A Plasma Humoral Factor of Extrarenal Origin Causing Release of Reninlike Activity in Hypotensive Dogs

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1 A Plasma Humral Factr f Extrarenal Origin Causing Release f Reninlike Activity in Hyptensive Dgs By E. De Vit, C. Wilsn, R. E. Shipley, R. P. Miller, and B. L. Mrtx ABSTRACT Plasma reninlike activity significantly increased in anesthetized and unanesthetized nrmal dgs after intravenus injectin f plasma frm nephrectmized and nrmal dgs that have been made hyptensive by cntrlled hemrrhage. The injectin f plasma frm nephrectmized, nnhyptensive dgs r the in vitr incubatin f nrmal plasma with calculated prprtins f plasma frm nephrectmized, hyptensive dgs did nt prduce any changes in plasma renin cncentratin r angitensin prductin. These results suggest the presence f renin-releasing factr f extrarenal rigin which is prduced and circulated in die plasma f dgs subjected t perids f hyptensin by means f cntrlled hemrrhage. A direct r indirect actin f the renin-releasing factr n the kidney in assciatin with r in cnjunctin with ne r mre f the ther mechanisms that have been hypthesized t explain the cntrl f renin release is nt as yet knwn. An alternative hypthesis shuld als be cnsidered. It is pssible that the renin-releasing factr may be an activatr f the cnversin f prerenin t renin and f its release frm the kidney int the bldstream. The surce and chemical nature f the renin-releasing factr are nt as yet knwn. KEY WORDS plasma renin-releasing factr hemrthagic hyptensin bilateral nephrectmy plasma renin activity angitensin renin-substrate cncentratin renin-angitensin system Several hyptheses invlving barreceptrs, the macula densa, and electrlyte shifts, as well as ther mechanisms, have been prpsed t explain the cntrl f renal renin release (1). Nne f the hyptheses appears t be cnsistent with all f the experimental findings, and the mechanism (s) causing renin release very quickly after the nset f hyptensin due t hemrrhage is still bscure. In 1966, Fascil et al. (2) suggested that the presence f a humral factr assciated with the actin f intrarenal barreceptrs culd be invlved in renin release. An extensive search fr substances that wuld accelerate the release f renin in anesthetized nrmal dgs (2) and frm kidney slices (3) was nt successful. Frm the Lilly Labratry fr Clinical Research and the Lilly Research Labratries, Indianaplis, Indiana Received February 16, Accepted fr publicatin August 27, In experiments designed t study the physilgical prperties f a "sustained pressr principle" described by Shipley et al. (4), anesthetized nrmal dgs were injected with plasma frm nephrectmized hyptensive dgs. Plasma reninlike activity increased substantially after the injectin, and the prbability that this increase was due t a humral factr was studied. The results f these experiments are reprted in this paper. Methds Male r female mngrel dgs weighing kg were anesthetized with secbarbital sdium (30 mg/kg iv). A bilateral nephrectmy was perfrmed thrugh a retrperitneal incisin under aseptic cnditins. Frty-eight hurs after nephrectmy, the dgs were anesthetized with phenbarbital sdium (150 mg/kg). ar d bld samples were drawn thrugh a plyethylene catheter inserted int the femral artery and cllected in cld tubes 446 Circulatin Resurcb, Vl. XXIX, Nvember 1971

2 HUMORAL RENIN-RELEASING FACTOR 447 cntaining heparin. Plasma was separated in a refrigerated centrifuge. The dgs were then subjected t acute hemrrhagic hyptensin (mean bld pressure, mm Hg) fr minutes. After hemrrhage, a bld sample was drawn, and plasma was separated by centrifugatin. Plasma btained under similar cnditins befre and after hyptensin frm nnnephrectmized dgs was used as a cntrl. All arterial bld pressures were recrded n a kymgraph with a mercury manmeter cnnected t the femral artery. Prtcl I. Experiments were perfrmed n 14 nrmal dgs (8 male and 6 female) weighing kg and maintained n standard labratry chw. Each was anesthetized with phenbarbital sdium (150 mg/kg), and arterial bld pressure was recrded as previusly described. A 15-ml bld sample fr cntrl determinatins f reninlike activity and angitensingen was taken frm the cartid artery thrugh a plyethylene catheter befre and several times after the injectin f ne f several plasma preparatins. Injectins were made thrugh a plyethylene catheter in the femral vein in accrdance with the fllwing experimental plans: 1. Plasma frm 48-hur nephrectmized dgs befre hemrrhage. 2. Plasma frm 48-hur nephrectmized dgs after hemrrhage. 3. Plasma frm nnnephrectmized dgs befre hemrrhage. 4. Plasma frm nnnephrectmized dgs after hemrrhage. 5. Plasma frm 48-hur nephrectmized dgs after hemrrhage and fllwing extensive dialysis f the plasma against nrmal saline. 6. Nrmal saline. Prtcl 2, In fur nrmal, unanesthetized dgs, trained t lie quietly n a bard, bld was taken by puncture f the femral artery. Plasma renin activity was measured befre and after the injectin f 15 ml f plasma cllected frm 48-hur nephrectmized dgs befre and after hemrrhage. Reninlike Activity. Reninlike activity was CtrcuUiH Rsseirch, Vl. XXIX, Nrimber 1971 measured accrding t the methd f De Vit and Fascil (5), and the results were expressed as ng angitensinlike activity/ml plasma. Angitensingen Determinatin Reninsubstrate cntent in the plasma was measured by incubating 0.5 ml f plasma fr 1 hur at 37 C in the presence f 0.2 Gldblatt units f hg renin btained by the methd f Miller et al. during the purificatin f sustained pressr principle, and free f this substance (6, 7), The vlume f each sample was made up t 2.5 ml with the fllwing buffer slutin: NaCL, 0.9 g/100 ml, 75 ml; 0.1M phsphate citrate buffer, ph 5.4, 25 ml; EDTA disdium salt, 150 mg. The reactin was stpped by placing the tubes in a biling water bath, and the precipitates were remved by centrifugatin. The supernatant fluid was brught t ph 7 and assayed against synthetic angitensin amide (Hypertensin, CIBA) in pentliniumtreated anesthetized rats. The results were expressed as ng angitensinlike activity/ml plasma. Results Figure 1 shws the results f 10 experiments carried ut accrding t prtcl 1. A significant increase in reninlike activity was fund in all the experiments after the injectin f plasma frm nephrectmized, hyptensive dgs (Ab, Bb, C, D, Eb, Fb). Als a significant increase was fund after the injectin f plasma frm a nnnephrectmized, hyptensive dg (G,H). N increases were fund in reninlike activity when the injected plasma was taken frm nrmtensive nrmal r nephrectmized dgs (Aa, Ba, Ea, Fa). Extensive dialysis against nrmal saline did nt remve the ability f plasma frm nephrectmized, hyptensive dgs t increase circulating reninlike activity in the recipient dg. Plasma frm a 48-hur nephrectmized dg subjected t hemrrhage f 37 ml/kg bdy weight was placed in Visking casing and dialyzed with gentle stirring against 4 liters f nrmal saline fr 36 hurs at 5 C. Nrmal saline was changed eight times. Twenty ml f

3 448 DE VITO, WILSON, SHIPLEY, MILLER, MARTZ I Renin Activity = R»nin Substrate ' 5,,n,. 2 6» u. O 0 I 600 j l O "i b I 1 i 7.3 kg wt. (7) b r? _»».»kg w». 0 Ui a. 600 ^ Z Ul t * z c. I j nil 14.4 = "Ok. wt. 1 ( rf 11.0 kg wt. 130 M ISO I N U T FIGURE 1 60 E _ 1*8.4 k ( wt. /lj.o k«wt. 2 Oz ) C Ul 300 \n Plasma renin activity and substrate cncentratin. A and B: Arrw a, injectin f 15 ml f nrmal dg plasma; arrw b, injectin f IS ml f 48-hur nephrectmized, hyptensive dg plasma btained 30 minutes after a hemrrhage f 24 ml/kg bdy weight. In A the plasma was btained the same day f the experiment, and in B, I week befre and then frzen. C: plasma (20 ml), frm a 48-hur nephrectmized, hyptensive dg that bled 21 ml/kg bdy weight was injected at arrw. D: Fresh plasma (30 ml) frm a 48-hur nephrectmized, hyptensive dg was injected at arrw. E and F: Dnr plasma was frm dgs nephrectmized 1 hur befre the experiment, and plasma was btained befre and 30 minutes after a hemrrhage f 30 ml/kg bdy weight in E and 19 ml/kg bdy weight in F. At arrws a and b, 15 ml f each f the tw plasmas was injected. G: Injectin f 25 ml f plasma frm a nnnephrectmized dg subjected t hemrrhage f 39 ml/kg and a bld sample drawn 20 minutes later. H: Injectin f IS ml f plasma frm a nnnephrectmized dg subjected t hemrrhage f 21 ml/kg and bld sample drawn 20 minutes later. I and J: Dialyzed plasma (20 ml) frm a 48-hw nephrectmized, hyptensive dg, bled 37 ml/kg bdy weight, was injected at arrw. dialyzed plasma was injected int each f tw nrmal anesthetized dgs (Fig. 1: I, J). The effects f plasma frm nrmal and frm nephrectmized dgs, nrmal dg plasma Circulaicm Risenrcb, Vl. XXIX, Nvtmbf 1971

4 HUMORAL RENIN-RELEASING FACTOR 11.4 kg wt kg wt. 600»/, E OS Hi i 300 O 6 i d*10.6 kg wt. «9. 8 kg wt. 600 m u. 3 a 300 a c 120 M I 180 Renin Activity N u FIGURE 2 T 60 E 120 s 180 = Renin Substrate Plasma renin activity and substrate cncentratin. A: Nrmal plasma (25 ml) was injected at the arrw. B: Plasma (30 ml) frm a 48-hur nephrectmized, nnhyptensive dg was injected at the arrw. C: Nrmal plasma (20 ml) dialyzed against saline was injected at arrw. D: Nrmal saline (30 ml) was injected at arrw. dialyzed against saline, and saline alne were tested in fur individual experiments. N increases in reninlike activity were fund (Fig. 2). Plasma-substrate levels were measured in each case, and n significant changes were fund. N significant change in arterial bld pressure was bserved in any f the recipient animals after injectin f any f the plasmas r saline. The renin activity befre hemrrhage in the nrmal dnr dg used in experiment G (Fig. 1) was 1.8 ng angitensin/ml plasma. After hemrrhage, it was 8.2 ng angitensin/ml plasma. The ttal renin injected int the recipient dg was equivalent t 205 ng f angitensin. The renin activity in 15 ml f plasma frm the dnr dg in experiment H (Fig. 1) was 157 ng. These renin activities are less than 0.05 Gldblatt units, and as was expected, n significant change in arterial bld pressure f the recipient dg was bserved. Additinally, dilutin f the dnr plasma in the recipient dgs wuld have caused an increase in renin activity n greater than 1 ng angitensin/ml plasma. Circulatin Reieerch, Vl. XXIX, tititmber 1971 The pssibility that the bserved increases in renin activity were due t an increase f substrate cncentratin r t the presence f an activatr f the renin-substrate reactin was ruled ut by adding, in vitr, a calculated prprtin f plasma frm nephrectmized, hyptensive dgs t the cntrl plasma (first cllected samples). Assuming that plasma vlume is 4.5% f the whle bdy weight, ml f plasma frm nephrectmized, hyptensive dgs was added t 5 ml f the cntrl plasma t btain the same apprximate dilutin as wuld exist when dnr plasma was injected int the bldstream f the recipient dgs. N difference in angitensin prductin was fund in the incubated samples with r withut the additin f plasma frm nephrectmized, hyptensive dgs in each f five experiments. An extrarenal surce f reninlike activity in the recipient dg was ruled ut when 20 ml f plasma frm nephrectmized, hyptensive dgs was injected int tw 48-hur nephrectmized dgs, and n reninlike activity was

5 450 DE VITO, WILSON, SHIPLEY, MILLER, MARTZ 6OO 3O 6O O 3O 6O M I N U T E S Renin Activity = Renin Substrate FIGURE 3 In fur nrmal, bard-trained dgs, plasma renin activity and renin-substrate cncentratin were measured befre and after the injectin f IS ml f plasma frm 48-hur nephrectmized, hyptensive dgs, fresh plasma in A and B and week-ld, frzen plasma in C and D. The dgs frm A and B were used 10 days later, but in these experiments, 15 ml f nrmal plasma and 15 ml f nephrectmized, nrmtensive dg plasma were injected in E and F, respectively. fund in their plasma either befre r after the injectins. Significant increases in reninlike activity were fund in the samples taken 20 and 60 minutes after injectin f 15 ml f plasma frm nephrectmized, hyptensive dgs int nrmal, unanesthetized, bard-trained dgs (Fig. 3: A, B, C, D). Plasma frm nrmal r nephrectmized, nnhyptensive dgs caused n change in the renin activity levels f the nrmal recipient dgs (Fig. 3: E, F). N significant changes in plasma-substrate levels were bserved. Discussin A significant increase in the plasma reninlike activity was fund in anesthetized and unanesthetized nrmal dgs after intravenus injectin f plasma frm nephrectmized and nrmal dgs that had been made Circilaticn Resurcb, Vl. XXIX, Nvtmttr 1971

6 HUMORAL RENIN-RELEASING FACTOR 451 hyptensive by cntrlled hemrrhage. An increase in renin-substrate cncentratin in the recipient dgs as the result f the injectin f plasma with high cncentratins f substrate cannt explain the increase f plasma renin activity because: (1) Plasma frm nephrectmized, nnhyptensive dgs with the same high substrate cncentratins did nt prduce any change in plasma renin cncentratin when injected int nrmal dgs. (2) The in vitr incubatin f nrmal dg plasma with plasma frm nephrectmized, hyptensive dgs in the calculated prprtin (cnsidering the dg's plasma vlume) prduced an amunt f angitensin similar t that prduced by nrmal plasma alne. The amunt f substrate injected was nt mre than 10? f the substrate riginally present in the recipient dg's plasma. The in vitr results als rule ut the presence f an activatr f the reninsubstrate reactin. The present experiments suggest that a renin-releasing factr is prduced and is circulated in the plasma f dgs that have been subjected t perids f hyptensin by cntrlled hemrrhage ver a perid f minutes. Plasma frm dgs that had previusly been nephrectmized and then subjected t hyptensin caused renin release, indicating that the surce f the renin-releasing factr was extrarenal. The finding that an extrarenal renin-releasing factr is elabrated during hyptensin des nt exclude r invalidate ther mechanisms that have been pstulated t explain the cntrl f renin release. It is pssible that the extrarenal renin-releasing factr may perate in assciatin with r in cnjunctin with ne r mre f the ther mechanisms that have been hypthesized. In the present experiments, the increase in plasma renin activity fllwing the injectin f plasma frm hyptensive dgs (nephrectmized r nt) was detectable abut 30 minutes after injectin and usually cntinued t increase ver at least 2 hurs. Furthermre, the actin f renin-releasing factr may nt be directly n the kidney, rather it may be mediated indirectly thrugh an effect n sme extrarenal Ctfctdtin Rtsttrcb, Vl. XXIX, Nptmber 1971 structure. Als the rate f renin release may increase as a result f a small, but persistent, direct effect n sme renal structure. An alternate hypthesis shuld als be cnsidered. Previus wrk (8) strngly supprts the cncept that there is an enzymatically inactive "prerenin" in kidney tissue that can be cnverted t an enzymatically active reninlike material. It is therefre pssible that the renin-releasing factr may be an activatr f prerenin and can accelerate its cnversin t renin and its release frm the kidney int the bldstream. That renin-releasing factr itself may be a small mlecule r active peptide can be ruled ut, because it retains full activity after extended dialysis. The surce f the renin-releasing factr and the factr's chemical nature are nt as yet knwn. Acknwledgment The authrs gratefully acknwledge the technical assistance f Mr. William Carr and Mrs. Rsa De Vit. References 1. PAGE, I. H., AND MCCUBBIN, J.W.: Renal Hypertensin. Chicag, Year Bk Medical Publishers, Inc., FASCIOLO, J.C., DE VITO, E., NOLXY, H., AND STANELONI, J.R.: Mechanism f renin release in adrenalectmized dgs and the reninreleasing activity f several substances. Prc 6th Pan-Am Cngr Endcrinl, Mexic City, 1965, p DE VITO, E., GORDON, S.B., CABHEBA, R.R., AND FASCIOLO, J.C.: Release f renin by rat kidney slices. Am J Physil 219: , SHIPLEY, R.E., HELMER, O.M., AND KOHLSTAEDT, K.G.: Presence in bld f a principle which elicits a sustained pressr respnse in nephrectmized animals. Am J Physil 149: , DE Vrr, E., AND FASCIOLO, J.C.: Methd fr the estimatin f renin activity in plasma. Acta Physil Lat Am 15: , MILLER, R.P., WILSON, C, AND SHIPLEY, R.E.: Extractin and partial purificatin f a sustained pressr principle frm kidney (abstr.). Fed Prc 28:330, MILLER, R.P., DE VITO, E., POPER, C, WILSON, C, AND SHIPLEY, R. E.: Purificatin and prperties f a sustained pressr principle frm kidney (abstr.). Fed Prc 30:432, DE VITO, E., CABRERA, R.R., AND FASCIOLO, J.C.: Renin prductin and release by rat kidney slices. Am J Physil 219: , 1970.