Downregulation of SPARC expression is mediated by nitric oxide in rat mesangial cells and during endotoxemia in the rat

Nitric oxide (NO) has been implicated in several forms of glomerulonephriti s. In this study, a low stringency reversed transcription/PCR protocol was used to evaluate the action of NO on the mRNA expression pattern in rat mes angial cells (MC). To mimic the state of glomerular inflammation, MC were s timulated by exposure to the cytokines interleukin-1 beta and tumor necrosi s factor-alpha into producing high levels of NO via expression of inducible nitric oxide synthase (NOS). To detect NO-mediated effects, the resulting expression pattern was compared to that of MC stimulated by the cytokines i n the presence of the NOS inhibitor N-G-monomethyl-L-arginine (L-NMMA). Com puter analysis of a differentially expressed cDNA fragment resulted in a 10 0% homology to the recently characterized mRNA of SPARC (secreted protein a cidic and rich in cysteine). Further characterization of SPARC regulation r evealed a cytokine- and cAMP-dependent decrease in SPARC mRNA and protein l evels. Blocking NO formation by L-NMMA reversed the effects of cytokines an d cAMP on SPARC expression, suggesting an NO-mediated mechanism. The NO don ors S-nitroso-N-acetyl-penicillamine and diethylenetriamine/NO further redu ced SPARC expression in cytokine-treated MC as well as in controls. Moreove r, downregulation of SPARC mRNA and protein expression in whole kidneys obt ained from rats treated with endotoxin was observed. This downregulation of SPARC was reversed by treatment with L-N-6-1 (iminoethyl) lysine dihydroch loride, a potent and highly selective inhibitor of inducible NOS. These dat a characterize SPARC as an NO-regulated gene. This observation may be impor tant in the context of tissue remodeling in chronic inflammatory kidney dis eases.