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Journal of Physiology (1991), 436, pp. 421-429 Wlith 2 fign re.s Prrinted in Great Britain

REGIONAL HETEROGENEITY OF ENDOTHELIUM-DEPENDENT VASODILATATION IN THE RABBIT KIDNEY

BY HUGH S. CAIRNS, MARY E. ROGERSON, JOHN WESTWICK* AND GUY H. NEILD From the Department of Renal Medicine, Institute of Urology, University College and Middlesex School of Medicine, University College London and the *Department of Pharmacology, Royal College of Surgeons, Lincoln's Inn Fields, London WC2A 3PN

(Received 11 July 1990) SUMMARY

1. Regional heterogeneity of endothelial function exists but its role in the local regulation of vascular tone is uncertain. This heterogeneity may be very important in the control of the glomerular filtration rate (GFR) in which the differential tone in the afferent and efferent arterioles is crucial. 2. When an endothelium-independent vasodilator, prostacyclin (PGI2) or nitroprusside, was infused into anaesthetized rabbits there were dose-dependent falls in both mean arterial pressure (MAP) and GFR; PGI2 (04 nmol kg-1 min-') altered MAP and GFR by -18 5 + 3-6 % (mean + S.E.M.) and -37-7 + 13-3 % respectively and nitroprusside (30 nmol kg-' min-') by -29 7+3 1% and -67-0+2-4%. In contrast infusion of an endothelium-dependent vasodilator, acetylcholine (ACh) or substance P, produced dose-dependent decreases in MAP but dose-dependent increases in GFR; ACh (10 nmol kg-1 min-) - 15 1 + 2 0 % and + 43 8 + 16-5 % and substance P (30 nmol kg-' min-') - 187+ 19% and +45-3+231% respectively. The effects of endothelium-dependent and independent vasodilators on GFR was significantly different (p < 0 005). 3. Simultaneous administration of indomethacin, Methylene Blue or NGmonomethyl-L-arginine (L-NMMA), inhibitors of cyclo-oxygenase and endotheliumderived relaxing factor (EDRF) respectively, attenuated or reversed the effect of ACh (10 nmol kg-' min-') on MAP and GFR. 4. These data suggest that endothelium-dependent vasodilatation in the kidney has a heterogeneous effect on the renal microvasculature, exerting a preferential effect on afferent glomerular arterioles and thereby preserving GFR despite the fall in MAP. If correct, this has important implications for the regulation of GFR. INTRODUCTION

The endothelium has a major influence on local vascular tone, both via the release of vasoactive factors and the metabolism of other, non-endothelial factors such as angiotensin. The major vasodilators produced by endothelial cells are endotheliumderived relaxing factor (EDRF; Furchgott & Zawadski, 1980; Griffith, Edwards, M,S S644

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Lewis, Newby & Henderson, 1984) and the prostaglandins PGI2 and PGE2 (Maclntyre, Pearson & Gordon, 1978; Gerritsen & Cheli, 1983). EDRF is probably nitric oxide (NO, Palmer, Ferrige & Moncada, 1987) derived from the amino acid Larginine (Schmidt, Klein, Niroomand & Bohme, 1988; Palmer, Ashton & Moncada, 1988); it activates soluble guanylate cyclase in vascular smooth muscle leading to cyclic GMP generation (Ignarro, Harbison, Wood & Kadowitz, 1986). The renal microcirculation releases EDRF in response to pharmacological stimuli, with a subsequent fall in renal vascular resistance (Bhardwaj & Moore, 1988). The prostaglandins PGJ2 and PGE2, in contrast, stimulate smooth muscle adenyl cyclase and cyclic AMP production (Kuehl, 1974). The renal sites of action, however, of both EDRF and endothelium-derived prostaglandins are unknown. The stimuli for their release are varied: rheological factors are important (Griffith, Edwards, Davies, Harrison & Evans, 1987) and many vasodilators such as acetylcholine, bradykinin, histamine and substance P are endothelium dependent and act by stimulating the release of EDRF and prostaglandins (Griffith et al. 1984). Heterogeneity of a number of endothelial cell functions has been demonstrated in different regions of the circulation. This regional heterogeneity includes different prostaglandin profiles (Gerritsen & Printz, 1981; Gerritsen & Cheli, 1983), thrombininduced generation of endothelial prostaglandins (Goldsmith & Kisker, 1982) and angiotensin converting enzyme activity (Caldwell, Seegal, Hsu, Das & Soffer, 1976). The functional relevance of this regional heterogeneity is still uncertain. The control of renal vascular tone is complex. Systemic influences, which are predominantly vasoconstrictor, include neural control (Kon & Ichikawa, 1983), circulating catecholamines and the renin-angiotensin system. Local factors may be either vasodilator, or vasoconstrictor such as endothelin (Yanagisawa, Kurihara, Kimura, Tomobe, Kobayashi, Mitsui, Yazaki, Goto & Masaki, 1988), angiotensin II and thromboxane. Although normally no single component is crucial to the autoregulation of GFR, individual, local factors can play a vital role in certain pathological conditions (Walshe & Venuto, 1979; Hricik, Browning, Kopelman, Goorno, Madias & Dzau, 1983; Curtis, Luke, Whelchel, Diethelm, Jones & Dustan, 1983). During autoregulation, the GFR is preserved, at least in part, by alterations in renal haemodynamics which maintain the transglomerular hydraulic pressure gradient (AP). Increases in tone of the efferent glomerular arteriole can preserve AP in response to a fall in systemic blood pressure; this probably occurs principally by angiotension 11-mediated constriction (Myers, Deen & Brenner, 1975; Edwards, 1983). Alternatively AP may be maintained by a selective reduction in afferent arteriolar tone. This paper addresses the hypothesis that regional heterogeneity exists within the kidney such that endothelium-dependent vasodilatation acts preferentially on afferent glomerular vessels. We have studied the effects of two known stimulators of EDRF and prostaglandin release, acetylcholine (ACh) and substance P, on renal function in the rabbit in vivo, comparing them with two vasodilators, PGI2 and nitroprusside, which act directly on vascular smooth muscle and which stimulate cyclic AMP and cyclic GMP generation respectively.

ENDOTHELIUM-DEPENDENT VASODILATATION

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METHODS

Acetylcholine and nitroprusside (Sigma) were dissolved in 0-9 % saline on the day of each experiment and stored at 4 'C prior to use. Light exposure of the nitroprusside-containing solutions was kept to a minimum. Substance P (Sigma) was stored in 0 9 % saline, 0 5 mg ml-', at -20 °C, aliquots being further diluted on the day of each experiment. Prostacyclin, a gift from Wellcome Laboratories, was dissolved in 500 mM-Tris pH 10-5 at 1 mg ml-' and then diluted to 0-04 mg ml-' in 10 mM-Tris pH 10-5. Further dilutions in 0-9% saline were made immediately prior to use. In order to assess the role of vasoactive eicosanoids, some animals received indomethacin (Sigma) which was dissolved in absolute ethanol, 10 mg ml-'. Indomethacin, further diluted in 0-9 % saline, was administered as a bolus of 2 mg kg-' i.v. at the start of the experiment and then 1 mg kg-' repeated every hour; this dose abolishes almost completely the renal generation of prostaglandins (Flower, 1974). Two EDRF inhibitors were also used: Methylene Blue which inhibits the action of guanylate cyclase (Ignarro et al. 1986) and NG-monomethyl-L-arginine (L-NMMA) which prevents the formation of nitric oxide from L-arginine (Palmer & Moncada, 1989). These agents were dissolved in 0 9 % saline and infused at rates of 0-8 mg kg-' min-' (Hogan, Lewis & Henderson, 1988) and 10 mg kg-' h-' (Rees, Palmer & Moncada, 1989) respectively, both at 0-3 ml min-'. New Zealand White (NZW) rabbits, 2-53-0 kg in weight, were anaesthetized with pentobarbitone, 15 mg kg-', and althesin, 2 mg kg-', and then anaesthesia was maintained with a constant infusion of althesin, 0-2 mg kg-' min-'. Prior to anaesthesia animals were volume expanded with 50 ml 0-9 % saline and given frusemide, 2 mg i.v., to increase urine flow. Animals were ventilated with air and oxygen via a direct surgical tracheostomy. Systemic arterial pressure (MAP) was measured via a cannula in the left carotid. A cannula was introduced into the aorta via the right femoral artery, the tip being one centimetre above the origin of the renal arteries; this was used for administration of vasodilators and cyclo-oxygenase and EDRF inhibitors. An electromagnetic flow probe (Gould Inc., Oxnard, CA, USA) was placed around the left renal artery to record renal blood flow (RBF). The right and left ureters were cannulated. A loading dose of 0-25 MBq "Cr-labelled EDTA was given at the start of the operation and then a further 0-25 MBq administered as a constant infusion over the period of the study. Timed urine collections were performed, volume being measured gravimetrically, and urine and plasma counts used to calculate GFR. After a 45 min recovery period, baseline measurements were performed. Each vasodilator was then infused at a rate of 0 3 ml min-' via the aortic cannula. After 10 min, when a stable systemic pressure has been achieved, measurements were made over a 10 min period and the infusion then stopped. Baseline measurements were then repeated 15 min later. This procedure was then repeated for the different vasodilators at varying doses. Each animal was studied for between 3 and 4 h. Infusion of 0-9 % saline alone at the same rate did not alter any of the baseline parameters. GFR of the left kidney was approximately 60 % of the right kidney although changes in response to infusion of vasodilators were parallel. GFR results shown are those of the right kidney. Results are expressed as percentage change from baseline measurements. Stati8tic. Multifactor analysis of variance was used to compare MAP, RBF and GFR responses between vasodilator agent and the Mann-Whitney U test to compare unpaired data with the inhibitors of cyclo-oxygenase and EDRF. P < 0-05 was accepted as indicating statistical

significance. RESULTS

Baseline MAP (diastolic blood pressure (BP) + 3 pulse pressure) was 75-2 + 2-7 mmHg (mean + S.E.M.; n = 37 animals). Baseline GFR of the right kidney was 5-54+0-26 ml min-' and baseline RBF was 21-4+ 1-6 ml min-'. Each of the vasodilators produced similar, dose-dependent falls in mean arterial pressure (MAP, Fig. 1 A). The effect on renal blood flow was more variable; nitroprusside and PGI2 both produced falls in RBF whereas RBF increased with ACh and did not change with substance P except at the highest dose employed (Fig. I B). There was a significant difference in the changes in GFR between

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endothelium-dependent and independent vasodilators; ACh and substance P both produced dose-dependent increases in GFR while nitroprusside and PGI2 produced dose-dependent falls in GFR (Fig. 1 C, ACh vs. PGI2 and nitroprusside P < 0 001 and P < 0-001 respectively, substance P vs. PGI2 and nitroprusside P < 0 005 and P < 0 005 respectively). A

Dose (nmol kg-1 min-1) 1.0 100

001

0.1

100.0

-30.rT A

-10 -T A

-20A -30

~~MAP

-40 B 50 )

o-o Acetycholine A-A& Substance P *-* Nitroprusside

*A-A Prostacyclin \iu

c

TT

A+ AA

P -50