Rüdiger Zart

Function and expression of endothelin receptor subtypes in the kidneys of spontaneously hypertensive rats.

OBJECTIVE The renal endothelin system has been implicated in the development and maintenance of hypertension in spontaneously hypertensive rats (SHR). However, little is known about the function and cellular distribution of endothelin receptor subtypes in the kidneys of SHR. METHODS We analyzed the expression of endothelin receptor subtypes in the kidneys of 16-week-old SHR using Scatchard analysis, receptor autoradiography, Northern blot analysis and in situ hybridization. Wistar-Kyoto rats (WKY) served as controls. Furthermore, we investigated the effects of the mixed (A/B) endothelin receptor antagonist bosentan and the ETA receptor antagonist BQ 123 on mean arterial blood pressure (MAP), renal blood flow (RBF) and glomerular filtration rate (GFR) in conscious chronically instrumented rats. RESULTS In SHR, we found by receptor autoradiography an overexpression of the endothelin A receptor (ETA) in the glomeruli (2.2 +/- 0.4-fold; P < 0.05) and smooth muscle cells of intrarenal arteries (1.9 +/- 0.2-fold; P < 0.05) compared to age-matched WKY. In addition, our study revealed a pronounced upregulation of endothelin B receptor (ETB) in the glomeruli of SHR (5.6 +/- 0.8-fold; P < 0.01). Blockade of endothelin receptors in SHR with bosentan (A and B receptor blockade) as well as with BQ 123 (A receptor blockade) led to a significant decrease in MAP (-18.6 +/- 2.1 and -19 +/- 1.3 mmHg, respectively; P < 0.05 in both cases) and a significant increase in RBF (+2.8 +/- 0.5 and +3.1 +/- 0.37 ml/min, respectively; P < 0.05 in both cases). The blockade of both ETA and ETB by bosentan had no further effect on MAP reduction or RBF increase in SHR compared to the ETA blockade by BQ 123. The ETA antagonist BQ 123 had no effect on GFR either in SHR or in WKY, whereas the combined blockade of ETA and ETB by bosentan significantly decreased GFR in SHR by about 50% but not in WKY. CONCLUSIONS Our data demonstrated a correlation between the overexpression of vascular ETA receptors and the pronounced upregulation of glomerular ETB receptors in the kidneys of SHR and their impact on the regulation of renal blood flow, glomerular filtration rate and blood pressure in these animals.

ETA receptor blockade induces fibrosis of the clipped kidney in two-kidney-one-clip renovascular hypertensive rats

Background In two kidney-one clip renovascular hypertension (2K1C), blood flow is reduced in the clipped kidney leading to ischaemia. The non-clipped kidney is characterized by increased shear stress. Circulating Ang II is elevated. All these factors are stimuli of the paracrine renal endothelin system. Indeed, we demonstrated an activation of the renal endothelin system in the 2K1C rat model. Methods We analysed the effects of chronic treatment with the ETA receptor antagonist BQ-123 on blood pressure, heart rate, plasma renin activity, and on the progression of glomerulosclerosis, interstitial fibrosis and vascular remodeling in the clipped and non-clipped kidney. Results Long-term treatment with BQ-123 led to a fibrotic atrophy of the clipped kidney characterized by a significantly reduced weight of the clipped kidney compared to the clipped kidney of the placebo-treated group. Computer-aided image analysis revealed a markedly enhanced interstitial fibrosis of these clipped kidneys after long-term ETA blockade. The effects of ETA receptor antagonists on the non-clipped kidney were less pronounced. Neither blood pressure nor plasma renin activity were significantly altered by BQ-123 treatment. Conclusions The present study indicates that long-term blockade of the activated endothelin system in the clipped kidney of rats with renovascular hypertension using an ETA receptor antagonist led to a fibrotic atrophy of the clipped kidney.

Paracrine renal endothelin system in rats with liver cirrhosis

1 Liver cirrhosis was induced in rats by CCl4 administration. We analysed the expression of endothelin receptor subtypes in the renal cortex and medulla using Scatchard analysis and receptor autoradiography, and measured plasma as well as renal‐tissue endothelin‐1 concentrations using a specific radioimmunoassay. Furthermore, we analysed the effects of the non‐selective (A/B) endothelin receptor antagonist, bosentan (6 and 100 mg kg−1 day−1) on mean arterial blood pressure, water and sodium excretion and glomerular filtration rate. 2 Our study revealed an overexpression of the endothelin B receptor (ETB) in the renal medulla of rats with liver cirrhosis (Cir: 2775 ± 299 fmol mg−1; Con:1695 ± 255 fmol mg−1; n = 8; means ± s.d., P < 0.01), whereas the density of ETB in the cortex and the endothelin A receptor (ETA) in the cortex and medulla were similar in both cirrhotic and control rats. Receptor autoradiography showed that the upregulation of medullary ETB in cirrhotic rats was due to an upregulation of ETB in the inner medullary collecting duct cells. 3 The tissue endothelin‐1 concentrations were increased in the renal medulla of cirrhotic rats (Cir:271 ± 68 pg g−1wet wt.; Con: 153 ± 36 pg g−1 wet wt., n = 8; means ± s.d., P < 0.01). 4 The glomerular filtration rate was slightly decreased in cirrhotic rats but not altered after bosentan treatment in either cirrhotic or control rats. Bosentan decreased sodium excretion to a similar extent in both cirrhotic and control rats, whereas water excretion was significantly reduced by both dosages of bosentan in cirrhotic rats only (Cir + vehicle: 12.5 ± 0.62 ml day−1, Cir + 6 mg kg−1 day−1 bosentan: 8.6 ± 1.0 ml day−1; Cir + 100 mg kg−1 day−1 bosentan:7.4 ± 0.6 ml day−1; n = 10; means ± s.e.mean). 5 We therefore suggest that the upregulation of the medullary ETB in cirrhotic rats is involved in the regulation of water excretion in rats with CCl4‐induced liver cirrhosis.

Distribution of endothelin receptor subtypes in the rat kidney. Renal and haemodynamic effects of the mixed (A/B) endothelin receptor antagonist bosentan.

The paracrine renal endothelin system has been implicated in acute and chronic kidney diseases. However, there are only few data about the expression of endothelin receptor subtypes and their impact on renal function in the normal rat kidney. Therefore, we analyzed the age-dependent expression of endothelin receptors (endothelin receptor A and B) using Scatchard analysis, in vitro and in vivo receptor autoradiography. Furthermore, we investigated the effects of the mixed (A/B) endothelin receptor antagonist bosentan on haemodynamic and renal function in conscious chronically instrumented rats. The renal endothelin receptor A and endothelin receptor B expression is age-dependent. The relative amount of endothelin receptor A significantly decreased with age, whereas the endothelin receptor B significantly increased with age. Compared to the other renal structures, a high endothelin receptor density (endothelin receptor B >> endothelin receptor A) was seen in the renal tubules and even more in the glomeruli. Bosentan blocks both the pressor and depressor response of endothelin. Blocking of both endothelin receptor subtypes using bosentan without application of endothelin, on the other hand, did not alter blood pressure, heart rate, renal blood flow, water excretion or glomerular filtration rate, but significantly decreased sodium excretion.

Significance of endothelin receptor subtypes in the kidneys of spontaneously hypertensive rats : renal and hemodynamic effects of endothelin receptor antagonists

Summary: The renal endothelin (ET) system has been implicated in the maintenance of hypertension in spontaneously hypertensive rats (SHRs). However, little is known about the expression and cellular distribution of the ET receptor subtypes in the kidney of SHRs. We therefore analyzed the expression of ET receptor subtypes in the kidneys of 16-week-old SHRs. Wistar-Kyoto (WKY) rats served as controls. Furthermore, we investigated the effects of the ETA receptor antagonist BQ 123 and the mixed (ETA/ETB) receptor antagonist bosentan on mean arterial blood pressure (MAP), renal blood flow (RBF), and glomerular filtration rate (GFR) in conscious, chronically instrumented rats. In SHRs we found overexpres-sion of the ETA in the glomeruli and smooth muscle cells of intrarenal arteries compared to age-matched WKY rats. Furthermore, our study revealed a pronounced upregulation of the ETB in the glomeruli of SHRs. Blockade of ETA and ETB receptors in SHR with bosentan as well as with BQ 123 led to a significant decrease in MAP and a significant increase in RBF, indicating that the ETA receptor plays a major role in the maintenance of high blood pressure and the regulation of RBF in SHRs. The blockade of both ETA and ETB receptors by bosentan has no further effect on MAP reduction or increase in RBF in SHRs compared to ETA blockade by BQ 123. In contrast, combined blockade of ETA and ETB receptors by bosentan significantly decreased GFR in SHRs, whereas no effect on GFR was observed in WKY rats, suggesting that the glomerular ETB overexpression in SHRs is of pathophysiologic relevance.

Role of the paracrine liver endothelin system in the pathogenesis of CCl4-induced liver injury

This study analyzed if the paracrine liver endothelin system participates in the pathogenesis of CCl4-induced hepatotoxicity. Wistar Kyoto rats were divided into four groups: a bosentan (mixed endothelin ETA and ETB receptor antagonist) treated group with CCl4 intoxication, a vehicle treated group with CCl4 intoxication, a nontreated control group and a bosentan treated control group. Hepatotoxicity was assessed by determination of alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH) followed by histopathological examinations. Tissue endothelin-1 concentrations and expression of endothelin receptor subtypes were analyzed. The tissue levels of endothelin-1 in the liver of rats with CCl4 intoxication were significantly higher than those in normal rats. Scatchard analysis revealed no differences in the density and binding constant of endothelin ETA and ETB receptor between rats with CCl4 intoxication and controls. Bosentan treatment of rats undergoing CCl4 inhalation resulted in a significant protection against elevation of ALT, AST, LDH and bilirubin. Histopathological examination of live sections for necrotic, swollen and lipid-laden cells revealed findings that were in agreement with the serum enzyme data. In conclusion, this study showed that the paracrine endothelin system is involved in the pathogenesis of CCl4-induced hepatotoxicity and that the blockade of the stimulated liver endothelin systems reduces CCl4-induced liver injury.

Regulation of the renal endothelin system in the two-kidney, one clip renal hypertensive rat.

Renovascular hypertension in the two-kidney, one clip (2K1C) model is characterized by initially elevated angiotensin-II (A-II) plasma concentrations, caused by ischemia in the clipped kidney and shear stress in the nonclipped kidney. These features are known stimuli of the endothelin (ET) system. The aim of this study was to analyze whether the renal ET system is activated in 2K1C renal hypertension in the rat. Wistar Kyoto rats (9 weeks old) were randomly assigned to four groups. Groups 1 and 3 underwent renal artery clipping, groups 2 and 4 were sham-operated. Groups 1 and 2 were used for analysis at 10 days after clipping, groups 3 and 4 12 weeks after clipping. We measured immunoreactive ET-1 renal tissue concentration as well as the ET(A)- and ET(B)-receptor density and affinity in renal cortex and medulla. We detected an increased immunoreactive ET-1 tissue concentration compared to the sham-operated control in the renal cortex of the nonclipped kidney 10 days (25.6 +/- 12.0 pg/g vs 12.5 +/- 5.0, 1 pg/g; p < 0.05) and in the renal cortex of the clipped kidney 90 days after clipping (92.4 +/- 47 pg/g vs 22.9 +/- 21 pg/g; p < 0.05), An increased ET(A)-receptor density was revealed in the renal medulla of the clipped kidney 10 days (624 +/- 130 fmol/mg vs 276 +/- 68 fmol/mg; p < 0.05) and 90 days (859 +/- 131 fmol/mg vs 493 +/- 93 fmol/mg; p < 0.05) after clipping. There were no differences in ET(B)-receptor density or binding affinity of either ET(A)- or ET(B)-receptors. In the 2K1C rat model of renovascular hypertension the renal ET system is activated. This activation is time-dependent and also dependent on the specific pathophysiological condition (clipped vs nonclipped). Increased ET-1 tissue concentration and upregulation of ET(A)-receptor density might lead to a synergistic activation of the ET system.

The endothelin system in polycystic kidneys of Han:SPRD rats

Polycystic kidney disease (PKD) is characterized by structural alterations such as thickening of the tubule basement membrane, interstitial fibrosis, and formation of cysts. Han:SPRD rats are a well-known rat model of human PKD. Interestingly, interstitial fibrosis, glomerulosclerosis, and cyst formation were also seen in human endothelin-1 (ET-1) transgenic mice. We therefore analyzed the tissue concentrations of ET-1 and the expression of ET receptor subtypes in the kidneys of young homozygous (cy/cy), heterozygous (cy/+) 6 week-old male Han:SPRD, and corresponding control rats. The kidneys of affected rats showed significantly elevated tissue levels of ET-1 compared to age-matched controls. Scatchard analysis, on the other hand, revealed markedly decreased ETA and ETB receptor density in all groups of affected rats. The binding affinity of both ET receptor subtypes was slightly decreased in Han:SPRD rats. These data show that the renal paracrine ET system is activated in PKD and might contribute to renal cyst formation and development of end-stage kidney disease.

Protective effects of the mixed endothelin receptor antagonist bosentan in rats with CCL4-induced liver injury

Summary: We determined whether the paracrine liver endothelin (ET) system participates in the pathogenesis of CCl4-induced hepatotoxicity. Wistar-Kyoto rats were divided into four groups: a bosentan-treated group with CC14 intoxication, a vehicle-treated group with CC14 intoxication, a nontreated control group, and a bosentan-treated control group. Hepatotoxicity was assessed by determination of serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH). Tissue ET-1 concentrations and expression of endothelin receptor subtypes were analyzed. The liver tissue levels of ET-1 in rats with CC14 intoxication were significantly higher than in normal rats. Scat-chard analysis revealed no differences in the density and binding constants of ETA and ETB receptors between rats with CCI4 intoxication and controls. Bosentan treatment of rats undergoing CC14 inhalation resulted in significant protection against elevation of ALT, AST, LDH, and bilirubin. In conclusion, this study showed that the paracrine ET system is involved in the pathogenesis of CC14-induced hepatotoxicity and that blockade of the stimulated liver endothelin system reduces CCl4-induced liver injury.