Hydrogen peroxide increases extracellular matrix mRNA through TGF-beta in human mesangial cells

Background. Reactive oxygen species (ROS) are excessively produced in patho logic states, including many renal diseases. Transforming growth factor-bet a (TGF-beta) may mediate renal fibrotic injury, and ROS may act through the TGF-beta pathway to exert a profibrotic effect. Methods. The expression of TGF-beta1 and extracellular matrix (ECM) compone nts were assessed in cultured human mesangial cells (HMCs) incubated with g lucose oxidase (GO), an enzyme that continuously generates hydrogen peroxid e from glucose. A neutralizing anti-TGF-beta antibody was added to test the hypothesis that hydrogen peroxide acts through activation of the TGF-beta pathway to stimulate ECM expression. Results. Northern blot analysis revealed significantly increased steady-sta te levels of TGF-beta1 and ECM proteins (collagen types I, III, and IV, and fibronectin) by approximately twofold. While no significant effect on mRNA stability after treatment with GO was observed, other studies employing pr omoter-reporter assays, competitive-quantitative reverse transcription-poly merase chain reaction, mink lung epithelial cell proliferation assay, and T GF-beta1 enzyme-linked immunosorbent assay all demonstrated significant sti mulation by GO (>1.5-fold) of TGF-beta1 promoter activity, mRNA level, bioa ctivity, and protein production, respectively. Catalase pretreatment preven ted the GO-induced stimulation of TGF-beta1 mRNA. When incubations were per formed with a panselective neutralizing anti-TGF-beta antibody, the GO-stim ulated expression of ECM molecules was prevented. Conclusions. GO-induced hydrogen peroxide production induces TGF-beta1 synt hesis and thereby increases ECM gene expression in cultured HMCs. These cel lular responses may underlie the development and progression of renal disea ses characterized by oxidative stress.