AT2 Receptor Activation Induces Natriuresis and Lowers Blood PressureNovelty and Significance
Rationale: Compound 21 (C-21) is a highly selective nonpeptide AT2 receptor (AT2R) agonist.
Objective: To test the hypothesis that renal proximal tubule AT2Rs induce natriuresis and lower blood pressure in Sprague-Dawley rats and mice.
Methods and Results: In rats, AT2R activation with intravenous C-21 increased urinary sodium excretion by 10-fold (P<0.0001); this natriuresis was abolished by direct renal interstitial infusion of specific AT2R antagonist PD-123319. C-21 increased fractional excretion of Na+ (P<0.05) and lithium (P<0.01) without altering renal hemodynamic function. AT2R activation increased renal proximal tubule cell apical membrane AT2R protein (P<0.001) without changing total AT2R expression and internalized/inactivated Na+-H+ exchanger-3 and Na+/K+ATPase. C-21–induced natriuresis was accompanied by an increase in renal interstitial cGMP (P<0.01); C-21–induced increases in urinary sodium excretion and renal interstitial cGMP were abolished by renal interstitial nitric oxide synthase inhibitor l-N6-nitroarginine methyl ester or bradykinin B2 receptor antagonist icatibant. Renal AT2R activation with C-21 prevented Na+ retention and lowered blood pressure in the angiotensin II infusion model of experimental hypertension.
Conclusions: AT2R activation initiates its translocation to the renal proximal tubule cell apical membrane and the internalization of Na+-H+ exchanger-3 and Na+/K+ATPase, inducing natriuresis in a bradykinin-nitric oxide-cGMP–dependent manner. Intrarenal AT2R activation prevents Na+ retention and lowers blood pressure in angiotensin II–dependent hypertension. AT2R activation holds promise as a renal proximal tubule natriuretic/diuretic target for the treatment of fluid-retaining states and hypertension.
Composed of multiple enzymes, peptide hormones, and receptors, the renin–angiotensin system (RAS) is a major regulatory element in the control of cardiovascular and renal function.1,2 Angiotensin II (Ang II), the pivotal peptide hormone of the RAS, directly binds to and activates 2 G protein–coupled receptors, the type 1 (AT1R) and type 2 (AT2R) angiotensin receptors that generally oppose each other. Activation of AT1Rs induces cellular dedifferentiation and growth, vasoconstriction, antinatriuresis, aldosterone secretion, and sympathetic activation that ultimately lead to hypertension. In contrast, AT2R activation induces cellular differentiation and growth inhibition, vasodilation, and natriuresis and potentially lowers blood pressure (BP).1,2 Because AT1R expression in cardiovascular and renal tissues is ubiquitously present and quantitatively greater than that of AT2Rs, AT1R actions generally predominate in vivo. However, AT2R actions can be demonstrated in vivo when the RAS is activated or AT1Rs are blocked, and several actions of AT1R blockade have been attributed, at least in part, to AT2R activation.3–5
The RAS is thought to be a fundamental driving force contributing to the development of hypertension in experimental animals and humans. According to the Guyton hypothesis, the capacity of the kidneys to excrete sodium (Na+) via the pressure-natriuresis mechanism is central in the regulation of BP. That is, to sustain a hypertensive process, the increase in renal perfusion pressure cannot be offset by an increase in Na+ excretion.6,7 Recent receptor cross-transplantation and selective renal tubule receptor knockout studies have validated this concept by demonstrating that renal proximal tubule (RPT) AT1Rs are required to sustain a hypertensive response to exogenous Ang II.8–10 Importantly, Li and Zhuo have recently demonstrated that RPT-dominant transfer of AT1aRs (short-term knock-in) induces increased BP responses to both extracellular and intracellular Ang II in AT1R-deficient mice.11 These observations underscore the importance of RPT AT1aRs in the control of BP through their effects to increase Na+ reabsorption.
AT2Rs are expressed in the adult kidney primarily in the RPT.12,13 We have recently demonstrated that RPT AT2Rs inhibit renal Na+ reabsorption and that, rather than Ang II, des-aspartyl1-Ang II (Ang III) is the predominant endogenous agonist for this response.14–17 The natriuretic actions of intrarenal Ang III were demonstrable, however, only when systemic AT1Rs were blocked, unless Ang III metabolism was also abrogated with an aminopeptidase N inhibitor.16
The present study was designed to explore the mechanisms of renal Na+ transport in response to both systemic and intrarenal AT2R activation with the highly selective nonpeptide AT2R agonist compound 21 (C-21)18,19 in vivo in the rat and mouse. We hypothesized that C-21 would increase Na+ excretion by promoting the internalization/inactivation of the 2 major Na+ transporters, Na+-H+ exchanger-3 (NHE-3) and Na+/K+ATPase (NKA), in the RPT. Here, we show that systemic C-21 administration activates RPT AT2Rs, inducing natriuresis in a bradykinin-, nitric oxide (NO)–, and cGMP-dependent manner in the absence of AT1R blockade. We also demonstrate that renal AT2R activation recruits RPT AT2Rs to the apical plasma membrane and retracts/internalizes RPT NHE-3 to the apical membrane base/subapical membrane region and NKA from the basolateral membrane to intracellular compartments. We further demonstrate that renal AT2R activation prevents Na+ retention and lowers BP in Ang II–dependent hypertension. Because no diuretic/natriuretic agents with predominant actions in the RPT are clinically available, these observations suggest that AT2R activation may be a novel approach to the pharmacotherapy of fluid-retaining states and hypertension.
Please see the Online Data Supplement for detailed methods (total renal cortical cell membrane and Western blot analysis, RPT cell [RPTC] apical membrane isolation and Western blot analysis, in vivo kidney perfusion and fixation, confocal immunofluorescence microscopy, immunoelectron microscopy, and specific experimental protocols).
All experimental protocols were approved by the Animal Care and Use Committee at the University of Virginia and performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The rat experiments were conducted on 12-week-old female (Harlan; protocols 1–5, 7) and male Sprague-Dawley (SD) rats (protocol 6). The mouse experiments were conducted on 12-week-old female wild-type (WT) C57BL/6 (Harlan) and AT2R-null mice (whole-body knockout) on a C57BL/6 background generously provided by Dr Tadashi Inagami of the Vanderbilt University Department of Biochemistry (protocol 8). All animals were housed in a vivarium under controlled conditions (temperature, 21±1°C; humidity, 60±10%; light, 8:00–20:00) and fed a normal Na+ diet (0.3% Na+; Harlan).
Standard Protocol for Rat In Vivo Studies
For studies that involved systemic AT1R blockade (protocols 1 and 6), a 24-hour osmotic mini-pump (Alzet Model 2001D) infusing candesartan (0.01 mg/kg per minute) was inserted 24 hours before experimentation. The rats were placed under short-term anesthesia with ketamine (100 mg/mL) and xylazine (20 mg/mL) via intraperitoneal injection, and the pumps were implanted in the interscapular region using sterile technique.
On the day of experimentation, the rats were anesthetized with inactin (100 mg/kg body weight) via intraperitoneal injection, and a tracheostomy was performed using polyethylene tubing (PE-240) to assist respiration. Direct cannulation of the right internal jugular vein using PE-10 tubing provided intravenous access through which 2% BSA made in 5% dextrose in water (protocols 1, 3–5, 7), 2% BSA made in 5% dextrose in water with inulin and lithium chloride (protocol 2), 2% BSA made in 0.9% saline (protocol 6), or C-21 made in either solution was infused at 50 μL/min. Direct cannulation of the right carotid artery with PE-50 tubing provided arterial access for monitoring mean arterial pressure (MAP). After a midline laparotomy, the right kidney was excised (so that substances infused directly into the kidney would not spill over to the opposite kidney confounding the results) and the ureter of the remaining left kidney was cannulated (PE-10) to collect urine for the quantification of urine Na+ excretion (UNaV).
Renal Cortical Interstitial Infusion
An open-bore microinfusion catheter (PE-10) was inserted under the renal capsule into the cortex of the left kidney to ensure renal interstitial (RI) infusion of vehicle 5% dextrose in water or pharmacological agent at 2.5 μL/min with a syringe pump (Harvard; model 55-222) as reported previously.14–17,20 When 2 agents were infused, separate catheters were used. Vetbond tissue adhesive (3M; Animal Care Products) was added to secure the catheters and prevent interstitial pressure loss in the kidney.
RI Fluid Microdialysis Technique
C-21 (Vicore Pharma), a highly selective, nonpeptide AT2R agonist (Ki=0.4 mol/L) was used to activate systemic and renal AT2Rs.17,18 C-21 demonstrates 25 000-fold selectivity at AT2Rs compared with AT1Rs.18,19 Candesartan (0.01 mg/kg per minute; AstraZeneca), a specific, potent, insurmountable inhibitor of AT1Rs (IC50>1×10−5 mol/L and 2.9×10−8 mol/L for AT2Rs and AT1Rs, respectively), was used for systemic AT1R blockade. PD-123319 (PD; 10 μg/kg per minute; Parke-Davis), a specific AT2R antagonist (IC50=2×10−8 mol/L and >1×10−4 mol/L for AT2R and AT1Rs, respectively), was used interstitially to block intrarenal AT2Rs. l-NG-nitroarginine methyl ester (L-NAME; 100 ng/kg per minute; Sigma) was used interstitially to block intrarenal NO synthase activity. Icatibant (HOE-140; 100 ng/kg per minute; Sigma), a specific, potent inhibitor of bradykinin B2 receptors, was used interstitially to inhibit intrarenal bradykinin B2 receptors. Ang II (200 ng/kg per minute; Bachem) was used to induce Ang II–dependent hypertension.
Data are presented as mean±1SE. Statistical significance was determined using 1-way ANOVA followed by multiple comparisons testing with the Student–Newman–Keuls test with 95% confidence. The level of significance was set at P<0.05.
Effects of Systemic C-21 Infusion±Intrarenal Infusion of AT2R Antagonist PD±Systemic AT1R Blockade on UNaV and MAP in Volume-Expanded Female SD Rats
UNaV (Figure 1A) was unchanged in response to vehicle infusion throughout the experiment (P=NS). In response to systemic C-21 infusion of 100, 200, and 300 ng/kg per minute, UNaV increased immediately from 0.24±0.06 μmol/min in a dose-dependent manner to 1.12±0.20 (P<0.001), 1.51±0.25 (P<0.001), and 2.04±0.21 μmol/min (P<0.0001), respectively (overall ANOVA F=18.9; P<0.0001 versus vehicle infusion). The C-21–induced natriuresis was abolished by concurrent intrarenal administration of PD (10 μg/kg per minute) at all C-21 infusion rates. UNaV was not enhanced by systemic pretreatment with AT1R antagonist candesartan at any C-21 infusion rate tested (P=NS). MAP (Figure 1B) was reduced by systemic infusion of candesartan (P<0.0001) but was otherwise unchanged by C-21 and PD.
Effects of Systemic C-21 Infusion on UNaV, MAP, Renal Blood Flow, Glomerular Filtration Rate, and Fractional Excretion of Na+ (FENa) and Lithium (FELi) in the Absence of Systemic AT1R Blockade in Volume-Expanded Female SD Rats
UNaV (Figure 2A) was unchanged by vehicle and was increased by systemic C-21infusion (F=7.8; P<0.01). MAP (Figure 2B), renal blood flow (Figure 2C), and glomerular filtration rate (Figure 2D) were unchanged by vehicle or C-21 infusion (P=NS). FENa (Figure 2E) increased from 0.44±0.05% to 0.99±0.18% (P<0.01), 1.0±0.21% (P<0.01), and 0.91±0.19% (P<0.05) with 100, 200, and 300 ng/kg per minute C-21infusion, respectively (overall ANOVA F=11.8; P<0.005). FELi (Figure 2F) also increased in parallel with FENa from 35.3±3.1% to 57.3±5.3% (P<0.01), 53.8±5.5% (P<0.01), and 52.6±6.8% (P<0.05) in response to C-21infusion of 100, 200, and 300 ng/kg per minute, respectively (overall ANOVA F=16.1; P<0.001).
Effects of Systemic C-21 Infusion±Intrarenal Infusion of AT2R Antagonist PD, NO Synthase Inhibitor L-NAME, or Bradykinin B2 Receptor Antagonist Icatibant on RI cGMP, UNaV, and MAP in the Absence of Systemic AT1R Blockade in Volume-Expanded Female SD Rats.
RI cGMP (Figure 3A) was unchanged in response to systemic vehicle infusion (P=NS). In response to systemic C-21 infusion, RI cGMP increased immediately from 4.92±0.83 pmol/mL to 13.0±2.0 (P<0.01), 13.0±2.4 (P<0.01), and 17.2±3.4 pmol/mL (P<0.01) at 100, 200, and 300 ng/kg per minute C-21 infusion, respectively (overall ANOVA F=10.6; P<0.0001). The C-21–induced increase in RI cGMP was abolished with concurrent intrarenal infusion of PD (10 μg/kg per minute), NO synthase inhibitor L-NAME (100 ng/kg per minute), or bradykinin B2 receptor antagonist icatibant (100 ng/kg per minute). UNaV (Figure 3B) was unchanged in response to vehicle infusion (P=NS). Systemic C-21 infusion induced an increase in UNaV from 0.26±0.05 μmol/min to 1.20±0.29 (P<0.05), 1.35±0.27 (P<0.01), and 1.42±0.22 μmol/min (P<0.01) at 100, 200, and 300 ng/kg per minute, respectively (overall ANOVA F=7.0; P<0.0005). Intrarenal administration of PD, L-NAME, or icatibant abolished C-21–induced natriuresis. MAP (Figure 3C) did not change in response to administration of any pharmacological agent.
Effects of Systemic C-21 Infusion on Total Cortical and Apical Plasma Membrane AT2R Density in the Absence of Systemic AT1R Blockade in Volume-Expanded Female SD Rats
To determine whether AT2R activation induces receptor recruitment to the apical plasma membranes of RPTCs, we used confocal immunofluorescence microscopy, immunoelectron microscopy, and Western blot analysis. Figure 4A to 4L demonstrates the subcellular distribution of AT2Rs as determined by confocal immunofluorescence microscopy from a representative set of rat RPTCs in response to systemic vehicle (Figure 4A–4F) and systemic C-21 (Figure 4G–4L) infusion (100 ng/kg per minute). Figure 4A and 4B, respectively, depicts the RPTC distribution of adaptor protein-2 (blue) marking the subapical membrane and phalloidin (red) marking the apical brush border. Figure 4C demonstrates the cellular distribution of AT2R protein using antibody (Chemicon) proven to be specific for AT2Rs by immunoblotting AT2R-null mouse adrenal glands (Figure 4P) that normally have a high degree of AT2R expression. Figure 4D demonstrates the merged adaptor protein-2, phalloidin, and AT2R image. As demonstrated in merged Figure 4D (vehicle infusion) and Figure 4J (C-21 infusion), C-21 administration induced a color shift in immunofluorescence from red to orange, indicating increased AT2R density in the apical plasma membrane in response to C-21. This color shift can be more easily visualized in Figure 4E and 4K, which are high-power images of the areas depicted in the squares from Figure 4D and 4J. Figure 4F and 4L depicts the AT2R immunofluorescence staining only in the apical plasma membrane area at higher magnification. These panels demonstrate increased apical membrane association of AT2Rs in response to C-21. Figure 4M shows the quantitative increase in relative AT2R fluorescence units in response to C-21 (n=4; P<0.01). Western blot analysis of AT2R total cortical and apical membrane levels are shown in Figure 4N and 4O, respectively. C-21 treatment (100, 200, and 300 ng/kg per minute) increased apical plasma membrane AT2R protein without changing total cortical AT2R protein expression. As shown in Online Figure I, similar results were obtained using Western blot analysis with another AT2R antibody (Alomone Laboratories) that also does not react with AT2R-null mouse adrenal glands (Online Figure IC). Figure 5 depicts high-powered electron photomicrographs of immunogold-labeled AT2Rs in apical plasma membrane brush border microvilli of RPTCs after systemic vehicle (Figure 5B) and C-21 (Figure 5C) infusion (100 ng/kg per minute). C-21 infusion significantly increases AT2R density in the apical plasma membrane. Figure 5D shows the quantitative increase in relative AT2R immunogold staining (P<0.01). Figure 5A provides a low-power micrograph of an RPTC. Collectively, these studies demonstrate the ability of C-21 to translocate AT2Rs to the apical plasma membrane.
Effects of Systemic C-21 Infusion on RPTC NHE-3 Apical Plasma Membrane Retraction and Cellular Internalization in the Absence of Systemic AT1R Blockade in Volume-Expanded Female SD Rats
To determine whether AT2Rs induce natriuresis by internalizing/inhibiting Na+ apical transporter NHE-3, we also performed immunofluorescence microscopy, immunoelectron microscopy, and Western blot analysis. Figure 6A to 6J demonstrates the subcellular distribution of NHE-3 as determined by confocal immunofluorescence microscopy from a representative set of rat RPTCs in response to systemic vehicle (Figure 6A–6E) and C-21 (Figure 6F–6J) infusion (100 ng/kg per minute). Figure 6A and 6F shows autofluorescence (blue) of the RPTC. Figure 6B and 6G shows NHE-3 (green) expressed in the apical brush border membranes of RPTCs. Figure 6C and 6H demonstrates subapical membranes visualized by adaptor protein-2 staining (red). In merged Figure 6D, there is no visible translocation of NHE-3 from apical to subapical membranes. In contrast, in response to C-21 infusion (Figure 6I), there is visible translocation from apical to subapical membranes, as demonstrated by the extensive yellow transformation. This C-21–induced color shift is more easily visualized in the high-power magnifications (Figure 6E and 6J) taken from the squares of Figure 6D and 6I, respectively. Figure 6K demonstrates the significant quantitative translocation of NHE-3 to subapical membranes in response to C-21 (n=4; P<0.01). Western blot analysis of NHE-3 total cortical distribution is shown in Figure 6L, where there was no change in response to C-21 (100, 200, and 300 ng/kg per minute). Furthermore, Figure 6M shows that systemic C-21 infusion significantly increased total cortical membrane phospho-NHE-3 (Ser 522) protein levels (P<0.001), an established indicator of NHE-3 retraction/internalization. Figure 7 depicts high-powered electron photomicrographs of immunogold-labeled NHE-3 in the apical brush border microvilli and apical plasma membrane base/subapical regions of RPTCs after systemic vehicle (Figure 7A and 7B) and C-21 (Figure 7C and 7D) infusion (100 ng/kg per minute), respectively. Figure 7E demonstrates that C-21 infusion did not affect the NHE-3 density in the apical membrane of RPTCs but dramatically increased the distribution of NHE-3 in the apical plasma membrane/subapical membrane region (P<0.01; Figure 7F). Collectively, these studies demonstrate the ability of C-21 to induce the retraction of NHE-3 from the apical to subapical region of RPTCs.
Effects of Systemic C-21 Infusion on Total Cortical Membrane Phospho-ERK1/2, ERK1/2, Phospho-Src (Tyr 416), Src, Phospho-αNKA (Ser 23), and αNKA Protein Expression in the Absence of Systemic AT1R Blockade in Volume-Expanded Female SD Rats
To determine whether AT2R activation can internalize/inhibit NKA and activate the Src/extracellular-signal–regulated kinase (ERK) signaling pathway, we performed Western blot analysis of renal cortical membranes. Western blot analyses of total cortical membrane phospho-ERK1/2 and ERK1/2 are shown in Online Figure IIA and IIB, respectively. C-21 treatment (100, 200, and 300 ng/kg per minute) significantly increased phospho-ERK1/2 protein (P<0.01) without changing total cortical ERK1/2 protein expression. Online Figure IIC and IID depicts Western blot analysis of total cortical membrane phospho-Src (Tyr 416) and Src, respectively. C-21 infusion also significantly increased phospho-Src protein (P<0.01), without changing total cortical Src protein expression. Online Figure IIE and IIF depicts Western blot analysis of total cortical membrane phospho-αNKA (Ser 23) and total αNKA respectively. Although there was no change in total cortical membrane αNKA protein (Online Figure IIF), C-21 infusion significantly decreased phospho-αNKA protein expression (Online Figure IIE; P<0.05), an established indicator of αNKA retraction/internalization. Collectively, these studies suggest that along with NHE-3, NKA is internalized/inactivated during C-21 infusion.
Effects of Systemic C-21 Infusion±Intrarenal Infusion of AT2R Antagonist PD±Systemic AT1R Blockade on UNaV and MAP in Na+-Loaded Female and Male SD Rats
In female rats (Online Figure IIIA), cumulative systemic C-21 infusion (100, 200, and 300 ng/kg per minute) increased UNaV from 1.49±0.17 to 8.07±0.71 μmol/min (overall ANOVA F=36.3; P<0.0001). This response was abolished with concurrent intrarenal infusion of AT2R antagonist PD (10 μg/kg per minute). Systemic pretreatment with AT1R antagonist candesartan increased UNaV to 10.7±0.70 μmol/min, a value significantly higher than with C-21 alone (P=0.02). In male rats (Online Figure IIIA), systemic C-21 infusion increased UNaV from 0.61±0.13 to 6.24±1.08 μmol/min (P<0.01); this response was also abolished by intrarenal PD. In the presence of systemic candesartan pretreatment, there was no difference in UNaV versus C-21 alone (P=NS). In contrast to female volume-expanded rats, UNaV in female Na+-loaded rats was enhanced by systemic pretreatment with candesartan (P<0.05). There was no significant sex difference in the natriuretic response to C-21 alone or C-21+PD (P=NS). However, the natriuretic response to C-21 in the presence of candesartan was significantly greater in female than male rats (overall ANOVA F=5.6; P<0.005). As shown in Online Figure IIIB, MAP was reduced (P<0.0001) by systemic candesartan administration but was otherwise unchanged in response to administration of pharmacological agents (P=NS).
Effects of Intrarenal C-21 Infusion±Intrarenal Infusion of AT2R Antagonist PD on UNaV and MAP in the Absence of Systemic AT1R Blockade in Volume-Expanded Female SD Rats
UNaV (Online Figure IVA) was unchanged as a result of intrarenal vehicle administration (P=NS). In response to intrarenal C-21 infusion, UNaV increased in a dose-dependent manner from 0.14±0.02 μmol/min to 0.50±0.11 (P<0.05), 0.68±0.13 (P<0.01), and 0.85±0.11 μmol/min (P<0.01) at 20, 40, and 80 ng/kg per minute, respectively (overall ANOVA F=15.2; P<0.0001). The natriuresis in response to intrarenal C-21 administration was abolished with concomitant intrarenal administration of PD (10 μg/kg per minute). MAP (Online Figure IVB) did not change in response to administration of any pharmacological agent (Online Figure IV).
Effects of Chronic Systemic C-21 Infusion on 24-Hour UNaV and MAP in Female WT (C57BL/6) and AT2R-Null Mice
In response to continuous systemic infusion of C-21 (300 ng/kg per minute), WT mice demonstrated increased 24-hour UNaV compared with WT mice infused with vehicle (Online Figure V; overall ANOVA F=14.0; P<0.0001). The C-21–induced natriuresis in WT mice was absent in AT2R-null mice, whose UNaV values were similar to vehicle-infused AT2R-null mice (P=NS). AT2R-null mice, whether or not they received C-21, demonstrated significantly lower UNaV values (antinatriuresis) than WT vehicle-infused mice (overall ANOVA F=14.3; P<0.0001). MAP, however, was not significantly affected by C-21 infusion in WT mice, albeit it was significantly lower than MAP of AT2R-null mice (Online Figure VI; overall ANOVA F=2.4; P<0.02).
Effects of Chronic Intrarenal C-21 Infusion on Mean Systolic BP and 24-Hour UNaV in Ang II–Dependent Hypertension in Female SD Rats
As shown in Figure 8A, systemic Ang II infusion (200 ng/kg per minute) increased systolic BP from 126±5 to 188±20 mm Hg during a 7-day period (ANOVA F=48; P<0.0001). Concurrent intrarenal administration of C-21 (60 ng/kg per minute) markedly inhibited the pressor effect of systemic Ang II infusion (F=12; P<0.0001). As shown in Figure 8B, consecutive 24-hour UNaV was reduced from 0.95±0.04 to 0.34±0.08 μmol/min (P<0.0001) on day 1 of systemic Ang II infusion. Ang II–induced antinatriuresis was inhibited by intrarenal administration of C-21 (F=23.3; P<0.0001) during the entire 7-day period of infusion.
The present study demonstrates, for the first time to our knowledge, that both systemic and direct intrarenal administration of highly selective AT2R agonist C-21 can induce a sustained increase in renal Na+ excretion in normal animals by activating RPTC AT2Rs. We show that systemic AT2R activation induces natriuresis both acutely in rats and chronically in mice, responses that were reversible with intrarenal administration of specific AT2R antagonist PD and genetic deletion of AT2Rs, respectively.
The importance of these findings is underscored by the absence of a requirement for concurrent AT1R blockade to unmask AT2R-mediated natriuresis. This is a novel finding, in that in past studies cardiovascular and renal responses to AT2R activation have been observed only when the RAS is activated or AT1Rs are concurrently blocked.2–4 Our results support the concept that potent, highly selective nonpeptide AT2R agonist administration may contribute to the future therapeutic management of fluid-retaining disorders and possibly hypertension.
This study also demonstrates that chronic renal AT2R activation prevents Na+ retention and lowers BP in an experimental model of Ang II–dependent hypertension. Intrarenal administration of C-21 not only abrogated the initial Ang II–induced antinatriuresis but also augmented Na+ excretion chronically in this model. Thus, intrarenal AT2R activation improved the pressure-natriuresis relationship in this model. Additional factors, other than natriuresis, may also contribute to BP reduction in this model. Future studies will include determination of the effect of sex and the route of C-21 administration on BP reduction in this model.
Previous studies have demonstrated the importance of the bradykinin, NO, and cGMP signaling cascade in the actions of AT2Rs in multiple cells and tissues, including the kidney.2–4,22–24 This signaling pathway can operate either by bradykinin B2 receptor activation or directly via NO and cGMP production without involving bradykinin.24 We explored the mechanisms of AT2R-induced natriuresis in the present study. The increases in renal Na+ excretion with AT2R activation were accompanied by increases in RI cGMP. The C-21–induced increases in both cGMP and Na+ excretion were abolished with intrarenal administration of either bradykinin B2 receptor antagonist icatibant or NO synthase inhibitor L-NAME, demonstrating dependence of the AT2R-induced natriuretic responses on the bradykinin, NO, and cGMP signaling cascade. Similar dependence of AT2R-induced natriuresis on cGMP signaling has been demonstrated for the major endogenous renal AT2R agonist, Ang III.17 Our previous studies identified a putative new signaling pathway by which cGMP released into the RI compartment may facilitate natriuresis, that is, by binding to the extracellular domain of αNKA inducing phosphorylation of downstream signaling molecules Src and ERK1/2.25 Here, we show that C-21 induces Src and ERK phosphorylation and internalization/inactivation of αNKA. This finding suggests, but does not prove, that αNKA internalization/inactivation may be induced by increased extracellular renal cGMP production secondary to renal AT2R activation. On the basis of these results, we hypothesize that extracellular renal cGMP is the major driving force for AT2R-mediated natriuresis by internalizing and inactivating the major RPT Na+ transporters. Additional studies will be required to determine definitively whether this signaling pathway mediates AT2R- and cGMP-induced natriuresis.
Because AT2Rs are encoded by a gene residing on the X-chromosome, sex differences in AT2R actions have recently been explored.26–28 Studies to date have shown that female rats have enhanced AT2R-mediated renal vasodilator and tubuloglomerular feedback responses compared with males.26 However, this difference does not translate to renal Na+ excretion, which was identical in male and female rats.26–28 In the present study, AT2R activation with C-21 induced natriuresis to a similar extent in male and female rats, and natriuresis could be attributed to reduced RPTC reabsorption as reflected by increased FENa and FELi without alteration in renal hemodynamic function. Lithium clearance studies have been shown to accurately identify RPT events with a maximum 4% error rate in Na+-replete animals.29 However, we did observe a significant augmentation of AT2R-induced natriuresis with concurrent AT1R blockade in female but not male rats. The reasons for this difference are not clear, and it is possible that differences in renal blood flow and glomerulotubular feedback may have occurred, which were not monitored during our experiments using candesartan. In addition to these studies,26–28 2 reports of C-21 induction of natriuresis have appeared in the literature. Kemp et al17 in 2011 first demonstrated that systemic administration of C-21 (0.3 μg/kg per minute) induced natriuresis (12-fold) that was blocked with intrarenal PD infusion in male uninephrectomized SD rats. In addition, Ali and Hussain30 reported that intravenous infusion of C-21 (5.0 μg/kg per minute, a 16-fold higher dose) increased UNaV, FENa, and FELi in obese Zucker rats and that this response was neutralized by systemic coadministration of PD.
In the present study, AT2R activation was accompanied by recruitment of AT2Rs to the apical plasma membranes of RPTCs without change in total cellular AT2R expression, as demonstrated by a combination of Western blot analysis and confocal and immunoelectron microscopy. This finding is consistent with previous results from our laboratory showing that natriuretic responses to endogenous renal AT2R agonist Ang III are also accompanied by translocation of AT2Rs from intracellular sites along microtubules to the apical plasma membranes of RPTCs.31 In addition, dopamine D1 receptor activation with fenoldopam induces natriuresis in an AT2R-dependent manner and translocates AT2Rs to the apical plasma membranes of RPTCs via cAMP and protein phosphatase 2A signaling mechanisms.32 Interestingly, hypertensive 12-week-old spontaneously hypertensive rats fail to recruit AT2Rs or mount natriuretic responses to Ang III, but both receptor translocation and natriuresis can be restored by inhibition of aminopeptidase N, increasing Ang III formation.20,31 Combined with our previous findings, the present results suggest that AT2R recruitment to the apical plasma membranes of RPTCs may be a critical common mechanism initiating and supporting sustained natriuretic responses to dopamine, Ang III, and C-21.
The RPT reabsorbs 67% of Na+ filtered into the nephron.33 NHE-3 is the principal apical Na+ transporter in the RPT, and flow-modulated NHE-3 activity is the mechanism for glomerulotubular balance.34 NHE-3 is expressed along the microvilli of the RPTC brush border but can also be detected in subapical, intracellular, and vesicular compartments, consistent with the regulation of its activity by membrane trafficking.35,36 Direct and indirect binding of NHE-3 to ezrin is required for its intracellular trafficking, and NHE regulatory factor-1 links NHE-3 to ezrin and the cytoskeleton, which are in turn regulated by RhoGTPase.37,38
In the present study, we hypothesized that AT2R activation with C-21 would internalize/inactivate not only NKA (discussed above) but also NHE-3 in the RPT. We demonstrated trafficking of NHE-3 from the tips to the bases of the apical microvilli and also into the subapical membranes of RPTCs in response to C-21. Retraction/internalization of NHE-3 is a marker of reduced NHE-3 activity.39 For example, acutely increased BP induces trafficking of RPTC NHE-3 from the tips to the bases of the apical microvilli (without internalization into the subapical domain), resulting in pressure-natriuresis.40 Activation of RPTC dopamine D1 receptors also inhibits proximal Na+ reabsorption by decreasing NHE-3 activity and protein abundance in the apical plasma membrane without changing total cellular NHE-3 expression.41 As discussed above, C-21 also induced internalization of αNKA, the major Na+ transporter at the RPTC basolateral membrane. Thus, AT2R activation internalizes and inactivates the 2 major transporters governing Na+ reabsorption in the RPT. Exploration of the detailed signaling mechanisms mediating transporter internalization and inactivation will be performed in future studies.
In summary, AT2R activation induces natriuresis by recruiting the receptor to the apical plasma membranes of RPTCs, stimulating a bradykinin–NO–cGMP–Src–ERK signaling cascade and internalizing NHE-3 and NKA. Renal AT2R activation prevents Na+ retention and lowers BP by improving the pressure-natriuresis relationship in Ang II–dependent hypertension. Because no clinically effective diuretic/natriuretic agents acting in the RPT are currently available, systemic AT2R activation potentially represents a unique opportunity for treatment of volume-overload/edema-forming states and hypertension in humans.
We thank Dr Tadashi Inagami of Vanderbilt University for providing the AT2 receptor-null mice for this study and Vicore Pharmaceuticals for providing compound 21.
Sources of Funding
This work was supported by National Institutes of Health grant R01-HL-095796.
In May 2014, the average time from submission to first decision for all original research papers submitted to Circulation Research was 14.87 days.
The online-only Data Supplement is available with this article at http://circres.ahajournals.org/lookup/suppl/doi:10.1161/CIRCRESAHA.115.304110/-/DC1.
- Nonstandard Abbreviations and Acronyms
- Ang II
- angiotensin II
- Ang III
- des-aspartyl1-Ang II
- AT1 receptor
- AT2 receptor
- blood pressure
- compound 21
- extracellular-signal–regulated kinase
- fractional excretion of lithium
- fractional excretion of sodium
- l-NG-nitroarginine methyl ester
- mean arterial pressure
- Na+-H+ exchanger-3
- nitric oxide
- nitric oxide synthase
- renin–angiotensin system
- renal proximal tubule
- renal proximal tubule cell
- urinary sodium excretion
- wild type
- Received April 2, 2014.
- Revision received June 2, 2014.
- Accepted June 4, 2014.
- © 2014 American Heart Association, Inc.
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- Catt KJ,
- Inagami T,
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- Griffiths R,
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Novelty and Significance
What Is Known?
Angiotensin II (Ang II) acts via one of 2 major receptors, type 1 (AT1R) and type 2 (AT2R).
AT2Rs generally oppose the actions of Ang II via AT1Rs.
Instead of Ang II, renal des-aspartyl1-Ang II activates AT2Rs, inducing natriuresis and opposing the usual antinatriuretic action of Ang II via AT1Rs.
What New Information Does This Article Contribute?
In the renal proximal tubule, compound 21 (C-21), a nonpeptide angiotensin AT2 receptor (AT2R) agonist, administered systemically induces natriuresis by activating AT2Rs via increasing renal production of bradykinin, nitric oxide, and cGMP.
Renal AT2R activation is accompanied by the recruitment of AT2Rs to the apical plasma membranes of proximal tubule cells and by internalization and inactivation of the major proximal tubule apical and basolateral membrane sodium (Na+) transporters.
C-21 administered directly into the kidney ameliorates Ang II–induced antinatriuresis and hypertension.
Ang II acts via 2 major receptors, type 1 (AT1R) and type 2 (AT2R). Most of Ang II actions are mediated via AT1Rs, including vasoconstriction, antinatriuresis, aldosterone secretion, and sympathetic activation that increase blood pressure and lead to hypertension. However, AT2R activation can dilate blood vessels and induce natriuresis by increasing renal production of bradykinin, NO, and cGMP. The endogenous ligand activating AT2Rs in the kidney seems to be des-aspartyl1-Ang II instead of Ang II.
We report that specific activation of renal AT2R with systemic nonpeptide agonist C-21 induces natriuresis in a cGMP-dependent manner by recruiting AT2Rs from intracellular sites to the apical plasma membranes of RPTCs and by inhibiting the major Na+ transporters, NHE-3 and NKA, in this nephron segment. We found that C-21 could ameliorate Ang II–dependent hypertension by renal activation of AT2Rs.
These findings suggest that proximal tubule AT2Rs could be a therapeutic target for hypertension and edema-forming states when the renin–angiotensin system is activated. Currently, no clinically effective diuretic/natriuretic agents acting at the proximal tubule are available, and therefore C-21 or similar agents could provide a complimentary nephron-specific site of diuresis/natriuresis for future clinical use.