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Updated: Jul 30, 2026
Phenotypic Characterization of Macrophages from Rat Kidney by Flow Cytometry
Published on: October 18, 2016
Cytotoxic effect of autocrine and macrophage-derived nitric oxide on cultured rat mesangial cells
Abstract:
Expression of the inducible form of nitric oxide synthase (iNOS) has been found to be up-regulated in cytokine-stimulated mesangial cells (MC) and in experimental glomerulonephritis. Since direct toxicity of nitric oxide (NO) has been implicated in damage of bacteria, neoplastic and intact pancreatic cells, we investigated whether NO is cytotoxic to cultured MC, which may be relevant to pathogenesis of glomerular injury. MC isolated from rat glomeruli generated substantial amounts of nitrite, the stable NO end-product, when cells were stimulated with IL-1beta and tumour necrosis factor-alpha (TNF-alpha). Total DNA synthesis was significantly reduced in the presence of IL-1beta and TNF-alpha, and this effect was completely reversed by N(G)-monomethyl-L-arginine (L-NMMA), an inhibitor of iNOS. Stimulation of MC with IL-1beta 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 prominent MC lysis (3H-thymidine release, specific cytolysis 11.5 +/- 5.3%). Toxic effects of cytokines were fully reversible by the iNOS inhibitor. Lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma), but not IL-1beta and TNF-alpha, induced rat peritoneal macrophages to produce large amounts of nitrite. In co-culture, such prestimulated macrophages had significantly cytotoxic (MTT test 62.9 +/- 19.9%) and cytolytic (3H-thymidine release 57.9 +/- 13.8%) effects on MC. Again, this toxicity was totally inhibited in the presence of L-NMMA. We conclude from these results that cytokine-stimulated generation of NO by MC or macrophages is directly toxic to MC, and may play a role in pathogenesis of glomerular injury involving mesangiolysis.
Insights
Nitric oxide (NO) produced by mesangial cells (MC) and macrophages is toxic to MC, contributing to glomerular injury. Inhibiting inducible nitric oxide synthase (iNOS) reverses these damaging effects.
Area of Science:
- Nephrology
- Immunology
- Cell Biology
Background:
- Inducible nitric oxide synthase (iNOS) expression is elevated in mesangial cells (MC) and experimental glomerulonephritis.
- Nitric oxide (NO) is known to be cytotoxic to various cell types.
Purpose of the Study:
- To investigate the cytotoxic effects of NO on cultured MC.
- To determine the role of NO in the pathogenesis of glomerular injury.
Main Methods:
- MC were isolated from rat glomeruli and stimulated with cytokines (IL-1beta, TNF-alpha).
- Nitrite production (NO end-product) was measured.
- Cell viability was assessed using MTT assay and 3H-thymidine release.
- Effects of N(G)-monomethyl-L-arginine (L-NMMA), an iNOS inhibitor, were evaluated.
- Co-culture experiments with activated macrophages were performed.
Main Results:
- Cytokine-stimulated MC produced nitrite and exhibited significant cytotoxicity and cytolysis, which were reversed by L-NMMA.
- Activated macrophages, stimulated by LPS and IFN-gamma, were cytotoxic and cytolytic to MC in co-culture, an effect inhibited by L-NMMA.
- NO generation by MC and macrophages is directly toxic to MC.
Conclusions:
- Cytokine-induced NO production by MC and macrophages is directly toxic to MC.
- This NO-mediated toxicity may contribute to the pathogenesis of glomerular injury, including mesangiolysis.
