Gzrdiovascular Research

EJ.SEVIER Cardiovascular Research 31 (19%) 499-510

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

Berthold Hocher a*bY* , Peter Rohmeiss ‘, Riidiger Zart b, Fritz Diekmann a, Volker Vogt ‘, Dieter Metz b, Myriam Fakhury a, Norbert Gretz ‘, Christian Bauer b, Klaus Koppenhagen d,

Hans H. Neumayer a, Armin Distler e a Department of Nephrology. Universitiitsklinikum Charite’ der Humboldt Universitiit zu Berlin, City, Germany

b In.stitute of Molecular Biology and Biochemistry, Free Univer,rity of Berlin, Berlin, Germany ’ Department of Nephrology, Klinikum Mannheim, University of Heidelberg, Heidelberg, Germany

d Department of Nuclear Medicine, Universitiitsklinikum Benjamin Franklin, Berlin, Germany (’ Department of Nephrology, Universitfitsklinikum Benjamin Franklin, Berlin, Germany

Received 7 April 1995; accepted 3 1 August 1995

Abstract

Objectiue: 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 f O&fold; P < 0.05) and smooth muscle cells of intrarenal arteries (1.9 + O.Zfold; 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 f O.&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 f 2.1 and - 19 f 1.3 mmHg, respectively; P < 0.05 in both cases) and a significant increase in RBF (+2.8 + 0.5 and + 3.1 f 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.

Keywords: Rat, spontaneously hypertensive; Rat, kidney; Hypertension; Endothelin receptor antagonist; Endothelin nxeptor

1. Introduction

Endothelin is a 21-amino-acid peptide that was origi- nally characterized in the supematant of vascular endothe- lial cells and is, by at least one order of magnitude, the

most potent vasoconstrictor known so far [ 1,2]. The biolog- ical effects of endothelin are mediated by plasma-mem- brane-bound receptors. Two endothelin receptor subtypes (ETA, ETB) have been cloned. They belong to the family of rhodopsin-like receptors and are G-protein-coupled. They differ in their binding affinity to endothelin isopep- tides (ETA: ET-l r ET-2 > > ET-3; ETB: ET-l = ET-2

l Corresponding author: Universituniversititklinikum Charitt? der Humboldt Universitit zu Berlin, Abteilung fir Nephrologie (Medizinische Klinik 5), Schumannstr. 20-21, D- I01 I7 Berlin, Germany. Tel.: ( + 49-30) 2802-5865; Fax.: ( + 49-30) 280:2-8471. Time for primary review 42 days.

0008-6363/%/$15.00 0 1996 Elsevier Science B.V. All rights reserved SSDI 0008-6363(95)00200-6

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= ET-31 as well as in their expression by different cell types [2,3]. ETA is the receptor responsible for endothelin-induced vasoconstriction in vascular smooth muscle cells and was also identified on glomerular mesan- gial cells and myocytes. Recently an ETB-like receptor mediating vasoconstriction has been described in vascular smooth muscle cells [4]. However, ETB is mainly found on vascular endothelial cells as well as on renal tubular cells and renal glomeruli. ETB activates phospholipase A, which leads to the release of arachidonic acid derivatives (especially prostacyclin). The synthesis of NO is another effect of endothelin action on ETB [5,6].

Endothelin-1 infusions increase blood pressure [3], and blood pressure is increased in patients with endothelin- secreting hemangioendothelioma [7]. On the other hand, endothelin-2 transgenic rats [8] are not hypertensive, but there is an increase in the blood pressure response to exogenous endothelin as compared to control rats. Surpris- ingly, heterozygous + endothelin-1 knock-out mice with reduced endothelin tissue levels showed an elevated blood pressure (beside the major finding of crania-pharyngial abnormalities in homozygous - / - endothelin knock-out mice, see Ref. [9]>. These findings indicate that the en- dothelin system is involved in the pathophysiology of hypertension, but an understanding of its functions and effects is far from being complete.

Our interest focused on renal endothelin receptors in SHR because there are some indications that the renal endothelin system is involved in the pathogenesis of hyper- tension in these animals. The pressor response to endothe- lin-1 is increased in renal arteries obtained from SHR [ 10,111. Furthermore, polyclonal endothelin antibodies in- crease the renal blood flow and glomerular filtration rate in SHR [ 121. The aim of our study was therefore to analyze the expression of endothelin receptor subtypes in the kid- neys of SHR using Scatchard analysis, receptor auto- radiography, Northern blot analysis and in situ hybridiza- tion. In addition, we used the ETA receptor antagonist BQ 123 and the mixed (A/B) endothelin receptor antagonist bosentan [ 131 to examine the functional importance of the paracrine endothelin system in the regulation of glomerular filtration rate, renal blood flow and blood pressure in this animal model of human hypertension.

2. Methods

2. I. Materials

Male Wistar rats (WKY) and male spontaneously hy- pertensive rats (SHR) were obtained from Mllegard (Den- mark) and fed a commercial diet (Altromin@, Altromin GmbH, Germany) and given water ad libitum. All animal experiments were conducted in accordance with local insti- tutional guidelines for the care and use of laboratory animals. [‘251]Endothelin-l (2000 Ci/mmol) was obtained

from Du Pont, Germany. 32 P-dATP and 35S-UTP was from Amersham (Braunschweig, Germany). Unlabeled ET-l was from Peninsula Laboratories, Inc. (Germany). The mixed (A/B) endoth 1 e in receptor antagonist bosentan (Ro 47- 0203, 4-tert-butyl-N-[6-(2-hydroxy-ethoxy)-5-(2-methoxy- phenoxy)-2,2’-bispyrimidine-4-yl]-benzenesulfonamide) was a generous gift from Dr. Martine Clozel (Pharma Division, F. Hoffmann-La Roche Ltd., Basel, Switzerland). The selective endothelin receptor ligands (BQ 123 and BQ 3020) were from California Peptides Inc., USA. Unless otherwise stated, all reagents were of analytical grade and were purchased from Merck (Darmstadt, Germany), Boehringer-Mannheim (Mannheim, Germany) or Sigma (Munich, Germany).

2.2. Membrane preparation

Membranes were prepared according to Nambi et al. (14). The rats were sacrificed, and the cortex and medulla were carefully dissected from each other. Approximately 150 mg of kidney medulla or cortex were homogenized at 4°C in 10 ml of 20 mmol/l NaHCO, using a motor-driven pestle homogenizer. The homogenate was centrifuged at 4°C for 15 min at 1000 X g. The supematant was decanted and centrifuged at 4°C for 30 min at 40000 X g. The pellet, consisting of crude plasma membranes, was resus- pended in assay buffer (1 mg/ml bacitracin, 100 mM Tris-HCl, 5 mM MgCl,, and 0.1 g% BSA, pH 7.4) at a protein concentration of 200 pg/ml.

2.3. Binding assay for ETA and ETB

Binding studies were performed as previously described [15,16] with some modifications. In order to analyse the expression of endothelin receptor subtypes (ETA, ETB) in the kidney, binding assays were performed in the presence or absence of the subtype-specific endothelin receptor ligands BQ123 (3 PM) and/or BQ3020 [14,16]. The assay buffer for binding studies contained 1 mg . ml-’ baci- tracin, 100 mM Tris-HCl, 5 mM MgCl,, and 0.1 g% BSA, pH 7.4, in a total volume of 150 ~1. The [ ‘251]ET-I tracer concentration was kept constant at 40000 cpm/tube, while the concentration of unlabeled ET-l was increased from 0 to 25 nM (competition studies with “cold saturation”). Samples from crude plasma membranes were used at a concentration of 0.53 mg protein . ml- ’ . Binding studies were performed at room temperature for 120 min. Non- specific binding was assessed in the presence of excess ET-l (5 FM). After adding 1 ml of cold binding buffer, free and receptor-bound radioactivity was separated by centrlfugation at 30 000 X g (4°C) for 15 min, and the pellets thus obtained were washed two additional times with 1 ml of cold binding buffer. [‘*‘I] was counted in a Packard Gamma Counter (78% counting efficiency for [ ‘25~]) (n = 6 in each group).

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2.4. Autoradiography of tissue endothelin receptor sub- types

Receptor autoradiography was performed as previously described [ 161. After anesthesia by intraperitoneal adminis- tration of pentobarbital, the inferior vena cava was cannu- lated. In order to block the ETA or the ETB receptor or both receptor subtypes, BQ 123, BQ 3020 or BQ 123 and BQ 3020 (control> were injected into the inferior vena cava followed by an injection of [‘2sI]ET-l -saline 15 min thereafter. The dosages of ET-l, BQ123 and BQ 3020 used did not alter the blood pressure during the experiment (data not shown). 15 min later, the rats were sacrificed, and the kidneys were perfused with 50 ml of ice-cold saline, followed by fixation with 2.5% glutaraldehyde/5% formalin. The kidneys were then removed and embedded in plastic material (Technovit@ , Kulzer GmbH, Germany). The plastic blocks were cut into 2.5-pm-thick sections, layered with photoemulsion (K5 nuclear trace emulsion, Ilford, UK) and stained with hematoxylin and eosin. Eval- uation was done by counting the silver granules. The number of silver grains in a given area was divided by the number of cell nuclei in the same area in order to consider possible differences in density of cells in SHR and WKY. (n = 6 in each group)

2.5. Northern blot

2.5.1. RNA extraction and Northern blot analysis Total RNA was extracted from frozen tissues using the

guanidinium isothiocyanate method and centrifuged through a CsCl gradient as described by Sambrook et al. [17]. Total RNA (15 pg per lane) was separated elec- trophoretically on a formamide/formaldehyde-agarose gel, blotted to Hybond N nylon membranes (Amersham, Ar- lington, UK) and irradiated with UV light. After prehy- bridization for 2 h at 42°C the membranes were hy- bridized with 32 P-dATP-labeled cDNA probes for 12 h at 42°C. Blots were washed at 65°C under high stringency conditions. Autoradiography was performed at - 80°C ac- cording to Sambrook et al. [17]. The autoradiographs were analyzed using a computer-aided densitometer (Scan- Pack’” Scanner-Densitometer). The ratio of endothelin receptor subtype mRNA//J-actin mRNA in WKY was defined as 100% and compared with the ratio of endothelin receptor subtype mRNA/@actin mRNA in SHR.

2.5.2. Hybridization probes We used a 2.5 kb Eco Rl/Not 1 rat-ETA fragment [18]

subcloned in pBluescript 2 + SK plasmid (Stratagene, La Jolla, CA, USA), a 2.5 kb Xba l/Pst 1 rat-ETB fragment [ 181 subcloned in pBluescript 2 + SK plasmid and a 0.5 kb rat p-actin fragment subcloned in pGem 72. The frag- ments were excised from PBS plasmid, labeled with 32P- dATP by the random priming method and had a specific activity of l-3 X lo6 cpm/ml hybridization solution (n = 6 in each group).

2.6. In situ hybridization

2.6.1. Tissue preparation Kidney samples were immediately frozen in liquid ni-

trogen and stored at -70°C. Cryostat sections (5 pm> were placed on siliconized slides treated with 3-amino-pro- pyltriethoxysilane for better adherence and dried on a hot plate at 80°C for 3 min. Tissue sections were fixed in 4% paraformaldehyde/phosphate-buffered saline (PBS, pH 7.4) for 20 min and dehydrated in graded ethanol.

2.6.2. Probes The following were used: a 2.5 kb Eco Rl/Not 1

rat-ETA fragment subcloned in pBluescript 2 + SK plas- mid (Stratagene, La Jolla, CA, USA), a 2.5 kb Xba l/Pst 1 rat-ETB fragment subcloned in pBluescript 2 + SK plasmid and a 0.5 kb rat actin fragment subcloned in pGem 72 [17-191. The plasmids were linearized with either Xhol or Sst2. Single-stranded RNA probes, complemen- tary (antisense probe) or anticomplementary (sense probe, negative control) to cellular RNA, were obtained by run-off transcription with T7 or T3 RNA polymerase (Transcrip- tion Kit from Boehringer Mannheim, Germany). 35S-UTP was used for labeling the RNA probes. The specific activ- ity of the probes was 1 .O-1.5 X lo9 cpm/mg RNA. Ap- propriate tissue penetration of the probes was achieved by controlled alkaline hydrolysis, reducing the RNA length to 50-200 bp.

2.6.3. Zn situ hybridization Prehybridization, hybridization, washing and RNAse A

digestion to remove non-specifically-bound probes as well as autoradiography were performed as described [20] with modifications. The tissue sections were treated with 0.2 mol/l HCl for 20 min, digested in 0.125 mg/ml pronase (Boehringer-Mannheim, Germany) for 10 min at 22”C, rinsed in 0.1 mol/l glycine/PBS, washed in PBS, fixed again in 4% PFA/PBS for 15 min, acetylated in a solution of acetic anhydide/O.l mol/l triethanolamine, pH 8.0 (dilution 1:400), rinsed again in PBS, dehydrated in graded ethanol and air-dried. Each slide was covered with 0.025 ml of a hybridization mixture containing l-3 X 10’ cpm of labeled RNA probe in 50% formamide/lO% dextran sulphate/lO mmol/l dithiothreitol (DTT)/lO mmol/l Tris-HCl, pH 7.5/10 mmol/l Na,HPO,/0.3 mol/l NaCl/S mmol/l EDTA/ 0.2 mg/ml yeast tRNA. Sec- tions were sealed with a siliconized coverslip. After 18 h of incubation at 50°C in a humid chamber, slides were washed for 4 h at 60°C in a solution of 50% formamide, 10 mmol/l D’IT, 1 X SALTS, and for 15 min in 10 mmol/l Tris-HCl, pH 7.5/0.5 mol/l NaCl/l mmol/l EDTA at 37°C then digested with RNAse A to reduce background caused by non-specific binding, washed again in 10 mmol/l Tris-HCl, pH 7.5/0.5 mol/l NaCl/l mmol/l EDTA (TES) for 30 min and finally rinsed in 2 x SSC, 0.1 X SSC and 0.05 X SSC for 20 min each at 22°C. The

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slides were dehydrated in graded ethanol, air-dried and dipped in Ilford K5 photoemulsion (Ilford, Mobberley, Cheshire, UK). After exposure for lo-28 days at 4°C sections were developed for 2.5 min using the Kodak D 19 developer (Kodak, Hemel Hampstead, UK), subsequently rinsed in 1% acetic acid, fixed in Kodak fixer for 2.5 min, washed in H,O, and counterstained by hematoxylin-eosin. All tissues were simultaneously processed using the same probes and reagents (n = 6 in each group).

2.6.4. Control experiments The following control experiments were performed: (i)

as a positive control for intact tissue, in situ hybridization (ISH) was done with sense and antisense actin probes, (ii) as a proof that the ISH signal obtained was due to RNA- RNA hybridization, some kidney sections were pretreated with RNAse A prior to hybridization. These slides were incubated in a solution of 50 mg/ml RNAse A in 2 X SSC for 30 min at 37°C subsequently washed in 2 X SSC for 15 min and then submitted to the same hybridization procedure as described above; (iii) sense and antisense ETA- and ETB-mRNA probes were used in each experi- ment.

2.7. Measurement of glomerular jIltration rate (GFR), mean arterial blood pressure (MAP), heart rate (HR), and renal blood flow (RBF) in conscious chronically instru- mented WKY and SHR

2.7.1. Surgical procedures One week prior to the acute experiments, the rats were

anesthetized with ether, and femoral artery and vein catheters were implanted [21]. Three days before the start of the experiments, flowprobes (1RB with implantable connector, Transsonic Systems Inc., Ithaca, NY, USA) were chronically implanted around the left renal artery. Briefly, the left kidney was exposed by a retroperitoneal access. The renal artery was carefully dissected using an operating microscope to avoid damage to the renal nerves. The flowprobe was then placed around the artery and, after the best signal had been achieved, the probe was fixed in proper position using a small envelope of Merocel Op-Wipe (Merocel Corp., Mystic, CT, USA) covering the probe and the artery at the point of reflector attachment. To improve signal conductance, the envelope was filled with ultra- sound gel. All catheters and cables were led subcuta- neously to the rats neck [21,22].

2.7.2. Circulatory measurements Mean arterial blood pressure (MAP) and heart rate (HR)

were measured via the arterial line with a Statham pressure transducer P23Db and a Gould pressure processor coupled to a Gould Brush 2600 recorder. Renal blood flow (RBF) was measured via the chronically implanted flowprobe with a transit time flowmeter (T106, Transsonic Systems Inc., Ithaca, NY, USA) and continuously recorded on a

Gould Brush 2600 recorder. The Transonic flowmeter systern determines absolute volume flow [23]. The flow- probes were precalibrated and measured absolute blood flow with an accuracy of *2%.

2.7.3. Measurement of glomerular filtration rate GFR was measured using the inulin single-shot method

[24]. The single-shot clearance was evaluated by display- ing a two-compartment model with resolution of the plasma inulin concentrations into two monoexponential functions. The rats received an intravenous i.v. bolus injection of 150 mg of inulin (Inutest”). Blood samples for determination of serum inulin concentrations were drawn at 0, 15, 30,90, 135 and 180 minutes after injection. Inulin was measured by a modified /3-fructosidase method [25]. GFR is ex- pressed as ml/min per 100s body weight (BW).

2.8. Effects of the mixed (A /B) endothelin receptor antag- onist bosentan and the ETA receptor antagonist BQ 123 on MAP, HR and RBF

To examine the effect of bosentan on resting MAP, HR and RBF in WKY versus SHR, the rats were divided into 6 groups: Group 1 (WKY; n = 6) and group 2 (SHR; n = 7) received cumulative i.v. bolus injections of bosentan (10 mg/kg) every 15 min up to a total load of 100 mg/kg; group 3 (WKY; n = 7) and group 4 (SHR; n = 7) received i.v. bolus injections of the selective ETA receptor antago- nist BQ 123 (1 mg/kg) every 15 min up to a total load of 10 mg/kg. This dose of BQ 123 completely blocked the ETA-mediated hemodynamic responses to ET-l i.v (data not shown). Group 5 (WKY; n = 6) and group 6 (SHR; n = 6) received i.v. bolus injections of vehicle.

2.9. Effects of the mixed (A/B) endothelin receptor antag- onist bosentan and the ETA receptor antagonist BQ 123 on GFR

The animals were divided into 6 groups: Group 1 (WKY, n = 6) and group 2 (SHR, n = 7) received 100 mg/kg bosentan, group 3 (WKY, n = 7) and group 4 (SHR, n = 7) received 10 mg/kg BQ123 and finally group 5 (WKY, n = 6) and group 6 (SHR, n = 7) received vehicle as an intravenous bolus injection, followed by an injection of 150 mg inulin 5 min later. At 0, 15, 30, 90, 135 and 180 min after injection of inulin, blood samples (200 ~1) were taken from the arterial catheter for determi- nation of serum inulin concentrations.

2.10. Analysis of data

The unpaired Student t-test was used for the determina- tion of statistical difference of group means. Analysis of variance followed by t-test was used if appropriate. Re- sults were considered significantly different at a value of P < 0.05.

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3. Results

3.1. ETA and ETB binding to the renal cortex

Scatchard analysis revealed only one type of ETA (data not shown) and ETB (Fig. 1) binding site in 16-week-old SHR and WKY. Cortical membranes of 16-week-old SHR exhibited a significantly higher density of ETA (P < 0.05) and ETB (P < 0.01) compared to age-matched WKY (Ta- ble 1). The ETA receptor density in the renal cortex of 16-week-old SHR was 255 + 41 fmol/mg and 174 + 19 fmol/mg in WKY (P < 0.05) of the same age, respec- tively. The ETB receptor density was 639 f 41 fmol/mg in SHR and 363 It 42 fmol/mg in WKY (P < 0.01).

3.2. ETA and ETB binding to the renal medulla

The density of ETA and ETB in renal medullary mem- branes was similar in 16-week-old SHR and WKY. As in the cortex, only one type of ETA and ETB binding site was found in the renal medulla; the binding constant (K,) did not differ between SHR and WKY of the same age (Table 1). Interestingly, the binding constants (K,) for ETB were higher in the renal medulla than in the renal cortex of both SHR and WKY (Table 1).

3.3. Autoradiography of tissue endothelin receptor sub- types

The expression of endothelin receptor subtypes differed between SHR and WKY. Differences were observed in glomerular and vascular endothelin receptor expression. We were able to demonstrate (by counting the silver grains, see “Methods”) that the ETA density was in- creased 2.2 f O.Cfold in the glomeruli of SHR compared to WKY (P < 0.05). In addition, we also found a pro- nounced ETB upregulation within the glomeruli of SHR (Fig. 2A,B). By counting the silver grains, we found that the ETB density was increased 5.6 f O.&fold in the glomeruli of SHR compared to WKY (P < 0.01). The ETA as well as the ETB receptor were homogeneously distributed throughout the glomeruli.

Differences in receptor density between SHR and WKY were also found in the renal vessels. All branches of the intrarenal arteries in SHR showed an increased ETA recep- tor density compared to WKY. The signals were located in the smooth muscle cells and were 1.9 f 0.2 times higher compared to WKY (P <: 0.05). No differences were seen between SHR and WKY with respect to the expression of vascular ETB receptors.

Non-specific binding was very low after blocking the endothelin receptors with the BQ 123 and BQ 3020, as shown in Fig. 2C.

3.4. Northern blot analysis

The same ETA and ETB messenger RNA transcripts of the predicted length (4.2 and 4.7 kb long for the ETA and

the ETB, respectively) were detected in all kidney sam- ples. ETA mRNA was overexpressed in the kidneys of SHR as compared to WKY. In contrast, ETB mRNA was similarly expressed in the kidneys of SHR and WKY (Fig. 3). The transcription levels of p-actin, a structural protein, were similar in the kidneys of SHR and WKY.

3.5. In situ hybridization

For the cellular localization of ETA and ETB mRNA in situ hybridization technique was used. 35S-labeled ribo- probes (sense and antisense probes) derived from the ETA and ETB cDNAs were used for the analysis of both endothelin receptor mRNAs. The specific hybridization of each probe under our experimental conditions was con- firmed by a very low hybridization signal in parallel experiments using sense probes. Furthermore, the two cRNA antisense probes for ETA and ETB mRNA showed different patterns of hybridization, indicating that the cDNA probes used did not cross-hybridize to the mRNA of the other ET-receptor subtype (Fig. 5B,D).

The ETA mRNA was mainly located in the glomeruli and the blood vessels. There was an increased ETA mRNA signal density in the glomeruli of SI-IR as compared to WKY (Fig. 4A and 4B). There was also an increased density of ETA mRNA in all branches of intrarenal arter- ies of SHR compared to WKY (Fig. 5A,B). The ETA signals in the vessels were located on the smooth muscle cells. The ETA-mRNA density in cortical and medullary tubules of SHR and WKY were similar.

ETB mRNA was mainly located in the glomeruli. We observed no differences between SHR and WKY in glomerular ETB mRNA expression. Only scattered ETB

0 1 2 3 4 5 6

Free (nmol/l)

Fig. 1. A representative experiment of [“‘]IET-1 specific binding in the presence of the subtype-specific ligand BQ123 to membranes isolated from the renal cortex of 16-week-old SHR (solid circles) and WKY (open circles) showing an increased density of ETB receptors in the renal cortex of SHR. Scatchard transformations of binding data (inserts) suggest only one binding site in both SHR and WKY. Non-specific binding was determined in the presence of unlabeled ET- 1 as described under “Meth- ods” and was always less than 10% of total binding.

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Fig. 2. Microscopic autoradiogram showing upregulation of ETB receptors in 16-week-old SHR compared to WKY of the same age. The ETB receptors ale equally disttibuted throughout the glomeruli. (A) WKY, ETB. (B) SHR, ETB. (C) Non-specific binding in SHR after blocking the ETA and ETB with BQ 123 and BQ 3020. Original magnification: X250.

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Table 1 Receptor density and binding constants of ETA and ETB derived from [ ‘zsIjET-I binding in the presence of the subtype-specific ligands to kidney membranes of It&week-old SHR and WKY

WKY WKY SHR

Kci (nmol/l)

CORTEX ETA 174LtlQ 255f41 a 0.47f0.02 0.46f0.02 CORTEX ETB 363f42 639f41 b O.Qf0.03 0.8&O&I MEDULLA ETA 121 k38 137*51 1 .o f 0.05 1.2 f 0.07 MEDULLA ETB 784*63 668*198 3.2*0.58 2.3kO.6

Data from Scatchard plots of (‘zsI)ET-l binding studies. To analyse expression of receptor subtypes, binding assays were performed in the presence of the subtype-specific ligands (BQ 123 or BQ 3020). Non- specific binding was assessed in the presence of 5 pmol.l- ’ of unla- beled ET-l. It ranges between 5 and 10%. Samples from crude plasma membranes were used at a concentration of 0.53 mg of protein. ml- ’ . For details see “Methods”. Values are means f s.d. of 6 separate animals in each group. a P < 0.05 compared to the density of ETA in the renal cortex of 16-week-old WKY. b P < 0.01 compared to the density of ETB in the renal cortex of 16-week-old WKY.

mRNA signals were detected on the endothelial cells of blood vessels in SHR and WKY. The smooth muscle cells showed no ETB mRNA expression in SHR or WKY (Fig. 5D). The ETB-mRNA expression in medullary tubules of SHR and WKY was strong, but did not differ between SHR and WKY (data not shown).

3.6. Effects of the mixed endothelin receptor antagonist bosentan and BQ 123 on mean arterial blood pressure (MAP), heart rate (HR) and renal blood flow (RBF)

Cumulative i.v. bolus injections of bosentan (10 mg/kg every 15 min up to a total load of 100 mg/kg) induced a progressive decrease in MAR in SHR, whereas no signifi- cant change was observed in WKY. RBF also increased only in SHR (Fig. 6). Cumulative i.v. bolus injections of a

0 Ii

Fig. 3. Bar diagram of Northern blot analysis showing the expression of ETA and ETB messenger RNA in the kidneys of WKY and SHR. The Notthem blots were analyzed using a densitometer. The ratio of endothe- lin receptor subtype mRNA (ETA or BTB)/ pactin mRNA in WKY was defmed as 100% and compared with the ratio of endothelin receptor subtype mRNA/ @-actin mRNA in SHR The relative amount of mRNA expression f s.e.m. is shown (n =* 6).

lower bosentan dose (3 mg/kg every 15 min up to a total load of 30 mg/kg) induced quantitatively and qualitatively similar effects as compared to the higher bosentan dose (data not shown). Cumulative injections of BQ 123 (1 mg/kg every 15 min up to a total load of 10 mg/kg) had also quantitatively and qualitatively similar effects on the maximal changes in MAR and RBF as compared to both bosentan dosages (Fig. 6). Injections of vehicle had no effects on these parameters (Fig. 6).

The ETA antagonist BQ 123 has no effect on heart rate, whereas the mixed endothelin receptor antagonist bosentan acts in a negative chronotropic manner. This effect is more pronounced in WKY than in SHR. The resultant heart rate reduction in WKY does not lower mean arterial blood pressure.

3.7. Effects of the mixed endothelin receptor antagonist bosentan and the ETA receptor antagonist BQ 123 on glomendar filtration rate (GFR) in 16week-old SHR and Wistar rats

Basal GFR did not differ between WKY and SHR. Bosentan induced an about 50% decrease in GFR in SHR, whereas no effect of bosentan was observed in WKY. BQ 123 did not alter GFR either in SHR or in WKY (Fig. 7).

4. Discussion

In the present study, we examined the expression of endothelin receptor subtypes in the kidneys and the effect of endothelin receptor antagonists on blood pressure and renal hemodynamics in spontaneously hypertensive rats and age-matched Wistar-Kyoto rats. Three main results were obtained, namely: (i) an ETA upregulation in the glomeruli and all branches of intrarenal arteries of SHR, (ii) a pronounced upregulation of ETB in the glomeruli of SHR; (iii) blockade of endothelin receptors in SHR using either the A and B receptor antagonist bosentan or the A receptor antagonist BQ 123 led to similar decreases in mean arterial blood pressure and similar increases in renal blood flow, suggesting that vascular ETA receptor overex- pression is important for the regulation of renal blood flow and contributes to high blood pressure in SHR. In contrast to renal blood flow, glomerular filtration rate was de- creased in SHR by bosentan only, whereas the ETA antag- onist had no influence on GFR.

4.1. Expression of ET receptor subtypes in the kidney

Receptor autoradiography revealed that the overexpres- sion of endothelin receptor subtypes, as detected by Scatchard analysis in the renal cortex of SHR, was due to an increased ETA and even more so to ETB density within the glomexuli. Glomerular endothelin receptors could be

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expressed either in mesangial cells or in glomerular en- dothelial cells. Our techniques do not permit differentiation between these two glomerular cell types. However, others have reported that glomerular ETA receptors are expressed by mesangial cells [26,27], whereas the ETB receptor seems to be located in the glomerular endothelial cell [28].

Our data showing an ETB overexpression in the renal cortex of SHR agree with those of Gellai et al. [29] who, using Scatchard analysis, also found a markedly increased cortical ETB expression. On the other hand, Gellai et al. [29] did not observe an overexpression of ETA receptors in SHR. The different results with regard to ETA expression could be due to the techniques used in their study. Using Scatchard analysis we found a slightly (but significantly) increased ETA expression in the renal cortex of SHR. Such a slight ETA overexpression may have been over- looked in the study by Gellai et al. [29] who adopted an indirect approach calculating ETA expression from total ET binding minus ETB binding. By using the more sensi- tive technique of in situ hybridization and receptor auto- radiography which allows the exploration of receptor ex- pression in different anatomical structures (i.e., blood ves- sels, glomeruli and tubules) we were able to demonstrate a pronounced ETA overexpression in vascular smooth mus- cle cells.

In addition, it is important to note that the normotensive controls are different in both studies. We used Wistar- Kyoto rats, whereas Gellei et al. [29] used Sprague-Dawley rats. This circumstance could also have contributed to the different results concerning ETA expression.

4.2. Renal ETA and ETB mRNA expression

The observed overexpression of endothelin receptor proteins in SHR as revealed by Scatchard analysis and receptor autoradiography could be due either to increased formation of receptor mRNA or to decreased receptor degradation. Using Northern blot analysis and in situ hy- bridization (Figs. 3 and 5) we were able to demonstrate that the increased number of ETA in the branches of renal arteries and glomeruli of SHR was due to increased mRNA formation.

On the other hand, the markedly increased ETB recep- tor protein density found in the glomeruli of SHR obvi- ously was not caused by an upregulation of ETB mRNA (Fig. 3) and could h ave been related to decreased ETB degradation possibly due to structural alterations of the glomerular ETB receptor itself (e.g., by altered glycosyla- tion or phosphorylation) leading to decreased degradation or to inactivation of enzymes involved in ETB degrada- tion.

4.3. Effect of endothelin receptor blockade on renal blood flow and mean arterial blood pressure

Gellai et al. [29] recently reported on a good correlation between the higher density of the ETB receptor subtype in the renal cortex of SHR and the increased potency of the exogenously administered ETB receptor agonist Sarafo- toxin 6c in mediating renal vasoconstriction and concluded that ET-induced renal vasoconstriction in SHR is mediated by ETB [29]. However, in our view, studies with an exogenously given ETB agonist examine pharmacological effects and do not reflect the functional importance of the endogenous endothelin system. As shown in our study by receptor autoradiography, ETB overexpression in the renal cortex of SHR was clearly due to overexpression of glomerular ETB, whereas renal vascular ETB expression was not altered in SHR. Therefore, our data do not support the view of Gellai et al. that the alteration of renal blood flow in SHR is linked to a higher proportion of ETB in the renal cortex.

Our study demonstrates that the decrease in blood pres- sure and the increase in renal blood flow achieved in SHR by blocking the ETA receptor with BQ 123 were of the same order of magnitude as the effects of the combined ETA and ETB receptor antagonist bosentan. The additional blockade of ETB obviously had no additive effect on blood pressure reduction or increase in renal blood flow. Thus, it appears that systemic blood pressure and renal blood flow in SHR are at least partially mediated by the overexpressed vascular ETA receptors.

Our finding of a blood-pressure-lowering effect of BQ 123 given to SHR confirms previous reports [29,30]. How- ever, chronic oral treatment of SHR with bosentan has recently been reported to have no effect on blood pressure [3 1 I. The reasons for this discrepant effects of acute intra- venous and of chronic oral blockade of endothelin recep- tors in SHR remain unclear. Possibly, the blood-pressure- lowering effects of endothelin antagonists are counteracted in the long run by increased activities of other vasocon- strictor mechanisms. In any case, the acute blood-pres- sure-lowering effects of BQ 123 and bosentan clearly show that the endothelin system contributes substantially to elevated blood pressure in SHR.

4.4. Effects of BQ 123 and bosentan on glomerular$ltra- tion rate

A bolus injection of the mixed (A/B) endothelin recep- tor antagonist bosentan decreased GFR (by about 50%) in SHR only, whereas GFR was not altered in SHR by the ETA antagonist BQ 123, indicating that the pronounced

Fig. 4. In situ hybridization of ETA receptor mRNA in the glomeruli of 16-week-old WKY and SHR showing an overexpression of ETA mRNA in SHR. (A) WKY, ETA. (B) SHR, ETA. (C) SHF-control with a 35S-labeled ETA mRNA sense probe. Original magnification: X 250.

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1 m SHR

0 WKY

* n

Bosenton BQ 123 Vehicle 100 n-g/kg 10 n-g/kg

Fig. 6. Maximal changes of mean arterial blood pressure (MAP, mmHg), heart rate (HR, bpm) and renal blood flow (RBF, ml/mitt) after treatment with bosentan, BQ 123 and vehicle in WKY (open bars) and SHR (filled bars). The rats (16week-old male WKY and SHR) received cumulative i.v. injections of bosentan (10 mg/kg every 15 min up to a total load of 100 mg/kg), cumulative i.v. irt$ctions of BQ 123 (1 mg/kg every 15 min up to a total load of 10 mg/kg) or vehicle. The maximal effects of bosentan and BQ 123 on increase of renal blood flow and decrease of mean arterial blood pressure were significantly different in SHR from those in WKY. * P < 0.05 (data are presented as means f s.e.m.1.

effect of bosentan on GFR observed in SHR was due to the blockade of the above-mentioned upregulated glomeru- lar ETB (Fig. 7). Several points should be addressed in this context: (i) Bosentan reduced the GFR although renal blood flow increased. (ii) It seems very unlikely that the reduction in mean arterial blood pressure following a single injection of bosentan would have influenced GFR, since a reduction of mean arterial blood pressure down to 100 mmHg does not alter the autoregulation of GFR in SHR [22]. This is further supported by our finding that the same reduction of mean arterial blood pressure following an injection of BQ 123 did not reduce GFR. (iii> Blocking of the upregulated glomerular ETB in SHR may decrease glomerular nitric oxide (NO) synthesis, which is mediated by ETB [ 10,111. Thus, it seems possible that a decreased glomerular production of nitric oxide contributes to a reduction of GFR in SHR. by reducing glomerular capillary pressure.

xfSEM

SHR WKY Fig. 7. Effects of i.v. bolus injections of either vehicle (0.9% NaCl) (black bars), bosentan (100 mg/kg) (white bars) or BQ 123 (10 mg/kg) (gray bars) on glomerular filtration rate (GFR, ml/min/lOO g BW) in 16-week-old male SHR and WKY. Bosentan significantly decreased GFR in SHR only. * P < 0.05 (data are presented as means + s.e.m.1.

In summary, our study revealed an overexpression of the ETA receptor in smooth muscle cells of the intrarenal arteries and a pronounced upregulation of ETB receptor in the glomeruli of SHR compared to age-matched WKY. 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 mean arterial blood pressure and to a significant increase in renal blood flow. The blockade of both ETA and ETB by bosentan has no further effect on blood pressure reduction or renal blood flow increase in SHR compared to the ETA blockade by BQ123, indicating that the ETA receptor plays a major role in the maintenance of high blood pressure and regulation of renal blood flow in SHR. In contrast, no effect on GFR was observed after BQ 123 treatment either in SHR or in WKY, whereas the combined blockade of ETA and ETB by bosentan significantly de- creased GFR in SHR but not in WKY, suggesting that the glomerular ETB overexpression in SHR is of pathophysio- logical relevance.

Acknowledgements

This study was supported by grants from the Fonds der Chemischen Industrie and grants from the Zentrum fur Medizinische Forschung, Mannheim. The technical assis- tance of Mr. P. Lima, Mr. 0. Chung and Mrs. S. Schiller is greatly appreciated.

Fig. 5. In situ hybridization of ETA and ETB receptor mRNA showing increased ETA mRNA expression in smootb muscle cells from intmrenal arteries of 16week-old SHR compared to age-matched WKY, whereas no ETB mRNA expression was detectable in smooth muscle cells of SHR. (A) WKY, ETA. (B) SHR, ETA. (C) SHR-control with a ‘5S-labeled ETA mRNA sense probe. (D) SHR with a 35S-labeled ETB mRNA antisense probe. The ETB mRNA was only detectable within the glomeruli. Original magnification: X 250.

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