Abstract 1487: Genetic Disruption of All Nitric Oxide Synthase Isoforms Causes Accelerated Renal Lesion Formation in Mice in Vivo
The roles of the nitric oxide synthase (NOS) system in renal lesion formation have been investigated with NOS inhibitors. However, because of the non-specificity of the NOS inhibitors, the ultimate roles of the NOS system are still poorly understood. Thus, we addressed this point in mice deficient in all three NOS isoforms (triply n/i/eNOS-KO mice) that we have recently developed (PNAS 2005). Renal injury was induced by unilateral ureteral obstruction (UUO) in 10-week-old male wild-type (WT) and triply-KO mice (n=6 – 8). At days 3, 7, and 14 after UUO, histological changes of the obstructed kidneys were examined by hematoxylin-eosin and Masson-trichrome staining. In the obstructed kidneys of both the WT and the triply-KO mice, significant renal tubular apoptosis and interstitial fibrosis were noted (both P<0.05). However, the extent of renal tubular apoptosis (incidence of apoptosis: 23±2% vs. 11±1%) and the extent of interstitial fibrosis (area of fibrosis: 44±4% vs. 23±3%) were both markedly greater in the triply-KO than in the WT mice (both P<0.05). Furthermore, macrophage infiltration (macrophage counts: 24±5 vs. 12±7/field, F4/80 immunostaining), a marker of inflammation, and levels of TGF-β (53±10 vs. 32±11 ng/g wet tissue, ELISA), an inducer of fibrosis, were also markedly enhanced in the obstructed kidneys of the triply-KO mice as compared with that of the WT mice (both P<0.05). Importantly, activity of angiotensin-converting enzyme was significantly higher in the obstructed kidneys of the triply-KO mice than in that of the WT mice (103±39 vs. 65±22 unit/ml, Kasahara method) (P<0.05), and long-term oral treatment with olmesartan (angiotensin II type 1 [AT1] receptor blocker, 5 mg/kg/day, 14 days) significantly reversed all these renal abnormalities of the triply-KO mice (tubular apoptosis [17±6 to 7±3], interstitial fibrosis [33±6 to 21±3], macrophage infiltration [23±5 to 13±3], and TGF-β levels [53±10 to 23±5]) (all P<0.05). These results provide the first evidence that genetic disruption of all NOSs causes accelerated inflammatory renal lesion formation in mice in vivo through the AT1 receptor pathway, demonstrating the critical role of the endogenous NOS system in preventing renal remodeling.