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Published in final edited form as: Gen Pharmacol. 2000 May;34(5):337–342. doi: 10.1016/s0306-3623(00)00079-3

Combined effects of AT1 and ETA receptor antagonists, candesartan, and A-127722 in DOCA–salt hypertensive rats

David M Pollock a,b,c,d,*, Vimal K Derebail a, Tatsuo Yamamoto e, Jennifer S Pollock a,d
PMCID: PMC3939804  NIHMSID: NIHMS408722  PMID: 11368889

Abstract

Several recent studies have provided evidence that many of the hemodynamic and mitogenic actions of angiotensin II (Ang II) are mediated by endothelin-1 (ET-1). We hypothesized that Ang II and ET-1 act synergistically to promote a decline in renal function and the development of renal fibrosis in the deoxycorticosterone acetate (DOCA)-salt model of malignant hypertension and renal dysfunction. Experiments were conducted to determine the effects of ETA receptor antagonism (A-127722) and AT1 receptor antagonism (candesartan cilexetil) on the development of renal fibrosis and the decline of renal function. Surgery was conducted on male, Sprague–Dawley rats to remove the right kidney and implant subcutaneously a time-release pellet containing DOCA. DOCA-treated rats were also given 0.9% NaCl to drink. After recovery from surgery, rats received one of four treatments via the drinking solution: (1) candesartan cilexetil (10 mg/kg/day), (2) A-127722 (10 mg/kg/day), (3) candesartan cilexetil plus A-127722, or (4) untreated controls. Over the course of a 3-week treatment period, systolic arterial pressure in all groups were elevated. However, this increase was significantly attenuated in the group given combined A-127722 and candesartan, but not with candesartan alone. Creatinine clearance, used as a measure of GFR, was significantly higher in rats treated with either or both drugs. At the end of the study, renal medullary tissue was harvested for determination of TGF-β and fibronectin content (ELISA). TGF-β levels were not reduced by either ETA, AT1, or combined ETA and AT1 receptor blockade. Likewise, fibronectin content was similar among groups. These studies indicate that combined ETA and AT1 receptor blockade may produce some improvement on hemodynamics, but have no effect on progression of renal damage in this non-renin-dependent model of hypertension.

Keywords: Endothelin-1, Angiotensin II, Deoxycorticosterone, Hypertension, Kidney

1. Introduction

Angiotensin II (Ang II) is responsible for producing hypertension and associated renal dysfunction in many pathological states. In addition to hemodynamic actions, Ang II contributes to vascular hypertrophy and fibrosis, i.e., overproduction of extracellular matrix (ECM) proteins (Laragh, 1995; Levy, 1998; Nicholls et al., 1998). Along these lines, blockade of AT1 receptors has been reported to decrease production of TGF-β and the subsequent deposition of ECM components such as fibronectin and collagen (Border and Noble, 1998; Kim et al., 1994). TGF-β has been reported to be elevated in the renal medulla of the DOCA–salt hypertensive rat and so it has been hypothesized that Ang II may be responsible for elevating TGF-β in this model (Kim et al., 1994). Several recent studies have also indicated that many of the hemodynamic and mitogenic actions of Ang II are mediated by endothelin-1 (ET-1) (d'Uscio et al., 1997; Herizi et al., 1998; Rajagopalan et al., 1997). In the deoxycorticosterone acetate (DOCA)-salt rat, blockade of ETA receptors reduces hypertension but not the associated decline in GFR (Allcock et al., 1998). Ang II blockade either by AT1 antagonists or ACE inhibition have no effect on arterial pressure or GFR but they do attenuate renal interstitial fibrosis (Kim et al., 1994). Similar to Ang II, ET-1 has actions similar to TGF-β in terms of promoting cell growth and synthesis of ECM proteins suggesting that these mediators may serve to exacerbate renal injury (Benigni and Remuzzi, 1998; Gomez-Garre et al., 1996; Hutchinson, 1998; Pönicke et al., 1997). Taken together, these findings have led us to hypothesize that Ang II and ET-1 act synergistically to promote a decline in renal function and the development of renal fibrosis in the DOCA–salt model of malignant hypertension and renal dysfunction. To test this hypothesis, experiments were conducted to (1) determine if ET-1 and Ang II act synergistically in the DOCA–salt model to cause a chronic decline in renal function and (2) determine if ET-1 and Ang II synergistically promote fibrosis in the kidney.

2. Methods

Male Sprague–Dawley rats (200–250 g, Harlan Laboratories, Indianapolis, IN) were housed under controlled light, temperature, and humidity while in metabolism cages to facilitate 24-h urine collection and monitoring of food and water intake. Systolic arterial pressure was determined on a weekly basis using a tail cuff pressure (TCP) measurement system as previously described (Pollock et al., 2000). Following baseline measurements, rats were anesthetized with methohexital sodium (50 mg/kg ip, Brevital, Eli Lilly, Indianapolis, IN) and the right kidney removed via a retroperitoneal incision. A time-release pellet containing DOCA was implanted subcutaneously at the same time. Following recovery from surgery, rats were given 0.9% NaCl rather than tap water to drink. In addition, rats were given one of four treatments over a subsequent 3-week period: (1) candesartan (10 mg/kg/day), (2) A-127722 (10 mg/kg/day), (3) candesartan plus A-127722, or (4) untreated controls (n = 6 in each group). Candesartan cilexetil, an AT1 receptor antagonist (Morimoto and Ogihara, 1994), and A-127722, an ETA-selective antagonist (Opgenorth et al., 1996), were kindly provided by AstraZeneca and Abbott Laboratories, respectively. Drugs were administered via the drinking solution to facilitate continuous administration and concentrations were adjusted daily to maintain constant dosing. TCP measurements and 24-h urine collections were taken weekly. At the end of the 3-week period, animals were anesthetized with Na pento-barbital and blood samples were withdrawn from the abdominal aorta for determining plasma creatinine. The kidney was removed, bisected, and one-half was frozen in liquid nitrogen and stored at –80°C for later quantitative determination of TGF-β and fibronectin while the other half was fixed in 10% neutral-buffered formalin and embedded in paraffin for histological analysis.

2.1. Assays

Urine concentrations of ET-1 were determined by radioimmunoassay (Amersham Pharmacia Biotech, Piscataway, NJ). Urinary sodium concentrations were measured using ion-selective electrodes (Synchron EL-ISE, Beckman Instruments, Brea, CA). Urinary and plasma creatinine was measured by the picric acid method adapted for microtiter plates (Allcock et al., 1998). Protein concentrations in the urine were determined by standard Bradford assay (BioRad, Hercules, CA) using bovine serum albumin as the standard. Sandwich enzyme immunoassays for TGF-β (R&D Systems, Minneapolis, MN) and fibronectin (Chemicon International, Temecula, CA) were used to determine tissue concentrations following protein extraction. Purified rat fibronectin was used as the standard in the fibronectin assay.

2.2. Protein extraction

Isolated renal cortex and renal medulla were weighed, then pulverized while frozen. The pulverized tissue was homogenized with hypotonic buffer (20 mM HEPES, pH, 7.4, 10 mM NaCl, 6 nM staurosporine, 1 mM vanadate, 10 mM NaF) in the presence of protease inhibitors (1 mg/ml PMSF, 1 μg/ml leupeptin, 1 μg/ml pepstatin A) at a ratio of 10:1 (w:v). The homogenate was centrifuged at 15,000 × g at 4°C for 20 min. The soluble extract was removed, aliquoted, and frozen at –80°C. Protein concentrations were determined by standard Bradford assay (BioRad) with bovine serum albumin as standard.

2.3. Histology

Standard hematoxylin and eosin staining was done with an automated Hacker slide stainer. Periodic acid Schiff reaction with hematoxylin was accomplished in the following manner. Slides were deparaffinized, and hydrated, treated with 1% periodic acid for 10 min, rinsed, treated with Schiff's reagent (Sigma, St. Louis, MO) for 10 min, rinsed, and counterstained with hematoxylin, washed, dehydrated, and coverslips mounted.

The levels of proliferative glomerular lesions were quantitated in a similar fashion as previously described (Yamamoto et al., 1998). A score of 1 for fairly normal glomerulus, 2 for mild proliferation, 3 for moderate proliferation, and 4 for severe proliferation or collapse. The mean value was calculated from 50 glomeruli, which were selected at random from each kidney.

2.4. Statistics

Statistical analysis of data was determined by either one-way analysis of variance or analysis of variance for repeated measures with post hoc contrasts (Super ANOVA, Abacus Concepts, Barkeley, CA). All data are reported as means ± S.E. with P < .05 being considered significant.

3. Results

Separate groups were treated with either ETA antagonist, A-127722, AT1 antagonist, candesartan cilexetil, or both. ETA blockade attenuated the development of hypertension in DOCA–salt rats, which was significant after 2 weeks but not after 3 weeks (Fig. 1). AT1 antagonism reduced arterial pressure after 3 weeks when given alone. The combination of ETA and AT1 blockade produced additive effects with arterial pressure being significantly lower than untreated controls at both Weeks 2 and 3.

Fig. 1.

Fig. 1

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Tail cuff measurements of systolic arterial in DOCA–salt rats. Separate groups were treated with ETA antagonist, A-127722, AT1 antagonist, candesartan cilexetil, A-127722 plus candesartan cilexetil, or untreated controls (n = 6 in each group). * P < .05 compared to the untreated control group at any given week.

In untreated controls, body weight increased during the first week, did not change from Weeks 1 to 2, and declined by the third week (Fig. 2). In rats given both ETA and AT1 receptor antagonists, body weight was significantly greater than controls at both Weeks 2 and 3. ETA or AT1 receptor antagonists given alone resulted in significantly increased body weight at Week 3 compared to controls.

Fig. 2.

Fig. 2

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Body weight in DOCA–salt rats treated with ETA antagonist, A-127722, AT1 antagonist, candesartan cilexetil, A-127722 plus candesartan cilexetil, or untreated controls. * P < .05 compared to the untreated control group at any given week.

Protein excretion increased in all four groups during DOCA–salt treatment (Fig. 3). Neither ETA nor AT1 receptor blockade had any significant effect on the development of proteinuria in these rats.

Fig. 3.

Fig. 3

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Urinary protein excretion in DOCA–salt rats treated with ETA antagonist, A-127722, AT1 antagonist, candesartan cilexetil, A-127722 plus candesartan cilexetil, or untreated controls.

Creatinine clearance was significantly increased in rats treated with either ETA or AT1 receptor antagonist (Fig. 4A). Consistent with an improvement in GFR, plasma creatinine concentrations were reduced in rats treated with A-127722 (0.88 ± 0.07 mg%), candesartan cilexetil (0.82 ± 0.06 mg%), or both (0.87 ± 0.07 mg%), compared to controls (1.45 ± 0.15 mg%). Urinary ET-1 excretion was unaffected by antagonist treatment (Fig. 4B).

Fig. 4.

Fig. 4

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Creatinine clearance (A) and urinary ET-1 excretion (B) in DOCA–salt rats treated with ETA antagonist, A-127722, AT1 antagonist, candesartan cilexetil, A-127722 plus candesartan cilexetil, or untreated controls. * P < .05 compared to the untreated control group.

Tissue concentrations of TGF-β and fibronectin in kidneys of DOCA–salt rats treated with A-127722, candesartan cilexetil, or both are presented in Fig. 5. Renal cortical TGF-β concentrations were not different between groups. However, candesartan treatment alone significantly increased renal medullary TGF-β levels. There were no differences in renal medullary fibronectin concentrations between groups.

Fig. 5.

Fig. 5

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Tissue concentrations of TGF-β (A) and fibronectin (B) in kidneys of DOCA–salt rats treated with ETA antagonist, A-127722, AT1 antagonist, candesartan cilexetil, A-127722 plus candesartan cilexetil, or untreated controls. * P < .05 compared to the untreated control group.

Histological sections of each kidney were examined and scored to evaluate the extent of glomerular damage. The renal histological findings were significant for focal necrotizing glomerulonephritis with interstitial arteriolar lesions. Some glomeruli show mild proliferative glomerular lesions, others display moderate lesions, while a small of glomeruli show severe changes including hypertrophy, proliferation, tuft necrosis, mesangiolysis, capillary thrombi, crescent formation, or collapse. Some interstitial arterioles showed vascular lumen narrowing, endothelial damage, medial hypertrophy, and fibrinoid necrosis. The mean levels of glomerular proliferative lesions were not remarkably different among treatment groups (Table 1).

Table 1.

Scores of proliferative glomerular lesions in DOCA–salt rats treated with ETA antagonist, A-127722, AT1 antagonist, candesartan cilexetil, A-127722 plus candesartan cilexetil, or untreated controls

Treatment group Lesion score n
Untreated controls 1.7 ± 0.1 6
A-27722 1.7 ± 0.1 6
Candesartan cilexetil 1.7 ± 0.1 6
A-127722 plus candesartan cilexetil 1.9 ± 0.1 4
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Urine volume and sodium excretion in all four groups of DOCA–salt rats are presented in Fig. 6A and B. Food and water intake are presented in Fig. 6C and D. Both ETA and AT1 receptor antagonist treatment groups were significantly greater than untreated controls for each of these variables. In addition, combined treatment with A-127722 and candesartan cilexetil resulted in higher urine sodium and water excretions, which were accompanied by increased food and water intake compared to untreated controls.

Fig. 6.

Fig. 6

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Urine volume (A), sodium excretion (B), water intake (C), and food intake (D) in DOCA–salt rats treated with ETA antagonist, A-127722, AT1 antagonist, candesartan cilexetil, A-127722 plus candesartan cilexetil, or untreated controls. * P < .05 compared to the untreated control group at any given week.

4. Discussion

Recent studies have established that blockade of ETA receptors can lower arterial pressure in salt-dependent models of hypertension (Pollock, 2000). In the DOCA–salt hypertensive rat, we recently reported that ETA blockade lowers arterial pressure, but has little effect on improving renal function (Allcock et al., 1998). In contrast to most animal models, the hypertension developed in the DOCA–salt model has been shown to have little dependence on the renin–angiontensin system as both angiotensin-converting enzyme inhibitors and angiotensin receptor blockers have no effect on arterial pressure (Kim et al., 1994). However, two studies have provided evidence that blockade of the renin–angiotensin system in this model may have beneficial effects in terms of preventing renal injury (Kim et al., 1994; Wada et al., 1995).

The current study was conducted to determine whether blockade of AT1 receptors provided additional benefit to DOCA–salt hypertensive rats. Our findings indicate that treatment of DOCA–salt hypertensive rats with candesartan cilexetil produced additive effects in reducing blood pressure in combination with the ETA antagonist, A-127722. When giving alone, ETA blockade produced a significant decrease in arterial pressure at Week 2 while AT1 receptor blockade reduced arterial pressure only at Week 3. In contrast to previous observations, ETA blockade did not produce a consistent blood pressure-lowering effect while AT1 blockade has been reported to have no effect (Allcock et al., 1998; Kim et al., 1994). We also observed that blockade of either receptor improved creatinine clearance and decreased plasma creatinine concentrations. These results that both ET-1 and Ang II are contributing to the tonic vasoconstriction observed in this model. The effect of ETA blockade on arterial pressure and creatinine clearance is in contrast with our earlier findings in which ETA blockade produced a sustained decrease in arterial pressure yet had no effect on creatinine clearance in the DOCA–salt model (Allcock et al., 1998). The reason for this discrepancy is not clear although rats in the current study had a more severe hypertension, suggesting that there may be a change in the contribution of ET-1 in control of the renal vascular resistance. In addition, body weight in the animals of the current study did not increase and actually began to decline in untreated controls indicating an apparently more severe response to DOCA–salt treatment.

Several laboratories have recently demonstrated that many of the vascular and mitogenic actions of Ang II are mediated by ET-1 (d'Uscio et al., 1997; Herizi et al., 1998; Rajagopalan et al., 1997). These studies have used endothelin receptor antagonists to inhibit the acute as well as long-term effects of Ang II. Since previous studies have shown that AT1 receptor blockade has a beneficial effect, we expected that ETA receptor blockade would produce an even greater benefit in terms of both hemodynamic as well as antifibrotic effects. However, since we observed very little beneficial effect of AT1 blockade beyond that ETA blockade alone, these studies suggest that Ang II activation of ET-1 synthesis in this model is not a major factor in the pathogenesis of renal dysfunction in this model. This is not completely surprising since the DOCA–salt rat is a model of low renin hypertension.

TGF-β plays an important role in regulating ECM and has been shown to contribute to a variety of fibrotic disorders in various organs including the kidney (Border and Noble, 1994). Development of tissue injury involves TGF-β-induced elevations in matrix components such as fibronectin and collagen. To assess whether any of our treatment regimens would reduce tissue injury in the DOCA–salt model, we measured tissue concentrations of TGF-β and fibronectin in the four groups of rats. Neither candesartan cilexetil nor A-127722 reduced the levels of TGF-β or fibronectin within the kidneys of DOCA–salt rats, which suggest that neither drug had any effect on the interstitial fibrosis associated with this model. These results are in contrast to the study by Kim et al. (1994). These investigators reported that increased mRNA expression for TGF-β, fibronectin, and collagen Types I, III, and IV in kidneys if DOCA–salt rats was reduced by either angiotensin receptor blockade or angiotensin-converting enzyme inhibition. The reason for this discrepancy is not clear although the previous study examined only mRNA expression and not protein. We observed that candesartan actually increased TGF-β in the renal medulla. The significance of this is not clear since the assay measured total TGF-β and cannot distinguish between active and latent forms. Neither antagonist had any significant effect on protein excretion, which suggests that ETA, and/or AT1 receptor blockade had no effect in terms preventing glomerular injury.

In summary, these findings indicate that both AT1 and ETA receptor blockade can produce at least small improvements in hemodynamic function in the DOCA–salt rat. Despite the slight reduction in arterial pressure and improvement of renal function, AT1 and ETA receptor blockade has no effect on renal interstitial fibrosis in this model of low renin hypertension.

Acknowledgments

The authors wish to express their appreciation for the expert technical assistance provided by Ms. Deborah Garner and Ms. Jean Roscow. The authors also wish to thank Dr. Peter Morsing of AstraZeneca Pharmaceuticals for kindly providing the candesartan cilexetil and Dr. Jerry Wessale of Abbott Laboratories for kindly providing the A-127722.

These studies were supported by grants from the National Institute of Health (HL 60653 and HL 64776), the American Heart Association (Scientist Development Grant), and AstraZeneca Pharmaceuticals.

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