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High glucose activates PKC- and NADPH oxidase through autocrine TGF-β₁ signaling in mesangial cells

¹University Health Network, ²Mt. Sinai Hospital, and ³Department of Medicine, University of Toronto, Toronto, Ontario, Canada

Submitted 25 January 2008 ; accepted in final form 17 September 2008

Conversion of normally quiescent mesangial cells into extracellular matrix-overproducing myofibroblasts in response to high ambient glucose and transforming growth factor (TGF)-β₁ 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) - activation. We investigated the role of TGF-β₁ in this action of high glucose on primary rat mesangial cells within 1–48 h. Both high glucose and exogenous TGF-β₁ stimulated PKC- 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-β₁ antibodies. Neutralizing TGF-β₁ antibodies, or a TGF-β receptor kinase inhibitor (LY364947), or a phosphatidylinositol 3,4,5-trisphosphate (PI3) kinase inhibitor (wortmannin), prevented PKC- activation by high glucose. TGF-β₁ also stimulated cellular membrane translocation of PKC-, -β₁, -, and -, similar to high glucose. High glucose and TGF-β₁ enhanced ROS generation by mesangial cell NADPH oxidase, as detected by 2,7-dichlorofluorescein immunofluorescence. This response was abrogated by neutralizing TGF-β₁ antibodies, LY364947, or a specific PKC- pseudosubstrate peptide inhibitor. Expression of constitutively active PKC- in normal glucose caused upregulation of p22^phox, a likely mechanism of NADPH oxidase activation. We conclude that very early responses of mesangial cells to high glucose include autocrine TGF-β₁ stimulation of PKC isozymes including PI3 kinase activation of PKC- and consequent generation of ROS by NADPH oxidase.

reactive oxygen species; transforming growth factor-β₁; PI3 kinase

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