INDUCTION OF MANGANESE SUPEROXIDE-DISMUTASE BY GLUCOCORTICOIDS IN GLOMERULAR CELLS

Our previous in vivo study demonstrated that methylprednisolone (MP) activates glomerular antioxidant enzymes and attenuates glomerular oxidant injuries, including those in experimental nephrosis. The present study investigates the cellular mechanism of the MP-induced activation of antioxidant enzymes and their contribution to the attenuation of cellular oxidant toxicity. When bovine glomerular endothelial cells (GECs) were treated with 10 muM MP, cellular manganese superoxide dismutase (Mn-SOD, 3.95 +/- 0.33 mu/mg protein, M +/-SE) and catalase (1.64 +/- 0.06 k/mg protein) activities were significantly (P < 0.05) elevated above control GECs (2.23 +/- 0.43 mu/mg protein and 1.06 +/- 0.09 k/mg protein, respectively). When GECs pretreated with MP (10 muM 24 hrs) were exposed to xanthine (0.1 mm) + xanthine oxidase (5 mU/ml) for four hours, levels of specific membrane lipid peroxidation products, that is, phosphatidylcholine- and phosphatidylethanolamine-hydroperoxides, remained at levels 10 to 25% of those measured in non-MP-treated (xanthine/xanthine oxidase-exposed) control cells. Moreover, the degree of cell damage following exposure to the superoxide generating system, assessed by Cr-51 release, was significantly attenuated in MP-treated cells (almost-equal-to 50% of MP-non-treated controls, N = 6). Thus, MP-treated GECs with elevated antioxidant enzyme activities by MP were more resistant to the toxic effect of reactive oxygen metabolites. The mechanism of antioxidant enzyme induction by MP was studied for Mn-SOD. MP was shown to enhance Mn-SOD mRNA in bovine GECs and rat glomerular mesangial cells (GMCs) in dose-dependent manners. The transcriptional activation of the Mn-SOD gene by MP was studied using a luciferase reporter gene containing a 1.2 kb fragment (-806 to +406 bp of the transcription initiation site) of rat Mn-SOD genomic DNA. GMCs transfected with the fusion gene construct demonstrated a significant increase (2.6-fold) in luciferase activity when treated with 10 muM MP. Therefore, the induction of Mn-SOD by MP involves transcriptional activation. Results with the fusion gene study predict the existence of a functional glucocorticoid responsive element(s) within the 1.2 kb fragment of the Mn-SOD gene DNA.