Reactive oxygen species alter gene expression in podocytes: induction of granulocyte macrophage-colony-stimulating factor.
It has been suggested that reactive oxygen radicals (ROS) play a crucial role in the pathogenesis of proteinuria and podocyte injury. It was investigated whether changes in gene expression might account for ROS-induced podocyte dysfunction. Differentiated podocytes were incubated with control media or with exogenous ROS from the xanthine/xanthine-oxidase reaction for 4 h. A PCR-based suppressive subtractive hybridization assay was applied to isolate and clone mRNAs that were differentially expressed by exogenous ROS. One differentially expressed clone was identified as the proinflammatory cytokine granulocyte macrophage-colony-stimulating factor (GM-CSF). Regulation of GM-CSF in podocytes was further studied by Northern analysis and enzyme-linked immunosorbent assay. Exogenous ROS caused a concentration-dependent, >10-fold induction of GM-CSF mRNA after 4 h. A >50-fold increase in GM-CSF protein release in podocytes that had been stimulated with ROS could be detected. Induction of GM-CSF protein was inhibited by actinomycin D, which indicated that increased mRNA transcription was involved. The ROS scavengers dimethyl-thio-urea and pyrrolidone-dithio-carbamate strongly inhibited increased GM-CSF production induced by ROS. GM-CSF release was also induced when internal ROS production was triggered with NADH, whereas H2O2 had only a small effect. GM-CSF release by podocytes was also stimulated by lipopolysaccharide (LPS), interleukin-1 (IL-1), and phorbolester (PMA). Dimethyl-thio-urea significantly inhibited the LPS-, IL-1-, and PMA-induced GM-CSF production. Activation of the transcription factor nuclear factor-kappaB (NF-kappaB) but not activator protein-1 was involved in the upregulation of ROS-induced GM-CSF production. The data indicate that GM-CSF is differentially expressed by ROS in podocytes. ROS also partially mediate the effects of PMA and IL-1 on podocyte GM-CSF production. Because GM-CSF can enhance glomerular inflammation and induces mesangial proliferation, these data might provide further insight into the mechanisms of ROS-induced glomerular injury.
Greiber
Stefan
S
Department of Medicine, Division of Nephrology, University Hospital of Freiburg, Freiburg, Germany.
Müller
Barbara
B
Department of Medicine, Division of Nephrology, University Hospital of Freiburg, Freiburg, Germany.
Daemisch
Petra
P
Department of Medicine, Division of Nephrology, University Hospital of Freiburg, Freiburg, Germany.
Pavenstädt
Hermann
H
Department of Medicine, Division of Nephrology, University Hospital of Freiburg, Freiburg, Germany.
D000818:Animals; D002478:Cells, Cultured; D016166:Free Radical Scavengers / Q000494:pharmacology; D015870:Gene Expression / Q000502:physiology*; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology*; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics* / Q000378:metabolism; D007375:Interleukin-1 / Q000494:pharmacology; D007668:Kidney / Q000166:cytology / Q000502:physiology*; D008070:Lipopolysaccharides / Q000494:pharmacology; D051379:Mice; D016328:NF-kappa B / Q000502:physiology; D011759:Pyrrolidines / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism* / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013859:Thiocarbamates / Q000494:pharmacology; D013890:Thiourea / Q000031:analogs & derivatives* / Q000494:pharmacology; D013997:Time Factors; D014158:Transcription, Genetic / Q000502:physiology; D015854:Up-Regulation; D019820:Xanthine / Q000378:metabolism; D014969:Xanthine Oxidase / Q000378:metabolism
D016166:Free Radical Scavengers; D007375:Interleukin-1; D008070:Lipopolysaccharides; D016328:NF-kappa B; D011759:Pyrrolidines; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D013859:Thiocarbamates; D019820:Xanthine; C020972:pyrrolidine dithiocarbamic acid; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; C038983:1,3-dimethylthiourea; D014969:Xanthine Oxidase; D013890:Thiourea; D013755:Tetradecanoylphorbol Acetate