DOI: 10.14457/cu.the.2016.158
title: Nongenomic actions of aldosterone on angiotensin receptor dimerization in rat kidney : role of mineralocorticoid receptor and NADPH oxidase

description: Previous in vitro studies demonstrated that aldosterone nongenomically elevates angiotensin II receptor (ATR) dimerization through transglutaminase type 2 (TG2) and reactive oxygen species (ROS). However, there is no in vivo study regarding this circumstance in the kidney. The purpose of the present investigation is to examine the nongenomic effects of aldosterone on protein abundances (dimeric and monomeric forms) of angiotensin II type 1 receptor (AT1R) and angiotensin II type 2 receptor (AT2R), TG2 (a crosslinking protein enzyme), and p47phox (a cytosolic part of NADPH oxidase activation to generate ROS), and localizations of TG2 and p47phox proteins in rat kidney.Male Wistar rats were received normal saline solution or aldosterone (150 µg/kg BW) by intraperitoneal injection for 30 minutes or pretreated with eplerenone [mineralocorticoid receptor (MR) blocker, 15 mg/kg BW] or with apocynin (NADPH oxidase inhibitor, 5 mg/kg BW) 30 minutes before aldosterone injection. Plasma membrane protein abundances of renal dimeric and monomeric forms of AT1R, AT2R, and p47phox as well as of tissue homogenated TG2 protein were measured by Western blot analysis. Protein localizations of TG2 and p47phox were examined by immunohistochemistry. Protein levels of AT1R/AT2R heterodimerization were analyzed by co-immunoprecipitation and Western blot techniques. Finally, the colocalization of AT1R and AT2R proteins was determined by double labelling immunohistochemistry. After 30 minutes of aldosterone injection, aldosterone enhanced renal dimeric forms of AT1R and AT2R (p &lt; 0.001) but monomeric forms of both receptors unaltered. Eplerenone inhibited only the dimeric form of AT2R while apocynin prevented the dimeric formation of both receptors. Aldosterone increased TG2 and p47phox protein abundances that were blunted by pretreatment with eplerenone or apocynin. Aldosterone stimulated TG2 protein expression mainly in the medulla whereas p47phox protein was increased in both cortex and medulla. Pretreatment with eplerenone or apocynin alleviated the expressions of both TG2 and p47phox. No significant changes in plasma membrane AT1R/AT2R heterodimerization were observed in all studied groups. The colocalization of AT1R and AT2R proteins was high in glomerulus, distal convoluted tubule, and cortical collecting duct meanwhile the colocalization was weak and diffused in the proximal convoluted tubule and peritubular capillary in all studied groups.In conclusion, the present data are the first document demonstrating that aldosterone nongenomically increases renal TG2 and p47phox protein expressions and then activates AT1R and AT2R dimerizations. Aldosterone-stimulated AT1R and AT2R dimerizations are mediated through activation of NADPH oxidase. However, aldosterone-induced AT1R dimer formation is MR-independent pathway whereas the formation of AT2R dimer is modulated via MR-dependent manner. Aldosterone-enhanced renal TG2 and p47phox protein expressions are depend on both MR and NADPH oxidase activations. The heterodimerization of AT1R/AT2R is maintained in all studied groups. The colocalization of AT1R and AT2R proteins is more prominent in the cortex region.