Angiotensin II type 2 receptor and receptor Mas are colocalized and functionally inter-dependent in obese Zucker rat kidney

Abstract

The actions of angiotensin II type 2 receptor (AT₂R) and the receptor Mas (MasR) are complex but show similar pro-natriuretic function; particularly AT₂R expression and natriuretic function are enhanced in obese/diabetic rat kidney. In light of some reports suggesting a potential positive interaction between these receptors, we tested hypothesis that renal AT₂R and MasR physically interact and are inter-dependent to stimulate cell signaling and promote natriuresis in obese rats. We found that infusion of AT₂R agonist C21 in obese Zucker rats (OZR) increased urine flow (UF) and urinary Na-excretion (UNaV) which were attenuated by simultaneous infusion of the AT₂R antagonist PD123319 or the MasR antagonist A-779. Similarly, infusion of MasR agonist Ang-(1-7) in OZR increased UF and UNaV, which were attenuated by simultaneous infusion of A-779 or PD123319. Experiment in isolated renal proximal tubules of OZR revealed that both the agonists C21 and Ang-(1-7) stimulated NO which was blocked by either of the receptor antagonists. Dual-labeling of AT₂R and MasR in OZR kidney sections and human proximal tubule epithelial cells showed that AT₂R and MasR are colocalized. The AT₂R also co-immunoprecipitated with MasR in cortical homogenate of OZR. Immunoblotting of cortical homogenate cross-linked with zero length oxidative (sulfhydryl groups) cross-linker cupric-phenanthroline revealed a shift of AT₂R and MasR bands upward with overlapping migration for their complexes which were sensitive to the reducing β-mercaptoethanol, suggesting involvement of –SH groups in cross linking. Collectively, the study reveals that AT₂R and MasR are co-localized and functionally interdependent in terms of stimulating NO and promoting diuretic-natriuretic response.

Introduction

Many receptors make homo- or heterodimers and thereby regulate downstream signaling and cellular events. Therefore, studying such receptor phenomena and the mechanism(s) behind them have been considered an important step for drug development in pharmacotherapy. G-protein-coupled receptors (GPCRs) have proven to be a successful therapeutic target accounting for ~30–50% marketed drugs. Angiotensin-II (ang-II) type 2 receptor (AT₂R)^1–4 and receptor Mas (MasR)^5–9 belong to non-classical GPCR family¹⁰ and mediate functions such as diuresis, natriuresis, vasorelaxation and blood pressure reduction in various animal models. The AT₂R³ and MasR^5,7,11,12 also show overlapping signaling in terms of nitric oxide (NO) formation and inhibiting Na⁺,K⁺-ATPase activity in renal proximal tubules. Overall, both these receptors are considered as part of the protective arm of the renin-angiotensin system and have been implicated in improving disease phenotypes such as obesity, hypertension and chronic kidney diseases, which are supported by pharmacological and knockout animal studies.^8,13–16

There is evidence showing that AT₂R¹⁷ and MasR^18,19 form hetero-dimers with AT₁R (ang-II type 1 receptor) and this hetero-dimerization provides a mechanism by which AT₂R and MasR attenuate AT₁R-induced signaling and cellular function. As it relates to an interaction between AT₂R and MasR, several studies suggest that these receptors may be functionally interdependent. For example, AT₂R antagonist PD123319 reduced vasodepressor effects of endogenous MasR agonist ang-(1-7).^20,21 Similarly, ang-(1-7) mediated endothelium-dependent vasodilation in cerebral arteries²² and aortic rings²³ of salt-fed animals was inhibited by AT₂R antagonist PD123319 as well as MasR antagonist A-779. In ApoE−/− mice, atheroprotective effects of ang-(1-7) were inhibited by PD123319 and A-779.²⁴ The cross-inhibition of ang-(1-7) mediated neuroprotection^22,23,25 and cardioprotection²⁶ by PD123319 and A-779 was also observed. In another study ang-(1-7) was not able to protect against intracranial aneurysmal rupture in AT₂R knock-out animals.²⁷

Although the AT₂R-knockout study indicates that an intact AT₂R may be needed for MasR to function,²⁷ pharmacological studies raise a question whether AT₂R or MasR ligands are specific or have overlapping affinity for these receptors.²⁸ Affinity profiles clearly suggest that MasR ligands have much higher affinity for MasR as compared to AT₂R. Similarly, AT₂R ligands have very high affinity for AT₂R as compared to MasR.²⁸ Considering that AT₂R and MasR ligands are preferential, a notion of physical interaction and thereby functional interdependence is a likely scenario. In support of such notion, molecular interactions between AT₂R and MasR were demonstrated by fluorescence resonance energy transfer and cross relation spectroscopy in human kidney-293 cells transiently transfected with vectors encoding fluorophore-tagged AT₂R and MasR.²⁹

Numerous studies from our laboratory and others suggest that AT₂R is upregulated and protective in the heart, kidney and other tissues during pathological conditions, including obesity.¹ In obese animals, we have reported that AT₂R stimulation promotes natriuresis¹ and protects against salt-sensitive hypertension.³⁰ Present study is designed to investigate whether AT₂R-mediated natriuresis/diuresis is dependent on functional MasR or vice-versa and whether these two receptors physically interact providing a potential molecular basis of functional interdependence. We observed that AT₂R and MasR are constitutively colocalized in renal cortex of obese Zucker rat kidney and may interact by forming disulfide bridges. Moreover, we have found that their signaling and function in terms of NO stimulation and diuretic-natriuretic response are interdependent.

Methods

Male obese Zucker rats (OZR, 9–10 weeks) were purchased from Harlan, Indianapolis, IN. After arrival, the animals were housed in the University of Houston animal care facility. Animal experimental protocols used in this study were approved by the IACUC at the University of Houston and adhere to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Details on various assay methods are available in the online-only Supplementary Information.

Statistical analysis

Data are presented as mean±SEM. The data were analyzed using GraphPad Prism 5 and differences were considered significant at p<0.05. Results were analyzed by One-way ANOVA with repeated measures followed by Fisher’s LSD test (each comparison stands alone) or Friedman test followed by Dunn’s multiple comparison test.

Results

Co-immunoprecipitation (Co-IP) experiment

Co-IP is a standard technique to identify if protein(s) of interest are colocalized. We isolated AT₂R and MasR complexes by immunoprecipitation with rabbit anti-MasR antibody in cortical homogenate of obese Zucker rat followed by immunoblotting with rabbit anti-AT₂R and goat anti-MasR antibodies. The data revealed that AT₂R was co-precipitated with MasR (Figure 1). Preliminary co-IP experiment using normal rabbit immunoglobulin (nonspecific) followed by immunoblotting of their complexes did not show presence of MasR or AT₂R in cortical homogenate supporting the specificity of CO-IP experiments (Figure S1A). In this set of experiment immunoprecipitates were eluted with glycine (pH 2.5). In subsequent experiments we used Laemmli buffer containing β-mercaptoethanol (β-ME) as an eluent, which provided better enrichment of immunoprecipitates. Co-immunoprecipitation was performed by two methods just to insure the validity of methods in our hand; (i) incubating homogenate with IP MasR antibody followed by protein A/G pulldown and (ii) incubating IP MasR antibody with protein A/G complex followed by incubation with homogenate (Figure S1B). Both techniques resulted in similar findings i.e., AT₂R- and MasR-specific immunoreactivity is observed above and below non-specific band of immunoglobulin subunit i.e., ~50 kDa, respectively. Approximate molecular size of immunoreactive bands of AT₂R and MasR was compared with bands directly measured in homogenate (lane 6, Figure S1B). Remaining immunoreactivity at ~ 100 and ~25 kDa are nonspecific and shows presence of immunoglobulin subunits. Specificity of rabbit anti-MasR antibody used to precipitate MasR in cortical homogenate was validated by knocking down MasR with siRNA in human proximal tubule epithelial cells (HK-2 cells) (Figure S2). The custom raised AT₂R antibody was validated by siRNA-knock down of AT₂R in left kidney of Sprague-Dawley rats as summarized in Figure S5. In summary, co-IP results show that AT₂R coexisted with MasR in obese Zucker rat kidney.

Figure 1.

Co-immunoprecipitation of AT₂R-MasR complex in renal cortical homogenate of male obese Zucker rat. Kidney homogenate was prepared as mentioned in SI Appendix, and supernatant was used for protein determination and Co-IP experiment. Renal cortical homogenate (500 μL, ~1.5 mg protein) was incubated overnight with gentle rotation at 4°C with rabbit 1° anti-MasR antibody, (~2 μg). Protein A/G agarose (20 μL) was added to antigen-antibody complex and incubated overnight at 4°C with gentle rotation. Protein A/G agarose was pulled down at 2500 rpm for 10 minute. Immunoprecipitates were washed with IP wash buffer (3 X 500 μL) and eluted with Laemmli buffer (50 μL), containing β-mercaptoethanol, separated on SDS-PAGE, 4–20% for MasR or 10% for AT₂R, transferred to PVDF membrane, blocked with 5% milk-TBST and immunoblotted with goat anti-MasR (1:1000) or custom synthesized rabbit anti-AT₂R (1:1000). Membranes were washed with TBST, re-probed with donkey anti-goat (1:4000) or goat anti-rabbit 2° antibody (1:3000), washed with TBST and analyzed via SuperSignal West Femto Maximum Sensitivity substrate. Results are representative of n=8. Only negative control sc-54848 showed non-specific immunoreactivity. Negative control ab56018 did not show any significant immunoreactivity.

Dual-labelling experiments

Findings of Co-IP experiments were confirmed with dual-immunolabelling experiment in cryo-sections of obese Zucker rat kidney tissue. AT₂R antibody used in western blot experiments did not yield clear results in immunohistochemistry experiment. Therefore, another commercially available AT₂R antibody was utilized (ab19134, Abcam Inc.) for immunohistochemistry experiments. Confocal laser-scanning microscopy showed immunohistochemical colocalization of AT₂R and MasR primarily in renal tubules, not in glomerulus of obese Zucker rat. The presence of colocalization as yellow puncta seems to be scattered around cell including in the cytosolic compartments (Figure 2). Such labelling is consistent to reports showing AT₂R expression in nuclear^12,31–33 and mitochondrial^31,34 compartment of the cell. Another study suggested that AT₂R predominantly are present in the cytosol and AT₂R agonist triggers their translocation to the plasma membranes.³⁵ Incubation with secondary antibodies alone did not show colocalization indicating specificity of experiment (Figure S3). Overall, the data support the concept of AT₂R-MasR colocalization.

Figure 2.

Colocalization of AT₂R and MasR in male obese Zucker rat kidney. Red pixels = AT₂R; Green pixels = MasR. Colocalization is evident as yellow pixels, marked with white arrow. Paraformaldehyde-perfused rat kidney was sectioned (5 μm) and blocked overnight. Antigen was retrieved via IHC-Tek epitope retrieval steam pretreatment (35 min). Sections were labelled with rabbit Anti-AT₂R (1:250) and goat Anti-MasR (1:250) antibody overnight at 4°C with gentle rotation. Sections were washed and re-incubated with Alexa Fluor 594 donkey anti-rabbit IgG and Alexa Fluor 488 donkey anti-goat IgG (both 1:400). Sections were dehydrated and mounted on Unifrost slides with fluorescent mounting medium with DAPI, sealed and analyzed using Leica SP8 confocal microscope using 40X/1.4NA oil objective lens (Z stack: 0.3 micron; Zoom 1; Smart gain: 800 (DAPI), 1150 (MasR), 1100 (AT2R); Smart offset −5%; pinhole 1.00 AU). Detailed method is provided in SI Appendix – “Dual labelling in rat kidney”.

We also measured co-localization of AT₂R and MasR in HK-2 cells. Unlike in the kidney, AT₂R-MasR are colocalized in both membrane and cytoplasm mainly in perinuclear region (Figure 3) and provides an overall better labelling. Moreover, the MasR specific siRNA knock-down in HK-2 cells significantly reduced MasR expression and accordingly reduced colocalization with AT₂R, showing specificity of the experiment. Notably, knockdown of MasR did not affect expression of AT₂R (Figure 3). Together these results confirm that AT₂R and MasR are colocalized in renal cortex of obese Zucker rat and in HK-2 cells.

Figure 3.

Immunocytochemical colocalization of AT₂R and MasR in human kidney-2 (HK-2) cells transfected with scrambled siRNA (A) or anti-MasR siRNA (B). Red pixels = AT₂R; Green pixels = MasR. Colocalization is evident as yellow pixels. Detailed method is provided in SI Appendix – “Immunocytochemistry of MasR and AT₂R in HK-2 cells transfected with scrambled or MasR siRNA”.

Cross-linking experiments

In order to examine the potential mechanism of physical interaction between AT₂R and MasR, we performed cross-linking experiment using cupric-phenanthroline complex (CuP, 1.5:4, 0–300 μM final concentration) in renal cortical homogenate of obese Zucker rat (Figure 4). CuP can enter transmembrane pocket and selectively oxidize free thiol (-SH) groups of cysteine located in close proximity (<7 angstrom) facilitating formation of cysteine-cysteine disulfide bridges. Immunoblotting of CuP cross-linked proteins revealed nearly identical electrophoretic migration of AT₂R and MasR-specific complexes which were apparent between 135–180 kDa and slightly above 245 kDa range as compared to their monomers of (AT₂R at 63 kDa; MasR at 54 kDa). These immunoreactive bands 2^nd and 3^rd from top appear to have same size for both antibodies and therefore may contain AT₂R and MasR. Addition of a reducing agent β-mercaptoethanol completely reduced all higher bands into monomeric form (lane 9). Samples without β-mercaptoethanol and Cu-P showed monomeric and higher order bands (lane 2). Addition of Cu-P seems to convert all monomeric to higher order bands (lanes 3–8).

Figure 4.

Zero-length oxidative cupric-phenanthroline (CuP) cross-linking of AT₂R and MasR in obese Zucker rat kidney homogenate (A) and their cross-linking efficiency (B). CuP-cross-linked samples (lane 2–8) were separated under non-reduced condition, i..e. without β-mercaptoethanol. In parallel another CuP (300 μM)-treated replicate was reduced with β-ME as negative control (lane 9). Reduced homogenate control (Ctrl, lane 1) was used as a marker to compare up-ward shift of protein complex after CuP treatment. Immunoreactive bands 2 and 3 from top represent plausible AT₂R-MasR heteromers. Detailed method is provided in SI Appendix – “Zero-length cupric-phenanthroline (CuP) cross-linking”.

We also performed cross-linking experiments using another amine-reactive cross-linker, disuccinimidyl tartrate (DST). It is a water soluble periodate-sensitive cross-linker containing amine (1°)-reactive NHS ester ends and 4-atom spacer arm. Cross-linking of renal cortical homogenate with DST (1–5 mM final concentration) followed by immunoblotting did not reveal AT₂R-MasR heteromers in cortical homogenate of OZR kidney (Figure S4).

Diuresis–natriuresis experiment

Diuretic (Figure 5A) and natriuretic responses (Figure 5B) in presence of AT₂R and MasR specific ligands were measured. Infusion of AT₂R agonist C21 (5 μg/kg/min) significantly increased urine flow (UF) and urinary sodium excretion (U_NaV) in OZR which was completely abolished by simultaneous infusion of MasR antagonist A-779 (50 μg/kg/min) or AT₂R antagonist PD123319 (50 μg/kg/min). Similarly, infusion of MasR agonist Ang-(1-7) (110 fmol/kg/min) significantly increased urine flow (UF) and sodium excretion (U_NaV) in OZR which was completely abolished by simultaneous infusion of AT₂R antagonist PD123319 (50 μg/kg/min) or MasR antagonist A-779 (50 μg/kg/min).

Figure 5.

Effect of infusion of AT₂R agonist C21 (5 μg/kg per min) in male obese Zucker rats on urine flow (UF) (A) and urinary Na-excretion (U_NaV) (B), which was attenuated by simultaneous infusion of the AT₂R antagonist PD123319 (50 μg/kg/min) or MasR antagonist A-779 (50 μg/kg/min). Effect of infusion of MasR agonist Ang-(1-7) (110 fmol/kg/min) in obese Zucker rats on UF (C) and U_NaV (D), which was attenuated by simultaneous infusion of the MasR antagonist A-779 (50 μg/kg/min) or the AT₂R antagonist PD123319 (50 μg/kg/min). Results are reported as mean ± SEM, analyzed by One-way ANOVA. *significantly different from basal; ^#significantly different from C21 (A, B) or Ang-(1-7) (C, D), p<0.05; n=3–5 rats per group. Detailed method is provided in SI Appendix – “In vivo renal function experiment”.

Experiments in isolated renal proximal tubules

To further support the renal functional results of diuresis-natriuresis, we measured cell signaling in isolated renal proximal tubules of OZR. We measured nitrites as a surrogate measure of NO formation, a common second messenger of AT₂R and MasR signaling. The AT₂R agonist C21 (1 μM) induced NO formation (Basal: 0.21±0.07, C21: 1.16±0.61) which was abolished with pre-treatment (10 min) of AT₂R antagonist PD123319 (1 μM) or MasR antagonist A-779 (1 μM) (PD+C21:0.29±0.12, A-779+C21: 0.41±0.19) (Figure 6). Similarly, nitrite formation by MasR agonist ang-(1-7) (1 μM) (Basal: 0.21±0.07, Ang-(1-7): 0.46±0.1) was abolished with pre-treatment (10 min) of MasR antagonist A-779 (1 μM) and AT₂R antagonist PD123319 (1 μM) (A-779+Ang-(1-7):0.19±0.04, PD+Ang-(1-7): 0.26±0.06).

Figure 6.

Effect of AT₂R agonist C21 or MasR agonist ang-(1-7) on stimulation of NO in proximal tubules of obese Zucker rat. Renal tubular suspension (1 mg/mL) was treated with agonists of AT₂R (C21 1 μM), MasR (ang-(1-7) 1 μM) and/or antagonists of AT₂R (PD123319 1 μM), MasR (A-779 1 μM) for 30 minutes at 37°C with gentle shaking. Samples were centrifuged at 3000 g for 30 minutes. Supernatant was deproteinized using Vivaspin 500®, 3 kDa MWCO filter. Total nitrites formed after agonist/antagonist treatment were measured by Griess reagent. Results are reported as mean ± SEM and analyzed by One-way ANOVA with repeated measures followed by Fisher’s LSD test (each comparison stands alone) (A) or Friedman test followed by Dunn’s multiple comparison test (B). *significantly different from basal; ^#significantly different from C21 (A) or Ang-(1-7) (B), p<0.05; n=5 rats per group. Detailed method is provided in SI Appendix – “Treatment of isolated renal proximal tubules”.

Discussion

We demonstrate by co-immunoprecipitation and confocal laser scanning microscopy that AT₂R and MasR are constitutively colocalized in renal cortex of obese rat and HK-2 cells. Disulfide bridge formation seems to be a mechanism involved in forming/stabilizing colocalization of these receptors. We also demonstrate that AT₂R and MasR are functionally interdependent in terms of NO formation and producing diuretic-natriuretic response in obese rats.

For many proteins which functionally interact, it’s important that such proteins are localized in close proximity. Present study utilizing confocal imaging and co-immunoprecipitation approach clearly suggest that AT₂R and MasR are expressed in close proximity and physically interact. Consistent with these observations, immunoblotting of CuP cross-linked renal cortical homogenate revealed significant overlapping of AT₂R- and MasR-selective heteromers which were not seen after cross-linking with amine-reactive cross-linker, disuccinimidyl tartrate (4-atom spacer arm). Crosslinking approach further suggests the role of cysteine residues in forming heteromers via disulfide bridges which are further enhanced in the presence of oxidative conditions. It appears that close proximity allows these receptors to make cysteine bridges, because absence of reducing agent marcaptoethanol shifted the bands of both the receptors from monomers to bands of higher order. This phenomenon is further enhanced by the oxidative agent Cu-P to the degree that all monomers were converted to the bands of higher order. AT₂R has multiple cysteine residues.³⁶ Leonhardt and co-workers recently suggested that C35 of AT₂R is essential for AT₂R-MasR heterodimerization as FRET efficiency was found significantly decreased in HEK-293 cells transiently transfected with vectors encoding mutant AT₂R-C35A-YFP and nonmutant MAS-CFP.²⁹ Particularly, Cys35 of the N terminus in one and Cys290 of exoloop 3 of other AT₂R played a role in constitutive homodimerization of AT₂R via disulfide bridge formation.³⁶ Another study demonstrated that deletion of residues 240–244 within intermediate portion of cytoloop 3 resulted in a complete loss of AT₂R-mediated effects.³⁷ While present study reasonably indicates a role of cysteine residues in AT₂R-MasR dimerization, mutagenesis experiments are required to precisely locate those cysteine residues and define their functional consequence on signaling and function of AT₂R-MasR dimers. Collectively, multiple methodological approaches taken in this study provide a clear evidence that AT₂R and MasR co-exist as dimer. Present study does not allow to understand sub-cellular distribution and functional relevance of the dimer that warrants further studies.

Renal function experiment and treatment of isolated renal proximal tubules of OZR with preferential ligands of AT₂R and MasR showed that they are functionally interdependent in producing diuretic-natriuretic response and NO formation. Functional inter-dependence observed in our study is in agreement with other studies showing abilities of AT₂R antagonist to prevent the effects involving MasR and vice versa.^{20–27,29,38,39} However, these in vivo and in vitro studies in isolated proximal tubule provide an interesting observation in that agonist activation of one receptor may not be critical for another receptor to function. This notion is supported by in vitro studies in isolated proximal tubules where either of the agonists alone AT₂R agonist C21 or MasR agonist Ang-(1-7) was sufficient to increase NO formation. Blocking of C21 response on NO formation by the AT₂R antagonist PD13319 is obvious that PD123319 displaces agonist binding,²⁸ but how MasR antagonist A-779 blocks C21 response is not clear. Because these ligands are quite selective, it may not be A-779 binding on AT₂R at the concentration used in our experiments. The likely scenario is that these antagonists PD123319 and A-779 do not simply occupy the binding sites, rather they cause conformation change affecting receptor downward signaling or they prevent agonist-induced active conformation. This notion is supported by our recent FRET studies in HEK cells expressing AT₂R and MasR that their agonists alone do not affect FRET suggesting that these ligands do not promote or dissociate dimerization.²⁹ Along the same line of notion, other studies suggested that pretreatment of agonist or antagonist did not affect interactions of AT₂R with AT₂R,³⁶ AT₁R⁴⁰ or B₂R.⁴¹

While numerous studies, including present study indicated functional interdependency of AT₂R and MasR, there are studies suggesting that these receptors may not be universally interdependent.^42,43 For example, AT₂R antagonist PD123319 did not abolish ang-(1-7) inhibition of Na⁺-ATPase activity in isolated basolateral membrane of pig kidney.⁴² The PD123319 did not affect ang-(1-7)-induced decrease in renal blood flow in anesthetized dog.⁴³ Similarly MasR antagonist had no effect on AT₂R-mediated pharmacotherapeutic actions.^44–46 Atheroprotective effect of AT₂R agonist CGP42112a were opposed by PD123319 but not by the MasR antagonist A-779.⁴⁴ Also A-779 did not affect AT₂R agonist CGP42112a-mediated secretion of atrial natriuretic peptide from isolated perfused rat atria.⁴⁵ It’s not clear which physiological conditions, anatomical location or cell types are conducive promoting AT₂R-MasR dimerization and functional interdependence, but nuances in the chemical and functional properties of these two receptors are evident. AT₂R and MasR are two emerging receptors in terms of their protective functions against AT₁R-mediated anti-natriuresis, hypertension, hypertrophy and inflammation, and understanding function of AT₂R and MasR at molecular level and nature of their functional synergy has a pathophysiological and translational significance.

In addition to providing cues of physiological functional interaction between AT₂R and MasR in kidney of obese rats, the present study offers means to develop ligands that can selectively stimulate AT₂R-MasR heteromers to enhance their ability in nitrite formation and diuretic-natriuresis response. In future it would be interesting to study whether AT₂R-MasR heterodimerization changed ligand affinity towards their preferential ligands. Since, activation of both, AT₂R and MasR, have been shown to lower blood pressure, it would be important to investigate whether co-treatment of AT₂R agonist C21 and MasR agonist ang-(1-7) produce synergistic reduction of blood pressure and renal injury in high-salt fed obese rats. While our studies clearly demonstrate functional interdependence of AT₂R and MasR, less obvious co-localization and dimerization aspects need further in-depth investigation, which can be considered a limitation of the current study.

Perspectives

Kidney is one of the prime targets which is adversely impacted by obesity and hypertension. Numerous therapeutic approaches are in use to alleviate kidney function. Based on our work and recent reports of heterodimerization of MasR with AT₁R and B₂R, and of AT₂R with AT₁R and B₂R, it is clear that AT₂R may constitutively heterodimerize in renal cortex of obese Zucker rat. Such interactions may be critical to the net physio- and pharmacological responses. Understanding of mechanism of their interactions and functional interdependence offers an opportunity for the development of tailored molecules that can specifically activate AT₂R-MasR heteromers in kidney. Such novel molecules modulating nitrite formation, diuresis and natriuresis would be of prime importance in treating pathologies including salt-sensitive hypertension and myriad kidney diseases in obesity.

Summary.

This study expanded and revised pharmacology of protective renin-angiotensin receptors – AT₂R and MasR, by showing that they are constitutively colocalized in renal cortex of obese rats. In renal proximal tubules, these receptors showed interdependency in NO formation, which is linked to diuretic-natriuretic response observed in obese Zucker rat kidney.

Novelty and Significance.

What is New?

• The AT₂R and MasR are constitutively colocalized in obese Zucker rat renal cortex and human proximal tubular epithelial cells.

• Thiol groups (–SH) of cysteine residues of transmembrane of AT₂R and MasR are located in close proximity and are freely available that they may form disulfide (–S–S–) bridges.

• AT₂R and MasR are interdependent in NO formation and diuretic-natriuretic response in obese Zucker rat kidney.

What is Relevant?

• Excessive sodium reabsorption is one of the mechanisms involved in pathogenesis of hypertension and renal injury in obesity. Understanding of mechanism of interaction and interdependency of AT₂R and MasR in NO formation and diuretic-natriuretic response is not only relevant to therapeutic applications of C21 and ang-(1-7) in chronic kidney diseases and comorbidities – obesity, hypertension and diabetes, but also to development of AT₂R-MasR heteromer-selective ligands.

• These studies involve naturally expressing AT₂R human kidney cells and obese Zucker rat kidney and in vivo studies which are closely applicable to clinical therapeutics as compared to in vitro transfection studies.