Differential Regulation of NADPH Oxidase Activity in Lipid Rafts in Renal Proximal Tubule Cells from Rats and Humans

Abstract

We tested the hypothesis that NADPH oxidase activity and Nox subunits are differentially regulated in lipid rafts (LRs) in renal proximal tubule cells (RPTCs) from humans and rats. Nox4 protein was expressed in LRs in rat RPTCs (47±3.1%) but not in human RPTCs (n=3); more Nox2 (40.3±3.7%) and Rac1 (58±3.1%) resided in LRs in human RPTCs than in rat RPTCs (18.2±4.4 and 29.5±4.4%, respectively). Cholesterol depletion with methyl‐β‐cyclodextrin (βCD/2%) decreased oxidase activity (42±7.1%), accompanied by decreased Nox2, Nox4 and Rac1 expression in LRs in rat RPTCs. In contrast, in human RPTCs, βCD treatment markedly increased oxidase activity (154±10.5 vs vehicle=103.1±3.4%, P<0.001 ANOVA, n=4), accompanied by a redistribution of Nox subunits into non‐LR fractions. The effect of βCD in both human RPTCs and rat RPTCs was reversed by cholesterol repletion. The stimulatory effect of βCD treatment on NADPH oxidase activity in human RPTCs was also reversed by inhibitors of NADPH oxidase, apocynin (10μM,100±3.2%) and diphenylene iodinium (5μM, 9.5±3.3%) (P<0.01, ANOVA, n=4–6/group). These studies demonstrate for the first time that NADPH oxidase subunits are differently targeted to LRs in human and rat RPTCs; and disruption of LRs produces opposite effects in humans (stimulation) and rats (inhibition). The results suggest that the integrity of LRs plays an important role in maintaining NADPH oxidase in the inactive state in human kidney cells.