Congestive heart failure (CHF) is characterized by left-ventricular (LV) dysfunction, increased systemic and renal vascular resistance, and neurohumoral activation (1). Plasma concentrations of endothelin-1 (ET-1), a potent vasoactive peptide produced by endothelial and renal epithelial cells, are increased in CHF (2-6). The effects of ET-1, the primary circulating isoform of the ET peptide family, are mediated via the ET-1-selective endothelin-A (ETA) receptor and the nonselective endothelin-B (ETB) receptor (3,6,7). The ETA receptor is expressed in vascular smooth-muscle cells and nonvascular tissues including the kidney, and the ETB receptor is preferentially expressed in endothelial cells (7-10). Because circulating concentrations of ET-1 are relatively low, it has been suggested that ET-1 acts primarily as an autocrine/paracrine hormone, and local tissue endothelin systems have been described in the kidney, the heart, the lung, and the brain (6,11). Studies measuring blood flow in renal, femoral, and coronary vessels under physiologic conditions have shown that the renal circulation is ∼10-fold more sensitive to the vasoconstrictor effects of endothelin than are other vascular beds (6). Administration of exogenous ET-1 at concentrations mimicking those observed in CHF is associated with systemic vasoconstriction, an increase in renal vascular resistance, and a decrease in renal blood flow, suggesting an important role for ET-1 in the pathophysiology of renal dysfunction associated with CHF (8,12,13).
However, local activation of the renal ET system as well as functional importance of ETA-receptor activation for renal hypoperfusion in CHF remains poorly defined. It was therefore the objective of this study to characterize local ET-1 as defined by tissue concentration and gene expression as well as functional integrity and gene expression of the renal ETA receptor and to define its role for renal vasoconstriction associated with CHF.
Male rabbits (Chinchilla Bastard, 3.0-3.5 kg, aged 3-4 months) were permanently instrumented with Doppler flow probes (DBF-120 A-M, Roditi, Hamburg, Germany) around the left renal artery via an abdominal midline incision under general anesthesia (ketamine, 50 mg/kg; diazepam, 6 mg/kg; pancuronium, 0.6 mg/kg; i.v.) and controlled ventilation (Rodent Ventilator 994601; TSE, Regensburg, Germany). Rabbits were allowed water ad libitum and free access to standard chow (Ringkanin) with the exception of 12 h before surgery. They were housed individually in a 12-h dark/light cycle-controlled room. All protocols were approved by the local standing committee and authority on animal research. The investigation confirmed with the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH Publication No. 85-23, revised 1985).
Two weeks later, when rabbits had fully recovered from surgery, CHF was induced by four repetitive infusions of epinephrine (300 μg/kg/60 min, i.v., each) administered at 12-day intervals. In pilot experiments, this protocol and dosage had been established and proved the best to produce reproducibly advanced CHF with renal vasoconstriction. With fewer infusions and lower dosages, only mild CHF without renal vasoconstriction was observed; with higher dosages, acute mortality was high. Previously our group reported the development of CHF after repetitive epinephrine infusions in rabbits (14).
All experiments were performed in conscious rabbits trained to stand quietly in examination boxes providing constant external conditions. Four days before the first (baseline) and 4 days after the fourth (CHF) infusion of epinephrine, cardiovascular hemodynamics were measured. Transthoracic echocardiographic measurements were obtained by left parasternal short-axis imaging by using a Hewlett-Packard Sonos 1500 system with a 5-MHz electronic probe (Andover, MA, U.S.A.). A two-dimensionally guided motion mode (M-mode) image of the left ventricle was recorded on VHS videotape and on paper at 100 mm/s with special care to optimize endocardial borders. From the recordings, LV end-diastolic (LVEDd) and end-systolic diameters (LVESd) were analyzed. Fractional shortening (FS) was calculated as FS = [(LVEDd − LVESd)/LVEDd] and expressed as a percentage. By using a standard pressure transducer (P23XL; Statham Instruments, Dusseldorf, Germany) mean arterial blood pressure (MAP) was continuously measured via a 24-gauge catheter placed in the central ear artery, which was also used for blood sampling. Heart rate (HR) and renal blood flow velocity (RBF) were measured via the Doppler flow probes by using a directional pulsed Doppler flowmeter (545C-4; Department of Bioengineering, University of Iowa, Ames, IA, U.S.A.). Renal vascular resistance (RVR) was calculated as RVR = MAP/mean RBF and expressed as arbitrary resistance units U (mm Hg/kHz). All hemodynamic parameters were recorded simultaneously on a multichannel recorder (Recomed; PPG Hellige, Freiburg, Germany). Serum osmolality was measured by the freezing-point method, and serum concentration of creatinine was measured with an automatic analyzer (SMAC; Technicon, Munich, Germany).
At baseline and CHF, blood samples were collected, and the acute systemic and renal hemodynamic effects of BQ-123 (Saxon Biochemicals, Bachem Inc., U.S.A.) administered as a bolus of 1 mg/kg, i.v., were measured online. BQ-123 (cyclo(D)-Trp-5-D-Asp-L-Pro-D-Val-L-Leu) is a peptidic selective ETA-receptor antagonist with a binding Ki of 17-25 nM for the ETA receptor and 11,100-31,000 nM for the ETB receptor (15,16). The dose of BQ-123 had been chosen based on the pharmacokinetic profile and according to the literature to provide short-term antagonism of the ETA receptor. At the end of the experimental protocol, rabbits were killed by methohexital administration. Tissues were rapidly excised, snap-frozen in liquid nitrogen, and stored at −80°C until analysis. Then 3-mm coronal sections of left ventricles were fixed in neutral buffered formalin and stained with hematoxylin-eosin to evaluate histopathologic changes.
Blood samples were collected into ethylenediamine tetraacetic acid (EDTA)-containing tubes and kept on ice until centrifugation at 976 g and 4°C for 15 min to separate the plasma. Plasma samples were stored at −80°C until analysis. Circulating ET-1 plasma concentrations were measured with commercially available radioimmunoassays (RK-023-01; Phoenix Pharmaceuticals, Inc., Belmont, CA, U.S.A.) according to the manufacturers' instructions after extraction by using Sep-Pak C18 cartridges (Waters Associates, Milford, MA, U.S.A.), as previously described (17) and expressed as picograms per milliliter.
For measurement of renal ET-1 tissue concentration, samples (300 mg) were homogenized as previously described (18), extracted by using Sep-Pak C18 cartridges, and the radioimmunoassay performed according to the previously mentioned procedures. Renal tissue protein content was measured according to the Folin phenol method of Lowry et al. (19). Immunoreactive ET-1 in renal tissue was measured as picograms per milliliter homogenate, normalized for protein content, and expressed as pg ET-1/mg tissue protein.
Renal total cellular RNA was isolated according to the method of Chirgwin et al. (20) and quantified by absorbance at 260 nm For Northern blot analysis, aliquots (40 μg) were size-fractionated by using the formaldehyde-agarose method (21) and stained with ethidium bromide to confirm the quantity and quality of RNA before and after transfer. After capillary transfer onto nylon membranes (GeneScreen Plus; NEN, Boston, MA, U.S.A.) with 10× SSC (1.5 M sodium chloride, 0.15 M sodium citrate, pH 7.0), the RNA was immobilized with a Stratalinker 1800 (Stratagene, La Jolla, CA, U.S.A.). Then after prehybridization for 4 h at 42°C in a buffer prepared according to Khandjian (22), the Northern blots were hybridized overnight at 42°C with a random-primed, α-[32P]dCTP-labeled, rabbit ETA-receptor cDNA probe in the same buffer. The rabbit ETA-receptor cDNA probe was a 514-bp fragment generated from rabbit renal total cellular RNA by reverse transcription and polymerase chain reaction with Superscript II (Gibco BRL, MD, U.S.A.), Taq polymerase (Boehringer Mannheim), and the primer pair 5′-GCCTCAGGGCATCCTTTTGG-3′ and 5′-GGAGGCAACTGCTCTGTACC-3′. Then the cDNA probe was electrophoresed through a 2% agarose gel extracted and purified with a commercially available kit (Qiaex II; Qiagen, Hilden, Germany) according to the manufacturer's protocol, and sequenced (Sequiserve, Munich, Germany). After hybridization, blots were washed in 2× SSC/0.1% SDS at room temperature for 20 min, followed by a 20-min wash in 0.2× SSC/0.1% SDS at 65°C. Blots were then exposed at −80°C to x-ray films (XAR-5; Eastman Kodak, Rochester, NY, U.S.A.) with intensifying screens for 15-30 h and the autoradiographs analyzed by laser scanning (Molecular Dynamics). All blots were stripped of the cDNA probe by 60-min incubation with 1% SDS at 95°C and also hybridized with a bovine 283-bp prepro-ET-1 cDNA probe and a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probe according to the previously mentioned procedures. The densitometric scores were normalized to that of GAPDH mRNA as an internal control.
All results are presented as mean ± SEM. Statistical analysis was performed by the Wilcoxon matched-pairs signed-ranks test by using the SPSS/PC+ Advanced Statistics program (SPSS, Chicago, IL, U.S.A.). Statistical significance was accepted at a value of p ≤ 0.05.
Characterization of epinephrine-induced CHF
Epinephrine-induced CHF was characterized by a significant increase in end-diastolic and end-systolic left-ventricular diameter and a reduction of fractional shortening (Table 1; Fig. 1A and B). MAP was significantly decreased, and HR was increased. Creatinine and osmolality were unchanged. CHF was further characterized by fluid retention and the presence of pleural effusions and ascites, as confirmed at autopsy. Hematoxylin-eosin staining of tissue slices from left ventricles showed extensive fibrosis and focal necrosis as compared with control.
Measurement of circulating and renal ET-1 concentration
CHF was associated with a significant increase of circulating ET-1 as compared with baseline (+68%; p ≤ 0.05; Fig. 2). Analysis of ET-1 protein concentration in renal tissue revealed a significant increase after induction of CHF (+37%; p ≤ 0.05).
Measurement of renal ETA-receptor and prepro-ET-1 mRNA
Renal rabbit ETA-receptor mRNA was detected with the major labeled band corresponding to 4.4 kb (Fig. 3). In rabbits with CHF, renal ETA receptor gene expression was unaltered as compared with control (+3%; NS). Prepro-Et-1 mRNA expression in renal tissue was not detected in both control and CHF.
Hemodynamic effects of ETA-receptor blockade
In control animals, selective ETA-receptor antagonism with BQ-123 (1 mg/kg) did not significantly change MAP (74 ± 4 vs. 75 ± 4 mm Hg; NS) or HR (217 ± 6 vs. 216 ± 8 beats/min; NS; Fig. 4). Infusion of BQ-123 had no significant effects on renal blood flow velocity or renal vascular resistance in these animals. Rabbits with CHF were characterized by a significant decrease in renal blood flow velocity (−56%; p ≤ 0.05) associated with an increase in RVR (+102%; p ≤ 0.05) as compared with baseline. Administration of BQ-123 had no significant effect on MAP (61 ± 5 vs. 59 ± 3 mm Hg; NS) or HR (234 ± 10 vs. 236 ± 11 beats/min; NS). However, in these animals, BQ-123 significantly increased RBF velocity (+94%; p ≤ 0.05) and reduced RVR (−40%; p ≤ 0.05). The magnitude of this effect was such that renal hemodynamics were almost completely restored to baseline conditions.
These studies were designed further to define local activation of ET-1 and the ETA receptor as well as the functional consequences of activated ET-1 for renal hypoperfusion associated with CHF. CHF was characterized by cardiac dysfunction and dilatation, systemic hypotension, and a profound increase in RVR, associated with a marked decrease in RBF velocity. Circulating and renal ET-1 concentrations were significantly elevated. Gene expression of the renal ETA receptor was unchanged, whereas renal prepro-ET-1 was below the range of detection. In contrast to control animals, short-term ETA-receptor antagonism produced a significant renal vasodilatation in rabbits with CHF.
The increase in circulating ET-1 in CHF is consistent with previous studies (4,5). The finding of increased renal ET-1 extends these studies and demonstrates local activation of renal ET-1 in CHF. The absence of significant renal prepro-ET-1 gene expression under control conditions is in accordance with Inoue et al. (23). Other studies could demonstrate low-level ET-1 gene expression in renal tissue, which was 2.2-fold higher in the renal medulla than in the cortex (24). The discrepancy of results may be due to technical reasons such as the separation of the renal cortex from the medulla in the latter study and/or the different amounts of RNA used for Northern analysis. The potential for regulation of ET-1 synthesis at the transcriptional level was previously demonstrated by studies that reported activated gene expression in the setting of renal artery stenosis and hypoxia (25). Our results, however, indicate that renal prepro-ET-1 gene expression is not regulated in CHF. Hence the augmented renal ET-1 protein concentration in CHF should represent enhanced binding of systemically delivered ET-1 to renal receptors rather than enhanced local production. These findings challenge the notion that ET-1 predominantly acts as a autocrine/paracrine peptide in the kidney in CHF and emphasize increased circulating ET-1 as an important mediator of renal hypoperfusion in CHF.
It has been suggested that ETA-receptor synthesis can be regulated at the transcriptional level (6). However, the regulation of renal ETA receptors in CHF has not been addressed. In our study, Northern analysis revealed an unchanged expression of ETA-receptor mRNA in renal tissue in CHF. In particular, renal ETA-receptor gene expression was not downregulated despite chronically elevated concentrations of ET-1 in plasma and renal tissue or upregulated, as was recently demonstrated for myocardial tissue (26). Furthermore, little is known about the effects of selective ETA-receptor blockade on renal hemodynamics in CHF. Indeed, the ETA receptor may be the receptor most important for renal regional hemodynamic adaptations to increased ET-1. Our study suggests that under baseline conditions, the ETA receptor does not participate in the control of renal vascular tone, as ETA-receptor blockade did not alter renal hemodynamics by itself. In contrast, this study provides strong evidence for an important role of the ETA receptor for renal vasoconstriction in CHF. Indeed, ETA-receptor antagonism in CHF resulted in a significant reduction in RVR associated with a significant increase in RBF velocity despite unchanged MAP or HR. The extent of this effect was of a magnitude, which almost completely restored renal hemodynamics to baseline conditions. In addition to a fundamental importance of the ETA receptor for renal vasoconstriction in CHF, these results further suggest a predominant role for ETA receptor-mediated renal vasoconstriction in CHF in comparison to other renal vasoconstrictors, such as norepinephrine and the renin-angiotensin system.
In this study, the contribution of the ETB receptor to renal perfusion in CHF was not studied. However, a preliminary study demonstrated no effect of ET-1 in CHF when infused in the presence of selective ETA-receptor blockade (14). These results suggested that the ETB receptor has neither significant vasodilatory or vasoconstricting effects on renal perfusion in CHF.
Because this study focused on the acute effects of ETA-receptor blockade on renal hemodynamics, it remains unclear whether the beneficial effects could also be achieved in a long-term setting. Because recent experimental studies suggested that ETA-receptor antagonism may also have beneficial effects if administered over the long term (27-30), it is reasonable to speculate that prolonged ETA-receptor blockade might also have continuous renal vasodilatory effects in CHF. If these effects were associated with an improvement of renal excretory function, they might also attenuate the progression of heart failure, which has been strongly linked to progressive renal dysfunction.
In conclusion, these studies provide further important insight into activation and functional significance of the renal ET system in CHF. These studies provide evidence for an activation of renal ET-1 independent of local production. They further provide evidence that local production and functional integrity of the renal ETA receptor is preserved in chronic heart failure. Whereas the ETA receptor does not seem to contribute to renal vascular tone under baseline conditions, ETA receptor-mediated renal vasoconstriction appears to be a principal mechanism of renal hypoperfusion in chronic heart failure. Further studies are warranted to study the effects of chronic ETA receptor antagonism on renal function in heart failure.
Acknowledgment: This study was supported by a grant from the NOVARTIS-Stiftung für therapeutische Forschung. The excellent technical assistance of Ingrid Kirst and Astrid Schieß1 is greatly appreciated.
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