Previous in vitro studies indicated that aldosterone nongenomically phosphorylates epidermal growth factor receptor (EGFR) through activation of upstream signals, heat shock protein 90β (Hsp90β), and cytosolic (c)-Src kinase. We demonstrated that aldosterone rapidly elevates EGFR phosphorylation in rat kidney. There are no in vivo data regarding renal Hsp90(α and β) and phosphorylated (p)c-Src protein expressions. The present study further investigates the expressions of these proteins. Male Wistar rats were intraperitoneally injected with normal saline solution or aldosterone (Aldo: 150 μg/kg BW). After 30 minutes, abundances and localizations of these proteins were determined. Aldosterone enhanced renal Hsp90β protein abundance (P < 0.001), but Hsp90α and pc-Src protein levels remained unaltered. Expression of Hsp90(α and β) was induced prominently in the proximal convoluted tubules (PCTs). Activation of Hsp90α was observed in vascular and outer medulla regions, whereas Hsp90β was induced in the cortex. Immunoreactivity of pc-Src was elevated in PCT with obvious staining at the luminal membrane. This in vivo study is the first to demonstrate that aldosterone nongenomically elevates Hsp90(α and β) protein expressions in rat kidney. Aldosterone had no effect on pc-Src protein levels but modulated localization. These results indicate that aldosterone regulates upstream mediators of EGFR transactivation in vivo.
Background: In vitro studies have demonstrated that aldosterone elicits nongenomic actions by enhancing protein expressions of phosphorylated epidermal growth factor receptor (pEGFR) and phosphorylated extracellular signal-regulated kinases 1/2 (pERK1/2). There are no available in vivo investigations regarding this action of aldosterone on renal pEGFR-pERK1/2 protein expressions. Methods: Male Wistar rats received normal saline solution, low-dose (LA: 150 µg/kg BW) or high-dose aldosterone (HA: 500 µg/kg BW) by intraperitoneal injection. After 30 min, protein abundances and localizations of renal pEGFR and pERK1/2 were determined by Western blot and immunohistochemistry. Results: Plasma aldosterone levels were increased in LA and HA groups (p < 0.001). Aldosterone enhanced renal pEGFR and pERK1/2 protein abundances (p < 0.001). HA showed a greater stimulation on pEGFR immunoreactivity than LA in the glomerulus, vasa recta, and thin limb of Henle’s loop in the inner medulla area. LA provided more reactivity of pERK1/2 in the thick ascending limb of Henle’s loop, outer medullary collecting duct, and proximal straight tubule, whereas HA illustrated more pERK1/2 activation in the glomerulus, peritubular capillary, and inner medulla region. Conclusion: This is the first in vivo study which demonstrates that aldosterone, via the nongenomic pathway, could elevate pEGFR and pERK1/2 protein abundances and expressions in the rat kidney. These results indicate that aldosterone induces phosphorylation of EGFR upstream of ERK1/2.
IntroductionPrevious in vitro studies demonstrated that aldosterone nongenomically induces transglutaminase (TG) and reactive oxygen species (ROS), which enhanced angiotensin II receptor (ATR) dimerization. There are no in vivo data in the kidney.Material and methodsMale Wistar rats were intraperitoneally injected with normal saline solution, or aldosterone (Aldo: 150 μg/kg BW); or received pretreatment with eplerenone (mineralocorticoid receptor (MR) blocker, Ep. + Aldo), or with apocynin (nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor, Apo. + Aldo) 30 min before aldosterone. Thirty minutes after aldosterone injection, protein abundances of dimeric and monomeric forms of AT1R and AT2R, and protein abundances and localizations of TG2 and p47phox, a cytosolic subunit of NADPH oxidase, were determined by Western blot analysis and immunohistochemistry, respectively.ResultsProtein abundances of dimeric forms of AT1R and AT2R were enhanced by 170% and 70%, respectively. Apocynin could block dimeric forms of both receptors while eplerenone inhibited only AT2R. Monomeric protein levels of both receptors were maintained. Aldosterone significantly enhanced TG2 and p47phox protein abundances, which were blunted by eplerenone or apocynin. Aldosterone stimulated p47phox protein expression in both the cortex and the medulla while TG2 was induced mostly in the medulla. Eplerenone or apocynin normalized the immunoreactivity of both TG2 and p47phox.ConclusionsThis is the first in vivo study demonstrating that aldosterone nongenomically increases renal TG2 and p47phox protein expression and then activates AT1R and AT2R dimerizations. Aldosterone-stimulated AT1R and AT2R dimerizations are mediated through activation of NADPH oxidase. Aldosterone-induced AT1R dimer formation is an MR-independent pathway, whereas the formation of AT2R dimer is modulated in an MR-dependent manner.
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