Inducible nitric oxide synthase induction in Thy 1 glomerulonephritis is complement and reactive oxygen species dependent

Thy 1 glomerulonephritis (GN) is a rat model of complement-dependent immune mesangial injury with induced glomerular nitric oxide (NO) synthesis. To e xamine mechanisms of inducible nitric oxide synthase (iNOS) induction, we s tudied the effects of treatment with the antioxidant N-acetyl-cysteine (NAC ) and soluble complement receptor 1 (sCR1). Thy 1 GN was induced by intrave nous anti-Thy 1 antibody. Glomeruli were isolated and kidney tissue taken f rom 30 min to 24 h after induction. Nitrite (NO2-) synthesis, luminol chemi luminescence for reactive oxygen species (ROS), and iNOS and cytokine mRNA were assayed in isolated glomeruli. Mesangial injury (mesangiolysis) and le ucocyte infiltration were quantitated on tissue sections. NAC (i.p. 1,000 m g/kg, 1 h prior to anti-Thy 1) reduced glomerular NO2- synthesis (3.5 +/- 0 .66 vs. untreated 8.2 +/- 1.1, p = 0.02), and iNOS mRNA expression, and abo lished enhanced chemiluminescence. In vitro incubation of nephritic glomeru li with 20 mM NAC also suppressed nitrite production (4.7 +/- 0.8 vs, untre ated 12.2 +/- 0.7 nmol NO2-/2,000 glomeruli/48 h, p = 0.003), and chemilumi nescence. In NAC-treated animals, neutrophil infiltration (0.5 +/- 0 vs, un treated 9.6 +/- 1.6 glomerulus, p = 0.0005), and macrophage infiltration (1 .7 +/- 0.4 vs. untreated 12.0 +/- 0.1, p = 0.006) were abolished, and mesan giolysis was significantly reduced (45.9 +/- 1.3 vs. untreated 34.4 +/- 2.1 cells/glomerulus, p = 0.009). NAC did not inhibit anti-Thy 1 antibody depo sition. C1q was unaffected, but C3 was reduced, sCR1 treatment prevented iN OS mRNA induction, the enhanced chemiluminescence, and the neutrophil infil tration at 1 h. IL-1 beta and TNF alpha mRNAs were not affected by either N AC or sCR1. These results show that NAC inhibits iNOS induction and NO synt hesis in this model, and suppresses ROS synthesis and injury. They suggest that complement-dependent ROS generation is the critical initiating event t hat follows fixation of anti-Thy 1 antibody.