Effects of endothelin-1 and endothelin-1-receptor blockade on renal function in humans

J. L. Tycho Vuurmans, Peter Boer and Hein A. Koomans

Department of Nephrology and Hypertension, University Medical Center Utrecht, the Netherlands

Correspondence and offprint requests to: Dr Peter Boer, PhD, Department of Nephrology and Hypertension, University Medical Center Utrecht, Room F03.226, PO Box 85500, 3805 GA Utrecht, the Netherlands. Email: p.boer{at}azu.nl



   Abstract
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. In patients with renal or cardiac failure, renal function may be endangered by elevated plasma concentrations of the vasoconstrictor endothelin-1 (ET-1). To mimic effects of pathologically increased plasma ET-1, we gave intravenous ET-1 in healthy subjects and examined whether simultaneous infusion of the ETA-receptor antagonist VML 588 would prevent the effects of ET-1 on the kidney.

Methods. Nine healthy men received on four separate days intravenous infusion of ET-1 (2.5 ng/kg/min) superimposed on vehicle (saline) or on VML 588 infusion (0.05, 0.20 and 0.40 mg/kg/h) in randomized order to assess the effects on renal function and renal haemodynamics.

Results. At resting plasma ET-1, infusion of VML 588 alone had no significant effects on renal function. Infusion of ET-1 alone decreased glomerular filtration rate by 11% and this reduction was not reversed by co-infusion of VML 588. ET-1 reduced renal blood flow by 35% and VML 588 reduced this decrease by one-third, in a dose-independent fashion. ET-1 increased the filtration fraction by 34% and VML 588 reduced this increase dose-independently by one-half. ET-1 increased renal vascular resistance by 59% and VML 588 reduced this increase dose-independently by one-half. Finally, ET-1 decreased sodium excretion by 58% and VML 588 reduced this decrease dose-independently by two-thirds.

Conclusions. ET-1-induced reductions in renal function were partially but not completely prevented in a dose-independent manner by the ETA-receptor antagonist VML 588.

Keywords: endothelin-1; endothelin-1-receptor antagonist; filtration fraction; glomerular filtration rate; renal blood flow; renal vascular resistance



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Plasma endothelin-1 (ET-1) exerts remarkable vasoconstrictive effects on renal function that consist of decreased glomerular filtration rate (GFR) and renal blood flow (RBF) as well as increased filtration fraction (FF), renal vascular resistance (RVR) and sodium retention [1–4]. During pathological conditions, such as renal failure [5,6], heart failure [7,8] and salt-sensitive hypertension [9], ET-1 levels may be strongly elevated. In patients with these conditions, ET-1 may endanger renal and cardiac function and treatment with ET-1-receptor antagonists may be helpful. ETA receptors having a high affinity for ET-1 are involved in the vasoconstrictive effects of ET-1, whereas ETB receptors have equal affinity for ET-1 and ET-3 and are involved in plasma clearance of ET-1 as well as vasorelaxation mediated by nitric oxide [10,11]. Studies on the effects of ET-1 antagonists on kidney function in humans have been scarce [3,4] and the results and conclusions from these studies have not been in agreement. In animal studies, results have been conflicting and differ from those in humans [12–16]. For these reasons, we studied the effects of VML 588, another ETA-receptor antagonist, on renal function in humans. This drug, formerly known as AXV-034343 and Ro 61-1790, is a competitive ET-receptor antagonist with ~1000-fold greater affinity for ETA than for ETB receptors [17]. Recently, we reported the effects of simultaneous infusion of ET-1 and VML 588 on cardiovascular function [18]. In the present study, we determined whether VML 588 by itself alters renal performance, whether it is able to prevent the effects of increased ET-1 on renal function and, if so, whether this prevention is complete or only partial. To do this, we investigated in the same subjects as in the cardiovascular study [18] the effects of ET-1 on kidney function without and with simultaneous administration of VML 588 at three doses that were sufficiently high to block all accessible ETA receptors.



   Subjects and methods
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 Subjects and methods
 Results
 Discussion
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Subjects and study design
The study was performed in nine healthy men (aged 18–34 years) having a sodium intake of 150–250 mmol/day. The protocol was approved by the institutional committee on ethics for study in humans and the subjects had given written informed consent. Each subject underwent four experimental sessions in a clearance setting on separate days with intervals of 7 days, in a randomized order. The studies were done after an overnight fast, with the subjects in supine position in a quiet room. Venous cannulae were placed bilaterally in the cubital fossa for infusion and blood sampling. To minimize urine collection errors, water diuresis was induced by intravenous infusion of 1 l 5% glucose. During the course of the study, diuresis was maintained by having the subjects drink amounts of water matching the urinary output. At 1 h after the glucose infusion, we administered a priming dose of a solution containing 2.5% inulin (Inutest®; Fresenius Pharma GmbH, Graz, Austria) and 2.5% p-aminohippurate (PAH). A continuous infusion of this solution was maintained for the remainder of the study. After 1 h of equilibration, three 30-min baseline urine collections were obtained by spontaneous voiding for determination of inulin, PAH and sodium. Then, an intravenous infusion was started with either 0.9% saline (vehicle study) or VML 588 dissolved in saline to deliver 0.05, 0.2 or 0.4 mg/kg/h and these were continued throughout the remainder of the experiment. Three more 30-min urine collections were made. Next, superimposed on these infusions, intravenous infusion of ET-1 (Clinalfa AG, Laufelfingen, Switzerland) dissolved in a plasma substitute (Gelofusine®; B. Braun AG, Melsungen, Germany) was started at 2.5 ng/min/kg [19] and four 30-min urine collections were made. At the midpoint of each urine collection period, blood samples for determination of plasma inulin and PAH were drawn from the contralateral forearm. Blood samples for determination of plasma ET-1 and VML 588 were collected at the end of each infusion period. Blood pressure was recorded at 5 min intervals with a Dinamap Compact T blood pressure monitor (Criticon, Tampa, FL, USA).

Chemical analyses
Inulin was converted to fructose by acid hydrolysis and determined spectrophotometrically by a chromogenic reaction with indoleacetic acid [20]. PAH was measured spectrophotometrically by a chromogenic aldehyde reaction [21]. Urinary sodium was measured by flame photometry. Blood samples for determination of immunoreactive ET-1 and VML 588 were collected in pre-chilled di-K-EDTA-containing tubes, centrifuged at 4°C and stored at –70°C until the assay. VML 588 was measured by high-performance liquid chromatography with mass spectrometry detection. ET-1 was extracted in duplicate from 1 ml plasma samples using Sep-Pak C18 solid-phase extraction chromatography cartridges (Waters, Etten-Leur, the Netherlands) and measured by radioimmunoassay (Nichols Institute, Wychen, the Netherlands), as described previously [22]. The recovery of ET-1 throughout the extraction procedure was 85%; the reported concentrations were corrected for procedural losses. Cross-reactivities of the ET-1 antibody for ET-2, ET-3, pro-ET-1 and VML588 were 52%, 96%, 7% and 0%, respectively. Between- and within-assay coefficients of variation were 15% and 9%, respectively. The detection limit was 0.9 pg/ml plasma.

Calculations and statistics
GFR and effective renal plasma flow (ERPF) were calculated as the ratios of urinary excretion rate to plasma concentration of inulin and PAH, respectively. FF was calculated as the ratio GFR to ERPF. RBF was calculated as ERPF divided by (1 – packed cell volume) and RVR as the ratio MAP:RBF. Data are presented as means±SEM. Statistical analysis comparing the means of the parameters as well as the means of changes in the parameters was performed by two-way analysis of variance for repeated measures. When statistically significant differences were found, the studentized Newman–Keuls test for multiple comparisons was used as the post-hoc test. Because plasma ET-1 levels were not normally distributed, they are presented as geometric means and the analysis was performed on logarithmically transformed data. A P-value ≤ 0.05 was considered to be statistically significant.



   Results
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 Subjects and methods
 Results
 Discussion
 References
 
Plasma ET-1 and VML 588 concentrations
Mean baseline ET-1 concentrations ranged from 4.1 to 4.6 pg/ml. Infusion of ET-1 alone caused plasma ET-1 levels to increase by 18.8 pg/ml (Table 1). The lowest dose of VML 588 had no effect on resting plasma ET-1, but the medium and highest doses caused statistically significant increments of 1.2 and 2.2 pg/ml, respectively (P<0.05). VML 588 dose-dependently enhanced the increases in plasma ET-1 induced by ET-1 infusion (P<0.05, both with respect to vehicle and to preceding VML 588 dose). Steady-state VML 588 concentrations, obtained after 1 h of VML 588 infusion, were 91±5, 383±16 and 752±29 ng/ml during the 0.05, 0.2 and 0.4 mg/kg/h infusion rates, respectively.


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Table 1. Plasma ET-1, MAP and renal function parameters during intravenous infusion of ET-1 and co-infusion of vehicle or VML 588

 
Mean arterial pressure
Infusion of ET-1 alone caused a significant increase in mean arterial pressure (MAP) (Table 1). VML 588 alone decreased MAP slightly but significantly, without a clear dose-response effect. During infusion of VML 588, the ET-1-induced increases in MAP were not only abolished, but were reversed into paradoxical decreases in MAP (Figure 1).



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Fig. 1. Changes in MAP and renal function parameters induced by intravenous infusion of ET-1 and co-infusion of vehicle or VML 588. Significances: *P<0.05 for changes induced by infusion of ET-1 and co-infusion of VML 588 (shaded bars) vs changes induced by infusion of ET-1 and co-infusion of vehicle (white bars). Na excr, sodium excretion.

 
Kidney function
Infusion of ET-1 alone caused decreases in GFR, RBF and sodium excretion and increases in FF and RVR (Table 1). VML 588 alone had no effect on these parameters, but reduced the ET-1-induced renal vasoconstriction by about one-half, as shown by the significantly smaller increases in FF and RVR compared with ET-1 alone (Figure 1). VML 588 did not reduce the ET-1-induced decrease in GFR, but the decrease in RBF was reduced by about one-third (Figure 1). The antinatriuretic action of ET-1 was reduced by about two-thirds (Figure 1). The effects of VML 588 were dose-independent and were similar for all three doses.



   Discussion
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Reports that ETA-receptor blockade has no effect on resting plasma ET-1 on the one hand, and that ETB-receptor blockade or non-selective ET-receptor blockade increases plasma ET-1 [23–25] on the other, indicate that the ETB receptor is involved in the clearance of ET-1 from the circulation. Although the affinity of VML 588 for ETB receptors is 1000-fold less than for ETA receptors [17], VML 588 nevertheless caused at the middle and highest doses small but significant increases in resting plasma ET-1 levels and dose-dependently enhanced the increase in plasma ET-1 following infusion of ET-1. A similar phenomenon has been reported for the ETA-receptor antagonist LU135252 [26]. A possible explanation for these and our observations is that the plasma concentrations of VML 588 and LU135252 were in the order of magnitude of the affinities of the ETB receptor for VML 588 and LU135252, respectively [17,27]. This suggests that our doses of VML 588 may have resulted in a progressive but not complete ETB-receptor blockade and that VML 588 did not act exclusively as a selective ETA antagonist.

Renal vasoconstriction and antinatriuresis are well-known effects of ET-1 infusion in humans [1–4] and even though ~70% of renal ET receptors in humans are of the ETB subtype [10], these renal effects are considered to be the result of ETA-receptor stimulation. In agreement with this, we previously found that infusion of ET-3, a specific ETB-receptor agonist, had no renal effects in healthy subjects [2]. In the present study, VML 588 reversed the ET-1 induced renal vasoconstriction only partially, whereas it completely blocked the effects of ET-1 on MAP (and even resulted in a small paradoxical decrease in MAP). Others have reported previously that ETA-receptor blockade with BQ-123 [3] and ABT-627 [4] cause effective and complete antagonism of ET-1-induced renal vasoconstriction in humans. However, a closer inspection of these data reveals only a partial prevention of ET-1-induced renal vasoconstriction [3,4], a lack of a dose-response effect [4] and a pattern of responses that was very similar to our study. The difference in interpretation may have been due to an incorrect explanation of the statistics. Conflicting or inconsistent results have also been reported in animal studies. In dogs, experiments with various ETA antagonists in doses that effectively blocked the systemic effects of ET-1, renal vasoconstriction effects were partly [12] or completely prevented [13] or were even reversed into vasodilatation [14]. In rats, ETA-receptor blockade prevented systemic but not renal ET-1-induced vasoconstriction, suggesting (at least in rats) that the effect is mediated through non-ETA receptors [15]. On the other hand, a recent study in rats showed that ET-1-induced renal vasoconstriction became stronger when the ETB receptor was blocked [16].

Compared with the effect of VML 588 on ET-1-induced vasoconstriction, the inhibition of antinatriuresis appeared to be more complete. A similar difference was found in humans with another ETA antagonist [4]. This suggests that vasoconstriction and antinatriuresis are not entirely mediated through the same (ETA) receptors. Studies in dogs have shown that infusion of ET-1 during ETA-receptor antagonism causes natriuresis [12–14]. It is unclear whether this effect was due to stimulation of the ETB receptor, which has been shown to mediate natriuresis in the rat [16]. Previous studies have shown that this effect does not occur in humans [4], which is compatible with our previous study showing that specific ETB stimulation by ET-3 in humans did not cause natriuresis [2].

The question may arise why the effects of ET-1 on the kidney were not completely prevented by ETA-receptor antagonists and why there were no dose-response effects. It is unlikely that the ETA receptors were not completely saturated, because the applied VML 588 dosages were sufficiently high to block the ETA receptors. The evidence for this is the complete normalization of MAP without any dose-response effect. In addition, the finding of progressive ETB effects on the plasma clearance of ET-1 despite the 1000-fold lower affinity of VML 588 for ETB than for ETA receptors indicates that the ETA receptors must have been blocked completely. Another possible explanation is that the renal ETA receptors are less accessible to ETA-receptor antagonists than the receptors in the peripheral circulation. This also seems unlikely since a dose-response effect in the kidney would again have been expected. Furthermore, it is unlikely that the amount of infused ET-1 was such that it could compete with VML 588 for binding at the ETA receptor, because the plasma levels of ET-1 were more than four orders of magnitude lower than the levels of VML 588. Alternatively, it is conceivable that part of the ET-1-induced renal vasoconstriction was mediated through ETB receptors. However, the ETB receptors were not completely saturated, since the VML 588 plasma concentrations were in the order of magnitude of the affinity of VML 588 for the ETB receptor. If ETB receptors were involved in the renal effects, a dose-response effect would have been expected in the kidney, comparable to the finding of dose-dependent enhanced increases in ET-1 in plasma during infusion of ET-1. Since none of these explanations is compatible with the present observations, we suggest that a yet unknown ET-1 receptor is involved in the actions of ET-1 on the kidney.

We infused ET-1 to obtain concentrations that could be found in pathological conditions. Endogenous ET-1 is secreted mainly from the abluminal side of endothelial cells [28] and increased plasma concentrations may reflect increased tissue concentrations. This might imply that tissue ET-1 concentrations in pathological conditions and during ET-1 infusion are different, even when plasma concentrations are similar. Although we administered ET-1 as an infusion instead as bolus, and part of the infused ET-1 may have reached the tissues, it remains possible that the ET-1 tissue-to-plasma concentration ratio during ET-1 infusion and pathological conditions are different.

In summary, we found that effects of high ET-1 plasma concentrations on renal function were partially but not completely restored by the ETA-receptor antagonist VML 588 in a dose-independent manner. We speculate that a yet unknown ET-1 receptor is involved in the actions of ET-1 on the kidney. Thus, current therapeutic interventions seem not yet able to completely block the potential harmful effects of elevated ET-1 concentrations on the kidney.



   Acknowledgments
 
We acknowledge the support of Vernalis Ltd, Wokingham, UK.

Conflict of interest statement. None declared.



   References
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received for publication: 22. 8.03
Accepted in revised form: 2. 6.04





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