Unique role of NADPH oxidase 5 in oxidative stress in human renal proximal tubule cells

NADPH oxidases are the major sources of reactive oxygen species in cardiovascular, neural, and kidney cells. The NADPH oxidase 5 (NOX5) gene is present in humans but not rodents. Because Nox isoforms in renal proximal tubules (RPTs) are involved in the pathogenesis of hypertension, we tested the hypothesis that NOX5 is differentially expressed in RPT cells from normotensive (NT) and hypertensive subjects (HT). We found that NOX5 mRNA, total NOX5 protein, and apical membrane NOX5 protein were 4.2 +/- 0.7-fold, 5.2 +/- 0.7-fold, and 2.8 +/- 0.5-fold greater in HT than NT. Basal total NADPH oxidase activity was 4.5 +/- 0.2-fold and basal NOX5 activity in NOX5 immunoprecipitates was 6.2 +/- 0.2-fold greater in HT than NT (P= < 0.001, n=6-14/group). lonomycin increased total NOX and NOX5 activities in RPT cells from HT (P<0.01, n =4, ANOVA), effects that were abrogated by pre-treatment of the RPT cells with diphenyleneiodonium or superoxide dismutase. Silencing NOX5 using NOX5-siRNA decreased NADPH oxidase activity (-45.1 +/- 3.2% vs. mock-siRNA, n=6-8) in HT. D-1-like receptor stimulation decreased NADPH oxidase activity to a greater extent in NT ( -32.5 +/- 1.8%) than HT (-14.8 +/- 1.8). In contrast to the marked increase in expression and activity of NOX5 in HT. NOX1 mRNA and protein were minimally increased in HT, relative to NT; total NOX2 and NOX4 proteins were not different between HT and NT, while the increase in apical RPT cell membrane NOX1, NOX2, and NOX4 proteins in HT, relative to NT, was much less than those observed with NOX5. Thus, we demonstrate, for the first time, that NOX5 is expressed in human RPT cells and to greater extent than the other Nox isoforms in HT than NT. We suggest that the increased expression of NOX5, which may be responsible for the increased oxidative stress in RPT cells in human essential hypertension, is caused, in part, by a defective renal dopaminergic system. (C) 2014 The Authors. Published by Elsevier B.V.