Purpose There is an increase in renal nitric oxide (NO) production in lupus nephritis (LN) that appears to be pathogenic, as pharmacologic inhibition of inducible NO synthase (iNOS) abrogates disease in mice. However, the mechanism through which iNOS activity is pathogenic in murine LN is not known. One mechanism may be via increased production of reactive oxygen species (ROS) by iNOS. We investigated the effect of inhibiting iNOS activity in murine LN on ROS production.
Methods Fifteen MRL/lpr mice having active LN were put into three treatment groups. Group one received distilled water (n = 6), group two NG-monomethyl-L-arginine (L-NMMA) (a nonspecific iNOS inhibitor, n = 5) and group three N6-(1-iminoethyl)-L-lysine, dihydrochloride (L-NIL) (a specific iNOS inhibitor, n = 4). Following two weeks of treatment, 24-hour urine sample were collected from each group. Urine samples were analyzed for nitrate + nitrite (NOX) using a chemiluminescence NO analyzer. Urine 8-isoprostane F2α (8-iso PGF2α, a surrogate marker for systemic ROS production) was analyzed by isotopic dilution, immunoaffinity extraction and gas chromatography-mass spectrometry-negative ion chemical ionization. Results were reported as mean ± standard error. A one-way ANOVA was used to determine the effect of drug on 8-iso PGF2α excretion.
Results The iNOS inhibitors significantly reduced urine NOX levels (control 10.4 ± 2.5, L-NMMA 1.3 ± 0.5 L-NIL 1.0 ± 0.4 μmoles/mouse/day respectively: p < .01 in all). INOS inhibitors also significantly reduced isoprostane levels (control 10.4 ± 0.1, L-NMMA 0.1 ± 0.05, L-NIL 0.1 ± 0.04 ng/mouse/day, p > .05 for all treatments vs. control). With iNOS inhibitor therapy, isoprostane levels were reduced in association with reductions in NOX levels (r = .85, p < .05).
Conclusion These findings are consistent with a majority of ROS production in this murine model of LN occurring via iNOS activation. Future studies will compare the effect of iNOS inhibitors to that of potent antioxidants in reducing the severity of LN in murine models.