Inducible nitric oxide synthase inhibitor SD-3651 reduces proteinuria in MRL/lpr mice deficient in the NOS2 gene

Abstract

Several studies have demonstrated the effectiveness of arginine analog nitric oxide synthase (NOS) inhibitor therapy in preventing and treating murine lupus nephritis. However, MRL/MpJ-FAS (MRL/lpr) mice lacking a functional NOS2 (inducible NOS [iNOS]) gene (NOS2) develop proliferative glomerulonephritis in a fashion similar to their wild-type (wt) littermates. This finding suggests that the effect of arginine analog NOS inhibitors is through a non-iNOS-mediated mechanism. This study was designed to address this hypothesis.NOS2 mice were given either vehicle or a NOS inhibitor (SD-3651) to determine if pharmacological NOS inhibition prevented glomerulonephritis, using wt mice as positive controls. Urine was collected fortnightly to measure albumin. At the time of full disease expression in wt mice, all mice were killed, and renal tissue was examined for light, immunofluorescence, and electron microscopic evidence of disease. Serum was analyzed for anti-double-stranded DNA antibody production.NOS2 mice had higher serum anti-double-stranded DNA antibody antibody levels than those of wt mice. SD-3651 therapy reduced proteinuria, glomerular immunoglobulin G deposition, and electron microscopic evidence of podocytopathy and endothelial cell swelling without affecting proliferative lesions by light microscopy.These studies confirm that genetic iNOS deficiency alone is insufficient to prevent proliferative glomerulonephritis and suggest that iNOS activity may inhibit autoantibody production. These results also suggest that SD-3651 therapy acts via a non-iNOS-mediated mechanism to prevent endothelial cell and podocyte pathology. Studies that elucidate this mechanism could provide a useful drug target for the treatment of nephritis.

FIGURE 1

Urine albumin with nitric oxide synthase (NOS)2 knockout and SD-3651 therapy over time. Urine albumin (milligram per mouse per day) was determined every 2 weeks from 24-hour urine collections of individual mice. The x axis depicts mouse age in days, whereas the y axis depicts the average level of proteinuria for each group at each time point. Urine albumin levels were significantly less in the older NOS2^−/− SD-3651–treated mice when compared with those of the NOS2^+/+ and the NOS2^−/− mice with active disease of the same age. *P < 0.05 versus NOS2^+/+ and NOS2^−/−.

FIGURE 2

Effect of genetic and pharmacological manipulation of inducible nitric oxide synthase (iNOS) on renal pathology. (A) Kidney sections from mice in each group were stained and scored for features of lupus nephritis (0–60). The x axis depicts the genotype and treatment group, whereas the y axis depicts the total score for each group. There were no significant differences in histological scores (P > 0.05). (B) Immunofluorescence scores for glomerular IgG and C3 deposition by group. Frozen kidney sections obtained at the time of killing (24/25 weeks) were immunostained for IgG and C3 and scored for the intensity and extent of coverage of fluorescence on a qualitative scale (0–3). The x axis depicts the genotype and treatment group, whereas the y axis depicts the qualitative score. The SD-3651–treated mice had significantly lower scores than those of the wt mice. *P < 0.05 for NOS2^+/+ versus NOS2^−/− SD-3651–treated mice.

FIGURE 3

Anti-dsDNA antibody titration curves for nitric oxide synthase (NOS)2^+/+, NOS2^−/−, and NOS2^−/− SD-3651–treated mice. Serum dsDNA antibody levels were determined at the time of killing (24/25 weeks). The x axis depicts the dilution of the serum before the analysis assay, and the y axis depicts the optical density at each dilution. The levels were significantly higher in the NOS2^−/− compared with those of the NOS2^+/+ mice and for the NSO2^−/− SD-3651–treated mice at lower dilutions (1:100 and 1:200). *P < 0.05 versus NOS2^+/+.

FIGURE 4

Electron micrograph (EM) scoring of endothelial cell swelling and podocyte effacement. Fixed kidney sections from a subset of mice (n = 3) were examined by EM and scored (0–3) by a renal pathologist for the extent of endothelial cell swelling and podocyte effacement. Both features were less in the SD-3651–treated mice, but differences were statistically significant only for endothelial cell swelling. P < 0.05 versus the remainder of the groups. NOS, nitric oxide synthase.

FIGURE 5

Representative immunoglobin G (IgG) immunofluorescence and electron microscopy. Representative IgG immunofluorescence micrographs of glomeruli from wild-type (A) and SD-3651–treated nitric oxide synthase (NOS)2^−/− mice (B) as described in Figure 2B. Representative electron micrographs of glomeruli from vehicle-treated NOS2^−/− (C) and SD-3651–treated NOS2^−/− mice (D) as described in Figure 4.