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·RENAL FUNCTION
Transactions of the First Conference
October 20 21 1949, Ney York, NY
Lined by
STANLEY E BRADLEY
DEPARTMENT OF MIDICINE COLLEGE OF PHYSICLANER SLIGEONS
COLL DIT LYRITY
JOSIAH MACY, JR FOUNDATION
565 PARK AVENUE, NEW YORK 21, NY

·Published 1950 by the
JOSIAH MACY, JR FOUNDATION
565 Park Avenue, New York 21, N Y.
Price $250
Printed in tbe United States of America
By Corlies, Mary & Company, Inc, NY

·PARTICIPANTS First Conference on Renal Function MICAIBERS ROBERT F PITTS Chairman Department of Physiology Correll Universty Medical College New York NY STANLLY E BRADLEY, Seaclary Department of Med Cine College of Physicians & Surgeons Columbua University New York, N) PHYLLIS A BOTT Department of Physiolog al and Clin cal Chemistry Womans Med cal College of Pennsylvanu Ph fadelphia Pa. DANIEL C DARROW Department of Pediatrics Yale Un versty School of Medicine New Haven Conn WILLIAM DOCK Department of Mel cine College of Medicine State University of New York Medical Center at New York City Brooklyn, NY ARTHUR M FISHBERG Department of Marche College of Medic nc New So k University New York, VY ROY P TORSTER Department of Zoology Dartmouth College Hanover NH ALLAN L. GRATTLIV Department of Anatomy School of Med ane Johns Hopkins Univers ty Baltimore Ma CHARLES JANEW AT Department of Pediatr ( Harvard Medical School Boston Mass JOIN A LUETSCHER JR Department of Med cene Stanford Insetety Shool of Mel cine San Francisco Call JEAN OLIVER Department of Paralogy College of NN CD State In xenty of Sex Ioch Maul Center at le lori Gry Franklyn NY WALD I SELKURT Department « My 1st Semel of Vaine Wetern Reserve Liversity Qersland Oho JA VES A SHANNOV Vstual Hein (4 tute at wal tas tutes of itsall LS THE Bleath note Tha M • Ahne ·NATHAN W SHOCK Section on Gerontology National Heart Jastitute National Institutes of Health and Balumore City Hospitals Baltimore Md JOHN V TAGGART Department of Medicine College of Physicians & Surgeons Columbia University New York NY GEORGE W THORN* Department of Medicine Harvard Medical School Boston, Mass GUESTS ROBERT W BERLINER Department of Medicine College of Physicians & Surgeons Columbia University New York NY FRANCIS BINKLEY Department of Pathology (Brochemistry), University of Utah College of Medicine Salt Lake City Utah ROBERT GAUNT Department of Zoology Syracuse University Syracuse NY HANS HELLER Department of Pharmacology University of Br stol Bristol England LOUIS LEITER Department of Medicine College of Physicians & Surgeons Columbia Univa New York NY GILBERT H MUDGE Department of Medicine College of Physicians & Surgeons Columbia Univer. New York NY HENRY A SCHROEDER Department of Medicine The School of Medicine Washington University St Louis MO JAMES V WARREN Departments of Physiology and Medicine Emory Un versity School of Medicine Atlanta GA LAURENCE G WESSON, JR Department of Phys ology College of Medicine New York University New York NY HARVEY LESTER WHITE Department of Physiology The School of Medicine Washington University St Louis Mo A WILHELMI Department of Physiological Chemistry Yale University School of Medicine New Haven Coon JOSIAH MACY JR FOUNDATION FRANK TREMONT SMITH, Aledical Director JANET TREED, Assistant for Conference Program • Abrent

·TABLE OF CONTENTS
1919 Conference on Renal Tunction
Conference Program Frank I remont Smith
9
Introductor Remuks Robert T Prits
12
The Vorphological Aspects of Renal Tubular Secrction
and Rebsorption Jcan Oluer
15
Discussion
42
Role of Glutaminase in Tubulır Processes I rancis Binklcy
Discussion
69
Biochemical Aspects of Renal Tubular Transport
John V Taggart
62
Discusson
05
The Tubular Secretion of Potassium and Icid Robert Berliner 102
Discussion
Ilster Rebsorption in the Renal Tulmiles
Laurence lesson ir
119
Dixension
131

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·OSIAH MACY, JR. FOUNDATION ONFERENCE PROGRAM FRANK FREVONT SVITH, Medical D rector With the accelerating rate at which new know ledge is accumulat ng and with the increasmg recogmtion that nature is of one piece t becomes cuident that the continued isolation of the several Branches of science from one another is a senous obstacle to scien fic progress Nowhere in science is the need for "combined operations" more vident than in medicinc Today to be effective medical rescarch and practice must cmbrice data from all the disciplines including nuclear physics at one end of the spectrım and cultural anthropology at the other for advances in onc field are frequently dependent upon knowledge derned from quite another Although the fertility of the multi-discipline approach is thus ecognized universities scientific societics and joumals have not ct made adequate provision for channels of interdisciplinary com muniention I am delighted to welcome you to the first meeting of this group on Renal Function and to gnc you as bnelly as possible an outline of what wc liope to accomplish by these conferences The foundation is interested in furthering knowledge about tena! function and is also interested in the broad aspects of problems of communication and integration which are important for the ad vancement of the whole of science It is our belief that scientific communications at scientific meelings and in the journals have been forced into a narrow mold in which logical sequence leading to Incvitable conclusions is substituted for the much more flexible and often unpredictable processes br uluch scientific inquin and the adunce of knowledge actualls take place All the creatnc the rolls crciting and interesting factors which are the soul and heart of science tend to be excluded today This unfortunatel discourages individuals who hne 1 creative and artistic tum of mind from cntcring the scientific fold and creates in the minds of students and of the pull e a profound misunderstanding of the nature and processes of science ·10 Renal Function The Foundations experience with the many research projects coming before it has led to the conviction that one of the greatest needs today is a re integration of science now artificially frag mented by the isolation of the several scientific disciplines and specialties Our Conference Program hopes to encourage this reintegration and to give in the published transactions of our con ferences a clenrer reflection of what takes place in the laboratory and what goes on in the minds of investigators than now appears in scientific literature Our eight groups on aging blood clotting and allied problems blood pressure biological antioxidants cybernetics infancy and childhood lner injury and metabolic interrelations (formerly meta bolic aspects of convalescence) which have been functioning for some years have held over 50 meetings in which more than 500 individuals have participated Recently five new conference groups have been organized on the following topics adrenal corter renal function nerve impulse connective tissues and problems of consciousness Each group will hold annual two day meetings for a period of five years It is our belef that only through continued association in an atmosphere of friendliness and mutual confidence can effective communication (exchange of ideas data methods and plans) across the barriers of the professions and specialties be promoted As a result of these meetings we have seen plans and ideas modi fied conclusions more clearly specified or placed in a broader perspective and spontaneous collaboration take place between investigators working in different departments or in different universities As a nucleus 15 scientists comprise the original group of members for any conference These are selected by the Churman of the Conference in consultation with the Foundation Every effort is made to include representatives from all pertinent disciplines From time to time new members are added by the group to fill gaps in viewpoint or technique A limited number of guests are invited to attend each meeting but for the purpose of promoting full participa tion of all members and guests attendance at any meeting is Limited to 25 A point which I should be to stress before closing is that between the disciplines there are real difficulties in communica tion - partly emotional and pıtly semantic Emotionally some of us accept only data coming from those methods or disciplines with which we are familar It is important that we do justice to the ·Conference Prograin validity of data and methods from other disciplines on the semantic Josel, the physical and biological sciences have little difficults, the medical psychnatric and social sciences an understand each other fairly well, but to bridge the gip from the physical and biological sciences to the psychological and social is very difficult llou ever, in the study of min all of the sciences must meet in medicinc which must be equally concerned with the psychological and social as with the biological and physical there is the greatest oppor. tunity, as well as necessity for mutual understanding among representases of all the sciences I believe that the hope for a umfication of science hics in the development of a Science of Man in włuch medicine must play a central role In closing I want to say that the Conference Program is an cxperiment and that you are part of that expenment We hope that at these conferences you will feel the freedom inbcrent in the scientific method and will belp us to improve our conference procedure ·INTRODUCTORY REMARKS ROBERT F PITTS, Chairman Let me add to Dr Fremont-Smith's welcome my own very cordial welcome to you all Some of you I have met only in a very impersonal way through your scientific writings, it is a real pleasure to meet you here and to exchange views with you directly Many of you I welcome as old frends from whom I have in the past derived a great deal of inspiration Basically we are gathered here because we have a common bond of interest in the kidney Some of us are biochemists or enzymologists, others are clinicians, and still others are morphologists I think we can find some common ound for free discussion in that we are all basically functionalists By drawing upon our respective disciplines, by freely exchanging our ideas, we can both learn from, and teach, each other As the Chairman of this discussion group, I feel that the confidence which you have shown by appearing here at this conference session demands some justification for the program which has been arranged The kidney by mutual agreement is a complex organ Yet there as no organ which can be studied functionally more completely in the intact organism We are indebted in large part to Dr Homer W Smith for the methods we use in such studies but we should not forget that we owe a great debt of gratitude to Dr A N Richards, Dr P B Rehberg D D D Van Slyke and others for fundamental contributions upon which these methods are based Within recent years there has developed an increasing interest in kidney function for in what other field of human physiology are we so directly and urgently faced with the ultimate question, of how an organ as a whole functions and how the individual cells perform their respective tasks? Even on a clinical level there is an attempt to rationalize therapy in terms of the fundamental nature of the disturbances in cellular function It seems to me, if I may be permitted a very hasty glance into my crystal ball, that we are on the threshold of major developments in renal cellular kinetics, and that in the not too distant future se may be able to look into the cell and understand what transpires there When We do, and when the rapid adiances which are sure to 12 ·Introduction 13 take place subsequently improve our understanding of the alterations of function in disease, we shall be a long way on the road towards more rational and more effectic therapy Because I am so convinced that the future of renal plıysiology lies in an understanding of cellular enzyme mechanisms, I have arranged a discussion of tubular absorption and secrction from the 1 in renul cellular junction ul I think we will all agree, that as one swallos does not make a summer neither does one enzyme make a functionally active renal tubulur cell We shall, therefore, progress later to a somewhat broader discussion of the enzymatic aspects of tubular sccretion and absorption We shall then consider to what extent we can fit these views into the challenging problems sosed by tubular secretion of potassium and acid and hy tubular absorption of chloride, sodium and water I personally am looking forward to leaming a grcat deal from this conference we all shall I am sure if we air our views and discuss our problems franWly Dr Oliver hins very kindly consented to discuss from the functionally minded morphologists point of view the problem of tubular secretion and absorption

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·16 Renal Function 1912) The dye is excreted in the urine and when he examined sections, not isolated nephrons, he saw that there was a tahing up, or absorption if you will, of the dye particles in certain renal tubules He came to the conclusion from the study of his sections that it was the proximal convolution in which this change occurred Moreover, he described a gradient of absorption in the convolution which seemed quite reasonable, that is, most of the dye was taken up near the glomerulus and faded out as one went down the tubule These comments refer to an examination of the situation in histological sections, at best a subjective and difficult procedure Three years later his finds were confirmed objectively by an examination of nephrons which had been isolated by microdissection (Oliver ) The histogenesis of chronic uranrum nephritis xth especial reference to epithelial regeneration) Exp Med 21, 425, (1915)) Such dissected nephrons are shown in Figure 1 On the extreme left is a nephron from a normal dog's kidney which was excreting trypan blue after intravenous injection and Jiere one sees the typical picture of the gradient beginning with a considerable amount of blue dje absorbed into the tubule cells of the proximal convolution starting at the glomerulus (A) The amount of dye in the cells decreases so that about halfway down the proximal convolution no more dye can be seen Of course, if we looked with a higher magnufication we would see some scattered down a little further but at no place else in the nephron are there appreciable amounts of trypin blue absorbed This particular process, as you all know, has been given a special name, athrocy. tosis I sometimes wonder if there is any need for setting off this process from some of the other mechanisms of tubular absorption Of course the absorption of a foreign body which is not metabolized into the economy of the organism certainly differs considerably from the absorption of something physiological which is actually used by the organism so that one would expect differences in appearances But after all it is absorption in both cases So, as I said I don't think this word athrocytosis is a happy one Pults I wonder if I could ask, for the sake of some of us, just hot mu menn hv athrocytosis? cases a foreign substance is used to demonstrale alus ja true that under physiological conditions some substances such as hemoglobin get into the tubule fluid They too are absorbed and

·Vorphological Aspects
17
FIGURE I
A

·18 Ronal Function of course are metabolized within the renal cells and I presume the products of the metabolic change are either used or at least maintained in the economy Some would call this process athrocytosis because the drops of hemoglobin can be seen for a certain period after the actual excretion of the substance has ceased, perhaps a few days or so Then, as we shall see a little later, there are other substances in which the period of storage in the cell, athrocytosis if you will, is even more reduced There is therefore a continuing series of phenomena from the handling of a substance like trypan blue where the foreign body remains a long time stored in the cell, to a substance like egg white where it only remains a few hours One might continue the series to include amino acids which are absorbed and metabolized or passed on to the blood in a matter of minutes Electrolytes, I suppose, pass through the cells into the blood in seconds with little or no mtracellular metabolic alteration Therefore it seems to me this special term athrocytosis, has a certain danger unless one understands exactly its significance The other three nephrons on Figure I are from dogs that had a chronic canine Bright's disease as we can perhaps call it (Oliver, Bloom, F, and MacDowell M Structural and functional transformations in the tubulur epithelium of the dog's kidney in chronic Bright's Disease and their relation to mechanisms of renal compensation and failure | Exp Med , 73, 141 (1941)) It is a queer chronic interstitial nephritis which is quite different from glomerular nephritis or any other human disease, but it does have the same ultimate effect on the architecture of the nephrons as the diseases which one sees in man These nephritic dogs were in what one might call the compensated state of the disease although their kidneys were far from normal and the nephrons were functioning in a different way Still they were functioning and the kidneys as a whole were adequate physiologically Clinically, the dogs were not uremic Certain interesting points come out as one looks at different nephrons For instance the second nephron to the right in Figure 1 shows what extreme hypertrophy and hyperplasia can occur in the well preserved or less badly damaged nephron of the chronic renal lesion As I remember we actually measured this one I forget the eract figure but there is 15 or 20 times as much proximal consolution in this nephron 45 there is in the normal one on the extreme left, and as you see, it takes up the dye in exactly the same way Although it takes up much more, the dye does not extend any further down the consolution The localization or the relatie localization at least remains the same I think this observation is ·Vorpolo Ical Asx cts 19 | some interest liccruise skeptics hase pointed out that lug! 517c nes not imph a better functioning nephron This preparation slions 11t they are latter it least in handling trypan hlue 1 null admit attlus may seems rather untecessary function perhaps to most ogs lyut still it is (nlener that it is proximil can do more than small onc The next two nephrow on the extreme right show the lunges which cur win actual arc luitectural dulicrcntes line ricloped You can see that the protimils ar dis arranged so that Laera k i stretch of llum arrus tubule and at other places the cural configuration of the tubul lins remained shore or less nchanged Ilie mtu restong points that were this remuned nchinged morphologicalls its function so far as the tn pan blue since mod also dis remained unchanLei Thic blur die is present out in the atrophic parts of the proximul con olution there is no lic it all Thiese cells are not dead 1 x Int to mile that point quite leur - they are quite alive Thic only difference is that they behar lifleruntli 2016 the function differentls Of course thut kads to a matter which lies been a subject of some de CUISSION and px thaps man controversy imeli low should also interpret certain func Nonni tests in normal hxiness slach fudk ses still resik from normal kidness in Lidncus in lich the ne plirons ure actuall unaged the mi ·22 Renal Function nephron that had been entered The kidney was then taken out and sent to us and we proceeded to get the marked nephron out Figure 4 shows the piece of kidney put into hydrochloric acid In (A) you can see that there is ink in one of the tubules That is the one we went after To the right above (B) you can see we are dissecting and isolating the nephron at the lower left (C) the nephron is isolated but the loops of its tubule are still entangled. To the right (D) it has been completely straightened out We could now make a camera lucida drawing and allowing for foreshortening, run a map tracer around it and so make measurements We could actually find the hole as indicated by the arrow in Figure 5 just above to the right where you see a break in the contour of the tubule, so that we knew where the pipet had entered Then making a camera lucida drawing we could determine exactly the source of the sample FIGURE 5 Figure 6 shows typical findings The ratio between the concentra tions of glucose in the tubular fud and the plasma (TF/PL) was - thun leonned to 08 further down the I Finally at fell to 04 and to in which nine tenths of the

·Vorphological Aspects
23
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·Renal Function ACTION OF PHLORHIZIN S 1 FIGURE 7 ment was luchi beciuse tuo samp'es were taken by chance from the same proximal tubule making possible two measurements of glucose ratios 16 and 20 Here the glucose concentration in creased because the sugir was not absorbed and water was These findings permit calculation of water absorption Figure 8 shows the same facts expressed in a more conventional was in the normal animals the concentration of sugar falls to a sery lou figure and therefore the values for glucose ratio are less than one Following phlorhızın, the values nse ·Sorphological Aspects 03 SA 134*812 Nyt * Gi วางมี nau LAZED AVUS O TATRANCE NOW FEREN) FIGURE 8 Ilete » annther interesting point If we look ngun at ligure 6 illustrating the nortni? alvorption *e find thirt aircr glucns is practically all remain from the tubular fnid halfwn down thic proximal tumle the friminal portion of the proumal convolutel tulul in the munini comes in contact with only a 11 lont com frsIm11091 (of sugar dunng norinni life In luman dulxctic of worse the situation is very different llere slucente rraloption 1* unetunplir and so the term i put of the provimit mulutun 2014) the reinuniler of the nephron come in contact with a cun sulerable mucentration of suzir push up for spits As San Lor in the old dus Ixcfyr du fos was tre steel with insulin sloten 75 tre time fortnd in the Indt in emble amount (ser Figure 9 (Oluet cw directions in tenul morphology Thir llorcy Loct (191115) 40, 102 (1915)) As in the sectiom slun Intar knuter picture the selle warr * filled with cherxen that there bullrin transformed practically info ninflung last four huiles of climgeu Thule prixess con le falural in dicuntal neplaris as 100 see on the left llere a pumil con alition is thout at a mugufication of 10 ke hair to mile illustrations of this kind ·26 Renal Function ? head FIGURE 9 precemeal because the long nephron won't fit under the microscope in one piece Prohably thirty or forty negalues have been cut out and fitted together to make this illustration Jou can sec the proximal con olution at the left in a sort of montage such as the astronomers use for making pictures of the Afulky Way, or the Air Force in making maps its first half looks entirely black That is because he preferred to stain it that way to bring out proper rclatre values What we are pirticularly interested in us the terminal part of the proximal convolution which is tremendously distortcd since glycogen has filled the cells and turned the tubule into a ·Vorphological Aspects swollen lump In other words, the part of thic prouimal convolution wuch is conditioned to or, one night sai is accustomed to the alsorption of sugar tales care of it quite normally in the dialxtı, but the portion of the proumni con olution which is not accustomed to absorlung sugar gets into difficulty a simple ctplanation perhaps too simple would be that the cells of the lower half of the prorimal lave alısorbed the sugar and then he not been ablc to piss it on and that there then has been 1 synthesis of glycogen in the cell The fact remains lower that the part of the prouml consolution which handles sugar normally does not get into trouble and the part that is not accustomed to handling sugir does I would like expressions of opinan regarding any other crplina tion for such an intracellular cumulation of glicogen in the renal cells There is glycogen all through the nephron it is only this extreme concentration to which I am cilling attention nos Is there any more likely use for an acmimulation of glycogen in the cells than loy absorption of sugar from the tubule fud and synthesis of glycogen in the cell? Certainly the diabetic has little glucogen in huis blocxl stream anci so son little could be passed on to the tubule cell l» cither secretion or absorption It seems unlikels to me therefore that these netuimulations en le of preformed glycogen Of course there is a general disturbance of the carlxhydrite metabolism in diabetes and queer deposits of glicogen are found elsewliete so that some geucri disturbance may lx enter. ing into the production of this senal pucture Sell it soms to me that the most likely replinition of such extremo intracellular deposits in the renal tutube is 7 loer synthesis of glycogent from glucose mtlier than absorption of prcformeel glicogen Whir Ders this depresition cutrnil Ixt me the prounil tubule Oltrer On the extreme night of ligure the clusta) tubular con Inlucion is shown in the upper part of tlus segment of the neplıron there is never an accumulation of gliocrn It is solut blick in the Figure 1xcruse its milk arr normal Just belou the solul bolach por tion there is a light area where the open in de moncentrated As I have said there is pliengen all thmugh this nephron just as thiete Is glicogen all through the last and even the lirit muncle initit is kallis ernsceralel is the proximal tuluule so much then for the localirition of cluctan al mirtim ind glici sitithesis so far mantling lors ret almmluxl from the fuluk The only dit ut i litle mti the friente form of transport pisage (mm hol to fulmile wuch I shall mall startam has to do asun uith an artifal die neutral 1o Tetr does not seem ·28 Renal Function to be much question as to the fact of secretion - showed these facts in Hoeber's laboratory a good many years ago Scheminsky (Schemınzky, F Uber die Harnbildung in der Froschniere XVII Die Farbstoffsekretion der 2 Abschnitte Arch ges Physiol 221, 641 (1929)) It just happened that at Stanford we were doing the same kind of work We had no idea they were working on this problem and within a month, I think the two papers appeared (Oliver, ) and Shevky E A comparison of excretion of neutral red and phenol red by the frogs kidney | Erp Med 50, 15 (1929)) Later our conclusions were confirmed by Dr Richards by other types of experiment (Richards, A N Urine formation in am phibian kidney The Kidney in Health and Disease Chap 2, Phuila delphia, 1935) In other words I believe it is quite firmly established that neutral red is secreted by the epithelium of the second part of the nephron of the frogs kidney, which is the analogue of the proximal con olution of the mummal We cimed our work on a httle further, bringing in the morphological aspect of this secretory process This extra vital method had certain adı antages for it al lou ed us to do things one could not do with the older form of in LILO experimentation (Oliver J and Lund, E M Cellular mechanisms of renal secretion a study by the extra vital method I The Structural Phase II The Functional Phase ) Exp Med 57, 435 (1933)) We studied the structural or morphological process of crcretion of neutral red in the perfused kidney of the frog In that way we could eliminate a lot of complications we could perfuse the frog's kidney with a Loche's solution which had sugar in it and which either did or did not have neutral red We could also take out one kidney and look at it before we started the perfusion or before we put the neutral red into the Loche's solution, so we could set kidney not secreting neutral red and look at the other kidnes who had been secreting neutral red We found that the renal cells these kidneys were remarkably well preserved after this artificia perfusion with Locke's In fact, they looked exactly like physio logical kidney cells So what we then did was this We took out one kidney and looked at it as a control Then the other hidncy of the frog wis perfused with Locke's solution, in which there was no neutral red The Loche's solution had sugar that was all It was certain I think that there was nothing present that a lidnei could secrete Then we could stain the cellular elements and see what had happened Figure 10 shows what we saw in the first lidney before he started the perfusion Without going into too much technical detail, ·Vorphological Aspirats 29 it is stained with iron licmitory in to show the roullets lou can sce the long filimentous batont ts slucli fill the proximal consolu tion Then the perfusion was started and after a certain length of time zyrhaps half an hour the kidney was fired The kidnei liad been functioning quite normalli that is it had been absorhung SICURT 10 unter and electrolites and it hund lxen lyxorlung suger Wien we loked at it at lewked for all practical purposes the sime as the first imperfused kids (by 11 You can still see the rexllets Tties are the long filamentous objects WS lict 11 11 Then we chil in the rinment in *lili u urtel ni il sume wille fritus! nel 1920* 1!!the pilluxhessalto aul sugar ani her kun 1 ser uul on hair sruth in tl ·28 Renal Function to be much question as to the fact of secretion - Schemir showed these facts in Hoeber s laboratory a good many years (Scheminzky F Uber die Harnbildung in der Froschniere XI Die Farbstoffsekretion der 2 Abschnitte Arch ges Physiol 2 641 (1929)) It just happened that at Stanford we were doing t same kind of work We had no idea they were working on th problem and within a month I think the two papers appeare (Oliver ) and Sherly E A comparison of excretion of ncutra red and phenol red by the frogs kidney | Exp Med 50, 15 (1929)) Later our conclusions were confirmed by Dr Richards by other types of experiment (Richards AN Urine formation in am phibıın kidney The kidney in Health and Disease Chap 2 Phila delphia 1935) In other words I beleve it is quite firmly established that ncutral red is secreted by the epithelium of the second part of the nephron of the frogs kidney which is the analogue of the proximal convolution of the mammal We cirried our work on a little further bringing in the morphological aspect of this secretory process This extra vital method had certain advantages for it al lowed us to do things one could not do with the older form of in erro experimentation (Oliver ) ind Lund EV Cellular mechanisms of renal secretion ? study by the extra vital method I The Struc tural Phase II The Functional Phase ) [rp Med 57, 435 (1933)) We studied the structural or morphological process of excretion of neutral red in the perfused kidney of the frog In that way we could eliminate a lot of complications We could perfuse the frogs kidney with 7 Loches solution which had sugar in it and which either did or did not hue neutral red We could also take out onc hidney and look at it before he started the perfusion or before we put the neutral red into the Loches solution so we could see a kidney not sccrcting neutral red and look at the other kidney wlucha had been secreting neutral red We found that the renal cells of these kidneys were remarhably well preserved after this artificial perfusion with Loches In fact they looked crictly like physio logical kidney cells So what we then did was thus We took out onc kidney and looked at it 15 7 control Then the other kidnes of the frog was perfused with Lockes solution in unch there was no neutrl red The Loches solution had sugar that was all It was certun I think that there was nothing present that a kidney could secrete Then we could stain the cellular ciements and see what Innd happened Figure 10 shows what we sast in the first kidnes before we started the perfusion Without going into too much technıcıl detail ·Morphological Aspects 29 it is stained with iron hematorylin to show the rodlets You can see the long filamentous batonets which fill the proximal consolu tion Then the perfusion was started and after a certain length of time perhaps half an hour the kidney was fixed The kidney had been functioning quite normally that is it had been absorbing FICURE 10 water and electrolytes and it had been absorbing sugar When we looked at it it looked for all practical purposes the same as the first unperfused kidney (Fig 11) You can still see the rodlets They are the long filamentous objects FICURE 12 Then we did another experiment in which we started out th same way le perfused one kidney with the plun Loches and sugar and here agun you see what you have ·30 Renol Function previous two Figures, that is, long filaments (Fig 12) They le on top of each other and they are not as clear as they would be if one could focus up and down, but they are certainly long filaments Then we put some neutral red into the Loche's solunon and the ludneys begin to secrete it is well as absorb the sugar, electrolyte, and water FIGURE 13 After a certain length of time we took out that kidney and as you see it looks qute different in that the rods have disappeared (in most places at any rate) and are replaced by big drops (Fig 13) If one looks at these drops in fresh material they are red They have a large amount of dye in them But here is an interesting point If one isolates these drops they remain discrete in the Loche's flud there is something in them that keeps them intrct for they do not immediately dissohe and dissipate in solution If onc prepares some of the isolated drops by crustung the tubule and treats them u2th Janus green the red drops tum a muddy green color In other words they take up the Janus green as well We will come bach to the significance of this fact later, for the present we can assume that wc hre seen a change in intracellular elements, the rodicts, that occurs in the secretion of a substance, neutral red, and which does not occur in the absorption of sates, electrolyte, and sugar fle therefore suggest that these appetrances are a part in some pay of the mecharusm of the secretion of neutral red. More recently we liase been interested in absorption and a situation, somewhat similar to that described for secretion of the dve, which seems to occur in the absorption of various proteins (Olses, The structure of the metabolic process in the nepturon ·Morphological Aspects 31 FIGURE 13 The Edward Gamaliel Janeway Lecture ! At Sınai Hosp 15, 175 (1948)) This work was started during the war with a study of various blood substitutes, including gelatin and plasma proteins We thus obtained a lot of kidneys to study morphologically from Dr Addis who was studying the functional way in which the kidneys were handling these proteins Figure 14 shows what we find when the Serum protein of the rat has been raised considerably by injecting large amounts of FIGURE 14 ·32 Renal Function rat serum intravenously or intraperitoneally If this is done i pro teinuria develops If one gets out the nephrons as can be seen in figure 14 one finds intracellular droplets ind these droplets show a very interesting localization They do not start out as one might expect in highest concentration near the glomerulus but there is a strip of tubule in which the absorption or the number of droplets is rather small and then in about the middle third of the proximal tubule the droplets begin to occur in great numbers and then again they peter out and disappear in the terminal part of the proximal convolution One does not see droplets in any cells elsewhere in the nephron Figure 15 shows egg white droplets in rit tubule cells We did not use egg albumin but native egg white which is convenient becuuse its proteins are of small molecular size and a lot of it comes through into the urine These droplets appear in the same part of the proumal convolution as the serum protein droplets If one injects enough this peculiar localization in the middle third dis appears The convolution begins to fill up and the distribution of .12 FIGURE 15

·Morphological Aspects
33
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·34 Renal Function drops extends so that almost the whole proximal tubule becomes filled. The morphologist might naively believe that they are in part, at least, simply absorbed egg white. You will notice that where one sees many drops the rodlets seem to have disappeared, but this is shown much better in Figure 16 At the top is a higher magnification of normal rods fused together as a result of the staining technique. The other two photographs show places where drops are forming and you see diffuse masses of material in the middle figure, and definite round drops in the lower figure. It seems very obvious to a morphologist that the long rods have "dissolved." They have swollen and are disintegrating and one sees a lot of blackish material about them. Now if one stains the same kidney that you see in Figure 16 with Gram's stain, he gets a different and much clearer picture of the drops as shown in Figure 17, because the rods do not take up the Gram's stain. However, the drops are strongly Gram positive and you see them very clearly outlined in great numbers in the proximal convolution. The same stain can be applied to the isolated nephron and we find them in the middle third of the Proximal convolution. 8 FIGURE 17 ·Morphological Aspects 35 What is the significance of the fact that they are Gram positive? As you know the bacteriologists have looked into this matter and have by means of luistochemical procedures involving the use of crystalline ribonuclease demonstrated to their satisfaction that the Gram positivity of certain bacteria is due to their content of ribonucleic acid so we did the same thing to the sections of these droplets (Tig 18) In the upper lefthand corner (A) is a section through a kidney field with egg white droplets stained with Grams FIGURE 18 ·36 Renal Function stain and you see the cells are filled with drops An adjoining section of the same bloch was put in a very low concentration of crystalline ribonuclease We used to prepirations, Armours and Worthingtons, and obtained similar results The section is treated with the enzyme for an hour or so, washed and then stained with Grum and you see the remarkable difference But you will notice the drops are still there The ribonuclease has not digested them, it has simply more or less and in some instances completely, romoved the staining affinity of the drop for the Gram's stain Perhaps I should produce sometlung here that might be called prima facie evidence At least it is temporary or provisional evidence that the Gram positivity depends upon the nbonucleic acid content in the drop There have been objections to such conclusions first, that the enzyme is not pure, that it has a mixture of protein dissolving enzymes in it, and also it has been clumed that although the enzyme may be pure, that the specificity of its action is not complete One way of answering these criticisms though I do not know that it is a complete answer, is to take a section such as (B) which is Gram negative, from which presumably the ribonucleic acid has been removed and pince it in a solution of a sult of ribonucleic acid magnesium ribonucleic acid In other words try to put back what was taken out That is what we did and in (C) you see the droplets are just as Grom positive as they were before the enzyme acted upon them Whether this fortifies the argument that there is a considerable content of nibonucleic acid in these droplets or not I will have to leave to the enzy mologists, but it seemed good evidence to tis I have not gone into the literature on this subject for it is a long story Other people have seen drops in renal cells particu larly the Belgian morphologists, Gérard and Cordier, and in this country Smetana and others (Gérard, P and Cordier, R Esquisse, d'une lustophysiologie comparee du rein des vertebres Biol Rio 9, 110 (1931)), Smetina, H and Johnson r R The origin of colloid ind lipod droplets in the epitſielial cells of the renal tubulc emer } Path 109, 1029 (1912)) They have examined the problem in different ways but I tlunk it has been the assumption on eseryone's part that the droplet was simply absorbed protein loweser, you ee our esperiment indicates that a very considerable part of the Irop is not protein but nbonucleic acid, a fundamental element of ·Morphological Aspects 37 the protoplasm of the renal cells In other words the droplet seems to be a sort of coacervate or combination of protoplasmic elements and the absorbed egg white One can perhaps get at this problem more exactly if one grinds up the kidney and prepares a suspension of the nuclei red blood cells and all the intracellular elements including the filimentous mitochondria and the droplets Such procedures have become stand ard methods with biochemical cytologists One can prepare suspen sions in sucrose or saline solution although there are advantages in using sucrose All this must be done at a low temperature so that FIGURE 19

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·Morphological Aspects 39 1 - That there is a further metabolism and degradation of the egg vhite, even the morphologist can show What I have been describng is the intracellular status 17 hours after injection Examinaion of Gram stained sections from kidneys at varying time in ervals after the injection of egg white shows what might be lescnbed as a cycle of change in the droplets At three hours they are few in number, small and only weakly Gram positive At 17 hours as I have described, they are large, deeply Gram positive and o numerous as to fill and distend the cell body At this time an occasional droplet can be found which either stains less strongly or is physically disintegrating as is evident by the broken contours of the droplet The intracellular “digestion of the droplets pro ceeds so that after 72 hours only a few persist Strangely enough, these few scattered droplets may be present for weeks In other words, we have morphological evidence of the fact that the egg white is being acted upon by enzymatic processes within the cell and that it is being broken down It therefore is not surprising that at 17 hours we found only five percent of native or original egg white What we are doing at the present time is in the earlier we can thus retabolism of the egg white within the droplets in the renal cell There is just one further tubular process which I wish to consider from the morphological viewpoint I cannot say whether it is absorption' or 'secre tion', only that it is an increase in the concentration of mineral salts in the cells of the nephron One can study this process very accurately by getting the nephrons out on a slide and incinerating them and so destroying everything but nonvolatile minerals The ash settles down on and adheres to the slide and produces the so called spodogram This is a replca of the original structure of the tissue which has been incinerated One can increase the mineral content of the kidney by giving calciferol and parathormone Whether this increase is ev of an "absorption" or "secretion" or just simply a "deposit" in the renal epithelium I don't know but at least there is something about the process which you can see This is the specific localization shown in Figure 20 (a) These are spodograms from a normal rat To the night is the blood vessel which is curving around and from it come afferents on which are glomerular tufts, the round objects There is not very much mineral in the Vessel or in the afferents which are therefore difficult to see Then below is a glomerulus and the first part of a proximal convolution ·38 Renal Function enzymes wont change everything One can then separate the par tacles into fairly pure fractions by repeated centrifugalizations In this way the drops may be separated from the rodlets or mitochon dria There is a supernatant left which contains what are usually called microsomes We have gone no further in trying to separate these intracellular particles Some examples of our preparations are shown in Figure 19 Above (A) is a mixture of everything lou can see short filamentous particles which are mitochondria and round drops that are con siderably larger Also you see debris and large objects that are probably red blood cells Below the suspensions are fairly pure At the right (B) is a relatively pure suspension of mitochondria though there are a few round objects that may be droplets Cer tainly the suspension of droplets on the left side (C) is I think remarkably pure Grafflın This is in sucrose? Olver Yes The procedure is simply an adaptation of other people s methods Hogeboom Schneider and Pallade were the men who established the particular methods we are using (Hogeboom G H Schneider WC and Pallade G E Cytochemical Studies of Mammalian Tissues 1 Isolation of intact mitochondna from rat liver , Brol Chem 172 619 (1943)) After the suspensions were prepared our biochemist Dr Werner Straus analyzed them Our interpretation of the biochemical analyses of the particulates is that the intracellular droplet at 17 hours consists of a small amount of egg white determined by immunological methods and a considerable amount of something resembling the mitochondrial substance of the rodlets in its lipid phosphorus and ribonucleic acid phosphorus This would seem to support the morphological sug gestion that the drop is not a drop of egg white but that it is omposed of cytoplasmic elements (mitochondra or rodlet ma erial) and absorbed protein Such a conclusion is interesting because it complicates the whole matter It also leads to a possible extension of our knowledge of ust what is going on in the renal cell when it absorbs protein It has been shown by others that certuin of the enzymes are concen Srated in the mitochondria One might suppose therefore that that we are seeing in this combination of cytoplasmic material mitochondria) and egg white is the bringing together of the ubtrate (egg white) and the enzyme and that the droplet forma ion is the mechanism by which the two operate in the intracellular metabolism of the absorbed egg white ·Morphological Aspects 41 . FIGURE 0() words there has been an increased concentration of nonvolatile minerals in the proximal convolution after the administration of calciferol Looking at the ascending limb and the distal below it can be seen that this is not the case There is no more than there n in it that one can hardly see its outline So here again there seems to be a definite localizat on of a metabolic process that can be seen by 1 parathormone is required to produce damige It is an interesting point that if one stuns these kidneys in which the nephrons con tain several times their normal calcium or total mineral ash by the ·40 Renal Function ind here the minerals are more heavily deposited I think that the irregularity is the result of our miceration We have to sonk these kidneys in hydrochloric acid in order to dissect them and thus takes out a large amount of the minern! We have acturlly determined how much is taken out and under our particular method of macera tion about nine tenths of the total mineral is removed by the preliminary treatment with acid So this higure shows only a part of the original total This is probably fortunate for if it were all present the preparation would be so dense and heavy that no detul would be discernible It is clearly seen that the proximal on the nght has much more mineral in it than the ascending limb and distal which is upside down and in part almost »nyssible from its low ash content on the left L FICURE 90(a) If one gnies an animal calaferol (or parathormone) and increases the mineral excretion (Tug 20 (b)) one obsenes a very definite change in appearances an overall change but the same sort of relative distribution At the top is the proumal consolution and in it one sees what one might call plates or caled solid masses of mineral ash vost of it of course is calcium but other elements are increased too It is quite different from that first prorimal In other ·minerals in the proximal consolution after the administration of in scieral times their normal calcium or total m.neral ash bi che dforphological Aspects 41 FIGURC Ob) words there has been an increased concentration of nonsolatile calciferol Looking at the ascending limb and the distal beloss it can be seen that this is not the case There is no more than there was in the normal control anımal Beginning below is the ascending limb Onc hus difficulty following the course of the tubule as it gets up tow ard the destul con olution because there is so little mineral in it that one can hardly see its outline So here again there seems to be a definite localization of 1 metabolic process that can be seen by the morphologist Pitts What would happen with prolonged treatment Oliver Damage in the hidney results Vluch more calciferol than arithormone is required to produce damage It is an interesting con ·42 Renal Function conventional methods which the pathologist employs to demon strate what he calls calcification One can see 10 increase in calcium at all There is no calcification in the old sense of the word I think therefore that here we have found a method of studying what might be called the preliminary stages of calafication before the actual deposit in its obvious histological form occurs Schroeder Can you tell where it is in the glomerulus? Oliver In the tuft we have some photographs in which one can see the tuft of the glomerulus quite clearly with its lobulations and in it the concentration is high These then are the examples which I wanted to show in which a morphologist can at least point and say "Here something is hap pening Often he cannot say much more than this at times by crude histochemical procedures he can guess but his hope is that now the biochemist and enzymologist can step in and say what really is happening in these places where structure has been dis turbed DISCUSSION Shannon It might be helpful for Dr Grafflın to comment on some work he did several years ago His work on the cellular dif Grafflın It has been adequately demonstrated that the proum.de tubule (ze the entire portion of the nephron provided with brush border extending from the glomerulus to the transition to the thin segment) of the cat and dog nephron characteristically contains fat under normal conditions and that this fat is regularly distrbuted in ? Darticular manner in the two species (Peter | Die Nieren 7 NA U Epithelzellen der Saugetierniere Arch mikros Asia (1911) Nakamura S Beobachtungen uber die Fette in den Hamnkanalchen von hatze und Hund Folia Anat Japon 13 45 (1935)) In the cat the entire pars convoluta and the upper portion of the pars recta are normally fat lnden while the terminal portion of the pars rccta is fat free In the dog the reverse is true the fut being confined exclusively to the terminal portion of the pars recta ·43 Vorphological Aspects In tubules teased from açıd maceration matenal the transition be tween the fat laden and fat free portions is characteristically abrupt (Peter Makamura loc cit) Zimmermann added greatly to our knowledge by demonstrating that the abrupt transition with respect to fat content is bkewise abrupt with respect to cell type In both species the luminal cell boundary pattern in the terminal portion of the pars recta - fat free in the cat fat laden in the dog is rectilinear In the remainder of the proximal tubule -- fat laden in the cat fat free in the dog - the lumınal cell boundary pattern is very complicated showing in marked degree the interdigitations usually considered characteristic of the mummalıın proximal tubule Despite the reversal in fat distribution in the two species these find ings seem to justify the conclusion that we are dealing here with two distinct segments of the proximal tubule Whether the bulk of the proximal tubule (exclusive of the terminal portion of the pars recta) can be further subdivided on cytological grounds must avait further work As is well known Suzuki has subdivided the mam maban proximal tubule into three or in the rabbit four segments (Suzuki T Zur Vorphologie der Nierensekretion unter physio logischen und pathologischen Bedingungen ſeni (1912)) but no critical cytological proof of his thesis has yet been adduced Since in both cat and dog the abrupt shift in fat distnbution demarcates so accurately the cytological transition between the upper and lower segments Foote and I have used isolated proximal tubules stained for fat as a means of obtaimg quantitative measurements of the relative lengths of the two segments (Foote ) and Grafilin A I Quantitative measurements of the fat laden and fat frec segments of the proximal tubule in the nephron of the cat and dog Anat Rec 72 169 (1938)) From each species sith complete proximal tubules - fifteen from each of four animals -hue been isolated from maceration matenal and measured In percentage of the total length of the protimal tubule the average length of the second segment is 195 in the cat 108% in the dog Olier in the cat we are quite certain that the gradient of fat deposit in the cells of the proximal convolution is not as it is in trypan blue The latter is an even gradient which is high at the glomerulus decreasing gradually doinu ards But with fat there is something rather similar to what is seen in the absorption of the proten It is first modernte scaches a peak in the mid proumal and then fades out us can be seen in Figure 21 Grafflin The distnbution of fat in the proumal tubule of the cat nephron was found to be relatisch constant The typical ·Renal Function FIGURC 21 may be summarized as follows At the glomerular end the fat begins abruptly with no apparent neck segment The fat droplets licre are of both small and medium size and are quite numerous Tor a short distance distally along the tubule they gradually increase in size their numbers remuning essentially the sime After the fat deposit has become heavy it remains so until near the distal end of the first segment, where the fat gradually diminishes At the end of the first segment the fat stops abruptly and completely, the second segment is wholly fat free If we drop back to the fishes and limit the discussion to four representative species with which I am personally well acquainted the proumal tubule ia the glomerular toadfish (Opsanus tau. marine teleost) exhibits only one cell type, whereas the proximal tubule in the glomerular sculpın (Afyoroccphalus octodecimspinosus marine teleost) eel (Anguila rostrata cury Inline telcost) and lung fish (Lepidosiren paradora fresh water) shows a differentiation into tuo histologically distinct segments The question iriscs as to what fundumental homologies if any, exist bctueen these differen trated portions in the four representative nephrons under considera ·Morphological Aspects tion in the sculpın and eel it seems perfectly clear on histological grounds that the lightly staining first portions of the proumal tubules in these two species constitute one homology and that the deeply stainag more lughly organized second portions constitute a second homology Furthermore, there can be no doubt that the proumal tubule (of one cell type) in the toldfish nephron is homologous with the second portions of this segment in the sculpın and cel and is markedly dissimilar in structure to the first portions in the latter two species If we attempt to extend these homologies to include the lungfish the problem is more complicated In Lepidosiren the order of the lightły staining and deeply staining portions (speaking in very general terms of the prouml tubule compared with the situation in the sculpin and eel as reversed the deeply staining portion following directly upon the neck segment Histologically and cytologically the structure of the second portion is so striking and unique that it most emphatically is not homologous with either of the differentiated portions of the proumal tubule in the sculpin and eel With respect to the first portion in Lepidosiren admittedly shows cert un distinctive features not encountered in th nephrons of teleosts Nevertheless if any fundamental homolog) exists and I believe that it does this first portion is homologous with the second portion of the proximal tubule in the sculpın eel (Grafln A L The structure of the nephron in fishes Anat Rec 68 287 (1937) this paper includes references to the important work of } C Edwards and other investigators in this field) The proximal tubule in the kidneys of amphibia and birds has not been adequately investigated with respect to segmental dif ferentiation In the mimmals we have the very sharp differentiation of the proumal tubule into tuo segments in the art and dog which we have alrendy discussed and the available evidence indicates that the proximal tubule in the rt is probably subdivided into at least two distinct segments Eduards has reported that the injection of iron salts into the rat leads to 1 deposition of iron exclusively in the termini third of the proymal tubule (Edwards J G Functional sites and morphological differentiation in the renal tubule Anat Rec 55 313 (1933)) In a group of five rats wluch ethibited a striking deposition of yellon to golden brown pigment in the cells of the proximal tubule the distribution of the pigment kas studied in complete proximal tubules isolated by tezsing after maceration with ly drochloric acid The major portion of the proximal tubule beginning at the glomerulus exhibited considerable vanations in the amount and chrracter of the pigment but these differences ·46 Renal Function were always achieved more or less gradually, with no suggestion of sharp intersegmental transitions On the other hand, in the majonty of the tubules studied the terminal portion of the proximal tubule was set off from the remainder of the proximal tubule by a rather abrupt alteration in pigment deposition and, in addition, by the presence of isolated pigment-laden epithelial cells The average length of this terminal segment was approximately 25 percent of the total length of the proximal tubule (Grafflın A L The storage and distribution of ron containing pigment and the problem of segmental differentiation in the proximal tubule of the rat nephron Am ] Anat, 70, 399 (1949) Adequate cytological evidence for such segmental differentiation of the proximal tubule has never been supplied for the other laboratory mammals or for man Shannon My que was prompted by Dr Oliver's comment about the deposition of glycogen in cells of the lower part of the proximal tubule when exposed to glucose He suggests that such an abnormal accumulation of glycogen occurs because the cells in the lower portion of the proximal segment are not normally exposed to an environment of glucose on their tubular lumen side He also suggests that glycogen does not occur in the more proximal portions of the segment since these cells are normally exposed to glucose It seems more likely to me that there is a functional differentiation within the proximal segment and that the abnormal accumulation of glycogen under Dr Oliver's particular experimental condition results from the different functional potentialities of the cells in the area concerned rather than from the circumstance that they are under his conditions, first being exposed to an abnormal environment This is merely to emphasize that in the teased total nephron one cannot consider the proumal segment to be made up of cells that are wholly homogeneous The difference in morphology certainly carries with at a suggestion of a difference in functional potentialities Grafflın I should like to ask Dr Oliver whether he sees a very sharp transition in the proximal tubules of kidneys exhibiting heavy deposits of glycogen Olwer Oh, yes We have dissected literally dozens of glycogen kidneys and they are all just the same one is the dead ringer for the next ercept in the amount of degree of swelling The tubule goes along quite even and straight and then suddenly swells where the glycogen has been deposited ·Morphological Aspects Shannon Anatomical appearances may vary radically under dif ferent conditions quite independently of functional characteristics Most people looking at the histological preparation of the brush border for example have the conception that it is just what it looks like that it is a stiff brush On the other hand it is my under standing that it is a very mobile area in Wfe capable of striking cellular activity Crafflın Tie brush border is a fascinating structure and can gne highly vanable pictures depending upon the fixatives em ployed The more brutal acıd fixates are most bhely to produce the picture of discrete kur like processes suggested by the term "brush" By the proper choice of fixatives one can obtain almost any variant between this extreme picture and that of a smooth almost homogeneous border with httle or no evidence of vertical stration Some years ago I made some studies of brush border in fresh spreads of the mammalan kidney and frequently I was able to follow the gradual development of striations in borders which were optically structureless when they first came under observation Wrslocka and Bennett have reported some very pertinent observations upon the varsable morphology and mobility of brush border in the placenta of man and monkeys (Wislocki CB and Bennett HS The histology and cytology of the human and monkey placenta with special reference to the trophoblast Am Anat 73, 335 (1913)) Twele years ago in Copenhagen Dr Wigglesworth demonstrated to me some almost incredible pictures of mobilty in the brush like border of the nephric tubules in an insect Rhodnius prolixus Oliver We have looked at these borders in rat lidneys with the phase microscope in the fresh or lung cell suspended m senim I do not know how quichly we looked at them that of course is a good point since their structure may vary with time - but as soon as ce did look which was within a minute or so they seemed to be definitely brush lhe more ble hurs The thing that astounded me yras their apperance in the isolated cell that is in a cell that had been completely isolated from its habitat and suspended in plasma or in Loches solution The cell is quite round and the brush border is a halo or crown around it that sticks out in every direction It looks much hke the corona of the sun in total eclipse It gres one a completely different picture than a microscopic section where the brush borders are stripes on the top of the cells The phase microscope might be a way of working with it Shannon just one other point I sould like to recall some obser tons made by Robert Chambers some vears ago Ile ·50 Renal Function Wilhelmi 10 000 to 16 000 Oliver Such as the basic proteins protamines for example? The trouble with them is that they are very toric Wilhelmi Another one not touc is cytochrome C which you could use Oliver Could we see it in the cells? Wilhelmi You can see it spectrophotometrically because it has a nice nucleus for absorption Schroeder Unfortunately it does not stay in the kidney as cyto chrome C There has been some work done by Beinert et al with cytochrome contuning radioactive iron and the iron is found in the ferrıtın fraction in the kidney and Inver (Beinert H Maier Leibnitz H Richey E O Reissmann . Further investigation on the injection of respiratory catalysts in an attempt to improve anonia toler ance School of Aviation Medicine Quarterly Rescarch Report 22 (July 1 to September 30 1949) Wilhelmi After you give cytochrome C? Schroeder it very rapidly disappears A considerable amount is excreted in the urine and the rest of it is apparently changed much of the iron being taken over into the ferritin fraction Olwer I know nothing about it as to the preparation of cyto chrome C can one prepare a considerable amount easily? Binhley You can buy it easily Oliter what is its size? Wilhelmi Between 10 and 16 000 so it would be roughlı onc half to one third the size of albumin Oliver It might in its pattern of absorption resemble more closelv trypin blue Bott I still have some of that PPD protein that Dr Florence Siebert let us take which seemed to be about 85 percent permeable and thrt has a molecular weight of about 14 000 I do not know how you can detect it but it does have many characteristics of a com plete protein Oliver It is really the Inrger particles that I would like to try something large rather than small Bott Did I understand Dr Oliver that I on think the glomerular membranes of the rats kidney are more permeable to serum pro tein than those of other mammals? Oliver That is my impression and it is hased on work done by Dr Addis and by Dr Gilson (Gilson S B Studies on protermina in the rit Proc Soc Exp Biol and 1(cd 72 609 (1949)) I do not think there is any question that serum albumin for instance goes ·51 Morphological Aspects through the rit glomerulus because the rat has a proteinura all the time Berliner Whipple and his co workers (Terry R Hawkins D R Church E H and Whipple GH Protemuna related to hyper proteinemia in dogs following plasma gn en parenterally J Exp Med 87, 561 (1948)) have found that if a normal dag is injected with farge doses of dog plasma a marked degree of albuminuria occurs when the plasma protein is sufficiently elevated Oliler I have never seen morphological evidence that globulin is absorbed by the tubule hence it should apper in the urine if it is filtered Bott Samples of fluid taken directly from the tubules in mammals by Dr Walker (Walker A V Bott P A Olner J and AlacDowell VC The collection and analysis of fluid from single nephrons of the mammalian kidney Am J Physiol 134 580 (19-17)) occasionallcontained some protein but many samples contained none by the method used - about 30 mg percent is the lowest detectable con centration Oliter That would mean 1 lot of protein in the urine by the time it gets to the bladder? Bott Yes Oltcr It would be a lot in the urine if none were absorbed in passing down the tabule The whole problem - this fact that we have the glomerulus putting protein in and the tuhule taking it out and our not knowing the relative degree of cithers very confusing White fins anyone ever tried to detect the presence of protein by any method other than just the cloud formed on adding a protoin precipitunt? Bott Dr Hudson working with Dr Walker and Dr Richards (Hudson CL and Mudd S An ultramicro technic for precipita tion and agglutination reactions J Immunol 28 34 (19351) used an immunological method which was very promising but so far as I know it has not been used since I think it should be reinsestigated especially in the light of some of the recent developments Dr Richards and I carried out 1 series of perfusion cyperiments some of which were made on nectun lle made glomerular punctures during the perfusion of various protein solutions and used a modi fication of the Folin and Ciocalteu method rently Greenbergs protein methed We were not able to measure much lower concen trations howeter than with the simple test But it was useful for these quantitatre comparisons of perfusion fiud sath glomerular ·Renal Tunction fluid (Bott P A and Richards A N The passage of protein molecules through the glomerular membranes y Biol Chem 141, 291 (1941)) Wesson Is it possible to interpret some of these morphological gradients down the proximal tubule which Dr Olner and Dr Grafflın hive described on a functional basis? What I have in mind is tlus The tubular fluid in one portion of the nephiron will have a different composition from the fluid in another portion Perhaps the differing apperrince of the cells represents the response to differing composstions of the tubular flud Both the concentration and the miss of any pirticular substance in the tubular fud may increase remain the same or decrease as the flud mores down the tubule In the case of colloids there my be 1 tendency for the tubular flud to separate out particles of different sizes in different regions according to the physical properties of the flow In the case of proteins it might be possiłłe that the rate of accumulation loy the cells is some function of the concentration of protein in the tubular fud Olver in the case of trypan blue that is the way I had rather vaguely thought of the process That is the amount available falls as the substance is absorbed and therefore one naturally expects an evenly filling gradient but when one considers those sulystances tint show first low absorption a peal of absorption further down and then a drop is the proteins do then I do not know how to explain the gradient on simple physical factors of relative concentra tion The rate of absorption of water is faster than the rate of absorption of protein and therefore the protein becomes more concentrated and therefore more is in contact with the cells so they take up more you can think of various passihilities which get terrifically comnlıcated I do not know any 11 23 of measuring the factors invoked Pitts I suppose change in jonic cnvironment might change the physical properties Oliver 1 muy siy with all due defcrence to my fellos morpholo gist Dr Cruin that though I hne nei cr had any doubt about the differences in cell pattern inc not thought of the problem from this angle rlone Ihre thought the gruent as duc probally to unknown physical conditions As the fund goes down the bule the ionic pattem changes and this might cause great physical dif ferences in the state of the protein u kuch is to be aliserhed The change in sonic pattern might be either more or less from able to cell actrities so that under some conditions they would absorb ·Morphological Aspects more and others less Without trying to go wto it, I had always thought of this sort of mechanism rather than that there were different cells that had a different job to do Pitts I wonder if in this connection Dr Forster might not want to comment on the neutral red ston Forster Yes, by way perkaps of forcing a definition out of Dr Shannon or some others of the word secretion* I would be to refer back to Dr Ouers clean (ut experiment in whuch he perfused the frogs kidneys frst sath I ches solution and then removed one of the kidneys for histological tuds The remainmg kidney was perfused with Locke s solution plus neutral red for a penod of time and then also examned hi tological So then he had tuo kidneys one of which had secrets i neutral red and the other one which had not secreted neutral ir I nor any other kind of substunce Then as you remember he puted out that as a concomntant of the secretion of neutral red there is a change in the nature of the cytoplasmic structures The filozi nt lis structures changed over into droplets or at least the droplets Ir present when a substance was being secreted and those dr pled were not present in the absence of secretion The mechanism of transport or secretion" if we can use the word of neutral red is quite unlike the transport of such substances as phenol red / phenolsulphonephthalein) diodrast pamino hip. puric acid and those compounds which we ordinand use for measurement of tubular function Teutral red us transferred only as long as the urine is more and than the fluid on the other side of the tubule cells Furthermore neutral red continues to be "secreted" by cells even after those cells have been treated bs such metabolic poisons as cianide whereas such erposure to clande or other inhibitors blocks the secretion of phenol red (phenolsulphonephthalein) As long as the urine is acid the moi cment of neutral red from the pertubular flmd into the lumen can be obserı cd cten in the presence of metabolic inhibitors A common laboratory crperiment in general physiolon which I believe, was suggested originally by Chambers pouts up again the need for a definition of this phenomenon shich I don't think can be called secretion Ile shoned that an excused frogs bladder containing acid urine qed up to form a sac and dropped into an alahae solution containing neutral sed will "secrete" neutral red across the inert membrone until a high concentration of the die is achieved Another difference between noutral red on one hand and such other substances as pamino hippure and dodrast and phenol ·54 Renal Function red, on the other, is that the former stains the more açıd vacuoles within the cell and becomes concentrated in intracellular droplets This is not the case with phenol red As an isolated renal tubule preparation, fish tubules may be placed in a balanced isotonic salt solution containing one milligram percent of phenol red Direct observation of these tubules in vitro reveals that phenol red is secreted until a concentration of dye is achieved in the lumen of as high as 100 milligrams percent Phenol red moves against a con siderable concentration gradient, irrespective of the pH of the urine without at any time an intracellular accumulation of dye becoming apparent I wonder whether we can make a distinction at this time between these two mechanisms of cellular transport as exemplified by the movement of neutral red on one hand, and by the passage of phenol red, p-amıno hippuric acid, penicillin diodrast, etc, on the other? Oliver I am sure that neutral red is a very special case I think everything that you have just mentioned is more or less implicit in some carly work we did For instance, the effect of acid on its excretion we considered before Dr Chambers and Dr Kempton became interested in the problem (Oliver and Lund E M Cellular mechanisms of renal secretion II The Functional Phuse I Exp Med 57, 459 (1933), Chambers, R and Kempton RT: The elimination of neutral red by the frog's kidney 1 Cell and Comp Physiol 10, 199 (1937)) We tried y few perfusion expen ments with phenol red and abandoned them just because phenol red did not behave in the same way as neutral red Morcover we could not see the things that we could see with neutral red and being morphologists we wanted to use something that we could sce I remember an amusing interchange of letters with Dr Chambers during his worh on the problem of the effect of acidity on the excretion of neutral red, in which we both wondered what "secretion" was and when we should use the word and when we should not My attitude has always been that I did not know and therefore I did not like to set up some particular "thing in itself" called "secretion" as different from other forms of cellular passage I think I suspected something utalistic about the term "secretion and therefore I just didn't like it No doubt I acted from pure prejudice You can even extend this sort of argument to include terms like "absorption" All these things go together so that when we begin talking about "secretion," "absorption" and about some "metabolic process” in the cell, we are making very artificial distinctions in our ·55 Morphological Aspects protein word ne used to think of it as a study of "absorption" Now We are thinking of it more as a study of "intracelular metabolism", that is the degradation of protero What we orginally thought of as an indication of "absorption" we are beguning to believe may not have so much to do with the entrance of material into the cell but rather with intracellular metabolic processes Pitts Dont you have some pertinent data on those points Dr Shannon? Shannon It may be worthwhile to emphasize at this point that the type of transport mechanism utilized in the transference of neutral red across the cell is similar to that used by a number of weak organic bases where apparently movement across the cell is determined largely by the dissociation constant of the base This kas demonstrated by Jailer and others (Jailer) W Rosenfeld V and Shannon } A Influence of orally administered alkali and acid on renal crcretion of quinacrme and chloroquine and santoquine I Clin Inuestigation 26 1188 (19-4") Jailer W Zubrod CG Rosenfeld M and Shannon A Effect of acidosis and anova on concentration of qunacrine and chloroquune in blood ] Pharma cal & Etper Therap 92, 345 (1948)) In these cases secretion" or "reabsorption apperred to be wholly determined by the hydrogen son concentration of the urine Furthermore it was possible to demonstrate that such a phe nomenon was not a distinguishing charactenstic of renal tubular cells but actually could be demonstrated in the body is a whole This is important since apparently one is dealing here with a very general phenomenon which to my mind has little to do with active tubular transport or tubular ereretion In the case of neutral red I heletet adsusible to view the total phenomenon 25 consisting of two discrete esents one of these is concerned with the more ment of neutral red cross cell membranes The other is concerned with what loappens to the neutral red once it has entered the cell When neutral rod moves across the renal tubular cells under cer tam circumstances it does at the same time behare in 7 ier peculi ir minner relative to its localization in thic cell That fact would appear to be a coincidence rither than cydence that one phenomenon is wholis dependent upon the other Some evidence for thus itew stems from the toucity studies on 4-aminoquinolines wuch behne relative to renal transport mechanisms in 7 minner qmte simular to neutral red These intinalınıls were administered to 1 variety of hosts in very high dosage actuilt to the point of death the animals being subsequently studied for evulence of ·56 Renal Function toxicity Morphologic studies of the kidneys of these animals re vealed little in the way of information which would indicate that incidental to the transport of these substances across cell walls abnormal accumulations occurred in the cells themselves In general my feeling is that it is quite important to differentiate between what we have felt is a non specific movement of material across cells and the biological mechanisms which are available within the renal tubular cell to produce diffusion gradients at the expense of energy These latter are commonly called secretory or transport processes Oliver What would be some of the bona fide secretory activities in contrast to those observed in the handling of neutral red? Shannon Phenol red is a good one A great deal of work has been done with it As Dr Forster brought out, there are apparently sharp gradients under the brush border because phenol red does not stain or concentrate in cells Ohuer I am not quite sure of that I have the recollection - it has been a long time - that we did see droplets of phenol red in some of our perfused kidneys Shannon You do if you use very high concentrations but not in the ordinary concentration Oliver These were in perfused kidneys Shannon In the chick mesonephric preparations of Chimbers, one does not find any degree of concentration of phenol red in the cells at ordinary concentration However, it is my recollection that when Dr Chambers went to such high concentration as 100 mg percent, there would occur cellular inclusions rather deeply stained Dr Chambers took these as an indication of toxicity of the phenol red to the cells The lack of such an occurrence at lower concentratron was taken to indicate that these are not an essential part of the transport mechanism Oliver Of course the morphologist does not like to be told that as soon as he sees something things are abnormal, although I will admit that this at times may be the case Bott Dr Shannon, do you make the same distinction between reabsorption of glucose and the so called reabsorption of protein? I don't feel that they are the same at all Shannon I feel that the reabsorption of glucose is quite different from the reabsorption of protein wholly apart from the difference in physicochemical characteristics of the two substances The tyto logical study of cells during an active secretory process on exposure of a renal cell to protein solution is difficult since the control kidneys ·Vorphological Aspects 57 or the control cells are not in a truly resting state Consequently it is not logical to conclude that the transport of protein creates a cytological picture which is specifically related to an actively secret ing cell as opposed to a resting cell The tubular cells in the control of kidney are far from resting since they are actively doing work with expenditure of energy in the reabsorption of salts and other solubles I suspect that when one imposes an abnormal variable the cytological picture must be a reflection of the cells reaction to that specific variable and the morphologic change with the protein is not one which can be interpreted to indicate the morphologic change which is to be expected with ? variety of transport processes as contrasted to what is present in the rest of the cell Olwer As I understand it then any morphological change which is seen is per se abnormal? Shannon You have changed the normal economy of the cell Oliver That leaves the morphologist in the situation where he is not much use Shannon No sir I would not say that It leaves the morphologist in the position where he has to carry that thought in mind in interpretation Oliver We have all that in mind Fremont Smith Abnormal with respect to what? With respect to the problem you are dealing with? It seems to me that a change can be looked upon either as a normal or abnormal change of function Shannon The point I wish to emphasize is that the so called 21 It can be used as a control but not as a so called resting control It is difficult for me to see actually how one can obtain an adequate control as to isolate one functional characteristic from her variables Fremont Smith you feel that the control should huve been the normal resting cell? Shannon To which you dont have access Olter No cell is "resting ercept in death Both morphologists und functionalists must accept living cells as actise and use them as "controls" under appropriately similar conditions Heller It may be difficult to me at a picture of resting cells in the frogs proximal tubule even when all erogenous substances ·58 Renal Function which are known to be secreted are excluded There is good evi dence in a paper by Marshall (Afarshall Ek Jr The secretion of urea in the frog ) Cell Comp Physiol 2 349 (1932)) that the frogs kidney secretes urea Your control kidney presumably was secreting urea all the time? Olwer Only that urea which might have been brought to it by the Loches solution washing through tissue I would agree with Dr Shannon fully What he describes is the ideal procedure for both the morphologist or the functionalist but it does not seem to me it is compatible with any realistic activity on the part of an investigator I have had a tremendous boost from Dr Opies cytological work of the last few years He has taken a cue I think from the biochemist Instead of trying to preserve tissues perfectly to fix them exactly without change he deliberately rips them to pieces by letting them macerate in fluids of various sorts In other words he does just what the biochemist does when he begins boilung leaching pre cipitating and what not No one doubts that by this method the biochemists have learned something They do not try to handle tissues or their problem in a physiological way They do quite the contrary They disrupt things and find out artificial” facts yes but from those artificial facts they can perhaps go back to physiological conditions I think the expenmental morphologist is in the same situation when he uses something like neutral red that is obviously a foreign material he is under no illusion that it bears any im mediate identity to the handling of some ideal physiological sub stance but at least he is finding out something Fremont Smith The physicists taught us when they tried to measure speed and mass of the electrons that you cannot obsere any phenomeni in nature without changing them because by virtue of observation you are changing them if we accept that premise then our job is to specify the nature of change we induce by observation and how it affects the interpretation of observation Shannon That is a perfectly valid point I think we do have an experimental approach to such a problem If one examines a series of other transport mechanisms such as glucose phenol red diodrast or the like one way or another and if one examines the cytology of the particulır part of the kidney involved at very low concentra tions of a perfusing flund and at very high concentrations where the mechanism is saturated one then has a controlled observation that has more validity for comparative purposes than when a completely new variable is superimposed upon a series of unknowns Presum ·59 Morphological Aspects ably the kidney can operate on these highly specific transport mechanisms without fundamental disturbance of many of the normal characteristics common to the cell Leiter Dr Shannon, what is the approximate size of neutral red and does the size have any beanng on the type of transport process? Shannon It is the same order of magnitude as that of phenol red However, it does undergo a varying degree of dissociation Oliler The size depends upon pl One can observe change 10 size As it becomes more acid or alkaline one can see this with the ultramicroscope Shannon it tends to form aggregates Leiter Does it reach the size of inulin? Oliter No We do not have absolute measurements but I do not think so Schrocder Does the normal kidney ever secrete anything that normally occurs in the body at usual concentrations? Shannon Ammonia Fishberg That is synthesized in the kidney but does the hidney tubule transport anything normally circulating in the blood? Pitts Presumably it would transport hippuric acid Fishberg Is any of the urinary hippuric acid derived from outide of the kidney? Binkley les Berliner Maybe somebody is enough a chemist to say whether or not this is a valid concept I wonder if the difference between secretion and the type of thing we hase with neutral red and atabrine, etc might not be copressed as follows any process which results in the transfer of a substance from a position where its activity is low to one where it is Iugh might be defined as secretion (By activity here I mean chemical potential or escaping tendencs ) On the other hand a transfer which does not in oke a change in the actity of the substance would not be called secretion The transfer of neutral red would fall in the latter category The activity of the free neutral red may be the same in the plasma and urine and the difference attributable to the amount of the salt present in one phase and in the other I am not certain of the physical chemistrs involved Somebody else might do better with it Pulls Dr Wilhelm will you take a crick at that? Wilhelmi It seems a reasonable point I am not enough of 2 physical chemist either to si positii eh that would be the case Certainls one could argue for ions that if there is an increase in actrits in one compartment greater than in another one rould ·60 Renal Function expect work to be done and you could describe that with a little elaboration as involving an active principle There are as we know from Donnan effects instances in which one can end with different concentrations of ions provided that you begin with the system at disequilibrium and allow it to come to equilibrium I think that this need not apply here since it is difficult to imagine that an organ like the kidney might arrive at a condition like equilibrium in physical chemistry Forster I would like to get back if I may to the question which was raised before whether materials the course of transport across the renal tubules accumulate in the cells or not Dr Oliver made the point that perhaps neutral red did not differ from phenol red in this regard In the passage of neutral red not only does one note accumulation of dye in the lumen but also in the intracellular droplets which become intensely stained This is a colored photograph (Frontispiece) of an isolated renal tubule taken from a fish and placed in a balanced isotonic salt solution which had orygen bubbling through it and contained a one milligram percent con centration of phenol red It is apparent that the cells of the renal tubule have not taken up any of the red dye Inasmuch as the protoplasm is slightly alkaline one would expect that the phenol red should stain the cell contents of the dye were present in any sig, nificant amount The dark line indicates the accumulation of phenol red in the lumen which in this instance approached a concentration one hundred times as great as that in the fluid bathing the tubule I think you showed Dr Oliver and I know Chambers did that phenol red accumulated in the cells only when instead of one milli gram percent or so the in vitro solution contained dye in concen trations of the order of twenty five milligrams percent The situation where dye is taken up in high concentrations intracellularly is quite different from that which prevails with the transport of phenol red at low plasma levels We hwe reason to believe that the mechanism ini olved in the latter is the same as concerned with the transport of diodrast p amino hippunc acid and the other substances cus tomarıly used for renal plasma flow measurements This is indicated by the observation that the addition to the in vitro solution of substances that compete for transport such as caronamide penicillin diodrast and p amino hippuric acid will block the transport of phenol red Presumably this dye is came across the tubules without accumulation in the cells and its transport is facilitated by some expenditure of metabolic energy on the part of the cells There are tuo basic differences between this transport mechanism and that ·Morphological Aspects 61 operating in the movement of neutral red First neutral red ac cumulates within the cell a fact which might account to a great extent for the transformation of filamentous structures into droplets The droplets may actually represent concentrations of neutral red The second point of difference is that the movement of neutral red does not involve the expenditure of energy So I think the situation which you described as concomitant of transport of neutral red that is the droplet formation is a very special case Didnt you say that in some of your experiments you actually had bserved phenol red secretion and noticed that you did not get the toplasmic transformations? Oliver That is true and I also should have sud that we used nous concentrations and times of perfusion of neutral red I have I thought about this neutral red work for so long that it comes i surprise that there is still any interest in it but I remember that made a very sharp distinction relatise to the passage of neutral from the capillary to the lumen in the perfusion etperiment ich in a simple minded way we called “secretion meaning only at was going in that direction nothing more) between wha alled direct secretion and indirect In the early stages of any perfusion experiment one gets considerable amounts of neutral red passing into the tubular lumen without any drops formmg in the cells and this passage en be affected by certain elecroly tes As I recall calcum stoned or decreased the passage Then as opposed to thus or added to it was an indircct method of passage in which we observed accumulation of the dye in droplets in the cells So neutral red certainly can pass through the tubule wall without causing droplets It commonly does in any perfusion experiment and then as time goes on accumulation begins and this other method proceeds I made qute a point of what we called direct secretion Perhaps it is not a good point We might have said immediate and delayed secretion the first having nothing to do with droplets and the second in oung the appearance of droplets Shannon it might be helpful were the phenomenon erannned in tissues other than lidney Perhaps Dr Graffin would be willing to comment on the behavior of macrophages in neutrl red solu trons more particularly in relation to the second type that is the intracellular type of handling this material by a nonrenal as well as a renul cell Graflin The simplest preparations for the study of this problem are fresh spreads of subcutincons connectic tissue which has been injected with a dilute solution of neutrl red In such preparations ·62 Renal Function the macrophages routinely segregate the dye in vacuoles or droplets which at least in the majority of instances, so far as one can tell, do not pre-exist in the cell The size and number of these vacuoles tends to increase with time, and many of them are very highly colored indeed Oliver By “segregate' you mean "concentrate'? Grafflın Yes Shannon This would appear to be an excellent example of what d occurring in would appear portant to ap preciate this and to appreciate further that when the phenomenon does occur in a cell capable of active secretion, the phenomenon itself is not necessarily related to the latter function Olwer These general facts do not make the situation different It simply means that what we are dealing with is the very fundamental process that living cells have of absorbing neutral red What happens then depends upon what the cell is The kidney cell passes it on to the tubule lumen ·THE ROLE OF GLUTAMINASE IN TUBULAR PROCESSES FRANCIS BINK Departments of Pall ology and Media Unn ersity of Ulub College of Medica Before I start on the discussion of my topic? I would like to brio up something that Dr Wilhelmi and I have been discussing la regard to the problem of glycogen deposition there as a possibility that the cells in the upper part of the tubules huse the herokinase system The glucose is phosphorylated by this reaction to gue glu cose 6 phosphate this reaction is thought to be the one blocked by phlorhızın and the one concerned with the reabsorption of glucose If this reaction is blocked then the glucose will pass down to the cells in the lower tubule and may be absorbed as glucose 1 phosphate a precursor of glycogen My interests are not distinctly in renal physiolog, but are concerned with the general processes of absorption by all tissues We have been driven to this concept loy our studies of hereditary diseases including muscular dystrophy Seteral theories concerning the creatinuria of dystrophy are possible Esther creatine is produced in great amounts so it is coming out in the urine or it is produced in normal amounts and is not absorbed or retained by the muscle tissue Therefore we have Mbeled creatine with hervy isotopes and have administered it to patients We find that within 48 hours all the administered creatine has been ercreted In other words the dystrophic muscle is unable to absorb or hold erogencous creatine presented to it So our problem would appeur to be simplified All we have to do is to determine how the muscle normally absorbs ats create through its membranes and how it holds it For some time I have been working on the metabolism of gluta thone and glutamine ile had found that an enzyme of the kidney his drolyzed glutathione at a very rapid rate so that the glutathione was broken down to an amino acid glutamic acid plus the dipeptide ysteinylglycinc (1) Since there was so little known about the meta ! From the Lahe atory for the Study of Hkrat up an 1 Metabolcsders 2 These studes vers supported by grants from the US Pullc Heslb Set ce A Nakamura J Watson C i Olson 13 Rimmel hale collaborate the arest *ton of var dus aspects of the problem ·64 Renal Function bolism of glutathione we thought it would be of some interest to isolate this enzyme from the kidney and study its behavior We are finally convinced that we have the enzyme in fairly pure form The enzyme is active in the hydrolysis of glutamine to glutamic acid plus ammonia As you are aware, this is the mechanism which has been suggested for the formation of urinary ammonia as reported by Van Slyke (2) But on the other hand this enzyme appears to constitute a very great part of the protein of the kidney The material which we isolate is a lipoprotein which by the critena we can apply to it appears to be homogeneous It is not soluble enough to permit studies in the electrophoresis apparatus The studies in the ultracentrifuge indicate a molecular weight of ab two million The nitrogen content of this material is about 1 percent It contains approximately 25 percent phospholipid Ti matenal constitutes 30 percent of the protein of a homogenate kidney tissue It had been our conclusion that this was a magnesium activatec enzyme but when we ran studies of activity of the enzyme as a function of the concentration of magnesium ion it was found that at concentrations of 0001 M magnesium there was activation As the magnesium ion was increased to or near 001 M, complete inhibition was observed Immediately we turned to a study of the effects of a large number of sons (Fig 22) The large number of the wonized materials of importance in renal physiology can be divided roughly into four groups In one group like urea and creatinine concentrations may be increased to 3 000 mEq per hter with no effect upon the activity of the enzyme Then we have another group represented by glycine and containing most of the amino acids There is an activation that approaches a maximum no further change is observed as the con centration is increused Another group represented by lithium chloride or mercuric chloride was found to inactivate the enzyme when present in small traces Then another large group of materials represented by sodium chloride was found with these materials a biphasic phenomenon was observed Activation was observed up to a certain concentration and then inhibition occurred Sodium sulfate behned roughly like urea and creatinine Sodium bicarbonate and potassium chlonde hue presented much the same behavior as sodium chloride We have tested a group of the so-called sro and have found them to be an- (Fig 23) One of the po ·Role of Glutaminase Glycine ACTA Urea, Creatinine LI CI Na CI 30 100 Meq per nter 250 . FICURE 22 renicillin is one of the best Phenol red, diodrast, annamide, and sulfanilamide nere found to be competitive inhibitors we tumed then to a study of quite a few of the various phenolphthaleins and sulfonphthaleins We find that these dyes inhibit, as competitie inhibitors only when the pll of our buffer is greater than theu pk For instance, phenol red is an ercellent inhibitor of glutamı nase at about 74 but phenolphthalein has no effect In fact it activates slightly as the pil is raised to about 85 at the higher pil it becomes a potent inhibitor There are some interesting effects that I can describe only in general terms For instance of this enzyme has the importance that I want to ascribe to it you may well erpect that muded hormonal effects should be obseried to have found that thyronin un more or less physiological concentrations is an activator of the enzyme Activations as great as 200-300 percent have been observed if howcier, the concentration of thyronn is increased far besond the physiological range, inhibition is obsen ed.

·66 Renal Function V/Activity Penicillin LI CI paminohippurate control V[Glutamine) FICURE 23 Since we are interested in absorption mechanisms in all tissues as well as in the kidney we have studied the enzyme in other tissues We find that the intestine has 2 system identical in so far as we can tell with that of the kidney It hydrolyzes glutamine and gluta thione and like the kidney enzyme is inhibited much in the same manner The phthaleins that are excellent Irratives at the pi of the intestine are good competitive inhibitors of the enzy me a higher values of pH Those that are not laxatives are not inhibitors The same system is not found in all tissues We feel we have two types of glutaminase the brain type and the kidney type The kidney type is found in the intestine but the nervous tissue type or brain type is found in muscle and all nen ous tissue In the brain type the activity toward glutamine is much greater than the activity toward glutathione whereas in the kidney and intestinal type they are about equal It is my thesis that this enzyme is a logical candidate as the mechanism for certain myjor aspects of absorption by the kidney We do not intend to imply that if the immediate source of energy for absorption is the hydrolysis of glutamine and glutathione such processes as the formation of hy drogen son in metabolism or phos ·Role of Glutaminase 67 phorylation are not of importance On the other hand we feel that we can erplain a lot of renal function of we are granted that this insoluble lipoprotein behases 25 1 self regenerating ion exchange resin Certain of the reactions are flustrated below Absorption of Sodium (2) Protein -COOH + Asci non conos Protein - Coola + HCI (2) Protes - Coola + glutamine Proteta COOVIL + glutamic acid (3) Protein -COOVIL + glutamic acid + energy Protein -COO)! glutamine In these reactions which are analogous to those that take place with a resin of the carborylic acid type the sodium is removed from solution by the resin cien though the equkbrium is in the opposite direction All that needs to be done is to lover the con centration of sodium ion at some site within the cell on the other side of the membrane and thus trick the sodium ions into moying into this area In other words there must be a reschon in which sodium ions are shoved off this is the reaction which we ascribe to glutaminase activats Hydrogen on might be expected to play a role in this process since it is a cation that can be produced by the ondation of substrates However since the ammonium ion can be produced and withdrawn under physiological conditions and without demands upon buffering mechanisms it would appear to be the more logical substrate The removal of the sodium son is pictured as its replacement by ammonia As the ammonit is resynthesized into glutamine resin carbonl is freed for further combination with sodium In this manncr 1 driving force for the pumping of sodium across the membrie would be maintuned This explanation does not give a very clear picture of the architecture of such a system as we propose and as a further explanation we propose a crude model of a membrane Protein COOH COOH соон COOH COOH COOH glutam cacd Protein + ammona The mobility of a sodium ion in un eschinge resın is known to be greater than its mobilitz in solution This might be emplained glutamine More ·68 Renal Function by a migration of the sodium ion from carboxyl to carboxyl grouping We have spoken of the system as one that requires glutamine as a source of energy It is quite possible however, that amide groupings are formed on the protein and glutamine is involved only as a donor of amide groupings to the protein In fact, such a mechanism is suggested by our observations that there is a loading of the enzyme molecule with amide groupings during the course of enzymatic activity It is obvious that a major consideration in any such mechanism is the architecture of the cell It is unfortunate that the kidney is so completely unidirectional in its activity for such behavior imposes many restrictions on any model that may be proposed Acidification of the Unine Pitts has shown that acıdıfication of the urine can be explained (3), by known reactions of the type of resin we are proposing as the absorptive unit When a salt of a strong base and a weak acid reacts with a carboxylic acid resin, the equilibrium lies well toward the left Protein -COOH + NapoPro en -COONa + HX It would be expected that sodium or potassium salts of acetate or pyruvate, or disodium phosphate would take part in such reactions Formation of Urinary Ammonia It is characteristic of carboxylic acid resins, such as IRC-50 that ammonia may be displaced by hydrogen ions in the range of pH developed by the reactions described above for the acıdıfication of urine It is difficult to visualize how penicillin and phenol red are transported across a membrane I frankly don't know, but what I can do is to give a suggestion For instance we have found that an inhibitor must have three qualifications (a) It must be an anon It must be capable of being an anion at the pH of the buller used most of them contain carboxylic acid groups (b) It must contain some type of resonating system ind (c) it must contun 1 large R group For instance the phthaleins are inhibitors Such tlungs as bicarbonate, acetate propionate and pyruvate are activators hut as the higher acids are reached they become indifferent and eventu ally became weak inhibitors That is an obser ation which is slightly reminiscent of some work that has been done in the organic chemis try of the amide groupings Steinhardt has shown that a large group of materials and or catalyze the hydrolysis of amide groupings (4) It has been suggested that amide groupings combine with these anionic ·69 2 Role of Glutaminase reagents to give an amide salt Perhaps we can assume that the agents for measunng renal blood flow or inhibitors have actually combined with the amide groupings of the protein or glutamine I am inclined to believe that the enzyme is the one with which it combines We have evidence that this is true because we can show that glutaminase will take up phenol red Phenol red attached to the glutaminase can be shown to be displaced from the enzyme by the addition of glutamine Thus it appears that the phenol red is combining with exactly the same groupings that combine with glutamine Since such agents do catalyze the hydrolysis of amude groupings this is possibly the mechanism by which they are carried through as the anions Some adaptation of the concept is undoubtedly necessary but that is our explanation as at stands at this time Jou may have gathered from this presentation that I feel that kidney is essentially a tube of ion exchange resin and that the strat:fication or localization of absorption which is observed is due 'o the effect of one son upon the other There is no real reason for ssuming that these cells are different in their absorptive behavior si a mixture of ions very much file that found in the glomerular filtrate, as nin through a column of IRC-50 a very similar stratifica tion of these various ions is observed There is one more point I would he to consider We started in with this program by sorrying about the behavior of creatme Unfortunately creatine has very little effect upon this enzyme Creatine phosphate hones er behaves very much like glicine Of some interest to us is that the glutaminase of muscle is associated in the actomy osin complex In so far as we can determine it is the actin of actomy osin that has the glutaminase activity REFERENCES 1 BINKLEY 5 AND NAMAMLRA K Metabolism of glutath one hydrolysis by tissues of rat 1 Bol Chem 173 411 (1948) 3 VAN SLYXE DD et al Glutamine 25 source material of uninary ammonia / Biol Chem 150, 481 (1943) 3 Parts, R F Renal excretion of acid red Prox 7, 418 (1948) 4 STEINHARDT ) The chemsstry of amino acids and proteins An: Ret Biochem 14, 145 (1945) DISCUSSION Mfudge Would you tell us fios sou prepare thus enzymes Binlicy Our preparation is from pig kidney It need not be fresh but can be stored frozen One hundred grams of pig didnes plus ·70 Renal Function 1000 ml of OOL V1 sodium barbiturate pH II is homogenized and heated at 45° for the hours This is centrifuged at 6 000 for twenty minutes Then the supernatant is dialyzed against distilled water for 24 hours The distilled water is changed about every four or five hours Then we pass in CO for a long time The enzy matically active material is precipitated As we punky it it becomes more insoluble We start with 100 grams of kidney and end up with the enzyme in two liters of OOL M sodium carbonate bicarbon ate mixture of pH of about 85 The next step is alcohol fractiona tion If we fractionate at this stage without changing the conic strength the active material is precintated between 3/10 and 6/10 volumes of ethanol In other words to two liters we would add 600 cc centrifuge discard that material and add another 600 cc. and centrifuge We have varied ionic strength many times for further fractiona tion If the material is dialyzed exhaustively one may add three volumes of ethanol and centrifuge to get rid of inactive material then pass in Co to precipitate the active matenal We lose at most about 10 to 15 percent actavity when treated with acetone and stored in the desiccator it is stable for months Leiter Have you extracted renal cortex and medulla separately? Binkley We have tried to We have not made much progress White Do you have any obser ations on red cells say of man where sodium is pumped out and potassium in? Binhley That is about the only species on which we hase any information This brings up one important point in the red blood cell glutathione predominates and in the plasma glutamine pre dominates As you know ammonia given to an animal disappears very quickly If ammonia is added to ordinary orygenated blood the ammonia disappears with the formation of glutamine and at the same time a decrease of glutathione it appears that the mechanism used to pick up the ammonia the glutaminase is in the cell stroma If we take the stroma through an alkalıne extraction we find the enzyme in solution White It would be interesting to know whether the mechanism 25 present in red cells which do not establish concentration gradients of sodium and potassium as well as in those that do Taggart One is impressed with the fact that this 30 percent i alue is fairly close to that which Schneider (Schneider WC Intra cellular Distribution of Enzymes II The oxidation of octandic acid by rat liver fractions ; Biol Chiem 176 259 (1948)) has re ported for the combined nuclear and mitochondrial fractions of ·Role of Glutaminase 71 liser and kidney cells The mitochondnal fraction of cell particles apparently contains all of the enzymes which are needed to implement the reactions of Krebs tricarboxylic acid cycle and a num ber of others besides Green (Green DE Loomis 1 F and Auerbach V H Studies on the Cyclophorase System I The com plete ondation of pyruvic acid to carbon dioxide and water | Biol Chem 172, 389 (1948)) applied the name "cyclophorase system" to this integrated compler of enzymes Is it not concen able that your glutaminase lies within such a complex and that the rather rigorous conditions of preparation have effectuely destroyed the other more labile activities? Binklcy I believe that is possible Taggart let you have spoken of the enzyme as being homo geneous Binkley I say "complex I wont say pure substance in the sense of a protein Taggart When one uses the term compler" a certain lack of homogeneity is often implied lou will agree then that the gluta minase may be just one of many enzymatic activities orginally present in the compler one which has surned the method of preparation Binhley That is why I was interested in Dr Olusers comments I hope to get at that by the use of staining materials Our enzyme stains quite beautifully by something like phenol red Schroeder Is this enzyme specific for glutamine? Does it attack hastamine or histadıne? Binkley It also attachs glutathione but has no activity on hista mine or histadıne Schroeder What about other primary and secondary amines as sources of ammonin? Binkley I have not tried them This enzyme is concemed with the hydrolysis of a peptide bond There are lots of things about the metabolism of glutamine und glutathione that puzzled people for a long time Every one has talked about glutathione as a possible activator of enzymes yet it has a turnover out of all order of magnitude for such an actn ator Certunk ecn from that data one might conclude that the biological utilization of glutathione is con comed with the hydrolysis In passing I would like to mention that bruin slices with the addition of glutamine will retam potassium Another very important hing that Krebs and his group have found is that i les totic wyroids have very lon glutathione izlues le hre confirmed that ·72 Renal Function This is in line with the activation of the hydrolysis of the glutamine by thyroxin Other work that may be explained partially on the basis of our theories is the work of Conn and his group on the effect of ACTH on the lowering of the level of glutathionine in blood We believe that the ACTH stimulates an increased utilization of glutathione for the pumping mechanism, probably to such a point that it is depleted and glycosuria develops The amount of glutathione normally supplied is not adequate to keep up with the load and more glutathione must be supplied as fuel for the pump You would probably be interested as to whether or not we have made any calculations as to the energy required by this mechanism, whether we can explain, for instance, within the observed oxygen consumption whether or not the kidney can use this as a pumping mechanism It can but not with too great a degree of efficiency Taggart What is the approximate efficiency required? Binkley About 30 percent Taggart Do you believe that the glutaminase catalyzes both the synthesis and hydrolysis of glutamine? Binlley There is evidence that this is so Taggart How do your views on glutaminc synthesis compare with those of Speck?' (Spech, J F The enzymatic synthesis of glutamine, a reaction utilizing adenosine triphosphate I Blo! Chem, 179, 1405 (1949)) Binhley Mly work is not in opposition to that of Spech It is a little bit different You will see that it is probably the same story We found if we lowered the pH down to about 7 in the hydrolysis of glutamine, the hydrolysis stopped rather abruptly at an equi librium point Instead of glutamic acid, pyrrolidonecarborylic acid, the anhydride of the glutamic acid was formed Pyrrolidonecar. boxylic acid with our enzyme, plus ammonia, gives glutamine, that is to say that the hydrolytic enzyme also catalyzes the reverse reaction What Speck has shown is that adenosine triphosphate plus glutamic acid and ammonia gave glutamine We find that pyrrolidonecarborylic acid plus ammonia gives glutamine Apparently his system contains an additional enzyme which is responsible for the formation of pyrrolidonecarboxylic acid from glutamic acid Wilhelmi Do you think that the activating effect of the amino acid, such as glycine, is due to its removing metallic ions? Binkley Glycine has a very great effect, histamine has practically no effect The amino acid story is fairly compler I don't know why ·Role of Glutaminase 73 some have such effects and some not Undoubtedly it has to do with the combination of other sons with the amino acids l'itts What is the effect of calcium salts? Binkley At low concentration activation at high concentration inhibition Schroeder Would you say something about the effect of potassium? Binhley If we get our enzyme free of sodium as we can bv constant dialysis against potassium buffers then the enzyme is inactive It requires sodium ion to be active and potassium docs not replace the sodium Potassium chloride does have an effect more or less like sodium chloride in the presence of sodium ions Schroeder Krebs in a recent symposium shows that shaking up a piece of muscle in a solution results in loss of potassium from muscle at a constant rate If glutamate is added to it it does not. If you added glutamate to the solution after the potassium had left the cells it went back into the muscle The concentration of the gluta mate was so small that it could not have been due to simple ion exchange It was more like an active process Binlicy The way we interpret the function of glutamine in the muscle is that the muscle is utilizing glutamine to pump out sodium and the potassium comes in to replace the sodium Thc pump is reasonably indifferent to potassium but not completely so Taggart Does phosphate have any effect? Binklcy I will have to explain several things about our phosphate effects You may recall that Dr Greenstein has been working with glutaminases He has described glutaminases I and II I is activated by pyruvate and II is activated by phosphate His evidence for calling them I and II is that the one activated by phosphate has a different pII optimum than the one activated by pyruvate and the activation is different in character The activation by phosphate shows a cut-off value whereas that by pyrui atc docs not lle have made the same observation with our punfied mitenals We think the glutaminases are identical the effect of phosphate is to sluſt the activity down towards neutrality Since we are working with the sodium salts of werk açıds in both cases ve may consider an exchange type of inction the phosphate concentration is too high the crelange will be stopped by inhubition of the enzymatic step Pitt's What is the difference in the in citro behavior of the enzyme to penicillin Crsus cinnamide $11 with the addition of glutamine? Binkley Not too different Penicillin is just a much more potent ·74 Renal Function inhibitor than carinamide, that is really the only test we have made Mudge Have you tried mercury or tetrathionate? Binkley Mercury is very potent as an inhubitor We have not tried the other Heller is the system inhibited by sulfanilamide? Binkley Yes Shannon I was wondering in calculating this as an ion exchange resin what the capacity of the resin is for sodium It is terribly important to calculate capacity You are trying to use a non specific ion exchange resin as a model to explain differences in the renal handling of specific ions If the capacity is as large as you suggest it should not differentiate between the sodium and potassium ions I believe it is only when the resin begins to reach the point of saturation that one may expect a differentul action on the uptake of specific ions with such a resin Binklcy I agree there are certain important things to be con sidered in a quantitative manner When I was showing you the effects of the sons on the enzymes (Fig 22), it was mentioned that the cut off values corresponded to the 'threshold" values If adjust our enzyme and the pH we can makc the cut off value con form to the sodium level of the blood stream but, if we vary the enzyme, the cut off value varies too It does not vary at as great a rate as might be expected For instance, if the enzyme is increased fivefold then the sodium concentration necessary for cut-off is increased ten or twenty milliequivalents In other words, the cut off is relatively insensıtıe to changes in enzyme concentration Shannon What is your gunde? Binhley We have three variables in finding the cut off value for an son One is substrate concentration, ie, how much glutamine or glutathione is present Two is the enzyme concentration and of course, the third one is the pH All these determine to some extent where the ion cut off comes Shannon Forgetting about the three, in considering this in rela tion to the kidney, we can remove some of these because certainly pH is constant in the area where most of these sonic changes the place So that there are left then substrate concentration and enzyme concentration One would assume if this is so important to the architecture of the kidney as such that the enzyme concentration would be constant The reason I am bringing this up is I find the theory very attractie I am trying to find some point of reality to permit bringing it into the kidney and out of the test tube If one assumes that the enzyme concentration is constant, then one should ·Role of Glutaminase 75 be able to tale kidneys under different states with a lugh and low capacity to reabsorb ions and find ery tight correlation between that function and the concentration of the substrate and the con centration of the glutamine contamed in the system so that the thesis is subject to experimental trial I think if that point were tied down and it again it were demonstrated that the capacity of the system were such as to permt the parallel of a simple con crchange resin of the type IRC-50 where one would get preferential handling of ions then we would have brought the system close to the kidnes closer than it is now at the present time Binhley We are working with intact anmals nou llc ve vrs interested in a group of expenments in which we are using hewy nitrogen to trace the uptake in the amdc groupings of protein und in the glutamine under different hormonal conditions to see if we cannot correlate these effects with the know effects of the hormones White In hypophy sectomized dogs where the Pall Tm is down to 20 percent of normal would it be possible or reasonable to try to determine whether the enzyme content of the hidnet of such 3 dog is normal? Binhlcy We want very much to do that type of experiment What we hope to do is use 7 dog with an explanted kidney so we can follow glutamine and glutathione in the renal and artenal blood But I am not proposing that this mechanism is at the mercy of the blood supply when the immons? goes out in the urine so that the kidney loscs ammonia then it must replerush the supply from the blood by removing glutamine from the blood Shannon The thing which bothers me more than anything else is that the renal mechanisms possess high hbility in terms of time I am thinking of such phenomena is occur when one adds a minute quantity of the antidiuretic hormone in the course of tuo or three minutes the characteristics of the urine change completely or if onc adds ser small amounts of phlorluzin as little as 19 mg per lg to a whole mun one drasticall upsets the reabsorpınc mechan ism for glucose Agun certain of the renal mechanismis possess a high degree of specificit as we live scen in indirect measure ments I have the feeling without being able to put my finger on it that dlus conception does not provide for the labfity and the specificits that are so characteristic of renal activity During the past two years ctchinge resins hoe been built up into industrial chemical processes in a side i ricts of situations One can do almnst anything with them I am quite prepared to acept De Birkley s sien hecause if you are limited to a simple ·76 Renal Function problem, regardless of what it is you can find an ion exchange resin to satisfy the particular need However when you use a resin to accomplish a highly specific end, then the conditions under which the process proceeds smoothly must be worked out in great detail with respect to the control of all variables Resins are used to extract penicillin from broth or streptomycin out of broth or crude solutions, and they are used in purification procedures of a wide variety in industrial procedures today, yet to obtain the control of variables to an extent where the specific operation has a high degree of efficiency requires extensive investigations Ion exchange resins are very susceptible in their action to minor changes in environment, such as in ion content, hydrogen ion concentration, and other variables They do have the potential of doing many specific jobs, but they do not possess the ability to perform many specific actions simultaneously Binhley I would like to think of a tubule as a tube packed with ion exchange resin The distal tubule would be another column of practically the same thing arranged, if you wish, with a different architecture Then I am asking if the thin segment could not pos sibly act as some type of automatic stopper, so that flow is stopped or retarded until a certain ion concentration is reached in the proximal tubule Grafflın In their extensive work upon fluorescence microscopy of the frog's kidney Ellinger and Hırt reported the jerky and rapid propulsion of the urine along the renal tubules Singer later confirmed this observation and spoke of a valve mechanism controlled by the cliated neck and intermediate segments Dr Eisenberg and I likewise observed this rapid propulsion of the urine upon the ventral surface of the frog's kidney and it is exceedingly easy to demonstrate (Grafflın, A L and Eisenberg, MJ, A micro fluoroscopic study of teleostean kidneys, Anat Rec, 59 449 (1931), this paper includes references to the publicatrons of Ellinger and Hırt and of Singer) I don't know of any evidence for the occurrence of such a valve mechanism in mammals Binkley I thought that such a valve might in some way change the whole character of more distal absorptive activity Heller Are these ion exchange resins substances shich shou a greater affinity for potassium than sodium? Binkley The carborylic acid ones do not Pitts I grant that you may doubt that the kidney is anion specific with respect to e But for the sake of argu. ment, let us - extent than 2.

·Role of Glutaminase 777 could be explained by differences in ion mobilty This exchange resin concept could not account for anion specificity in absorption, could it? Binhley I dont know I am surprised to find that the physiologisks speak of absorption of sodium ions and water as separate processes It would appear to me that it is much more reasonable to think of the ions as absorbed in the hydrated form so that each ion carries along with it ? quot of water as water of hydration In the first place the energy insoh cd in the dehydration of an ion such as sodium would be considerable and thus an improbable mechanism In the second place it is possible that some mechanism may be ai uhble for the modification of the hydration of an ion 17tkun wide limits and thus bring about changes in the amount of reabsorption of water Patts Is it not true that the energy produced in this reaction comes fundamentall from the hidrolisis of glutamine? lou would dissociate sodium on the basis of this hy drolysis Energy would be consumed in the resynthesis of glutamine would it not? Benllcy That is correct at least initially The hydrogen son is a source of energy and can be used in the resynthesis of glutamine Pitts lion would you get around that if there were not? Binkley I can think of two sources One is the production of hy drogen ion and the other is the production of glutimine If enough hydrogen ions are produced then they can be exchanged for sodium Pitts And by passing the glutamine? Binkley Just coming through In such a case as sodium chloride the culorile must be following closek behind the sodiun But if on the other hand an anion cinnot follow to the same extent that the chlorile can follow it must there and electroneutrilits will be achused br the movement of an ly drogen son Shannon It does not have to move at all Binllcy ly guess is at tres to but cannot Shannon if you consider a lattice of cubor groups solut can move across the lattice moſsed without disturbing electric neutrality or osmotic pressure Binhley I think of the lattice as buffered so that at any one time there we not many acid groups Shannon You knock one sodum lon off at onc end of the lattice creating a capacits for a sodium ion at the other end lou dont create a situation wherein an anion has to move Binkley Ercent to presene ncutralt if there is an accumul tion of positae ions on the other side ·78 Renal Function Shannon You lose 1 hydrogen ion in the initial exchange so you dont disturb the system electrically Binkicy We are saying the same thing except that I am taking the other way around I am suying that the sodium ion is being moved and does create a charge The way to neutralize that charge is for hydrogen ion to go out Putts Do you interpret the effect of sulfanilamide on the acidity of the urine as being its effect on the glutaminase? Binhley I am not too familır with the details of the effects but do believe it can be explained on the basis of this mechanism Willielmi If I understand you correctly there is a very intimate relationship between which son happens to be on your resin and the actual cnzymatic activity of the resin? Binhley les I ilhelmi How do you imagine that that is established? Bınkley The sodium ion and ammonium ion are competing with other ions this is one explanation I can offer Shannon in relation to this sodium versus ammonium effect I would hhe to ask Doctor Potts how long it takes his animals to come to equilibrium with the excretion of 1 maximal amount of ammonia Pitts I cannot give you details of an experiment nor 7 cry accurate time schle However I think there is a real time lng in the output of ammonia If you reduce the bicarbonate of the serum ncutely from 25 to 10 milimols per liter by the intravenous infusion of hydrochloric acid over a period of two or three hours ummonia output will by no means have approached the maximum in that length of time If the same degree of acidosis is produced by giving hy drochloric acid by mouth over a period of several days ammonia cxcretion will reach much higher levels The greater ammonia out put following oral administration of acid may in part be due to a time lag in ammoniz output or it may be due to the grenter depletion of stores of body base Shannon I mean intravenous experiments where you bring it down abruptly und hold Pitts Unfortunately we never carried those on past about four hours Shannon Compared to chronic animals they did not liave the same excretion rate of ammonia? Pitts Not any here near Wute That is not the experience of the Scandinavian vorher Ryberg who found that ammonia production reached a plateau within 2 or 3 hours with no further increase in the nert 24 or 45 ·Role of Glutaminase 79 hours (Ryberg, C On the formation of ammonia in the kidneys dunag acidosis Acta physiol Scandinat 15, 114 (1948)) Wesson Is it possible to predict from the properties of this system the effect of mercury on the excretion of these sons sodium potas sium hydrogen and ammonium? What would be the cffect on ammonium excretion for examples Binhley I think I am logically obligated to say that it would reduce ammonia ercrehon Leiter te huve found that mercury has no effect on ammonia production in human subjects with or without heart fulure Vudge We found that mercunais had no apparent cffect on ammonia excretion in the dog Pitts We found no effect either Wesson Ke found no significant change in one experiment Pilts That is true also of the hydrogen ion concentration Berliner We have not found mercurs ils to live ans appreciable effect on either acidification or ammonia formation Patts That is what we found too Binlicy We have not tested whether the mercunal duuretics inlubit in the same fashion as mercuric chloride Pitts I think the general concensus is that an organic mercurial diuretic acts by virtue of its hvemtion of sonic mercun that if one measures the duretic response to fuchloride of mercury it is essen talks comparable to the organic mercunals in terms of mereurs Furthermore there is not a great deal more damige done by bichloride than by the organic mercunals Smull doses of huchloride of mercury zre somewhat more ne phrotoxic than the orgme com pounds but not of a different order of magnitude entirely Taggart Certuin of the obsen t1905 watch Dr Forster und I hase made on isolated fish tubules oppose the ider that the orgime mercurials are actie onks through the bleration of mercuric ions Ilhen tested as inlubitors of phenol red irinsport mercting chloride and mercupurin were found to be approximately cqual in actity on 1 molecular b3515 Such 1 finding would imply that the meren INITID *75 completek dissociated an unlikel possiblits in uick of the arbon mercur lond present in the organic mercunal In addition lfellering and others have shown that one of the most cloctie inhibitors of the sufin dnl onzı mes is another orginc mercurial p-chloromercurilenzoite I citer llould so is willing to print the mercun icted on the pmnrimal tubule rather than on the distal? This sonld cuplun the situation in regard to mmmoma formation ·BIOCHEMICAL ASPECTS OF RENAL TUBULAR TRANSPORT JOHN V TAGGART Department of Medicine College of Physicians and Surgeons Columbia Unters ty During recent years the development of micropuncture and clear ance techniques has enabled the physiologist to describe various discrete renal processes in a very precise manner Through the use of these techniques abundant evidence has been obtained for a number of active secretory and reabsorptive mechanisms in the renal tubule The term active transport implies the movement of a solute across the tubular epithelium against a concentration gradient a process which necessarily entails the expenditure of energy derived from metabolic reactions within the cell Despite the recent progress which has been made in the fields of inter mediary metabolism and renal physiology we know relatively little about the metabolic events which underhe active transport The only noteworthy exception is to be found in the case of glucose reabsorption The studies of Verzır and Laszt on intestine and of Lundsgaard and Kalchar on kidneys are too well known to need description here It is sufficient to say that a fairly strong case can be made for the stepwise phosphorylation and dephosphorylation of glucose in the cells of the proximal convoluted tubule The dis tribution of phosphatase in the luminal margins of the cells of this tubular segment as revealed by histochemical studies provides additional evidence in support of this concept When our attention is directed to other transport systems such as those responsible for the secretion of diodrast phenol red or p aminohippurite no obvious mechanism is suggested by available biochemical information However it is reasonable to assume that the substance being transported undergoes a series of two or more reactions and that at least one of tliese involves the participation of an energy donating system Consequently certain importance attaches to the mechanisms by which metabolic energy is translated into osmotic work. The utilization of phosphate bond energy in various biological syntheses in muscular contraction the propaga tion of the nerve impulse etc suggests its possible importance in 82 ·Biochemical aspects 83 renal transport processes The implication of phosphate bond energy in selected transport mechanisms appeared to be a reasonable point of departure in the senes of studies which I shall present this eiening it has long been recognized that at least certain of the aerobic oxidation reictions of the Krebs citrc acid cycle are coupled with the generation of energy nch phosphate bonds og adenosinctn phosphate Such aerobic phosphorylation can be readily demon strated with the washed water insoluble particles of kidney homogenates The enzymes contained in the particles catalyze all of the reactions of the citric acid cycle and provide a final commor ondutive pathway for the complete combustion of pyruvate fatty acids and certain amino acids Thus complex of enzymes has been quen the name cyclophorase system (i) Vore recently it has veen conclusively demonstrated that this enzyme complex is con 'ned to the so called mitochondrial fraction obtuned from tissue homogenates by differential centnfugation (2) The coupling of phosphorylation with scrobic ovdation provides a mechanism whereby energy hherited by catabolic processCS is made nilable for biosynthetic reactions and for the work of the cell The efficiency of this mechanism is quite remari able During the oxidation of letoglutarate in the rabbit didney ciclophorase system almost 3 molecules of inorganic phosphate disappear for eich atom of oxygen consumed The inorganic phosphate has been incorporated into energy rich adenylpyrophosphates (ATP) The ATP generated from adeny lic acid and inorganic phosphate can be measured b the addition of lierokinnse and glucose to the system as eich molecule of ATP is formed the herokinase catalyzes the transfer of the terminal phosphate group onto the herose to form herose monophosphinte stable end product sn this enzime sistem By the use of this technique it is possible to approximate the PO mtio (moles of inorganic P esterified ntom of orygen) for each of the 5 ondatric reactions of the citne acid cycle The P/O nito appears to be 3 for cich of the reactions ercept that of succinate to fumirate in which it is 2 Tlus at least 11 molecules of inorganic phosphinte tre rused to the energy nich level in the course of the file oxidine reactions such constitute the complete combustion of one molecule of purus ate The minimal amount of energi nuclı can be hberated on this series of reactions is theo retically sufficient to generate about 23 energy rich plosphate bonds Consequenth clie efficiency of the enzime sistem in <stro is approtimatek 11/23 or almost 60 percent (3) ·84 Renal Function Of particular importance to the present discussion is the finding that 2 4-dıntrophenol (DNP), in extremely low concentrations, is capable of interrupting the coupling of ondation and phosphoryla tion in such an enzyme system Essentially complete blocking of aerobic phosphorylation is obtained with M/10,000 DNP, while partial inhibition is observed with concentrations as low as M/100,000 Within this range of concentrations, the rate of orygen consumption in the cell free system remains unchanged This should be contrasted with vanous other inhibitors of aerobic phosphorylation (eg, cyanide, azıde arsenite or phlorhızın) which act by interfering with the electron transport system utilized in respiration We were first attracted to the study of DNP by the observ? tions of Clowes and Krahl (4a, b) These investigators noted that DNP exerted a pronounced stimulatory effect on the respiration of fertilized sea urchin eggs, but at the same time arrested cell division Hotchkiss (4c) later made similar observations with suspensions of respiring staphylococci and yeast and noted in addition that the uptake of inorganic phosphate from the medium was blocked Thus, DNP produces an unusual combination of effects in intact cell systems, viz, respiratory stimulation and impairment of inorganic phosphate assimilation A high degree of structural specificity underlies these effects of DNP The 2 and 4 mononitrophenols and even 2 amino-4 nitro phenol are completely inactive The phenolic group appears to be indispensable Various substitutions in the 6 position of ? 4- dınıtrophenol have rather marhed effects, alkyl and certain aromatic substituents enhance the activity of the parent compound, whereas a third nitro group (picric acid) abolishes activity In an attempt to uncover the role of phosphate bond energy in renal processes it seemed reasonable to examine the effects on tubular transport of a series of closely related nitrophenols, some of which had previously been shown to be active and some inactive in uncoupling aerobic phosphorylation The technique devised by Forster (5) by which the transport and accumulation of phenol red in isolated renal tubules of the flounder is observed microscopically, appeared to offer a very suitable test object for such studies Consequently, Dr. Forster and I joined forces at the Mt Desert Island Biological Laboratory during the summer of 1918 and carried out the following studies (6) Long segments of renal tubules are easily teased from the kidneys of freshly caught founder When the tubules are suspended in 3 balanced isotonic saline solution containing dilute phenol red (1 mg ·Biochemical Aspects 85 percent), a definitely detectable concentration of dye within the tubular lumina is usually apparent in 5 minutes The concentration of dye increases during the following 30 to 60 minutes to color intensities corresponding to 100 mg percent or higher The degree of phenol red accumulation was graded 0 to ++++, 0 indicating no accumulation, + a barely detectable concentration, and ++++ an intense red color corresponding to 100 mg percent or more Solutions of the vanous agents to be tested were prepared in the usual suspending medium and, when necessary, the pH was readjusted to that of the control Each agent was tested in concentrations ranging from M/I0 000 to M/160 000 and comparison was made with an inhibitor-free control Only those expenments were considered satisfactory in which a ++++ concentration of dye was observed in the control within 30 to 60 minutes The effects of the various nitrophenols on phenol red transport are summarized in Table I DNP inhibited transport completely at M/10,000, graded effects were obtained between M/20,000 and CONCENTRATION X 10 CONTOOND TESTED 10 OS 025 0125 0.0625 7 mitrophenol 4 natrophenol 3-amino-4 nitrophenol 2,4-daaminophenol 4,4-dichlorophenol 1,4-dinicroanisole 1.4 A-Linstrophenol 3,4-dimitrophenol 2,4-dinitro-6-methylphenol 3,4-dinitro-6-phenylphenol 1,6-diestro-r-chlorophenol 3,6-dichloro-4 nutropenol ++++ ++++ +++ +++ ++++ +++ Achny hralod trossport ++++ ++++ ++++ ++++ ++++ ++ ++++ +++ ++++ ++++ ++++ o + ++ +++ ++ +++ o ++ ++++ +++ + +++ o + + ++ +++ ++ TABLE I Efect of various substituted plunols on phenol red transport in Lsolated renal tubules of the founder (Am J Physiol 161 16- 1950). M1/80,000, lule no inlubition was apparent at W/160,000 This gradation of effects was found to be remurkably reproducible It is of interest that the degree of inhibition obsen ed at each level of DNP corresponded closels to that previousl obtuned with the aerobic phosphorylation system of mbbit kidney In addition the inlubiton activities of the other mtrophenols of the senes paralleled their effectiveness as inhibitors of acrobic phosphorylation with ·86 Renal Function one outstanding exception Trinitrophenol, or picric acid, which is inactive in the phosphorylation system, proved to be a very potent inhibitor of phenol red transport Picric acid may be recognized on chemical grounds to be an exceptional member of this series in another respect Substitution of a third nitro group in the 6 position greatly increases the dissociation of the phenol (pKa of 038 as compared with 40 for DNP) Therefore, it seemed possible that picrate bloched phenol red transport in some manner other than by interfering with aerobic phosphorylation It was suggested that picrate might differ from the other active mitrophenols by irre versibly inactivating some component of the transport system However, such is not the case When phenol red transport has been completely blocked by either DNP or picrate for as long as 20 minutes, transfer of the tissue to an inhibitor free solution results in a prompt uptake of dye It then occurred to us that the action of picrate might be differentiated from that of the other active nitrophenols by an examination of the respiratory effects of the various agents These studies were performed in the Warburg apparatus with a crude mince of flounder kidney suspended in the usual salıne medium Each agent was examined at a concentration which had previously been found to block completely phenol red transport, or in the case of the inactive agents at M/10,000 The addition of either phlorhızın, arsenite or sodium azide markedly depressed the rate of respiration On the other hand, DNP increased respiration to 75 percent above the control value Thus the characteristic stimulatory effect of DNP on fertilized sea urchin eggs, staphylococci and yeast was also obtained with fish kidney Examination of the 12 nitrophenols yielded the following results The 6 compounds inactre as inhibitors of either phosphorylation or phenol red trunsport had no significant effect on respiration the 5 compounds active in both systems stimulated respiration 39 to 75 percent Pıcrate, the previously noted exception had no effect on respiration One may reasonably conclude that the action of picrate on phenol red transport differs from that of the other active nitrophenols and is probably not primarily attributable to an interference with essential phosphoryla tion reactions It is obviously impossible, on the basis of these preliminary observations, to assign a definite role to aerobic phosphorylation in the overall mechanism of tubular transport However, the generally excellent correlation between the results obtained with a mammalian kidney enzyme system and with the isolated renal tubules of the ·Biochemical Aspects 87 founder suggests that phosphate bond energy is utilized in the cellular transport of phenol red Encouraged by these results, Dr Gilbert Vudge and I then examined the effects of DNP on various tubular transport mechanisms in the dog by the clearance technique (7) In each experiment three control period intravenous dose of DNP The study was continued penods This dose of DNP always provokes some panting and a temperature elevation of about 1° C, but can be administered repeatedly at two week intervals over a period of months without causing weight loss or evidence of renal damige A typical experi ment in which the maximal rate of Pal transfer (Tmexii) was measured is presented in Table II Administration of DNP resulted DAMNOE SYURATZ TER CE V DECTAL TEXT PLARLA DWT P UY F T MA AM/L. 0 24 74-49 40-63 64-78 35 3 37 7 37 3 "C 38 S 30 O 39 0 10 8 128 12 0 24 9 29 3 O 64 46 0 57 8 o 96 $6 S 077 448 Dintitrophenol 10 mg/kg 586 o 68 483 59 7 o 76 49 7 65 4 52 5 48 76-97 97-120 120-140 331 34 6 38 6 S3 29 7 39 3 39 5 JO 4 36 14 O 78 45 TABLE I Effect of a single intras cno is injection of d n trophenol on Tmran Dog B w 120 kg Glomerular filtration rate (Cr) was measured by creatinine clearance Tubulur transport (T) represents the dfference between the amount ex steted (U1) and the amount filtered (F) PAII was infused at a rate of 38 mg/min (Am Physiol 161 173 1950) in a fairly prompt depression of the Tm to approximately 40 percent of the control value The plasma level of DWP as measured spectrophotometrically by a specific method fell from V/4000 to 11/5000 dunng the penod of obsers ation Thus only partial impairment of PAII transport is apprent at plasma les els considerably above those required to block completels phenol red transport in the fish tubules Iloncier dialysis studies reveal that approximatek 90 percent of the plasma DVP is protein bound Consequenth the level of D\Pın plasmı water is actually about 1/40 000 1 concentration which exerts only partnl effects on the two other sistems previously mentioned Very similar results were obtained in number of experiments Studies performed at low plasma levels of Pull such as arc uscd in estimating renal plasma flow showed a similar degree of transport plubition ·86 Renal Function one outstanding exception Trinitrophenol, or picric acid, which is inactive in the phosphorylation system, proved to be a very potent inhibitor of phenol red transport Picric acid may be recognized on chemical grounds to be an exceptional member of this series in another respect Substitution of a third nitro group in the 6 position greatly increases the dissociation of the phenol (pKa of 038 as compared with 40 for DNP) Therefore, it seemed possible that picrate blocked phenol red transport in some minner other than by interfering with aerobic phosphorylation It was suggested that picrate might differ from the other active nitrophenols by irte- Versibly inactivating some component of the transport system However, such is not the case when phenol red transport has been completely blocked by either DNP or picrate for as long as 20 minutes, transfer of the tissue to an inhibitor free solution results in a prompt uptake of dye It then occurred to us that the action of picrate might be def ferentiated from that of the other active nitrophenols by an examination of the respiratory effects of the various agents These studies were performed in the Warburg apparatus with a crude mince of flounder kidney suspended in the usual saline medium Each agent was examined at a concentration which had previously been found to block completely phenol red transport, or in the case of the inactive agents at M/10 000 The addition of either phlorhızın, arsenite or sodium azide markedly depressed the rate of respiration On the other hand, DNP increased respiration to 75 percent above the control value Thus the characteristic stimulatory effect of DNP on fertilized sea urchin eggs staphylococci, and yeast was also obtained with fish kidney Examination of the 12 nitrophenols yielded the following results The 6 compounds inactie as inhibitors of either phosphorylation or phenol red transport had no significant effect on respiration the 5 compounds active in both systems stimulated respiration 39 to 75 percent Pocrate, the previously noted exception, had no effect on respiration One may reasonably conclude that the action of picrate on phenol red transport differs from that of the other active nitrophenols and is probably not primarily attributable to an interference with essential phosphoryla- It is obviously impossible, on the basis of these preliminary observations, to assign a definite role to aerobic phosphorylation in the overall mechanism of tubular transport However, the generally ercellent correlation between the results obtained with a mammalian kidney enzyme system and with the isolated renal tubules of the tion reactions ·Biochemical Aspects 87 flounder suggests that phosphate bond energy is utilized in the cellular transport of phenol red Encouraged by these results Dr Gilbert Mudge and I then examined the effects of DNP on various tubular transport mechanisms in the dog by the clearance technique (7) In each experi- 1 periods This dose of DNP always provokes some panting and a temperature elevation of about 1° C, but can be administered repeatedly at two week intervals over a period of months without causing weight loss or evidence of renal damage A typical experi ment in which the maximal rate of PAH transfer (Tmpau) was measured is presented in Table II Administration of DNP resulted PAINOKITURATI TIME C Y LCTAL TEMP PLASNA DNP P UY F т m/s 24 5 24 9 153 35 3 37 7 37 3 231 °C 38 5 39 O 39 O IO & 12 8 I20 o 24 24- 49 49-63 64- 71 76-97 97-120 120-140 532 o 64 46 O S7 8 96 56 5 o 77 44 8 2 4 Danstrophenol to mg/kg 10 58 6 o 68 48 3 $9 7 076 49 7 65 4 o 98 37 5 23 3 4 8 O 16 33 34 6 38 6 29 7 39 3 39 $ 394 34 1 45 O 25 TABLE IL Effect of a single intravenous injection of dintrophenol on Tmpau Dog B wt_120 kg Glomerular bitration rate (Ce) was measured by creatinine clearance Tubular transport (T) represents the difference between the amount er creted (UV) and the amount filtered (F) PAH was infused at a rate of 36 mg/min (Am J Phynol 161 173 1950) in a fairly prompt depression of the Tm to approximately 40 percent of the control value The plasma level of DNP, as measured spectrophotometrically by a specific method fell from M/4000 to 1/5000 dunng the period of observation Thus only partial impairment of PAH transport is apparent at plasma levels considerably above those required to block completely pheno) red transport in the fish tubules However dialysis studies reveal that approximately 90 percent of the plasma DNP is protein bound Consequently, the lesel of DNP in plasma water is actually about M/40 000 a concentration which exerts only partial effects on the tuo other systems previously mentioned Very similar results were obtained in a number of experiments Studies performed at low plasma levels of PAH, such as are used in estimating renal plasma flow showed a similar degree of transport inhibition ·86 Renal Function one outstanding exception Trinitrophenol or picric acid which is inactive in the phosphorylation system proved to be a very potent inhibitor of phenol red transport Picric acid may be recognized on chemical grounds to be an exceptional member of this series in another respect Substitution of a third nitro group in the 6 position greatly increases the dissociation of the phenol (pha of 038 as compared with 40 for DNP) Therefore it seemed possible that picrate blocked phenol red transport in some manner other than by interfering with aerobic phosphorylation It was suggested that picrate might differ from the other actre mitrophenols by irre versibly inactivating some component of the transport system However such is not the case When phenol red transport has been completely blocked by either DNP or picrate for as long as 20 minutes transfer of the tissue to an inhibitor free solution results in i prompt uptake of dye It then occurred to us that the action of picrate might be dif ferentiated from that of the other active nitrophenols by an examina tion of the respiratory effects of the various agents These studies were performed in the Warburg apparatus with a crude mince of flounder kidney suspended in the usual saline medium Each agent was examined at a concentration which had previously been found to block completely phenol red transport or in the case of the inactive agents at M/10 000 The addition of either phlorhızın arsenite or sodium azide markedly depressed the rate of respiration On the other hand DNP increased respiration to 75 percent above the control value Thus the characteristic stimulatory effect of DNP on fertilized sea urchin eggs staphylococci înd yeast was also obtained with fish kidney Examination of the 12 mitrophenols yielded the following results The 6 compounds inactne as inhibitors of either phosphorylation or phenol red transport had no significant effect on respiration the 5 compounds active in both systems stimulated respiration 39 to 75 percent Picrate the previously noted exception had no effect on respiration One may reasonably con clude that the action of picrate on phenol red transport differs from that of the other active nitrophenols and is probably not primarily attributable to an interference with essential phosphoryla tion reactions It is obviously impossible on the basis of these preliminary observations to assign a definite role to aerobic phosphorylation in the overall mechanism of tubular transport However the generally ercellent correlation between the results obtained with a mammalian lidney enzyme system and with the isolated renal tubules of the ·Biochemical Aspects 87 lounder suggests that phosphate bond energy is utilized in the ellular transport of phenol red Encouraged by these results, Dr Gilbert Mudge and I then ramined the effects of DNP on vanous tubular transport mecha. disms in the dog by the clearance technique (7) In each experiperiods This dose of DNP always provokes some panting and a temperature elevation of about 1° C, but can be administered repeatedly at two week intervals over a period of months without ausing weight loss or evidence of renal damage A typical experiment in which the maximal rate of PAH transfer (Tmpat) was measured is presented in Table II Administration of DNP resulted DASAXDELTOLATE TIME C, V PICTAL TERE MLAMA PNP P UV F T . &#1 24 5 24 9 5 3 10 8 128 377 37 3 12 O 28 3 8 49 0 26 O 24 21 "C M/ 0-24 53 2 46 35 3 38 3 14- 49 57 8 O 06 23 39 0 1963 56 S 0 77 44 8 39 0 54-71 2,4 Dinitrophenol 10 mg/kg 76-97 586 68 483 33 + 39 3 07-120 597 o 76 49 7 34 6 29 7 39 $ 20-140 654 098 sas 38 6 394 TABLE 11 Effect of a single intravenous injection of dimtrophenol on TMAX Dog B wt 120 kg Clomerular filtration rate (CF) was measured by creatinine clearance Tubular transport (T) represents the difference between the amount et Creted (UV) and the amount filtered (F) PANI was infused at a rate of 36 mg/min (Am 1 Phjol 161 173 1950) in a fairly prompt depression of the Tm to approumately 40 percent of the control value The plasma level of DNP, as measured spectro. photometrically by a specific method, fell from M/4000 to M1/5000 during the penod of observation Thus only partial impairment of PAH transport is apparent at plasma levels considerably above those requred to block completely phenol red transport in the fish tubules However, dialysis studies reveal that approximately 90 percent of the plasma DNP is protein bound Consequently, the level of DNP in plasma water is actually about 1/40,000, a concentration which exerts only partial effects on the two other systems previously mentioned Very similar results were obtained in a number of experiments Studies performed at low plasma levels of PAH, such as are used in estimating renal plasma fow, showed a similar degree of transport inhibition ·88 Renal Function Apparently a number of tubular excretory processes can be depressed by DNP administration, since very similar results were obtained with both diodrast and phenol red Studies on certain tubular reabsorptive mechanisms yielded very different and somewhat surprising results Glucose Tm remained unaltered after the usual dose of DNP, nor was there any appreciable effect on the tubular reabsorption of glycine, inorganic phosphate, sodium or potassium We are unable to explain adequately why the inhibitory activity of DNP appears to be limited to tubular excretory mechanisms Certainly one should expect that the impairment of aerobic phosphorylation would result in a significant reduction of the glucose transport capacity However, it should be emphasized that doses of DNP tolerated by the intact animal probably produce only a partial depression of aerobic phosphorylation We may be dealing simply with a quantitative rather than a qualitative difference The energy required to support either type of transport at a maximal rate remains unknown There is the additional possibility that DNP is selectively localized at the peritubular margins of the renal cells and, consequently exerts its most striking effects on the transport processes which are initiated at that site In this connection I can add only that limited analyses of renal cortex have failed to reveal any significant concentration of the drug Finally, it is quite possible that phosphate bond energy is utilized in quite a different manner in PAH and diodrast transport as compared with that of glucose Since there is little reason for believing that either of the former compounds undergoes phosphorylation during transport, it appears much more likely that it is one of the cellular components of the secretory mechanism which must be ‘primed through interaction with an energy rich phosphate Certain of the studies which follow support this idea Throughout the course of our studies we felt the need for relatively simple in vitro techniques to facilitate biochemical studies on transport, techniques which would be more readily available in our city 1 mammali early in the accumulation of glutamate by brain slices (0) Vue vi members of our group Dr Richard Cross, soon found that this technique could be readily adapted to serse our purpose (9) Thin shces (03 mm) of rabbit kidney corter are prepared as for respiration studies Approvmately 300 mg of tissues are suspended in a Warburg vessel in 27 ml of a suitable isotonic saline medium mnth ܐܐܘ ܙ. M ·Biochemical Aspects 89 containing dilute PAH The gas space is slled with orygen and the vessel is shaken i a 250 water bath for the desired time Oxygen consumption is measured and recorded as a 00, (cmm of O per milligram of wet wt of tissue) At the termination of the experiment the slices are quickly removed blotted on filter paper weighed and extracted with 3% trichloracetic acid a filtrate of the medium as also prepared for estimation of PAH Figure 24 shows the results obtained with varying periods of time It is evident that the concentration of PAH in the kidney slices rises rapidly at the expense of that in the medium To express the gradient established we have em ployed simply the concentration ratio slice/medium (SA) If 06 05 SLICE TW 809/ 04 23 TIME 0 15 30 60 90 120 SLICE / MEDIUM 10 51 87 13.5 174 202 02 PAH CONCENTRATION 011 MEDIUM 90 120 30 60 TIME IN MINUTES FIGURE 24 ·92 Renal Function this group of inhibitors Carinamide, examined because of its known capacity to inhibit a number of tubular excretory processes, was effective at M/5000 Two representative substances, diodrast and penicillin, which are excreted in part by the renal tubule, blocked PAH accumulation at concentrations of M/1000 and M/250 respectively Inasmuch as the last three compounds have no appreciable effect on respiration, it seems probable that they compete with PAH for the transport system Incidentally, I was interested in Dr Binhley's earlier statement that penıcıllın is much more effective than carinamide as an inhibitor of glutaminase We have found quite the reverse, carinamide is approximately 20 times as active as penicillin G The slice technique, therefore, has yielded results which are in good agreement with those obtained by clearance methods Another series of observations provides some evidence of the usefulness of this experimental procedure Since acetate had been shown to stimulate PAH accumulation in a striking and consistent manner, Dr Mudge examined the effect of acetate infusions on Tmpar in the dog (10) Table IV presents a typical experiment in which the TIKE ܝ M -46 -44 39 D-AMONOHTYPURATE Tm C AVERAGE P UV CyXP Tm CONTIOL //MIN ME NaClinfusion started, 140 mEq/1 at 10 ml/min i v 3gm creatinine and 3 gm PAH in 7, ml water i v Sustaining infusion started 75 mg creatinine and 33 mg PAH per ml in isotonic NaClat 2 ml/min 69 3 52 47 I 46 1 37 7 13 4 70 2 S5 44 o 44 3 30 9 96 79 41 1 46 4 31 4 IS O TOS NaCl infusion replaced with Na acetate ipfusion 140 mEq/l at 10 ml/minute 72 3 8 40 5 29 4 17 O 111 71 2 6o 36 5 45 3 200 197 138 71 3 47 33 8 464 24 I 22 3 161 67 9 63 34 1 46 3 23 1 23 3 167 96 76 5 0-10 10-21 21-31 31 31-41 41-5 55-75 75-95 46 4 TABLE IV Effect of sodium acetate infusion on TAYAR Dog S wt 170 kg Experiment demonstrates an increase in Tmp without significant change in the filtration rate (CF) or urine volume (V) erum bicarbonate rose from 22 1 mEq/1 in period 3 to 282 m period 7 (An Physiol, 161, 191, 1950) infusion of 82 PM of sodium acetate per kg per min resulted in elevation of the Tmpan to 67 percent above the average control values The increase in Tm occurred independently of any significant change in either the glomerular filtration rate or urine volume In 9 other experiments in which the rate of acetate infusion ranged ·Biochemical Aspects 93 from 66 to 210 xM/kg/min Tmrse was increased 44 to 85 per cent Lactate infusions produced a similar increase On the other hand succinate or fumarate administered in the same manner de pressed the Tm 20 to 47 percent below the control values confirm ing the results with the slice technique Studies of the plasma elec trolytes during admmistration of the various substrates revealed that acetate is metabolized at a very rapid rate whereas significant amounts of lactate succinate and fumarate accumlate in the plasma In view of the possibility that acetate might be effective because of its alkalınızıng effect additional studies were performed with sodium bicarbonate infusions in one of these there was a slight depression of Tmpan in two others a slight stimulation It was quite clear from these experiments that bicarbonate administration did not produce effects quantitatively similar to those of acetate despite a comparable degree of alkalosis It is also improbable that the increase in Tmpau induced by acetate is due to the opening up of previously inactive nephrons since acetate had no demon strable effect on the reabsorptive I'm of glucose These last observations serve two functions First they establish the usefulness of the slice technique in exploratory studies on the biochemical mechanisms involved in renal transport Second they focus our attention more sharply on the possible importance of acetate as a rate limiting cellular component of the PAH transport system The very fact that there are maumal rates of tubular transport implies that certain cellular components of the system are available in limited quantities Acetate is not an unlikely candi date for such a role Although isotope dilution studies have shown that the daily turnover of acetate is relatively tremendous its concentration in cells and body fluds at any given time is vanish ingly small In addition the biochemist has become increasingly aware in recent years of the multitude of biological reactions in which acetate participates The analysis of certain of these reactions such as the acetylation of foreign amines or choline and the forma tion of acetoacetate or citrate reveals that acetate as such is not involved but a more reactive C compound derived from acetate or its precursors through interaction with energy nch phosphates Though the nature of this active acetate is not yet known it is quite certainly not acetyl phosphate in mammalian systems Nor can we describe the manner in which acetate enters into the re actions of PAH transport All as aulable evidence points away from acetylation of the p amino group as being an important reaction If active acetate does play an important role in this ·94 Renal Function it must be regarded as a potential donor of either acetyl or phos phate groups You must be quite aware at this point that the studies presented do not provide a very complete description of any one of the active transport mechanisms We are encouraged however by the fact that there has been such good agreement between the observations obtained with 1 cell free enzyme system with isolated tubules and kidney slices and with the intact animal By approaching the problems of transport at each of these levels of cellular organization we feel that it is reasonable to anticipate further progress in un covering certain of the biochemical reactions involved REFERENCES (1) GREEN DE LOOMIS W F and AUERBACH V H Studies on the cyclophorase system I The complete oxidation of pyruvic acid to carbon dioxide and water | Biol Chem 172, 389 (1948) (2) SCHNEIDER, WC Intracellular Distribution of Enzymes III The oxidation of ocatanoic acid by cat liver fractions | Biol Chem 176, 259 (1948) (3) Cross R J Taggart ſ V Covo G A and GREEN DE Studies on the cyclophorase system Vi The coupling of oxida tron and phosphorylation Biol Chem 177, 653 (1919) (4) a Al mata b KRAHL ME and Clowes GH A Studies on cell meta bolism and cell division II Stimulation of cellular oxidation and reversible inhibition of cell divis on by dihalo and tri halophenols Gen Physiol 20 173 (1936) C HOTCHKISS Å D Gramıcıdın Tyrocidin and Tyrothricin Adrances in Enzymology 4 153 (1944) (5) FORSTER R P Use of thin kidney slices and isolated renal tubules for direct study of cellular transport kinetics Science 108 65 (1948) (6) TAGGART J V and TORSTER RP Renal tubular transport Effect of 2 4 denitrophenol and related compounds on phenol red transport in the isolated tubules of the flounder Am ) Physiol 161, 167 (1950) (7) MUDGE G H and Taggart J V Effect of 2 4 d a trophenol on renal transport mechanisms in the dog Am | Physsoſ 161, 173 (1930) (8) STERN J R EGGLESTON L V HEMS Rand Kress H A Accumulation of glutamic acid ja isolated brain tissue Brochem }, 44, 410 (1949) ·Biochemical Aspects 95 (9) CROSS R J and TAGGART J V Renal tubular transport Ac cumulat on of p am nohippurate by rabbt kidney sl ces Am Physiol 161 181 (1950) (10) MUDGE GH and TAGGART J V Effect of acetate on the renal excret on of p-aminohippurate in the dog Am J Physiol 161 191 (1950) DISCUSSION Pitts There is a point upon which I would like your reaction In connection with the general problem of competitive inhibition is inhibition on the basis of competition for some cell component present in limited amounts or is there some limitation of energy available to the cell? Do your results have any direct beanng on this question? Taggart The inhibitors which we have studied thus far would appear to fall into three categories First there are those which interfere with one or another step in electron transport phlorhızın arsenite azıde and cyanide are representative By restricting the oxidative activities of the cell they limit the amount of energy which can be liberated Second the active nitrophenols permit oxidative reactions to proceed at an accelerated pace but they paralyze the mechanism whereby the energy liberated diverted into useful work Lastly there are such compounds as diodrast PAH phenol red and penicillin which appear to compete with one another for some cellular component of the transport system Since we know so little about the energy requirements of the various transport systems it is indeed difficult to say whether the available energy ever repre sents the limiting factor Heller By what mechanism is dinitrophenol excreted by the ladney? Taggart We have done a limited number of clearance studies with dinitrophenol It is cleared at a rate of considerably less than 1cc per minute Taking into account the protein binding of plısmı DNP the small amount which is filtered must be fairly completely reabsorbed There is little reason to believe that DNP competes with PAH for the excretory mechanism Warren In your studies with dintrophenols did you find in creased orygen consumption? Taggart We have never measured oxygen consumption in the intact animal but studies by Tainter have shown that it is appre cably increased in dogs following the administration of doses such as we have employed We have also observed as did Tainter that ·96 Renal Function venous blood shows a lower oxygen content after DNP administra. tion Incidentally, in one experiment in which the nght renal vein was catheterized the renal venous blood also appeared less saturated than usual In that experiment, DNP lowered the clearance of PAH at low plasma levels approximately 50 percent, but the renal plasma flow calculated by the Fick principle had decreased only 10 percent In short, the effect of DNP was primanly on the extraction of PAH rather than renal hemodynamics Leiter Do you know whether under the conditions of the renal clearances there is actually an increase in oxygen consumption? Taggart I do not know of any data on renal oxygen consumption during active tubular transport of a foreign substance Binkley I find a little bit of concern over this proposed mecha. nism of action of dinitrophenol I was wondenng, for instance, if anyone has tested the simple idea that there might be an activation of phosphatases Taggart I believe that such studies have been done in Con's laboratory It is my understanding that no effect on the generation of ATP was observed during anaerobic glycolysis In the present studies we would be primanly concerned with the possible activation of adenosinetriphosphatase by DNP We have found that DNP does not have this effect Binkley We find that the small acıds are activators of glutaminase We find that the larger acids are inhibitors and most, if not all, dicarboxylic acids are inhibitors I don't know what significance may be attached to these findings Taggart We have been hard pressed to find an adequate explanation for the inhibitory actions of so many substrates, especially those of the citric acid cycle All of the latter stimulate respiration and their oxidation should provide additional available energy for transport We have considered the possibility that an acceleration of the cycle depletes the cells of available acetyl groups through the formation of tricarboxylic acids, but that is not a very attractive idea Moreover, we find it impossible to reverse the effect of a ketoglutarate by the addition of an excess of acetate Of course, the rate at which acetate diffuses into the cell may be a limiting factor in this situation Sclkurt Since one can increase PAH Tm with acetate or depress it with dinitrophenol, have you ever tried using both in a single experiment? Taggart Acetate cannot completely reverse the effect of dimtro- ·Biochemical Aspects 97 administers dinitrophenol the final rate of transport is considerably below the control values Pitts Suppose you were to give p aminohippurate and then depress that Tm with penicillin and then raise it again with acetate what have you done with a concept of Tm and the factor which causes limitation? Taggart Of course we too wonder just was Tm means In the past we have regarded any particular Tm as a sort of fingerprint for each individual Is it not probable that the relative constancy of a Tm as usually measured simply reflects a fairly constant set of experimental conditions? Pitts It is now surprising to me that Tm should be in an animal or man on successive measurements as nearly constant as it is Mudge We measured Tm in one dog 12 times in 8 months and were impressed by the fact that the value was not very constant Tin varied from 9 to about 16 Pitts We have a series of experiments in which we have been testing in dogs the toxicity of two mercurial compounds One of our measures has been p aminohippurate Tm I dont think Tm varied more than plus or minus lo percent in a large series of measurements Forster Is that the case with glucose Dr Shannon? Shannon That was our experience Taggart It has occurred to us that the different values of Tm obtained from time to time may reflect spontaneous variations in the concentration of acetate in the cells Pitts Would you go so far as to say although it is very low it is maintained under usual conditions at a fairly stable concentration? Taggart I would think so Pitts Constancy of Tm would be a function of the constancy of whatever cellular mechanism maintained cellular acetate Wilhelmi There is another possibility It may Auctuate with the nutntional state of the animal If one considers the possibility that the so called active acetate is a rather complex substance then it might not be so much the concentration of acetate in the tissue at any particular time as the rate of production of acetate together wzh the quantity of material that can form the active acetate com pler so that the two things together might easily determine for any given instance - If this mechanism is specific for the p aminohip Purate transport - just what the Tm would be Taggart That is exactly what we would like to believe - that the ·100 Renal Function Berliner In that connection, it is of some interest that urate reabsorption is depressed by diodrast but not by hippurate (Tal bott, J H Gout Oxford Unversity Press, New York (1943)) Wilhelme With respect to the point that Dr Shannon made a while bach that the mechanism of transport might in some instances be the limiting factor in transport, there are some experiments done long ago by Auchinachie, Macleod and Magee (Auchunachie, DW, Macleod, I J R, and Magee, H E Studies on diffusion through surviving isolated intestine, J Physiol 69, 185 (1930)) on the absorption of glucose from isolated loops of rabbit intestine They noticed that at the concentrations of glucose which in the active aerated intestine led to maximal absorption, the absorp tion of sugar after the intestine had been killed with hot water was in fact much greater so that in that instance too the transport mechanism seemed in fact to be a limiting factor in the regular departure of glucose from the lumen of the rabbit loop to the outside fluid There might be another instance comparable to that which Dr Shannon mentioned, in which the mode of the movement through the cell, whatever mechanism is, provides an obligatory pathway which can be filled only to a certain extent In such an instance the rate of energy supply would not necessarily be a limiting factor in the transport A comparable instance of competition arises in the sense that if one administers glucose and galactose simultaneously the total absorption is not surnmated but each sugar is absorbed at a rate rather less than that at which the sugars individually would pass into the blood stream Forster Would you say, Dr Taggart, that the dependence upon oxygen as an energy source is pretty well established for lidney function in contrast, for example, to nerve action or muscle action? Taggart There is certainly little doubt in my mind that acuve tubular transport depends primarily on aerobic processes for its energy supply If anaerobic glycolysis could provide the energy needed dinitrophenol should not have such a pronounced effect on transport In addition, we have already noted that the maintenance of relatively bugh oxygen tension is required for transport in both isolated fish tubules and rabbit kidney slices One wonders whether this is not related to the fact that the kidney is perfused throughout its vascular bed by blood of relatively high orygen content Pitts As A renal physiologist I have always operated on an as sumption which I took finally to be almost law, namely, if a substance is reabsorbed, it is pot secreted, if a substance is secreted, ·Biochemical Aspects 101 it is not reabsorbed In reality this assumption underlies all measure ments of either secretory or absorptive capacity Reasoning teleo logically, zf the body considers it suficiently worth while to get nid of a substance to develop a secretory mechanism to do so why should it develop a mechanism to reabsorb that substance Not too long ago Dr Berliner and his colleagues and Dr Nudge and his colleagues began tapping on what we thought were firm foundations for renal physiology and found them not so solid Just how much of those foundations have been nddled and wormholed I don't know Dr Berliner is to introduce the topic of tubular secretion of potassium and I hope will point out some of the discrepancies in our basic assumptions ·THE TUBULAR SECRETION OF POTASSIUM AND ACID ROBERT BERLINER Department of Medicine College of Physicians and Surgeons, Columbia Unitersity I have been asked to introduce a discussion of the excretion of açıd and potassium by the kidney The matter of the secretion of acid is one about which Dr Pitts is much better qualified to speak and anything I may say will be more or less a summary of his work with which you are all familiar I will, for the most part, confine myself to the excretion of potassium with which we have worked I should like to say at this point that the material for this presentation represents the joint effe of Drs Thomas J Kennedy, Jr, Dr James G Hilton, and myself (2) Although the excretion of acid and the excretion of potassium may not be particularly closely related, certain similarities may warrant their being considered jointly The first similarity is concerned not so much with the mechanisms involved but with our thinking about them for a long time it was assumed, and not unreasonably, that the excretion of both was regulated by filtration and reabsorption In both cases it had been shown that the amount excreted did not exceed the amount filtered under ordinary circumstances The amount of acid excreted could be explained by the filtration of buffers and the reabsorption of the more basic com ponents - bicarbonate and dibasic phosphate, the average daily excretion of potassium is only about 10% of the amount filtered, and therefore easily accounted for by filtration reabsorption However in both cases when conditions were modified so as to produce marhed increases in excretion, it became apparent that the amount filtered was not sufficient to yield the amount ercreted You are all familiar with Dr Pitts' work which showed that the reabsorption of bicarbonate and phosphate was inadequate to explain the titratable acid in the urine under properly chosen erperimental conditions and that hydrogen ions must therefore be secreted We will for the present, omit consideration of the subsequent proposal of Menaker (7) that reabsorption of carbonate might account for the acıdıfication of the urine since this would make available as hydrogen ion, the hydrogen of bicarbonate I personally don't think this is a very likely mechanism but perhaps someone would like to bring 102 ·Tubular Secretion of Potassium and Acid 103 it up in the subsequent discussion Turning to potassium we find a similar situation The fact that potassium might be secreted was suggested by isolated observations of McCance (6) and of Leith Osterberg and hing (4) which were more or less completely over looked by workers in renal physiology including ourselves VicCance observed a potassium clearance of 11 with an inulin clearance of 9 in a patient with severe alkalosis Some question as to the vahdity * Ու ut 6 bttle data but stated that in a normal subject who developed diarrhea and a drop in filtration rate after an oral dose of Cl they observed a potassium clearance 32% greater than the inulin clear ance Our conclusion that there is a secretory mechanism for potas sium was based on experiments similar to that shown in Table V Time Umno Flow Amin Creol Clearance imin Cheopono win Pleima Potova meyd Finered Polim Errow Polartium nin Mooto Exo/FIN 0 Sa union of can win and th Oru Mato 0:3 N KO I Immi 68 90 3.85 56 56 52 279 350 I 25 112 5 45 63 64 51 313 454 145 128 589 71 71 50 340 513 150 146 545 72 70 53 356 525 147 150 167 Intus on of 75 mM al 100 um bco boneto 500 nt of well 670 184 1118 77 75 43 38 476 150 199 7 87 80 78 4.5 346 510 40 214 640 76 76 45 328 484 148 229 480 82 BO 45 359 487 1.37 TABLE V Thus expenment was performed on a normal female dog which had received by mouth 5 gms of Cl twice a day for one week Well consıder the significance of the oral (CI later The dog recened an infusion of creatinine muln and thiosulfate in Cl throughout the study potassium being infused at a rate of 05 mEq per min lou can ·101 Renal Function see that the potassium was excreted at just about the rate it was infused, which is some 40% or more greater than the product of plasma potassium and creatinine clearance ſhe dose of bicarbonate was given to test a suggestion that creatinine might be reabsorbed from alkaline urnes The comparison with inulin does not suggest that this was the case In fact we have, during a number of studies of potassium excretion in dogs, measured the filtration rate with two or more substances always including creatınıne with uniformly good checks It is clear from this expenment and similar experiments in 13 other dogs that there must be a tubular mechanism for adding potassium to the urine The question now arises as to the role of secretion in the normal excretion of potassium Are we to assume that secretion plays a part only when the excreted potassium exceeds the filtered? Certainly this is the only time at which it can be unequivocally demonstrated Here the situation again resembles that for acidfication of the urine - under all normal circumstances bicarbonate reabsorption is adequate to explain the excreted titratable acıd, but it is not safe to assume that there is no hydrogen ion secreted unless it can be unequivocally demonstrated Dr Pitts has in fact, offered evidence for acid secretion even when the urine is highly alkaline The same might be true for potassium secretion There is at present, no way of arriving at a certain conclusion All we can measure is the net effect of opposing reabsorption and secretion, so that quantitative evaluation of either process alone is not feasıble Let us turn to a consideration of some of the factors which may determine the rate at which potassium is excreted You may recall that in Table V shown a short time ago the plasma potassium was only very slightly elevated in the first group of periods and close to the average normal value after the dose of bicarbonate, yet the excretion of potassium was at a very high level – say some ten times as high as if this dog had, with the same plasma potassium, been receiving sodium chloride rather than potassium chloride Shannon Do you have the sodium concentrations, Dr Berliner? Berliner I did not have room in this Table for all the data but the plasma sodiums were well within the norma) range which, in the dog is a little higher than in man, 140 to 145 mEq per lter This will yllustrate, then, one of the most striking features of potassium excretion -a iery marled dissociation of the rate of excretion from the plasma concentration, or perhaps more important from the standpoint of our usual thinking in renal physiology, a dissociation of the rate of excretion from the filtered load This, and ·Tubular Secretion of Potassium and Acid 105 an additional point, may be further illustrated by the data plotted in Figure 25 This represents a number of experiments in the same dog in each of which potassium was infused, but in combination with different anions Before every experiment the dog had received 175 150 1ood 125 900 o 800 100 ht 700 EXCRETED K+ weg /min 600 500 SALT INFUSED • KCI OK,S,', x K, Fe(CN). 400 300 300 400 500 700 800 900 600 FILTERED K peqmn FIGURE 25 oral KCl for at least one week You can see that at any filtered load there may be a wide range of amounts excreted a part of the variation, but by no means all, may be explained by the differences ·106 Renal Function obtained with different anions The highest ratios of excreted to Altered - and the greatest absolute excess of excreted over filtered - are regularly obtained by the infusion of potassium thiosulfate, the lowest with the chloride, while ferrocyanide generally gives intermediate values although the difference between thiosulfate and ferrocyanide is not so well shown here The explanation of this effect of the anion lies, we believe, in the demand they create for cation excretion by the obligatory excretion of anion Chloride can be almost completely reabsorbed by the tubules so that the extent to which it holds cation in the tubular fluud is subject to variation Ferrocyande, on the other hand, is not reabsorbed so that an amount of cation at least equal to the filtered and actually equal to the amount of ferrocyanide infused must be excreted Thiosulfate is excreted without reabsorption, but urines obtained dunng the infusion of thiosulfate contain about as much sulfate as thiosulfate, and the required cation excretion is greater than the amount infused We have found incidentally, that the infusion of p aminohippurate facilitates the demonstration of K secretion in man We may say, then, that the rate of excretion of potassium is at least partly determined by the rate of excretion of anion Another factor which accounts for part of the variability in the relationship of excreted to filtered was alluded to when it was men. tioned that the experiments were preceded by a week of oral KCI This is the factor of tolerance, the results of which are illustrated by Figure 26 This represents two exactly similar experiments on the same dog The first of these was the initial experiment on the dog, the second was preceded by the oral administration of 4 gms of KCl twice a day for two weeks In each KCI was infused at a rate of 500 Eq per minute, one hour allowed for equilibration and eight 20 minute clearance periods obtained in the first experiment, potassium excretion lags, falling well below the amount infused during the greater part of the experiment Consequently the plasma potassium becomes moderately elevated and the amount excreted remains well below the amount filtered When tolerance has been developed, the rate of excretion has, within the hour's equilibration), become equal to the rate of infusion The plasma potassium is considerably lower than in the previous study and the excreted exceeds the filtered throughout Bradley How long does it take for tolerance to develop? Berliner I don't know Five days is plenty, but we have not really explored it to find out We were at the time not particularly inter.

·Tubular Secrction of Potassium and Acid 107 5001 400 w soon 20 2001 70 PASama 1001 60 K O ECHETED X FLTERED K • PLASMA mer PERIOD so 1 2 $ EXP'T 3 6 7 2 I I FIGURE 26 ested in the tolerance except so far as it got us where we wanted to go The nature of this tolerance has not been clanfied It is not Attributable to a continuously high potassium excretion The rate of potassium excretion before a dose is administered is not greater in the tolerant animal than in the dog which has previously received no potassium We have tried, without success, to reproduce the tolerance by the administration of desoxycorticosterone Given in large doses for a short period before an experiment the DOCA was without demonstrable effect Given over longer periods such potassium depletion was produced that the administered potassium was almost quantitatively soaked up and very little was excreted When the K excreted in excess of intale in response to DOCA was readministered, the results were inconclusie During the infusion of potassium salts, large doses of desorycorticosterone acetate in oil intramuscularly, or in propylene glycol intravenously, or large doses of desor corticosterone glucoside intravenously had no effect on potassium ercretion These findings are in accord with those of Thatcher and Radike (12) who first descnbed the phenomenon of tolerance to K in rats and found that they were unable to reproduce the tolerance with DOCA While there remains no question of the fact that adrenal steroids affect potassium etcete- ·108 Renal Function the effect is not clear, nor is there any clear relationship between the adrenals and the development of tolerance As to the nature of the factors which determine potassium excretion and their quantitative relationships we can draw no final conclusions It seems likely that potassium excretion is determined at least partly by intracellular potassium concentration and possibly by the renal cellular potassium concentration Determination of these concentrations does not at present seem feasible I believe it is Dr Mudge's interpretation, that the evident secretion of potassium in their experiments with forced osmotic diuresis with urea, is attributable to the increased intracellular potassium concentration attendant upon cellular dehydration Leaving these most important questions more or less unsettled, another aspect of the mechanism of excretion warrants consideration You will recall that Dr Pitts suggested that the acidification of the urine is accomplished by an exchange of hydrogen ions for sodium ions The reciprocal relationship between sodium and potassium excretion under certain circumstances suggested that a similar process might be involved in potassium secretion If a urine could be obtained which contained essentially one anion which could not be secreted and if this urine contained potassium in excess of that filtered, then the potassium would have to be present in place of Bool 600 400 EXCRETION 200 IN peg /mn SECRETED MCO, 200 PO. 400 FIGURE 27 ·Tubular Secretion of Potassium and Acid 109 some filtered cation It seemed desirable to use an anion which is excreted at the level of glomerular filtration since there would then be some assurance that this anion was not secreted or reabsorbed For this purpose ferrocyanide was selected Figure 27 will illustrate one of a number of experiments in which potassium ferrocyanide was infused After an hour and a half a series of 20 min clearance periods were obtained The excretion of + Na+ NH + ferro cyanide, Cl- bicarbonate so, and phosphate were measured as well as the creatinine clearance and plasma potassium In plotting this figure the excreted potassium has been divided so that the amount filtered is above horizontal kne the minimum amount secreted below On the anion side the ferrocyanide is plotted above the line since there is far evidence that it cannot be secreted The other anuons are plotted below although at least for sulfate and phosphate there is fairly good evidence that they are likewise not secreted It is worthy of note that the sum of the measured canons checks fairly closely with the total of measured anions the anions being in slight ercess possibly partly accounted for by Ca and Mlg which were not measured in any case it seems unlikely that there is any large amount of some aruon which has been overlooked You can see that throughout the experiment the secreted potassium s far in excess of the total of anions which for present purposes e may concede might have been secreted with it In other words any anion has been secreted with the potassium it is not appearing the fully elaborated urine Three possible mechanisms which might account for these findings are presented in Figure 28 Those are analogous to those suggested for the secretion of acid The first represents secretion of the potassium salt of some acid such as hydrochloric with subsequent reabsorption of the anon as the sodium salt If this were the case one might expect to find a con sistent increase in the excretion of the anion The second represents secretion of the bicarbonate with subsequent reabsorption of sodium bicarbonate by the mechanism suggested by Ds Pitts Were this mecharusm operatze one mght anticipate a consistent increase in bicarbonate excretion The last represents a direct erchange of potassium ions for sodium ions It has been well known for a long time (Loeb et al (5)) that in creasing excretion of potassium is usually associated with a tendency for the urine to become alkalme An investigation of this phe nomenon seemed indicated for the light it might throw on the mechanism for the secretion of potassium An attempt was made to establish conditions in which the results obtained would be relate ·110 Renal Function TUBULAR CELL URINE K+A- → K+ A-+Na+ Na+ A- No+ A + K+ O*A*** K+HCO; HO+CO-HCO3+H+ → K+H00; +NG No+HCO:+ H.CO, -CO,+HOSTI NaHCO Kit Na+ Not K+ FIGURE 28 to the administration of potassium and independent of the anion administered with it to accomplish this experiments were done which started with the infusion of the sodium salt of one of several anions Sırılar results were obtained with the ferrocyanide the thiosulfate and the phosphate The expenment illustrated in Figure 29 was done with ferrocyanide Sodium ferrocyanide was infused at a rate of 1200 microequivalents a minute After 90 minutes to reach a more or less steady state, clearance studies were begun and the infusion was arranged so that the sodium was progressively replaced by potassium at equivalent concentration keeping the ·Tubular Secretion of Potassium and Acid 111 ferrocyanide constant The change from sodium to potassium was started at the end of the first 20 minute clearance period and was 90% complete two hours later Soon after the infusion of potassium was started there was a sharp rise in chloride excretion and in bicarbonate ercretion as indicated here by the pH These changes were not associated with changes in the amount of these anions iltered since the chlonde filtered remained con tant and the bicar bonate fell progressively They were therefore in some way related to the change in potassium excretion The increase in anion excre tion however was maximal before the excretion of potassium had reached the amount filtered The excreted potassiurn exceeded that filtered by a small margin in the sixth period Vote that the sodium excretion remained at its initial high level until the sixth period and that its fall is associated with stabilization of the excretion of chloride and bicarbonate 1200 food 800 ELECTROLYTE EXCRETION . Not O K+ weq/ m600 CI хрен 400 80 2001 70 PH 60 - ! 2 3 4 6 PLASMA CO MM./L 7 PERIOD 8 9 JO 50 22 21 FIGURE 29 19 17 16 ·112 Renal Function No conclusive interpretation of these experiments can be made, but we can narrow it down to two alternatives We may start by assuming that reabsorption and secretion cannot proceed simul taneously in the same segment of the tubule, and from what is known of the site of various tubular processes (Richards (11), Walker, et al (13)) we may place the reabsorptive process as most probably in the proximal tubule The secretory process must then be distal Distal secretion of the potassium salts of chloride and bicarbonate seems unlikely especially in view of experiments like that shown in the previous Figure since a still more distal site for reabsorbing the sodium salts would be required The observed findings could be explained by a) a decrease in the proximal reab sorption of potassium and the associated anions b) a diversion of distal reabsorption from reabsorption of sodium with chloride and bicarbonate to exchange of potassium for sodium One further possibility remains, however, concerning the localızation of the secretory process This is the possibility that both reab sorption and secretion are functions of the proumal tubule, secretion representing a reversal of the reabsorptive process In this case, secretion and reabsorption could not occur simultaneously so that the increased chlonde and bicarbonate excretion could be due only to decreased reabsorption It might throw into question the theory of direct cation exchange since it would leave room for reabsorption in the distal tubule of any secreted anion as the sodium salt However if we consider the effect of mercurial diuretics on the excretion of potassium it would seem very unlikely that secretion is simply a reversal of the reabsorptive process I don't want to go into detal now as to the nature of the effects of mercurials on potassium excretion Perhaps Dr Mudge would like to comment on this later I would like to refer to one type of experiment which is pertinent to this question If a dog is given an infusion of some potassium salt, so that a large excretion of potassium 15 obtained with an appreciable excess of excreted over filtered, and a dose of some mercurial diuretic 1$ then administered, there invanably follows a marked drop in potassium excretion which usually reduces the ratio of excreted to filtered to well below unity Such a drop might be due to either decreased secretion or increased reabsorption If the effect of mercurial were on a single alteratzvely reabsorphye and secretory mechanism, a change in the ratio to one but not past st might be anticipated, since reduction to one would represent inhibition of the active process and past one an enhancement of the actre process On this basis, it seems reasonable to conclude that the ·Tubular Secretion of Potassium and Acid 1 reabsorptive and secretory mechanisms are functionally and pre sumably anatomically separate To summarize bnefly this rather involved argument there is reason to believe that two separate tubular mechanisms are con cemed with the ercretion of potassium With the assumption that the reabsorptive mechanism is located in the proumal tubule the secretory process is in all probability a distally located mechanism for exchanging potassium ions The role played by secretion in the excretion of low and moderate amounts of potassium is not clear but high rates of excretion are probably achieved by a combination of diminished reabsorption and increased secretion The factors con trolling the rate of excretion are likewise not clearly understood but it is striking that the amount filtered is a relatively unimportant factor I have rather neglected the subject of the excretion of acid and vould like to return to it briefly before concluding There is some agmentary information concerning the intermediary processes volved which should be brought up for consideration in their ork with the mechanism of urine acıdıfication Dr Pitts and his ociates (10) showed that the acidification could be at least par ly inhibited in vivo by the administration of sulfanilamide They sented this as evidence that carbonic anhydrase played some central role in this process suggesting that the hydrogen ions were made available from carbonic acid formed by the hydration of CO, Davies and others (3) have suggested that the process of acid secretion in the stomach is analogous to that in the kidney and proposed a slightly different role for carbonic anhydrase They suggested that the hydrogen ions are derived from the reduction of aron porphyrins and that the carbonic anhydrase catalyzes the combination of CO with hydroryl ions formed on the re-oudation of the son porphyrin One further observation may be of interest although its significance is not entirely certain we have found that acidification of the urine can be completely abolished in acıdotic dogs with CO s as low as 10 ml per iter and blood pH below 73 by a dose of malleic acid of about 04 ml per llogram Valleic acid you will recall is the cis isomer of fumiric acid. The system affected is not certain since a number of different effects have been ascribed to malleic acid A point of interest however is that mal leate at a concentration of 100 mV per liter applied to the outer surface did not inhibit the secretion of acid by the isolated mucosa of the frogs stomach Be this as it may the most reasonable erplana tion so far offered would seem to be that secretion of ·114 Renal Function attributable to a fixed spatial arrangement of some enzyme or series of enzymes yielding hydrogen ions on reduction and hydroryl ions on ondation that the hydroxyl ions are combined with CO, to form bicarbonate which is returned to the interstitial fluid with the cation for which the acid has been exchanged REFERENCES 1 BERLINER, RW and KENNEDY, T J JR Renal tubular secre tion of potassium in the normal dog Proc Soc Exper Brol & Afed 67, 542 (1948) 2 BERLINER, RW, KENNEDY, T J, Jr, and HILTON, J G Renal mechanisms for the excretion of potassium (In press) 3 DAVIES REHydrochlor acid production by isolated gastric mucosa Biochem 42, 609 (1948) 4 KEITH NM OSTERBERG, À E and KING HE The excre tion of potassium by the normal and diseased kidney, Tr A Am Physictans 55, 129 (1940) 5 LOEB KF ATCHLEY DW, RICHARDS DW, BENEDICT, EM, and DRISCOLL ME On the mechanism of nephrotic edema ] Clin Intestigation 11, 621 (1932) 6 McCANCE RA and WINDOWSON, E M Alkalosis with dis ordered kidney functions, Lancet 2, 247 (1937) 7 MENAKER, W Buffer equilibrium and seabsorption in the pro - 1*174 71948) 8 enal excre 15 (1948) 9 MUDGE G H FOULKS JAMES A Ill, and GILMAN, A Effect of drugs on renal secretion of potassium in the dog Am 1 Physiol (in press) 10 PITTS RF Renal excretion of acid Federation Proc 7, 418 (1948) 11 RICHARDS AN Urine formation in the amphibian kidney, Harvey I ectures 30, 93 (1934 35) 12 THATCHER IS and RadixC AV Tolerance to potassium intoxication in the albino rat Am Physiol 151, 138 (1917) 13 WALKER A M and Bort P A, OLIVER ) and MacDOWELL, MC The collection and analysis of fluid from single nephrons of the mammalian kidney Am | Physiol 134, 362 (1911) 1 TO) DISCUSSION Bradley To what extent do mercurial diuretics affect potassium excretion in the presence of low serum potassium concentrations? Berliner I would say - and this is only a very tough approxima- En 'n NPT it what higher than that, say 50 to 15 uby pri su uw ·Tubular Secretion of Potassium and Acid 115 be much change If it is much higher than that initially, potassium excretion always goes down after the mercurial Bradley How do you interpret that? Berliner The changes after mercurial drureties have been inter preted in two ways The obvious explanation would be a decrease in potassium transport in either direction (Vudge G H et al (8) (9)), reduced reabsorption and reduced secretion We have some data (the results have not been sufficiently reproducible to satisfy us entirely) which suggest that mercurials might increase reab sorption These findings originally gave us the idea that potassium might be secreted we found in several experiments (we were not giving potassium at the time) that following a dose of mercurial the excretion of potassium mght go up or down but then reached some valuc at which it remained constant despite considerable changes in the amount filtered Now that suggested to us that there was some secretory mechanism working at a fairly constant rate and not dependent on the amount filtered This could produce the observed potassium excretion of all of the filtered potassium were reabsorbed sny in place of non reabsorbed sodium so that the potassium coming out in the urine was only that secreted When that hypothesis paid off in our being able to demonstrate that there uas secretion of potassium we thought it must have been ? good adea (Berliner RW and Kennedy TI Ir (1)) I think now that there may be good reasons for questioning it It seems to be a rather extraordinary effect for an agent which usually depresses active processes to increase the reabsorption of potassium In any case there is the problem of why potassium excretion sometimes goes up and sometimes down If potassium secretion is a distal erchange for sodium it would not be unlhely that the rate of secretion might be related to the amount of sodium dehvered to the distal tubules An effect of mercurials on sodrum reabsorption in the proximal tubule might increase the sodium deltered to the distal tubules and consequently increase secretion of potassium Schroeder Does not a mercunal sometimes increase the excretion of magnesium and calcium as well as potassium and sodium? That has been reported Berliner Dr Famsworth reported such changes We have not measured the ercretion of calcium or magnesium Schroeder I think she found chlonde always came out but that the cations might be any one of those four Berliner There is one experiment which might gue conclusive evidence as to whether the effect of mercunal diuretics is diminished ·118 Renal Function Are we truly depressing reabsorption of potassium or are we stimu lating secretion? Berliner Until you have ratios of excreted, filtered over 1, one cannot say We don't know any way of telling whether the change is due to changes in reabsorption or changes in secretion I would be inclined to say both, but that is purely personal prejudice and not based on any logical thesis is that what say, Dr Mudge? you would 1.4 1.2- UREA 10 1.0 CCR Ck/ CK HYPERTONIC NACL 35 .6 ISOTONIC NACL SO WATER 100 30 lo up CREATININE FIGURE 30 ·Tubular Secretion of Potassium and Acid 119 Mludge We have studied this problem with Dr Gilman With the possibility that two processes are occurring simultaneously, as has been indicated one cannot make a conclusive statement about what is happening in the tubules However, by studying a number of vanables we have obtained data which seem best interpreted as showing that the increase in potassium excretion with hypertonic infusions is due to secretion In Figure 30 we have plotted the Ck/Cr ratio against the U/P ratio of creatinine obtained during different types of diuresis The experiments with urea show that ratios, significantly over unity can be obtained without the ad ministration of potassium These animals were hypertonic, we have obtuned similar evidence of secretion with hypertonic sodium bicarbonate infusions, but our highest ratio with sodium chloride is about 08 Berliner Do you consider that an elevated urea makes the animal hypertonic? Mudge Not directly, but in our experiments the serum sodium was always elevated We have not re studied the effects of urea with normal or low blood sodium levels As the figure shows, the results with urea and hypertonic saline are so similar that we have considered them both as representing the effects of hypertonıcıts It should be noted that, at any U/P creatinine ratio (10 for in stance), more than twice as much potassium was excreted with hypertonic infusions than during isotonic diuresis Isotonic saline increased potassium cxcretion to about 20 percent of the filtration 10 ISOTONIC SALINE HYPERTONIC SALINE 75+ CK CCR Sof -100 0 +100 -100 TIME MINUTES HG +10o FIGURE 31 ·120 Renal Function rate water diuresis on the other hand was associated with a slight fall in potassium output In Figure 31 some experiments on the effects of mercurials given during salıne diuresis are shown With isotonic infusions mercury produced httle change in the potassium clear'ınce However when the animals were made hypertonic the potassium clerrınce sose in every case and this was immediately depressed by mercury The experiments are comparable as far as the values for filtration rate serum potassium and total electrolyte excretion rc concerned The concentration of serum sodium and the rate of potassium er cretion were the only differences between the two sets of experi ments Thus there are two points which these experiments establish First hypertonicity increases the potassium clearince which under extreme conditions can exceed the filtration rate and secondly the increased excretion is readily reversed by mercurials We inter pret these as indicating that the potassium excretion of hypertonic dehydration is due to secretion Pitts Would you feel that the secretion of potassium is going on at all times and that the infusion of hypertonic salıne depresses absorption perhaps proximi absorption if Dr Olver will excuse the physiologist from talking about sites without looking Perhaps this reduction of proximal absorption could be an osmotic action Uncovering what is going on all the time Would that at all be your conception? Mudge If we were to say that hypertonic infusions depressed the proximal renbsorption of potassium becouse of the resulting increase in the rate of urine flow we would have great difficulty in explaining two related observations Why isnt there a similar effect from isotonic salıne diuresis when urine flow and total electrolyte excretion are the same? And secondly if the increased potassium excretion were considered as depressed reabsorption then mercury would have to increase the rate of reabsorption For us at least it is difficult to conceive of a transport mechanism whose activity would be enhanced by un agent such as mercity Selkurt What happens to the unne flow when you give mercury? Mudge in four of five expenments it increased in one it did not Shannon What is the total cation excretion that is sodium and potassium before and after mercury Mudge in terms of molecular equivalents the changes in sodium are far greater than those in potassium For example in one of the ·Tubular Secrction of Potassium and Acid 121 experiments with hypertonic saline sodium excretion was increased from 550 to 1400 pEq per minute while potassium fell from about 120 to 60 pEq per minute Berliner Is that associated with a marked increase in chloride excretion? Mfudge Yes chloride excretion increased Berliner We have wondered whether the effects of mercurials are exerted primarily on anion of cation reabsorption - whether it is chlonde reabsorption that is primarily depressed and the in creased sodium ercretion brought about just because some cation has to go along or whether the situation is the reverse Our original hypothesis is a very useful one but Im now not so sure of its valıdıty I would not be prepared to defend either position There are some observations on man for which we dont have detailed studies which may be contnbutory Sometimes when a nercurial diuretie is given to a cardiac or cirrhotic there may be o increase in potassium and chloride ercretion with practically o change in sodium excretion The sodium may go from 1 veq per ry to 2 or 3 while the excretion of potassium may go up an extra o Eq per 24 hours Schroeder is that dependent upon the plasma level of sodium? Berliner Practically all our cardiacs and cirrhotics who are edem atous and retaining flud have low plasma sodiums so it is difficult to say Luetscher Have you observed tubular secretion of potassium in patients with chronic glomerulonephritis? Could not this mechan ism become a very important factor in the elimination of potassium when glomerular filtration is much reduced? Berliner We have studied potassium excretion in a number of patients (I might say that all the work I ve been talking about has been done in dogs ) You can demonstrate potassium secretion in normal man but it is hard to do because the injection of large amounts of potassium intravenously is so painful However if a patient with impaired renal function whether a cardiac hyperten sive cirrhotic or nephritic has even moderate impairment of renal function it is easy to demonstrate potassium secretion Many patients with marked depression of renal function have what appears to be potassium secretion all the time with the qualification that the inulin clearance is equal to the filtration rate under these cir cumstances ·122 Renal Function Wesson Dr Anslow and I have recently done some experiments which confirm the findings of Drs Berliner and Mudge Like Dr Mudge we employed urea diuresis Our experiments bear on his data because where he was working at elevated plasma sodium levels we made considerable effort to keep the plasma sodium at normal or subnormal levels Consequently the occurrence of the excretion of potassium at a greater rate than it was being filtered cannot in these experiments be attributed to a high plasma sodium We felt that since the urea penetrates the entire body fluid space cellular dehydration does not exist The admunstration of urea does not necessanly interfere with a water diuresis While under a urea load you can have a clearance ratio of potassium to creatinine well in excess of 10 at the same time that the animal is water diuresing Another thing which has been mentioned today is the change in urine pH when the rate of potassium excretion is rising We have performed the experiment in reverse When we administered a mercurial diuretic to an animal which had a previous high level of potassium excretion we got the same thing that Dr Mudge did a profound drop of the potassium excretion to very low levels but there was no change in the pH of the urine From an entirely different line some data obtained by Dr M F Levitt in Dr Smith s laboratory bears out the concept of potassium excretion by the use of radioactive isotopes This is a field in which I don t feel qualified to offer criticism so I will just repeat the data Dr Levitt found the same effect which Dr W O Fenn has previously described without particular reference to renal function The procedure was this a single large dose of radioactive potassium was administered the dose was large in terms of activity but small in terms of total potassium in the injection Following the injection the plasma level of radioactivity rose abrup then declined steadily During the hours following the injection Dr Levitt found that the ratio of activity to total potassium coming out in the urine was con siderably in excess of the ratio of activity to total potassium present in the circulating plasma The only explanation thus far advanced to explain this finding is thus the administered radioactive potas sium will be piched up by highly vascular and metabolically active liver and kidney much faster than by other parts of the body. Then while the activity of the liver and kidney are in excess of the rest of the body they will tend to lose their activity to muscle and other tissues If the activity of the potassium in the renal parenchyma es greater than that in the circulating plasma then the only way you ·Tubular Secrction of Potassium and Acid 123 can get the more active potassium into the urine would be for at to come through the cells This we took as substantiating the concept of potassium secretion Leiter Were any similar experiments carried out after increasing he tolerance of the dog to ordinary potassium? Wesson The doses were well below those customanly assumed to have no biological effect Pitts Under those circumstances the actual increase in the rate of excretion of potassium was very small? Wesson It was not detectable Berliner Was a large amount of potassium coming out in the urine? Wesson No The experiments under which the animals might have been potassium loaded with large amounts excreted in the urine, were not done They might be very interesting Berliner Have you any data on specific activity of any tissues in relation to specific activity of the plasma? Wesson Dr Fenn has published a great deal of data on that subject I do not recall his exact figures Tissue analyses were not made in Dr Levitt's experiments Mludge I believe that a similar finding has been reported with phosphate (Friedlander, H D and Wilde, W S Can transfer rate be calculated from urine isotope ratio? Federation Proc, 8, 51 (1949) Wesson I could not understand the data The same experiment which I have described for potassium was done by Dr Levitt with sodium, but no significant differences between plasma and unne were noted Berliner The sodium in extracellular fluid does not differ very much from that of plasma so that there is no store of high activity Wesson Since there is no place to store the sodium it is not real evidence against secretion of sodium Darrow Dr Berliner, you might be creatng an abnormal animal with high extracellular potassium lou might load the animal and get some evidence However, the amount of filtrate is not so large as to give an answer Ordınırıly potassium is not stored We tned to see if we could raise the muscle potassium by giving large amounts of potassium I think we gave water having a concentration of around 400 fLg of potassium per liter and the rats must have been excreting a unne having about 700 \of potassium per lter The muscle composition changed but curiously potassium went down instead of up It went down about 10 percent in the ·124 Renal Function muscle and there was a sught mcrease in muscle sodium That would be us line with what you spoke of as the intracellular com position altering this mechanism This fits in with the increase in potassium excrehon associated with alkalosis or after the animal has been loaded with potassium for several days Mfudge You gave potassium chlonde and the muscle potassium fell? Darrow Yes Berliner Those calculations are based on the chloride space? Darrow The tissue analyses are interpreted in terms of chlonde being largely extracellular You can check the calculation in terms of sodium plus potassium in water but the evidence is that po tassium left the cells Mudge About 10 years ago didnt you report that muscle po tassium rose when potassium was guten? Darrow We get muscle potassium to rise of serum potassium nises if the conditions are such that serum potassium does not risc in other words of the animals reach a steady state under a Joad of potassium muscle potassium will go down to normal or below normal Do you see what I meu? I don t believe we have gotten high muscle potassium except in the presence of high potassium in the serum (Am Physiol 130 747 (1940) - See page 757) Lester Are these animals being dehydrated by the procedure? Darrou ! ni mara natassium chloride Then they refu drated. We were trying to see it we could raise intracellular potassium while leaving the animals in a more or less tolerable state If we got potassium too lugh they decided they would rather Ive on body fluids than tahe the stuff we were giving them Pitts - the means by włuc! tissues? Darrow I dont thunk we know any thing about it as wall su intracellular composibon is concerned I analyzed some lidneys years ago but they gase me such a headache that I put the results in the wastebasket lle have some recent preliminary analyses of ladneys of rats subjected to alkalosis or potassium deficiency There seems to be a considerable change in the ratio of chlonde to sodium and potassium in these rats as compared to normals The changes suggest that there is more sodium in the cells I have a 2. W ·Tubular Secretion of Potassiumn and Acid 125 מע 1 whole row of bottles awaiting analysis when I can get a flame photometer working I beleve we can get some methods permit accurate ani U !! Lurge changes The w wok as though the changes will be large enough to permit them to be interpreted to indicate that larger amounts of sodium and small amounts of potassium are staying in tubular cells I dont know how you would interpret such a finding in terms of absorption. It might mean greater reabsorption of bicarbonate or potassium I dont know what effect the change in cell composition has on enzymes Leiter I don t see why renal reabsorption and secretion of potassium cannot be parts of the same mechamsm dependent on the same transfer system but in dynamic equilbrium as to direction of transport If you depress one process the other might be stimu lated the reabsorptive if you depress the secretory and vice versa Berliner From what you say it seems to me that you are re ferring to two mechanisms this is in agreement with our hypothesis Leiter I am suggesting one mechanism working in both ways nt either end Binkley It comes down to a matter of definition Shannon Dr Binkleys system works two ways Binkley There should be no difficulty in explaining such ob servation on the basis of such a system Olwer May I ask why do you think the separation between the site of the mechanism is so great s from proumal to distal? Why could it not be upper proximal and lower medullary prouimals We know definitely that those tuo parts of the proximal hw some metabolic differences Is there any reason to jump fron proximal to distal? Berliner No Selkurt I have some data on the effect of renal venous obstruc tion in which you might be interested It appears to indicate the operation of a dunt mechanism for reabsorption and secretion of potassium The procedure used is probably famılar to all since it is similar to that used by Blake and his associates (Blake WD Hegna R Reating R B and Hard II P Effect of increased renal venous pressure on renal function Am į Physiol 157,1 (1919)) The clearances of electrolyte by the nght and left ladneys were studied separatels The left renal vein vas occluded in varying ·126 Renal Function degree so as to elevate the venous pressure unilaterally Under these circumstances we may use the clearance of the night kidney as control, thus eliminating the effect of systemic factors which presumably operate equally on both kidneys In Figure 32 the ratio between creatinine clearances of the left to the nght kidney remains close to unity throughout an experiment in which the left renal venous pressure was raised stepwise Two clearance values were obtained prior to imposition of pressure in each stage of pressurization, and finally, on release of pressure The pressure levels are given at the bottom of the figure in centimeters of water Except in the third perod of pressure elevation where the creatınne clearance dropped on an average of 10 percent below the average control figure, the filtration rate remained farly constant NO I7 H ATTEET பாயARTILITED 2.35 280 1 42 WERED.T.11.17 CK UK 1.78 147 RATIQ EXP/CONT CCR ITALIA TITUTO yo YA 090 099 049 093 121 I $1 அபமாயாயை TUIKUT MUURAUN ago 12. W ow und uch os 097 103 092 096 I 15 133 to hirutem HEMU DOO BH Da | CNA 00 OF 30 och I OI 102 097 101 104 100 20- DULUNU 11 UR NE Yo Un od 570 ws 930 100 1 06 098 097 Ditkorn CC LLUPITEL. LUNA PIESE AVP MW 70 74 37.97 1717 27.8 272 19 TIME IN MINUTES 40 10 FIGURE 32 The sodium clearance ratio was well maintained until the second period of elevation where the clearance of sodium dropped to about 80 percent of control Finally during the period of highest pressure elevation, the sodium clearance dropped to about 30 or 40 percent of the control On release of pressure the sodium clearance came back to about 80 percent of control almast immediately The uninary sodium concentration ratio (U\a) remained constant near ·Tubular Secretion of Potassium and Acid 127 unity throughout thus erpenment Note also the very close correla tion between sodium clearance and urine volume the lowest figure it appears that reabsorption of sodium under these con ditions is perfectly isosmotic We have studied this effect in animals Teceiving isotonic as well as slightly hypertonic salıne priming In the study shown here hypertonic saline solution was used Nearly all of Blakes studies were made on animals receiving slightly hyper tonic salne solution though he does mention a couple of expen ments in which he used isotonic solutions He does not give the relative data and one does not know what happened Returning to potassium we find it does not behave like sodium During the first period of pressure elevation the potassium clear ance remained fairly high but began to fall in the second period was lowest in the third period of elevation and returned almost immediately to control upon release of the venous obstruction In most of our experiments the potassium clearance never drops as much as sodium or putting it another way the potassium urinary concentration ratio nses during this procedure from about 10 to is much as 17 We may assume that any potassium reabsorptive nechanism should resemble and behave like that for sodium ince potassium does not follow sodium and since it is somewhat incentrated in the urine it is possible that we are unmasking here secretory mechanism which maintuins a high unnary concentra 'n and excretion of potassium despite increased reabsorption we that of sodium The other possibility of course is that po tassium is not as well reabsorbed as sodium under these circum stances I would welcome comment on these points Blake does not present data in his paper on the behavior of potassium Bradley A similar dispinty in the renal ercretion of sodium and potassium ions appears in man when renal venous pressure is elevated by application of a tight abdominal binder under a pres sure of 80 mm Hg Dr Blake Dr Vudge and I have studied this response in five normal subjects and in seven patients with uncon trolled diabetes insipidus In nearly every subject elevation of renal Venous pressure by abdominal compression resulted in a signifi cant and equal decrement in renal plasma flow and glomerular filtration rate The excretion of water sodium and potassium also decreased Water and sodium output usually fell markedly as in the patient with diabetes isipidus presented in Figure 33 As a nile unne flow decreased less than the sodium excretion so that the sodium concentration of the unne diminished both fell more than filtration and the ratio between output and filtration de- ·130 Ronal Function time relationships Rats can be trained as it were to excrete water faster than normal Obviously that is in some measure a renal phenomenon but we thought it was in part a manifestation of er. trarenal changes - although we didn't know what they were We did not think it had to do with increased adrenal cortical activity If I remember correctly the cytological evidence of Greep and Deane indicated that the administration of potassium will cause detectable adrenal changes interpreted as stimulation of the glomerulosa over a long period of time Even if that be true I think it would not necessanly follow that an adaptation of the renal tubule to handling potassium is due simply and solely to increased adrenal cortical activity Perhaps we can draw an analogy to what happens in a muscular systern If an animal is subjected to severe exercise without any cortical hormone it will go into shock If it is subjected to exercise in the presence of cortical Hormone it will adapt to the exercise and become capable of increased amounts of muscular activity This Inay be associated with an increased adrenal cortical activity but the adaptation occurs in the muscle and the cortical hormones while necessary to support the altered cellular activity, are probably not its direct cause Another example is that seen in Jactation Cortical hormones are necessary for the synthetic pro cesses of milk production and the amount of milk produced is to some degree dependent upon the amount of cortical hormone present Yet the hormones themselves have no lactogenic activity, ie they do not themselves stimulate milk synthesis They only permit other factors to do so I would guess that this same sort of thing might happen as far as the kidney is concerned as it adapts itself to handling specific substances That doesnt mean that the adrena) hormones do not effect specific kidney functions directly I am talking about adaptational phenomena Luetscher I would like to back up Dr Gaunts viewpoint Dr Deming and I have been using adrenalcctomized rats in 1saying desorycorticosterone We thought at first that potassium cxcretion might be a useful end point but the results were so varinble that we returned to the time honored effect on sodium cycretion Lien at doses fifty or a hundred times those necessary to produce de tectable retention of sodium the increase in potassium cxcretion was small and regularly related to dosage Extricts of urinary corticoids from a vanety of patients have produced little or no increase in potassium ercretion in a five hour period I wonder of the effect of DOCA is not primanly a sodum retention with a later displacement of potassium Perhaps our experiments are too ·Tubular Secretion of Potassium and Acid 131 short to see the full effect Patients who recerve an overdosage of DOCA for long periods lose potassium but there is usually an increase in serum sodium concentration or some edema at the same time In studying patients with renal dysfunction due to disease the problems become even more complex In the nephrotic syndrome the retention of an excess of sodium in the body seems to be related both to a reduction in glomerular filtration and to an overactivity of the tubules in reabsorbing sodium even when the glomerular filtration rate is increased Assay of unnary corticoids in the severe forms of nephrosis shows an increased ercretion of sodium retaining steroids At the same time the retention of sodium may produce a change in muscle sodium and potassium content similar to that observed in animals after prolonged overdosage of DOCA This leads to a situation in which one would have to balance the ob servations very carefully in order to distinguish what is primarily a renal action and what is a metabolic or displacement effect in the body cells Heller Speaking about the effect of corticoids on potassium and sodium excretion I should like to mention a senes of experiments which have just been completed (Heller H and Stephenson R P Effect of posterior pituitary extract and its fractions on renal clcc trolyte excretion Mature 165 189 (1950)) When small doses o pitocin or pitressin were injected into rats it was found that both posterior pituitary fractions increased the excretion of potassium after pıtressin sodium ercretion was depressed and after pitocin not significantly altered These results may be of interest as suggesting that renal potassium and sodium ercretion are not invariably inter dependent Shannon What dose? Heller Three milliunits per 100 gm rat injected subcutaneously Similar results in rats were obtained by O Schaumann (Naturf u Mcd in Dcutschland 61 192 (1939/46)) who however used much larger doses (40 milliunits per 100 gms) His findings agree with ours insofar as increases of potassium ercretion were noted after both posterior pituitary fractions and less sodium was eliminated hen the pressor fraction had been injected On the other hand he found that the injection of his dose of orasthın (=ory tocic fraction) resulted in a markedly increased excretion of sodium in the urine Gaunt He used only pitressin and the antidiuretic substance from serum We will have to try pitocin ·134 Renal Function cortical hormones in the control of water and sodium excretion 15 well worth while if for no other reason than that it may give a Ley to experimental preparations which in turn may permit the more critical studies of the kidney at normal water and electrolyte loads It is frequently essential in the initial phases of any investigation to produce wholly abnormal circumstances in order to clearly define the absence or presence of a certain mechanism However, one cin take quite another view in terms of normal economy of the body at a later date For example we have a clear demonstration that the renal tubules both absorb and secrete potassium, still our data do not preclude the possibility that all filtered potassium is reabsorbed in the proximal segment, and potassium that appears in the urine under conditions of normal filtration rate, and normal electrolyte composition appears there as the result of secretory processes of the distal segment In other words control under normal potassium loads is a function of the distal process Gaunt I can only say, both from our own expenence and from that of helping write a recent review on the subject that it is almost impossible to make any definite statement about the effects of cortical hormones on salt and water excretion except in terms of a precisely defined experimental situation It is certainly casy in sorting out experimental literature to see that what happens in the animal is dependent upon various conditions of salt load, water load and - as Dr Shannon suggested - with the balance of steroid hormones present We are accustomed to thinking of DOCA as a sodium retainer There are several reports showing that DOCA can increase sodium excretion of the conditions are right It is well known that cortisone may cause either sodium retention or sodium excretion depending on the conditions Dr Pitts and his group have even recently shown us that the adrenalectomized animal can actually reabsorb more sodium than normal in certain situations Generalizations are obviously difficult Fremont Smith Are we not clinging to an old philosophical concept, now outworn of single causality? It seems to me that we will have to accept as basic to our thinking that the effect of any given stimulus on an organism is not a uniform response, but that the nature of the response is determined by the state of the organi isin I was so glad that Dr Gaunt and Dr Pitts, as well as Dr Wesson brought out the relativity of the effect of pitressin 14, that the antidiuretic hormone may be either diuretic or antidiuretic, the response depends upon the state of the animal when the hor- ·Tubular Secretion of Potassium and Acid 135 of circumstances Instead of being bothered by the relatnity of re sponse we should try to specify the situation in terms of pertinently related sets of circumstances which influence or determine the response to the particular stimulus under study I believe it is both impractical and philosophically unsound to seek a single characteristic response to a stimulus Shannon 1 I think that, one knows al system will always give the same result Oliver But one never knows all that. Shannon The point is when one gets variable results then one has to re-examine the system in order to determine or discos er the additional conditioning factors That is not the same thing as saying that the same stimulus may produce different results in the same system at different times Fremont Smith There is no point where one can get down below a concatenation of events If you go into the cell or into the nucleus or into the atom you are invoked in the same situa tron! When you say you hase to know all the variables and must hold all the variables constant in order that a given stimulus should invariably produce the same effect you are saying exactly the same thing that i am The stimulus has effect A under conditions A but the sumulus would also have effects B C or D under different conditions The concept of single causalty has outwom its use fulness I feel it is more in accordance with reality and with the scientific method and less likely to get us into troublc if ne accept and male part of our thinking the concept of multi causility and concatenations of esents Our emphasis then will be to specify as miny of the sanables as we can indicating those which are con trolled and to what extent they are controlled leaving also plenty of room for unknown variables Aciertheless I would he to emphasize again that I believe Dr Shannon and I are saying the same thing from different angles Shannon I am not sure ·138 Aonal Function monkey wrench into the situation and made Newton'un concepts invalid with respect to certain other areas That is exactly the situa tion here I cannot for a minute believe that the concept of clear ance will not be fundamental with respect to certain problems but I also believe that there will be situations in which the present con cept of clearance will provide at best only a first or perhaps a second approximation to the facts I want to ask you to expose yourselves, and this seems a good chance to expose myself, to an idea which is quite contrary to the basic concepts by which many of us are operating I think it is generally believed today that the capillary glomerular bed is being used at close to maximum capacity most of the time There is evi dence, of course, that glomerular filtrate does increase under certain circumstances The vascular blood flow through the kidney does go up and certainly good evidence exists that it can be sharply reduced as in anxiety reactions or in traumatic shock. We have been talking about tubular reabsorption today and my concept has to do with tubular reabsorption It is generally agreed that when concentrated urine is being formed or when antidiuresis takes place there is an increased amount of tubular reabsorption It is assumed that such an increased tubular reabsorption is due primarily to an increase in the activity of certain tubular cells 10 all the tubules I would like to suggest the possibility of a simple and more flexible mechanism which would allow for changes in the amount of reabsorption by the tubules by varying the surface of the tubular cells to which the filtrate is exposed Thus if a given amount of filtrate were exposed or allowed to flow over a large tubular surface much more reabsorption would take place than if the same amount of filtrate were in contact with a much smaller area of tubular cells The basic assumption underlying this hypothesis is that the normal blood flow through the kidney Occupies only a fraction of the glomerular capillary bed at any one time and the fraction of glomerular capillaries which are open can be varied by renal vasoconstrictor mechanisms which allow for a redistribution of the functioning glomerular capillaries It is further assumed that the total amount of glomerular filtrate is approximately unchanged when the kidney shifts from producing a concentrated urine to a mad mum winter diuresis This I believe is in accordance with observa tions of Rehberg on mın (Rehberg P B Studies on Kidney Funch tion 1 The rate of filtration and reabsorption in the human kidney II The ercretion of urea and chlorine analysed according to a ·Tubular Secrction of Potassium and Acid 139 modified filtration reabsorption theory Biochem 20 447-182 (1926)) and was bome out by the observations of my colleagues and me on man in which glomerular filtrate as measured by endogenous creatinine clearance did not vary significantly from a concentrated urine to a water diuresis If we consider the mammalian glomerulus as reconstructed by Vimtnip (Krogh A The Anatomy and Physiology of Capillanes Revised Edition New Haven Yale University Press 1929) as given in the frontispiece we see that each glomerulus is composed of approximately 50 non anastomosing capillary loops. Thus on theo Joop open in each But note that in such redistribution of blood flow from one glomerulus with 50 capillary loops open to 50 glomeruli with only one capillary loop open in each there need be no change in total blood flow 10 a redistribution of a constant volume of blood flow through the kidney could provide two contrasting situa tions One in which the blood was evenly distributed through all glomerlı but with approximately only one capıllary loop open in each In this case the Gltrate would be distnbuted to all the tubules but only a small fraction to eich This would result in 7 inaximum reabsorption of filtrate and a concentrated urine In the second situation the samnc blood flow would be distributed only to approxi mately one in every 50 glomerul Each of these honerer would have its 50 capillary loops wide open There would be the same amount of total renal blood flow as in the first situation and the same amount of total filtrate would be formed but in this case the filtrate would be distributed to only one in every 50 tubules and these tubules would receive approximately fifty times as much filtrate per unit of time as they receive in the first situation The filtrate would be exposed to only 1/50 of the total surface of tubular reabsorbing cells and the contact with these cells for each unit of filtrate would be of very much shorter duration This would be the situation of mimimal tubular reabsorption and manmum diuresis I have two figures to illustrate tlus hypothesis In Figure 31 on die teſt is shown a diagram of a glomerulus with only one capillary loop open to illustrate the suggested condition of all the glomerul when i concentrated unne is being formed on the nght is shown a diagram of a glomerulus with all ffty capillary loops open to illustrate the condition of these glomerul which are open (only alsout one in 50 would Le open) during a water diuresis Tigrire 35

·140
Renal Function
illustrates the same two conditions on the left a diagram shoung
a large number of glomeruli with only one capillary loop open in
ench and on the right a diagram showing a large number of
glomeruli with only a few open but with these few wide open re,
UR NE CONCENTRATED
VOLUNE SNALL
VR NE D LUTE
V UNE LANGE
FIGURE 34
Dagrammatic picture of glomerulus on left with only one capillary open and
on the right with 50 capillaries open It would require 50 glomeruli with only one
capillary open to make as much filtrate as would be produced by one glomerulus
with 50 capillaries open
UR NE CONCENTRATED
VOLUNE SMALL
UR NE D LUTE
VOLUME LARGE
ODD
0000000000
DOO
ΦΦΦΦΦΦΦΦΦ
DO
FIGURE 35
dahinn of hod flow in glomerular capullary bed
c
50
in
u

·Tubular Secretion of Potassium and Acid 141 1 es + גדו with all 50 capillary loops receiving the blood Thus by a red str bu tion of the same volume of renal blood flow in the one case a ry limited flow through each glomerulus and in the other a mi w ry flow through only a few glomeruli, the same volume of glemplar filtrate would be delivered in the first instance to all tht and in the second instance to only about one in 50 tubulos If h a mechanism does in fact exist one would anticipate a con € trated unne from the first situation because there would be maximum opportunity for tubular reabsorption while in the second situation druresis would take place since all the filtrate would be pourrg rapidly down only about one in every 50 tubules at anv one moment An hypothesis has virtue only if it will explain data more logically than any other hypothesis, if it provides for useful generalizations or if it acts as a challenge to new experimental work which other wise might not be carried out I proposed this hypothesis ofore the American Society for Clinical Invest ation 1930 (Fremont Smith F, Fremont Smith M, Dailey M E et al St idem Edema I The mechanism of water diuresis in min 1 Clin 1 1 gation 9,7 (1930)) without a real expectation that it wonld out to be correct but in the hope that it would stimulate new unrk Since that time I have discussed it informally whenever possible and have as yet to find what seems to me adequate evidence to dis pose of this theory I would like to take a few minutes to show a number of physiological and clinical phenomena which perhaps can be correlated by this theory and which so far as I know are not yet correlated by any other theory At present there is no theory to explain what happens in the kidney in the shift from a concentrated urine to a water druresis other than the concept of increased reabsorptive activity of tubular cells nor is there any theory to explain the two contrasting clinical conditions ie edema without uremia and uremia without edema The proposed concept has the virtue of correlating the known renal pathology underlying these tso contrasting conditions with their major differences in kidney function In other words one would predict that in condition of edema without uremia the kidney is in a condition of constant maximum tubular reabsorption and yet has sufficient filtration surface to clear the non protein nitrogen from the blood and furthermore that the kidney is fired in this condition at least temporuly From the viewpoint of the proposed theory one would erpect the vast majority of the glomerul to be functioning but almost none of them to be functioning fully 14, one would anticipate 2 diffuse injury insolving all glomeruli but ·142 Renal Function allowing some blood flow through all or nearly all The actual pathology of glomerular nephritis, of nephrosis, and of amyloid disease of the kidney, in fact corresponds to just this condition All three show edema often without uremia In contrast is the pathology of the kidney in uremia without edema, as in the arteriosclerotic kidney Here the kidney is unable to concentrate the urine and a diuresis takes place continnously, in this situation the theory would require that there be a reduction of glomerular filtration surface to the point where the blood cannot be adequately cleared of non protein nitrogen but that those glomeruli which are functioning should be, in the main relatively normal, ie, be able to receive a maximum blood flow through all the capillaries Aguin this is in fact the case In the arteriosclerotic kidney there is a progressive loss of whole glomeruli - not a diffuse injury of all As more and more glomeruli are destroyed, the blood flow through the kidney must be diverted to the remaining normal ones which will now have to open more and more capillaries in order to accom modate the renal blood flow and get rid of the non protein nitrogen This will mean that the filtrate is formed in progressively fewer glomerul and is shunted down fewer and fewer tubules and one would expect and does in fact find a progressively increasing polyurea Eventually the filtration surface is insufficient to get rid of all the non-protein nitrogen, uremia progressively increases and the remaining glomeruli continue to function at maximum capacity Polyuna continues and edema does not take place The kidney at autopsy shows destruction of the majority of glomeruli but of the remainder many are not only normal in appearance but actually show dilation as if they were receiving constantly a maximum blood flow and making a maximum amount of filtrate It should be pointed out that a quite different clinical condition 1e, congenital poly cystic kidney shows the same clinical picture of progressive diuresis followed by uremia but never any edema, and again at autopsy shows massive destruction of the majonty of glomeruli but those remaining appear quite normal und dilated In animals it is well known that if the mass of the kidney is reduced by surgical ercision, the urine flow does not decrease but increases! Thus if the kidney is reduced to one quarter its size the urine formed may be considerably greater than from the normal kidney This aguin could be explained on the assumption that the blood flow in the kidney of reduced size is pos diverted through the relatively few remaining glomeruli and the filtrate formed pours into relatively fewer tubules with a resulting reduction of reabsorp- ·Tubular Secretion of Potassium and Acid 143 tive surface and time for reabsorption with a resulting increased urine volume Diabetes insipidus according to this theory would be explained by the kidney functionally fivated in the condition of diuresis ie with blood flowing through a relatively few glomerulı which are wide open The antidiuretic action of pitressin would be explained not by an increased activity of all tubular reabsorbing cells but rather by redistribution of the blood flow in the kidney only a minimum glomerulus The but in such small volume that there is ample time for maximum reabsorption in each tubi le This concept therefore appears to offer a correlation of kidney function and pathology in the major clinical conditions of edema without uremia of uremia without edema of normal water diuresis of diabetes insipidus of the antıdıuresis of pitressin and also of the increased volume of urine formed when the kidney is experi mentally reduced in size I would welcome the opinion of the group regarding this hypo thesis Is there cleancut evidence at present to make the theory untenable? Can it be dismissed outright or on the other hand are there experiments that could be done which would either prove or disprove it? My own suggestion of an experiment would be in an unanesthetized dog with the kidney previously brought outside the abdominal wall to inject the renal artery with India ink This would be done in one group of animals during maximum water diuresis and in another group when a concentrated urine was being formed or after an injection of pitressin in each case during the first few seconds of India ink injection the pole of the ladney would be sliced off and immediately fixated Careful serial section recon Injecteu should shox Curton in all capillaries of the glomerulus winle in the animals producing concentrated unne all glomerul should show India in, but only one or two capillanes in each glomerulus would be injected It would be necessary to slice off the Liddes pole almost immediately after starting the India ink injection for there is nothing in the proposed thcon which would climinate "intermittince" re during the formation of concentrated urine althoigh only one capillars would be open in each glomerulus ·144 Renal Function at any one time the same capillary would not necessarily be open continuously Therefore after some minutes of injection all the capıllary loops might show some carbon particles Similarly, in water druresis with only one glomerulus in fifty open at any one time there would be the possibility - even the probability - that one glomerulus after another would take its turn in becoming wide open thus shifting the burden of tubular reabsorption from one tubule to another and avoiding fatigue or exhaustion of the cells which are actively reabsorbing water This concept can be wrong but I have for nineteen or twenty years challenged everyone that I could from Rehberg on to bnng forward any good evidence to show that this is impossible and I have not been able to have the pleasure of getting that evidence yet So I throw it back to you Shannon I think Dr Forster has some data that might bear very directly on the problem Forster I was originally interested in Richards techniques which were developed of course for the direct examination of glomerular and tubular activity Later when I turned to clearance studies I chose to work on the frog for the specific purpose of comparing the results obtained by using clearance techniques on this animal with those previously made by others using microdissection procedures One of the checks on the clearance technique was to see whether such alterations in clearance would be obtained as could be correlated with observations on glomerular intermittency ob served directly in the frog The problems Dr Fremont Smith has posed soon presented themselves Are variations in urine flow accounted for by opening and closing of the glomerul? Is glomerular activity in all or none phenomenon? Does a glomerulus function all the time in its full capacity or are there gradations of circulation through an individual glomerulus? Dr Fremont Smiths Figure 34 7 digram of an individual glo merulus presents the possibility that blood in coming through the afferent uteriole can truerse all of these 50 channels one of the channels or any intermediate number of them Back in 1937 or so I discussed with Dr Shannon the possibility of using clearance techniques to examine the problem of glomerular inter mittency and it was decided to measure maumal rates of glucose reabsorption (glucose Tm) by the tubules under conditions which radıcally altered glomerular filtration rates in frogs The concentra tion of glucose in the plasma was maintained trio or three times as high as was necessary to insure saturation of the tubular reabsorp ·Tubular Sccrction of Potassium and Acid 145 five mechanism In any single nephron unit then with the glucose plasma level several times higher than that needed to insure satura tion it would be possible to reduce the filtration rate of its glo merulus by 50 percent or more without altering the rate of glucose reabsorption in the associated tubule We would expect then that of the situation illustrated in this slide prevails the filtration rate could luctuate considerably without resulting in corresponding variations in the rate of glucose reabsorption As far as clearance determinations are concerned the glucose Tm would not very directly with the glomerular filtration rate This was shown not to be the case That is the maxmal rate of glucose reabsorption always exactly paralleled the glomerular filtration rate measured as the inulin clearance Fremont Smith Did your experiment prove that you were using at any time all the capillines in the kidney or that under a water diuresis which is quite different from a glucose druresis you could not have a shift? Aly theory does not attempt to explain sodium chloride diuresis vrea or caffeine diuresis but only water diuresis Different mechanisms are invol cd in these other forms of druresis Forster I just wanted to say that the urine flows in these frogs cre vanied at least tenfold cren with the animals full of sugar y alternately taking them out of water and immersing them Filtra tion rates saned as well as urine floss Alterations in the filtration rates as great as tenfold in the frog were accompanied by exactly the same alterations in glucose Tm under these conditions Fremont Smith If any glomerulus opened it did so completely? Forster If it had been otherwise it would have been possible you see to close down 50 percent of the circulation reduce the filtration rate by 50 percent and have no change in the glucose Tm If this situation prevailed that is of instead of completely closing down glomerul 50 percent of the channels through individual glomerul were closed the filtration rate could krie been reduced by 50 percent without necessanly altering the glucose Tm rremont Smith Then am I right that what your experiment proved was that under the conditions of the vinations in glomerular filerte due to this glucose situation glomerul either opened or slut in an "all or none" way? Torster That is nght Fremont Smith. But it did not gie any evidence as to the impos sibility of their shutting and opening part way under other con dations Forster That is entirely truc ·146 Renal Function Shannon Dr Forster really should have emphasized that al these experiments were carried on pretty much with the same plasma glucose level He changed the glucose load by changing glomerular filtration rate not by changing plasma glucose Fremont Smith Would it be assumed then from this that in the frog and in perhaps mammals if a glomerulus is open the blood flow per unit of time through that glomerulus is constant? Shannon No sir Fremont Smith It seems to me if you can have variations in the blood flow per unit of time through a glomerulus it gives oppor tunity for essentially the same variation in rate of filtrate flow down the tubules as suggested above Shannon I think you have to draw on Homer Smith s observa tions with p aminohippurate for that if you accept the valdity of his measurements I believe this can be done with safety since direct measurements by catheter techniques applied to the renal vein are in essential agreement Smith found in the human that one can change the blood flow up or down but there is no change in reabsorptive capacity This would imply that the reabsorptive surface has not changed Grafflın Some years ago in a course in physiology for medical students many frogs were carefully (though still crudely) prepared for continuous observation of the living kidney in a selected micro scopic field The phenomenon of intermittence in the glomerular circulation was regularly observed in the first 20 or 30 minutes after the prepiration was completed If the frogs skin was then bathed with hypertonic salt solution all glomeruli became com pletely inactive within a very short time On the other hand of the skin was freely bathed with distilled water all glomerul in the field soon attained what appeared to be maximal activity and retained it for prolonged periods Fremont Smith Is that water diuresis associated with an enormous increase in glomerular filtrate? Grafflın Measurements were never made White Glomerular filtration comes up to normal when the frog is properly or normally hydrated If each loop were open there would be 50 times as much filtrate as on the left of your figure so that with all of them open there would be 6000 cc a minute instead of 120 Shannon The frog does just that HI hute Dr Bott knows more about this than I do I dont know how Dr Richards feels about this since I have not talked with hm ·Tubular Secretion of Potassium and Acid about it for a good many years but I believe he would say that when a frog is normally hydrated you dont see any intermittence You take the absence of intermittence as evidence of nornal circula tion the more normal he is the less likely is intermittence to occur A number of years ago I did an experiment which in my nille way of looling at things had some bearing on this If you inject a little India ink into a dog s renal artery and clamp the pedicic four or five seconds later all the glomerult will be injected Departures from that I am sure can be explained on chance distribution of the injected material You dont see intermittence you may see an uninjected wedge one whole interlobular artery or group of inter lobuhr arteries may fail to receive it but where injected all of their glomeruli ure injected. That is true whether the dog has been dehydrated or whether he had an abundance of water or was es en zaven water by stomach tube before you do this I might say the ame thing is also true in the rabbit He can be hydrated or deydrated I believe recent work by Brod and someone else from miths laboratory accepts the vies now that the glomerular filtra in rate is not parallel with unine flow in the rabbit any more than is in the dog or man (Brod J and Sirota JH Effects of emo tional disturbance on water diuresis and renal blood flow in the rabbit Am | Physiol 157 31 (1949)) Fremont Smith Would you gie us the reference to your experi ments on the dog? 11 / ite They were published (Whute II L Observations indicat ang absence of glomerular intermittence in normal dogs and rabbits Am J Physiol 128 159 (1939)) Pitts Dr White I would like to ask if you think the frog abnormal every time it crawls out on a lily pad Does diuresis continuie till the frog dnes up? 1 hite I don't believe he will stay on that pad long enough I believe he will see that he gets lumself moistened The question of how dehydrated a frog can get and still be considered normal is of course open to debate I would like to turn the floor oser to Dr Bott Bott I dont pretend to be able to answer that question but I do know that when we were doing punctures on mammals there was the difficulty of finding the glomerult on the surface Thien there was another difficulty of collecting from some that were on the very surface It was casier to puncture those just shghth below and collect from them Dr Walker sau a number of them on the surface from which he could not collect and watched these for a

·148
Renal Function
time never
however, t
and still be
This was no
it erther, although salıne infusions were being made

·WATER REABSORPTION BY THE RENAL TUBULES LAURENCE G WESSOV Jr Department of Physiology, New York Unnersity College of Medicine Sodium falls into that group of substances uliose rate of excretion by the kidney under normal or physiological conditions is subject to facultate control by the orgamsm as a whole this control being exercised through functional vanation in the level of activity of specific reabsorptive or excretory cells of the tubules or else through changes in the magnitude of the load delivered to the tubules Once a facultative control of electroly te excretion is assumed the problem is to discover the sequence of events in the bods wherely an increase in body sodium chloride is followed by excretion In the case of water cxcretion the primary event appears to be a change in the osmotic pressure of the extracellular fluid In the case of sodium the primary eient appears be a physical increase in the extracellular fud volume or somc fraction of it but the mecha nism lying between fluid volume increase and increased electrolyte etcrction is still unknown Whatet er the nature of this mechanism ats action remains almost unique among substances encreted by the kidneys since the full range of body salt control from complete retention to the excretion of the greatest endurable rate of salt ingestion is accomplished by an increase or decrease of only a few percent of the total amount of salt filtered at the glomerulus The ability to adjust body salt balance by fractional percentage changes in the total amount reabsorbed imples an extraordinarily sensitie mechanism and the immediate problem of salt reabsorption becomes that of determining how the percent tubular reabsorption can be vined within that small range Furthermore the importance of small percentage changes in total reabsorption greatly enhances the potential difficulty of studying sodum excretion for it is not sur prising that a great niet of factors are known which will change at least temporanly by a few percent the percent of sodium reab sorbed by the tubules and therefore will produce is great a percent change as mn be observed under so-called normal conditions by increased sodium chloride feeding A change in the diet innumcrible 149 ·150 Renal Function drugs renal oxygen supply renal venous pressure other excreted ions are some of these factors If we include with these endocrine and metabolic agents we have i group which might be called the minor vanables to be distinguished from the major variable of filtered salt load with its two components concentration and volume The difficulty of controlling all of these variables is tremendous And any one or several of these minor variables although capable of affecting total salt reabsorption by only a few percent may be critical in determining salt excretion under physiological or pathological conditions The mechanism by which sodium is reabsorbed by the renal tubules is almost certainly complex reflecting the sum of two o. more separate reabsorptive mechanisms The evidence supporting the yeu that 3/4 to 9/10 or more of the filtered sodium chloride is reabsorbed in the proximal tubule of mammals as good In addition there appears to be good evidence for the existence of ion exchange mechanisms whereby hydrogen ammonium and potassium ions are secreted into the tubule in exchange for sodium ions reabsorbed such mechinisms together accounting for perhaps 1 to 2 percent of the total sodium filtered But there is also a philosophical reason for seeking the existence of still another reabsorptive mechanism for sodium and that is the belief that it is more probable to find the body controlling the excretion of sodium by large vanations in ? reabsorptive mechanism receiving small amounts of the filtered lord of sodium than by small variations in a mechanism which reabsorbs the bulk of the filtered load We may then tentatively propose the following system The bulk of the filtered sodium is reabsorbed in the proximal tubule in large and variable amounts and without being under critical facultate control Distal to the proximal reabsorptive area but not necessarily located in the anatomical distal tubule is ? distal reabsorptae area which has these properties a) its maximal reabsorptne capacity is equal to 10 to 20 percent of the normal filtered sodium load b) the magni tude of this distal process is subject to facultate variation by the body over a wide range c) the amount of sodium escaping from the proumal area is normally equal to or less than the maximal distal reabsorptive capacity Regions in which ion exchange takes place may be located either proximally or distally but it should be noted that if such exchange mechanisms exist in the distal tubule it is essential that un adequate supply of sodium remun to enter the distal tubule after proumal transfer is completed I inations in sodium excretion may then take place in either of 2 ways by ·Water Reabsorption 151 increasing the load of sodium escaping from the proximal process so that it exceeds the distal process or by decreasing the rate of distal reabsorption Either or both of these processes may be con ceived to be under body control It is by no means evident that it is possible to prove or disprove the existence of a third tubular reabsorptive mechanism It would seem theoretically possible to do so under direct examination by micropuncture but this may not be technically feasible so that we are at present limited to indirect methods Here the only technique which has offered any hope of success is a possible correlation between sodium and water reabsorption According to all available evidence water is reabsorbed in large quantities in the proximal tubule The driving force for this reab sorption appears to be obligatory passive and due to osmotic pres sure differences between intratubular and peritubular fluids which have been set up by the prior active transfer of solute If 50 percent of the filtered solute measured as osmols is transferred then 50 percent of the filtered water is capable of moving until isotonıcıtı across the epithelium is restored Conversely if 50 percent of the water diffuses from the proximal tubule then it follows that at least 50 percent of the osmols has been transferred also Since sodium salts account for 90 to 95 percent of the total filtered osmols the amount of water transferred measures 18 7 first approximation the amount of sodium transferred According to the micropuncture studies the proximal tubular flud remains very nearl isosmotic with plasma as far down as the investigators have been able to go In this case the measure of solute transfer is a precise one It does not follow however that osmotic equilibrium across the proximal epithelum is so nearly complete under more physiological condi tions The flud reaching the end of the proximal tubule may be quite hypotonic to plasma due to the inability of water to keep pice suth solute transfer Accordingly these possibilities may exist at the end of the proti mul tubule 1 A small volume of flud representing virtually complete trans fer of electrolyte and isosmotic transfer of water 2 A larger volume of strongh hypotonic fluid representing Virtually complete transfer of electrolyte und incomplete trans fer of water 3 A large volume of fluid neurly or completely isosmotic to plasma and therefore carning considerablc quantities of electrolyte ·150 Renal Function drugs renal oxygen supply renal venous pressure other excreted ions are some of these factors If we include with these endocrine and metabolic agents we have a group which might be called the minor variables to be distinguished from the major variable of filtered salt load with its two components concentration and volume The difficulty of controlling all of these variables is tremendous And any one or several of these minor variables although capable of affecting total salt reabsorption by only a few percent may be critical in determining salt excretion under physiological or pathological conditions The mechanism by which sodium is reabsorbed by the renal tubules is almost certainly complex reflecting the sum of two or more separate reabsorptive mechanisms The evidence supporting the view that 3/4 to 9/10 or more of the filtered sodium chloride is reabsorbed in the proximal tubule of mammals is good In addition there appears to be good evidence for the existence of ion exchange mechanisms whereby hydrogen ammonium and potassium ions are secreted into the tubule in exchange for sodium ions reabsorbed such mechanisms together accounting for perhaps 1 to 2 percent of the total sodium filtered But there is also a philosophical reason for seeking the existence of still another reabsorptive mechanism for sodium and that is the belief that it is more probable to find the body controlling the excretion of sodium by large variations in a reabsorptive mechanism receiving small amounts of the filtered lord of sodium than by small variations in a mechanism which reabsorbs the bulk of the filtered load We may then tentatively propose the following system The bulk of the filtered sodium is reabsorbed in the proximal tubule in large and variable amounts and without being under critical facultative control Distal to the proximal reabsorptive area but not necessarily located in the anatomical distal tubule is a distal reabsorptive area which has these properties a) its maximal reabsorptive capacity is equal to 10 to 20 percent of the normal filtered sodium lord b) the magn țude of this distal process is subject to facultative variation by the body over a wide range c) the amount of sodium escaping from the proximal area is normally equal to or less than the maxima distal reabsorptive capacity Regions in which ion exchange takes place may be located either proximally or distally but it should be noted that if such exchange mechanisms exist in the distal tubule it is essential that an adequate supply of sodium remain to enter the distal tubule after proximal transfer is completed Variations in sodium excretion may then take place in either of 2 ways by ·Water Reabsorption 151 increasing the load of sodium escaping from the proximal process so that it exceeds the distal process, or by decreasing the rate of distal reabsorption Either or both of these processes may be concened to be under body control It is by no means evident that it is possible to prove or disprove the existence of a third tubular reabsorptive mechanism It would seem theoretically possible to do so under direct examination by micropuncture, but this may not be technically feasible, so that we are at present limited to indirect methods Here, the only technique which has offered any hope of success is a possible correlation between sodium and water reabsorption According to all available evidence, water is reabsorbed in large quantities in the proximal tubule The driving force for this reabsorption appears to be obligatory, passive, and due to osmotic pressure differences between intratubular and pertubular fluids which have been set up by the prior active transfer of solute If 50 percent of the filtered solute, measured as osmols, is transferred, then 50 percent of the filtered water is capable of moving until isotonicity across the epithelium is restored Conversely, if 50 percent of the water diffuses from the proximal tubule, then it follows that at least 50 percent of the osmols has been transferred also Since sodium salts account for 90 to 95 percent of the total filtered osmols, the amount of water transferred measures, as a first approximation, the amount of sodium transferred According to the micropuncture studies, the proximal tubular fluid remains very nearly isosmotic with plasma as far down as the investigators have been able to go In this case, the measure of solute transfer is a precise one It does not follow, however, that osmotic equilibrum across the proximal epithelium is so nearly complete under more physiological condi tions The Aud reaching the end of the proximal tubule may be quite hypotonic to plasma due to the inability of water to keep pace with solute transfer Accordingly, these possibilities may exist at the end of the proszmal tubule 1 A small volume of fluid representing virtually complete transfer of electrolyte and isosmotic transfer of water 2 A larger volume of strongh hypotonic Auid representing virtually complete transfer of electrolyte and incomplete trans fer of water 3 A large volume of fluid, nearly or completely isosmotic to plasma and therefore carrying considerable quantities of electrolyte ·152 Renal Function 4 A varying combination of the other three Whether or not water may be used as an index of sodium reabsorp tion depends upon which of these conditions is the thic one A second fraction of water reabsorbed is that represented by the production of a hypertonic urine This might represent in man 3 to 5 cc per minute and necessanly requires the direct expenditure of cellular energy as osmotic work The locus of this activity may be tentately referred to the distal tubule A third fraction of water reabsorption is that which appears as an increase in urine fow dur ing water druresis or as a result of interruption of neurohypophyseal function and represents about 15 percent of the total filtered It is generally assumed that this water arises from the depression of a specific reabsorptive mechanism brought about by a decrease in the concentration of circulating antidiuretic hormone Two theones have been advanced to account for this facultative water fraction According to one theory the facultative water arises from the thin limb It is postulated that an abundant hypotonic fluid enters the thin limb Under the stimulus of the antidiuretic hormone water passes through the cpithelium of the thin limb untıl osmotic equilibrium is attained and 2 scanty flud enters the distal tubule to be further concentrated by the active seabsorptive process In the absence of the antidiuretic hormone the thin limb remains impermeable to water so that an abundint hypotonıc Auid enters the distal tubule in excess of the desta) reabsorptive capacity and escapes from there into the bladder According to the second theory the facultative water is reab sorbcd actively by the same distal mechanism which produces 2 hypertonic urine In this regard it is a simplification over the fore going in the absence of stimulation by the antidiuretic hormone active reabsorption is depressed so that an increased volume of fluid escapes into the collecting tubules It is not essential to this theory that the fund entering the distal tubule und from which the facultative water is abstracted be isotonic although proposing that the tubular urine comes to osmotic equilibrium in the thin limb is an intriguing auxivary hypothesis Nevertheless in order that water reabsorption may serve as a key to the eastence of an additional sodium rebsorptive process it must have just these properties that osmotic equlibrium be attuned in the proximal tubule or thin limb (and it makes no difference u hich) thus affording a measure of the amount of sodium reabsorbed in the proximal tubule And further that the facultatie water be reabsorbed actively in the distal tubule thus affording a measure of the volume of electrolyte ·Water Reabsorption 153 entering the distal tubule Subtracting the amount excreted from the amount entering measures the magnitude of reabsorption by the distal tubule Measuring hydrogen, ummonium and perhaps some fraction of potassium excretion will determine the sodium reabsorbed by transfer If this or an analogous situation does not descnbe the behaviour of water, then water cannot be used as a measure of sodium movements in a complex reabsorptive system of the type described The mechanism of water reabsorption must be solved before much further progress can be made with sodium Until it can be determined whether there are one or two specific sodium reabsorptive processes, and if multiple whether they can be measured, investigations on such things as the relationship of filtration rate and plasma concentration of sodium to sodium reabsorption, the actions of mercury, of pituitary and adrenal hormones, of osmotic diuretics and other ions and the mechanisın through which changes in body salt content are corrected by changes in the rate of excretion must rest upon a very tentative basis There are so many problems associated with salt and water reabsorption and excretion that I would prefer to save them for general discussion I would like to conclude this introduction by listing some viewpoints which have been conditioning our appronch to the problems associated with water and salt excretion We feel that investigation of the mechanisms of sodnim and water reabsorption should be initiated in the following order 1 Nature of the components of the water reabsorption system 2 Nature of the components of the sodium reabsorption system 3 The relationship of load, and its two components, concentra tion and volume to reabsorption by each component of the sodium reabsorption system of particular interest is the relationship of filtration rate, the volume factor 4 Which of the variables relating to which component of the sodium reabsorption system is normally employed by the organism to adjust body sodium content to variable rates of sodium ingestion 5 The details of the mechanism by which the body controls this variable or vanables 6 Elucidation of the effects of other factors, such as pH vanable anions, hormones ete upon sodium and water excretion in terms of their relation to specific components of the sodium system ·154 Renal Function DISCUSSION Pitts There is a question which has worried me all the way through this presentation I would like to sk all of you your opinions and Dr Bott especially We all have the tendency I think to accept the work of Walker Bott Oliver and MacDowell as the basis or starting point for the interpretation of all data on salt and water excretion Their experiments on the mummal were performed presumably under 1 given set of constant conditions which were neither those of marked osmotic diuresis nor those of water diuresis I wonder if Dr Bott would care to comment whether she feels that it is valid to assume that fluid is absorbed isosmotically in the proximal segment and that 68 percent or more of the filtrate is returned to the blood streim in that segment under conditions of water diuresis and osmotic diuresis as well as under the conditions of her experiments Bott I must admit as we certainly had to from the beginning that these were not absolutely normal animals in that they had to be anesthetized Certain procedures had to be done on them in order to be able to take the samples and I think that we do have to keep that in mind constantly in interpreting the results I bcheve We have all admitted that However I feel that they are certainly a good basis from which to start I dont know what other one would be better As far as the osmotic pressure is concerned I think that surprised us as much as it did anyone to find these low osmotic pressures of tubular flud all the way down and by the way I believe the three determinations on the distal tubule are perfectly real results They are very often not considered because we were hesitant to put too much stock in them having only three that Dr Walker felt were reliable collections Just how much the anesthetic might influence the results I cannot say for sure I have been ashed something about the chloride determinations and whether they fit in with the low osmotic pressures I feel there is nothing in those chloride values that interferes with the acceptance of those osmotic pressures because the calculated osmotic pressure of the known constituents still leaves some room to spare Pitts I am afraid I created the wrong impression by my com ments I did not expect you to defend those specific experiments becluse we all accept them The fact that disturbs me is not that those experiments were performed necessarily under abnormal con ditions I would consider they were a whole lot more normal than are those experiments performed under high osmotic loading But ·Water Rcabsorption 155 can we assume that the same type of proximal osmotic relationshups will obtain under osmotic loadıng? Also can we accept the premise that the proximal tubule cannot perform osmotic work on water necessarily? Is that absolute? Bott I think the only way to be sure about the osmotic loading question is to try it in experiments of this kind I think that there is no reason why they cannot be done with time and trouble though Dr Walker is not working in the field just now Selkurt Will you clarify what you mean by osmotic loading? Extraneous substances like mannitol? Pitts Any type of osmotic loading whether by urea sodium or mannitol I am getting down to the point of ashing Dr Bott whether she contemplates at all providing the data herself Bott Some of our experiments were run during sucrose injections so there must have been quite a bit of sucrose present in the tubule fluid Unfortunately I dont remember which were which but in going back over my notes I may be able to find out where we did have a lot of sucrose present and if we still got the low osmotic pressure ? Shannon Could Dr Bott tell us the order of magnitude of the osmotic pressure difference? Bott In many cases it was within experimental error of the osmotic pressure of the plasına through the first third or half I think of the provmal tubule There was one definitely lower one I tlunk I would liesitate to say how much low er but it was definitely lower It would seem to be out of line with the others Those three on the distal tubule were still lower than most of those in the proximal tubule Then when the osmotic pressure of bladder urine was taken that was very high comparatively Of course there is a big gap in between The part that struck me in that too is that the chlondes were higher than the chloride of the plasma in the proximal tubule where the osmotic pressure was about equal to that of plasma The chlonde in the urine was lower than that of plasma in spite of greatly increased osmotic pressure In other words the increase was not due to an increase in chloride Il csson What was the osmotic pressure in the distal tubule in the three or four simples you obtained 1(A search of my does revealed that osmot c pressure of tubule su d was not deter mined after sucro C At As but that tubule fu do mot c pressur as low n the p of cons derable glucose after phlo h zn) ·156 Renal Function Bott It was plotted as related to plasma I cannot remember exactly but I think it was 20 percent lower I would have to check that Wesson Further on down the urine was found to be hypertonici Bott That is night Wesson Suggesting that water reabsorption took place somewhere between the point of puncture and the bladder Bott Early distal Shannon We always talk about the mammalian kidney being characterized largely by its ability to form a hypertonic urine This potentiality is strikingly limited Under the most extreme conditions of dehydration without adding unusual constituents it is unusual for freezing point depressions to overrun about 13 14°C freezing point depression That occurs at low flows of urine in the dog in the order of 02 milliliters per minute Should one accept the data of Drs Bott and Walker as indicating isotonic tubule urine in the proximal segment then in the normal animal without any osmotic loading perhaps one might expect there to be osmotic pressures that are significantly lower than those obtained in the rat It is pure speculation Pitts Getting back to your own experiments with dogs in diabetes insipidus when they were dehydrated they did form as I recall urine which was considerably hypertonic- Shannon Not considerably The plasmı reached a freezing point depression in the order of 0 7°C the urines were a maximum of about 10°C While all the dogs studied had diabetes insipidus as ordinarily described some had some residual posterior pituitary tissue The striking thing as far as we were able to determine was the lack of correlation between the amount of apparently normal tissue remaining and the animals ability to produce hypertonic Pitts I would like to ask Dr Wesson and Dr Mudge if they have any indication of what the relatie magnitudes of absorption of sodium or chloride might be in either the proximal or the distal segment under normal conditions Wesson We have no direct expenmental indication of these magnitudes Any estimate that we might make would be only a deduction from a current working hypothesis It is our feeling that of tubular water reabsorption has certain properties then we have a useful clue to the magnitudes of proumal and distal sodium reabsorptive processes If for instance the flud reaching the end of the proumal tubule is measured by the urine volume of diabetes urine ·Water Reabsorption 157 insipidus and if further thus flwd us isotonic with plasma then the amount of sodium reabsorbed in the proumal process is readily calculated Shannon May I ask a question? Any assumption is reasonable of at leads to the design of a cntcal experiment Hou ever in retro spect you say the first assumption is that the tubule unne at the end of the proxmal segment is 150tonic or essentially so and the total argument really from there on depends upon that one point Wesson The kitle evidence available seems to support that view Otherwise it is only a useful workoag hypothesis which we wish we could subject to experimental test Putts Basically it goes back to Dr Botts demonstration that half way or more down the tubule it is isotonic By extrapolation we maintain that it is still isotonic at the end That is the reason for my initial question is that reasonable? Wesson Isotonic ty throughout the profunal tubule seems reason able under the abnormal circumstances of heavy osmotic loading At least the amount of water which must be transferred to assure isotonicity of prodimal urine with plasma is quite small and there is no reason to suspect that the osmotic agent opposes a barrier to equilibration However we do not feel justified in extending this reasoning to normal conditions where the osmotic load is very small I would appreciate Dr Shannons comments on this series of experiments Suppose it is observed that the total increment in urine flow when antidiuretic hormone is withdrawn from animals under a heavy water load is constant and independent of the magni tude of a simultaneous osmotic load Could that be interpreted as evidence that the volume of the increment mensures a specific reabsorptie process? Slannon I should think so However the setup is so complicated that it is diff cult to make use of the information Let us start with a simple system with a normal glomerular filtration rate and remove the antidiuretic hormone either operately or through adminis tration of water One obtuns a une high in volume essentially low in electrolyte concentration I believe everybody has assumed unui recently that this situation is due to the absence of the posterior pituitary hormone in which case the distal segment does not actn ciy reabsorb water What appears as unne may be essentially the same type of flud as lenses the proximal segment I am wonder ing if we can take the newer information that we me now and add onto such a rather simple working hypothesis If we zecept Dr Hessons view and accept as probable an additional senes of ·158 Renal Function reabsorptive processes we may complicate the working hypothesis unnecessanly We have before us as of today undoubted evidence of secretion of potassium I wonder if it is not reasonable to suppose that this may not be unique for postassium It is conceivable that the nephron can also secrete sodium I find it not at all difficult to accept the possibility With such i system it is possible to assign to the proximal the reabsorption of sodium potassium and water and assign to the distal segment the further reabsorption of water and the final definition of electrolyte composition to three processes that take place in the distal segment the secretion of potassium the secretion of sodium and the adjustment of the hydrogen son concentration through 2 hydrogen ion mechanism If you accept the reasonableness of such a conception an experimental hypothesis emerges which is capable of being tested in a fairly direct way Pitts It seems to me what we need basically is to be able to measure either the proximal or the distal reabsorption of either sodium or water I see no way under the sun to do it to do either one of those Wesson An advantage to a four vanable system is that you can explain almost anything with it Shannon That is the difficulty Bott I would like to correct an impression that I may have made a while ago when I was saying that I thought that these things can be done I was not thinking particularly that I would do them However I do have available now an ultramicromethod for inulin I felt the necessity for having a measurement of water reabsorption in all of these things In fact if we had had that inulin method at the time we certainly would have used it We also have now the sodum method and a iery excellent chloride method and I am beginning on a potassium method which I have been thinking about for years and to which I am finally getting around It may be that with group work we cin get along much faster than I could by myself and I dont pretend that I am promising to do all this myself but I think we hue a number of methods as ailable for doing work of thuis kind that we did not have a few years bach With all the preliminary work that Dr Waller did on finding out what inimal was best and what he had to do to get the best condi tions - with all of that accomplished I think that it should not be too difficult to go on with some punctures on mammalian kidness The thing I have been wondering is whether some preliminary information on the imphibian lidney would be good at all lou ·Water Reabsorption 159 remember that some of these things show up in the amphibian kidney fairly well For instance that mounting up of chloride in the proximal tubule - we thought perhaps it was not even real in the amphibian because it was comparatively small Yet if you study the diagrams closely you will see that in many cases it went up almost 20 percent higher than the concentration of the plasma and in the mammal that was exaggerated and the tubule Aud at the same time was approximately isosmotic Of course I realize you dont get hypertonic urine but I wonder whether we could learn anything from the amphibia before going to mammals Fishberg I wonder if one could not further the knowledge of tubule physiology by a study of chronic renal insufficiency in man There you have in man a urine isosmotic with plasma and the freezing point does not vary at all it not glomerular filtrate because various constituents may be present in higher concentra tions than in the plasma but it is isosmotic I have seen such patients in the last days of life with a urinary solume of 50 cc in 24 hours and urea in the blood ten times normal and the urine was still isotonic with the plasma This urine must be very similar to the fluid issung from the provmal tubule I wonder if a study of such patients would not reveal a perpetuation of one stage of urin formation So far as I know it has never been studied in detail Shannon There is another point from quite a different point view that might be mentioned A fair amount of work has bel.. done on the composition of fresh water and marine teleost fish urine By and large the freezing point depressions are low In such kidneys absorption of solute proceeds at a more rapid rate than absorption of water and the rate of flow down the nephrons is rather slow the urine flow being in the manne teleost about 4 cc per hg per day This is the result of activity of a single proximal segment in the healthy sculpın During diuresis as the result of skin injury the fish begin to excrete sodium chloride and urine is formed at a Very high rate I dont recall the composition of such urines Wesson Dr Anslow and I have some experimental material which might be of interest in this general problem We have at tempted the expenments which I descnbed earber to Dr Shannon that is the superimposition of a water druresis on top of an osmotic duresis In several experiments the increase in urine flow due to the unter diuresis is about the same size as the water diuresis in the same animal when not under an osmotic load Fremont Smith Could you get any greater urine flow than that which occurs with a maximum water diuresis?

·160 Renal Function Wesson No, we could not get a larger increment than the control The relative constancy of the results thus far are inclining us to the view that the water of water diuresis measures some fairly specific reabsorptive process I am interested in criticism of this viewpoint since acceptance or rejection of this type of data could be critical to the interpretation of other experiments Pitts Was there any change in osmotic pressure of the urine during the superimposed water diuresis? Wesson The control urine, during osmotic diuresis alone, was essentially isotonic With the onset of water diuresis, it became strongly hypotonic Pitts What is your concept then? Is it that the fluid delivered into the distal segment is hypotonic and water is absorbed to bring at to isotonicity: Wcsson We cannot see anything in these experiments which vould answer that question For no other reason than simplicity, ve have been inclined to the view that the water of water diuresis reabsorbed during oligunc conditions in the same region where e unne is concentrated Until we have data to the contrary, it is simpler to think in terms of 2 fractions of water reabsorption rather than three Gaunt How did you induce a water diuresis under these circumstances? Wesson We put water into an osmotically loaded animal The water was hept in the body by the continuous administration of pitressin When urine flow and composition stabilized, the pitressin infusion was stopped Shannon What was the urine flow before the water diuresis? Wesson Eighteen cc per minute and it rose to about 23 cc per minute Shannon Did the change of osmotic pressure reflect just that difference? Wcsson Yes Aludge Did the sodium excretion change? Wesson There was frequently a slight drop in sodium excretion when the pitressin infusion was stopped Shannon I did not think that that experiment was not feasible because I assumed you did it when you described the result I believed it was difficult to devise an expenment to isolate a single segment for examination on the functional basis If one accepts this experiment as a vald prototype of a number of expenments which you probably are going to do, would not they argue for a hypo- ·Water Reabsorption 161 toncity of tubular urine before it reaches the site of secondary water reabsorption? Wesson It could argue for hypotonicity at that point but I dont think it is a necessary corollary Shannon No Wcsson If we exclude from consideration the secretion of water it is evident that at some point the reabsorption of solute sodium chlonde mostly from the tubular urine has outstripped water reab sorption in order to forin hypotonic urine On the basis of the few experiments which we have done the hypotonicity seems to be measured better in absolute terms than u terms relative to some other variable such as filtration rate osmotic load or total sodium or water reabsorption Hence we would inſer tentatively that the magnitude of water druresis is more closely related to a certain small fraction of solute than to a hypotonicity developing from the beginning of proximal reabsorption of solute Whether this small fraction of solute and the water are normally reabsorbed in the same segment or whether on the contrary the solute is normally reabsorbed in one region leaving a hypotonic fud to be facultatively reabsorbed in a lower region is something we have no idea of I think that Dr Shannons data on the effects of water and osmotic diuresis on urea clearance are still pertinent sith their suggestion that the perme ibility to urea of that portion of the tubule normally reabsorbing facultative water is low It is tempting to infer that this may indicate a low permeability to water also whence it would follow that the normal reabsorption of facultate water is an active instead of a pussie process Berliner We were rather disappointed in the fish kodney for the A7 2 in the urine TVhitc You spole about the assumption of a more complete reabsorption of sodium and potassium both as I understood you in the proumal tubule assuming quite hypotonie unine and then the distal putting back whatever salts were needed I was not clear how you proposed to put thus to erpenmental test Shannon Dr Wesson gave one such erpenment which would fit in with the concept that there are tiso general weas in the nephron one that is priminly concerned with reabsorption of solumn and potassium and one distal to that reabsorptite aren ·162 Renal Function and influenced by the osmotic gradient where a sizable proportion of Water is reabsorbed One may speculate that reabsorption of water is not complete to the point of osmotic equilibrium The more distal portion of the nephron may be assigned three specific functions the active secretion of potassium perhaps the active secretion of sodium and third the facultatue reabsorption of water li hite Even though the flud issuing from the proximal tubule were quite hypotonic it still would not necessarily follow that all of the sodium is gone Shannon Oh no! White There could still be more than enough sodium to account for what appears in the urme I dont see why uc have to have any secretion of sodium Shannon One does not have to assume it However it is difficult to be certain it does not occur md equally dificult to design an expenment to demonstrate the presence of such secretion White Until there is some reason for believing it does take place we can only speculate that it might Of course that is permissible Pitts I think you have cleared up the question I directed at Dr Afudge Dr Wesson I was not questioning necessınly isos mosity of the proximal flud but rather the argument that since the urine is isosmotic the distal tubule is doing nothing It may very well be ahsorbing water and whatever else it absorbs more or less isosmotically too As your own experiment would indicate it is absorbing some five to six cc of water what you get out in the unne is not what entered the distal tubule volume has been reduced by activity of the distal tubule and since osmotic pressure is unchanged solute must be removed. Both processes apprrently occur in osmotic diuresis Mfudge I would like to ask Dr Wesson how he explains the fact that in acute erperiments such as those with salıne infusions the increased excretion of sodium is almost always 25sociated with an increase in the amount reabsorbed The amounts filtered rerbsorbed and excreted all increase I do not see how the increased excretion can be erplained on the basis of saturation of a limited reabsorptive mechanism Sclhurt Perhaps our work may throu some light on this question We infused dogs with hypertonic sodnm chlonde and related the excretion to the load in the sense it can be done for glucose This was an attempt to see whether an active mechanism such as that postulated might be saturated ·Water Reabsorption 163 Iines) and sodium excretion (lower lines) expressed in millions per minute are plotted against the filtered loads of sodium expressed in millimols per minute The solid circles or triangles repre. sent the ascending loads as we rapidly infused the sodium chloride The first two values on the left are the control values After stop- TAT anumal No 26 where reabsorption is less during the phase of decreasing load A. INCREASING LOAD AO DECREASING LOAD 20 MM MIN 10F REABS. 20 5 20 EXCR. 0 15 20 LOAD MM MIN. FICURE 36 Figure 37 plotted as it umuting fact but in others it continues to rise continuously Shannon Is there anything pecular in the way the en on dog 26 was set up?

·164 Renal Function 26 мм. MIN REABS To 20 26 25 -24 5 20 24 EXCR. 5 15 20 10 LOAD MM MIN. FIGURE 37 Selkurt No, dog 25 shows a similar response We are wondering of this does not indicate some limitation of the transfer mechanism If it does, of course all the explanations that might be given here would have to include the possibility of changes in hormonal balance resulting from the hypertonic infusion Shannon What was the time relationship in the experiment? Selkurt This has to be done rapidly As you know, when you load with sodium, sooner or later the plasma sodium level begins to stabilize It is very difficult to increase the sodium load sufficiently o produce elevated plasma sodium concentrations Each experi nent takes about two hours about an hour for each phase We infuse five percent sodium chloride at a rate of about 10 cc er minute giving from 500 cc to 1 000 cc Then we half the infu ion and follow the descending relationship in the same manner Mudge Were any dogs studied twice? Selkurt No These dogs were anesthetized with pentobarbital ind sacrificed at the end Wesson Dr Selkurt and I have compared notes and we find that rur data agree very well He has been studying the earlier phases of one of these infusions and we have been interested in some of he later phases These are curves drawn from pooled data on ·Water Reabsorption 16S several dogs with several experiments on each dog (Figure 38) All of the data has been put together in order to gue some idea of the order of variation observed The figures do not represent the consistency with which data can be obtained in a single animal continuous am AO MTS ** KO MONTES PHASE 3 60 MINUTES > Aast USON WONIN 041 047903 150 MO 76 EU o con Contacte A 2.30 57 PCA CENT 110 + PCA CENTU en HCC22 LE23 A3612 MO MINUTES sto FICURE 38 Average 1 alues for une low (V) Sltration file (UCF) renal plasma flow (CPA) aort kltrafion fraction (FF) following crpansion of the extracellular Aud yolume in 18 esperiments on sit normal dogs Numbers are means and mean devia tions from the mein Bctore averaging se sndividual cuties the time scales of the experiments on each animal were adjusted so that their infections conesde with the average inflection comes for all experiments since repeated esperiments on a single animal will show far less variation Our procedure wis to administer sakne in the form of Locke's solution, and as fast as the animl excreted water and electrolyte in the unne se put back into it more flusd of the same volume and composition as the urine so that plasma volume and composition remained essentially unchanged from the beguoning to the end of the crpermes ·166 Renal Function about sixty minutes after beginning the infusion we find that the increase in urine flow (V) is of the same order of magnitude, in general as the increase in creatinine clearance (wCr), but averages somewhat less Since thus urine is rich in sodium, most of the increased filtered sodium load has appeared in the urine Consequently, if we plot filtered load against reabsorption during this period, we find a fattening out, just as Dr Selkurt, suggesting a resistance to increased reabsorption If, however, one follows these dogs 6 or 8 hours after the infusion, then one finds a different picture The creatınıne clearance goes through a dip and about 5 hours PNA FILTEAED LOAD 10 OH EXCRETED 수아는 EXCRETED EXCRETED MEO/ MINUTES TOTAL REABSORPTION 70- CONTA 2 IN FUSION للطلابلد 400 CO 0 40 120 183 MINUTES 24 200 320 380 FIGURE 39 The filtered load of sodium (W/w PN, XCF) and rate of total sodrvm Teabsorption in two dogs (two experiments per dog) folkywing fast infusion of Lockes a + on The mterval betsveen Inad and reabsoryi son measures the magnitude of i pa np tubulir reabsorpt on B and C Clomerular than Eq/L The end to Figure 38 ·Water Reabsorption 167 after the beginning of the experiment has risen again very nearly to the maximum (Figure 35) The late unne fow, howeier is quite different from u hat it is at the peak of creatine clearance early in the expenment Figure 39 shows that sodium reabsorption was following a course to a considerable degree independent of the glomerular filtration rate At the points A B and C the plasma sodium conceptration and the filtration rate and therefore the filtered load all have essen tually the same magnitude let the rate of excretion of sodium at these points of equal load progressi ely diminishes from A to C CONT9 Test PSC 32 180- MOLTO 3 4440 120 A MO KAO 105 DO 1001 KUTATOM RATE 1PER CENT OF CONTROL DO 10 $ U Suow CC 60 WATES 100 so FIGURE 40 Leine sodsum concentration (Ex} sodium scabsorption per sosit of glofierular bitrate in care and nomunsi concentration of sodium in the total mabsolute Troy caluhred from the meaa values in the six longest experiment hy die same procedure for the con is in Figure 37 Lrine Box and filtration rate are repeated from Fogue 5 for con prison umbers are means and mesa deviatsons from the or ·168 Renal Function Reciprocally the rate of reabsorption of sodium at constant load was increasing What is probably a consequence of increasing tubular reabsorp hon of sodrum is the interesting observation that it was usual in our experiments to detect a transitory period when the urine is hypotonic to plasma (Figure 40) The urine may have an osmotic U/P raho as measued by freezing point as low as 085 Infusion of pitressin does not prevent hypotonicity With three semi independent variables filtration rate sodium reabsorption and water reabsorption it is hardly surprising that the ratio of sodium reabsorption to filtration rate (TNA/CF) and the ratio of sodium reabsorphon to water reabsorption (TNA/TH O) (Figure 40) show peculiar curves It is hard to escape the conclusion that elevation of the filtration rate represents an important means of excreting sodium in the dog The work of Ladd and Rarsz (Ladd M and Raisz LG Response of the normal dog to dietary sodium chlonde Am ) Physiol 159 149 (1949)) in our laboratory showed that 50 grams of sodium given by mouth to a 15 kilogram dog were at least 90% excreted in 20 hours The process of excretion was accompanied by a great rise in filtration rate and urine flow Furthermore this rate of salt ingestion for a week resulted in no change in weight Snce the available data for man does not indicate any comparable in crease in filtration rate under Isotonic salt lord one wonders whether a profound species difference does not exist and whether man may not rely for control of sodium excretion primanly on changing Teabsorptive capacity rather than changing load Shannon I want to comment on certain feeding experiments where the only thing measured was the weight of patients placed on high or low salt drets The patients returned to their initial weight about three days after the change in sodium load and it would appear that the time required for the patient to accommodate himself to the new situation in terms of excretory mechanisms is of that order though it depends in part upon the preceding salt in take Actually we were led to do those experiments because we observed pretty much what Dr Wesson has described here that is a five to ten percent expansion of extracellular fluid in the dog led to a five or ten percent dilution of plasma and that in turn was accompanied by increase in the glomerular filtration rate All varı ables returned to normal at about the same time we thought se had finally found a mechanism that was responsible for the control ·169 Water Reabsorption of volume of extracellular flud In setting up similar experiments in the human we were quite disappointed I think one ought to bring out the fact that these studies vere performed in what is commonly called a standard state and did not permit examination of such variables as posture mild exerc se sleep etc any or all of which may have important effects on the functions we were interested in It appeared likely however that extrarenal factors were of importance The extent of these is perhaps brought out very well by some data that Dr White collected some years ago It would be interesting if he told us about it White If one changes from standing up to lying down there is about a threefold increase in the rate of output of water and chloride a safold or sevenfold increase of the output of bicarbonate and about a 10 percent increase in glomerular filtration rate as measured by inulin or creatinine clearances and intermediate increases in the substances which are partly but not so nearly completely reabsorbed as is sodium This was done a long time ago we did not do sodmm and potassium but sulfate and phosphate increase 30 40 or 50 percent as does urea I must ask anyone who may consult the paper in which the findings are presented (White HL Rosen IT Fischer SS and Wood G H The influence of posture on renal activity Am J Physiol 78 185 (1926)) to disregard our labored attempts at interpretation and consider only the data I now think of it in this way with substances which normally are almost completely reabsorbed if you bring about even 1 slight increase in filtration rate as on changing from standing up to Iving down and if there is no change or a lesser increase in the amount reabsorbed there will be of course a great increase in the amount excreted while the substances which are intermediate as sulfate and phosphate will show intermediate degrees of change and sub stances which are not at all reabsorbed will show the least change These changes in outputs are the direct and immediate result of changes in filtration rate in the absence of immediate corresponding in the extent of tubulır reabsorption They do not mean that the capacity of the tubules to reabsorb has been overwhelmed they mcan simply that nothing has happened set to inform the tubules that sodium or chlonde is being excreted in ercessive amounts so they continue to reabsorb the same amounts as before I cannot behese that even a doubling of the filtration rate over tates the reabsorptue capacity of the tubules One can double the filtration rate in the dog for miny days by giving certain anterior lobe preparations but this does not deplete him of salt kuus tubules changes ·170 Renal Function will soon increase their reabsorption so that the amount excreted is essentially the same as before the filtration was doubled The increases in salt output seen with pure increases in filtration rate do not last very long When a man lies down he begins to put out 3 times as much salt as while standing but so far as I know there is no evidence that one goes into any meastırable negative sodium balance merely by going to bed for a week although there is negative balance for the first few hours The sequence of events is that on lying down one quickly gets to the point where the plasma sodium level drops a little bit, the tubules recognize this and start taking back a little more sodium I dont know how the tubules know, but they will reabsorb the amount they need to reabsorb to keep plasma levels about constant if they didn't, you would soon die If they fail for a short time, as they do when filtration rate is first increased, to reabsorb sodium in the proper amount, the plasma level will soon fall and that signals them, through hormonal influ. ences or in some more direct way, to increase the amount taken back Fluctuations in filtration rate are not the primary cause of fluctuations in reabsorption Reabsorption continues unchanged for a while after filtration rate is increased it is the resultant slight drop in plasma level which soon arouses the tubules to increased reab sorption The change in plasma level need not be great, just as the change in plasma hydrogen ion concentration necessary to effect changes in respiration need not be great A further point is that such changes in plasma level in order to be effective, must be acutely produced, the rate of change may be the decisive factor The plasma sodium level today may be 4 percent higher than a week ago with no change in output, but so may the plasma hydrogen ion concentration show changes that are great from one week to the next without any change in respiratory volume, although such changes acutely produced might show great effects To sum marize these remarks while it is trie, in the long run that changes in filtration rate are accompanied by corresponding changes in the amount of sodium reabsorbed the changes in reabsorption are not the direct result of the changes in filtration but an indirect result brought on by the slight change in plasma level which is the direct result of changed filtration rate with temporarily unchanged reabsorption Berliner There is one thing about this that disturbed me as I understand it Dr Wesson's hypothesis is that as long as there is any excretion of sodium in the unne there is a saturation of some mechanism ·Water Reabsorption 171 Wesson That would be our viewpoint with the qualification that ercretion should be greater than in trace amounts and not caused by non reabsorbable ansons Berliner You can explain that with the hypothesis of reabsorptron by tuo mechanisms -one varying with the amount of filtered the other with a fired capacity Entirely aside from that it would seem to me that this would be a unique situation in renal physiology if such were the case that 15 of sodium were ercreted on when the reabsorptive capacity is saturated The measurement of Tm falls I think in the category of what Dr Shannon calls finding out what tncks the kidney can do Actually if we consider all the various reabsorptive mechanisms characterized by measurable Tms there is no reason to bebere that any of them except possibly sulfate (Dr Pitts probably knows a lot more about this than I do is aturated when the substance in question begins to be excreted in le urine I see no particular reason why the situation should be fferent in the case of sodium especially when we have to force ourselves and force the datı quite a bit to make it come out that way Have you any comments on that Dr Pitts? Pitts I am becoming convinced that I dont know anything about sodium I have been working on it for several years and i know much less than I thought I did when I sturted Berliner When amino acids phosphate and even glucose appear in the urine it is usually not because the Tms are saturated It is Very strikingly true of unc acid I dont know why we should expect something different in the reabsorption of sodrum whether there is a Tm for sodrum or not Bott Jou say that includes glacose? Berliner Most glucose excretion occurs at those plasma glucose concentrations examined only in "atration experiments The indi vidual who puts out a little glucose after 2 meal does not do so because hus Toi is saturated Usually we expect people to spill glucose at a les el of 175 200 mg percent and calculating from the average filtration rate and glucose Tm a normal individu should not spill until his les el got to 300 Actually ta saturate the glucose reabsorptive capacity you have to go up to 400 mg percent Putts One thing a hich I might add to confuse the issue somer hat is the fact that if you load 2 normal dog with sodium chloride by infusing a 35 percent solution at 10 ce per minute and measure his capacity to eliminate or expressed in the other way his capacity to absorb take out the adrenals and do the same erpenment a couple weeks later the adrenalectomized animal sorb more ·172 Renal Function than the normal animal He excretes less and thus has excretory tolerance Luetscher We heard from Dr Taggart that Tm is not sacred and fired point that it has to stand still under all con Not only may there be vanations below a certain load, but es maximum may change with the action of various stimuli on ti I think that we should turn more of our thought to some factors which somehow got down at the end of the list of var for our consideration Endocrine secretions are known to in renal functions and it seems almost certain that there are ch in endocrine function during experiments which involve str of the types described We still need a great deal of fundam data, and we need better methods for the assay of endocrine ? tion so that we can measure these factors which can shift the and remove the fixed point of calculations Pitts As the Chairman of this group I want to thank all of for your participation in the conference Your contributions to have made it a success

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ADRENAL CORTEX
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INFANCY & CHILDHOOD
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