Diabetic Endothelin B Receptor–Deficient Rats Develop Severe Hypertension and Progressive Renal Failure
Thiemo Pfab*,,
Christa Thöne-Reineke*,
Franziska Theilig,
Ines Lange*,
Henning Witt*,,
Christiane Maser-Gluth¶,
Michael Bader||,
Johannes-Peter Stasch**,
Patricia Ruiz*,,
Sebastian Bachmann,
Masashi Yanagisawa and
Berthold Hocher*
* Center for Cardiovascular Research/Institute of Pharmacology and Institute of Anatomy, Charité Mitte, Department of Nephrology, Charité Campus Benjamin Franklin, Max Planck Institute for Molecular Genetics, and || Max-Delbrück Center for Molecular Medicine, Berlin, Germany; ¶ Institute of Pharmacology, University of Heidelberg, Heidelberg, Germany; ** Bayer AG, Wuppertal, Germany; and Howard Hughes Medical Institute, University of Texas, Dallas, Texas
Address correspondence to: Prof. Berthold Hocher, Center for Cardiovascular Research/Institute of Pharmacology, Charité Mitte, Hessische Strasse 3-4, 10115 Berlin, Germany. Phone: +49-30-450-514098; Fax: +49-30-450-514938; E-mail: berthold.hocher{at}charite.de
Received for publication August 9, 2005.
Accepted for publication January 18, 2006.
The endothelin (ET) system has been implicated in the pathogenesisof diabetic nephropathy. The role of the ET-B receptor (ETBR)is still unclear. The effect of ETBR deficiency on the progressionof diabetic nephropathy in a streptozotocin model was analyzedin four groups: (1) Homozygous ETBR-deficient (ETBRd) diabeticrats, (2) ETBRd rats, (3) diabetic controls, and (4) wild-typecontrols. BP and kidney function were measured for 10 wk, followedby biochemical and histologic analysis of the kidneys. The studydemonstrates that ETBRd diabetic rats on a normal-sodium dietdevelop severe hypertension, albuminuria, and a mild reductionof creatinine clearance. The strong BP rise seems not to becaused by activation of the renin-angiotensin-aldosterone systemor by suppression of the nitric oxide system. Elevated plasmaET-1, possibly reflecting a reduced ETBR-dependent clearance,seems to cause the severe hypertension via the ETA receptor.The results do not support the hypothesis that a reduction ofETBR activity inhibits the progression of diabetic nephropathy.The study demonstrates for the first time that the combinationof diabetes and ETBR deficiency causes severe low-renin hypertensionwith progressive renal failure.
Diabetic nephropathy is the leading and most costly cause ofESRD and dialysis treatment in industrialized countries (1,2).Strategies to stop its progression are still not effective enough.The endothelin (ET) system might be an interesting novel targetfor the treatment of diabetic nephropathy (3–6). Besidestheir potent vasoconstrictive properties, ET are profibrotic-actingparacrine hormones, especially in the kidney (7). The ET systemseems to be particularly important for the pathogenesis of diabeticnephropathy: (1) The renal ET system is activated in patientswith diabetic nephropathy as well as in animal models of diabetes-inducedkidney damage (8–10), (2) a primary activated renal ETsystem causes kidney fibrosis in ET-1 transgenic mice (7,11)as well as in ET-2 transgenic rats (12), and (3) blocking theET system using ET-A receptor (ETAR) or combined ETAR/ETBR antagonistsimproves protein and albumin excretion and reduces pathologicmatrix protein synthesis in diabetic animals in a BP-independentmanner (3–6).
There is evidence that the ETBR might play a role in remodelingprocesses of the kidney (13). This led us to the hypothesisthat reduction of ETBR activity might be protective againstdiabetic nephropathy. A widely used ETBR-deficient (ETBRd) ratmodel, developed by the group of Yanagisawa (14–16), servedas a tool to test this hypothesis. In this study, we analyzedthe effect of ETBR deficiency on the progression of diabeticnephropathy in rats with streptozotocin (STZ)-induced diabetes.
Animals and Study Protocol
ETBRd rats (ETBRsl/sl) and wild-type (WT) controls were providedby Yanagisawa (16). All animal experiments were conducted inaccordance with local institutional guidelines for the careand use of laboratory animals. Genotyping was accomplished bystandard PCR of genomic DNA as described previously (15,16).Animals were kept on a standard diet (0.2% sodium) and waterad libitum. ETBRd rats are known to be normotensive on a sodium-deficientdiet and mildly hypertensive on a standard diet and exhibitsevere hypertension on a high-sodium diet (15). We analyzedfour groups of 6-mo-old male rats: Homozygous ETBRd rats withSTZ-induced diabetes (ETBRd-STZ; n = 6), homozygous ETBRd nondiabeticrats (ETBRd; n = 9), diabetic controls (STZ; n = 8), and WTcontrols (n = 6). The animals received a single tail-vein injectionof STZ (65 mg/kg) or vehicle as described previously (9). OnlySTZ-treated animals with plasma glucose concentrations >15mmol/L after 48 h were included in the study. Systolic BP andheart rate were measured in weeks 0, 5, and 10 by tail plethysmographyas described previously (17). All animals were weighed and placedin metabolic cages in weeks 0, 5, and 10. Blood was taken onEDTA from the retro-orbital vein plexus. Plasma was obtainedafter centrifugation (4000 x g, 3 min) and stored at –20°C.The animals were killed after week 10, and heart and kidneyswere excised, washed in ice-cold saline, blotted dry, and weighed.The left kidney was frozen immediately in liquid nitrogen. Theright kidney and the heart were formalin fixed (4%, 24 h).
Plasma and Urine Measurements
Glucose, osmolality, sodium, potassium, creatinine, urea, andalbumin were measured using an automated analyzer (Hitachi 717,Boehringer Mannheim, Germany). The endogenous creatinine clearancewas used as a surrogate for the GFR. Plasma renin concentration(PRC) and activity (PRA) were determined by an indirect enzyme-kineticassay based on the generation of angiotensin I according topublished methods (18). Measurement of urinary aldosterone wasperformed by RIA after organic solvent extraction and chromatographicpurification on celite columns (19). Urinary excretion of cGMPwas determined in triplicate using a commercial RIA kit (IBL,Hamburg, Germany) as described previously (20). Urinary excretionof nitric oxide (NO) metabolites (nitrite and nitrate) was measuredusing a commercial test kit (Boehringer Ingelheim, Mannheim,Germany). Briefly, nitrate is reduced to nitrite, which thenreacts with sulfanilamide and Griess chromophore. The resultingdiazo dye is spectrophotometrically quantifiable at 540 nm (21).Measurement of ET-1 concentration in plasma and urine was performedas described previously (22) using a commercially availableELISA-Kit (Immundiagnostik, Bensheim, Germany).
Histology
All samples were embedded in paraffin; cut in 3-µm sections;and submitted to periodic acid-Schiff (PAS), elastica, and Siriusred staining. The analysis was performed on the basis of a previousreport (7). Glomerulosclerosis was defined by the presence ofPAS-positive material within the glomeruli. A semiquantitativescore was used to grade a minimum of 80 glomeruli per specimen.A score from 1 to 4 was assigned according to the percentageof PAS-positive material within each glomerulus (<25%, >25%,>50%, and >75%). All samples were evaluated blinded andindependently by two investigators (23). A glomerulosclerosisindex was calculated by averaging the grades assigned to allglomeruli. The media/lumen ratio of all cross-cut intrarenalarteries that were found in one histologic section per kidneywas analyzed on the elastica-stained specimens using a videomicroscope connected to a Macintosh computer. The images obtainedwere processed using the NIH Image 1.61 program (Bethesda, MD).Interstitial matrix deposition was evaluated after Sirius redstaining. Thirty random microscopic images from interstitialregions of each specimen (not showing any glomeruli or vessels)were taken by digital camera. The percentage of red-stained(fibrotic) interstitial area was calculated using the ImageJ1.30 program.
Immunohistochemistry
Paraffin sections (3 µm) were dewaxed and rehydrated,and antigen retrieval was carried out by heating in a microwaveoven in 2.9 g/L citrate buffer (pH 6.0, 20 min) or by incubatingin a pressure cooker (5 min). After blocking in 5% skim milk,sections were incubated with primary rabbit antibodies againstrenin (gift from Prof. Dr. A. Kurtz, Regensburg, Germany) andneuronal NO synthase (nNOS) (Axxora, Grünberg, Germany)for 2 h at room temperature followed by an overnight incubation.Detection of bound antibodies was performed using biotinylatedsecondary anti-rabbit antibodies and a catalyzed signal amplificationsystem (DakoCytomation, Hamburg, Germany) based on the streptavidin-biotin-peroxidasereaction, according to the manufacturer’s instructions.A signal was generated by incubation with diaminobenzidine andH2O2. For evaluation of the arteriolar renin status, the numberof renin-positive sites at the juxtaglomerular apparatus andat upstream locations in preglomerular vessels was determinedwithin an area of 100 glomeruli. The evaluation was based onthe well-established fact that, with varying stimuli, a metaplastictransformation occurs between renin-containing and typical smoothmuscle cells of the afferent arteriolar wall, thereby displayinga length shift of the immunoreactive portion of the vessel inan up- or downstream direction of the blood stream (24). Thechanges correspond to the levels of renal renin synthesis andto plasma renin levels under various conditions (24). The amountof renin mRNA and immunoreactive renin-containing arteriolesare known to be largely co-localized and to vary in parallel(25). Quantification of nNOS was performed similarly, as describedpreviously (25).
Western Blot
The frozen left kidney was pulverized in liquid nitrogen andthen homogenized by ultrasonic homogenizer (20 s) in ice-coldbuffer (1 µl/mg) that contained 250 mmol/L sucrose, 10mmol/L triethanolamine, and 1 tablet of protease inhibitor cocktailper 50 ml (Roche, Grenzach-Wyhlen, Germany). The homogenatewas centrifuged (4000 x g, 4°C, 10 min), and the supernatantwas ultracentrifuged to obtain a fraction enriched for membranes(200,000 x g, 4°C, 60 min). The pellet was resuspended andstored at –80°C. Protein concentrations were determinedusing the BCA Protein Assay Kit (Pierce, Rockford, IL), andthe samples were diluted accordingly with modified Laemmli bufferto ensure equal loading. Membrane proteins (25 µg/lane)were separated by SDS-PAGE (10%) and wet-blotted onto nitrocellulosemembranes. Coomassie staining of gels and Ponceau staining ofmembranes confirmed equal loading of proteins. Membranes wereblocked with 5% skim milk (60 min) and then incubated overnight(4°C) with primary rabbit antibodies directed against ETBR(1:200; gift from Prof. Dr. W. Müller-Esterl, Frankfurt,Germany), the subunit of the epithelial sodium channel (ENaC,1:200; Biotrend, Köln, Germany), and primary sheep antibodiesagainst ETAR (1:200; Axxora, Grünberg, Germany). Afterextensive washing, blots were incubated with a horseradish peroxidase–linkedanti-rabbit (anti-sheep for ETAR) IgG (60 min, 1:3000; DakoCytomation).Immunoreactive bands were detected using an enhanced chemiluminescencekit (Amersham Pharmacia, Freiburg, Germany) and were subsequentlyquantified with the Bio-Profil Bio-1D 97.04 software (Froebel,Wasserburg, Germany).
Statistical Analyses
Data were analyzed with SPSS 11.5 (SPSS, Inc., Chicago, IL).Results are expressed as mean ± SD. Differences betweengroups were compared by the nonparametric Kruskal-Wallis andthe Mann-Whitney U tests. All tests were two-sided, and P <0.05 was considered significant.
Basic Characterization of the Model
As expected, a strong rise of plasma glucose and osmolality,urinary glucose excretion, fluid intake/excretion, weight loss,and bradycardia is observed after the induction of diabetes,independent of the genotype (Table 1).
Table 1. Plasma/urine glucose, osmolality, fluid balance, body weight, and heart rate (week 10 after induction of diabetes)a
BP
As described previously (15) ETBRd animals are hypertensivewhen on a normal-salt diet. Baseline systolic BP levels are164 ± 12 mmHg in ETBRd versus 131 ± 13 mmHg inWT rats (P < 0.001). After induction of diabetes, there isa marked and significant rise of systolic BP to a maximum of188 ± 12 mmHg in the ETBRd-STZ group, whereas BP fallsslightly but significantly in the STZ group (Figure 1).
Figure 1. Systolic BP over the course of the experiment. *P < 0.05, **P < 0.01 versus nondiabetic group of same genotype. Data are mean ± SD.
Kidney Function and Morphology
Kidney function deteriorates faster in the diabetic ETBRd animals.After 10 wk, plasma creatinine (53 ± 3.4 versus 39 ±3.4 µmol/L; P < 0.01; Figure 2A) and albuminuria (10.4± 5.0 versus 2.3 ± 3.9 mg/d; P < 0.01; Figure2B) are increased in the ETBRd-STZ group as compared with theSTZ group. The same applies for the decrease of creatinine clearance(ETBRd-STZ 3.2 ± 0.8 versus STZ 4.3 ± 0.9 ml/minper kg; Table 2), whereas there is only a trend toward an increaseof plasma urea (ETBRd-STZ 17 ± 3.9 versus STZ 13 ±4.7 mmol/L; P = 0.09; Table 2). Morphologic data are summarizedin Table 2. The media/lumen ratio of the intrarenal arteriesis significantly elevated in the ETBRd-STZ group as comparedwith the STZ group (P < 0.01; Table 2). Changes of the media/lumenratio of the intracardiac arteries follow the same pattern asin the kidney (Table 2). Representative micrographs showingsignificantly enhanced glomerulosclerosis in the diabetic groupsare shown in Figure 3. There is a trend toward an increasedglomerulosclerosis index in the ETBRd-STZ group as comparedwith the STZ group (P = 0.12).
Figure 2. Plasma creatinine (A) and urinary albumin excretion (UAE) (B) over the course of the experiment. UAE of the streptozotocin-induced diabetic (STZ) group in week 10 is not yet significantly elevated compared with wild-type (WT) rats. However, UAE per kg body weight is significantly elevated in the STZ group (data not shown). *P < 0.05, **P < 0.01, ***P < 0.001 versus nondiabetic group of same genotype; P < 0.05, P < 0.01 versus STZ; P = 0.08 versus WT. Data are mean ± SD.
Figure 3. Representative glomeruli from all groups. Enhanced glomerulosclerosis is present in the diabetic groups (STZ-induced diabetic endothelin B receptor–deficient [ETBRd-STZ] and STZ). For glomerulosclerosis index, see Table 2. Magnification, x1000, periodic acid-Schiff staining.
BP-Regulating Systems
To identify mechanisms that are involved in the diabetes-inducedhypertension in ETBRd rats, we further analyzed several candidatesystems: The renin-angiotensin-aldosterone system (RAAS), theNO system, and the ET system. There is no evidence of the circulatoryRAAS being upregulated in the diabetic ETBRd group. On the contrary,PRA, PRC, and urinary aldosterone excretion is significantlylower in the diabetic ETBRd group as compared with the STZ group(Table 3). A similar tendency (n = 3) is present as far as theimmunohistochemical evaluation of renin- and nNOS-positive glomeruliis concerned. Evaluation of nNOS was performed because studiesin the in vitro perfused juxtaglomerular apparatus give someevidence that nNOS is acting as a positive modulator of reninsecretion (26). The quantity of the aldosterone-inducible subunitof ENaC is not significantly different among the four groupsin the Western blot analysis (data not shown). Electrolyte excretionis not significantly different between both diabetic groups(Table 3).
Table 3. Evaluation of possible mechanisms that lead to hypertension in the ETBRd-STZ group (week 10)a
Because it is widely known that ET-1 via the ETBR activatesendothelial NO synthase (eNOS), we analyzed urinary excretionof NO metabolites and its second messenger cGMP as overall markersof NO production. Nitrogen intake was similar in all groupsas calculated from food intake (data not shown). Diabetic ETBRdrats show an elevated urinary nitrite/nitrate excretion (183%;P = 0.07; Table 3) and cGMP excretion (Figure 4).
Figure 4. Urinary excretion of cGMP. *P < 0.01, **P < 0.001 versus nondiabetic group of same genotype; P < 0.05, P < 0.01 versus STZ. Data are mean ± SD.
The urinary ET-1 excretion (as an approximation of renal ET-1synthesis) is strongly elevated in both diabetic groups at theend of the study (Table 3). Plasma ET-1 concentrations are significantlyelevated in the ETBRd-STZ group as compared with the ETBRd group5 wk after induction of diabetes, whereas there is no elevationin the STZ group at any time (Figure 5).
Figure 5. Plasma ET-1 concentration over the course of the experiment. *P < 0.05 versus nondiabetic group of same genotype; P < 0.05, P < 0.0001 versus STZ; P < 0.05, P < 0.001 versus WT. Data are mean ± SD.
To quantify renal ET receptor expression, we performed a Westernblot analysis. Figure 6 depicts the regulation of renal ET receptors.ETBR deficiency causes a moderate downregulation of renal ETAR,whereas diabetes leads to a 57% increase of renal ETBR.
Figure 6. Western blot for renal ETAR and ETBR; n = 3 per group. Band size is approximately 41 kD for ETAR and approximately 37 kD for ETBR. *P < 0.05 versus nondiabetic group of same genotype; P < 0.05 versus STZ; P < 0.05 versus WT. Data are mean ± SD.
This study demonstrates for the first time that ETBRd diabeticrats that are on a normal-sodium diet develop severe low-reninhypertension and albuminuria and a mild reduction of creatinineclearance. The strong BP rise in the ETBRd-STZ rats seems notto be caused by activation of the circulating RAAS or by suppressionof the NO system. The elevated plasma ET-1, possibly reflectinga reduced ETBR-dependent elimination of ET-1, seems to causethe severe hypertension via the remaining ETAR.
The STZ model in our setting reflects an early stage of diabeticnephropathy. The STZ-treated rats do not yet develop significantalbuminuria and show only a modest rise of plasma creatinineand
vascular kidney damage after 10 wk. In contrast, the simultaneouspresence of ETBR deficiency and diabetes causes severe hypertensionand enhanced functional renal impairment within this period.We therefore created a rat model of a more advanced stage ofdiabetic nephropathy than the pure STZ model. This is similarto other STZ models with concomitant BP elevation such as spontaneouslyhypertensive rats (27) and (even more pronounced) transgenic(mRen-2)27 rats (28). In contrast to those models, a furthersignificant BP rise happens after the induction of diabetesin ETBRd rats, which enhances the kidney damage mainly duringthe course of the experiment.
There are two possible explanations for the finding of enhancedalbuminuria in the ETBRd diabetic rats. On the one hand, thesevere hypertension on top of hyperglycemia certainly acceleratesits occurrence. On the other hand, increased renal ET-1 activity(reflected by increased urinary ET-1 excretion in diabetic rats)and reduced clearance (because of ETBR deficiency) might contributeto its development. Renal ET-1 has been implicated in podocytedamage and development of proteinuria (29), and ET receptorantagonists are known to reduce proteinuria and renal matrixprotein expression in rats with STZ-induced diabetes (3).
The decrease of kidney function and the BP rise in the ETBRd-STZgroup go in parallel. Although we cannot rule out that the BPrise results secondary to the kidney damage, we assume the opposite,because the differences of renal impairment between both diabeticgroups do not seem strong enough to explain the pronounced differencesof BP.
Because recent literature about renin in diabetes is controversial,we used several independent methods to evaluate its possibleinvolvement in BP regulation in our model. We could not detectchanges in PRA, PRC, and renin-positive glomeruli in the STZgroup after 10 wk. Reports of an elevated PRA (30) contrastwith others reporting reduced PRA in patients with type 1 diabetes(31). A study with STZ-treated rats showed a reduction of PRAafter 4 wk (32), whereas an older study observed a biphasicreaction with an increase in the first week followed by a reductionof PRA until week 8 (33). Our results show a highly significantreduction of circulating renin only in the ETBRd-STZ group.This downregulation supposedly can be attributed to a negativefeedback mechanism as a result of severe hypertension. Our resultsdo not rule out a diabetes-induced activation of local tissueRAAS, e.g., in the kidney or the heart. Conversely, urinaryaldosterone excretion is elevated in both diabetic groups. However,aldosterone excretion in ETBRd rats is much lower than in thediabetic WT animals and therefore cannot explain the BP rise.It has been demonstrated in rats that ET-1 exerts a secretagogueeffect on adrenal aldosterone secretion that seems to be mediatedprimarily by ETBR (34). The reduced aldosterone excretion inthe ETBRd groups might be attributed to the lack of ETBR inthe adrenal zona glomerulosa. We assume that (pseudo)hyponatremia,hyperosmolality, and increased urinary potassium excretion aredue to diabetes-induced electrolyte disturbances and acidosisindependent of the genotype (35).
Another important BP-regulating system is the NO-cGMP system.This is especially interesting because it is widely known thatET-1 via the ETBR activates eNOS (36). Our results suggest thatthe system is not downregulated and support the concept of asecondary counterregulation of the NO system. Excretion of NOend products and even more excretion of its second messengercGMP is upregulated in ETBRd-STZ rats. This suggests that inour model, the NO system seems activated by mechanisms thatdo not involve the ETBR. However, because urinary nitrate/nitriteand cGMP concentrations give only a limited reflection of theendogenous NO system activity, definite conclusions cannot bedrawn.
The mechanisms underlying the (reversible) bradycardia thattypically is found in the STZ model are not fully understood(37). Studies in isolated cardiac preparations indicate thatSTZ-induced diabetes is associated with a depression in basalspontaneous pacemaker rate (38). However, chronotropic responsivenessto catecholamines was found to be intact (38) or even increased(39). Catecholamine action as a cause of hypertension thereforeis unlikely as we would expect an associated rise of heart rate,which in contrast does not differ between both diabetic groups.
In the diabetic ETBRd rats, plasma ET-1 is strongly elevated.ET-1 is one of the most potent vasoconstrictors known and viathe remaining ETAR possibly causes the severe hypertension.An exogenous two-fold increase of plasma ET-1 already causesa significant rise of systemic vasoconstriction and BP in vivo(40–42), whereas there is a six-fold increase of plasmaET-1 in this study. However, exogenous administration of ET-1might not be directly comparable to conditions of this study.Different mechanisms might be involved in the rise of plasmaET-1 in ETBRd-STZ rats.
The ETBR acts as a clearance receptor, eliminating circulatingET-1 (43,44). Consistently, our study confirms elevated ET-1plasma concentrations in the ETBRd group (+76% versus WT atstudy end) as described previously (15). The concomitant presenceof diabetes causes an even stronger rise of circulating ET-1(+503% versus WT at study end), which is not present in diabeticanimals with intact ETBR. The observed upregulation of ETBRin diabetic animals might be necessary to eliminate the increasedamount of ET-1 produced in diabetes (8,45). The rise of plasmaET-1 in ETBRd-STZ rats therefore might be attributed to thesynergistically acting effects of diabetes-induced activationof the ET system and an impaired ability to eliminate the increasedquantity of ET-1 via the ETBR.
The renal ET system is activated in diabetic animals as describedpreviously (8,9). Urinary ET-1 excretion as an approximationof renal ET-1 synthesis is equally elevated in both diabeticgroups only at the end of the experiment. This indicates thatthe elevated plasma ET-1 is not caused by an enhanced diabetes-inducedrenal ET synthesis in ETBRd animals.
Another, however speculative, hypothesis to explain our findingsis that of an activated vascular ET system as the source ofthe increased plasma ET-1, because the endothelium is the mostprobable structure that is able to liberate large amounts ofET-1. A generalized endothelial dysfunction in patients withdiabetes leading to the liberation of ET-1 has already beendiscussed (46), and the liberation of ET-1 in a state of endothelialdysfunction was observed previously (47). Moreover, ETBR seemto play a role in this context as they have been shown to beupregulated in vessels of diabetic rats (45). However, furtherstudies are needed to clarify those issues.
Study Limitations and Outlook
In the diabetic ETBRd rats, BP, plasma ET-1, creatinine, andalbuminuria rise in parallel. Because of the study design chosento answer the initial hypothesis, it is difficult to reconcilewhat occurs first: Hypertension and renal damage followed byET-1 increases or ET-1 activation as a result of decreased clearancefollowed by BP rise and renal damage. For addressing the roleof ET-1 in mediating the hemodynamic and structural changes,further study designs should consider two approaches. One istreating the hypertension with a non-ET modulator such as avasodilator to determine whether this abolishes structural changesin the kidney. The second is to administer long term an ETARantagonist to sort out the direct contribution of the ET-1/ETARcomplex.
Our data do not support the initial hypothesis that a reductionof ETBR activity inhibits the progression of diabetic nephropathy.However, there might be differences between our ETBRd animalmodel and pharmacologic antagonism. This assumption is supportedby the finding that diabetes does not affect BP in rats thatare heterozygous for the ETBR defect (T.P. and B.H., unpublishedobservations), thus indicating that ETBR antagonists that blockonly part of the receptors (as do most available substances)might have different effects. However, BP and kidney functionshould be monitored closely if ETBR antagonistic drugs are administeredto patients with diabetes in future studies.
Acknowledgments
This study was supported by the Deutsche Forschungsgemeinschaft(DFG) to Dr. Berthold Hocher (DFG Ho 1665/5-2).
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
T.P. and C.T.-R. contributed equally to this work.
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Top
Abstract
Introduction
subunit of the epithelial sodium channel (ENaC, 1:200; Biotrend, Köln, Germany), and primary sheep antibodies against ETAR (1:200; Axxora, Grünberg, Germany). After extensive washing, blots were incubated with a horseradish peroxidase–linked anti-rabbit (anti-sheep for ETAR) IgG (60 min, 1:3000; DakoCytomation). Immunoreactive bands were detected using an enhanced chemiluminescence kit (Amersham Pharmacia, Freiburg, Germany) and were subsequently quantified with the Bio-Profil Bio-1D 97.04 software (Froebel, Wasserburg, Germany). 
