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
Received7 April 1995;accepted
31 August1995
Abstract
Objectiue: The renal endothelin system has been implicated in the development and maintenanceof 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) comparedto
age-matchedWKY. 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 receptorsin SHR with bosentan(A and B receptor blockade) as well as with BQ 123
(A receptor blockade) led to a significant decreasein MAP (- 18.6f 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 bosentanhad no further effect on MAP reduction or RBF increasein SHR comparedto 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
bosentansignificantly decreasedGFR in SHR by about 50% but not in WKY. Conclusions: Our data demonstrateda correlation between
the overexpressionof vascular ETA receptorsand the pronounced upregulation of glomerular ETB receptorsin the kidneys of SHR and
their impact on the regulation of renal blood flow, glomerular filtration rate and blood pressurein these animals.
Keywords:
Rat, spontaneously
hypertensive;Rat,kidney;Hypertension;
Endothelin receptorantagonist;Endothelinnxeptor
1. Introduction
Endothelin is a 21-amino-acid peptide that was originally characterized in the supematant of vascular endothelial cells and is, by at least one order of magnitude, the
Correspondingauthor: Universituniversititklinikum Charitt?der
HumboldtUniversitit zu Berlin, Abteilungfir Nephrologie(Medizinische
Klinik 5), Schumannstr.
20-21,D-I01I7 Berlin, Germany.Tel.: ( + 49-30)
2802-5865; Fax.:( + 49-30)280:2-8471.
most potent vasoconstrictor known so far [ 1,2]. The biological effects of endothelin are mediated by plasma-membrane-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 isopeptides (ETA: ET-l r ET-2 > > ET-3; ETB: ET-l = ET-2
l
0008-6363/%/$15.000 1996ElsevierScienceB.V. All rights reserved
SSDI 0008-6363(95)00200-6
Time
for primary review 42 days.
500
B. Hocher et al./ Cardiovascular
= 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 mesangial 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 phospholipaseA,
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 endothelinsecreting 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
exogenousendothelin as comparedto control rats. Surprisingly, 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 endothelin 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 systemis involved in the pathogenesisof hypertension in these animals. The pressor responseto endothelin-1 is increased in renal arteries obtained from SHR
[ 10,111.Furthermore, polyclonal endothelin antibodies increasethe 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 kidneys of SHR using Scatchard analysis, receptor autoradiography, Northern blot analysis and in situ hybridization. In addition, we used the ETA receptor antagonist BQ
123 and the mixed (A/B) endothelin receptor antagonist
bosentan[ 131to examine the functional importance of the
paracrine endothelin systemin 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 hypertensive rats (SHR) were obtained from Mllegard (Denmark) and fed a commercial diet (Altromin@, Altromin
GmbH, Germany) and given water ad libitum. All animal
experiments were conductedin accordancewith local institutional guidelines for the care and use of laboratory
animals. [‘251]Endothelin-l (2000 Ci/mmol) was obtained
Research 31 (1996) 499-510
from Du Pont, Germany. 32P-dATP and 35S-UTPwas from
Amersham(Braunschweig, Germany). Unlabeled ET-l was
from Peninsula Laboratories, Inc. (Germany). The mixed
(A/B) endothe1in receptor antagonist bosentan (Ro 470203, 4-tert-butyl-N-[6-(2-hydroxy-ethoxy)-5-(2-methoxyphenoxy)-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 supematantwas decanted
and centrifuged at 4°C for 30 min at 40000 X g. The
pellet, consisting of crude plasma membranes,was resuspended 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 assayswere 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-’ bacitracin, 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 increasedfrom 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. Nonspecific binding was assessedin 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).
B. Hocher et al./ Cardiovascular
2.4. Autoradiography
of tissue endothelin receptor
sub-
Research 31 (19%) 499-510
501
2.6. In situ hybridization
types
Receptor autoradiography was performed as previously
described[ 161.After anesthesiaby intraperitoneal administration of pentobarbital, the inferior vena cava was cannulated. 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 pressureduring 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. Evaluation 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 samearea 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 electrophoretically on a formamide/formaldehyde-agarose gel,
blotted to Hybond N nylon membranes (Amersham, Arlington, UK) and irradiated with UV light. After prehybridization for 2 h at 42°C the membranes were hybridized with 32P-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 according to Sambrook et al. [17]. The autoradiographswere
analyzed using a computer-aided densitometer (ScanPack’” Scanner-Densitometer). The ratio of endothelin
receptor subtype mRNA//J-actin mRNA in WKY was
defined as 100% and comparedwith 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
[ 181subclonedin pBluescript 2 + SK plasmid and a 0.5 kb
rat p-actin fragment subcloned in pGem 72. The fragments were excised from PBS plasmid, labeled with 32PdATP by the random priming method and had a specific
activity of l-3 X lo6 cpm/ml hybridization solution (n =
6 in each group).
2.6.1. Tissue preparation
Kidney samples were immediately frozen in liquid nitrogen and stored at -70°C. Cryostat sections (5 pm>
were placed on siliconized slides treated with 3-amino-propyltriethoxysilane for better adherenceand 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 plasmid (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 subclonedin pGem
72 [17-191. The plasmids were linearized with either
Xhol or Sst2. Single-stranded RNA probes, complementary (antisenseprobe) or anticomplementary (sense probe,
negative control) to cellular RNA, were obtained by run-off
transcription with T7 or T3 RNA polymerase (Transcription Kit from Boehringer Mannheim, Germany). 35S-UTP
was used for labeling the RNA probes. The specific activity of the probes was 1.O-1.5 X lo9 cpm/mg RNA. Appropriate 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, dehydratedin 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. Sections were sealed with a siliconized coverslip. After 18 h
of incubation at 50°C in a humid chamber, slides were
washedfor 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
502
B. Hocher et al./ Cardiovascular Researcii 31 (19%) 499-510
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
sectionswere developedfor 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 counterstainedby hematoxylin-eosin.
All tissues were simultaneously processedusing 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 senseand antisenseactin probes, (ii)
as a proof that the ISH signal obtained was due to RNARNA 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 experiment.
2.7. Measurement of glomerular jIltration rate (GFR),
mean arterial blood pressure (MAP), heart rate (HR), and
renal blood flow (RBF) in conscious chronically instrumented 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
catheterswere 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 damageto 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 ultrasound gel. All catheters and cables were led subcutaneously to the rats neck [21,22].
Gould Brush 2600 recorder. The Transonic flowmeter
systern determines absolute volume flow [23]. The flowprobes were precalibrated and measured absolute blood
flow with an accuracy of *2%.
2.7.3. Measurement of glomerular filtration
rate
GFR was measuredusing the inulin single-shot method
[24]. The single-shot clearance was evaluated by displaying a two-compartmentmodel 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 samplesfor determination
of serum inulin concentrationswere drawn at 0, 15, 30,90,
135 and 180 minutes after injection. Inulin was measured
by a modified /3-fructosidase method [25]. GFR is expressedas ml/min per 100s body weight (BW).
2.8. Effects of the mixed (A /B) endothelin receptor antagonist bosentan and the ETA receptor antagonist BQ 123
on MAP, HR and RBF
To examine the effect of bosentanon 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 antagonist 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 responsesto 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 antagonist 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 determination of serum inulin concentrations.
2.7.2. Circulatory measurements
Mean arterial blood pressure(MAP) and heart rate (HR)
were measuredvia the arterial line with a Stathampressure
transducerP23Db and a Gould pressureprocessorcoupled
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
2.10. Analysis of data
The unpaired Student t-test was used for the determination of statistical difference of group means. Analysis of
variance followed by t-test was used if appropriate. Results were considered significantly different at a value of
P < 0.05.
B. Hocher et al. / Cardiovascular
3. Results
3.1. ETA and ETB binding to the renal cortex
Scatchardanalysis revealed only one type of ETA (data
not shown) and ETB (Fig. 1) binding site in 16-week-old
SHR and WKY. Cortical membranesof 16-week-old SHR
exhibited a significantly higher density of ETA (P < 0.05)
and ETB (P < 0.01) comparedto age-matchedWKY (Table 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, respectively. 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 membranes 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
types
of tissue endothelin receptor sub-
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 increased2.2 f O.Cfold in the glomeruli of SHR compared
to WKY (P < 0.05). In addition, we also found a pronounced 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 increasedETA receptor density comparedto 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 respectto 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 sameETA and ETB messengerRNA transcripts of
the predicted length (4.2 and 4.7 kb long for the ETA and
503
Research 31 (19%) 499-510
the ETB, respectively) were detected in all kidney samples. ETA mRNA was overexpressed in the kidneys of
SHR as compared to WKY. In contrast, ETB mRNA was
similarly expressedin 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-labeledriboprobes (senseand antisenseprobes) 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 confirmed by a very low hybridization signal in parallel
experiments using sense probes. Furthermore, the two
cRNA antisenseprobes for ETA and ETB mRNA showed
different patternsof 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 increasedETA 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 arteries 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 representativeexperiment of [“‘]IET-1 specific binding in the
presence of the subtype-specific ligand BQ123 to membranes isolated
from the renal cortex of 16-week-oldSHR (solid circles) and WKY (open
circles) showing an increaseddensity of ETB receptorsin the renal cortex
of SHR. Scatchardtransformationsof binding data (inserts) suggestonly
one binding site in both SHR and WKY. Non-specific binding was
determinedin the presenceof unlabeled ET- 1 as describedunder “Methods” and was always less than 10% of total binding.
B. Hocher et al./Cardiovascular
Research 31 (1996) 499-510
Fig. 2. Microscopic autoradiogramshowing upregulation of ETB receptorsin 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.
B. Hocher et uI./Cardiovascukw
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 membranesof It&week-old SHR and WKY
WKY
SHR
WKY
Kci
(nmol/l)
ETA 174LtlQ
CORTEX
ETB 363f42
CORTEX
MEDULLA ETA 121k38
MEDULLA ETB 784*63
255f41 a
639f41 b
137*51
668*198
0.47f0.02
O.Qf0.03
0.46f0.02
0.8&O&I
1.o f 0.05
3.2*0.58
1.2f 0.07
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). Nonspecific binding was assessedin the presenceof 5 pmol.l- ’ of unlabeled ET-l. It ranges between 5 and 10%. Samples from crude plasma
membraneswere used at a concentrationof 0.53 mg of protein. ml- ’ . For
details see “Methods”. Values are means f s.d. of 6 separateanimals in
each group.
a P < 0.05 compared to the density of ETA in the renal cortex of
16-week-oldWKY.
b P < 0.01 compared to the density of ETB in the renal cortex of
16-week-oldWKY.
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 decreasein MAR in SHR, whereas no significant 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 messengerRNA in the kidneys of WKY and SHR. The
Notthem blots were analyzed using a densitometer.The ratio of endothelin receptor subtype mRNA (ETA or BTB)/ pactin mRNA in WKY was
defmed as 100% and compared with the ratio of endothelin receptor
subtype mRNA/ @-actinmRNA in SHR The relative amount of mRNA
expressionf s.e.m. is shown (n =*6).
Research 31 (1996) 499-510
505
lower bosentandose (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 antagonistBQ 123 has no effect on heart rate,
whereasthe mixed endothelin receptor antagonistbosentan
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% decreasein GFR in SHR,
whereas no effect of bosentanwas 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 decreasesin
mean arterial blood pressureand similar increasesin renal
blood flow, suggestingthat vascular ETA receptor overexpression is important for the regulation of renal blood flow
and contributes to high blood pressurein SHR. In contrast
to renal blood flow, glomerular filtration rate was decreasedin SHR by bosentanonly, whereasthe ETA antagonist had no influence on GFR.
4.1. Expression of ET receptor subtypes in the kidney
Receptor autoradiography revealed that the overexpression of endothelin receptor subtypes, as detected by
Scatchardanalysis in the renal cortex of SHR, was due to
an increasedETA and even more so to ETB density within
the glomexuli. Glomerular endothelin receptors could be
506
B. Hocher
et al./Curdiovascular
Research
31 (1996) 499-510
B. Hocher et al./Cardiovascular
expressed either in mesangial cells or in glomerular endothelial cells. Our techniques do not permit differentiation
between these two glomerular cell types. However, others
have reported that glomerular ETA receptorsare expressed
by mesangial cells [26,27], whereas the ETB receptor
seemsto 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 Scatchardanalysis, also found a markedly increased
cortical ETB expression. On the other hand, Gellai et al.
[29] did not observe an overexpressionof ETA receptorsin
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 overlooked 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 sensitive technique of in situ hybridization and receptor autoradiography which allows the exploration of receptor expression in different anatomical structures(i.e., blood vessels, glomeruli and tubules) we were able to demonstratea
pronounced ETA overexpression in vascular smooth muscle cells.
In addition, it is important to note that the normotensive
controls are different in both studies. We used WistarKyoto rats, whereasGellei 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 hybridization (Figs. 3 and 5) we were able to demonstrate
that the increasednumber of ETA in the branchesof renal
arteries and glomeruli of SHR was due to increasedmRNA
formation.
On the other hand, the markedly increased ETB receptor protein density found in the glomeruli of SHR obviously was not caused by an upregulation of ETB mRNA
(Fig. 3) and could have been related to decreasedETB
degradation possibly due to structural alterations of the
glomerular ETB receptor itself (e.g., by altered glycosylation or phosphorylation) leading to decreaseddegradation
or to inactivation of enzymes involved in ETB degradation.
Research 31 (!996) 499-510
507
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 Sarafotoxin 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 overexpressionin 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 demonstratesthat the decreasein blood pressure 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 antagonistbosentan.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
overexpressedvascular ETA receptors.
Our finding of a blood-pressure-lowering effect of BQ
123 given to SHR confirms previous reports [29,30]. However, chronic oral treatment of SHR with bosentan has
recently been reported to have no effect on blood pressure
[3 1I. The reasonsfor this discrepant effects of acute intravenous and of chronic oral blockade of endothelin receptors in SHR remain unclear. Possibly, the blood-pressurelowering effects of endothelin antagonistsare counteracted
in the long run by increased activities of other vasoconstrictor mechanisms. In any case, the acute blood-pressure-lowering effects of BQ 123 and bosentan clearly
show that the endothelin system contributes substantially
to elevated blood pressurein SHR.
4.4. Effects of BQ 123 and bosentan on glomerular$ltration rate
A bolus injection of the mixed (A/B) endothelin receptor antagonist bosentan decreasedGFR (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.
508
B. Hocher et al./Cardiovascular
Research 31 (1996) 499-510
B. Hocher er al./ Cardiovascular
Research 31’ (1996) 499-510
509
xfSEM
1
m
SHR
0
WKY
*
n
Bosenton
100 n-g/kg
BQ 123
10 n-g/kg
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-oldmale SHR and WKY. Bosentansignificantly decreasedGFR
in SHR only. * P < 0.05 (data are presentedas means+ s.e.m.1.
Vehicle
Fig. 6. Maximal changesof 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 decreaseof
mean arterial blood pressure were significantly different in SHR from
those in WKY. * P < 0.05 (data are presentedas meansf s.e.m.1.
effect of bosentan on GFR observed in SHR was due to
the blockade of the above-mentionedupregulated glomerular ETB (Fig. 7). Several points should be addressedin this
context: (i) Bosentan reduced the GFR although renal
blood flow increased. (ii) It seemsvery 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 pressuredown 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 seemspossible that a decreased
glomerular production of nitric oxide contributes to a
reduction of GFR in SHR.by reducing glomerular capillary
pressure.
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-matchedWKY.
Blockade of endothelin receptorsin SHR with bosentan(A
and B receptor blockade) as well as with BQ 123 (A
receptor blockade) led to a significant decreasein mean
arterial blood pressureand to a significant increasein renal
blood flow. The blockade of both ETA and ETB by
bosentanhas no further effect on blood pressurereduction
or renal blood flow increase in SHR comparedto 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
treatmenteither in SHR or in WKY, whereasthe combined
blockade of ETA and ETB by bosentan significantly decreasedGFR in SHR but not in WKY, suggestingthat the
glomerular ETB overexpression in SHR is of pathophysiological 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 assistance 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 increasedETA mRNA expressionin smootb muscle cells from intmrenal arteriesof
16week-old SHR compared to age-matchedWKY, whereasno 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 senseprobe. (D) SHR with a 35S-labeledETB mRNA antisenseprobe. The ETB mRNA
was only detectable within the glomeruli. Original magnification: X 250.
510
B. Ho&r
et al./Cardiouascular
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