CYTOTOXIC EFFECT OF AUTOCRINE AND MACROPHAGE-DERIVED NITRIC-OXIDE ON CULTURED RAT MESANGIAL CELLS

Expression of the inducible form of nitric oxide synthase (iNOS) has b een found to be up-regulated in cytokine-stimulated mesangial cells (M C) and in experimental glomerulonephritis. Since direct toxicity of ni tric oxide (NO) has been implicated in damage of bacteria, neoplastic and intact pancreatic cells, we investigated whether NO is cytotoxic t o cultured MC, which may be relevant to pathogenesis of glomerular inj ury. MC isolated from rat glomeruli generated substantial amounts of n itrite, the stable NO end-product, when cells were stimulated with IL- 1 beta and tumour necrosis factor-alpha (TNF-alpha). Total DNA synthes is was significantly reduced in the presence of IL-1 beta and TNF-alph a, and this effect was completely reversed by N-G-monomethyl-L-arginin e (L-NMMA), an inhibitor of iNOS. Stimulation of MC with IL-1 beta and TNF-alpha caused remarkable toxicity to these cells, measured by the MTT test (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide cleavage, specific cytotoxicity 41.5 +/- 20.3%), and much less promin ent MC lysis (H-3-thymidine release, specific cytolysis 11.5 +/- 5.3%) . Toxic effects of cytokines were fully reversible by the iNOS inhibit or. Lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma), but not IL-1 beta and TNF-alpha, induced rat peritoneal macrophages to produc e large amounts of nitrite. In co-culture, such prestimulated macropha ges had significantly cytotoxic (MTT test 62.9 +/- 19.9%) and cytolyti c (H-3-thymidine release 57.9 +/- 13.8%) effects on MC. Again, this to xicity was totally inhibited in the presence of L-NMMA. We conclude fr om these results that cytokine-stimulated generation of NO by MC or ma crophages is directly toxic to MC, and may play a role in pathogenesis of glomerular injury involving mesangiolysis.