THE EFFECTS OF AN ANGIOTENSIN BLOCKER (SARALASIN) ON KIDNEY FUNCTION IN DEHYDRATED SHEEP

Transcription

1 Quarterly Journal of Experimental Physiology (1982) 67, Printed in Great Britain THE EFFECTS OF AN ANGIOTENSIN BLOCKER (SARALASIN) ON KIDNEY FUNCTION IN DEHYDRATED SHEEP NANCY E. YESBERG, MYRNA HENDERSON, KATHLEEN WILSON, SANDRA LAW AND R. B. CROSS Department of Physiology and Pharmacology, University of Queensland, Brisbane, Australia (RECEIVED FOR PUBLICATION 9 MARCH 1981) SUMMARY Saralasin, an angiotensin II analogue and receptor blocker, was infused at 7 and 15,ug. min-' into dehydrated conscious Merino ewes. This caused mean arterial blood pressure, cardiac output, heart rate and renal vascular resistance to fall, and central venous pressure to rise. Renal plasma flow was unaffected but there were significant reductions in glomerular filtration rate, filtration fraction, urine flow, sodium and potassium excretion, solute clearance and solute-free water reabsorption. It is suggested that saralasin produced these effects by inhibiting endogenqus angiotensin II activity, and in particular by causing a reduction in renal post-glomerular resistance. This in turn caused a fall in glomerular filtration rate and filtration fraction. While saralasin might have had effects on renal tubular function and perhaps on vasopressin secretion, the observed effects on renal function can be explained by the decrease in glomerular filtration rate and filtration fraction. INTRODUCTION Arginine vasopressin (AVP) infused into a hydrated, diuresing sheep has the expected antidiuretic effect, but in the non-hydrated or dehydrated sheep it causes an increase in water and electrolyte excretion associated with an increase in glomerular filtration rate (G.F.R.). The diuresis occurs despite an increase in solute-free water reabsorption (Tc,H,o) (Yesberg, Henderson & Budtz-Olsen, 1973). The diuretic and salt-losing effects of AVP are reversed by simultaneous infusion of angiotensin II (A II) (Yesberg, Henderson & Budtz-Olsen, 1979), an effect which appears to be dependent on a reduction in G.F.R. rather than on tubular processes. This paradoxical water-losing effect of AVP is not seen during severe water deprivation either in the field or under laboratory conditions where endogenous AVP levels are high (Macfarlane, Morris, Howard, McDonald & Budtz-Olsen, 1961; Yesberg, Henderson & Budtz-Olsen, 1970). It may be that a concurrent rise in A II, which also occurs during water deprivation (Blair-West, Brook & Simpson, 1972), counteracts the diuretic effect of AVP. To test this hypothesis, dehydrated sheep were treated with the A II blocker, sarcosine-1, alanine-8 A II (saralasin) in order to remove the antagonistic activity of endogenous A II in the hope of exposing the effect of the high endogenous AVP on kidney function. METHODS Eleven Merino ewes of body weight kg (mean + S.E. of mean) were used in a total of fifteen experiments (two experiments were conducted on each of four sheep, allowing a recovery period between experiments of at least 4 weeks, and one experiment was conducted on each of the other seven sheep). Each sheep was dehydrated for 3 d prior to the experimental period. It was then 4 EPH 67

2 98 N. E. YESBERG AND OTHERS supported on a comfortable sling and infused via the left jugular vein with an aqueous solution containing 1-3 g inulin, 0 4 g sodium para-aminohippurate and 0 9 g sodium chloride per 100 ml at a rate of 1 5 ml. min-1 throughout the entire experimental period. After a priming period of 1 h, during which no samples were collected, urine flow (V), sodium and potassium excretion (UNa V and UK V), urine osmolality (Uosm), G.F.R., renal plasma flow (R.P.F.), filtration fraction (F.F.), solute clearance (Cosm) and Tc,H2O were measured each 10 min during a 40 min control period, a 60 min saralasin (Norwich-Eaton Pharmaceuticals) infusion period at either 7 /ug. min-1 (five sheep) or 15 ug. min-' (ten sheep), and a 60 min post-saralasin infusion period. Methods of analysis and calculation were as described previously (Yesberg et al. 1979). Plasma renin activity (P.R.A.) was estimated in one or two blood samples from each period in eight experiments using the NEN Angiotensin I (126I) Radioimmunoassay Kit. In four sheep, arterial blood pressure was measured by direct puncture of the exteriorized carotid artery from which mean arterial blood pressure (M.A.B.P.) and heart rate (H.R.) were recorded by a Devices Instrument. Cardiac output (c.o) was also computed from thermal dilution of 10 ml cool saline delivered via a catheter in the right atrium and detected by a thermistor in the pulmonary artery. The right atrial catheter also allowed measurement ofcentral venous pressure (c.v.p.). Total peripheral resistance (T.P.R.) and renal vascular resistance (R.V.R.) were calculated in peripheral resistance units (p.r.u.) as M.A.B.P. (mmhg) d M.A.B.P. (mmhg) (100-Haematocrit) c.o. (ml. sec-') R.P.F. (ml. sec-') 100 respectively. The effectiveness of an A II blocking action of saralasin at both 7 and 15 /ug. min-1 was tested in preliminary experiments by injecting A II before and after saralasin and observing M.A.B.P. Analysis of results The effect of saralasin at 7,ug. min-' in five sheep was qualitatively and quantitatively similar to the effect of saralasin at 15 jug. min-' in the last ten sheep, so the results for all sheep were combined for analysis. Results for the 40 min control period and the last 40 min of both the saralasin and post-saralasin infusion periods were averaged for each sheep. The changes between one period and the preceding period were statistically assessed by the paired t test, or where indicated by the sign test (S.T.) (Snedecor & Cochran, 1967). Responses in individual sheep were assessed by the simple t test. The table shows the mean+s.e. of the mean of fifteen experiments for each parameter in each infusion period, while the graphs show the mean + S.E. of mean for each of the 10 min clearance periods. Significance of the change between one period and the preceding period is shown as * (P < 0 05), ** (P < 0 01) and *** (P < 0 001). RESULTS Saralasin at both 7 and 15 jug. min-' blocked the arterial pressor responses to injected A II at up to four times the threshold dose of 0.1 jug A II. The pressor response was at least partially restored within 20 min after stopping the saralasin infusion, though in some sheep its effect was more prolonged. The effects of saralasin on blood composition, the circulation and renal function are shown in Table 1 and Figs. 1 and 2. The infusion of saralasin caused small decreases in M.A.B.P. and H.R., a statistically significant fall in c.o. and a rise in c.v.p. Although the fall in M.A.B.P. was not significant for the pooled data of the group of four sheep tested, in three of the four individual experiments the decreases were statistically significant (P < 0 01, 0-01 and 0-001). Similarly the decreases in H.R. which occurred in all four sheep were statistically significant in three cases (P < 0 05, 0 05 and ), and the increases in c.v.p. which occurred in all four sheep were significant in two cases (P < 0 05 and 0 001). T.P.R. tended to rise (in one case significantly, P < 0 01), but R.V.R. fell significantly in three sheep (P < 0-05, 0-05, and 0-001).

3 ANGIOTENSIN BLOCKADE IN DEHYDRATED SHEEP 99 Table 1. The effect of saralasin infusion at 7 or 15,sg. min-' in dehydrated conscious Merino ewes a b c M.A.B.P. (mmhg) H.R. (beats. min-) * c.o. (l.min-') * c.v.p. (mmhg) T.P.R. (p.r.u.) R.V.R. (p.r.u.) P.R.A. (ng.h-' ml-) PN (mol.ml-') PK (umol. ml-,) PO1m (iosm.ml-,) V (ml. min-') ** (S.T.) ** (S.T.) UNa V (mol. min-') * 40+7*** UK V (mol. min') *** 46+5 Uosm (uosm.ml-,) *** G.F.R. (ml.min-') * 64+4* R.P.F. (ml. min-') F.F * * I Cosm (ml.min-') l10*** * TC,H,O (ml.min-') *** 1 17i+01*** Results of fifteen experiments are expressed as the mean + S.E. of mean for a 40 min control period (a), a 60 min saralasin infusion period (b), and a 60 min post-saralasin infusion period (c). Statistical significance of the change between one period and the preceding period was assessed by the paired t test (or sign test (S.T.) where indicated), and shown as * (P < 0 05), ** (P < 0 01) and *** (P < 0 001). P.R.A. tended to rise, but none of the individual changes was statistically significant. Plasma electrolytes and osmolality (PNa, PK and P1sm) were unaffected by the saralasin infusion. The effects of saralasin on kidney function were more clear cut. R.P.F. was unchanged but G.F.R. fell significantly, resulting in a significant fall in F.F., and there were highly significant reductions in Cosm and Tc,H,O. There was a significant fall in V (P < 0.01, S.T.) when pooled data from all animals were considered but in individual experiments the decreases were generally much more significant (P < in seven sheep), although in one case there was an uncharacteristic rise (P < 0-001). There were significant reductions in UNa V and UK V, and a slight fall in Uosm. On withdrawal of the saralasin, most of the above changes were reversed. M.A.B.P. was restored almost to control values and H.R. increased significantly. c.o. continued to fall and c.v.p. and T.P.R. to rise while R.V.R. remained virtually unchanged at its lower value. P.R.A. tended to fall to control values and again there was no change in PNa, PK or Posm. R.P.F. tended to rise, though not significantly, but the increases in G.F.R. and F.F. were statistically significant. Significant increases in Cosm and Tc H2, occurred, also in V, UNa V and Uosm. UK V also rose though the increase was not significant. The decrease in G.F.R. induced by saralasin showed a significant correlation with the decrease in Cosm (r = 0.68, P < 0.01), and again when saralasin was withdrawn the increase in G.F.R. correlated well with the increase in Cosm (r = 0.85, P < 0-001). 4-2

4 100 N. E. YESBERG AND OTHERS 105 M.A.B.P. 1 (mmnhg) [ 9~~ 100 r I HR. [ (beats.min') [ (p.r.u.) g15[ 4 4N%,4.t,, (ng..ml) H -*- (Iosmo. mni ) [ -n - Time (mi) Fig. 1. The effect of saralasin infusion on cardiovascular function and plasma composition showing the mean and S.E. of mean for fifteen sheep. The significance of any difference between the means for each infusion period is shown as * (P < 0 05). DISCUSSION The results indicate that saralasin produced changes in cardiovascular and renal function which were generally reversed when saralasin was withdrawn. Some parameters, however, were not restored to control values within an hour of stopping the saralasin infusion, and it may be that the analogue has a variable rate of removal from the various tissues affected. The dose of saralasin was effective in blocking pressor doses of injected A II, and it is likely that it caused the fall in M.A.B.P. by blocking endogenous A II. The decrease in c.o. and

5 ANGIOTENSIN BLOCKADE IN DEHYDRATED SHEEP (,umol." mi*l UN V (Mmol. min-') S/ 20 L F UK V (ymol min-' L 40L IFW A uosm.-mp') (posm. mll') T T 1500 G.F.R. (ml. min-') R.P.F. (ml. min_1) F.F. 65 K cosm 17 (ml. min-') 1*5 TC, H20 (ml. min-') 0-5 L L ~*** **- - Saralasin III Time (min) Fig. 2. The effect of saralasin infusion on kidney function showing the mean and S.E. of mean for fifteen sheep. The significance of any difference between the means for each infusion period is shown as * (P < 0 05), **(P < 001) and *** (P < 0001). H.R. with a consequent increase in c.v.p., the decrease in R.V.R. and the rise in P.R.A. are all consistent with a reduction in effective endogenous A II activity, although, as indicated in the results, only some of these changes appeared to be statistically significant, namely C.O., M.A.B.P., H.R., R.V.R. and c.v.p. in individual sheep. There was an apparent rise, not statistically significant, in T.P.R. which is not consistent with a fall in effective A II. It may have been due to a sympathetic reflex response to minimize the reduction in M.A.B.P., initiated by the arterial baroreceptors. Similarly, the continued rise in T.P.R. after withdrawal of saralasin may have been due in part to a continued reflex response since M.A.B.P. was not fully restored to control levels, and in part to an increase in A II activity.

6 102 N. E. YESBERG AND OTHERS The effects of saralasin on kidney function are generally explicable in terms of the effects on the renal vasculature. R.P.F. remained fairly constant over-all, presumably because the decrease in M.A.B.P. was matched by a fall in R.V.R. In three sheep R.P.F. was significantly raised despite decreases in c.o. and M.A.B.P., and in two sheep there were significant falls in R.P.F. The qualitative effect of saralasin on R.P.F. therefore seems to depend on the relative changes in M.A.B.P. and R.V.R., with an over-all net result of no significant change in R.P.F. The effect on G.F.R. was more marked and consistent, and the over-all decrease, with consequent rise in F.F., can be explained most simply by a fall in post-glomerular resistance. It has previously been shown that infused A II reduced both R.P.F. and G.F.R., but a significant rise in F.F. indicates relatively more efferent than afferent arteriolar vasoconstriction. It is not surprising that if saralasin has an A II blocking effect it is most apparent at the efferent site, thus causing G.F.R., and F.F. to fall while not affecting total R.P.F. The decrease in G.F.R. can explain other observed renal effects of saralasin, namely the reductions in V, UNa V, UK V and Cosm. The fall in TC,H2 would simply be a consequence of the smaller distal tubular fluid flow, and it is not necessary to postulate changes in the plasma vasopressin level. The reduction in TC,H20 could account for the slight dilution of the urine, if V were reduced relatively less than Cosm. The possibility of saralasin acting on water and salt excretion by inhibiting a tubular effect of A II, or even by its own inherent tubular activity, cannot be excluded. It is not likely that saralasin had a stimulating effect on aldosterone secretion (Beckerhoff, Uhlmschmid, Furrer, Nussberger, Schmied, Vetter & Siegenthaler, 1975), but even if it did have a steroidogenic effect, it seems unlikely that this would have contributed to the reduction in UNa V since this occurred fairly rapidly, and there was a simultaneous decrease in UK V of even greater magnitude. Although the reduction in UK V cannot be explained solely by the reduction in filtered K load, it may be that the small filtered Na load reduced distal tubular Na-K exchange thus reducing the usual amount of K secretion. It has been suggested earlier that the changes induced by saralasin withdrawal might not be as marked and significant as the effects of saralasin itself if there were a variable rate of analogue wash-out from the different tissues. Nevertheless the significant rise in G.F.R. and F.F. towards control levels despite R.V.R. remaining at its lower value is puzzling. The tendency for M.A.B.P. and R.P.F. to rise after saralasin does not explain the significant increase in F.F. More R.V.R. measurements are obviously needed. The failure of UK V to return to its control rate after the saralasin infusion was stopped also invites speculation. It may be that saralasin, rather than being steroidogenic, was in fact anti-steroidogenic (Stephens, Davis, Freeman, Watkins & Khosla, 1977). If this were so it would probably be an hour or so before its effects on electrolyte excretion were observed, that is, an expected reduction in K excretion and an increase in Na excretion which also happens to be enhanced by the rise in G.F.R. While saralasin produced effects which for the most part can be satisfactorily explained, it did not reveal any diuretic and G.F.R.-elevating effect which could be ascribed to the presumably unopposed high levels ofendogenous AVP which could be expected under these experimental conditions. A rise in G.F.R. and Vmight have been prevented if saralasin caused plasma AVP to fall, or because of the fall in M.A.B.P. or interference with the vasopressor activity of the AVP on the efferent arteriole. We are grateful for the generous supply of saralasin by Dr K. 0. Ellis of Norwich-Eaton Pharmaceuticals, Norwich, New York.

7 ANGIOTENSIN BLOCKADE IN DEHYDRATED SHEEP 103 REFERENCES BECKERHOFF, R., UHLSCHMID, G., FURRER, J., NUSSBERGER, J., SCHMIED, U., VETTER, W. J. & SIEGENTHALER, W. (1975). In vivo effects of angiotensin antagonists on plasma aldosterone in the dog. European Journal of Pharmacology 34, BLAIR-WEST, J. R., BROOK, A. H. & SIMPSON, P. A. (1972). Renin responses to water restriction and rehydration. Journal of Physiology 226, MACFARLANE, W. V., MORRIS, R. J. H., HOWARD, B., MCDONALD, J. & BUDTZ-OLSEN, 0. E. (1961). Water and electrolyte changes in tropical Merino sheep exposed to dehydration during summer. Australian Journal of Agricultural Research 12, SNEDECOR, G. W. & COCHRAN, W. G. (1967). Statistical Methods, p Iowa State University Press. STEPHENS, G. A., DAVIS, J. O., FREEMAN, R. H., WATKINS, B. E. & KHOSLA, M. C. (1977). The effects of angiotensin II blockade in conscious sodium-depleted dogs. Endocrinology 101, YESBERG, N. E., HENDERSON, M. & BUDTZ-OLSEN, 0. E. (1970). The excretion of vasopressin by normal and dehydrated sheep. Australian Journal ofexperimental Biology and Medical Science 48, YESBERG, N. E., HENDERSON, M. & BUDTZ-OLSEN, 0. E. (1973). Hydration and vasopressin effects on glomerular filtration rate in sheep. Australian Journal of Experimental Biology and Medical Science 51, YESBERG, N. E., HENDERSON, M. & BUDTZ-OLSEN, 0. E. (1979). Inhibition by angiotensin II of some vasopressin effects on renal function in sheep. Quarterly Journal of Experimental Physiology 64,