In high glucose protein kinase C-ζ activation is required for mesangial cell generation of reactive oxygen species

Abstract

Background. We postulated that in mesangial cells exposed to high glucose, protein kinase C-ζ (PKC-ζ) is necessary for the generation of reactive oxygen species (ROS) by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and that the requirement of PKC-ζ for filamentous (F)-actin disassembly may involve ROS. To identify signaling mechanisms relevant to PKC-ζ activation and ROS generation, including phosphoinositide 3 kinase (PI3 kinase), we examined mesangial cell stimulation with platelet-derived growth factor (PDGF). Methods. In primary rat mesangial cells cultured in 5.6 mmol/L or 30 mmol/L D-glucose, PKC-ζ expression was identified with immunoblotting and activity was analyzed in cell membrane immunoprecipitates and by confocal immunofluorescence imaging. ROS generation was measured by dichlorofluorescein fluorescence using confocal microscopy and was inhibited by transfection of antisense against NADPH subunits p22phox or p47 phox or with Tempol. F-actin disassembly was observed by dual-channel confocal fluorescence imaging. PI3 kinase activity was detected by immunoblotting of phosphorylated Akt. Results. In high glucose, generation of NADPH oxidase-dependent ROS was dependent on PKC-ζ. Conversely, sustained PKC-ζ activity was dependent on ROS generation, suggesting a positive feedback. PKC-ζ-dependent F-actin disassembly in high glucose required ROS generation. PDGF stimulated NADPH oxidase generation of ROS through a PKC-ζ mechanism that was independent of Akt phosphorylation and remained unchanged in high glucose. Conclusion. In high glucose, mesangial cell PKC-ζ is required for ROS generation from NADPH oxidase similar to PDGF stimulation of PKC-ζ-dependent ROS generation through a pathway independent of PI3 kinase. F-actin disassembly in high glucose also requires ROS. A positive feedback loop occurs between ROS and the activation of PKC-ζ in high glucose. © 2005 by the International Society of Nephrology.