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(Hypertension. 2008;51:481.)
© 2008 American Heart Association, Inc.

Original Articles Part 2

Lipid Rafts Keep NADPH Oxidase in the Inactive State in Human Renal Proximal Tubule Cells

Weixing Han; Hewang Li; Van Anthony M. Villar; Annabelle M. Pascua; Mustafa I. Dajani; Xiaoyang Wang; Aruna Natarajan; Mark T. Quinn; Robin A. Felder; Pedro A. Jose; Peiying Yu

From the Department of Pediatrics (W.H., H.L., V.A.M.V., A.M.P., M.I.D., X.W., A.N., P.A.J., P.Y.), Georgetown University School of Medicine, Washington, DC; the Department of Veterinary Molecular Biology (M.Q.), Montana State University, Bozeman, Mont; and the Department of Pathology (R.A.F.), University of Virginia, Charlottesville, Va.

Correspondence to Peiying Yu, MD, Division of Pediatric Nephrology, Department of Pediatrics, Georgetown University Medical Center, 4000 Reservoir Rd, NW, Washington, DC 20057. E-mail yup{at}georgetown.edu

Recent studies have indicated the importance of cholesterol-rich membrane lipid rafts (LRs) in oxidative stress-induced signal transduction. Reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidases, the major sources of reactive oxygen species, are implicated in cardiovascular diseases, including hypertension. We tested the hypothesis that NADPH oxidase subunits and activity are regulated by LRs in human renal proximal tubule cells. We report that a high proportion of p22^phox and the small GTPase Rac1 are expressed in LRs in human renal proximal tubule cells. The D₁-like receptor agonist, fenoldopam (1 µmol/L per 20 minutes) dispersed Nox subunits within LRs and non-LRs and decreased oxidase activity (30.7±3.3%). In contrast, cholesterol depletion (2% methyl-β-cyclodextrin [βCD]) translocated NADPH oxidase subunits out of LRs and increased oxidase activity (154.0±10.5% versus control, 103.1±3.4%), which was reversed by cholesterol repletion (118.9±9.9%). Moreover, NADPH oxidase activation by βCD (145.5±9.0%; control: 98.6±1.6%) was also abrogated by the NADPH oxidase inhibitors apocynin (100.4±3.2%) and diphenylene iodonium (9.5±3.3%). Furthermore, βCD-induced reactive oxygen species production was reversed by knocking down either Nox2 (81.0±5.1% versus βCD: 162.0±2.0%) or Nox4 (108.0±10.8% versus βCD: 152.0±9.8%). We have demonstrated for the first time that disruption of LRs results in NADPH oxidase activation that is abolished by antioxidants and silencing of Nox2 or Nox4. Therefore, in human renal proximal tubule cells, LRs maintain NADPH oxidase in an inactive state.

Key Words: NADPH oxidase • ROS • dopamine receptors • lipid rafts