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THE OUAIIffITATION Al,lD SIGNIFICAIICE 0F RENIN IN BIOLOGICAL FLUIDS

A THESIS

Submitted for the degree

of

DOCTOR OF MEDICINE

of

The Uníversity of Adelaide, South Australia

by

EUGENIE RUTH LIMBERS, M.8., B.S.

L969 DECI.ARATI ON AND ACKNO/,ILEDGEIVIENTS

r declare thaË this Èhesís is of my or'Jrl cornposition and

Èhat ít is a record of orígínal work conducÈed in the Department of Human Physíology and Pharmacology, at the uníversíËy of Adelaíde, duríng the years 1966, L967 , 1968 and L969. The experiments described hereín have not been submítted for any other degree or díp1oma.

I would like to express my gratitude to my supervísors,

Dr s" L. skinner and Professor R. F" I¡ühelan for their guidance and interesÈ, and r should also líke to thank my coJ-leagues, Dr R" L.

Hodge and Dr R. c. Harr, for Èheír ínteresÈ; Míss F" Katic and Mrs M. Deutrom for Èhe excellenË Èechnícal assísüance they províded; and those colleagues and sÈudenÈs who were willíng to act as subjects for the investigaÈions descríbed.

I would also like t,o Èhank F.B"A" PharmaceuEicals for Èheir donatíons of Trasylol, cíba co" Ltd (Australia) for supplyíng Angiotensín acid (33 902 BA), and Searle LÈd for providíng Aldactone-4"

Finally, r should like Èo thank the Nationar Health and Medical Research councÍI, and Èhe Mr "x" Fund of the universíty of Adelaide, for supporËíng me finaneíally during the four years of study that has been undertaken.

z s/ PREFACE

Renin is an enzyme whíeh acÈs ori an cÌ2-globulin substrate present, in plasma Èo produce the decapeptíde angiotensín I. Angiotensin r ís converted Ëo the octapept,ide, angíotensin rr, by the aetíon of a chlor:ide-dependent enz)¡me present in the blood and the lungs. Angiotensín is a faeüor ín the control of the secretion of -aldosterone and is a powerful vasoeonstríctor. Therefore, renin ís indírectl-y concerned wíth cireulatory homeost.asís.

Thís thesís deseríbes the experímental techniques and results obtaíned in studies on Èhe presence of renín in human urine and factors influencing íts excretion; the presence of renín in human maternal and foetal tissues; and the contributíon of renín and renin subsËrate levels to Èhe changes in renín acÈivíty induced by natrj-uretíe agents, the hormonal varíatíons of a nor-mal menstrual cycJ-e, oral- contraceptíves and pregnancy.

The role of Ëacþyphylaxis ín the constricÈor response Èo angiotensin in normal subjects has been ínvestigated. Stud.ies have also been carrled ouÈ on the perJ-pheral vaseular response Èo angíotensín in Èhe pregnåD.t woflÉin; CONTENTS

Page

HISTORICAL SUR\EY 1

INTRODUCTION 29

SECTION ONE

METHODS 31

SECTION TWO 47

(A) THE OCcURRENcE Æ{D ASSAY oF RENIN IN HI]MAN URINE 48

(B) OBSERVATIONS ON THE ORIGIN OF RENIN IN HIN,IAN URINE 68

SECTION THREE

THE EFFECT OF THERAPY !üITH THE NATRIURETIC AGENTS, SPIRONOLACTONE AND CHLOROTHIAZIDE, ON PI,ASMA RENIN

ACTIVITY' PLASMA RENIN CONCENTRATION AND RENIN SUBSTRATE

LEVELS 86

SECTION FOUR

RENIN CONCENTRATION IN HUMAN FOETA]. A\ID MATERNAL TISSUES 95

SECTION FIVE

CI{AÌ{GES IN PLASMA RENTN ACTIVITY, PLASMA RENIN CONCENTRATION AND RENIN SUBSTRATE LEVELS: 108

(A) DURING THE MENSTRUAL CYCLE 109

(B) DURING ADMINISTRATIoN oF oRAI coNTRACEPTIVES LL2

(C) DURING PREGNANCY L2L

SECTION SIX

THE SENSITIVITY OF TIIE HAND BLOOD VESSELS TO ANGIOTENSIN

AI{D NORADRENALINE IN PREGNANT AND NON-PREGNÆ{T I^IOMEN 130 SECTION SEVEN Page

TACITYPHYLAXIS TO ANGIOTENSIN IN MAN t4r

APPENDIX

(A) STATISTICALMETHODS

(B) TABLES (1-17)

BIBLIOGRAPHY t 0FI ift

r¡¡ ) HISTORICAL SURVEY /iD t A\ol

In 1836, Ríehard BrighË described the association beÈween hypertension manífested by left ventrieular hypertrophy and díseases of Èhe kidney. The recognít,íon of this associaÈíon and the concept of inËernal glandular secretíon by various organs, including Èhe kidney, as proposed by Brown-séquard (1889, LB92) and Brown-séquard and d'Arsonval (L892), led TigersÈedt and Bergmarrn(f89S) to investigate the presence of subsÈances in the kídney affect,íng the cardiovascul-ar system" rn renal tíssue they found a non-díalysable, heat-labile substanee, soluble in waÈer but not in alcohol, which they cal-led frenínt. They concluded Ehat the síÈe of actíon of renín was Èhe perípheral vascurar apparatus, more partíeularry the peripheral ganglia of the vascular nerves" TígerstedË and Bergmannfs work was the first definítíve study of Ëhe pressor raisíng propertíes of renal extraets to meet with real success. Other workers reporÈed a varieÈy of results. oliver

(1897) failed t,o demonstrate a constrictor rnat.erial, in renal- exËracËs using isolated frogts mesenÈery as an assay preparation. vineenÈ and

Sheen (1903) studíed the pressor and depressor properties of a number of tíssues and coneluded that, of all the tíssues studied, the kidney was the most likely Ëo produee å pressor response on íntravenous injectíon and that thís pressor response \^ras converted to a depressor 2 response by prior boí1ing of the extract.

Shaw (1906) and Bingel and Strauss (1909) likewise obtaÍned pressor responses on j,ntravenous ínjection of renal exËracts, buË

Miller and Miller (1911) and Collip (1928) failed to confirm these

findings " In Ëhe líght of presenÈ day knowledge of the actíons and properties of renín iÈ is not surprisíng that these early workers, using a variety of Ëechniques for extracÈion and assay, produced such

conflicting results " To study the association between the kidney and hypertensíon, ít is desirable to produce, by experímental manipulatíon of the kídney, a persistent hyperÈension r¿hich can be directly attríbuÈed to the experimental procedure performed. Untí1 L934, no reliable technique exísted which produced persistent renal hyperÈension.

Bílateral nephrecEomy \{as not associated with persistent hypertension

(Backman, L9I6; Cash , L926), whereas manípulatíons of the renal vessels produeed varying degrees of hypertensíon" For exanple, KatzensËein (1905) observed a transíent, ríse in blood pressure followíng complet,e occlusíon of the renal vessel-s; so díd Baekman

(1916) and Cash (1926). Backman and Cash Èhus showed that, whereas bilateral nephrecÈomy failed to induce hypertension, hyperÈensíon did occur followlng reduction in functioning renal tissue plus lígatíon of Èhe renal vessels. 3

Brídgman and Hírose (1919), however, faíled to índuce

hypertension wíth partial occlusíon of the renal vessels of the dog usíng aluminium bands to produce constriction of the renal vessels.

Techniques applied by other workers meË with varying degrees of

success (references cited by Braun-Menéndez, Fascíolo, Lel0ir, Munoz and Taquíni, L946).

In 1934, Goldblatt, Lynch, HanzaL and Sunrnerville described

a method for the production of renal hypertensíon using a screw-clamp which partially occluded the renal vessels. This technique for produetion of renal hypertension was both dependable and reproducíble. The hypertensíon produced with Èhe Goldblatt clamp was studíed ín

detail by Goldblatt, Lynch, HanzaL and summerville (1934) and. other workers (GoldblaÈt, L937; Goldblatr, 193g; Goldblarr, Kahn, and

HanzaT, 1939; Blalock and Levy, 1937; Blalock, Levy and Cressman,

1939). rt was found to be índependent of ínnervation of the kidney

(nlalock and Levy, Lg37; Goldblatr, L937; page, 1935), alÈhough Glenn' chíld and Huer (L932), on the basis of transplant studies, suggested thaÈ Èhe renar nerves may play a role ín the maintenance of renal hypertension. Removal of the clarnp on the renal vessel or r:emoval of Èhe c1ípped kidney led to remíssíon of the hypertension induced by clampíng. Actual- necrosís of the renal Ëissue r^ras not necessary for the onset of hypertension (coldblatt,, Lynch, Hanzal and. summerville, L934), a fíndinþ conËradictory to the earlíer findings 4

of cash (1926). occlusion of the renal vessels, or of the aorËa

above Èhe renal arteríes (Goldblatt, Kahn and Hanzal, 1939) was thus found to be a specific mechanísm for the índuction of experimental hypertension, and \^ras not reproduced by clarnping or íschaemia of

vessels to other organs such as Ëhe spleen (Goldblatt, Lynch, Hanzal

and Summerville' 1934) or Íntestine (nlalock, Levy and Cressman, 1939). Partial clarnping of one renal artery produced hypertension which was not persistent. Severe and persistent hyperËension depended upon Lhe clarnping of both renal art.eries or unilateral clamping plus

contralateral nephrectomy (GoldblaÈt, 1938) . In 1939, Page described another model for the production of persistent hypertension in the experímental animal due to

constriction of the whole renal parenchyma. The consËríction was índuced by wrapping the kidney ín celrophane which caused the formaLíon of a fibrocollagenous hu1l.

sinee these experiments showed a lack of nervous facÈors in Èhe inítiation of renal hyperÈension, and remíssíon of hypertensíon occurred followíng removal of the ischaemíc kídney, a humoral mechanism as the predominant facEor ín the initíation of renal hypertension became a possíbítity. rnterest revived, Èherefore, ín the action of renal extracts on the cardiovascular system, and sÈudies of Èhe changes in the content of pressor materíal in Ëhe kidney induced by renal arÈery clamping were undert,aken. 5

Prínzmetal and Fríedman (L936), æd Harrison, Blalock and

Mason (1936) found Ëhat extracts of elípped kídneys produced a greater constríctor response when ínjected íntravenously into dogs Èhan did extracts of normal kidneys. They also found thaE preparations of renal tissue from patíents wíth hypertension possessed more powerful pressor actions than díd extracts of kidneys from normotensíve patients " The most important point to establísh was wheËher or not the íschaemic kidney released a vasoconstrictor material into the círculation following clamping of the kídney. TransplanÈ studíes performed by Houssay and Fascíolo (1937, 1938), Fascíolo, Houssay and Taquini (1938) and Fasciolo (1938a, b, c), ín whích clipped kídneys transferred from a donor dog to the neck of a recípient inft¡ced hypertensíon in the recipient, suggested that mechanical and nervous factors \^rere not responslble for the íníÈiatíon of the hypertension and Ëhat a humoral mechanism was involved. Moreover, in 1938,

Fasciolo, Houssay and Taquíni showed that the venous effluent of an íschaemic kidney contained a powerful vasoconstríctor substance whích was non-dialysable" Mason and Roze11 (1939) r^rere, however, unable to repeat these experímenÈs" Further evídence for the release of a hypertensíon- producing substance by the kidney was obtaíned by Taquiní Ln 1940"

Taquini found thaÈ following release of a clamp whích had completely 6

arresËed the blood supply to the kídney, marked hypertensíon ensued.

Such a fínding suggested that a vasoconstricËor material was "washed

outil l-nto the circulation after the elamp was released. FurËhermore,

Page, in 1940, found Èhat Èhe peripheral plasma from renal hyper- tensíve dogs was more po\^rerfully vasoconst,ricÈor than was renal venous plasma, and also more powerfully vasoconstrícÈor than the peripheral plasma from normal dogs. sínce he used the ísolated ear

artery as an assay preparation, he may have been lookíng at angio- tensín raËher than renin Ítself. This could possíbly account for the greater levels of vasoconstrictor maÈeríal ín the peripheral plasma as compared to renal venous plasma"

In 1941, Goldblatt, Kahn and Lewis, ín an ínteresting study of the effecÈ of ureÈeríc clampíng on the production of hype.rtensíon by renal artery clipping, confirmed the findings of Houssay and

Fasciolo that venous effluent from a clípped kídney contained a vasoconstricËor material. Therefore, it appeared that Èhe ínitiatíon of renal hypertension was based on a humoral mechanism, the substance involved beíng renin, since ít possessed some of the characteristics of renín first descríbed by Tigerstedt, and Bergmann (Prínzrnetal and

Fri-edman, 1936; Píckering and PrínzmeËal, L937; prínzmetal, Lewís and Leo, .L940 a and b). Considerable enthusiasm \¡ras expressed ín support of the role of renín ín Ëhe iníEíation of noÈ only renal hypertension, but 7

also of essentíal hypertension (Goldblatt, 1958), a role experimental

observation has yeÈ t.o justífy. The absence of sígnifícant or consístent changes in renin levels in essential hyperÈensíon (Fascíolo,

de Vito, Romero and CucchL, L964), or in all cases of renal arÈery

stenosís (Brown, Davies, Lever and Robertson, 1964a), has cast a shadow on these original expectaÈions, and even in the fíeld of experímental hypertension all is not clear.

The fíndíngs of Regolí, Hess, Brunner, Peters and Gross (1962),

Regolí, Brunner, Peters and Gross (1962), and Gross, Brunner and ZíegIer (1965) on the relationship between renin content of the kidneys and renal hypertension and, in parÈicular, the action of the contra-

lateral unclamped kidney ín maíntaíníng high renin levels in the clípped kidney, and also Èhe role of neurogenic factors in chronic

renal hypertension (ltcCubbin and Page, 1963; Taquini, 1963), emphasize

that the relatíonshíp beÈween renin and renal hypertensíon is complex. Moreover, the occurrence of renal hypertension ín rabbits successfully ímmunízed against angiotensin following clippíng of the kidney (Hedwall, L968; Hutchinson and Johnston , 1969) further confuses the pícture, especially since antirenin has proved successful ín reducíng blood Pressure in acute and chroníc renal" hypertensíve dogs (Iniakerlin, 1958; Haas and Goldblatt, 1963; Deodhar, Haas and Goldblatt, 1964). perhaps a break through of the effective antibody titre by endogenous angio- tensin production results in the fail-ure of remissíon of renal 8 hypertension follor¿ing immunizaEíon of experimental animals to angíotensín.

A recent statement by Piekering (1968) comments on the present knowledge of the mechanism of productíon of experímental renal hyperËensíon: "The mechanísm by whích renal artery constríction produces hypertension thus remains quíÈe obscure. rhislsan extra- ordínary position, considering the relative símplicíty of the procedure, íts replicability and íts reproducibility""

Renín ís not, the active vasoconstricÉor or pressor compound. It ís a proteolyLic enzyme which acts upon an clr-globulin substrate to form the decapeptíde angíotensin r, whích ís split by a chloride- dependent enzyme ín plasma to the octapeptíde angíotensin rr, the actíve pressor agent.

Kohlçtaedt, Helmer and page (1938); KohlstaedÈ, page and

Helmer (1940) found that plasma r^ras necessary for the vasoconstrictor actíon of partially purífied renín on ísolated, perfused preparations.

In 1940, t\^ro groups, Helmer and page, and Braun-Menêndez, Fasciolo, Leloir and Munoz, publíshed papers describing the formation of a pressor compound from the action of renín on prasma. Helmer and

Page described thís pressor compound as rangiotonin!, and Braun- Menéndez, Fasciolo, Leloír and Munoz descríbed it as thypertensinr.

These t\¡ro terms remained in use until 1958 (Braun-Menéndez and page,

1958) when the joínÈ name of fangíotensinr was agreed. upon. 9

Braun-Menéndez, Fasciolo, Leloir and Munoz (1940) concluded

ÈhaL renín hras an enzyme which acËed on a pseudoglobulín fractíon jn plasma to produce angiotensín. Plentl-, Page and Davis (L943a) sírnilarly concluded that renín r^ras an enzyme. It ís therefore apparent that Ëhe formatíon of angiotensín in plasma would be dependent not only upon the concenÈraËíon of enzyme Present, but also upon the concentratíon of substraLe present" In facË, Braun-

Menéndez, Fascíolo, Leloir, Munoz and Taquíni (L946) showed that variaEions in the amount of renin subsÈrate in plasma influenced Èhe rate of formation of angiotensín in the presence of a constanÈ excess of renín. They also demonstrated the specífícity of the renin-renin substrate reaction on a species basís; for example, píg renin faíls to react wiLh hurnan substrate (Fasciolo, Leloír, Munoz and Braun- Menéndez, L940), a fínding charaeteristíc of renins other than those deríved from primaÈes (Braun-Menéndez, Fascío1o, Leloir, Munoz and

Taquini, 1946). Moreover, renin substraÈe is relaËively specific Èo the proteolytic action of renín, although under certain conditíons of pll iÈ can be splít by pepsín to produee pepsiÈensin, a compound wíÈh pharmacologícal properties símí1ar Lo angioEensín (Croxatto and

CroxaÈto, L942; B1air, L962) and Ëhe same amino acíd sequence as angioËensin I (de Fernandez, Paladíni and Delíus, 1965).

Dífficulties in the assayìof angíotensin produced as a result of the actíon of renin on substraËe were encountered due to the 10 presence of angíoËensínases " Page and Helmer (1940) thought Èhat renin itself destroyed angioÈensin, However, Brar.m-Menéndez,

Fasciolo, Leloir and Munoz (1940) showed that Ëhe removal of angio-

Èensinases abolished Èhe fall ín concentration of angíotensin which

\^ras for:ned on incubatíon of renín with plasma" Renin substrate was found to be present in the clr-globulín fraction of plasma (PtenÈl, Page and Davis, L943b) and formed ín the liver (Page, McSwain, Knapp and Andrus, L94L; Leloir, Munoz, Taquini,

Braun-Menéndez and Fasciolo, L942) .

Skeggs, Kahn, LenEz and Shumr¡ay (L957) undertook the isolation and purífícation of renin subst.rate, fírst isolatíng a polypeptide substrate r¿hich would react with renin, and later synthesízing a tetradecapepËíde (Skeggs, Lerrtz, Kahn and Shumway,

1958) which liberated actíve pressor compor:nds ídentícal Ëo angiot.ensín I and angíoÈensín II when reacted with renín" Skeggs , Ler.Lz, Hochstrasser and Kahn (1963, 1964) purifíed hog renin substrate, isolating several forms of substrate - A, 81r 82, CL and C, - of which A, Cl and C, were glycoproteins, differing only in glucosamíne and hexose eontenË. Cook and Lee (1965) found no evidence of multiple peaks of rabbit renín substrate actívíty followíng elution from Díethylamíno- ethyl cellulose column and suggested that the mulÈípl-e peaks found by

Skeggs, Lentz, HochsÈrasser and Kahn (1963, L964> were the resulÈ of 11 partial acíd degradation of the renin subst,rate molecule (Lee, L969).

Helmer and Griffith (L952) showed thaÈ renln substrate levels ín plasma eould be íncreased by treating animals wiËh oesËrogens. Helmer and Judson (1967), and Skínner, Lumbers and

Symonds (L969) showed thaË renin substrate levels ín plasma ínfluenced the rate of formation of angiotensin" ElevaËions ín renin subst,raËe were thought by oËher workers (Pickens, Bumpus, Lloyd,

Smeby and Page, 1965; Haas and Goldblatt, L967; Ayers, L967) Èo be unímportant ín determining the rate of formaËion of angíotensin in plasma since the concentration of substrate normally found ín plasma was ín relative excess. This ís now known not to be so; therefore, elevations in renin substrate can influence plasma renin act.ivity"

Skeggs, Marsh, Kahn and Shumway (1954) found that Èwo forms of angiotensín exisÈed - angiotensin I (the decapepËide) and angíoÈensin II (the ocÈapeptide) - the conversion of angíotensin I t,o angiot.ensin II being dependenË upon the preserice of a chloride- dependent. enzyme in plasma" They extracted and purified rhe chloride- dependent enzyme from horse plasma and studied íts properÈies (Skeggs,

Kahn and Shumway, 1955) " More recently, it has been shown by Ng and Vane (1967) that large concentrations of thís convertíng enzyme exist in the lungs.

Det.erminatíon of Èhe amino acid composítíon of angiotensin I

(Peart, 1955, L956; Skeggs, Marsh, Kahn and Shumway, 1955) and of L2 angíotensín II (Lentz, Skeggs, tr{oods, Kahn and Shumway, 1956) and of the amino acid sequence of angiotensin I (¡tliot and Peart , 1956,

Lg57) led to Èhe successful synthesis of angiotensÍn, isoleucyls angioÈensín beíng syntheÈízed by Bumpus, Schwarz and Page (1957 , 195S) and asparågínyl isoleucy15 angiotensín by Schwyzer, Iselín, Kappeler, Riníker, Rittel and Zuber (L957). Successful synthesis of angíotensín enabled determinatíon of amíno acid groupings necessary for biological actívíty to be made

(eage and Bumpus, 1961). Tn L962, Smeby, Arakawa, Bumpus and Marsh suggested an cr-helical conformaËíon for angioÈensin based on optical roÈatory dispersal studies, internal hydrogen bondíng beíng necessary for the tertiary structure of the molecule and hence for bíologícal actívity, However, Èhe cl-helical conformaÈion of angiotensin II ín aqueous solutions, as described by Smeby, Arakawa, Bumpus and Marsh, (1962), has not been substantiated by Paíva, Paíva and Scheraga (1963) who suggest a random conformatíon of the peptíde ín aqueous solutions. Renin ítself has been subJected to intensive ínvesEigaÈion" Early studies on the purífication of renín \^tere carried out by Helmer and Page ín 1939. Haas, Lamfrom and GoldblatÈ (1953) purified hog renin a¡rd obtained a preparatíon of high acËívity, but noted the ínsËability of purified renín at a neutral pH" Renin is a proteolytíc enzyme which is remarkably stable over a wide pH range in ímpure preparatíons (Haas, Goldblatt and Gipson, 1963). This property makes 13 iÈ possible to separate renÍn from renín substrate by acid denatur- (Skinner ation of the latter, leaving the renin molecule íntact , 1967) " Renín specifically attacks the leucyl-leucyl bond of the renin substraÈe molecule to liberate angioËensín I. However, iÈ does not depend (as do other specifíc proteolyÈic enzymes) on Ëhe presence of adjacent aromatíc or basic amino acíds (Skeggs, Kahn, Lentz and

Shumway, L957).

The molecular weíghÈ of hog renin (Kemp and Rubin, 1964) and hurnan renin (tr{arren and Dolinsky, 1966> ís in the region of

40,000 to 501000. Peart, Lloyd, ThaÈcher, Lever, Payne and Stone

(1966) extracted and purified hog renin and on electrophoresis agaínsÉ a specífic antíserum, obtained one major and two mínor precípitin lines.

AnËibodíes to renin hrere first produced by Johnson, üIakerlin and Goldberg (1941) and by Goldblatt, Katz, Lewis, Ríchardson,

Guevara-Rojas and Gollan Ln 1942, and have been used wldely ín Èhe investígatíon of the role of renin ín experimental hypertension (Helmer, 1958; Ialakerlin, 1958) as a criteríon for the identificatíon of renin (Skinner , L967) and in studíes on the location of renín r^7íthin Èhe kidney (Edelrnan and llartroft, 1961). The anËígeníc and caÈalytíc sites of renín appear to be separate, as shown by the studíes of Haas, Goldbl-att and Gipson , L963. The exact locatíon of renin in the kídney ie still not L4

clear" Tigerstedt and Bergmann, ín 1898, concluded that renín \¡ras

located in the renal cortex and a considerable number of sËudies have

been carried out to determíne the frenj-n contaíning cellst of Èhe kídney,

In the renal cortex, renin occurs in assocíation with Èhe

glornerulí. The deeper the glomerulus from the ouÈer surface of the

kídney, the lower the renin content (Brown, Davíes, Lever, Robertson

and Tree, 1963). cook and Pickering (L962) assayed Èhe renin content of the uPper and lower halves of indivídual glomerulí and found that renín was located in the vascular pole of the glomerulus, i"e. in

associatíon wíth the juxtaglomerular apparaÈus. Edelman and Hartroft (1961) and Hartroft (1963) found fluorescent antirenin localized in Èhe jtrxtaglomerularcells liníng the afferent arteríole, supportíng the hypothesis of Goormaghtigh (L945) rhaÈ the granules of the juxtaglomerular cells contaín renín" rndirect evidence for

Goormaghtíghrs hypothesÍs r^/as also obtained by Marx and Deane (1963)

and verniory and Potuliege (L964) who showed that a correlation exísted between renin content and the degree of granularity of the juxtaglomerular cells.

However, microdisseetion studies done by Bíng and Kazmierczak (1959, 1960 , L962, L964) and Bíng (1964) found rhar renin was located rnainly in those fragmenÈs of renal tissue contaíníng

tubular remnants, raËher than the vascular portíons of Ëhe 15

juxtaglomerular apparatus, and furthermore, thaË renín r^râs present

in nephrogenie renal Lissue devoid of both grarrular and non-granular epithelioíd cells,

An inËeresting observatíon r^ras made by Hess and Pearce (1959) and Marx and Deane (1963) " They observed that the glucose-6- phosphate dehydrogenase content of the macula densa parallels the changes in granulaÈíon of the juxtaglomerular cell-s indueed by trenal cLip hyperÈensiorit (Hess and Pearee, 1959), low sodium diet

(Marx and Deane, 1963) and infusions of angiotensin (Marx, Dèane, - Mowles and Shephard, L963> " lrlhether renín is formed ín Ëhe maeula densa and stored in the juxtaglornerular cells (Bing and Kazmiexezak, L962) or whether the macula densa aets only as a tsensíng siter which controls Èhe release of renín from Èhe juxtaglomerular eells remaíns to be determined.

Renin ís not exclusively confíned to Ëhe kidney. tr'lerle, Vogel and Goldef (1957) and l^lerle, Baumeíster and Schmal (L962)

described a renin-líke enzyme in the submaxillary gland of Èhe whíte mouse. This enzyme released a vasoactíve prínciple whích was simílar to angiotensín on íncubaËion wíth plasma. Turrian (1960) found, however, that it was not subjeet to control by sodiurn loadíng or

eortexone admínísÈratíon. Dengler (1956) found a renín-like enzyme in extracLs of hog aorÈa. Goul-d, Skeggs and Kahn (1964) made a more extensíve sËudy of the dísÈríbution of pig extra-renal renin, 16

descríbing an enzyme of simílar biochemical properËies Eo renal renín in a varíety of Èíssues. They found only mínuÈe amounÈs of renín in the placenta and myomeÈríum of the pregnant pig.

Renin-like enzymes oecur ín the plaeenta of Ehe rabbít (Gross, SchaeteLín, Zíegler and Berger, L964; Bíng and Farrup,

L966), Ëhe cat (Stakeman,1960), Èhe dog (Hodarí, Bumpus and Page, L967) and ín human arnníot,ie fluid (Brown, Davies, Doak, Lever,

Robertson and Tree, L964). Furthermore, renin is extractable from a varíety of body fluids such as lymph (Lever and PearÈ, 1962), urine (Brown, Davies, tever, Lloyd, RoberÈson and Tree, 1964) and blood

(Helmer and Judson, 1963; Lever, Robertson and Tree, L964; Boucher,

Veyrat, de Champlain and Genest, L964; Píckens, Bumpus, Lloyd,

Smeby and Page , L965; Gould, Skeggs and Kahn , 1966; Haas, Gould and

Goldblatt, 1968) .

The development of assay techniques sensítive enough Èo detect renin or angiotensín and to correlate these levels to boÈh the physiologícal and pathologícal síËuatíons has been of considerable imporËance,

Assays of renín ín blood can be broadly dívíded into two groups. First, those Ëhat measure the so-called trenín acLivityr of plasma (e"g. methods deseríbed by Boucher, Veyrat., de Champlaín and Genest, L964>. Renin aet.ivity is deLermined from the amount of angiotensin formed per unit time as a result of the reaction beEween t7

renín and endogenous renín substraËe. Thís is a qualiÈaËive assay,

sínce it does not take into account Èhe varíatíons ín the levels of

endogenous substrate ÈhaË may occur. The second group of renin assays are Èhose which determine the co-called plasma rrenin

concentrationr (e"g. methods described by Lever, Robertson and

Tree, L964; Skinner, L967). The endogenous substrate ís removed or

denatured and a const,ant amount of an artíficial substrate added to the system prior to incubatíon. In this system, Ëherefore, the

anount of angiotensin formed refleets the levels of enzyme present"

An angiotensin assay was developed by Scorník and Paladini (1961), but suffered from the dísadvantage thaÈ relatively large

volumes of blood had to be taken for extractíon of angíotensin" A more recenÈ development has been the radíoimmunoassay of angíotensin, using antíbodíes raised agaínst synthetic angíotensín II (Boyd,

Landon and Peart, L967; Catt, Caín and Coghlan, L967). Thís method

increases the ease of assaying whilst Èhe adaptatíon of radioinununo- assay to determine Ëhe amount of end-producË formed followíng in uitro incubaÈion of renin ¡¿ith subsËrate (Boyd, Adamson, Fítz and Peart, L969) allows for greater ease in determining renin levels ín

cerÈaín experímental situations " The eonsíderable varíations ín the methods used for the

assay of renin make it díffícult Ëo ínterpret and correlate results obtained from various laboratories (Haas, Gould and Goldblatt, 1968). 18

The biological role of renin is dependenÈ upon the actions of angiotensin. Angiotensin, weight for weight, is the most powerful pressor substance known" IÈ has both direct and índirect actions in raísíng blood pressure. The pressor effect of angíotensín was demonstrated in man by l,lilkins and Duncan, I94L; Bradley and Parker, L94l; Corcoran, Kohlstaedt and Page, 194I, Direct perfusíon of vascular beds has shown that angiotensin is vasoconstrictor in all situations studíed, constríctíng the hand vessels (üIilkins and Duncan, L94L; De Bono, Lee, Mottram, Piekeríng, Brown, Keen, Peart and Sanderson, L963; Scroop, üIalsh and l^Ihelan, 1965), muscle vessels (De Bono et aL, 1963; Scroop, trlalsh and trrlhelan, 1965) and the splanchnic vasculature (De Bono et aL, 1963). Further studies have revealed that it. also has a central indirect action, the efferent pathway beíng the sympathetic nervous system (Bickerton and

Buckley , I96L; Laverty, 1963; Scroop, trrlalsh and l,tlhelan, 1965) . Angiotensin does not appear t,o sÈímul-ate the perípheral sympathetic nervous system in man (!ühelan, Scroop and tr{alsh, 1969), but potent-

íation of the constríctor response to ínfused noradrenalÍne by angíoÈensin has been observed (Scroop and trlalsh, 1968). Although animal studíes have revealed that angioÈensín can release catechol- amines from the adrenal meduLla of the cat (Feldberg and Lewís , 1964) and the dog (Peach, Cline and l¡tratts , L966), thís has not been satisfactorily demonstrated in man (Vincent, Kashemsant, Cuddy, Fríed, L9

Smulyan and Eich, 1965; trrÏhelan, 1967) " Angiotensín also stimulates the eontraction of uterine muscle (Gross and Turrian, 1960; Paiva and Paiva, 1960), guinea pig íntestíne (Gross and Turrían, 1960) and rat colon (Bisset and Lewís, L962). The latter tissue has been used for continuous assay of changes in angíotensin blood levels (Regoli and Vane, 1964). There is little convíncing evidence for a direct myocardial acÈion of angíotensin. The effects of angíoÈensín on the myocardium described in the literature can be attributed to one or more of the following: effect of reductíon in coronary blood flow; st,imulation of sympathetíc nerves; or inítíatíon of baroreceptor reflexes as a result of the pressor action of the drug. For example, the negatíve inoÈropic effect observed by Downing and Sonnenblíck (1963) ís probably due to the effect, of a reductíon in coronary blood flow. Koch-l,rIeser (L964) did, however, describe a positíve ínotropíc effect of angiotensín on kítten myocardíal preparations " In the intact aninial , angiotensin causes a reflex bradycardia (t"tiddleton and lrliggers , L944; Johnson and Bruce , L962; Nishith, Davís and Youmans, 1962; Scroop, I,Ialsh and Inlhelan, 1965). In the experímental animal and ísolated tÍssue preparatíons a reduction ín responsiveness to large doses of angíotensin has been observed. This phenomenon ís deseribed as "tachyphylaxis'r. Khairallah, Page, Bumpus and Turker (L966) attributed this loss of 20

responsiveness to angiotensin to sat,uratíon of reeeptor sites and found that addítion of angiotensínases to perfusíng fluids reversed rachyphylaxis. Kaplan and Sílah (1964) have descríbed a clinl-cal t,est for detecÈion of high endogenous renin levels in hypertensive patients which is based on a reduction in sensiËivity to intravenous angiotensin. A similar reductíon in pressor sensítivíty has also been observed with pregnant vromen (chesley, Talledo, Bohler and Zuspan'

1965) in whom high endogenous renin levels oceur' one of the most important actions of the renin angiotensín

system is its role as a factor in the control of aldosterone secretíon from the zona glomerulosa of the adrenal cortex. Aldo- sterone was fírst isolated by Simpson, Taít, tr{ettstein, Neherr von

Er¡^r and Reichsteín in 1953. A humoral mechanism as a factor in the control of aldosterone secretion was implícated by the cross- circulation experíments of Yankopoulas, Davis, Klínman and Peterson

(1959) and Denton, Goding and !üright (1959a and b). The work of Davis, Ayers and Carpenter (1961); Davis, Carpenter, Ayers, Holman

and Bahn (1961); Mulrow and Ganone (1961); Mulrow, Ganong, cera and Kuljían (Lg62); and Ganong and Mulrow (L962) showed that the kidnev'

and more particularly renín, I^las involved in Èhe release of aldo- sterone. It has been further shown that it is angiotensin which regulaËes aldosterone secretíon, not renin (Carpenter, Davís and Ayers, 1961; Biron, Koíw, Nowaczynskí, Brouillet and Genest, 1961; 2T

Blair-l{est, Coghlan, Denton, Godíng, Munro, PeÈerson and !üíntour, L962; Davis, L962; Ganong, Mulrow, Boryezka and Cera, L962; Mulrow,

Ganong and Boryczka, L963). The aeËion of angioLensín on the zona glomerulosa ís to stimulate the biosynthesis of aldosterone (Kaplan 1964; Bledsloe, Island and Liddle, L966)" The renin angíotensin system ís therefore clearly ímplí- eated as a factor in the cont,rol of sodium homeostasis through its role as a regulator of the secretion of aldosterone. It is also possíble that the renín angiotensin syst,em is ínvolved íntrarenally Ín the control of sodium excretíon. Thurau, Schnermann, Nagel,

Horster and tr'lahl (1966) suggested that this system may acÈ as an íntrarenal sodium conserving system. This hypothesis remains to be proved. Angíotensín in low doses admínístered to both man and anlinals is antidiuretÍc and anÈinatriuretic, but when administered to animals in large doses, it is diureÈic and natríuretíc (Louis and Doyle, L964> " A similar reversal- of the antínatríuretic resPonse to angíotensin is seen ín human subjecËs in whom hígh endogenous levels of angiotensín are present (Laragh, Cannon, Bentzel, Sícínski and Meltzer, 1963) or following ínfusíons of very hígh doses of angiotensin (Louís and Doyle , Lg64>. S,r"f, a bíphasic action of angiotensín on sodium and water excretion is dífficulË to ínterpret. Louis and Doyle (1964) postulate that ín low doses angíotensín ís antínatríuretic by virtue of íts vasoconstríctor 22 action which reduces glomerular filtration rate. They posËulate that it also has an inhibítory action on the tubular transPorÈ of sodium" I,lith increasing doses of angiot.ensín, Èhis inhibiËory act.ion becomes relatively greater and overrides the acÈíon of angiotensin in reducing sodíum excretíon as a result of the reductíon in filtratíon rate which, moreover r progressively dirnínishes with increasing dose levels of angiotensin" Angiotensin has been'implicated as an inhíbítor of the proxímal Èubul-ar transport of sodium by Leyssac

(1965), alËhough this \^ras not, substant,iated by Thurau, Schnermann'

Nagel, Horster and trrlah1 (L966). using stop-flow techníques, vander (1963) demonstrated inhibition by angiotensín of sodíum transport in the distal Èubule, and more recently an ínhibítory action of angiotensin on the ascending limb sodíum pump has been described by Healey, Ellíott and Pearce (i969). since the renín angíotensín system is involved ín sodium homeostasís, it is not surprísing to fínd that it is ínfluenced by changes in sodium balance. Sodium depletion and repletion (Veyrat, de Champlain, Boucher and ä"rr""t , Lg64; Genest, de Champlaín, Veyrat, Boucher, Tremblay, SÈrong, Koiw and Marc-Aurèle, L964; Brown, Davíes, Lever and Robertsonr L966, respectively) are assocíated wíth elevations and depressions ín plasma renín levels. Tobian (L964, suggested that renin release ís stimulated by changes ín afferent arteriol-ar tone, Many of the faeÈors kno\'m to 23 effect renin release could be ¡nediated by such a baroreceptor mechanísm; for example, release of renín by a reduction ín renal perfusíon pressure (Skínner, McCubbin and Page, 1963, 1964) and sympathetic nerve sÈímulatíon (Vander, 1965) could affect afferent arteriolar tone and thus release renin" Sodium depletion by reducing círculating blood volume, and sodium loading by expandíng blood volume, would also affect the degree of streÈch of the afferent arÈeriole (Vander and Luciano, L967a). However, not all experímental findíngs can be explained on the basís of such a baroreceptor mechanism as has been postulated by Tobían. In Particular' Ehe action of diuretics in suppressing the release of renín índuced by aortic clamping (Vander and Miller, L964) and Ëhe dissociatíon of the haemodynamic changes induced by norepinephrine from sodlum excretion (Nash, Rostorfer, Bailie, I,{athen and Schneider, 1968) are not satísfactorily explained on the basís of a barorecePtor mechanism alone" Moreover, those changes which woul-d stímulate a barorecePtor mechanism would alter eiËher Èhe sodium load or the sodium concen- tration at the macula densa" This applíes also Èo the potentiaÈing acËion of the íntact sympathetic nervous system on renín release

(Vander, L965; Vander and Luciano, L967b3 Bunag, Page and McCubbin,

L966; Hodge, Lowe and Vane, L966), alLhough a dírect action of the renal nerves on rrenin-secretíngr cells cannot be elíminated" The proxírniËy of the macula densa to the juxtaglomerular cells and the 24

realísation that Èhe sodíum load and concentration in the Ëubular fluíd varíes wíth variatíons in glomerular filtratíon rate has led. to the hypothesis Ëhat renin release is controlled by a t sensÍng devicer at the macuLa densa. This remains to be proved, as does the mechanisrn by which renín release is stímulated. It is not osmolalíÈy, since both hypertoníc mannitol, which increases the distal tubular osmolaliËy, and hypertonlc salíne, whích decreases the distal tubular osmolality (Gottschalk and Mylle, 1959), both suppress renín release (Vander and Miller, 1.964). Vander (1967) suggested Èhat it is the sodíum load at the macula densa which stimulates renin release. This hypothesis would explain the suppression by díuretics of renin release induced by aortíc clampíng" Diuretics which inhibiÈ reabsorption of sodíum proxlmal Èo the macula densa and so increase the load of sodium reachíng the macula densa Ëherefore suppress renin retrease. Recent studies by Landwehr, Schnermann, Klose and Giebisch (1968) on t,ubular fluid compositíon during a reduction of glomerular fí1tratíon rate suggest that an inverse relaEionship exísts between sodíum concentration of disEal tubular fluid and the glomerular filtration rate; Ëhat is , íf the glomerular fíltration rate falls , the sodíum concenÈration of the early dístal Èubular fluid is high as a result of íncreased abstracÈíon of wat,er from the loop of Henle

(Schnermann, 1968). Such elevatíons ín sodíum concentraÈíon may then 25

be postulated to stimulaÈe the release of renín. Thurau, Schnermann, Nagel, Horster and I^Iahl (1966) have suggested, on Èhe basis of proximal tubular collapse tíme studies, that when high concentraÈions of sodium are perfused through the dístal Èubule, Ëhe release of renín whích occurs acts via angiotensín to reduce glomerular filtration raÈe as measured by proxímal tubular collapse time. However, this evídence is indírect" A recent hypothesís for auto- regulatíon by renin also based on changes in sodíum concent,ratíon at the macula densa as a stimulus for renin release has been suggested by BritÈon (1963). Britton suggests that a high flow rate along the loop of Henle increases macula densa sodír¡n concentration act,ívating a carríer molecule which facílitates transPort of renin to the afferent art,eríolar cytoplasm, initiating local autoregulatíon" He postulaËes Èhat the release of renin into the renal venous effluent

may not reflect the cytoplasmic changes in renín content "

It can be seen, therefore, that theories concerníng control of renin release from the kídney are controversial and the íntrarenal role of the renín angíoÈensín system remaíns purely speculative" 26

SUMMARY

The association between the kídney and hypertensíon was recognised by Bright in 1836 who suggested that the diseased kidney may produce a circulatíng pressor substance.

The discovery of such a pressor subsÈance was made by

Tígerstedt and Bergmann in 1898. They called thís substance renin" In the early part of this century it was found difficult to isolate a pressor compound from the kídney. A revival of interesË ín the role of the kidney in experímental hypertension followed the advenÈ of a reproducible experimental model (ColdU1att, Lynch, HanzaL and Summervílle , L934). Subsequently renin was ísolaÈed from the kídney and its pressor actions studied. Renin was found to be an enz)¡me (Braun-Menêndez, Fasciolo, Leloír and Munoz, 1940; Plentl and Page, 1943) which acted on an crr-Blobulin in plasma to produce the active pressor compound, angíotensín " FurÈher work revealed that renín splits the leucyl-leucyl bond in the renín substraÈe molecule, líberatíng a decapeptíde (angiotensin I) whích ís splít by a chloríde-dependent enzyme present, in plasma to the active octapeptíde

(angíotensín II). Amino acid analysís and deËermínation of Èhe amino acid sequence of angiotensín led to its synÈhesis (Bumpus, Schwarz and Page, 7.957; Schwyzer, IseLin, Kappeler, Riníker, Rittel and

Zuber, L957).

Angiotensin was found to have both direcË and indirect 27 acEíons in raising blood pressure and also to be an imporÈant factor in the control of aLdosterone secretion. Renin -is located in the juxtaglomerular apparatus, possibly in the granulated cells lining Ëhe afferenÈ arÈeriole or in the macula densa portíon of the early distal Èubule. The release of renin into renal venous blood is sÈimulated by experímental sítuations which threaten renal perfusíon" Therefore, renín is released under situations of sodium depletion, haemorrhage and constriction of the renal vessels.

Two theories have been proposed t,o explaín the mechanism of renin release. Tobian (1964) has suggested Ëhat the granular cells lining the afferent art.eriole are stretch receptors, and a reducÈion ín Èhe degree of stretch in the afferenÈ arteriole, such as ís induced wíÈh small changes ín renal perfusíon pressure, releases renín" The work of Vander and Míller (L964) led Eo Èhe conclusion that such a st.retch receptor mechanism as proposed above could not accourit for the inhibiÈion of renín release that occurs when diuretics are administered in the presence of a reduced renal perfusíon pressure (induced by aorÈic clampíng) " Vander (L967) suggested that the toÈal sodíum load delívered Èo the macula densa region of the distal tubule provides the mechanism controllíng renín rel-ease, Increases in sodíum load would inhibit renin release; decreases in sodium load would sÈímulate it. Thurau, Schnermann, 28

Nagel, Ilorst,er and lüahl (1966) proposed Èhat an íncrease in sodíum load to the macula densa may be inversely relaËed Ëo sodium concentration, as has been demonstrated when glomerular flltratíon rate ís reduced (Landwehr, Schnermann, Klose and Gleblsch, 1968),

Èhís phenomenon beíng due to the increased reabsorptíon of water in the ascending Iímb of the loop of Henle aÈ 1ow flow rates" The presence of renín-like enz)rutes in tíssues other than the kídney has been descríbed. Renin is present in arÈeríal walls,

Èhe submaxÍllary glands of the whiÈe mouse and in the foetal membranes

and maÈernal reproductíve tíssues. 29

INTRODUCTION

The work described in this thesis is based on the methods of assay of plasma renin activity, plasma renin concentratíon and renín substrate descríbed by Skínner (L967). Using these methods, ít has been possíble to sËudy the distríbutíon of renin in tíssues and body fluids deficient ín renin substrate. Thís ís possible sínce levels of renin can be detected and quant,ítated using an artificíal substrate prepared from nephrectomized sheep plasma" Since ít ís also possible to símultaneously estímate trenin actíviËyr (which, as described prevíously, is dependenË upon both the levels of endogenous renín and renin substrate) , plasma renín concentration, and renin substrat,e levels, studies have been done to determíne the relatíve contríbution of both enzyme and substrate to the íncreased levels of renin act,ivíty observed ín several physíological sítuations. Fina11y, studies were undertaken Èo deËermíne the role of tachyphylaxís in the normal- constríctor response to angiotensin and to see whether Èhe peripheral vessels contríbuted Ëo the reduced pressor response seen in pregnant rÂromen followíng the administratíon of exogenous angíotensin" The thesis is therefore dívided into the following sect,ions:

(1) A descríption of the meÈhods used for Èhe preparation and 30

and bío-assay of renín and the methods for measuremenÈ of the vascular response to induced drugs.

(2) The quantíÈation of renin leveLs ín human uríne and factors controlling the excretlon of renín inÈo human urine" (3) The role of renln and renin substraËe levels in the increases in plasma renin act.ivity observed following adminístration of the natríureÈic agents chlorothíazide and spíronolactone¿

(4) The presence and quanËítation of renín in human maternal and foetal tfssues. (5) The role of renln and renin subsÈrate levels fn the changes in plasma renin activíÈy induced by: (a) the normal Ílenstrual cycle; (b) administrat,ion of oral contraceptíve agents; (c) pregnancy. (6) The índucÈíon of Èachyphylaxís to angiotensín ín normal sub ject,s. (7) PerÍpheral vascular reaetivity to angiotensin in normal pregnant üromen as compared Èo normal non-pregnant l,rromen" 31

SECTION ONE

METHODS 32

METHODS

TNTRODUCTION:

Since renin is an enzyme, Èhe símplest \^lay to determine the amount of renín present in a sample ís to measure the reacÈion velocíty. Thls is the rate at which angíotensín is formed from renin acËíng on renín substrate under st,andard eonditions of temperature t pH, subsÈraÈe concentratíon, and freedom from end-product ínacÈívaÈion.

Angiotensin is mosÈ commonly measured by bio-assay" The blood pressure response of an anímal to r¡nknown concenÈraÈions of

angiotensin is compared with Èhe blood pressure response obtained to known concent.rations of angiotensín" Animals used include Èhe rat

(Peart, 1955) and the dog (Haas and GoldblatÈ, 1967) . Angíotensin I is formed from the action of renín on renin substrate (Skeggs, Marsh, Kahn and Shurm,uay, 1954). Therefore, íf

comparison is Ëo be made usíng angíotensin II as sÈandard, eiÈher

converting enzyme must be presenÈ in the incubaÈíon míxÈure ín

excess, or an animal preparaËion musÈ be used whích will convert injected angioÈensín I to angiotensin II. Estimations of renln ín plasma can be broadly dívided into

tr^ro groups: Ehose whích measure Èhe renin f aeÈivítyt of plasma, and those which measure Ëhe plasma renin 'concentratíont" Plasma renín actívíÈy is defíned as the rate of formatíon 33

of angiotensín from the action of endogenous renín on endogenous substrate in a system in which angiotensinase activity is inhíbíted.

The amount of angioËensín formed, Èherefore, depends upon the concenÈraËíons of both enzyme and substrate and does not provide an accurate quantit.ation qf renin levels, although ít may be a reflection of the in uíuo ability of plasma to form angíotensín. Plasma renín concentratíon refers to the raËe of formatíon of angiot,ensín formed from the acÈíon of renin on a constanÈ (excess) substrate concenÈration. IË permíts, therefore, accurate quantítation of the levels of enzyme present, províded there are no essential unaccounted cofacÈors in Èhe system. The methods described ín this Èhesis for the assay of renín in plasma, urine, amníotic fluíd and tíssue extracts are based on the simplífied methodology described by Skinner in 1967. Renin levels are det.ermined indirectly by determl-naÈion 'of the amount of end-product formed following incubation of renin, either wíth endogenous substraÈe, giving an estimaËion of the so-called trenín activityt, or the íncubaÈíon of renin wíth a constanË level of an artificíal substrate (prepared ín this system from nephrectomized sheep plasma) , ín which case the 'renín concentratiod of the sample is determined. To avoíd Ëhe interferíng aclion of endogenous s'ubstrate ín determíníng renin concentratíon, endogenous substrate

ís denaÈured by a low pH treatment which does not adversely affect 34 renin" End-product ínact,ívatíon is prevented by low pH Èreatment and the addition of ethylenediamine-tetra-aceEíe acíd, dísodíum salË, to the system. The technique, as described by Skinner, is simple and does not ínvolve gornplic4ted exÈraction procedures. The recovery of renin is high, Pyeparation of pLasma for pLasma renin eoncentration and aetiuity estímations: Heparin (10 or 20 units/rnl-) was used as anÈícoagulant and added to samples ímmediately aft,er withdrav¡al- of blood from an antecubiÈal vein. These concent,ratíons of heparín do not interfere with renin-renin substrate reaction (Sealey, Gerten, Ledingham and Laragh , Lg67) in this system (Skinner, unpublíshed observatíons) . The bl-ood was chilled and spun at 3O0O r.P.m. for 30 minutes at lOoC"

The plasma obt,ained was dialysed f.ox 24 hours (Figure 1-1) in size 8/32 Visking cellophane casings agaínst 5 lítres of Buffer I or II'

wíth one change, aË a ÈemPerature of 3oC.

To estimate pJ-asma renín concentration, samples were heated

to 32oC after díalysis to pH 3.3 (Buffer I) for one hour. This procedure írreversibly denatures the renín substrate, but renín ís unaffected (Skinner, L967). Followíng dialysis for 24 hours to pH 7.5 against 5 litres of Buffer III with one change, Ëhe samples were decanted, and neomycin sulphate (2 ng/nL), a bacteriostatíc agent, and Trasylol (1OO units/m1), a kallikrein inhíbítor, added" FIGURE 1-1

Flow sequence for handl-ing plasma for estimation of plasna

(PRA) renín concent,ration (PRC) and plasma renin aeÈivlty "

5 ml cold p lasma

Dialyse agalnst pH 3.3 Dialyse against pH 4.5 amino-acetic acl-d-HCl ctrÈrlc acid-phosphate buffer and EDTA 0"005 M buffer and EDTA 0.005 M

t at 32o

60 ¡nins mlns

Dialyse agalnst pH 7.5 disodlun phosphate-dfhydrogen phosphate buffer (0.001 M EDTA)

I Add lrasylol (1OO untt,s/mf) and NeomyeJ.n (2 mg/m1)

I Add standard substrate I

I Ineubate at 37oC and measure veloclty of ångiotensln formatton by bf,o-aesay 1n the rat

Plasma RenLn ConcenÈration Plasma Renln ActfcftY 35

Restoration of the original volume \¡ras accomplíshed by the addition of cold Buffer III.

Nephrectomízed sheep substrate was added to the samples (ratio of two parts of plasma to one part of substrate) and the

samples íncubated at 37oC, or stored at -zOoC.

Plasma for renin acLívíty estimatíon üras dialysed in a

similar manner to an initíal pH of 4.5 (nuffer tt) and heated to 32oC at thís pH for 30 minutes. Thís treatmenÈ denatures angíotensín- ase, but does not, denature eiÈher renin or renín subsÈrate (skinner,

1967). Plasma was then díalysed to pH 7.5 against Buffer rrr and decanted; neomycín sulphate (2 ne/nL) and Trasylol (100 unirs/m1) were added and the voh¡ne corrected by the addition of cold Buffer

III. The sarnples \¡rere then incubated at 37oC, or stored at -20oC. Estimatíon of nenin çubstz,ate LeueLs:

Endogenous renín substrate l_evels were determined by íncubation of pH 4.5 rreated plasma dialysed ro pH 7.5 (i.e" ractivity plasmar) with €m excess of human renal renin. The concen- trat,ion,. of renin added was sufficient to drive the reaction to completion 10 minutes in " Four parts st.andard renín were added to one part of plasma and Èhe mixture íncubated for 30 minutes at 37oc. For estimation of low substrate concenÈrations as may be found in amníotic fluíd and urine, t\^7o parÈs of renín were added Ëo one part of substraÈe, giving 36

a sensítívity limiÈ of 30 nglml of angiotensín of Èhe origínal plasma.

Detev,mí,natíon of the renðn actitsity of renin coneentTatíon: Both renín acÈivity and renín concentraÈíon were determined by the anount o{ pressor materíal (angiotensin) formed following incubatíon of Èhe sarnples at 37oc for varyíng lengths of Èíme"

The pressor matería1 formed hlas assayed by bío-assay in the

ganglion-blocked rat (Peart, 1956) "

Bio-as s ag preparat'íon : Al-bíno male rat,s (200 gn) were used" Anaesthesia was

Índueed with sodium pentabarbítone (Nembutal) 60 mg/kg Ínjected intraperítoneally. Ganglíoníc blockade was effected by penÈoliníum tartraËe (Ansolysen) 2.5 nLg/L00 gm injected subcuÊaneously (Peart, 1955) " A tracheal cannula was ínserted Ëo ensure an adequaEe aírway. A nylon catheter (No. 1, Portex tubing, bore 0.75 rnm, exËernal

diameter 0.94 rnrn) was ínserÈed ínto the jugular vein for ínjectíon of

standard and unknown solutions (Figure L-2). ArÈerial pressure responses \^Iere recorded with either a saline-fíllqd glass eannula ínserted into the carotíd artery and

connected to a Condon manometer (Figure 1-3), with an ink-wríting pen reeording on a Palmer kymograph (speed 8 mur/min), or wiËh a saline- filled nylon catheÈer (tqo. 1, PorËex tubíng) inserted ínto the carotíd artery whích hlas connected through a three-way taP to a Southern InsËruments capaciËance transducer (A) (Figure 1-4). The ouÈput of olln ryrlngo

vrln csnnulo

Fíg. 1-2 Díagram of a raL prepared for bío-assay. Catheters were inserËed into the jugul-ar vein for ínjection of standard angíotensín and test solutíons, and into the carotid artery Èo record the blood pressure respon.se to intravenous injecÈíon of samples. \ -¡¡l-r,i

re Ð' <=æ'-}-a

I-ig-:-l--3 A bío-assay preparation in whích the blood pressure responses \{ere measured with a saline-filled glass cannula connected to a Condon manometer. By means of an ínk-r¡ríting pen, Ëhe resPonses \,vere recorded on a Palmer kltnrograph. ¿,

: f?,

I

Fíe. I-4 A bio-assay prepaîation ín whích the blood pressure responses r¡Iere recorded by means of, a SouËhern Instruments capaeít.ance transducer (A). The ouÈput of the transducer varies Ëhe frequency of an oscillator (B) and the frequency change is detected by a discriminator. The output of the discríminator circuít passes through an íntegrating circuít (C) and is recorded by a Texas recorder (D). 37 the transducer varíes the frequency of an oscíllator (B). Ttris frequency change was detected by a discrímínator. The output of the discrtminator circuit goes through an íntegrating círcuit (C) before beíng fed into a Texas recorder (D). The íntegraÈíng circulÈ damps out Èhe pulse pressure and so gíves a mean Pressure Ërace' The recording system was calibrated to record a one nm Pen deflectíon

for a one mm Hg ríse in pressure. The paper speed was 6 mm/min. Assay of pressor materLaL: The pressor contenÈ of each sample following incubation hTas

deÈerntined by bracket assay, the amount of pressor maÈerial in each

sample being bracketed beÈween two doses of standard synthetic p-asparaginyl-angiotensin II (Hypertensin, clba) (Figure 1-5). All

ínjectíons \^7ere 0.1 rnl or less and were folLowed by a wash in of 0.05 mt of 0.15 M salíne.

Eæpz,ession of resuLts : The pressor conEent of each sample after incubation for

known períods was determined. The íncubatíon mixture was sampled at three appropriaÈe Èime intervaLs duríng íncubatíon and, wíthout further extractíon, each a|íquoÈ \^las assayed for the pressor material generated. At least t\^lo rats were used for assay' Following deÈermination of the pressor conÈent in Èhe three aliquots, a velocíty curve of the amount of angíotensin formed to the time of íncubation \¡Ias drawn and the iníËial velocíty was I 'v5nNtãovl¡rll svx3l'Nol.snoH'o!IVUo¿aO3Nlllll ÊrN3n nu.tsNt vx¡1.

-- -

I I I I I I u 6 u I u to u I

Fls. 1-5 BrackeÈ assays of plasma (U) whích conÈained unknor¡n amounts of angiotensin, usíng $-asparaginyl angiotensín II as standard. (tlumbers refer to nanograms of standard angíotensin a ÍnjecËed. ) Upper trace: Bracket assay obtained usíng capaciËance Ëransducer and Texas recorder' Lower trace: Bracket assay obÈaíned usíng a Condon mercury manomeÈer and a kymograph Ëo record the blood pressure resPonse' 38

caleulated (Fígure 1-6) (Lever, Robertson and Tree, L964; Brown,

Davies, Lever, Robertson and Tree, L964>. The results are expressed as follows:- PLasma yení,n actí.uity Ís expressed as the rate of for- mation of angiotensín ín nanogramslmt/hr, aË pH 7"5, and 37oC.

PLasma renín eoneentratíon is expressed ín unÍts/ml, where one unít of renin ís defined as the amounÈ of renín whíe.h, when lncubated at 37oC and pH.7.5, generates angÍoËensín at Ëhe raÈe of one nglmlfhr from a eoncentratíon of standard sheep substrate of 530 nglrnl.

Sínee the addítion of sÈandard sheep substrate diluÈes Èhe renín exÈTact by 1lS, to obtaín the orlgi.naJ- renl-n eoncenËratÍon the measured veloeíry is multipLLed by 3/2" Renin subsLrate ís expressed as nanograms of angíoÈensin/ml, generated fol-lowíng incubaÈion of pl"asma (pret,reaÈed Èo pH 4"5) wíth an excess of human renal renín at pH 7"5 and 3ToC" Stqrñayd buffens:

Buffer I - AminoaceËie acfd - HCj- buffer (pH 3.3)

Aminoacetíc aeíd - 0.05 M

HCl - 0.01 M

EDTA - 0"0051 M

NaCl - 0.0949 M I

^ 80 -E \ u, tr l, óo z -tl t¡lz40 Il- 920z

o 24 6 8lot2l4 TIME (HouR,sl

FÍs. I-6 DetermínaËíon of initial velocíty, í.e. the amount of angÍoÈensin formed Ln ng/mL/hr following bracket assay of the concefitration of angiotensin ín a sample formed aftet varying periods of íncubatíon aË 37oc. 39

Buffer II - CiËric aeíd-phosphate buffer (pH 4.5)

Cítric acid - 0.00278 M Na^HPo,z4¿ "12H^o - 0.045 M EDTA - O"OO51 M

NaCl - 0"0821 M Buffer III - Phosphate-phosphate buffer (pH 7.5)

O.OL22 M NaH^PO,¿4¿ .12H^O -

Na^HPo,"12H^o24¿ - 0.0867 M EDTA - O"OO1 M

NaCl - 0"075 M

Preparatíon of human v,enaT venin (Fi,g" L-7): The Èechnique used to extract renin from the kídney ís essentially that described by Brown, Davíes, Lever, Robertson and Tree (1964) . Approxímately 6 Kgms of kídneys were demedul-lated, decapsulated, sliced and minced in a tr{aring blender. The mínced tissue was taken up into 10 liUres of dístilled water, sÈirred con- tínuously for 24 hours , aE a t,emperaEure of 8oC and then deep fxozen"

The I¡Ias Ëha\^7ed filtered, and Ëhe pH of the suspension ' fíltrate adjusted to 2"9 with 20% trichloroacetic acíd. The filtrate was sÈirred continuously during additíon of NaCl 52 gn/L, and Èhe pH was readjusted Eo 2.9 wíth 207" ExLchloroaeetíc acid. The mlxture was then stored for 24 hours at 8oC" It was laEer centrifuged, filtered through l,,Ihatman (No. 50) filter paper Ëwice, and suffícíent ammoníum FIGURE L-7

Preparation Hr¡nan Renal Renín

6 Kgms kidney, demedullate, decapsulaÈe Slice and mínce

I

I 10 Litres dístílled l¡later, sÈír for 24 hrs at 8oC; deeP frozen

I Thaw, I filter pH 2.9 wíÈh 20% ttríchloroaceÈl-c acid

I 52 gmll. NaCl, pH 2.9

I

I

Stir overnight Add (NH4) z so4 r, 757. saÈuratíon I

I Precipítate taken up'ínto distílled \^later Dialyse,agaínsl pH 3.0, amíno:acetíc acfd - HC1 buffer, 0.005 M F,DTA

l I Heat 30oi, 30 míns

I I Dialyse against pH 7.5, disodium phosPhaËet dihYdrogen PhosPhate buf fer Decant; add Trasylol (100 uníts/ml) , Neomycín sulphate (2 me/ml) t+O

sulphate added Ëo give a 5A7" saturated solutíon" Thís was stírred overníght, aÈ 8oC, eentrifuged and ammc¡níum sulphate added to the supernatant to bríng Ëhe saÈuratíon up Eo 757.. The precípitate thâÈ formed was taken up into dístÍlled vüater, díalysed in 8132 Viskíng cellophane easíngs againsÈ pH 3"0 amino-acetíc aeid-HCl- EDTA buffer

(0"005 M EDTA, made 0.16 molar with NaCl) and heated at thís pH for

30 minutes. It was then díalysed against pH 7.5 (Buffer III) , deeanted, and neomyein sulphate (2 me/mI) and Trasylot (100 unfts/mt) added.

Renin prepared in this way had a protein eonceneratíon of 4 mg/mlrwas free of endogenous pressor aetivity, but exhausted plasma subsÈrate levels during 10 mínutes íncubaÈion at 37oC. AngÍotensinase was present, suffíeienL to produce 507" destruction of, added synÈheÈie p-asparaginyl angiotensin II at the end of 3 hours íncubatíon aÊ 37oC" However, greater than 802 reeovery of added angíoËensinwasobËained at the end of 30 ml-nutes íneubaÉion, making Èhe renín preparatíon adequate for renín subsËrate det,erminat,ions. Preparatí"on of renin substrate:

Renín substrate r¡ras prepared aecording to Ëhe method of

Skínner (L967). An er^re !üas heparinized (7500 unÍts fntravenously) and exsanguinated 6 days after bílaeeral- nephreetomy.

The plasma hras separated by eentrífugation aË 3000 r,p,m. for 30 minutes and díalysed to a pH of 3"9 agaínsÈ cÍËric acíd- 4L

phosphate buffer made 0.16 M wíËh NaCl and conËainíng 0,005 M EDTA,

The plasma \Âras then heated for 45 minutes at 32oC, dialysed agaínst pH 7 "5 buffer (¡uffer III) , and srored ar -2OoC after addítíon of neomycin sulphate (2 nglnL) and Trasylol (100 units/rnl).

A second batch of renin subst,rate vras prepared in a manner sirnílar to that described above, except Ehat heparin (10 uníts/m1) was added to Ëhe blood followíng removal from the sheep, and the plasma obtained was dialysed to pH 4"5 (Buffer II) and heated at pH 4.5 for 30 mins prior ro díalysis ro pH 7.5 (Buffer III)"

Al1 renin subsËraÈe preparaËions \,\rere angíoÈensinase free and free of endogenous pressor activíty up to and íncludíng 100 hours of íncubat,ion. VariaÈions ín actual concenËration of the renin substrate were obtained, but all results expressed in thís t,exÈ are corrected t,o Èhe rate obtaíned usíng a substraËe concenÈratíon of 530 ng/ml, unless stated oËherwíse.

Preparation of qntirenin :

Antirenín \¡ras prepared according to Ëhe method used by Skinner (1967). Rabbíts I^/ere immunízed wiÈh twice-weekly injections of 1:1 emulsíon of Freundrs íncomplete adjuvant and concenÈrated renal renin. A third rabbit was immunized wíth Freundrs íncomplete adjuvant alone, and 20 ml of blood was Èaken from the rabbit duríng an ínfusíon of normal saline Ëo suppress endogenous renÍn release"

Immunoglobulíns r^rere precipitated by the addition of 16%, 42

and then 142 sodíum sulphaËe, then dial-ysed against pH 4.1 cítric-

acid phosphate EDTA buffer (0.001 M EDTA made Eo 0.16 M wíth NaCt).

The plasma üras warmed to 32oC príor to dÍalysls to pH 7.5 against

standard Buffer III, centrifuged and. stored at -2OoC following the addítion of neomycín sulphate.

Prepatatíon of hwnan øngíotensin:

Human renal renin (4 mL) was added to angíotensinase-free human subsÈrate (i.e. plasma,treated by dialysis to pH 4.5, heated

and dialysed to a fínal pH of 7.5) and the mixture íncubated at 37oC

for 7 hours. UltrafiltraÈion yielded a filtrate contaíníng I Ug/rnl of

angíotensin, which \Áras stable when incubated alone but rapidly destroyed on íncubatíon wíth normal plasma at pH 7.5.

Detection of angiotens.Lnase actíuíty :

Human angiotensín and synthetíc g-asparaginyl angioÈensin II were added to EesE plasmas, urines and Èíssue exLracts ín concen-

trations of 100-200 ng/mL" Test solutions hrere consídered Èo be angiotensinase-free if greater than 807" of the added angiotensin was still- present after 24 hours incubatíon when tested agaínst the uníncubated control.

Venous occLusion pLethy smognaphg :

The technique of venous occlusion plethysrnography was used to measure hand and forearm bl-sod fl-ow and Eo deÈermíne the effects of angiotensin and other drugs on the peripheral vessels in man. 43

MeasuremenÈ of blood flow to a segment of a lirnb ís deter- mined by enclosing the segment in a rígid contaíner and occluding,

íntermíttenÈly, the venous drainage" Duríng the period of venous oeclusíon the increase in volume of the segment is proportíonal to the raÈe of arterial blood flow (Brodíe and Russell, 1905) " The upper limb can be partitíoned ínto either the hand (Lewis and GranË,

L925) or the forearm (Freeman, 1935), and the flow in each segment measured. Measurement of forearm flow requíres the exclusion of hand blood flow by arterial occlusíon at the wrisÈ (Grant and Pearson,

1938).

I¡Jater-filled pleÈhysmographs \^rere used in most experiments

(l'igure 1-8). These are simílar to the plethysmograph descríbed by

Greenfield (1954) and Greenfíeld, I,rlhitney and Mowbray (L963). The plethysmograph is sealed by means of an atÈached rubber sleeve or glove (Lewís and Grant, L925). It is temperature controlled, Ëhe correct \¡rater temperature being determíned to allo\¡r measuremenË of restíng flow (Barcroft and Edholm, L946). Intermittent inflation and deflation of the venous collectíng cuff is auËomated by means of a sequence Ëímer (Paton Industries, Adelaíde), electrically operated solenoid valves releasíng aír from reservoírs at constant pressure, the resefvoirs beíng filled from compressed air cylinders using consËant pressure valves.

The volume changes \¡rere recorded on a Brodie-Starling OC

r,ig.1-SAtemperaturecontrolled,waËer-fil]-edplethysmograph showing rubber sleeve and sealíng plates used in the rneasurement of forearm blood flow. 44

kymograph (l'igure 1-9), or by means of a P23 BC Statham transducer connected to a Ríkadenki pen writing servo recorder (nigure 1-10).

Calibration of the system r^ras carríed ouÈ by introduction of known volumes of aír into the sysÈem, or in experíments where Ëhe transducer \^ras used to record blood flow, by the íntroduction of known volumes of water into the plethysmograph, with the subject ín posítion. The flow was calculated by Ëhe method of Barcroft and

Swan (1953) (tr'igure 1-11) and expressed as ml/lOO m1 tissue/minute. In a few experímenÈs, a capacitance pleËhysmograph

(l.iilloughby, 1965; Fewíngs and l,,Ihelan, L966) was used to measure forearm blood flow (Figure 1-12).

Intz.a- ay teriaL {n fus ions : In order to examine the direct effects of drugs on the hand and forearm vessels, ínfusíons of the drug to be studíed were gíven into one arm vía intra-arterial infusion inÈo the brachíal artery at the elbow. Sínce the drugs are infused ínÈra-axEerLaLIy, concen- traÈíons of the drug sufficient to produce a local vasoconsErictlon or a vasodílatation are obtaíned, whilst dílution or destruction of the drug in Èhe peripheral tíssues prevents any systemíc effect. Therefore r âny cenÈral actíon of the infused drug is avoided as well as any compensaÈory acLíon of the cardiovascular sysLem Èo prèssor or depressor effects of ínfused drugs. By using the opposite non- infused hand or forearm as a control, varíatíons in flow due to *'ùj-

Fíe. L-g General- laboratory set-up showíng a subject lying on a couch prepared for measuremenË of hand and forearm blood flow' In the background is the Brodie-starling kymograph for recording the blood flows obËaíned, infusion plmp and sequence timer for moniËoríng venous occlusíon cuff inflaÈion and deflation' Fie. 1-10 A record of hand blood flow measured by venous occlusion plethysmographyusingwater-filledp1-eËhysmographs,andrecordedwith a P23 BC SÈatham Èransducer connected to a Rikadenkí rnulti-pen recorder. f--a{.' ..-I

Fie. 1-11 Flow measuríng apParatus superimposed on a venous occlusíon plethysmographíc kymograph Ëracing' a ql -t -)o-i' a f. / I

/

Fis.L_lzAcapacitanceplethysmographusedforrecordingforearm blood flow. 45 sympatheÈic activlty can be accounted for (lurr, L952). Infusíons were admÍnistered through a 22-gauge needle ínserted cenËripetally into the brachial arÈery at the elbow under local anaesÈhesLa (2% Lignocaine), Ëhe needle being taped into positíon and remaining in eitu for the duraÈion of the experiment.

The needles used were shorË bevel, but,tless needles, 3.2 cm long. They were connecÈed by a 30 cm length of polyÈhene Ëubing

(external díameter 0.75 mm, inÈernaI diameter 0.5 mm) to a 50 ml syringe driven by a constant ínfusion purnp.

Infusíons were given at a rate of 2 rnl/mín. The total dead space of the system between the arËery and Èhe syrínge \^7as approxí- mat,ely 0.17 ml, which represent,ed a delay of 5-6 seconds before the drug reached Èhe art,ery.

During control periods when no drugs were infused, a

coristant infusíon of NaCl (0.15 M) was gíven. NaCl (0.15 M) was also used as the vehícle for the drugs infused.

Eæpression of z,esuLts :

trrlhen the dose-response relationshíp was'required for

comparisons between the acËíons of dtfferent drugs on subJecÈs, Èhe percentage change in blood flow was determíned. This was calculated from the dífference between the flow during the two minutes príor to

the infusion of Èhe drug and the last two minutes of ínfusíon of Èhe drug, by which tíme the response to the drug had become stable. 46

Moreover, an additíonal correction could be made with regard to flow changes. Sínce the smaltr doses of the drugs lnfused Èo obÈain a response díd noË have any systemic action, the non-lnfused síde was used as a control. Thus, ¡{hen calculating the Percentage change produced by infusion sf a drug, allowance for spontaneous varíatíons

1n flow coul-d be made by assuming ËhaÈ ín the absence of the drug the t\,rro sides would mAínËain the same relat,ionship as in the pre- infusion period (lutf, L952). 47

SECTION Tl^lO

(A) THE OCCURRENCE AND ASSAY OF REN]N ]N NORMAL HUMAN URINE

(B) FACTORS CONTROLLING THE EXCRETION OF RENIN INTO URINE 48

(A) THE OCCURRENCE A}ID ASSAY OF RENIN IN NORMAL HIMAI{ URINE

Tn L942, Houssay, Braun-Menéndez and Dexter deËected renin- like aoÈivity in the urlne of dogs following inÈravenous infusions of large doses of hog renín.

In L964, Brown, Davies, Lever, Lloyd, RoberËson and Tree claimed to have identífied a renín-líke enzyme ín normal human urlne. Extrapolation of their results revealed that relatívely high concen- trations of this renin-like enzyme occurred ín normal human uríne. Thls fÍnding raísed the possibility of renín being secreted from the macula densa into the tubular fluid, and since angíotensin has been ímplícated ín control of sodium reabsorption by both the proxímal (Leyssac, 1965) and the dístal t,ubule (Vander, 1963), secretion of renín ínto tubular uríne could subserve some role in the intra-renal regulatíon of sodium reabsorptíon. A study was undertaken to ídentífy and quantitate the renín- like enzyme present in human uríne.

IUETHODS:

Renín in urine vüas quantítatívely assayed usíng the renin concentratíon meËhod developed for plasma by Skinner in L967. Figure

2-1 shows the flow sequence for the handling of a uríne sample. Up Èo 200 ml or more of freshly voided urine hrere concen- trated. by ultraflltratíon at 750 mm Hg , at a temperature of 3oC, FIGURE 2-L

Uríne samp le (ultrefíl-Èered íf neceésary)

Díalyse Èo pH 3. 3 againsË E.D.T"A. (0.005 M) overníght

Heat at 32o C for 60 mins

Dialyse to pH 7.5 agaí:nsÈ E.D.T.A. (0.001 M) overníght

Add Trasylol and Neomycin

I I

I Add standard substraËe

I

I Incubate at 37oC .rrd *.t"rrre velocity of angiotensín formation by bioassay in the rat 49

in síze B/32 Vískíng cellophane casíngs (Fígure 2-2>. The uríne was reduced to a volume of 10 ml or less. Followíng concentration, ít was subjected to the dialysis sequence described for the handling of plasma for renin concenÈration determínation (figure 1-1, 2-1).

Followlng dialysts to pH 7.5, the uríne samples were decanÈed, neomycin sulphate (2 mg/mL) and Trasylol (100 uníts/rnl) added, and Èhe volume corrected, íf necessary, by addítion of standard

Buffer III. Samples r^rere stored at -2OoC. Nephrectomízed sheep subsÈrate was added to samples ín the ratio of one part of substrate to thro parts of urine. These were íncubated aE 37oC. Alíquots of incubation mixËure were taken at three Èime intervals, the durat,íon of which ranged from 15 minuËes to

100 hours. These \^rere assayed for pressor actívity withouÈ further extraction on Èhe ganglíon blocked raÈ, using synthetíc $-asparaginyl angíotensin II as sÈandard. The initial velocíty of the reaction was deÈermined and the concentration of renín expressed in units/ml (vlettrods).

Deteetion of angiotensinase actíuitg in urine, Ehe preparation of standand sheep substTate, the estimat,íon of. r,enin substz,ate in uríne and preparatíon of antí-renin are described ín Sectíon I of this thesis.

Acid-denatured renin substrate \¡ras prepared by díalysing standard sheep substraLe to pH 3.3 against Buffer I and heating for \ ti

Fj'g. 2-2 Apparatus used for concentration of urine for renin estimation by ultrafiltration at a negatíve Pressure of 750 nrrn Hg. 50

30 minutes at this pH prior to dialysis ts Èhe required pH. Pepsin (Sigma, 3 tímes crystallized) was sÈored at 3oC in

0.01 M HCl 1n a concentration of I mg/rnl.

PoLy-L-Lysíne hydnobromíde (100 mg/ml, Sígma) MI¡tr 150,000

Ì,ras prepared and sÈored fn 0.15 M HCl.

u-ehymotnypsín (trIorthington, 3 times crystallízed) was prepared as requlred in pH 7.5 buffer.

RESULTS: oeewrence of renin in nozrnaL uyine: Príor to the addftion of renln substrate to urfne,

lncubation of. 26 normal urines, concenÈrated up to 44 tlnes and passed through the handlíng sequence (Figure 2-L), failed to generate

any pressor or depressor actívity over 72 hours incubation at 37oC.

Additlon of standard substrate, however, ?rovoked the línear generation of pressor materlal at rates of 0.5 to 22.4 ng angiotensln/ ml of incubate/hour. The rates varied wlth the exËenÈ of iniÈial conceRtratíon by ultrafilËratíon. Expressed as unlLs of renin rel-atíve to the unconcentrat,ed sarnple, normal uríne contained on the average 0.6 ! 0.4 unfËs/ml

(mean t SD) (range 0.1-1 .9, n=26). These levels r^rere approxímaËely 1/15 those descrlbed for pl-asma. Only urínes contafning less than 0.75 uníts/ml required concent,ration by ultrafil-t,ration. 51

Chav,acteristics of the end-prodaet prodszeed, by tþte aeti,on of renin in wine on nephreetomized sheep substv.ate: The materLal formed on íncubation of dlalysed urine with standard sheep substrate gave a pressor response on intravenous injection ínto the ganglion blocked rat which was simíIar in shape Èo that seen wíÈh injections of the standard B-asparaginyl angiotensin II.

The pressor material was díalysable, noL destroyed on boílíng, buË totally destroyed by íncubation with o-ch¡rmotrypsin at pH 7.5 and pepsin at pH 5.7 (fable 1, Appendix). It formed at a línear rate on incubation up to and íncluding 100 hours and during eonsumption of L20 ng of substrate (Figure 2-3).

The reactíon rate íncreased with a rise in temperature up.Èo (Figure and including 5Ooc 2-4). Above this temperature no Þressor , material formed. This was due to inactivatíon of both the enzyme contained in uríne and the sÈandard sheep substraÈe, sínce non-heated uríne faíled to generate pressor material on íncubatíon with standard substrate prevíously heated to 55oC for one hour, and uríne heated t.o

55oC simílarly faí1ed to generate pressor materíal when íncubated wiÈh non-heated standard substrat,e.

Pz.opertíes of the enzume present in uyine: Effeet of stbstt ate concentz,atíon: Similar curves were obtained for the effect of substrate changes on Ëhe reaction rate for renín extracted from plasma and 130

100 j = z(9 lrl É (JJ =, 50 =

=. (t) -.lJ-J oF (Ð =.

0

0 5 10 TItYlE (HOURS)

Figj 2-3 The linear formation of Pressor materíal wíth Ëirne following incubation of urine ¡nríLh standard sheep subsËrate at 370C. 70

L s\ \E o E 35 z -râ z l¡l ¡- o (!)- z

o 30 40 50 óo TET}IPERATURE of INCUBAl I ON

of Fie. 2-4 The effect of tenPerature on the rate of formation angiotensin by urlnary renin actíng on standard sheep substrate at pII 7.5 (APPendix Table 2) ' 52

uríne" The plot for urinary renin is the mean derived from two urine samples containing 9"6 and 4.2 renin units/ml (rigqre 2-5). Attempts to determine the Michaelis consÈant, (Kn), using eiËher a l¡loolf or a Líneweaver-Burk plot (Dixon and l,trebb, L964), yielded consíderable variatíon. The mean Km of 5 determinations on urine from 4 indíviduals was 247 ng/ml, the range beíng 140 to 347 r,g/mI (Fígure 2-6, TabLe 3,

Appendíx). The cause of this spread may be largely accounted for by the error incurred in measuring ínítíal velocíty at low substraËe concenËratipn. I,rÏith low concentrat,ions of the substrate, greaÈer than

30"/. of the substrate must be consumed in order to generate sufficlenÈ pressor material for accurate bío-assay. Effeet of enzyme concenty,atíon upon the rate of reaction beü¡een uninaty renin qrtd støndny,d substrate:

The concentration of renin, after serial dilution of enzyme prepared from urine containing both hígh and low concenÈratíons of enzyme, showed a linear relat,íonship Èo the vel-ocíËy of formation of angíotensin (Figure 2-7>. Such a díreet relatíonship exísts for plasma renin extracted by the same method, but not for renal renÍn (Skinner , L967). Effact of pH on the rate of forrnation of angiotensin by uz'ínarn¿ y,enin q,cting on standaz,d substyate:

Urínary renin used to investígate the effect of pH on the reaction betr^reen renin and sÈandard substrate r^ras exposed to a pH of 100 8 E oo o oo F (-) o % o _Jo l¡J 9O =¿ o =x o = o b.e E

0

0 550 1100

SUBSTRATE CONCENTRATION ¡¡e /mt,

Fis.2-5TheeffectofÈheconcentratíonofstarrdardsheep substrate on Ëhe rate of formation of angíotensín by urínary renin ( O ) and p1-asma renin ( O )' exPressed as a percentage renín of the maximr:m velocíty obtaíned. The plot for urinary 4'2 is the mean obtained from 2 urines containing 9.6 arlð' uníts /ml. o r9 o

o

vl

o

Fig. 2-þ lJoolf plots derived from the effect of standard sheep subsÈraÈe on the rate of formaËion of angíotensin (V) by 5 urines plotËed against the rate of formation of angíotensín divíded by substraËe concenrraLÍon. (Appendix Table 3(a), 3(b)) É, 100

(9= z, Oz,

Oæ.= tJ- 50 A =, z,ct) trJ A O (9 A z, A

IJ. O o o 0 o oo o(J LU 0 50 100

7" OF ORIGINAL RENIN CONCENTRATION

FiÉ. 2-7 The effect of the concentratíon of renín on reactíon velocity, usíng 4 separate uríne extracts conËaíning L2.0r 30.0' 63.0 and 147 ruríts/ml. OrdinaÈe : uncorrected measured rate. (Appendix Table 4). 53

9.6, after pH 3.3 treatmenÈ, to achieve denaturaÈíon of an angio- tensinase active below pH 7.0 and to denature pepsin. The effect of pH on reaction rate was found to be símílar to that of plasma renín (Skinner, 1967) (fígure Z-8>. The optimum pH of the system r^ras 7.8-8.5, the curve beíng flat between pH 7.0 and pH 9.0.

The actíon of ættibody to z.enaL z,enin on the aetiuity of the u.z,ínary enzame:

Incubation of uríne, Èo which one part of anÈibody to human renal renin had been added to four parts of urínerwíth standard sheep substrate, faíled to produce any pressor material, whereas the control dilut,íons of urine produced varyíng amounts of pressor material as,. díd urines incubaÈed with immunoglobulin conËaíníng no antíbody titre to human renal renin (11 experíments).

Accurate assay of renín in uríne necessitates that Ëhe followíng críÈería be fulfílled: (a) That no co-factors shoul-d be presenÈ in urine ín rate- lírniÈíng anounts.

(b) That the recovery of renin should be high and cons is ten È .

(c) fhat the syst,em is free of substances which wíll inactivat,e the end-product formed. (d) That the system ís free of the interfering action of 100 o o o o o o o o o o o o È o o (J o o o J o o lrl o o =¿ 50 o = x o = be o

o

0

4,0 5,0 6,0 7,0 9.0 9,0 10,0

PH OF INCUBATION

I.tg.'2-8 The effect of the pH of lncubation on the rate of format,ion of angÍoËensin by urinary renín ( O ) and plasma renin ( O ) actíng on standard sheep substrate. Results are expressed as a per cenË of Èhe maxímum velociËy (obtained at pH 8.0) (Appendtx Table 5). 54

pressor and depressor s-ubstances formed by the actíon of other enzymes duríng íncubation.

(d Laek of eo-faetor influence on uninary renin-yenin substz,ate reactíon:

The línear relaÈíonshíp beËween the concentratíon of urÍnary reni-n and Ëhe rate of formatíon of angíotensín made íÈ unlíkely that errors in quantitaÈíon due Eo co-factor influences were incurred. Further evidence of lack of co-factor ínvolvemenË in the formatíon of angiotensin from ufl-nary renin and standard sheep sub- strate was obtained by addíng a constant amount of urinary renín extracted from a ur:ine contaíning a high concentraÈíon of renin to 3 urínes conLainíng low corrcerrËlations of renin. The added enzyme ïras assayed aE 2.6, 2,7 and.:.r ,rrrlt"/ml, whíte the expected concentraEion was 3.3 units/ml . These differences r^rere not significant. FurÈher- more' the addition of renal renin ts 3 other uríne exËracts generaÈed. angíotensín at 40, 42 ar'd 44 ng/nL/hr, which \^ras noË signíficantly dífferent from the control sample díJ-uted wíth standard Buffer III

(40 nglm1 /nr¡ . (b) ReeoueyA of renin and z,epLicate estímations:

Although plasma renín was found to be stable on exposure to the pH treatment used in thís study .to denaÈure endogenous subsËrate and angíotensinase (skínner , L967), it was necessary Ëo ensure that the urínary enzyme was also sÈable and, furthermore, that r.r,ir, r." 55

not lost by ultrafilÈration undef pressure " Stability of the enzyme Èo low pH treatment was established

by recyclíng 4 tímes a pooled sample of urine Èhrough the entíre neLhod, excludíng ulÈrafiltraÈion, and estlmating the fall in velocity of angíotensin formation after each eycle of the díalysis sequence. velocity measuremenËs obtained were 2"8, 2.5 and 2.3 ng/nr/ hr after the first, thírd and fourth cycles, respectívely. Possíble loss during ulËrafíltratíon was ínvesËígaÈed in

two ways. Firstly, a sample of poored urine r^ras concentraÈed, dialysed and treated as in Figure 2-L, and then díluted to the original volume, re-concenÈrated and retreaÈed. The velociÈy of angiotensín formation was the same after each step, beíng L.2 ng/n]-/lnr.

Second1y, I ml of stock human renal renín was added to 200 rnl of urine and, afËer saving an aliquot, the remainder \^ras concen- Èrated, and both the concentraÈe and the ultrafiltrate assayed for renín content.

The ultrafílÈrate failed to generate any pressor materíal on incubatíon wiÈh renin substraËe, whilst, the concentrate generated angiotensin at the raÈe of. 366 ng/ml/hx. After correctlon for Èhe

6.2-foLd concentration step, the actual rate of 59 nglmL/hr was not signlficantly different from Èhe raÈe of the unconcentrated aliquoÈ

(56 ng1rnl/hr) , s6

DupLieate estimations (TabLe 6, Appendiæ):

(a) Rat assay variatíon:- Repeat assays of the same

samples on the same rat êfter short íntervals of tÍme produced a mean dif ference in velocitl-es of 4.77. (t 6.0%, n=8).

(b) RepeaÈ assays of the same sampLe on dl_fferent rats

after storage for r¿eeks or months at -20oC gave a rnean difference 1n velocities of 16% Q. ttZ, n=13).

(c) Duplicat,e assay of the s¿rme s¿rmples, usíng stored uríne and passing it through the method for the second tírne after intervals of days or weeks, gave an assay variabilÍÈy of 26% (!L9%, n=l 3) .

Thís rather large assay varíability may be a reflection of the extremely 1ow levels of renin found in urine which makes accurate quantítatíon of these levels more díffícu1t. ( e). Ereedotn from end-pz,oduet inaetiuation: ' Angiotensínq,ses present in norrnaL w,ine: The followlng experíments demonsËrate that the angíotensinase actívíty in uríne is due to Èhe presence of at leasË thro separate enz)rmes.

Urine samples to be tested for angÍotensínase activíty were dialysed against a range of buffers of pH 7.5 to 3"3, both ín the presence and absence of EDTA (FÍgure 2-9). The samples r¡ere heated for one hour at Èheir respectlve pH and then dialysed to a fínal pH 100 cJ)É. OJ J 1 C\¡ 11 + F E.D.T.A,

=. ct) lrl=, 50 oF (D z.

L¡- - E.D.T,A. o

É. lJ.J o(J U.JÉ. 0

be 3,0 6,0 8.0 PH OF INITIAL DIALYSIS

Fis. 2-g The effect of the pH of initial dialysÍs and the presence of EDTA on Èhe activity of angiotensinase ín uríne, tesËed by íncubation of urine with ß-asparaginyl angíotensín II ar pll 7.5 (Appendíx Table 7). 57 of 7,5. A concehtration of S-asparaginyl angiotensin II (250 ng/m1) was added to the samples and an aliquoÈ of the mixture incubated for

24 hours. Comparíson wíËh Ëhe unincubated control showed that an angiotensinase \^ras present ín urine, acÈíve aÈ pH 7.5 and resístanÈ to exposure to a pH greater than 3.3. This enzyme I¡Ias, however, inacEivated by a pH of 3.3 or by Ëhe addítíon of 0.001 M EDTA to Ëhe sys tem.

Although an iniÈíal pH treaÈment of 3.3 or the addition of

EDTA to the sysËem inactivated angíotensínase in samples íncubated at pH 7.5 ar above, this hras not, the case with samples íncubated at a fínal pH of less than 7.0. Thís fínding suggested the presence of a second angioËensínase in urine. Thís second enzyme was found t,o be denatured, however, by dialysis and heaÈing for one hour at plt 9.6 against 0.16 M glycine-NaOH buffer príor to díalysls Èo the fínal pII for incubation wíËh 250 nglml of B-asparaginyl angiotensin II (rigure 2-10). The above fíndings were obtaíned with normal dílute urine, but it was anËicipated that with ultrafiltration the angiotensínases present would be concentrated ín parallel. This was confirmed by the finding Èhat only 20% - 802 survival of S-asparagínyl angiotensin II was obtained in 13 of 20 normal urines concentrated 2O-fold and incubated at pH 7.5 followíng prior díalysis to a pH of 3.3 and treaÈment with EDTA. 100 É.ct't -)o I

(\l (B) A

-, ct) -.lrJ 50 t- (A) O (9 -,

É. UJ (-)O U.JÉ

B.€ 0

3.0 6,0 8,0 PH OF INCUBATION

Fie. 2-IO The effect of the pII of incubat,ion on angíotensÍnase activíty in uríne incubated with ß-asparaginyl angiotensin II. A : Urine íncubaËed wÍth ß-asparaginyl angioËensin II wÍthout prior dialysis and heaLing aË pli 9.6. B : Urine íncubated with $- asparaginyl angíotensin II following dialysís and heaËíng at pH 9.6 (Appendix Table B). 5B

üIhen, however, the angioÈensin which forms as a result of incubaËion of renal renin on human substrate was used as a substrate to test angiotensínase activity, greater than 80% recovery \¡ras,reeorded afÈer 24 hours of incubatíon in the 15 urínes tesÈed. Further evldence for freedom from end-product inactivatíon is contained ín the fínding that the formation of the end-producË of

the reaction between urínary renin and sÈandard substrate bears a linear relationship Èo time.

(il Fy,eedom of the system for the assay of nenin ín humart urine from the forrnatíon of depnessoz' artd pz'essov' matev'íaLs by othez' enzAmes: (1) Presence of kíntn-forvning enzymes in the system: Trasylol was used rouÈinely to block Èhe formaÈion of, kinins ín the presenÈ system. It has been shown to be an effective antagonist of urínary kaltr-ikreín actíng on plasma globulin (Prado, Prado, Brandi and Katchburían, 1963) and does not influence the rate of reactíon between renin and renin substraÈe in a concentration of 100 uníts/mI, nor does ít have any effect on rat blood pressure (Skínner , 1967). Trasylol was added routínely to all samples tested. However, ít was also found that no depressor maÈeríal díd form duríng incubaËion of 5 concenËrated urine samples (to whích Trasylol

r^ras not added) wíth tTrasylol-freer sËandard substrate. (z) fhe presenee of enzymes in uv'ine othez' thart renin

uhíeh ate eapabLe of fonmì,ng pnessor materiaL on ínettbation uith 59

stdndqrd eubstz.ate:

Since pepsín is known Èo act on renin substraÈe Èo produce a pressor materíal identícal pharmacologícally and structurally to angíotensin I (Alonso, Croxatto and Croxatto, L943; Blaír, 1962; de Fernandez, PaLadini and Delius, L965) and, furthermore, is known to occur ín normal uríne (Bucher, L947; Mirsky, Block, Osher and Broh-Kahn, 1948), it was of interest to deÈermine whether pepsin ín uríne was capable of acting on standard substrate r¡nder the right pH condítions and i furthermore, wheÈher ít was capable of forming Pressor subsËances which would ínterfere wiÈh Èhe accurate assay of renín ín urine.

IncubaËion of urinary renin wíth standard sheep substrate at pH 7.5 ensured denaturation and desÈructíon of pepsín (HerrioÈt, L962). However, íts importance ín systems íncubated at a pH of less than 7.0 was emphasized by Èhe folLowing experiments.

(a) Pooled uríne r^ras concentrated 5O-fold and divided ínto two parts. One alíquot was dialysed as usual Èo a pH of 3.3 wíth

EDTA and heating prior to dialysis to a final pH of 7.5" ,A second aliquot was dialysed to an initíal pH of 2.0 and símílarly treated.

0n incubation for 2 hours rttith standard substrate the results seen in Figure 2-11 (top trace) were obÈaíned. The pH 3.3 treated aliquot diluted 3-fold showed the anticipated pressor acÈivíty for this degree of concentration (46 ng/nL/hr), whereas Ëhe pH 2.0 treated 60

urine \Âras lnactive. These findíngs are characteristic of the denaturation properties of renín (Skinner, L967). llhen, however, a further aliquot of the pH 2"0 treated urine was incubated with standard sheep substrate at a pH of 5,6 ínstead of pH 7.5, pressor materíaL formed at the remarkable rate of greater than 1000 ¡g/mL/nr. Formation of this pressor maËerfal was totally ínhibited by dialysis to pII 7.5 príor to dialysís back to a final pH of 5.6 (l'ígure 2-lL, bottom trace). These findings are characteríst,ic of the denaturatíon of pepsin (Herriott, L962), buÈ not renín (Brar:n-Me¡êndez, Fasciolo, Leloir, Munoz and Taquini, 1946). (b) The propertÍes of the acid-stable, alkali-labile.enzyme ín urine were furÈhef investígaËed by studying its abillty to form pressor material from acíd-denatured substrate. A further alíquot of the 5O-fsld pH 2.0 Èreated concentrate of uríne \^ras reacted wíth both normal and acid-treated. substrate at pH 5.6 at 37oC. It was capable of formíng pressor material from both substrates aË the same fast rate (figure 2-L2).

The same concentrate treated by dlalysís to pH 3.3 and then díalysed to a final pH of 7.5 faíLed to generate pressor mateçial from the acíd-denatured substrate, whereas the expected amount of pressor maËeríal \^ras generaËed'from normal substrate (Fígure 2-L2). These flndings are consistent with the known abílity of a; j = t O

AB C A

t

A D E A

renin Fie; 2-11 Top trace: DenaÈuration characteristÍcs of urínary rnl 3'3 treated at pH 2.0. A : 10 ng angiotensin (0.1 ml)' B :0'l PII uríne after 2 hr íncubatíon wíÈh substrate at pH 7'5' C : 0'1 m1 pH 2.0 Ëreated urine; otherwise as for B' Bottom trace: Pepsitensín formation aË pH 5'6' treaEed urine A: 10 ng angiotensín (0.1m1)' D : O'05 m1 pII 2'O same sample íncubated 30 rnin with substrate at pli 5'6' E : 0'05 ml of otherwíse as for D' afËex díalysis Ëo pH 7.5 and reÈurn to pli 5'6; ¿a9_r =l o=l FI

I I I I A B c D AEFA

and pepsin Fís. 2-L2 Se paration of the effecËs of urÍnary renin by action on denatured renin substrate' A : 10 ng angioËensín (0. I m1-) BzpH2.0treatedurine(0.O4mr)íncubatedfor30mín wiÈhouÈ subsÈraËe at PH 5'6' C;O.05rnlofBíncubated30minwiËhstandardsubstraÈeat pH 5. 6. D:O.05mlofBincubated30minwithdenaturedsubstraËe at PII 5 .6. E:O.lmlofPH3.3treatedurineincubated30ninwíth denatured subsËraËê at PH 7'5' F : 0.1 mt of pH 3.3 treated urine íncubated 30 min wíËh standard substrate at PH 7'5' 6t pepsÍn, but not of renín, to form pepsitensln or angiotensín from denatured renín substrate (Braun-Menêndez and Paladíni, 1958). üIe confírmed the ability of pepsin to act on acid-denatured renin subsËrate by incubatíng crystalline pepsin with acid-denatured renín substrate at 37oC and pH 5.6. A concentratíon of 15 ug/ml exhausted the subsÈrate wíthín one hour under these conditions of incubatíon, whereas a concentration of renal renin which produced substrate exhaust,íon withín 10 mínut,es when reacted with standard subsÈrate

formed an equivalent amor¡nt of angiotensín from acid-denatured substrat.e only after incubat.ions for 7 days or more.

Some characteristies of the action of pepsin on ren'in substv'ate: The reactíon between pepsín and renin substrate ís not línear wíth time (figure 2-L3>, a curved velocity plot being produced whích is probably a reflectíon of the concomiÈant destructíon of the angiotensin by pepsin as ít ís produced. !üith low concentrat,íons of pepsin added to renin substrate and íncubated at pH 5"6, the amount of angíoËensin formed is not dírectly related to the concenÈratíon of the enzyme. At. concentrations of pepsin of less than 8 ug/ml (tr'ígure 2-L4) little pressor material is generated. This rnay be due to the concomítant destruction of pressor material as it is formed, or may be due to pepsin-ínhibitor present in the renin substrate preparatíon which would tend to

combíne with pepsin at a pH of greater than 5.4 (HerríotÈ, 1940; t40

12o

roo - E I g, 80 ¿c z I,l- óo 2 l¡l l- tâ- À 40 t¡t À 20

o 5 to T IIUIE tminutesl

Fie. 2-L3 The formation of pressor material with time by pepsin concentrations of 15, 10 and B Ug/ml actíng on standard sheep substrate at pll 5.6 (Appendix Table 9). 200

- -E \ u) ¿c z tlI Ioo z l¡l F tâ- À lr| È I III o 20 p¡plft.l (,rglml I

on Ëhe rate rirÉ.: 2:L4 The effect of Ëhe concentratíon of pepsín of formarion of pepsirensin (ng/rnr) aÈ pH 5.6 (Appendix Table 10 ). 62

Van Vunakis and Herriott, 1956).

(c) poly-l-lysine has been shown to be an effectÍve antagonist of the prot,eolytic activíËy of pepsln (Katchalski, Berger and Neuinann, L954). An almost compleÈe inhíbition of the action of pepsin on renin-substraËe and the pressor material liberated follow- ing incubatíon of uríne with renin subsÈraËe at pH of 5.6 was achieved by the addítion of poly-l-lysíne at a concentratíon of 0.5 rng/rnl to the incubation míxtures. This concentraËion had no effect on the pressor material formed as a resulÈ of incubation of urine with renin substrat,e at pH 7.5" It has been suggested Ëhat amylase is capable of reactlng to produce a pressor material. However, this occurred with ímpure preparations of amylase and its actual sígnificance is obscure, Èhere being a progressive loss of actívity wíth purification of the enzyme

(i{alaszek, Bunag and Huggins, L962). Lack of interference of urínary

amylase in the present system ís suggested on the basis of immuno- logical data since antibodies to renal renin inhibtted Lhe enzyme in urine"

DTSCUSSTON:

The experiments described demonst,raÈe thaL there is ín

uríne at leasÈ trrTo enzJrmes capable of reacËing r^rith standard nephrectomized sheep to produce pressor on .substrate "rrb"trrr"." íntravenous injecËion inÈo the ganglíon-bl-ocked rat" FortunaËely, 63

Ëhese two enzymes can be separated on the basis of susceptibilíty Èo denaturatíon by low or high pH; by the optimun pH for íncubation; by differences 1n the raËe of reaction of the enzymes on acid- denatured substrate; and by inhibiÈlon by specifíc inhíbitor or antibody.

An enzyme which has sírnílar actions Èo Ëhose descríbed for pepsln, in that it acts upon renín substraÈe Èo produce a pressor materíal at a pH of 5.6, ís present in urlne. This enzyme ís resistant to a pH Èreatment of.2.0, but ís denatured by an alkaline pH of 7.5. Like pepsín, ít is inhíbíted by Ëhe addíÈion of poly- l-lysíne to the íncubation míxture.

The second enzyme ín urine appears in all respects studied to be identical to plasma and renal renin. This enzyme can be distinguished from urínary pepsín, in that, ít ís denatured by low pH treatment (pH 2.0), although ít ís stable to a pH of 3.3. 0n incubatlon wíÈh standard substrate at pH 7.5 a pressor material is generat,ed, the formaËion of whích ís linear wíth time. Urinary renin is ínhibited by anÈibody raísed ín the rabbíÈ t,o human renal renin, but not Ëo rabbit plasma obÈaíned from anímals

ímmunízed wíÈh Freundfs íncomplete adjuvant alone. The pressor material formed as a result of incubation of urine wíth renín'substrate at pH 7.5 ís similar to angíotensin in that it ís día1-ysable, heat sÈabLe, destroyed by the action of 64

proteolytic enzymes such as chymotrypsin and, furthermore, the pressor response on intravenous injectíon is ídentical to Èhat obtaÍned wíÈh angiotensin II. The identity of the end-product obtaíned from íncubatíon of urínary renin wiËh renin substrate has not been establíshed beyond idenÈificaÈion of its peptíde nature. It ís possible that the peptíde formed is angíotensin I whích ís rapidly converted on ínÈra- venous injection into the rat Èo angíotensin II, since EDTA used ín the present sysÈem to inhibtE angíotensinase would also Èend to inhibit the action of any coriverting enzyme Èhat may be present in the incubatíon míxture (Skeggs, Kahn and Shumway' 1956). The enz;rmatic nature of Ëhe reaction between urine and renin substrate at pH 7.5 is apparent from the findings that the productíon of p_ressor material ís línear with tíme; that the reactíon is temper- aÈure dependent; influenced by substrate concentraÈíon and also by pH.

Suppressíon of the reacÈíon by antí.body to renal renin and the similarity in the effects of pH and substrate concentration on the rate of reaction beËween enzyme and substrate suggest a common identíty of plasma and urinary enzymes. Although Èhe relatíonshíp of angiotensín formaÈíon to concenÈration ís linear for both plasma and urinary renin, this is not, the case for renal renín actíng on standard sheep substrate (Skínner , L967). 65

Treatment of urine to a PH of 3.3 was thought ínitially to be necessary t,o ensure denaÈuratíon of any endogenous substraÈe that may be presenË in urine. Although no substrate ï7as found in normal urine, Ëhe presence of small amounts of,substrate in several urines dísplaying hearry proteinuria necessítated Èhe rouËine use of the low pH treatment in these situations. AÈ least two enzymes are present ín normal urine which are capable of ínactívating angiotensin as it is formed. Angíotensínase activiÈy was slightly fasÈer agaínsË the synÈhetíc ß-asparaginyl angiotensin II than ít was against the end-product of lncubaÈion formed in the present system. Thís may be accounÈed for by the fínding Ehat the s-asparÈyl amide is hydrolysed more rapidly than ís lhe nalural-ly ',occurring acid (Regoli, Riniker and Brunner, L963; Nagatsu, Gillespie, Folk and Glenner, 1965).

Inhibition of angíotensinase acting at PH 7"5 was achíeved by eiËher pH 3.3 treatment or the addítion of EDTA to the system. This treatment failed to inacÇivate the angioÈensinase active at a pH of less than 7.0. However, thís angiotensinase ís inhibíÈed by prior díalysís and heating at pH 9.6, a findlng that suggest,s that at least part of this act,ivity could be due to the action of pepsín present ín urine (Munoz, Braun-Menérrdez, Fasciolo and Leloir, L9403 Plentt and Page, L944). The linear formation of pressor materíal on íncubatíon of 66 urine with substrate further suggests that the presenL system is free from end-product inactivation, whilsË the linear relatíonshíp beËween concentratíon of the enzyme and formatíon of the pressor materíal suggest.s that there is no co-factor or ínhibítor present in

Ëhís system.

The levels of renín in normal uríne are low, beíng of the order of 0.6 uníts/rnl. Thís necessitated the use of a concenËrat,ion sÈep (ultrafiltration) to achieve levels hígh enough for accurate assay of renín in uríne. The low levels found \^rere not consequent upon the loss of renin duríng treatmenÈ of tTre uríne samples r nor were Èhey exaggerated by any loss duríng ultrafiltratíon, sínce recyclíng of urine samples Èhrough the method did not cause any sígnífícant fall ín the concentration of renin presenÈ. The results presented by Brown, Davies, Lever, Lloyd,

Robertson and Tree (1964) suggest ÈhaÈ urinary renin concênÈration is of a similar order to that found in plasma, whílst the concentratíon of renin in urine as determíned by the present assay system is only

1/15 ttrat found in plasma. Thís discrepancy ín results led to an

ínvestÍ-gatíon of the'properties of the enzyme actíve at pH 5.6, and it was concluded that this enzyme \¡ras very símílar to pepsín. Thís was êstablished by the dífferentíal pH trealment of urine described in the text which showed clearly that there are t\^ro enzymes in uríne capable of producing pressor mat,erial on íncubaËion with sÈandard 67

renín substrate. Since the pressor material- formed by the acÈion of pepsín on renín substrate is ÍndísÈinguíshable from angiotensin r

(Alonso, croxatto and croxatÈo, L943; Blair, 1962; de Fernandez,

Paladíni and Del_ius, 1965) , díf ferenÈiation of the tr^ro enzymes ís

dependent upon ídentificatíon of the propertíes of the enz)rmes.

It was concluded thaÈ some of the rrenín-liker enzyme activity in normal uríne incubated at pH 5"6 could be due to the

actíon of urinary pepsin on renín substrate. sínce pepsínogen would be presenË ín uríne, Èreatment of urine to a pII of.2.0, whích, in the present assay sysÈem, results ín the denaturatíon of renin

(skinner, 1967), would convert the urínary pepsinogen Èo pepsin and

subsequent incubatíon at pH 1evels of less Ejhan 7.0 míght produce

pepsít,ensin, especially if the enz)rme r^ras concentrat.ed during extraction procedure.

Brown, Davíes, Lever, L1oyd, Robertson and Tree (L964)

found Ëhat the renin-lik" in normal human "rr"y*" urine resembled renal renin on sÈarch-gel electrophoresis (peart, Lloyd, Thatcher,

Lever, Payne and sÈone, L966), but an enzyme such as pepsín, with an

ísolectric point of less Èhan 1.0 and a molecular weight of 35r0oo, nlght also be expected t,o migrate wíÈh renin in the pre-albumín range. 68

(B) OBSERVATIoNS ON THE ORTGIN OF RENIN IN HI]MAN URINE

The development of an assay sysËem for the quantíÈatíon of

renin in human urine provídes a method for studyíng the renal handlíng

of renin. Although the levels of renin found ín uríne hrere consider- ably less than those descríbed previously, the renal handling of

renín is ímportant ín view of the suggestion made concerníng the

action of angiotensin on proximal.Èubular sodium (Leyssac, L967) and distal sodium transport (Vander, 1963). Furthermore, in anímals, renín appears to induce proteínuria (Píckering and Prinzmetal, 1940;

Deodhar, Cuppage and Gableman, L964; KaEz, Sellers and Bonnot'ís,

L964), and since proteinuría was found to be augmented in condíÈions

assocíated wiÈh elevation in plasma renin Levels (fobian and Nason, L966), iÈ was of ínterest to ínvestígate the effects of elevation of plasma renín levels on the excretíon of protein in normal subjectsi, In order to assess the díagnostíc potential of urínary renin estímation, urinary renin and plasma renin levels were estimated ín a varíety of pathological situations.

METHODS:

Experíments \,yere performed on 19 nonhal males , 8 normal females and 19 hospital paÈíents. Unless otherwíse stated, the subjects r¡/ere requesËed to pursue theír normal pâtLern of daíly

activíty and no deliberate conËrol \¡ras exercísed over díetary sodium 69

or fluid íntake.

All blood samples for plasma renin esÈimatíon were taken from an antecubítal vein with the subject in the sítËíng position.

sanples were taken at 1100 hours or 1600 hours as índicated in the text..

Díurnal varíations in urinary renin excretion were d.eter- mined over 12 hour collection períods between 0900 and 2loo hours.

Blood for plasma renin estímatíon hTas taken at the times indicated above. voided uríne r^ras stored at 4oc duríng collect,íon períods.

Ttre 24 hour renin clearance r^7as determÍned from the formula: crenír, = urerrin (units/m1) x v (nL/24 hours) P renín (uníts/m1)

Urinary and plasma renin levels were estimated before and

duríng ÈreaÈment of 7 normal male subJecÈs wíth spíronolactone, an

ardosterone antagonist (Aldactone, searle), 50 mg twíce daily for L2 days which was combined wiËh chlorolhíazíde (chlotríde, Merck, sharp and Dohme) o.s grams daily duríng the lasÈ 5 days of therapy. rn thís study, the blood samples were all taken at 1lo0 hours and the urinary renín outPut calculat,ed in the 24 hour urine sample collected over the previous 24 hours.

Methods of estimation of par,øneters measu.z.ed:

Urinary renín and renín subst,rate r¡rere assayed quantitatively by the methods descríbed previously. Plasma renin coricentratíon was 70

determíned by the method of skinner (L967). plasma renín concen- tration, and urínary renin concentration and output are expressed ín Èhe same trniËs.

Sodíum r^ras estímated by fJ-arne phoÈometry; urinary creatinine, plasma creatínine, and pJ-asma proteíns by the Technicon Autoanalyser (Technícon, Ardsley Ny, 1965). Total protein excretion was determíned by a modification of the Lowry method, using the Folín-CíocalÈeu reagent (Lowry, Rosebrough,

Lewis Farr and Randall, 1951). A calíbration curve r¡ras constructed usÍng bovíne serum aLburnin as sËandard (Fígure 2-L5). SÈandard protein solut.íons and uríne samples were read aE 750 mu agaínst a saline blank cont,aíníng reagents alone.

In order to avoíd errors due to reacÈive groups in amíno acíds and peptides, uríne samples for urínary proteín estimation were dialysed for a toÈal of 48 hours in 8/32 vísking cellophane casíngs agaínst runníng tap hTater and Èhen against ísotoníc saline. Thís díarysis was performed at atmospheric pressure and loss of proteín was therefore avoided (Rowe and Soothill, L96r3 Kíng and Boyce, 1963).

Qual-ítative esÈímatíons of protein in urine vrere made using

Albustíx (Ames) and the results graded from negatíve Èo f-H+ by col-our matching wíth the Albustíx chart.

Urínary and plasma albumin levels were estimated by the soothíll modifícation of the OuchterJ-ony technique (soothill, 1962). o.3

t E o o tô È >o.2 o E laz olg o ¿

IÞ Ëor

o 50 loo t50 200 CONCENTRAÍ¡ON PROTEIN (ug/ml I

Fie. 2-15 Calibratfon curve constructed with standard coneentrat.lons of protel-n, using a calorimetric technígue. The changes ín optfcal denslty were read aË 750 mu (Appendix Table 11). 7I

RESULTS:

NorrnaL renin output í,n uzine:

The fíndínþs lllustrated ín Figure 2-16 and Table 2-1 show that the normal renin outpuË estímated in 19 normal males ranged widely fxom L62 to 7500 ur.íts/24 hours (mean 1196). Despíte the wide

variatíons in urinary renin output beÈween individuals, the renin

output within one individual Èended to remaín constant (Figure 2-L6). Renin output into urine in 8 normal females ranged from 1013 to 2L,730 units/24 hours (mean 6811) (figure 2-16, Table 2-2).

The levels of renín found ín. the urine of normal females were therefore signíficanÈl-y higher than those found ín normal males of

comparable ege (0.01>P>0.002) . No rel-ationship was found between plasma renin concentration and the 24 hour excret,ion rates of renin ínto urine (fatte Z-t) (r = -0.I7rn.s.), nor was there any relationship between plasma renin

concentration and urínary sodium (r = 0.33, n.s.) wíthin the 19

normal males studied. There r^ras no índícatíon that further estimations would have elevated these relationships to a sígnifícant leve1. RenaL cLeayance of z.enin: The renal clearance of renin in 16 normal males ranged from

L2 to 893 nL/24 hours (mean 138). The renal clearance of renin in 8 normal females was sígnificantly higher (0.01>P>0.002) than that

found ín males, ranging f.rom 79 to 3149 nL/24 hours (mean 950 ml/ 3ün o

o a,r, o s ? a\ o C\5- (n F o O-----ì:.¡ = a

O

F LJJ o É o x(_) a o lrJ o z. -a .ta lrl .t------a" É a a

ffi __-__at__ &;,, _-_ J a -'o o--

TVIALES FEIVIALES

Fie. 2'L6 RenÍn ourput (units 124 hours) into urine of 19 joÍn normal mal-es ( o ) and 8 normal females ( o )' LÍnes repeat estimaÈíons on indivídual-s performed at intervals of weeks or months. TABLE ,' 2-L

Renin and sodíum excreÈion in normal males

PRC Renin excretíon Renin clearance Sodium excreËíon No (units lml) (units /24 nrs) (mL/24 hrs) (mEq/24 hrs)

I 783 2 706 3 614 4 268 J 4a 9.3 257 27 5 6.4 596 93 288 5a r1.5 s67 49 6 6.8 2L0 31 319 t 6a 5.3 490 92 7 24.0 846 35 347 8 8.3 L,g7g 226 340 9 9.5 L,234 130 205 10 14.0 L62 L2 235 11 LL .4 273 24 189 L2 15.0 67s 45 202 13 8"4 27L 32 35' L4 10"0 815 82 101

15 L2 "4 287 23 261 L6 8.4 7 ,500 893 L7 10 .3 432 42 18 9.8 4,442 453 t9 13.5 6rs 46

Mean LL "2 L,Lg6 138 229

s 4a, 5a, and 6a are repeaÈs on subjecÈs 4, 5, and 6, respecÈively. Repeats \¡/ere averaged in calculatíng group means. PRC = plasma renín concentraÈion. Correlatíon coefficíents, renín excretion : PRC (r=-0.17) n.s., renin exc,retíon : sodium excretíon (r=0.33) n.s. TASLE 2-2 Renín excretíon in normal. females

PRC Renln excreÈíon Renín clearance No (units/ml) (units /24 hrs) (nL/24 hrs)

1 6 1750

2 L,643

3 8.8 L0,754 L 1222

4 6"9 2L,730 3,L49

5 9.5 1,098 LL6

6 L2.8 1 ,013 79

7 9.6 9,435 983 I L4.T 2;072 148

Mean 10. 3 6 ,811 950

PRC = plasma renin concenÈratíon 72

24 hours). Assuming a mean normal creatiníne clearance of l8O litres/

24 hours , the renal clearance of renin for males was only O '077"/" that of the creatinine clearance, and for females 0.53% that of the clearance of creatinine.

Effect of pLasma ven¿n Leuels on renin eæcretion: Elevatíons in plasma renin levels were induced in 7 normal male subjects by administration of Èhe díureÈic spíronolactone for

L2 ð.ays combined wíth chloroEhíazide for the last 5 days. Plasma and urine samples r¿ere collecÈed prior to commencement of natriuretic therapy, on the 7th day of therapy with spíronolactone and on the L2tt. ð,ay (after 5 days of combined therapy). By the 7Ëh day of therapy plasma renin concenÈration was elevated Eo 2.5 times the

control levels. Thís \^7as noÈ assocíated with any significant change in urínary renín levels (rigure 2-L7, Tabl-e 2-3). However, by the

L2th ð,ay of therapy, when the plasma renin levels were elevated to 4.7 times the conÈrol (P

43% of conrrol levels by the L2th day of therapy (rígure 2-L7, Table 2-3). creatínine clearance did not aLter sígnificanÈly throughout the course of natriuretic therapy (table 2-3), whilst Ëotal protein clearance fell Èo 85% of. conËrol levels by the 12th day of therapy 500 PRC J o ú. F 400 o=. (J 30 o= ay, lJ- 200 UnV lJ-l r.9

¿ 100 (J UnV be PRC 0 ALDACTONE + CHLOTRIDE

7 I2 DAYS

ELg: 2-L7 The effecÈ of Aldactone (spÍronolactone) and Chlotride (chlorothiazíðe) on p1-asma renin concenÈration (PRC), urinary output (UnV¡ and renín clearance (H) on days 7 arrd, L2 of natríureÈíc therapy. Results from 7 normal males are exPressed as Ëhe mean ! SD of control of 100. TABLE 2-3(a) Absolute values in plasma renin coricent,raÈÍon, and the clearances of renin, creaÈinine, alb"'nin and protein,on days T and L2 of natriuretic therapy SUBJECT

Day I 2 3 4 5 6 7

PRC 0 8.4 9.3 10.3 9.8 13. s 5.3 11 .5

(units lurl) 7 L9.5 23,8 27 ,5 20 .0 23.8 L7 .3 34.2 L2 3t.7 38.4 51;3 55 .0 61.3 31. 3 46.3

Rênin i 0 7500 257 t+32 4442 615 490 56V excretíon 7 69 83 3s4 839 346A 613 3r7 938 (unitsl24 hrs) L2 5000 962 1.062 6000 L628 s68 855

Renin 0 893 27 42 453 46 92 49 1 27 clearance 7 358 15 3L 173 +6 18 (nLl24 hrs) L2 158 25 2L 109 27 18 18

Creatínine 0 193 L6L 153 2L3 163

clearance 7 156 180 91 1s1 t66 (t/24 hrs) L2 240 L74 138 220 L48

Albumin 0 0.095 0"102

J clearance 7 0.075 (mLl24 hrs) L2 0.085 0 .069

Protein 0 18. I 24.6 10 .6 20,2 L4.5

ctrearance 7 14.s 18. 5 8.9 L4.2 11 .9 (mLl24 hxs) t2 l3;3 24.1 8.6 15. 3 L4.L

* No detecËable albr:min TABLE 2-3(b)

Percentage ehange from conÈrol of IOO% in plasma renin concent.ratíon, and the cleara¡rces of, renin, creatinine, albumín and proËein on days 7 and 12 of natriureËic therapy SUBJECT Yfean 7" P Day I 2 3 4 5 6 7 change

PRC 0 (units lml) 7 232 256 267 204 L76 326 297 +151 <0"001 L2 377 413 498 561 454 59L 4A3 +37L <0 .001

Renin 0 excreÈion 7 93 L38 L94 78 100 65 L6s +19 n"s (r¡nits 124 hrs) L2 67 374 246 135 265 LL6 1s1 +93 0.05

Renin 0 clearance 7 40 56 74 38 57 20 55 -sL.4 <0.001 .A <0 002 (rn1/24 hrs) L2 18 93 50 2l+ 59 20 37 -57 "

Creatiníne 0 clearance 7 81 LL2 59 7L L02 -15 .0 n S (t 124 urs¡ L2 L24 108 90 103 9L + 3"6 n c

Albr.rnin 0 & clearance 7 73 (m1124 hrs) L2 89 68

Protein 0 -l clearance 80 75 84 70 82 -2L.8 0"001 (mLl24 hrs) L2 73 98 81. 76 97 -15.0 0.05 * No deËectable albr¡nin 73

(Table 2-3). The slight fall in clearance is due to Èhe rise in plasma proteín, presumably consequent upon reduced extracellular volume. There \^7as rro significant relationship between the changes in renin clearance and glomerular fíltratíon rate (as measured by clearance of creatínine) either between índivíduals or within the same indivíduals duríng sodium depletion, nor was there any sig- nifícant relationship between the renal- clearance of renin and the total proteín clearance. Thís was partícularly emphasízed in subjects

4 and 5 ín whom the control renín clearances díffered widely, being 453 and 46 nL/24 hours, respectively, whílst the albumin clearances were simílar (0.095 and 0.L02 nLl24 hours), as \^rere Èotal protein clearances (10.6 and 20.2 nL/24 hours) (Table 2-3).

The effect of eLeuations in pLasma renin on totaL protein eæeretion: Although natríuretic t,herapy índuced marked elevations ín plasma renín concentration, there \^ras no sígnificant rise in total protein excretion (fígure 2-L8) or Èotal proteín clearance during the course of Èhe therapy.

DiuvmaL uaríations in renin eæez,etion:

Renín excretion r^7as measured over the 12 hour períods (0900 Èo 2100 hours and 2100 to 0900 hours). It was found that there r^7as no signifícant díurnal variatíon ín renin excretion ín the 15 normal male subjects studied (¡'ígure 2-L9, left panel). The consÈancy of renín output within the one indívidual is also 60

EJ (n 50 z. = 40 -.(-) -O L) 30 - -. u-J 20 É. ct)= 10 o- 0 0 100 200 PROTEIN EXCRETION (me/24 rns)

Fíe"2-lsRelationshipbetweenplasmareninconcentrationand proteín excreËion during treaÈment with spironolactone' Control values (O). Spironolactone therapy ( f )' Splronolactone wíth chlorothíaeíde ( A ). (Appendíx Table 12) ' RENIN (urulrs/l2Hns) S0DIU¡v¡ (Neo/l2Hns) V0LU¡v¡E 0qud2sns) 1000 1500 B 7

200 6

J 5 9 14 1000 500 7 12 I 11 10 2 15 3 10 5 12

14 il 15 500 4 13 6 10 0 0

DAY NIGHT D N D N

Fie. 2-L9 Day (D) and night (N) excretíon of renín, sodíum and vraËer in 15 normal males. Numbers refer to índividual subjects (Appendix Table 13). 74

aPParent frorn thís data. Although there r,ras no díurnal variatíon in urínary renin output, urinary sodium showed the anticipated

(Stanbury and Thomsonr l95l) nocturnal decrease ín ten ouÈ of twelve

subjects (figure 2-L9, middle panel, P<0"05). Urine volune, however,

did not alter signífícantly (Fígure 2-L9, ríght panel). There was

consíderable varíation in both urínary renin output and sodíum output

between individuals. However, no altempt r¡ras made in these experímenÈs to control dietary sodium intake.

No relationship beËween urinary sodium and urinary renín output was observed in these subjects. rn order to detect any occult

relatíonshíp, Èhe results vüere expressed as the day/night raËíos of renin and sodium excretíon wíthín índividuals. Again, no sígníficant

relatíonship emerged, nor \^ras there any signifícant relationshíp between the day/night ratíos of renin output to day/ni.ght raÈio of volume. I,rlhen, however, the ratios of. day/ni-ghË renin concentratíon hrere correlated wiÈh the day /nLg]r,| ratío of volume, a signifícant negative correlatíon \,\ras found (tr'igure 2-20) , T = 0.53, P<0.05. This ís a findíng characteristic of any substance, the excreÈion of which is independent of urine volume., Uz.inaz,y renin Leuels ín disease states:

Table 2-4 shows the data from 17 patienÈs suffering from a variety of pathological eondítíons. In fíve of eight severe hyper-

tensives, renin excretíon was withín normal límíts for their sex. o o 5

rl

(J =,o (J 3 =.

u-l= É. oo ooo É, o 1 o oo =, o o o ú, o o -t 0 0,5 1,5 2,5 VOLUfvlE RATIO

rj!e. 2-20 Relatíonshlp between the day-night ratlo of renÍn concenÈratíon and urinary volume (Appendix Table 13) ' TABLE 2-4 Renin excretíon, plasma renin concenÈratíon, creatiníne clearance and renín clearance ín patíenÈs

Renin Creatinine Renín excretíon PRC Urinary clearance clear¡nce No. Sex Disease r¡niËs 24 }:-rs uníts rotein L 24 lnrs lz4 urs J I M Essential hypertension 49 8.4 Negative 8I+ 5"8 2 M EssenËial hypertension 3,300 15 "7 + 2LO 3 Essentíal hyperËensíon LL ,87 4 29.5 # 403 M t 4 M Essentíal hypertension I ,710 26.8 Negatíve 64 J ¿ 5 M Essentíal hypertensíon 135 28.L Negative 4.8 6 F Malignant hypertensíon 217 9"6 {# 49 22,6

7 F Malignant hypertension (renal artery thrombosís) 48,000 384.0 "{-# 86 L25 M MalignanÈ hyperËension (polyarteritis nodosa) 55 ,86 3 360 .0 39 155

9 M Cirrhosis wiÈh ascites 200 30"0 NegaÈíve 6.7 10 Acute nephritís 14,000 13. 5 Negatíve t067 M I .{-*#v 11 I'l AcuËe nephriËís l r25r 15. 3 109 82

J,2 F Chronic pyelonephritís 2,687 9.8 H 107 274 13 F Chronic pyelonephritis 12,750 L2.l ffi 1045 I4 M Terminal chronicr tenêl faílure 22,L50 70 .5 i.# 314 15 M Teminal chroníc renal 20,025 31"5 # 636 failure & J .i:# T6 M Nephrotíc syndrome I r23O LI.7 26 105 49 L7 FNe roÈic TOme 6 360 130 "0 ì-# g 2 x l2-hour urinary renÍn output and c eerance. t ProËeín excreËíon, 1.6 hours " I Protein excretíon, 5 gl24 hours" PRC = pl-asma renin concentration" 75

Two pat,ients (n'rnbers 7 and 8) had the highest levers of renín excretion and plasma renin levels yet encountered in this laboratoryr yet the clearance of renin was wiËhin normal límits.

Both of these patients suffered from gross proteínuria. patíent 7

suffered from severe polyarterítís nodosa, whilst patíent g had a

traumatíc stenos'ís of the right renal artery. Ríght nephrectomy subsequenÈly resulted Ín a remissíon of the hypertension in patient

8 and plasma renin levels fel1 to wíthin the normal range. Since the right kídney was completely anuric and the source of the hígh renin

levels in plasma (because its removal resulted in a fall ín plasma renin levels), íË ís suggested that the hígh levels of renin in urine

resulted from the fíltration of renín produced from Èhe ischaemic

kídney through the glomerulí of Èhe normal kidney. Four ot.her patients had elevated urínary renín excretions:

numbers 3 (hypertensíon), 10 (acute nephritis) 14 and 15 (terminal chronic renal failure). Three of these patients also had elevated

plasrna renin levels, the exception beíng patient lO.

Although in normal subjects ít was found that there \^ras no relatíonshíp between urinary renín ouÈput and proteín excretion, it was thought Ëhat, sínce renín r¡ras a proteín, its excretion would be

increased ín patients with heavy proteinuría. Thís was only the case when the paËíenÈs also had elevatíons in plasma renín levels as well

as proteinuria. Patíents 6, LL ) L2, 13 and 16, who had normal plasma 76

renin Ievels, also had normal renín excretíon and renín clearances

despíte the presence of heavy proÈeinuría. patíents 3, 7r g, 14 and 15 had elevated plasma renin levels, and renin excretion was also

elevated above the lírnits of normaL for theír sex. patient 17 was the only exception to thís rule. rn thÍs patíent, heavy proteinuría

and elevatíons in plasma renin levels \^rere not associated with an

excess renin ouÈput. rn sunmary, therefore, iÈ did not seem that

pathologícal proteinuria \^ras a sígnificant factor ín influencing the ouËput of renin into urine.

Renín subety,ate in uz,íne:

The urine of three patíents wíth heavy proteinuria was

examined for renin substrate. rn only one of these paÈients was

renÍn substrate for¡nd, the concentratíon being o.15 ug/nl , L4% of Ehe 1eve1s normally found in plasma (Skínner , Lg67). Renin in u.retez,ic uyine:

rn two patíents, one male and the other female, urine was

collected for one hour from catheters passed up the ureters Èo the renal pelvis. The renin output was 180 uníts/24 hours and 117 units/ 24 houxs, respectively.

DTSCUSSION:

rn order to study the renal handlíng of a substance the followíng críteria must be fulfílled Firstly, quantitative measurement of the substance, both ín 77

plasma and urine, must be possÍble, and, secondlY, ít must be esÈab- líshed that there is no contríbution by the lower urinary tract' to uríne of the substance beíng studied. The methods of assay of:renín ín plasma and uríne described in Èhís thesis províde for the quantltation of renin in plasma and urine. There is no evidence in the liÈerat,ure to suggest thaÈ renín ís secreted by the cells 1-ínlng the urínary tracËr Furthermore, renín r^ras presenÈ in uríne collecÈed et the level of the renal pel-vis by ureteríc catheters ín the thTo strbjects. The levels found were lower than those found ín normal subjects. Hourever, in these patlenÈs uríne csllections r¡tere made for only one hour following anaesthesia for lnvestigat,íon of url-nary tract abnormalitíes. Moreover, the subjects were elderly: hence, a strícÈ comparíson with young, acÈíve' heatthy subJects is probably noÈ Justífíable. In concluslon, ít seems that the lower urinary tract, is not a source of renin in human uríne. Renin outpuË ín normal urine showed a wíde índivídual varíation for which no obvious reason \^las apParenÈ. However, the

renin excreËion in one Person tended to be constanÈ. There I^7as no correlation between the renín outpuÈ and plasma renin levels, or

between renin ouÈput and glomerular fíltration rate, sodíum' \¡Tatert or protein excreÈion that would accoun¿ for the wide variations in

renín output seen. 78

It was surprísÍng to find that renin excretion in females was hígher than Èhat observed for normal males. There is no apparent reàson for this firidíng. Since the plasma renin levels were of the same order as those observed ín normal males, the clear-

ance of renín into uríne \^Ias also greater ín females than ín males. Since t"tttl. has been found ín the reproductíve tíssues of man and other animals (Brown, Davies, Doak, Lever, Robertson and Tree, L964; Gross, Schaechtelin, ZiegLer and Berger, L964; Ferris, Gorden

and Mulrow, 1967; Skinner, Lumbers and Symonds, 1968), ít ís possible thaÈ there is a contribution to urínary renin from the reproductive tract of the female of a renín molecule of dífferent size or charge, or dífferent bíochemical propert.ies. Although ít has been shown that, renin from the reproductive Èract has simílar molecular weight and propertíes to renal renín in the rabbít

(Anderson, Herbert and Mulrow, 1968), the I(m for uterine renin is

1ower, índícating an increased affínity of the enzyme for the substrate.

If urinary renín ín the female were deríved from Èhe reproductive

tract, and if it showed a similar reductíon in Km as does rabbit

renin, then the amount, of renin estimated by reaction velociÈy aÈ a given substrate concenÈraÈion would appear to be greater. The renal clearance of renin in normal males ís low, beíng 0.0777" that of the clearance of creatinine. It varíed in a manner

that was not related to total protein clearance. Although an error 79

is incurred by assaying only one plasma renín sample durlng Eh.e 24 hour collectíon, since some diurnal fluctua'tion in plasma renín levels has been reported (Gordon, i,lolfe, Island and Líddle, 1966) and the renin levels appear to be much lower in recumbent subJects (Lumbers, unpublíshed observaÈíons) , thls would noË account for the wide individual differences ín clearance seen, sínce the time of collecÈion of plasma samples was standard ín all subJects and differences in plasma levels r^7ere cempared Èo varíaËions in renín excretion. The clearance of renin fell markedly during elevaÈion of

Èhe plasma renin level by admínisËraÈion of natriuretic agents. This hras not consequent upon a fall Ín glomerular fíltration rate, nor did proteín exeretion suffer the same degree of suppression. Such a finding indicates that Èhe olearance of renÍn is not dependenÈ upon filtratíon of renín alone and suggest,s thaÈ an increased proportion of the renín must be removed from Èhe tubular fluid by reabsorption during sodium depletion. Another possibí1iÈy is that there is a constant secreÈion of renín into urine from tubular cells, possíbly from the macula densa, and that Èhis is the only source of renín in urine. Hence, when an elevatíon of plasma renín is índuced, this ís not associated with any change ín. the tubular secretion of renín and therefore Èhe clearance of renin apparently faLls. However, although the clearance 80

of renín falls duríng sodium depletíon, the ouÈput of renin inÈo uríne rises and, by the Èwelfth day of therapy, iË ís 1.9 tímes eontrol levels, a findíng whíeh suggests that renin in urine ís derived from filtration and subjected to reabsorpËion from the tubular fluíd" This is further substantíated by fínding a hígh renin outpuÈ ín the uríne of one pat,ient, wíth eomplete anuría of one kídney due to renal artery sÈenosís. In this patíent, sínce the renín contenL of Lhe normal kídney can be expected to be low (Regoli, Hess,

Br¡rrrner, Peters and Gross, L962> and it is this kidney Èhat is responslble for Èhe formatíon of urine, the finding of a high renin ouÈput in thís patíent suggests that renin released into the círculation by the anurie, íscþaemíc kidney is fíltered lnto urine by the normal kidney"

Sínee renin ís a protein, one could posÈulate that Èhe renal handlíng of renín would be símil-ar to Ëhat of oÈher proteins in uríne" The renal handlíng of proteins appears to vary accordíng to the size of the proteín molecul-e excreted. Ilardwíeke and Squíre (1955) concluded, from a study of the clearanee of proteins ín proÈeínuria, that proteín reabsorption was non-selective. Their data applies to the reabsorptíon of prot,ef-ns of large molecular weight' and reeent studies by llarríson and Blainey (L967) suggest thaË the elearance and exeretion of low molecular weighË proteíns (15 to '000 20r000) ís not elevaÈed eonsístently ín,heavy proteinuria" Such a 81 fínding indicaÈes thaË the tubular reabsorpÈíon of low molecular weight -proteins is differenË from that of proteíns of molecular weíght greaÈer than thaL of albumin.

The lack of assoeiation beÈween renin exereÈíon and protein excreËion in pat,íents with hearry proteínuria suggesÈs thaÈ the tubular reabsorption of a proËeín of moLecular weíght of 42 r000 (Kemp and Rubin, L964, and l,rlarren and Dolínsky, L966) ís similar to that of low molecular weight proÈeíns. This finding is subsËantiated by the data of Blainey and Northan (1967) who showed rhat rhe el-ear- ance of amylase (moleeular weÍght 45 1000) also remaíns normal in the presence of heavy prot,einuría.

ThaÈ molecular size alone is not the only deÈermínant of the reabsorpÈion of these enzymes ís indíeated from a eomparison of the clearance rates of Èhe enzymes renín, amyl-ase and pepsínogen.

The molecular weight.s of these three enz)rmes are símílar, beíng 42,300 (renin - tr{arren and Dolínsky, 1966>, 451000 (arnylase - Blaíney and Northam, L967> and 42,600 (pepsinogen - Hírsehowitz, L957>, yeÈ Èhe renal elearances are remarkably dífferent, beíng, respeetívely, 0.077%1 3% ar'd 30% that of creat,i.nine. Such a wíde variation ín clearance of enzymes of símilar mol-eeular weíght ís unlikely to be due to filtraÈion differences al-one, nor to small differences ín molecular weight affeetíng the rate of tubular reabsorpLion" This data suggests, therefore, that a fílÈration-selectíve- 82

reabsorpÈion system for renín ís operatíng ín the renal tubules.

since injections of renin into experímental animals have

been shown to induce proteinuria (píckering and prinzmetal, 1940;

KaEz, Sellers and Bonnoris, 1964; and Deodhar, Cuppage and

Gableman, 1964) and rhe findíngs of Tobian and Nason (1966) are

consistent \,üíth the associaÈíon between high levels of renin ind.uced

by sodíum depleÈion and proteínuria ín Èhe rat, it has been suggested

that orthostatíc proteínuria ín man may be explaÍned on Èhe same basis. However, the present findíngs do not support thís possíbí1íty, sínce no relationshíp was found between the levels of plasma renín and total protein excretion in normal subjects and, secondry, a rise

in plasma renín induced by natriuretic therapy r¡ras associaÈed with a

fal1 in protein clearance, no rise in protein excretion beíng observed, The possibility Ëhat high levels of renín may induce proteinuría in man ís not excluded in this study, but ít seems unlikely that small

fluctuatíons in renín levels due to postural changes are Èhe mechanism responsíble for the assocíated íncrease in protein excretion seen on assumpÈion of the upright posture (Robinson and Glenn , Lg64). SÈudies of urínary renin output in disease states revealed that there \¡/as no díagnostic advantage to be gained from urinary renin estímations, sínce elevations in plasma renin vÍere not consístently assocíaÈed wíth increases ín urínary renin excretíon

(patíents 4, 5 and 9) . rn patíents wíth very high 1eve1s of plasma 83

renin Èhere \^ras an assocíated increase in renin excreËion. The independence of urinary renin excretion and the presence of heavy proteinuria was clearly demonstrated ín these patients" The urinary renín levels only increased ín patienÈs with elevated plasma renín levels and not in paÈients with proteinuria alone. These findings support the conclusíons made as to the handling of renin by the kidney.

SAMMARY TO SECTTON 2

1" A system for the quanÈítatíve assay of renin 1n human urine has been developed. It is based upon Èhe incubaÈion of uríne with nephrectomized sheep subsÈraÈe aÈ pH 7.5. Angiotensínase activity ís abol-íshed by pH 3.3 treatment of urine samples prior to dÍalysis to pH 7.5 and the presence of EDTA in the íncubation mixture. The alrount, of renin present ís determíned by an inítíal velocity technique, the concentration of renin being expressed in units/m1. The inËerfering actísn of pepsín is prevented by incubation of enzyme-substraËe mixture at pH 7.5.

2. Identifícation of the urínary enzlrme with plasma renin has been established in ír¡munological sÈudies, kínetíc studíes and the pI{ dependence of the two enz)rmes.

3. ExperimenÈs have shown thaÈ pepsin in urine can form a pressor material indistinguíshable from angiotensin on incubat.ion with renin substrate. 84

4" The presence of at least two separate anglotensínases in urÍne was established by studying the denaturatíon characteristics of angiotensinase in urine. 5. SÈudies on renin output and renin clearance in normal subjects and hospital patíents Led to the followíng findíngs:

(a) Urinary renín output varled wídely among normal subjects but wíthín Èhe one subject Ít remained relatively consËant. Renin output was higher in females than ín males.

(b) There riras no relationship between urínary renin levels and plasma renin levels, glomerular fiLtration rate, urÍnary sodium, \^rater or protein excretion. The negative correlatíon beÈween renín concentration in urine and uríne volume índicated an índependence between renin excretion and urínary volume. (c) Although elevatíons of plasma renin levels induced by natríureÈíc therapy elevated urinary renín excretion, renin clear- ance fell, whilst creatiníne clearance and total protein clearance remained relatively constant. Excess renin excretion did not occur in prot,eínuríc patients unLess plasma renin levels were also elevated. These resul-ts and consideraÈion of the clearances of renín, pepsín- ogen and amylase into urine (enzymes of símilar molecular weíght) led to the concluslon that renin ís fíltered and subjected to a selectíve reabsorption process. (d) The absence of any signíficanÈ change ín total proteín 85 excretionintourineduríngelevationofpl-asmareninlevelstofive levels t,ímes control levels makes it unlikely that changes in renín induced by posture are responsíbIe for the concomitant postural changes ín Protein excretíon' 86

SECTION THREE

THE EFFECT OF ADMINISTRATION OF THE NATRIURETIC AGENTS.

SPIRONOLACTONE A}ID CHLOROTHIAZIDE ON PLASMA RENIN ACTIVITY

PLASMA RENIN CONCENTRATION AND RENIN SUBSTRATE LEVELS. 87

THE EFFECT OF ADMINISTRATION OF THE NATRIURETIC AGENTS.

SPIRONOLACTONE AND CHLOROTHIAZIDE ON PLASMA RENIN ACTIVITY

PLASMA RENIN CONCENTRATION AI{D RENIN SUBSTRATE LEVELS.

In a previous sect,íon of thís thesis, experiments are described ín which the natriuretíc agents, spironolactone and chloro- thíazíde, \^rere admínistered to normal man to study the effects of elevaËíon of plasma renin levels on Èhe output of renin into Ëhe urine.

Thís sÈudy \.üas expanded in order to correlate the actions of such natrÍuretíc agents on the plasma renín actívity and plasma renín concentration, and to see whether natríuretic therapy índuced a qualitative or quanÈitaLive change ín renin subsLrate levels.

A rise in renin actívíty consequent upon sodíum depletion induced either by dietary deprivatíon or admínÍstration of natriuretics

ís a well descríbed phenomenon (VeyraÈ, de Champlain, Boucher and

GenesÈ, 1964; Conn, Cohen and Rovner, L964). Thís elevaÈíon ín plasma renin activity is consequent upon a rise in plasma renin concentratíon (Brown, Davies, Lever and RoberËson, L963, 1964). It ís not possible, however, to correlat,e the rise in renin activíty wíth the rise in renin coricentratíon observed by these workers sínce dífferent techniques T¡rere used ín the preparation of plasma for estimaÈion of renín activity and conc,entratíon" 88

METHODS:

Fíve normal male subjects r^rere treated wíÈh 0.5 Gn daíly of chlorothíazide (chlotríde: Merck, sharp and Dohme) for 4 days. on the fifth day the dosage of chrorothíazíde was increased to I.o Grn. Seven normal subjecÈs l^7ere treated as prevíous1y described,

í"e. they were given spironolactone (Aldact,one, searle) 50 mg twice daíly r.or L2 days. For the last 5 days thís was combined with chlorothlazíde 0.5 Gm daily. rn none of these subjects was any control exercised over díetary sodír-un íntake.

Twenty-four hour urine coll-ectíons \^rere taken prior to the commencement of therapy, on the 5th day of therapy wiÈh chloroÈhi azid.e, on the 7th day of spironolacEone therapy, and on the 5th day of comblned spíronolactone and chlorothíazide therapy.

All venous blood samples were Èaken at 11OO hours following compleÈion of Èhe 24 hour urine colrection, the subject being seated. Blood was wíthdrawn from an antecubital vein and heparín (20 units/ rn1) was used as anÈicoagulant.

Plasma renin actÍvity, plasma renin concentration and renin substrate r¡rere estínated by Èhe methods described (skínner, L967).

serum sodíun, potassir¡n and creatinine \^rere measured by the Technicon Aut,oanalyser method (Technicon, Ardsley, N.y. , 1965). To'determine whether díuretic therapy caused any qualítatíve change ín renín substrate, a constant amount of renal renín was added 89

to pooled plasma sampl-es Èreated as for trenln activítyt, i.e. dialysed to an initíal pII ol 4.5 prior to díalysís to pH 7.5. These plasma samples were obtained from subjects príor to commencement of therapy; on tlne 7 th day of therapy wíÈh spíronolactone, and on the 5th day of combined therapy with spironolactone and chlorothiazíde. The velocíty of formation of angíotensín was deÈermíned ln the samples followíng Èhe addítíon of a constant amounÈ of renal renin, and ín control dílutíons which \nlere set up in parallel to allow correctíon for the endogenous velocity of the samples.

To determíne wheÈher there \^ras a linear relatíonship between the concentration of plasma renin treated to pH 3.3 acting on hrrnan substrate and Èhe rate of formation of end-product, serial dilutfons of pooled pH 3.3 treated plasma were added to pH 4.5 Ereated plasma.

The endogenous velocity of a control dilution of pH 4.5 plasma was again determíned to allow for correctíon of Èhe velociËy obtained.

RESULTS:

Treatment r^7ith chloxoEhíazide alone for 5 days produced a ríse ín plasma renin activity and plasma renin concenÈratíon ín all subjects (l'igure 3-1, Table 3-1). Plasma renin activity rose to 7.1 times control levels (0.01>P>0.002), whilst the plasma renin concen- tratíon rose Èo 2.8 tírnes Ëhe control levels (0.01>P>0.002).

Treatment wíth spíronolactone alone f.or 7 days produced a 4.5 tímes control rise in plasma renin activit,y (P<0.001) and a 2.5 7o/g

lrl (, z - I 35o l- z l¡l I ú lrl È

roo

o DAYS

Fig. 3-1 Mean per cent changÇ from conËrol of 100 Ín plasma renin acËívity, plasma renin concentration and renin-substrate ín 5 normal subjects treated with chlorothiazíde. TAsLE I

Absslute values in PRC, PRA and renín substrate before and after chlorothiazLde therapy for 5 days"

PRC PRA RENIN SUBSTRATE Normal N Normal 1 . 710 . 5 ng /rnl lhr 1"1t0"3 elnL 9"1l3"5 unitsfml e 6 0 0 0 0"8 0.6 1 8.3 29.2 0.6 6.0 0.4 2 20.0 31" 3 0.95 4,L 0"6

L.2 3 L2.3 37.5 0"8 5.0 t.2

1"0 4 9.9 28.8 0.92 5.4 1.0

L.2 5 11 .9 34 .2 0,7 6.4 I"2

Mean 7" +17 8 +611 -L2 Change

0 .0 1>P>0 .002 P>0"L P 0 .0 1>P>0 " 002 90 tímes con¡rol rise in plasma renín concentration (P<0.001) (Fígure 3-2, Table 3-2), whilst combined therapy wíËh spironolactone íncreased the plasma renín activity to 10.2 tímes control (P<0.001) and the plasma renín concentration to 4.7 tímes the control (P<0.001) (Fígure 3-2, Table 3-2).

Serum sodiurn, serum potassium and creaEiníne clearance remaíned unchanged duríng therapy wíth spironolactone alone and spironolactone plus chlorothiazíde (Table 3-2) . Renin substrate leve1s díd not change signifícanÈly through-

out Èhe course of diuretic therapy. It was thought that the greater íncrease in plasma renin

activíty, as compared wíth the íncrease in p1-asma renin concentraÈion, might be due to a qualitative change in renin substrate induced by diuretic therapy, resulting in a greater affíniÈy of the enzyme for the substrate. This could be due to a change in the substrate mole- cule or to the addition of a cofactor to plasma. However, when this hypothesís hlas tested, by adding human renal renin to plasma treated as for esËímatíon of renin activity, the plasmas being pooled control plasmas, pooled plasmas collected after treaÈment with spironolactone

and pooled plasmas collected after Èreatment wíth spironolactone

and chlorothiazide, angiot,ensín was generated at rates of 65 ¡ 65 and 54 nglnl/hr, respecÈively, The slower rate of generation of angio-

Èensin in the plasma sample colLected after combined therapy \,\tas a rooo

l¡l (, z - I l- PRA z soo t¡t I ol u¡ À P

roo R-s

o t2 D AYS

100 1n plasma Fie. 3.'2 Mean per cenÈ change from control of reninactivity,plasmareninconcenLratÎonandrenin.substTate leve]-sinTnormalmalestreatedwíthspironolactonealoneforT for days and spironol-actone and chlor olíníazide (in combinaË1on) a further 5 daYs. TABLE 3-2

values in plasma renin concentraÈion, plasma renín acËívity, renin substrate' serun sodir:rnt Absolute therapy' serum potassí'' and creatinine clearances before and on the 7th and 12th days of natriuretic

CreaÈinine PRA Renin substrate Serum Na Serum K clearance Subjects PRC mllrnin units lrnl nzlmLllnr uelml rneq lL meq lL 0 712 o7 0 7 0 712 0712 07L2

1.5 r37 L36 1s5 4"3 4"6 4.4 193 156 1 8.4 19.5 3r.7 1.4 4.8 10.0 2.0 1.4 r.2 L36 i35 r34 4.6 4"5 4.0 161 180 2 9.3 23.8 38.4 L .4 6.0 L5 .6 r.4 L.4

r.6 139 L36 135 4.3 4"7 4,5 J 10.3 27 .5 51 .3 1.0 6.4 15.0 1.2 r.4

L"4 L4L l3s 4"1 4.8 4"2 1s3 91 4 9.8 20.0 5s.0 1.3 6.0 L4,7 L.9 1.9 r37

L.2 137 r40 r37 4"3 4.7 4" 8 2L3 151 5 13.5 23.8 61 . 3 1.4 6.7 r4.1 r.6 1.3

r.6 139 135 133 t+.3 4.5 4,L 6 5.3 L7.3 31.3 1.4 s.5 t2"8 1.8 1.8 2.O r.4 r37 136 140 5.2 5"0 4.5 163 166 7 1l-"s 34.2 46.3 1.3 5.6 9.5 1.3

Mear. % 0+l +6 -1 -15 +15 t +37 L +354 +922 +3 -9 Change

<0.001 n.s n.s. fl .s n.s. n"s n.s. P <0.001 <0.001 <0.001 n"s. 91

reflectÍon of the lower concentratíon of renin substrate in Èhis sample (I.2 yglnl, as compared with 1.4 vg/nL ín the two other plasma samples) .

I,rlhen this experíment \^ras repeated with a lesser concen- tratíon of amníotic fluid renin, the rate of forrnation of angiotensín was 11.1, 11.5 and 9.6 ng/mL/hr, respectívely. These fÍndíngs indicate

Èhat there \^ras no increased reactívity of plasma duríng naÈríureÈic therapy due to íncreased substrate reactivity or cofacLors.

Figure 3-3 shows the linear relatíonshíp between the coÍrcentratíon of pH 3.3 treaÈed plasma renin added to pH 4.5 treated plasma and the rate of formation of angíotensin after correction for endogenous velocíty has been made.

DISCUSSTON:

Both chlorothiazide and spíronolactone produced strikíng increases in plasma renín actívíty and concentration ín the absence of any sígnifícanÈ changes in serum sodium, serum potassíum and creatinine clearance. Combíned therapy with chloroÈhíazíde and spíronolactone produced a further rise in p1-asma renin actívity and concentrat,íon. This ís probably a reflecÈion of Èhe augmentation of the natríuretíc acÈion of these drugs when used ín combínation

(Kagawa, 1960), since the drugs have different sites of action on the renal tubule. Chlorothiazide acts on the cortical diluting segment (Earley, Kahn and Orloff, 1961) and spironolactone acts as a ro o sL \ I E \ Ðc

z 5 ItD-z TI lr o o I (' z o o o so too "Á INITIAT RENIN coNcN

Fie. 3-3 Effect of serial dilution of pH 3'3 treated plasma reninontherateofformatíonofangiotensinfromnormalhrrnan subs trate. 92

specific aldosÈerone antagonf-sÈ (Vander, WíIde and Malvin, 1960).

Both drugs can produce a natríuresís ín Èhe absence of signíficant changes in glomerular fílÈratÍon rate (Kagawa, L96O; Fuchs and Marlin, 1960).

The elevation of plasma renin activity to above normal levels by the adminístratíon of chlorothiazíde alone is ín contra- distínctíon to the findings of Bourgoignie, Catanzaro and Perry (1968) who found, using comparable doses of hydrochlorothíazide, that arr elevatíon in plasma renín activity in essenÈial hypertensíves occurred only when díureÈíc therapy was eombíned with restrícEion of dietary sodíum ínÈake to less than 20 meq/day. This may lndicate that a hypertensive subject ís refractory to moderate sËímu1i whích cause Èhe release of renin in normal subjeets. The explanatíon for the relat,ívely greater increase in plasma renin actívít,y over plasma renin concentration observed in all- subjects sÈudied ís not clear. It ís not due to any quantítative change ín the renín subsÈrate, nor ís ít due to any qualitatíve change

ín renin substrate, resul-ting in an íncreased affinity of the enzyme for Èhe substrate. The linear relatíonshíp between the concentraÈíon of renin ín plasma treated to an inítíal pH of 3.3 and the raËe of formatíon of end-product from human substrate makes it unlikely that there is any co-factor actíng ín normal- plasma" 93

However, t\^ro further possíbi1Íties exist. Firstly, thaÈ there ís a cofactor present, the release of which is sËímulated by natriuretic therapy which is not capabJ-e of cross-reacËing \riËh elther hr¡nan renal renin or amníoÈic fluid and which ls desËroyed by pH 3.3 treatment. Secondly, it is possible that sodíum depletion produces some st,ructural or spatíal rnodífication to Èhe renin molecule

ítself, resultíng ín an increased affíníÈy for substrate. Such a spatía1 modification of the renín molecule could be easily damaged by exposure to a low pH, such as pH 3.3, whílst being retaíned duríng pH 4.5 treatmenÈ" This hypoÈhesis would explain the fíndíngs observed

ín the experiments described and would ímp1y that natriuretíc Èherapy induces intracellular changes whích are refLected ín the productíon of a modifíed form of renín.

SUMMARY TO SECTION 3 Spironolactone and chlorothiazide, admínistered separately or in combination, produce sÈriking elevaËions in plasma renín activity and plasma renin concentration. In all subjecÈs studí-ed the rise ín plasma renin acÈíviLy r/üas greater than the concomitant changes in plasma renin concentration. Thís was not due to an associated increases ín renín substraÈe levels, nor ltras there any evidence of increased cofacÈor actívíÈy in plasmas from patienÈs receíving nat,ríuretíc agents. The formatíon of pressor material from the acËion of plasma renín on human subsËrate bears a 94 línear rerationship to concent,ration of the enzyme. A hypothesis has been described to explain these findings. 95

SECTION FOUR

RENIN CONCENTRATION IN I{UMAN FOETAL AND MATERNAI TISSUES 96

IN CONCENTRAT ON IN HUMAN FOETAL AND MATERNAI TISSUES

Renin-like enzymes have been found ín a diverse group of

Èíssues, íncludíng Èhe submaxlllary gl-and of the whíte mouse (Werle, Vogel and Goldel, 1957; Turrian, 1960), the arterial wall of the pig (Dengl-er , 1956; Gou1d, Skeggs and Kahn , L964) and the reproductíve tíssues of varíous animals. Renín has been found in the placenta of the cat (Stakeman,

1960) and the dog (Hodari, Bumpus and Srneby, L967). Gould, Skeggs and

Kahn (1964) found very low concenÈratíons of renin-like enzymes 1n the reproductive tíssues of the pig, the concentrations of enzyme present being only 0.6 to 0.4 x 10-4 units/gm wet tissue for placenta and myometríum, respectively. The renal concentration of renin exÈracted by these workers was of the order of I unít/g* of wet tissue. lJhen Gross, Schaechtelin , ZiegLer and Berger (1964) studied the reproductíve tíssues of the rabbit, they found hígh concentratíons of renin-líke enz)¡mes present, concentratíons of the order of I/10th of those found ín the renal cortex.

Very high concentraËíons of renÍn have been found ín human amníotíc fluid (Brown, Davíes, Doak, Lever and Robertson, L964).

Ttte levels found were 100 times maternal pJ-asma levels artd 20 tímes

Èhe l-evels found in cord pJ.asma. On the basís of these results, a study was undertaken to quanÈitate the l-evels of renín found ín Èhe 97 reproduct,íve tissues of the pregnant female and Èo locate the source of renin ín amniotic fluíd.

METHODS: Tissue ettTacts:

Tissues were obtaíned from 6 healthy r^7omen undergoing elect-

íve caesarean section. In none of these vromen was there any evidence of preeclamptíc toxaernía. Tissues were also obtained from 4 normal l\romen followíng spontaneous vagínal delivery" Fígure 4-l is a diagrarnmatic representaÈion of the sites from which tissues were taken. Chorion laeve was sËripped from amnion ín regions distant from the placenta. Chorlon plate was taken from the foetal surface of the placenÈa, after dissecting off the amnion and strípping the choríon off the underlying placenÈa. Placental tissue iËself was díssected free from the maÈernal surface of the placenta, whílst, uterine muscle and decidua ülere obÈaíned by sharp dissectíon during caesarean section. Renal cortex was obtained from 4 adults withín 24 hours of death, the paËients having no evidence of renal disease. PLasma øtd amniotíc fluid sampLes:

Arnníotíc fluid was collected from 6 women at vaginal delívery t caesarean sectíon or during therapeutic abortion. MaÈernal venous plasmas and venous and arterial cord plasmas \^rere also obtained from these 6 women. FurÈher sampl-es of amniot,ic fluid were taken seríally etrium

I chorion Plate

'/'/ placenta decidua

chorion

amnron

Fis. 4-.l Diagran showing sítes of sarnpling of maternal and foetal, tissues for estination of renin contefit' 98

from 5 \^romen following artíficíal rupture of the membranes, the last sampl-es being Ëaken at the end of the first stage of labour. Preparation of tissue sønpLes and ønniotíc fLuid for assay of renín: Antniotíc fLuid: AmnioËíc fluid was chílled immedíately following collectíon. Since quantítatíon of the concentratíon of renín was desired, the amníotíc fluíd obÈained \^/as treated as for estimatíon of plasma renin concenÈratíon (Methods). It was díalysed to a pH of 3.3, heated for one hour at 32oC at this pH and dialysed to a fínal pH of 7.5. Fol1owíng addítion of neomycin sulphate

(2 ng/nL) and Trasylol (100 units/m1) and correctíon of the volume, the samples r^rere díluted Èo enable accurate quantitatíon of Ëhe large amounts of renin present. Standard sheep substrate was added (ratio of two part,s of amniotic fluíd to one parË of sheep substrate) and the mixture incubated at 37oC, wiÈh sampling and assay of angíoËensín by the meEhods descríbed previously.

Eætraetion of renín fnom tissues: The tissues obtained were washed 5 Èírnes ín cold phosphate buffer, at pH 6.8'excess fluíd was absorbed into paper, and 0.5 to

I.0 gram of tissue r"" r"igned out and stored at -15oC. Renín was extracted by lysis of the cells ín distílled \^raÈer, followed by

fxeezíng and thawing twice, and then homogenizatíon for 30 seconds

(Serval Omnimix). The whole tissue homogenate r¡ras placed in B/32

Visking cellophane casings and subjected to the díalysis sequence as 99

for estimat,ion of plasma renin concentraËíon (Methods). Following

the díatysÍs sequence, the samples I,ìIere decanted and centrífuged. This step yielded a clear tíssue extract to whích neomycín sulphate, Trasylol and standard sheep subsÈraËe were added in the concentrations

described as for the ÈreaÈment of amniot,ic f1uíd. Tíssue extracts which contained large anounts of renin were diluted before addítíon of substrate t,o allow accuraÈe quantítaËion of the amounts of renín

present to be made. To determíne the effecEs of concenËration of amnioÈíc fluid renín on the rate of formatíon of pressor maÊerial, serial díluÈíons of amniotic fluíd (contaíning 96 units/ml of renin) were íncubated wíth both standard sheep subsÈrate and human substraËe. Correction

for an endogenous velocíty of 0 .5 ng/nLlhr was made when human

substraÈe preparations I^rere used. The effect of substrate concentration (using substrate from

both non-pregnanË and pregnant women) on the rate of formation of angíotensin at consÈant concentratíon of renín in amniotíc fluid was

determined. This was compared wíth Ehe effect of variatíons in human

substrate on Èhe rate of formation of angiotensin aÈ a constant

concentratíon of pH 3.3 treated renín. Control dílutíons of human

substrate hrere set up ín parallel to correct for the endogenous

velocity of human subsÈrate preparatíons. 100

RESULTS:

Chæacterí,sties of the enzAme in ønnì.otic fLuid ond tíssue eætv'aeLs: The ámniotic fluid enz)¡me and the enzyme present in tíssue extracts formed angíotensín at, a linear rate wíth relatíon to tíme. The formation of pressor materíal was parallelled by a concomíÈanÈ consumption of renin substrate" One extract of choríon and one sample of amníotic f1uid, each wíth a high renín contentr \^7ere incu- bated wíth sËandard subsËrate until bio-assay índicated substrate exhaustion. Followíng thís, addítion of renal renin in excess faíled

Ëo generate angiotensín from these incubates. The forrnaËion of pressor material bore a linear relatíonship to Èhe concentration of the enzyme following serial dílution of àvrníotic f1uÍd when tesLed against both sheep (fígure 4-2a) and human

substrate (Figure 4-2b) " Specificity of the enzyme r¡ras indícated from studíes using antíbody t,o human renal renin, the erì.zyme in amniotic fluid being totally inhíbited by Èhe additíon of one part of antibody Ëo 4 parts of amniotíc f1-uíd, whilst the control sample generated 250 ng of angíoËensin during a 16 minute incubatíon.

The i(m (Figure 4-3) of amniotic f1uíd on substrate from pregnant and non-pregnant vtomen was 3.0 and 4"0 Ug/ml, resPectively.

The Ifu for plasma renin on human substrate was 7.6 ug/ml . o t2

L ¡ \ - \E Ð c z o Ilnó z l¡¡ l- o I (Ð 2 o

o o o roo % INITIAT REN¡N CONCN

Fis.4-2(a)EffecÈofserialdílutíonofamnioticf]-uid renín on the rate of formatíon of angiotensín from standard sheep substrate (Appendix Table 15) ' 70 o

L s\ - Ê \ U' C

tl =35z t¡l l- o o (Ð- z o o o o 5 roo 7" rNITrAt R ENIN coNcN

Fie. 4-2$) Effect of seríal- dilution of arnniotíc fl-uÍd renin on the rate of formatíon of angiotensÍn from human substraËe (APPendix Table 15). t50

roo

v

o 50

o

25 lto v s

Fig.4-3![oolfplotsderivedfromtheeffecÈofsubstrateofi the rate of fornatíon of angiotensin (v) plotLed agaínst the rate offormationofangiotensíndividedbysubstrateconcentraËíon. ( o amnioËíc ( I ) pII 3.3 treated plasma renin on hrmnn substrate. ) 16(c)) fluid renin on human substrate' (Appendix Tabl-e 16(a) ' ' 101

The pzreeenoe of eubetrate tn øtmåotic fLuid: Renin substrate was found in 2 amniotic fluíd sampl-es tested, the concentratíon beíng of the order of 100 to 150 ng/ml.

Curved velocity plots were obtaíned when these amniotíc fluid samples were treated to pH 4.5, príor to dialysís to PH 7.5 and íncubated at

37oC without the additíon of sheep substraÈe (l'igure 4-4). Since the

samples were free of angioEensinase, the non-linearíÈy of Ëhese curves is probably associated with consumptíon of greater than 50% of the substrate. Three other amníotíc ftuid samples contaíned no detect- able renín substraËe.

Angiotensinase actì,ÐitA : No evidenee of angioÈensinase actíviÊy ûras present ín any of

Èhe Ëíssue extracËs Ëested. Angiotensínase activíty was tesÈed by incubation of amniotíc fluid and tissue extracts with the end-product

formed as a result of incubation of human renal renin utíth hurnan substraEe (Methods). Greater than 907. of. angíoËensin activíÈy

remained following incubation of the samples fot L2 to 24 hours at

37oc.

Absenee of inhíbitov,s ín tissu.e eætracts:

One part. of amníotic fluid renín was added to níne parts of pooled ext,racts of myometrium, decj-dua and placenta. After correctíon

for endogenous renin conÈenÈ of these extracÈs, the observed renin levels, 57r 60 and 60 units/ml, were not significantly dÍfferent from rro

\E ul ec z -râ o zl¡l F 9 t9 z

L- o 40 80 rNcuBATtoN lnns I

Fíe.4-4Endogenousplasmareninactívityof2amnioticfluíd samples treated to PIt 4.5 prior Èo incubation at pII 7'5' LO2 the expected rate of 65 ng/ml, These findings suggest that the variations ín leveLs found in the tíssues Ëested are riot due to the presence of inhíbitors. Coneentratíons of rendn in tíssue eætracts qnd ø¡vtiotic fLuid: Renin eontemt of ti.ssue eætTq,cts: Table 4-1 shows the reletíve concentratíons of renin in tíssue exËracts obtained from 6 patients undergoing elective caesarean secËíon and from 4 paÈíenÈs following normal vaginal dellvery. In any one subjecÈ, choríon contained from 1.5 to 12 tímes as much renín as amnion (P<0.001) and 2 to 48 tímes as much renin as decídua (P<0.001).

The concentratisn of renin in choríon plate whích r^ras díssect,ed off the placenta (Patíents 7-LO, Table 4-1) was less Èhan in perípheral chorion (P<0.05) but more than in tissue dísseeted from

Ëhe maternal síde of the placenta (P<0.05). In order t.o compare the renin content of tíssues extracËed from the reproductive tract to the renín content of Ëhe renal cortex, 4 cadaver kídneys rirere assayed for their renín contenL. The concen- traÈion of renin was considerably higher than the levels found ín choríon, the actual- values being 721800, 33,600, 78r4OO and 44'800 units/gm wet tissue. Renín LeaeLs in ørmiotic fLuid, foetaL and matevmaL pLasma" The renin concenEråtion in amniotie fluid aÈ term ranged Renín concentratíon (r¡nits/G wet weight) in foetal and maternal tissue collected at caesarean secËíon and vagínal delivery

Caesarean Arnníon Choríon DecÍdua Myometritm Chorion PlaÊe Placenta

I 1 400 6050 L270 80 80

2 770 9500 L270 32 64

3 2020 31 80 1 700 42 42

4 1330 6 800 420 42 74

5 8500 177 37 T2

6 680 1400 48

Vaginal

7 r270 2L2 53

I 2L20 740 48

9 960 297 64

10 500 2L

Me¿rr L240 4420 967 46.6 437 50.6 103

from 160 Eo 2650 unirs/ml (mean l05g unirs) and displayed a 40 ro I gradíent wíEh respect to foetal (mean 26 units) and maternal plasma (mean 27 units) (table 4-2). There üras no consistent arteriovenous

difference recorded across the cord; Moreover, foetar and maternal plasma 1evels vrere not signífícantly dífferenÈ" The amniotic ftuíd

renÍn content of draíníng líquor díd not change consistently over períods of up to 7 hours (Table 4-3).

Comparison of the group data ín Tables 4-1 and 4-2 shows that

the concentraËion of renín in choríon is signíficanÈly higher (rnean 4420 units/gm) rhan thar in amníoric.fluid (mean 1o5g uníÈs/e*) (P<0.02), whilsÈ decidua (mean 967 units/gm) and amnÍon (mean 1240 units/gm) have levels sirnilar to those found ín amníotic fluid. These results are sunmarj,zed ín Figure 4-5.

DTSCUSSTON:

The híghest conËent of renin ín the inÈra-uterine tíssues studíed was found in chorion laeve. only in this tíssue was the concentraÈíon of renin greater than Èhat found ín amniot,íc fluid"

This fíndíng would suggest that renin díffused from chorion through the relatívely simple membrane barrier constituted by the amníon into amníotíc f1uid.

since the gradíenÈ between amniotíc fluíd and maternal plasma is of the order of 40 to 1, there may be some barríer Ëo the outward diffusion ínto maternal prasma of renín produced by the TASITE 4-2 Renín concenÈratíon (unÍts/m1) in amnioËíc fluíd maternal and cord plasma

Amniotic MaÈernal- Cord Plasma PatíenÈ Delivery fluíd plasma Art. Veno

1 Caesarean 2650 39 26 43

2 Caesarean 160 30 42 42

3 Caesarean 285 2L L7 L2

4 Vaginal 75A J.2

5 Vaginal 27 26

6 Vaginal 11 11

7 Vaginal 2t+0 35

8 Vagínal 2400 2L 22

9 Vaginal 700

10 Vagínal I 800

11 Vaginal 540

L2 IlysteroËomy (16 weeks) 560

l3 HysËerotony (14 weeks) 105

& Mean 105 I 27 24 26

* In ealeulating mea¡r of amniotic fluid levels paËÍents 12 and L3 were excl-uded TABLE 4-3 Renín concenÈratíon (unitslml) ín draíníng liquor

Patíent Hours after Renin A"R.M" Coneentration

7 0 240

4 385

5 5 240

8 0 2400

5 I 900

9 0 700

7 800

l0 0 I 800

1.5 I 800

11 0 540

2 "25 550

A"R"M" = artifícial rupture of membranes /- - -\

ium r 058 myometf \ 47 I \ decidua I \ 967 / I I plac,enta chorion 4420 I \ I \ \ \ amn¡on clrorion plate 1240 437

\___-,

ríe. 4-s Diagram of the concentrations of renin found in the various maternal and foetal Ëissues. 104

chorion. The decidua contaíns as much renin as is found in amnion and amníotíc fluíd, whíIst the steepest gradíent of renin is between the decídua and the rnyomeÈrium. Therefore, iÈ seems likely thaÈ the decidua, whích ís a thick and relatlvery avascular layer, constitutes a barríer to Èhe outward díffusion of renín. These findíngs suggesÈ that renín ín amníoÈíc fluid ís derived from the choríon. This impJ-íes Èhat the chorion must syn- thesíze and release renin, a concept which has received substantiaÈíon from Èhe ín uitro culÈure studies of symonds, stanley and skÍnner (1968). Moreover, since the levels of renin in a¡nníoÈic fluid do not fall during prolonged draínage of líquor at a tlme when amníotíc fluíd ís thought to be conÈínuously formed at a slow rate, there is apparently a contínuous producËion of renin and secretion ínto amniotíc fluid.

The presence of hígh renín levels early ín pregnancy makes ít unlíkely that the foetus ís a source of the renin for¡nd ín amniotic.fluid, sínce the level-s obtained are símílar to those found at ' term" Furthermore, foetal plasma renin levels are of Èhe same order as maternal plasma levels, and since renín ouËput ínto human urine has been found to be low and renin levels during draíning of liquor remain constant ín amníotic fluj-d, íÈ is unlikely that Èhe foeËus contributes renín in any significant amount Ëo anniotíc fluid. The identíËy of the enzyme ín the reproductÍve tíssues 105

appears Ëo be similar Èo thaÈ of plasma renin, since iË has Èhe s¿rme specíficity Èowards antíbody inactivation and Ëhe generaËion of pressor material is accompanied by a fall in renín substrate. Sínce all tÍssue ext.racts \^7ere Èreated ín the same manner and no inhibítors or non-specifíc depressor subsËanees r¡rere found, ít seems líkely that a quantitative estimation of the actual conÈent of renin ín the different Èíssues examined has been obtaíned"

The Krn of amniotíc fluíd actíng on human renín substrate

ís lower than that for plasma renín acting on human substrate. Thís índicates a slíghtly higher affinity of amniotíc fluíd renin for human substrate, a simílar fínding to that observed wíÈh rabbit ut,eríne renin and rabbít renal- renin (Anderson, Herbet and Mulrow, 1e68).

The intra-uterine disËríbution of renin wíÈh relatively lor¿ concentrations ín Ëhe myometrium and placenÈa r¡ras in conÈra- distinctíon to the findíngs of Ëhe distríbut,ion of renín in rabbit reproductive tíssues, However, this species dífference r^7as confirmed by assay of renin content in the tissues of the rabbit. Tíssue extracts were taken from a rabbit with 3 foetuses ín one cornu and none in Èhe other. Myometrium from each cornu conËained a high renín content (400,000 unit,s /gram). PlacenEa contaÍned relatívely little renin (100 units/gram) and amniotie fluid only 30 uníËs /gxam. The foetal membranes contaíned more renín than amniotíc f1uíd (67,000 106

uníts /gxam), but less Èhan the myometrium. There Ì¡ras no gradient apparent, between the amníotÍe fluíd and maternal plasma levels of

renin, sínce Èhe levels ín these tr^ro fluids \^rere approxímately the

same "

A preliminary study ín the sheep has revealed hígher renin levels in maternal plasma than ín amníotíc fluid. These fíndings indicate that Èhere is a consid.erable differ-

ence in the dístribuÈíon of renin ín the ínt,ra-uterine tíssues and suggest,, furthermore, that care must be taken ín extrapolating results

obtaíned ín experimental animals to Èhe sítuatíon ín man. The acÈual functíon of renín ín the reproductíve tract ís

not el-ear. rt ís not known whether it subserves some intra-

uterine funcËíon. Renín derived from amniotíc fluid appears to have

the same propert,ies as regards molecula:: size and charge, al_though

Ëhe Michaelis constant appears Èo be dífferent (Anderson, Herbet and Mulrow, 1968).

It ís also noÈ known whether renin produced in the repro-

ductive tissues contributes to Èhe circuLaÈing mat.ernal plasma renj-n

levels and hence affects sodíum homeostasís in the pregnant \^romen.

Ferrís, Gordon and MuLrow (L967) have shown that Èhe Pregnant animal maínÈains a hígh plasma renín level after bílateral nephrectomy" However, these results were obtained ín the rabbit, and, as already mentíoned, there appears to be a species dífference in the 107

distríbution of renín ín the reproductÍve tíssues" Moreover, in man

the source of renin, Èhat is the chorion, ís separated from Èhe

maternal círculation by the Èhick and avascular decídua.

Histological examínatÍon of the choríon has revealed two layers, a deep trophoblast.ic layer r¿ith some attached decidual cells

and a superfícial fibroblastíc 1ayer, but whích of Ehese celr Èypes ís the síte of synthesís of renin is not yeE apparent.

SUMMARY TO SECTTON 4

1 ' High levels of renin were found in the reproductíve tíssues of the normal pregnant female.

2, The highest levels were found ín Èhe chorion laeve. Renin 1eve1s in the amnion, decÍdua and amníotic fluíd were similar.

3' The steepest gradíent ín renin concentratÍon exísts beËween the decídua and the myometríum. These flndings suggesÈ that renin is formed in the chorion and diffuses into the amníotíc f1uíd, the ouÈward díffusion of renin into Èhe maternal plasma being prevented by the avascularíty of Èhe decídua.

4" The results obËaíned also índícate that renÍn is formed and continuously secreted ínto amníotic fluíd.

5. The levels of renin ín the choríon are 7 .i% of the levers of renin found ín the renal_ cortex. 108

SECTION FIVE

CHANGES IN PLASMA IVITY PI,ASMA RENIN

CONCENTRATION A}¡D RENIN SUBSTRATE LEVELS

(A) DURING THE NOR}4AL MENSTRUAL CYCLE

(B) DURING ADMINISTRATION OF ORAL CONTRACEPTIVES

(C) DURING PREGNANCY 109

(A) CHANGES IN PLASMA RENIN ACTIVITY. PLASMA RENIN CONCENTRATION

AI{D RENIN SUBSTRATE LEVELS DURING THE NORMAL MENSTRUAL CYCLE

Brown, Davies, Lever and Robertson (L964) reporÈed that a

ríse ín plasma renin levels occurred during the second half of the normal menstrual cycle.

Elevations ín aldosterone secretion have also been reported

duríng the luÈea1 phase of the menstrual cycle (neich, 1962). These fíndings suggest Èhat there is a physiological

elevatíon in rsodíum-retaíning actívítyr in order to compensate for a tendency to an increase ín sodium loss from the body during the late

menstrual cyc1e. The secreÈion of progesterone during the second half

of the menstrual cycle would provide such a sodium losing mechanism, sínce progesterone can apparenÈly act as a specífic aldosterone antagoníst, acting ín a competítíve manner to ínhíbit the actíon of aldosterone at a tubular level (Landau and Lugibíhl, 1961). Therefore,

the elevatíons ín renín levels descríbed by Brown, Davies, Lever and

Robertson may be necessary to maintaín normal sodíum homeostasis during the menstrual cycle"

In order to study the aetions of oral contraceptives on the renin angiotensín system, iË was first necessary to deËermíne the normal variaËíons ín plasma renin acËívity, plasma renín concentration

and renín substrate levels oecurring during Èhe course of a menst,rual 110

cycle.

METTTODS:

Venous blood samples were taken from 6 normal vlomen at,

íntervals during the course of a normal menstrual cyele. 0n each occasíon the samples r^rere taken from an antecubital vein at 1100 hours, with the subjecÈ seated. Ileparin (20 units/ml-) was used an ant,i- coagulant. No control hras exercised over dietary sodíum or fluíd lntake" The duration of the individual cycles ranged Lrom 24 to 28 days. Samples were taken on three occasíons during the menstrual cycle: at the end of the menstrual b1eed, at the esÈímated ml-d-poinÈ of the cycle, and during the luÈeal phase of the cycle. To establish whether ovulation had occurred, urinary preg- nanediol levels were esÈimated serially ín three normal subjects (Cox, 1963, 1968).

RESULTS:

All síx \romen showed a ríse ín plasma renin actívíty (P<0"01) in the second half of Ëhe menstrual cycle. This was consequent upon a ríse in renin concentratíon in Èhe seeond half of the cycle (P<0.001).

Renín substraÈe levels remained unchanged (l'ígure 5-1, Table 5-1). In one subject (subject 6, Table 5-1), a rnid-cycle elevat,ion

ín both renin activity and renin concenEration occurred for no apparent reason. However, in the IuÈeal phase, the rise ín both renín activíty and renin concentration above the levels found in the 6 20

5 s I E I ¡ at E 2 I c El E f r- C¡ c c o c, 3 =r^ôrv o L -t E ú, o 4¡ () J I c UI -s o oG () c Ê. .c oc oE Ê, É.

o to 20 o to 20 ro 20 Doy of menstruol cycle

I'ig. 5-1 Variations in plasma renin acËivity, plasma renin concentratíon and renin-substraËe levels during the course of a normal menstrual cYcle in 6 women. TA3LE 5-1 VarLatlons ln plasma renin actlvlty (fnn¡, plasma renin concentration (PRC) and renín substrat,e levels

dur:1ng the course of a normal menstrual cyc1e.

Days Actlvity Concentratíon Subst,rate Subj ect Menstrual cycle (nelmUhr) (uníts /rnl) (ug /rnl)

I 3 1.0 6.8 0.8 10 L.4 8.3 0.8 16 2.9 11.3 0.8

2 4 0.8 4.8 L.2 10 0.8 6.3 o.7 t9 1.1 9.8 0.9

3 4 0.4 7.9 1.3 11 0.6 8.3 1.3 20 1¡5 L2,5 1.3

4 5 1.6 6.9 1.3 L2 1"6 8.0 L.2 I9 3.2 13.5 1.0

5 5 0.6 6.5 1;0 T2 1.5 8.3 o.7 r9 3.8 14.0 1.0

6 5 L.4 9.3 1.3 T2 5.0 17 .0 1.3 19 3. r L2.I L.6 111 fírst half of the cycle was evident (figure 5-1). In Èhe three subjects ín whom the excretlon of pregnanediol

ínto the urfne l^las measured serfally, 1,5, 4.0 and 5.O-fold elevations occurred between days 12 and 19, índicaÈing ovulation'

DISCUSSION: These fíndings supporË Èhe results obtained of Brownt Davies, Lever and Robertson (1964) and show that Èhe elevation in renin activity is due to an elevatlon in renín concentration, sínce renín substrate levels are unchanged. As stated previously, Ëhis rÍse in renÍn activity is associated wíth elevaËions in aldosÈerone secretion, suggesting thaË a homeostaÈie mechanism {s beÍng ínvoked to offset the natriuret,ic actíon of progesEerone which ís secreËed followlng release of the ovum and formaÈíon of the corPus luteum' Sínce renin substrate levels remaín unchanged' one r¡ould have anticípated thaÈ the relative rise l-n renín activíty and renín concentraËíon would be the same. In fact,, Èhe renín activity rose Èo 2"6 times pre-ovulaËíon levels, whilsÈ renin concentration rose to only 1.75 tímes pre-ovulation levels" This finding ís slmílar Èo Èhat described ín a prevíous section in whích the relative increases in plasma renin actívity and

plasma renin concentratíon were eompared followíng natriuretíc

therapy " LL2

(B) CHANGES IN PLASMA RENIN ACTIVITY. PLASMA RENIN

CONCENTRATION AND RENIN SUBSTRATE LEVELS

DURING ADMINISTRATION OF ORAL C ONTRACEPTIVES

Sínce plasma renín activity is influenced by Èhe varíatl-ons

in levels of those hormones responsíble for mainÊaíning the normal menstrual cycle, it míght be anticipat,ed thaÈ oral contracepÈíves which are a combinaÈion oestrogen-progestin Èherapy would modify this cyclical rhyÈhm in the renin angíotensin system.

rÈ has been reported that boÈh oestrogens and oestrogen- progestín therapy do, ín facË, cause a rise in plasma renin ectivíty

(crane, Heítsch, Harrís and Johns, 1966; Helmer and Judson, 1967) and that thís is associated with a rise ín aldosterone secretion (Layne, Meyer, vaíshwanar and píncus, 1962). of partieular interest, therefore, are the recent reports of hypertension occurríng in normo- tensíve hTomen followíngthe administrat,íon of oral contraceptive agenÈs (Laragh, sealey and Ledíngh¿ì:n, 1967; I,troods , Lg67; cLezy, personal communicatíon), although the actual íncidence of hypertension in women taking the "pi11" ís not yet defíned (Goodlln and In/aech¡er,

L96e) "

Sínce elevation of renin substrate levels have been induced by administraÈíon of diethyl stilboestrol- to experimenËal anímals and man (Helmer and Griffith, rg52), it is important to determine the 113

relative contríbution made by changes ín renin concenÈration and renín

substrate levels to the eLevations in plasma renín activlty observed

in women takíng oral contraceptive agenËs.

METHODS:

control venous blood sampres were taken durfng the course

of a normal menstrual cycle frorn 8 normal r^romen who had noË received

oral conÈraceptives príor to the commencemenÈ of this study. samples were taken from an antecubital veín at 1r0o hours, with the subject seated. No deliberate control \^ras exercised over dfetary sodium or fluid intake"

Followíng administration of an orar conÈraceptive prepar-

ation' a second blood sample was colrected during Èhe subsequent contraceptive cycle,

Four women received Anovlar (ethínyl oesËradÍal o.05 mg and noreÈhisterone acetaÈe 4 rg, scheríng A. G. Berrin); one received Noracyclin (mestranol 0.15 mg and lynoestror 5 rg, cíba, Basle) and three received crr-44L (E1í-Lí11y, sydney) , whích ís a seríal form of therapy, 0.1 mg of mestranol- being adrnínistered. for 7 days followed by an oesËrogen-progestin combínation, chlormadínone acetate I.5 mg and mestranol 0"1 *g for a further 13 days). Heparia (20 units/ml) was added as anticoagurant Èo samples taken for renin esÈímation. The effect of oral contraeeptíves on the renín angiotensín system 6 women in wíth hypertensi-on (not of renovascular origín) was 114

studied. All patíenËs had been taking oral conÈraceptives for at least 6 months. AlL patients \¡irere receiving both a thiazide

diuretíc and guaneËhidine sulphate (cíba, Basle). Renin levels were sÈudied during conËraceptíve therapy and r^rithín Èwo weeks of stoppíng

therapy " Enzyme assaA and köneLíe studies:

Plasma renín actívity, plasma renín concenËraEíon and renín subst,rate levels were esËimated by the method of skínner (L967).

Possible kíneÈic dífferences between renín substraÈe from normal hTomen and women taking oral contraceptives \¡rere studied by constructing standard subsÈraÈe curves at constânÈ enzyme concen- tration. Pooled plasma diarysed to an íniËial pH of 4"5 was used ín thís study. This Ëreatment inactivated angíotensínases present.

Plasma renin (pH 3.3 treated) was the source of renin used. control dílutions r^rere set up in parallel to allow correct,ion for endogenous velocíty"

RESULTS:

Table 5-2, Fígure 5-2, show the changes índuced in Èhe renín angiotensin system by admínísËratíon of oral conLraceptíves to

I normal I^7omen. All preparations used had the same qualitaËíve effect. DespiÈe the normal menstrual cycle rise, which ínfluenced. the leve1 of the contror samples, a further rise in renin actívity was observed ín all women studied" However, the tine of control PLqSIYIA RBUN H.¡St\',lA Rt'llN ACTIVTTY û]NCB{TRATIO'I EIIN SIINTRATE HeÁùsn n¡rs/m- 6

5 n ,

4 16 4.

3 n 3-

2 8 2

4 I 1

EL E L E L

fvt PC rvlc PC rvlc PC

îj-e. 5-2 Effect of administratíon of oral conÈracePtíves on plasma renín actívity, plasma renin concenÈration and renín- substrate levels in B normal women. MC : mensLrual cYcle PC : contracePtive cYcle E : control samples taken during the first half of the menstrual cYcle L : conËrol samples taken duríng Èhe second half of the menstrual cYcle. TABLE 5-2

Changes in plasma renín actívfty, concênÈratíon a4d substrate induced by oraL contraceptives

Activity Concentration SubsÈrate Subject Cycle Day (nelml/hr) (uníts /ml) (Pelnl)

I MensÈrua1 24 2,7 7.O 1.5 Anovlar 23 4.s s.3 4.0

2 MensÈrual 2I 3.3 8.3 2.0 Anovlar 19 3.5 5.3 3.2

3 Menst,rual L6 2.2 6.3 0.9 Anovlar L4 5.8 6.3 >2.6

4 Menstrual 4 0.6 8.0 L.6 Anovlar L4 5.9 8.0 3.4

5 MensÈrua1 20 3.8 16. s 1.3 CTT_44L 8 4.9 9.8 5.3

6 Menstrual I 0.8 5.1 1.0 CTI-44L L2 2.4 4.0 3.7

7 Menstrual 9 2.4 8.1 L.6 CTT-44L 2L 3.4 4.2 2"8

8 Menstrual L4 L.9 13.5 1.1 Noraeyclln 10 3.8 8.4 2.3 115

sampling during the previous menstrual cycle did affect Ëhe degree rise in renín activity obtaíned. Thus, ín subjects 4,6, 7 arrð.8, ín whom the contror samples were taken in the fírst half of the normal menstrual cycle when the renin acËivíty ís lowest, a mean four-ford elevatíon in renin activity was obtained followíng Èhe íntroducÈion of contraceptive therapy, whilst the mean elevatíon in activity in subjects Ir 21 3 and 5 ís 1.7 times control levels. In these subjects, control samples were taken duríng the second half of the menstrual cycle when the renin activíËy is at its highest level. In the normal menstrual cycle the rise in renín actívíËy ís associated wiÈh a ríse in renin concentraËíon. In the contra- cept,ive cyc1e, on the oLher hand, the rise ín renin activíty is associated with a ríse in renín substrate.levels whílst Ëhe renin concentrat,íon remaíns the same or fa1ls. Thís elevaÈíon in renin substrate levels is due to the oestrogeníc component of the "pí11", since Èhe ríse ín renin substrate levels were observed in subjects

5 and 6 who, at the tíme of sampling, \^rere receiving mesËranol alone. Fígure 5-3 shows dísÈribuÈion of the levels of renin acËíviËy, renín concentration and renin subsËraËe in 8 normotensíve r¡lomen taking oral contraceptive preparations compared to 9 luteal phase normal hromen and 16 normal males. The dífferences in Èhe variations in renin actívity, renín concentratíon and renín substrate in luteal phase l¡/omen and normal males is non-signíficant, whilst the 6

20 4

5 L 1 E T L-c, E E .È3t5 oll 3 s J. Ct! J + = c q, .9t E' E' 't ! tn C -ot I Ø 2 .s c8to C, o .s o g &.2 (¡) .Ec rE (1) É. 5 t tl

o o o MFOC MFOC MFOC

Fig.5-3Levelsofplasmareninact'ivíty,plasmarenin concenËraËion and renín substrate ín 16 normal males (M), 9 females, luËeal phase of menstrual cycle (f) , and B females takÍng oral contraceptives (OC). Values are shown as mean + I SD. LL6 elevation in renín acÈívity duríng oral contraceptive adminístratÍon is sígnificanÈ (P.0.002), as is the elevation in subsÈrate (p<0"001) and the suppressl-on of renin concentration leve1s (p<0.01).

Hypertensiue uomen:

The elevatíons in renin substrate levels observed Ín hyper- tensive women taking oral contraceptíve preparations ïrere as high, or higher than, Ëhose observed in normal.hromen takíng oral contra- ceptíve preparatíons. rn pat,ients 1-4 (table 5-3), Ëhe erevatíon ín renín actívíÈy was hígher than Èhat, Índueed in normal- women wÍth contraceptive therapy. cessaÈion of contracepÈive therapy resulted in a fall ín renin substraÈe levels in all r¡romen.

The changes in renin activity therefore became a reflectíon of wheÈher or not cessation of therapy r^ras assocíated with changes in renín concentration. In patÍ.enÈ 1, the concentration of renin díd not change markedly and Ëhe fall in renín substrate was Ëherefore assocíated with a fall ín renin activiÈy to within normal límits. In paËíenË 2, renin concentraÈíon rose to above normal levels, although the renín activíÈy fell to withín Ëhe normal range, whereas ín patients 3 and 4 the relative rise Ín renín concent.ration was sufficíent Èo offset the fall in renín substrate, hence the renin activity rose or remaíned elevated. Patients 5 and 6 were studied whílsÈ on conËracepÈíves only. They showed the previously described changes, i.e. a rise in renín activity, consequent upon a rj.se in TAsLE 5-3 Plasina renin acEivíty, concentrat,ion and substrate ín six hyperËensive women. on and off .oral contraceptives

AcÈívity Concentrat,Íon SubsÈrate' Seri¡m K (ne/r¿Llhr) (uníts/ml) (uelml) (nrq/l) PatíenË 0ra1 (normal.range (normal range (normal range (normal range contraceptives 0.5-3.5) 4-f8) 0,7-2.5) 4-5.5)

1 0n, 10 6.3 5.0 4.3 0ff 1.8 8.0 1.5

2 On 8.5 16 .5 >2.2 3.0 0ff 3.3 30 .0 1.1

3 On 7.2 TL.7 7.0 1.8 off 11 .0 23.0 1.8

4 On 13.0 32.5 3.9 4.3 0ff 13.5 57.0 2.L

5 On 3.7 3.6 4.5 3.6

6 On 3.1 4.4 6.0 3"7

$

¿i LL7 renin substrat,e, whilst the renin coricentratíon levels were suppressed.

Enzyme kínetíe etudtes :

In Fígure 5-4, the reaction velocíty at constanË enzyme concentraËion is ploEted against the concentration of substrate using substrate from normal women and women takíng oral contraceptíve preparations. At equal substraËe concenÈratíon boÈh substrates yíeld identical velocíties, indicatíng that no qualitative change has occurred in the substraÈe molecule as a resulÈ of contracepËive therapy, the affíníty of the enzpe for the substrate molecule being unchanged. Even aË substrate concentratíons of 4.5 ug/ml, maximum velocíty had not been obÈaíned. Recíprocal plots (Lineaweaver-

Burk, Dixon and trrlebb , L964) show Ëhat, the maximum velocity would be obtained aË concentraËions of substrate of the order of 10 Ug/ml. Therefore, in all plasmas studied so far substrate levels are rate-

limiting "

DISCUSSION:

Helmer and Judson (1967) have recentLy found Èhat renin substrate is a rate-lirníting factor ín Èhe formatíon of angiotensín by normal plasma. The results descríbed above substanËíate these fíndíngs. Other workers have concluded Èhat renín substraÈe ís presenË in excess (Píckens, Bumpus, Ll-oyd, Smeby and Page, 1965;

Haas and GoldblatÈ, 1967; Ayers, 1967). 30

2o I,L î-c E cctr .= (J Io r0 o

o 23 4 5 Substrote (¡.g ml-r)

Fie. 5-4 Effect of substrate concentration usíng humart substrate from normal females ( O ) and substrate from females takírrg oral- contraceptives ( o ). 118

There is no apparenË reason for this discrepancy in findfng.

However, ít may be dependent upon the dífferenÈ condít.ions as regards ionic strengÈh and pH to whích plasmas are exposed when treated in differenE laboratories.

The results of the present experiments demonstrate thaÈ sítuations can occur in which the íncreased formation of angioÈensín by plasma is not due to variatíons ín the amount of renín released from the kidney but Èo increased circulatíng lqvels of renin substraÈe. Such ís the action of the oral conÈraceptive" That Ëhe íncreased rangíotensin forming abilityr ís unwanted is suggested by the finding that, associated with the íncrease in renin substrate levels there ís a concomitant fall in renin concentration,

This suppression of renin release could be medíaÈed either direcÇly by the increased levels of clrculatíng angiotensin formed as a result of elevation of subst,rate levels, or indírectly as a resulE of the effects on sodium homeostasís due-:"to Ëhe increased círculaËíng levels of angiotensín. In conÈradistinctíon to the findíngs described in this present study, Newton, Sealey, Ledingham and Laragh (1968) found that part of the increase ín renin act,ivíty csuld be due to ríses in renin concentratíon as well as elevatíons ín renín subsÈrate.

However, due to differences ín methodology, it is not possible Èo explain this discrepancy. Newton, Sealey, Ledingham and Laragh 119

laÈer suggest ÈhaE the normalízaÈion of endogenous renín act.ivlty levels followíng long-Èerm admínisÈratíon of oral contraceptives ís evídence of suppression of the tÈrue renin concentratíonf.

0ra1 contraceptives produce a sÍmilar elevation ín subsÈrate

levels Èo that seen ín pregnancy. In pregnancy, however, Èhe renín concentraÈíon is also elevated, whereas wíth oral contraceptíve adminístration it is suppressed"

In the course of a normal menstrual cycle a ríse ín plasma renín activíty is observed. This ís consequent upon a ríse in plasma renin concentratíon. During the pitt cycle, however, the ríse ín plasma renin activíty is now consequenË upon an elevation ín renín substrat,e levels. As explained previously, Ëhís rise in renín substrate ís compensaÈed to some extent by a fal1 in renin concen- tratíon. It is therefore ínteresting to postulate Èhat the

aggravatíon or inítiation of hypertensj-on in ¡¡omen taking oral

contracepÈíves is due, in part, to failure of Èhis feed back suppression. There is litËle evídence at, thís stage to prove such a hypothesis" However, ít is interesEíng to observe thaÈ in two hypertensíve women studied the elevaEíons ín plasma renin acÈivity were such thaË they could account for the aggravatíon of pre-exísÈing hypertension. cessation of contraceptíve Èherapy resulted in a fall in plasma renín act,ivity to rÀriÈhÍn normal levels. rt is suggested that ín these hromen an inability Èo suppress renin release L20 adequately in the Presence of hígh levels of renín substrate resulted ín r.¡ndue elevatíons ín plasma renfn acttvity. Although plasma renín actívlty was further elevaÈed l-n tr^ro other hypertensive subjects, the persisÈence of an elevated leve1 of plasma renln activity due to a compensatory ríqe in renin concent,ration followíng cessaËíon of coritraceptive therapy makes it unlÍkely that oral contraceptíves would aggravate hypertension in Èhese cases.

rÈ is inÈerestÍng to noÈe that hypertenslon has been induced by adrninistration of oestrogenic compounds to experímental animals, including the rat (Grollman, Harrison and tr{illiams, r94o;

Leatham and Drill, 1960) and Ëhe Èoad (segura, Lascano and d'Agostíno, 1967). The mechanism for the ínductíon of thís hyper- tension is not clear, but the use of experlmental anímal-s *"y rr"rp to elucídate the mechanism of hypertension occurring in ¡vomen taking oral conÈracepËive preparations . L2I

(C) CHANGES IN PLASMA RENTN ACTIVITY, PLASMA REN]N

CONCENTRATION AND RNNIN SUBSTRATE LEVELS DURING PREGNANCY

High levels of aldosterone are assoclaËed wíth pregnancy, the secret.íon of aldosterone beíng accounted for partly by the

increased actívity of the renin angíoÈensín system (Genest, de Champlain, Veyrat, Boucher, Tremblay, Strong, Koiw and Marc-Aurèle,

Lg65). Genes t et aL described progressive increases in plasma renín activity up Èo 22 weeks. Sínce renin actívíty is influenced by both renín levels and substrate levels, a study of the relatíve contrí- bution of changes in renín concentration and renin substrate to Èhís

elevated maternal plasma renin actívity in pregnancy !üas underÈaken.

Evidence obtaíned from the líterature suggests Ehat boEh enzyme and

substrat,e levels are elevated in pregnancy. Brown, Davíes, Doak, Lever and Robertson (1963) described íncreases in plasma renin concenÈration in pregnancy, the highest levels beíng obtained ín

early pregnancy, whilst Helmer and Judson (L967) described a progressive elevatíon in renin substrate levels duríng pregnancy"

METHODS:

Venous blood samples were taken from an antecubiÈal vein

ín 25 normal rromen at various sËages of gestation" All samples vrere

taken at 1100 hours wíth the subjeet seat,ed" No conËrol was exercised over díeÈary sodíum or fluid íntake. Heparín (20 units/m1) L22

I^7as used as anÈicoagulant.

Plasma renin actívity, plasma renin concenÈration and renin subsËrate levels were estímated by Èhe methods descríbed.

Possible kinetíc differences in plasma from pregnant \oomen,

compared with plasma from normal subjecLs, r^rere studíed by construct-

ing standard subsËrate curves of pregnant and normal plasma, using a constant. concentration of renin. Amniotic fluid was used as the source of renin, a consËant amounË being added to seríal dilutions of pregnant and normar plasma, and the rate of formatíon of angío- tensin at any partícular substrate concentraÈion determíned. Control

dilutíons I^7ere set. up in parallel to a1low correcËíon for endogenous velocity.

The range of plasma renin actívíty in normal males ís 1.7

! 0.5 ng/mL/hr (mid-afternoon sarnpring), for plasma renín concen-

tration ít is 9.1 units/mr t 3.5, and for renín subsËrate it is 1.1 1 0"3 nglml (Skínner, L967).

RESULTS:

PLasma renín aetöui,ty:

The plasma renín actívíty was elevated to above normal, non- pregnant levels in all subjects studied (Figure 5-5, 5-B; Table

5-4). There I^las no significant posítive correlation between plasma renin activíÈy and the duraÈion of gesEatíon. t5 a O¡ a o o ¡L I a o E a OO o a .:lo- O a a o Þ a o ! a l-I t o ( o a o =z- 4r¡lÐ

r,lf ÀJ

o PREGNANCY (weeksl

FiE. 5-5 The level-s of plasma renin acÈlvity ín 25 normal Pregnant r^tomen. I¿II'L.tr )-+

PRA, PRC and renin substrate in normaL pregnanÈ women.

Duration PRCt (uníts /rnl) PRA (nglnLlhr) Reni-n subst,raÈe (uglml) Pregnancy PRC* Normal range: Normal range: Normal range: Subj ect (weeks) (units lrnl) 9.1!3.5 1.7r0.5 1"110.3

I 4 50.0 47, 7 9.0 1.3 2 6 82.5 68. I r0.2 3.0 3 10 66 55. 0 8.4 3.0 4 10 76.5 63" 8 8.3 3.3 5 11 34.5 28. 8 L4.8 3.0 6 T2 136.5 113. 8 11.3 4.4 7 L2 105 87. 5 5"8 L"2 8 L4 99 82.5 L2 "2 6.0 9 18 81 67. 5 9.5 5.7 10 20 103.5 86" 3 14. s 4.2 11 22 4s 37.5 t4.4 3.6 12 22 36 30. 0 10.6 9"0 13 23 34.s 28. 8 6.8 6,0 L4 24 33 27. 5 7.8 4.2 15 36 25 "5 2L, 3 12.0 5"4 t6 36 82.s 68. 8 14.4 4.2 L7 36 44.r 36. 8 11.0 2.7 18 37 43.s 36. 3 9.2 7.0 I9 37 52.5 43, 8 5.7 7"0 20 38 49 .s 4r. 3 LT.2 4"8 2l 4A 33 27. 5 8.0 6"0 22 40 36 30. 0 LO.2 7.0 23 40 25.2 2L 0 14.0 6.0 24 40 42.0 35 0 t3.7 3.8 25 40 46:5 38 75 6.4 4.2

* Values used in Figures 5-6 and 5-8.

f Corrected for subsÈrate concentration 530 ng/url. L23

Chøtges in pLasma y.enín eoneenty,ation:

The plasma renín concenËration was elevated to above

normal' non-prêgnant levels in all üromen studied. The highest values occurred duríng Èhe firsÈ 20 weeks of pregnancy (trigure 5-6r 5-g;

Table 5-4), wíth a peak value ín one subjecÈ occurring aE L2 weeks

gestation. The plasma renin concent,ration had fallen by 22 weeks of pregnancy, alÈhough Èhe levels were st.ill above the normal, non-

pregnanÈ range. After 22 weeks pregn¿mcy, the levels tended Èo remain constent. Renin substpate:

Renin subsÈrate levels r¡rere normal in two of the twenÈy-

five women studied, these \^romen beíng 4 and 12 weeks pregnant. rn al-l other subjecÈs renin subsÈrate levels were above those found ín

normal, non-pregnant indívíduals. There \¡ras a progressíve íncrease ín renin substraËe levels duríng pregnancy (r = 0.51, o.ol>p>O.001) (l'igure 5-7, 5-B; Tabte 5-4).

tr{hen the reactíon velocíty at consÈant renin concentraÈion

ís plotted againsË Èhe substrate concentratíon, the símílaríty between curves for pregnanÈ prasma and normal pLasma ís apparent, indicating that Ëhere has been no qualítative change in renín substrate levels

ín pregnaney (fígure 5-9, 5-10) .

DlSCUSSTON:

IË has recenËly been shor^¡n that, renin substrate levels ín E ùIt I e, Ð (,z ¡ I 2 o70 rJ z z ¡ ¡¡¡ t t 4 I I - I I tl I ÀJ

o to 20 30 40 PREGNANCY (weeksl

Fis. 5-6 The levels of plasma renin concentratíon in 25 normal pregnant hromen. 9.O

t A

E A o ^ I t A a þ¡I ( ^ Fú 4.3 I,l A A A É Ð A |a ^ z 2 A AA ¿E ^

^

o t 20 30 PR,EGNANCY (wcek¡l

Fig. 5-7 The levels of renin substrate ín 25 normal Pregnant r¡7omen. PRA PRC MIIN SIINÏRATE rua/mu/nn urrrs/r'n- ISlML n

5 10 1m

4 8 m

3 6 60

tl 40 2

2 20 1

010nn 40 010n n q0 010nn q0 PREGNANCY (l,lEEKS)

Fíe. 5-B Mean changes ín plasma renín activíÈy, plasma renin concentration and renin substrate leve1s ín 25 normal Pregnant \¡tomen. Values represent means of values from women wíthín each 10 week period of pregnancY. roo ^sL o

=E \o E Y a z 50 o I o zrâ ¡¡t o l- o o oI 2 oo o a o I 3 SUBSTRATE t.rsr-11

Fig. 5-g Effect of non-pregnant ( O ) and pregnant ( O ) substrate on Èhe rate of formatíon of angiotensin by amniotíc fluid renin. t50

t v a

o 50 o

o

ao a o v s

Fie.5-10 !Íoo1f plot derived from Ëhe effect of non-pregnant ( O ) and pregnant ( O ) plasma on raÈe of formaËíon of anglotensin by amníotic fluíd renín. V = angíotensin (ng/nl-/nr) V iotensin r -=S substrate concentration Þo (Appendíx Table 16(a), 16(b)) 124

plasma are present in raÈe-límitíng amounts (Helmer and Judson, 1967; Skínner, Lumbers and Symonds, 1969). Therefore, increases in renin substraÈe, such as are observed ín pregnancy, could increase the renin acÈivity of plasma independent of changes ín concentratíon of the enzyme" The íncrease in renin activit.y in pregnant plasrna is

Èherefore consequenÈ upon boÈh an increase ín substrate and an

íncrease in Èhe eírculating levels of renín, there beíng no evidence of any qualitative ehange ín renin substrate. The cont.ríbuËion made by each to the renin activíty varies accordíng to the stage of pregnancy, so that Èhe renin activiÈy of plasma remaíns relaÈively constant Èhroughout pregnancy. The release of renin appears to be híghest in early pregnancy; later, renin levels tend to fall"

Renín substrate levels, on the other hand, show a progressíve íncrease as Ëhe duratíon of pregnancy increases.

The elevation ín renin substrate levels is consequent upon the íncreases in the circulating level-s of oestrogens that occur

ín pregnancy (Síiterí and MacDonald , L966), sinee it has been shown that oestrogens stimulate producÈíon of renín substraLe ín man and other anj-mals (Helmer and Grif f íth, L952) . A ríse in renin concentratíon ín early pregnancy has also been described by Brown, Davies, Doak, Lever and Robertson (1963)"

The cause of this early rise in renin release is noË known. It is possíble Ëhat íncreases ín progesÈerone stímulate release of renin L25

from Èhe kídney, since progesterone has a natriuretic action ínhibit-

íng the actíon of aldosterone on Ehe dístal- tubule (Landau and

Lugíbihl, t96I) " Increase ín renin concentration, and hence renin actívity, may thus be necessary to offset thís díuretíc action of progesterone. If progesÈerone ís the stimulus for renin release early in pregnancy, sínce levels of progesterone rise progressívely throughout pregnancy, it would be anticipated that renín concentration would also rise progressively. However, iÈ does not: in fact, after 22 weeks the renín levels fall, suggesÈíng feed-back suppression of renin release

from the kídney by increased anglotensín levels assoclated wiÈh increasing renín substrate levels. The hormonal control of sodium metabolísm ín pregnancy ís

complex. Firstly, oestrogens have a sodíum ret.aíning actíon (Ihowlton, Kenyon, Sandford, LoÈwin and Fricker, L942), and sínce the maternal levels of oest¡ogen íncrease duríng pregnancy, it míght anticipaÈed be that their sodíum-retaíníng effect also increases " Secondly, progesÈerone appears to play an active role ín sodíum metabolísm during pregnancy. EhrLíeh, Laves, Lugibihl and Landau

(L962) showed that Ëhere is a very close interrelationship beÈween

aldosterone and progesËerone levels in the pregnant r¡roman. They found that both urlnary pregnanediol and urínary aldosËerone levels

fell concurrently with sodium loadíng in the pregnant hroman and rose L26

r,rríËh cessaÈion of the high sodium diet. They also showed that Èhe secretion of progesterone (as measured by urinary pregnanedíol) was dírectly related t.o the amount of míneralocortícoíds gíven to

Addisonían pregnant r¡romen. In r¡romen receiving no mineralocorticoíd supplemenÈ, the pregnanediol levels were very low. These results imply that progesÈerone is therefore actívely concerned in the control of sodium homeostasis in Èhe pregnant hToman.

As regards the relaÈíonship between aldost.erone excretion and maternal renin levels ín the pregnanÈ woman, thjsrelationship appears to be obscure. Venning, Prímrose, Calígarís and Dyrenfurth

(1957), Rinsler and Rigby (1957), and Martín and Mills (1956) noted progressive ríses in aldosÈerone secreÈion duríng the course of pregnancy. However, \^re have found Èhat the plasma renín activity, although elevated, does not ríse progressively duríng pregnancy.

GenesË, de Champlaín, Veyrat, Boucher, Tremblay, Strong, Koiw and Marc-Aurèle (1964) noted a progressive rise ín renín actívíty up to Ëhe Ëwenty-fírst week of pregnancy, the levels then stabílízing or dropping slightly. Therefore, the progressive ríse ín aldosterone excretion contrasts wíth Èhe high, buÈ comparatively stable, levels of renín actívity" If renín actívity ís a true reflectíon of the ín uíuo abilíty of plasma to form angíotensín, a dissociaÈion Ëherefore exísts between the renin angiotensín system and aldosterone secreÈíon, L27

suggest,ing that other factors are ínvolved ín Èhe control of aldo- sÈerone secretion ín the pregnant woman" Obviously, more studíes are requíred, in particular application of radioimmunoassay of angiotensin levels in the pregnant woman would be helpful. It ís possíble Èhat there ís a contribut,ion to maternal plasma renín levels of renin from some extrarenal source, since renín has been found ín high concentratíons in Èhe human reproductíve tract (Skinner, Lumbers and Symonds, 1968). In this regard, iË ís inÈerestl-ng Lo noËe Ëhat the varíatíons ín maternal- plasma renín levels thaË occur Ín pregnancy show a símilar pat,tern to the varl- ations Ëhat occur ín maÈernal plasma levels of chorionlc gonado- trophín (Jones, Delf and Stran, 1944). This ís excreted only from the pregnant, reproductive EracË. It, like renín, ís a protein hormone and the tissue of origín is, líke renín, believed to be Èhe choríon.

Therefore, ít is not unlíkely that those factors whích resulË ín Èhe release of choríoníc gonadotrophín ínto the maternal circulatíon in early pregnaney also resulË in the rel-ease of renín from the reproductive tract.

SAMMARY TO SECUON 5

1" Plasma renin actívity, plasma renín concenËration and renin subsËrate levels have been studíed during the course of the normal menstrual cycle following adminístraEíon of oral conËraceptíve agenÈs and duríng normal pregnancy. L28

2. A rise ín plasma renín activity consequent upon an elevation in plasma renín concentrat.ion occurs in Ëhe second half (luteal phase) of the menstrual cycle. Thís is probably the resulÈ of the natrÍuretíe action of progesÈerone which ís secreted during the luteal phase of the normal menstrual cycle"

3. Admínistratíon of oral contraceptíves to normal vromen produced an elevation in plasma renin actívíty which r^7as greaÈer than the rise in renin acËÍvity seen during the luteal phase of a normal menstrual cyc1e. Thís elevatíon in plasma renin acËívity was due to a concomíÈant increase in renin substrate levels induced by the oestrogeníc component of the pi1l. Plasma renin concentratíon levels hTere suppressed below Èhe levels found príor to admínisÈratíon of the oral conËraceptÍ-ve agent. The changes in renin subsËrate índuced by oral contracepÈíves are quantitaËíve; no qualíÈative change in renin substrate was observed. An hypothesis based on failure of feed-back suppression of renin release from the kidney has been advanced to explaín Èhe rhypertensíve effect of the píllr. 4" Plasma renin activity levels are elevated in early preg- nancy and remain elevated ÈhroughouË Èhe course of pregnancy. The erevaÈed levels of plasma renin aetivíty ín early pregnancy are associated with increased levels of plasma renín concentration. As pregnancy progresses renín substrate levels increase, whílst the renin concentratíon leve1s fall", although Ëhey remaín elevated to L29

above normal non-pregn¿nË levels. The.se findíngs suggest that the elevatÍon in renin substrate as pregnancy progïesses eonËributes to

a greater exËehÈ Ëo the íncreased plasma renin actívíty and thus

invokes a supptession of the ínitfally high renin secretion from the kÍdney. No explanaüíon for Ëhe earJ-y marked íncreases fn renín 'l-evels ís apparent" The control of sodíum homeostasis in pregnanc)r is eomplex and humoral agenes sueh as progesterone may be actlvely

ínvolved' lt, ís arso possibi"e that renín may be released from the reproduetÍve traet ínt,o the maüernal eireulaÈion duríng pregnancy. 130

SECTION SIX

THE SENSTTIVITY OF THE HAND BLOOD VE SSELS TO ANGIOTENSIN

AND NORADRENAIINE IN PREGNANT AND NON- PREGNANT I^IOMEN 131

THE SENSITIVITY OF THE HAND BLOOD VESSELS TO ANGIOTENSIN

AND NORADRENAI INE IN PREGNANT AND NON_PREGNÆ{T I,{OMEN

A reductíon in the pressor response to inËravenous l-nfusions of angiotensín is characterisÈíc of pathological conditíons assoc- iated with high circulating levels of renín and presumably angiotensin

(Kaplan and Sílah, L964) " A simílar assocíaÈion occurs in pregnancy. The sensltivíty to intravenous infusions of angiotensín ís reduced in the pregnant \¡roman when the pressor response obtained fot a given dose of angíotensin is compared to that obtaíned ín non-pregnarit

r^romen (Chesley, Talledo, Bohler and Zuspan, 1965) " Elevations in renin 1eve1s and renin activity ín pregnancy are well deseribed (Genest, de Champlaín, VeyraÈ, Boucher, TrembJ-ay,

Strong, Koiw and Marc-Aurèle, L965; Hel-mer and Judson, 1967;

Lumbers, Skinner and Symonds, unpublíshed observatíons). Since the response to angiotensin by the hand vessels was for:nd Èo be reduced in patíents wíth renovascular hypertensíon (Scroop and tr'Ihelan, 1968) t a situation in which there ís an associated deerease in the pressor response to angiot,ensin as well as hígh renín levels, it was of interest to see whether Ëhe decreased pressor resPonsiveness of the pregnarit r^roman was also ¿rssocíaÈed rttith a reduction in the sensítivíty of the peripheral vessels to the constrictor actíon of

Èhe drug. t32

METHODS:

Nine pregnant female volunteers I^Iere sËudied. Their ages ranged from 18 to 26 years, all were nulliparous, more than 32 weeks pregnanÈ and had no clinical evídence of toxaemia of pregnancy. Six female voh¡nteers, who r^Iere rIoE pregnant and not taking oral contraceptíve preparations, r,\rere also studied. Thelr ages extended over the s¿rme range as that, descríbed for pregnant women.

Hand blood fl-ow \¡ras measured by intermíttenÈ venous occlusion plethysmography" The laboratory temperature htas maíntained at 24-25oC. The subjects resÈed recumbenË on a couch for at leasÈ

30 mínutes príor to ínfusíons of drugs. Duríng this time recordíng apparatus was applied and a Z2-gauge, buEËless needle inserted centripetally inÈo the brachial arÈery under local anaesthesía with

27" xyLocaLne.

Measurements of flow were made wíth the subject in the lefÈ lateral position Èo avoid compressíon of Èhe ínferíor vena cava by the pregnant uEerus.

Hand blood flow \^ras recorded 2-4 tímes every minute, using water-filled, temperature controlled plethysmographs (Greenfield,

L954; Greenfield, trühiËney and Mowbray, L963). A P23BC StaÈham t,ransducer connected to a Rikadenkí mulÈípen recorder \^ras used to record blood f1ow. The plethysmograph temperature \^las maínËained at

32-340c. 133

NaCl (0.15 M) was ínfused intra-ar.teriral-1y vÍ:a a constanÈ ínfusÍon pump aÈ a rate or 2 mllmin durÍng conËrol perfods and also used as the vehicle for the infused drugs " Drug concentraÈlons were calculated so that the minut,e doses of the drugs r^rere cont.ained in 2 ml" AngioÈensin was ínfused for 5 mínutes and noradrenaline for 4 mínutes " The doses of drugs infused were such Èhat Ëhe vessels of the ínfused límb ¡¿ere exposed to high concent.ïations, whilst the

concentraÈion of drug reaching the general círculatÍon hras ínsuffíeient Èo have any systemíc action. Therefore, the non-infused hand was used as a control, æd allowance for spontaneous variation ín flow could be made by assuming that ín the absence of the ínfused drug the ratio of flow in the control and ínfused hands would remain the same (Duff, L952).

The drugs used were ß-asparaginyl angíotensin II

(Hypertensin, Ciba) and noradrenalíne bítartrate (Levophed, I^línËhrop).

Doses of angÍotensín are expressed as weight of the amide and noradrenalíne as weíght of the base. Ascorbíc acíd (1:50r0oo) was added to all noradrenaline sol_uÈions " AIJ- non-pregnant subjects received ínfusions of lOO, 200,

400 and 800 nglmin of both noradrenal-ine and angíotensin. one pregnant subject received 200 and 400 ng/min of angiotensín and noradrenalíne, whilst 8 pregnant women received 1oo, 200, 400 and

800 ng/min of angiotensin and at least Èwo of the followíng d.oses r34 of noradrenaline - 100 , 2OO, 400 or 800 ng/mín"

Blood for plasma renin estímatíon \^ras taken from an ante- cubítal veín aË the end of the experfment. Samples were Ëaken from 7 of the 9 pregnant subjects and all non-pregnant subjects. All subJects had been reeumbent for 3-4 hours príor to samples beíng taken. Heparin (20 units/m1) was used as anticoagulanË. Plasma renín acEivíty, plasma renín concentration and renín subsÈrate levels were estÍmated according to the method of Skínner (1967> as descríbed prevíously.

RESULTS:

Figure 6-1 shows the individual log-dose response curves to both noradrenalíne and angíotensin which were obtained frorn both pregnant and non-pregnant \^romen (fígure 6-1a and 6-lb, respectively). The mean per cent falI ín hand blood flow, ploËted agaínst the log-dose of angioËensin and noradrenaline, respecÈively, is shown ín Fígure 6-2.

The per cent fall in hand blood flow obtained in pregnant

üromen was sígnificantly less than that obtained in non-pregnanÈ

I^Iomen for the followíng doses of angíotensin - 100 (0.01>P>0 "02), 2OO (P>0.001) and 400 ng/min (0.02>P>0.05) - bur noÈ for doses of 800 ng/min. There r¡ras no significant difference in the per cent fall in flow obtaíned with noradrenalíne in pregnant, as compared with non-pregnant, r^romen . m ANGIOTE1'lSIN NORADRE1'lALINE

F

EJ cô

50 =

*

0 mmmSm lmmqmm ¡ç/mn ne/mlru

- -

Fig. 6-1(a) Per cent fal1 in hand blood flow duríng intra- arterial infusions of angiotensin and noradrenaline ín 6 normal non-pregnant r^romen. (Appendíx Table 17 (a) ) ANGIOTENSIN NORADREI{ALINE lm lm

dË E EI_J

50 z.=f) f

0 0 lm m4m m lm 2m qm m rr/NrH rr/r.uH

Fíg. 6-1(b) Per cent fall ín hand blood flow duríng intra- arterial infusions of doses of angiotensin and noradrenaline ín

9 normal pregnant \^romen. (Appendix Tab le 17 (b ) ) ANGIOTENSIN 1{()RATIREI{ALINE

dt E ãd 3 =, d ù.4 E

lm rn q0 8m lm 2m m m

Fie' 6-2 Mean per cent fall (! 1 SD) in hand blood flor^r obtaíned on infusion of angiotensín and noradrenaline to non- pregnant ( o ) and pregnant ( o ) \^romen. 135

Plasma renin aclf-vÍty, p1-asma renÍn concentratj.on and renin

substrate levels were signifícanÈ1y hfgher fn pregnant subjects Ëhan in non-pregnant subjects. rn one pregnant subject, the renin activity

and renin substrate levels were wíEhin normal lj.míts. Renin

concenÈration was also suppressed, but a quantitative assay was not completed. No explanation for thís fíndíng was appârent (Fígure 6-3, Table 6-1).

DISCUSSTON:

The reduction Ín Ëhe sensítívíty of Èhe hand vessels to inÈra-arterial ínfusions of angíotensín índícates that the reductíon in pressor sensitívity to the íntravenous adminístratíon of angio-

tensín in pregnant women (Chestey, Talledo, Bohler and Zuspan, 1965)

is a reflect,ion of decreased responsíveness of the vessels and not the result of a modífication of the central action of angioÈensin or

of baroreceptor mechanisms.

The presence of el-evated plasma renin levels ín all_ but one of the subjects studied suggests thaÈ reduced vascular sensí- Èivíty may be a eonsequence of the raised levels of círculatl-ng angíotensin and ís ín keeping with the resulËs obtained in paÈients with renovascular hypertension (Scroop and l,,Ihelan, 1968). The mechanism responsíble for the reductíon ín sensit,ívíÈy to angiotensin in pregnancy and other situations asseciated wíth elevated círculatíng levels of angiotensin ís noÈ cl_ear. RA PRC ffiNIN SIßSIRATE rc/mu/un wrrs/ru ¡re/ru- o o a 8 o 40- ¡8 3 o 6 o n 6' o o T 4 2ù q. o -

o 2 2 10- o o o O ð & o o 0 "þ

N-P P N-P P N+P

Fíg. 6-3 Plasma renín activity, plasma renín concentration and renin substrate levels in non-pregnant ( O ) and pregnant ( O ) \^/Omen. TABLE 6-1

Plasma renLn aclivíty, concenÈratlon and renin subsÈrate

levels ín pregnant and non-pregnanÈ \^romen.

Plasma renin P renin Renín activity concentration subs trate Sub iects (n,s,lmL/t,r) (r¡nits /m1) (ue/m1)

I 0.5 3.5 0.8 2 3.5 1"6 3 0.3 2.5 0.6 4 0.2 lr3 0"6 5 0.4 2.8 0.9 6 1.1 5.2 1.1

7 7.8 28. B 8.8 8 6.8 46.O 2.I 9 7.5 38.4 3.9 10 5.6 24.2 3.6 11 0.5 L"2 L2 7.4 75;2 1.5 15 6.0 48.0 3.6

1-6 Non-pregnant 7-15 PregnanÈ 136

The reducÈion ín sensítívity to angioEensín was specific, since the sensitivíty to noradrenalíne remained unchanged. It ís therefore unlíkely that non-specl-flc factors, such as dllutlon of

the drug as a resulË of increased blood flow (rnean blood flow: non- pregnant subjects 9"3 nl-l 100 m1 of tissue/mín, pregnanË subjects 2I.3 nL/I00 ml tissue/min) or a decrease in vascular tone are res- ponsible for the reduct.ion ín sensiE,ivity to angiotensín. Pregnancy is associated wiËh elevations in the círculatíng 1evels of progesterone and oestrogens. A1Ëerations ín vascular responsiveness induced by these compounds is probably not responsíble

for the reductíon ín sensitivity to angiotensín observed, since such

an effect would Èend to be non-specífic and hence the sensítivíty to noradrenaline would be affected ín a simílar manner. Furthermore,

the admínistratíon of oestrogens Èo man and experímental animal-s does noË alter either the pressor response (Hettíaratchi and Píckford,

1968) or the renal response Èo angíotensín (Chesley and Tepper, L967>.

Admínístration of progesterone does alter both the pressor response to angiotensín ín anírnals (HettiaraËchi and Pickford, 1968) and the renal response ín man (Chesley and Tepper, L967). However, progesterone ís an aldosterorie antagonist and therefore is natriuretíc

(Landau and Lugíbíhl, 1961). This acÈíon r¿ould probably stímulate reilease of renin from the kidney and give rise to elevaÈions in renín Ievels such as are seen ín the luteal phase of the menstrual cycle L37

(Brown, Davies, Lever and Robertson, 1964; Skínner, Lumbers and

Symonds, 1969). Therefore, the reductíon in pressor and renal responses to angiotensin followíng the administraÈion of progesterone may be consequent upon the increased production of endogenous angiotensin.

In all situations in whích there is a reductíon ín the sensítívíty to angiotensin there ís an elevation of plasma renin levels and presumably endogenous angiotensín levels (Kaplan and

Silah, 1964; Scroop and !'Ihelan, 1968). Elevatíons in plasma renín activity were found ín all but one of the pregnant r^romen studied. In this subject, Èhere hras no apparenÈ reason for the normal renin activity levels obtained as she did not have any clinical evídence of pre-eclamptic toxaemia. The levels of renin activity found in pregnant \^romen were 12 times the levels seen ín non-pregnant controls and due t,o elevations in both renín concentration and renín subsÈrate levels (table 6-l). The levels of renin actívíty and renin concenËration in normal non-pregnant subjects r',rere Low compared to the levels obtaíned by Skínner (1967) (talle 6-1). This rnay be attributed Èo the fact thaÈ Skinnents normal range ís for mid-afternoon ambulant subjects, whílst the non-pregnant, cont,rols had been recumbent for 3 to 4 hours prior to blood samples being taken. I,rlhether such a postural effect occurs ín pregnant subjects ís not cLear at this sËage. 138

Tachyphylaxis to angiotensin implíes a saturatíon of receptor sítes for angíotensin so that no further sítes are available

for association wíth the infused drug (page and Mccubbin, 196g).

Adrnínistration of hígh doses of angíotensin to Èhe hand or forearm

for periods of up to one hour faíls to resulË in a reductíon ín the degree of constriction obÈained (Lumbers and hlhelan, to be publíshed).

Tachyphylaxis is a complex problem, since it ís dependent not only on the doses of the drug infused, but also on the levels of angíotensin- ases present. !ühether or not it is responsible for the reductíon ín

vascular sensitívíty to angiotensin ín pregnanÈ \¡romen cannot be

decided wíth certaínty, since iÈ may not be justifiable Ëo extrapolaEe

Ëhe results obtained in the normal subjecÈ (Lurnbers and trühe1an, to be

published) to pregnant \^romen where endogenous levels of angiotensín have been elevaÈed for months.

Talledo, Rhodes and LívingsËone (1968) and page (L947) have described elevations in circulatíng angíotensinases in pregnant r¡romen. The physiological role of circulating angiotensínases as

regulaÈors of Èhe action of angiot,ensín ís uncertain. The half lífe

of angiotensín in the blood of pregnant \^romen ís, however, reduced, buÈ is of suffícient duration to make it unlíkely that a reduct,íon in angíotensin concentration had occurred in the blood reaching the hand ín pregnant. hromen, as compared to non-pregnant r^romen, followíng

the infusion of angiotensin int,o the brachíal artery aË the elbow. 139

Hodge, Ng and Vane (L967) coneluded ÈhaË circulating angiotensinases play a minor role ín the desÈruction of angiotensín II, as compared to the removal of angíotensín across the perípheral vascular bed. It is possible that. círculatíng levels of angíotensin- ase may reflecÈ tissue angiotensínase activíty. Therefore, the

íncreased levels observed in plasma in pregnancy (Talledo, Rhodes and Lívíngstone, L967; Page, L947) may reflect an increase in tissue angíotensinase activíty whích c,ould be interpreEed as a protecÈive mechanism against the constrictor potentíal of the increased leveLs of endogenous angioËensin.

Fínally, it could be hypothesized that Éhe reduction ín

sensiÈívity to angiotensín ís the result of reduced sympatheÈic tone.

Ginsburg and Duncan (I967) menÈíon the possibílity thaË reduced sympathetic tone ís responsible for the íncreased hand blood flow

seen ín pregnant \¡romen" Although there ís no evidence of augmenÈ- atíon of perípheral sympatheÈic actívíty by angiotensin in man (I,,Ihelan, Scroop and ü/alsh , 1969), there ís evidence of poÈentiation of the

action of released noradrenaline by angíotensín (Scroop and Wa1sh, 1968). It is eonceivable that a reducÈíon Ín sympathetic tone could

lead to a reduct.ion in the amounË of noradrenalíne released into Ëhe vessel wall which would account, ín part, for Èhe reduced sensítivity

to angiotensin observed in pregnanË women" 140

SUMMARY TO SECTTON 6

The hand vascular sensitivity to angioÈensin is reduced ln pregnant \^7omen as compared to non-pregnant \^tomen. Thls reducÈion ín sensítivÍty ls specific to angloÈensin, since lt does not occur wíÈh noradrenallne" It suggests that the reduction ln sensiÈlvity to the pressor response Èo anglotensín seen Ín pregnant r^romen l-s due to a reduction 1n perípheral vascular sensítivity. Non-specifíc factors such as ínereased hand blood flow, or changes in vascular responsíveness lnduced by oesÈrogens or Pro- gesterone are riot responsible for thís reduct,íon in sensítivity to angioÈensÍn as the sensitivity Èo noradrenaline would be símllarly affected. Elevations Ín plasma renín activity, renín concentration and renín substrat.e levels are assoclated wlth the reducÈíon in sensitívity to angiotensin observed. L4I

SECTION SEVEN

TACHYPHYLAXIS TO ANGTOTENST.N IN },IAN 142

IACHYPHYLÆ(IS TO ANGIOTENSIN IN MAN

Tn L964, Kaplan and Silah observed that the blood pressure response'to inÈravenous angíotensín was dimlníshed in patients wíth renovascular hypertension. Scroop and l,{helan (1968) reported a reductÍon in sensitÍvity of the hand blood vessels Ëo anglotensín ín patients with renovascular hypertensíon. A diminution in the pressor response Ëo intravenous angiotensín has also been described ín preg- nant r^romen (Chesley, Talledo, Bohler and Zuspan, 1965) and there ís also a reduction ín the sensitívity of hand vessels to angiotensin

(Lurnbers, to be published) . In all Èhese cases, the reduced sensiÈivity to angiotensin is associated \^/ith elevaÈions in plasma renin levels and presumably circulating levels of angiotensín. It has been suggesÈed Ëhat the reduced vascular sensitívíty to angiotensin in these subjects may be a manifestaÈion of taehyphylaxís of the vessels to angiotensin. The present study was undertaken to determine whether tachyphylaxis of human vessels could be índuced to angiotensin and other drugs.

METHODS:

The subjects urere volunteer medícal students.

Laboratory ÈemperaÈure r^ras mainËained, at 23-26oC and the subjects rest.ed recumbent on a couch for at least 30 rnínutes príor 143

to infusion of drugs. During thís time recording apparatus \,rras applíed and a needle insert,ed ínto the brachíal artery.

Blood flow through the hand and forearm r¡ras measured by inÈermíÈtent venous occlusion plethysmography using water-filled

plethysmographs. The temperature of the plethysrnograph was maint,aíned

at 32oC for hand and 34oC for foreann measurements.

Records of blood flow were obËaíned 3-4 tímes every minuÈe. Blood flor¿ was recorded by a straín gauge transducer (Statharn P23Bc) connected Èo a RÍkadenkí multí-channel servo recorder., In some of the early experiments forearm blood flow was recorded wíth capacitance plethysmographs (willoughby, 1965; Fewíngs and tr'Ihelan,

1966) .

Int,ra-arterial infusions were admínísËered through a 22- gauge buttless, short bevel needle inserted l-nLo the brachial artery centripetallyr under local anaesthesía wíÈh 27" xyLocaine. The needle was connected Ëo a constant ínfusíon pump which delivered the infusions aç a rate of 2 ml/mín.

. Saline (0.15 l"t) was infused duríng control períods and also used as a vehicle for the drugs ínfused.

The doses of drugs infused were such thaÈ, wíth the excep- tion of vasopressin, they díd not produce any systemíc effects. Therefore, the uninfused límb could-be used as a control.and correction for spontaneous varíatíons in flow made by assuming Èhat 144

in the absence of the ínfused drug the ratio of flow between the two

límbs would remain the same.

PercenÈage change in blood flow was calculated as des- cribed prevíously.

Dnutgs infused:

ß-asparaginyl angiotensín II (angíotensin amide), Hypertensín,

Ciba, ß-,aspartyl angioËensin II (angíorensín acíd) 339028a, Cíba, noradrenarine bitartrate monohydrate (Levophed, I,,linthrop), aceÈyI- cholíne chloríde (Roche), vasopressín (pítressin, park Davis), isoprenaline hydrochloride (Isuprel, !ùínthrop) and propranolol hydrochloride (Inderal, I.C.I.) were administered. The doses of noradrenalíne are expressed as weights of the base, of angiotensín as weights of the acíd or amide, respectively, aceÈylcholine as weights of Èhe salt, and vasopressin as unlts/ml. Drugs were administered eíther as repeated, short infusions of L-2 mínutest durat,íon, wíth a 2-5 mínute ínterval between eaeh infusíon, or as prolonged infusions contínuing for períods of 30 minutes to one hour.

RESULTS: Angiotensin: Repeated infusions:

Angiot,ensín acid (B experiments) and angíot,ensin amide

(3 experíments) were given as repeated, short infusíons ínto the L45

brachíal art.ery, and the hand or forearm blood flow recorded. The intervals beEween infusíons were of 2 to 4 minutes and were suffícient to allow the blood flow Eo return Ëo near control levels before the next infusíon began.

Infusl-ons of 16, 32, 100, 200 or 400 ng/min were ínfused Ínto the forearm (10 experínents) and doses of 2OO, 400 or 800 ng/mín were infused ínto the hand. The reduction in response obÈaÍned was the same with successive infusísns (Fígure 7-L).

Pz,oLonged infusions: In thírteen experíments, each on a dlfferent subject

(6 with forearm blood flow measurement and 7 wíËh hand blood flow recordíng), angiotensín amíde was ínfused conÈinuously for 30 to 60 rnínutes. Doses of 16 and 32 ng/min were infused fnËo Ëhe fore- arm, and 2001 400 and 800 ng/min fnto the hand. The per cent reduction ín flow from the restíng level was calculaËed for each 5 minute period of the infusíon and the resulÈs of all experítnents are shown in Figure 7-2.

During all infusions ínto Ëhe hand of doses less than 800 ng/min, and all but one,of these ínto the forearm, the constrlctor response r^ras sustaíned throughout the ínfusíon period. Símílarly, the constrictor response was maíntained wíÈh prolonged infusíons of angíoËensin acid (32 ng/nLn into the forearm and 2oo ng/min ínto the hand). Infusíons of 800 ng/mín of angíotensin amide into the hand I.A.ANGIOTENSIN

5 ) o J4 l¡' .: o\ÂE o:rt.È ÉO o^ fFz 4: l¡l Él o IL .Fl

o MINUTES

Fis. 7-l The effect of repeated l-minute infusions ínto the brachíal artery of angíotensin amÍde (16 ng/min) on forearm blood flor¿. ( I ) infused síde. Ì o J l¡ I 4

t¡¡ 4, o l¡ 50

=¡¡¡ (, z T () s Ì o lr ê z ¡

t¡¡= c' z

I s 30

MINUTES Fíg. 7-2 Per cent fall in forearm blood flow (upper frame) and hand blood flow (lower frame) during continuous ínfusíons of angiotensin amide. Doses of 16 ng/rnin ( f ) artd 32 ng/min ( O ) were administered and forearrn blood flow recorded. Doses of 200 ne/mj-:n ( ), 400 nglmÍn ( r ) and 800 nglmin ( O ) were adminístered and hand blood flow recorded. r46 resulted ín a progressive diminutíon of the constríctor effect ín

Èhree out sf four subjects. In view of the reduced constríctor response of the hand vessels to sustaíned infusíons of 800 ng/mín of angÍotensín, it was deeíded to administer larger doses of angiotensín ínto the forearm vessel-s to see whether a simÍlar reducËion ín constrictor resPonse could be obEained. The forearm vessels are more sensitíve to angl-otensín than are the hand vessel-s (Scroop, I^Ialsh and l{helan,

1965), and doses of Èhe order of 100 , 2OO, 4OO and 800 ng/rnin of angíotensín would be expeeted to caqse cOmplete vasoconstríction. In fact; both flow and arterial pulsation were abolíshed wfËhin one mínuÈe of the onset of Èhe infusion, but, Èhen the flow began Èo recover slowly and after 5 minuÈes had returned to 30-50i4 of the pre- infusíon value. The flow remained at, this level for Èhe remaínder of the infusion. Thfs pattern of response was observed with doses of lOO to 800 ng/min ín 4 infusíons of the acíd and 3 lnfusions of Èhe amide of angíotensin (l'ígure 7-3) .

Vasopressin:

Repeated ì,.nfusions:

RepeaÈed, short ínfusions of vasopressin were adminísÈered to t\^ro subJects. In one, hand blood flow was measured and doses were admínistered whích ranged from 0.0025 to 0.1 unitsfmin, infused for 2 to 3 mínut,es. The hand blood flow did not alt,er sígníficantly 5 Angiotensin ccld eoo nglmin

4

3 .:2 Arr¿t¡*rl1 E :Êr o : F----rtrttit çttttttttt ¿.L%aaagagggl ¿gaggggrÐ :o o to óo E

Angiotensin omide soo ã7 lr ô6 o o E5 f I

t¡t 4 ¡¡3o

2

ffifitttft??

o ¿¿glag:.!.g o to 55 MINUTES

Fig. 7-3 Effect of cont,inuous íntra-atbexíal ínfusions of angíotensín acid (800 ng/min) (upper frame) and amide (lower frame) on forearm blood flow. ( O ) ínfused síde. r47

with any of these doses, although wíÈh hígher doses paJ-lor and brady- cardÍa was sbserved, indfcaÈing that sufficient was being gíven for a systemic effect to be produced. sinee contínuous blood pressure

recordings r^rere not made, it was not, possible to ascertaín wheÈher a signífícant rise in blood pressure had occurred. rntra-arterial noradrenal-ine was subsequently fnfused ínto Èhís subjecÈ and the fall ín hand blood flow that occurred indícaÈed that the infused drugs \^/ere, ín fact,i reaching the hand vessels. rn one sther subject, vasopressín (0.0025 uníts/min) was ínfused ínto the brachíal artery and Ëhe forearm blood flow recgrded. Repeated 2 minute ínfusíons, were given, with a period of 2 Eo 4 mínutes between each infusíon

(Figure 7-4>. A progressíve reducÇíon ín the constríctor response was obtaíned. Trlhen, after the seventh infusíon, a period of L2 minut,es was allowed to erapse, duríng which saline only was ínfused, the response t.o the sarne dose of vasopressin was restored (l'ígure

7-4, lower frame). After a further control period of 17 mínuÈes, 0.0025 uníts of vasopressín per minute r^rere ínfused contínuously for a períod of 10 minuËes. The ínítial constrictíon lasÈed for 3 to

4 minuÈes, after which blosd flow rose rapidry, and by Èhe sevenÈh mínute of infusion r^ras well above the pre-infusion.Ievel (lígure 7-4, lower frarne). The flow through the opposit,e conËrol forearm also showed a rise during the ínfusíon period. t. A. vAsoPR E 55l N

3

.: 2 E : E C) : : o E Ì o e J l¡

C¡ o o J o 4- I ¿ lr¡ 4 o E 2-

o- MINUTES Fig; 7-4 Effect of j-nfusion of 0.0025 units/min of vasopressin into the brachial artery on forearm blood flow. ( O ) infused síde. Upper frame: repeated 2-minute infusions. Lower frame: a 2-minute infusion following a resË period of 12 minutes. After a further períod of 17 minutes vasopressin was infused eontínuously for 10 minutes. 148

Nov,adyenaLine:

Repeated ínfusions:

Frequent shorÈ lnfusfons of noradrenallne 64 and 400 ng/ mín, respecÈively, r^rere adminístered Lntra-arterially ínËo t\^ro subjects ín one of whom the forearm and ín Èhe oÈher Èhe hand blood flow were measured (l'ígure 7-5). No reduction of the constricÈor response r^Ias seen in eíther of these subjects.

Pz.oLonged infasíons:

An hour-long infusion of 800 ng/min of noradrenallne was given intna:arterially ín one subJecË., and forearm blood flow recorded. The flow fell to a very low level wíthin 1-2 mínutes of the onset of the infusion, but sver the nexÈ 3 to 5 mínutes gradually rose Ëo a val-ue of 507 of Èhe pre-infusi-on levels and remaíned at this leveL throughout the remalnder of the inf,usíon period (figure 7-6).

Sínilar results were obtained ín three other subjects in whom nor- adrenelíne (800 ng/min) was infused for 20 minutes. In three subjects, 800 ng/min of noradrenaline was infused intra-arterialLy for 15 mínuÈes. Following thís, isoprenaline 0.05 Ug/mín was ínfused intra-arterially for 2 minutes. Propranolol hydrochloride 100 fg/min for 8 minutes abolíshed the ditatation observed \^riËh 0.05 Ug/min of ísoprenalíne, but only part,ially abslished Èhe reduction in con- stríctor response seen with noradrenalíne 800 ng/min in two out of three subjects. I.A.NORADRENALINE

5 ì o l¡-¡4 C ô h

JE3s3 o $ o I:2 4\

4l o ¡¡ o

) to o JE ô\ E o oÊ Jo É:5 z:ô\ t

o f; MINUTES

Fíe. 7-5 Effect of repeated infusíons of 64 ng/mí:n of. noradrenalíne into the forearm (upper frame) and of 400 ng/nin into the hand (lower frame). ( l) infused síde. I.A. NORADRENATINE eoo ng/min .: \E - E o o l- E ì o J l¡ ct o o J o : 4 ívú.\rry IT É o lr o o to óo MINUTES

Fig. 7-6 Effect of a prolonged infusíon of 8OO ng/mín of noradrenaline ínto the forearm. ( O ) infused side' L49

AcetyLchoLine:

Acetylcholíne was gíven ínÈra-arteríally into the forearm of one subjeet in both frequent, short lnfusions of 4 mg/mín and also

in a contínuous l5-minute ínfusion of 1 mg/mln (Fígure 7-7). There

r^7as no reduction in the vasodllator response obtalned ín eíÈher case,

DTSCUSSION:

Haas and Goldblatt (L964) point ouÈ rhat the term tachy- phylaxís was fírst used by champy and Grey (1911) to descríbe the

rapidly developíng refracÈory state of an animal against Èhe Èoxic

effects of crude Èissue extracts contaíníng foreign proteins by the previous adminístraËion of small- amounts of the same extract. Ttre phenomenon r^ras thus a manifestatíon of the immune response, and ít was considered by Haas and Goldblatt (1964) noÈ jusËifiable to apply the term t,o Èhe refractory staÈe t,o renÍn evoked by previous adminístratíon of Èhe same substance"

However, the term has gained considerable usage ín the pharmacologlcal world, beíng used Èo descríbe the development, of a stat,e of refracÈoríness to a drug by an isolated tíssue preparation or by the whole anímal.

A more recenÈ defínítion in accerd wíEh the current usage of the term ís "the loss of response to a drug following íts repeated or contínuous administration ín large amounts" (page and MccubbÍn,

196B). Tachyphylaxís to renín was firsr described by Tígerstedt and a a to

a c €t ,fi a "\ w , o ¿ E o o o ¡J t- g a o E

to

o MINUTTT

Fj:e: 7-7 Repeated infusions of acetylcholine (4 mg/min) (upper frame) and a contínuous infusion (15 minut,es) of 1 mg/mín. (o)ínfusedsíde. 1s0

Bergmann in 1898. Followíng the recognítíon Ëhat renín r¡ras an enz)rme and acted on a substrate Èo produce angiotensin r, ít was thought

that the loss of the pressor actíon of renín was due Èo consumption

of circulatíng substrate. However, in 1962, Gross and Bock demon-

sËrated that the development of tachyphylaxís to renín üras concomítant wíth the development of tachyphylaxís to pressor doses of angioÈensín. These findings indicated that the refract,oríness to renín was due not only Èo consumption of substrate, but also to refractoriness Eo the actíon of the peptlde at the receptor. Tachyphylaxís, Èherefore, can be explained on the basis of the recepÈor concept of drug actíon, as the resuLt of filling all recepËor sites so that noreremaín aceessíble. rf, as paton

(1961) states, the stímulant action of a drug ís proporÈÍonal to Èhe

rate of association of the drug wíth iEs receptor, attachmenË and. fílling of all receptor sítes by angíotensin would result ín loss of constríctor action of the drug. Such a basis for the mechanism of production of tachy- phylaxis to angíotensin has been postulated by page and Bumpus (1961) and Khaírallah, Page, Bumpus and Turker (L966), and for tachyphylarís to noradrenaline ín the cat by Burn and Rand (1959).

The present experíments were designed to determine whether the development of taehyphylaxís to angiotensín played any part ín the normal constricËor response to angíotensin, and to deÈermine ls1

whether the adrninistratíon of doses of angiotensin comparable wiÈh the levels of angÍotensin seen ín paÈíenÈs with renovascurar hyper- tensfon !íêre associaÈed wíth a reduction in constricçor potency on repeated or continuous admínistraÈíon. The develoPment of a progressíve reductíon ln constrlctor resPonse Èo vasopressin with either repeated or cont,inuous ínfuslons indícated that tachyphylaxis was demonsÈrable ín human forearm vessels with doses of aidrug which reduced blood flow by only 5o%. However, progressive reductions in the const,rlcÈor response to angioÊensín símllar to that seen with vasopressín or in Èfssue preparatíons wiËh a4giotensín could not be demonstrated with either repeated or continuous ínfusíons of angiotensin Èo normal subjects.

The reÈurn of forearm blood flow to 30 to 5oy" of resting levels when very hígh doses of angíotensín (800 ng/min) were ínfused into the forearm may represent tachyphylaxís Ëo the drug, buË lt was by no means a complete phenomenon, and the fact thaÈ a simflar pattern was seen wíth an infusion of a large dose of noradrenaline suggests Èhat Ëhe recovery of flow may be non-speclfíc and represent a:physiologícal dílator response to íschaemía which opposes the constrict,or actíon of the drug.

The doses of angíotensin which can be safely adminístered to a nsrmal- subjeet may be ínsufficient to produce tachyphylaxls to the drug. However, infusions or. 32 ng/min ínto the forearm vessels L52 r^Iould result in an arríving concentration of L.6 ng/mL, and infusíons of 800 ng/mín into the forearrn and hand would result ín arrívíng concentraÈj-ons of 40 and 16 ng/mL, respectively. Such levels are in exeess of those observed for normal subjecËs (0.021 ng/ml) and pathologÍcal sÈaLes such as renal artery sÈenosis (0.218 ng/m1), chronic nephritis (0.251 nglml), and mallgnant hyperËensLøn (L,727 ng/ml) (Catt, Cain and Coghlan, L967). Abolitíon of Èhe pressor response ín animals requires ínÈravenous doses of the order of. 20 to 50 ue/Kg by injectl-on and 5 to 15 ug/KC by contínuous infusíon (Gross and Bock , L962; Bock and Gross , L96l; Doyl-e, Louis and Osborne, L967). These doses cannot be safely admÍnistered to normal- subjeets and indicate the difficulty of inducíng tachyphylæris in the whole anímal. In isolated artery preparat,íons, concentratíons of Èhe order of 100 Ug/rnl ín the baÈhíng solutÍons and injectíons of 0.25 pg ínt,o perfusíng solutíons are used to induce tachyphylaxis

(Bohr and Uchida, 1967; Khaírallah, Page, Bumpus and Turker, 1966). AddíÈíon of angÍotensínase to perfusíng soluËíons of ísolated ear arËery preparations reverses the tachyphylaxís índuced by prior injectíons of angioËensin.

FacÈors contaíned ín plasma, such as noradrenaline, albumin and serotonín, also cause apparent reversal of Èachyphylaxís

(Freckelton and Speden, Ëo be published; Teh, to be published). Therefore, in both the whole anímal and the normal subject the 153

developmenÈ of tachyphyLaxís is inhibited by the above menÈíoned factors.

The. present, experiments have shown Èhat. tachyphylaxis to angiotensín does not play a role in the response to angiotensín ín physiologícal situaËions. !ühether ít plays a role in Èhe reductíon ín sensltivíty of the vessels of paÈíents with renovascular hyper- tensíon ís less certaín. Although the doses adminisËered were in excess of those seen ín arteríal blood ín pathological situations

(Catt, Cain and Coghlan, L967) and venous plasma (Boyd, Landon and PearÈ , L967), factors such as duration of exposure of vessels to angíotensín and alteratíon ín plasma factors may play a role ín Índucing tachyphylaxis. It 1s unlíkely that tachyphylaxls is dependent upon reducÈion of angiotensinase levels ín such pathologícal conditions, since pregnancy, which ís assocíated wíth reduced pressor responsiveness, and hand vascular sensitívity to angíotensín is also assocíated hrlth increased levels of circulaËíng angiotensinase

(Page, L947; Talledo, Rhodes and Lívíngstone, 1967>, 154

SUMMARY TO SECTTON 7

Tachyphyl,axls of the hand and forearm vessels Èo angio- tensin could noÈ be demonst,rated.

Tachyphylaxis to vasopressin was, however, readily demon- strated

It is unlikely Èhat 1n normal subJects tachyphyJ-axis Ëo anglotgnsin plays a role in the normal vascular response to anglotensln. If tachyphyl-axls plays a role ín the reduced vascular r'esponsíveness Ëo an€-í€Ëensin 1n paflents wíth renovascular hypertension, it is probably attribut,able not only Èo the el-evaÈed levels of angíot,ensln, but, ln addítíon, Èo other facÈors such ás duration of exposufs--Èo angioËensin and al-Èeration of plasma cons tituents . STATISTIGAI METHODS

1 Calculation of mêan, varíance and standard deviatíon: Mean=I =1Xx "^')) variance =sz = x)ç'-!(xx)" n

Standard deviation S /s2

2 Studentrs t-test was used as a test for sígníflcance, using elther t = x - u* (n-1 degrees of freedom) Sñ.- for palred data

or Olívettl Programrna 101, Nunber 136 for unpaired data.

3. CorrelaÈion coefficÍente'rrere calculated using the fo::nula:

T lxy - Xx -ry. n (Eo)', (ryr,)'} /{t*2 - {Ly2 -

with n-2 degrees of freedom t = 7ær/n-2 or usíng Èhe Bravais Pearson coefficient of linear

correlation, OllveÈti Prograrnma 101, Nurrber 132.

* Reference: SÈatísÈical Methods in Biology Bailey N. T. J; (1959) The English UniversíÈles Press Ltd. TASLE 1

Urine No: 1 2 3 4

L7 30-50 0 50-70 0

20 20-40 0 20-40 0

22 50-70 0 70-90 0

24 80-100 100-120 0

1 Amount of pressor material formed followÍng incubatlon of

allquots of 4 urfnes incubated at 37oC wtth standard substnate;

2 Effect of boiling príor to íncubation on the formation of

pressor,material by 3 of 4 urines lncubated at 37oC wíth standard substrate,

3 Concentratfon of pressor materl.al (ng/mL) following boiling of

samples (coh¡nn 1) for t hour¿ -.

4. Concentration of .pressor materiaL (ng/ml) remainlng fol-lowing

incubatíon of samples (coltrnn 3) with ol-chyruot,rypsfn at, 37oC for t hour. TA3LE 2 Effeet of temperature on the rate of formatíon of

angíouensin (nglmllfrr) by urínary renin acting on standard sheep substrate at pH 7.5.

Temperat.ure Angiotensin (oc) (ng1mllhr)

31 33.0

37 4s.0

40 50. 1

43 60 .0

47 66.0

50 66.0

55 0

60 0 TABLE 3

Effect of the concenËration of sËandard sheep substrate on the rate of,formatíon of angíotensín (ng/nLlt,r) by 5 urines.

TABLE 3(a)

Substrate, (nelml/hr) (V) concentrat,íon Angior,ensín nelml (s) UrÍne I Urjrne 2 Urine 3

L200 18.4 2.7 6"0 -2.6 1000 19"8 5.7 800 2.5 5.8 600 16. I 1.8 5.1 500 10.0 1.9 5.3 400 10 .0 L.6 4.8 300 7.2 1"3 4.0 200 7.4 r.2 3.2 160 4.2 1.1 3.3 100 3.0 1.8 50 3.0

Km 298 ng/mLo 34, ng/rl* 238 ng/ml*

* (1) Km - calcuLated from l¡'Ioolf plot V : V/S (2) Slope (b) = -Krn (3) (b) calculaÈed from Bravais-Pearson coefficient of linear correlaÈíon. ,b=n Xxy-TxTy z z Xx - (rx) n

x= VIS, Y V TABLE 3(b)

Substrate Angiot,ensin (ne/mL/nr) (V) concentration ml S Urine 4 Urine 5

1066 3.2 L,4

533 2.8 1.3

480 1.1

400 2.7 L.07

266 1.9 0.9

133 L.4 1.0

80 1.0 0.6

53 0"8 0.4

Kn 2L2 nglmL 140 nglml TABLE 4 Effect of the concentratíon of urinary renln on the.rate of formatíon of angiotensin (ng/ml/hr) uslng 4 urínes.

% i¡LELaL l_n I ¡* concentration Urine A Urine B ine C Urlne D

100 8.0 20 .0 42,0 9 8.0

75 5.8 16.7 70 .0

50 4.3 10.0 25.5 51.0

37 .5 8.6

25 10.3 4.0 11 .0 28.O

12.5 2.8 6.0 L2.O

6.25 2.6

Urlne A (12 units/m1) Urine B (30 units/ml) Uríne C (63 uníts/m1) Urine D (L47 uníts/ml-) * measured velocity TABLE 5 EffecÈ of the pH of lncubaÈion on the rate of formatlon of angiotensl-n by renin in 3 urÍnes (4, B, C) acting on sÈandard sheep substrate.

Anglotens xUIea¡ % Plasma renÍn pH of ne/ml/hr velociÈy maximal % maxinal , , incubatlon A c A B c ve vel-o

4.0 0 0.8 0 28.6 14.5 29

4.5 1.5 s3. 6 s4.s 59

5.0 L2.0 7 .5 1.5 52.2 37.5 53.6 48.6 65

5.5 - 12,5 1.8 62.5 64.3 64.4 7L

6.0 19 .5 2.2 84. 8 78.6 83.0 76

6.5 2.5 89. 3 90.8 7L

7.0 2r.o 19 .0 2.5 91.3 95.0 89. 3 91.2 76

7.5 22.6 20 .0 2.7 98.3 100 96.4 99 .8 83

I 8.0 23.0 18.7 2.8 100 93.s I00 100 88

8.5 - 18.0 2.7 90 .0 96.4 94.7 83

9.0 2L.0 L7 .5 2.8 91.3 87.5 100 96,2 100

* CorrecÈed for a meen 7" value of LÙO% at pH 8.0 TA3LE 6 Varlabllity of repeat determinatlon sf the coricentration of renin ln urfne.

TABLE 6(a)

Duplicate assays of, the same samples using the sane rats.

Concentratíon sf renin Mean 7" DLfference. (x) units /ul of ofAandB B AandB from

4,63 4.3 4,47 7. 38

20.0 20.0 0 0

10 .5 10.5 o 0

7.5 7.5 0 0 5.0 4.8 4.9 4.0

9.6 8.7 9.15 9.84

1.3 1.3 0 0

2.3s 20.O 2.175 16 .09

x 4.66

SD 6 2 32 Xx d n TAsLE 6(b)

DupJ-ícate estimations sf samples following sËorage

of one sample at -ZOoC and assay ssme weeks laÈer.

Con centratíon of renín Mean 7" Difference (x) uniÈs /mI of ofAandB A B AandB from mean

3.3 4.63 3.97 33.5

3.3 4.3 3.8 26.3

22.4 20.o 2L "2 LL.32

l0¡6 10 ,5 10 .5s 0.95

8.8 7.5 8" 15 ls.95

5.2 5.0 5.1 3.9

5"2 4.8 5"0 8.0

10. 8 8"7 9.75 2r.5

10. B 9.6 to.2 tL.76

I .68 1.25 L ,47 29.25

1. 68 L,27 L,4B 27 .7

2.3 2.35 2.325 2.15

2.3 2.0 2.L5 13.95

x L5.9 , SD 10.9 TABLE 6(c)

DupJ-fcate estimations of samples, one alÍquot of which has

been díaLysed afÈer a long lnterval followlng storage at -2OøC.

Concentration of renin Mean % Dlfference (x) units /ml of ofAandB A B AandB from mean

18.0 12.0 15.0 40 .0

30 .0 30.0 30 .0 0

30.0 39.9 34.95 28.3L

30.0 29.25 24.63 2.5

30 .0 39.9 34.9s 28.3

30.0 29.25 29.63 2.5

39.9 29.25 34 .58 30.8

97 .s 63.0 80.25 43.0

97 .5 4s .0 7L.2s 73,68

63.0 45 .0 54.0 33.3

2"49 2.L45 2.32 ls .09

24.O 17.0 20.5 34.L4

4"2 4.20 4;2 0

0 "26 0.36 0. 31 32.3

0.69 0 .50 0.595 31 .9

0.27 0.33 0. 30 20 "0

x 26.0 , SD 19. 3 TA¡LE 7

Effeet of the pH of initlal dialysts and the additlon of EDTA (0.005 M) sn.the destruction of p-asparaginyL-angloÈensin II by url-ne íncubated at pH 7.5 Í.or 24 trrs (l 0.001 M EDTA, respecËívely)

pH of lnftlal Angfotensin remaÍnlng followfng incubatlon dfalysís + EDTA - EDTA

7.5 80-100 0-20

6.6 80-100 0-20

5.6 80-100 0-20

4.7 80-100 0-20

4.0 80-100 20-40

3.3 80-100 80-100

a TA3LE 8

Destruction of p-àsparaginyl-angfotensin II by urlne on

incubat,Íon at a pH of 7 .5 or less, r¿ith and wÍÈhout prfor dlalysis and heating to pH 9.6.

7" lmg tensin remaíning hrs 1n at Urine not treated Urine treated pH.of incubatÍon to pH 9.6 at pH 9.6

7.5 20-40 80-100

7.0 20-4A 80-100

6.0 2A-40 80-100

5.0 20-40 80-100

4.0 70-90 80-100

3.0 80-100 80-100 TA3LE 9

Effect of time on the rate of formatlon of pepsitensln by pepsin acting on standard sheep substrate at pH 5.6.

Concentration Concentrat,lon o f Concentration o pepsin pepsítensfn after pepsltensin after 5 ml-n l-ncubaÈíon 10 cubaÈion

15 80-100 100- 1 40

10 40-60 60-80

I 30-50 30-50 TABLE 10 Effect of the concentratÍon of pepsin (Ug/rl) on the

anount of pepsitensin formed from sÈandard sheep substrate after lncubatlon for 10 mln at.pH 5.6.

Csncentration PepslËensin pepstn ug/ml nglml

20 1 60-200

15 100-140

10 80-100

I 60-80

6 10-30

5 10-30

4 r0-30

2 10-30 TAsLE 11 Calíbration curve for standard protein solutions

(ug/mf) related to optícaL density at 750 mu.

Protein ug/ml o. D.

20 0.06

40 0"08

60 o.12

80 0.165

100 o.20

L20 0.225

140 o.24

160 0.28

200 0.35 TA3LE L2 The effeet of elevations in plasma renín concentration (PRC) on the urínary exeretisn of proËeJ-n (mg/24 hrs).

S ub i e c t I 2 4 5 7

PRC 0 8.4 9.3 9.8 13.5 11.5 (r:nlts /ml) 7 19 .5 23.8 20.0 23"8 34,2

L2 3L,7 38. 4 55.0 61.3 46 "3

Protein 0 137.8 182 .0 80. 7 L43.2 11.0. 4 exeretion (me/24 nrs) 7 115 .9 140. I 72.6 103.96 100.1

T2 10 3.5 185.6 66.4 t22.5 108.9 TABLE 13

Day and night urínary renín concentrations (unítslml), urínary renín outputs (rxríts /L2 hrs), volumes (m1/12 hrs) and urínary sodlums (mEq/12 hrs).

n. I Kenl-n conc Renin output Volume uríne Na,' No r:nits /nl uníts/l2 hrs mL/L2 lnrs nEq/12 hrs D N D N D N D N

I 1.1 0.73 449 334 410 455

2 0.32 0,23 374 332 1170 L47s

3 0.27 0.43 336 287 1245 660

4 0,20 0.20 133 135 680 690

5 0.26 0.51 275 32L 10 80 631 162 L26

6 0.13 0.13 L22 88 968 700 193 r26

7 0 "27 0.6 325 52L 1205 869 217 130

8 0.7 1 .88 904 975 I 310 520 262 78.0

9 0. B4 o .57 64L 593 763 1040 153 52

10 0,22 0 .04 125 37 .4 569 936 84.2 151 .6

11 0.25 0.22 156 rL7 630 525 lL7 ;3 72.2

L2 0 .47 o .47 384 29L 825 625 130 72.2

13 0. 43 0.25 131 L40 302 550 29.r 6.2

L4 0.8 1.3 262 553 327 440 51.1 50.1

15 0.24 0.14 136 L52 575 10 75 92.0 L69 .4 TABLE L4 Effect of serial dllutíon of plasma renin (pH 3.3 treated) renin on Ëhe formatlon of angiotensín (¡¡g/nL/t¡.x) from hunan subst::aÈe;

% Tr;^l-tLaL renin Angiotensín conclentraÈlon nslm1/hr*

100 9.3

50 3.9

25 1.8

12.5 1.0

* Csrrected for EV = 0 .5 ng/mL/hr TABLE 15 The effect of seríal dilutlon of amníotic fluíd on the rate of formaËíon of angfoÈensin (ng/nL/hr) from

standard sheep substrate and human, substraÈe.

% Initial Anglotensin ng/ml/hr concentrat,ion SÈandard lluman otí fl_ renl_n subs trat,e subs tr g*

100 64.0 12.7

50 27.0 6.5

25 L2.4 2.6

L2.5 6.7 L.2

6.2s 3¿0 0.9

* Gorrect,ed for endogenous veloclty of 0 .5 ng/mL/hr TABLE 16 The effeet .of substrate concentratíon on .the rate sf for.natísn of angiotensin (ng/rnl/hr) by amniotlc fLuid renÍn actlng on plasma

fro¡t normal and pregnant subjects, and plasma renín acÈ1ng on plasma from nor¡ral and t'oral contracepÈive treated" subJects.

TABLE 16(a)

Effect of the concenÈratlon substrate.ín normal hr¡nan plasma on the formatlon of angiot,ensin by amnlotlc fluíd renin.

Substrate (S) Angíot,ensln concent,ratlon ng/mL/hr V V S

L.75 60. 3 34.46

1.313 45 .0 34.27

0.875 34.4 39. 31

0.438 L5.7 35"84

0.2L9 10.0 45,66

Ktrr 3;0 pg /mI TABLE 16(b)

Effect of substrate céficentratíon ln.pregnarit plasma on Èhe rat,e of format,íon of anglotensin by amnioÈic fluid renfn.

Substrate (S) Angiotensln concenÈratlon ng/mL/hr m1 V S

3.0 87 .6 29.2

2.O 53.0 26.5

1.0 37 .O 37 .0

0.5 L7 .0 34 .0

0.2 6.4 32.O

Km 4.0 ug/ml TABLE 16(c) EffecÈ of substrate concenLration in normal and "oral

contraceptíve ÈreaÈed" pJ-asma on the formaËion of arigíotensín by pH 3.3 treaÈed plasma renfn.

Substrat,e (S íotensln concenÈraÈion ng/mL/br uelml (v) V/S

4. 31 28.3 6.57

3.24 22.2 6 .85

2.16 L6.5 7 .64

1.08 10. 5 9.7

0.54 4.7 8.7

0.27 2.3 8.5

0. 135 1.2 8.9

Km 7.6 ve/mL TABLE L7

Dose response curves obÈained wíth íntra-arÈerial infusions of

angíoÈensin in (a) tton-pregnant a¡rd (b) Pregnant r^romen.

TABLE L7 (a)

Non-pregnant women

Per cent constrictÍon Subj ect o in t enaline 800 0 200 40

1 61.1 70 .0 8L.7 87. I 51.8 58.9 67.3 74.8

2 65.7 7 4.3 7L.6 65.6 54;4 77 .O 72 "r 78. B

3 62.L 77 .5 83. I 84. 8 85.2 77 .6 9r.4 94.3

4 5.6 34.5 29.4 30.2 42,;0 71 .0 82.4 95 ;0

5 62 .3 4s. I 73.0 77.5 53.3 70.7 7 3,5 82.9

6 L7 .7 39 .5 72.8 82.4 49.s 47.3 93.4 97.3

Mearr 45.8 56.9 68.6 71.4 56.0 67.1 80.0 87.r

SD !26.7 r19.1 !19.8 !2L.6 !I4.96 111"8 !10.8 !9.6 TABLE L7

Pregnant \¡Iomen

Per cenË constríction Subject An íotensin Noradrenaline 100 200 00 0 00 200 4 800

7 18. 9 8.0 7 4.0 82.L 14.5 90.8

8 0 18. 6 9.0 47 .B 63.3 81 .9 93.6

9 10.9 29.0 63.4 77 .4 33"0 36.9 95 "L \ 10 24 "9 4L .4 62.2 82.5 82.3 81.1 93.6 96.L

1tr 28.8 29.7 4L.7 63.5' 68. s 76.6 74.7 91.0

L2 0 11 .0 24.8 s2.4 25 "9 4L.I 91 .6 96.4

13 6.3 L3.2 51 .6 4s.9 4L.9 54.8 90 .0

L4 24.0 23.L 29.0 29.L 20.2 79.6 95 .9

15 9.9 o.6 27 .7 82.4

Mean 14.2 20 .4 39 .6 60. 1 s5 .6 56 .8 87 .6 94 -8

SD r11.5 111.3 !25.5 t19.5 t2L.9 !26,5 t 7.6 ! 2,6 BISLIOGRAPHY

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