Reduced 22Na+ Uptake and Dysregulation of NHERF2/Akt/NOS Pathway in Rat Renal Proximal Tubule Cells Stably Overexpressing p22phox

Abstract

We showed that superoxide (O2−) reduced renal proximal tubule (PT) reabsorption (Hypertension. 2009 Oct.), but, the molecular mechanisms are unknown. We tested the hypothesis that O2− impairs Na+‐H+ exchanger 3 (NHE3) activity. A stably p22phox transfected rat PT cell line (s‐p22phox), compared to empty vector transfected wild type cells (Wt), had increased p22phox mRNA and protein by 4.1‐ and 1.59‐fold, and O2− by 2.2‐ fold but reduced 22Na+uptake by 36% (all P<0.05). Immunoblot assay of s‐p22phox cells showed no change in NHE3 but increased expression of inhibitory Na+–H+ exchanger regulatory factor 2 (NHERF2), neuronal and inducible nitric oxide synthase (nNOS & iNOS) by 183%, 293% and 372%, respectively (all P<0.05). Since activated Akt regulates NOS and NHERF2, we compared the Akt in both cell types. Total Akt (t‐Akt) protein abundance was similar but the phosphorylated Akt at ser473 (p‐Aktser473) was markedly increased leading to a 207% higher p‐Akt/t‐Akt ratio in s‐p22phox cells. Knockdown of NHEFR2 by specific siRNA in s‐p22phox cells increased significantly (25±4.9%; P<0.05) 22Na+uptake. In conclusion, overexpression of p22phox in PT cells inhibits NHE3 transport activity and upregulates NHERF2, nNOS and iNOS, and phosphorylated Akt. This dysregulation of NHERF2/Akt/NOS signaling pathways by O2− impairs PT function. This provides a novel insight into the molecular mechanisms underlying the effects of oxidative stress on ion and fluid homeostasis in the kidney.