Rosiglitazone Prevents High Glucose-Induced Vascular Endothelial Growth Factor and Collagen IV Expression in Cultured Mesangial Cells

Peroxisome proliferator-activated receptor (PPARγ), a ligand-dependent transcription factor, negatively modulates high glucose effects. We postulated that rosiglitazone (RSG), an activator of PPARγ prevents the upregulation of vascular endothelial growth factor (VEGF) and collagen IV by mesangial cells exposed to high glucose. Primary cultured rat mesangial cells were growth-arrested in 5.6 mM (NG) or 25 mM D-glucose (HG) for up to 48 hours. In HG, PPARγ mRNA and protein were reduced within 3 h, and enhanced ROS generation, expression of p22phox, VEGF and collagen IV, and PKC-ζ membrane association were prevented by RSG. In NG, inhibition of PPARγ caused ROS generation and VEGF expression that were unchanged by RSG. Reduced AMP-activated protein kinase (AMPK) phosphorylation in HG was unchanged with RSG, and VEGF expression was unaffected by AMPK inhibition. Hence, PPARγ is a negative modulator of HG-induced signaling that acts through PKC-ζ but not AMPK and regulates VEGF and collagen IV expression by mesangial cells

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

In high glucose protein kinase C-ζ activation is required for mesangial cell generation of reactive oxygen species.BackgroundWe 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).MethodsIn primary rat mesangial cells cultured in 5.6mmol/L or 30mmol/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 p47phox or with Tempol. F-actin disassembly was observed by dual-channel confocal fluorescence imaging. PI3 kinase activity was detected by immunoblotting of phosphorylated Akt.ResultsIn 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.ConclusionIn 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