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Renal control of renin secretion and some actions of the angiotensins in the kidney of the teleost Salmo… Bailey, John Richard 1979

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RENAL CONTROL OF RENIN SECRETION AND SOME ACTIONS OF THE ANGIOTENSINS IN THE KIDNEY OF THE TELEOST SALMO GAIRDNERIi by JOHN RICHARD BAILEY B.S c , Un i v e r s i t y of Ottawa, 1969 M.Sc, University of Ottawa, 1973 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Faculty of Graduate Studies (Zoology) We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA May 1979 @ John R. Bailey, 1979 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an' advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the l i b r a r y s h a l l make i t f r e e l y a v a i l a b l e for reference and study. I further agree that permission f o r extensive copying of t h i s t hesis f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s representa-t i v e s . I t i s understood that copying or p u b l i c a t i o n of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of Zoology The'University of B r i t i s h Columbia Vancouver, B. C., Canada i i ABSTRACT The renin-angiotensin system i s a hormonal system found throughout the vertebrate phylogenetic scale with the exception of elasmobranchs and c y c l o -stomes. This sytem has only been extensively studied i n mammalian species to date. Renin secretion, i n the mammals studied, i s influenced by a number of factors such as renal perfusion pressure, renal tubular sodium l e v e l s , c i r c u l a t i n g catecholamines and the sympathetic nervous sytem. In f i s h e s factors which influence renin secretion had not been investigated but i t was known that de-hydration of f i s h e s caused a depletion of renal renin stores. This action may be due to two possible causes either a decline i n blood volume with a consequent decrease i n blood pressure or an increased blood osmotic pressure due to water l o s s . Therefore the e f f e c t s of changing either blood pressure or blood osmo-l a r i t y (using sodium as the osmotically a c t i v e p a r i c l e ) on renin release were investigated. Further some actions of the angiotensins on the trout kidney were investigated to determine the action of the renin-angiotensin system i n combatting dehydration. Since i t i s very d i f f i c u l t to assay renin per se, i t i s necessary to assay renin a c t i v i t y , i . e . , to determine the amount of product formed from the renin-renin substrate r e a c t i o n . This has been done by means of a bioassay but since the bioassay technique tends to be time consuming, a radioimmuno-assay technique was developed. A s i g n i f i c a n t c o r r e l a t i o n between blood lo s s and plasma renin a c t i v i t y was established. Further work using an i s o l a t e d n o n - f i l t e r i n g perfused kidney preparation showed that a decline i n renal perfusion pressure caused an increase i n renin release as evidenced by an increase i n renin a c t i v i t y . This appeared to be a d i r e c t e f f e c t on the renin-secreting c e l l s as sympathetic blocking agents had no e f f e c t on the response. Increasing renal perfusion i i i pressure has l i t t l e or no e f f e c t on renin secretion but the end product of the system, angiotensin I I , w i l l apparently i n h i b i t further renin secretion by a short-loop negative feedback type system. Plasma sodium l e v e l s do not appear to a f f e c t renin secretion i n the trout as hypertonic sodium perfusion of the i s o l a t e d n o n - f i l t e r i n g kidney had no e f f e c t on renin release. S i m i l a r i l y , hypertonic sodium perfusion of the i s o l a t e d f i l t e r i n g kidney had'no apparent e f f e c t on r e n i n release, which indicates that r e n a l tubular sodium loads have no e f f e c t on renin secretion i n t h i s species. Angiotensin II i s known to have several diverse actions i n mammalian kidneys but i t s action i n the f i s h kidney was*.obscure. I t was found that angiotensin I has l i t t l e or no e f f e c t on urine flow rates but has an apparent a n t i n a t r i u r e t i c e f f e c t on the renal tubules. Angiotensin II on the other hand has both a d i u r e t i c and an a n t i d i u r e t i c e f f e c t , depending on which part of the r e n a l vasculature i s stimulated. In addition, the angiotensin I I has an a n t i -n a t r i u r e t i c e f f e c t which i s of s i m i l a r magnitude to that exerted by angiotensin I arid t h i s may also be a d i r e c t e f f e c t on the renal tubule. These data ind i c a t e that the angiotensins may have a r o l e i n the maintenance of blood volume and blood osmolarity i n the trout. They also i n d i c a t e that the angiotensin I may have been the p r i m i t i v e messenger of the system as t h i s compound i s not highly a c t i v e i n mammalian species. i v TABLE OF CONTENTS P a g e S e c t i o n I THE RENIN-ANGIOTENSIN SYSTEM General I n t r o d u c t i o n 1 Sec t i o n I I RENIN ANALYSIS I n t r o d u c t i o n 6 Methods 1 4 Results and D i s c u s s i o n 2 0 Se c t i o n I I I CONTROL OF RENIN SECRETION: PRESSURE EFFECTS I n t r o d u c t i o n 40 Methods 47 Res u l t s and D i s c u s s i o n 54 Sec t i o n IV SODIUM AND RENIN RELEASE I n t r o d u c t i o n 84 Methods 9 5 Results and D i s c u s s i o n 9 8 Se c t i o n V ANGIOTENSIN AND THE KIDNEY I n t r o d u c t i o n 1 0 8 Methods 1 1 2 Results and D i s c u s s i o n 1 1 5 General D i s c u s s i o n 6.0 RENIN IN FISHES 1 5 0 Summary 1 6 5 L i t e r a t u r e C i t e d 167 Appendix 1 1 8 2 Appendix 2 1 8 3 Appendix 3 1 8 3 Appendix 4 1 8 8 V LIST OF TABLES Page TABLE 2.1 E f f e c t of i s o t o n i c s a l i n e and plasma extracts on mean pressure increase following various treatments of plasma. TABLE 3.1 E f f e c t of changes i n renal perfusion pressure on renin release. 26 60 TABLE 3.2 E f f e c t of alpha receptor blockage on the renin response to hypotension. TABLE 3.3 E f f e c t of beta receptor blockage on the renin response to hypotension TABLE 3.4 E f f e c t of combined alpha and beta receptor blockage on baroreceptor induced renin secretion. 69 73 77 TABLE 4.1 E f f e c t of sodium perfusion on renin release i n n o n - f i l t e r i n g and f i l t e r i n g kidneys. TABLE 4.2 E f f e c t of hypertonic s a l i n e perfusion on urine flow rates i n the i s o l a t e d perfused kidney. 101 106 v i LIST OF FIGURES Page FIGURE 2.1 Cross s e c t i o n of t r o u t kidney. 22 FIGURE 2.2 A. Cross s e c t i o n of s k i l f i s h kidney. 24 B. Cross s e c t i o n of p i r a r u c u kidney. 24 C. Cross s e c t i o n of j e j u kidney. 24 FIGURE 2.3 The e f f e c t of temperatures on r e n i n a c t i v i t y . 30 FIGURE 2.4 The e f f e c t of pH on r e n i n a c t i v i t y . 33 FIGURE 2.5 C o r r e l a t i o n between a c t i v i t i e s obtained from RIA and BA. 36 FIGURE 3.1 Diagrammatic view of the kidney p e r f u s i o n p r e p a r a t i o n and apparatus. 50 FIGURE 3.2 C o r r e l a t i o n between haemorrhage and plasma r e n i n a c t i v i t y . 56 FIGURE 3.3 The e f f e c t of a d e c l i n e i n r e n a l p e r f u s i o n pressure on r e n i n r e l e a s e from the i s o l a t e d perfused t r o u t kidney. 59 FIGURE 3.4 The c o r r e l a t i o n between r e n a l p e r f u s i o n pressure and r e n i n s e c r e t i o n i n the i s o l a t e d perfused t r o u t kidney. 62 FIGURE 3.5 Changing r e n a l p e r f u s i o n pressure and r e n i n r e l e a s e i n the i s o l a t e d perfused t r o u t kidney. 64 FIGURE 3.6 E f f e c t of a d d i t i o n of alpha b l o c k i n g agent on the baroreceptor response i n the i s o l a t e d perfused t r o u t kidney. 68 FIGURE 3.7 The e f f e c t of a d d i t i o n of beta bl o c k e r and changing r e n a l p e r f u s i o n pressure on r e n i n r e l e a s e i n the i s o l a t e d perfused t r o u t kidney. 72 FIGURE 3.8 The e f f e c t of combined b l o c k i n g agents on r e n i n r e l e a s e caused by changing r e n a l p e r f u s i o n pressure i n the i s o l a t e d perfused t r o u t kidney. 76 FIGURE 3.9 The i n v i t r o e f f e c t of a n g i o t e n s i n I I on r e n i n r e l e a s e . 81 v i i Page FIGURE 4.1 FIGURE 4.2 FIGURE 4.3 FIGURE 5.1 FIGURE 5.2 FIGURE 5.3 FIGURE 5.4 FIGURE 5.5 FIGURE 5.6 FIGURE 5.7 FIGURE 5.8 FIGURE 5.9 FIGURE 5.10 FIGURE 5.11 FIGURE 5.12 Diagrammatic representation of.the juxta-glomerular apparatus of mammals. 86 The e f f e c t of hypertonic s a l i n e perfusion on renin release from.the i s o l a t e d perfused trout kidney. 100 The e f f e c t of hypertonic s a l i n e perfusion on renin release i n the i s o l a t e d perfused f i l t e r -ing trout kidney. 103 Urine flow rate and perfusion pressure i n the i s o l a t e d perfused trout kidney. 117 The e f f e c t of i n j e c t i o n of angiotensin I into the a r t e r i a l side of the renal c i r c u l a t i o n on urine flows. 119 Urine flow rates following angiotensin I i n j e c t i o n i n t o the venous side of the re n a l c i r c u l a t i o n . 121 Urine flow rates following angiotensin II i n j e c t i o n into the a r t e r i a l side of the renal c i r c u l a t i o n . 125 The e f f e c t of i n j e c t i n g angiotensin II into the venous c i r c u l a t i o n of the kidney on urine flow rates. 127 Urinary sodium l e v e l s following i n j e c t i o n of angiotensin I on the a r t e r i a l side of the renal c i r c u l a t i o n . 130 Urinary sodium l e v e l s following angiotensin I i n j e c t i o n into the venous c i r c u l a t i o n of the kidney. 132 Urinary sodium l e v e l s following i n j e c t i o n of angiotensin II into the renal a r t e r i a l c i r c u l a t i o n . 135 Urinary sodium l e v e l s following i n j e c t i o n of angiotensin II into the renal venous c i r c u l a -t i o n . 137 Urinary potassium l e v e l s following i n j e c t i o n of angiotensin I into the renal a r t e r i a l c i r c u l a t i o n . 140 Urinary potassium l e v e l s following i n j e c t i o n of angiotensin I into the renal venous c i r c u l a t i o n . 142 Urinary potassium l e v e l s following i n j e c t i o n of angiotensin II into the renal a r t e r i a l c i r c u l a t i o n . 145 v i i i Page FIGURE 5.13 Urinary potassium l e v e l s following i n j e c t i o n of angiotensin II into the renal venous c i r c u l a t i o n . 147 FIGURE 6.1 Diagrammatic summary of renin release and the actions of the renin-angiotensin system i n the trout. 152 i x Acknowledgements. V ^ I would l i k e to acknowledge the assistance, both ph y s i c a l and moral, that I recieved from a number of people, both friends and colleagues, during the course of t h i s study. I would e s p e c i a l l y l i k e to thank my supervisor, Dr. D.J. Randall, f o r h i s patience. I would also l i k e to thank; Steve Haswell for h i s a i d i n cannulating trout, Dr. A.M. Perks for h i s c r i t i c a l comments during the writing of t h i s thesis and Dr. H. Nisftimura f o r her a i d i n developing the radioimmunoassay technique which was used during t h i s study. This work was p a r t l y supported by U.B.C. teaching a s s i s t a n t s h i p s and p a r t l y by NSERC operating grants to Dr. D.J. Randall. 1 SECTION I GENERAL INTRODUCTION 1.0 THE RENIN-ANGIOTENSIN SYSTEM. 1.1 Renin i n Mammals. Renin i s an enzyme found i n modified smooth muscle c e l l s i n preglomeru-l a r a r t e r i o l e s of the kidney of mammals (De Muylder, 1945; Barajas and Latta, 1963; H a r t r o f t et al., 1964; Bing et al. , 1967). The enzyme' acts on a protein precursor, angiotensinogen, found i n blood plasma and s p l i t s o f f a decapeptide moiety c a l l e d angiotensin I (Oparil et a l . , 1973). Another enzyme, which i s simply c a l l e d a converting enzyme, s p l i t s o f f two more amino acids from angiotensin I ( A l ) . The r e s u l t i n g octapeptide, which i s c a l l e d angiotensin I I , has various e f f e c t s on both s a l t and water balance as w e l l as being a potent pressor substance (Peters and Bonjour, 1971). In most mammals, angiotensin II (hereafter r e f e r r e d to as A l l ) acts on the zona glomerulosa of the adrenal cortex to stimulate aldosterone secretion (Peters and Bonjour, 1971). There are numerous factors which influence the rate of renin secretion and the renin-angiotensinogen reaction i n mammals. The e a r l i e s t known factor i s a decline i n systemic blood pressure or, more s p e c i f i c a l l y , i n t r a - r e n a l blood pressure. This r e s u l t s i n an increase i n renin secretion (Tobian et a l . , 1959; Skinner ejt a l . , 1963). In j e c t i o n of v a s o - d i l a t i n g drugs r e s u l t s i n an increase i n plasma renin a c t i v i t y which i s dose-dependent (Pe l l i n g e r et a l . , 1973) and maximum afferent a r t e r i o l a r d i l a t i o n r e s u l t s i n a maximum rate of renin release i n dogs which i s unaffected by other factors known to stimulate renin release (Eide e_t _al. , 1978) . Thus i t appeared that renin secretion was con t r o l l e d by stret c h receptors i n the kidney preglomerular afferent a r t e r i o l e s , the l o c a t i o n of the juxtaglomerular c e l l s . 2 Varying concentrations of sodium i n the renal tubular f l u i d can r e s u l t i n an e f f e c t on renin secretion and so i t was postulated that the sodium concentration reaching the macula densa of the d i s t a l convoluted tubule c o n t r o l l e d renin secretion. Results have been obtained from a number of stud-ies to s u p p o r t or disprove t h i s rather c o n t r o v e r s i a l hypothesis. Vander and M i l l e r (1964) found that increasing sodium concentrations i n renal per-fusion f l u i d i n i n t a c t kidneys caused a decreased renin release into the blood. The converse was demonstrated by Davis et a l . (1967), i . e . , a decline i n perfusion sodium concentrations leads to an increase i n renin secretion. Unfortunately, other workers showed that an increase i n sodium concentrations causes renin release (Thurau ^t_ a l . , 1967) . J-n v i t r o studies have shown that when renal c o r t i c a l s l i c e s are incubated with a high sodium medium, renin release i s i n h i b i t e d i n an almost l i n e a r fashion (Michelakis, 1971a). A more recent study has shown that increasing renal plasma osmolar-i t y with either sodium, urea or dextrose r e s u l t s i n an increase i n renin release and that t h i s increase ends within 15 seconds a f t e r the increased renal plasma osmolarity begins to decline (Young and Rostorfer, 1973). The authors postulate a d i r e c t e f f e c t of the increased osmolarity on the juxta-glomerular c e l l s as the cause of the renin release. Calcium was found, by Michelakis (1971a), to f i r s t increase and then decrease renin secretion. A l a t e r study by Lester and Rubin (1977) showed that e x t r a - c e l l u l a r calcium had l i t t l e e f f e c t on the mechanism of renin secretion but an increased i n f l u x of the cation was needed for either synthesis or m o b i l i z a t i o n of renin. These authors proposed that i n t r a -c e l l u l a r release of calcium may be the s i g n a l which t r i g g e r s renin secretion. Baumbach and Leyssac (1977), however, found that decreasing e x t r a - c e l l u l a r 3 calcium resulted i n an increased renin secretion which was graded and revers-i b l e . A calcium ionophore had a s i m i l a r but slower e f f e c t while lanthanum caused a s i g n i f i c a n t depression of renin release. These authors suggested that basal renin release i s a function of a c t i v e , calcium-dependent c e l l volume regulation, i . e . , swelling caused an increase i n renin release. They further suggested that membrane-bound calcium has a d i r e c t e f f e c t on the c e l l membrane permeability to renin. Another factor which has been- i n v e s t i g a t e d i n recent years i s some form of neural c o n t r o l . Vander and Luciano (1967) found that the sympathetic nervous system, which innervates the juxtaglomerular apparatus, plays a modi-fying r o l e i n renin secretion i n i t i a t e d by s a l t depletion. Infusion of catecholamines can r e s u l t i n an increase i n renin secretion. Stimulation of the renal nerves has a s i m i l a r e f f e c t (LaGrange et al., 1973). E a r l i e r work had shown that blocking both the alpha and beta adrenergic receptors i n h i b i t e d renin release (Winer ejr a l . , 1969) but the authors concluded that these receptors were renal i n o r i g i n and not dependent on the renal nerves as transplanted human kidneys showed a normal renin response. Vandogen et a l . (1973) found that the beta receptor blocking agent, d,l-propranolol, i n h i b i t e d renin release during catecholamine perfusion of i s o l a t e d r a t kidneys but phenoxybenzamine, which blocks alpha receptors and reduces catecholamine uptake by c e l l s had no e f f e c t on renin secretion during the perfusion. The beta receptor blocker can i n h i b i t renin release caused by the action of v a s o - d i l a t i n g drugs ( P e l l i n g e r e_t a l . , 1973). Later studies concluded that the vascular, tubular and sympathetic adrenergic mechanisms which govern renin release ( i n dogs) are capable of functioning independently (Osborn et_ a l . , 1977). 4 There are various factors which can influence the renin-angiotensinogen reaction once renin i s released into the plasma. A l l has been shown to i n h i b i t renin release i n r a t s , by a d i r e c t e f f e c t on the juxtaglomerular c e l l s i n a negative feedback type mechanism (Michelakis, 1971b). Since A l l r e s u l t s i n l i b e r a t i o n of aldosterone from the adrenal cortex, i t may be expected that aldosterone would also feedback to i n h i b i t renin release. However, t h i s i s not apparent as aldosterone stimulates renin secretion. Adrenal i n s u f f i c i e n c y causes an increase i n renin secretion but dexamethasone, a synthetic g l u c o c o r t i c o i d , abolishes t h i s increase (Reid et_ a l . , 1973). An increase i n angiotensinogen (renin substrate) i n blood plasma w i l l r e s u l t i n an increase i n plasma renin a c t i v i t y but t h i s soon f a l l s o f f , probably due to an e f f e c t on sodium balance or angiotensin feedback i n h i b i t i o n (Menard e_t a l . , 1973). Mammalian kidneys also contain a n a t u r a l l y - o c c u r r i n g i n h i b i t o r of the renin-angiotensinogen reaction, t h i s i n h i b i t o r being a phospholipid. Osmond et a l . (1973) have a t t r i b u t e d t h i s i n h i b i t i o n to lysophosphatidylethanolamine. In vivo i n h i b i t i o n of renin r e a c t i v i t y i s caused by phosphatidylethanolamine but t h i s compound has no e f f e c t i n v i t r o , which suggests that i t has to be converted to the other form. The i n vivo conversion i s rapid and has been at t r i b u t e d to a blood phospholipase which deacylates the phosphatidylethano-lamine at the C2 p o s i t i o n . A l a t e r study has shown that the pressor response to renin may be i n h i b i t e d by f a t t y acids ±n vivo but response to A l l i s unaffected (Kotchen ej; a l . , 1978). This study suggests that the f a t t y acids may modify the renin-renin substrate reaction i n vivo as well as in v i t r o . In addition, Buftag and Walaszek (1973) have synthesized two synthetic phos-p h o l i p i d i n h i b i t o r s , one an o l e y l and the other a p a l m i t y l d e r i v a t i v e . 5 1.2 Renin i n Non-Mammalian Vertebrates. The preceding has been a very b r i e f overview of some of the actions of the renin-angiotensin system and some of the factors a f f e c t i n g i t i n various mammalian species. However, the renin-angiotensin system i s not unique to mammals. Both renin and angiotensin have been i s o l a t e d from b i r d s , r e p t i l e s , amphibians and some species of bony fishes (Capreol and Sutherland, 1968; Sokabe ej: a l . , 1969). I t has not been found i n cyclostomes or elasmobranchs (Nishimura et a l . , 1970). The r o l e of the renin-angiotensin system as well as the c o n t r o l mechan-ism i s obscure i n the bony f i s h e s . P a r t i a l dehydration of Japanese e e l s , e i t h e r by exposure to seawater or by exposure to a i r , r e s u l t s i n an increase i n plasma renin a c t i v i t y as w e l l as a decline i n kidney renin content. This observation has also been made for T i l a p i a mossambica (Sokabe et a l . , 1968; Sokabe et a l . , 1969; Sokabe et a i l . , 1973). I t has been suggested that the renin-angiotensin system plays a r o l e i n s a l t adaptation i n euryhaline species but t h i s does not explain the r o l e of renin i n stenohaline species, e s p e c i a l l y marine t e l e o s t s . In summary, the renin-angiotensin system has been extensively studied i n mammals with much les s work done on t h i s system i n non-mammalian verte-brates. However, t h i s hormonal system apparently arose i n p r i m i t i v e bony f i s h e s . Therefore, the subject of t h i s study i s some of the factors i n f l u -encing renin secretion i n f i s h e s . More s p e c i f i c a l l y , the questions asked were did the s t r e t c h receptor-induced renin secretion evolve i n f i s h e s and i s there a sodium-induced renin response i n these animals? In addition, some of the renal e f f e c t s of the angiotensins w i l l be examined to determine a portion of the p h y s i o l o g i c a l r o l e of t h i s system. 6 Section I I . Introduction. 2.0 RENIN ANALYSIS. 2.1 Renin Measurement. The renin-angiotensin system appears to have evolved i n the p r i m i t i v e bony fis h e s (Nishimura et^ a l . , 1973) and i s found throughout the animal kingdom with the exception of cyclostomes and elasmobranchs (Nishimura, -1970). In humans the renin-angiotensin system has been implicated i n various hypertensive diseases and consequently the vast majority of work has been done on mammals with a view towards solving a purely medical problem. One of the problems associated with woxk on . t h i s s y s t e m i s the i n a b i l i t y of researchers to obtain a pure renin. Murakami and Inagami (1975) have published a report which shows a method f o r p u r i f y i n g hog renin but the y i e l d i s low (25%) and requires a 180,000-fold p u r i f i c a t i o n . Consequently, r e l a t i v e l y large quantities of kidney material are required, but even so the p u r i f i e d product s t i l l shows some contamination or at l e a s t minor components are present; the function of these components i s unknown. 2.1.1 Bioassay. Since renin displays enzymatic a c t i v i t y , the most commonly used proced-ure i s determination of the product, angiotensin I ( A l ) , formed from the renin-renin substrate reaction. This determination was usually c a r r i e d out by means of a bioassay, the rat vaso-pressor assay, as elucidated by Boucher jet _al. (1964). The major disadvantage of t h i s form of assay i s the r e l a t i v e -l y large quantities of blood plasma required for the incubation and extrac-t i o n steps. In a c l i n i c a l s i t u a t i o n , however, these volumes are r e a d i l y a v a i l a b l e without causing any serious i n j u r y to the subject and so t h i s d i s -advantage i s not apparent. But i n small animal work the problem becomes 7 obvious. Consequently, the assay of Boucher was modified by various groups so that i t could be used f o r assorted species. Plasma renin a c t i v i t y has been measured i n marine t e l e o s t s , both glomerular and aglomerular species (Malvin and Vander, 1967; Mizogami et^ _al . , 1968), freshwater teleosts and holocephalins (Nishimura et^ al., 1973) , amphibians (Sokabe et_ al. , 1972) , and cetaceans (Malvin and Vander, 1967). In addition, an alternate method for bioassay of mammalian renin has been described by Brown et a l . (1969). These modifications generally involve reducing the amount of blood plasma required f o r the assay. However, even with the large reduction i n volumes required, the amount of blood necessary would s t i l l produce a s i g n i f i c a n t reduction i n blood volume for some of the species under study. This f a c t then circumscribed the nature of experiments which could be c a r r i e d out. 2.1.2 Radioimmunoassay. With the advent of e a s i l y obtainable radioisotopes and commercially made antibodies a new technique has come into prominence and t h i s i s the radioimmunoassay. The f i r s t radioimmunoassay procedures developed were for angiotensin II ( A l l ) due to the e a r l i e r a v a i l a b i l i t y of the octapeptide as an antigen. As a r e s u l t of various problems with t h i s technique a l a t e r development of a radioimmunoassay for A l was c a r r i e d out and t h i s i s the technique that i s most commonly used, again i n a c l i n i c a l s i t u a t i o n . 2.1.2.1 A l l Radioimmunoassay- Attempts to measure plasma A l l by radioimmunoassay i n unextracted plasma have produced somewhat e r r a t i c r e s u l t s as plasma proteins tend to i n t e r f e r e with antigen-antibody binding i n an unpre-d i c t a b l e fashion (Gocke et. a l . , 1968). Unfortunately, extraction procedures frequently r e s u l t e d i n v a r i a b l e recoveries of A l l and concomitant concentra-8 t i o n of s a l t s , small proteins, and peptides that i n t e r f e r e d with the ra d i o -immunoassay (O p a r i l , 1977). The extraction procedure can be improved by using an u l t r a f i l t r a t e of plasma which gives an average recovery of A l l of 98%. The extraction proced-ure does not t o t a l l y remove a l l i n t e r f e r i n g substances and so the amount of u l t r a f i l t r a t e used i n the radioimmunoassay becomes c r i t i c a l (Ruiz-Maza _et a l . , 1974). One group of workers has reported a d i r e c t radioimmunoassay procedure f o r A l l on unextracted plasma. This procedure obtained an excellent c o r r e l a -t i o n between bioassay and radioimmunoassay (Mion et a l . , 1974) although the mean A l l concentrations obtained i n t h i s study did not agree with those obtained i n s i m i l a r studies but d i f f e r e n t assay methods (Oparil and Haber, 1974). This may be due to i n d i v i d u a l v a r i a b i l i t y between subjects and extraction techniques or may be due to interference by plasma protein. Attempts to measure A l l secreted i n urine have been successful (Fubuchi, 1974). However, as an a n a l y t i c a l t o o l t h i s assay i s f a r from successful since there i s a poor c o r r e l a t i o n between simultaneous a r t e r i a l and urinary A l l concentrations, the l a t t e r depending more on renal perfusion than plasma A l l concentrations ( O p a r i l , 1977). Thus i t would appear that while an antibody for A l l i s r e a d i l y a v a i l a b l e , the usefulness of such for a radioimmunoassay procedure i s l i m i t e d by the interference of various plasma components i n the antigen-antibody binding phase. Also extraction procedures tend to be time consuming and not very reproducible. 2.1.2.2 A l Radioimmunoassay - A somewhat more r e l i a b l e , technique which has been developed i s a radioimmunoassay for A l . B a s i c a l l y t h i s tech-nique involves the formation of A l from the renin-renin substrate r e a c t i o n 9 and allowing t h i s formed A l to incubate with a radioisotope l a b e l l e d A l and an Al-antibody. The bound and unbound f r a c t i o n s of A l are then separated and quantified. Various groups have reported on the success of t h i s tech-nique and indeed have worked on comparing commercially a v a i l a b l e k i t s (Goldberg and Spierto, 1973; Michelakis jet a l . , 1974; Poulsen and Jorgensen, 1974). Controversy s t i l l e x i s t s over the use of t h i s technique and most of t h i s revolves around the choice of the incubation medium for the renin-renin substrate r e a c t i o n . Problems e x i s t with s t a b i l i z i n g such f a c t o r s as pH, since CC>2 i s apparently l i b e r a t e d during the reaction. This requires the use of concentrated buffers to avoid d i l u t i n g the reaction mixture (Oparil, 1977). In addition the optimum pH of the reaction mixture appears to be i n the range of 5.5 to 6.5 rather than p h y s i o l o g i c a l pH (Heise, 1975). The absolute amount of A l generated under these conditions appears to be two to four f o l d greater than those generated under p h y s i o l o g i c a l pH and so t h i s pH range i s the preferred range for the i n v i t r o incubation-step. Another problem revolves around the choice of converting enzyme i n h i b i t -ors. As A l i s r a p i d l y converted to A l l (Boucher et a l . , 1974; Gagnon et a l . , 1974; Oakes and Stakes, 1974) t h i s r e a c t i o n must be i n h i b i t e d i n order to measure l e v e l s of A l . Beckerhoff e t a l . (1975) reported that a mixture of dimercaprol,. 8-hydroxyquinolihe and EDTA worked well at p h y s i o l o g i c a l pH while O p a r i l et a l . (1974) preferred d i i s o p r o p y l fluorophosphate with EDTA at acid pH. Poulsen and Jorgensen (1974) did not use converting enzyme i n h i b i t o r s but rather added the antibody to the reaction mixture. Since the antibody had a higher a f f i n i t y for A l than the converting enzyme, a peptidase,,. the A l bound p r e f e r e n t i a l l y to the antibody. Later l a b e l l e d A l was added to the reaction mixture i n a large volume to d i l u t e and thus end the renin-renin 10 substrate reaction. The mixture was then allowed to e q u i l i b r a t e and the normal radioimmunoassay procedure c a r r i e d out. The v a r i e t y of procedures and enzyme i n h i b i t o r s tends to make compari-sons of studies d i f f i c u l t . However by comparing the data obtained from a radioimmunoassay and from a standard bioassay, some of the d i f f i c u l t i e s may be removed. 2.1.3 Summary. In summary, there are three possible means of measuring plasma renin a c t i v i t y and these are a radioimmunoassay for A l l , a radioimmunoassay for A l , and a bioassay f or A l . The d i r e c t measurement of c i r c u l a t i n g plasma renin l e v e l s i s not p r a c t i c a l at t h i s time due to the absence of a r e a d i l y a v a i l -able standard pure renin preparation and so renin a c t i v i t y i n terms of i t s enzymatic function must be measured. This requires blood plasma i n varying amounts and may cause experimental d i f f i c u l t i e s depending on the plasma volumes required. 2.2 Characterization of Renin. 2.2.1 Mammals. The juxtaglomerular c e l l s of the kidney have been shown to be the source of renin i n those animals which produce t h i s .enzyme: (Pitcock et a l . , 1959; Tobian et a l . , 1959; Hartr o f t et a l . , 1964). These c e l l s appear to be modified smooth muscle c e l l s containing granules which are homogenous, dense and osmiophilic. In addition, these granules bear a close resemblance to the beta c e l l granules of the pancreatic i s l e t s (Barajas and La t t a , 1967). These juxtaglomerular c e l l granules s t a i n s p e c i f i c a l l y with Bowie's s t a i n and weakly with aldehyde-fuchsin while p e r i o d i c a c i d - S c h i f f reagent stains a l l granules of the juxtaglomerular c e l l s (Krishnamurthy and Bern, 1969). The juxtaglomerular c e l l s are part of a c e r t a i n anatomical arrangement 11 known as the juxtaglomerular apparatus. This consists of the aff e r e n t and efferent a r t e r i o l e s , the macula densa (a s p e c i a l i z e d area of the d i s t a l convoluted tubule) and a network of c e l l s which t i e the other two components together; t h i s network being v a r i o u s l y known as the l a c i s or the Polkissen or colour cushion (due to i t s s t a i n i n g properties) or the extraglomerular mesangium (Sokabe et a l . , 1969). Renin has c e r t a i n properties which aid to d i s t i n g u i s h i t from the peptide hormones. These properties are as follows: i t i s h e a t - l a b i l e , n o n - d i f f u s i b l e through cellophane and destroyed by a c i d i f i c a t i o n to pH 2.0 (Skeggs ej; a l . , 1967). Renin i s also c o n s i d e r e d . , present i n a kidney i f s a l i n e extracts of that kidney produce a potent pressor substance upon incubation with homologous plasma (Chester,Jones et . a l . , 1966). While renin i s most c l o s e l y associated with the kidneys, recent work has shown that renin, or at l e a s t r e r i i n - l i k e substances, are found i n many other tissues throughout the body. Renin has been located i n the sub-maxillary glands of mice (Menzie et a l . , 1974), i n various parts of the brain (Sakai et a l . , 1974), and i s thought to o r i g i n a t e i n the chorionic membrane of the foetus (Symonds et a l . , 1968). In addition, Boucher et a l . (1974) have postulated the existence of a new enzyme which leads to the d i r e c t formation of A l l from renin substrate. These authors c a l l t h i s enzyme tonin (located i n the r a t sub-maxillary glands) and base t h e i r hypothesis on the fa c t that i t i s not i n h i b i t e d by any renin i n h i b i t o r s . 2.2.2 Fishes. Renin has been demonstrated i n various species of bony fis h e s by a number of workers. Capr£ol and Sutherland (1968) c a r r i e d out a comparative h i s t o l o g i c a l study on a number of f i s h species and were able to demonstrate renin granules by means of the Bowie's st a i n i n g technique, i n afferent 12 glomerular a r t e r i o l e s i n a l l species tested, with the exception of salmonids and elasmobranchs. Incubating kidney s a l i n e extracts with homologous plasma has resulted i n the formation of a pressor substance i n a large number of species and f a i l u r e to do so i n a number of others. Cyclostomes do not appear to possess any hormone resembling renin as defined by these c r i t e r i a (Nishimura et a l . , 1970) although incubating lamprey kidney extract with p u r i f i e d dog substrate resulted i n the formation of a pressor substance (Nishimura and Ogawa, 1973). These data-result i n the p o s s i b i l i t y that the lamprey may possess a r e n i n - l i k e substance but i s lacking the substrate upon which i t could act. Elasmobranchs do not have renin or renin substrate (Nishimura et a l . , 1970). Dogfish show a pressor response to A l l but t h i s appears to be mediated by epinephrine, as dogfish vascular beds which lack chromaffin ti s s u e respond to epinephrine but not to A l l (Opdyke and Holcome, 1978). Both freshwater and marine osteicthyes have a renin-angiotensin system (Malvin and Vander, 1967; Mizogami et a l . , 1968). This apparently includes salmonids despite the lack of h i s t o l o g i c a l evidence (Sokabe et a l . , 1968). Renin also has been demonstrated i n l u n g f i s h and coelacanths (Nishimura and Ogawa, 1973; Blair-West et a l . , 1977). Extra-renal sources of renin have not been extensively reported i n t e l e o s t s . The main work i n t h i s area has been concerning the corpuscles of Stannius, glands which are generally located on the kidney of t e l e o s t s (Stannius, 1839). The c e l l s of these glands display granules which appear s i m i l a r to renin granules. However, attempts to show that these glands are part of the renin-angiotensin system have given equivocal r e s u l t s (Chester-Jones et a l . , 1966; Chester-Jones et^ a_l. , 1969). Indeed, Bailey and Fenwick (1975) showed that the blood pressure e f f e c t exerted by these glands was 13 probably an i n d i r e c t e f f e c t as a r e s u l t of di s t u r b e d calcium homeostasis i n which the corpuscles of Stannius are d i r e c t l y i n v o l v e d . 2.2.3 Summary. To summarize, r e n i n i s found i n the kidneys of mammals and f i s h e s . Mammals a l s o d i s p l a y many e x t r a - r e n a l sources of r e n i n but t h i s has not been e x t e n s i v e l y i n v e s t i g a t e d i n f i s h e s . Renin d i s p l a y s c e r t a i n p r o p e r t i e s : n o n - d i f f u s ' i b l e through cellophane, h e a t - l a b i l e and destroyed by a c i d i f i c a t i o n to pH 2.0 or lower. Also i n c u b a t i n g s a l i n e kidney e x t r a c t s w i t h homologous plasma should r e s u l t i n the formation of a pressor substance. 14 Section I I . Methods and Materials. 2.3 L o c a l i z a t i o n and Characterization of Renin i n Trout. 2.3.1 Experimental Animals. Adult rainbow trout (Salmo gairdneri) of eit h e r sex were used throughout t h i s study. These were obtained from the Sun Valley Trout Farm i n Haney, B. C. and ranged i n length from 30-45 cm and weight from 250-500 gm. The f i s h were maintained i n dechlorinated tap water i n large c i r c u l a r outdoor tanks. Water temperature ranged from 6° to 14°C and photoperiod was not c o n t r o l l e d . Food consisted of Clarke's New Age High E f f i c i e n c y V f i s h p e l l e t s (Clarke-Moore Co. Inc., Salt Lake C i t y , Utah). 2.3.2 Histology. Adult rainbow trout of either sex were k i l l e d by a sharp blow to the head. A l o n g i t u d i n a l i n c i s i o n was made i n the mid-ventral l i n e stretching from the vent to the transverse septum. Blocks of kidney ti s s u e were excised and f i x e d for either 24 or 48 hours i n Zenker-formol f i x a t i v e . The fi x e d t i s s u e blocks were then dehydrated i n a serie s of alcohol baths, cleared i n x y l o l and prepared f o r sectioning by i n f i l t r a t i n g with molten Paraplast (Sherwood Medical Industries) for 24 hours. The blocks were then cut into 7 mu sections on a microtome and the sections f i x e d to glass s l i d e s by albumin. The sections were rehydrated and treated with Lugol's iodine solu-t i o n to remove excess mercury. Following t h i s treatment the sections were stained by the Bowie's st a i n i n g technique (Bowie, 1935-1936) and covered with glass cover s l i p s which were fixed to the s l i d e by Permount (Fisher S c i e n t i f -i c ) . The s l i d e s were then allowed to dry and examined microscopically f o r the presence of renin granules. 15 In addition to trout, kidney blocks from several other species of f i s h were examined for the presence of renin granules. These f i s h were s k i l f i s h , E r i l e p i s z o n i f e r , pirarucu, Arapaima gigas, and j e j u , Hoplerythrinus  unitaeniatus. 2.3.3 Characterization of Renin. 2.3.3.1 D i a l y s i s E f f e c t s - Plasma samples were treated as i n the bioassay procedure described i n Section 2.4.2 to determine i f the pressor a c t i v i t y could be removed by d i a l y s i s . Other plasma samples were assayed by the bioassay procedure except d i a l y s i s of the plasma was not c a r r i e d out to determine i f the pressor a c t i v i t y was present before d i a l y s i s . 2.3.3.2 pH E f f e c t s _ Plasma samples were a c i d i f i e d by adding commercial stock HC1 u n t i l a pH of 2.0 or le s s was reached. These a c i d i f i e d samples were then assayed by bioassay to determine i f pressor a c t i v i t y was s t i l l present. 2.3.3.3 Temperature E f f e c t s - Plasma samples were heated to 80°C i n a water-bath for 15 minutes. These samples.were then assayed by bioassay to determine i f pressor a c t i v i t y was s t i l l present. 2.4 Assay Techniques. 2.4.1 Plasma C o l l e c t i o n . Blood was c o l l e c t e d from a dorsal aorta cannula which was i n s t a l l e d as follows. The f i s h was anaesthetized i n a 1:20,000 aqueous s o l u t i o n of MS-222 (Sandoz) u n t i l i t could be picked up without any evidence of struggling. A nose-cone of Intramedic PE-200 (Clay-Adams Inc.) was inserted through a hole punctured through the snout anterior to the external nares. The dorsal aorta was then punctured at the l e v e l of the f i r s t g i l l arch by means of a Medicut (Sherwood Medical Industries Inc.) #16 intravenous cannula. The 16 needle was withdrawn leaving the p l a s t i c sleeve i n place and a cannula of Intramedic PE-60, which was f i l l e d with heparinized (2 I.U. heparin/ml) Cortland (Wolf, 1963) s a l i n e , was inserted into the dorsal aorta through the sleeve which was then withdrawn. The cannula was sutured to the roof of the mouth, the free end passed through the nose-cone and a length of s u r g i c a l s i l k was t i e d t i g h t l y around the nose-cone where i t exited from the snout. The purpose of t h i s length of thread was to secure both the nose-cone and the cannula from s l i d i n g back and f o r t h i n the mouth. Blood was withdrawn through the cannula as r a p i d l y as possible and c o l l e c t e d i n a heparinized 10 cc Plastipak (Becton, Dickinson Co. Canada Ltd.) syringe and then transferred to an i c e - c o l d polystyrene disposable culture tube (Fisher S c i e n t i f i c ) . I t was then centrifuged i n a r e f r i g e r a t e d c e n t r i -fuge at l,400g and 0°C for 15 min, the plasma c o l l e c t e d and the c e l l s discarded. 2.4.2 Bioassay. One m i l l i l i t e r of plasma was dialyzed at 2°C for 24 hours against 0.22% disodium EDTA. The dialyzed plasma was then added to 1 ml of Dowex (Dow Chemical Co.) 50W-X2 r e s i n , NH^ form, and 0.1 ml ammonium EDTA. This reaction mixture was then incubated at 20°C for 24 hours. Generated angiotensin was eluted from the r e s i n by f i r s t t r a n s f e r r i n g the r e a c t i o n mixture to a water-jacketed chromatography column where i t was washed with ammonium acetate buffer (pH 6.0), 10% a c e t i c acid and d i s t i l l e d water. E l u t i o n was c a r r i e d out with a 0.2 N aqueous diethylamine s o l u t i o n and 0 . 2 N ammonium hydroxide. The eluate was evaporated to dryness under vacuum and washed four times with 80% ethanol which was evaporated under vacuum. The residue was then resuspended i n 0.9% aqueous NaCl so l u t i o n which contained 0.001% Tween 20 ( J . T. Baker Chemical Co.). To test the accuracy 17 of the extraction procedure known quantities of synthetic angiotensin I (Calbiochem) , type A s p \ Ileu~*, dissolved i n 1 ml of Cortland s a l i n e were added to 1 ml of the r e s i n and treated as a plasma sample. Recoveries were found to range from 90 to 104% i n a seri e s of 8 t r i a l s with 5 runs per t r i a l . This i s a modified procedure of Boucher et_ al_. (1964). A s e r i e s of angiotensin II (Hypertensin-CIBA) solutions, 0, 10, 20, 40, 60, 80, and 100 ng/0.2 ml, was prepared i n 0.9% aqueous NaCl and used as standards i n the rat vasopressor bioassay. Male 250-300 gm Wistar albino r a t s were anaesthetized with an i . p . i n j e c t i o n of urethan ( A l d r i c h Chemical Co.) and the c a r o t i d artery cannulated with Intramedic PE 50 for blood pressure measurement and the jugular vein cannulated with Intramedic PE 50 for i n j e c t i o n purposes. Blood pressure was measured with a Statham P23AC s t r a i n gauge pressure transducer and recorded on a Beckman RS Dynograph recorder. A standard angiotensin II pressor dose (1 ug/kg body weight) was administered and the preparation discarded i f the mean pressure increase was l e s s than 30 mm Hg . 2.4.3 Radioimmunoassay. Plasma renin a c t i v i t y was assayed d i r e c t l y i n t h i s procedure, .there were no extraction procedures. For the radioimmunoassay a NEN Angiotensin I Radioimmunoassay K i t (New England Nuclear Canada Ltd.) was used. In the recommended procedure, two aliquots of plasma to which had been added dimercap-r o l , 8-hydroxyquinoline sulphate-, (which act as converting enzyme and angio-tensinase i n h i b i t o r s ) and maleate b u f f e r , pH 6.0, are taken. One aliquot i s incubated at 37°C and the other at 4°C for one hour. Following the incuba-t i o n time 100 u l of each aliquot i s added separately to 100 y l of an aqueous 125 I - l a b e l l e d angiotensin I-Tris-acetate (Sigma Chemical Co.) buffer, pH 7.4 18 at 20°C, s o l u t i o n and 500 y l of an aqueous angiotensin I antiserum-Tris-acetate buffer s o l u t i o n . This reaction mixture was allowed to incubate for 24 hours at 4°C a f t e r which 1 ml of an activated charcoal suspension was added, the mixture centrifuged and the supernatant saved and counted. A standard curve i s run by adding known quantities of synthetic angiotensin I to the l a b e l l e d angiotensin I and the antiserum, incubating at 4°C for 24 hours, adding the charcoal suspension, c e n t r i f u g i n g and counting the super-natant. Both the standard and unknown counts were compared to the counts obtained from a known quantity of l a b e l l e d angiotensin I to determine the percent angiotensin I bound to the antiserum. This procedure was modified as the k i t was designed f o r human plasma renin a c t i v i t i e s and repeated tests with f i s h plasma showed that following the recommended procedure gave highly inconsistent r e s u l t s . The f i r s t m odification was i n the incubation procedure, that of the k i t being replaced by the following procedure (Nishimuro et a l . , 1977). One m i l l i l i t e r of plasma was dialyzed against 0.22% disodium EDTA at 2°C f o r 24 hours and then 200 y l of t h i s dialyzed plasma were incubated with 20 y l of a combined aqueous sol u t i o n of 1% neomycin sulphate (Sigma Chemical Co.) and 1% thimerosal (Sigma Chemical Co.) and 20 y l of 2 M ammonium acetate buffer (pH 6.0) and 0.1 mg of phenylmethylsulfonylfluoride (dissolved i n ethanol -Sigma Chemical Co.) for 2 hours at 20°C i n a polystyrene disposable culture tube (Fisher S c i e n t i f i c ) . A further modification was made by d i l u t i n g the standards and tracer by ha l f of what the k i t recommends. The exceptions to t h i s were the Tr i s - a c e t a t e buffer, the charcoal suspension and the antiserum s o l u t i o n which were made up to the recommended concentration. 1 9 buffer, pH 7.4 at 20°C, and 50 y l of "'"^I-labelled angiotensin I (New England Nuclear Canada Ltd.) were added to each tube except the f i r s t two which had 1600 y l of buffer and 50 y l of tracer only, these tubes weje. the t o t a l count controls. Also, tubes 3 and 4 had 500 y l buffer, 50 y l tracer and 50 y l bovine serum albumin s o l u t i o n . The r e s t of the tubes had either 50 y l of standard angiotensin I s o l u t i o n (NEN Canada Ltd.) or 50 y l of reac-t i o n mixture added. At t h i s point 500 y l of antiserum (NEN Canada Ltd.) were added to a l l the tubes with the exception of tubes 1 through 4. A l l tubes were then incubated at 2°C for 24 hours to allow competitive binding between the tracer angiotensin and the generated angiotensin with the antiserum. A 1 ml volume of charcoal suspension (NEN Canada Ltd.) was added to each tube with the exception of tubes 1 and 2. The charcoaled tubes were then c e n t r i -fuged for 15 minutes at 1500g i n a r e f r i g e r a t e d centrifuge at 0°C. The supernatant was c o l l e c t e d and counted by l i q u i d s c i n t i l l a t i o n counting i n an Isocap Programmable S c i n t i l l a t i o n Counter (Nuclear Chicago L t d . ) . The f l u o r used was R i a f l u o r (NEN Canada Lt d . ) . A l l counts were then compared to the t o t a l count controls. The accuracy of the assay was checked at frequent i n t e r v a l s by using plasma samples of known a c t i v i t i e s (NEN plasma renin a c t i v i t y standards) and the assay was found to be accurate within ±5%. 2.4.4 Determination of Optimum pH. Dialyzed plasma samples were incubated at a seri e s of d i f f e r e n t pH's, 5.5, 6.0, 6.5, 7.0, 7.5, and 8.0. A c t i v i t i e s were then measured by RIA. The purpose of t h i s was to determine which pH gave optimum renin a c t i v i t y for f i s h plasma. 2.4.5 Determination of Optimum Temperature. Dialyzed plasma samples were incubated at a s e r i e s of d i f f e r e n t tempera-tures to determine optimum temperature for f i s h renin a c t i v i t y . These 19 a F o l l o w i n g t h e i n c u b a t i o n p r o c e d u r e a numbered s e r i e s o f d i s p o s a b l e p o l y s t y r e n e c u l t u r e t u b e s was s e t up i n an i c e b a t h and 50 y l o f T r i s - a c e t a t e t e m p e r a t u r e s were as f o l l o w s : 4°, 10°, 15°, 20°, 25°, 35°, and 37°C. The RIA p r o c e d u r e was used t o measure t h e a c t i v i t i e s a t d i f f e r e n t t e m p e r a t u r e s . 2.4.6 A c t i v i t i e s . A l l a c t i v i t i e s a r e e x p r e s s e d as plasma r e n i n a c t i v i t y and t h e u n i t s used a r e ng o f a n g i o t e n s i n I I g e n e r a t e d p e r m i l l i l i t e r o f plasma p e r hour o f i n c u b a t i o n t i m e f o r t h e b i o a s s a y and ng o f a n g i o t e n s i n I g e n e r a t e d p e r m i l l i -l i t e r o f plasma p e r hour o f i n c u b a t i o n t i m e f o r t h e radioimmunoassay. 2.4.7 Comparison o f B i o a s s a y and Radioimmunoassay. P o o l e d plasma samples were d i a l y z e d as p r e v i o u s l y d e s c r i b e d and a l i q u o t s were t a k e n f o r a n a l y s i s o f r e n i n a c t i v i t y by b i o a s s a y and radioimmunoassay, sample s t a n d a r d c u r v e s o f each may be found i n A p p e n d i x 3. 2.5 S t a t i s t i c a l Methods. 2.5.1 C o r r e l a t i o n s . The b i o a s s a y and radioimmunoassay were compared by d e t e r m i n i n g t h e c o -e f f i c i e n t o f c o r r e l a t i o n ( r ) . A p r o b a b i l i t y v a l u e o f l e s s t h a n 0.05 was r e g a r d e d as s i g n i f i c a n t and a p r o b a b i l i t y v a l u e o f l e s s t h a n 0.01 as h i g h l y s i g n i f i c a n t . 2.5.2 Comparison o f Means. The S t u d e n t ' s t t e s t was used t o d e t e r m i n e i f d i f f e r e n c e s between mean p r e s s o r a c t i v i t i e s were s i g n i f i c a n t . A p r o b a b i l i t y o f l e s s t h a n 0.05 was r e g a r d e d as s i g n i f i c a n t and a p r o b a b i l i t y o f l e s s t h a n 0.01 as h i g h l y s i g n i f i c a n t . 20 Section I I . Results and Discussion. 2.6 Characterization of Renin. 2.6.1 Histology. Figure 2vtl shows a d e t a i l of the juxtaglomerular apparatus of the trout. Renin granules may be seen iri the wa l l of the a r t e r i o l e and below the blood vessel may be seen the glomerular c a p i l l a r y bed and the Bowman's capsule. The remaining c e l l s i n the fi g u r e are probably either i n t e r r e n a l , chromaffin or haematopoietic tissues which are also found i n the trout kidney (Hickman and Trump, 1969). Renin granules were also found i n the kidney sections of the other species of f i s h examined. In the pirarucu and j e j u renin granules tended to be farther away from the glomerulus than i n - t h e trout."and s k i l f i s h (Figure 2.2). No structure resembling the macula densa was observed i n any of the species examined. However t h i s i s not su r p r i s i n g as the macula densa i s apparently r e s t r i c t e d to mammals although an intermediate type structure has been found i n b i r d kidneys (Sokabe e_t a l . , 1969) . The granules observed i n the wa l l of the a r t e r i o l e f u l f i l l the require-ments for renin, that i s to say they s t a i n w i t h the Bowie's s t a i n and are located i n the wa l l of the afferent a r t e r i o l e close toz'the..lumen of the vessel (Krishnamurthy, 1969). The granules are small and somewhat d i f f i c u l t to locate as they are not always found close to the glomerulus. Capr£ol and Sutherland (1968) i n t h e i r comparative study noted that these granules are frequently located d i s t a n t from the glomerulus. Unfortunately, while Sokabe e_t a l . (1968) were able to i d e n t i f y ( v i a bioassay) renin i n salmonids, Caprdol and Sutherland i n t h e i r study were 21 Figure 2.1. Cross section of trout kidney showing glomerulus (G), renin granules (RG) and afferent a r t e r i o l e (AA). The remaining c e l l s are probably supportive and glandular tissues which are found i n the te l e o s t kidney. X1000. 23 Figure 2.2 (A). Cross section of s k i l f i s h kidney showing the glomerulus (G), renin granules (R.G.) i n the walls of the afferent a r t e r i o l e (A.A.). X1000. Figure 2.2 (B). Cross section-.of pirarucu kidney showing renin granules (R.G.), glomerulus (G), afferent a r t e r i o l e (A.A.) and nephrons (N). The remaining -tissue i s probably either supportive or glandular. X1000. Figure 2.2 (C). Cross section of j e j u kidney showing renin granules (R.G.) i n the wall of the afferent a r t e r i o l e (A.A.) and sections of the nephron (N). X1000. 24 25 unable to i d e n t i f y renin granules i n the trout kidney as they apparently did not s t a i n with the Bowie's s t a i n for t h i s group. Accordingly, i t was neces-sary to carry out further tests to confirm the h i s t o l o g i c a l r e s u l t s . 2.6.2 Chemical Characterization of Trout Renin. Table 2.1 shows the e f f e c t of ..dialysis,~ heating, and acid-treatment of plasma on renin a c t i v i t y . Using i s o t o n i c s a l i n e as a c o n t r o l to determine the e f f e c t s of volume i n j e c t i o n on the mean pressure increase, i t may be seen that the plasma extract possessed pressor a c t i v i t y following incubation whether or not the plasma was dialyzed. However, i t should be noted that the dialyzed plasma sample apparently possessed a greater concentration of pressor a c t i v i t y than the undialyzed sample. A c i d i f i c a t i o n of the plasma p r i o r to incubation apparently destroyed the plasma's a b i l i t y to form the pressor substance as did heating the plasma, since there was no s i g n i f i c a n t increase i n mean a r t e r i a l pressure following i n j e c t i o n of extracts from these treated plasmas. Mammalian renin i s known to be h e a t - l a b i l e , destroyed by a c i d i f i c a t i o n to pH 2.0 or lower and i s non-dialyzable through cellophane (Skeggs et a l . , 1967). E e l (Anguilla anguilla) renin also has s i m i l a r properties (Chester Jones _et al.,'1966) and so trout renin should possess these properties. Therefore, i f the plasma forms a pressor substance upon incubation and i f the formation of t h i s pressor may be i n h i b i t e d by the aforementioned treatments, then the conclusion which may be drawn from t h i s evidence i s that i t i s the action of renin which forms the pressor substance. There are two possible reasons f o r the di f f e r e n c e i n a c t i v i t y manifested, between the dialyzed and undialyzed plasma samples. D i a l y s i s of the plasma before incubation i s known to i n h i b i t angiotensinases which break down the product angiotensin I (Al) (Nishimura e± a l . , 1978). Therefore i t i s possible Table 2.1 Ef f e c t of Isotonic Saline and Plasma Extracts on Mean A r t e r i a l Pressure Increase (a) Following Various Treatments of Plasma I n j e c t i o n Dialyzed Plasma Undialyzed Plasma A c i d i f i e d Plasma .Heated Plasma Isotonic S a l i n e b 2.9 ± 0.38 mm Hg C 1.7 ± 0.26 mm Hg d 3.7 ± 0.41:. mm Hg 1.89 ± 0.11 mm Hg (10) (10) (8) (9) Plasma Ex t r a c t ^ 6.5 + 0.50 mm Hg C 3.8 ± 0.33 mm Hg d 3.5 ± 0.50 mm Hg 2.78 ± 0.83 mm Hg (10) (10) (8) (9) K3 a - Comparisons for each treatment made between i s o t o n i c s a l i n e and plasma extract, b - A l l values are means ± S.E.M. (N). c - P<0.01 d - P<0.01 27 that i n the undialyzed condition the generated A l i s p a r t i a l l y i n a c t i v a t e d by the angiotensinases before i t can adsorb on the chromatography r e s i n thus giving a lower t i t e r of a c t i v i t y . On the other hand there may be a d i f f e r -ence i n concentration of ei t h e r renin or renin substrate i n the plasma samples as i n t h i s s e r i e s of tests d i f f e r i n g plasma samples were used rather than aliquots from a pooled source. The most l i k e l y p o s s i b i l i t y i s a combin-ation of the two factors with the lack of d i a l y s i s being the major factor as a c t i v i t i e s from the undialyzed samples were cons i s t e n t l y lower than those from the dialyzed samples. The t e s t does prove that whatever i s generating the pressor substance i n the plasma i s neither added nor removed from the plasma following d i a l y s i s , which i s one of the properties of renin. Renin, being a large protein molecule, i s susceptible to denaturation by extreme pH l e v e l s (Skeggs e_t a l . , 1967) . Indeed when concentrated HC1 i s added to the f i s h plasma a c e r t a i n amount of coagulation i s observed. There-fore the acid treatment should not only degrade the renin molecule i t s e l f but w i l l probably act on the renin substrate as w e l l , since the substrate i s a plasma protei n , i n mammals an alpha g l o b u l i n (Oparil et_ a l . , 1973). The same reasoning would apply to heat treatment. Large proteins are denatured by extremes of heat and as i n the acid treatment when the f i s h plasma was heated i n .the water bath coagulation was observed i n the plasma sample. Therefore, one would expect l i t t l e or no a c t i v i t y i f both renin and renin substrate are destroyed by the heat treatment. The reason for the s l i g h t pressor a c t i v i t y , above that of the s a l i n e c o n t r o l l e v e l , i s that some angiotensin may have been formed as the plasma heated before the large proteins denatured;... a.ng i o t ens i n s are known to be more heat stable than renin (Nishimura et: al. , 1973) . 28 2.6.3 Summary. In summary, granules are found i n the walls of afferent glomerular a r t e r i o l e s . These granules s t a i n w i t h t h e Bowie's s t a i n i n d i c a t i n g that they are renin granules. Incubation of plasma produces an extractable vasoactive substance. D i a l y s i s of plasma does not remove the source of t h i s pressor substance but heat treatment and acid treatment of the plasma do. The conclusion to be drawn from these data i s that the renin-angiotensin system e x i s t s i n trout, pirarucu, j e j u , carp and s k i l f i s h . 2.7 Renin A c t i v i t y . 2.7.1 Temperature Optimum. When trout plasma samples were incubated at various temperatures, range 4° to 37°, and the angiotensin measured by the radioimmunoassay, a steady increase i n plasma renin a c t i v i t y was observed which apparently peaked at 35°C (Figure 2.3). The diffe r e n c e i n a c t i v i t y between the 25° and 35° l e v e l s was not s i g n i f i c a n t (0.1>P>0.05) but the diffe r e n c e between the a c t i v i t y at 35° and 37° i s s i g n i f i c a n t (P<0.05). Therefore the optimum temp-erature for trout renin i s apparently 35°C. This compares with the mammalian renin temperature optimum of 37°C (O p a r i l , 1977), a temperature at which trout renin begins to lose a s i g n i f i c a n t amount of a c t i v i t y . I t appears unusual that trout renin would have such a temperature optimum for renin a c t i v i t y as these animals generally prefer much cooler temperatures i n t h e i r habitat and indeed do not survive at 35°C (Fry, 1971). The most probable reason for t h i s optimum t e m p e r a t u r e v i s t h a t i n the in v i t r o .• protocol other factors such as pH are kept constant by the use of various buffers which are not n a t u r a l l y occurring and thus those factors which may i n h i b i t the reaction in vivo do not come into play. The f a c t that at 4°C trout renin displays approximately 30% of the a c t i v i t y at 35°C shows that i n 29 Figure 2.3. The e f f e c t of temperature on renin a c t i v i t y . The y-axis i s plasma renin a c t i v i t y i n ng of A l formed per ml of plasma incubated per hour of incubation time. The x-axis i s temperature i n °C. Data points are the means plus or minus the standard error of the mean and represent the mean of 6 separate determinations. The a c t i v i t y at T = 35°C i s s i g n i f i c a n t l y d i f f e r e n t from a l l other points with the exception of the a c t i v i t y at T = 25°C. R E N I N A C T I V I T Y { r i g A - 1 / m l / h r } 1 ro ro _1_ c o -o m > 73 'O n O CO o o " 31 structure i t varies from mammalian, s p e c i f i c a l l y human, renin which i s i n h i b i t e d at t h i s lower temperature l e v e l ( O p a r i l , 1977). Thus the plasma blank for the analysis must be run at a lower temperature than that advised i n the k i t and i t was found necessary to keep the plasma frozen as much as possible to prevent any development of a c t i v i t y . Since 35°C i s an u n r e a l i s t i c temperature f o r trout, the assays were gener-a l l y run at a much lower temperature. The chosen temperature was 20°C as t h i s was closer to the preferred habitat temperature yet s t i l l gave good a c t i v i t y (Figure 2.3). I t was assumed that at t h i s temperature l e v e l plasma renin a c t i v i t i e s from the i n v i t r o protocol would be much closer to those found i n the n a t u r a l l y occurring i n vivo s i t u a t i o n . 2.7.2 pH Optimum. From Figure 2.4 i t may be seen that the pH optimum for trout renin i s i n the range of 6.0-6.5 pH u n i t s . This indicates that renin functions best i n an acid medium. This i s s i m i l a r to mammalian renin which also functions best i n s l i g h t l y a c i d i c conditions. Indeed the pH optima of the two species of renin appear the same, as mammalian renin has an optimum pH of 5.5-6.5 ( O p a r i l , 1977) . The amount of angiotensin formed at t h i s pH i n mammals i s apparently two to four times that which can be generated at p h y s i o l o g i c a l pH (Oparil, 1977). The reason for t h i s pH optimum being at a lower l e v e l than p h y s i o l o g i c a l pH i s obscure. There i s a p o s s i b i l i t y that since kidney c e l l s are known to be somewhat a c i d i c (Koch, 1974), that blood within the renal c i r c u l a t i o n may become s l i g h t l y a c i d i c and renin evolved to function i n a s l i g h t l y acid environment. On the other hand a r t e r i a l blood i s high i n oxygen and low i n carbon dioxide, as compared to venous blood (Eddy, 1976) and thus tends to be s l i g h t l y more basic, again when compared to venous blood. Thus renin which 32 Figure 2.4. The e f f e c t of pH on renin a c t i v i t y . The y-axis i s plasma renin a c t i v i t y i n ng of A l formed per ml of plasma incu-bated per hour of incubation time. The x-axis i s pH i n pH u n i t s . The a c t i v i t i e s at pH 6.0 and 6.5 are s i g n i f i -cantly d i f f e r e n t from a l l other points with the exception of the a c t i v i t y at pH 7.0. The points on the graph are the means + S.E.M. of 7 determinations. 34 i s released into the a r t e r i a l side of the renal c i r c u l a t i o n would be somewhat i n h i b i t e d u n t i l the venous c i r c u l a t i o n was reached. But, the bulk of pub-l i s h e d reports indicates that the primary s i t e of action of the renin-renin substrate reaction products are the afferent and efferent glomerular a r t e r i o l e s . The p o s s i b i l i t y e x i s t s that the acid i n v i t r o medium i s not a f f e c t i n g the renin-renin substrate reaction per se but rather i s a c t u a l l y a c t i v a t i n g renin which has been released i n an i n a c t i v e form.known as "prorenin", as t h i s prorenin i s known to be acid-activated (Peach, 1977). Thus, i n the i n v i t r o system there i s simply a greater concentration of a c t i v e renin at acid pH than at p h y s i o l o g i c a l pH giving the impression that the renin-renin substrate reaction has a s l i g h t l y a c i d i c optimum pH. Also a s l i g h t l y a c i d i c pH i n the in v i t r o system may cause some s l i g h t deformation of the structure of the renin substrate making the leucine-leucine bond that the renin cleaves more a v a i l a b l e . However, th i s i s s t i l l "in the realm of speculation as studies on prorenin and pH of renal blood are s t i l l i n the preliminary stage and consequently there are not much data on t h i s subject. 2.7.3 Comparison of Assay Techniques. I n i t i a l experiments showed that trout A l apparently bound to human A l antiserum and so i t appeared possible to use a radioimmunoassay technique for measuring plasma renin a c t i v i t y . However i t was necessary to compare the data obtained for renin a c t i v i t y from the radioimmunoassay (RIA) with a c t i v i t i e s which were obtained v i a bioassay (BA) to ensure that what was binding to the antibody was i n f a c t trout A l and that i t was competing equally with the l a b e l l e d exogenous A l . The r e s u l t s of t h i s study may be seen i n Figure 2.5. From t h i s f i g u r e i t may be observed that there i s a good c o r r e l a t i o n between plasma renin a c t i v i t y (PRA) obtained v i a RIA and BA. The c o r r e l a t i o n c o e f f i c i e n t of 0.93 i s s i g n i f i c a n t (P<0.05). However the 35 Figure 2.5. Correlation between a c t i v i t i e s obtained from RIA and BA. The y-axis i s plasma renin a c t i v i t y obtained from the radioimmunoassay and i s measured i n ng A l formed per ml of plasma incubated per hour of incubation time. The x-axis i s plasma renin a c t i v i t y obtained from the bioassay and i s measured i n ng A l l per ml of plasma per hour. The l i n e equation i s y = 0.09x + 0.04 and the c o r r e l a t i o n c o e f f i c i e n t of 0.93 i s s i g n i f i c a n t at the 0.05 l e v e l . co-o B I O A S S A Y Cng A - l l / m l / h r ) 37 fac t that the BA gave PRA's which are approximately ten f o l d greater than those PRA's obtained as a r e s u l t of RIA suggests that the trout A l i s only p a r t i a l l y bound to the human A l antiserum. This i s not s u r p r i s i n g as Nakajima et a l . (1971) found that t e l e o s t angiotensins appear much more basic than the mammalian forms. But since the c o r r e l a t i o n between the two i s quite good, RIA was used as the method of choice for determining PRA's. While the r e s u l t s using the NEN antibody are quite good, those obtained from using the Squibb antibody were better, giving a c o r r e l a t i o n c o e f f i c i e n t of 0.984 (Nishimura, pers. comm.). But the NEN method was used as i t gave good r e s u l t s and was r e a d i l y a v a i l a b l e . The reasons for using a RIA technique over a BA technique are many and varied. P r i m a r i l y the RIA technique i s fa s t e r i n terms of actual time spent on the incubation and extraction procedures and le s s expensive i n terms of number of animals required. That i s the BA technique requires both trout and r a t s , the trout being the experimental animals and the r a t s being used to measure the amount of angiotensin formed. In addition there i s a c e r t a i n amount of v a r i a b i l i t y between the animals i n the BA which can complicate i n t e r p r e t a t i o n of r e s u l t s . I t i s true that t h i s v a r i a b i l i t y can be overcome i n a large part by a r t i f i c i a l l y depressing the rat's blood pressure so as to render the assay more s e n s i t i v e but t h i s procedure also adds to the complex-i t y of the BA. One other problem may occur during the extraction procedure as recovery from the chromatography r e s i n of the generated angiotensin may vary from 90-104%, although i n t h i s study the recovery of a c t i v i t y from the r e s i n averaged 95%, using either synthetic A l or A l l as controls. One main a d v a n t a g e of" the RIA'over-the BA i s that the RIA involves only one enzymatic step, i . e . , the generation of A l , while the BA involves two. 38 A l i s generated during the incubation period, absorbed on the chromatography r e s i n and extracted and redissolved i n i s o t o n i c s a l i n e . I t i s t h i s s o l u t i o n which i s injec t e d into the r a t and there i s a lag phase between time of i n j e c t i o n and increase i n mean blood pressure which indicates that the A l must be converted to A l l . I t i s e n t i r e l y possible that some of the a c t i v i t y may be l o s t by alternate metabolic routes during :the conversion as these are known to e x i s t (Freeman et a l . , 1978) or by binding of the rat-generated A l l to some other receptor than the vascular smooth muscle receptor. The RIA which i s car r i e d out i n an e n t i r e l y i n v i t r o s i t u a t i o n would not face these problems. Problems with the RIA system do e x i s t however. When using an antibody made for human A l to bind trout A l there i s the d e f i n i t e p o s s i b i l i t y that the l a b e l l e d exogenous A l may out-compete the trout-generated A l for binding to the antibody. Indeed t h i s has happened when the RIA i s used to measure ee l (Anguilla sp.) PRA, i . e . , e e l AT w i l l not bind to the antibody (Nishimura et a l . , 1978). In addition i t has been observed by other workers that the choice of converting enzyme i n h i b i t o r must be based on more than the pH optimum of the i n h i b i t o r ; . i n addition the e f f e c t of the i n h i b i t o r on the renin-renin substrate reaction must be taken into account as some i n h i b i t o r s w i l l adversely a f f e c t the renin reaction (Nishimura, pers. comm.). On the other hand, the technique i s generally quick and easy to carry out, which are d e f i n i t e facts ; i n i t s favour. 2.7.4 Summary. In summary, trout renin has i t s temperature optimum, jin v i t r o , of 35°C and a pH optimum, again i n v i t r o , of 6.0-6.5 pH u n i t s . Trout plasma renin a c t i v i t y may be measured by radioimmunoassay using a commercially a v a i l a b l e 39 kit as trout Al is similar to human Al in i t s binding characteristics to an antibody which is made specifically for human Al. 0 40 S e c t i o n I I I I n t r o d u c t i o n . 3.0 CONTROL OF RENIN SECRETION : PRESSURE EFFECT. 3.1 I n t r a r e n a l C o n t r o l of Renin S e c r e t i o n . 3.1.1 Renal Ischemia. I t has long been known that c e r t a i n kidney diseases r e s u l t i n chronic sustained hypertension. Attempts to e x p l a i n t h i s phenomenon were not very s u c c e s s f u l u n t i l the work of G o l d b l a t t et^ a l . i n 1934. This group devised a technique whereby a v a r i a b l e clamp was placed on e i t h e r one or both of the r e n a l a r t e r i e s to render the kidney(s) ischemic. They found that when the r e n a l a r t e r i e s were p a r t i a l l y occluded, there was a sustained increase i n systemic blood pressure and when the clamps were removed the systemic blood pressure would soon r e t u r n to normal (pre-operative) l e v e l s . As a r e s u l t of t h i s type of study i t was i n i t i a l l y p o s t u l a t e d that the hypertension was a r e s u l t of r e n a l ischemia, p o s s i b l y due to anoxemia and/or hypercapnia caused by t h i s ischemia. Then, i n 1942, Huidobro and Braun-Menendez examined the e f f e c t on r e n i n output in<dogs when the animals were breathing 7-8% oxygen and 5% carbon d i o x i d e and un f o r t u n a t e l y f o r t h i s hypothesis d i d not f i n d a s i g n i f i c a n t change i n r e n i n s e c r e t i o n . These authors concluded that r e n a l anoxemia and hypercapnia were not major c o n t r o l l i n g f a c t o r s i n r e n i n r e l e a s e . Reduction of a r t e r i a l oxygen l e v e l s , from 96% to 56%, had no d i s c e r n i b l e e f f e c t on r e n i n s e c r e t i o n (Skinner et a l . , 1963; 1964a). As a r e s u l t of these s t u d i e s the hypothesis that r e n a l ischemia c o n t r o l l e d r e n i n s e c r e t i o n was p a r t i a l l y d i s c r e d i t e d . 3.1.2 Pulse Pressure Hypothesis. Concurrent w i t h the ischemia s t u d i e s other groups were examining the p o s s i b i l i t y of a haemodynamic s i g n a l f o r r e n i n r e l e a s e . When i s o l a t e d dog 41 kidneys were perfused, an increase i n r e n i n output was observed, t h i s i n a s s o c i a t i o n w i t h a decreased pulse pressure (Kohlstaedt and Page, 1941). I t was then concluded that decreased r e n a l blood flow was a consequence of increased r e n i n production due to a decrease i n pulse pressure. However, l a t e r work showed that r e n a l a r t e r y s t e n o s i s r e s u l t e d i n an increase i n r e n i n r e l e a s e , r e g a r d l e s s of p u l s a t i l e or n o n - p u l s a t i l e flow ( K o l f f , 1958). In 1964-1965, Skinner et a l . examined the pulse pressure hypothesis during t h e i r s t u d i e s on r e n a l ischemia. They found that compression of the kidney, by means of an oncometer, r e s u l t e d i n an increased r e n i n r e l e a s e without any s i g n i f i c a n t change i n renal, blood flow. A l s o c o n s t r i c t i o n of the r e n a l v e i n so that r e n a l blood flow was decreased by 50% had no e f f e c t on r e n i n s e c r e t i o n although an increased r e n i n output occurred during suprarenal a o r t i c c o n s t r i c t i o n even when the r e d u c t i o n i n r e n a l p e r f u s i o n pressure d i d not produce a change i n r e n a l blood flow. In a d d i t i o n , increased r e n i n output i s observed i n dogs w i t h high-output heart f a i l u r e as a r e s u l t of a l a r g e arteriovenous f i s t u l a (Davis et a l . , 1964). A l l these s t u d i e s argued against a decreased pulse pressure being the cause of r e n i n r e l e a s e but r a t h e r some other f a c t o r , such as mean r e n a l p e r f u s i o n pressure, e s p e c i a l l y s i n c e i n the l a t t e r study the animals d i s p l a y e d a marked Widening of the pulse pressure. 3.1.3 Renal P e r f u s i o n Pressure Hypothesis. Tobian et a l . (1959) observed decreased g r a n u l a t i o n i n the juxtaglomerular (JG) c e l l s w i t h a r i s e i n r e n a l p e r f u s i o n pressure ( i n the i s o l a t e d r a t kidney) and they suggested that the JG c e l l s may a c t as s t r e t c h receptors and thus change t h e i r r a t e of r e n i n s e c r e t i o n as the a r t e r i o l a r w a l l changes i t s degree of s t r e t c h . But as these authors pointed out, changes i n r e n a l blood flow and/or glomerular f i l t r a t i o n r a t e were probably 42 associated with t h i s increased perfusion pressure, so there was a p o s s i b i l i t y that the increased renin secretion may have been a r e s u l t of these factors as w e l l . The work of Skinner ej: a l . (1963; 1964a) supported the perfusion pressure hypothesis but t h i s work has been c r i t i c i z e d for the authors' f a i l u r e to control such factors as changes i n renal tubular sodium and glomer-ular f i l t r a t i o n rate which could possibly have an e f f e c t on the rate of renin secretion (Blaine et a l . , 1970; Davis et_ a l . , 1971; Davis and Freeman, 1976). As a r e s u l t of the r e a l i z a t i o n of the l i m i t a t i o n s of t h i s e a r l i e r work a new approach was developed, that of the n o n - f i l t e r i n g kidney model (Blaine et a l . , 1970). This model thus prevented any tubular e f f e c t s on renin secretion or so i t was assumed as the macula densa was rendered non-functional. Experiments were done to determine the e f f e c t of haemorrhage and a o r t i c con-s t r i c t i o n (Blaine and Davis, 1971; Blaine et a l . , 1971); plasma renin a c t i v i t y was observed to increase markedly a f t e r these experimental manoeuvres. These authors then concluded that there was d e f i n i t e l y an i n t r a r e n a l vascular receptor which c o n t r o l l e d renin release. The n o n - f i l t e r i n g kidney model was l a t e r improved so as to cancel a l l possible neural and humoral f a c t o r s . This was done by denervation of the kidney and adrenalectomy " to exclude adrenal steroids and catecholamines (Blaine et a l . , 1971; Davis et^ a l . , 1971). These authors then found that the renin response to haemorrhage or a o r t i c c o n s t r i c t i o n was not abolished but rather more marked than i n the simple n o n - f i l t e r i n g model. Therefore, by the i s o l a t i o n of the JG. c e l l s and renal afferent a r t e r i o l e s from the influence of the macula densa, the renal sympathetic nerves and the catechol-amines, the presence of a renal vascular receptor which responds to changes i n renal perfusion pressure i s convincingly demonstrated. 43 A d d i t i o n a l studies have provided further evidence to support the renal vascular receptor hypothesis. Isolated dog kidneys respond to u r e t e r a l clamping by increased renin release while a subsequent increase i n renal a r t e r i a l pressure decreased renin release to the control l e v e l . Conversely, an i n i t i a l increase i n renal perfusion pressure decreased renin which was returned to the c o n t r o l l e v e l by u r e t e r a l clamping (Kaloyanides et a l . , 1973). High perfusion pressures i n i s o l a t e d rat kidneys have depressed renin release while low perfusion pressures have stimulated renin release i n t h i s preparation (Hofbauer et^ al., 1974). C h u r c h i l l et a l . (1974) found an inverse r e l a t i o n s h i p between changes i n renal perfusion pressure and plasma renin a c t i v i t y when a o r t i c clamping i s superimposed on u r e t e r a l occlusion, which also supports the renal vascular receptor theory. Having determined that a renal vascular receptor e x i s t s , the next problem i s to determine the l o c a t i o n of t h i s receptor, i . e . , i n what part of the vascular tree i t i s located. The studies designed to a s c e r t a i n t h i s l o c a t i o n used papaverine which i s known to block renal autoregulation, an afferent a r t e r i o l a r function (Davis et a l . , 1972). These authors infused papaverine into the renal artery of a deriervated n o n - f i l t e r i n g kidney, then haemorrhaged the experimental animal i n the amount of 20 ml/kg and found that the renin response to haemorrhage was completely blocked. However, i n denervated f i l t e r i n g kidneys the renin response to haemorrhage s t i l l p e r s i s t s despite i n f u s i o n of papaverine. Davis et a l . (1972) then concluded that the data supported the hypothesis of the renal vascular receptor and that t h i s vascular receptor was probably located i n the afferent a r t e r i o l e s since papaverine i s known to d i l a t e the renal afferent a r t e r i o l e s as i t prevents renal autoregulation. Furthermore i n dogs with a thoracic vena caval c o n s t r i c t i o n and e i t h e r . a f i l t e r i n g or n o n - f i l t e r i n g kidney, papaverine 44 produced a s t r i k i n g f a l l i n renin secretion, an increase i n renal blood flow and a decrease i n renal vascular resistance (Witty et a l . , 1971). In sodium-depleted dogs with a denervated kidney, papaverine i n f u s i o n caused an 85% f a l l i n renin secretion and a decrease i n renal resistance while papaverine had no d i s c e r n i b l e e f f e c t i n normal control animals (Gotshall ej: a l . , 1974). A l l of these observations point towards an afferent a r t e r i o l e locus for the renal vascular receptor. C o r s i n i and B a i l i e (1973) (reported i n Davis and Freeman, 1976) found that furosemide increased renin secretion i n adrenalectomized dogs with a denervated n o n - f i l t e r i n g kidney, i n a s s o c i a t i o n with an increase i n renal vascular resistance. Papaverine alone produced a maximal decrease i n renal resistance as renin output doubled. But furosemide did not have any e f f e c t above that produced by papaverine on the renal a r t e r i o l e s and renin secretion did not increase further. These observations are consistent with the hypothe-s i s that renin response i s mediated by a r e n a l vascular receptor which responds to decreases i n renal perfusion pressure. This hypothesis i s also known as the baroreceptor response hypothesis. 3.1.4 The C.N.S. and the Renal Vascular Receptor. Having established that there i s a renal vascular receptor i n the a f f e r -ent a r t e r i o l e s which causes renin release i n response to decreased renal perfusion pressure the next question to be resolved concerns the r o l e of the sympathetic nervous system i n t h i s response. The kidney appears well inner-vated by sympathetic f i b r e s (Ham, 1965; Barajas and Latta, 1967) and thus there e x i s t s the p o s s i b i l i t y that the vascular receptor i s under neural c o n t r o l . Direct stimulation of the renal nerves r e s u l t s i n an increase i n renin secretion (Vander, 1965; L o e f f l e r at a l . , 1972). However, since i n those 45 studies the stimulating electrode was placed around the renal artery and nerves, there existed the p o s s i b i l i t y that the p a r t i a l occlusion of the artery was causing the response rather than the stimulation. Therefore, Johnson et a l . (1971) dissected the nerve free from the artery and stimulated the freed nerve. They found that renin secretion increased as a r e s u l t of t h i s stimulation. In addition, c i r c u l a t i n g catecholamines have been shown to cause renin release i n denervated kidneys (Johnson e_t a l . , 1971) . Bunag et a l . (1966) showed that the acute response i n renin release to haemorrhage was not affected by blocking ganglia or by using a l o c a l anaes-t h e t i c on the renal nerves. Another group (Hodge et a l . , 1966) found that renin response to non-hypotensive haemorrhage was blocked by blocking the renal nerves. This seemed to i n d i c a t e the presence of a neural-hormonal l i n k . Weber ejt a l . (1974) have suggested that renin response to a non-hypotensive haemorrhage i s a r e s u l t of sympathetic a c t i v i t y rather than an action of the r e n a l vascular receptor. Johns and Singer (1974) found that propranolol would only block renin release i f t h i s release was caused by adrenergic stimulation. In another study, methoxamine, an alpha adrenergic stimulator, could not be shown to stimulate renin release (Vandogen and Peart, 1974). In a more recent study, Osborn e_t a l . (1977) found that propranolol would only p a r t i a l l y block furosemide-induced renin release i n the denervated non-f i l t e r i n g kidney preparation. These authors concluded that the vascular response and sympathetic response were capable of acting independently. Davis and Freeman (1976) i n t h e i r review concluded that the acute renin response to haemorrhage i s mediated only i n part by the sympathetic nervous system, e s p e c i a l l y as a denervated kidney i s capable of responding to a decrease i n renal perfusion pressure. 46 3.1.5 Summary. In summary, renin secretion from the kidney appears to be mediated by an i n t r a r e n a l vascular receptor. This receptor appears to be located i n the afferent a r t e r i o l e and does not require innervation i n order to function. It responds to a decrease i n renal perfusion pressure rather than ischemia or a change i n pulse pressure. 3.2 Vascular Control of Renin i n Fishes. There i s l i t t l e published information a v a i l a b l e on the vascular control of renin release i n f i s h e s . The only i n d i c a t i o n that t h i s i s a p o s s i b i l i t y i s that dehydration of eit h e r Japanese eels or T i l a p i a mossambica causes an increase i n plasma renin a c t i v i t y and a decrease i n juxtaglomerular c e l l renin content (Sokabe e_t -al. , 1966; Sokabe et a l . , 1968; Sokabe et a l . , 1973). This could be a r e s u l t of a decline i n renal perfusion pressure due to water loss and a consequent f a l l i n blood volume. Therefore the question was asked, i s there a renal vascular receptor which controls renin release i n fishes? 47 Section I I I M a t e r i a l s and Methods. 3.3 Kidney P e r f u s i o n P r e p a r a t i o n . The animals used i n t h i s study were a d u l t rainbow t r o u t obtained and maintained as p r e v i o u s l y described (see Secti o n 2.3.1). The f i s h were anaes-t h e t i z e d i n a 1:20,000 aqueous s o l u t i o n of MS-222 (Sandoz) and the d o r s a l a o r t a cannulated as p r e v i o u s l y described (Section 2.4.1). The caudal peduncle was then severed and a cannula of Intramedic PE 60, f i l l e d w i t h h e p a r i n i z e d s a l i n e (10 I.U. heparin per m l ) , was i n s e r t e d i n t o the caudal a r t e r y and pushed a n t e r i o r l y as f a r as the vent. The cannula was then secured i n place by means of s i l k sutures which encompassed the a r t e r y and the v e r t e b r a l column. A l o n g i t u d i n a l i n c i s i o n was made i n the mid - v e n t r a l l i n e of the animal which s t r e t c h e d from the vent to the p o s t e r i o r border of the heart. The u r e t e r s were then l o c a t e d and t i e d o f f by means of s i l k sutures. The v i s c e r a i n c l u d i n g the swim bladder were then freed from the body w a l l by bl u n t d i s -s e c t i o n and the blood v e s s e l s l e a d i n g to the:swim bladder were t i e d o f f and cut, the purpose of t h i s being to prevent leakage from these severed v e s s e l s . The v i s c e r a were then removed and the blood v e s s e l s l e a d i n g to the v i s c e r a from the aorta were t i e d again to prevent leakage. The removal of the v i s c e r a exposed s e v e r a l segmental v e s s e l s i n the body w a l l ; these v e s s e l s were then c a u t e r i z e d by hot-wire cautery. The p e r i c a r d i a l c a v i t y was then opened and the heart exposed. Ah Intramedic PE 90 cannula was then implanted i n the v e n t r i c l e v i a the v e n t r a l a o r t a and bulbus a r t e r i o s u s and t i e d i n t o place by means of s i l k sutures. During the e n t i r e s u r g i c a l p r e p a r a t i o n the animal's g i l l s were kept i r r i g a t e d w i t h a 1:200,000 c h i l l e d , aerated, aqueous s o l u t i o n of MS-222. 48 The completed preparation was transferred to a bath of Cortland s a l i n e which was placed i n an ice-bath so that the preparation could be kept cold. The dorsal aorta cannula was used as a manometer to measure perfusion pressure, the caudal artery cannula was used as the perfusion cannula and the heart cannula as the c o l l e c t i n g cannula as i s diagrammatically represent-ed i n Figure 3.1. The preparation was then perfused with aerated Cortland s a l i n e under constant pressure and flow for at l e a s t 60 minutes. This preliminary per-fusion period was the c l e a r i n g period, that i s a l l the blood remaining i n the preparation would be replaced by perfusate. Also, i f during t h i s time the perfusion pressure, as measured by the dorsal aorta cannula, f e l l below 15 cm H^ O the preparation was discarded. During the experimental period, perfusion pressure could be a l t e r e d by changing the height of the perfusion b o t t l e and r e s e r v o i r (see Figure 3.1). 3.4 Experimental Procedures. 3.4.1 E f f e c t of Blood Withdrawal. Varied amounts of blood were withdrawn from i n t a c t animals as described previously (Section 2.4.1). The blood was then centrifuged and the plasma assayed for renin a c t i v i t y by bioassay as previously described. The f i s h were anaesthetized and weighed to e s t a b l i s h a ground for comparison of plasma renin a c t i v i t i e s . The data were then examined to determine i f blood loss and plasma renin a c t i v i t y were r e l a t e d . 3.4.2 E f f e c t of Perfusion Pressure. A kidney perfusion preparation was set up as described i n Section 3.3 and the kidney perfused for 90 minutes, following the clearance period, at a pressure head of 35 cm H^O. Outflow perfusate samples were c o l l e c t e d at 0, 30, 60, and 90 minutes i n disposable polystyrene culture tubes (Fisher 4 9 Figure 3.1. Diagrammatic view of the kidney perfusion preparation and apparatus. The trout carcass was held i n the upright p o s i t i o n by a small piece of sponge wedged between the f i s h and the wall of the bath. In addition, the c o l l e c t -ing cannula led over the side of the bath and not the end as i t appears i n the diagram. •Dorsal Aorta Cannula L-Ae ration Reservoir Perfusion-Bottle .Meter Stick Meter Stick-o ice Collecting Cannula ,C ortland Saline Perfusion-Cannula k e 51 S c i e n t i f i c ) ; the tubes were sealed with Parafilm (American Can Co.), frozen and stored f or l a t e r assay. At t h i s time the perfusion pressure head was lowered 10 cm and the kidney perfused at the lower perfusion pressure for a further 90 minutes. Outflow perfusate samples of 2 ml each, same volume as the e a r l i e r samples, were c o l l e c t e d at 100, 130, 160, and 190 minutes i n disposable tubes, the tubes sealed with Parafilm, frozen and stored frozen f or l a t e r assay. Perfusate renin a c t i v i t y was measured by adding 0.5 ml of perfusate to 0.5 ml of pooled plasma stock and mixing thoroughly. This mixture was then treated with the radioimmunoassay procedure. Control samples were simply 0.5 ml of Cortland s a l i n e and 0.5 ml of the plasma stock. 3.4.3 Alpha Receptor Blockers. The alpha adrenergic blocking agent, phenoxybenzamine (Smith K l i n e & French I.A.C.), was added to the perfusate and the experimental procedure outlined i n the previous section c a r r i e d out. 3.4.4. Beta Receptor Blockers. The beta adrenergic blocking agent propranolol (Ayerst, McKenna & H a r r i -son Ltd.) was added to the perfusate and as before, the experimental procedure previously described c a r r i e d out. 3.4.5 Combined Receptor Blockage. In t h i s s e r i e s of experiments both propranolol and phenoxybenzamine were added to the perfusion s o l u t i o n . The kidney was then perfused as i n the e a r l i e r d e s c r i p t i o n . 3.4.6 Increasing Perfusion Pressure. In t h i s s e r i e s of experiments the kidney perfusion preparation was used.. The preparation was allowed to clear and s t a b i l i z e for 60 minutes at the 35 cm perfusion head. The perfusion head was then lowered 10 cm and the kidney 52 perfused for 90 minutes. The time when the perfusion head was lowered was regarded as zero time. Samples were taken at times 0, 30, 60, and 90 minutes. At t h i s time the perfusion head was raised 10 cm and the kidneys per-fused for a further 90 minutes. Samples were taken at times 100, 130, 160, and 190 minutes. The perfusate renin a c t i v i t y was assayed as described i n Section 3.4.2. 3.4.7 Angiotensin II and Renin Release. Adult trout of eit h e r sex were k i l l e d by a blow to the head, a mid-vent-r a l i n c i s i o n was made i n the body w a l l and the kidney quickly excised. The excised kidney was placed i n i c e - c o l d Cortland s a l i n e and the posterior t h i r d portion divided into s i x equivalent blocks. One piece of tiss u e was then placed i n each of s i x p e t r i dishes containing 2.0 ml aerated Cortland s a l i n e and 0, 50, 100, 150, 200, and 250 ng of angiotensin II (Hypertensin -CIBA) r e s p e c t i v e l y . The tissue blocks were allowed to incubate for 15 minutes and a 0.5 ml sample of f l u i d was taken from each p e t r i dish. These samples were assayed for renin a c t i v i t y as previously described. 3.4.8 Perfusion Solutions. Perfusates consisted of Cortland s a l i n e with 4% polyvinylpryolidinone (PVP), average molecular weight - 40,000 (Matheson, Coleman and B e l l Manu-facturing Chemists), added to simulate the oncotic pressure of the plasma proteins. In the alpha blocking experiments phenoxybenzamine was added to make a f i n a l concentration of 2 x 10 ~* grams per m i l l i l i t e r perfusate. In the beta blocking experiments propranolol was added to the perfusate to make a f i n a l —6 concentration of 10 grams per m i l l i l i t e r perfusate. I d e n t i c a l concentra-• -5- . tions were used i n the combined blocking experiments, i . e . , 2 x 10 g phenoxy-53 —6 benzamine and 10 g propranolol per ml of perfusate. In the jm v i t r o experiments Cortland s a l i n e with 4% PVP added was used as the incubation medium. In a l l cases the perfusate or incubation medium was f i r s t f i l t e r e d through Whatman No. 1 (W, & R Balston Ltd.) f i l t e r paper and then through a M i l l i p o r e f i l t e r , type HA and pore s i z e 0.45 uM ( M i l l i p o r e Corporation) to remove any p a r t i c u l a t e matter which could clog c a p i l l a r i e s and thus increase resistance to flow. 3.4.9 S t a t i s t i c a l Methods. Linear regression analysis was c a r r i e d out to determine the c o r r e l a t i o n c o e f f i c i e n t (r) and the Student's t test was used i n comparison of means to determine s i g n i f i c a n t d i f f e r e n c e s . A P value of l e s s than 0.05 was taken as s i g n i f i c a n t and a P value of less than 0.01 was taken to be highly s i g n i f i -cant . 54 Section I I I Results and Discussion. 3.5 E f f e c t of Haemorrhage and Changes i n Renal Perfusion Pressure. 3.5.1 Haemorrhage. There i s a s i g n i f i c a n t (P<0.05) p o s i t i v e c o r r e l a t i o n between amount of blood l o s t and plasma renin a c t i v i t y i n the trout as may be seen i n Figure 3.2. While i t i s true that t h i s does not n e c e s s a r i l y define a cause-effect r e l a t i o n s h i p , the c o r r e l a t i o n does i n d i c a t e that haemorrhage or a decrease i n blood volume r e s u l t s i n an increase i n plasma renin a c t i v i t y . Because the blood was withdrawn as r a p i d l y as possible v i a the cannula, the plasma renin a c t i v i t i e s observed i n t h i s study may be somewhat exaggerated. Rapid blood withdrawal i s necessary, however, so as to minimize time l o s t before the plasma i s frozen. I t w i l l be r e c a l l e d that the f i s h renin-renin substrate r e a c t i o n proceeds f a i r l y r a p i d l y even at 4°C (Section 2.7.1) and thus i t i s desirable to minimize the amount of time i n order to minimize the reaction before assay. This response has been observed i n other species. C h u r c h i l l (1973) found that moderate to severe haemorrhage caused a proportionate increase i n plasma renin a c t i v i t y i n r a t s . This response may also be observed i n rabbits (Weber et^ al., 1974) but minor haemorrhage, les s than 10% loss of blood volume, had no e f f e c t on plasma renin a c t i v i t y i n man (Goetz et a l . , 1974). Two possible reasons may be found for the renin response to haemorrhage. Since renin i s produced i n the kidney vascular bed of f i s h e s (Capreol and Sutherland, 1968; Nolly and F a s c i o l o , 1972) there i s a d i s t i n c t p o s s i b i l i t y that an i n t r a r e n a l vascular receptor, such as may be found i n mammals, could e x i s t . The other p o s s i b i l i t y i s that there may be a systemic receptor which influences renin release v i a some sort of neural pathway. Accordingly, before 55 Figure 3.2. Cor r e l a t i o n between haemorrhage and plasma renin a c t i v i t y . The y-axis i s plasma re n i n a c t i v i t y i n ng All/ml/hr while the x-axis i s blood loss i n terms of ml blood loss per kg body weight. For the l i n e equation y = 0.89x + 0.17 the co r r e l a t i o n c o e f f i c i e n t r = 0.81 i s s i g n i f i c a n t at the 5% l e v e l . The number of animals used i n t h i s experiment was 15,and plasma renin a c t i v i t y was measured by bioassay. 57 any conclusions may be drawn from the e f f e c t s of haemorrhage on renin, i t i s necessary to examine the r o l e of renal perfusion pressure i n renin release. 3.5.2 Changes i n Renal Perfusion Pressure. When renal perfusion pressure i s decreased by approximately 50% renin release, as measured by renin a c t i v i t y , shows a 60% increase. Figure 3.3 shows the o v e r a l l pattern of renin secretion during the en t i r e perfusion period. As may be seen during the f i r s t 90 minutes of perfusion (at high pressure) the renin a c t i v i t y shows a s l i g h t n o n-significant increase followed by a small decline. When the perfusion pressure i s lowered (at 90 minutes) the renin a c t i v i t y immediately increases to a point greater than that which i t had reached up to t h i s time. Renin a c t i v i t y shows a steady increase from 100 min to 190 min and t h i s increased a c t i v i t y i s s i g n i f i c a n t l y higher than the a c t i v i t i e s at 0 min, 90 min, and 100 min. This shows that the renin secre-t i o n from the kidney has s i g n i f i c a n t l y increased. In Table 3.1 may be found the average values for renin a c t i v i t y during t h i s period. Renin a c t i v i t y • increases from 0.36 ng Al/ml/hr to 0.58 ng Al/ml/hr, an increase of 60%, while the perfusion pressure decreases from 21.7 cm H^O to 13.1 cm H^O, a 40% decrease. Indeed there i s a highly s i g n i f i c a n t c o r r e l a t i o n between renin a c t i v i t y and renal perfusion pressure as may be seen i n Figure 3.4. Increasing renal perfusion pressure from a low l e v e l to a high l e v e l apparently had no e f f e c t on renin a c t i v i t y as may be seen i n Figure 3.5 and Table 3.1. In t h i s case the i n i t i a l perfusion period i s done at low pressure and seems to r e s u l t i n an apparent'decline i n renin a c t i v i t y . However t h i s decline i s not s i g n i f i c a n t . When the perfusion pressure i s increased at 90 minutes the renin a c t i v i t y does not either increase or decrease but remains at approximately the same l e v e l as that found at 90 minutes. In Table 3.1 i t may be seen that when perfusion pressure increases from 12.9 cm H„0 to 58 Figure 3.3. The e f f e c t of a decline i n renal perfusion pressure on renin release from the i s o l a t e d perfused trout kidney. The y-axis represents renin release as measured i n perfusate renin a c t i v i t y which i s i n ng Al/ml/hr while the x-axis represents time of perfusion as measured i n minutes. The break i n the l i n e between 90 and 100 minutes shows the point at which the perfusion pressure was changed from a high (35 cm R^O) to a low (25 cm H^O). Each point on the figu r e is/the mean ± S.E.M. of 10 ex-perimental animals. PLASMA RENIN ACTIVITY (ng A-i/ml/hr) TABLE 3.1 Ef f e c t of Changes i n Renal Perfusion Pressure on Renin Release 0 Experiment Perfusion Period Perfusion Pressure (cm H 20) Renin A c t i v i t y (ng AI/ml/hr) High to Low Pressure I n i t i a l Second 21.7 ± 0.38 (10) 13.1 ± 0.71C (10) 0.36 ± 0.07 (10) . 0.58 ± 0.07C (10) Low to High Pressure I n i t i a l Second a - A l l values are means ± S.E.M. (N) 12.9 ± 0.77 (14) 19.6 ± 0.72* (14) 0.68 ± 0.07 (14) 0.58 ± 0.08 (14) b - I n i t i a l r e f e r s to the f i r s t 90 minutes of perfusion while second r e f e r s to the remaining 90 minutes of perfusion c - P<0.01 d - P<0.05 e - P<0.01 61 Figure 3.4. The c o r r e l a t i o n between renal perfusion pressure and renin secretion i n the i s o l a t e d perfused trout kidney. The y-axis represents renin release as measured by perfusate (plasma) renin a c t i v i t y and i s i n ng Al/ml/hr. The x-axis i s renal perfusion pressure as measured i n cm H^O. For the l i n e equation y = -11.77x + 23.93 the c o r r e l a t i o n c o e f f i c i e n t r = -0.92 i s s i g n i f i c a n t at the 1% l e v e l . 2-0 o P E R F U S I O N P R E S S U R E ( c m H 2 0 ) 63 Figure 3.5. Changing renal perfusion pressure and renin release i n the i s o l a t e d perfused trout kidney. The y-axis repre-sents the renin release from the kidney and i s measured i n perfusate renin a c t i v i t y , i . e . , ng Al/ml/hr. The x-axis represents the time of perfusion i n minutes. The break i n the graph between 90 minutes and 100 min-utes i s the point at which the perfusion pressure was changed from the low (25 cm R^O) to the high (35 cm R^O). Each point on the graph represents the mean ± S.E.M. of 14 determinations. 64 0 l b 9b ibo 3o" T I M E (min) ioo iFo" 65 19.6 cm R^O, a highly s i g n i f i c a n t increase, renin a c t i v i t y shows only a s l i g h t n o n - s i g n i f i c a n t decline from 0.68 ng Al/ml/hr to 0.58 ng Al/ml/hr. These data show that renal perfusion pressure plays an important r o l e i n renin release i n the trout. However i t i s only a decline i n the perfusion pressure which stimulates renin release as increasing the perfusion pressure has no e f f e c t on the rate of renin release. Therefore, i t appears that once renin release has been i n i t i a t e d , the juxtaglomerular (JG) c e l l s w i l l continue to secrete renin u n t i l i n h i b i t e d by some, as yet unknown, s i g n a l . This response i s very s i m i l a r to the baroreceptor response found i n mammals (Liard et a l . , 1974). The nature of t h i s response was e l u c i -dated by C h u r c h i l l at a l . (1974) when they combined s a l t - l o a d i n g which would i n h i b i t renin release and a decline i n renal perfusion pressure to show that the baroreceptor response overrode the sodium response. On the other hand, other groups have recently shown that the baroreceptor response i s not n e c e s s a r i l y l i m i t e d to the i n t r a r e n a l receptor as c a r o t i d sinus hypotension causes renin release (Jarecki jit al., 1978). Also reductions i n r i g h t a t r i a l pressure can cause an increase i n renin secretion (Brosnihen and Bravo, 1978). It would appear that i n the trout the receptor i s an i n t r a r e n a l one, since i n t h i s preparation the only vascular c i r c u i t r y l e f t s u b s t a n t i a l l y i n t a c t i s the renal c i r c u l a t i o n and to some extent the c r a n i a l c i r c u l a t i o n . The recep-tor i s pressure-sensitive as the changes i n renal perfusion pressure were not accompanied by a change i n the perfusate outflow. This shows that the flow rate through the renal vascular bed was constant. The most probable reason for the constant perfusate flow through the renal vascular bed i s the pres-ence of an i n t a c t dorsal and l a t e r a l segmental c i r c u l a t i o n . These vessels could not be cauterized as they e x i t d i r e c t l y from the dorsal aorta into the muscle mass. It thus appeared that these vessels acted as a v a r i a b l e r e s i s t o r 66 i n the perfusion c i r c u i t . Therefore the change i n renin secretion would not be due to a change i n perfusate flow rates. The next question which has to be asked i s the nature of the receptor. These experimental data show that the s i g n a l received by the receptor i s a decline i n renal perfusion pressure. However, how i s t h i s s i g n a l acted on, i . e . , i s i t a d i r e c t e f f e c t on the JG c e l l s , or i s i t mediated by some sort of neural r e f l e x mechanism? The l a t t e r i s a p o s s i b i l i t y , for the kidney i s w e l l innervated by sympathetic f i b r e s i n both mammalian (Barajas, 1964; Barajas and Latta, 1967) and piscean species (Sokabe and Ogawa, 1974). In order to examine t h i s p o s s i b i l i t y sympathetic receptor blocking studies were c a r r i e d out while a l t e r i n g the renal perfusion pressure. 3.5.3 Blocking Studies. Figure 3.6 and Table 3.2 show the e f f e c t s of the alpha receptor blocking agent, phenoxybenzamine, on renin secretion induced by the baroreceptor response. During the period of high pressure perfusion the renin a c t i v i t y shows a s l i g h t , non-significant increase but when the perfusion pressure decreases the renin a c t i v i t y shows a massive increase u n t i l the f i n a l l e v e l i s approximately 10 f o l d higher than the i n i t i a l l e v e l . Table 3.2 shows that when the average perfusion pressure declines from 21.3 cm ^ 0 to 14.7 cm H 20, a s i g n i f i c a n t (P<0.05) decrease, then the average renin a c t i v i t y increases f i v e f o l d , from 0.02 ng Al/ml/hr to 0.10 ng Al/ml/hr. These data would indi c a t e that the baroreceptor response of renin secretion i s not c o n t r o l l e d or effected through the sympathetic alpha receptors, since e a r l i e r work has shown that phenoxybenzamine i s an e f f e c t i v e alpha receptor blocking agent i n salmonids (Randall and Stevens, 1967). 67 Figure 3.6. E f f e c t of addition of the alpha blocking agent phenoxybenzamine on the baroreceptor response i n the is o l a t e d perfused trout kidney. The y-axis i s renin a c t i v i t y i n ng Al/ml/hr while the x-axis i s time of perfusion i n minutes. The break i n the l i n e represents the point at which the perfusion pressure was changed from the high to the low. Each point on the l i n e represents the mean ± S.E.M. of 10 determinations. The dose administered was 2 x 10 ^ gm phenoxybenzamine per m i l l i l i t r e of perfusate. •15H 68 loO iTo" 77 TIME 1 3 0 (min) TABLE 3.2 Ef f e c t of Alpha Receptor Blockage on the Renin Response to Hypotension Perfusion Period^ Perfusion Pressure Renin A c t i v i t y (cm H 20) (ng Al/ml/hr) I n i t i a l 21.3 ± 1.48° 0.02 ± 0.013d (10) (10) Second 14.7 ± 1.33° 0.10 ± 0.04 d (10) (10) a - A l l values are means ± S.E.M. (N) b - I n i t i a l refers to the f i r s t 90 minute perfusion period while second refers to the second 90 minute perfusion period c - P<0.01 d - P<0.05 70 In mammals, stimulation of the renal alpha receptors generally r e s u l t s i n an i n h i b i t i o n of renin secretion (Vandogen and Peart, 1974). This may not occur i n fishes as blocking the alpha receptors has no e f f e c t on renin release, and i f these receptors i n h i b i t renin secretion, blockage should r e s u l t i n an increased renin secretion during the control period. Figure 3.7 and Table 3.3 show the e f f e c t s of beta receptor blockage on hypotension e l i c i t e d renin release and as may be seen there i s l i t t l e or no e f f e c t . Renin a c t i v i t y declined s l i g h t l y then rose s l i g h t l y during the period of high pressure perfusion. As before, the decline i n the perfusion pressure produced a s i g n i f i c a n t increase i n renin a c t i v i t y as i s seen i n Figure 3.7. The average perfusion pressure decreased from 19.2 cm R^O to 14.2 cm E^O, a 26% decrease, and renin a c t i v i t y increased from 1.61 ng Al/ml/hr to 3.35 ng Al/ml/hr, a 100% increase (Table 3.3). These data i n d i c a t e that the beta receptors do not play a major r o l e i n hypotension induced renin secretion as blocking these receptors has no.effect on the renin response. These receptors have been implicated i n control of renin secretion i n mammalian species. Pettinger e_t aJL. (1973) found that blocking beta receptors i n h i b i t s the increase i n plasma renin a c t i v i t y caused by the va s o d i l a t i n g drugs, minoxidil and hydralazine. Renin secretion i s increased when the i s o l a t e d r at kidney i s perfused with catecholamines but t h i s increase i s i n h i b i t e d by propranolol (Vandogen et_ al., 1973). I n t r a -muscular i n j e c t i o n of propranolol, i n ra b b i t s , w i l l cause a decrease i n 71 Figure 3.7. The e f f e c t of addition of the beta blocking agent propranolol and changing renal perfusion pressure on renin release i n the i s o l a t e d perfused trout kidney. The y-axis represents renin release measured i n terms of perfusate renin a c t i v i t y as expressed i n ng AI/ml/ hr. The x-axis represents time of perfusion and i s measured i n minutes. The break i n the l i n e between 90 and 100 minutes represents the point at which the perfusion pressure was changed from high to low. Each point on the l i n e represents the mean + S.E.M. of 9 determinations. The dosage administered was 10 ^ gm propranolol per m i l l i l i t e r perfusate. 72 5 H 3 0 3) 9 * 0 1 £ ) 0 ~ i5o 1 6 0 ~ 1 9 0 T I M E (min) TABLE 3.3 E f f e c t of Beta Receptor Blockage on the Renin Response to Hypotension Per f u s i o n P e r i o d ^ P e r f u s i o n Pressure Renin A c t i v i t y (cm H 20) (ng Al/ml/hr) I n i t i a l 19.2 ± 1.35° 1.61 ± 0\36 d (9) (9) Second 14.2 ± 1.20 C 3.35 ± 0.48 d (9) (9) a - A l l values are means ± S.E.M. (N) b - I n i t i a l r e f e r s to the f i r s t 90 minutes of p e r f u s i o n and second r e f e r s to the second 90 minutes of p e r f u s i o n c - P<0.01 d - P<0.05 74 plasma renin a c t i v i t y and indeed there was found an inverse c o r r e l a t i o n be-tween plasma l e v e l s of propranolol and plasma renin a c t i v i t y (Forman and Mulrow, 1974). Stress-induced renin release may also be i n h i b i t e d by propranolol (Leenen and Shapiro, 1974). These studies were concerned p r i m a r i l y with the e f f e c t s of the sympathetic system on renin release and generally do not involve the baroreceptor response. In a more recent study c a r r i e d out by Osborn et a l . (1977), i t was i n the denervated n o n - f i l t e r i n g kidney model, beta blockage could blunt furosemide induced renin release although the response could not be e n t i r e l y abolished. Therefore, i n mammalian species i t would appear that the baroreceptor response may i n part be mediated v i a the sympathetic nervous system, s p e c i f i c a l l y the beta receptors. This does not appear to be the case i n trout for as previously mentioned beta receptor blockage had no major e f f e c t on the baroreceptor response. I t i s possible that the alpha and beta receptors may have a secondary e f f e c t , possibly as a r e s u l t of an action on vascular tone. This would explain the considerable d i s p a r i t y i n the l e v e l s of renin a c t i v i t y between the two experi-ments, con t r o l 0.02 ng Al/ml/hr for alpha and 1.61 ng Al/ml/hr for beta at s i m i l a r perfusion pressures while following a decline i n perfusion pressures the values were 0.1 ng Al/ml/hr for alpha and 3.35 ng Al/ml/hr for beta, again at s i m i l a r perfusion pressures. If vascular tone does e x i s t i n f i s h kidney, then while the drugs would not have any d i r e c t e f f e c t on the JG cells*, there may be an e f f e c t as a r e s u l t of the loss of vascular tone due to the blocking agents. Accordingly the next experiment was to block both receptors and observe the e f f e c t on renin release. Figure 3.8 demonstrates the pattern of renin a c t i v i t y , and consequently renin release, over the t o t a l perfusion period when both alpha and beta r e -75 F i g u r e 3.8. The e f f e c t o f combined b l o c k i n g a g e n t s on r e n i n r e l e a s e caused by c h a n g i n g r e n a l p e r f u s i o n p r e s s u r e i n t h e i s o -l a t e d p e r f u s e d t r o u t k i d n e y . The y - a x i s r e p r e s e n t s r e n i n r e l e a s e as measured by p e r f u s a t e r e n i n a c t i v i t y and i s e x p r e s s e d i n ng A l / m l / h r . The x - a x i s r e p r e s e n t s p e r f u s i o n p e r i o d and i s measured i n m i n u t e s . The b r e a k i n t h e l i n e between 90 m i n u t e s and 100 m i n u t e s i s t h e p o i n t a t w h i c h t h e p e r f u s i o n p r e s s u r e head was l o w e r e d . Each p o i n t on t h e l i n e r e p r e s e n t s t h e mean ± S.E.M. o f 9 d e t e r m i n a t i o n s . I n t h i s c a s e t h e dosage o f the a l p h a b l o c k i n g agent phenoxybenzamine was 2 x 10 ^ gm/ml p e r f u s a t e and t h e b e t a —6 b l o c k i n g agent p r o p r a n o l o l was 10 gm/ml p e r f u s a t e . 75 o" % — cT) 90 1*00 L5O lJo l5o TIME (min) TABLE 3.4 Ef f e c t of Combined Alpha and Beta Receptor Blockage on Baroreceptor Induced Renin Secretion Perfusion Period** Perfusion Pressure Renin A c t i v i t y (cm H 20) (ng Al/ml/hr) I n i t i a l 22.2 ± 0.87° 0.191 ± 0.034 (9) (9) Second 14.2 ± 1.27c 0.52 ± 0.06d d (9) (9) a - A l l values are means ± S.E.M. (N) b - I n i t i a l refers to the f i r s t 90 minutes of perfusion while second r e f e r s to the second 90 minutes of perfusion c - P<0.01 d - P<0.05 78 ceptors were blocked. Up to 60 minutes of the i n i t i a l (high pressure) per-fusion period renin release rose s l i g h t l y , t h i s being followed by a small decrease. When the perfusion pressure declined from an average of 22.2 cm E^O to 14.2 cm R^O (36%), renin a c t i v i t y increased from 0.191 ng Al/ml/hr to 0.52 ng Al/ml/hr, a nearly 3 f o l d increase. The l e v e l s of renin a c t i v i t y obtained i n t h i s study appear to be approximately midway between those obtained when only one blocking agent i s used. This experiment serves to confirm the hypothesis that the sympathetic receptors do not play a s i g n i f i -cant r o l e i n the response to a decline i n renal perfusion pressure because blocking both receptor types had no d i s c e r n i b l e e f f e c t on the hypotension induced renin release. This was to be expected as blocking e i t h e r receptor type s i n g l y had no e f f e c t . But the l e v e l s of renin a c t i v i t y obtained i n t h i s experiment were s i m i l a r to those found i n the i n i t i a l experiments i n t h i s s e r i e s (see Figures 3.3 and 3.4 and Table 3.1). Thus there may exi s t some sort of vascular tone i n the renal c i r c u l a t i o n which i s modified by action on eit h e r the alpha or beta receptors. But t h i s i s somewhat specula-t i v e at t h i s time as there i s l i t t l e information a v a i l a b l e on the nature of the vascular bed i n the f i s h kidney. I t i s equally a p o s s i b i l i t y that these v a r i a t i o n s i n o v e r a l l l e v e l s may simply r e f l e c t a seasonal v a r i a t i o n or changes i n some other uncontrolled v a r i a b l e , as these experiments were ca r r i e d out over a 6-month period. Since these receptors do not play any s i g n i f i c a n t r o l e i n the release of renin, caused by hypotension, the decline i n perfusion pressure must a f f e c t the J,G c e l l s i n some d i r e c t fashion. As the J G.. c e l l s are part of the a r t e r i o l e i t i s probable that when renal perfusion pressure decreases the transmural pressure seen by these c e l l s decreases. Thus the degree of st r e t c h sensed by these c e l l s changes. This i n turn r e s u l t s i n a deformation of 79 these c e l l s followed by the release or active secretion of renin from the c e l l . The secretion of renin following the membrane deformation may be an active process which i s p a r t i a l l y calcium dependent (Baumbach and Leyssac, 1977). But e x t r a c e l l u l a r calcium does not play an e s s e n t i a l r o l e i n the mechanics of renin secretion although i t may be necessary for synthesis and/or storage (Lester and Rubin, 1977). More recently, Park and Malvin (1978) found that renin secretion i s mediated i n part by i n t r a c e l l u l a r changes i n calcium concentration. While these experiments were done on mammalian subjects i t does not appear u n l i k e l y that a s i m i l a r mechanism could be found i n f i s h e s . 3.5.4 „ I n h i b i t i o n of Renin Secretion. Once renin secretion has been i n i t i a t e d , what i s the s i g n a l which causes an i n h i b i t i o n of renin secretion? There are two p o s s i b i l i t i e s , one being a r i s e i n perfusion pressure and the other being a hormonal negative feedback system. The former would be a r e s u l t of the action of renin i n causing a vaso c o n s t r i c t i o n and a r i s e i n perfusion pressure. The l a t t e r involves again the formation of angiotensin I I but t h i s time the generated hormone feeds back to i n h i b i t further renin release from the JG. c e l l s . Angiotensin I could be the compound which i n h i b i t s further release but i t i s more l o g i c a l to assume that i t i s the end-product which i s the i n h i b i t o r , presupposing the existence of a negative feedback system of t h i s type. Figure 3.5 and Table 3.1 i l l u s t r a t e the e f f e c t of increasing perfusion pressure on renin and as previously noted, the increased perfusion pressure had no s i g n i f i c a n t e f f e c t on renin a c t i v i t y ; a n d consequently renin release. However as may be seen i n Figure 3.9, incubation of kidney s l i c e s with angiotensin II r e s u l t s i n a steady dose-dependent decline i n renin secretion, from these kidney s l i c e s . Therefore, since t h i s i s an ±n v i t r o s i t u a t i o n and 80 •Figure 3.9. The i n v i t r o e f f e c t of angiotensin II on renin release. The y-axis represents the renin release as measured by renin a c t i v i t y and i s expressed i n ng Al/ml/hr. The x-axis represents the dose of angiotensin II added to the incubation medium and i s i n ng. For the l i n e equation y = -0.005x +1.62 the c o r r e l a t i o n c o e f f i c i e n t r = -.88 i s s i g n i f i c a n t at the 1% l e v e l . RENIN ACTIVITY (ng A-l/ml/hr) + 82 there i s no longer an i n t a c t renal vascular bed, the decreased renin a c t i v i t y i s a r e s u l t of a d i r e c t e f f e c t on the JG c e l l s by the angiotensin I I . Thus renin secretion i s i n h i b i t e d i n a short-loop negative feedback system inv o l v -ing angiotensin I I . This system i s not unique to f i s h e s and has been found i n various mammal-ian species. Vander and Geelhoed (1965) found that renin secretion i n dogs could be i n h i b i t e d by low doses of angiotensin I I . This phenomenon may also be observed i n sheep (Blair-West et a l . , 1971), humans (DeChamplain et a l . , 1966) and rats (Vandogen et a l . , 1974). In addition, blocking angiotensin II by use of s p e c i f i c angiotensin antagonists such as l-Sarcosine,8-Alanine,an-giotensin II and converting enzyme i n h i b i t o r s r e s u l t s i n an increase i n plasma renin a c t i v i t y i n a l l species examined. These data are consistent with the hypothesis that renin secretion i s i n h i b i t e d by a short-loop nega-t i v e feedback type mechanism (Davis and Freeman, 1976). 3.5.5 Model Mechanism for Renin Secretion. Based on the experimental evidence previously discussed the following model for renin secretion i n teleosts i s proposed. A decline i n renal per-fusion pressure r e s u l t s i n a decrease i n the transmural pressure i n the afferent a r t e r i o l e s . This causes a deformation of the J G. c e l l s which are located i n the walls of the afferent a r t e r i o l e s and there i s a consequent secretion of renin. The c i r c u l a t i n g renin then acts on the renin substrate i n the blood plasma to form angiotensin I which i s converted into angiotensin I I . This angiotensin II thus formed exerts two e f f e c t s . One i s on the a r t e r i o l e s causing v a s o c o n s t r i c t i o n which probably has l i t t l e or no e f f e c t on the release of renin. The other i s a d i r e c t e f f e c t on the J.G= c e l l s to i n h i b i t further renin release i n a short-loop negative feedback type mechan-ism. The evidence which supports t h i s hypothesis that renin release i s a 83 r e s u l t of a d i r e c t e f f e c t on the J G c e l l s i s the lack of e f f e c t of eit h e r alpha or beta receptor blockage. The i n v i t r o i n h i b i t i o n of renin secretion by the addition of angiotensin II to the incubation medium supports the negative feedback hypothesis as does the lack of e f f e c t of increasing renal perfusion pressure. 3.5.6 Summary. In summary, renin i s secreted i n response to a decline i n renal perfusion pressure. This i s not mediated by the sympathetic nervous system. Renin secretion i s i n h i b i t e d i n a short-loop negative feedback type mechanism. 84 Section IV Introduction. 4.0 SODIUM AND RENIN RELEASE. 4.1 Macula Densa Hypothesis. 4.1.1 Relationship of Macula Densa and JG C e l l s . The macula densa i s an area of s p e c i a l i z e d c e l l s i n the d i s t a l convoluted tubule of mammalian nephrons (Ham, 1965), as i s diagrammatically i l l u s t r a t e d i n Figure 4.1. This area appears to play a r o l e i n renin release but the nature of that r o l e has to t h i s time been somewhat c o n t r o v e r s i a l . The c e l l s of the macula densa are highly s p e c i a l i z e d c e l l s i n close contact with the granular juxtaglomerular (JG) c e l l s (Hartroft and H a r t r o f t , 1961). In f a c t , these c e l l s are only separated from the JG c e l l s by an incomplete basement membrane with cytoplasmic extensions (Barajas and Latta, 1967). In addition, there i s a p o s i t i v e c o r r e l a t i o n between glucose-6-phosphate dehydrogenase a c t i v i t y i n the macula densa and renal renin or JG c e l l granulation i n several experimental conditions i n rats such as sodium-deficient d i e t and adrenalectomy ( C a p e l l i et^ _al. , 1968) . Barajas (1971) has found that the macula densa i s more c l o s e l y a s s o c i -ated with the afferent a r t e r i o l e and mesangial c e l l s than with the JG c e l l s i n the r a t . This author hypothesized that the degree of contact between the macula densa and the JG c e l l s i s a function of tubular volume and sodium load and that as sodium and tubular volume decrease the distance between the macula densa and the JG c e l l s increases. This then r e s u l t s i n an increase i n renin secretion. Further evidence which r e l a t e s the macula densa to the JG c e l l s i s provided by the work of Vander and M i l l e r (1964) who found that renin secre-85 Figure 4.1. Diagrammatic representation of the juxtaglomerular apparat-us of mammals. AA - afferent a r t e r i o l e , 'EA. - efferent a r t e r i o l e , G - glomerulus, RG - renin granules, MD - macula densa, DCT - d i s t a l convoluted tubule, EGM - extraglomerular mesangium. 86 87 t i o n a f t e r a o r t i c c o n s t r i c t i o n could be blocked by simultaneous administra-t i o n of d i u r e t i c s . Also renin secretion can be i n h i b i t e d by hypernatremia and hyperkalemia i n the normal i n t a c t f i l t e r i n g kidney but not i n the non-f i l t e r i n g kidney model. This leads to the conclusion that the macula densa i s r e l a t e d to the J G. c e l l s i n some fashion as the macula densa has been rendered non-functional i n the n o n - f i l t e r i n g kidney and renin s e c r e t i o n i s not i n h i b i t e d by either hypernatremia or hyperkalemia (Shade et a l . , 1972). Retrograde microperfusion studies have provided other evidence of the r e l a t i o n s h i p between the macula densa and the J.G. c e l l s . Retrograde per-fusion of the macula densa with solutions of sodium chloride has provided two pieces of evidence; one being that renin a c t i v i t y i n a s i n g l e J.G. apparatus was increased and the other being that single-nephron glomerular f i l t r a t i o n rate decreased which could be at t r i b u t e d to afferent a r t e r i o l a r c o n s t r i c t i o n due to increased l o c a l formation of angiotensin II (Thurau, 1971; Thurau et a l . , 1972). 4.1.2 Role of the Macula Densa i n Renin Release. Having established that there i s a r e l a t i o n s h i p between the macula densa and the J G c e l l s , the next problem to confront i s what i s the r o l e of the macula densa i n renin release, i . e . to what does the macula densa respond? Administration of d i u r e t i c s blocks the renin response to a o r t i c c o n s t r i c t i o n and i t was suggested that the blockage was due to increased sodium load at the macula densa caused by the d i u r e t i c s (Vander and M i l l e r , 1964). They then suggested that a decrease i n macula densa sodium leads to an increase i n renin secretion. A low sodium intake r e s u l t s i n sodium and volume depletion, decreased glomerular f i l t r a t i o n r a te, and, i n severe cases, hyponatremia so that the f i l t e r e d load of sodium i s decreased with a concomi-tant increase i n plasma renin a c t i v i t y (PRA) i n man (Brown jet al., 1963; 1964). 88 Similar e f f e c t s have been shown i n other mammalian species, notably the r a t and the dog (Vander and Luciano, 1967; Pickens and Enoch, 1968; Vander and Carlson, 1969). While i t would appear that a low sodium load at the macula densa causes renin secretion as shown by the previous studies, Davis and Freeman (1976) i n t h e i r recent review have suggested that these studies may not be as d e f i n i t i v e as once thought. They point out that to extrapolate the value for f i l t e r e d sodium to the concentration of sodium at the macula densa, i . e . , macula densa sodium load, may not be v a l i d as there i s the a d d i t i o n a l i n f l u -ence of the loop of Henle on tubular sodium and chloride before i t reaches the macula densa. This area i s rather i n a c c e s s i b l e to micropuncture and so l i t t l e i s known about the changes that occur i n t h i s area. Also, these authors point out that there i s evidence for a chloride pump rather than a sodium pump i n the ascending limb of the loop of Henle. Another group of workers have held to the hypothesis that i t i s an increased sodium load which the macula densa senses (Thurau et a l . , 1967; Thurau _et a l . , 1972). This group used retrograde perfusion of the nephron and observed that i s o t o n i c or hypertonic sodium solutions injected into the d i s t a l tubule resulted i n either a decrease i n proximal tubule diameter or proximal tubule collapse. These observations were interpreted as a r e s u l t of reduced glomerular f i l t r a t i o n rate. Control experiments conducted with hypotonic sodium chloride or with hypertonic and/or i s o t o n i c choline.chloride and mannitol solutions did not show any changes i n proximal tubule diameter. They then proposed the following mechanism: an increased release'of renin i n response to increased sodium concentration at the macula densa, l o c a l angiotensin II formation i n the afferent a r t e r i o l e , afferent a r t e r i o l a r con-s t r i c t i o n , decreased glomerular f i l t r a t i o n rate and thus proximal tubule 89 collapse. Thurau (1971) o r i g i n a l l y proposed that the increased sodium load caused or resulted i n an increase i n renin release. However i n a l a t e r paper (Thurau et a l . , 1972) i t was maintained that only increased renin a c t i v i t y i n the J.G c e l l s occurred and t h i s r e f l e c t e d increased renin a c t i v a t i o n from a preformed molecule. This was due to the f a c t that the time course of the response to the retrograde perfusion was too rapid to allow for renin synthesis. This hypothesis has been supported by a number of other groups. Rabbits given furosemide showed an increase i n PRA despite the f a c t that sodium and volume depletion were prevented by r e i n f u s i n g u r e t e r a l urine into the femoral veins (Meyer ejt a l . , 1968). As the ascending limb of the loop of Henle i s the major s i t e of action of furosemide (Glapp and Robinson, 1968) and the concentration of sodium i n the early part of the d i s t a l tubule i s increased, Meyer et a l . (1968) suggested that the increased sodium concentration at the macula densa was causing an increase i n renin secretion and consequently the increased PRA. Cooke et a l . (1970) found that intravenous i n f u s i o n of ethacrynic acid during r e i n f u s i o n of u r e t e r a l urine into the femoral veins caused an increase i n renal vein renin a c t i v i t y while chlorothiazide did not. Since chlorothiazide acts on the d i s t a l convoluted tubule while ethacrynic acid acts on the ascending limb of the loop of Henle, they suggested that a sodium r i c h tubular f l u i d was reaching the macula densa i n the f i r s t case but not i n the second. I t was then proposed that the increased sodium load was causing the increase i n renal vein renin a c t i v i t y . This response has also been observed i n another study ( B i r b a r i , 1972). In v i t r o studies have also supported the Thurau hypothesis i n that increasing sodium concentrations r e s u l t i n increased renin secretion while the addition of oubain, a sodium-potassium ATPase i n h i b i t o r , causes a decrease i n renin secretion under these 90 conditions (Lyons and C h u r c h i l l , 1974). Another group, Blair-West et a l . (1977), observed that dehydrated sheep had plasma renin contents two to three f o l d greater than normal l e v e l s . They found that changes i n plasma renin content were rel a t e d to sodium consumption, excretion and plasma content but not to plasma volume insofar as plasma volume i s r e f l e c t e d by a l t e r a t i o n s i n plasma proteins. They concluded that the changes i n renin release were mediated by the increased sodium l e v e l s and not by a l t e r a t i o n s i n body f l u i d volume and were under the influence of the macula densa mechanism. This hypothesis has been attacked by a number of other workers i n the f i e l d . Gottschalk and Leyssac (1968) pointed out that the proximal tubular, collapse could be due to f l u i d leaking from s i t e s where the proximal tubule was punctured for i n j e c t i o n of the lissamine green dye i n the Thurau prepara-t i o n . Also the proportion of angiotensin I to angiotensin II conversion i n the kidney appears to be low and t h i s i s further evidence against the Thurau hypothesis (Vane, 1974). In addition Davis and Freeman (1976) noted that the observation that choline chloride f a i l e d to influence the mechanism proposed by Thurau must be reconciled with the evidence for an a c t i v e chloride pump i n the ascending limb of the loop of Henle. This l a t t e r c r i t i c i s m may not be v a l i d as Stephens et a l . (1978) have found that sodium or potassium l a c t a t e i n h i b i t e d renin secretion i n the dog. This evidence supports the theory that i t i s sodium l e v e l s at the macula densa which a f f e c t renin and not chloride l e v e l s . C o r s i n i et a l . (1974) have noted that i n v i t r o studies may not n e c e s s a r i l y be giving u s e f u l information as renin secretion by the kidney s l i c e s i n v i t r o may be proceeding at a maximal or near-maximal rate and thus respond only weakly at best to s t i m u l i which would usually e l i c i t renin release i n vivo. 91 The o r i g i n a l hypothesis of Vander and M i l l e r (1964) was that increased sodium load at the macula densa leads to a decrease i n renin secretion. This hypothesis also has been supported by a number of other groups. Freeman jit a l . (1974) found that when renin secretion and renal vein renin a c t i v i t y were compared during i n f u s i o n of ethacrynic acid there was a marked decline i n renin secretion but not i n renal vein renin a c t i v i t y . This decline i n renin secretion was associated with a marked increase i n urinary sodium concentra-t i o n and rate of secretion. Thus a high tubular sodium l e v e l i n h i b i t e d renin secretion but not the a c t i v i t y of prereleased renin. DiBona (1971) found that renin secretion increased i n a s s o c i a t i o n with a decreased d i s t a l tubular sodium d e l i v e r y and concentration which supports the o r i g i n a l Vander and M i l l e r hypothesis. Intravenous i n f u s i o n of sodium chloride or sodium s u l f a t e revealed that sodium s u l f a t e i n h i b i t e d renin secretion to a greater extent than sodium chloride which suggested that i t was sodium l e v e l s rather than sodium and chloride which i n h i b i t e d renin release ( C h u r c h i l l et^ _a_l. , 1975),. This was confirmed by the work of Stephens at a l . (1978) who used sodium l a c t a t e to i n h i b i t renin release. However, Kotchen et a l . (1978) found that choline chloride i n h i b i t e d renin to a greater extent than sodium chloride while choline bicarbonate was t o t a l l y without e f f e c t . These r e s u l t s suggest-ed that chloride may have a very important r o l e to play i n regulating responses to sodium loading i n the r a t . Nash e_t a l . (1968) presented e v i -dence for a sodium-sensitive mechanism located i n the kidney and suggested that the s i g n a l which mediated renin release was a r e s u l t of decreased sodium transport by the macula densa c e l l s . Vander and Carlson (1969) explained that the e f f e c t of furosemide on renin release i s probably due to a d i r e c t action of the drug on the c e l l s of the macula densa; t h i s action being to decrease sodium transport by the macula densa. 92 4.1.3 Summary. In summary, there i s an area of s p e c i a l i z e d c e l l s i n the d i s t a l convol-uted tubule of the nephron of mammals which i s c a l l e d the macula densa. This area i s anatomically and f u n c t i o n a l l y r e l a t e d to the . JG. c e l l s and plays a r o l e i n renin release from the 'JG c e l l s . The nature of the s i g n a l to which the macula densa responds i s somewhat c o n t r o v e r s i a l . There are two main hypotheses, the Thurau hypothesis which states that an increased tubular sodium load at the macula densa causes an increase i n renin release or renin a c t i v a t i o n and the Vander hypothesis which claims that the macula densa stimulates renin release only when faced by a decrease i n tubular sodium concentration. At t h i s time i t i s not possible to r e c o n c i l e these two hypoth-eses as both are backed by good experimental evidence and each may explain away the e f f e c t s of the other. 4.1.4 Macula Densa i n Non-Mammalian Vertebrates. As was shown previously, the macula densa i s an important part of the juxtaglomerular apparatus i n mammals and t h i s leads to the question i s such a structure present i n non-mammalian vertebrates? Sokabe and Ogawa (1974) have reviewed the evolution of the juxtaglomerular apparatus i n vertebrates. It appears that a structure homologous or, analogous to the macula densa i s not found i n f i s h e s , amphibians or r e p t i l e s . However, there i s a structure i n the d i s t a l convoluted tubule of birds which resembles the macula densa of mammalian species. Examination by electron microscopy reveals that these c e l l s do not possess a l l of the c h a r a c t e r i s t i c s of the mammalian type and therefore are probably an intermediate of some sort as the b i r d kidney i s generally considered to be intermediate between that of the r e p t i l e s and of mammals. 93 Since a macula densa i s lacking i n the lower vertebrates, i s the renin response to changing sodium loads equally lacking? Sokabe ^ t _al. (1972) have examined PRA i n b u l l f r o g s which were i n various dehydrated states and found that dehydration decreased PRA w h i l e I n t r a v e n o u s i n f u s i o n of i s o t o n i c s a l i n e or 2% glucose resulted i n an increase i n PRA. This response then appears to be a response to changing plasma osmolarity rather than a sodium response. However, where marine te l e o s t s were adapted to a d i l u t e medium, PRA declined i n American eels but not i n toadfish (Nishimura et a l . , 1976). Sokabe et al.. (1973) found that PRA showed a transient increase when Japanese eels were transferred from freshwater to seawater. When T i l a p i a mossambica were adapted to seawater, i t was observed that the number of ,JG • c e l l s i n the kidney increased over those i n freshwater adapted fi s h e s and appeared to be larger i n s i z e (Krishnamurthy and Bern, 1973). On the other hand, Malvin and Vander (1967) did not observe any changes i n plasma renin i n two species of tuna and T i l a p i a mossambica when these f i s h were adapted to a d i l u t e medium despite changes i n plasma sodium. Therefore, the evidence for a renin response to changing sodium l e v e l s , of various f i s h species, i s at best c o n t r o v e r s i a l . 4.1.5 Sodium as a Humoral Agent. Sodium may act as a humoral agent i n various mammalian species. Hart-r o f t and Hart r o f t (1961) were the f i r s t to suggest that plasma sodium l e v e l s influence renin secretion from the .'J. G c e l l s . The most convincing evidence for sodium acting as a humoral agent comes from a seri e s of experiments which involved a l t e r i n g sodium concentrations i n the plasma perfusing the i s o l a t e d dog-kidney (Yamamoto et^ _al. , 1968). Another group found that i n t r a r e n a l infusions of hypertonic s a l i n e to dogs with a n o n - f i l t e r i n g kidney had no e f f e c t on renin secretion for the f i r s t 45 minutes of i n f u s i o n but then a 94 s i g n i f i c a n t decrease i n renin secretion occurred a f t e r 60 minutes of i n f u s i o n which most probably r e f l e c t s a secondary mechanism (Shade et a l . , 1972). Saline loading of dogs decreases renin a c t i v i t y ( C h u r c h i l l et_ al., 1974), while dehydrated sheep show an increase i n PRA about 2-3 times greater than c o n t r o l l e v e l s (Blair-West et_ al., 1977). In both cases the authors concluded that t h i s was due to a macula densa mechanism. Therefore the evidence for sodium acting as a humoral agent i s scanty and i t i s quite probable that such an e f f e c t would not function i n the-..normal physiology of mammals as the macula densa mechanism i s more than adequate. 95 Section IV Materials and Methods. 4.2 Sodium Perfusion of Kidney. 4.2.1 Non-Filtering Kidney. Adult trout were prepared for kidney perfusion as was previously describ-ed. The perfusion apparatus was modified by s e t t i n g up two perfusion b o t t l e s and r e s e r v o i r s connected by a 3-way polyethylene stopcock so that perfusion f l u i d s could be changed without a l t e r i n g perfusion pressure. The kidney was perfused with Cortland s a l i n e , sodium concentration 138 meq/1, for 90 minutes post-clearance time and samples c o l l e c t e d as described i n the preceding chapter. The stopcock was then switched so that the kidney was being perfused with a hypertonic sodium Cortland s a l i n e (198 meq Na +/1) for a further 90 minutes, samples being taken for every 30 minutes. These perfusate samples were then analyzed for renin a c t i v i t y by adding 0.5 ml sample to 0.5 ml homologous plasma, vortex mixing and then using the radioimmunoassay procedure. 4.2.2 F i l t e r i n g Kidney Preparation. A kidney perfusion preparation was modified i n two respects . to create a f i l t e r i n g kidney preparation. These were as-follows: f i r s t the caudal vein as w e l l as the caudal artery was cannulated with Intramedic PE 60 so as to perfuse a portion of the renal p o r t a l c i r c u l a t i o n of the kidney and second the ureters were not t i e d o f f but rather cannulated with a length of In t r a -medic PE 90 to allow urine c o l l e c t i o n . The preparation was then perfused for 90 minutes (post-clearance time) with Cortland s a l i n e and perfusate samples c o l l e c t e d . In addi t i o n urine samples were c o l l e c t e d i n a disposable micro-pipet for the f i r s t 30 minutes 96 of perfusion and the l a s t 30 minutes of perfusion. Hypertonic sodium Cort-land s a l i n e was then used to perfuse the kidney for a further 90 minutes, perfusate samples and urine samples were c o l l e c t e d for the f i r s t 30 minutes and the l a s t 30 minutes i n disposable micro-pipets. The pipets were then sealed with c l a y and stored frozen f o r l a t e r a n a l y s i s . Perfusate samples were then assayed for renin a c t i v i t y and urine flow rates calculated from the urine volumes c o l l e c t e d . In addition the kidney was examined to determine i f the perfusate was a c t u a l l y passing through the a r t e r i a l and. venous c i r c u l a t i o n . This was c a r r i -ed out by means of the Batson's corrosion casting technique as described by Murakami (1971) and Nowell et a l . . (19.72) and Gannon (1974) . Separate casts were done to determine the extent of the a r t e r i a l and venous c i r c u l a t i o n but i n each case the compound was i n j e c t e d through the respective cannula u n t i l i t was observed to appear i n the heart cannula. 4.2.3 Perfusion Solutions. Two types of perfusion solutions were u t i l i z e d during t h i s study. The f i r s t was Cortland s a l i n e with 2% PVP added and secondly Cortland s a l i n e with a d d i t i o n a l sodium i n the form.of sodium chloride and 2% PVP. This l a t t e r s o l u t i o n i s referred to as hypertonic sodium Cortland s a l i n e . In both cases chlorophenol red (B.D.M. Canada) was added to the perfusate i n the content r a t i o n of 250 mg/1. The presence of chlorophenol red i n the urine was taken as evidence that the tubular c e l l s of the kidney were v i a b l e and capable of a c t i v e transport as t h i s dye i s known to be a c t i v e l y transported by t e l e o s t nephrons (Hickman and Trump, 1969). 97 4.2.4 S t a t i s t i c a l Methods. The Student's t test was used throughout t h i s s e r i e s of experiments to test the s i g n i f i c a n c e of a differ e n c e between two means. A P value of less, than 0.05 was considered s i g n i f i c a n t and a P value of les s than 0.01 was considered highly s i g n i f i c a n t . 98 Section IV Results and Discussion. 4.3 Renin Release Following Sodium Perfusion i n Trout Kidneys. 4.3.1 Non-Filtering Kidney. Hypertonic s a l i n e perfusion of the i s o l a t e d n o n - f i l t e r i n g kidney has l i t t l e or no e f f e c t on renin release as may be seen i n Figure 4.2 and Table 4.1. The renin a c t i v i t y o s c i l l a t e s up and down over the e n t i r e perfusion period, both during i s o t o n i c and hypertonic s a l i n e perfusion. At the end of the t o t a l perfusion period renin a c t i v i t y has shown a s l i g h t increase above the beginning l e v e l . Table 4.1 shows that the average renin a c t i v i t y of 0.07 ng Al/ml/hr obtained during the i s o t o n i c s a l i n e perfusion has increased s l i g h t l y to 0.074 ng Al/ml/hr during the hypertonic s a l i n e perfusion. The purpose of these experiments was to determine i f plasma sodium exerted a d i r e c t humoral e f f e c t on renin release i n the trout. The data i n -dicate that there i s no such e f f e c t . s i n c e the renin-:activity obtained i n : these experiments did not show any s i g n i f i c a n t trend following hypertonic s a l i n e perfusion with constant perfusion pressure. But dehydration of f i s h causes a s i g n i f i c a n t increase i n plasma renin a c t i v i t y (Sokabe et a l . , 1966; Sokabe et_ a l . , 1968) and t h i s increase may be mediated by changes i n sodium, concentration. Although plasma sodium has no e f f e c t on renin secretion there s t i l l e x i s t s the p o s s i b i l i t y that tubular sodium loads may a f f e c t the rate of renin release from the J.G. c e l l s . 4.3.2 F i l t e r i n g Kidneys. In t h i s preparation the kidney i s i n t a c t and capable of both passive f i l t r a t i o n and ac t i v e transport as i s evidenced by the f a c t that chlorophenol red from the perfusate appears i n the urine. Figure 4.3 shows the pattern of 99 Figure 4.2. The e f f e c t of hypertonic s a l i n e perfusion on renin release from the i s o l a t e d perfused n o n - f i l t e r i n g trout kidney. The y-axis represents renin release as measured i n terms of perfusate renin a c t i v i t y , and i s i n ng Al/ml/hr. The x-axis i s time of perfusion and i s i n minutes. The f i r s t 90 minutes of perfusion represent the period of i s o t o n i c s a l i n e perfusion. The break i n the l i n e between 90 and 100 minutes represents the time at which the perfusion solutions were changed and the l a s t 90 minutes of perfusion, time 100 to time 190, represent the period of hypertonic s a l i n e perfusion. Each point i s the mean + S.E.M. of 10 determinations. 100 oi-0 3D 60 9b 100 l5o l o 1^ 0 TIME (min) TABLE 4.1 3. E f f e c t of Sodium Perfusion on Renin Release i n Non-Filtering and F i l t e r i n g Kidneys Perfusate Non-Filtering F i l t e r i n g Isotonic 0.07 ±0.02 1.4.± 0.113 Saline (10) (7) Hypertonic 0.074 ± 0.014 1.22 ± 0.1 Saline - (10) (7) a - A l l values are means ± S.E.M. (N) of renin a c t i v i t y i n ng Al/ml/hr 102 Figure 4.3. The e f f e c t of hypertonic s a l i n e perfusion on renin release i n i s o l a t e d perfused f i l t e r i n g trout kidney. The y-axis represents renin release as measured i n terms of perfusate renin a c t i v i t y i n ng Al/ml/hr. The x-axis i s time of perfusion and i s i n minutes. The f i r s t 90 minutes of perfusion represent the i s o t o n i c s a l i n e perfusion, and the break i n the l i n e between 90 and 100 minutes i s the time at which the perfusion solutions were changed. The remain-ing 90 minutes represent the period of hypertonic s a l i n e perfusion. Each point i s the mean + S.E.M. of 7 determin-ations. 103 104 renin secretion during i s o t o n i c s a l i n e perfusion and hypertonic s a l i n e per-fusion. Renin a c t i v i t y shows an approximately 10% decrease during the hypertonic s a l i n e perfusion at one point but following t h i s appears to be increasing back to the con t r o l l e v e l . This decrease i s not s i g n i f i c a n t . Table 4.1 shows the average e f f e c t of both perfusions on renin secretion and as may be seen renin a c t i v i t y shows a s l i g h t n o n-significant decrease from the con t r o l l e v e l . The macula densa i s the tubular receptor for sodium loads i n the mammal-ian kidney (Davis and Freeman, 1976) but t h i s structure may be peculiar to mammals as i t i s not found i n f i s h , amphibians, or r e p t i l e s but may e x i s t i n a rudimentary form i n birds (Sokabe and Ogawa, 1974). Therefore one may expect that sodium would not exert any e f f e c t on renin secretion i n non-homeothermic vertebrates and t h i s appears to be so i n fishes as there was no s i g n i f i c a n t change i n renin secretion following hypertonic s a l i n e perfusion of the f i l t e r i n g kidney preparation. The decline i n renin a c t i v i t y observed during the hypertonic s a l i n e perfusion appears to be a continuation of the decline observed during the i s o t o n i c s a l i n e perfusion period. Since s u r g i c a l trauma and/or stress stimulate renin release i n mammalian species (Reid e_t a l . , 1978) , i t i s possible that a s i m i l a r s i t u a t i o n e x i s t s i n f i s h and that the observed decline i s a r e s u l t of abnormally high trauma-induced renin release. While i t i s possible that such an e f f e c t , i f present, could mask a renin response to the high tubular sodium l e v e l s obtained during hypertonic s a l i n e perfusion (see Appendix 1), t h i s i s u n l i k e l y . Other authors have found that i n h i b i t i o n of renin release by one set of circumstances may be overridden by a stimulus which causes renin release ( C h u r c h i l l et a l . , 1974). The fac t that the observed decline i n renin secretion i s only 10%, that i t appears to be part 105 of an o v e r a l l trend and as w e l l renin a c t i v i t y seems to be on the r i s e at the end of the hypertonic sodium perfusion period argues against masking of a sodium e f f e c t . Therefore the conclusion which may be drawn from these experiments i s that sodium has no e f f e c t on renin release i n the trout. 4.3.3 Summary. In summary, sodium perfusion of the n o n - f i l t e r i n g trout kidney has no e f f e c t on renin secretion. Hypertonic s a l i n e perfusion of f i l t e r i n g trout kidneys i s equally without e f f e c t . Therefore the conclusion drawn i s that renin secretion i n the rainbow trout i s probably not d i r e c t l y affected b y either plasma or .tubular sodium l e v e l s . 4.4 Sodium Perfusion and Urine Flow Rates. One of the i n i t i a l responses to a t r a n s i t i o n from freshwater to seawater i n euryhaline species i s a decline i n the GFR and urine flow rates (Oide and Utida, 1967). Various groups have attempted to explain t h i s immediate re-sponse of the kidneys as being mediated by various humoral agents or by some form of neural c o n t r o l . Table 4.2 shows the e f f e c t on urine flow rates when perfusing the f i l t e r i n g kidney preparation. Urine flow undergoes a s i g n i f i -cant decrease, from 0.163 ml/kg/hr to 0.097 ml/kg/hr. During each perfusion the urine flow decreases slightly,:'.i.e. , during i s o t o n i c s a l i n e , 0.163 to 0.157 ml/kg/hr, and hypertonic s a l i n e , 0.097 to 0.07 ml/kg/hr, and t h i s i s not a s i g n i f i c a n t decrease. The o v e r a l l decrease i n flow i s s i g n i f i c a n t i n d i c a t i n g that the increased sodium or more probably osmotic pressure causes a decrease i n flow rates. Since the bladder has been removed from consider-ation, as the ureters are cannulated, the urine flow rate r e f l e c t s the glomerular f i l t r a t i o n rate. These data i n d i c a t e that an increased plasma sodium load w i l l cause a decrease i n the glomerular f i l t r a t i o n rate. How-TABLE 4.2 E f f e c t of Hypertonic Saline Perfusion on Urine Flow Rates i n the Isolated Perfused Kidney 3 Perfusate Begin Perfusion End Perfusion Isotonic 0.163 ± 0.01 b 0.157 ± 0.011 C Saline (7) (7) Hypertonic 0.097 ± 0.019b 0.07 ± 0.02 C Saline (7) (7) a - A l l values are means ± S.E.M. (N) and units are ml/kg/hr b - P<0.01 c - P<0.01 107 ever, further work i s required to determine i f t h i s i s a d i r e c t e f f e c t of the sodium ion or an e f f e c t due to increased osmotic pressure of the per-fusate which would lead to an increased f l u i d (water) reabsorption from the nephron. 108 Section V Introduction. 5.0 ANGIOTENSIN AND THE KIDNEY. 5.1 Mammals. The renal e f f e c t s of the renin-angiotensin system may be divided into d i r e c t e f f e c t s and i n d i r e c t e f f e c t s . That i s , the e f f e c t of angiotensin on the renal vasculature and tubule and e f f e c t s mediated by another hormone which i s l i b e r a t e d from another gland by the ac t i o n of angiotensin. 5.1.1 Direct E f f e c t s of Angiotensins. Angiotensin I (Al) has long been regarded as the i n a c t i v e precursor of angiotensin II ( A l l ) although i n i s o l a t e d myocardial preparations i t displayed approximately 50% of the a c t i v i t y of A l l . However the a c t i v i t y could be abolished by pretreatment of the preparation with converting enzyme i n h i b i t o r s obtained from Bothrops jararaca venom which indicates that i t s a c t i v i t y on myocardial preparations i s due to conversion to A l l (Peach, 1977). Infusions of A l into the perfused kidney i n the presence or absence of a converting enzyme i n h i b i t o r induce a s e l e c t i v e decrease of the inner c o r t i c a l and medullary blood flow without apparently a f f e c t i n g the outer c o r t i c a l blood flow. A l l c o n s i s t e n t l y decreased the outer c o r t i c a l blood flow but had v a r i a b l e e f f e c t s on the inner c o r t i c a l flow ( I t s k o v i t z and McGriff, 1974; Vane and McGriff, 1975). These authors have proposed that A l may be the major determinant of the p a r t i t i o n i n g of the i n t r a r e n a l blood flow. Osborn ejt a l . (1974) found that A l could a l t e r renal blood flow i n sheep but that A l l had a much greater e f f e c t . In t h i s study, i t was not determined i f A l had a d i r e c t e f f e c t or one mediated by conversion of A l to A l l . A l l on the other hand i s known to have both d i u r e t i c and a n t i d i u r e t i c 109 actions as well as n a t r i u r e t i c and a n t i n a t r i u r e t i c actions on the mammalian kidney. I t i s known that A l l i s a vasoconstrictor (Sokabe, 1974) and i t i s t h i s action which has been used to explain the often c o n f l i c t i n g e f f e c t s of A l l on the kidney. Small doses cause both d i u r e s i s and n a t r i u r e s i s i n r a t , ra b b i t , and dog (Healy at al. , 1965; Langford and Pickering, 1965; Cannon et a l . , 1966; Barraclough et a l . , 1967; Malvin and Vander, 1967), while Davis et a l . (1974) consider that A l l acts d i r e c t l y . o n the afferent a r t e r i o l e s to reduce renal blood flow to aid i n maintenance of systemic blood pressure. Since renin i s found i n the kidney and mammalian kidneys are known to autoregulate blood flow, i . e . , keep renal blood flow constant over a wide range of pressures, there i s a p o s s i b i l i t y that angiotensin may be involved i n t h i s autoregulatory process (Fojas and Schmid, 1970; Gagnon et a l . , 1970). More recent work has resulted i n a c e r t a i n amount of controversy. Potkay and Gilmore (1973) found that renal autoregulation was unaffected by renin depletion i n dogs. Also, renal autoregulation was maintained even when A l l was not present (Gagnon et: a l . , 1974) . Sokabe (1974) does not believe that the renin-angiotensin system i s involved with renal autoregulation as renin i s released i n s i t u a t i o n s which would c a l l for a v a s o d i l a t i o n i f autoregula-t i o n i s to be maintained. However, when dogs are maintained on desoxycortico-sterone (DOC) and a high sodium d i e t to deplete the kidneys of renin, renal autoregulation i s impaired across a large range of either high or low a r t e r i a l pressures (Kaloyanides eit a l . , 1974). This supports a s i m i l a r study by Brech et al. (1973) who found that renal autoregulation and d i s t r i b u -t i o n of renal blood flow were severely affected i n dogs which were" kept on a high sodium d i e t and DOC which causes renin depletion. Thus there are argu-ments both for and against a r o l e of the renin-angiotensin system i n renal autoregulation and further work i s required to s e t t l e t h i s controversy. 110 Recent work has shown that A l l may have a d i r e c t e f f e c t on the r e n a l tubules. Melton and F r a z i e r (1976) observed that angiotensin infused into the l e f t kidney of dogs produced consistent reductions in-the excretion of sodium, potassium and ch l o r i d e . These changes could not be a t t r i b u t e d to a l t e r a t i o n s i n glomerular f i l t r a t i o n rate or renal:plasma flow. E l e c t r o l y t e excretion by the r i g h t uninfused kidney was constant. These data are consis-tent with the hypothesis that A l l may function as an i n t r a r e n a l a n t i n a t r i u r -e t i c hormone. This hypothesis i s supported by the work of Johnson and Malvin (1977) who observed that A l l exerts a d i r e c t stimulatory e f f e c t on renal tubular sodium reabsorption independent of changes i n glomerular f i l t r a t i o n r a t e, renal plasma flow, f i l t r a t i o n f r a c t i o n , or i n t r a c o r t i c a l d i s t r i b u t i o n of blood flow. 5.1.2 Indirect E f f e c t s of Angiotensins. The i n d i r e c t e f f e c t of A l l on the kidney involves sodium retention and i s mediated by aldosterone. A l l stimulates the f i r s t step i n the biosynthesis of aldosterone, the conversion of c h o l e s t e r o l to pregnenolone (Aguilera and Marusic, 1971). I t would appear that i t has no e f f e c t on the conversion of corticosterone to aldosterone (Haning et; al., 1971) and the stimulatory e f f e c t of A l l on the zona glomerulosa of the adrenal cortex may be i n h i b i t e d by either high sodium or low potassium concentrations (Dufau et a l . , 1969; Boyd jet a l . , 1973). Further work has shown that the stimulus for aldosterone biosynthesis may not be A l l but rather a n a t u r a l l y occurring metabolite, des-aspartyl-AII, which has been t e n t a t i v e l y c a l l e d angiotensin III (Campbell jet a l . , 1974). Williams et a l . (1974) have observed that A l l i s le s s active than angiotensin I I I (AIII) i n the adrenal cortex. These authors have suggested that A l l binds to the zona glomerulosa receptor where i t i s convert-ed to the heptapeptide which then acts on the c e l l s to stimulate aldosterone I l l b iosynthesis. On the other hand, more recent work has tended to disagree with t h i s hypothesis and has found that i n the dog, A l l was not converted to AIII i n i s o l a t e d zona glomerulosa c e l l s and that aldosterone was s t i l l produced (Douglas e_t a l . , 1978) . A l l has been implicated i n the release of ADH i n rats (Claybaugh and Share, 1972). This work has also been duplicated by other groups ( K e i l jet a l . , 1975; Gregg and Malvin, 1978). 5.1.3 Summary. In summary, A l l has both d i r e c t and i n d i r e c t e f f e c t s on the kidney. The d i r e c t e f f e c t s include an a l t e r a t i o n in. renal blood flow, an a n t i d i u r e t i c e f f e c t and a d i u r e t i c e f f e c t as a r e s u l t of the vasopressor function. Other d i r e c t e f f e c t s are n a t r i u r e t i c or a n t i n a t r i u r e t i c functions which may be a r e s u l t of an e f f e c t on the renal tubules. In addition, A l l has i n d i r e c t e f f e c t s on the kidney which are mediated by some other hormone(s) released by the action of A l l at t h e i r s i t e s of o r i g i n . 112 Section V Materials and Methods. 5.2 Renal E f f e c t s of Angiotensins. 5.2.1 Perfusion Solutions. Perfusion solutions consisted of Cortland s a l i n e with 2% PVP. The s o l u -t i o n used to perfuse the venous c i r c u l a t i o n of the kidney also contained chlorophenol red (B.D.H. Canada Ltd.) at a concentration of 250 mg/1. A s e r i e s of angiotensin II (Hypertensin - CIBA) solutions were made, each con-t a i n i n g either 50, 100, 150, 200, or 250 ng angiotensin Il/ml and these were used for i n j e c t i o n purposes. A s i m i l a r series of angiotensin I (Calbiochem) solutions was used for i n j e c t i o n purposes; the concentrations were i d e n t i c a l with the angiotensin II solutions. The angiotensins, both I and I I , were dissolved i n Cortland s a l i n e to avoid any possible osmotic e f f e c t s . 5.2.2 Angiotensin Injections. The kidney of adult trout of e i t h e r sex was prepared f o r perfusion as previously described. This preparation was of the f i l t e r i n g kidney type thus allowing the e f f e c t s of e i t h e r angiotensin I or II on urine flow rate to be determined. To determine the e f f e c t s of angiotensin I on urine flow rates, two s e r i e s of experiments were c a r r i e d out. The protocol was as follows. Urine flow rate was measured for 30 minutes (post-clearance time) with constant perfusion pressure. A 1 ml dose of an angiotensin I s o l u t i o n was injected v i a the perfusion l i n e and urine flow rate measured for another 30 minutes. A second angiotensin I dose was i n j e c t e d , urine flow measured, and the pro-cedure repeated u n t i l one dose of each of the standards had been in j e c t e d . In the f i r s t s e r i e s of experiments the angiotensin was i n j e c t e d into the a r t e r i a l perfusion l i n e and i n the second ser i e s i t was i n j e c t e d into the 113 venous perfusion l i n e . The e f f e c t s of angiotensin II on urine flow rates were determined by using the same protocol as described above except that angiotensin II was injected. In a l l experiments urine was c o l l e c t e d i n glass disposable micro-pipets (Becton, Dickinson and Company), the ends sealed with p l a s t i c clay (Clay-Adams, Inc.) and stored frozen u n t i l l a t e r a n a l y sis. The urine was protected from contamination by the clay by allowing a small a i r bubble to remain at each end of the pipet between the surface of the f l u i d and the clay. The presence of chlorophenol red i n the urine indicated that the kidney prepara-t i o n was v i a b l e . If chlorophenol red did not appear the preparation was discarded. 5.2.3 Ion Analysis. The frozen urine samples were thawed, the ends of the micro-pipets cut o f f and the sample blown gently onto a piece of Parafilm (American Can Co.) where the sample formed a small bubble. Aliquots of each sample were quickly taken and d i l u t e d 1:1,000 with d i s t i l l e d water and analyzed for sodium concen-t r a t i o n by flame emission spectrophotometry on a flame, photometer. For urinary potassium a n a l y s i s , aliquots of the urine were d i l u t e d 1:1,000 by a 500 meq/1 Na + s o l u t i o n . This "sodium-swamping" technique allows any interference by sodium to be cancelled out when c a l i b r a t i n g the machine. Urinary potassium l e v e l s were then measured by flame emissionrspectrophotomet-ry using a flame photometer. 5.2.4 S t a t i s t i c a l Methods. Linear regression analysis was used to determine the c o r r e l a t i o n coef-,". f i c i e n t (r) while the Student's t test was used to compare means. In each 114 case a P value of le s s than 0.05 was considered s i g n i f i c a n t and a P value of l e s s than 0.01 was considered highly s i g n i f i c a n t . 115 Section V Results and Discussion. 5.3 Renal E f f e c t s of Angiotensins. 5.3.1 Urine Flow Rates. Figure 5.1 shows the e f f e c t of perfusion pressure on urine:flow rate i n the i s o l a t e d perfused kidney. As may be seen, urine flow rate varies d i r e c t l y with perfusion pressure. In the e e l , A n g u i l l a r o s t r a t a , there i s a l i n e a r c o r r e l a t i o n between systemic blood pressure, glomerular f i l t r a t i o n rate and urine flow rate (Rankin ejt a l . , 1967; Butler, 1969). These data simply show that the same phenomenon e x i s t s i n the trout kidney as there i s a l i n e a r c o r r e l a t i o n between renal perfusion pressure and urine flow rates. Further-more an a l t e r a t i o n i n perfusion pressure w i l l be r e f l e c t e d i n the urine flow rate. When angiotensin I (Al) i s injec t e d into the a r t e r i a l side of the kidney vascular tree, urine flow rate shows a s l i g h t increase over the dose range as may be seen i n Figure 5.2. These data imply that A l has some a c t i v i t y i n the trout kidney and indeed A l appears to have an e f f e c t on renal blood flow i n mammals (Peach, 1977). However, since t h i s increase i s not s i g n i f i c a n t t h i s implies that i f A l does have some a c t i v i t y i n the trout kidney i t i s only weakly a c t i v e . Further evidence for A l having some a c t i v i t y i n trout kidney may be seen i n Figure 5.3. This f i g u r e shows the e f f e c t of i n j e c t i n g A l into the venous side of the kidney vascular tree and as i n the previous case, the urine flow rate shows a s l i g h t n o n - s i g n i f i c a n t increase. The lack of a s i g n i f i c a n t response may be due either to a lack of a c t i v -i t y of A l on the kidney or that A l i s only weakly a c t i v e i n the kidney. The l a t t e r hypothesis appears more l i k e l y as i t i s known that A l w i l l compete, to a c e r t a i n extent, with angiotensin II ( A l l ) f o r the l a t t e r compound's recep-116 Figure 5.1. Urine flow rates and perfusion pressure i n the i s o l a t e d perfused trout kidney. The y-axis represents the urine flow rates i n ml urine formed per kg body weight ,per hour. The x-axis represents perfusion pressure and i s measured i n cm H^O. :: For the:.line equation y = O.Olx + (-0.02) the c o r r e l a t i o n c o e f f i c i e n t r = 0.969 i s s i g n i f i c a n t at the 1% l e v e l . (N = 10.) P E R F U S I O N P R E S S U R E ( c m H,0) 118 Figure 5.2. The e f f e c t of i n j e c t i o n of angiotensin I into the a r t e r i a l side of the renal c i r c u l a t i o n , o n urine flows. The y-axis i s urine flow rates i n ml urine formed per kg body weight per hour and the x-axis i s does of angiotensin I adminis-tered i n ng. Each point on the graph represents the mean ± S.E.M. of 6 determinations. I j — I y — j r — U 50 100 lio 200 250 DOSE (ng A-l ) 120 Figure 5.3. Urine flow rates following angiotensin I i n j e c t i o n into the venous side of the renal c i r c u l a t i o n . The y-axis repre-sents the urine flow rates i n ml urine formed per kg body weight per hour and the x-axis represents the dosage of angiotensin I injected i n ng. Each point on the l i n e rep-resents the mean ± S.E.M. of 6 determinations. oH 121 L 50 100 150 250 250 DOSE (ng A - I ) 0 2 ~& T. 1 122 tors (Needleman ert al., 1972) and thus have a weak vas o c o n s t r i c t i v e e f f e c t . In addition, A l may be converted to A l l i n the kidney as Haber et a l . (1972) have found that there i s a net conversion of A l to A l l i n the ;kidney a l -though the majority of the A l appears to be degraded to smaller i n a c t i v e peptides. These authors also found that the rate of conversion was too rapid to be accounted f o r by plasma enzymes alone which implies that the renal vasculature possesses the converting enzyme. There are two possible explanations for the increase i n urine flow rates, one being a va s o c o n s t r i c t i v e e f f e c t and the other being a d i r e c t tubular e f f e c t . In the ra b b i t , A l injected into the i s o l a t e d perfused kidney causes an increase i n perfusion pressure, glomerular f i l t r a t i o n rate and urine volume (Regoli, 1972). This was taken as evidence that A l had a vasoconstric-t i v e e f f e c t on the efferent glomerular a r t e r i o l e but not on the afferent v e s s e l . This s i t u a t i o n may e x i s t i n the f i s h kidney so that A l w i l l cause efferent a r t e r i o l a r c o n s t r i c t i o n thus increasing the renal perfusion pressure and thus eventually urine flow rate. Since A l i s only weakly active the change i s not s i g n i f i c a n t . In addition to smooth muscle, f i s h renal veins also contain groups of chromaffin c e l l s (Gannon, 1972). It i s known that either A l or A l l can cause release of either epinephrine or norepinephrine from chromaffin t i s s u e (Opdyke and Holcome, 1978). Therefore i n j e c t i o n of A l into the venous c i r c u l a t i o n would r e s u l t i n release of epinephrine from the chromaffin t i s s u e which i n turn could r e s u l t i n some degree of vasoconstric-t i o n (probably very s l i g h t ) leading to an increased glomerular perfusion pressure. This increased perfusion pressure would be a r e s u l t of increased downstream resistance and would r e s u l t i n an increase i n urine flow rates. Since veins are thin-walled vessels t h i s would explain why there was no s i g n i f i c a n t e f f e c t from A l . I t i s possible that the A l could have a d i r e c t 123 e f f e c t on the tubule such that water loss from the peritubular vessels i n -creased due either to increased tubular, secretion of water or increased secretion of an ion, such as sodium, and water follows passively. This p o s s i b i l i t y w i l l be examined more c l o s e l y i n Section 5.3.2. Injec t i o n of A l l into the a r t e r i a l system r e s u l t s i n a massive decrease i n urine flow rates which i s dose-dependent (Figure 5.4). The most probable reason for t h i s decrease i s that the A l l i s causing severe vasoconstriction of the afferent glomerular a r t e r i o l e s which would r e s u l t i n a decreased perfusion pressure and a decreased urine flow rate. On the other hand, Nishimura and Sawyer (1976) found that i n f u s i o n of A l l into freshwater adapted and s a l i n e loaded eels (Anguilla.rostrata) produced a d i u r e t i c e f f e c t which these authors concluded was the r e s u l t of an increase i n systemic blood, pressure. Regoli (1972) concluded that A l l has as i t s primary target the efferent a r t e r i o l e to increase perfusion pressure and thus, urine volume. Brown et a l . (1972) concluded that studies with exogenous A l l were pharma-c o l o g i c a l rather than p h y s i o l o g i c a l i n nature as i t i s the l o c a t i o n of the converting enzyme a c t i v i t y which determines the target for A l l . A s i m i l a r s i t u a t i o n may e x i s t i n f i s h as i n j e c t i o n of A l l into the renal venous c i r c u l a -t i o n r e s u l t s i n an increased urine flow rate (see Figure 5.5) which i s apparently dose-dependent. This action may be explained as either the r e s u l t of a d i r e c t v a s o c o n s t r i c t i v e e f f e c t of A l l on the venous smooth muscle or by an i n d i r e c t e f f e c t mediated by epinephrine or norepinephrine released from venous chromaffin c e l l s or possibly a combination of the two f a c t o r s . This would explain the r e l a t i v e l y greater e f f e c t of A l l as compared to A l . In addition, the A l l may have a d i r e c t e f f e c t on the renal tubule such that ion transport i s affected which i n turn could cause an increase i n urine flow. Another possible s i t e of action of A l l could be on the neck and intermediate 124 Figure 5.4. Urine flow rates following angiotensin II i n j e c t i o n into the a r t e r i a l side of the renal c i r c u l a t i o n . The y-axis represents the urine flow rates i n ml urine formed per kg body weight per hour and the x-axis represents the dosage of angiotensin II administered i n ng. Each point on the l i n e represents the mean ± S.E.M. of 6 determinations. 125 126 Figure 5.5. The e f f e c t of i n j e c t i o n of angiotensin II into the venous c i r c u l a t i o n of the kidney on urine flow rates. The y-axis represents the urine flow rate i n ml urine formed per kg body weight per hour and the x-axis represents the dosage of angiotensin II administered i n ng. Each point on the l i n e represents the mean ± S.E.M. of 6 determinations. 128 segments of the nephron which are c i l i a t e d and may function as a means of pro p e l l i n g the f i l t r a t e down the tubule (Hickman and Trump, 1969). A l l may cause an increase i n a c t i v i t y i n these regions thus increasing the rate at which the f i l t r a t e i s propelled down the tubule and therefore the urine flow rate. However, th i s i s speculative at t h i s time as there i s some controversy over the function of these segments. 5.3.2 Urinary Sodium Levels. When A l i s injec t e d into the a r t e r i a l and venous sides of the renal vascular tree, there i s an e f f e c t on urinary sodium l e v e l s as i l l u s t r a t e d i n Figures 5.6 and 5.7. It may be seen that when A l i s injec t e d into the a r t e r -i a l side there i s a decrease i n urinary sodium l e v e l s (Figure 5.6) but t h i s decrease i s not s i g n i f i c a n t at any dose l e v e l . When injected into the venous side, however, there i s a s i g n i f i c a n t dose-dependent decline (Figure 5.7). These data would indi c a t e that A l has a d i r e c t e f f e c t on the nephron to increase sodium reabsorption. This e f f e c t appears to be independent of the e f f e c t on urine flow rate as A l causes an increase i n t h i s parameter (see Section 5.3.1), and i n addition there does not appear to be a s i g n i f i c a n t c o r r e l a t i o n between urine flow rate and urinary sodium l e v e l s . Therefore an increased urine flow rate i s not due to eit h e r i n h i b i t i o n of sodium reabsorp-t i o n or an increase i n sodium secretion with water following passively. There i s l i t t l e published information, on the e f f e c t of A l on the r e n a l t u b u l e s and a l l i s concerned with mammalian kidneys. Peach (1977) has suggested that t h i s decapeptide may be considered the f i r s t p o t e n t i a l and most p r i m i t i v e message of the renin-angiotensin system as i t i s the i n i t i a l peptide formed v i a the p r o t e o l y t i c action of renin and the data obtained i n th i s study seem to support t h i s hypothesis. However, since there 129 Figure 5.6. Urinary sodium l e v e l s following i n j e c t i o n of angiotensin I on the a r t e r i a l side of the renal c i r c u l a t i o n . The y-axis represents urinary sodium l e v e l s and i s i n meq/1 Na + and the x-axis i s dosage of angiotensin I administered i n ng. Each point on the l i n e represents the mean ± S.E.M. of 6 determinations. 131 Figure 5.7. Urinary sodium l e v e l s following angiotensin I i n j e c t i o n into the venous c i r c u l a t i o n of the kidney. The y-axis represents urinary sodium l e v e l s i n meq/1 and the x-axis represents the dosage of angiotensin I administered i n ng. Each point on the l i n e represents the mean ± S.E.M. of 6 determinations. 132 133 was no . i n h i b i t i o n of converting enzyme a c t i v i t y i t i s possible that the A l i s being converted to A l l which i s then exerting the observed e f f e c t . Figure 5.8 shows the e f f e c t of A l l on urinary sodium l e v e l s when.the octapeptide i s injected into the a r t e r i a l side. There i s a s i g n i f i c a n t dose-re l a t e d decrease i n urinary sodium l e v e l s . Again t h i s decrease i s not r e l a t e d to the urine flow rate since there i s no s i g n i f i c a n t c o r r e l a t i o n between-the two parameters. These data are apparently i n d i r e c t opposition to the findings of Nishimura and Sawyer (1976) who observed a n a t r i u r e t i c response correlated with the d i u r e t i c response i n A l l infused eels. This d i f f e r e n c e could be a r e s u l t of A l l not reaching the nephron receptor i n the infused e e l and there-fore sodium retention i s not affected. These data would indi c a t e that A l l has a d i r e c t e f f e c t on the nephron to cause an increase i n sodium reabsorption from the tubular f l u i d . This hypothesis i s supported by the data obtained when A l l i s injected into the venous side of the renal c i r c u l a t i o n as i s shown i n Figure 5.9. Again there i s a s i g n i f i c a n t dose-related decrease i n urinary sodium l e v e l s following i n j e c t i o n of A l l . This e f f e c t has also been observed i n mammalian species where the i n j e c t i o n of A l l caused consistent reductions i n the excretion of sodium and chloride which could not be a t t r i b -uted to decreases i n glomerular f i l t r a t i o n rate or renal plasma flow (Melton and Fraz i e r , 1967; Johnson and Malvin, 1977). One other p o s s i b i l i t y , i s that the injected angiotensins are causing a release of C o r t i s o l or some other c o r t i c o s t e r o i d from the i n t e r r e n a l t i s s u e i n the trout kidney. C o r t i s o l i s the major adrenocorticosteroid i n trout i n t e r r e n a l t i s s u e , while aldosterone i s not present i n detectable quantities (Columbo at a l . , 1971). The renin-angiotensin system may have l i t t l e e f f e c t . on C o r t i s o l release as Nishimura et a l . (1976) were unable to f i n d any c o r r e l a -134 o Figure 5.8. Urinary sodium l e v e l s following i n j e c t i o n of angiotensin II into the renal a r t e r i a l c i r c u l a t i o n . The y-axis represents the urinary sodium l e v e l s i n meq/1 and the x-axis represents the dosage of angiotensin II administered i n ng. Each point on the l i n e represents the mean ± S.E.M. of 6 determinations U R I N A R Y S O D I U M L E V E L ( m e q / l ) 136 Figure 5.9. Urinary sodium l e v e l s following i n j e c t i o n of angiotensin II into the renal venous c i r c u l a t i o n . The y-axis repre-sents the urinary sodium l e v e l s i n meq/1 while the x-axis represents the dosage of angiotensin II adminis-tered i n ng. Each point on the l i n e represents the mean ± S.E.M. of s i x determinations. 138 t i o n between plasma renin a c t i v i t y and C o r t i s o l l e v e l s i n either the e e l or the toadfish (Opsanus tau) upon adaptation to d i l u t e media. In addition, i t i s known that the r o l e of A l l i n aldosterone release i n mammals i s to increase the i n i t i a l step i n the biosynthesis of aldosterone, i . e . , the con-version of c h o l e s t e r o l to pregnenolone (Aguilera and Marasic, 1971), but the major biosynthetic pathway from c h o l e s t e r o l to C o r t i s o l proceeds v i a 17a-hydroxypregnenolone i n f i s h (Sandor et a l . , 1966). Therefore the A l l c a t a l y z -ed step i s apparently lacking i n t e l e o s t s . In addition the s i m i l a r i t y between the magnitude of the response pro-duced by A l and A l l (Figures 5.7 and 5.9) on the venous side of the c i r c u l a -t i o n suggest that either there i s no differ e n c e i n a c t i v i t y of the two compounds or that a l l of the A l i s being converted to A l l . The l a t t e r sugges-t i o n appears u n l i k e l y based on the evidence provided by the e f f e c t of A l and A l l on the urine flow rate (Figures 5.3 and 5.5) as the response produced by the A l l i s approximately double that produced by the A l . This suggests that A l and A l l have equal a c t i v i t y when concerned with sodium transport and indeed i n some ti s s u e s , adrenal medulla and medullary blood vessels of mammals, the a f f i n i t y of A l and A l l i s about equal (Peach, 1977). The p h y s i o l o g i c a l r o l e of t h i s equivalent a f f i n i t y i s at present obscure. 5.3.3 Urinary Potassium Levels. Urinary potassium l e v e l s are also affected by either A l or A l l but i n th i s case the e f f e c t s are much less c l e a r - c u t . Figure 5.10 shows the e f f e c t of i n j e c t i o n of A l i n the a r t e r i a l side on potassium l e v e l s and as may be seen there i s a s l i g h t decrease but t h i s i s not s i g n i f i c a n t and does not appear to be dose-related. However, i n j e c t i o n of A l on the venous side produces a s i g n i f i c a n t dose-dependent decline i n urinary potassium (Figure 5.11). These data ind i c a t e that the A l has a d i r e c t e f f e c t on. the nephron 139 Figure 5.10. Urinary potassium l e v e l s following i n j e c t i o n of angioten-s i n I into the renal a r t e r i a l c i r c u l a t i o n . The y-axis represents the urinary potassium concentrations i n meq/1 while the x-axis represents the dosage of angiotensin I administered i n ng. Each point on the l i n e represents the mean ± S.E.M. of s i x determinations. URINARY POTASSIUM LEVEL tfneq/l) 141 Figure 5.11. Urinary potassium l e v e l s following angiotensin I i n j e c -t i o n into the renal venous c i r c u l a t i o n . The y-axis represents the urinary potassium concentrations i n meq/1 while the x-axis represents the dosage of angio-tensin I administered i n ng. Each point on the l i n e represents the mean ± S.E.M. of s i x determinations, with the exception of the i n i t i a l point where the standard error was smaller than the s i z e of the symbol. DOSE (ng A - 0 143 to increase the reabsorption of monovalent cations as i t does r e s u l t i n an increase i n sodium reabsorption as was indicated i n the previous section. This would explain the apparent lack of e f f e c t of the A l on the a r t e r i a l side as i n t h i s case i t i s possible that the hormone i s simply not reaching the s i t e of action. A l l apparently does not have an e f f e c t on urinary potassium l e v e l s for while i n j e c t i o n of A l l on the a r t e r i a l side r e s u l t s i n a decrease i n urinary potassium (Figure 5.12) which i s not s i g n i f i c a n t , i n j e c t i o n of A l l on the venous side r e s u l t s i n a s l i g h t r i s e in.urinary potassium (Figure 5.13). I t i s possible that the angiotensin may act on the tubule to cause potassium reabsorption as i t i s known to do i n mammals (Melton and F r a z i e r , 1976). This does not appear l i k e l y as there i s l i t t l e e f f e c t on urinary potassium l e v e l s following A l l administration. However, these data do show that sodium retention r e s u l t i n g from either A l or A l l administration i s not a r e s u l t of sodium-potassium exchange as urinary potassium l e v e l s do not increase s i g -n i f i c a n t l y . The lack of e f f e c t of A l l on the urinary potassium l e v e l s , however, i s not s u r p r i s i n g . Potassium i s abundant i n both plant and animal c e l l s and thus r e l a t i v e l y large quantities of the ion are obtained i n the d i e t . In addition, there i s very l i t t l e potassium, when compared to sodium, found i n the e x t r a c e l l u l a r f l u i d and therefore f i l t e r e d potassium l e v e l s are low. The end r e s u l t i s that potassium loss to the environment does not pose a major problem for the organism. What i s s u r p r i s i n g i s that A l should cause potassium retention at the tubular l e v e l . This may be due to a c t i v a t i o n of a non-specific transport protein i n the c e l l s of the nephron by A l which w i l l pick up either sodium or potassium. I t i s also possible that t h i s e f f e c t i s 144 Figure 5.12. Urinary potassium l e v e l s following i n j e c t i o n of angioten-s i n II into the renal a r t e r i a l c i r c u l a t i o n . The y-axis represents the urinary potassium concentrations i n meq/1 while the x-axis represents the dosage of angiotensin II administered i n ng. Each point on the l i n e represents the mean ± S.E.M. of s i x determinations. URINARY POTASSIUM LEVEL flneq/l) 146 Figure 5.13. Urinary potassium l e v e l s following i n j e c t i o n of angio-tensin II into the renal venous c i r c u l a t i o n . The y-axis represents the urinary potassium concentrations i n meq/1 while the x-axis represents the dosage of angiotensin II administered i n ng. Each point on the l i n e represents the mean ± S.E.M. of s i x determinations U R I N A R Y P O T A S S I U M L E V E L ( m e q / O 148 merely an a r t e f a c t of the flame photometry although t h i s seems u n l i k e l y when viewing the other data. The potassium data a l s o i n d i c a t e that the d i u r e t i c e f f e c t of A l l ( S e c t i o n 5.3.1) i s a r e s u l t of the v a s o c o n s t r i c t o r a c t i o n . Potassium l e v e l s do not change s i g n i f i c a n t l y d e s p i t e the increased u r i n e flow r a t e s and t h i s shows that the flow r a t e increase i s not a r e s u l t of e i t h e r decreased water reab s o r p t i o n or increased water s e c r e t i o n . Rather i t i s due to an increase i n G.F.R. which i s most l i k e l y brought about by an increased f i l t r a t i o n pressure due to post-glomerular v a s o c o n s t r i c t i o n . 5.3.4 Summary. Therefore, i n summary, both A l and A l l have a d i u r e t i c e f f e c t although the d i u r e t i c e f f e c t of A l l depends on which s i d e of the v a s c u l a r t r e e A l l i s a c t i n g and the d i u r e t i c e f f e c t of A l i s i n s i g n i f i c a n t . Both hormones cause sodium r e t e n t i o n and t h i s appears to be a d i r e c t e f f e c t on the nephron. This probably occurs e i t h e r i n the d i s t a l segment or the c o l l e c t i n g tubule both.of which are s i t e s of sodium rea b s o r p t i o n (Hickman and Trump, 1969). These data a l s o i n d i c a t e that i n f i s h A l and A l l are e q u a l l y e f f e c t i v e as an a n t i n a t r i -u r e t i c hormone. These data, as w e l l , i n d i c a t e that A l has an e f f e c t on potassium e x c r e t i o n but that A l l does not. In c o n c l u s i o n , the hormones, e i t h e r A l or A l l , are i n t i m a t e l y i n v o l v e d i n water and i o n r e g u l a t i o n i n f i s h . The data which support t h i s hypothesis are the a n t i n a t r i u r e t i c e f f e c t of both A l and A l l and the d i u r e t i c and a n t i -d i u r e t i c e f f e c t of A l l . A lso e i t h e r A l or A l l may act as l o c a l hormones, i . e . , without a systemic a c t i o n , as there appears to be converting enzyme a c t i v i t y present i n the kidney. This a c t i v i t y may not be necessary to b r i n g about sodium r e t e n t i o n as A l and A l l appear to have equivalent e f f e c t s on the 149 tubule where sodium excretion is concerned. Another conclusion is that the Al is the primitive messenger of the renin-angiotensin system. This is based on the facts that Al is formed as a direct action of renin and is necessary for the formation of A l l . Also the equivalent effects of Al and A l l on natriuresis are additional support for this hypothesis. 150 Section VI General Discussion. 6.0 RENIN IN FISHES. • 6.1 Renin i n Freshwater Fishes. The p h y s i o l o g i c a l r o l e of the renin-angiotensin system i n fishes i s obscure at the present time and unfortunately the c l a s s i c a l endocrinological approach of removing the source of the hormone and observing the resultant e f f e c t s i s in a p p l i c a b l e due to the fac t that the hormone i s produced i n the kidney. The system plays a r o l e i n the renal handling of ions and water, as i t i s known to do i n mammalian species (Peach, 1977). The data discussed i n the previous section (Section 5) i n d i c a t e that angiotensin I i s an a n t i n a t r i -u r e t i c hormone while angiotensin II i s both a d i u r e t i c and a n t i d i u r e t i c as we l l as an a n t i n a t r i u r e t i c hormone. (See Figure 6.1 for a summary of the co n t r o l and actions of renin.) Accordingly, i t i s possible that i n freshwater fi s h e s the renin-angiotensin system plays a r o l e i n osmoregulation both through d i r e c t e f f e c t s on the renal tubules and i n d i r e c t e f f e c t s on the vascular supply to the kidney. The primary problem faced by freshwater bony fi s h e s may be found i n the fact that these animals are hyperosmotic to the medium i n which they l i v e and thus are faced with both water i n f l u x and ion l o s s . The function of the kidney i n these animals i s therefore that of conserving ions and excreting water, rather than nitrogenous waste excretion as i s found i n mammals. Therefore, a hormone which acts to cause conservation of ions and to increase water excretion would be of great value to these animals. It i s possible that the renin-angiotensin system i s acting i n the follow-ing fashion i n osmoregulation. A stimulus i s received by the juxtaglomerular 151 Figure 6.1. Diagrammatic summary of renin release and the actions of the renin-angiotensin system i n the trout. 152 Decreased Renal Perfusion Pressure. Plasma Sodiuni Levels No Ef f e c t ^ Feedback . i n h i b i t i o n -stimulates* Alpha and Beta Adrenergic Receptor Blockers have no E f f e c t on t h i s Response. ->-RENIN -release Increased Sodi Retention by Tubular E f f e c t . Al. Formation : converting enzyme AIT Formation -Vasoconstriction Afferent A r t e r i o l e s A n t i d i u r e t i c Action Efferent A r t e r i o l e s D i u r e t i c Action 153 (JG) c e l l s causing release of renin. The stimulus may be a decline i n plasma sodium or a decrease i n the osmolarity of the plasma and may be either a d i r e c t e f f e c t on the JG c e l l s or transmitted v i a the nervous system from a receptor located elsewhere i n the animal . The renin then acts to form angiotensin I (Al) i n the plasma. There are two alternate pathways at t h i s point. E i t h e r the A l d i r e c t l y a f f e c t s the tubule to increase sodium reabsorp-t i o n or the A l i s converted to angiotensin II ( A l l ) which w i l l a f f e c t both the vascular system and the tubule i n such a fashion as to cause an increase i n the urine flow rate and an increased sodium retention. The net e f f e c t therefore i s to r i d the animal of excess water while conserving ions. This mechanism i s based on the decreased urinary sodium l e v e l s obtained by i n j e c t i n g A l or A l l into the i s o l a t e d perfused kidney (Section 5.3.2). In addition, A l l has been found to increase urine flow rate when injec t e d into the venous side of the renal vascular tree (Section 5.3.1). Unfortunately the nature of the s i g n a l perceived by the JG c e l l s i s obscure. Published data i n d i c a t e that a hypoosmotic plasma has no e f f e c t on renin release as seawater adapted T i l a p i a mossambica showed no increase or decrease i n plasma renin a c t i v i t y on e i t h e r acute or chronic adaptation to freshwater despite marked changes i n plasma sodiumMevels (Malvin and Vander, 1967). In addition, perfusing i s o l a t e d kidneys with hypertonic sodium perfusate appears to have no e f f e c t on renin release e i t h e r stimulatory or i n h i b i t o r y (Section 4.3). The renin-angiotensin system may be more important i n the freshwater f i s h e s , since Mizogami et a l . (1968) have shown that marine teleosts possess less renin per unit weight of kidney ti s s u e than freshwater t e l e o s t s . The p o s s i b i l i t y e x i s t s t h a t m a r i n e t e l e o s t r e n i n i s more a c t i v e "..than- f r e s h w a t e r t e l e o s t r e n i n and t h e r e f o r e l e s s r e n i n per u n i t weight, kidney mass i s required by marine t e l e o s t s . Chromatography studies however show that teleost angiotensins appear to have the same structure and that there i s l i t t l e d i f f e r e n c e between those produced i n marine teleosts and those from freshwater teleosts (Nakajima et a l . , 1971). Renin may be continuously released i n low l e v e l s from the JG c e l l s and a c e r t a i n l e v e l of c i r c u l a t i n g hormone i s required for kidney function i n freshwater t e l e o s t s . The h a l f l i f e of c i r c u l a t i n g renin i n humans i s i n the order of 120 minutes (Hannon j2t a l . , 1969) and i t i s e n t i r e l y possible that an equivalent h a l f l i f e may be found i n f i s h e s . This would i n turn r e s u l t i n a c e r t a i n l e v e l of c i r c u l a t i n g A l and A l l and these hormones may be the actual substances required for the maintenance of kidney function. Also, Park jet _al. (1978) have proposed that two pools of renin e x i s t i n the kidney of dogs, one pool releases renin at a constant low rate and the other responds to s t i m u l i which are associated with renin release. I t i s e n t i r e l y possible that a s i m i l a r s i t u a t i o n e x i s t s i n f i s h e s , that i s one pool for constant low l e v e l release and the other f o r response to extraphysiological conditions. One argument against t h i s hypothesis i s that renin release i s i n h i b i t e d by A l l i n a negative feedback loop i n trout (Section 3.5.4) as we l l as i n mammals (Michelakis, 1971). Thus continuous production of A l l should i n h i b i t further renin release from the kidney. On the other hand, Nakajima et a l . (1971) have found that t e l e o s t angiotensins are very susceptible to proteases. Thus, the formed A l l may be degraded before i t can reach the JG c e l l s to i n h i b i t renin release. The feedback system may only be operative when r e l a t i v e l y large quantities of renin are released into the c i r c u l a t i o n and A l l i s formed faster than i t can be broken down so that some may reach the JG c e l l s to i n h i b i t further renin release. Unfortunately, i t 155 i s extremely d i f f i c u l t to determine i f there i s a c i r c u l a t i n g plasma l e v e l of renin as removal of blood to measure l e v e l s of the hormone causes renin release as was demonstrated i n Section 3.5.1. The renin-angiotensin system may also.play a r o l e i n response to haemorrhage i n t e l e o s t s . A f a l l i n blood volume i s usually accompanied by a decline i n systemic blood pressure which i n turn r e s u l t s i n a decline i n renal perfusion pressure. Renin i s then released i n response to the f a l l i n r enal perfusion pressure (Section 3.5) and the end r e s u l t i s the formation of A l l . There are two possible actions of t h i s hormone, one being an a n t i -d i u r e t i c e f f e c t and the other an a n t i n a t r i u r e t i c e f f e c t . The former action would be a r e s u l t of afferent a r t e r i o l a r c o n s t r i c t i o n and would r e s u l t i n water conservation to ultimately aid i n r e s t o r i n g plasma volume. The latter, a c tion i s the r e s u l t of a d i r e c t tubular e f f e c t and would be necessary for the following reason. In the trout one response to blood loss i s an increase i n v e n t i l a t i o n volume (Smith, pers. comm.) which could r e s u l t i n an increased water uptake at the g i l l s . While t h i s increase aids i n r e s t o r i n g plasma volume, the plasma i s rendered hypoosmotic. Thus sodium conservation at the kidney l e v e l would aid i n r e s t o r i n g the osmolarity of the plasma. During periods of prolonged exercise i n trout, c e r t a i n p h y s i o l o g i c a l changes occur and these changes may, i n part, be mediated by the renin-angiotensin system. Exercise i s accompanied by an increase i n v e n t i l a t i o n which i n turn r e s u l t s i n an increase i n both water uptake and sodium loss at the g i l l s (Wood and Randall, 1973a). In addition, dorsal a o r t i c blood pressure increases but t h i s increase appears t r a n s i t o r y . A further e f f e c t i s a prolonged d i u r e s i s which begins sh o r t l y a f t e r the onset of exercise and t h i s increase i n renal water e f f l u x i s observed over the e n t i r e period of 156 exercise (Wood and Randall, 1973c). The decline i n dorsal a o r t i c blood pressure could r e s u l t i n a decline i n renal perfusion pressure and thus release of renin. An increase i n plasma renin l e v e l s could thus explain the prolonged d i u r e s i s as A l l i s known to have a d i u r e t i c e f f e c t i n whole animals as a r e s u l t of increased systemic blood pressure (Nishimura and Sawyer, 1976). In addition, A l l has a d i u r e t i c e f f e c t on the.kidney i t s e l f when present i n the renal venous c i r c u l a t i o n (Section 5.3). Also, Wood and Randall (1973b) have found that over prolonged exercise sodium e f f l u x i s i n i t i a l l y greater than sodium i n f l u x but a f t e r 2-3 hours t h i s s i t u a t i o n i s reversed and disturbances i n renal function tend to disturb the observed pattern. Since both A l and A l l have an a n t i n a t r i u r e t i c e f f e c t on the kidney (Section 5.3), i t i s possible that angiotensin-mediated sodium conservation at the kidney i s aiding i n the maintenance of t h i s balance. Thus, the renin-angiotensin system may play a r o l e i n exercise by causing water excre-t i o n and sodium conservation. I t must be pointed out, however, that the d i u r e s i s may be due to the increase i n dorsal a o r t i c blood pressure and consequently f i l t r a t i o n pressure. But.the dorsal a o r t i c blood pressure increase i s t r a n s i t o r y and therefore the d i u r e s i s may be a r e s u l t of a combination of these f a c t o r s . Therefore to summarize b r i e f l y , the renin-angiotensin system probably plays a r o l e i n osmoregulation i n freshwater t e l e o s t s . It i s established that both A l and A l l have equivalent a n t i n a t r i u r e t i c e f f e c t s on trout kidney and that A l l may have a d i u r e t i c e f f e c t i n normal p h y s i o l o g i c a l conditions. These hormones may be necessary for the maintenance of kidney function i n the t e l e o s t . In order to f u l f i l t h i s r o l e renin may be continuously.released, i n low l e v e l s , from the J G c e l l s . Renin may also play a r o l e i n the r e -sponse to haemorrhage and i n recovery from exercise. 157 6.2 Renin i n E u r y h a l i n i t y . In addition to i t s r o l e i n freshwater t e l e o s t s , the renin-angiotensin system may also play a s i g n i f i c a n t r o l e i n the adaptation to seawater. Renin content of e e l (Anguilla japonica) kidneys has been observed to decrease when these animals were transferred from freshwater to seawater (Sokabe et a l . , 1966) and t h i s phenomenon has also been observed i n a number of other euryhaline species (Sokabe ej; al., 1968). The decreased renin content of the kidney may indic a t e that the hormone i s being released from the JG c e l l s into the blood plasma but the stimulus for t h i s release i s unknown. On the other hand, the decreased renal renin content may simply be a r e s u l t of decreased renin synthesis. When a freshwater adapted euryhaline f i s h i s transferred to seawater several changes i n blood constituents, kidney function and urine constituents are observed. The main considerations here are a decrease i n dorsal a o r t i c blood pressure (Chester Jones _et a l . , 1969), an increase i n plasma sodium l e v e l s (Sokabe et a l . , 1973) , a decrease i n glomerular f i l t r a t i o n rates (Chester Jones et a l . , 1969), and a decrease i n urine volume (Hickman and Trump, 1969). The decreased dorsal a o r t i c blood pressure would r e s u l t i n a decreased renal perfusion pressure which i n turn causes renin release. The increase i n c i r c u l a t i n g l e v e l s of renin would then r e s u l t i n an increase i n the formation of A l and consequently of • A l l . Since A l l i s a vasoconstrictor t h i s could r e s u l t i n afferent glomerular a r t e r i o l e c o n s t r i c t i o n . The blood flow to the glomeruli i s now decreased and there i s a f a l l i n the glomerular f i l t r a t i o n rate and consequently urine flow and urine volumes. The net e f f e c t therefore i s to decrease water loss through the kidney and aid i n combating dehydration induced by transfer to seawater. The increase i n plasma 158 sodium may also be p a r t l y a r e s u l t of the increased plasma renin a c t i v i t y as both angiotensins have been shown to cause a decrease i n urinary sodium l e v e l s which would i n turn r e s u l t i n a s l i g h t increase i n plasma sodium. The mechanism for the decrease i n dorsal a o r t i c blood pressure on trans-fer to seawater remains obscure. This may be a r e s u l t of a simple i n i t i a l water e f f l u x across the g i l l s following transfer to the hyperosmotic medium. On the other hand the decrease may be a t t r i b u t a b l e to some other stimulus such as a neural r e f l e x . In any case the drop i n dorsal a o r t i c blood pressure would d e f i n i t e l y r e s u l t i n a drop i n renal perfusion pressure. This decreased perfusion pressure would r e s u l t i n an increased renin release (see Section 3.5.2) and t h i s action i s a r e s u l t of a d i r e c t e f f e c t on the J G c e l l s rather than through some neural-humoral r e f l e x (see Section 3.5.3). The increase i n plasma renin a c t i v i t y r e s u l t i n g from t h i s stimulus could a c t u a l l y remove the source of stimulation by the vasoconstrictive action of A l l causing an increase i n the renal perfusion pressure. However t h i s does not appear to be the case as renal perfusion pressure has no e f f e c t on hypo-tension-induced renin release i n the trout (see Section 3.5.2) and renin release i s only i n h i b i t e d by a short-loop negative feedback system involving angiotensin II (see Section 3.5.4). The short-loop negative feedback system should i n h i b i t further renin release and thus show only an acute reduction i n glomerular f i l t r a t i o n rates and urine flow rates. But t h i s does not apparently occur. This could be explained by a f a i r l y long h a l f l i f e for c i r c u l a t i n g renin, or i n h i b i t i o n of angiotensinases or by some other mechanism. The most l o g i c a l p o s s i b i l i t y i s that the generated A l l c o n s t r i c t s the afferent a r t e r i o l e s above the l e v e l of the J G c e l l s . This would r e s u l t i n a further drop i n perfusion pressure being perceived by the J G c e l l s and a further increase i n the amount of 159 renin released by these c e l l s . The apparent decline i n renal renin content observed by Sokabe at a l . (1966) indicates that some such mechanism i s oper-ating as t h i s i s a long- l a s t i n g decline, up to 11 weeks i n some species. The a n t i n a t i u r e t i c e f f e c t of A l and A l l would seemingly be of disadvant-age to an animal faced with an increased i n f l u x of sodium r e s u l t i n g from freshwater to seawater transfer. However, t h i s e f f e c t may not be of any s i g n i f i c a n c e due to the decreased glomerular f i l t r a t i o n rate. That i s , there i s le s s u l t r a f i l t r a t e formed, due to afferent a r t e r i o l a r c o n s t r i c t i o n , and although there i s le s s sodium excreted the absolute amount of sodium retained by the kidney would i n t h i s case be n e g l i g i b l e since there i s so l i t t l e f l u i d passing through the tubules. Thus, an increased sodium reten-t i o n by the tubule i s not going to have much e f f e c t i f there i s l i t t l e sodium to be retained. S i m i l a r l y , the d i u r e t i c e f f e c t of the angiotensins would count for l i t t l e i f the kidney i s shut down. Therefore the net e f f e c t of the increased plasma renin a c t i v i t y would be water conservation due to renal.shutdown. Indirect support for t h i s hypothesis may be found i n another species (Gasterosteus aculeatus) where a decrease i n renal perfusion pressure r e s u l t s i n a decreased s i z e of glomeru-lus to Bowman's capsule r a t i o (Bonga, 1976). This could be due to afferent a r t e r i o l a r c o n s t r i c t i o n caused by an increased renin release r e s u l t i n g i n a decline i n blood flow and consequent loss of blood volume of the downstream vessels. In addition, the renin-angiotensin system i s known to a f f e c t other organs than the kidney i n mammalian species (Oparil, 1977) and i t i s possible that the system may be a f f e c t i n g i n t e s t i n a l absorption of water and e l e c t r o -l y t e s during adaptation to seawater. I t may also have an e f f e c t on the g i l l vessels under these conditions to ensure further conservation of water and excretion of undesirable ions. However, t h i s remains purely speculative at 160 the present time as there i s l i t t l e or no published information concerning, the e f f e c t s of the renin-angiotensin system on target organs other than the kidney i n the tel e o s t f i s h e s . 6.3 Evolution of the Renin-Angiotensin System. The renin-angiotensin system i s found throughout the vertebrate kingdom with the exception of cyclostomes and elasmobranchs (Sokabe jet a l . , 1969) but the p h y s i o l o g i c a l r o l e of t h i s system has been studied extensively only i n the mammals. Renin release i s known to be affected by a large number of factors i n mammals such as catecholamines, changes i n renal perfusion pressure, changes i n plasma sodium and the sympathetic nervous system .(Davis and Freeman, 1976). The angiotensins also have a m u l t i p l i c i t y of functions i n mammals ranging from acting as a l o c a l hormone at the kidney l e v e l to a systemic vasoconstrictor (Peach, 1977). But since t h i s hormonal system f i r s t appeared i n the p r i m i t i v e bony fishes (Nishimura et al., 1971) the question that remains to be answered concerns the evolution of the system. Hormonal systems evolve through either modifying the structure of the hormone or modifying the receptor such that a d d i t i o n a l functions may be added or the system may assume an e n t i r e l y d i f f e r e n t r o l e i n the physiology of organisms throughout the phylogenetic scale. From the data gathered i n t h i s study, one may conclude that there i s a good p o s s i b i l i t y that the most p r i m i t i v e s i t u a t i o n had A l as an end,product of the system.' . This conclusion i s based on f i r s t , the a n t i n a t r i u r e t i c e f f e c t of A l which i s equivalent to that exerted by A l l ; second, the potassium r e t a i n i n g e f f e c t of A l which i s not exerted by the A l l ; and t h i r d , the fac t that A l i s necessary for the formation of A l l . Another conclusion that may be drawn from these data i s that the e a r l i e s t function of the system was to cause sodium retention, a d e f i n i t e advantage for animals which are evolving 161 i n a hypoosmotic medium. The next probable step i n the evolution of these hormones can be found i n the d i u r e t i c e f f e c t exerted by A l l . Again t h i s i s an advantage for the p r i m i t i v e f i s h e s f or not only are they faced with ion loss to the environ-ment but i n addition they are faced with water i n f l u x . A d i u r e t i c hormone thus becomes quite desirable to aid the .animal i n ri d d i n g i t s e l f of excess water. Since the A l i s not very e f f e c t i v e as a d i u r e t i c (Section 5.3.1) t h i s involved modifying either the receptors or the hormone and i t would appear that i t was the hormone which was modified, as i t changed from a decapeptide to an octapeptide. This change probably promoted the vasocon-s t r i c t o r action which i s probably the main cause of the d i u r e t i c e f f e c t . As the bony fis h e s p r o l i f e r a t e d some groups began to invade the marine environment. This imposed a new s e r i e s of stresses on the animals' osmo-regulatory mechanisms. Since fishes are unable to excrete a concentrated urine, then the next best way to prevent water loss through the kidney i s to reduce the f u n c t i o n a l volume of t h i s organ. While t h i s would have severe repercussions on nitrogenous waste excretion i n mammals, i t would not have a s i m i l a r e f f e c t i n fishes as these animals excrete nitrogenous wastes i n the form of ammonia across the g i l l s . I t i s possible at t h i s point i n the evolutionary time scale that the a n t i d i u r e t i c action of A l l became more prominent although t h i s function may have evolved as a response to blood volume loss such as haemorrhage. Therefore the p r i m i t i v e functions of the angiotensins are to act as an a n t i n a t r i u r e t i c , a d i u r e t i c and an a n t i d i u r e t i c hormone. The d i u r e t i c and a n t i d i u r e t i c actions of A l l are probably brought about as a r e s u l t of an e f f e c t v i a a vascular receptor. Renin i s released into 162 the afferent glomerular a r t e r i o l e and presumably A l i s formed quite r a p i d l y . The action of A l l which i s formed from t h i s A l now depends on two f a c t o r s , the l o c a t i o n of converting enzyme a c t i v i t y and the amount of renin released. Low l e v e l s of plasma renin a c t i v i t y and converting enzyme a c t i v i t y i n the efferent side of the renal c i r c u l a t i o n w i l l r e s u l t i n efferent vascular con-s t r i c t i o n , a r i s e i n f i l t r a t i o n pressure due to increased efferent resistance and thus d i u r e s i s . On the other hand, high l e v e l s of plasma renin a c t i v i t y w i l l r e s u l t i n afferent a r t e r i o l a r c o n s t r i c t i o n as large amounts of A l l w i l l be formed. High l e v e l s of the hormone i n the blood w i l l increase the prob-a b i l i t y of the hormone reaching the afferent glomerular a r t e r i o l e s . Here i t would cause vaso c o n s t r i c t i o n , which decreases glomerular f i l t r a t i o n and the r e s u l t i s a n t i d i u r e s i s . It i s i n the mammals that the system apparently a t t a i n s the greatest degree of complexity. I t has not only a d i u r e t i c , an a n t i d i u r e t i c and an a n t i n a t r i u r e t i c action but also displays a n a t r i u r e t i c a c t i o n , probably due to the vasoconstrictor action of A l l (Peach, 1977). Angiotensins are also known to induce behavioural modifications i n mammals. Intracerebral i n j e c -tions of A l l induce a drinking response (Regoli et a l . , 1974). Such e f f e c t s have not been investigated i n other vertebrates to any great extent but i n t r a p e r i t o n e a l i n j e c t i o n of A l l w i l l cause drinking responses i n the common iguana, Iguana iguana (Fitzsimmons and Kaufman, 1977). In addition, the hormonal system may also be linked to other hormones which are known to a f f e c t the kidney. A l l may be responsible for the increased biosynthesis of aldosterone that occurs when mammals are faced with a hypotonic sodium plasma, t h i s action leading to sodium retention at the kidney l e v e l (Peach and Chiu, 1974). On the other hand, the A l l may be further modified, to carry out t h i s function, into a heptapeptide form known 163 as angiotensin- III or AIII (Williams e_t al_. , 1974) . While t h i s e f f e c t has not been extensively examined i n the other vertebrates, Nishimura et a l . (1976) were unable to show a r e n i n - c o r t i c o s t e r o i d axis i n f i s h . Also, A l l has been shown to r e s u l t i n an enhanced vasopressin release from the poster-i o r p i t u i t a r y which could p a r t i a l l y account f o r the a n t i d i u r e t i c action of A l l but the p h y s i o l o g i c a l s i g n i f i c a n c e of t h i s i n t e r a c t i o n i s doubtful as the a n t i d i u r e t i c response of A l l may also be a t t r i b u t e d to i t s vasoconstric-. tor action (Peters and Bonjour, 1971; Claybaugh and Share, 1972). Thus as the renin-angiotensin system evolved, i t can be seen that the angiotensins have been altered from a decapeptide, with a s p e c i f i c action, to an octapeptide, with much more general e f f e c t s , to a heptapeptide, again with a very s p e c i f i c action. But the question remains, have the receptors for t h i s system been altered? In fishes there appear to be two types of angiotensin receptors, a re n a l tubular receptor and a vascular receptor. The renal tubular'receptor appears to be the more p r i m i t i v e of the two as i t w i l l accept either A l or A l l while the vascular receptor w i l l only accept A l l . In mammals, a t h i r d type of receptor has apparently evolved and t h i s i s the adrenal c o r t i c a l receptor. This l a t t e r type was believed to accept only A l l but more recent work has shown that i t w i l l p r e f e r e n t i a l l y accept the AIII form (Williams et a l . , 1974). Mammals have the vascular receptor and i n addition may s t i l l possess the tubular receptor as A l l has a d i r e c t e f f e c t on tubular sodium reabsorp-t i o n i n dogs (Melton and F r a z i e r , 1976; Johnson and Malvin, 1977). But the tubular e f f e c t s of the angiotensins may not be of any p h y s i o l o g i c a l s i g n i f i -cance i n mammals as the primary sodium retention hormone i s aldosterone. Thus the receptors have not appreciably changed i n nature over the evolution-ary tree. 164 The next question i s what i s the basic stimulus for renin release? Since the angiotensins are involved i n sodium retention, i t might be expected that a f a l l i n plasma sodium or a decrease i n blood osmolarity would i n i t i a t e renin release. In f i s h there are l i t t l e or no data to support t h i s hypothesis. A f a l l i n plasma osmolarity or a decreased e l e c t r o l y t e concentration oppose a reduction i n renin release caused by i s o t o n i c volume expansion i n lung f i s h (Blair-West et a l . , 1977). But transfer of seawater adapted teleosts to freshwater does not always produce an increase i n plasma renin a c t i v i t y de-^ -sp i t e marked changes i n plasma sodium and blood osmolarity (Malvin and Vander, 1967; Nishimura e_t aJL., 1976). This suggests that the hypoosmotic stimulus may have existed i n pr i m i t i v e f i s h e s but was l o s t i n the more modern animals. In addition perfusion of trout kidneys with a hypertonic sodium sol u t i o n has no e f f e c t on renin release, which suggests that there i s no i o n i c stimulus for renin release i n t e l e o s t s . Renin release i s correlated, however, with blood loss and the actual stimulus for release i s a f a l l i n renal perfusion pressure. This s i t u a t i o n would be useful i n freshwater to seawater transfer as there i s a f a l l i n dorsal a o r t i c blood pressure i n these conditions. In mammals, renin secretion i s co n t r o l l e d by several mechanisms and these are, the c e n t r a l nervous system, renal perfusion pressure and the macula densa. The l a t t e r two mechanisms appear to be independent of the ce n t r a l nervous system (Winer je_t al. , 1969). The macula densa mechanism probably evolved as the renal p o r t a l system was l o s t since f i s h e s , amphibians and r e p t i l e s which possess a fu n c t i o n a l renal p o r t a l system,have no macula densa. The b i r d s , which possess a remnant of the p o r t a l system, have an intermediate type structure and the mammals, which do not have a p o r t a l system, have a f u l l y developed macula densa (Sokabe and Ogawa, 1974). This 165 loss of the p o r t a l system and the evolution of the macula densa probably came about as mammals are faced with the problem of water loss rather than ion l o s s . This then requires a system whereby water may be conserved and excess ions excreted. To f u l f i l t h i s r o l e the mammals have evolved a kidney which i s capable of producing a blood hypertonic urine and thus water may be conserved while r i d d i n g the animal of excess ions and nitrogenous wastes. The structure of t h i s mechanism i s such that the presence of a p o r t a l system would preclude production of the concentrated urine. I t i s poss i b l e , however, for mammals to lose ions as well and t h i s event must be guarded against. This i s achieved through the macula densa mechanism. The macula densa appears i n h i b i t e d by high renal tubular sodium and stimulated by low renal tubular sodium l e v e l s . Thus the renin-angiotensin system may now respond to changes i n blood volume, as r e f l e c t e d i n changes i n renal perfusion pressure, and changes i n plasma osmolarity, as r e f l e c t e d i n changes i n renal tubular sodium load. Therefore, i t seems e n t i r e l y probable that the renin-angiotensin system evolved from a blood volume maintenance hormonal system to a much more complex system as i s evident i n the mammals. Unfortunately there are several gaps i n the scheme as presented and further work must be done with t h i s system to ascertain exactly what r o l e the renin-angiotensin system plays i n the physiology of the non-mammalian vertebrate species. 6.4 Summary. 1. The rainbow trout has been found to produce renin i n i t s kidneys. This renin forms a product s i m i l a r enough to mammalian angiotensin I to allow i t to bind to a human anti-angiotensin I and thus renin a c t i v i t y i n the trout may be measured by a radioimmunoassay technique. 166 2_. A decrease i n renal perfusion pressure causes an increase i n renin release and t h i s increased renin release i s a r e s u l t of a d i r e c t e f f e c t on the J.G. c e l l s . Renin release i s not i n h i b i t e d by removing the stimulus but rather by a hormonal short-loop negative feedback system involving angiotensin I I . 2L Hypertonic plasma sodium l e v e l s have no d i r e c t e f f e c t on plasma renin l e v e l s i n the trout as perfusion of the trout kidney with hypertonic sodium solutions has no e f f e c t on renin release either stimulatory or i n h i b i t o r y . 4_. Angiotensin II may be a d i u r e t i c hormone or an a n t i d i u r e t i c hormone depending on what part of the renal vascular tree i s stimulated. Both angiotensin I and angiotensin II have a d i r e c t a n t i n a t r i u r e t i c e f f e c t on the r e n a l tubule i n the trout. _5. The p h y s i o l o g i c a l r o l e of the renin-angiotensin system i s obscure i n freshwater t e l e o s t s but i t may play a r o l e i n blood volume maintenance i n response to haemorrhage and sodium retention during exercise. 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(meq/1) Isotonic Saline 8 . 4 7 ± 1 . 4 3 (7) 8 . 7 4 ± 0 . 3 9 (7) Hypertonic Saline 12.8 ± 1.43 C7) 1 5 . 4 ± 2 . 0 9 (7) a- A l l values are means ± S.E.M. (N) >>••• P" 0 / 0 5 bV-P<0.05 c- P<0.01 APPENDIX 2. Urinary Potassium Levels Following Hypertonic Saline Perfusion of the Isolated F i l t e r i n g Trout Kidney. 3 Perfusate Begin Perfusion. (meq/1) End Perfusion. (meq/1) Isotonic Saline 0.17 ± 0.03 (4) 0.2 ± 0.061 , (4) Hypertonic Saline 0.29 ± 0.05 (4) 0.38 ±0.015 (4) a- A l l values are means ± S.E.M. (N) b- P<0.01 183 Appendix 3 Figures A . l and A. 2 are sample standard curves for the bioassay and radioimmunoassay techniques respectively. The bioassay was the standard rat vasopressor assay which involved cannulation of the caroti d artery and the jugular vein. The artery was used to record blood pressure while the vein was used for i n j e c t i o n purposes. The procedure followed involved i n j e c t i o n of a standard dose of angiotensin II, the animal was allowed to recover as evidenced by the return of the blood pressure to pre-injection levels and then a measured amount of the unknown was injected u n t i l the blood pressure showed a 5 mm Hg pressure increase. An equivalent volume of isotonic saline was injected as a contro l . The mean a r t e r i a l pressure was calculated by the formula: d i a s t o l i c pressure plus one t h i r d of the s y s t o l i c - d i a s t o l i c pressure difference. The mean a r t e r i a l pressure difference was calculated by subtracting the pre-injection mean a r t e r i a l pressure from the post-i n j e c t i o n mean a r t e r i a l pressure. 184 Figure A . l . Standard curve for the r a t vasopressor bioassay. The y-axis represents the dose of A-II administered, i n nanograms while the x-axis represents the calculated mean a r t e r i a l pressure increase. 186 F i g u r e A.2. S t a n d a r d c u r v e f o r a n g i o t e n s i n I r a d i o i m m u n o a s s a y . The y - a x i s r e p r e s e n t s t h e p r o p o r t i o n o f r a d i o a c t i v e t o n o n - r a d i o a c t i v e a n t i g e n ( a n g i o t e n s i n I) b o u n d t o t h e a n t i b o d y a n d i s i n a p e r c e n t a g e o f t h e t o t a l amount o f r a d i o a c t i v i t y u s e d p e r t u b e . The x - a x i s i s t h e amount o f n o n - r a d i o a c t i v e a n g i o t e n s i n I and i s i n aanograms. L8T 188 Appendix 4 L i s t of Abbreviations A l Angiotensin I A l l Angiotensin II AIII Angiotensin III BA Bioassay CNS Central Nervous System DOC Desoxycorticosterone EDTA Ethylenediaminotetraacetic a c i d JG Juxtaglomerular JGA Juxtaglomerular Apparatus JGC Juxtaglomerular C e l l MD Macula Densa PRA Plasma Renin A c t i v i t y PVP Polyvinylpryolidinone RG Renin Granules RIA Radioimmunoassay SEM Scanning Electron Microscope. 

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