Endothelin receptor A blockade reduces proteinuria ... - Clinical Science

2 downloads 0 Views 91KB Size Report
diet in a blood-pressure-independent manner. Jo$rg TRENKNER*†, Friedrich PRIEM‡, Christian BAUER†,. Hans-Hellmut NEUMAYER*, Manfred RASCHAK§ ...
Clinical Science (2002) 103 (Suppl. 48), 385S–388S (Printed in Great Britain)

Endothelin receptor A blockade reduces proteinuria and vascular hypertrophy in spontaneously hypertensive rats on high-salt diet in a blood-pressure-independent manner Jo$ rg TRENKNER*†, Friedrich PRIEM‡, Christian BAUER†, Hans-Hellmut NEUMAYER*, Manfred RASCHAK§ and Berthold HOCHER*† *Department of Nephrology, Charite! , Humboldt University of Berlin, Schumannstrasse 20-21, 10098 Berlin, Germany, †Institute of Molecular Biology and Biochemistry, Free University of Berlin, D-14195 Berlin, Germany, ‡Department of Pathobiochemistry, Charite! , Humboldt University of Berlin, D-10117 Berlin, Germany, and §Knoll AG, D-67008 Ludwigshafen, Germany

A

B

S

T

R

A

C

T

The renal endothelin (ET) system is involved in the pathogenesis of kidney fibrosis as well as blood pressure control by regulating tubular sodium excretion. Long-term effects of ETA receptor blockade on blood pressure and kidney function in spontaneously hypertensive rats (SHRs) on a high-salt diet are unknown. We treated SHRs on a 6 % (w/v) NaCl sodium diet (SHR-S) for 48 weeks with the ETA antagonist LU 135252 (whose selectivity for ETA is 150 times greater than for ETB) with 10, 30 and 100 mg : kg−1 : day−1 or placebo. The ETA antagonist had at no time-point any effect on blood pressure. Glomerular filtration rate was normal in SHR-S and not altered by LU 135252. However, urinary albumin excretion was markedly reduced by the ETA antagonist (SHR-S, 145p50 mg/day; SHR-Sj10 mg : kg−1 : day−1 LU 135252, 33p11 mg/day, P 0.05 versus SHR-S; SHR-Sj30 mg : kg−1 : day−1 LU 135252, 55p16 mg/day and SHR-Sj100 mg : kg−1 : day−1 LU 135252, 32p11 mg/day, P 0.05 versus SHR-S at both concentrations). Total urinary protein excretion was likewise significantly reduced by treatment with 10 mg : kg−1 : day−1 LU 135252 (SHR-S, 0.25p0.06 g/day; SHR-Sj10 mg : kg−1 : day−1 LU 135252, 0.089p0.01 g/day, P 0.05 versus SHR-S). The higher dosages of LU 135252 showed only a trend towards reduction of total urinary protein excretion. Computer-aided image analysis after haematoxylin/eosin and periodic acid-Schiff staining revealed that treatment with 10 mg : kg−1 : day−1 LU 135252 significantly reduces the media/lumen ratio of intrarenal arteries. Higher dosages of LU 135252 were less effective. Renal matrix protein synthesis in SHR-S was not altered by LU 135252. In conclusion, the renal ET system contributes in a blood-pressureindependent manner to the regulation of urinary protein excretion and renal vascular hypertrophy in SHR-S. Lower doses of the ETA antagonist were more effective, indicating that a potential additional blockade of the ETB receptor using higher doses of LU 135252 seems to oppose the beneficial effects of a sole ETA blockade. Urinary protein excretion is an independent risk factor of chronic renal failure, thus ETA antagonists might be a therapeutic tool to prevent proteinuria-induced chronic renal failure.

Key words : blood pressure, ETA antagonist, protein excretion, salt. Abbreviations : ET, endothelin ; ETA, ET receptor A ; ETB, ET receptor B ; SHR, spontaneously hypertensive rat. Correspondence : Dr B. Hocher (e-mail berthold.hocher!charite.de).

# 2002 The Biochemical Society and the Medical Research Society

385S

386S

J. Trenkner and others

INTRODUCTION Studies in experimental models of chronic renal failure suggest an important role of the renal endothelin (ET) system during development and progression of renal disease [1]. ET-1 is a potent vasoconstrictor peptide and can be synthesized by numerous cells, including renal cells. Besides its effects on renal and systemic haemodynamics, ET-1 elicits many of other biological actions [2]. ET-1 induces mitogenesis in vascular smooth muscle cells as well as in mesangial cells [3,4]. ET-1 promotes cell proliferation and accumulation of extracellular matrix proteins [5]. Antagonists for the ET receptor A (ETA) have been shown to be effective in abrogating proteinuria and kidney fibrosis in various models of rats with kidney damage [6]. Proteinuria is one of the major risk factors for disease progression in patients with chronic nephropathies and, moreover, albuminuria is associated with a greater incidence of vascular events in hypertensive patients [7,8]. We therefore analysed the effect of long-term treatment with an ETA receptor antagonist (LU 135252) on protein and albumin excretion, kidney function and morphology, and blood pressure in salt-loaded spontaneously hypertensive rats (SHRs).

METHODS Animals and experimental design SHRs aged 9 weeks received a high-salt diet containing 6 % (w\v) NaCl in drinking water. The animals were divided in four groups : a placebo group and three groups with different doses (10, 30 and 100 mg : kg−" : day−") of the selective ETA receptor antagonist LU 135252. All rats were treated for 48 weeks. Systolic blood pressure was measured monthly by the tail cuff method. Body weights were measured monthly. Glomerular filtration rate was determined in week 44 by the endogenous creatinine clearance.

haematoxylin\eosin, Sirius Red and periodic acid–Schiff (PAS) staining and analysed as described previously [10]. The media : lumen ratio of intrarenal arteries were analysed using a video microscope connected to a personal computer. The data obtained were analysed using the Image 1.61 program. Glomerulosclerosis was defined by the presence of PAS-positive material within the glomeruli [10].

Data analysis Differences between groups of data obtained at the same time point were compared by analysis of variance using the SPSS software. For serial measurements of blood pressure, analysis of variance for repetitive measurements was used. The non-parametric Kruskal–Wallis test for multiple comparison was used for analysis of statistical differences after scoring glomerulosclerosis. Results were considered significantly different at P 0.05.

RESULTS AND DISCUSSION The rats tolerated treatment with LU 135252 very well. No side effects (reduced growth, elevated liver enzyme levels etc.) were observed. The ETA antagonist had no effect on blood pressure and glomerular filtration rate (Figure 1 and Table 1). Body weight as well as heart and kidney weight were not altered after long-term treatment with LU 135252. The ETA antagonist had also no effect on glomerular matrix deposition (Table 1). The lowest dose of LU 135252 reduced significantly the media : lumen ratio of intrarenal arteries (Table 1). Long-term treatment with all dosages of LU 135252 reduced the daily excretion of albumin (Figure 2a). Total urinary protein excretion, on the other hand, was only

Urine measurements Protein concentration in urine was measured with a Pyrrogal Red\molybdate complex reagent (Analytic testkit) in a Hitachi 717 automated analyser [9]. Albumin concentration in urine was measured using a nephelometric analyser (Behring). We used an anti-rat albumin antibody from goat antiserum; rat albumin was dissolved in 0.9 % (w\v) NaCl and used for preparation of the albumin calibration curve.

Histological evaluation For pathohistological evaluation, all samples were embedded in paraffin, cut in 3 µm sections and submitted to # 2002 The Biochemical Society and the Medical Research Society

Figure 1

Systolic blood pressure in SHRs on high salt-diet

Rats were treated with placebo ( ), 10 mg : kg−1 : day−1 LU 135252 (#), 30 mg : kg−1 : day−1 LU 135252 (>) or 100 mg : kg−1 : day−1 LU 135252 ($). N l 6 in each group. Data points are mean values and the error bars represent the S.D.

ETA blockade and urinary protein excretion in rats

Table 1 Heart and kidney weight, glomerular filtration rate, glomerulosclerosis score and media : lumen ratio of intrarenal arteries in SHRs on a high-salt diet

Rats received placebo or different dosages of LU 135252 (10, 30 and 100 mg : kg−1 : day−1). N l 6 in each group. Results are given as meanspS.D. FP compared with placebo-treated SHRs on a high salt diet. LU, LU 135252; GFR, glomerular filtration rate.

Number of rats Left kidney weight (g) Left kidney/body weight (%) Right kidney weight (g) Right kidney/body weight (%) GFR ( µl : min−1 : 100 g−1 of body weight) Glomerulosclerosis score Media : lumen ratio of intrarenal arteries Heart weight (g) Heart/body weight (%)

0.05

SHRj6 % NaCl

SHRjLU 10 mgj6 % NaCl

SHRjLU 30 mgj6 % NaCl

SHRjLU 100 mgj6 % NaCl

6 1.43p0.02 0.40p0.01 1.40p0.06 0.39p0.01 400p88 3.42p0.26 8.45p3.91 1.48p0.16 0.42p0.04

6 1.52p0.04 0.39p0.02 1.45p0.08 0.37p0.03 400p101 3.52p0.35 4.59p1.13F 1.69p0.17 0.44p0.02

6 1.62p0.09 0.42p0.01 1.52p0.08 0.39p0.01 438p43 3.4p0.24 7.94p4.43 1.58p0.14 0.41p0.02

6 1.44p0.06 0.35p0.01 1.43p0.03 0.35p0 347p92 3.6p0.27 9.11p7.75 1.61p0.06 0.4p0.01

(a)

(b)

Figure 2 Effects of LU 135252 on urinary albumin and total protein excretion

Urinary albumin (a) and total protein (b) excretion were measured after 44 weeks of treatment with 10, 30 and 100 mg : kg−1 : day−1 of LU 135252. FP 0.05 compared with placebo treated SHRs on a high-salt diet. N l 6 in each group. Values are means and the error bars represent the S.D. reduced significantly by treatment with the lowest dose (10 mg : kg−" : day−") of LU 135252 (0.089p0.01 g\day versus 0.25p0.06 g\day for untreated SHR-S; P 0.05). The higher dosages of LU 135252 (30 mg : kg−" : day−" and 100 mg : kg−" : day−") showed only a trend towards reduction of total urinary protein excretion (Figure 2b).

Taken together, the lowest dose of LU 135252 was most effective with respect to reducing albumin excretion, protein excretion and the media : lumen ratio of intrarenal arteries in SHR on high-salt diet. Because the receptor selectivity of LU 135252 is about 150 : 1 (ETA : ETB), it seems possible that the higher dosages of the ETA antagonist resulted in a potential additional blockade of the ETB receptor. An additional blockade of the endothelial ETB receptors could slow the generation of endothelial-derived nitric oxide or prostacyclin, which in turn counteracts the effect of an ETA receptor blockade by LU 135235 [11]. The effects of LU 135252 on albumin and protein excretion are obviously blood-pressure-independent and the drug had no influence on systolic blood pressure (Figure 1). However, an effect of LU 135252 on intraglomerular pressure could not be excluded by our study design. It appears that chronic ET antagonism is only effective in lowering blood pressure in those models of hypertensive renal damage characterized by a vascular overexpression of ET-1 is found (such as deoxycorticosterone-salt-treated rats and deoxycorticosterone-salttreated SHRs) [12]. The blood pressure effect of LU 135252 on albumin and total protein excretion might be important clinically, since it was shown that proteinuria is an independent risk factors for disease progression in patients with chronic nephropathies and moreover albuminuria is associated with a greater incidence of vascular events in hypertensive patients [7,8].

ACKNOWLEDGMENTS We thank Ines Mu$ ller and Christine Lehmann for their technical assistance. This study was supported by the Deutsche Forschungsgemeinschaft (Ho 1665\2-2). # 2002 The Biochemical Society and the Medical Research Society

387S

388S

J. Trenkner and others

REFERENCES 1

Kedzierski, R. M. and Yanagisawa, M. (2001) Endothelin system : the double-edged sword in health and disease. Annu. Rev. Pharmacol. Toxicol. 41, 851–876 2 Hocher, B., Thone-Reineke, C., Bauer, C., Raschack, M. and Neumayer, H. H. (1997) The paracrine endothelin system : pathophysiology and implications in clinical medicine. Eur. J. Clin. Chem. Clin. Biochem. 35 (3), 175–189 3 Hocher, B. and Paul, M. (2000) Transgenic animal models for the analysis of the renal endothelin system. Nephrol. Dial. Transplant. 15 (7), 935–937 4 Ruiz-Ortega, M., Gomez-Garre, D., Alcazar, R. et al. (1994) Involvement of angiotensin II and endothelin in matrix protein production and renal sclerosis. J. Hypertens. 12 (Suppl. 4), S51–S58 5 Eddy, A. (1996) Molecular insights into renal interstitial fibrosis. J. Am. Soc. Nephrol. 7 (12), 2495–2508 6 Bruzzi, I., Remuzzi, G. and Benigni, A. (1997) Endothelin : a mediator of renal disease progression. J. Nephrol. 10 (4), 179–183

# 2002 The Biochemical Society and the Medical Research Society

7

Remuzzi, G., Perna, A. and Benigni, A. (1996) Proteins abnormally filtered throughout glomerular capillary have an intrinsic renal toxicity. Contrib. Nephrol. 118, 164–172 8 Remuzzi, G. (2000) A unifying hypothesis for renal scarring linking protein trafficking to the different mediators of injury. Nephrol. Dial. Transplant. 15 (Suppl. 6), 58–60 9 Watanabe, N., Kamei, A., Ohkubo, M. et al. (1986) Urinary protein as measured with a pyrogallol redmolybdate complex manually and in a Hitachi 726 automated analyzer. Clin. Chem. 32, 1551–1554 10 Hocher, B., Thone-Reineke, C., Rohmeiss, P. et al. (1997) Endothelin-1 transgenic mice develop glomerulosclerosis, interstitial fibrosis, and renal cysts but not hypertension. J. Clin. Invest. 99, 1380–1389 11 Clozel, M. and Breu, V. (1996) The role of ETB receptors in normotensive and hypertensive rats as revealed by the non-peptide selective ETB receptor antagonist RO 46–8443. FEBS Lett. 383, 42–45 12 Schiffrin, E. L. (2000) Endothelin : role in experimental hypertension. J. Cardiovasc. Pharmacol. 35 (4 Suppl. 2), S33–S35