unne

·Utilization of Glutathione
145
50-N
Hematocrit
0 0.5 2 5 7 24
HOURS
FIGURE 21
Effert of glutamine upon the hematocrit of a dog 18 gm glutamine in 500 ml
sahne was given intravenously to a 40 kg dog la iwelve experiments the average
hemodilution was 32 + 12 percent at 4 hs following admunistration of the glutamine
Since glutami lcysteine is glutathione less glycine glycine may
have been used in the synthesis of the extra creatine Studies of
another patient are given in Figure 20 The dosage of ACTH was
larger and glutathione disappeared from the blood
4

·146 Renal Function 250 100- Spleen Kidney 200- 50- Wha Zelda MG PER CENT L ver -100- 50 с 20 40 60 120 C 20 40 60 120 MINUTES FIGURE 27 Effect of adm n trat on of glutath one on tle levels of glutath one and the products of its hydrolyss n t ssues of the rat Vean values and standard dev at ons for cyste ne and glutath one of a control group respect ely are ind cated the enously at the f gluta 001) When glutamine was infused in the dog there was a sharp drop of the hematocrit most of the decrease occurred in the first fifteen minutes In the experiment shown in Figure 21 infusion was stopped in about an hour and the drop of hematocrit continued for several hours and returned to normal within twenty four hours Many of our studies have been handicapped by lack of knowledge of the fate of glutathione and the factors influencing the utilization of glutathione Therefore a rather extensite group of studies were undertaken in the first experiments glutathione was given intra found that the glutathione of muscle disappeared following the administration of ACTH and it appears that the glutathione had ·Utilization of Glutathione 147 Blood 25- Skeletal Muscle Chhillit lol. Cordiac Muscle 25- с 8 24 4 HOURS FIGURE 23 TA AT neally ervals f the (C) olumn Lysteinylglycine and glutamylcysteine are found in those tissues (blood and muscle) but are not found in liver kidney tissue or the spleen The decrease of glutath one and the increase of y glutamylcysteine in muscle tissue were found to be highly significant No significant changes were found in spleen kidnev or Liver t ssue been converted to glutamylcysteine (Figure 23) There was some increase of the y glutamylcysteine in the blood vith the short term administration of ACTH In cardiac muscle there was very much the same type of reaction (Figure 23) Shorr flow much ACTII was given? Binkley He gave 2 mg to each rat Thorn You believe that ACTH increuses the utilization of gluta thione by the kidney?

·150
Henal Function
BLOOD
25
SKELETAL MUSCLE
25-
manh
CARDIAC
MUSCLE
MO PER
NMG PER CENT
25-AV
30 60 90 120
MINUTES
FIGURE 28
Effect of adm mistration of insulin on the levels of glutathione and the products
of its hydrolysis in the tossues of rats Two unuts insulin were given subcutaneously
to rats and the groups (five or more) were sacrificed at the intervals indicated
Levels of cysteinylglycine y glutamylcysteine and glutathuone (mean and standard
error of controls mean of expenmental groups) Bre indicated the changes of
glutathione in cardiac muscle were bighly signifcant The increase of glutathione
in blood was found to be highly significant when smilar experiments were carried out
with rabbits (Serial Samples)

·151
Utilization of Glutathione
SPLEEN
50-
KIDNEY
100-
MG PER CENT
II
100-
LIVER
50-
C 30 60 90 120
MINUTES
FICURE 27
Effect of adınınıstration of insulin a continuation of the study of Figure 26
Levels of cysteine and glutathione are indicated the changes of glutathione in
When amounts of insulinas great as 100 units were given there was a decrease of
cysteine in kidney at 120 minutes

·152 Renal Function and tissues other than muscle start building up higher and higher levels of glutathione The administration of insulin appears to mobilize this extra glutathione from the liver (Figure 27) All the glutathione in the liver may be removed within one hour after administration of larger amounts of insulin At the same time, the kidney was obviously utılızıng larger amounts of glutathione since the concentrations of cysteine were increasing It would appear that insulin does mobilize glutathione first, by increasing the utilization by muscle tissue, and second, by stimulating the removal from the liver so that all tissues are allowed to take up excess glutathione very much as when glutathione was administered Thorn You have not tried to balance ACTH and insulin on Inver glutathione? Do you suppose you would observe antagonistic action of these hormones on this system? Binhley We have not tried balancing the effects Janeway Do you think glutathione is synthesized? Is it synthesized and built up in the liver and then taken from the liver and utilized in other places? Or is it built up in the kidneys or any site, such as the muscles and other tissues? Binhley Our concept is that it can be synthesized in all tissues except the reticuloendothelial system The adaptation in crisis depends upon how much there is in the Iner and the syntheti ability of the liver There is definitely transportation in the rec blood cell Thorn I wonder whether the hematocrit chmge in part might not reflect change in red blood cell size Binhley They tell me not Thorn There is a real entry of flud into the vascular bed Darrow Is there any change in the electrolyte concentration of the serum? Binhley A slightly reduced concentration of sodium Shannon What would be the average plasma dilution you ohtained with these various doses? Binhley Something around 15 to 20 percent Dock Did you give glutathione and glutamine together? Binllcy In the experiment in Figure 21, glutamine was given Somewhat better effects were obsers ed with glutamine plus glut? thione ·Utilization of Glutathione 153 Shannon \Vhat are the normal concentrations of these substances m plasma? Binkley Of glutamine, it carries approximately 10 mg percent, of glutathione 25 to 40 mg percent Shannon What changes result from the dosages you admostei? Binkley We cannot change the glutathione les el by the adminis tration of glutathione, the tissues take it up very promptly I should not say we cannot affect blood levels in conditions like operative shock on rats the levels of blood glutathione are found to fall to quite low levels We can bring those les els back to but not beyond, normal levels by the introduction of glutathione Thorn Have you ever tried raising the level in a nephrectomized animal by administration intravenously under these circumstances? Binhley. No Thorn I just wondered whether you thought the lner is the organ which defines the level Binlicy The liver I think to a large extent is a reservoir of gluta throne The liver hydrolyzes glutathione at a low rate Shannon Have you made any calculations on what happens to the total dose administered? You have data on muscle, liver spleen and blood Binkley I believe what you are asking is how well do we account for the glutathione we give Shannon Yes Binhley We account for 20 to 30 per cent Shannon Twenty to thirty percent? Binkley Yes Shannon When you add in fragments derned from glutathione, how much further does that go up? Binkley It brings it up perhaps to around 50 to 60 percent Shannon What happens to the remainder? Buhley We have not analyzed certain tissues Shannon You Jiave the ball, the spleen, liver, kidney, inuscle and blood

·151
Renal Function
Binhley I think 60 percent
Shorr How rapid is the rate of glutathione hy droly sıs?
Binkley Extremely rapid
Shorr Is it more ripid under anzerobic condihons?
Shorr Yes
Shannon It is surprising you can account for so much of such
lubile substance in a fairly long interı al
Binkley We are determining the fragments Cystine although
degraded is not degraded too fast by tissues other than liver
Thorn Did you get any appreciable fall in blood sugar levels
when you administered your gluththione?
Binhley Actually there is an increase of blood sugar
levels
Thorn What about man? Isnt it true that most of the nausea
from the amino acid preparation is related to glutamic acıd? Can
you give very much of your combination to man without producing
serious symptoms?
Binkley We have not given any of the materials to the human
as yet There is the problem of the pyrogens
REFERENCES
1 GRUNERT RR and PHILLIPS PH Sodium and its relation
to alloxan diabetes and glutathione 1 Biol Chem 181 821
(1949)
2 BINKLEY F TUJI S and KIMMEL J R Metabol sm of gluta
thione II Determination of glutathione and products of its
hydrolysis in blood Ibd 186 139 (1950)

·BIOASSAY OF SODIUM RETAINING CORTICOIDS AND SOME CHANGES IN EXCRETION OF THESE SUBSTANCES IN DISEASE* JOHN A LUETSCHER JR and QUENTIN B DEVING Department of Medicine Stanford Unnersity Salool of Medicine 11 HEX DR PITTS asked me to present this material I emphasized to him that our work is in a preliminary stage and that many funda mental questions remain to be answered This work started with some observations on nephrosis in col laboration with Dr A D Hall(1,2) Dr Fremont Smith expressed carler his dissatisfaction with the time honored concepts of the sequence of events in hypoproteinenuia as being due to loss of pro tein leading directly to hypoproteinenua and edema In studies of treatment of nephrosis with albumin "e found that replacement of the circulating albumin of patients with nephrosis was regularly followed by modest increases in albumin concentration and in collaid osmotic pressure but little change in total protein concentr. tion A large increase in plasma volume ensued accomprnied by increased clearances of muhin and creatinine accelerated water Output and rise in serum sodum concentration in about half the patients the whole thing stopped there and no effective elmination of edema was achieved in the other half of the patients the urme sodium increased and an effective druresis followed The total serum protein concentration rose as diuresis progressed These observations suggested that urine sodium excretion was the limiting factor in the diuresis after the initial stage of water dniresis had been passed and that the serum protein concentration could be raised only after diuresis of sodium had been established We then attempted an analysis of this data on the basis of pos sible causes of excretion of sodium to fajl under these circumstances and came to the conclusions that a large number of factors were involved and that our experiments were not adequate to define any single factor responsible for sodium elimination or its fulure Albu min deficiency a lou plusma volume a low serum sodium concen Tlus in est git on was supported by a research grant from the National Heart 155 ·156 Renal Function tration, and a low glomerular filtration rate were frequently asso ciated with sodium retention, but the return of any one of these factors to normal was not necessarily followed by inci cased sodum excretion Furthermore, when the sodium excretion was increased, it could not necessarily be correlated in time with any one of these factors It appeured that a low glomerular filtration rate could impair sodium excretion or even, at extremely low levels of filtra tion rate, conld prevent sodium excretion But at higher rates of glomerular filtration or, we should say, inulin clearance, the tubule could still restrict the output of sodium to very low levels You ale familiar with the nephrotics with supernormal filtration rates, which are another reflection of the same phenomenon(3) In casting about for a reason for the impairment of sodium excretion in these edematous patients, it seemed worth investigating further the possibility that something outside of the kidney might be playing a role It seemed to us that the deficiency in albumin in the nephrotic might lead to a low plasma volume which in turn might stimulate both posterior pitutary-like activity, as one mght see it after hemorrhage ou fainting, and adrenal activity like that due to any comparable stress Some information has been published during the course of our studies which indicates that there are increased levels of total corticoids in the urine in heart failure and in certain patients with toremias of pregnancy with edem1(45) These data did not seem conclusive, since we know that reducing and glycogenic corticoids vary greatly in their sodium retaining effect So we started out in the simplest way to measure the sodium retaining activity of the unnary corticoid fraction We have also attempted to measure antıdıuretic activity in urine and in serum Dr Quentin B Deming has carried the bulk of the work in these - nrnnorlures would like to say that he has really done a most 1 Our antıdıuretic hormone assays have been limited by probleints connected with the available methods Assays of urine of feeble activity by intraperitoneal injection in rats have given us serv questionable results The concentration of urine before injection leads to all sorts of errors Dr Deming has not been able to elute the material quantitatively from any chromatographic adsorbent so far Assiys of serum according to Dr Gaunt's method have given us occasional positive results of some interest, which I may mention ·Bioassay of Sodium Retaining Corticoids 15 later in connection with one case We have done no really sys tematic studies of antidiuretic hormone excretion 01 the serum levels The sodrum retaining activity of the corticoid fraction is measured by bioassay in adrenalectomized rats(6) We started off in the measurement of this fraction following the method of Dorfman lesing radio-sodum(7) It soon became obvious that if one did not strictly control the rats intake of water and sodium there were large spontaneous variations in output So ve put them on sodium free diets and gave them measured quantities of salt and of water by injection The sodrum chloride was given as an isotonic solution intraperitoneally the night before the test and an injection of water was given intraperitoneally at the beginning of the test to stimulate urine flow Unfortunately using adrenalectomized animals with this water load we turned it into a sort of water test at the same time This has one ment in that it discriminates gute clearly the activity of some of the 11 oxysteroids which produce a diuresis and a loss of sodium in this test as compared with desoxy corticosterone for instance which causes sodium retention We gave up radioactive sodium in favor of the flamc photometer for reasons of convemence The solvents employed in the administration of these extracts to rats are of great importance because of the considerable activity of some of the conventional solvents in searching for a vehicle which did not affect the sodium excretion in the rats and still would be a good solvent for the urinary extracts we fell back on ethyl alcohol in very minute quantities This does not bother the rats as much as a larger injection of some other substances which you might think would be much less irritating It is an cxcellent solvent and does not affect the rats output of sodium A twenty four hour specimen of urine is collected to avoid pos sible variations from day to night This is acıdified with hydro chloric acid to p!I 15 and extracted immedintely with chloroform The days urine extract in chloroform is dried evaporated and taken up in a minute quantity of ethyl alcohol A twenty minute sample is then injected into each adrenalectomized int We have expressed all of our doses in terms of the time during which the patient excreted the urine equivalent to the dosage rather than in terms of volume of urine in order to avoid the influence of urine flow on the assay The rat is then placed on a metabolism cage und urine is collected for the next five hours measured ind analvzud for sodium content ·156 Renal Function tration ind a low glomerular filtration rate were frequently asso ciated with sodium retention but the return of iny one of these fructors to normal was not necessarily followed by increased sodrum excretion Furthermore when the sodium excretion was increased it could not necessarily be correlated in time with my one of these factors It appeared that a low glomerular filtration rate could impair sodium excretion or even at extremely low levels of filtra tion rate could prevent sodium excretion But at higher rites of glomerular filtration or we should say inulin clearance the tubule could still restrict the output of sodium to very low levels You are familiar with the nephrotics with supernormal filtration rates which are another reflection of the same phenomenon (3) In casting about for a reason for the impairment of sodium excretion in these edematous patients it seemed worth investigating further the possibility that something outside of the kidney might be playing a role It seemed to us that the deficiency in albumin in the nephrotic might lead to a low plasma volume which m turn might stimulate both posterior pituitary like activity as one might see it after hemorrhage or fainting and adrenal activity like that due to any comparable stress Some information has been published during the course of our studies which indicates that there are increased levels of total corticoids in the urine in heart failure and in certain patients with toxemias of pregnancy with edema (45) These data did not seem conclusive since we know that reducing and glycogenic corticoids vary greatly in their sodium retaming effect So we started out in the simplest way to meisure the sodium retuning activity of the urinary corticoid fraction We have also attempted to measure antidiuretic activity in urine ind in serum Dr Quentin B Deming has carried the bulk of the work in these assay procedures I would like to say that he has really done a most difficult job with the greatest crre and with a highly critical attitude proper to such troublesome methods Our antıdıuretic hormone assays have been limited by problems connected with the available methods Assays of urine of feeble activity by intraperitoneal injection in rats have guentis serv questionable results The concentration of urme before injection leads to all sorts of errors Dr Deming has not been able to elute the material quantitatively from any chromatographic idsorbent so far Assays of serum according to Dr Gaunts method have gn en us occasional positive results of some interest which I may mention ·- Bioassay of Sodium Retaining Corticoids 159 between 20 and 40 minutes of urinary extract from a patient from ormals as well as the niore active extracts from the cardınc patients ind the nephrotics Below this level the response of the rats de reased in the expected way When more than this dose of extract was given no further increase in sodium retention was caused by he higher dose even though the response was well below the maximum effect that could be elicited from those animals by larger doses of desorycorticosterone for this reason we can speak only of the activity of these crude extracts under the specific conditions of the experiment and cannot sperk in more general terms I think this data emphasizes the need for fractionation and for further shady of interfering substances or whatever is the limiting factor in the response of the rats to these extracts We studied the effects of a small series of crystalline steroids Desorycorticosterone or its acetate was used as a standard The rats were sensitive down to approximately one microgram of DOC 01 DCA with a fairly straight line response from there up to five micrograms Above that dose the response to further incieises in dose falls off until n platenu is reached at a dose of about 10 to 25y Cortisone at 10 micrograms gives no response At 100 microgrims cortisone produces a striking diuresis of inter in these adren dlec tomized animals and more sodium appears in the urine We have not run any other adrenal steroids and have not run competitive studies between desoxycorticosterone and cortisone Testosterone estradiol and progesterone are wenk sodium retriners active only at high dosage(6) In a series of individual and pooled specimens of human urine we determined the activity of twenty minutes equivalent of urine from normal individuals and found a me in activity equivalent to 15 micrograms of desoxycorticosterone per twenty minutes of extract Eleven out of twelve individual assays have shown levels of 23 microgrims or less for twenty minutes. There has been no dfference in activity of unne from men and women as far as we can make out in this small sample The effect of the menstrual cycle on this assay is apparently small although this has not been sys tematically tested In one group of patients tested by the original method in which we simply estimated the factor by which the sodum excretion was reduced the crude assays indicated that there was relatively little activity in the extracts from normal patients and control patients ·158 Renal Function The assay takes three days to perform Each rat serves as its own control On the control day it receives no injection or a control injection of alcohol On the test day it receives the extract to be tested on the stundard day it receives five micrograms of desorycorticosterone acetate in order to assign a quantitative measure of activity we have used two measures The first one is the factor the ratio between the output of sodium by the rat on the control day and the output on the test day In other words an active extract might reduce the excretion of sodium by a "factor" of two compared with the control day Groups of nine rats are used for each assay and the logarithms of the individual factors rre averaged to obtain an average factor The log of the factor obtained is approximately proportional to dosages of DCA between 1 and 5 micrograms for each rat group If a line is drawn on semi log paper from the origin (fictor 1 dosage 0) to the point represent ing the response to the standard injection (factor determined dosage 5v) the activity of the unknown can be assigned an eguna lent in micrograms of DCA When we started to test this assay procedure we encountered another disturbing variable If one uses rats immediately after adrennlectomy there are changes from day to day If one w uts too long one has a number of sich or dying rits We have worked in the intermediate stage of about three to five days after adrenalec tomy In order to avoid the day to day variation we adopted the suggestion of Dr A G Spencer who was working on a very similar method last year at Columbia(8) We divided each group of nine rats into three subgroups włuch received the control standard and test injection respectively on each day The subgroups were rotated each day so that at the end of three dys each subgroup had recen cd the three different injections on different days The reproducibility of these assays has been surprisingly good since the assay has been going smoothly If for any reason an assay is suspected of being erroneous it is repeated The second assay is within plus 10 or minus 10 percent of the duplicate assay if we are lucky and as badly off as 20 percent on an occasioni bad day With crude extracts of human urine one does not obtain a knear response as the dosage is increased Larger doses did not make the rits visibly sich nor did they impair the survival of these rats but it was obvious in plotting the dose of unne extract against its activity that a peah uns reached when each individual sat received ·Broassay of Sodium Retaining Corticoids 161 Burncit These nephrotic patients all have edema? Luctscher There has not been a strict correlation between the presence or absence of edema and any given level of actuity of the unnary extract in general those patients showing the Bughest assays have been uniformly edematous and those patients with the lowest assays have been almost free of edema However I dont Want to suggest in any way that this is the only possible cause of edema in nephrosis or any other condition All we are trying to do as to introduce another complexity into your thoughts on this subject The series of untreated nephrotics now comprises tuele patients In these patients the mean assay is 38 micrograms with 1 range of 17 to 84 micrograms Eight of these tu clve patients have been above 23 micrograms You will remember that eles en of twehe controls assayed 23 microgrims or less Shortly after we became convinced that these levels were in usually lugh in the nephrotics cortisone became available If the activity which we were measuring arose in the adrenal cortisone might be expected to reduce the excretion of this abnormally actite material of course there are a great number of other possible explanations of the various effects of cortisone in nephrosis lle wondered whether it might have some infuence on the discuse process itself We wondered whether we might see some intagonism of antidiuretic activity of the posterior pituitary was involved in the phenomenon We could speculate at length but first let us consider some data from patients with nephrosis under treatment with cortisone In Figure 2* the assiy levels are indicated in micrograms per twenty minutes as before Here is a five year-old girl who has a very moderate amount of edema She is only about a kilo above her ideal weight She has a modest proteinuria about a gram and a half a day she has a lou secum protein level of 4 gm percent She is putting out a small urine olume containing modest amounts of sodium With the administration of 100 mg of cortisone z da for sur days one sees an increase in the level of proteimurin to double the original low level 1 complete shutting off of urinary excretion of sodium and an increase in body weight uhich had been stationary before treatment After the discontinuance of treatment there is a release of water and sodium in the urine a sharp fall in the See 22 ·160 Renal Function from the hospital wards We encountered much more actre extracts in patients with cardiac failure and m edematous patients with the neplırotic syndrome Some patients with hypoproteinemi hyper cholesterolemui, and proteinuria, but without edemt did not gie a positive assas So there was a rough correlation suith the presence or absence of edema(6) When the assay was improved by the modifications described we begin to express the results in microgrims of DCA We can nou compare twelve patients with nephrosis with an equal number of normals and hospital controls (Figure 28) At a level of about 23 micrograms one can draw a fine artificial line which roughly separates our group of normals and patient controls from the edematous nephrotics with the high assays The most striking in creases in activity has e been observed in patients with the edema of nephrosis and in those with heart failure Levels above average normal have been noted in one case of acute pericudites with effusion and signs of tamponade, one patent with chronic constric tive pericarditis and in two hypertensne patients uncomplicated by other factors One hypertensive in cardie failure and a patient with malignant nephrosclerosis and mild cudac fahre Irve gnen a high assay P<1% > 9 8 SO 109 SD: 106 DOCA 7 LIKE 6 EFFECT 3 NEPHROTICS NORMALS FIGURE 38 Comparison of sodium retarning artwity of urinary corticoid fraction in mepl ros* and in normals ·Bioassay of Sodium Retaining Corticoids 163 the administration of 100 mg of cortisone day we observed the diphasic response which has been referred to before First there was an increase in body weight and proteinuria Then after a week of treatment there was an increased urine volume r fall in body weight a diminution in proteinuria and small increases in serum protein and albumin concentrations A small increase in urine sodium also appeared At this time an assay indicated some reduc tion below the control level of activity in the urinary extract Then following the withdrawal of cortisone there was a great increase in urinary volume and sodium The body weight fell 12 kilos below the control weight Proteinuria became less than 200 mg per day At this time there was a fall in the assay to a normal level Improvement seemed to occur regularly in these patients at about the time the eosinophils were rising after the end of cortisone administration We interpreted this coincidence in time as evidence SSVP NOK COR SONE POR DAN GM MC 0900 Dhe EMP 39-30 PROTEINURIA $ - BODY WE GHT - 3 DOC ASSAY Y 701 L. PLASMA TOTA 11 PROTEINSSO 900 41 M ALBUM 31 100 93 UR NE VOLUME AND SODIUM 40 EOS NOPHES 10 ITO po Dars 2451 812345678910 2 3 4 5 FICURE 29 Bref rem ss on interrupted by infection M he In press) ·162 Renal Function proteinttria down to levels of 40 mg a day in this instance and an increase in the serum protein and albumin concentrations to levels approaching normal In this particular instance, we were unable to demonstrate any effect on the level of sodium retaining activity, which was within normal limits before treatment Figure 3* shows some changes in a seven year old boy with the nephrotic syndrome, in this case with massive edema a proteinuria of 4 gm a day, a very small urine volume, essentially no sodium in the urine, and very low levels of total protein and albumin concentrations This patient's urine gave the highest assay we have observed in any patient We have been impressed with the very high levels of activity in many of the children with nephrosis and having no sound basis for the correction for body weight, ie prese these data as observed We were enced, too, by the coincidence that at this time Dr Harold Faber asked us to see a baby three months old with Lawson Wilkins (9) syndrome of congenital adrenal hyperplasia of the androgenic type with the lack of the sodium retaining hormone of the adrenal and excess of androgenic hormones I was astonished to discover that this infant required just as much desorycorticosterone for maintenance as an adúlt In a casual search of the literature, this appeared to be the rule Jancutay That is a peculiar situation, isn't it Dr Thorn in which you don't hue adienal itisufficiency in the ordinary sense but you might say you have adrenal dysfunction with disproportion in the production of different hormones, the adrenal putting out huge amounts of androgen We find they need two or three times the ordinary dose for maintenance I don't know that you can extra polate from that to the needs of normal children Thorn No Dock Thus condition persists throughout life? Jancuay They are born with it Thorn The 11 oxy methods give pretty close correlation of sma}} children and adults on the excretion basis This docs not of necessity of course refer to secretion of hormone Luetscher In any case, here was a youngster with the severe form of nephrotic syndrome, with a very high control assay During # Sec p23 ·Buoassay of Sodium Retaining Corticoids 165 tintil eosinopenia was noted There was a very sinall output of urine, and the weight was steadily increasing Treatment was intei rupted because of the rise of the serum potissium to 75 mEq per L After treatment, the weight gain proceeded as before Tiere was no effect on proteinuna, and no release of sodium There was some increase in serum protein concentration The appetite im proved greatly Increasing effusions caused respirator embarrass ment and Southey's tubes were used to draw off much of the edem. The administration of concentrated luman serum albumin was followed by duuresis and by a fall in the activity of the urinary corticoids Figure 31 shows a simular response in a twenty year old man with the nephrotic syndrome Neither diuresis nor reduction in sochum retaining activity followed a brief course of cortisone but the lea 1 of the assay fell with the diuresis after the administratio of albumin Pitts Could you summarize there? Lý do CONT SOM NU AO-20 ALBUM N V KODY BODY WEIGHT 22 PROTEINURIA DOC ASSAY Y 308 437 327 1BY PLASMA TOTAL PROTEINS su 09 N n3 h to URINE VOLUME - AND SODIUM OLE LOSS 11 19 200 220 DATS 2 23 TU 9 10 2 FIGURE 31 ketaining corticoids with subsequent response to albumin Fa fure of a brief course of cortisone to alert edema or lesel of yod un ·164 Renal Function of a release from the effects of cortisone, not only on the eosinopluls but on the kidneys and on the excretion of salt-actie corticoids Such a good clinical response has followed cortisone treatment in approximately half of the patients Even when it does occur, it may be very rapidly interrupted by an intercurrent infection Figure 29 shows the couse of a three-year-old girl who developed a urinary tract infection after her diuresis There was an immediate rise in proteinuria and a disappearance of urinary sodium At this time the assay, which had decreased after the end of treatment, increased to a les el higher than that in the control period In those patients who failed to respond to cortisone, we have seen if anything a slightly higher sodium retaining activity in th urine after treatment than we saw during the control period Figure 30 shows the course of a three year old boy treated with cortisone We were uncertain as to the proper dose, and increased the dose LA 3. MEP Moss EN COATSON W CDA Co to 4 880 SUNN 10 10 13 BODY WEIGHT 10 KG 11 rer PROTEINURIA 20 . DOC ASSAY Y 757 >5 98Y 20Y PLASMA TOTAL PROTEINS OG GM/100 ALBUM URINE VOLUME - AND SODIUM 10 2- EOS NORMES DAS 1 2 3 34 S&23 FIGURE 30 Failure of a brief course of cortisone to affect edema or Insel of solum retaining corticoids with subsequent response to albumin Note increased serum protein and albumin leveh during cortisone administra tron ·Falure of 2 br f course of cort sone to affect edena or le el of 1 1 ( - t 1 I t Bioassay of Sodiun Retaining Cont until eosinopenia was noted There was unne and the weight was sterdily increasing 7 rupted beciuse of the rise of the serum pot L'After treatment the weight gain proce ! was no effect on proteinuria and no releas M some increase in serum protein concentrat prored greatly Increasing effusions caused ment and Southeys tubes were used to draw i! The adminstration of concentrated humus followed by diuresis and by a fall in the att corbicoids Figure 31 shows a similar response in a twenty Veu the nephrotic syndrome Neither diuresis nor red icti retamıng activity followed a brief course of cortisor e b tti of the assay fell with the druresis after the admin tat 1 1 1 albumin Pitts Could you summarize there? 1C 20 EPOS RR 1600 ALBUM N POR DA BODY WEIGHT 72 PROTEINURIA DOC ASSAY Y 30Y 437 328 18Y PLASMA 19 A PROTEINS wone * h24 yo UR NE VOLUME DE . SOO(UM 3 NA 2015 TOOLS 2 N DAS TIGURF 31 Uud ning cort o ds sth subsequent respon e to album n.

·166 Renal Function Luetscher Eight of fourteen patients have had a diuresis after cortisone treatment in three of the eight there is some doubt that cortisone was responsible for the improvement All except one of the eight had a reduction in proteinuria Two patients had a nearly complete remission in the sense of a fall of proteinuria to a few milligrams per day and a rise in serum protein concentration to essentially normal levels in the other six patients diuresis has failed to occur after the administration of cortisone We have not been able to determine why this failure occurred It is not related to the apparent etiology or activity of the renal lesion occurring both in patients with so called pure nephrosis ind in those with evident glomerular nephritis We have not been able to divide the successes from the failures in terms of dosage or duration of tre: ment To summarize the assays Of ten patients on whom assays have been done five patients have hnd 1 diuresis after treatment and three of these had hnd high assays before treatment Assays on these three patients fell after treatment The other two patients who had diureses gave assays in the normal range before trertment and these were unchanged after treatment Five patients who failed to show a diuresis after cortisone treatment all showed higher assay levels after the administration of cortisone thin before Two of these cases showed a fall in assay after a subsequent diuresis with albumin Two questions immediately arise in connection with these results The first one is What is the effect of a low sodium diet on the activity of the corticoid fraction? We have not yet set up systematic experiments to clarify this point Three patients on low sodium diets comparable to those in the nephrotics have shown normal levels of sodium retaining activity One of our nephrotics on a normal diet gave one of the highest assays that we have seen Sodium intake was not a major factor in these patients On the other hand we cannot rule it out in the other patients The second question is whether diuresis per se might account for these results If one thinks of it in terms simply of 2 mgh water output we can say conclusively that this is not the case since we have seen situations in which the water ontput has increased while the assays remained fugh although sodium excretion remained quite low So simply a large urine volume is not the re son for the assay level to fail ·Bioassay of Sodium Retaining Corticoids 167 There have been other important changes during the treatment of these patients in addition to the changes in the ussay figures The creatinine clearance is usually increased during tre itment with cortisone or ACTH This increase may begin when the urine sodium is very low The creatinine clearance is often falling when the peal of diuresis is reached after the end of treatment There has often been elimination of water during the latter days of treatment which bas sometimes resulted in a considerable loss of weight and increased senim sodium concentration There have also been increased con centrations of serum albumin ind in some cases of total protein All of these changes are in the direction of normal but at the end of treatment it is very difficult to look at one of these patients and predict from the extent of these changes whether he is going to be one of the 50 percent who are going to have a druresis or of the 50 percent who are not going to show a diuresis However when you look at the groups as a whole there is a Inghei werage glomerular filtration rate and a higher average serum sodium at the end of treatment in those patients who are going to respond I should like to mention briefly the effects of ACTH in the nephrotic So far five out of six patients have had diuresis There as little question from these results and those reported by othei students of the disease that ACTH is probably i more effectie agent for clinical treatment However from an experimental stand point it leads to a much more complicated situation We started off with cortisone largely to try to keep our experiments as simple as possible We dont have enough data as yet from the bioassay to indicate with any certainty what the trend of the bioassays in patients treated with ACTII is going to show In attempting to explain the diuresis which occurs during ACTII treatment we have insufficient data to form a satisfactory basis for hypothesis Figure 4* shows some changes in a sixteen year old girl with the nephrotic syndrome who was treated with cortisone with albumin and later with ACTH This is the only one of fourteen patients treated with cortisone and with albumin in whom no Significant amount of edema was eliminated There was loss of edema during and after the subsequent administration of ACTH Flere one can comprre the effects which occurred during these different treatments We like to emphasize the diphasic changes in edema during cortisone and ACTH administration with the • Ser p 24 ·168 Renal Function corresponding changes in urine volume Urine flow is depressed during the enrly stages but usually increases gain on later days of treatment In Figure 4* you can see this later release of water without change in sodium excretion with corresponding fall in body weight and rise in serum sodium concentration A similar increase in urine volume occurred during ACTH therapy at first without excretion of sodium Then as the serum sodium concentra tion approached normal significant quantities of sodium appeared in the urine Finally there was a moie profuse diuresis after cessa tion of ACTH The changes in serum sodium are in the direction of 7 dilution and subsequent concentration of the serum sodium levels but this may not be the only factor involved Attempts to corelate the changes in sodium excretion with glomerular filtration rate alone have met with little success In this patient as in most of our other patients there were considerable increases in creatinine clearance which have been checked from time to time with inulin indicating that considerable increase in glomerulır filtration rate has occurred during treatment with corti sone without any effect on the urine sodium There is also in increase in creatinine clearınce during the administration of ACTH but the peak is slightly out of phase with the perk of sodium excretion The serum sodium level appears to exert an effect on the appearance of sodium in the urine but there is only 1 rough cor respondence between the tubular lond of sodium and the quantity of sodium in the urine There seems to be a number of factors involved and it is unlikely that any one factor will explain all the changes In summary the assay data so far indicate that we have a very high control level in a number of edematous patients with heart failure or with the nephrotic syndrome We are not suggesting that this is the whole story of edem, or a cause of disease It is simply a contributory factor which appears to play a role in many of these patients We wondered whether perhaps this might be a com pensatory factor evoked by the deficiency of circulating protein in the nephrotic syndrome or by the reduced cardiac output in heart failure If increasing such activity is followed by increased proteinuriz(10) in the nephrotic syndrome it is conceivable that a vicious circle might be set up in which the attempt to retain salt and water and to ruse the circulating blood volume might . See p 24 ·Bioassay of Sodium Retaining Corticoids 169 aggravate the proteinuria This is mere speculation but might explain the prolonged remissions which have followed therapy Much longer observation of patients will be necessary to determine possible effects on the underlying disease We have emphasized the correlation of the activity of these urinary extructs with the edema of nephrosis without meaning in any way that this is the only factor involved in the production of edema It is obvious that protein deficiency a low plasma volume low serum sodum concentration and low glomerular filtration rate are all important We would apprecirte suggestions about where to go next We have much systematic work which we must do such as the effect of dietary sodium We feel that it is important to go on to some fractionation of these extracts Being interested in whether this was desorycorticosterone or not we partitioned one of these active extracts between water and benzene We found that the abnormal activity just as Dr Pincus reported to your Conference on Adrenal Cortex last year on the activities of normal urine appcared in both the benzene and water fraction It does not go entirely into benzene as one might expect if it were desory corticosterone Dr Edward Hyman worhing with our group has done some preliminary chromatographic studies He has not found any material resemblınm desoxycorticosterone in his chromatogram of the active extracts When one of these active extracts is put on the column most of the components appear to be in the more polar fractions of the adrenal steroids Moreover we have failed to detect much activity in the extracts from unine of patients who are receiving DCA We would like to work on blood but hue hesitated because of the very small quantities of circulating steroids We plan to do some studies on adrenal cortical extract and on more of the crystalline adrenal steroids to compare with the material which we recover from urine If it turns out that the activity which we measure comes from the adrenals we may remember that there is still a lot of sodium retaining activity in whole adrenal extracts which apparently is not related to the crystalline compounds in the concentrations in which they are descnbed in extracts One might rationalize the unusual activity by the concept of some autonomous iu the glomerulir zone if you think that is where the hormones are produced what is the stimulus to this Ana ·170 Renal Function sive activity or excessive secretion of the sodium retaining lior mones? Under the cucumstances I will say no more about these points but simply refer to the recent review of Sayers(11) on the regulation of adrenal activity Much more work is necessary before we can be sure that we are dealing with an adrenal secretion DISCUSSION Doch What is the level of excretion in Addisons disease? Luetscher One case untreated with DCA fell in the low normal range The problem is that with the bioassay a very low activity is meaningless Let us say when we inject a microgram and a half of desorycorticosterone into each of a group of rats that it may influence this group of rats to retain sodium equivalent to a factor of let us say 15 But we know that given a control injection con taining no active material some rat groups may give a factor as high as 14 Therefore in the lowest levels of activity it is not possible to talk about the presence or absence of activity or to make an accurate quantitative essay Ralli Dr Pincus has perfused isolated beef ndrenals with 11 desoxycorticosterone Its perfusion produced material which when isolated in sufficient amount was characterized chemically as corti costerone or compound B in Kendalls list Pincus infers that this indicated the ability of the gland to oxygenate in the 11 position and to produce the 11 hydroxy group(12) This may or may not have some bearing on what you have been doing Another point is that the method of assay that you are using mght not detect what was being excreted Thorn Stahl in Strasbourg is actively at work on the metabolsm of desoxycorticosterone in patients with liver disease Such patients handle DCA very differently from the normal It is also true that many arthritic patients reveal impaired liver function and some even exhibit amyloid disease It might be that one obtuns much more complete metabolism of DCA under these circumstances than in a normal subject It would also be desirable to check an Addisonian maintained on a constant dose of DCA and also an Addisonian on 100 or 200 mg of cortisone to be certain that there is no conversion to DCA An absence of DCA in patients with intact adrenals treated with cortisone could reflect an inhibition of endogenous DCA formation ·Broassay of Sodrum Retaining Corticoids 171 Luetscher He have studied some patients who have been on high doses of cortisone for the treatment of arthritis Some of these patients have shown high normal les els but we have seen no extru agantly high levels in answer to Dr Rallis question I think Dr Pincus observed that the adrenal could convert 11 desory corticosterone to corticos terone That is presumably not the normal method of degradation of desorycorticosterone in the body The fact that very little 11 oxysteroid is demonstrated after the administration of desoxycorta costerone indicates that in 7) probability very little goes in that direction Thorn We have observed one patient with a mitral and tricuspid stenosis in marked edem? with very lou sodium level who on cortisone therapy developed normal sodium levels much like the nephrotic This was the first time in a long period of hospitalization the normal serum was obtained It is to be noted however that He administered potassium chloride to the patient with cortisone Burnett Before or after? Thorn The serum sodum returned to normal after cortisone therapy Did you ever notice chloride excretion trenty four hours before an increased sodium excretion of either ACTH or a des sociation of those tuo as a preliminary to sodurn excretion? Luctscher We have not measured chloride excretion that is on the agenda which has become much longer than the things we have been able to do Nalle Is it possible that the diuresis that occurred during the administration of ACTH was due to substances in the pituitary extract other than ACTH? Luetscher We assumed there were two phases the first one the druresis of water without sodum We had thought of it as a possible anti antidiuretic action of the type described by Dr Gaunt In the sodum release during trentment we were interested to observe in one of our patients that the eosinophils rose simultaneously while in another one there was no convincing "escape" to judge by the eosinaphıls We are continuing to look for hints that the adrenal somehow shifting gears at this point le plan to measure the 17 hetosteroids and some other contentional indices of adrenal activity during these changes to see whether we could show any ·172 Renal Function change in adrenal activity coinciding with the changes in urinary output White Do I understand correctly that in these nephrotic cases during the period of administration of cortisone the proteinuria jisually increases or does not change and then drops after you stop giving it? Is that the usual thing? Luetscher Unfortunately, the usual picture is that nothing linp pens to the proteinuna after cortisone When the protemuria changes, there is usually an increase during the first few days of cortisone administration, followed by a decrease in protein loss at the same time that one sees release of water and sodium If a real improvement is to occur, it occurs in the two or three days following the end of administration of cortisone Wlute How long does the protein output stay down? Luctscher In some patients, the proteinung reappears very quickly and returns to the pretreatment level, either after an infec tion or without obvious cause On the other hand we have two patients who have now gone between eight and nine months with no evidence of a return of proteinuria to the pretreatment level White I jas pusst wondering if there is any eydence of an increased production of cortisone in measles, more than in most other diseases Luctscher I would like to say only one thing Our one failure with ACTH treatment confounded us by getting chicken por one month later and having a magnificent diuresis Fishberg In one patient I had a remarkable diuresis with measles and that patient relapsed I give ACTH and agun obtained a very fine result Janeu ay Our group has been interested not specifically in the hormones, but in the problem of nephrosis for the past six or seven years I think the first point which I am sure Dr Luetscher will admit, is that anything that you do with a chronic disease like diers has to be done against the background of what you know about its natural history If you get a nephrotic in a bed in a hospital and leave him alone, very often you will see regular fluctuations in body weight 17thout any treatment A few times we have been able to get complete balance studies during such weight changes It looks 25 z fairly constantly you are having stufts of water into cells and ·Bioassay of Sodium Retaining Corticoids 173 then out of the cells without any change in sodum excretion Apparently you can have changes in intracellular water going on which may account for these rather minor fluctuations The second point is spontaneous druresis which can occur at the most unexpected and embarrassing moments I was interested in looking at your Figure 4* Dr Luetscher where ACTH appeared to initiate a very remarkable diuresis llas that it? It had 211 the characteristics of the type of diuresis which may occur in these patients out of the blue There was a much sharper drop in pro teinuria than we have usually observed with administration of ACTH I think it is not beyond the realm of possibility that such a druresis might have happened whether the patient got ACTH or not certainly these patients are in a condition where the slightest stimulus of almost any sort can set in motion so miny mechanisms we dont understand that it is terribly difficult to interpret any thing that is not repeatable again and ag un We have probably fallen into the trap of confusion but have restricted ourselves entirely to ACTH in these patients because we wanted to learn as much as we could about one agent and thus have not yet used cortisone at all We put all patients on 100 mg 1 day for ten days and they have a dheiresis with I should say about 60 to 75 percent regulanty just prior to or on withdrawal of ACTH One patient had a slight druresis cach time the drug u as stopped as the cosinophils rose Following the third and most intensie course the cosinophils only returned to normal very gradually but this is when the weight came down the urine volume rose and the sodium excretion rose Sery sharply In that patient the changes in proteinura were not Very striking While there is some reduction the diuresis proceeded with continuing proteinuria It was only following the diuresis that proteinuria dropped off to much smaller amounts I should say the one difference we have seen in patients who recented ACTII from those to whom we have given measles - and we have a series of about ffteen of those children now - is that following measles proteinurin diminishes iery sharply just at the time or before the diuresis is initiated whereas in general in our patients who have been given ACTH the total output of protein per twenty four hours in the urine has really changed relativel little I dont think that is a terribly significant fact but it is about the only aspect of the response to these two stimul that apperrs to be somewhat dif ferent We have treated twenty one patients nou for total of • Sec p *1 ·Renal Function 174 twenty nine courses and the response would appear to be dependent more than anything else on the duration of the disease Dr Luetscher spoke about pure nephrosis and glomerular nephritis I wont get into that controversy except to say the more we get into studying the disease in children the more we are in clined to believe it is one disease that it is merely a matter of the intensity and duration of the process which gives you the different clinical picture This statement is based on work by Dr Vetcoff and Dr Kelsey(13) who have made many observations of glomeru lar filtration rate by inulin and thiosulfate clearance both done in checking each other and agreeing fairly well at different stages of the disease in these patients The two striking findings hne been first that glomerular filtration rate is always reduced in the patients that we have observed in the first few months of the disease and second that supernormal clearances may be observed in some of these patients from two to six months after onset Subsequently clearances in these patients may fall to low levels and this has beer associated with the appearance of greater numbers of red cells in the urine whereas they are in a relatively normal range during the high clearance phase The main thing that I wanted to point out is the magnitude of the effect of ACTH on glomerular filtration rate in these nephrotic children In one patient it rose from 40 ml per minute up to 85 ml In another patient in whom clearance had fallen from the supernormal phase to quite low levels in ten days at has come back to nearly normal and clinically at least that patient is nou doing extremely well What the explanation for these changes in glomerular filtration is is certainly far from clear These are a good deal more striking than the changes which Dr Burnett has seen in his normals with doses that are somewhat greater than the ones we have given Another thing which to me is very striking is that in seieril patients who have failed to respond with any diuresis to the administration of ACTH there has been a very striking chiange in the abilty of the kidney to carry out its biochemical functions Just the other day I saw a patient whose NPN had risen from 45 over 2 three months period to a level of 98 who was becoming acıdotic and for whom we give a very had prognosis ACTH was given without any effect on water balance whatsoever but ever since that time his NPN has been normal lns acidosis has been repaired and his tubules are apparently able to carry out the regulatory job which they could not do before Burnett Did the hematocrit fall?

·Bioassay of Sodium Retaining Corticoids 175 Janeway ACTH tends to increase the red cell mass but the plasma volumes remain almost exactly the same 11 hite The behavior of the filtration rate in dogs on ACTH is like that seen by Dr Burnett in the normal humans Neither normal nor hypophysectomized dogs have shown any increased filtration rate on ACTH We gave much smaller doses than those mentioned here One out of three of our hypophysectomized dogs did show a 30 percent increase in filtration with ACTH but so did one out of two adrenalectomized dogs show the same increase on ACTH Thorn We have been interested in this mechanism and you might be interested in some of our observations When we first gave ACTH we gave it for a short period ie five to seven days The druresis we observed occurred following the withdrawal of ACTH und we felt that in part at lerst the inactive adrenal following withdrawal of hormone was responsible for the increased sodium chloride and water loss However in later experiments with pro longed ACTH administration a diuresis was observed on ACTII in the presence of a very active adrenal secretion Another interesting point is that during the period of diuresis of the patient is tested with pitressin or with desorycorticosterone the kidney tubule appears to be relatively refractory to these agents just the reverse of adrenal insufficiency in other words a standard dose of desorycorticosterone 20 mg or standard dose of putressin did not give the sodium or water reduction which we usually see in patients with adrenal insufficiency and we wondered from that whether there was not as a result of the ACTH treatment in in capacity on the port of the kidnev itself to react normally to these two ngents Having studied a number of patients with acute nephritis sub acute nephritis and nephrotic syndrome I have come to the con clusion that ACTH and cortisone hue little beneficial effect on kidney disease and I think that this may also be true in liver dis ease It strikes me that this is very interesting when one considers the beneficial effects of these hormones in such a wide variety of nonspecific inflammatory diseases With nephrotic patients the re sults are different Here the diuretic actions of ACTH and cortisone are very satisfactory I do not beliese the course of the disease is really modified specifically however we recognize that in many patients any agent which will induce the disappearance of edema Une ·Renal Function 176 is likely to be followed by 1 decrease in protein excretion und a more or less prolonged remission Burnctt Our experience with cortisone in nephritis is that it may actually make the disease process worse Although renal function was improved by cortisone proteinuria and hemiturri were also marhedly increased during the administration of this steroid(14) Lauson I would like to remark as long as we have come back to the nephrotic syndrome again that it seems possible to achieve a certain amount of unification of concept from the various studies on ACTH albumin therapy and nitrogen mustard I have been impressed by the fact that when both nitrogen mustard and ACTH therapy result in diuresis there is a concomitant reduction in pro teinuria an expansion of plasma volume and an improvement of the circulatory status - in other words a more adequate circulation As far as I know there has been a rise in glomerular filtration rate ench time that there has been a successful diuresis Albumin does the same thing except that duuresis occurs after direct expansion of blood volume and rise of filtration rate without decrease in proteinuria On the contrary the bulk of the administered albumin goes out in the urine I think it is possible at present to look upon the nephrotic syndrome as a consequence of a single initial event nimely proteinuria resulting in a condition of circulatory inade quacy Reversal of that circulatory inadequacy may well be the basis of spontaneous diureses or those induced by ACTH nitrogen mustard or albumin I had a question I would like to ask Dr Luetscher on the tech mcal aspects of making a chiloroform extract of highly protemaceous urine I wonder whether you have done the control experiments of adding to normal urine both plasma proteins and an adrenal steroid like pure desorycorticosterone for example and then put that through the extraction procedure and assay to see whether in the presence of all the protein you can get out that little bit of steroid? Is there perhaps 1 possibility that you are getting some thing nonspecific out of this protein mixture that may be a salt retaining effect in the assay rat? Luctscher This possible source of error is ruled out I believe by the low activity of the urines of those patients who have recen ed concentrated human serum albumin li diuresis occurs in these patients the sodium retuning activity returns to normal These ·Bioassay of Sodium Retaining Corticoids tehtively inactive extracts come from urines which contain more protein than the control urines which were highly active Pitts You spoke Dr Luetscher about certain difficulties in find ing in your solents themselves some sodium retaining activity and your eventual choice of ethyl alcohol as being the least disturhing Could you amplify that a little more? Luctscher We started out with the conventional method of suspending the extract in bland oil and encountered two problems In the first place many of the more polur steroids are quite insoluble in oil and it is very difficult to get uniform suspensions of urinary extracts We liad to work with unstable suspensions in oil wlich seemed very unsatisfactory Furthermore if you are taking up chloroform extracts int) oil it is easy to contaminate the oil with trices of chloroform which may produce renal injury in the rats So we tried 7 numlic of vehicles which we thought would be better solients and would be caster to work with in esery way lle run into a lot of nexpected activity in control experiments with injections of the solvents(6) of the solvents that were tried only polyethylene glycol and alcohol appeared to be both nontoxic to the rats and to have a lack of effect on urmary excretion of sodium Me tested carbowax propul ene glycol glyceune and carbstol When injected in the furly large quantities necessary to dissolue the extracts the adrenalectomized rats showed either some disturbance of renal function suppression of water output or suppression of water and sodum output The only two that we found that were decent solvents for the ringe of compounds in which we were interested ind which had no effect on the excretion of sodum or of water by the adrenalectomized rat were minute quantities of ethyl alcohol and polyethylene glycol Caunt Propylene glycol can be i toric substance in the adrenn! ectomized rat Luctscher There are a lot of interesting points that we tend to overlook. Certain bland solvents have been observed to increase the liver gh cogen in the adrenalectomized rat There was one moment When we thought we could work out a furly complete treatment for adrenal insufficiency from a mixture of ordinary solvents but unfortunately the pathological studies of some of these ruts after Wards indicated that the large doses w buch we used us ere quite toxic ·178 Renal Function REFERENCES 1 LUETSCHER JA JR HALL A D and KREMER V L Treat ment of nephrosis aith concentrated human serum albumin 1 Effects on the proteins of body fuds Cl Instigation 28 700 (1949) 2 Treatment of nephrosis with concentrated human serum albumin 11 Effects on tenal function and on excretion of water and some electrolyzes but 29, 896 (1950) 3 EATERSON K JR and DOLE V P Diodrast and inulin clear ances in nephrotic chuldren with supernormal urez clearances Ibid 22, 447 (1943) 4 PARRISH A E The bioassay of adrenal corticoids in the urine of patients with congestive heart failure Ibid 28 43 (1949) TONIAN L JR Cortical steroid excretion of edema of pres nancy pre eclampsia and essential hypertension Clere Eruda irmal 2, 319 (1949) 6 DEMING Q Band LUETSCHER, ) A Je Biorssay of desoxy corticosterone like material in urine Proc Soc Exper Biol & Kred 73 171 (1950) 7 DORFMAN RI POTTS AM and Fril ML The use of radiosodium for the detection of small quantities of desoxycor ticosterone Endorsmology 41, 464 (1947) 8 SPINCER A G Biological assay of small quantities of desoxycor ticosterone acetate Nature 166, 32 (1950) 9 WILKINS L The Dragnosis and Treatment of Endocrine Drs orders » Cildhood and Adolescence Springfield 111 Charles C Thomas 1950 10 Addis T et al Effect of adrenalectomy on spontancous and induced proteinuria in the rat Proc Soc Exper' Brol & Aler 74 43 (1950) 11 SAYERS G The adrenal cortex and homeostasis Physiol Rev 30 241 (1950) 12 PINCUS G Regulation of Adrenal Cortical Secretion Arexial Cortex Rall, EP Bloch K and Pincus G, Editors Trans Tiest Conf Nea York Josiah Macy jr Foundation 1947 13 METCOFF J KELSEY V and JANFTAY C. A The nephrotic syndrome in children a physiological interpretation based on clearance methods Am Di Child 80 524 (1950) 14 BURNETT CH dd The effects of cortisone on the course of acute glomerulonephtites report of a case New England ] Med 213 jozs (1950)

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·JOSIAH MACY, JR,
FOUNDATION
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ADRENAL CORTEX
AGING
OLOGICAL ANTIOXIDANTS
BLOOD CLOTTING
BLOOD PRESSURE
CLINICAL PSYCHOLOGY
CONNECTIVE TISSUES
CONSCIOUSNESS
CONVALESCENCE
CYBERNETICS
INFANCY & CHILDHOOD
LIVER INJURY
:TABOLIC INTERRELATIONS
NERYE IMPULSE
RENAL FUNCTION
JOSIAH MACY, JR.
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