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1 University Health Network, Canada; Department of Medicine, University of Toronto, Toronto, Ontario, Canada
2 Mt. Sinai Hospital, Canada; Department of Medicine, University of Toronto, Toronto, Ontario, Canada
* To whom correspondence should be addressed. E-mail: catharine.whiteside{at}utoronto.ca.
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 p22phox and p47phox mRNA and protein, respectively. Quantitative real-time RT-PCR analyzed collagen IV mRNA. Using confocal microscopy, ROS were detected with dichlorofluorescein and intracellular collagen IV by immunofluorescence. In HG, ROS were generated within 1h, sustained up to 48h and prevented by a NADPH oxidase inhibitor, diphenylenechloride iodonium (DPI), or a conventional PKC isozyme inhibitor, Go6976. In NG, phorbol myristate acetate stimulated ROS generation that was inhibited with DPI. In HG, expression of p22phox and p47phox was increased within 3 to 6h, inhibited by Go6976. In HG, Go6976 or transfection with antisense against p22phox reversed the 1.8-fold increase of collagen IV mRNA. In HG, the antioxidants Tempol or Tiron, or transfection with antisense against p22phox or p47phox prevented ROS generation and the 2.3-fold increase of 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.
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