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 oxid e (NO) after stimulation with cytokines or lipopolysaccharide (LPS). A s the role of NO within the glomerulus may be important in conditions such as glomerulonephritis, we have studied the effect of dexamethason e (DX) and pirrolidine dithiocarbamate (PDTC), an inhibitor of the nuc lear transcription factor, NF-kappa B activation on the induced synthe sis of NO in rat mesangial cells (RMC). LPS, tumor necrosis factor-alp ha (TNF-alpha) and the combination of both were able to induce NO synt hesis in a dose-dependent manner as measured with the determination of NO2- levels. Treatment with LPS (10 mu g/ml) + TNF-alpha (100 ng/ml) for eight hours was the most potent stimulus for iNOS activity. DX (1 mu M) 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 bioa ssay, whereas cGMP was measured as a reflection of bioactive NO. DX in hibited induced NO synthesis when RMC were exposed to this agent befor e (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 hour s 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 mu M) was able to in hibit the iNOS activity induced by LPS and TNF-alpha independently (56 .8 and 49.9% inhibition, respectively), and in combination (79.1% inhi bition). PDTC (1 to 100 mu M) inhibited LPS + TNF-alpha-induced NO syn thesis 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 di fferent combinations of inductive stimuli for NO synthesis were employ ed. Our results establish DX and PDTC as useful tools to study the reg ulation of NO synthesis in the mesangial cell and glomerulus, and sugg est that NF-kappa B is involved in the transcriptional regulation of i NOS RMC.