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(Hypertension. 2005;45:270.)
© 2005 American Heart Association, Inc.

Scientific Contributions

Renal Angiotensin II Type-2 Receptors Are Upregulated and Mediate the Candesartan-Induced Natriuresis/Diuresis in Obese Zucker Rats

Amer C. Hakam; Tahir Hussain

From the Department of Pharmacological and Pharmaceutical Sciences, University of Houston, Texas.

Correspondence to Tahir Hussain, PhD, Assistant Professor of Pharmacology, Department of Pharmacological and Pharmaceutical Sciences, Science and Research Building 2, University of Houston, 4800 Calhoun, Houston, TX 77204-5037. E-mail thussain2{at}uh.edu

	Abstract

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Recently, there has been a growing interest in studying the role of angiotensin II type-2 (AT₂) receptor in renal/cardiovascular function in pathological conditions. The present study was designed to determine the functional role of the AT₂ receptors on natriuresis/diuresis and compare the level of the tubular AT₂ receptor expression in obese and lean Zucker rats (12 weeks old). Under anesthesia, candesartan (angiotensin II type 1 [AT₁]–specific antagonist; 100 µg/kg bolus) produced natriuresis/diuresis to a greater degree in obese than in lean rats. The specific AT₂ antagonist PD123319 (50 µg/kg per minute) after candesartan administration abolished the natriuretic/diuretic effects of candesartan in obese rats but not in lean rats. Infusion of AT₂ receptor agonist, CGP-42112A (1 µg/kg per minute), produced greater increase in sodium and urine excretion over basal in obese than in lean rats. The presence of the AT₂ receptor expression in the brush-border and basolateral membranes was confirmed by Western blotting using specific antibody and antigen-blocking peptide. Densitometric analysis of the bands revealed 1.5- to 2.0-fold increase in the AT₂ receptor proteins in both membranes of obese compared with lean rats. Our results suggest upregulation of the AT₂ receptors, which play a role in mediating the natriuretic/diuretic effects of AT₁ receptor blockers in obese Zucker rats. We speculate that AT₂ receptors, by promoting sodium excretion, may protect obese Zucker rats against blood pressure increase associated with sodium and water retention.

Key Words: angiotensin II receptors • obesity • angiotensin II • kidney • angiotensin I • rats, Zucker

	Introduction

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Angiotensin II (Ang II), a major effector hormone of the renin-angiotensin system (RAS), signals via 2 major receptor subtypes: Ang II type-1 (AT₁) and Ang II type-2 (AT₂).¹ The AT₁ receptor is predominant in adult tissues, and most of its mediated effects, such as vasoconstriction, hypertrophy, and sodium retention,² are well documented. Recently, there has been a growing interest in studying the role of the AT₂ receptor in renal/cardiovascular function in pathological conditions. Contrary to the initial notion, the presence of AT₂ receptors in various tissues of adult animal models has been reported. Recent evidence implicated AT₂ receptors in the regulation of renal and cardiovascular function, including vasodilation and natriuresis.² Also, the AT₂ receptors have antiproliferative and antigrowth effects, and they promote apoptosis.^2–3 All of these characteristics are very important in preventing tissue remodeling and, therefore, disease progression. It has been shown that AT₂ receptor knockout mice have higher blood pressure and exaggerated response to Ang II infusion on blood pressure.⁴ Acute and chronic blockade of AT₂ receptors prevented the hypotensive effects of AT₁ receptor antagonist in normal rats.⁵ Cardiac overexpression of AT₂ receptors diminished the AT₁ receptor–mediated pressor response.⁶ AT₂ receptors are present in adult rat kidneys⁷ and are involved in interstitial fluid cGMP modulation under low-salt condition.⁸

Zucker rats, a genetic model of obesity and hyperinsulinemia, exhibit hyperactive RAS^9–11 that is manifested in a mildly elevated blood pressure.¹⁰ Obese Zucker rats have greater reduction in blood pressure when treated with AT₁ receptor antagonist compared with lean rats.¹⁰ The natriuretic/diuretic response to AT₁ receptor antagonist is exaggerated in obese Zucker rats when compared with lean rats.¹² The expression and functional role of AT₂ receptors, especially on AT₁ receptor antagonist–mediated renal effects in obese Zucker rats, are not known. Therefore, the present study was designed to determine the expression of AT₂ receptors in proximal tubular membrane and their functional role on natriuresis/diuresis in obese Zucker rats. We observed that AT₂ receptors were upregulated and mediated the candesartan-induced sodium excretion in obese Zucker rats.

	Methods

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Animals
Male obese and lean Zucker rats (10 weeks of age) were purchased from Charles River Laboratories. Animals were housed in the University of Houston animal care facility and had free access to standard rat chow and tap water. The institutional animal use and care committee approved animal experimental protocols.

Urinary, Plasma, and Hemodynamic Parameters of Zucker Rats
Fasting blood glucose was measured using a glucometer (BioScanner 2000; Polymer Technology Systems). An RIA kit (Linco Research) was used to determine plasma insulin levels. Urinary and plasma creatinine levels were determined using a creatinine analyzer (model 2; Beckman). Plasma and urine levels of Na were measured using a flame photometer (Ciba Corning Diagnostics).

Kidney Function Experiment Protocol
Rat surgery and kidney function were preformed as described previously.^12,13 Briefly, rats were anesthetized using Inactin (100 to 160 mg/kg IP). The left jugular vein and carotid artery were cannulated for saline/drug infusion and blood pressure measurement, respectively. The ureter is cannulated for urine collection. After a stabilization period of 1 hour, we collected urine in 30-minute intervals. The first 2 periods were used to compute the basal parameters. After the second period, we delivered a bolus dose of candesartan (100 µg/kg), an AT₁ receptor antagonist, followed by a 30-minute period of urine collection. After this period, we started an intravenous infusion of PD123319 (50 µg/kg per minute), an AT₂ receptor–specific antagonist, and collected urine for 2 periods. Urine samples were placed on ice. Blood pressure was monitored throughout the experiment. At the end of the protocol, kidneys were excised, patted dry, and weighed. A schematic representation of the different protocols we used is shown in Figure 1.

View larger version (21K): [in this window] [in a new window]   Figure 1. Schematic representation of protocols used in the study.

At the end of each urine collection period, urine volume was measured, and urine flow rate (UF) was calculated (µL/min). The urinary sodium excretion rate (urinary sodium volume [U_NaV]; µmol/min) was computed as UFxurinary sodium concentration (µmol/µL). The glomerular filtration rate (GFR; mL/min) was calculated based on creatinine clearance. The U_NaV (µmol/min) was divided by the plasma sodium concentration (mg/dL) to compute the fraction of sodium excreted in urine (FE_Na; percentage)

Proximal Tubule Membrane Preparation
Animals were anesthetized using sodium pentobarbital (50 mg/kg IP). After a midline incision, kidneys were excised and cut sagitally. Outer cortices were used for preparation of basolateral membrane (BLM) and brush-border membrane (BBM) as described previously.¹⁴ The BLM and BBM were characterized with the presence and absence of Na-K-ATPase, respectively. Protein estimation of these samples was done using bicinchoninic acid protein assay kit (Pierce).

Western Blot Analysis
Equal amounts (40 µg protein) of BBM and BLM proteins from lean and obese Zucker rats were used for Western blotting using AT₂ receptor antibody. Anti-rabbit IgG–horseradish peroxidase conjugate and chemiluminescent substrate were used to detect the signal that was recorded on x-ray film. The band was densiometrically quantified. Antigen peptide was used to determine the AT₂-specific band.

Enzymatic Deglycosylation of the AT₂ Receptor Protein
The deglycosylation experiment was preformed as recommended by the kit manufacturer (ProZyme). Briefly, samples from BBM were incubated with denaturing solution and heated at 100°C for 5 minutes. Once cooled to room temperature, the samples were incubated with N-glycanase at 37°C for 3 hours. After incubation, samples were used for Western blot analysis as described above.

¹²⁵I-Sar¹–Ang II Binding
Binding of ¹²⁵I-Sar¹–Ang II to BBM and BLM was performed according to the method described previously.¹⁵ Briefly, 50 µg of protein was incubated with the ligand (25 fmol/L) in a 200 µL (final volume) of binding buffer at 25°C for 15 minutes. The radioligand was displaced with varying concentration of unlabeled Sar¹–Ang II (500 pmol/L to 10 nmol/L). The nonspecific binding was determined using 10 µmol/L Sar¹–Ang II. Losartan (1 µmol/L) was used to determine the AT₁-specific binding. Binding data were subjected to Scatchard analysis for B_max and K_d calculation.

Chemicals
Antibody for AT₂ receptor and its antigen-blocking peptide were purchased from Alpha Diagnostics. Anti-rabbit IgG was purchased from Santa Cruz Biotechnology. ¹²⁵I-Sar¹–Ang II was purchased from Perkin-Elmer Life Sciences. PD123319, CGP-42112A, and all other chemicals were purchased from Sigma Aldrich. Candesartan was a generous gift from AstraZeneca (Wilmington, Del).

Statistical Analysis
Data are presented as mean±SE. One-way ANOVA with post hoc tests (Newmann–Keuls) was used to analyze variation within the group. Student t test was used to compare variation between groups. Binding data were analyzed using computer software by Biosoft. All other statistical analyses were done using Graph Pad Prism, version 3.02 (GraphPad Software). A value of P<0.05 was considered statistically significant.

	Results

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General Parameters
As shown in Table 1, obese rats had significantly higher body weight and significantly lower kidney-to-body weight ratio when compared with lean rats. Fasting blood glucose and plasma insulin levels were significantly elevated in obese rats compared with lean rats. Plasma creatinine level was greater in obese rats when compared with lean rats, suggesting possible kidney damage. Obese rats had significantly higher blood pressure (mean arterial pressure [MAP]) and similar heart rate when compared with lean rats.

View this table: [in this window] [in a new window]   TABLE 1. General and Hemodynamic Parameters in Zucker Rats

Effects of PD123319 on Candesartan-Induced Natriuresis/Diuresis in Obese Rats
In the initial set of experiments, we determined the effect of candesartan and PD123319 alone on UF and hemodynamic parameters. As shown in Table 2, a bolus dose (100 µg/kg IV; protocol 1) of candesartan increased UF in lean and obese Zucker rats that was sustained for 2 hours. The increase in UF over basal was statistically significant in obese rats but not in lean rats. The GFR and MAP were measured for 2 hours after candesartan infusion. MAP and GFR were not altered in lean or in obese rats (Table 2), suggesting a tubular effect of candesartan. In a separate set of experiments, PD123319 infusion (50 µg/kg per minute IV; protocol 2) did not alter UF significantly in lean or obese rats and also caused no changes in GFR and MAP (Table 2). PD infusion did increase the U_NaV in lean rats, but the change did not reach statistical significance.

View this table: [in this window] [in a new window]   TABLE 2. Effect of Candesartan (100 µg/kg bolus) Alone and PD123319 (50 µg/kg per minute infusion) Alone on Kidney Function in Zucker Rats

As indicated in experiment protocol 3, we assessed the effect of PD123319 on candesartan-induced changes in UF and U_NaV. Candesartan (100 µg/kg IV bolus) produced a significant increase in UF, U_NaV, and FE_Na over basal values in obese rats. Similarly, bolus dose of candesartan modestly increased UF and FE_Na over basal in lean rats (Figure 2A through 2C). However, the natriuretic/diuretic response to candesartan was more exaggerated in obese compared with lean Zucker rats. The infusion of PD123319 (50 µg/kg/min IV) almost abolished the natriuresis/diuresis produced by candesartan in obese rats, whereas in lean rats, PD123319 did not alter the candesartan-induced natriuresis/diuresis. Basal UF, U_NaV, and FE_Na were higher in obese than in lean Zucker rats (Figure 2A through 2C).

View larger version (18K): [in this window] [in a new window]   Figure 2. Effect of candesartan (Cand; 100 µg/kg bolus) and PD123319 (PD; 50 µg/kg per minute infusion) on UF (A), UNaV (B), and FENa (C) in Zucker rats. Values are represented as mean±SEM; *P<0.05 vs basal within the same group (ANOVA followed by Newman–Keuls multiple comparison test); #P<0.05 vs candesartan within the same group (ANOVA followed by Newman–Keuls multiple comparison test); $P<0.05 vs lean within the same treatment (Student t test).

Effect of CGP-42112A on Natriuresis and Diuresis in Obese Zucker Rats
In this experiment, we determined the effect of the specific AT₂ agonist^5,16 CGP-42112A on natriuresis and diuresis in lean and obese Zucker rats. The UF and U_NaV in response to CGP-42112A infusion (1 µg/kg per minute; protocol 4) were more increased in obese rats than in lean rats (Figure 3A and 3B). Similarly, FE_Na in response to CGP-42112A infusion was greater in obese than in lean rats (Figure 3C). This dose of CGP-42112A did not alter GFR significantly (lean 0.56±0.06 versus 0.57±0.03; obese 0.71±0.04 versus 0.69±0.08). The MAP was not changed by CGP-42112A in lean (92±5.8 versus 91±4.3). The MAP in obese rats was modestly but insignificantly decreased by CGP-42112A (113±9 versus 103±9). The basal U_NaV and FE_Na were lower in this experiment compared with the previous one; however, the magnitude of difference between lean and obese rats was similar.

View larger version (15K): [in this window] [in a new window]   Figure 3. Effect of CGP-42112A (1 µg/kg per minute) on UF (A), UNaV (B), and FENa (C) in Zucker rats. Values are represented as mean±SEM; *P<0.05 vs basal within the same group (Student t test); #P<0.05 vs lean rats after CGP-42112A infusion (Student t test).

AT₂ Receptor Expression
We determined the expression of the AT₂ receptor protein by Western blotting analysis of the BBM and BLM of lean and obese Zucker rats. The AT₂ receptor antibody detected 3 bands at 50, 45, and 30 kDa (Figure 4A, lane 1). All 3 bands were displaced by antigen peptide of the AT₂ receptor (Figure 4A, lane 2). The densitometric analysis of all 3 bands revealed a significant increase in the density of the AT₂ receptor protein in BBM (50%) and BLM (85%) of obese Zucker rats compared with lean rats (Figure 4B).

View larger version (34K): [in this window] [in a new window]   Figure 4. A is a representative Western blot of the AT2 protein before and after deglycosylation and in the presence and absence of blocking peptide. B, Expression of the AT2 receptor protein in the BBM and BLM of obese Zucker rats as percentage of lean rats (12 weeks old). Bars in B represent the mean intensity of all 3 bands. Values are represented as mean±SEM. *P<0.05 compared with lean of the same group (Student t test; n=6).

It has been reported that the AT₂ receptor has 5 different potential sites for N-glycosylation.¹⁷ We investigated whether a different level of glycosylation is responsible for the different bands, as detected in Figure 3. Treatment with N-glycanase, specific N-linked deglycosylation enzyme, shifted all 3 bands to 1 band (28 kDa), as detected with AT₂ antibody (Figure 3A, lane 3). This band was displaced by the AT₂-blocking peptide (Figure 3A, lane 4), suggesting the band is AT₂ receptor specific.

¹²⁵I-Sar¹–Ang II Binding
Scatchard plot analysis of the binding data revealed no change in total receptor number (Figure 5A) in the BBM and BLM of obese compared with lean (B_max BBM 257±8.6 in lean versus 203±38 in obese rats; B_max BLM 360±11 in lean versus 373±32 in obese rats). K_d values were similar in lean and obese in BBM and BLM (Figure 5B). B_max and K_d values were calculated using Sar¹–Ang II, a nonspecific antagonist. We also determined whether there is a difference in the AT₁ binding between lean and obese. We used losartan (1 µmol/L) to determine the non-AT₁ binding. We found similar AT₁ binding in lean and obese in BBM and BLM (Figure 5C).

View larger version (19K): [in this window] [in a new window]   Figure 5. 125I-Sar1–Ang II binding to the BBM and BLM obtained from lean and obese Zucker rats. Binding data were subjected to Scatchard analysis using LIGAND computer program. The maximum binding sites (Bmax; A) and dissociation constant (Kd; B) were calculated. C represents total (AT1 and AT2) and AT1-specific binding using Sar1–Ang II (10 µmol/L) and losartan (1 µmol/L) for nonspecific binding, respectively. Values are represented as mean±SEM of 5 experiments. *P<0.009 compared with lean and obese of the same group (Student t test). #P<0.05 compared with lean and obese BBM (Student t test).

	Discussion

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Ang II is a potent antinatriuretic and antidiuretic hormone¹⁸ and, thereby, plays an important role in the maintenance of sodium and fluid homeostasis.¹⁹ Increased function of Ang II, either attributable to increased production of the hormone or increased activity of the AT₁ receptor, contributes to development of hypertension.¹⁰ The use of selective AT₁ receptor antagonists leads to reduction in blood pressure in human as well as in experimental animal models, including obese Zucker rats.^5,10,20,21 The greater reduction in blood pressure and greater natriuresis in the absence of enhanced renin activity in the plasma or the kidney of obese Zucker rats^10,22 may be attributed to increased activity of the AT₁ receptors. However, there has been a growing interest to understand the role of the AT₂ receptors, which are known to counteract the effects of the AT₁ receptors, in the renal cardiovascular regulation.^1,3,5,6,18 In the present study, we found that intravenous infusion of PD123319, an AT₂ receptor antagonist, abolished the candesartan-induced natriuresis/diuresis in obese Zucker rats. On the other hand, PD123319 had no effect on candesartan-induced natriuresis/diuresis in lean rats. We also found that direct stimulation of the AT₂ receptors with CGP-42112A produced a significant natriuresis/diuresis in obese but not in lean rats.

Candesartan, PD123319 alone or in combination, and CGP-42112A did not alter the GFR significantly in lean or obese rats. These drugs also did not alter MAP in lean rats and obese rats, except for a modest but insignificant effect of CGP-42112A on MAP in obese rats. The decrease in MAP could be attributed to the effect of CGP-42112A on AT₂ receptors in the blood vessels where AT₂ receptor upregulation has been shown previously.²³ Despite a decrease in MAP, CGP-42112A continued to promote natriuresis in obese rats; therefore, the natriuretic effects may be attributed to the activation of the AT₂ receptors in the tubular membranes. In these experiments, inactin was used as anesthetic agent. Although it has minimal effects on the cardiovascular system,²⁴ we should still be cautious when interpreting the results.

The complete reversal by AT₂ receptor antagonist of candesartan-induced natriuresis and diuresis in obese and not in lean rats indicates a role for AT₂ receptors in obese but not in lean rats. The obvious role of AT₂ receptors in obese and not in lean rats could be attributed to the level of AT₂ receptor expression in lean and obese rats. AT₂ receptor expression in the cortical membranes BBM and BLM of obese rats was 1.5- to 2-fold greater than in lean rats. Although alone, PD123319 infusion had no significant effect on urine and sodium excretion in lean and obese rats, the blockade of AT₁ receptors leaves AT₂ receptors to function unopposed. It is likely that the level of the AT₂ receptor expression in lean rats is not enough to produce natriuresis in lean rats as in obese rats, in which the AT₂ receptor expression is greater. This notion is further supported by the results of the AT₂ receptor agonist–induced natriuresis/diuresis in obese and not in lean rats. The use of losartan, an AT₁ receptor antagonist, showed that there was no difference in the AT₁ receptor–binding sites on BLM or BBM between lean and obese rats.

It was surprising that whereas PD 123319 did not change the basal tone of urine/sodium excretion significantly in obese rats, the AT₂ agonist does show an increase in the urine/sodium output in obese rats. The response to the AT₂ agonist in lean rats was minimal but insignificant compared with the basal levels. The reason for the inability of PD123319 to affect urine/sodium output is not clear. However, it may be extrapolated from other hormonal responses such as dopamine. Dopamine, which activates D₁ receptor present on the proximal tubules, is an autocrine/paracrine natriuretic hormone.²⁵ It is reported that D₁ receptor agonists, and not the antagonist, promotes natriuresis/diuresis in rats. The effect of D₁ antagonist is reported only under acute volume expansion.²⁵ Based on these reports, it may be speculated that under conditions such as sodium load, the effect of AT₂ antagonist becomes obvious; however, that is yet to be investigated.

The mechanism by which AT₂ promotes natriuresis/diuresis in obese Zucker rats is not known. However, it has been shown that renal AT₂ receptors in Sprague-Dawley rats can mediate the production of bradykinin and NO and therefore increase the levels of cGMP.^1,8 In rabbit proximal tubule, it has been shown that AT₂ receptors on the BBM mediate activation of phospholipase A2 and increase the release of arachidonic acid.²⁶ In vitro studies have shown that AT₂ receptors on proximal tubules mediate inhibitory effects on sodium and bicarbonate absorption.²⁶ It is likely that the increased expression of AT₂ receptors on the proximal tubular membranes of obese Zucker rats stimulate the above-mentioned mechanisms affecting the tubular sodium transport when the AT₁ receptors have been blocked by candesartan or the AT₂ receptors were selectively activated by an agonist. However, the functional status of the AT₂ receptors in terms of the second messenger and cellular mechanisms is yet to be determined in the kidney of obese Zucker rats.

The increased renal function of AT₂ receptors on sodium metabolism may have important physiological consequences in obesity-related hypertension. Obese Zucker rat is a model of insulin resistance and development of mild hypertension.¹⁰ It has been reported that treatment with losartan, an AT₁ receptor antagonist, lowers blood pressure to greater extent in obese than in lean Zucker rats.¹⁰ On the basis of our results, it can be speculated that overexpression of the tubular AT₂ receptors might have contributed to the greater reduction in blood pressure of obese rats treated with losartan. However, the direct role of AT₂ receptors in blood pressure regulation in this model of obesity with insulin resistance is yet to be determined. Previous studies in animal models have shown that the AT₂ receptor plays a role in altering blood pressure.^4,5 AT₂ receptor knockout mice have elevated basal blood pressure and exaggerated pressor response to exogenous Ang II infusion when compared with wild-type mice.⁴ In spontaneously hypertensive rats, the AT₂ receptor agonist produced vasodilatation in the presence of AT₁ receptor antagonist, a response that was abolished by the AT₂ receptor antagonist PD123319.¹⁶ Carey et al reported that the AT₂ receptor stimulation using specific AT₂ receptor agonist decreases arterial pressure in normal rats, an effect that was abolished in the presence of the a specific AT₂ receptor antagonist.⁵

Perspectives
Present studies demonstrate a functional role of the renal AT₂ receptors in obese Zucker rats. The enhanced expression of the tubular AT₂ receptors mediates the natriuresis/diuresis induced by the AT₁ receptor blocker (ARB) or by the selective activation of the AT₂ receptors. ARBs and angiotensin-converting enzyme inhibitors (ACEIs) are used to improve renal function in diabetes and to treat hypertension. Recent scientific data have initiated a debate of preferential use of ARBs over ACEIs. The basis of such preference lies in that ARBs selectively block AT₁ receptors and leave the AT₂ receptors unopposed to function, whereas ACEIs lower Ang II production, leading to reduction in the functions of AT₁ and AT₂ receptors. The present study supports the notion that the use of ARBs will leave the AT₂ receptors intact, which mediates the beneficial effects on renal sodium excretion and in lowering blood pressure in obesity.

	Acknowledgments

 
This work is supported by National Institutes of Health grant R01-DK61578. Candesartan was a generous gift from AstraZeneca.

Received July 1, 2004; first decision July 22, 2004; accepted November 13, 2004.

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