High glucose activates PKC-zeta and NADPH oxidase through autocrine TGF-Px signaling in mesangial cells

摘要

High glucose activates PKC-zeta and NADPH oxidase through autocrine TGF-beta signaling in mesangial cells. Am J Physiol Renal Physiol 295: F1705-F1714, 2008. First published September 24, 2008; doi: 10.1152/ajprenal.00043.2008.--Conversion of normally quiescent mesangial cells into extracellular matrix-overproducing myofibroblasts in response to high ambient glucose and transforming growth factor (TGF)-(Bi is central to the pathogenesis of diabetic nephropathy. Previously, we reported that mesangial cells respond to high glucose by generating reactive oxygen species (ROS) from NADPH oxidase dependent on protein kinase C (PKC) -zeta activation. We investigated the role of TGF-beta in this action of high glucose on primary rat mesangial cells within 1-48 h. Both high glucose and exogenous TGF-betai stimulated PKC-zeta kinase activity, as measured by an immune complex kinase assay and immunofluorescence confocal cellular imaging. In high glucose, Akt Ser473 phosphorylation appeared within 1 h and Smad2/3 nuclear translocation was prevented with neutralizing TGF-betai antibodies. Neutralizing TGF-beta antibodies, or a TGF-P receptor kinase inhibitor (LY364947), or a phospha-tidylinositol 3 ,4,5-trisphosphate (PI3) kinase inhibitor (wortmannin), prevented PKC-zeta activation by high glucose. TGF-betai also stimulated cellular membrane translocation of PKC-a, -beta, -8, and -e, similar to high glucose. High glucose and TGF-betai enhanced ROS generation by mesangial cell NADPH oxidase, as detected by 2,7-dichlorofluores-cein immunofluorescence. This response was abrogated by neutralizing TGF-p! antibodies, LY364947, or a specific PKC-zeta pseudosub-strate peptide inhibitor. Expression of constitutively active PKC-zeta in normal glucose caused upregulation of p22phox, a likely mechanism of NADPH oxidase activation. We conclude that very early responses of mesangial cells to high glucose include autocrine TGF-fJi stimulation of PKC isozymes including PI3 kinase activation of PKC-zeta and consequent generation of ROS by NADPH oxidase