Identification of a Primary Renal AT₂ Receptor Defect in Spontaneously Hypertensive Rats

Abstract

Rationale:

Previous studies identified a defect in angiotensin III (Ang III)-elicited AT₂ receptor (AT₂R)-mediated natriuresis in renal proximal tubule cells (RPTCs) of spontaneously hypertensive rats (SHR).

Objective:

This study aimed to delineate in pre-hypertensive SHR kidneys the receptor and/or post-receptor defect causing impaired AT₂R signaling and renal sodium (Na⁺) retention by utilizing the selective AT₂R agonist Compound 21 (C-21).

Methods and Results:

Female 4-week-old Wistar-Kyoto (WKY) and SHR rats were studied after 24h systemic AT₁ receptor (AT₁R) blockade. Left kidneys received 30 min renal interstitial (RI) infusions of vehicle followed by C-21 (20, 40, and 60 ng/kg/min, each dose 30 min). Right kidneys received vehicle infusions. In WKY, C-21 dose-dependently increased urine Na⁺ excretion (U_NaV) from 0.023±0.01 to 0.064±0.02, 0.087±0.01, and 0.089±0.01 μmol/min (P=0.008, P<0.0001, and P<0.0001, respectively) and RI fluid levels of AT₂R downstream signaling molecule cGMP from 0.91±0.3 to 3.1±1.0, 5.9±1.2, and 5.3±0.5 fmol/mL (P=NS, P<0.0001, and P<0.0001, respectively). In contrast, C-21 did not increase U_NaV or RI cGMP in SHR. Mean arterial pressure was slightly higher in SHR, but within the normotensive range and unaffected by C-21. In WKY, but not SHR, C-21 induced AT₂R translocation to apical plasma membranes of RPTCs, internalization/inactivation of NHE-3 and Na⁺/K⁺ATPase and phosphorylation of AT₂R-cGMP downstream signaling molecules Src kinase, extracellular signal-related kinase (ERK), and vasodilator-stimulated phosphoprotein (VASP). To test whether cGMP could bypass the natriuretic defect in SHR, we infused 8-Br-cGMP. This restored natriuresis, Na⁺ transporter internalization/inactivation, and Src and VASP phosphorylation, but not apical plasma membrane AT₂R recruitment. In contrast, 8-Br-cAMP administration had no effect on natriuresis or AT₂R recruitment in SHR.

Conclusions:

The results demonstrate a primary RPTC AT₂R natriuretic defect in SHR that may contribute to the development of hypertension. Since the defect is abrogated by exogenous intrarenal cGMP, the renal cGMP pathway may represent a viable target for the treatment of hypertension.

Graphical Abstract

The renin-angiotensin system is a hormonal cascade critical in the regulation of Na⁺ and fluid balance and blood pressure (BP). Angiotensin II (Ang II), the major effector peptide of the system, acts via renal AT₁ receptors (AT₁Rs) to increase Na⁺ reabsorption and BP, a response that is normally opposed by renal AT₂Rs. The preferred endogenous AT₂R agonist increasing U_NaV is Ang III. SHR are routinely employed as a model of human hypertension, and recent studies from our laboratory have shown that, in contrast to WKY, SHR have reduced natriuretic responses to Ang III, due to an AT₂R/post-receptor signaling defect or accelerated Ang III metabolism, or both. The present study identifies for the first time a primary RPT AT₂R natriuretic defect in young pre-hypertensive SHR. This defect is characterized by failure of exogenous intrarenal C-21 to (1) increase U_NaV, (2) recruit AT₂Rs to the apical plasma membranes of RPT cells, (3) inactivate RPT Na⁺ transporters, (4) generate renal cGMP and (5) activate AT₂R/cGMP downstream signaling molecules. The AT₂R natriuretic response and downstream signaling in SHR can be fully restored, however, by administration of intrarenal 8-Br-cGMP. Consequently, renal cGMP may provide a novel target for the treatment of hypertension in humans.

INTRODUCTION

The renin-angiotensin system (RAS) plays a critical role in the regulation of body fluid, electrolyte balance, and blood pressure (BP) both in health and disease (1–3). The RAS acts via two major receptors: angiotensin (Ang) type-1 (AT₁R) and Ang type-2 (AT₂R). Although the actions of AT₁Rs are relatively well understood, functions mediated by AT₂Rs have been more enigmatic. Nevertheless, previous studies have confirmed that, while renal AT₁Rs induce sodium (Na⁺) retention, AT₂Rs increase urinary Na⁺ excretion (U_NaV) at the level of the renal proximal tubule (RPT) and that, instead of angiotensin II (Ang II), des-aspartyl¹-Ang III (Ang III) is the predominant endogenous agonist for this response (4–7). In the normal kidney, AT₂Rs induce natriuresis by a bradykinin (BK)-nitric oxide (NO)-cyclic guanosine 3’,5’monophosphate (cGMP)-dependent signaling cascade accompanied by AT₂R translocation to RPT apical plasma membranes and internalization/inactivation of major RPT Na⁺ transporters Na⁺-H⁺ exchanger-3 (NHE-3) and Na⁺/K⁺ATPase (NKA) (7,8).

Spontaneously hypertensive rats (SHR) develop hypertension at approximately 6 weeks of age and are widely employed as a model of human hypertension. A proposed mechanism of initiation of hypertension in SHR is a primary increase in renal Na⁺ reabsorption. Over time, this defect requires an increase in BP to normalize U_NaV, an adaptation that is central to the development and maintenance of hypertension (9,10). In previous studies we have shown that hypertensive 12-week-old SHR lack natriuretic responses to renal interstitial (RI) Ang III administration, whereas Ang III induces robust natriuresis in 12-week-old Wistar-Kyoto control rats (WKY) (11). Recently, we also demonstrated that pre-hypertensive 4-week-old SHR already have absent natriuretic responses to intrarenal endogenous agonist Ang III together with impaired AT₂R translocation and Na⁺ transporter internalization/inactivation (12). The Ang III/AT₂R defect in SHR did not appear to be due to accelerated Ang III metabolism (12). These results suggested that the Ang III/AT₂R/NHE-3 signaling pathway is a RPT-specific, natriuresis-promoting mechanism, maintaining normal body salt and fluid balance and BP, that is defective in hypertension (12,13).

In the present study, we hypothesized that the defect in AT₂R-mediated natriuresis in SHR is primarily due to impaired signaling at the receptor or post-receptor level. To address this hypothesis, we infused highly selective non-peptide AT₂R agonist Compound 21 (C-21) intrarenally in 4-week-old WKY and pre-hypertensive SHR and determined natriuretic responses and AT₂R-based signaling mechanisms. Here we report a primary AT₂R natriuretic defect affecting virtually all known signaling pathways distal to the receptor in SHR kidneys. Importantly, natriuresis and downstream signaling, but not AT₂R recruitment, are restored in SHR with exogenous intrarenal cGMP administration.

METHODS

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Please see the Online Data Supplement for detailed methods (total cortical homogenate preparation and Western blot analysis, RPT cell (RPTC) apical membrane isolation and Western blot analysis, In vivo kidney perfusion and fixation procedure, and confocal immunofluorescence microscopy). Please also see the Major Resources Table in the Supplemental Materials. 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.

BP measurements.

Mean arterial pressure (MAP) was measured by the direct intra-carotid method with the use of a digital BP analyzer (Micromed, Inc). MAPs were recorded every 5 min and averaged for all periods. Experiments were initiated at the same time each day to prevent diurnal variations in BP.

Renal cortical interstitial infusion.

An open bore micro-infusion catheter (PE-10) was inserted under the renal capsule into the cortex of each kidney to ensure the RI infusion of vehicle (VEH) 5% dextrose in water (D₅W) or pharmacological agent at 2.5 μL/min with a syringe pump (Harvard; model 55–222). When more than one agent was simultaneously infused, separate interstitial catheters were employed. Vetbond tissue adhesive (3M Animal Care Products) was added to secure the catheter(s) and prevent interstitial pressure loss in the kidney.

RI Fluid (RIF) microdialysis technique.

RIF cGMP levels were measured using microdialysis probes that were constructed and utilized as described previously in our laboratory (7) and measured using an enzyme immunoassay (Cayman Chemical).

Animal preparation.

The experiments were conducted on 4-week-old female WKY (N=39) and SHR (N=39; Envigo) rats 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 (0.30%). Previous studies have shown that natriuretic responses to C-21 are virtually identical in male and female Sprague-Dawley rats (8). Also, we did not see differences between 4 week-old male and female SHR when AT₂Rs were activated with Ang III (12). In the future, experiments will need to be repeated in young male WKY and SHR to confirm absence of sexual dimorphism in response to C-21.

Our previous studies in Sprague-Dawley rats demonstrated that C-21 induces natriuresis via similar signaling mechanisms in the absence of AT₁R blockade, indicating that AT₂R-induced natriuresis is physiologic (8,14). The current experiments in young WKY and SHR were conducted in the presence of AT₁R blockade to ensure that the defect in SHR is restricted to AT₂Rs. For studies that involved systemic AT₁R blockade (Protocols 1–4), a 24h osmotic mini-pump (Alzet Model 2001D) infusing candesartan (CAND; 0.01 mg/kg/min) was inserted 24h prior to experimentation. While the rats were under short-term anesthesia with isoflurane, 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) and a tracheostomy was performed using polyethylene tubing (PE-60) to assist respiration. Direct cannulation of the right internal jugular vein using PE-10 tubing provided intravenous access through which 1% bovine serum albumin (BSA) made in VEH D₅W was infused at 20 μL/min. Direct cannulation of the right carotid artery with PE-10 tubing provided arterial access for monitoring MAP. Following a midline laparotomy, both ureters were cannulated (PE-10) to collect urine for the quantification of urine Na⁺ excretion (U_NaV). Rats were randomly assigned to the various treatment groups. Power calculations for the number of rats for each treatment group were based on similar results using C-21 obtained from previous studies.

Pharmacological agents.

Candesartan (CAND; Alomone Labs), a specific, potent, insurmountable inhibitor of AT₁Rs (IC₅₀>2.9×10⁻⁸ and 1×10⁻⁵ for AT₁Rs and AT₂Rs, respectively), was used for systemic AT₁R blockade. Compound 21 (C-21; 20, 40, and 60 ng/kg/min; Vicore Pharma), a highly selective, non-peptide AT₂R agonist (K_i=0.4 mol/L) was employed to activate intrarenal AT₂Rs. C-21 demonstrates 25,000-fold selectivity for AT₂Rs over AT₁Rs. PD-123319 (PD; 10 μg/kg/min; 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 to block intrarenal AT₂Rs. 8-Bromo-cAMP (8-Br-cAMP; 72 μg/kg/min; Tocris) and 8-Bromo-cGMP (8-Br-cGMP; 72 μg/kg/min; Tocris) were used as the cell permeable analogs of cAMP and cGMP, respectively.

Specific protocols.

All studies involved a 2-kidney model where the right kidney served as a time control receiving RI infusions of VEH D₅W while the left kidney received RI infusions of pharmacological agents.

(1) Effects of acute intrarenal C-21 infusion on U_NaV, MAP, and RIF cGMP levels in the presence of systemic AT₁R blockade:

Following a 1h equilibration period in which VEH was infused into the RI space of each kidney, the following groups of rats were studied: (1) WKY C-21 (N=7): right kidney received RI infusion of VEH for four 30 min periods and the left kidney received RI infusion of C-21 (20, 40, and 60 ng/kg/min; each dose for 30 min) following a 30 min RI infusion of VEH. (2) WKY C-21 + PD (N=6): right kidney received RI infusion of VEH for four 30 min periods and the left kidney received RI infusion of C-21 (20, 40, and 60 ng/kg/min; each dose for 30 min) + PD (10 μg/kg/min) following a 30 min RI infusion of VEH. (3) SHR C-21 (N=7): right kidney received RI infusion of VEH for four 30 min periods and the left kidney received RI infusion of C-21 (20, 40, and 60 ng/kg/min; each dose for 30 min) following a 30 min RI infusion of VEH. U_NaV and MAP were measured for each period. In order to prevent potential renal trauma associated with the insertion of both a RI catheter and RI microdialysis probe, a separate set of rats for group 1 (N=8), group 2 (N=8) and group 3 (N=6) were studied to measure RIF cGMP levels.

(2) Effects of acute intrarenal C-21 infusion on RPTC apical plasma membrane AT₂R and NHE-3 or total cortical homogenate AT₂R, NHE-3, pSer⁵⁵²-NHE-3, α-NKA, pSer²³-NKA, Src, pTyr⁴¹⁶-Src, ERK 1/2, pThr²⁰²/Tyr²⁰⁴-ERK 1/2, VASP, pSer²³⁹-VASP, and PKG-1 in the presence of systemic AT₁R blockade:

Kidneys were harvested at the end of the study in Protocol 1 (WKY VEH and C-21 and SHR VEH and C-21) and processed for Western blot analysis (12). N=6 for each condition. In a separate set of animals, WKY and SHR were perfused separately following the second RI infusion dose of C-21 (40 ng/kg/min) and processed for confocal microscopy.

(3) Effects of acute intrarenal 8-Br-cAMP infusion on U_NaV and MAP in the presence of systemic AT₁R blockade:

Following a 1h equilibration period in which VEH was infused into the RI space of each kidney, the following groups of rats were studied: (1) SHR 8-Br-cAMP (N=6): right kidney received RI infusion of VEH for four 30 min periods and the left kidney received RI infusion of 8-Br-cAMP (72 μg/kg/min) for three 30 min periods following a 30 min RI infusion of VEH. (2) SHR 8-Br-cAMP + C-21 (N=6): right kidney received RI infusion of VEH for four 30 min periods and the left kidney received RI infusion of 8-Br-cAMP (72 μg/kg/min) + C-21 (20, 40 and 60 ng/kg/min, each dose for 30 min) for three 30 min periods following a 30 min RI infusion of VEH. U_NaV and MAP were measured for each period.

(4) Effects of acute intrarenal 8-Br-cAMP infusion on RPTC apical plasma membrane and total cortical homogenate AT₂R in the presence of systemic AT₁R blockade:

Kidneys were harvested at the end of the study in Protocol 3 and processed for Western blot analysis (12). N=6 for each condition.

(5) Effects of acute intrarenal 8-Br-cGMP infusion on U_NaV and MAP in the absence of systemic AT₁R blockade:

Following a 1h equilibration period in which VEH was infused into the RI space of each kidney, the following groups of rats were studied: (1) WKY 8-Br-cGMP (N=9): right kidney received RI infusion of VEH for four 30 min periods and the left kidney received RI infusion of 8-Br-cGMP (72 μg/kg/min) for three 30 min periods following a 30 min RI infusion of VEH. (2) SHR 8-Br-cGMP (N=13): right kidney received RI infusion of VEH for four 30 min periods and the left kidney received RI infusion of 8-Br-cGMP (72 μg/kg/min) for three 30 min periods following a 30 min RI infusion of VEH. U_NaV and MAP were measured for each period.

(6) Effects of acute intrarenal 8-Br-cGMP infusion on RPTC apical plasma membrane AT₂R and total cortical homogenate AT₂R, NHE-3, pSer⁵⁵²-NHE-3, α-NKA, pSer²³-NKA, Src, pTyr⁴¹⁶-Src, ERK 1/2, pThr²⁰²/Tyr²⁰⁴-ERK 1/2, VASP, and pSer²³⁹-VASP in the absence of systemic AT₁R blockade:

Kidneys were harvested at the end of the study in Protocol 5 and processed for Western blot analysis (12). N=6 for each condition.

Statistical analysis.

Data are presented as mean ± 1 SE. For U_NaV, RI cGMP, and MAP measurements, statistical significance was determined by using a two-way ANOVA followed by a post-hoc Tukey analysis with multiple comparisons with 95% confidence using Prism Graphpad 8 software. All columns were compared to each other and P-values are provided for comparisons to own control period. For confocal immunofluorescence and Western blot analyses, statistical significance was determined by using a one-way ANOVA followed by a post-hoc Tukey analysis with multiple comparisons with 95% confidence using Prism Graphpad 8 software. A weakness is that we did not correct for multiple comparisons across the study. All columns were compared to each other and P values were provided for comparisons to the corresponding time control kidneys of the rats (right kidney). P values <0.05 were considered statistically significant. Rats that produced a U_NaV value less than 0.005 μmol/min and/or a urine flow rate value less than 0.003 mL/min at the onset of experimental urine collections were excluded due to the unreliability of future experimental urine collections. As a result, 12 WKY and 9 SHR rats were excluded. Values that were greater than 2 standard deviations above or below the mean were also excluded. During Western blot analysis the following values were excluded: 1 value for SHR VEH and SHR C-21 for pERK and the ratio of pERK/ERK; 1 value for WKY VEH and WKY C-21 for pVASP and the ratio of pVASP/VASP; 1 value for SHR 8-Br-cGMP for total AT₂R. All data sets were analyzed for normality using the Shapiro-Wilk test and were found to be normal.

RESULTS

Effects of RI C-21 Infusion on U_NaV, RI cGMP, and MAP (Figure 1):.

Figure 1:

Panel A. Urine Na⁺ excretion (U_NaV) under the following conditions: WKY right (RT) kidney renal interstitial (RI) vehicle infusion (N=13), WKY left (LT) kidney RI C-21 (20, 40, and 60 ng/kg/min) infusion (N=7), WKY LT kidney RI C-21 + PD (10 μg/kg/min) infusion (N=6), SHR RT kidney RI vehicle infusion (N=6), and SHR LT kidney RI C-21 infusion (N=7), all in the presence of systemic AT₁R blockade. Panel B. RI fluid (RIF) cGMP measurements under conditions described in Panel A in a separate set of rats (N=15, N=8, N=8, N=6, and N=6, respectively). Panel C. Mean arterial pressure (MAP) under conditions described in Panel A. Data represent mean ± 1 SE. Overall 2-way ANOVA analysis for Panel A; P<0.0001, Panel B; P<0.0001, and Panel C; P=0.23.

Natriuretic, RI cGMP, and MAP responses of 4-week-old WKY and SHR to RI infusion of C-21 (20, 40, and 60 ng/kg/min) are shown in Figure 1, Panels A-C. In WKY, C-21 significantly increased U_NaV and RI cGMP without affecting MAP. Co-administration of AT₂R antagonist PD abolished U_NaV and RI cGMP responses to C-21 in WKY. In contrast, natriuretic and cGMP responses to C-21 were absent in pre-hypertensive SHR. MAP was significantly increased but was still within the normotensive range in SHR and remained elevated after intrarenal C-21 infusion.

Effects of C-21 on RPT Cell (RPTC) Apical Plasma Membrane AT₂R Density (Figure 2).

Figure 2:

Confocal micrographs (600x magnifications) showing AT₂R localization in WKY and SHR renal proximal tubule cell (RPTC) thin sections (5–8 μm) after renal interstitial (RI) infusion of vehicle (VEH) or Compound-21 (C-21; 20 and 40 ng/kg/min). As indicated, rows of images show a representative set of RPTCs from (top-to-bottom) from WKY VEH, WKY C-21, SHR VEH, and SHR C-21 treatment groups. As indicated, columns (left-to-right) depict brush border membrane staining with phalloidin (Panel A), subapical area staining with adaptor protein-2 (AP-2) (Panel B), AT₂R staining (Panel C), merged image (Panel D), enlarged merged image (4x) of the square section in Panel D (Panel E) and enlarged image with only AT₂R staining of the brush border membrane area quantified for AT₂R (Panel F). The encircled areas in Panels E and F encompass brush border apical membranes quantified for AT₂R intensity. Scale bars in the first and sixth columns represent 10 and 2 μm, respectively. Panel G. Quantification of RPTC apical membrane AT₂R fluorescence intensity performed on 6 RPTCs with 4 measurements per cell from one rat for WKY VEH (), WKY C-21 (), SHR VEH (), and SHR C-21 () treatments. Panels H and I depict Western blot analysis of AT₂R in RPTC apical membranes and total cortical homogenate, respectively, in WKY and SHR after VEH or C-21 (20, 40, and 60 ng/kg/min) infusions (N=6 for each condition). RPTC apical membrane signals were normalized to villin, a brush border apical membrane marker. Total cortical homogenates were normalized to β-tubulin. Data represent mean ± 1 SE. Overall 1-way ANOVA analysis for Panel G; P<0.0001 and Panel H; P<0.0001.

To determine whether AT₂R activation with C-21 induces receptor translocation to the apical plasma membranes of RPTCs of WKY and SHR, we employed confocal immunofluorescence microscopy and lectin pulldown of RPTC apical plasma membranes followed by immunoblotting. Figure 2, Panels A-F illustrates the subcellular distribution of AT₂Rs in a representative set of RPTCs from WKY and SHR after intrarenal VEH or C-21 infusion (40 ng/kg/min) as determined by confocal immunofluorescence microscopy. Panels 2A and B show the RPTC distribution of phalloidin (red) marking the apical plasma membrane and adaptor protein-2 (AP-2) (blue) marking the subapical region, respectively. Panel 2C demonstrates the subcellular distribution of AT₂Rs (green) using an antibody (Millipore; Cat # AB1554) specific for AT₂Rs as previously demonstrated by immunoblotting AT₂R-null mouse adrenal glands that normally have a high degree of AT₂R expression (8). Panels 2D and 2E at higher magnification of the area depicted in the respective box of Panel 2D, show merged phalloidin, AP-2, and AT₂R images. Panel 2F depicts a higher power image of only AT₂R staining in the brush border area, thus representing AT₂R in the apical plasma membrane. This panel demonstrates substantially higher staining in the apical plasma membrane of WKY in response to C-21, but no change in AT₂R staining in response to C-21 in SHR. Panel 2G with the quantitation of immunofluorescence images shows increases in relative AT₂R fluorescence units in response to C-21 (P<0.0001) in WKY but not in SHR. As further corroborated by lectin pulldown of apical membranes followed by immunoblotting (Panel 2H), C-21 increased apical plasma membrane AT₂R density (P<0.0001) in WKY without changing total cortical homogenate AT₂R protein expression (Panel 2I). On the other hand, in SHR apical plasma membrane AT₂R density increased only slightly (P=NS) in response to C-21. The AT₂R trafficking response to C-21 was thus significantly less for SHR than for WKY (P<0.0001). Total cortical AT₂R expression was similar in WKY and SHR kidneys under all conditions (Panel 2I).

Effects of C-21 on RPTC NHE-3 Apical Plasma Membrane Retraction and Cellular Internalization (Figure 3 and Online Figure I).

Figure 3:

Confocal micrographs (600x magnification) showing NHE-3 localization in WKY and SHR renal proximal tubule cell (RPTC) thin sections (5–8 μm) after renal interstitial (RI) infusion of vehicle (VEH) or Compound-21 (C-21; 20 and 40 ng/kg/min). As indicated, rows of images show a representative set of RPTCs from (top-to-bottom) from WKY VEH, WKY C-21, SHR VEH, and SHR C-21 treatment groups. As indicated, columns (left-to-right) depict confocal auto-fluorescence (Panel A), subapical area staining with adaptor protein-2 (AP-2) (Panel B), NHE-3 staining (Panel C), merged image (Panel D), enlarged image (4x) of the square section in Panel D (Panel E) and an enlarged image with only NHE-3 staining in the subapical area quantified for NHE-3 (Panel F). The encircled areas encompass the subapical area that was quantified for NHE-3 fluorescence intensity. Scale bars in the first and sixth columns represent 10 and 2 μm, respectively. Panel G. The quantification of RPTC subapical area NHE-3 fluorescence intensity performed on 6 RPTCs with 4 measurements per cell from one rat for WKY VEH (), WKY C-21 (), SHR VEH () and SHR C-21 ()treatments. Panels H and I depict Western blot analysis of RPTC apical membrane and total cortical homogenate NHE-3 expression respectively in WKY and SHR after VEH or C-21 (20, 40, and 60 ng/kg/min) infusions (N=6 for each condition). RPTC apical membrane signals were normalized to villin, a brush border apical membrane marker. Total cortical homogenates were normalized to β-tubulin. Data represent mean ± 1 SE. Overall 1-way ANOVA analysis for Panel G; P<0.0001, Panel H; P=0.0004, and Panel I; P=0.023.

To delineate whether AT₂R activation with C-21 induces natriuresis by internalizing/inhibiting RPTC Na⁺ apical membrane transporter NHE-3, we proceeded as described above for AT₂R localization. Figure 3, Panels A-F demonstrates the subcellular distribution of NHE-3 as determined by confocal immunofluorescence microscopy from a representative set of WKY and SHR RPTCs after intrarenal VEH or C-21 infusion. Panel 3A depicts renal auto-fluorescence (blue) and Panels 3B and 3C depict AP-2 (green) and NHE-3 (red) staining, respectively. Panels 3D and 3E show merged and higher power images of the area depicted in the respective box of Panel 3D. Panel 3F shows a higher power image of NHE-3 staining only in the subapical region. WKY, but not SHR, demonstrate increased subapical NHE-3 density in response to C-21 (P=0.0003), which was quantified in Panel 3G. In Panel 3H, Western blot analysis of RPTC apical plasma membranes demonstrates a reduction of NHE-3 density in response to C-21 infusion in WKY (P=0.004), but not in SHR. Panel 3I shows that total cortical NHE-3 expression did not change in response to C-21 in either WKY or SHR. However, total renal cortical NHE-3 was significantly higher in SHR compared to WKY VEH (SHR VEH vs. WKY VEH, P=0.026).

Phosphorylation of NHE-3 (pNHE-3) at serine 552 (pSer⁵⁵²-NHE-3) after activation of cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) is required for maximum inhibition of NHE-3, as mutation of this amino acid reduces the inhibitory effect of cAMP on NHE-3 (15,16). Consequently, pNHE-3 is considered a marker of NHE-3 retraction/internalization and inactivation (17,18). The subcellular distribution of pSer⁵⁵²-NHE-3 was thus evaluated (Online Figure I). Panels A-C show RPTC auto-fluorescence (blue), phalloidin (red; marking apical plasma membranes) and pSer⁵⁵²-NHE-3 (green), respectively. As demonstrated in Panels D-F, C-21 infusion increased pSer⁵⁵²-NHE-3 density in the subapical region of the RPTC in WKY. There was no change in pSer⁵⁵²-NHE-3 in response to C-21 in SHR. Quantification of results show that C-21 increased the relative pSer⁵⁵²-NHE-3 fluorescence intensity (P=0.014) in RPTC subapical regions of C-21-infused WKY, but not in C-21-infused SHR (P=NS; Panel G). In total kidney cortical homogenate, C-21 increased pSer⁵⁵²-NHE-3 and the ratio of pSER⁵⁵²-NHE-3/total NHE-3 in WKY (P=0.005 and P=0.018, respectively), but not in SHR (Panels H and I, respectively).

Effects of C-21 on RPTC NKA Basolateral Membrane Retraction and Cellular Internalization (Online Figure II and Figure 4).

Figure 4:

Confocal micrographs (600x magnification) showing pSer²³-NKA (pNKA) in WKY and SHR renal proximal tubule cell (RPTC) thin sections (5–8 μm) after renal interstitial (RI) infusion of vehicle (VEH) or Compound-21 (C-21; 20 and 40 ng/kg/min). As indicated, rows of images show a representative set of RPTCs from (top-to-bottom) from WKY VEH, WKY C-21, SHR VEH, and SHR C-21 treatment groups. As indicated, columns left-to-right) depict confocal auto-fluorescence (Panel A), subapical area staining with adaptor protein-2 (AP-2) (Panel B), pNKA (Panel C), merged image (Panel D), merged image showing only auto-fluorescence and pNKA (Panel E), and an enlarged image (4x) of the square sections in Panel E (Panel F). Scale bars in the first and sixth columns represent 10 and 2 μm, respectively. Panel G. The quantification of RPTC intracellular pNKA fluorescence intensity performed on 6 RPTCs with 4 measurements per cell from one rat for WKY VEH (), WKY C-21(), SHR VEH (), and SHR C-21 () treatments. Panel H. Western blot analysis for total cortical homogenate pNKA in WKY and SHR after VEH or C-21 (20, 40, and 60 ng/kg/min) infusions (N=6 for each condition). Total αNKA is shown in Online Figure II. All blots are normalized to β-tubulin. Panel I. Ratio of (pNKA/β-tubulin)/(α-NKA/β-tubulin) for all 4 conditions. Data represent mean ± 1 SE. Overall 1-way ANOVA analysis for Panel G; P=0.0005, Panel H; P=0.0014, and Panel I; P=0.003.

AT₂R activation with C-21 also induces natriuresis by internalizing/inhibiting RPTC Na⁺ basolateral membrane transporter NKA. Online Figure II, Panels A-C, shows RPTC auto-fluorescence (blue), apical plasma membrane marker phalloidin (red) and α-NKA (green), respectively. α-NKA localizes mainly to the RPTC basolateral membrane in VEH-infused WKY (merged Panel D), and is internalized in response to C-21. In VEH-infused SHR, α-NKA is distributed largely in the basolateral membrane and the distribution is unchanged after C-21 infusion. NKA internalization can be best appreciated in high power views of the areas depicted in the respective box of Panel E (Panel F). α-NKA staining was measured within the small boxes in Panel F that were placed 4 μm from the basolateral membrane to ensure measurements of intracellular α-NKA. As quantified in Panel G, in WKY, but not in SHR, C-21 induced a higher intracellular α-NKA density compared to VEH-infused controls (P=0.002). Panel H shows no significant difference in total cortical expression of α-NKA in WKY or SHR in response to C-21 infusion.

Phosphorylation of NKA (pNKA) at serine 23 (pSer²³-NKA) serves as an established indicator of α-NKA retraction/internalization wherein a reduction signifies retraction (19). The subcellular distribution of pSer²³-NKA is shown for representative confocal micrographs in Figure 4, Panels A-F, and the relative RPTC pSer²³-NKA fluorescence intensities are quantified in Panel 4G. RPTC auto-fluorescence (blue), AP-2 (green), and pSer²³-NKA (red) are shown in Panels 4A-C, respectively. Merged Panel 4D shows a reduction in RPTC pSer²³-NKA in response to C-21 in WKY, but not in SHR. This difference between WKY and SHR is more easily observed in Panel 4F, a high power view of the area depicted in the respective box of Panel 4E. The response is quantified in Panel 4G, demonstrating that intracellular pSer²³-NKA fluorescence is reduced by C-21 in WKY (P=0.002) but not in SHR (P=NS). Panels 4H-I show that total cortical homogenate pSer²³-NKA and the ratio of pSer²³-NKA/total α-NKA are reduced by C-21 in WKY (P=0.008 and 0.034, respectively), but not in SHR.

Effects of C-21 on Renal Downstream AT₂R Signaling Molecules (Figure 5 and Online Figures III and IV).

Figure 5:

Western blot analysis of total cortical homogenate tissue for WKY vehicle (VEH;), WKY C-21 (20, 40, and 60 ng/kg/min;), SHR VEH (), and SHR C-21 () treatments (N=6 for each condition). Panels A and B depict pTyr⁴¹⁶-Src (pSrc) and the ratio of (pSrc/β-tubulin)/(Src/β-tubulin), respectively. Total Src is shown in Online Figure IIIA. Panels C and D depict pThr²⁰²/Tyr²⁰⁴-ERK 1/2 (pERK) and the ratio of (pERK/β-tubulin)/(ERK/β-tubulin), respectively. Total ERK is shown in Online Figure IIIB. Panels E and F depict pSer²³⁹-VASP (pVASP) and the ratio of (pVASP/β-tubulin)/ (VASP/β-tubulin), respectively. Total VASP is shown in Online Figure IIIC. Data represent mean ± 1 SE. Overall 1-way ANOVA analysis for Panel A; P<0.0001, Panel B; P=0.0005, Panel C; P=0.004, Panel D; P=0.009, Panel E; P<0.0001, and Panel F; P<0.0001.

To determine whether the absence of C-21-induced natriuresis in SHR is related to defects in one or more specific downstream AT₂R signaling pathways, we measured the phosphorylation of several renal cortical signaling proteins by Western blot (Figure 5 and Online Figures III and IV). In WKY, C-21 increased renal cortical pTyr⁴¹⁶-Src (pSrc; P<0.0001; Figure 5, Panel A) without changing total Src (Online Figure IIIA), thus increasing the ratio of pSrc/Src (P=0.0007; Figure 5B). In SHR, C-21 did not change pSrc or total Src densities (Figure 5A and Online Figure IIIA, respectively). In WKY, C-21 increased the density of renal cortical pThr²⁰²/Tyr²⁰⁴-ERK 1/2 (pERK; P=0.008, Figure 5C), did not alter total ERK (P=NS; Online Figure IIIB), and increased the ratio of pERK/ERK (P=0.016; Figure 5D). In SHR, C-21 did not alter pERK, ERK, or the pERK/ERK ratio (P=NS; Figure 5C, Online Figure IIIB and Figure 5D, respectively).

Protein kinase G-1 (PKG-1) mediates downstream actions of NO-stimulated cGMP, and vasodilator-stimulated phosphoprotein (VASP) serves as a biomarker for PKG-1 activity (20,21). C-21 increased renal cortical pSer²³⁹-VASP (pVASP) density (P<0.0001; Figure 5E) in WKY but not in SHR. Total VASP was unchanged in response to C-21 in either WKY or SHR (P=NS; Online Figure IIIC). The ratio of pVASP/VASP increased in response to C-21 in WKY (P<0.0001) but not in SHR (Figure 5F). PKG-1 expression did not change in response to C-21 in either WKY or SHR (P=NS; Online Figure IV).

Effects of Intrarenal 8-Br-cGMP Infusion on U_NaV and MAP in the Absence of Systemic AT₁R Blockade (Figure 6).

Figure 6:

Panel A. Urine Na⁺ excretion (U_NaV) under the following conditions: WKY right (RT) kidney renal interstitial (RI) vehicle infusion (N=9), WKY left (LT) kidney RI 8-Br-cGMP (72 μg/kg/min) infusion (N=9), SHR RT kidney RI vehicle infusion (N=13), and SHR LT kidney RI 8-Br-cGMP infusion (N=13), all in the absence of systemic AT₁R blockade. Panel B. Mean arterial pressure (MAP) under conditions described in Panel A. Data represent mean ± 1 SE. Numbers 1, 2, and 3 refer to the three 30 min infusion time periods. Overall 2-way ANOVA analysis for Panel A; P=0.037 and Panel B; P=0.30.

cGMP is a crucial mediator of natriuresis (22). To test whether cGMP could restore natriuresis in SHR, we performed RI infusions of 8-Br-cGMP (72 μg/kg/min). In WKY, 8-Br-cGMP did not alter U_NaV or MAP (P=NS). In contrast, in SHR 8-Br-cGMP engendered a significant natriuretic response in the last two experimental periods (P=0.0012 and 0.0003, respectively) without affecting MAP (Panels 6A and B). In SHR, the natriuretic response to 8-Br-cGMP was not significantly different from that observed in response to C-21 in WKY (Figure 1, Panel A). Baseline MAP was slightly higher in SHR compared to WKY, but normotensive.

Effects of Intrarenal 8-Br-cGMP Infusion on RPTC Apical Plasma Membrane AT₂R Density and Na⁺ Transporter Retraction and Cellular Internalization (Figure 7 and Online Figure V).

Figure 7:

Western blot analysis of renal proximal tubule cell (RPTC) and total cortical tissue homogenates for WKY vehicle (VEH;), WKY 8-Br-cGMP (72 μg/kg/min,), SHR VEH (), and SHR 8-Br-cGMP (72 μg/kg/min,) treatments (N=6 for each condition). RPTC apical membrane signals were normalized to villin, a brush border apical membrane marker. Total cortical homogenates were normalized to β-tubulin. Panels A and B depict AT₂R and NHE-3 RPTC apical membranes, respectively. Total AT₂R is shown in Online Figure VA. Panels C and D depict total cortical homogenate pSer⁵⁵²-NHE-3 (pNHE-3) and the ratio of (pNHE-3/β-tubulin)/(NHE-3/β-tubulin), respectively. Total NHE-3 is shown in Online Figure VB. Panels E and F depict total cortical homogenate pSer²³-NKA (pNKA) and the ratio of (pNKA/β-tubulin)/(α-NKA/β-tubulin) respectively. Total α-NKA is shown in Online Figure VC. Data represent mean ± 1 SE. Overall 1-way ANOVA analysis for Panel B; P=0.011, Panel C; P=0.002, Panel D; P=0.045, Panel E; P=0.029 and Panel F; P=0.06.

Analysis of RPTC apical plasma membrane AT₂R (Panel 7A) and total cortical homogenate AT₂R (Online Figure VA) demonstrates no significant change in response to RI 8-Br-cGMP infusion in either WKY or SHR. NHE-3 density decreased in response to 8-Br-cGMP infusion in RPTC apical plasma membranes of SHR (P=0.022), but not in WKY (Panel 7B). Total cortical homogenate pSer⁵⁵²-NHE-3 increased in SHR (P=0.007), but not WKY, in response to 8-Br-cGMP (Panel 7C). Total cortical NHE-3 expression did not change in response to 8-Br-cGMP in either WKY or SHR (Online Figure VB) and the ratio of pSer⁵⁵²-NHE-3/NHE-3 was significantly increased in response to 8-Br-cGMP only in SHR (P=0.037; Panel 7D). Panel 7E shows a significant reduction in total cortical homogenate pSer²³-NKA in response to 8-Br-cGMP in SHR (P=0.027), but not in WKY. Since 8-Br-cGMP did not alter total cortical homogenate α-NKA (Online Figure VC) in either WKY or SHR, the ratio of pSer²³-NKA /α-NKA decreased significantly only in SHR (P=0.043) (Panel 7F).

Effects of Intrarenal 8-Br-cGMP Infusion on Renal Downstream AT₂R Signaling Molecules (Figure 8 and Online Figure VI).

Figure 8:

Western blot analysis of total cortical tissue homogenate for WKY vehicle (VEH;), WKY 8-Br-cGMP (72 μg/kg/min,), SHR VEH (), and SHR 8-Br-cGMP (72 μg/kg/min,) treatments (N=6 for each condition). Panels A and B depict pTyr⁴¹⁶-Src (pSrc) and the ratio of (pSrc/β-tubulin)/(Src/β-tubulin), respectively. Total Src is shown in Online Figure VIA. Panels C and D depict pThr²⁰²/Tyr²⁰⁴-ERK 1/2 (pERK) and the ratio of (pERK/β-tubulin)/(ERK/β-tubulin), respectively. Total ERK is shown in Online Figure VIB. Panels E and F depict pSer²³⁹-VASP (pVASP) and the ratio of (pVASP/β-tubulin)/(VASP/β-tubulin), respectively. Total VASP is shown in Online Figure VIC. Data represent mean ± 1 SE. Overall 1-way ANOVA analysis for Panel A; P=0.003, Panel B; P=0.002, Panel E; P=0.029, and Panel F; P=0.0004.

Phosphorylation of cGMP downstream signaling molecules Src, ERK, and VASP in response to 8-Br-cGMP was evaluated in WKY and SHR (Figure 8). While 8-Br-cGMP, did not increase pSrc or the pSrc/total Src ratio in WKY, it increased pSrc and the pSrc/total Src ratio in SHR (P=0.008, Panel 8A and P=0.009, Panel 8B, respectively). Infusion of 8-Br-cGMP did not alter phosphorylated or total ERK or their ratio (P=NS) in either WKY or SHR (Panel 8C, Online Figure VIB, and Panel 8D, respectively). However, while not increasing pVASP or the pVASP/total VASP ratio in WKY, 8-Br-cGMP increased VASP phosphorylation (P=0.027) and the ratio of phosphorylated to total VASP (P=0.005) in SHR (Panels 8E and 8F, respectively).

Effects of Intrarenal 8-Br-cAMP Infusion on U_NaV, MAP, and RPTC Apical Plasma Membrane AT₂R Density (Online Figure VII).

cAMP is a major downstream signaling molecule regulating both dopamine D₁ receptor (D₁R) and AT₂R cellular trafficking (23). In SHR, RI 8-Br-cAMP infusion (72 μg/kg/min) either alone or in combination with C-21 (20, 40, and 60 ng/kg/min) did not significantly alter U_NaV (Panel A) or MAP (Panel B). Apical membrane AT₂R density (Panel C) did not change in response to RI 8-Br-cAMP and there was no change in total cortical homogenate AT₂R expression (Panel D). There was a slight increase in apical membrane AT₂R density in response to RI 8-Br-cAMP + C-21 (P=0.047), which was most likely a result of C-21 treatment.

For comparison, Panel E demonstrates the significant C-21-induced increase in apical membrane AT₂R density (P<0.0001) in WKY and the marked reduction in 8-Br-cAMP + C-21-induced AT₂R translocation to apical membranes in SHR (P<0.0001).

DISCUSSION

This study demonstrates, for the first time, an unambiguous early RPTC AT₂R defect in SHR. Previous studies from our laboratory have shown that hypertensive as well as pre-hypertensive SHR, compared to age-matched WKY controls, have defective natriuretic responses to endogenous peptide agonist Ang III together with disruption of AT₂R translocation to apical plasma membranes and internalization/inactivation of Na⁺ transporters in RPTCs (11,12). Although Ang II and Ang (1–7) are AT₂R ligands, past studies have shown that only Ang III has the capability of inducing natriuresis by a selective renal tubule mechanism (7). The Ang III/AT₂R/NHE-3 signaling defect within the kidneys of SHR did not appear to be due to accelerated Ang III metabolism, because Ang III-induced natriuretic responses were absent even when renal Ang III levels were high (12), but changes in peptide metabolism could not be completely excluded (12). Thus, the possibility remained that SHR manifested either a primary defect at the receptor/post-receptor level or a secondary receptor defect due to lack of availability of its preferred endogenous agonist.

The present study, showing absent natriuretic and downstream signaling responses to exogenous non-peptide AT₂R agonist C-21 in pre-hypertensive SHR, in contrast to age-matched WKY, conclusively demonstrates a primary RPTC AT₂R signaling defect in SHR that is independent of the presence of hypertension. Similar to Ang III, the C-21 natriuretic defect in SHR is accompanied by (1) marked reduction/absence of AT₂R translocation to the apical plasma membranes of RPTCs and (2) failure to retract/internalize and inactivate major RPTC apical membrane Na⁺ transporter NHE-3 and basolateral membrane Na⁺ transporter NKA.

In addition to AT₂R translocation and inactivation of NHE-3 and NKA, which are elicited in response to both endogenous AT₂R preferred agonist Ang III and synthetic small-molecule AT₂R agonist C-21, we explored the ability of C-21 to activate signaling molecules associated with the established AT₂R downstream mediator cGMP. The BK-NO-cGMP signaling cascade is the best-studied and accepted signaling pathway mediating AT₂R actions in the kidney (3,24–26). Previous studies have demonstrated the importance of the terminal signaling molecule of the pathway, cGMP, in AT₂R-induced natriuresis (8,27,28). Indeed, the cellular production and export of cGMP into the extracellular RI compartment is an important modulator of both NO- and pressure-induced natriuresis (29–31). The present study demonstrates that the primary RPTC AT₂R signaling defect identified in SHR is accompanied by the failure to increase RI levels of cGMP in response to C-21, in contrast to significant cGMP responses to C-21 in age-matched WKY controls. The precise mechanisms of this defect remain to be explored, among them whether there is impairment of renal cGMP formation or cellular export, or both.

Our study further documents cGMP-related downstream signaling responses to C-21 in WKY and SHR. Src family kinase is an important downstream signaling molecule in both cGMP- and pressure-induced natriuresis (22). In the cGMP-induced natriuretic response, Src activation occurs early in the cGMP signaling cascade and is independent of PKG as well as reactive oxygen and nitrogen species (22). Past studies have shown that PKG-1 is a substrate of Src and may be phosphorylated and activated by this kinase (32). Here we demonstrate C-21-induced Src phosphorylation in the kidneys of WKY, but consistent with the defective RI cGMP response, absence of Src phosphorylation in SHR. Both Src and ERK are components of a putative signaling cascade that, as we previously showed, may facilitate cGMP-induced natriuresis after cGMP binding to the extracellular domain of NKA (22). Previous studies have shown that pressure- and cGMP-induced natriuresis are dependent on PKG because administration of PKG inhibitors abolishes both natriuretic responses (33). The current study did not demonstrate any significant change in renal PKG expression with C-21 administration in WKY or SHR. While direct biomarkers for PKG activation are unavailable, the phosphorylation of VASP is considered an established marker of PKG-1 activity (20,21). Consistent with the RIF cGMP responses to C-21 in WKY, we show here C-21-induced VASP phosphorylation in WKY kidneys but not in SHR. We thus have identified an early primary renal AT₂R defect in SHR that is present before hypertension develops and is associated with reduced intrarenal cGMP and impaired downstream signaling (Online Figure VIII).

To determine whether the above-described renal AT₂R defect in SHR can be circumvented, we infused the cGMP analog 8-Br-cGMP directly into the RI compartment of SHR kidneys while monitoring U_NaV and RPT Na⁺ transporter trafficking. In contrast to cGMP, 8-Br-cGMP traverses cell membranes and readily enters RPTCs (31). We employed an 8-Br-cGMP infusion rate (72 μg/kg/min) that increased RI cGMP levels to a maximum in a rat pressure-natriuresis model (22). Contrary to the absent U_NaV responses to C-21, SHR responded to RI 8-Br-cGMP by increasing U_NaV to levels comparable to those of WKY infused with C-21. The natriuretic response to cGMP in SHR was confirmed by internalization/inactivation of NHE-3 and NKA, as indicated by the significant reduction in apical membrane NHE-3 and increase in total cortical pSer⁵⁵²-NHE-3 density as well as basolateral membrane NKA retraction and reduction in renal pSer²³-NKA density, respectively. Furthermore, SHR kidneys responded to exogenous RI 8-Br-cGMP with increases in Src and VASP phosphorylation, established downstream cGMP signaling molecules, comparable to WKY in response to C-21.

Studies from our laboratory have demonstrated that renal D₁Rs and AT₂Rs cooperatively inhibit RPTC Na⁺ reabsorption, functioning together to strengthen and provide redundancy for natriuretic responses that oppose AT₁R-mediated Na⁺ reabsorption (23,34–36). Indeed, our studies have shown that natriuresis induced by D₁R activation can be abolished by intrarenal AT₂R blockade (34). The cooperativity of D₁Rs and AT₂Rs in natriuresis appears to be reinforced by the recruitment of both receptors to the RPTC apical plasma membrane by a common signaling molecule, protein phosphatase 2A (PP2A), which can be activated by either cAMP via PKA (D₁Rs) or cGMP via PKG (AT₂Rs) (23,36). Therefore, we tested whether 8-Br-cGMP could restore AT₂R recruitment in SHR. The finding that natriuretic and downstream cGMP signaling responses were restored, while AT₂R recruitment was not, reinforces the concept that a primary (upstream) AT₂R defect exists in SHR and can be circumvented, but not corrected, by cGMP. Furthermore, we demonstrated that the defect in AT₂R recruitment also could not be restored with intrarenal 8-Br-cAMP stimulation of PP2A through PKA.

We were surprised to find that 8-Br-cGMP did not induce a significant increase in Na⁺ excretion in WKY, which was confirmed by failure to internalize/inactivate NHE-3 and NKA. This may be due to the adequate bioavailability of endogenous renal cGMP at baseline or the relative immaturity of young WKY RPTs, or both. In contrast, reduced endogenous cGMP availability may have increased the sensitivity of natriuretic responses to exogenous 8-Br-cGMP in SHR. This will be investigated in more depth in the future.

We have previously shown that there is no renal AT₂R defect in the Ang II infusion model of experimental hypertension (14). To our knowledge, however, no studies have addressed the possibility that the AT₂R defect as described here in SHR also exists in other rodent models of hypertension. This remains to be explored.

The rescue of the SHR AT₂R natriuretic defect with cGMP indicates that downstream signaling processes remain intact, at least at or distal to cGMP, and suggests that the renal cGMP pathway may constitute a viable therapeutic target for the treatment of hypertension in experimental animals and possibly also in humans. In support of this possibility, polymorphisms in PKG-1 are associated with loss of pressure-natriuresis in a Src-NKA-dependent manner and salt-sensitive hypertension in humans (37).

In summary, this study identifies, for the first time, a primary AT₂R natriuretic defect in the RPTC that may lead to renal Na⁺ retention and thereby hypertension in SHR. SHR are widely employed as a model of human primary hypertension, due at least in part to increased activity of the renal RAS mediated by AT₁Rs. AT₂Rs serve as a major counter-regulatory mechanism for AT₁R-induced Na⁺ retention. Defective AT₂R signaling in SHR includes failure to translocate the receptors to the apical plasma membranes of RPTCs, impaired renal cGMP generation, and absent downstream Src, ERK, and VASP phosphorylation (Online Figure VIII). This renal AT₂R defect is circumvented by direct intrarenal administration of cGMP, which restores downstream signaling leading to natriuresis, but does not re-establish upstream AT₂R translocation. The results suggest that the renal cGMP pathway by circumventing the AT₂R defect may represent a novel therapeutic target for hypertension.

NOVELTY AND SIGNIFICANCE.

What Is Known?

• Spontaneously hypertensive rats (SHR) are commonly employed as a model of human hypertension.

• The mechanism of initiation of hypertension in SHR is thought to be a primary increase in renal tubule sodium (Na⁺) reabsorption.

• Angiotensin AT₂ receptor (AT₂R) activation with its endogenous agonist des¹-aspartyl-Ang II (Ang III) increases renal Na⁺ excretion (U_NaV) in normal Wistar-Kyoto rats (WKY), but this natriuretic response is reduced in SHR.

What New Information Does This Article Contribute?

• We identified a primary renal proximal tubule (RPT) AT₂R natriuretic defect in SHR.

• In response to AT₂R activation with exogenous non-peptide agonist Compound-21 (C-21), this defect is characterized by (1) absent natriuretic responses, (2) failure to translocate AT₂Rs to the apical plasma membranes of RPT cells, (3) impaired renal cyclic guanosine 3’,5’ monophosphate (cGMP) generation, and (4) absent Src family kinase (Src), extracellular signal related kinase (ERK) and vasodilator-stimulated phosphoprotein (VASP) phosphorylation.

• This renal AT₂R defect in SHR can be circumvented by direct intrarenal administration of 8-Br-cGMP, which restores downstream signaling leading to natriuresis, but does not re-establish upstream AT₂R translocation.

Nonstandard Abbreviations and Acronyms:

8-Br-cAMP

8-bromo-adenosine cyclic 3’,5’-monophosphate

8-Br-cGMP

8-bromo-guanosine cyclic 3’,5’-monophosphate

Ang II

angiotensin II

Ang III

angiotensin III (des-aspartyl¹-angiotensin II)

AP-2

adaptor protein-2

AT₁R

angiotensin type-1 receptor

AT₂R

angiotensin type-2 receptor

BP

blood pressure

BSA

bovine serum albumin

C-21

Compound 21

cAMP

adenosine cyclic 3’,5’-monophosphate

CAND

candesartan

cGMP

guanosine cyclic 3’,5’-monophosphate

D₅W

5% dextrose in water

ERK

extracellular signal-related kinase

MAP

mean arterial pressure

NHE-3

sodium-hydrogen exchanger-3

NKA

sodium-potassium adenosine triphosphatase

PD

PD-123319

pERK

phosphorylated extracellular signal-related kinase

PP2A

phosphoprotein-2A

pSrc

phosphorylated Src family kinase

pVASP

phosphorylated vasodilator-stimulated phosphoprotein

RI

renal interstitial

RIF

renal interstitial fluid

RPT

renal proximal tubule

RPTCs

renal proximal tubule cells

SHR

spontaneously hypertensive rats

Src

Src family kinase

U_NaV

urinary sodium excretion

VASP

vasodilator-stimulated phosphoprotein

VEH

vehicle

WKY

Wistar-Kyoto rats