Endothelin induces an increase in renal vascular resistance and a fall in glomerular filtration rate in the rabbit isolated perfused kidney

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1 Br. J. Pharmacol. (1989), 98, Endothelin induces an increase in renal vascular resistance and a fall in glomerular filtration rate in the rabbit isolated perfused kidney Hugh S. Cairns, Mary E. Rogerson, Lynette D. Fairbanks, Guy H. Neild & *'John Westwick Dept of Nephrology, Institute of Urology, St. Philip's Hospital, Sheffield St., London WC2A 2EX and *Dept of Pharmacololgy, Royal College of Surgeons, Lincolns Inn Fields, London WC2A 3PN 1 The effects of endothelin infusion on renal vascular resistance (RVR), glomerular filtration rate (GFR) and the interaction with locally generated endothelium-derived relaxant factor (EDRF) were studied in the rabbit isolated perfused kidney (IPK). For comparison the effects of infusions of angiotensin II (All) and noradrenaline (NA) were also assessed. 2 Each kidney was perfused at a constant rate of 1Oml minm and alterations in RVR determined by measuring changes in perfusion pressure. GFR was determined by the clearance of [31Cr]- EDTA, using timed urine collections. 3 Endothelin (1O-1`-1109M) produced a dose-related increase in RVR. Endothelin was approximately 30 times more potent in molar terms than All and 500 times more than NA at inducing a 50 mmhg increase in perfusion pressure. 4 Endothelin appeared to be a weak inducer of EDRF release in the IPK as EDRF inhibitors methylene blue (10uM) or haemoglobin (10yM) only slightly augmented the increase in RVR at a given concentration of endothelin. In contrast the effect of NA on RVR was significantly increased by methylene blue (1OpM) whereas that induced by All was not affected. 5 Endothelin infusion produced a significant, dose-dependent decrease in GFR of the IPK, con- of NA on GFR. This trasting with an increase in GFR during All infusion and a minimal effect supports evidence that All is predominantly a constrictor of efferent glomerular arterioles and that NA constricts both afferent and efferent glomerular vessels. We suggest that the vasoconstrictive effect of endothelin in the kidney is predominantly preglomerular, which explains its effect on GFR. Introduction The importance of the endothelium in the regulation of vascular tone has become increasingly evident in recent years (Vanhoutte, 1988). The endothelium releases a variety of vasoactive factors including the prostaglandins prostacyclin (PGI2) and prostaglandin E2 (PGE2) (Maclntyre et al., 1978; Ali et al., 1980), endothelium-derived relaxant factor (EDRF) (Furchgott & Zawadski, 1980) and the recently described vasoactive peptide endothelin (Yanagisawa et al., 1988). The precise role these factors play in regulating vascular tone in health and disease is, as yet, unclear. Author for correspondence at: Dept. of Pharmacology, Hunterian Institute, Royal College of Surgeons, Lincoln's Inn Fields, London WCA 3PN The 21 amino acid peptide endothelin is a very potent vasoconstrictor in vitro (Yanagisawa et al., 1988); in molar terms it is times more potent than angiotensin II which was previously one of the most potent vasoconstrictor peptides known. Its effect in vivo is less clear as, in some vascular beds, it is a vasoconstrictor whereas, in others, it is a vasodilator (Lippton et al., 1988; Wright & Fozard, 1988). The precise site of origin of endothelin is unclear as the endothelium of the microcirculation seems to be unable to synthesize endothelin (Yanagisawa et al., 1988). However, endothelin is capable of producing an effect distal to, and therefore downstream of, its site of release (Yanagisawa et al., 1988), suggesting that it may have a role in the control of vascular tone within the microcirculation. The Macmillan Press Ltd 1989

2 156 H.S. CAIRNS The role of endothelin in the control of blood flow within individual organs and its interaction with other endothelium-derived vasoactive factors such as EDRF is unknown. We have investigated the effects of endothelin infusion on renal vascular resistance and glomerular filtration rate (GFR) in a rabbit isolated perfused kidney model (IPK), and compared it with the known vasoconstrictors angiotensin II (All) and noradrenaline (NA). We have also studied the interaction between these vasoactive factors and EDRF generated within the kidney. Methods Isolated perfused kidney Each rabbit was anaesthetized with pentobarbitone (Rhone Poulenc) 40-60mgkg-' i.v. and ventilated with air following surgical tracheal intubation. Frusemide (4 mg), which exerts a protective effect against hypoxic damage to the kidney (Myers & Moran, 1986), was administered intravenously at induction of anaesthesia. Laparotomy via a midline incision was performed and the left kidney exposed. The left renal artery was cannulated directly with polythene tubing (Portex 800/100/260/100), such that the average warm ischaemic time was 30-60s. The kidney was initially perfused in situ at 40C with 100ml of KHB/AA which contained indomethacin 5.6 gm. Cold perfusate was used as we found that cooling the kidney prior to handling and removal improved function of the IPK. The left ureter was cannulated with polythene tubing (Portex 800/100/ 340/100) and the kidney was then removed and placed in an organ chamber where it was perfused with KHB/AA (containing indomethacin 5.6 pm) warmed to 37 C and gassed with 95% 02/5% CO2. Venous effluent was discarded, i.e. a non return system was used. Each kidney was perfused at a fixed rate of 10ml minm throughout each experiment and the perfusion pressure at the renal artery was monitored continuously via a Statham pressure transducer. Drugs were infused at 0.12 ml min into the circuit prior to the main perfusion pump. Changes in perfusion pressure were measured as the maximal increase in pressure (mmhg) during infusion of each agent and results expressed as mean + s.e.mean. In some kidneys a vascular tone was induced with a continuous infusion of noradrenaline 150 nm (Edwards, 1983) and the perfusion rate adjusted to produce a perfusion pressure of 90 mmhg. The responses to infusion of endothelin (range M) were assessed. Measurement of renalfunction in IPK GFR of each isolated kidney was determined by clearance of [5"Cr]-EDTA. [51Cr]-EDTA (0.5 MBq) was added to the KHB/AA and urine was collected over 5 min intervals, volume being measured gravimetrically. Urine and perfusate counts were determined and GFR calculated (Ross et al., 1973). Each agent was infused until a steady perfusion pressure had been achieved for S min and then GFR was measured for a further 5 min. After each agent was stopped baseline perfusion pressure was reached and after a further 5 min GFR was again measured. Results are calculated as percentage change in GFR during infusion of each agent (GFR during infusion compared with the mean of GFR pre and post infusion) and expressed as meaft + s.e.mean. Materials The isolated perfused kidney (IPK) was perfused with Krebs-Henseleit buffer supplemented with 20 essential and non-essential amino acids (KHB/AA) (Epstein et al., 1982). Albumin or other oncotic agents were not added to the perfusate as a non return system was employed; large quantities of perfusate were used in each experiment. Hypo-osmolar perfusate does not significantly affect vascular responses of GFR in the IPK, osmolality of the perfusate being more important for tubular function (Ratcliffe et al., 1986). The perfusate had the following final electrolyte concentrations (mmol -'): NaCl 118, KCl 4.75, MgSO4 1.19, NaHCO3 25, K2HPO3 1.19, CaCl2 1.9, glucose The amino acids were added by using 100ml of Vamin 14 Electrolyte Free solution (Kabi-Vitrium) per 101 of final solution (KHB/AA); this produced the required final concentrations (Epstein et al., 1982). Indomethacin (Sigma) was dissolved in absolute alcohol to a concentration of 2mg ml-1 and then added to KHB/AA to a final concentration of 5.6 ym (2 ug ml- 1) which is sufficient to inhibit completely cyclo-oxygenase activity (Ferreira et al., 1971). Buffer was then filtered through a 0.45 pm filter (Millipore, UK). Porcine endothelin (Peptide Institute, Osaka) was dissolved in 0.9% saline to a final concentration of 5OgM and stored in aliquots at - 20 C. For each experiment an aliquot was diluted in 0.9% saline to appropriate concentrations, samples being stored on ice before use. Methylene blue (Sigma) was dissolved in 0.9% saline for each experiment. Haemoglobin (Sigma) was dissolved in distilled water to a concentration of 1 mm, converted to the ferrous form by addition of sodium dithionite 10 mm and then dialysed twice against 100 volumes of distilled water for 2 h to remove the sodium dithionite (Martin et al., 1985). The solution was then stored at - 20 C for up

3 ENDOTHELIN, VASCULAR RESISTANCE AND GFR IN RABBIT IPK 157 to 2 weeks before use, further dilutions were made in 0.9% saline. All (Sigma) was dissolved in ethanol to a concentration of 1 mm and stored at - 20'C, further dilutions were in 0.9% saline. NA (Sigma) was dissolved in 0.9% saline to appropriate concentrations on the day of each experiment. All concentrations refer to final concentration in the KHB/AA in the IPK. Statistics Multifactor analysis of variance with a least squares difference test was used to compare responses with and without the EDRF inhibitors methylene blue and haemoglobin. P < 0.05 was accepted as indicating statistical significance. X lo10-l x 9 1 X 10-8 Constrictor dose (mol I-1) Figure 1 Maximum change in perfusion pressure of the rabbit isolated perfused kidney induced by the constrictor agents (El) endothelin (n = 6), (A) angiotensin I1 (n = 8) and (0) noradrenaline (n = 8). Each point represents the mean and vertical lines show s.e.mean. Results Basal perfusion pressure, prior to infusion of vasoactive agents, was mmhg (mean + s.e.mean). Endothelin was a potent vasoconstrictor (Figure 1), producing a detectable increase in perfusion pressure at a concentration of 10"M (range to 10-9M). In comparison AII and NA, given alone, produced similar vasoconstrictor responses in the concentration ranges 5-50 x 1O-9M and x 10-9M respectively (Figure 1). The calculated dose of endothelin required to produce a 50mmHg increase in perfusion pressure was 3.4 x 10-1OM whereas those for All and NA were 9.8 x 10-9M and 170 x 1O-9M, respectively. The duration of effect of equipotent doses of endothelin, All and NA was similar. Simultaneous infusion of the EDRF inhibitor methylene blue 1OpM did not affect the constrictor response to endothelin (Figure la), although haemoglobin 10pM did significantly increase the constrictor response to endothelin (P < 0.01) (Figure 2a). In our model, methylene blue 10 M significantly increased the constrictor response to NA (P < 0.01) but not to All (Figure 2c,b). Figure 2 a 150,T 0) W.' E a) E 100± CO" W'- Coa 0. E c X.2 Itn 0) a Endothelin (nmol 1-') b T I g I T I Angiotensin 11 (nmol l-1) Noradrenaline (nmol 1-') Maximum change in perfusion pressure of the rabbit isolated perfused kidney induced by the constrictor agents in the absence and presence of inhibitors of EDRF. (a) (C1) Endothelin alone (n = 6), (M...U ) endothelin + methylene blue (1OpM) (n = 6), (N-- --) endothelin + haemoglobin (1OyM) (n = 5) (P < 0.01 vs endothelin alone). (b) (A) Angiotensin II alone (n = 8), (A) angiotensin II + methylene blue (1OpM) (n = 6) (c) (0) noradrenaline alone (n = 8), (0) noradrenaline + methylene blue (1OpM) (n = 6) (P < 0.01 vs noradrenaline alone). Each point represents the mean and vertical lines show s.e.mean.

4 158 H.S. CAIRNS U 60 T A 0 l X 10-I0I X 19 T 1.0 X Co CC Dose of constrictor (mol 1-1) Figure 3 Effect of the constrictor agents endothelin (El) (n = 6), angiotensin II1(A) (n = 8) and noradrenaline (0) (n = 8) on the glomerular filtration rate (GFR) of the rabbit isolated perfused kidney. Each point represents the mean and vertical lines show s.e.mean. B 70 I c E 60 E 50 Co (L -X - 40 a) x.0 10 z O 0/~~ 0/ Endothelin dose (mol l-1) Figure 5 Effect of endothelin on perfusion pressure of rabbit isolated perfused kidneys preconstricted with noradrenaline (150nM). Each point represents the mean (n = 6) and vertical lines show s.e.mean. T I Endothelin infusion produced a marked and dosedependent decrease in GFR of the IPK; 10- M induced a 52% fall in GFR (Figure 3). Higher doses of endothelin (e.g. 10-8M) produced a virtual cessa- *tion of urinary flow associated with a marked and sustained increase in the perfusion pressure, with no recovery of urine flow during a subsequent hour. NA altered GFR only at higher doses, inducing a slight fall, whereas All, in contrast, produced a marked dose-dependent increase in GFR (Figure 3). Simultaneous infusion of the EDRF inhibitors methylene blue and haemoglobin did not affect the changes in GFR that occurred with infusion of endothelin (Figure 4). When vascular tone was induced in the IPK by a continuous infusion of NA 150nm, the vasoconstric- u.20-2 Endothelin dose (mol 1-') 1 X l l 10-9 U 'o-so C.- A Figure 4 Effect of the EDRF inhibitors methylene blue and haemoglobin on the change in glomerular filtration rate (GFR) induced by different doses of endothelin. (l ) Endothelin alone (n = 6), (O.-) endothelin + methylene blue (10.uM) (n = 6), (O-- -U) endothelin + haemoglobin (10pM) (n = 5). Each point represents the mean and vertical lines show s.e.mean. tive effect of endothelin was detectable at concentrations as low as M; lower concentrations of endothelin, down to M, resulted in neither vasoconstriction nor vasodilatation (Figure 5). Discussion Our results indicate that endothelin is a very potent vasoconstrictor in the rabbit IPK, being approximately 30 times more potent in molar terms than AII and 500 times more potent than NA. Experiments in vivo suggest that endothelin may be a vasodilator in some vascular beds, including, at least initially, the kidney (Lippton et al., 1988; Wright & Fozard, 1988). We detected no evidence of vasodilatation due to endothelin in the IPK. Isolated organs, including the kidney, have a very poor vascular tone when a cell-free perfusate is employed and, in such a model, any vasodilator response may not be detected. However, when a vascular tone was induced in the IPK with NA, which constricts both pre- and post-glomerular vessels (Edwards, 1983), endothelin, at concentrations as low as 10-15_ 10-13M, failed to produce a vasodilator response. This suggests that, at least in the rabbit IPK, vasodilatation is not an effect of endothelin. The short lived vasodilatation to endothelin that occurs in vivo may reflect a reflex response of the kidney which is lost in the IPK model. Although endothelin in vivo has a prolonged duration of action, in the IPK we did not find that its effect was any more longer lasting than either AII or NA. This may merely reflect a washout effect in a non return perfusion system in vitro. However, the plasma half-life of endothelin in vivo is probably very short as approximately 60% is removed by the lungs

5 ENDOTHELIN, VASCULAR RESISTANCE AND GFR IN RABBIT IPK 159 during a single pass (de Nucci et al., 1988). Therefore the effect in vivo may be the result of an interaction of endothelin with some blood-derived or neural component, both of which are absent in the IPK. It is unlikely that the prolonged effect in vivo is due to a sustained transduction mechanism in vascular smooth muscle as this would presumably also be seen in the IPK. Endothelin also produced a substantial fall in GFR in the IPK, in contrast with the increase in GFR that occurred with equipotent concentrations of All. This effect of endothelin is in agreement with recent evidence in a rat IPK model where endothelin was a renal vasoconstrictor and lead to a substantial fall in GFR (Firth et al., 1988). All is both a contractor of rat mesangial cells (Ausiello et al., 1980) and a constrictor predominantly of the efferent glomerular arteriole in the rabbit (Edwards, 1983) and the rat (Myers et al., 1975). These two actions of All have opposing effects on the GFR; mesangial cell contraction leads to a fall in the filtration coefficient (Kf) and therefore GFR, whereas increased efferent arteriolar tone increases the transglomerular basement membrane pressure (AP) and therefore GFR. As GFR increased with All infusion, the effect of increased AP must outwiegh the fall in Kf. NA increases symmetrically the tone in both pre and post glomerular arterioles (Edwards, 1983), which should maintain AP at a relatively constant level. The minor fall in GFR of the IPK that occurred only with higher concentrations of NA supports this. As the GFR fell substantially with endothelin infusion, this suggests that endothelin is predominantly a constrictor of preglomerular vessels, resulting in a fall in AP and therefore in GFR. This is particularly true in our IPK model where the perfusate flow rate is fixed and therefore unable to influence GFR. Recent evidence in cultured mesangial cells of the rat and an in vivo rat model suggests that endothelin both contracts mesangial cells and in vivo produces a substantial fall in Kf (Badr et al., 1989). This is somewhat surprising since All, as already stated, is a potent contractor of mesangial cells and yet does not References ALI, A.E., BARRETT, J.C. & ELING, T.E. (1980). Prostaglandin and thromboxane production by fibroblasts and vascular endothelial cells. Prostaglandins, 20, AUSIELLO, D.A., KREISBERG, J.I., ROY, C. & KARNOVSKY, M.J. (1980). Contraction of cultured rat glomerular cells of apparent mesangial origin after stimulation with angiotensin II and arginine vasopressin. J. Clin. Invest., 65, BADR, K.F., MURRAY, J.L., BREYER, M.D., TAKAHASHI, K., INAGAMI, T. & HARRIS, R.C. (1989). Mesangial cells, glomerular and renal vascular responses to endothelin in the rat kidney. J. Clin. Invest., 83, produce a fall in GFR, presumably indicating that Kf does not fall substantially. This suggests that the effect of endothelin in reducing Kf may in some way be separate from its ability to contract mesangial cells. Alternatively, endothelin may be stimulating intrarenal All generation, thereby increasing efferent arteriolar resistance and contracting mesangial cells, possibly in synergy with endothelin. Although endothelin inhibits activation of the renin angiotensin system (RAS) by rat isolated cortical cells (Takagi et al., 1988) and isolated glomeruli (Rakugi et al., 1988), it activates the RAS in vivo, producing an increase in plasma renin activity (Miller et al., 1989). The effect of angiotensin converting enzyme inhibitors and angiotensin II antagonists on the response to endothelin both in the IPK and in vivo would therefore be of particular interest. EDRF is involved in the control of renal vascular resistance in the IPK (Bhardwaj & Moore, 1988) and in vivo (Conger et al., 1988), although the distribution of its effect and its site of origin is unclear. The relative inability of the EDRF inhibitors methylene blue and haemoglobin to augment the vasoconstrictor response to endothelin suggest that endothelin, like All and unlike NA, does not, to any great extent, induce release of EDRF. In the rat mesenteric vascular bed endothelin induces the release of EDRF (de Nucci et al., 1988). The interaction between these two potent endothelium-derived vasoactive factors is obviously important and may well vary between vessel beds and species. In summary, endothelin, in the rabbit IPK, is a potent vasoconstrictor, probably predominantly of preglomerular arterioles, and produces a significant fall in GFR. As such it may play a causal role in the pathogenesis of a variety of conditions where reductions in renal blood flow and GFR occur together, for example acute renal failure (Firth et al., 1988) and cyclosporin nephrotoxicity (Thiel, 1986). 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