Regulation of inducible nitric oxide synthase expression in rat mesangial cells and isolated glomeruli

The presence of the inducible isoform of nitric oxide synthase (iNOS) in glomerular mesangial cells facilitates the synthesis of nitric oxide (NO) after stimulation with cytokines or lipopolysaccharide (LPS). As the role of NO within the glomerulus may be important in conditions such as glomerulonephritis, we have studied the effect of dexamethasone (DX) and pirrolidine dithiocarbamate (PDTC), an inhibitor of the nuclear transcription factor, NF-kappa B activation on the induced synthesis of NO in rat mesangial cells (RMC). LPS, tumor necrosis factor-alpha (TNF-alpha) and the combination of both were able to induce NO synthesis in a dose-dependent manner as measured with the determination of NO2- levels. Treatment with LPS (10 micrograms/ml) + TNF-alpha (100 ng/ml) for eight hours was the most potent stimulus for iNOS activity. DX (1 microM) had an inhibitory effect on LPS-, TNF-alpha- and LPS + TNF-alpha-induced NO synthesis (51.2, 42.5 and 68% of inhibition, respectively). The inhibitory effect of DX was confirmed using a reporter cell bioassay, whereas cGMP was measured as a reflection of bioactive NO. DX inhibited induced NO synthesis when RMC were exposed to this agent before (16 hr of pretreatment, 75.7% inhibition) or at the same time (8 hr of cotreatment, 61.2% inhibition) as TNF-alpha + LPS but not four hours after the stimuli. Northern blot analysis showed marked blunting of mRNA expression in RMC treated with DX, in concordance with functional studies. Both actinomycin D and cycloheximide significantly inhibited NO synthesis and iNOS mRNA expression. PDTC (100 microM) was able to inhibit the iNOS activity induced by LPS and TNF-alpha independently (56.8 and 49.9% inhibition, respectively), and in combination (79.1% inhibition). PDTC (1 to 100 microM) inhibited LPS + TNF-alpha-induced NO synthesis and iNOS mRNA expression in a concentration-dependent fashion (69 to 86% inhibition of NO synthesis and 50 to 100% inhibition of mRNA expression). Addition of PDTC four hours after exposure to TNF-alpha + LPS was still able to markedly inhibit NO synthesis. The effects of DX and PDTC were also demonstrated in isolated glomeruli, where two different combinations of inductive stimuli for NO synthesis were employed. Our results establish DX and PDTC as useful tools to study the regulation of NO synthesis in the mesangial cell and glomerulus, and suggest that NF-kappa B is involved in the transcriptional regulation of iNOS in RMC.