o... ....,,, .’ .- av.-. .- .... -~.-. ..__,.. <_ ...- ..... EN‘FF 337.5% GL‘LTEGFF: SF L‘LF FLFLFLFRLF'RC FL“ “FEW? EN 3‘? WELLS: F3 FLULD LLLLLL LL LL LLLLLLL LL‘LLLLLLLLLLL ---- i I O V; I A . _ 4 v - I E . ’ A L .,.a ’ 2 I . ~ V This is to certify that the thesis entitled Investigation of Antinatriferic Activity in Hemodialysis Fluid, Blood, and Plasma Ultrafiltrate presented by James Murray Terris has been accepted towards fulfillment of the requirements for Ph .D . degree in PhYSiOlogy LLWJ/L/gefié A Major professor Date February 19, 1975 0-7639 M8 & SONS L 330x BLNLLRLLLL -~BR}1 L’lY EI‘HJ-Lt: ‘ smmom. women m ABSTRACT Cb\\ INVESTIGATION OF ANTINATRIFERIC ACTIVITY IN HEMODIALYSIS FLUID, BLOOD, AND PLASMA ULTRAFILTRATE By James Murray Terris An investigation of the literature of the last few years dealing with the renal excretion of sodium reveals that at the present time there are a number of theories available to describe the manner in which the excretion of this ion is regulated by the kidney. Most of the hypotheses deal with alterations in intrarenal hemodynamics. However, evidence since l960 has been accumulating suggesting the existence of a presently unidentified natriuretic hormone which appears in the urineand/or blood of man, rats, cows, dogs, and cats, under certain conditions of extracellular fluid volume expansion. Plasma demonstrating natriuretic activity has also been reported to inhibit sodium transport across frog skin and toad bladder membranes (anti- natriferic activity). Whether the material responsible for these activities is a single substance, or has a definite physiological function. has not yet been determined. I Antinatriferic and natriuretic activities have been reported to occur in the plasma of humans with chronic renal disease. Since the possibility exists that an antinatriferic substance might be dialyz- able in vivo from humans, as appears to be the case with dogs, a study James Murray Terris was undertaken to determine if antinatriferic activity could be iso- lated from plasma ultrafiltrates and spent hemodialysis fluid following maintenance hemodialysis of patients with end-stage chronic renal failure. Methods are described for processing and concentrating these large volumes of fluid which can be as much as 180 liters in a given dialysis treatment. The assay employed to detect this substance uti- lized isolated ventral frog skins held in a Ussing-type chamber. Potential difference and short circuit current across the membranes were alternately monitored before and after addition of a test sample. There was no reproducible antinatriferic activity observed in the specimens obtained from the patients in this study. All patients had been maintained by hemodialysis for 2 weeks or more, and none demonstrated a fluid retention greater than 5% of their body weight since the previous dialysis treatment. Plasma samples with and without trichloroacetic acid deproteinization were studied. In addition, dialysis fluid samples were concentrated 3600-fold by ultrafiltration and plasma ultrafiltrates obtained from the artificial kidney were similarly concentrated 8 to 27 fold and assayed for antinatriferic activity. It is concluded that patients who have a fluid retention which is less than 5% of their body weight, and who are undergoing chronic maintenance hemodialysis, do not possess measurable quantities of a previously described natriuretic hormone which others have sug- gested has antinatriferic activity. James Murray Terris In addition to the uremic human studies, dogs were expanded with saline to examine the antinatriferic activity of expanded plasma. No reproducible antinatriferic activity was demonstrated in expanded dog plasma with or without trichloroacetic acid deproteinization. Also, evidence is presented suggesting that direct application of trichloro- acetic acid treated samples to frog skin membranes may yield erroneous results due to the presence of small amounts of residual trichloro- acetate anion not removed by conventional extraction procedures. From these studies it is concluded that the natriuresis seen in acute expansion of the extracellular fluid volume with isotonic saline cannot be attributed to an antinatriferic substance. INVESTIGATION OF ANTINATRIFERIC ACTIVITY IN HEMODIALYSIS FLUID, BLOOD, AND PLASMA ULTRAFILTRATE By James Murray Terris A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Physiology 1974 DEDICATION Yb my uncomplaining wife, whose patience, hard work, and unselfish dedication to her family during my years as a graduate student were my source of strength when times were tough; to Jimmy, John, and Jason, who make life worth its struggles. ii ACKNOWLEDGMENTS The author wishes to express his most sincere thanks to Dr. Ronald Easterling, Associate Professor of Internal Medicine and Director of the Hemodialysis Unit, University Hospital, Ann Arbor, Michigan, for his cooperation, suggestions, and assistance with the human studies of this thesis. A very special thanks is also extended to those twelve patients who consented to participate in this project. I would also like to express my gratitude to the members of my committee, Drs. Burnell Selleck, Jerry Scott, William Frantz, w. Doyne Collings, and David Rovner for their critical review of this manuscript and their suggestions for its improvement. TABLE OF CONTENTS Page LIST OF TABLES ....................... . vii LIST OF FIGURES ........ p . . . . . . . . . . . . . . . x INTRODUCTION ......................... l REVIEW OF THE LITERATURE ................... 6 A. Circulating Hormonal Factors in Volume Expanded States ...... . . ................ 6 Chemical Characteristics of Natriuretic- Antinatriferic Factors ..... . ........ ll Source of Hormonal Natriuretic-Antinatriferic Factors ...... . ............... 18 Possible Natriuretic Hormone Involvement with Reduced Nephron Populations and Renal Disease. . . 22 B. Physical Factors Affecting Tubular Reabsorption of Sodium Independently of Factor I and Factor II. . . . 24 PURPOSE OF INVESTIGATION ................... 30 METHODS. , .......................... 31 A. Sodium and Potassium Determination ......... . 31 B. Chloride Determination ................ 36 C. Glucose Determination ................ 38 D. Ammonium Ion Determination .............. 43 E. Ether Extraction of Trichloroacetic Acid (H-TCA) from Samples ..................... 48 iv TABLE OF CONTENTS-~c0ntinued RESULTS A. B. C. D. . Removal of TCA by Fractionation on Sephadex Resin. . . Short Circuit Current Determinations . . ...... . Column Chromatography ................ . Collection and Handling of Uremic Human Specimens. . . Ultrafiltration Procedure .............. . Acute Volume Expansion with Dogs . ......... . Sample Preparation for Short Circuit Current Measurement. .................... Fractionation of Specimens on Sephadex Resin . . . . Effects of ADH, TCA', and Ammonium Ion on Frog Skin Short Circuit Current, Membrane Potential, and Resistance . . ................... Human Uremic Studies . . . ..... . . . . . . . . Acute Volume Expansion Experiments with Dogs . . . . DISCUSSION .......................... SUMMARY AND CONCLUSIONS ................... APPENDICES. ......................... I. II. Effects of lyophilized uremic fraction IV plasma samples, with and without H-TCA deproteinization, on frog skin short circuit current (SCC), membrane potential (MP), and resistance (R) . . . . . . . . . Effects of lyophilized uremic fraction IV from di— alysis fluid concentrates on frog skin short circuit current (SCC), membrane potential (MP), and resis- tance (R) ..... . ....... . . . . . Page 69 74 79 83 88 93 100 106 106 121 139 148 I63 I74 T76 176 I79 TABLE OF III. IV. VI. VII. VIII. BIBLIOGRAPHY ....................... . . CONTENTS--continued Effects of lyophilized uremic fraction IV from plasma ultrafiltrates, obtained from the artificial kidney, before and after concentration by ultrafil« tration at 4°C on frog skin short circuit current (SCC), membrane potential (MP), and resistance (R). . Effects of H-TCA deproteinized dog plasma samples from four of the 3% total body weight expansion experiments on frog skin short circuit current (SCC), membrane potential (MP), and resistance (R) befor fractionation on Sephadex GZSF resin ..... . . . . . Effects of lyophilized fraction IV from H-TCA depro- teinized dog plasma samples obtained in the 3% total body weight expansion experiments on frog skin mem- brane potential (MP), short circuit current (SCC), and resistance (R) ...... . ........... Effects of H-TCA deproteinized dog plasma samples from the 6% total body weight expansion experiments on frog skin membrane potential (MP), short circuit current (SCC), and resistance (R) before fractiona- tion on Sephadex GZSF resin . . . . . . . . . . . . . Effects of lyophilized fraction IV from H-TCA depro- teinized plasma samples from dog 9 (6% total body weight expansion) following fractionation on Sephadex GZSF resin .......... . ...... Effects of lyophilized fraction IV from non H-TCA deproteinized dog plasma samples from the 6% total body weight expansion experiments on frog skin mem- brane potential (MP), short circuit current (SCC), and resistance (R). . . . . . . . . . . . . . . . . . vi Page 180 182 184 187 189 190 193 TABLE 10. 11. LIST OF TABLES . Effects of various 'natriuretic hormones' and other known substances on renal sodium excretion and sodium transport across amphibian membranes ................ . Summary of the effects of various treatments on natriu- retic-antinatriferic activities obtained during carotid artery occlusion in cats and from humans with chronic renal disease ..................... . Procedure for the preparation of the sodium and potassium solutions utilized for the preparation of standard curves . Procedure for the preparation of the glucose standard solutions utilized for the preparation of standard curves . Determination of the molar extinction coefficient (5) of HwTCA in distilled water at 2l0 mu ............ . Composition of the frog buffer utilized throughout the studies concerned with short circuit current measurements on frog skins ......... . ............ . Summary of the experimental conditions under which the 3% (total body weight) 0.154 M NaCl volume expansion experiments on dogs were carried out ........... . Summary of the experimental conditions under which the 6% (total body weight) 0.154 M NaCl volume expansion experiments on dogs were carried out .......... . Effects of ADH on frog skin short circuit current, mem- brane potential, and resistance ............. Preparation of Na-TCA samples employed to determine the effects of TCA'on frog skin short circuit current, mem- brane potential, and resistance ............. Preparation of samples employed to determine the effects of ammonium ion on frog skin short circuit current, mem- brane potential, and resistance . ............ vii Page 12 13 33 39 59 79 97 103 124 127 134 LIST OF TABLES--c0ntinued 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. Blood pressure data and change in dry body weight of patients participating in this study ........... Summary of the values of the short circuit current during the control period (C), following addition of lyophilized fraction IV from uremic samples (E), and during the re- covery period (R) after replacement of the test sample by fresh frog buffer ..... . . . . . . ...... . . Summary of the effects of lyophilized fraction IV from uremic specimens on frog skin short circuit current (SCC), membrane potential (MP), and resistance (R) . . . . . . . Results of ammonium ion determinations on lyophilized fraction IV samples obtained from human uremic specimens following elution of specimen from Sephadex GZSF resin with 10 mMolar ammonium acetate ............. Initial volumes and magnitude of concentration of plasma ultrafiltrates obtained from the artificial kidney. . . . Summary of chemical analyses of plasma samples obtained from dogs prior to (CONTROL) and following a 3% total body weight volume expansion with 0.154 Molar NaCl. . . . Summary of the values of the short circuit current during the control period (C), following the addition of unfrac- tionated H-TCA treated plasma samples from the 3% ex- panded dogs (E), and 20-30 minutes after replacing the test sample by fresh frog butter (R) ........... Summary of the effects of unfractionated H-TCA treated plasma samples from the 3% expanded dogs on frog skin short circuit current (SCC), membrane potential (MP), and resistance (R) ...... . ............. Summary of the effects of lyophilized fraction IV from H-TCA treated plasma samples from the 3% expanded dogs on short circuit current (SCC), membrane potential (MP), and resistance (R). . . . . . ............. . . . Summary of the values of the short circuit current during the control period (C), following the addition of lyophilized fraction IV from H-TCA treated plasma samples from the 3% expanded dogs (E), and 20~30 minutes after replacing the test samples by fresh frog buffer (R) . . . viii Page 140 141 144 146 147 149 150 151 153 154 LIST OF TABLES--continued 22. 23. 24. 25. 26. 27. Summary of chemical analyses of plasma samples obtained from dogs prior to (CONTROL) and following a 6% total body weight volume expansion with O.l54 Molar NaCl. . . . Summary of the values of the short circuit current during the control period (C), following the addition of unfrac- tionated H-TCA treated plasma samples from the 6% ex- panded dogs (E), and 20-30 minutes after replacing the test samples by fresh frog buffer (R) .......... Summary of the effects of unfractionated H-TCA treated plasma samples from the 6% expanded dogs on frog skin short circuit current (SCC), membrane potential (MP), and resistance (R). . . ................. Summary of the values of the short circuit current during the control period (C), following the addition of lyophilized fraction IV from the H-TCA treated plasma samples from dog 9 (E), and 20-30 minutes after replacing the test samples with fresh frog buffer (R) ....... Summary of the values of the short circuit current during the control period (C), following the addition of lyophilized fraction IV from dogs lO-lS (non H-TCA treated plasmas)(E), and 20-30 minutes after replacing the test samples by fresh frog buffer (R) ........ Summary of the effects of lyophilized fraction IV from dogs lO-lS (non H-TCA treated plasmas) on frog skin short circuit current (SCC), membrane potential (MP), and resistance (R) ...................... ix Page 155 156 158 159 161 162 FIGURE 10. 11. 12. 13. 14. LIST OF FIGURES . Standard curves for sodium and potassium as determined with a Baird—Atomic Model KY—3 combination clinical and research filter flame photometer .......... . Standard curve for glucose as determined with a Beckman Model DB spectrophotometer at 400 mp. . . . . ..... . O'Brink modification of the Conway microdiffusion unit. . Summary of repeated ether extractions of HvTCA from cat plasma samples ............... . . . . . . . Ultraviolet absorption spectrum of l mMolar H—TCA in distilled water and 0.09 N HCl ............. . Standard curves for H—TCA in approximately 0.6 N HCl at 205 mu and 210 mu .................. . Standard curves for H—TCA in distilled water at 205 my and 210 mp ....................... . Ether extraction of H—TCA from dog plasma with and with— out acidification with HCl after extraction number 2. . . Ether extraction of Na—TCA added to dog plasma ultra- filtrate ........................ Ether extraction of H-TCA from dog plasma. TCA“ ab— sorption at 2l0 mu was corrected for plasma ultrafil» trate absorption at 210 mp ............... Ether extraction of H-TCA from dog plasma ....... Results of fractionation of 10 ml of Na—TCA frog buffer on Sephadex GZSF resin at 4°C ............. Frog skin short circuit current setuup ......... Construction of Sephadex resin column ......... Page 35 42 45 51 53 56 58 61 64 68 71 73 77 82 LIST OF FIGURES--continued 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. Diagrammatic representation of attachment of patient to hemodialyzer ...................... Construction of the ultrafiltration chambers employed to concentrate dialysis fluid and plasma ultrafiltrate and prepare protein free dog plasma ultrafiltrate . . . Protocol for dogs 2-8 receiving an initial 3% (total body weight) volume expansion with 0.154 M NaCl . . . . Protocol for dogs lO-lZ receiving an initial 6% (total body weight) volume expansion with O.l54 M NaCl . . . . Protocol for dogs l3-l5 receiving an initial 6% (total body weight) expansion with 0.154 M NaCl ........ Elution pattern at 280 mu of a sample of plasma from patient GS ....................... Elution pattern at 280 mu of a sample of plasma from patient CH. Also included is the elution pattern of plasma ultrafiltrate, obtained from the artificial kidney, before and after an 8-fold concentration by ultrafiltration .................... Elution pattern at 280 mu of plasma ultrafiltrate from patient GS after a l5-fold concentration by ultrafil- tration ........................ Fractionation of dialysis fluid concentrate from patient GS ....................... pH elution pattern of plasma and plasma ultrafiltrate obtained from the artificial kidney, before and after an 8-fold concentration, from patient CH ........ Major components of resin eluate samples demonstrating osmotic activity following fractionation of plasma ultrafiltrate concentrate (concentrated l5-fold by ultrafiltration) obtained from patient GS ....... Calibration of the 2.5 X 95 cm column packed with Sephadex GZSF resin utilized in the studies described in this thesis ..................... Effect of ADH on frog skin short circuit current (SCC), membrane potential (MP), and resistance (R) ...... xi Page 86 90 96 99 102 108 110 112 115 118 120 123 126 LIST OF FIGURES--continued 28. 29. 30. 31. 32. Effects of TCA on frog skin short circuit current and resistance ....................... Effects of TCA' on frog skin membrane potential . . . . Effect of 0.9 mMolar Na-TCA on frog skin short circuit fiugrent (SCC), membrane potential (MP), and resistance R .......................... Effect of 2.0 mMolar ammonium ion on frog skin short circuit current (SCC), membrane potential (MP), and resistance (R) ..................... Effect of 0.4, 0.8, and 2.0 mMolar ammonium ion on frog skin short circuit current, membrane potential, and resistance ....................... xii Page 129 131 133 136 138 Flu h 1 .1. (6 Fl 1‘ INTRODUCTION In the maintenance of the volumes of the various fluid compart- ments of the body, the most important inorganic constituent is the sodium ion. The volume of the extracellular fluid compartment is determined primarily by the total amount of osmotically active solute which it contains. Since sodium and chloride are by far the most abundant osmotically active solutes in the extracellular fluid, and since changes in chloride are largely secondary to changes in sodium, the amount of sodium in the extracellular fluid is the most important determinant of the extracellular fluid volume. Therefore, the mechan- isms that control sodium balance are the major mechanisms 'defending' this volume. Prior to 1961 ideas concerning sodium excretion by the kidney were dominated by considerations of glomerular filtration rate (GRF or factor I) and the adrenal hormones, primarily aldosterone (factor II). Selkurt et al. (153), in 1949, showed that reduction of the GFR to 63% of the control value resulted in almost total sodium reabsorp- tion, an observation confirmed more recently by others (22,101,107). Simpson and Tait (156), in 1952, isolated a new adrenal steroid which was to be called aldosterone and was shown to be the most potent of the naturally occurring sodium retaining steroids. With these observa- tions it was felt that the primary factors concerned with sodium control by the kidney were established. 1 One of the first indications that there is a renal mechanism which operates independently of factor I and factor II for the excre- tion of excess sodium evolved during the period from 1941 to 1960. Investigations by several workers (6,63,75,143) demonstrated the ex- istence of a phenomenon now referred to as 'mineralocorticoid escape'. When deoxycorticosterone acetate (DOCA) or aldosterone was chronically administered to humans (6,143), it was noted that there was a dramatic fall in sodium excretion. Following an initial period of sodium retention and weight gain (fluid retention), weight gain ceased and sodium excretion returned approximately to control levels despite con- tinued administration of these compounds. Davis and Howell (63) demonstrated, in the dog, that even with an increased GFR there was only a transient retention of sodium with continued use of DOCA. DeWardener et al. (65) established another line of evidence for a sodium excreting factor not related to GFR or mineralocorticoids when they demonstrated that saline diuresis could occur in the presence of a reduced GFR during acute volume expansion with saline infusion in dogs treated with 9-alpha-fluorohydrocortisone. With the findings that natriuresis could still occur in volume expanded states despite an appreciable fall in GFR and high mineral- ocorticoid levels, some other mechanism, or 'third factor', had to be sought which would presumably affect tubular reabsorption of sodium independently of factor I and factor II. That such a 'third factor' exists is not disputed. The nature of this factor, on the other hand, has been the subject of extensive studies, prompting one investigator to ask "Which factor j§_third (l6)f? The result of these studies has led to the conclusion that the renal handling of the sodium ion is a very complex interrelationship of many factors, both physical and hormonal, and that the 'third factor' natriuresis seen following extra- cellular fluid volume expansion may in fact be the result of changes in many factors. During the early 1960's investigations began to suggest that this 'third factor' natriuresis observed in volume expanded states may in part be due to an unidentified 'natriuretic hormone' ('NH'). One study suggested that this 'NH' may induce natriuresis by inhibiting active reabsorption of sodium in the nephron by inhibiting Na-K-ATPase (99). However, this is not a consistent finding (97,166). Other studies have demonstrated that plasma obtained from volume expanded animals and humans not only induces natriuresis but also inhibits sodium transport across frog skin and toad bladder membranes (an anti- natriferic activity) (19,20,21,32,33,35,49,53,137). In addition, using an Ussing-type short circuit current preparation, Nutbourne et a1. (37) demonstrated that following volume expansion in the dog there was a reduction in sodium transport across frog skins incorporated into the dog's circulation. The expansion in these studies was performed with blood equilibrated with that of the dog undergoing expansion. Concomi- tant with the decreased sodium transport in the frog skin there was an increase in urinary excretion of sodium in the expanded animal. These observations, and others to be discussed in a later section, suggested that the natriuretic and antinatriferic activities observed in plasma following volume expansion may be due to a single substance. The present studies were designed to further evaluate the con- tribution of a 'natriuretic hormone' (with antinatriferic activity) to the third factor natriuresis seen in volume expanded states. A natriuretic and antinatriferic activity has been demonstrated to be present in the serum of chronically uremic humans (19,20,21), who are characteristically volume expanded. Since an antinatriferic substance has been reported to be dialyzable ifl_V1VO from dogs exhibiting natri- uresis as a result of saline expansion (36), the possibility that the material observed in humans with end-stage renal failure might also be dialyzable jn_vivo was considered. If the material is dialyzable from humans undergoing maintenance hemodialysis, plasma ultrafiltrates and spent dialysis fluid obtained from the artificial kidney could serve as an unlimited source for this substance. With such a source suffi- cient quantities of material might be obtained permitting its identifi- cation. The newness of the concept of this 'natriuretic hormone', and the manner in which it was viewed as late as 1969, is perhaps best described by Cort and Lichardus (55) in reference to a symposium held in June of that year at Smolenice Castle in Czechoslovakia: "Most symposia tend to stress conceptual and theoretical advances in a delimited field of research. The present meeting had quite different goals--to discuss whether a given field exists or not and in detail the methods applied to try to find out." The following discussion first reviews the literature which has provided evidence for the existence of this 'natriuretic hormone' in situations of extracellular fluid volume expansion. This is followed by a summary of the information currently available which suggests that this same 'natriuretic hormone' may also be antinatriferic with regard to sodium transport across toad bladders and frog skins. Finally, a consideration of the source of this natriuretic—antinatri- feric substance is given, in addition to a review of some of the physical factors which have been shown to contribute to the 'third factor' natriuresis seen with volume expansion. REVIEW OF THE LITERATURE A. Circulating Hormonal Factors in Volume Expanded States The first evidence suggesting a circulating sodium excreting hormone in volume expanded states evolved from the experiments of DeWardener et a1. (65). These workers demonstrated that natriuresis occurred not only in dogs volume expanded with isotonic saline, but also in an isolated kidney being perfused with blood from the expanded animal. This transmitted natriuresis also occurred in a cross perfused intact recipient animal. Administration of exogenous aldosterone ruled out mineralocorticoid dilution (which could cause natriuresis by decreasing sodium retention), and a complex system of perfusion pres- sure regulation was employed to keep the perfused intact animal at a constant body weight. There was thus no volume stimulus from cross circulation in the recipient animal. Changes in hematocrit, plasma prote1n oncotic pressure, and serum sodium were also not responsible for the observed natriuresis. Levinsky (103) and Levinsky and Lalone (104) later substantiated these findings. Johnston and Davis (91) and Johnston et a1. (92), with cross perfusion experiments performed on intact dogs, also provided evidence for the involvement of a hormonal substance in the natriuresis accompany- ing saline loading, In a control study blood was cross circulated between DOCA escape donors and normal recipients. There was no sig- nificant increase in sodium excretion in the recipient dogs due to the cross circulation itself. When the donor dogs were expanded with 1 liter of 0.9% saline there was a significant increase in sodium excretion in the recipient dogs. In these experiments it was also noted that there was a significant increase in sodium excretion during periods of cross circulation when the filtered load of sodium in the recipient was significantly decreased by aortic constriction above both renal arteries. Using a Ringer Locke solution containing canine red blood cells and 6% bovine albumin to expand a donor dog, Lichardus and Pearce (114) also demonstrated a natriuresis in a cross perfused recipient animal when the GFR was decreased by clamping the arterial perfusion line. This recipient natriuresis was not due to changes in hematocrit, oncotic pressure, or increased renal plasma flow. Similar results were reported by Bahlmann et a1. (9) in cross circulation studies with dogs in which the donor dog was expanded with whole blood. Martinez-Maldonado et al. (123) report a 38% decrease in proximal tubule reabsorption, as measured by micropuncture techniques, when plasma from saline expanded animals was perfused into isolated kidneys. Kaloyanides and Azer (94) demonstrated an increased sodium excretion in an isolated kidney being perfused with blood from a second dog under- going volume expansion with equilibrated blood in a reservoir to which had been added 5% albumin in saline. The natriuresis in these studies occurred in spite of a decreased renal blood flow, decreased arterial pressure, and in the absence of any change in plasma protein concen- tration or packed red cell volume. The donor animal had been pre- treated with DOCA. As a result of these cross circulation experiments the use of the term 'natriuretic activity' began to make an appearance in the literature in reference to a potential substance in the blood respons- ible for the natriuresis being observed. Rector et al. (142), in 1968, became one of the first to Fefer to this postulated circulating substance as 'natriuretic hormone' ('NH'). Stronger evidence for the existence of a 'natriuretic hormone' is provided by studies snufl1 as those of Sealey and Laragh (151) and Sealey et al. (150). Plasma and urine from salt-loaded humans and sheep, as well as patients with primary aldosteronism and essential hypertension, demonstFated an inhibitory effect on sodium reabsorption in rats. Similar results were obtained by Kruck (100) utilizing dialysates and ultrafiltrates prepared from urine of orally hydrated humans. Glomerular filtration rate, renal plasma flow, and blood pres- sure were not affected. Buckalew and Lancaster (33) demonstrated in dogs undergoing DOCA escape that a substance appeared in plasma ultra- filtrates which inhibited the short circuit current (SCC) in toad bladders. It was also noted that variations in urinary excretion of sodium coincided with oscillations in the inhibitory activity of the plasma samples. These authors suggested that a natriuretic hormone (with antinatriferic activity) may thus be involved in the day-to-day regulation of sodium balance. Clarkson et a1. (44). using whole blood to volume expand dogs. demonstrated that renal tubule fragments incubated in plasma obtained after expansion were less able to maintain a sodium gradient or accumulate para-aminohippurate (PAH) than when incubated in plasma obtained before expansion. PAH transport has been shown to be a sodium dependent process (19). Individuals carrying out the transport studies did not know the identity of the plasma samples. Extracts of urine were prepared by Clarkson and DeWardener (43) from salt-depleted and salt-loaded humans. The extracts from salt-loaded sobjects in- hibited sodium transport in tubules prepared from rabbit kidneys. Extracts from salt—depleted subjects had no effect. Finally, Lichardus and Nizet (113) expanded the blood volume of dogs with whole blood from a donor dog in which the hematocrit and protein concentration were matched. Prior to the expansion, one of the dog's own kidneys was tied off, transplanted to the neck and anastomosed to the carotid artery and jugular vein in order to eliminate afferent and efferent renal nerves. Although there were no significant changes in GFR, renal blood flow, post-glomerular hematocrit, or plasma protein concentration following the transfusion, there were moderate but sig- nificant increases in urine output and renal sodium excretion in the transplanted kidney. The animals had been pretreated with DOCA and ADH prior to the experiment. The authors interpret the results as being consistent with the proposition that a specific factor ('NH') plays a role in the mechanism of natriuresis after blood volume expan- sion. 10 Although a considerable number of investigators have reported evi- dence in favor of 21 circulating salt excreting hormone during periods of blood volume expansion, a few have reported negative findings (7,10, 18,147,170). However, it might be of interest to note that with only two exceptions (147,170), all of these experiments reporting negative findings involved attempts to elicit the substance in rats. Wright et al. (170) failed to show the presence of a dialyzable inhibitor of proximal sodium reabsorption from plasma of dogs undergoing DOCA escape, and Schrier et al. (147) failed to demonstrate it with hypo— tonic volume expansion in dogs. Lichardus and Ponec (116), commenting on their own negative find- ings in the rat, suggest that a species difference may exist and that the natriuretic response mechanism in the rat could be more dependent on a mutual interplay of the hormonal and physical factors than in the dog. They noted that diuresis due to blood volume expansion with isov tonic or iso—oncotic blood in rats leads to a significant increase in plasma protein concentration. When the urine of a donor rat was re« turned intravascularly through a catheter connecting the bladder with the jugular vein, the protein concentration in the cross circulated blood to a rec1pient did not change. Under these conditions a signifi« cant diuresis and natriuresis took place in the recipient during donor expansion. Utilizing thlS procedure, Sonnenberg et al. (160) substan— tiated these findings in cross perfused rats. .-—l n...— 11 Chemical Characteristics of Natriuretic- Ahtinatriferic Factors Although the chemical nature of the natriuretic-antinatriferic factors studied is unknown, comparison of the available information regarding these factors reveals some interesting similarities. For example, a natriuretic activity has been reported to appear in the blood of animals during mineralocorticoid escape (32,33,35,l37), volume expansion in dogs (37), carotid artery occlusion in cats (49,53), and humans with chronic renal disease (19,20,21). This same blood has also been shown to be antinatriferic when tested on frog skins and toad bladders in these experiments. Table 1 lists these various natriuretic- antinatriferic substances and their effects on renal sodium excretion (natriuretic-antinatriuretic activity) and sodium transport in frog skins and toad bladders (natriferic-antinatriferic activity). Also included are other known compounds or classes of compounds which have been considered as possible candidates for 'NH'. From the table it can be seen that the only substances which demonstrate natriuretic as well as antinatriferic activity are the so-called natriuretic hormones. The possibility exists, of course, that there are actually two substances involved, one of which is natriuretic and the other antinatriferic. Further comparisons, however, tend to suggest that if the activities are not due to a single substance, they are at least due to similar substances. 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Bricker (27), however, does state that he is able to inactivate the substance with a Specific peptidase, although the nature of this peptidase was not revealed. The human substance is not, therefore, completely immune to enzymic degradation. Cort et al. (49,58) and Cort (47) have concluded that the sub- stance released during carotid artery occlusion in cats is a peptide, small enough not to be precipitated by trichloroacetic acid (H-TCA). They suggest that the substance has at least one basic amino acid since both natriuretic and antinatriferic activities are destroyed by trypsin. The partial loss of both activities following incubation with aminopeptidase suggested a free terminal group, and the partial loss of both activities following incubation with chymotrypsin sug« gested one or more aromatic amino acid residues existed somewhere in the structure. Incubation of nondeproteinized plasma at 370C resulted in complete loss of both activities within 20 minutes. A similar lnCU* bation at 0°C had no effect. The fact that there were parallel changes in natriuretic and antinatriferic activities with the treatments described above and listed in Table 2, suggested that a single substance was responsible for both of’ the observed activities. Although no known purified naturally occurring substance possesses both activities, the observa— tion that synthetic (4-leu)-oxytocin (152) does possess all the proper« ties attributed to ‘NH' provides evidence that such a naturally 15 occurring substance could exist. In cats, (4-leu)-arg-vasotocin is even more natriuretic than (4-leu)-oxytocin (60), Furthermore, oxytocin inhibitors (i.e., 2-0-methy1tyrosine-oxytocin) inhibit not only the natriuretic action of oxytocin, but also the natriuretic response to carotid artery occlusion (144). This suggests that the 'NH' in cats may be similar in structure to oxytocin. Comparing the natriferic (enhanced sodium transport in frog skin and toad bladder) effects of known concentrations of oxytocin with the antinatriferic activity of carotid artery occluded cat plasma, Cort et al. (58) estimate the maximum plasma concentration of this -1] to 10']5 antinatriferic substance to be 10 molar. The assumption made in arriving at this conclusion, which is open to criticism, was that one molecule of oxytocin and antinatriferic material have equal but opposite effects on frog skin sodium transport. Finally, use of dibenzyline to counteract circulating catecholamines had no effect on the natriuretic response to carotid artery occlusion or 20% blood volume expansion with 6% dextran in saline (51). This suggested that the natriuretic substance is not a catecholamine. Bricker et a1. (29) observed that, following Sephadex fractiona- tion of plasma from chronically uremic humans, one fraction was obtained that inhibited the accumulation of PAH by rabbit kidney cortical slices. A similar fraction from normal humans had no effect. Inter- estingly, the same fraction obtained from saline loaded dogs and cows also inhibited PAH transport whereas that from non-expanded animals did not. In subsequent studies, this same Sephadex fraction was shown to 16 inhibit the SCC in frog skin (20) and enhance sodium excretion in rat kidneys (19). These observations, and others listed in Table 2, suggest that the antinatriferic«natriuretic activities observed with acute volume expansion in animals and uremia in humans are due to substances which are at least similar. Utilizing Sephadex chromatography and ultrafiltration studies, Bourgoignie et al. (20) estimated the molecular weight of this human, cow, and dog natriuretic-antinatriferic material to be approximately 500 to 1000 Daltons. Other studies demonstrated that the natriuretic activity was not destroyed by boiling or freeze drying (19,20,29), and, in contrast to Cort's findings in the cat, was also not susceptible to degradation by pronase and chymotrypsin. Boiling the active Sephadex fraction for 10 minutes at pH 10.5 destroyed the natriuretic activity in the rat assay (19). This treatment was not performed on the samples used in the frog skin and PAH studies. Also, these authors noted in the frog skin studies that the antinatriferic activity was stable for at least 4 weeks if stored frozen. If the specimen was kept at —80°C it could be stored for ten weeks before Sephadex fractionation without a detectable loss in antinatriferic activity. Ultrafiltration studies by Buckalew et a1. (36) led these authors to conclude that the molecular weight of an antinatriferic factor obtained from volume expanded dogs could not exceed 3000 Daltons. Fractionation on Sephadex G10 resin suggested that the minimum molecular weight was approximately 500 to 700 Daltons (34), although in further studies the molecular weight appeared to be less than 500 to 700 Daltons (37). Similar ultrafiltration studies with an antinatriferic 17 factor obtained during mineralocorticoid escape suggested that this material had a molecular weight less than 10,000 Daltons (35). No attempts were made to determine how much less. Of interest was the observation that the antinatriferic substance was dialyzable 1 vivo from dogs undergoing volume expansion with saline, as it suggests that it might also be dialyzable in_vivo from other species including humans. Martinez-Maldonado et al. (123) report that a natriuretic sub- stance obtained by them from saline loaded rats and dogs was small enough to be dialyzable, could be stored at 4°C, but was destroyed when freeze dried. Cort et a1. (56) describe a bovine material ob- tained by volume expansion of cows with dextran. From Sephadex chromatographic separation studies the molecular weight was estimated to be from 800 to 1000 Daltons. Sealey et al. (150) and Sealey and Laragh (151) report that a natriuretic substance obtained in the urine from salt—loaded humans appeared to have a molecular weight between 10,000 and 50,000 Daltons based on ultrafiltration and gel filtration studies. Attempts to demonstrate a lower molecular weight sodium transport inhibitor proved to be unsuccessful due to technical problems. The material was not destroyed by boiling, a finding consistent with Bourgoignie and Cort, but, in contrast to Cort, Sealey reports that her material is destroyed by H-TCA. In summary, a few studies have attempted to characterize the substances responsible for the natriuretic and antinatriferic activ1ties observed in plasma following various modes of extracellular fluid l8 volume expansion. These studies suggest that both of these activi« ties may be due to a single substance, or at least to substances which are very similar chemically, irregardless of the type of expansion or species studied. Source of Hormonal Natriuretic-Antinatriferic Factors If a 'natriuretic-antinatriferic hormone' exists, and accumulat- ing evidence would seem to suggest that it does, then it should be possible to identify the source of such a hormone. Lichardus (109) and Lichardus et al. (ll5) are of the opinion that the main cause of difficulties in the identification of such a substance is the lack of knowledge concerning this production site. Studies to date have in- volved body fluids in which the concentration of the 'hormone' may be very low. In efforts to overcome this difficulty cows have been used in an attempt to secure larger plasma volumes without significantly decreasing the total blood volume in expansion experiments (29,ll5). However, if the substance is dialyzable jn_viyg_from humans, another alternative to secure larger quantities of material would be to isolate it from spent dialysis fluid from uremic patients undergoing maintenance hemodialysis. A report which appeared May, l97l, by Sealey and Laragh (l50) did not contribute to the clarification of this question. These authors found that, of ten different organs investigated in salt-loaded sheep, 8 possessed a natriuretic activity. It seems unlikely that all of these organs can be responsible for the secretion of such a factor. A more 19 probable explanation would be that blood circulated to these organs during imposed natriuresis contained the natriuretic factor which was then extracted by the investigators. Particularly disturbing in Sealey's study was the lack of activity found in the brain, since other investigators have found evidence indicating this organ to be the primary source of an antinatriferic and/or natriuretic factor. One of the first indications that a sodium transport inhibiting substance may be elaborated from some area of the brain came from observations that jugular venous blood produced a greater sodium diuresis in the kidney and a greater inhibition of sodium transport across frog skins and toad bladders than blood from other areas of the body (36,47,54,57). Cort and Lichardus (54) observed that during bilateral common carotid artery occlusion in cats, jugular venous samples decreased frog skin short circuit current by 26.3%, while femoral arterial blood decreased it only l0.6%. Femoral venous blood and renal venous blood increased the short circuit current l0.8% and 5.8% respectively. All samples obtained from these anatomical loca— tions before occlusion increased the SCC from 6.5 to l2.5%. Buckalew et al° (36) observed that ultrafiltrates prepared from jugular venous blood inhibited the SCC of toad bladders significantly more than did similar ultrafiltrates from femoral venous blood following saline expansion in dogs. From l4 rats, a total of 8000 posterior, ventromedial, dorsomedial, and arcuate nuclei were histologically examined in 2 groups of animals by Lichardus et al. (ll2). One group of rats was given 2% saline to 20 drink ad libitum, and a second group drank tap water. A statistically significant decrease in nuclear volume was noted only in the posterior hypothalamic nuclei of the saline group. This was taken as evidence that the posterior nucleus has a neurosecretory function in the elimination of a saline load. In addition, it has been noted that electrolytic lesions of the posterior hypothalamus eliminate the natri- uresis seen with carotid artery occlusion (52,53,59) and iso—oncotic blood volume expansion in cats (50). Acute hypophysectomy markedly decreased sodium and urine output compared to nonhypophysectomized rats during blood volume expansion (llO). Homogenates of anterior pituitary tissue were ineffective in restoring the ability of these hypophysectomized rats to excrete sodium after infusion of a saline load (ll7). Homogenates from the posterior pituitary, on the other hand, were effective. Incubation of anterior and posterior bovine hypothalamic extracts with proteolytic enzymes such as chymotrypsin, trypsin, and swine kidney aminopeptidase, resulted in the appearance of an antinatriferic activ- ity from posterior extracts (47,57,58). However, continued incubation with these enzymes resulted in a progressive loss of antinatriferic activity. The conclusion from these studies was that a small antinatri- feric substance was first released from a larger 'hormonogen' form dur- ing the incubation. Further incubation resulted in a proteolytic degradation of the active material. Anterior hypothalamic material gave rise only to natriferic activity. Since no differences were found in the natriuretic response to carotid occlusion in intact and adrenalectomized cats, Licardus and 21 Cort (lll) concluded that the adrenals are not the source of a poten- tial natriuretic hormone. Bourgoignie et al. (20) report that 2 of 3 anephric chronically uremic patients studied demonstrated in their serum an inhibitor of sodium transport in frog skin. Thus they con- cluded that the kidney was not responsible for the synthesis of this antinatriferic material. Evisceration and nephrectomy before saline loading in rats and dogs did not abolish the inhibitory activity of plasma on proximal tubule sodium reabsorption as measured by micro- perfusion techniques (l23). Finally, Levinsky (103) found that the natriuresis observed in iso-oncotic blood volume expansion in dogs persisted after adrenalectomy and removal of the spleen, liver, and intestines. Elimination of the head and brain did not prevent a natriuresis. However, this is a rather drastic procedure, changing many parameters, and rendering rigid controls difficult. Andersson et al. (l) noted that injection of 0.85 M NaCl into the third ventricle of unanesthetized goats resulted in a 5 to lO-fold increase in sodium excretion and a 3-fold increase in urine flow. Although depression of aldosterone secretion could not be ruled out in these experiments, further studies the following year (2) demonstrated that aldosterone administration did not prevent the increase in electro- lyte excretion and that injections into the lateral ventricle had no effect. In l969 Born and Porter (68) perfused the third ventricle of rat brains with several substances and was able to induce natriuresis. Nhen 0.85 M NaCl was perfused at a rate of 0.7 ul/min he noted an 22 insignificant increase in urine flow but a lO-fold increase in sodium excretion over the control rate. Infusion of 0.l54 M NaCl and l.7 M glucose into the ventricle and intravenous infusion of 0.85 M NaCl at the same rate produced no natriuresis. One explanation offered for the observed natriuresis was that hypertonic saline solution may pass from the ventricle into adjacent hypothalamic tissue and stimulate a neural or hormonal mechanism which mediated the natriuretic response. Similar injections by Born at al. (67) into third ventricles of anesthetized dogs also induced a significant increase in urinary sodium excretion which was not related to alterations in renal plasma flow, GFR, or filtered load of sodium. The authors interpret all of these findings as being consistent with the action of a cerebral natriuretic hormone. They comment that "... the identicalness of an 'NH' whose secretion is stimulated by systemic volume loading and one whose re- lease follows injection of the third ventricle with hypertonic saline is conjectural. It is conceivable that 2 hormones exist, one respon- sive to alterations in volume and one to changes in concentration of sodium. These hormones acting in concert could serve as a hormonal modulator of total body sodium." It would seem, therefore, that a natriuretic-antinatriferic factor, or factors, has its origin in the posterior hypothalamus. This, however, remains to be proven unequivocally. Possible Natriuretic Hormone Involvement with Reduced Nephron Populations and Renal Disease Third factor hormonal activity has been demonstrated under situa- tions of acute and massive expansion of extracellular fluid volume and 23 carotid artery occlusion. An interesting question which arises is whether or not this factor is available only as an emergency mechanism or does it play a role in the day-to-day regulation of sodium balance as has been suggested (33,35,l50). This is difficult to ascertain in normal individuals with a full complement of nephrons because changes in salt excretion per nephron required by the usual range of salt in- gestion are of such a magnitude that it is difficult to distinguish between glomerular and tubular factors. With a decreased number of nephrons, however, the distinction might be more readily determined since each nephron is required to excrete a greater fraction of the filtered load in situations of imposed saline loads. This type of assay system was used by Bourgoignie et al. (l9), in rats, to test for a natriuretic substance in the plasma of uremic humans. These authors (19,l49,l57), and others (l68), feel that the response to a given natriuretic stimulus is increased under these circumstances. Schultz et al. (l48) decreased the nephron population of one kidney of dogs approximately 80% by ligating terminal branches of the’ renal artery. The residual nephrons retained their normal blood supply. Prior to the removal of the contralateral kidney, the remnant kidney reabsorbed 99% of the filtered load of sodium. 0n the same salt intake, with the contralateral kidney removed, sodium excretion increased in the remnant kidney and fractional reabsorption decreased. Renal artery constriction ruled out hyperfiltration as the cause of the natriuresis. Since the dogs were on supramaximal doses of fluorohydrocortisone, mineralocorticoid insufficiency was also disregarded as the cause of the elevated saline diuresis. These authors conclude that the volume 24 control mechanism becomes more responsive in uremia, with the relative contribution of an 'NH' versus changes in intrarenal physical factors needing further investigation. Similar conclusions from experiments with dogs were reported by Men at al. (168). Hayslett et al. (80,8l), in rats, reduced renal mass by progres— sively excising renal tissue. Following uninephrectomy these authors noted a 2-fold increase in sodium excretion per nephron with a 75% increase in GFR per nephron. Nith further removal of renal tissue, sodium excretion per nephron increased 5 to 6 times normal with no further rise in GFR per nephron. However, the halfvtime of fluid reabsorption in proximal tubules blocked with oil was unchanged by renal ablation. The authors therefore concluded that a 'third factor' ' hormone did not participate in the adjustment made to experimental renal insufficiency in these studies since this substance has been proposed to exert its effect in the proximal tubule. Here again is a negative finding with attempts to elicit 'NH' in the rat. This finding is in contrast to the observation of Bourgoignie and co—workers (l9,20, 2l,27,28,29), discussed previously, who have found evidence for a natriuretic and antinatriferic activity in the serum of patients with chronic renal disease. These individuals do have a reduced population of functional nephrons. B. Physical Factors AffectinggTubular Reabsorption of Sodium Independently of Factor I and Factor II In 1963 Blythe and Welt (l7) infused 5% saline into dogs and de« creased the GFR by inflation of a balloon inserted via the femoral vein 25 into the inferior vena cava to a point distal to the renal veins. Noting that urinary excretion of sodium could change independently of the filtered load of sodium, they concluded that the excretion of sodium is somehow related to the plasma levels of the ion and not necessarily to the filtered load. Dirks et al. (66), infusing isotonic and hypertonic saline into dogs, also noted a marked depression of proximal tubular fractional reabsorption which was independent of GFR and not blocked by reduction in GFR. Glabman et al. (76), however, concluded from micropuncture studies on proximal tubules of nonexpanded rats that filtered load of sodium is an important determinant of the rate of sodium reabsorption by the proximal tubule. Compounding the difficulties of detecting decreased proximal sodium reabsorption by measuring the urinary excretion of sodium is reabsorption of the ion in the distal portions of the nephron. Higgins (84), for example, showed that infusions of 600 ml of 5% albumin or dextran in 5% glucose into normally hydrated dogs caused only moderate increases or even decreases in sodium excretion. Similar expansion with blockage of sodium reabsorption distal to the proximal tubule by ethacrynic acid and chlorothiazide resulted in large increases in sodium excretion. He concluded, therefore, that during plasma volume expansion the ultimate sodium excretion rate is determined by distal reabsorption in spite of a decreased proximal reabsorption. Simdlar conclusions were reached by Buckalew et al. (38) and Davis et al. (64) with acute saline loading in dogs and Sellman et al. (l54) and Hayslett et al. (79) in the rat. During mineralocorticoid escape in normal man, 26 Martino and Earley (126) report that most of the proximally rejected sodium is recaptured by reabsorption at a site distal to the medullary loop of Henle, with Sonnenberg (158) placing it in the collecting duct during deoxycorticosterone escape in the rat. Studies of McDonald et al. (128), in which dogs underwent volume expansion with Ringer's lactate solution or saline plus 6% dextran, and Martino and Earley (127) using isotonic saline or saline plus 5% bovine albumin, suggested that decreases in blood and plasma viscosity accompanying saline infusion may potentiate the natriuretic response by decreasing renal vascular resistance and increasing capillary hydro- static pressure. This 'pressure natriuresis' was also observed by Kaloyanides et al. (96) when the arterial pressure was increased in an isolated kidney being perfused with blood from a nonexpanded intact dog. Similar observations were made by MacDonald and DeWardener (121) in isolated kidneys perfused at constant pressure with blood from an intact dog receiving an intravenous infusion of saline. Lewy and Nindhager (105) and Windhager et a1. (l69), from micro! puncture studies in the rat, suggest that proximal tubular reabsorption is partly controlled by the rate of vascular removal of epithelial reabsorbate. Capillary removal of reabsorbate is influenced by the hydrostatic pressure gradient between renal interstitium and capillary lumen. Increased peritubular capillary hydrostatic pressure could lead to a decrease in the rate of capillary removal of reabsorbate. This would decrease the rate of sodium reabsorption by increasing renal interstitial pressure. Decreases in renal interstitial hydrostatic pressure would have the opposite effect (55, pp. l7«25). 27 Earley (69) demonstrated that vasodilatation of the kidney with infusions of acetylcholine into the renal artery resulted in an in- creased sodium excretion. Increasing the arterial pressure in this vasodilated kidney resulted in an additional increase in sodium excre- tion, which occurred even when renal blood flow and GFR were decreased. These observations provided additional evidence that changes in arterial pressure or vascular resistance may be involved in determining the natriuretic response to saline loading. Other investigators have shown that postglomerular plasma protein concentration, either with (3,5,88,124,l36) or with (4,23,25,l05,l6l,165) blood volume expansion, is an important determinant in sodium excretion. Martino and Earley (125), for example, showed that when hyperoncotic albumin solutions (30%) were infused into animals previously loaded with isotonic saline, sodium excretion decreased despite an increase in GFR, renal blood flow, and arterial pressure. Spitzer and Windhager (161) found that perfusion of capillaries with colloid-free Ringers decreased reabsorption of sodium 49%, but inclusion of dextran (8%) in the Ringers produced results similar to those during normal blood perfusion. These observations support the Windhager theory that renal interstitial pressure affects sodium reabsorption. Another factor which appeared to affect sodium reabsorption, both with (90,130,13l,l46) and without (24,26,39,42,98,l32,l33,145) blood volume expansion, is the hematocrit° Brenner et al. (26) and Brenner and Galla (24), as a result of micropuncture studies in rats, observed that although changes in hematocrit lead to corresponding changes in proximal sodium reabsorption, parallel changes in peritubular capillary 28 protein concentration also occur. It was concluded that this change in postglomerular protein concentration may in fact be responsible for the changes observed in sodium reabsorption which accompany changes in hematocrit. Substantiating this conclusion, Bahlmann et al. (8) isovolemically decreased the hematocrit in the dog 28% with Hartmann's solution containing bovine albumin and found no appreciable change in sodium excretion. A similar result was reported by Ponec and Lichardus (139) in the rat. Burke et a1. (39), with recollection micropuncture techniques in the dog, suggest that changes in viscosity which alter capillary hydrostatic pressure may also account for hematocrit changes on proximal sodium reabsorption. In addition to the physical factors previously discussed, others have been advanced to explain the mechanisms controlling sodium excre- tion. These include changes in renal plasma flow (70,71,72,169) and redistribution of blood flow from medullary to more superficial high sodium excreting nephrons both with (40,103) and without (11,85,87) saline loading. For example, Windhager et al. (169) demonstrated with micropuncture techniques in rat kidneys that the decreased proximal reabsorption of sodium seen with experimentally increased renal venous pressure was proportional to the decrease in renal plasma flow. With saline loading in the dog, Earley and Friedler (70,72) suggested that the increased renal plasma flow seen in their experiments may be one factor that contributes to the decreased tubular reabsorption of sodium. 133 Using Xe washout techniques, Hollenberg et al. (85) found that 67% of the total renal blood flow in humans on a 10 mEq/day salt intake 29 was cortical. In subjects on a 200 mEq/day salt diet 84% of the total renal blood flow was cortical. Barger (11) suggested a similar redis- tribution in dogs with right sided congestive heart failure. Other investigators, however, have reported that there is no significant redistribution of blood flow following saline loading (120,129,155). In summary, it is apparent that the 'third factor' natriuresis which occurs following extracellular fluid volume expansion is a compli- cated interrelationship of many factors. In vjyg_experiments attempting to evaluate the role of a 'natriuretic hormone' must be carefully de- signed in order that the physical factors just described, which con— tribute to this 'third factor' natriuresis, do not change significantly. PURPOSE OF INVESTIGATION From the preceding review it is evident that the 'third factor' natriuresis observed with volume expansion may in fact prove to be multi-factoral, with the importance of individual physical and/or hormonal factors being dependent upon the mode of expansion. The cur- rent study was undertaken to l) substantiate the observation that an antinatriferic substance exists in the plasma of expanded chronically uremic humans, and 2) to investigate the potential of utilizing spent hemodialysis fluid as a source of this substance. To substantiate Cort's finding that an antinatriferic substance remains in plasma following trichloroacetic acid deproteinization, the antinatriferic activity of H-TCA deproteinized and non H-TCA deproteinized plasma from uremic humans and moderately volume expanded dogs was also studied. 30 METHODS A. Sodium and Potassium Determination Sodium and potassium were determined by flame photometry using a lithium internal standard (double beam technique) which has a reson— ance line at 671 mu. The resonance doublet employed for sodium occurs at 590 mu and that for potassium at 767—769 mu. In practice, the ratio of the emission intensity of the analysis line to that of the internal standard line is recorded and plotted against the concentra— tion of the analysis element to prepare a calibration curve for a series of standards. Unknown concentrations of test element are then deter- mined from the standard curve. Some advantages of this method over direct determination (single beam methods measuring absolute light intensities rather than ratios) in the flame are: . Better precision Compensates for effects of irregularities of atomization Compensates for variable loss of liquid in the spray chamber Reduces systematic errors due to possible differences in viscosity and surface tension of samples (affects rate of delivery of sample into the flame) 5. Reduces errors due to some radiation interferences. wa-H The instrument used was a BairdvAtomic Model KY~3 combination clinical and research filter flame photometer equipped with a gravity feed spray chamber aspirator (flow rate 3—6 ml/min) and Meker burner. A gas air_mixture (air pressure 10 psi) yielding a low flame tempera- ture of approximately 2100°K was utilized. 31 32 Reagents I) 2.00 mMolar sodium chloride and potassium chloride stock solution: Weigh 11.688 mg of dried reagent grade NaCl and 14.912 mg of dried reagent grade KCl into a 100 ml volumetric flask. Dilute to volume with 90 ppm lithium water. II) Stock lithium water: Dilute 5.0 m1 of 18,000 ppm lithium solution to 1000 ml with deionized water. Final lithium ion concentration is 90 ppm. This solution is to be used to prepare all samples and standards to be analyzed. Preparation of Samples Pipette 0.020 ml of unknown sample to be analyzed into 10 ml of 90 ppm lithium water for the determination of sodium. For the deter- mination of potassium, pipette O 020 ml of unknown sample into 2 ml of 90 ppm lithium water. Mix by inversion or vortexing and read in flame photometer. Preparation of Standards In Table 3 is the procedure for the preparation of the sodium and potassium standard solutions utilized for the preparation of the standard curves. The designated volumes were pipetted into 15 ml Pyrex test tubes and mixed by inversion. All solutions to be stored for indefinite periods should be stored in plastic bottles to prevent leaching of ions from the glass (especial- ly new glassware), and refrigerated to minimize bacterial growth. The procedure for the preparation of the standards and samples was adapted from Terris (163). 33 Table 3. Procedure for the preparation of the sodium and potassium solutions utilized for the preparation of standard curves. Standard Reagent I Reagent II Na:K Number (m1) (ml) (mEq/l) l 0.12 9.87 0.024 2 0.25 9.75 0.050 3 0.50 9.50 0.10 4 0.75 9.25 0.15 5 1.00 9.00 0.20 , 6 1.50 8.50 0.30 Calculation of Unknown Na and K Concentrations Na(mEq/1) = Instrgmgg: Reading X Dilution Factor = Instrument Reading Slope X 500 K(mEq/1) = Instrgmggz Reading X Dilution Factor _ Instrument Reading - Slope X 100 If the samples are prepared as described, the dilution factor53 employed in the calculations are as given above. The slope is obtained from a standard curve prepared by plotting the instrument reading (ordinate) versus the concentration of the standards (abscissa) 1" mEq/l. An example of such a curve is given by Figure l. Figure l. 34 Standard curves for sodium and potassium as determined with a Baird-Atomic Model KY-3 combination clinical and research filter flame photometer. Instrument reading (arbitrary units) is plotted on the ordinate, with known concentrations of sodium and potassium in mEq/l being plotted on the abscissa. 35 800i 700 * Instrument — K 8 Reading 1100 2983 1‘8” [’8‘ Ins-mum READING «92 1V 39.9 . " ' Instrument Na’ = 329.125.... x 1.00 1090 322 . s 3 1E / ‘ s I’,/’/’ /: -. I l 00‘ 0.2 003 IEq/l Figure I . 36 B. Chloride Determination Chloride was determined by a coulometric-amperometric titration with silver ions utilizing a Buchler-Cotlove chloridometer. The method is based on the coulometric generation of reagent (silver ions) and amperometric indication of the end-point. A constant direct current is passed between a pair of silver generator electrodes in the genera- tor (coulometric) circuit, causing release of silver ions into the titration solution at a constant rate. The end-point is indicated, after all chloride has been precipitated, by the increasing concentra- tion of free silver ions which cause a rising current to flow through a pair of silver indicator electrodes. At a preset increment of indica- tor current a relay is actuated, stopping a timer which runs simul- taneously with the generation of silver ions. Since the rate of genera- tion of silver ion is constant, the amount of chloride precipitated is proportional to the elapsed time. Reagents I) Nitric-Acetic Acid Reagent (0.1 N HNO3 and 10%, V/V, glacial acetic acid): To 900 m1 of distilled water add 6.4 ml of concentrated reagent grade nitric acid and 10 m1 of reagent grade glacial acetic acid. Store in glass container with a glass stopper. II) Gelatin Reagent: To 6.2 grams of 60:1:1 dry mixture (gelatin: thymol blue (water soluble): thymol (reagent grade crystals)) add approximately 1 liter of hot water and stir gently until the solution is clear. Store refrigerated in glass tubes in volumes sufficient for a set of analyses. A new tube should be used for each day's analyses. To liquify the gelatin immerse the tube in hot water. Do not freeze (as this destroys the effectiveness of the gelatin). Do not use the gelatin if it has been at room temperature for more than a day or two. III) Sodium Chloride Reagent (160 mEq/l): Dissolve 9.3520 grams of dried-reagent grade NaCl in distilled water and dilute to exactly 1 liter. 37 Preparation of Samples, Standards, and Blank§_ A) Unknowns: To 0.1 ml of sample in a titration vial add 4 ml of nitric-acetic acid reagent and 4 drops of gelatin reagent. B) Standard Sample: To 0.1 ml of NaCl standard solution (160 mEq/l) in a titration vial add 4 m1 of the nitricvacetic acid reagent and 4 dr0ps of the gelatin reagent. C) Blank Sample: To approximately 4 ml of the nitricvacetic acid reagent in a titration vial add 4 drops of gelatin reagent. Samples and reagents should not be stored in contact with rubber as sulfhydryl groups may be released which may combine with silver ions leading to inaccurate determinations. In addition, do not titrate a sample unless the solution is acid and the gelatin reagent is present (indicated by a red color of the thymol blue indicator). Calculation of Chloride Ion Concentration a) Gross seconds = timer reading b) Average net seconds of standard_= average gross seconds of standard minus average blank seconds c) Calibration factor = K K = (m1 of NaCl reagent) (Lle] in mEq/l) average net seconds of standard d) Net seconds of unknown - gross seconds minus average blank seconds K (net seconds of unknown) ml of unknown Unknown Cl- concentration If the samples, standards, and blanks are prepared as previously described, the above equation for the concentration of chloride ion in the unknown samples (in mEq/l) reduces to the following: 38 [C1’] = (159) (O 1) if‘ net seconds for unknown net seconds for standard T~ m1 of unknown T'TT“ 160 X net seconds for standard" net seconds for unknown _ net seconds for unknown _ net seconds for standard'(]60) mEq/l C. Glucose Determination Glucose was determined by a glucose oxidase method, which combines the following two reactions: l) Glucose + 02 + H20 glucose oxidase >. H202 + gluconic acid 2) H202 + o-dianisidine (reduced) Perox‘dasee> o-dianisidine (oxidized) + H O 2 Glucostat, a prepared reagent for the quantitative colorimetric deterv mination of glucose containing peroxidase, glucose oxidase, and reduced o-dianisidine, is available in a lyophilized form from Worthington Biochemical Corp., Freehold, N. J. This preparation was used in these analyses. Glucose oxidase is highly specific for beta«0«glucose, and since glucose in solution is usually 36% alpha and 64% beta, complete oxida« tion requires mutarotation of the alpha to the beta form. Complete oxidation is not necessary for the success of the method. If, however, the glucose preparation does not contain mutarotase to accelerate this reaction, standard solutions prepared from dry glucose should stand at least 2 hours to insure that mutarotation has reached a state of equi« librium. Reagents I) II) III) 39 Stock glucose: 200 mg percentnwDissolve 200 mg of reagent grade glucose in 100 ml of distilled water in a 100 m1 volumetric flask. Store refrigerated. Glucostat reagent: Dissolve l vial of 4X chromogen (o-di- anisidine) and 1 vial of 4X enzyme in 200 ml of distilled water. Prepare fresh for each assay, or if storage of the prepared reagent is desired, place in an amber bottle and keep refrigerated for no longer than 1 month. 4 N HCl: Dilute 33.33 ml of concentrated HCl (12 N) to 100 ml with distilled water. Preparation of Standards In Table 4 is the procedure for the preparation of the standard solutions utilized for the preparation of the standard curves. The designated volumes were pipetted into 15 m1 Pyrex test tubes and mixed by inversion. Table 4. Procedure for the preparation of the glucose standard solu- tions utilized for the preparation of standard curves. Standard Reagent I H O Glucose Glucose Number (ml) (m1) (mg/ml) (mMolar) l 0.25 10.00 0.49 0.27 2 0.50 10.00 0.98 0.54 3 0.75 10.00 0.14 0.78 4 1.00 10.00 0.18 1.01 5 1.25 10.00 0.22 1.22 6 1.50 10.00 0.26 1.45 40 Preparation of Samples and Standards for Analysis Dilute 1/2 ml of unknown(s) to 10 ml with distilled water. Pipette 1/2 m1 of this diluted sample to 2 ml with 1.5 ml of glucostat reagent (Reagent II above). To prepare the standards for analysis, pipette 1/2 ml of the above solutions to 2 ml with 1.5 ml of the glu- costat reagent. Exactly 10 minutes after the addition of the gluco- stat reagent add 2 drops of the 4 N HCl to halt color development (the 10 minute incubation having been performed at room temperature). Following the addition of the acid, mix by inversion or vortexing immediately. Standards and unknowns should be analyzed simultaneously under conditions such that the rate of oxidation is proportional to the glucose concentration. In some methods the final mixture is acidi- fied slightly to stop the reaction and the yellow color developed is measured at 400 mu (as is the case here). In stronger acid, the color becomes pink with maximum absorption at 540 mu (where both sensitivity and stability are improved). Introduction of the enzyme peroxidase and a chromogenic 02 acceptor (reduced o-dianisidine) provides the color development. Calculation of Unknown Glucose Concentration Glucose (mg/ml) = (A/Slope) (dilution factor) A = absorbance of unknown at 400 mu Slope = slope of standard curve, with absorbance of standards plotted on the ordinate and concentration of standards on the abscissa Dilution Factor = 20 if the above procedures are followed. A representative standard curve is given in Figure 2. 41 Figure 2. Standard curve for glucose as determined with a Beckman Model DB spectrophotometer at 400 mu. Absorbance at 400 mu is plotted on the ordinate against known concentra« tions of glucose in mg/ml on the abscissa. A (too Ind 0.9 0.1 0.2_ 0.6 0.5 0.4 o.g_ 0.2 0.1 q 42 A 3.281 Glucose a X Dilution Factor l I 0.1 0.2 eLuooss (lg/ml) Figure 2. 0.3 43 D. Ammonium Ion Determination The determination of ammonium ion as ammonia utilized the general principle of microdiffusion of volatile substances. A small aliquot of the sample to be tested is placed into a special microdiffusion apparatus originally described by Conway and Byrne (45). The sample is mixed with a concentrated alkali solution and the volatile ammonia thus liberated diffuses from an inner chamber to an inner well which contains an indicator solution. The concentration of alkali (sodium carbonate) in these studies was approximately 16%, adopted from Brown et a1. (30). Titration of the absorbed ammonia with an acid of known concentration permits the quantitative determination of the ammonium ion contained in the original sample. The apparatus employed for the determination of ammonium ion in these studies was a modified Conway unit described by O'Brink in 1955 (138). Figure 3 shows the construction of the unit. Reagents 1) II) III) IV) 20% sodium carbonate: weigh 20 grams of anhydrous sodium carbonate into a 100 m1 volumetric flask and dilute to volume with distilled water. The final concentration in the diffu- sion unit will be approximately 16% if 0.2 ml of sample to be tested is used. 0.004N HCl: dilute 0.40 ml of 1.0 N HCl (Acculute) to 100 ml with distilled water. Tashiro's reagent (162): to 200 m1 of a 0.1% alcoholic solu- tion of methyl red add 50 m1 of a 0.1% alcoholic solution of methylene blue. If stored in a brown bottle the solution will keep indefinitely. 1% boric acid: weigh 1 gram of boric acid into a 100 ml volu- metric flask and dilute to volume with distilled water. 44 cmnEmgo copso Am Langmcu coccp Am _~mz coca? A” .pwc: cowmzwwaOLUwE XQZCOU on“ $0 :OwumuwwaOE waLm.O .m mczmwm 45 .n 35mg 46 V) Indicator solution: to 35 ml of distilled water add 15 m1 of the 1% boric acid prepared as described above. To this mixture pipette 5 ml of Tashiro's reagent and mix thoroughly. For best results prepare this solution fresh just prior to use. Procedure Into the inner well of the microdiffusion apparatus pipette 1 m1 of the indicator solution and place the lid onto the unit. Gently rotate the apparatus to completely wet and distribute the solution over the entire inner well. Observe the indicator color for 5-10 minutes to insure that the well is not contaminated (indicator will turn from violet to brown or green if a contaminant is present). Remove the lid, and into the inner and outer chambers pipette 1 ml of the 20% sodium carbonate solution. Into the inner chamber pipette 0.2 m1 of sample to be tested in such a manner that it does not come into contact with the alkali. Replace and rotate the lid to completely distribute the alkali between the lid and outer chamber areas of contact. This must be done very carefully to insure a complete seal between the lid and diffusion apparatus. An incomplete seal will permit the ammonia liberated to diffuse out of the unit and lead to erroneous results. With the establishment of a complete seal between the lid and outer chamber, the entire unit is then rotated several times to mix the test sample with the sodium carbonate in the inner chamber. Care must be taken not to allow spillage of the contents of the inner and outer chambers into the inner well. If this occurs the determination must be repeated. With the above volumes this danger is minimal. 47 Once the solutions have mixed, the unit is allowed to sit for 90‘120 minutes to allow diffusion of the liberated ammonia into the inner well. An occasional swirling of the solution during this period will facilitate the process. Following the incubation period the lid is very carefully re- moved. Care must be taken to avoid splashing of the sodium carbonate from the outer chamber and lid into the inner well. With gentle swirling the inner well is titrated with 0.004 N HCl to the original color of the indicator solution. For a blank, prepare the apparatus as described using 0.2 ml of water in place of the test solution. Titrate the blank and subtract this volume of HCl from that required to titrate the test solutions. As an alternative, known concentrations of ammonium ion can be titrated and standard curves established. To clean the units after use do not use detergent. Allow them to soak in dilute acid overnight and rinse several times with tap water. Follow the tap water rinses with several distilled water rinses and allow the units to dry before reuse. Calculation of the Unknown Ammonium Ion Concentration At the equivalence point in any titration the number of mEq of standard is exactly equal to the number of mEq of substance being deter- mined. To calculate the mEq of ammonia, therefore, one need only determine the mEq of HCl utilized during the titration. The final value can then be expressed as desired. mEq HCl utilized = (vol of HCl in ml) (N of HCl) + _ (m1 HCl) (N HCl) NH4 (mEq/1) - ml of unknown X 1000 48 E. Ether Extraction of Trichloroacetic Acid (H-TCA) from Samples As previously discussed, Cort et al. (49,58) and Cort (47) have reported that plasma from carotid artery occluded cats which had been deproteinized with approximately 6% H-TCA exhibits both antinatriferic and natriuretic activities. The procedure which Cort describes for the removal of the added H-TCA involves repeated extractions with diethyl ether until the sample attains a pH between 5.0 and 6.0. At this point Cort et al. (58) report that the H—TCA is no longer an acid and that nearly all of the added acid has been removed. This procedure for the removal of plasma proteins was adopted in some of the studies reported here, since work by Cort and others suggests that rapid removal of proteins is desirable to retain maximum activity of any salt losing hormone which might be present in a given sample. Once proteins have been removed, samples have reportedly been stored up to 7 months (36) at -5°C with apparently no loss of activity. To determine the number of ether extractions required to obtain a pH of 5.0 to 6.0 following addition of H-TCA to plasma, blood samples were obtained from several cats.h To ten m1 of plasma was added 5 ml of 20% H-TCA. Following the removal of the protein precipi- tate by centrifugation, the deproteinized plasma was decanted into a 60 ml separatory funnel and approximately 3 volumes (30 m1) of ether added to it. The separatory funnel was shaken for either 5 or 10 minutes, the layers allowed to separate, and the ether removed and dis- carded. A fresh 30 m1 aliquot of ether was added to the aqueous layer 49 in the separatory funnel and the process repeated until a pH between 5.0 and 6.0 was obtained. Since there was no difference between the samples which had been extracted for 5 and 10 minutes, the results were combined and averaged. Figure 4 summarizes these results, and as can be seen at least 9 such extractions were required to obtain a pH of approximately 5.0, with several more being required if one is to obtain a pH of 6.0. This procedure is not only arduous, but as will be shown, apparently does not effectively remove all of the TCA" anion. At pH 5.0 to 6.0 the TCA" is perhaps no longer in the acid form, as Cort et a1. (49) have stated, but in this form it is no longer ether soluble and may lead to erroneous results as it exhibits 'NH'-like activity on amphibian membranes. Since TCA' demonstrates antinatriferic activity on amphibian membranes (RESULTS--Section B), it became necessary to quantify the levels of this ion remaining in samples following ether extraction. A colorimetric procedure was developed in order to determine the effi- ciency with which ether extraction, as described by Cort et a1. (49), removes H-TCA from plasma samples. As a result of these studies, Cort's procedure was modified to more effectively remove the H-TCA. Figure 5 illustrates the ultraviolet absorbance spectrum of l mMolar H-TCA in water and 0.096 N HCl. In each case the H-TCA sample was referenced against the corresponding solvent. Samples were prepared by diluting 0.2 ml of 25 mMolar H-TCA to‘5 ml with the appropriate sol- vent. There is an absorption maximum in the vicinity of 202-206 mp when the diluting solvent is made acidic. In HCl at 205 mu, however, 50 .mmmwumnm exp :0 conga: :owpomcuxm umcwmmm mpmcwnco asp co umppopa m. In .2mm.H mmFQEmm m mo mommcm>m ocm NF Op FF mpcwoa cowpomcpxo tam .2mm.H mm_asmm e mo mommcm>m mcm op 0p _ mpcwoq cowpomcpxm .cmcwmpno mm: 0.0 ow o.m AFmpmewxocaam we :a w Free: umpmmgmc mm; mmmooca mxgh .uonchmwu mam cm>oeoc cmcgw mg“ use .mpmcmqwm ow vmzoppm mcmxmp mg» .mmpzcws o— co m com coxmsm mm: Pmcczm xcoumcmamm one .u. on umvum cospm mo APE omv mmaspo> m >qumswxocaam vcm chcze xcoumcmamm F5 on m ops? umpcmomu mm; mamm_a um~wcwmgocamu asp .cowpmmzewcpcmo An mumpwawumcg cwmwoca mgp mo Fm>osmc mg» mcwzoppom .cowmcm>cw >3 noxwe cam mammfia mo FE op ow venue mm: m one .comm saw; mums «no: meowuacmscmgmu “noncommucw mpmowpawg» new .umcmaoca xppcmucoaoucw ago: .F\ams cw cowumcucmucou oEmg mmpmowncw A-“ .Fomz z qmF.o sup; copmcmaxm mE:_O> Augmrmz xcon ”mpOpv fie m mcwsrmumc a mom Lou FOUOPOLQ mew m? umnsfiocw cmfi< .Fumz z ¢m_.o spa; cormcmaxm wE:_o> Apsmwmz xuon _mu0pv fim "crows? cm m:w>wmumc m-m moon cow "QUOpOca .m_ manned 96 i .Ppmz z,empwo to am om,:uwz IsomFQuL mm: um>osmg moo—m «spasmm mpzcws cm can mp Lmu$< .cowmcmaxm Pmpuwcw .3 530328 g3: nova—E me can .om .mp .o mu? cw umpomppou quFn mFosz PE ow DP 1 .NP aazmwa mgcmEmgzmooE pwzocwu pcosm Lee cmgmamgq + tapaz_;uwz Fe OF on naszpwu + mmmcxgu cu umNWFmgaoxp + mco_pomcuxm Apzcawwuv gmgpm cmpmmamc Amgapmgmaemu soon pm .ucwuv .uaa :wmuoga Aug + “tapas; cos »_anms.xocaaa co uam.o .coa <0» ~a=_cv oom n av awoo< m==Ao> . mmzuk zoamaazH zoamaazH zoamz Fonz z emp.o Apcmwmz xuon pmpouv.um on» corn: Love: mcovuwucou Fupcmsrcmaxm us» we zguesam .u spam» 98 .A.pcmuv screamscwcpcmu mg museum iazm mo Fm>OEmL mmwcwcmwm Aiv .Fumz z va.o spwz cowmcmaxm mE:_o> Apcmwmz xuon Fmbouv x0 mepvcw cm mcw>wwomg m~-o_ mmou Low _000¢oca .m_ masmaa 99 oposom gmaogcp :omocpwc ocammogo moo mm Loos: coo no oogoupwwogpF: osmopo so FE mm .xogooo ». .m, mason; woos mucoeocamooe pcogazo proogwo “Logo Laces; mace new: F2 o_ on oop=p_um=ooog cowuoogm cowgo + mmocago op oa~_Fr;aox_ >H =o_pascc oov no :5=_oo ammo xooogoom o» Vow~ooo osmopo ,2 op access» cases » mazuxwe ocopoooioow ago cw :oNogm osmopo —2 NF “Vi. mucoeocamoos poocgzo “Soto taco 3% 3.333 can”: cap_wom_o not: Fe op on oaozpwu A, mmocxso o» oo~wpwcoo>F muwwpoogpxo 2.335 .55“ oopooooc Aogopogoosou Eco; no .pcoov .uoo cwooogo fiiv 9 ASE coo .xogoao Lo mmo.m .gou H colossal » goo um cszpoo ammo xooonoom op oowpooo osmopo FE o— +, _ (V 8.2.. .33.: no .283 0mm 3 cowmcooxo 2.5.2: mcfioposoo 4 Loose mopzcws om go mF mow cw mow cw oouooppoo uaoaazoo 8°: 22.: E 8. 8oz afiz to E 8 # n<~zmzammaxu _ — nomnzooiii a 2.2.: Mg: 103 .cowmcooxo Fowuwcw mg» no cowuopo isoo Lopes coxop mum: moPoEom Foucoswgooxo cog: moswp o» some; mos?» gocpo .oposom Fogpcoo op mascot us m.m coop mp.o mm.” o om“ o.- m, 0.0 cc“ om.o cm.” 8 one m.“ e, o.oF omm om.o um.P o oem o.m m. N.“ mmm me.o mm.o o own o.NP N_ _.N oeo m_.o ¢N.o o oem o.m __ ¢.~ ooa m_.o NF._ 8 cum m.m o_ me.om m.m omm .mp.o.os mo.o o cam m.m m broom: Apsv A=Pe\ox\_ev A=Pe\mX\_sv Apev >aom u no omoo< u:=no> mmzH» onmDqu onmsqu onmz Fonz z emF.o Angora; soon _sooov no we» gave: have: mcoppwocoa Pmocaswcaaxa ago to scaeszm .m aFaep 104 The individual 10 ml aliquots obtained from samples which were fractionated on Sephadex G25F resin were pooled into 5 fractions as shown in Figure 23. These pooled fractions were lyophilized to dryness and fraction IV prepared for short circuit current measurements by one of the following methods: Method I (20): Plasma samples, both H-TCA treated and non-H-TCA treated, were diluted with distilled water to 1/10 the volume which was applied to the resin column. For short circuit current determinations 0.3 ml of this concentrated sample was mixed with 0.15 ml of a 3-fold concentrated frog buffer. Removal of 0.45 ml of frog buffer from the serosal side of the frog skin and injection of the 0.45 ml of sample resulted in an approximate lO-fold dilution of the reconstituted sample. Thus the concentration of any plasma antinatriferic substance in contact with the frog skin would be nearly equal to that of the original sample obtained. Plasma ultrafiltrate and dialysis fluid concentrates were handled in a similar fashion, except that the concentration of an anti- natriferic material in these samples relative to plasma was unknown. Ammonium ion concentration determinations were performed on all samples, as it has been shown that low levels of this ion have an antinatriferic effect on amphibian membranes (78)--O.5 mMolar ammonium ion decreasing the short circuit current by 12%, 1.0 mMolar 22%, and 2.0 mMolar 30%. Method II (20,36): Following lyophilization to dryness, fraction IV samples were diluted with frog buffer to a volume equivalent to the volume of sample 105 applied to the resin column. Ammonium ion concentration, milliosr molality, and pH of the sample were measured before short Cir U7t current measurements were made. RESULTS A. Fractionation of Specimens on Sephadex Resin Elution of a sample of plasma (dog or human) with ammonium acetate (10 mMolar) from Sephadex G25F resin produced an optical dens- ity pattern of 280 mu similar to that observed by Bourgoignie et a1. (20). The pattern obtained from patient GS is illustrated in Figure 20, showing the 3 major absorption peaks used by Bourgoignie et a1. (20)ix)loca1ize the region of natriuretic-antinatriferic activity. This region, believed to contain these activities, occurs between the latter two peaks in Figure 20 in an approximate elution volume of 500 to 780 ml. Figure 21 compares the fractionation of a plasma sample from patient CH with that of an aliquot of plasma ultrafiltrate obtained from the artificial kidney before and after an 8-fold concentration by ultrafiltration at O-4°C. It can be noted that the latter 2 peaks of the concentrated plasma ultrafiltrate have an increase in absorbance at 280 mu. Conspicuously absent from these ultrafiltrate patterns is the absorbance due to protein in the region of approximately 150 to 300 ml. This demonstrates the effectiveness with which the hemodialysis membranes used in the hemodialyzer retain these large molecular weight substances. Figure 22 depicts the elution pattern of plasma ultra- filtrate after a 15-fold concentration from patient GS. 106 107 .copooFFoo :owpomgw >3 oopoonoo mam: mpooov_o FE op .muopooo Ezwcoeao Lo_ozs OF cur: swoon mmmo xooocomm soc» oou:_o mm; oposom .mu “cowpmo soc; osmo_o wo oFoEom o wo as omm no ccmpuoo co_p:_m .om magmas 108 .om shaman Aaao mxpoo> zonaoou (a. 092) v 109 9:83 4 compmcucmocoo opomiw co Lopwo opochWeocp_: oEmo_m I cofiobcoocoo mgomoo 332C853 9583 e .Lopoofiyoo cowpoocm x3 oopom__oo mam: mpODUWFo PE OF .opooooo EswcoEEo LmFozs op got: cvmoc mew xooogoom Eon» oop:_o ocoz mofioEom .uom_ pm cospocowpooce woo woo om ooEcoeLoo mm: cospocpp_eocp_: .covpocu -FWcoLHFD >2 cowpocpcoocoo oFomim co coowo oco ocowon .xocowx _mwovwepco ogp Eon» no:_opoo .opocppwwocp_: osmo_o we scoppmo cowpzpo on“ m? omo:_o:+ om_< .xu pcowuoo Eoce oEmoFo wo oposom m we as omm pm :coppoo cowp:_m .FN masons 110 . wm 93mg Ad: 249w .8ng 005 Sn (til 09?.) v 111 .coooo__oo corpoocm xo ooooopfioo mam; moosowFo _E o_ .opopmom SascoEEo LmFOZE OF not; :vmmc ammo xooogoom Eoce oop:_o mo: oFQEom .cowoocppwmoLHF: x3 compocpcoocoo o_ow-m_ a copes mu pcowooo Eocm opocp—weocpfis osmopo mo as omw pm :cmppoo co_p:_m .NN ac:m_n H2 Jmeflfifl Adav mzsgo> ZOHBDQM 00m CON. com con 00— H _ _ _ . _ _ _ i- _ ANT _ ole/e e'eI. e\e‘eIel/n\ / 1\\ If: \ 113 Figure 23 indicates the absorbance at 280 and 360 mu of the individual 10 ml fraction collector samples obtained following elution of an aliquot of concentrated dialysis fluid obtained from patient GS. To be noted first is the presence again of the 2 major absorbance peaks at 280 mp and the absence of a protein peak at about 180 m1. During the concentration of dialytis fluid and plasma ultrafiltrate, the specimens become progressively pigmented. The nature of the pig- ment is at present not known. The color varies from a light yellow to brown and can occasionally be noted to be present in as much as the first 900 m1 of the elution volume (depicted in Figure 23 by the horizontal line above the elution volume axis). An absorbance spectrum of the pigment demonstrated a maximum absorbance at approximately 360 mp. The individual 10 ml fraction collector samples exhibited 3 ab- sorbance peaks at 360 mu, the latter 2 corresponding to the maximum visible coloration as indicated by the darkened area on the horizontal pigment line. In addition to the absorbance at 280 and 360 mp, the pH and milliosmolality of the individual 10 ml fraction collector samples are also shown in Figure 23. The pH of the eluant is approximately 6.5. As noted earlier, the 36 liters of dialysis fluid is acidified with HCl to retard bacterial growth over the period of 4 days required for concentration by ultrafiltration. This added hydrogen ion can be seen to be eluted in a volume very closely correlated with the major osmotically active substances, the lowest pH occurring at a volume of approximately 400 ml. Plasma, and plasma ultrafiltrates obtained 114 .Looooppoo corpooce xo oooooPPoo mam: mpozowpm FE ooh .mpwuooo EowcosEo LoFozE op new; :Fmoc ammo xooocoom soc» oops—o mo: oFoEom och .oogczooo cowpmcoFoo opowmw> ummxcoo ogp cowcz cw mosspo> mg“ 3ozm mow, asp co mcowucoo oocoxcmo och .omumwxo cowuoocosmwo o_oooowpoc o cows; :? mmso_o> ogu moowooo mwxo mE=Fo> cowp:_o on“ o>ooo pmon oCTF PopcoNWLo; och .mzuogoooo “coccoo uwooc_o ucosm :wxm mos» on“ cw oozommo xFoc_u:og mo; >H corpomgm .Amocw_ Poowuco>v > oco .>H .HHH .HH .H mcowpoocm o>wm op omFooo mosspo> cowp:_o oz» one :3osm om_< .xpvpo_osmo_FFwe ocm .1o .15 com oco 0mm po mocoocomoo oco mazFo> :owpzpo um:_omo oouquo .mw p:o_poo soc» mpocpcmocoo ow:_e mwmxfiowo wo cowpoco_poogm .mm magmas 115 .mm ouSMfim “Hay QZDAO> onaqu 8: 8m 8. _ i _ Li. _ // \x. \ [to < lo.— la; lo.~ > E H: H» H mN I (Hoioa It}? \\li. - i .w. m 8. n \ / ./.N/ O 1'- m l \ / m m . I1 e\e/\x e ,.|Ie’e.|e|uele\e oom I no xix M. 116 directly from the artificial kidney, are not acidified with HCl. As shown in Figure 24 these specimens exhibit no decrease in pH in this region but rather an increase. The presence of proteins in the plasma imparts an additional pH peak in an elution volume of approximately 220 to 300 ml (protein buffering of the eluant perhaps) not seen in the ultrafiltrate samples. Closer inspection of the composition of the osmotic peak reveals that sodium, chloride, potassium, and glucose are the major contribu- tors. These substances do not appear in any other region in measurable quantities. Elution of glucose with the inorganic ions was unexpected. Based on its molecular weight (180 Daltons) one would have expected it to appear in the volume containing small amino acids (approximately 850 m1). Figure 25 illustrates the' elution pattern of these substances from a sample of plasma ultrafiltrate concentrate obtained from patient GS. The importance of these observations is the demonstration that there is no sodium, potassium, chloride, glucose, inorganic acid or base, or any other osmotically active substance in the fraction reported to contain antinatriferic-natriuretic activity (Fraction IV). There also is an absence of substances with a measurable absorbance at 280 mp in Fraction IV. To estimate the approximate molecular weights of substances in the various fractions obtained, compounds of known molecular weight were applied to the resin column and eluted with 10 mMolar ammonium 6 acetate. Substances employed were Blue Dextran (M.W. 2 X 10 Daltons), bovine albumin (M.W. 69,000 Daltons), -inu1in (M.W. 5000 Daltons)1| 117 .LopooF_oo cowoomce xo oopooppoo ocoz mpozoPFo _E OF .mpopooo Ezwcoeso LmFozs OF cow: :wmog mmmw xooocoom Eoge oouopo mam; moPQEom ogp .UOo no cowpocppweocppo oco QOmF om omEcoccoo mo; cowpocovpoogm .Io powwpoo soc» .cowpocpcoocoo opoeim cm Loose oco ococoo .xocowx meowewpcm on“ Eoce oo:_opno opmcppwwoch: osmo_o oco osmopo to :Loppoo coves—o Io .em at:m_a 118 . 5. Bang AHHV §DAO> onaafiw oom o2. oom oon oow _ _ _ _ _ _ _ . _ .‘.I.Io|oI-.Iollluolulcl-\.I.\usu\-l-Iu c\.I-II.JI\.IIIIVII'I./. I I O .I. \ W W onaéazmozoo «may: /. . 1o. a maamSEéSo «me—3m / \ ./. Tffi. . . I’Olel\\ Tl o.w \ce’eI-loielo\oIllelo’ls‘leIi-leI-I.I us \‘Ie‘aeIIIsee‘lelele Ila :Ow onaészmozB mmemm X. .\ mafiSEES: 5.3% x. Iw. n ,. \ ’ . .1 2.;.(.\‘ Jen. lo.» eluI-IeIele\ele\./o Ielelel lonlllole‘alolelee- el sa\a:eI|-|elle Inlouew as :. . . r s .. . / ..:;.\ l x. .\ ooh sass ,... \ 1m. s ,... .c. 'fell\ To” 119 V. ... G Pu u .AV oz u mu omoozpm u G moWFmFoEmoprwe u o .Lopoo—Foo cowpoocm an omuooppoo mew; mpoooWFo FE op .mpooooo EowcoEEo Lopozs o_ gov: :Pmog ammo xooocoom sogw ompopo mo: oposom ogh .mw “corona gone oocwopoo AcovpocuPWw locupo >3 opomimF ooomcpcoocoov opocpcoocoo opocp_wmogppz osmmpo mo cowpocowpooae mcwzoppom xpw>_uoo owoosmo mcwpocpmcoEmo mo_oEmm mpmopo :wmo; mo mucocoosoo coho: .mm aa=m_a 120 com — LII )~ 2% r elnll .3 sm 1 waiimnm m C (II n Kw com Hi8 .mN enough 25 3.33 zoned 8: Endowzmoag com 81 9‘31 81 (Joan/ban) 1'5 "x vanou El 121 bacitracin (M.W. 1400 Daltons), and tryptophan (M.W. 204 Daltons). Figure 26 demonstrates that Blue Dextran, albumin, and inulin are all eluted with the column void volume of approximately 180 ml. Bacitracin is eluted with a volume of approximately 300 ml, and tryptophan at approximately 850 ml. Substances eluted with volumes between 300 and 850 ml (Fraction IV) might therefore be expected to have molecular weights on the order of 1400 to 200 Daltons, a molecular weight range consistent with that reported for 'NH'. It should be stressed, however, that although there may be a cor- relation between molecular weight and elution volume, a more accurate relationship exists between the three-dimensional configuration of a molecule and its elution volume. Non-linear substances, for example, are excluded from the resin particles to a greater extent than are linear molecules of similar molecular weight and are therefore eluted from the column sooner than would have been expected. Heterocyclic and aromatic substances are often abnormally retarded in their passage through the resin and therefore are eluted later than would have been expected based on their molecular weight. This phenomenon is affected by such parameters as ionic strength and pH of the medium being employed. Column calibration must be cautiously interpreted therefore. B. Effects of ADH,_TCA', and Ammonium Ion on Frog Skin Short Circuit‘Current, Membrane Potential, and Resistance As mentioned in METHODS, Sections E and L, ammonium ion and TCA" exhibit antinatriferic activity with frog skin preparations. As these 122 :_Fscw cmcpxmo msfim ll ID II “ awasnfim .meagb wasp cw nanwaaman mowvgpm ago at Uan_ru= gamma ammo xaUMLQmm Law; taxama :Eg_oa 20 mm x m.N 0:0 co coppaan__mu .8 $ng 123 Aeom .a- mqqpomaoxv zammoamuma 0|. .8 .55 38v “HZ—50> 205.5 a... _ a Aoo:., .a- mzow Aomzmv 0 0 O O O o w m w m. _ m. _ _ _ _ O ... m ...... x L: /- 4.. _ ... c x _ mm m . R/w. .. m m .. _ /. ._ .. x. .‘ mx .\ D .... i... ...... IO my ,. x a H . .\ mm /_ x. /. m .. m .c m /. .. /. <\ m .\. .I/. m III .Illillillilulnu/VIII- A _._ o _. .__ _ __ _ .__ ..o _ __ a. _ .__ rm . m_ %_ w. m. wTw. w. .m— m. m. 1 A25 m: can :3 com. Figure 2?. 127 To ascertain the effects of the trichloroacetate anion (TCA') on the frog skin, the sodium salt of the acid (Fischer Scientific) was utilized. Two buffer solutions were prepared, one as usual (METHODS--Section G), and the second with Na-TCA replacing NaCl. Buffers with various concentrations of Na-TCA were then prepared according to Table l0. Following a period of equilibration, one of the Na-TCA buffer solutions was applied to the serosal side of the skin; measurements obtained for 40 minutes; and the Na-TCA buffer then re- placed with the normal NaCl buffer. Figure 28 summarizes the results of the TCA‘ anion on the short circuit current and resistance of the frog skin, with Figure 29 depicting the effects on the membrane po- tential. Figure 30 illustrates the time course of the effects of 0.9 mMolar TCA- on these parameters. Table 10. Preparation of_Na-TCA samples employed to determine the effects of TCA on frog skin short circuit current, membrane potential,and resistance. Buffers were prepared as described in METHODS-—Section G. The designated volumes were pipetted into l5 ml Pyrex test tubes and mixed by vortexing. TCA- CONCENTRATION m1 Na-TCA m1 NaCl (mMoles/liter) BUFFER BUFFER 0.l0 0.009 9.99 0.20 0.018 9.98 0.30 0.027 9.97 0.40 0.036 9.96 0.50 0.045 9.96 0.60 0.054 9.95 0.70 0.064 9.94 0.80 0.073 9.93 0.90 0.082 9.92 1.10 0.100 9.90 l.50 0.l36 9.86 2.00 0.182 9.82 128 Figure 28. Effects of TCA" on frog skin short circuit current and resistance. PERCENT CHANGE FROM CONTROL PERIOD 129 RESISTANCE SHORT CIRCUIT CURRENT I I 0.5 1.0 1.5 2.0 TCA? (uncles/1) j l Funum 38. 130 Figure 29. Effects of TCA' on frog skin membrane potential. 1% 6 18 If. 0 131 Ii. 0 l'.“ O PERCENT CHANGE FROM CONTROL PERIOD 0 _1‘_é 0.5 MEMBRANE POTENTIAL I 1 I 1.0 1.5 2.0 TCA“ (afloles/l) Figure 29. Figure 30. 132 Effect of 0.9 mMolar Na-TCA on frog skin short circuit current (SCC), membrane potential (MP), and resistance (R). Following a 25 minute control period, the frog buffer was drained from both sides of the membrane and replaced by regular frog buffer on the mucosal side of the skin and frog buffer containing 0.9 mMolar Na-TCA on the serosal side. After 40 minutes the medium was drained from both sides of the membrane and flushed 3 times with fresh frog buffer. Fresh frog buffer was added to both sides of the membrane and measurements continued for another 30 minutes. The skins were con- tinuously short circuited, with MP and SCC being recorded at 5 minute intervals throughout the control and experimental periods. 133 0.9 mMolar Na-TCA R} O O ( NHO) aonvmsmaa added I Na-TCA 100 I .-....\ removed I \g I I . 80 I A - >8 I R v I g3 60 I \ . u — I . .\ \o 5 V °\ ' A \,/ -°":::/ .\ .... MP < . v. " . .... .....’ 3... 7 \ v \ . .. 8 "i \.- -.-..y" u: I I «29. I I I ! I I I I I I I -k0 -20 0 +20 +40 +60 +80 TIME (MINUTES) Figure 30. 134 From these figures it can be seen that the TCA- produces an inhibition of the SCC and MP which is reversible. The effects of the TCA- on the SCC and MP result in a reversible increase in the calculated resist- ance. To determine the effects of ammonium ion on the frog skin, a stock solution of 20 mMolar ammonium acetate was prepared by dissolving 77 mg in 50 ml of frog buffer. Solutions to be tested on the skins were prepared according to Table 11. Table 11. Preparation of samples employed to determine the effects of ammonium ion on frog skin short circuit current, membrane potential, and resistance. Buffers were prepared as* described in METHODS-—Section G. The designated volumes were pipetted into 15 ml Pyrex test tubes and mixed by vor- texing. ml AMMONIUM FREE ml 20 mMolar AMMONIUM AMMONIUM ION BUFFER BUFFER (mMoles/l) 9.80 0.20 0.40 9.60 0.40 0.80 9.00 1.00 2.00 The results of 2.0 mMolar ammonium ion on the frog skin are illustrated in Figure 31. Figure 32 summarizes the percent change in short circuit current, membrane potential, and resistance produced by 0.4, 0.8, and 2.0 mMolar ammonium ion. Comparison of Figures 27, 30, and 31 illustrates that in contrast to ADH, TCA' and ammonium ion result in a decrease in short circuit current and membrane potential with an increase in resistance. Figure 31. 135 Effect of 2.0 mMolar ammonium ion on frog skin short circuit current (SCC), membrane potential (MP), and resistance (R). Following a 25 minute control period, the frog buffer was drained from both sides of the membrane and replaced by regular frog buffer on the mucosal side of the skin and frog buffer containing 2.0 mMolar ammonium acetate on the serosal side. After 40 minutes the medium was drained from both sides of the membrane and flushed 3 times with fresh frog buffer. Fresh buffer was added to both sides of the membrane and measurements continued for another 30 minutes. The skins were continuously short circuited, with MP and SCC being recorded at 5 minute intervals throughout the control and experimental periods. scc (pA) and MP (mV) 136 2.0 mMolar NH. ion 1500 0"}? . ./’\ . g . SCC i000 9:22:21- \. ._,//-"\. I l. addid 1800 I . m." 1.. I ”'/\i/ removed 1700 :u I \\ —- on 5’3 ' e; I § ' 1600<7 I —- a: I / 3 I - ' E I \J I I I I ‘\ ‘~—-’"'i . I “S. ;,u/ I \m ”"I 1300 I l l I r l l l T -40 -2o 0 +20 +40 +60 +80 TIME (MINUTES) Figure 31. 137 I. I. .LmFozs o.m vcm m.o Low 2mm + mcowum>cmmno m ucm empozs ¢.o com 2mm + mcowum>cmmno m we mommcm>w mew mucwom .mucmpmmmmg new .meucwpoa acmanmE .ucmcgsu u_:ucwu “Legm :wxm meg» co cow Ezwcossm empoze o.m vcm .w.o .e.o mo pummem .Nm aesmwa 138 .mm 8&2 AQuoHozav o.~ ma o._F n._o Emma HHBGHU Scum. S AdHEBom 2mg: pod Pod 35.565 4\_ ON+ (10183:! 10811100 nous HOWE!) 1.150836 139 C. Human Uremic Studies Patients involved in this study were selected on the basis of a change in their dry body weight follow1ng the previous dialysis treatment, an increase being indicative of fluid retention and possible volume expansion. The dry body weight for a given individual is de- fined as the total body weight at which the patient develops hypoten- sion (a decrease in diastolic pressure of 30 mm Hg or orthostatic hypotension). This change in dry body weight for the patients studied, as shown in Table 12, ranged from -0.54 to +4.17%. Table 13 presents a summary of the types of speCimens (plasma, plasma ultrafiltrate from the artificial kidney, and/or dialysis fluid) obtained from each of the individuals studied. Non H—TCA treated plasma specimens were obtained from 9 patients, with H-TCA being used to precipitate proteins from the plasma of 4 patients. Dialysis fluid was obtained from 5 patients, and plasma ultrafiltrate from the artificial kidney was obtained from 8 patients. Non-TCA treated plasma from patients 052, CdeB, and DH was frozen in a dry ice-acetone bath until fractionated on Sephadex GZSF resin. All other specimens, excluding dialysis fluid, were placed in an ice bath until Sephadex fractionation. Table 13 also summarizes the effects of lyophilized fraction IV from each sample on frog skin short circuit current. This fraction (elution volume of approximately 550 to 780 m1) has been reported to contain natriuretic and antinatriferic activities following elution of human uremic plasma and plasma of volume expanded dogs and cows (l9,20,21,29). 140 mm.m+ om\mqp oo_\oqp Io mm.m+ om\om_ oop\ooF mmuu PN.N+ Nm\¢oF om\omp mmn ~_.¢+ om\o¢p oN\omF mu mm.F+ ON\om~ ou\o¢~ mmn mm.m+ oo\om_ om\om~ a; oo.o+ czocxcs ooF\omF ma m~.F+ om\ooP om\oo_ A: mo.m+ ¢m\mmp om\ooF oo «0.0+ oo\moF oo\omp z: mm.F+ om\oop ou\omp Io mo.P+ ow\oo_ om\om_ Nu em.o- czocxcz oo\ON_ gw Hzm24mma mqum pszmz pzmzpoom >mo 2H mwz0000 A<00 0200000 0000000 00000 .00000 000 00 000000000 00 000000 000000000 00000 00000 N 00 0 00 000000 0002 0000000 .000 000 00000000 00 000>00 0000 000000000 00 000 000 00000000 0000 0000000 00000 -000000 0000000000000 000000 .000000 0000 00 000000 0000 000 00 00000000000 00000 0000000 00-0N 0000 0002 00000> >00>ou00 .000000 0000 000000 000000000000 0 0000 000 0000000>0 00 00000000 0003 00000> .000000 0000 00000 00 000000 0000 000 00 00000000000 00000 “00 000000 000>ou00 000 000000 000 .000 0000000 000000 0000 >0 00000000 00~00000000 00 00000000 000300000 .000 000000 0000000 000 000000 0000000 0000000 00000 000 00 00000> 000 00 0000000 .m_ 00000 OLD (0(1) 00 MG) Q’m 0(1) CNN [\N md' OOQ' NM kDN ...." oo 09 om oo Cato mm 0000 mix Q‘N 0<00 .00000 000 00 00000000000 000 00 00000000 000 0003 0000 .000>00000000 .0000_\000 0.0 000 0.0 00 0000000000000 0 00 000 00000000 000000000 00 000 000 00000000 000 00000000000 0000000000000 000000 000 00000« .00000m00 00 0000 0000 000 0000000000 000 .0000000 0000000 00000 .000000000 00000000 000 00 :00 + 00000 00000 000 000 00000 000 00 00000000 000< .00000000000 0000 0000 000 00 000000 0000 00 00000000 00000 000000000 00 000000 0000000 000 000000 .3: 0000000000 000 ..EE 0000000000 00000000. .303 0000000 00000.8 00000 0000 0000 00 000000000 000000 0000 >0 00000000 00N00000000 00 0000000 000 00 0000000 .00 00000 146 00.0 00.0 :0 00.0 00.0 0 0000 0N.0 00.0 00.0 N00 00.0 00.0 0N.0 00 00.0 00.0 000 00.0 00.0 00 00.0 00.0 00 00.0 00.0 02 0N.0 00.0 00.0 00 00.0 0N.0 00.0 0: 0N.0 N0.o 00.0 :0 00.0 N0 00.0 00 000<000 <00I: <00I: oz,.. . .000<00zm0zo0,_ . ,0000002002002: 00:00 000>0<00 0200000 000000 , w .: ‘I,-; 0000000000000 0000000 2000000200000 200 00020000 .0000000 000 I00000 000020 00 0003 00000 00N0 x0000000 0000 00000000 00 0000000 000300000 000000000 000000 00000 0000 00000000 0000000 >0 00000000 00N00000000 00 00000000000000 000 00000000 00 0000000 .00 00000 l47 Table l6. Initial volumes and magnitude of concentration of plasma ultrafiltrates obtained from the artificial kidney. The initial volumes of plasma ultrafiltrate were reduced and concentrated by ultrafiltration at O-4°C using an Amicon UM05 ultrafiltration membrane. Following ultrafil- tration the specimen remaining in the ultrafiltration cell was eluted from Sephadex GZSF resin with l0 mMolar ammon- ium acetate, fraction IV lyophilized to dryness, and diluted in frog buffer. INITIAL VOLUME 0F PLASMA MAGNITUDE 0F CONCENTRATION ULTRAFILTRATE OBTAINED INCREASE AFTER ULTRAFILTRA- FROM ARTIFICIAL KIDNEY TION AND SEPHADEX FRAC- PATIENT (ml) TIONATION CH 400" 8 HM 240 9 CO 180 18 JSe 275 27 J52 265 26 CdeB 250 25 DH 150 15 GS 150 15 These procedures resulted in an 8— to 27—fold concentration of the original sample. From Tables l3 and l4 and Appendix III it can be seen that, even after these concentration procedures, the plasma ultra- filtrates did not contain marked reversible antinatriferic activity in the absence of ammonium ion° A similar result was obtained with dialysis fluid specimens which had been concentrated 3600-fold. As previously described (METHODS—~Section L), samples for frog skin measurements were prepared in two ways. Samples from patients CZ, JK, CH, ML, and CO were diluted in distilled water to approximately l48 l/lO of the volume applied to the Sephadex column. A 0.3 ml aliquot was then diluted to 0.45 ml with a 3-fold concentrated frog buffer; 0.45 ml of frog buffer removed from the serosal side of the frog skin; and the 0.45 ml of sample added (Method 1, Section L--METHODS). All other specimens were assayed on the frog skins according to Method II described in Section L of METHODS. D. Acute VolumeLExpansion Experiments with Dogs Dogs numbered 2 through 8 underwent an initial volume expansion with 0.154 M NaCl equivalent to 3% of their total body weight. All plasma samples obtained were deproteinized with H-TCA, the H-TCA being removed by ether extractions as previously described in METHODS-- Section E. Samples from dogs 2, 3, 7, and 8, were then prepared for short circuit current assay before fractionation on Sephadex GZSF resin. The results of chemical analyses of these samples are tabulated in Table 17. Sodium in these samples averaged 110 i 0.3 SEM mEq/liter; potassium, 2.9 :_0.0 SEM mEq/liter; and milliosmolality, 228 :_0.3 SEM mOsmol/liter. Samples from dogs 2-8, following elution from Sephadex resin and lyophilization, exhibited a range of ammonium ion concentra- tion from 0.00 to 62.0 mEq/liter (Table I7). Table 19 and Appendix IV summarize the effects of the unfrac- tionated 3% expansion samples from dogs 2, 3, 7, and 8, on frog skin short Circuit current, membrane potential, and resistance.“ With the exception of a plasma sample from dog 7, obtained 30-minutes after expan- sion, there is no evidence for a reproducible antinatriferic substance. 149 oo. No NNN N. 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N N.oN o.mN N.oN N N moo o.oN N.NN o.NN N.NN N.NN N.NN N.NN N.NN N.NN N N.oN o.No N.No N.NN N.NN N.NN N.NN o.oN N.oN N N moo N.NN N.oN N.oN N.No N.NN N.NN N.NN N.oN o.oN N N.NN N.NN o.mN N.NN N.NN N.oN o.mm N.NN N.oN N o moo N.NN N.oN o.NN o.NN N.NN N.No . o.No N.NN N.oo N N.NN N.NN N.No o.oN N.NN N.No o.mN N.NN o.NN N N moo I N.No o.No N.NN N.NN o.NN o.NN o.oo o.No o.No N N.NN o.mN o.oN N.oN N.NN N.NN o.oN N.NN o.mo N o moo N.NN N.No N.mo N.NN N.No N.oN N N.NN N.NN N.NN N.NN o.NN o.NN N N moo o.oN N.NN N.NN N.oN o.oN N.NN N.NN o.NN N.NN N N.NN N.NN N.NN N.NN N.NN N.NN N.oN o.No N.oN N N moo N m o N NI o N m o N m o N m m zNNN NNNozNz No NoNozNz oN NNNozN: mN I ‘ NmNozNz o ooNNzoo NmzNN moozmm Nmoo szNNom NNooNNo NNoIN .ooNomo zoom chooo NNcmEmoomomE N NNoN mop chNoom>o No chNoNoo mom: NmoNo> .NNV omNNoo mooN cmmoN No NmNoEoN NNmN mop chuoNomo omNNo mmpocNE ONION oco .NNV Noon nmocooxm NN mNN EooN NmNoEom oeNoNo ompomop N coNNuooN NmNNNNoooxN No :oNNNooo mop NchoNNoN .Nuv ooNomo Noopcom mop NcNooo pcmoooo “NoooNo Nooom mop No NmoNo> mop No NooEEom .NN mNooN 155 oo.o NNN o.N oNN NN moo oo.o NNN N.N NNN m moo. "NmNozNz No oo.o III III III oN moo oo.o III III III NN moo I oo.o NNN o.N oNN m moo "NmNozNz oN oo.o III III III NN moo IIII NooN Noo NooN NN moo oo.o mNN m.N NoN oN moo oo.o NNN N.N NNN m moo "NNNozNz NN oo.o NNN N.N oNN m moo ”NmNozNz o oo.o III III III NN moo oo.o III III III oN moo oo.o III III III NN moo oo.o oNN N.N moN NN moo NN.o NNN m.N oNN NN moo oo.o . NNN N.N oNN oN moo oo.o NNN N.N oNN m moo ”ooNNzoo NNNmNoo NNN NNNosooo NNNmmoo N NNNmmoo oz zoNNozoNNooNN NNNNN zoNNmzoNNNNNN mNonN .Nommo :NNN mooN ooN :oNN Ioooomoo oco .coNNoNNNNoooNN .coNNmooNxm omoum No No>oEmo I NgmNmz Noon Nope» NN o NchoNNoN coo NNNNNzouv oN ooNoo Nooo sooN uchopoo NmNoEoN osNoNo No mmNNNoco NooNemso No Noose=m .NN mNooN 156 o.No N.No N.mo N N.NN o.No N.No N.oN o.NN o.NN N NN moo N.Nm o.No,o.No N N.NN o.oN N.oN N NN moo N.NN N.NN N.No w N.oN N.NN N.oN N N.NN o.mN N.NN o.om o.No N.oN N oN moo o.NN N.NN N.NN N.NN o.NN N.NN N N.oN o.NN N.No N.NN.N.oN o.NN N.NN N.NN N.NN o.NN N.NN N.NN N m moo N N o N N m N N o N N o N N m zNNN NNNozNz No NNNozNz oN NNNozNz NN NNNozNN o I, NoNNzoo NNNNN NNNNNN Nmov NZNNNoo NNooNNo NNoNN .prmN mmmv ImN ompo>mNm :Nopcoo op :zoom mom: NNION Noon sooN NmNoEoN NN< .ooNomo :uom choou mNcmEmoomoms N NNoN mop NoNOoom>o No ochoNoo mom; mmoNo> .NNV omNNoo NooN :NmoN No mmNoEoN ammo mop chmoNomo omNNo NmNoch ONION woo .NNV NOON omooooxm NN mop EooN NmNoEom oENoNo umpomoN mop No Nooesom .NN mNnoN l57 H-TCA treated plasma samples from the 6% expanded dogs (E), and 20-30 minutes after replacing the test samples by fresh frog buffer (R). The control samples from dogs 10, ll, and 12, contained TCA' at a con- centration of 2.9, 1.2, and l.4 mEq/liter, respectively, following ether extraction. The 45 minute sample for dog l2 contained TCA' at a concentration of approximately 1.3 mEq/liter, and the l5 minute sample from dog l0 contained 0.82 mEq/liter. Quantitative data on the levels of TCA- in the plasma samples from dog 9 are not available. The TCA' concentration in each of the above samples would be expected to be somewhat less in the final sample following lyophilization. This is because there is some sample loss during the ether extraction procedure and the specimens become slightly overdiluted when made up in the frog buffer. Nonetheless,-the reversible inhibition of the short circuit current seen with these samples was probably due to TCA- contamination. Table 24 and Appendix VI summarize the effects of these samples on frog skin short circuit current, membrane potential, and resistance. As can be seen the effects were variable. Table 25 summarizes the values of the short circuit current dur- ‘ ing the control period (C), following addition of lyophilized fraction IV from H-TCA treated 6% expansion plasma samples from dog 9 (E), and 20-30 minutes after replacing the sample with fresh frog buffer (R). Only with the 45 minute sample is there an indication of a reproducible reversible inhibition of short Circuit current. Also in this table (and Appendix VII) is a summary of the effects of these samples on short circuit current, membrane potential, and resistance, and it can be seen that the 45 minute sample had a variable effect on the membrane potential. 158 — o N _ N __ _ N o N NNN“ o m I mh N + oNflwoNI N + N + N + N + NNI S. N + NNI N I 25: 2+ NNI o I oNI o I NNI NN+ m I N + N NN moo o o o N N I N I o I N NN OOO 2+ 2. NI o I N I NNI N N I N + o + NN+ NNI NNI N oN moo N + N I N I m + NNI N I N N + NNI NNI N I m + N + N + NNI oNI NN+ NNI NNI N m moo N NNN o: N NNN o: N NNN o: N mom o: N mom oz zNNN NNNozNz No NNNozNz oN NNNozN: NN NNNozNz o NoNNzoo NNzNN Noozom .zNN.H Nooms oooom moo moo mNooN ms» :N umooncN oNN< .prmu mmNV ImN oNN; :Nopcou op czoom mom; NN ON ON Noon Eco» NmNoENN NN< .mNoEoN uNmN »o :oNNNooo mop omu»o omumsoooo mg» :N mmcocu Ncmmomo moo NmoNo> .NNV mucoNNNNmo oco .Nozv NoNpcmNoo mcooosms .NOONV pcmooou “NouoNu Noogm chm moo» am Noon umocooxm RN mop eoo» NmNoEoN oENoNo omuomop .NNNNV ooooumNNmo ooo .Nozv NoNuompoo mooooeme .NOONN oomoooo uNoooNo uoooN oNNN moo» oo NmNoEom mNmou Eoo» >N coNuooo» omNNNNoooNN »o Noom»»m moo »o NooEE=N o NN mNooN moo oN oNN< .ooNomo ooom moNooo Nuomsmoomoms N NNoN moo monoom>o No omoNoooo mom: NmoNo> .NNN om»»=o moo» ono» oNN: NmNoEoN ammo moo moNooNomo omo»o NmpooNE ONION ooo .NNV m moo soo» NmNosoN oENoNo ompomo» N ooNpooo» omNNNNoooNN »o ooNoNooo moo chonNo» .NOV ooNomo Noouooo moo moNooo uomooso NNoooNo uooom mop »o NmoNo> mop »o Noossom .NN mNooh 160 H-TCA treated plasma samples from dogs l0, ll, and l2, were not frac- tionated Table 26 summarizes the values of the short circuit current during the control period (C), following the addition of lyophilized fraction IV from non H-TCA treated plasma samples from dogs lO-lS (E), and 20-30 minutes after replacing the test samples by fresh frog buffer (R). Table 27 and Appendix VIII summarize the effects of these samples on frog skin short circuit current, membrane potential, and resistance. Inspection of these tables again reveals no consistent evidence for a reversible inhibition of short circuit current which is accompanied by a reproducible decrease in membrane potential and increase in resist- ance. l6l N NNNN.NN o.NoN N.oN,N.NN N.oN N N.NN o.oN o.NN N.oN N.oN N.oN N NN moo o.No N.No o.oo N.NN N.NN o.NN N o.oN N.NN N.NN o.Nm o.oN o.NN N oN moo o.oo o.No o.oN .. o.No N.NN o.oN N N.oN N.NN N.oN N.No N.No N.NN N NN moo o.NN N.NN N.NN N.NN N.No N.oN N N.NN o.NN N.oN o.NN N.NN o.NN N NN moo o.NN N.NN N.NN o.NN N.oN N.NN N N.mm NNN o.NN N.oN N.oN N.No N NN moo N.NN o.NN N.oN o.NN N.NN N.oN ‘N o.NN N.NN N.oN N.NN N.NN N.NN N oN moo N N o N N o N N. o N N m ZNNN NNNozNz No NNNozNz oN I NNNozNz NN ooNNzoo NNzNN Noosz NNNO NZNNNoo NNooNNo NNoIN mom: NmoNo> .ooNomo ooom moNooo NoomEmooNome N NNoN moo monoom>o No omoNoNoo .NNV om»»:o moo» ono» No NmNoEoN Nmmo mop moNooNomo omN»o NmoooNE ONION ooo .NNV NNoENoNo omoomoo N ooNNooo» omNNNNoooNN »o ooNNNooo moo moNzoNNo» .NOV ooNomo Noooooo moo moNooo oomoooo uNoooNo pooom moo »o NmoNo> moo »o Noose:m .NN mNooN 162 N ON o NN mN NN N m NN anH oNI Nm+ NN+ N I ON+ o + ON+ NN+ ON+ N I NN+ NN+ z .NNV moooNNNNmo ooo .Nozv NoNNomooo mooooeme .NOONV Nomoooo NNoooNo NoooN oNNN moo» co NNoENoNo omoomoo N :oNNooo» omNNNNoooNN »o NNom»»m mop »o Noossom .NN mNooN DISCUSSION It has been reported that an antinatriferic-natriuretic sub- stance, which appears to be similar to that found in experimental acute and chronic volume expansion, occurs in the serum of patients with end-stage renal disease (l9,20,2l). This observation, plus the finding that an antinatriferic substance is dialyzable jn_vivo from acutely volume expanded dogs (36), prompted an investigation for this substance in spent hemodialysis fluid and plasma ultrafiltrates from patients with chronic renal failure undergoing maintenance hemodialysis. Studies were also conducted to measure the antinatriferic activity of plasma, with and without deproteinization with trichloroacetic acid, obtained from these patients and saline expanded dogs. The membranes currently employed in artificial kidney hemodi- alyzers have been shown to have solute clearances of approximately 30 ml/min for substances with molecular weights of 500 and approx1mately l0 ml/min for substances with molecular weights of 3000 (77) (and per- sonal communication with Cordis Dow Corp., Walnut Creek, Calif.). In addition, approximately 3 to 13 ml/min of plasma ultrafiltrate appear in the dialysis fluid depending on the transmembrane pressure gradient developed between the blood and dialysis fluid line (Gambro, Inc., Wheeling, Ill.). This plasma ultrafiltrate would increase substantially the yield of any antinatriferic substance which might be dialyzable. l63 164 In the present studies the yield of plasma ultrafiltrate was from 150 to 400 ml during a 30 minute collection period. It seems, therefore, that if a small molecular weight antinatri- feric material is present in uremic serum it should appear in the dialysis fluid and/or plasma ultrafiltrate in vivo. However, evidence that this substance is dialyzable from humans can only be inferred from the work of others. Bourgoignie et al. (20) observed, in non- dialyzed patients, that a fraction obtained following elution of serum from Sephadex GZSF resin produced a 24.9% (N=l8) inhibition of sodium transport across frog skins. The same fraction from dialyzed patients produced a significantly smaller inhibition of l6.2% (N=l3), and that from normal subjects 5.3% (N=ll)° In the studies of this thesis a 2.5 X 95 cm column, packed with Sephadex GZSF resin, was employed to fractionate the uremic specimens and samples obtained in the expansion studies performed with dogs. This column was similar to that employed by Bourgoignie et al. (20). Comparison of elution patterns at 280 mu suggests that the column used by Bourgoignie was similar to the one used in the present study. Further evidence for the similarity of these systems was obtained with electrolyte determinations in the individual l0 ml fraction collector samples. Electrolytes in the present studies were found to be eluted in a volume similar to that in which Bourgoignie et al., above, noted a high specific conductance. Therefore, a similar elution volume (fraction IV--approximately 550-780 ml) would be expected to contain any antinatriferic material which might be present in the specimens of this investigation. 165 To be noted in the elution patterns of plasma ultrafiltrate and dialysis fluid (Figures 21, 22, and 23) is the absence of a protein absorbance at 280 mu in the region of approximately lSD to 300 ml. As proteins are not dialyzable or ultrafilterable through the artificial kidney hemodialyzer membranes, this is to be expected. However, in these fluids there is an absorbance maximum at approximately 400 and 850 ml which is seen with elution of plasma. Both of these regions show an increased absorbance following concentration. These observao tions are important because they indicate that l) substances with molecular weights larger than the postulated antinatriferic substance are being dialyzed and ultrafiltered from the patient's blood (absor« bance peak at 400 ml). and 2) that it may be being retained and concen« trated by the ultrafiltration procedure (greater absorbance at 850 ml than is seen with unconcentrated plasma). The absence of measurable amounts of sodium, potassium, chloride, osmotically active substances, and no unusual pH‘s beyond an elution volume of 500 ml, indicates an efficient desalting by the resin column of fraction IV (Figures 23, 24, and 25). Elution of substances with known molecular weights from the column suggested that materials with elution volumes from 300 to 850 ml might be expected to have molecular weights from approximately l400 to 200 Daltons (Figure 26), a range consistent with that reported by others for natriuretic and anti“ natriferic substances (20,27,28,29,36,37,56). This conclusion must be viewed with caution, however, since as pointed out in RESULTS—-Section A, some substances may be abnormally retained by or excluded from the l66 resin particles during elution. Glucose appeared to be an example of such a substance in these studies since it was eluted from the resin much earlier than would be predicted on the basis of its molecular weight (Figure 25). In addition to decreasing the short circuit current, fraction IV from uremic serum was also reported to decrease the membrane potential and increase the resistance in frog skin preparations (20). These effects were reversible when the test sample was removed and replaced with fresh frog buffer. Similar findings were reported by Buckalew et al. (36) using plasma dialysates and ultrafiltrates from volume expanded dogs. In the present studies there were no uremic samples, either plasma with or without H-TCA deproteinization, dialysis fluid, or plasma ultrafiltrates, that reproducibly demonstrated these effects that could not be accounted for by ammonium ion contamination (Tables 13 and 14). The dialysis fluid sample from patient CZ produced a l3% de- crease in short circuit current which was reversible, a second deter- mination producing a l% decrease which was not reversible. Plasma ultrafiltrate from patient C0, which had been concentrated lB-fold, produced a l0% reversible decrease in one frog skin run but a non- reversible 9% decrease in a second determination. Dialysis fluid from patient ML inhibited the short circuit current 7% reversibly in one determination, but had no effect in a second determination. The effect of these specimens on membrane potential and resistance were variable. H-TCA treated plasma from patient ML was without effect. Although non 167 H-TCA treated plasma from patient DH reversibly inhibited the SCC 7% and 13%, the membrane potential decreased 5% and increased 15%, respectively. The plasma ultrafiltrate from this patient, which had been concentrated lS-fold, inhibited the SCC an average of 6% non- reversibly. The only examples of a marked reversible inhibition of both the SCC and MP, which were accompanied by an increase in resistance, occurred with plasma ultrafiltrates from patients JSe and GS. However, the ammonium ion concentrations in these samples were 4.40 and 1.10 mEq/liter, respectively. This level of ammonium ion is sufficient to account for all of the inhibition seen (refer to Figure 32--RESULTS). In the experiments of this study, in which dogs underwent an acute volume expansion with 0.154 M sodium chloride equivalent to 3% of their total body weight, all plasma samples were quickly deproteinized with H-TCA to eliminate possible degradation of antinatriferic activity. Blood samples were obtained prior to the expansion and 0, 15, 30, and 45 minutes after completion of the expansion. Before being fractionated on Sephadex GZSF resin, only 2 samples (Table 19) demonstrated an in- hibitory effect of frog skin short circuit current and membrane poten— tial. One determination with H-TCA deproteinized plasma taken 15 minutes after expansion from dog 3 decreased the SCC 17%, the membrane potential 7%, and increased the resistance 12%. As can be seen in Table 18 this effect was partially reversible upon sample removal. A second determination with this sample on another skin was without effect. Plasma sampled from dog 7, 30 minutes after expansion, reversi- bly inhibited the SCC and MP but caused both an increase and a decrease 168 in resistance in two separate runs (Table 19). Following fractionation of the 3% expansion samples on Sephadex GZSF resin, several exhibited inhibitory effects on the short circuit current (Table 20). However, the effects on membrane potential and resistance were variable. Only one determination with a plasma sample from dog 2, taken 15 minutes following expansion, exhibited a reversi— ble (Table 21) inhibition of SCC of 8% (Table 20). Since most samples did not inhibit the SCC prior to Sephadex fractionation, it is con- cluded that the levels of TCA- obtained following ether extraction were low enough not to interfere with the determinations. It is also concluded that there was very little, if any, antinatriferic material recovered as a result of the expansion followed by H-TCA deproteiniza- tion of the plasma at any of the sample times. This is in contrast to the findings of Cort (47) and Cort et al. (49,58) with H-TCA de- proteinized plasma from carotid artery occluded cats. Although the inhibition of SCC seen by these authors following occlusion could have been the result of TCA- contamination, it is difficult to explain why other samples treated similarly were not inhibitory. In addition to the 3% total body weight expansion experiments, several animals were expanded with a volume of 0.154 M saline equiva- lent to 6% of the total body weight. Blood samples were obtained prior to the expansion and O, 15, 30, and 45 minutes after the expansion. There were several examples of inhibition of SCC with the samples before fractionation on Sephadex resin, including inhibition with control samples (Table 24). However, the effects on membrane potential and A 169 resistance were variable. Only with the control samples from dogs 9, 10, and 12, and the 45 minute sample from dog 12, are found examples of reversibility of the effect (Table 23). Since H—TCA was found to be present in the samples from dogs 10 and 12 (0.82 to 2.90 mEq/liter), inhibition due to H-TCA cannot be discounted. Quantitative determina- tions of H—TCA could not be made for the samples from dog 9. . Following fractionation of the samples from dog 9 on Sephadex resin, inhibitory activity was found with fraction IV plasma samples obtained immediately following (0 minutes) and 45 minutes after com- pletion of the expansion (Table 25). Only with the 45 minute sample was the inhibition reversible (Table 25). With both determinations there was an increase in resistance, but the effect of the specimen on MP was variable. In addition to the samples from dogs 10, 11, and 12, which were deproteinized with H-TCA, samples were also obtained which were not treated with H-TCA but were deproteinized by fractionation on Sephadex GZSF resin. Although there were several samples which resulted in a decrease in frog skin short circuit current (Table 27), none were reversible (Table 26). The effects on the membrane potential and resistance were variable. Similar samples from dogs l3, l4, and 15, including the control samples, also exhibited inhibitory activity on the SCC (Table 27). However, in only one instance (30 minute sample from dog 13) was the effect a reversible one (Table 26). A second determination with this sample produced an increase in the SCC. Effects on MP and R were variable. 170 These studies indicate that hemodialyzed uremic patients, who exhibit a fluid retention which is less than 5% of their dry body weight, possess no reproducible evidence of an antinatriferic activity in their plasma. Plasma ultrafiltrates which were concentrated from 8— to 27-fold, and dialysis fluids concentrated 3600-fold, also did not demonstrate an antinatriferic activity previously described by others. Dogs which were acutely volume expanded with saline, equivalent to 3% and 6% of their total body weight, also exhibited no antinatriferic activity which was similar to that reported by others. If a natri- uretic hormone exists which is also antinatriferic, it is concluded that such a substance was not present in the specimens of this investi- gation. The lack of antinatriferic activity in the uremic plasma samples might have been due to the long delay between sample collection and fractionation. Cort (47) and Cort et al. (49,58) report that an anti- natriferic substance from carotid artery occluded cats is destroyed by a 30 minute incubation in the presence of plasma protein at 37°C. A similar incubation at O“C resulted in no loss of active material (as measured by its effect on frog skin SCC). Although the material from cats is stable at 0°C for at least 30 minutes in nondeproteinized plasma, the human material in the present studies may not have been stable in an ice bath for the 2 to 4 hours which elapsed between col- lection and fractionation. However, plasmas from JSe, CdeB, and DH were frozen in a dry ice-acetone bath immediately after removal of the red blood cells by centrifugation. Only fraction IV from the plasma 171 from OH resulted in a reproducible reduction in short circuit current, but a variable effect on membrane potential was obtained. Also, none of the plasmas deproteinized with H-TCA was inhibitory. A lack of antinatriferic activity in the dialysis fluid and plasma ultrafiltrate samples could indicate that the substance is not dialyzable from humans i vivo. Bourgoignie et al. (20), for example, observed that when whole uremic serum was ultrafiltered through a membrane with a molecular weight rejection of 50,000 Daltons, no anti- natriferic activity was present in the ultrafiltrate. On the other hand, ultrafiltration of an active fraction (fraction IV) from Sephadex GZSF resin through the same membrane did result in anti- natriferic activity in the ultrafiltrate. It was suggested that per- haps the active material was released from a larger molecule (i.e., bound to a plasma protein) on passage through the resin. Nondeproteinized uremic plasma samples, as discussed previously, have been shown to retain an antinatriferic activity. Also, plasma samples from carotid artery occluded cats have been shown to possess an antinatriferic activity following H-TCA deproteinization. Uremic plasma samples in the studies reported here, which were Similarly treated, exhibited no such activity. This observation further substantiates the conclusion that the patients studied did not possess an antinatri- feric material in their plasma, which would also account for the lack of this activity in the concentrated dialysis fluid and plasma ultrafiltrate specimens. One possible explanation for the lack of antinatriferic activity in the volume expansion experiments may be that none of the animals 172 studied were prehydrated, pretreated with mineralocorticoids, or given excess salt in their diet prior to the studies. These maneuvers are frequently done in experiments of this type (9,32,65,71,9l,94,104,131, 147,148,165). AS a result there may not have been an adequate expan- sion of the extracellular fluid volume with isotonic saline to require the release of an antinatriferic substance. Other factors previously discussed (REVIEW OF THE LITERATURE--Section B) may have been suffi- cient to eliminate the imposed saline load. Higgins (83), for example, observed that dogs which were in a state of positive sodium balance, as a result of DOCA administration and a high salt diet, showed a more rapid rate of sodium excretion dur- ing saline loading than did dogs that were salt depleted. It was postulated that the interstitial fluid volume may play an important role in the control of sodium excretion. In salt depleted dogs, with a plasma volume significantly below normal, influsion of 6% dextran in 5% glucose expanded the plasma volume from 3.5% to 5.4% but failed to increase the rate of sodium excretion (84). Even in dogs on a normal salt diet, infusion of up to 600 ml of albumin or dextran caused only moderate increases (and also decreases) in sodium excretion. In support of Higgens‘ observations, Schrier et al. (147) ob- served that the stimulus to natriuresis in the dog seemed to be an increase in the total extracellular fluid volume, including the inter- stitial space, rather than an increase in just the intravascular volume. Also, Sonnenberg and Pearce (159), investigating the natriuretic re- sponse to measured blood volume expansion in differently hydrated dogs, 173 observed that there was a significantly greater response in animals prehydrated with saline than was true in normally hydrated or de- hydrated animals. These authors suggested that the renal regulation of the extracellular fluid volume in the dog in response to intra- vascular expansion is determined by the existing extravascular volume. SUMMARY AND CONCLUSIONS The possibility of using spent hemodialysis fluid and plasma ultrafiltrates from uremic humans undergoing maintenance hemodialysis as a source of antinatriferic activity was investigated. Uremic plasma, from the patients studied, either with or without H-TCA deproteiniza- tion, demonstrated very little if any reversible antinatriferic activ- ity in fraction IV following Sephadex fractionation. Fraction IV from spent hemodialysis fluid and plasma ultrafiltrate concentrates also demonstrated no reversible antinatriferic activity which could not be attributed to ammonium ion. It is concluded from these studies that patients who have a fluid retention which is less than 5% of their dry body weight, who are being maintained by chronic hemodialysis, do not possess measurable quanti- ties of a previously described antinatriferic material. It is also concluded that the spent hemodialysis fluid and plasma ultrafiltrates from these patients do not contain measurable amounts of this substance as determined by the methods used in this investigation. Plasma samples from acutely volume expanded dogs, with or without H-TCA deproteinization, demonstrated no reversible antinatriferic activity in fraction IV following Sephadex fractionation. Plasma samples from these dogs which had been deproteinized with H-TCA, but had not been fractionated on Sephadex resin, demonstrated no reversible 174 175 antinatriferic activity that could not be attributed to TCA' anion contamination. From these results it is concluded that the natriuresis seen in situations of acute expansion of the extracellular fluid volume with isotonic saline cannot be attributed to an antinatriferic sub- stance in the plasma. If a natriuretic hormone possesses antinatri- feric activity, as previously described by others, it is also concluded that there was no natriuretic hormone present in any of the uremic or dog specimens of this study. APPENDICES 176 .mNooo moo oN ozooN No omooNooNoo No3 mooooNNmmo moN .NooNomo NoocmeNomoxm ooo Nooooom moo oooomooooo NNo>omooN moooNE N oo omoooomo chmo oz ooo OON ooNz .omoNoooNo oooom NNmooooNoooo mom: moNNN mooN .ooNomo NooomeNomoxm moo moNooo omoNoooo Nooocoo Eoo» mmoooo oomoomo moo NN mmoomN» mNmoo 3ono NNmooNomEEN .NNV mooooeme moo oo mNoEoN moo »o ooNoNooo omo»o omNoo NoomEmoomomE N oNoN moo »o mmoom>o moo No omzoNNo» NN NOV ooNomo Noooooo moo moNooo omoNoooo NoomEmoomomE N oNoN moo »o mmoom>o moN .mNosoN ooom ooNz mooe mom: NoNNN mooooomm oo NooNoooNEomomo moooNNooo .mooomoo EoNooEEo ooNozs ON ooNz oNNmo NNNO xmoooomm Eoo» omoon mom: ooo NNNNNoNooEmo moooomooNoE moNomomoo: NoomNooo Eoo» omoNoooo mom: NmNoEoN .NNV mooooNNNmo ooo .Nozv NoNoomooo mooooeme .NOONV oomooom oNoooNo ooooN oNNN moo» oo .ooNooNNonoooomo (ONI: oooooNz ooo ooNz .NmNoEoN oENoNo >N ooNoooo» oNEmo: omNNNNoooNN »o Noom»»N N xNozmoo< 177 omocNoooo NN+ NNI no- NN+ NNI NNI ooN _NNo N.NN N.NN_ N.NN_ N.NN mNN .NNN o.NN_ N.NN N.NN_ N.NN N .NN- NNN+ NNN+ NoN+ no NNN+ oNNN _oomN o.NN_ N.oN N.NN_ o.oo oNNN _omNN N.NN_ N.NN o.NNNIo.NN N NN NNN- NNN+ NNN+ NNN- INN+ NN+ ooN“ _ooN o.oN. N.NN N.NN_ N.No on _oNNN o NN_ N.NN o.NN_ o.NN N NNN- NoN+ NNN+ NNN- Noo+ NNN+ ooNN oNNN o.No N.mN N.NNN N.No NNN Nom o.oo o.oN o.No o.NN N No NONI NN+ NNI oNNN _oomN o.NN_ N.NN N.oo_ o.No N .NoI No NNI oNNN oNNN o.No o.No N.NN o.NN N N: I NONI. N N N. NN+ Ii , NoN _NNo N.NN _o.NN N.m _ N.N N NNN+ NNI NN+ NoN _NoN N.oN _o.NN o.oN_ N.NN N om NN+ NoN- NNI .NNM I oNNN _oNoN o.NN .o.oN o.oN_ o.NN N o: NN+ NNI No N o o NNN .NNN N.NN _o.NN o.o _ o.o N o: N oo oIoN mom I Nooooo No NN+ II NN+ NNI NNoNoo N-oNO oz NNN _oNN N.oN _o NN o.NN_ ooN N NNI NNI No- oNoN ooNN o.oN NN.NN o.NNNIo.NN N No III A Li N .I o N o N o N o N FIN N N o zNNN NZNNNNN Nooooo NN Ioooo ooN Noso oz Nosoo. NN «(no ooN.. .Nooo oz NomNII INN: oNNNzNNNoNoNo <=N IomooN moooNe N oo omoooomo mono oz ooo OON ooN: .omoNoooNo oooom NNmooooNoooo mom: NoNoN moom .ooNomo NooomsNomoxm moo moNooo omoNoooo Noooooo goo» mmoooo oomoomo moo NN mmoomN» mmmoo zono NNmooNomEEN .NNV mooooeme moo oo mNosoN moo »o ooNoNooo omo»o omooo NoomEmoomome N oNoN moo »o mmoom>o moo No omzoNNo» NN NOV ooNomo Noooooo moo moNooo omoNoooo Noomsmoomoms N oNoN moo »o mmoom>o moN .mNosoN ouom ooN: moos mom: NoNNN mooooomm oo NooNoooNsomomo moooNNooo .Ooo oo ooNooooNN»oooN= No omooooomoooo mono omo»o mooomoo Eooooeso ooNozs ON ooN: oNNmo NNNO xmoooomm soo» omoon mom: ooo NNNNNoNoosmo moooomooNoe moNom Iomooo NoomNooo Eoo» omoNoooo mom: NmNoEoN .NNV mooooNNNmo ooo .Nozv NoNoomooo mooooeme .NOONV oomoooo oNoooNo ooooN oNNN moo» oo mmooooomoooo oNoN» NNNNNoNo soo» >N ooNoooo» oNsmo: omNNNNoooNN »o Noom»»N NN xNozmoN< 180 . a . . I A. NNI NN+ .No mNN _NNN N.ON _N.mN o.N _ O.N N NNN+ NNI NoN+ NNN+ Nm+ NmN+ NNN NNN N.NN N.NN N.N _ o.N NNN _NNN o.oN _o.NN N.N _ o.o N o: NoN+ NNNI _ NNI fl oNNN oNNN N.No o.NN o.oN. o.NN N NNI NN+ No NN+ NNI NNI NNNN NmN N.oN_ o.NN o.NN o.NN oNNN _oNNN o.oN NN.NN o.NNN N.NN N IN N _ o N _ o N o N _ o N _ o N N o zNNN NZNNNoo NNooov N. Nmov ooN NNE No Noooov N Noov ooN NNEO oz ozoNNoNNZNozoo NNNNNO NNNNNNNNNNNNN NzoNNomooN moooNE N oo omoooomo mono N2 ooo OON ooN: .omoNoooNo ooooN NNmooooNoooo mom: NoNNN mooN .ooNomo NooomENomoxm moo moNooo omoNoooo Noooooo soo» mmoooo oomoomo moo NN mmoomN» mmmoo 3ono NNmooNomEEN .NNV mooooEmE moo oo mNoEoN moo »o ooNo INooo omo»o omxoo Noomsmoomome N oNoN moo »o mmoom>o moo No omzoNNo» NN NOV ooNomo Noooooo moo moNooo omoNoooo Noomemoomome N oNoN moo »o mmoom>o moN .NooNooooomoooo omo»o z: oomNooo ooo ooNooooomoooo moo»mo IO oomNooo oomoxmv mNoEoN ooom ooN: moos mom: NoNNN mooooomm oo NooNoooNEomomo moooNNoao .mooomoo EoNooeso ooNoze ON ooN: oNNmo NNNO xmoooomm Eoo» omoon mom: ooo NNNNNoNooEmo moooomooNoE moNomomoo: NoomNooo Eoo» omoNoooo mom: NmNoEoN .NNO mooooNNNmo ooo .Nozv NoNoomooo mooooEmE .NOONV oomoooo oNoooNo ooooN oNNN moo» oo Ooo oo ooNooooNN»oooN= No ooNooooomoooo omo»o ooo moo»mo .NmooNN NoNoN»Nooo moo Eoo» omoNoooo .NmooooNN»oooN: oENoNo Eoo» >N ooNoooo» oNEmoo omNNNNoooNN »o moom»»N NNN xNozmoN< 181 L NN+ N- NN+ oNNN oNNN N.NN o.NN o NN_ N.NN NNN+ NNI N N+ NNN _NNo o.No _o NN N.NN N.oN Io NN+ NNN+ NoN+ NNo _mNo o.NN _o.NN N.NN_ N.NN NNN+ NNN+ N N+ NNN _NoN N.NN _N.NN o.oN N.NN Nooo NNI No- NNI I NmN _Noo N.NN _o.oN N.NN_ N.NN NoN+ NNN- No- NNN _NNN N.Nm o oNN o.oN_ N.NN NNo No+ NNI NNN- omN NNN N.NN _o.oN N.NN. o.oN NoNI NNN- NNI omoN oNN N.Nm o moN NoN. NoN Nmo NN+ NNN- NoN- oooN oooN N.NN _N.NN o.NN_ o.No NNNN+ NNN- NNoI omNN ooN N.m _N.NN o.NNNN.NN oNoI NNN+ NoN- No NN+ NNII No NNN _NmN N.NN .N.NN o.NN_ o.NN NNN _oNN N.mN _o.oo o.NN _o.NN NNI om- NoN- No+ NNI NN+ NoN NNoN N.mN _N.NN o.NNaNN.NN I NNN _NNN N.mN _N.oN .N.oN._N.oN om ........... I I p.’ /\ I... 8 1.4, flfll I / OIWWTIOIg.‘ OIwn MINI-m r m a 8¢i a IlIl o.NN N.oN N.NNNNmNN 4/ flO¢+ NmN+ N.No_ N.NN N.NN_ N.NN N . ‘ ‘1 N moo NNNN NNNN I! lilIII N~+ . o o . ooN+ omoN+. .Mm N.NN ooNN oNNN N NN\ N NN N NW N NN N onN oNNN N.oN N N N NN+ NmN+ ooNN. NN+ NN? , NNN» NNN_ NNN N.NN N.NN o.NN o.oN NNNNNNN o.NNNIo.mN N.NN_ o.NN N N moo NNN- NN+ NNN- NmN+ NNNN+ NNN+ - I NNN mNN N.mN. N.mN o.NN N.NN oNNN NNm N.NN_ o.N o.NN_ N.N N NNI NNo+ NNo+ NNN+ NmN+ NNoNI mNN ooN N.oN N.NN N.NN N.NN oNoN NmN N.NN o.NN N.NN o.NN N N moo NW N o N o N o N m NI o N m ZNNN .I NNoooO N No o ooN N>oo oz NNoooO N N ooN N> oz NNNozNo o I ooNNzoo NNzNN Noozmm oNNZNzNNNNxN .N xNoomoo< oN o3ooN No omooNooNoo No3 mooooNNNmo moN IomooN moooNe N oo omoooomo mono oz ooo OON ooN: .omoNoooNo ooooN NNmooooNoooo mom; NoNNN mooN .NooNomo NooomENomoxm ooo Noooooo moo oooomooooo NNo> .ooNomo NooomeNomoxm moo moNooo omoNoooo Noooooo Eoo» mmoooo oomoomo moo NN mmoomN» mmmoo 3ono NNmooNomEEN .NNN mooooems moo oo mNoEoN moo »o ooNoNooo omo»o omxoo Noomsmoomome N oNoN moo »o mmoom>o moo No omzoNNo» NN NOV ooNomo Noooooo moo moNooo omoNoooo Noomemoomome N oNoN moo »o mmoom>o moN mNoEoN ooom ooN: mooe mom: NoNNN mooooomm oo NooNoooNEomomo moooNNooO Iooo» moo»mo NNN mooooNNNmo ooo .Nozv NoNoomooo mooooEmE .Nmmmoo oNoe oNN .oNNmo NNNN xmoooomm oo ooNooooNo .NOONV oomoooo oNoooNo ooooN oNoN moo» oo Noome INomoxm ooNNoooxm oomNmz Nooo Noooo NN moo »o ooo» soo» NmNoEoN oENoNo moo omNNonoooomo N xNozmoo< 183 ooo+ om- om+ Noo ooo. o.oo o.oq o.om o.oN o ooo mNoosz No 1‘ om- om¢+ oom+ ooo Nmoo m.mm o.om o.om o.oN o moo ooo+ oNN- owe- oooo .omoo «.mo_ o.oN o.NN N.N¢ ooN- ooN- ooN- mom omo o.No o.om N.m o.oN o ooo o¢+ oo+ ooo‘ woo woo o.oN o.oN m.o__ o.oo o~o+ ooo- oo- moo oom o.oo o.N. o.o _ o.o m ooo o¢~+ omo+ nooo+ oo¢o_ oooo o._o_ m.o m.o__ m.o goo- oNo+ omo+ omo _ooo o.o¢,_m.mm o.ook_o.¢m N ooo mmhzsz om.— mmhzzoz mm § 184 .o xooomoo< oo ozoom mo omoooooooo mo: muooomommo ,moooomo pooome -oomoxm ooo ooooooo moo oooooooooo moooomooo mooooe m on omoooumo moomo o: ooo oom oooz .omooooooo oooom oomoooooooou mom; mooom mooN .oooomo _ooomeoomoxm moo mooooo omoooooo _oooooo sooo mooooo oomoomo moo mo mmoooom mmmoo zoomo oomoooomeso .Amv moooosms moo oo moogom moo oo oooooooo omooo omooo moomE -moomome m omoo moo oo mmoom>o moo zo omZooooo mo on uooomo _oooooo moo mooooo omoooooo moomEmoomomE m omoo moo oo mmoom>o moo umooaom ooom oooz moms mom; mooom mooooomm oo moooooooEomomo moomooooo ”mooo -mmo EooooEEo ooooze oo oooz oommo Nmmo xmoooomm goo» omooom ommo woo mmooEom oo< .Amv moooomommo woo .Auumv oomooom ooooooo oooom .Aozv ooooomooo mooooEmE ooom mooo oo moomsoomoxm ooomoooxm oooomz xooo Foooo om moo co omoooooo mmooEom ogmooo moo omNooomoooomo H oooooooo omNooooooxo oo moomoom > xoazmoo< 185 omoooooou NNo+ Noo+ Nmo+ ooNo oNNo N.NN_ NgoN N.NN_ Noon N NN+ o- No+ No N o.oo N.oo N NN_ o.NN o N Noon NNN+ NM- NN¢+ NNN NNN N Nm_ o,NN o.NN_ N.NN N No NN+ NN+ oNNo oNNo‘ N.Nm. NNON o No_ N.NN o‘ N ooo NN+ Noo+ N _+ NNN _ NNN N.oN NDNN N.Now N.oN N NN+ Noo+ NNN+ ooN _ NNN m NN. o.NN N.NN_ N.NN o N ooo NN+ No- on- NoN _ oNN o.NN_ o.oN o.oN_ o.oN N N N+ NN- N4o+ NNN NNN‘ N.NN_ o.NN o.NN o.oo _ N ooN Noo+ NN- Noo+ 4N+ No- No oNN . NON N.No_ N.oo o.___ o.o_ NNN NNN N.NN_ N.om o ON_ o.ON N NNN+ No- N¢N+ NNN+ mo- NNo+ omm . ooN N.NN. N.NN o.NN. N.NN NNN _ ooN o.NN o.NN N.oN. o.0o o N ooo NN- NN- om- NN- NNN+ N N+ oooo. oooo o.oN N.oN N.oN .N.NN omoo_ oomo o.NN N.NN o.omo N.NN N NN- omo- NNo- NN+ N N+ NNN+ “mom _ coo N oN N NN o No ”o mNi‘ oNN _ NNN o NNN N on o or“ N No o N ooN NIMH o N o N o N o N _ o N .o o Zoom omoooo N oooo Now >2 N owe ooxo oomo:pom Noso No.1 mNoooo: o NoNozoo zoo Noozom NooZNooNNNXN \ N _NN- A No_. NNo; o.N NNN N.NN_ ooNN N oN_ o.NN N NNN+ NN+ NNN+ Now. NNN ,N . N. N o. N o.N _ N moo NoN+ NN- NNN+ NNN NNN N.oN. N.oN N.N . o.N N NN+ N- NN- moo. oo NNN N.NN o.N N.NN. o.NN N {N ,io... .., w , ,MMFDZHNNN N¢ NoN+ No NoN+ NNN _ NNN N.NN_ N.NN N.oN N.No N NN- NN- NN+ oNNoo oNNN! o.NN_‘N.oN N.NN. o NN+ N N ooo NN+ Nmo- No- NNN _ N.N N.NN_ N.NN N.NN o.oN N NN_+ Noo- No- NNN _ NNN N.N: N.N: MIN». o.N N, N 8o Nmo- NN+ NNo- , _ooN__ oN_N N.NN_ N.oN o.oo N.__ N Noo+ Noo- NN+ N.NoorolmomulNluwpwwpowoowNuhmhmwuohmm N NNNNNPN NN- N+ Noo+ NNN+ NoN+ NNN+ NNN _ NoN N.NN o.NN N.NN_ N.NN ooN NNN N.NN. N.NN .o.NN o.oN N., NWP+ NJF+ NWN+ NNN+ Nmm+ Nmo+ STE N.Nm N.NN N.N N.m NE. E. N.NN N.NN N.N Po _ E NN+ NN+ NNF+ Noo+ 4N+ NNo+ oo___ oNoo o.NN_ N.NN N.NN N.NN NNN _ NNN o.NN o.NN o.NN_ o.oN N NNN- NN_+ NNN- NNo- NNN+ Noo+ Noo, NNN, owom. o.NN o.N o.N ooN N NNN oNNN. N.Nm N.N.NN_ N.NN o N ooo NN- NN- NNo- NNN _ NNN N.NN_ N.NN N.No_ o.N. N NNN+ N- NN+ NNN_ NNN N.N: o.oN N.N: No P NINE. N o N o N o N Xo| Now N o o N zoom .. Nmeooomol NNN. ooN N>oo No omeooo.N .omoo ooN Hoowo N: NNoozoz om NooozNz No mNzNo NNNNNN NooZNzNNNNXN omooooooo--> xNNZNooo 187 umoooooom Noo+ NNN- NN- — — NNN- NNN N.NN _N.NN N.NN _N.NN N No_- NN- Nmo- NNo+ No- NN+ oNN NNN o.NN _N.NN N.Nm _N.NN ooN . NNN o.NN_-oNNN o.N _ N.N _ No moo» . No No No NNoozNz NN NoN . NNN o.NN_ o.NN o.NN_ o.NN N NN- NN- - NN- omm _ .NN o.oN_ N.oN N.oo_ o.oo _ _o ooNN NN- NN- Noo- NNN _ NNN N.NN_ N.oN N.N _ N.oo N NNN+ NNN- oNo- NNN . NoN o.NN_ N.NN o.NN_ o-NN o oo moo» NN+ NNo- NN- omm . _NN o.NN N.NN o.N__ N.NN N NN+ Nmo- Noo- Noo+ N N- Noo- ooN_ oNNN N.NoN N.NN o.NN _N.NN NNN N NoN N.No_ N.NN N.N _ N.N _ N moo N-u o N _ N N N o N-IN o N _ o N-N o ZNNN NmEomo N ooNN-onm moo» No Nmsooo N oNNN ooN N>EN om:- NNoozNz N Nox_zoo NNzNo NNNNNN NooNNzNNNNXN .moom—om>m ooo mom m moo goo» mmooEmm oo -msz:o.oo moooomooEomomo m>oomoooom=o .-mo omoo oomooom oo ozoom mom: mmooENmN .H xooomoo< o» czoom mm omomoomomm mm: moooomommo mo» .moooomo omoomsoomoxm mom Fooooom moo oooomooooo mom>omooo moooos m om omogommo moomo a: mom oum oooz .omooomoom oooom Nomoooooooom mom: mooom mooN .oooomo Pmoomeoomoxm moo moooom mmoomooo _ooooom Eco» mmomom oommomo moo m» mmoomo» mmmoo zoomo Nomomoomeso .ANV momooEmE moo oo moosmm »o ooooooom omo»o omomo moomemoommms m ommo moo »o mmoom>o moo No umzoooo» m» Nov mmoomo oooooom moo mooooo mmoomooo moomsmoommms m ommo moo »o mmoom>o moo .omm: mom: mooom oomom»»»u .mome mom: moooomooaomomu momuooooo momoz .commo Nmmm xmomoomm oo oooomoooommo» moo»mo omv moomomommo mom .ouomv oomooom ooomoom oooom .Aozv omooomooo momooEme zoom moo» oo moomeoomoxm ooomomoxm oomomz Nooo omooo mm moo Eco» mmoosmm mammoo moo om~ooomoooomo xoozmmm< 188 Noo+ Noo- No- NNN . oNN N.NN. N.NN o-NN _N.NN N No- NN+ NN+ NNN _ NNN o.oN. N,NN o.oo _N.o o oo moo» NN+ le fiMI — _ 1— ooo _oNo N.No_ N.No N.No. N.NN N No+ NNN- NNo- NN- NN+ NN+ NoN-oNNN o.NNNN.NN N.Nr o.NN oNNo-Nom N.waowNN N.oNNo.oN N o moo N _ o N N o N o N o o N _ o N -N o zNNN moooo N oNoN ooN N>NN No NmEooN N oNoN-Dom -ooso N: NNoozNz om NNooNNz No NNNNN Noooom _NNZNNNNNNNN omooooooo--N> NNozNNNN 189 APPENDIX VII Effects of lyophilized fraction IV from H-TCA deproteinized plasma samples from dog 9 (6% total body weight expansion) following fraction- ation on Sephadex GZSF resin. Duplicate determinations were made using different skins. The average of the last 3 measurements obtained dur- ing the control period (C) is followed by the average of the last 3 measurements taken after addition of sample to the membrane (E). Imme- diately below these figures is the percent change from control obtained during the experimental period. Frog skins were continuously short circuited, with SCC and MP being recorded at 5 minute intervals throughe out the control and experimental periods. The resistance was calculated as shown in Appendix I. H-TCA deproteinized plasma samples from dogs l0-l2 were not fractionated. MP (mV) SCC (pA) R (ohms) DOG 9‘ SKIN c E c 1 E c E I CONTROL 1 22.0 27.0 35.0 l39.8 628 678 +23% +14% +8% 2 6.5 111.2 48.7 180.7 133 1 140 +72% +66% +5% 0 MINUTES 1 25.7 125.0 48.7 139.0 528 1 641 -3% -20% +21% 2 47.5 142.2 28.3 123.0 1670 1l840 -11% -19% +10% 15 MINUTES 1 7.8116.0 43.2 159.3 182 1 269 +105% +37% +48% 2 21.0 129.0 59.3 168.0 345 1 426 +38% +15% +24% 30 MINUTES 1 19.5 22.7 46.3 155.3 421 1 410 +1 % +19% -3% 2 8.8 10.0 68.0 176.7 130 1 131 +14% +13% +1% 45 MINUTES 1 7.3 1 7.0 42.7 134.7 171 1 202 -4% -1 % +17% 2 13.2 14.0 27.7 ‘25.8 476 1 542 +6% -7% +14% 190 .o xooomoo< co oxeom mm omomoomomu mm; moomomommm .moooomo omoomeoomoxm mom Nooooom moo oooomooooo mom>omooo mooooE m om omooommo moomo oz mom mom oooz .omooom -oom oooom Nomooooooooo mom: Nooom mooN .oooomo omoomsoomoxm moo mooooo mmoomooo Foooooo goo» mmomom oommomo moo mo mmoamo» mmmoo zoomo homomoomeso .ANV momooEmE moo oo moosmm »o ooooooom omo»o omomo moomEmoommmE m ommo moo »o mmoom>o moo No omzoooo» mo on mooomo _ooooom moo mooooo mmoomooo Noomemoommms m ommo moo »o mmoom>o moo .mooom oomom»»oo moomo moms mom; moooomoosomomo momuooooo .ANV mmomomommo mom .Auumv oomooom ooomoom oooom .Aozv omooomooo momoosms ooNN moo» oo moomaoomoxm ooomomoxm oomomz Nooo omooo on moo Eco» mmoosmm ooNN—o moo omNooomoooomo N oooommo» omNooooooxo »o momm»»N HHH> xHozmoo< 191 omoooooou NoN+ NNN- -woo+ 4N+ NN- NN+ NNN_ NNN N.NN_ moo o.oN N.NN 2N oNN o.NN_ N.oN N.NN_ooNo N NNN+ NoN- N o+ Noo+ No- omo+ NNN _ NNN o.oo_ o.NN N.m o.m NoN _ NNN N.oom N.oN N.m _N.N o No moo . - NNN- o- NNo NNN _ oNN oNoN. o.NN o.No. N.oo N “N. NN- No- NNN _NNN o.oN_ o.NN N.oN—,N.NN o No moo NN+ NNN- NN- NNN _ oNN N.NN_ o.NN N.NN_ N.NN N N _+ Noo+ NNN» NNN M NNN N.NN_ N.NN o.NN_ N.oN o No moo 4N- NNN+ NNN+ oNN NoN N.NN. N.Nm N.NN. N.NN N NN+ NN+ NN+ oNN _ NNN N.NN_ o.NN N.NN_ N.Nm o No moo Noo- NNN+ Nmo+ No NN+ NN+ oNNN. oNNN N.NN_ N.NN N.NN. N.NN oNooM oNoN N.oN. N.NN _.N.mN_ o.NN N NN- NNN+ NNN+ N+ NNN+ NNN+ oN_ _ NNN No__ o.oN N.NN_ N.N moo _ .NN o.oN_ N.NN to.N _ o.N o oo moo No+ No- do NNN- Nwo+ NNN+ oNN _ _Nm o.NN_ N.oN o.oN o.oN oNoN. oNNN N.oN N.oN o.NN_ N.NN N No- No- NNN- om- NNN+ NomoN ooN _ NNN N.NN N.oN o.NN_.N.NN NoN _ NNN N.NN_N.NN N NNN o.oN o oo moo N .N o N m N . _ o N _Tmo -Noi _ o zNNm -Nweooo N, No -oom oNoN Mo mmsoooN -NNNN oom -Nooooo .mmwoooz.oo ooNozom mozoo Noozom No ZNNHNNNNN 192 No- NN+ No NNN _ NNN N.oN .o.oN N.NN .N.NN N no- NNN+ Nmo+ NNN . NNN N.Nm _N.NN N.NN _o.oN _ No moo of. N? am NoN . NNN o.NN .o.om o.N. _N.NN N NNN- Nom+ om+ oNN oNoo N.Nm N.oN N.NN N.NN _ No moo NNN- ooNo+ Noo+ omm _ oNoN N.Nm _N.NN N.NN_ N.NN N NoN- NNN+ Noo+ Noo _oNoo o.NN _N.NN o.oN_-N.Nm _ No moo N _ o N _ o N .NN o N o N o N o zoom Nmsooo N ooNN ooN Nos oo- omoooo N oso o: NNoozNz NN - NNooNoz om o xHozwam< BIBLIOGRAPHY BIBLIOGRAPHY Andersson, B., M. Jobin, and K. Olsson. Stimulation of urinary salt excretion following injections of hypertonic sodium chloride into the third brain ventricle. Acta Physiol. Scand. 67:l27, l966. Andersson, B., M. Jobin, and K. Olsson. A study of thirst and other effects of an increased sodium concentration in the third brain ventricle. Acta Physiol. Scand. 69:29, 1967. Andreucci, V. E., J. Herrera-Acousta, F. J. Rector, Jr., and D. M. Seldin. Effective glomerular filtration pressure and single nephron filtration rate during hydropenia, elevated ureteral pres- sure, and acute volume expansion with isotonic saline. J. Clin. Invest. 50:2230, l97l. Aperia, A. C., C. 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