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Hypertension. 1997;30:1578-1584

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(Hypertension. 1997;30:1578-1584.)
© 1997 American Heart Association, Inc.

Articles

Role of Endothelin in Hypertension of Experimental Chronic Renal Failure

Gregg S. Potter; Ron J. Johnson; ; Gregory D. Fink

From the Department of Pharmacology and Toxicology, Michigan State University (East Lansing).

Correspondence to Gregory D. Fink, PhD, Department of Pharmacology and Toxicology, Michigan State University, B-440 Life Sciences Bldg, East Lansing, MI 48824-1317. E-mail finkg{at}pilot.msu.edu


*    Abstract
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 *Abstract
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 down arrowMethods
 down arrowResults
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Abstract Surgical ablation of renal mass leads to a reduction in kidney function and commonly to the development of hypertension and chronic renal failure (CRF) in rats. The objective of this study was to determine whether endothelin (ET)-1 is involved in the maintenance of the hypertension that accompanies loss of renal mass. First, we demonstrated the antihypertensive efficacy of PD 155080, a selective, orally active ETA receptor antagonist, in a group of rats made hypertensive by continuous intravenous infusion of ET-1 (2.5 pmol · kg-1 · min-1) for 7 days. ET-1 produced a sustained hypertension and PD 155080 (56.4 µmol/kg [25mg/kg] BID PO) normalized blood pressure (BP) during the 5 days of drug administration. In a second experiment, Sprague-Dawley rats underwent a 5/6 reduction in renal mass (RRM); 4 weeks later, PD 155080 administered for 7 days resulted in a sustained reduction in BP. Sham-operated rats also showed a slight hypotensive response to PD 155080 administration. Plasma urea nitrogen, plasma creatinine, urinary protein excretion, and creatinine clearance were not altered by PD 155080 administration in RRM or sham rats. In a third experiment, we investigated the contribution of the renin-angiotensin system to BP control in RRM rats given PD 155080. In these rats, PD 155080 reduced BP during 5 treatment days, and this antihypertensive effect was not altered by coadministration of the angiotensin-converting enzyme inhibitor enalapril in the drinking water (508 µmol/L [250 mg/L]). These results demonstrate that (1) ET-1 plays a role in established RRM hypertension through activation of the ETA receptor subtype, (2) lowering blood pressure with PD 155080 in RRM rats does not adversely affect renal function, and 3) the antihypertensive effect of ETA receptor antagonism is not opposed by the renin-angiotensin system.


Key Words: angiotensin-converting enzyme inhibitor • renal ablation • reduced renal mass • endothelin


*    Introduction
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 up arrowAbstract
 *Introduction
down arrowMethods
 down arrowResults
 down arrowDiscussion
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Chronic renal failure is a gradual deterioration of renal glomerular function manifested by progressive proteinuria, azotemia, and impaired renal handling of water and various electrolytes. As CRF worsens, the majority of affected individuals also exhibit hypertension, which then becomes an important risk factor for an accelerated decline in renal function1,2 as well as other cardiovascular diseases. Several mechanisms have been proposed to explain the high incidence of hypertension in patients with CRF: body fluid volume expansion,3 abnormalities of the renin-angiotensin system in relation to body fluid status,4 increased sympathetic neural activity,5 and abnormalities of vascular smooth muscle cell ion transport and distribution.6 More recent evidence has drawn attention to the endothelium-derived peptide ET-1 as a likely contributor to the pathogenesis of hypertension in experimental7,8 and clinical9 CRF.

The ETs are a family of structurally related 21–amino acid peptides (ET-1, ET-2, and ET-3). ET-1 is the best characterized and is known to have potent vasoconstrictor10 and mitogenic11 activity. Vascular endothelial (and other) cells release ET-1 in response to a variety of stimuli. The peptide causes vascular relaxation by ETB1 receptor–mediated release of endothelial cell nitric oxide and prostacyclin12,13 and vascular contraction through activation of smooth muscle cell ETA or ETB2 receptors.14,15 Several studies provide evidence that the ET system is activated in patients with CRF. Both blood9,16 and urine17 concentrations of ET-1 were shown to be elevated in uremic, hypertensive individuals, although these are not universal findings.18,19

More direct evidence implicating ET-1 in the hypertension associated with CRF has come from experimental models of renal failure.8,20 One commonly used model created in laboratory animals is the RRM model. The RRM is accomplished through two nonequivalent procedures: ligation of branches of the renal artery or excision of the poles of the contralateral kidney, either of which is followed by uninephrectomy. The ligation method causes severe and immediate hypertension due to renal ischemia with high intrarenal renin concentrations and is unaffected by changes in sodium intake.21 ACEIs have been shown to control hypertension and limit renal deterioration in the ligation variant of RRM.22,23 In the excision method of RRM, BP rises progressively over a period of weeks to months after renal ablation, more rapidly if the animals are eating a diet high in sodium chloride.24,25 Low intrarenal renin concentrations are measured, and ACEIs only reduce BP in the presence of salt restriction when plasma renin levels rise.21 Because of the differences in the development and progression of hypertension between the two methods, results of drug studies in one model cannot be readily extrapolated to the other.

In the ligation model of RRM, intraperitoneal administration of a selective ETA receptor antagonist beginning 1 week after subtotal nephrectomy was shown to significantly slow hypertension development.20 Although this result suggested that ET-1 contributes to hypertension associated with RRM in rats, the mechanism remains unclear. For example, ETA receptor antagonist treatment also improved renal function relative to that of untreated RRM animals.20 Thus, interference with possible deleterious effects of kidney-derived ET-1 on renal function could account for the ability of the drug to slow hypertension development. Another possibility, however, is that endothelium-derived ET-1 exerts a direct and sustained systemic vasoconstrictor effect in RRM rats. If so, then administration of an ET-1 receptor antagonist should not only impair gradual hypertension development but also rapidly lower BP in RRM rats in which hypertension is already well established.

The involvement of ET-1 in the hypertension of rats undergoing the excision method of RRM has not been previously reported. The objective of this study was to determine whether ET-1 is involved in the maintenance of RRM hypertension when the reduction in nephron mass is accomplished via the excision method. Here, we describe experiments designed to test the antihypertensive effectiveness of oral administration of the selective ETA receptor antagonist PD 155080 (Parke-Davis Pharmaceutical Research) in RRM rats at 4 to 6 weeks after partial excision of renal mass.


*    Methods
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 up arrowAbstract
 up arrowIntroduction
 *Methods
down arrowResults
 down arrowDiscussion
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Animals
On arrival at our facility, male Sprague-Dawley rats (Sasco) were maintained according to protocols approved by the Michigan State University All-University Committee on Animal Use and Care. The rats were housed in a light- and temperature-controlled room and maintained in strict accordance with Michigan State University and National Institutes of Health animal care guidelines. Rats were housed individually in clear plastic boxes with free access to standard rat chow and tap water before surgery. After catheterization, rats were individually housed in metabolism cages to allow daily monitoring of water intake and urine output.

Drug Administration
PD 155080 (56.4 µmol/kg [25 mg/kg]) was mixed in {approx}7.5 g of powdered sodium-deficient rat chow (0.002 mmol Na+/g) and given to rats twice daily: in the morning (8 to 10 AM) and late afternoon (4 to 6 pm). Rats usually consumed all of the food provided within a 20-minute period. Enalapril maleate (508 µmol/L [250 mg/L]) was dissolved in distilled drinking water, and doses were calculated based on the amount consumed multiplied by the concentration.

Surgical Procedures
All surgical procedures were performed after the administration of pentobarbital sodium (181.5 µmol/kg [45 mg/kg] IP; Abbott Laboratories). If necessary, anesthesia was supplemented during surgery with methohexital sodium (17.6 to 35.2 µmol/kg [5 to 10 mg/kg] IV; Eli Lilly). Postoperative analgesia was provided by a single injection of butorphanol tartrate (1.1 µmol/kg [0.5 mg/kg] SC; Apothecon).

Renal Mass Reduction
Male Sprague-Dawley rats weighing 200 to 250 g were used in these procedures. Surgical renal reduction was performed in two stages. Initially, a left midflank incision was made, and the left kidney was exteriorized. The renal artery was temporarily occluded, and both poles of the kidney (two thirds of the mass) were excised with a scalpel. The incision sites were electrocauterized, and bleeding was controlled with thrombin topical powder (Parke-Davis) and pressure. The remnant kidney was returned to the abdominal cavity. Rats were allowed 1 week of recovery before the right kidney was exposed; the right renal artery, vein, and ureter were ligated with silk; and the entire kidney was removed. Both stages of the sham operation involved exteriorizing the kidney and subsequently replacing the intact kidney back into the abdominal cavity. Here, in the first stage, the renal artery was occluded for 2 minutes with no excision taking place before replacement. These RRM and sham-operated rats were kept in clear plastic boxes for 4 weeks before catheter implantation.

Arterial and Venous Catheterization
Arterial and venous catheters were surgically implanted with the animals under pentobarbital anesthesia. A silicone-rubber/polyvinyl catheter was fed into the abdominal aorta via the external iliac artery and sutured into place. This catheter was used to record MAP and to draw blood samples from conscious, freely moving animals. Another silicone-rubber/polyvinyl catheter was inserted into the posterior vena cava via the external iliac vein. This catheter was used to administer ET-1 (Peninsula Laboratories) and saline. Both catheters were tunneled subcutaneously to the top of the head, where they were exteriorized and fed through a stainless steel spring to protect the catheters from the rat. One end of the metal spring was attached to the skull with dental acrylic and jeweler's screws, and the other end was attached to a plastic swivel mounted above the cage. This allowed the rat free movement within the cage. The venous line was attached to a syringe-type Harvard infusion pump via the swivel for continuous 24-hour infusions of sodium chloride or ET-1. Rats were fed sodium-deficient rat chow (Teklad) during experimental protocols and received their entire daily sodium intake by intravenous infusion of sodium chloride in 5 mL of distilled water. Arterial lines were filled with a heparin-sucrose solution and occluded when not in use. Animals were allowed 3 days of recovery after surgery and received ticarcillin disodium (SmithKline Beecham) at a dose of 35 µmol/kg (15 mg/kg) IV BID prophylactically before measurements were begun.

Analytical Methods
MAP
BP was recorded daily between 9:00 and 12:00 AM by connecting the arterial catheter to a pressure transducer (model P10EZ; Gould) attached to a digital BP monitor (model BP2; Stemtech) and a polygraph (model 7B; Grass).

Plasma Assays
Blood samples (0.7 mL) were taken once per experimental period from the exteriorized arterial catheter into a syringe containing heparin (5 USP units/mL [32 µg/mL]). The samples were spun in a refrigerated microcentrifuge; 0.35 mL of plasma was drawn off and frozen at -70°C until assays were performed. All samples were analyzed in the same assay to control for interassay variability. PUN was determined with a prepared colorimetric assay kit (kit 640; Sigma Chemical) involving ammonia production.26 Pcr was determined with a prepared colorimetric assay kit (kit 555; Sigma Chemical) using the alkaline picrate method.27

Plasma ET-1 concentrations were determined using a solid-phase ELISA kit (Parameter; R&D Systems). Blood samples (2.0 mL) were taken once per experimental period on days alternate to other blood sampling and prepared as before except EDTA (5.3 µmol/mL [2 mg/mL]) was added to the inhibitor solution containing heparin. Red blood cells were immediately resuspended in isotonic saline and reinfused through the arterial catheter. ET-1 was extracted from 1 mL of plasma with 1.5 mL of extraction solvent composed of acetone/1 mol/L HCl/water (40:1:5). The mixture was centrifuged for 20 minutes at 3000 rpm at 4°C. The supernatant was dried down with a centrifugal evaporator, and the pellet was reconstituted in sample diluent and assayed. Optical density readings of unknown samples were plotted against a standard curve of synthetic ET-1 spiked rat plasma samples over a range of 0.4 to 45.4 fmol/mL [1 to 113 pg/mL]. The recovery from the extraction procedure was 36±3%. The interassay variation was 8.2%, and the intra-assay variation was 10.1%.

Blood samples (0.5 mL) for assay of PD 155080 (sodium 2-benzo[1,3]dioxol-5-yl-3-benzyl-4(4-methoxy-phenyl)-4-oxobut-2-enoate) were taken once during the treatment period from the exteriorized arterial catheter into a syringe containing heparin (5 USP units/mL [32 µg/mL]). The samples were centrifuged and stored as before. Plasma concentrations of PD 155080 were determined through high-performance liquid chromatography.

Urine Assays
Urine from 24-hour urine collection samples was put into calibrated cylinders. All samples were spun in a refrigerated microcentrifuge to separate any particulate matter and then stored at -70°C until assayed. All urine samples were analyzed in the same assay to control for interassay variability. Upro was determined with a prepared colorimetric assay kit (kit 541, Sigma Chemical) involving the biuret method.28 Ucr concentration was assayed using the same methods for rat plasma. Ccr was calculated using the standard formula.

Fluid and Electrolyte Measurements
Daily water intake and urine output were measured and WB was calculated by subtracting urine output from the sum of water intake and infusion volume. Urinary sodium concentrations were measured with an Instrumentation Laboratory IL943 flame photometer. UNaV was calculated by multiplying urine output times urinary sodium concentration.

Experimental Protocols
Study 1: PD 155080 in ET-1–Induced Hypertension
Two groups of male Sprague-Dawley rats weighing 350 to 400 g were catheterized for hemodynamic measurements, and the continuous IV infusion of ET-1 at 2.5 pmol · kg-1 · min-1 in a saline solution calibrated to deliver a sodium intake of 6.0 mmol/24 h. The experiment lasted a total of 12 days—2 control days followed by 10 days of continuously administered ET-1. After 3 days of ET-1 infusion alone, half the rats were given PD 155080 (n=5) for 5 days and the other half served as control (n=5). Plasma samples for PD 155080 were drawn 12 hours after the last dose of PD 155080 on the third day of drug administration. Treatment with PD 155080 was withdrawn for the final 2 days of ET-1 infusion.

Study 2: PD 155080 in Established RRM and Sham-Operated Rats
Both RRM (n=5) and sham (n=5) rats were maintained on normal rat chow and distilled drinking water for 4 weeks after completion of the renal ablation, while awaiting catheterization. After arterial and venous catheterization, all rats were fed a sodium-deficient rat chow and received a sodium intake of 2.0 mmol/24 h through the venous catheter. The experiment lasted a total of 15 days and was divided into three experimental periods: 3 control days followed by 7 treatment days and ending with 5 recovery days. PD 155080 was given as described above during the treatment days.

Indices of glomerular function (PUN, Pcr, Ccr, and Upro) were monitored once per experimental period. WB and UNaV were measured daily. Samples for ET-1 plasma concentrations were taken once at the end of each experimental period. During the treatment period, samples for plasma PD 155080 and ET-1 determinations were drawn 12 hours after the dose of PD 155080 on alternate days.

Study 3: Antihypertensive Effect in RRM Rats of Coadministration With PD 155080 and Enalapril
RRM rats (n=5) were maintained on normal rat chow and distilled drinking water for 6 to 7 weeks after completion of the renal ablation. After arterial and venous catheterization, these rats were fed sodium-deficient chow and received a saline infusion measured to deliver a sodium intake of 2.0 mmol/24 h through the venous catheter. The experiment lasted a total of 9 days: 2 control days, followed by 7 experimental days. PD 155080 was given alone as previously described for the first 2 experimental days. For the next 3 days, enalapril maleate (Sigma Chemical) at a dose of 508 µmol/L [250 mg/L] was added to the drinking water. On the final 2 days of the experiment, the rats received only enalapril maleate.

Statistical Analysis
Results are expressed as mean±SEM. For all data, within- and between-group differences were analyzed by mixed-design analysis of variance. Posthoc within-group comparisons were performed using the method of contrasts. The criterion for statistical significance was a probability level of <.05. All analyses were performed with a computer software package for statistics (CRUNCH Version 4).


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 *Results
down arrowDiscussion
 down arrowReferences
 
Study 1: PD 155080 in ET-1-Induced Hypertension
Continuous infusion of ET-1 at a rate of 2.5 pmol · kg-1 · min-1 in normal rats produced a slowly developing, sustained hypertension (Fig 1Down). Oral administration of PD 155080 for 5 days resulted in a complete normalization of BP. After discontinuation of PD 155080, MAP returned to hypertensive levels within 1 day. Plasma concentrations of PD 155080 were 13.6±2.3 nmol/mL).



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  		Figure 1. MAP in normal rats receiving PD155080 ({blacksquare}) or sodium-deficient rat chow alone ({square}). On control days C1 and C2, all rats received only intravenous saline infusion. On experimental days E1 through E10, all rats received an intravenous infusion of ET-1 at a rate of 2.5 pmol · kg-1 · min-1. The treated rats (n=5) were given PD155080 (56.4 µmol/kg [25 mg/kg]) BID on days E4 through E8, mixed in with their chow. The untreated rats (n=5) were kept on sodium-deficient chow. *Significant (P<.05) difference from the average of 2 control days within each group.

Study 2: PD 155080 in Established RRM Hypertensive and Sham-Operated Rats
Fig 2Down shows the effect of PD 155080 on BP in RRM and sham rats. Untreated RRM rats demonstrated a sustained, gradually progressive hypertension throughout the experiment. Hypertensive RRM rats receiving PD 155080 exhibited a significant and well-maintained decline in BP throughout the treatment period. This antihypertensive effect was reversed by 24 hours after discontinuation of PD 155080. In fact, during the recovery period, BP was significantly higher than during the pretreatment control period. Normotensive sham rats given PD 155080 showed a slight, inconsistent hypotensive effect during the treatment period compared with BP during the 3 control days. As expected, untreated sham rats showed no progression of BP over the course of this experiment.



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  		Figure 2. MAP in RRM and sham rats receiving PD155080 ({blacksquare}) or sodium-deficient rat chow alone ({square}). After 3 days of control measurements (C1 through C3), PD155080 (56.4 µmol/kg [25 mg/kg]) BID PO was given to RRM (n=5) and sham (n=5) rats for 7 treatment days (T1 through T7), and recordings were continued for 5 recovery days (R1 through R5). The untreated RRM (n=5) and sham (n=5) rats were kept on sodium-deficient chow alone. *Significant (P<.05) decrease from the average of 3 control days within each group. {dagger}Significant (P<.05) increase from the average of 3 control days within each group.

Indices of glomerular function and plasma ET-1 concentrations during PD 155080 treatment in sham and RRM rats are shown in the TableDown. As expected, PUN, Pcr, and Upro were elevated and Ccr was decreased in RRM rats compared with sham rats. In sham rats, 7-day treatment with PD 155080 caused no significant changes in glomerular function. Likewise, PD 155080 administration in RRM rats did not cause significant changes in glomerular function, despite a strong antihypertensive effect observed during the treatment period. ET-1 plasma levels were not significantly different between any groups during the experiment except for a slight elevation in nontreated RRM rats during the recovery period (control, 0.40±0.04 fmol/mL; recovery, 0.68±0.04 fmol/mL). It is noteworthy that PD 155080 administration in both sham and RRM groups did elevate plasma ET-1 concentrations from the control levels, but these changes did not reach statistical significance. Plasma concentrations of PD 155080 in sham and RRM rats were not different (sham, 17.1±4.3 nmol/mL; RRM, 14.3±9.3 nmol/mL).


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  		Table 1. Renal Parameters and ET-1 Concentrations in Sham-Operated and RRM Rats

There were no measurable differences in WB or UNaV throughout the experiment in untreated or PD 155080–treated sham rats. In RRM rats, the first day of PD 155080 administration resulted in a significant decrease in UNaV compared with the average of the 3 control days (treatment, 1.22±0.1 mmol Na+/24 h; control, 1.68±0.2 mmol Na+/24 h). A concomitant increase in WB was observed on the first day of PD 155080 treatment (treatment, 20.2±3.6 mL/24 h; control, 11.0±2.1 mL/24 h), but this did not reach statistical significance. On discontinuation of PD 155080 in RRM rats during the first recovery day, a significant increase in UNaV was observed compared with the average of 3 control days (recovery, 2.20±0.3 mmol Na+/24 h; control, 1.68±0.2 mmol Na+/24 h). This significant increase in UNaV was coupled with a decrease in WB during the first recovery day, which did not reach statistical significance (recovery, 8.2±2.1 mL/24 h; control, 11.0±2.1 mL/24 h).

Study 3: Antihypertensive Effect in RRM Rats of Coadministration of PD 155080 and Enalapril
Fig 3Down shows MAP responses to PD 155080 and enalapril administration in RRM rats with established hypertension. In these RRM rats, 6 to 7 weeks after completion of the reduction in renal mass, PD 155080 treatment lowered BP for all 5 days given (days E1 through E5). The addition of enalapril in the drinking water (508 µmol/L [250 mg/L]) for 3 days did not elicit an additional antihypertensive effect during coadministration with PD 155080 (days E3 through E5). On discontinuation of PD 155080, while the rats were still receiving enalapril, MAP increased to control levels within 24 hours.



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  		Figure 3. MAP in RRM rats (n=5). Two control days (C1 and C2) were followed by 7 experimental days (E1 through E7). On days E1 through E5, rats were given PD155080 (56.4 µmol/kg [25 mg/kg]) BID PO. On days E3 through E7, rats were given enalapril (508 µmol/L [250 mg/L]) dissolved in their drinking water. *Significant (P<.05) difference from the average of 2 control days.


*    Discussion
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 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
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Proper interpretation of the results from the main experiment reported here depends on the selectivity and efficacy of PD 155080 as an antagonist at ETA receptors in the rat. Concerning selectivity, in vitro screens indicate that drugs in this series have low affinity for a variety of endogenous receptor types other than those for endothelins.29 Furthermore, affinity of PD 155080 for ETA receptors appears to be {approx}1000-fold higher than for ETB receptors as estimated using binding and functional assays in human and a variety of animal tissues.30,31 In vivo evaluations suggest that plasma PD 155080 concentrations ranging from 11.2 to 135.4 nmol/mL (5 to 60 µg/mL) exert selective antagonism of ETA receptors in the rat (J. Keiser, personal communication).

Traditional tests of antagonist drug efficacy usually entail comparison of acute responses to administration of appropriate agonists in the presence and absence of the antagonist. However, because the main goal of our study was to uncover evidence for a chronic influence of ET-1 on arterial pressure regulation, initial experiments were performed to demonstrate the efficacy of PD 155080 in reversing long-term cardiovascular effects of ET-1. To this end, rats were made hypertensive by continuous intravenous infusion of ET-1 for several days and then treated with PD 155080. The results showed that PD 155080 at 56.4 µmol/kg (25 mg/kg) BID was able to fully and reversibly inhibit the hypertensive response to exogenous ET-1; thus, this dose was used in additional experiments designed to evaluate the effect of endogenous ET-1 on BP regulation in RRM rats.

In RRM rats studied 4 to 5 weeks after reduction in renal mass by the excision method (study 2), BP was significantly higher than that of sham-operated rats, but plasma ET-1 concentrations were similar in the two groups, confirming an earlier report.8 Nevertheless, 1-week treatment with PD 155080 caused a significant and sustained decrease in BP in RRM rats while producing only a modest hypotensive effect in normotensive sham-operated animals. Concerning the latter observation, a physiological role for endothelin in the maintenance of basal vascular tone in humans has been suggested by Haynes et al.32 Other investigators have not found a significant role for ET in the normal hemodynamic status of rats33 and dogs.34

This difference in BP response was not due to impaired elimination of PD 155080 in rats with remnant kidneys because plasma levels of the drug at the time of BP measurements were similar in the two groups. The results from this study suggest that endogenous ET-1, acting at ETA receptors, exerts a tonic pressor effect in hypertensive RRM rats, although a nonselective antihypertensive effect of PD 155080 at non-ET receptors cannot be ruled out.

Increased ET-1 gene expression in the kidney and elevated urinary excretion of ET-1 occur in RRM rats, indicating enhanced intrarenal synthesis of the peptide.7 Renal ET-1 causes sodium retention,35 so blockade of renal ET receptors by PD 155080 could cause a fall in BP by promoting fluid excretion via the kidney. Recent work36,37 suggests, however, that most actions of ET-1 in the rat kidney are mediated through ETB receptors, making this explanation unlikely. Our results also do not support such an explanation in that PD 155080 administration to RRM rats was associated with sodium and water retention rather than with diuresis and natriuresis.

Release of ET-1 from endothelial cells in the systemic vasculature causes vasoconstriction and smooth muscle cell growth by stimulating ETA receptors.38 The time course of the antihypertensive response to PD 155080 in these experiments was too short for reversal of vascular structural changes; therefore, inhibition of ET-1–induced vasoconstriction probably accounted for the BP-lowering effect of ETA receptor blockade in the RRM rats studied here.

It is not possible on the basis of our data to determine whether the synthesis and release of ET-1 from vascular endothelial cells (or other tissues) are increased in RRM rats. Schiffrin and colleagues39,40 reported increased vascular ET-1 gene expression in several models of hypertension in rats, but this has not been investigated in the RRM model. Failure to observe elevated plasma levels of ET-1 in the RRM rats in our study does not rule out increased ET-1 release from endothelial cells because most of this secretion probably occurs abluminally.41 It is worth reemphasizing that our results demonstrate clearly the risk of evaluating physiological functions of ET-1 based on only measurement of plasma concentrations. The increased ET-1 plasma levels observed in both sham-operated and RRM rats during treatment with PD 155080 may result from increased vascular ET-1 production due to inhibition of a negative feedback mechanism. An alternative explanation is that PD 155080 at the dose used may bind ETB receptors normally involved in the clearance of ET-1. Increases in plasma ET-1 levels during nonselective ETA/ETB receptor blockade have been reported in other experimental models of hypertension.42 However, with selective blockade of ETA receptors, increases in circulating ET-1 may stimulate endothelial cell release of vasodilatory modulators through the ETB1 receptor. This mechanism may in part explain the BP-lowering effects of PD 155080.

Increased pressor sensitivity to ET-1 could account in part for the apparent hypertensive effects of ET-1 in RRM rats. We have shown that the BP response to chronic, low-dose IV infusion of ET-1 in rats is markedly augmented when the animals are salt loaded.43 Also, RRM is associated with an impaired ability to excrete sodium chloride.25 Therefore, even normal rates of ET-1 release in RRM rats might be sufficient to increase BP in a setting of excess body sodium content.

We have reported,44 and others have confirmed,45 that increased BP caused by long-term infusion of ET-1 is reduced by ACEI. Substantial additional evidence suggests an important interaction between ET-1 and the renin-angiotensin system.46,47 To explore a potential interaction in RRM rats, we tested whether concomitant administration of an ETA receptor antagonist and an ACEI would produce synergistic effects on BP. In RRM rats studied 6 to 7 weeks after RRM by the excision method, the hypotensive effect of PD 155080 was not altered by the addition of enalapril to the treatment regimen, and enalapril alone did not reduce BP in this setting. Thus, the antihypertensive effect of ETA receptor blockade in RRM rats does not appear to be enhanced or opposed by the renin-angiotensin system.

The lack of an additional lowering of BP by enalapril was not due to inadequate inhibition of the RAS. Water intake in these RRM rats averaged 45.6±3.2 mL/d; therefore, the calculated average consumption of enalapril was 33 mg · kg-1 · d-1. This dose of enalapril has been shown to block angiotensin I–induced pressor responses in the rat, indicating that ACE activity is significantly inhibited.48 The lack of an antihypertensive effect due to enalapril administration alone in RRM rats could be masked by the level of salt intake in these rats. Terzi and coworkers49 demonstrated in RRM rats prepared by the excision method that ACEIs attenuate only the progression of hypertension under conditions of dietary sodium restriction. Rats in this study were maintained on a normal sodium intake of 2.0 mmol Na+/24 h.

Our findings indicate that ETA receptor blockade may be an effective therapy for the hypertension associated with CRF. It is obviously important, however, that any such therapy not further impair renal glomerular function. It is therefore noteworthy that the antihypertensive response to PD 155080 in RRM rats caused only transient sodium retention and was not accompanied by any measurable decrease in creatinine clearance or an increase in Pcr, PUN, or urinary protein excretion over the short time course of our experiment. In fact, chronic treatment with an ETA receptor antagonist was shown to slow hypertension development and progressive deterioration of glomerular function in RRM rats prepared through use of the ligation method.20 Thus, the ET system may be a good target for therapeutic interruption of multiple pathophysiological processes occurring in CRF.


*    Selected Abbreviations and Acronyms
 
ACE = angiotensin-converting enzyme
ACEI = angiotensin-converting enzyme inhibitor
BP = blood pressure
Ccr = creatinine clearance
CRF = chronic renal failure
ET = endothelin
MAP = mean arterial pressure
Pcr = plasma creatinine
PUN = plasma urea nitrogen
RRM = reduction in renal mass
UNaV = urinary sodium excretion
Ucr = urinary creatinine
Upro = urinary protein concentration
WB = water balance


*    Acknowledgments
 
This work was supported by grant HL-24111 from the National Heart, Lung, and Blood Institute. PD 155080 was a generous gift from Parke-Davis Pharmaceutical Research (Ann Arbor, Mich). We would like to acknowledge the technical assistance of James Shavrnoch in helping with animal care. We would like to thank Joan Keiser, Steve Haleen, Hussein Hallak, and Edie Quenby-Brown for their assistance.

Received March 3, 1997; first decision March 25, 1997; accepted June 18, 1997.


*    References
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 up arrowDiscussion
 *References
 

  1. Brazy PC, Stead WW, Fitzwilliam JF. Progression of renal insufficiency: role of blood pressure. Kidney Int. 1989;35:670–674.[Medline] [Order article via Infotrieve]
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