Angiotensin II AT2 receptors act as a functional antagonist for the AT1 receptors in various tissues. We previously reported that activation of the renal AT2 receptors promotes natriuresis and diuresis; however, the mechanism is not known. The present study was designed to investigate whether activation of AT2 receptors affects the activity of Na+-K+-ATPase (NKA), an active tubular sodium transporter, in the proximal tubules isolated from Sprague-Dawley rats. The AT2 receptor agonist CGP-42112 (10−10-10−7 M) produced a dose-dependent inhibition of NKA activity (9–38%); the inhibition was attenuated by the presence of the AT2 receptor antagonist PD-123319 (1 μM), suggesting the involvement of the AT2 receptors. The AT1 receptor antagonist losartan (1 μM) did not affect the CGP-42112 (100 nM)-induced inhibition of NKA activity. The presence of guanylyl cyclase inhibitor ODQ (10 μM) and the nitric oxide (NO) synthase inhibitor Nω-nitro-l-arginine methyl ester (l-NAME; 100 μM) abolished the CGP-42112 (100 nM)-induced NKA inhibition. ANG II (100 nM), in the presence of losartan, significantly inhibited NKA activity; the inhibition was attenuated by PD-123319. CGP-42112 also, in a dose-dependent manner, stimulated NO production (∼0–230%) and cGMP accumulation (∼25–100%). The CGP-42112 (100 nM)-induced NO and cGMP increases were abolished by the AT2 receptor antagonist PD-123319, ODQ, and l-NAME. The data suggest that the activation of the AT2 receptor via stimulation of the NO/cGMP pathway causes inhibition of NKA activity in the proximal tubules. This phenomenon provides a plausible mechanism responsible for the AT2 receptor-mediated natriuresis-diuresis in rodents.
- sodium transport
the renin-angiotensin-system (RAS) is a major regulator of sodium and water homeostasis. The octapeptide ANG II is the primary mediator of the RAS effects. ANG II induces its effects by binding to two major receptor subtypes, AT1 and AT2 (4). The AT1 receptors are ubiquitously expressed and mediate ANG II-induced vasoconstriction, sodium reabsorption, aldosterone secretion, and cell growth and proliferation (7). The functional responses associated with AT2 receptors are less understood, however, the AT2 receptors have recently been of great interest as functional antagonist of the AT1 receptors. The AT2 receptors are expressed in adult rat tissues (14, 24) and are implicated in mediating vasodilation, apoptosis, and antiproliferation (17, 23). Recently, we have shown that the renal AT2 receptors promote natriuresis in Zucker rats (14). Most of the in-vivo effects mediated by the AT2 receptors in the kidney seem to involve the nitric oxide (NO)/cGMP pathway (5).
Within the kidney, sodium homeostasis is controlled via many sodium transporters, some of which are present in the proximal tubules. ANG II via its action on the AT1 receptors stimulates the activity of the Na+-K+-ATPase (NKA) (3), Na+/H+ exchanger (NHE) (2), and the Na+/HCO3− cotransporter (NBC) (11), thereby leading to an increase in sodium reabsorption. Of these sodium transporters, the NKA, a basolateral membrane protein, is an active sodium transporter and plays a major role in pumping sodium out of the tubular cells against its concentration gradient. Others and we have shown that the AT2 receptors are expressed on the proximal tubular membranes (14, 15, 24). Also, recently, we have shown that the AT2 receptors promote sodium excretion (14). However, it is not known whether activation of the AT2 receptors causes direct inhibition of the NKA activity; therefore, this study was designed to investigate the AT2 receptor-mediated effects on the NKA activity in the isolated proximal tubular suspension from Sprague-Dawley rats. Also, we determined whether AT2 receptor stimulation leads to increases in NO formation and cGMP accumulation that participate in AT2 receptor-mediated NKA inhibition in the proximal tubular suspension. Here, we report for the first time that the renal AT2 receptors have an inhibitory effect on the proximal tubular NKA activity via a NO/cGMP-dependent pathway.
Age-matched male Sprague-Dawley rats, weighing 200–250 g and purchased from Harlan (Indianapolis, IN), were used in this study. The 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 the animal experimental protocols.
Experimental protocol for renal function.
Rat surgery and measurement of kidney function were performed as described earlier (14, 15, 20, 28). Briefly, rats were anesthetized using Inactin (100–160 mg/kg ip). The left jugular vein and carotid artery were cannulated for saline/drug infusion and blood pressure measurement, respectively. The ureter was cannulated for urine collection. Normal saline was continuously infused at a fixed rate of 1% body wt to maintain constant hydration. After a stabilization period of 1 h, we collected urine in 30-min intervals. The first two periods (30 min each) were used to compute the basal parameters, the second two periods (30 min each) were used to compute the candesartan effect, and the third two periods (30 min each) were used to compute the PD-123319 and candesartan effects. The following is the schematic representation of the protocol.
At the end of each urine collection period, the urine volume was measured and urine flow rate (UF) was calculated (μl/min). The urinary sodium excretion rate (UNaV μmol/min) was computed as UF × urinary sodium concentration (μmol/μl). The glomerular filtration rate (GFR) (ml/min) was calculated based on creatinine clearance. The UNaV (μmol/min) was divided by the plasma sodium concentration (μmol/μl) and GFR (μl/min), the quotient was then multiplied by 100 to compute the fraction of sodium excreted in the urine (FENa, %). 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, Norwood, MA).
Preparation of renal proximal tubular suspensions.
Renal proximal tubular suspensions were prepared as described previously (6). Rats were anesthetized with pentobarbital sodium (50 mg/kg ip). After a midline incision, selective perfusion of the kidneys was performed with modified Krebs-Hensleit buffer containing collagenase type IV (230 U/ml) and hyaluronidase type III (250 U/ml). Kidneys were excised and the outer cortex was removed that was minced into fine pieces and digested with collagenase-hyaluronidase solution under a 95% O2-5% CO2 atmosphere until uniformly dispersed. Enrichment of proximal tubules was carried out using a 20% Ficoll gradient. Trypan blue exclusion test was used to determine tubule cell viability (12). More than 95% of the tubules excluded Trypan blue, indicating viable tubular preparation. Protein in the proximal tubular suspension was assayed using a kit (Pierce Products, Rockfort, IL).
The proximal tubule suspensions (1 mg/ml) were incubated without (basal) and with CGP-42112 (10−10-10−7 M) in the presence and absence of the AT2 receptor antagonist PD-123319 (1 μM) for 30 min at 37°C in a shaking water bath. The AT1 receptor antagonist losartan (1 μM), the nitric oxide synthase (NOS) inhibitor Nω-nitro-l-arginine methyl ester (l-NAME; 100 μM) (26), and the NO-dependent soluble guanylyl cyclase (sGC) inhibitor 1H-[1,2,4] oxadiazolo- [4,3-a] quinoxalin-1-one (ODQ, 10 μM) (10) were added with the AT2 agonist CGP-42112 (100 nM) in the proximal tubule suspension. These various inhibitors were added to the tubule suspension 10 min before the agonist was added. In a different set of experiments, the proximal tubule suspensions were incubated for 30 min at 37°C in a shaking water bath with ANG II (100 nM) in the presence and absence of the AT1 receptor antagonist losartan (1 μM), the AT2 receptor antagonist PD-123319 (1 μM), or both. After incubation, the proximal tubules were permeabilized by rapid freezing on a dry ice/acetone mixture and thawed, and were used for the NKA activity assay, as described previously (18, 25). Briefly, the samples (100 μl each) were suspended in 1 ml of reaction mixture A (mM: 70 NaCl, 5 KCl, 5 MgCl2, 6 NaN3, 37.5 imidazole, 1 NaEGTA, 75 Tris·HCl; pH 7.4) for total ATPase activity, and reaction mixture B (mM: 5 MgCl2, 6 NaN3, 37.5 imidazole, 1 NaEGTA, 150 Tris·HCl, pH 7.4) with 1 mM ouabain, for ouabain-insensitive ATPase activity. The reaction was initiated by the addition of 4 mM ATP and incubated for 15 min at 37°C. The reaction was terminated by addition of 50 μl of ice-cold trichloroacetic acid solution (50%). The tubes were transferred onto ice and kept for a few minutes. Coloring reagent (1 ml) (5% FeSO4 in 1% ammonium molybdate in 1 N H2SO4) was added. The liberated inorganic phosphate (Pi) was determined by measuring the absorbance at 740 nm. The NKA activity was measured as a function of liberated Pi. The total ATPase activity minus the ATPase activity in the presence of ouabain (non-specific) represents the specific NKA activity.
The proximal tubules (1 mg/ml) were incubated without (basal) and with CGP-42112 (10−10-10−7 M) at 37°C for 30 min in a shaking water bath. Various inhibitors, PD-123319 (1 μM), l-NAME (100 μM), and ODQ (10 μM) were added to the tubular suspension 10 min before the agonist (100 nM). After incubation, samples were boiled for 5 min to stop the reaction. The samples were acidified by adding 10 μl of 0.1 N HCl and centrifuged for 10 min at 600 g. The supernatant was aliquoted and stored at −20°C for cGMP determination using an ELISA kit (R&D Systems, Minneapolis, MN). A set of standards (0.4–500 pmol/ml) was assayed in duplicate along with the samples. Nonspecific binding and the background were subtracted from each reading and the average optical density was calculated. The data were processed using GraphPad Prism. Values were presented as picomoles of cGMP per milligram protein.
For measuring urinary cGMP, urine samples from the functional study were diluted 100-fold according to the manufacturer's recommendation. The cGMP was assayed in duplicate using an ELISA kit, as described above. The concentration was extrapolated from the standard curve, and then the 100-fold dilution was accounted for. The final concentration was multiplied by the UF to calculate the concentration per unit of time.
Total nitrite/nitrate was measured using an enzymatic kit (R&D Systems). The proximal tubules (1 mg/ml) were incubated without (basal) and with CGP-42112 (10−10-10−7 M) for 30 min at 37°C for 30 min in a shaking water bath. When applicable, the proximal tubules were incubated with PD-123319 (1 μM) and l-NAME (100 μM) 10 min before CGP-42112 (100 nM) was added. After incubation, samples were boiled for 5 min to stop the reaction. After boiling, the samples were filtered by centrifuging at 5,000 g for 1 h in 10,000 MW protein cutoff centrifuge tubes (Millipore, Bedford, MA). The filtrate was aliquoted and stored at −20°C for nitrite/nitrate measurement. A set of standards (3.25–100 μM) was assayed in duplicate along with the samples. The background was subtracted from each reading, and the average optical density was calculated. The data were processed using GraphPad Prism. The values are represented as nmol of nitrite/nitrate per mg protein.
CGP-42112, PD-123319, l-NAME, ODQ, ANG II, and all other chemicals were purchased from Sigma (St. Louis, MO). Losartan was a generous gift from Merck Sharp & Dohme. Candesartan was a generous gift from AstraZeneca.
Data are presented as means ± SE. One-way ANOVA with post hoc tests (Neumann-Keuls) was utilized to analyze variation within the group. Student's t-test was used to compare variation between groups. All statistical analyses were done using GraphPad Prism, version 3.02 (GraphPad Software, San Diego, CA). A value of P < 0.05 was considered statistically significant.
Effect of AT2 receptor antagonist on the AT1 receptor antagonist-induced natriuresis-diuresis and urinary cGMP.
The administration of the AT1 receptor antagonist candesartan (100 μg/kg iv bolus) generated significant diuresis and natriuresis that were abolished by the AT2 receptor antagonist PD-123319 (50 μg·kg−1·min−1; Fig. 1, A and B). The FENa was significantly increased by the administration of candesartan and the increase was partially but significantly decreased by the AT2 receptor antagonist, suggesting that the natriuresis observed was a tubular effect of the AT2 receptors (Fig. 1). Neither the AT1 nor the AT2 antagonists altered the GFR (basal: 0.25 ± 0.009 vs. candesartan: 0.29 ± 0.008 vs. candesartan & PD-123319: 0.28 ± 0.017 ml/min) or the mean arterial pressure (MAP; basal: 99 ± 3.6 vs. candesartan: 96 ± 2.6 vs. candesartan & PD-123319: 94 ± 2.4 mmHg), suggesting a tubular effect of these antagonists. In a separate set of experiments, we have established that the AT2 receptor antagonist PD-123319 alone does not alter kidney function parameters and that the AT1 antagonist candesartan alone produces a significant diuresis-natriuresis that lasts for 3 h without altering GFR or MAP (data not shown).
In the same set of experiments, the administration of the AT1 antagonist candesartan (100 μg/kg iv bolus) caused a significant increase in the urinary cGMP excretion that was abolished by the administration of the AT2 antagonist PD-123319 (50 μg·kg−1·min−1; Fig. 1D), suggesting that AT2 receptor activation by endogenous ANG II leads to cGMP production.
Effect of AT2 receptor agonist on NKA activity.
The AT2 receptor agonist CGP-42112, in a dose-dependent manner (10−10-10−7 M), inhibited NKA activity (Fig. 2A). The minimal inhibitory effect of ∼9% was observed at 100 pM, and the maximal inhibitory effect of ∼38% was observed at 10 nM of the agonist. The ouabain-insensitive (Mg+-ATPase) was not affected by the CGP-42112 treatment (Fig. 2B). The inhibitory response to CGP-42112 was inhibited at all concentrations by PD-123319 (1 μM), suggesting that the CGP-42112 effect was AT2 receptor mediated (Fig. 2A). PD-123319 by itself did not affect the basal NKA activity (basal: 192 ± 15.8 vs. PD-123319: 195 ± 8.2 nmol Pi·mg protein−1·min−1). We tested various inhibitors to investigate the potential pathway involved in mediating the CGP-42112 (100 nM)-induced inhibition of the NKA activity. The AT1 receptor antagonist losartan (1 μM) did not alter the inhibitory effect of CGP-42112 (100 nM) on the NKA activity (Fig. 3). The nitric oxide synthase inhibitor l-NAME (100 μM) and the sGC inhibitor ODQ (10 μM) abolished the AT2 receptor agonist-induced inhibition of NKA activity (Fig. 3). These inhibitors on their own had no effect on basal NKA activity (basal: 192 ± 15.8 vs. l-NAME: 200 ± 7.9 and ODQ: 189 ± 9.7 nmol Pi·mg protein−1·min−1), suggesting that the AT2 receptor-mediated inhibition of the NKA activity is NO and cGMP dependent.
Effect of ANG II on NKA activity.
ANG II is reported to produce a biphasic effect on NKA activity, stimulation at pM and inhibition at nM/μM concentration of the peptide (3). In the present study, we used nM concentration, so that a modest inhibitory response to ANG II may be examined in the presence of AT2 and AT1 receptor antagonists. Incubating the proximal tubule suspension with ANG II (100 nM) produced a significant inhibition of NKA activity (Fig. 4). The presence of a selective AT1 antagonist losartan (1μM) significantly augmented the ANG II (100 nM)-induced inhibition of NKA activity (Fig. 4), suggesting that the AT1 receptor activation was counteracting the AT2 receptor-mediated inhibition. The presence of the AT2 antagonist (PD-123319, 1μM) abolished the augmentation of NKA activity inhibition observed with the presence of the AT1 antagonist alone (Fig. 4). However, the simultaneous presence of the AT1 and AT2 receptor antagonists did not restore NKA activity to the basal level (basal was significantly higher then ANG II + PD-123319 and ANG II + PD-123319 + losartan groups), suggesting that ANG II is acting via AT2-dependent and AT2-independent pathways to exert its inhibitory effect on NKA activity.
Effect of AT2 receptor agonist on cGMP accumulation.
The AT2 receptor agonist (CGP-42112) in a dose-dependent manner (10−10-10−7 M) increased cGMP accumulation in the proximal tubules (Fig. 5A). The maximal stimulatory effect of ∼100% was observed with 10 nM, and the minimal stimulatory effect of ∼30% was observed with the 100 pM of the agonist. Preincubating the proximal tubules with the AT2 receptor antagonist PD-123319 (1μM) abolished the CGP-42112 (100 nM)-induced cGMP accumulation, suggesting the involvement of the AT2 receptors. PD-123319 by itself did not affect the basal cGMP levels (basal:0.59 ± 0.067 vs. PD-123319: 0.51 ± 0.062 pmol/mg protein). The NOS inhibitor l-NAME (100 μM) and the NO-dependent sGC inhibitor ODQ, both abolished the CGP-42112-induced cGMP accumulation, suggesting that it is NO and dependent and sGC mediated. Both inhibitors alone did not significantly alter the basal cGMP levels (basal: 0.59 ± 0.067 vs. ODQ: 0.51 ± 0.11 and l-NAME: 0.67 ± 0.1 pmol/mg protein).
Effect of AT2 receptor agonist on nitrite/nitrate formation.
Total nitrite/nitrate levels, a measure of NO production, were stimulated in the proximal tubules by the AT2 receptor agonist CGP-42112 (10−10-10−7 M) in a dose-dependent manner way (Fig. 6). The maximal stimulatory effect of ∼240% was observed with 10 nM, while the minimal stimulatory effect of ∼90% was observed with 1 nM agonist. Preincubating the proximal tubules with the AT2 receptor antagonist PD-123319 (1 μM) abolished CGP-42112 (100 nM)-induced nitrite/nitrate formation, suggesting the involvement of the AT2 receptors. The NOS inhibitor l-NAME (100 μM) also abolished the AT2 receptor-mediated stimulation of nitrite/nitrate, suggesting that it is NOS mediated. PD-123319 or l-NAME alone did not significantly affect the basal nitrite/nitrate levels (basal: 3.67 ± 0.6 vs. PD-123319: 4.3 ± 1.3 and l-NAME: 2.84 ± 0.5 nmol/mg protein).
Recently, renal AT2 receptors have been shown to mediate physiological effects in whole animal studies (5, 14, 15). In a recent report, we showed that renal AT2 receptors promote natriuresis/diuresis in obese Zucker rats (14). In that report, we demonstrated a direct effect of the renal AT2 receptors on sodium metabolism using the AT2 agonist CGP-42112. We also showed that the AT1 receptor antagonist-induced diuresis-natriuresis is mediated by the AT2 receptors because infusing the AT2 receptor antagonist PD-123319 abolished it (14). Similar findings are observed in the present study showing that the natriuresis-diuresis induced by candesartan was inhibited by the infusion of the AT2 receptor antagonist PD-123319 in Sprague-Dawley rats. Since infusion of these antagonists did not affect GFR or MAP, the changes in natriuresis may be attributed to the changes in tubular sodium transport. Although these studies demonstrated that renal AT2 receptors promote natriuresis-diuresis and therefore act as functional antagonist to the renal AT1 receptors, the question remained to be answered of whether the AT2 receptor activation modulates the tubular sodium transport, leading to increase in tubular sodium excretion.
In the present study, we investigated the effect of AT2 receptor activation on NKA activity in the isolated proximal tubule, the site of maximum sodium reabsorption and the site of the AT2 receptor expression (14, 15, 24). We found that the AT2 agonist CGP-42112 produced concentration-dependent inhibition of NKA activity. The presence of the AT2 antagonist PD-123319 diminished this inhibitory effect, while the AT1 antagonist losartan did not affect the CGP-42112 (100 nM)-induced inhibition, suggesting the involvement of the AT2 receptors. The inhibition of NKA activity was not associated with any significant changes in the ouabain-insensitive ATPase.
We also found that ANG II (100 nM) significantly inhibited NKA activity. The presence of the AT1 receptor antagonist losartan in the incubation buffer augmented the ANG II-induced inhibition of NKA activity. This augmentation in NKA inhibition was abolished by the presence of the AT2 receptor antagonist PD-123319, suggesting the role of the AT2 receptors. There have been reports suggesting that the nanomolar concentration of ANG II causes inhibition in sodium transport (3); however, the issue is not settled as to the receptor subtype (AT1 or AT2) involved in the inhibitory effect. In the present study, the simultaneous presence of both the AT1 and the AT2 receptor antagonists could not completely abolish the ANG II-induced inhibition of NKA activity. This suggests that ANG II induced an inhibitory effect on NKA activity via the AT2 receptor as well as via an unknown mechanism, which is insensitive to losartan and PD-123319, that is yet to be investigated. We have previously reported the expression of AT2 receptors on both brush-border and basolateral membranes (14, 15). Since we performed the assay in tubular segments and the access of the drug (CGP-42112) to the luminal AT2 receptors may be hindered, it is likely that the CGP-42112-mediated NKA inhibition is mediated by the basolateral AT2 receptors.
Recently, it has been reported that ANG II via AT2 receptors causes inhibition on the Na+-ATPase in the basolateral membranes isolated from pig kidney; however, they showed no effect of the AT2 receptors on NKA activity (8). The authors acknowledged that the lack of any response to ANG II on NKA activity could be attributed to the fact that they used isolated membranes and not whole intact cells and therefore, the intracellular machinery required for any effect of the AT2 receptors on NKA activity was absent. Haithcock et al. (16) showed an inhibitory effect of ANG II via the AT2 receptors on NBC in rabbit proximal tubule cells in culture. The inhibitory effects of the AT2 receptors on tubular NKA and NBC activities could explain the role of the AT2 receptors on natriuresis reported in our previous studies (14, 15).
Most of the studies on the renal AT2 receptors suggest that NO is the intracellular mediator of their physiological effects (5). It has also been shown that the renal AT2 receptors stimulate cGMP accumulation, the second messenger of NO (5). We investigated the role of NO and cGMP in the CGP-42112-induced inhibition of NKA by utilizing the NOS and sGC inhibitors l-NAME and ODQ, respectively. The CGP-42112-induced inhibition of NKA activity is abolished by l-NAME and ODQ, suggesting that the CGP-42112-induced inhibition is NO and cGMP dependent. NO also can directly influence fluid absorption independently of cGMP (9, 13). In our preparation, our data suggest that the effect of NO is cGMP dependent since the sGC inhibitor abolished the inhibitory effect of CGP-42112 to the same extent as the NOS inhibitor. The changes in urinary excretion of cGMP also support the role of this molecule in AT2 receptor-mediated sodium excretion. We found that the systemic infusion of the AT1 receptor antagonist candesartan increased urinary cGMP levels associated with an increase in natriuresis. The increase in urinary cGMP and Na excretion was abolished by infusing the AT2 antagonist, suggesting that in the presence of the AT1 receptor antagonist, the endogenous ANG II acting via the AT2 receptors is mediating natriuresis-diuresis in a cGMP-dependent manner.
Various reports exist describing the inhibitory effect of cGMP on the NKA (21, 27). The cGMP is known to interact with various downstream signaling pathways (19) to mediate its effect, and of interest is the cGMP-dependent protein kinase (PKG). The inhibitory effect of the cGMP on the NKA is abolished by the PKG inhibitor and not by the cAMP-dependent protein kinase inhibitor as demonstrated by McKee et al. (21). cGMP can phosphorylate the protein phosphatase inhibitors inhibitor 1 and DARPP-32 via a PKG-dependent pathway in the rat brain and plexus (21). cGMP, the protein phosphatase inhibitors inhibitor 1, and DARPP-32 are present in the kidney and are implicated in mediating the dopamine inhibition of the NKA (1, 22). It is likely that this downstream signaling pathway is also a part of the AT2 receptor-linked inhibition of NKA activity.
Activation of the AT2 receptor in proximal tubules may lead to the inhibition of other transporters involved in sodium metabolism such as the NHE or the NBC via the signaling pathway described above or via a different pathway. Any influence of the AT2 receptors on these transporters may affect intracellular sodium homeostasis and therefore, influence NKA activity. However, NO, which is a mediator of AT2 action on NKA in the present study, is known to inhibit NHE (9). It has also been shown that NO can inhibit the NKA activity independently of a NHE effect or luminal sodium entry in renal medullary slices (21). NO has also been shown to inhibit NKA activity in a transformed mouse proximal tubular cell line (SV40). The NO-mediated NKA inhibition in SV40 cells was not affected by the presence and absence of nystatin, a cation ionophore that eliminates cation gradients across the plasma membranes. This study suggests that the inhibitory effect of NO on NKA activity is independent of intracellular sodium concentration (13). However, it is likely that in our preparation the AT2 receptors via a NO/cGMP pathway are involved in modulating the activity of other sodium transporters such as NHE. Such an effect can influence intracellular sodium concentration, leading to changes in NKA activity. We should also acknowledge that the AT2 receptors may directly affect NKA activity. These possibilities are yet to be investigated.
In summary, this study demonstrates, for the first time, that the activation of the ANG II AT2 receptors via a NO/cGMP-dependent pathway causes inhibition of NKA activity in the proximal tubule isolated from Sprague-Dawley rats. This NKA-inhibitory effect can explain the physiological role of the renal AT2 receptors in mediating the AT1 receptor antagonist-induced (14, 15) or the AT2 receptor agonist-induced diuresis-natriuresis observed in Zucker rats (14). This observation is of great therapeutic importance since it supports the argument for the use of AT1 receptor antagonists for the treatment of hypertension, especially salt-sensitive hypertension.
This work is supported by National Institutes of Health Grant R01-DK-61578. Losartan was a generous gift from Merck Sharp and Dohme. Candesartan was a generous gift from AstraZeneca.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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