Clinical Science (1979) 56,105-108

Inactive renin in rabbit plasma: effect of haemorrhage

H. K. R I C H A R D S , S. A. G R A C E , A. R. N O B L E A N D K . A. M U N D A Y Department of Physiology and Pharmacology, Uniuersilyof Southampton, Southamptom, Hants., U.K.

(Received 7 September 1977; accepted 7 September 1978) Summary 1. Renin activity in rabbit plasma increases after acidification (pH 3.3), probably due to activation of an inactive form of renin. 2. Both active and inactive renin in plasma increase after haemorrhage. This stimulus does not change the relative proportions of the two forms. 3. After ligation of the renal blood vessels neither form of renin increases in response to haemorrhage. 4. One day after bilateral nephrectomy no inactive renin could be demonstrated in plasma. 5. In the rabbit, therefore, the kidney is a major source of the inactive renin in plasma.

Key words: haemorrhage, renin. Introduction

Kidney extracts from a number of species including the rabbit (Leckie, 1973), pig (Boyd, 1974) and the rat (Morris & Johnson, 1976) contain a form of renin that is considered inactive at physiological pH but which can be activated by acidification. Analogous inactive forms of extrarenal renin forms exist in human amniotic fluid (Morris & Lumbers, 1972), rabbit uterus (Jorgensen, 1976) and mouse submaxillary gland (Bing & Poulsen, 1976). Two important questions arise therefore: does inactive renin in the plasma change in response to changed physiological conditions, and does it originate from the kidney? Day, Luetscher & Zager (1976) conCorrespondence: Dr A. R. Noble, Department of Physiology and Pharmacology, School of Biochemical and Physiological Sciences, Medical and Biological Sciences Building, Bassett Crescent East, Southampton, SO9 3TU, Hants., U.K.

sidered that the high-molecular-weight, enzymatically inactive ‘big renin’ in human plasma did not respond to altered physiological states. Other authors report altered concentrations of inactive renin in hypertension and in pregnancy. This might suggest a role for inactive renin in the regulation of plasma renin activity (Sealey, Moon, Laragh & Atlas, 1977; Leckie, McConnell, Grant, Morton, Tree & Brown, 1977). The contribution of extrarenal sources to circulating amounts of inactive renin was indicated by the demonstration of large amounts of this form in the plasma of anephric man (Leckie et al., 1977; Sealey et al., 1977; Weinberger, Wade, Aoi, Usa, Dentino, Luft & Grim, 1977). Studies with an isolated, perfused rat kidney, by Vandongen, Poessee, Strang & Birkenhager (1977), also suggest an extrarenal source for inactive renin. We have investigated the response of circulating concentrations of active and inactive renin in the rabbit to two levels of haemorrhage, a stimulus well known to elevate plasma active renin. Methods

All experiments were performed on New Zealand White rabbits of both sexes, weighing 34-4.5 kg. Group 1: mild haemorrhage with sham operation. Ten rabbits were anaesthetized with urethane (1.25 g/kg body wt: intraperitonolly) and in each the left common carotid artery was cannulated. A loose tie was placed around the vascular pole of each kidney as a sham operation. The animals were left for 30 min before removal of blood samples. These rabbits were subjected to a ‘mild‘ haemorrhage, by the withdrawal of four blood samples (8 ml each: used for renin 105

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estimations) from the carotid cannula at 15 min intervals. This approximately 10% reduction in blood volume gave a fall in arterial blood pressure of about 10 mmHg. Group 2: severe haemorrhage with sham operation. Ten rabbits were anaesthetized and prepared as described for group 1. These animals were bled more severely by the removal of an additional 15 ml of blood (discarded) after each of the first two 8 ml samples. This ‘severe’ haemorrhage was estimated to be approximately 20-25% of blood volume, and caused a fall in blood pressure of 3040 mmHg. Group 3: severe haemorrhage after ligation of the renal blood vessels. This group of 10 animals was prepared as described for group 2, but the renal blood vessels were ligated 30 min before a ‘severe’ haemorrhage. Group 4: severe haemorrhage 24 h after bilateral nephrectomy. Bilateral nephrectomy was performed on six rabbits under halothane (3% in nitrous oxide/oxygen, 2 :1) anaesthesia. Twenty-four hours later, these animals were subjected to the ‘severe’ haemorrhage procedure previously described. Each 8 ml sample was collected into 50 pl of disodium EDTA (50 mmol/l) and plasma separated. Half the plasma sample was dialysed to pH 3-3 over 24 h against ‘buffer A’ (Skinner, 1967), heated to 32OC for 1 h and then dialysed to pH 7.5 against ‘buffer C’. The other half of the plasma was dialysed against buffer C for 48 h. Both sets of

samples were then assayed for renin activity. Recovery of added renin (already fully activated) through the two protocols was 91.3% (acidified) and 9 1.4% (non-acidified). Renin ‘concentration’ was measured by radioimmunoassay based on the method of Stockigt, Collins 8c Biglieri (197 I), with excess of exogenous pig renin substrate, prepared as described by Skinner (1967) for sheep substrate. The pig was bilaterally nephrectomized 72 h before exsanguination. Mean values of plasma renin ( ~ S E M )for each group of rabbits as a whole are given, and differences were assessed statistically by Wilcoxon’s test. Results

Groups 1 and 2: mild or severe haemorrhage with sham operation Table 1 shows the predictable progressive rise in plasma renin (non-acidified) after a mild haemorrhage. Renin increased to a mean value of 89% above the initial value in each animal. After acidification there was a further rise in plasma renin in all samples, which was usually of the order of 15-20% of the non-acidified value. A similar pattern is seen for the group of animals with a ‘severe’ haemorrhage. For non-acidified samples renin increased to 139% above the first sample

TABLE1. Plasma renin in acidiJied ( p H 3 . 3 ) and conlrol (non-acidified samples Results shown are mean values & SEM. Plasma renin (pmol of angiotensin I h-‘ ml-’) Time (min) ... 30 Control

45 Acidified

Control

60 Acidified

75

Control

Acidified

Control

Acidified

24.9 f 4.9

29.8 f 5.8

31.3 f 6.9

37.3 f 7.4

Group 1: mild haemorrhage with sham operation (n = 10)

16.7 f 2.9

19.2 f 3.4

P > 0.05

20.4 f 3.9 22.7 f 3.9 P < 0.05

P < 0.02

P < 0.02

Group 2:severe haemorrhagewith sham operation (n = 10)

18.5 f 3.0

21.9 f 4.4

P < 0.02

20.1 f 3.5 23.7 f 4.1 P < 0.01

25.6 2 4.6

29.1 5 5.1

P < 0.05

44.2 f 12.3

50.9 f 13.0

P < 0.02

Group 3:severe haemorrhage starting 30 min after ligation of renal blood vessels (n = 10)

4.9 f 1.0

5.8 f 1.4

P < 0.05

4.2f 1.0

4.72 1.1

P > 0.05

4.1 f 1.2

3.7 5 1.1

P > 0.05

2.0 f 0.4 2.6 f 0.6 P > 0.05

Group 4:severe haemorrhage 24 h after bilateral nephrectomy (n = 6)

0.59f 0.09

0.54f 0.07

P > 0.05

0.58 f 0.10 0.57 f 0.09 P > 0.05

0.52f 0.07

0.56 f 0.09

P > 0.05

0.49 f 0.09

0.56 f 0.16

P > 0.05

Inactive renin in rabbits after haemorrhage taken and acidification gave increases in renin comparable with those hthe group 1 animals. In both of these groups of animals haemorrhage caused a rise in plasma renin and there were equivalent changes in both active and inactive renin. There was no evidence for a differential increase in the amount of one form of renin compared with the other over the time course studied.

Group 3 : severe haemorrhage after ligation of the renal blood vessels In the first blood sample, collected 30 min after ligation of the renal blood vessels, plasma renin (non-acidified) was only 25% of that found at a comparable time in the sham-operated groups. Plasma renin further declined, despite the severe haemorrhage imposed, until 75 min after ligation of the renal blood vessels mean plasma renin (nonacidified) was 67% of that in the sample taken after 30 min in each animal. In this series of rabbits only the first sample taken showed significant activation of renin after acidification. Against the background of falling plasma renin after ligation of the renal blood vessels there is no evidence to suggest increased secretion of either active or inactive renin from extrarenal sources in response to haemorrhage. Group 4: severe haemorrhage 24 h after bilateral nephrectomy In this group of six rabbits the plasma renin (non-acidified) in the initial sample taken was only 5% of the value in the sham-operated groups. These values did not change when the animals were subjected to a severe haemorrhage and plasma renin did not increase after acidification. One day after bilateral nephrectomy therefore there was no evidence of extrarenal sources secreting inactive renin into the circulation.

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Vandongen et al. (1977) suggested that the low proportion of renin in the inactive form in their study could be an effect of the anaesthetic. Support for this idea comes from the work of Derkx, Wenting, Man in t’Veld, Van Gool, Verhoeven & Schalekamp (1976). However, James & Hall (1974), in a study of responses to haemorrhage and frusemide infusion in anaesthetized dogs, found rises of 50% or more in renin activity on acidification. The possible role of anaesthetic agents in causing a selective secretion of the different forms of renin needs clarification, but species differences may also be responsible. Two levels of haemorrhage in rabbits increased the plasma concentrations of both active and inactive renin, although the two forms remained in the same relative proportions. If it is proposed that inactive renin has a specific physiological role to play, with a discrete secretion control mechanism, then one might expect to observe differential secretion of the two forms in response to some stimuli. In the present study the absence of such a change couid reflect either the short time course of the experiment or the nature of the stimulus chosen for investigation. Both possibilities must be evaluated. In the group of rabbits subjected to severe haemorrhage after ligation of the renal vessels, acidification did not produce activation of renin in samples taken 45 min or longer after interruption of the blood supply to the kidneys. No inactive renin could be detected in the plasma of rabbits 24 h after bilateral nephrectomy. This suggests that, in the rabbit, inactive renin is of renal rather than extrarenal origin. Although inactive renin has been isolated from the kidneys of several species, including the rabbit (Leckie, 1973), other authors (Vandongen et al., 1977; Leckie et al., 1977; Sealey et al., 1977; Weinberger et al., 1977), in studies on the rat and man, have proposed that inactive renin in plasma is primarily of extrarenal origin. Our results do not support this conclusion.

Discussion

Acknowledgments

In rabbit plasma 15-20% of renin is acid-activatable. This compares closely with the average value of 15% reported by Vandongen et al. (1977) for the rat but is substantially less than the 62% in normal human plasma found by Skinner, Cran, Gibson, Taylor, Walters & Catt (1975). Similar values to these were also reported by Leckie et al. (1977) and Boyd (1977). These surveys relate to samples taken from conscious subjects and

We are grateful to the British Heart Foundation for financial support. S.A.G. was supported by an MRC Research Studentship. References BUG, J. & POIJLSEN,K. (1976) Vast and apparently paradoxical continuous rise in plasma renin after removal of gently manipulated submaxillary glands in nephrectomised mice. Acta Pathologica et Microbiologica Scandinavica, Section A, 84,285-290.

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BOYD, G.W. (1974) A protein-bound form of porcine renal renin. Circulation Research, 35,426-438. BOYD,G.W. (1977)An inactive higher molecular weight renin in normal subjects and hypertensive patients. Lancet, i, 215218. DAY,R.P., LUETSCHER, J.A. & ZAGER,P.G. (1976)Big renin: identification, chemical properties and clinical implications. American Journal of Cardiology, 31,667-674. DERKX, F.H.M., WENTING,G.J., MAN IN T’VELD,A.J., VAN Goor, J.M.G., VERHOEVEN,R.P. & SCHALEKAMP, M.A.D.H. (1976)Inactive renin in human plasma. Lancet, ii, 496-498. JAMES,S.K. & HALL, R.C. (1974)The nature of renin released in the dog following haemorrhage and frusemide. Pfugers Archiv, 341,323-328. JORGENSEN, J. (1976)Acid activation of renin in rabbit uterus. Acta Pathologica el Microbiologica Scandinavica, Section A, 84,123-129. LECKIE,B. (1973)The activation of a possible zymogen of renin in rabbit kidney. Clinical Science, 44,301-304. LECKIE,B.J., MCCONNELL, A., GRANT, J., MORTON,J.J., TREE, M. & BROWN,J.J. (1977)An inactive renin in human plasma. Circulation Research, 40 (Suppl. I), 1-46-1-51. MORRIS, B.J. & JOHNSON,C.I. (1976) Isolation of renin granules from rat kidney cortex and evidence for an inactive form of renin (prorenin) in granules and plasma. Endocrinology, 98,1466-1474.

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Inactive renin in rabbit plasma: effect of haemorrhage.

Clinical Science (1979) 56,105-108 Inactive renin in rabbit plasma: effect of haemorrhage H. K. R I C H A R D S , S. A. G R A C E , A. R. N O B L E...
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