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Reduced 22Na+ Uptake and Dysregulation of NHERF2/Akt/NOS Pathway in Rat Renal Proximal Tubule Cells Stably Overexpressing p22phox
Author(s) -
Luo Zaiming,
Panico Carolina,
Artigiano Francesca,
Gutweiler Alex,
Jose Pedro A.,
Palm Fredrik,
Welch William J.,
Wilcox Christopher S.
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.1059.4
Subject(s) - protein kinase b , chemistry , transfection , phosphorylation , microbiology and biotechnology , gene knockdown , pi3k/akt/mtor pathway , nitric oxide synthase , nitric oxide , signal transduction , biology , biochemistry , apoptosis , gene , organic chemistry
We showed that superoxide (O 2 − ) reduced renal proximal tubule (PT) reabsorption (Hypertension. 2009 Oct.), but, the molecular mechanisms are unknown. We tested the hypothesis that O 2 − impairs Na + ‐H + exchanger 3 (NHE3) activity. A stably p22 phox transfected rat PT cell line (s‐p22 phox ), compared to empty vector transfected wild type cells (Wt), had increased p22 phox mRNA and protein by 4.1‐ and 1.59‐fold, and O 2 − by 2.2‐ fold but reduced 22 Na + uptake by 36% (all P<0.05). Immunoblot assay of s‐p22 phox 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‐Akt ser473 ) was markedly increased leading to a 207% higher p‐Akt/t‐Akt ratio in s‐p22 phox cells. Knockdown of NHEFR2 by specific siRNA in s‐p22 phox cells increased significantly (25±4.9%; P<0.05) 22 Na + uptake. In conclusion, overexpression of p22 phox 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 O 2 − 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.