Mesangial cell NADPH oxidase upregulation in high glucose is protein kinase C dependent and required for collagen IV expression

Abstract

Excess collagen IV expression by mesangial cells contributes to diabetic glomerulosclerosis. We hypothesized that in high glucose reactive oxygen species (ROS) generation by NADPH oxidase is PKC dependent and required for collagen IV expression by mesangial cells. In rat mesangial cells cultured in 5 mM (NG) or 25 mM d-glucose (HG), RT-PCR and Western immunoblotting detected p22phoxand p47phoxmRNA and protein, respectively. Quantitative real-time RT-PCR analyzed collagen IV mRNA. With the use of confocal microscopy, ROS were detected with dichlorofluorescein and intracellular collagen IV by immunofluorescence. In HG, ROS were generated within 1 h, sustained up to 48 h, and prevented by a NADPH oxidase inhibitor, diphenylenechloride iodonium (DPI), or a conventional PKC isozyme inhibitor, Gö6976. In NG, phorbol myristate acetate stimulated ROS generation that was inhibited with DPI. In HG, expression of p22phoxand p47phoxwas increased within 3 to 6 h and inhibited by Gö6976. In HG, Gö6976 or transfection with antisense against p22phoxreversed the 1.8-fold increase in collagen IV mRNA. In HG, the antioxidants Tempol or Tiron, or transfection with antisense against p22phoxor p47phox, prevented ROS generation and the 2.3-fold increase in collagen IV protein. Increased mitochondrial redox potential in HG was unaffected by transfection with antisense against p22phox. We conclude that in HG, mesangial cell ROS generation by upregulated NADPH oxidase is dependent on conventional PKC isozymes and also required for collagen IV expression.