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

• Published in: Hypertension (Dallas, Tex. 1979) Tex. 1979), 2008-02, Vol.51 (2, Part 2), p.481-487
• Main Authors: Han, Weixing, Li, Hewang, Villar, Van Anthony M, Pascua, Annabelle M, Dajani, Mustafa I, Wang, Xiaoyang, Natarajan, Aruna, Quinn, Mark T, Felder, Robin A, Jose, Pedro A, Yu, Peiying
• Format: Article
• Language: English
• Subjects: Acetophenones - pharmacology; Arterial hypertension. Arterial hypotension; beta-Cyclodextrins - pharmacology; Biological and medical sciences; Blood and lymphatic vessels; Blood vessels and receptors; Cardiology. Vascular system; Cell Membrane - enzymology; Cholesterol - metabolism; Dopamine Agonists - pharmacology; Enzyme Activation - drug effects; Enzyme Inhibitors - pharmacology; Fenoldopam - pharmacology; Fundamental and applied biological sciences. Psychology; Humans; Immunoblotting; Isoenzymes - genetics; Kidney Tubules, Proximal - cytology; Kidney Tubules, Proximal - enzymology; Medical sciences; Membrane Glycoproteins - antagonists & inhibitors; Membrane Glycoproteins - genetics; Membrane Microdomains - physiology; NADPH Oxidase 2; NADPH Oxidase 4; NADPH Oxidases - antagonists & inhibitors; NADPH Oxidases - genetics; NADPH Oxidases - metabolism; Onium Compounds - pharmacology; Reactive Oxygen Species - metabolism; Receptors, Dopamine D1 - agonists; RNA, Messenger - metabolism; RNA, Small Interfering - pharmacology; Vertebrates: cardiovascular system
• ISSN: 0194-911X; 1524-4563
• DOI: 10.1161/HYPERTENSIONAHA.107.103275

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 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%; control98.6±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 βCD162.0±2.0%) or Nox4 (108.0±10.8% versus βCD152.0±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.