Aims The receptor for advanced glycation end products (RAGE) contributes to the development and progression of diabetic nephropathy. In this study, we examined if the protective effects afforded by RAGE blockade are via modulation of the renal AT-2 receptor. Methods Control and streptozotocin diabetic mice, wild type (WT) or deficient in either the angiotensin II type 2 receptor (AT-2 KO) or RAGE (RAGE KO), were studied for 24 weeks. Albumin excretion rate (AER), creatinine clearance (CrCl), renal cortical RAS components and RAGE expression were assessed. Results With diabetes, RAGE deficient mice had lower AER and attenuation of hyperfiltration, as compared with both diabetic WT and AT-2 deficient mice. Gene expression of RAGE was elevated in diabetic kidneys, while elevations in renal membranous RAGE expression were evident in WT and AT-2 KO mice with diabetes. Diabetic RAGE KO mice had an amelioration of diabetes induced increases in renal AT-2 gene expression. Although renal AT-2 protein expression was increased by diabetes in WT mice, it was further elevated in diabetic RAGE KO mice. Diabetes-induced increases in renal superoxide, were prevented in diabetic RAGE KO but not diabetic AT-2 KO mice. Adenoviral overexpression of RAGE or AGE treatment, decreased cell surface AT-2 expression, resulting in superoxide generation which was reversed by apocynin in primary mesangial cells. Conclusions We postulate that RAGE appears to be a common and key modulator of AT-2 receptor expression, which implicates a newly defined RAGE/AT-2 axis in the development and progression of diabetic nephropathy.

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.

Objective Obesity is a known risk factor for various metabolic disorders and cardiovascular diseases. Recently we demonstrated that angiotensin female AT2 receptor (AT2R) knockout mice on high-fat diet (HFD) had higher body weight and adiposity with a parallel reduction in estrogen (17,β-estradiol/E2). The present study investigated whether the anti-adiposity effects of the AT2R are estrogen-dependent in female mice. Methods Female C57BL/6 ovary-intact (Ovi) mice were treated with an AT2R agonist (C21, 0.3 mg/kg, daily i.p.). Ovariectomized (Ovx) mice, supplemented with E2 (5 μg/day, pellets implanted subcutaneously), were treated with an AT2R agonist (C21, 0.3 mg/kg, daily i.p.) or vehicle. After 4-days of pre-treatment with C21, Ovi and Ovx mice were placed on either normal diet (ND) or HFD while the C21 treatment continued for the next 10 days. For a long-term study, Ovi mice were placed on HFD and treated with C21 for 12 weeks. Results Ovi mice fed the HFD had increased parametrial white adipose tissue (pWAT) weight, plasma free fatty acid and triglycerides compared to Ovi mice on ND. Ovariectomy alone caused similar changes in these parameters which were further increased by HFD-feeding. C21 treatment attenuated these HFD-induced changes in Ovi as well as Ovx mice. HFD also, increased the liver/body-weight ratio and decreased the liver expression of the β-oxidation enzyme, carnitine palmitoyltransferase 1 (CPT1-A). C21 treatment attenuated these changes as well. The long-term C21 treatment of Ovi mice lowered the HFD-induced body weight gain, increase in pWAT weight, parametrial adipocyte size and hyperinsulinemia induced by HFD. Finally, HFD drastically reduced urinary estrogen and the beneficial metabolic changes in response to C21-treatment occurred without significantly increasing urinary estrogen. Conclusion We suggest that the pharmacological activation of AT2R by the agonist C21 reduces adiposity and body weight gain independent of estrogen in female mice. Improvement in fatty acid metabolism is a potential mechanism by which the AT2R exerts anti-adiposity effects.

Obesity is a major disease condition, in turn leading to pathological changes collectively recognized as metabolic syndrome. Recently angiotensin receptor AT2R has been associated negatively with body weight (BW) gain in male mice. However, the gender differences in AT2R and BW changes have not been studied. To understand the gender based role of AT2R involving BW changes, we fed male and female wild type (WT) and AT2R knock out (AT2KO) mice with C57BL6 background with high fat diet (HFD) for 16 weeks. The male AT2KO had higher HFD calorie intake (WT: 1280±80; AT2KO:1680±80 kcal) but gained less BW compared with the WT (WT: 13; AT2KO: 6 g). Contrary to the male animals, the female AT2KO mice with equivalent caloric intake (WT: 1424±48; AT2KO:1456±80 kcal) gained significantly more BW than the WT mice (WT: 9 g; AT2KO: 15 g). The male AT2KO on HFD displayed lower plasma insulin level, less impaired glucose tolerance (GT), and higher plasma T3 compared with WT males on HFD; whereas the female AT2KO mice on HFD showed elevated levels of plasma insulin, more impaired GT, lower plasma T3 and higher free fatty acid and hepatic triglycerides compared with WT females on HFD. Interestingly, compared with WT, AT2KO female mice had significantly lower estrogen, which was further reduced by HFD. These results suggest that AT2R in female mice via potentially regulating estrogen may have protective role against BW gain and impaired glucose tolerance and lipid metabolism.