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-FASlpr (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.

Lipopolysaccharide induces inducible nitric oxide synthase-dependent podocyte dysfunction via a hypoxia-inducible factor 1α and cell division control protein 42 and Ras-related C3 botulinum toxin substrate 1 pathway

“…On the other hand, mice given the inhibitor did not develop proteinuria throughout the study [9], suggesting a protective role of NOS inhibition in preventing podocyte injury or dysfunction. This protective effect of NOS inhibition was also supported by a study utilizing N 6 -(1-iminoethyl)- l -lysine, dihydrochloride (L-NIL; a partial, selective inhibitor of iNOS), although the dose used was not selective for inhibition of iNOS [7]. In this study, proteinuria was absent in LNIL-treated MRL/lpr mice lacking the NOS2 gene.…”

“…Several studies showed a strong link between inducible nitric oxide synthase (iNOS) expression and development and severity of the disease phenotype in several murine models of lupus nephritis [6,7]. Both MRL/MpJ-Fas lpr /J (MRL/lpr) and (New Zealand Black × New Zealand White) F1 mice develop spontaneous proliferative lupus nephritis.…”

The biology of reactive intermediates in systemic lupus erythematosus

“…NOS2−/− mice had elevated anti-dsDNA antibody levels and had, as observed in the past, no significant reductions in glomerular pathology or proteinuria. However, iNOS inhibitor therapy significantly reduced proteinuria and podocyte flattening/eNOS cell swelling by electron microscopy [51]. This suggests that iNOS inhibitor therapy reduces pathologic changes in podocyte and endothelial cell pathology in this model in an iNOS-independent (and possibly eNOS-dependent) fashion.…”

NADPH oxidase and nitric oxide synthase-dependent superoxide production is increased in proliferative lupus nephritis

“…However, the importance of increased inducible nitric oxide synthase (iNOS)-mediated nitric oxide (NO) production in LN was questioned when genetic deletion of functional iNOS in lupus mouse models failed to reduce the onset of classic pathologic features of LN except vasculitis (6, 7). However, ROS production in this murine model of lupus nephritis was significantly reduced by inhibitors of iNOS (8), suggesting that driving pathogenic consequence of iNOS expression in LN is the production of SO rather than the production of NO.…”