Inducible nitric oxide synthase inhibitors reduce urinary markers of systemic oxidant stress in murine proliferative lupus nephritis

Abstract

Background: Proliferative lupus nephritis (PLN) is characterized by increased expression of inducible nitric oxide (NO) synthase (iNOS). Inhibition of iNOS with NG-monomethyl L-arginine (L-NMMA) abrogates renal disease in two models of murine PLN, but the mechanism of this effect is unknown. Reactive oxygen species have both direct and indirect pathogenic effects in inflammatory lesions and are therefore potentially an important therapeutic target in PLN. We hypothesized that inhibition of iNOS activity would reduce ROS production in murine PLN.

Methods: A dose escalation of L-NMMA (0, 20, 100, and 500 mg/kg/day) was performed in New Zealand Black x New Zealand White F1 (NZB/W) mice with active renal disease. Twenty-four-hour urine nitrate + nitrite (NOX) was measured with a chemiluminescence NO analyzer. Twenty-four-hour urine 8-isoprostane F2alpha (8-iso-PGF2alpha) was measured by gas chromatography-negative ion chemical ionization mass spectrometry. MRL-MpJFASlpr (MRL/lpr) and NZB/W mice were divided into three groups and given either L-NMMA, L-N6-iminoethyl-lysine (L-NIL), or distilled water for 2 weeks. Urine NOX and 8-iso-PGF2alpha were determined after 2 weeks.

Results: L-NMMA reduced both urine NOX and 8-iso-PGF2alpha levels in a dose-dependent fashion in NZB/W and MRL/lpr mice. Urine NOX and 8-iso-PGF2alpha levels were highly correlated. Both specific (L-NIL) and nonspecific (L-NMMA) iNOS inhibition reduced urine NOX and 8-iso-PGF2alpha levels in both models of murine PLN.

Conclusion: These findings suggest that iNOS activity is a major source of reactive oxidant stress in these models of murine PLN. Future studies will address the pathogenic role of reactive oxygen stress in PLN.

FIGURE 1

Effect of increasing doses of N^G-monomethyl l-arginine (l-NMMA) on urine nitrate + nitrite (NO_x) and 8-isoprostane F_2α (8-iso-PGF_2α) levels in New Zealand Black × New Zealand White (NZB/W) mice. NZB/W mice with active disease (n = 4) were given increasing doses of l-NMMA (an inducible nitric oxide synthase inhibitor; 0, 50, 100, and 500 mg/kg/d). After 4 days at each dose, urine was collected and analyzed for NO_x (a surrogate marker of systemic NO production) and 8-iso-PGF_2α (a surrogate marker for systemic oxidant stress). l-NMMA reduced both NO_x and 8-iso-PGF_2α levels in a parallel, dose-dependent fashion. Urine NO_x and 8-iso-PGF_2α were significantly associated (r = .709, p = .002).

FIGURE 2

Effect of inducible nitric oxide synthase (iNOS) inhibitors on systemic nitrate + nitrite (NO_x) levels and oxidant stress in MRL/lpr mice. Either a specific iNOS inhibitor (l-N6-iminoethyl-lysine [l-NIL], n = 4), a nonspecific iNOS inhibitor (N^G-monomethyl l-arginine [l-NMMA], n = 6), or distilled water (n = 6) was administered for 2 weeks to MRL/lpr mice with active disease. Urine NO_x and 8-isoprostane F_2α (8-iso-PGF_2α) levels were significantly lower in the combined treatment groups when compared with the control group. *p < .05.

FIGURE 3

Effect of inducible nitric oxide synthase (iNOS) inhibitors on systemic nitrate + nitrite (NO_x) levels and oxidant stress in New Zealand Black × New Zealand White (NZB/W) mice. Either a specific iNOS inhibitor (l-N6-iminoethyl-lysine [l-NIL], n = 5), a nonspecific iNOS inhibitor (N^G-monomethyl l-arginine [l-NMMA], n = 9), or distilled water (n = 7) was administered for 2 weeks to NZB/W mice with active disease. Urine NO_x and 8-isoprostane F_2α (8-iso-PGF_2α) levels were significantly lower in the combined treatment groups when compared with the control group. *p < .05.