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Nongenomic effect of aldosterone on angiotensin II type 1 receptor dimerization in human renal proximal tubular cells: Implications for endoplasmic reticulum stress

Kittisak Sinphitukkul^a,g, Krissanapong Manotham^b, Somchai Eiam-Ong^c, Masaomi Nangaku^d, Reiko Inagi^e, Somchit Eiam-Ong^f,*

 
ABSTRACT:     In vitro studies have showed that aldosterone increases oxidative stress molecules through a nongenomic effect. Oxidative stress induces angiotensin II type 1 receptor (AT1R) dimerization and endoplasmic reticulum (ER) stress, leading to renal tubular damage. However, the nongenomic effect of aldosterone on AT1R dimerization and ER stress in renal cells has not been determined. Here, we examined the nongenomic action of aldosterone in renal proximal tubular epithelial cells (PTECs) to better understand the underlying mechanisms. HK-2 cells, human renal PTECs, were exposed to vehicle or aldosterone for 30 min. In two additional groups, the cells were pretreated with eplerenone, a mineralocorticoid receptor (MR) blocker or apocynin, an NADPH oxidase inhibitor, for 30 min before aldosterone incubation. Protein abundances of dimeric/monomeric forms of AT₁R, p47phox (a cytosolic part of NADPH oxidase), and activating transcription factor 4 (ATF4), a transcription factor responsive to ER stress, were determined by Western blotting. Aldosterone nongenomically increased plasma membrane protein expression of AT₁R dimeric forms in a time- and dose-dependent manners. The levels of the cytosolic p47phox protein declined while the membranous protein level was enhanced following aldosterone treatment. The aldosterone induced alteration in these two proteins was abolished by pretreatment with eplerenone or apocynin. In addition, aldosterone (100 nM) induced nuclear ATF4 protein accumulation in a time-dependent fashion, which was blocked by apocynin and partially attenuated by eplerenone. Aldosterone nongenomically increased AT₁R dimerization and nuclear ATF4 protein accumulation dependent on MR and NADPH oxidase activation. Hence aldosterone could induce AT₁R dimerization and activate the endoplasmic reticulum stress response.

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* Corresponding author, E-mail: eiamong@yahoo.com

Received 6 Jan 2018, Accepted 17 Sep 2018