Tetrahydrobiopterin reverses the inhibition of nitric oxide by high glucose in cultured murine mesangial cells

Alterations of intrarenal nitric oxide (NO) synthesis play an important role in the pathogenesis and progression of diabetic nephropathy. We tested the hypothesis that hyperglycemia modulates intrarenal NO synthesis, which might mediate the mesangial cell proliferation and matrix production. Murine mesangial cells were grown in media containing varying glucose concentrations, and cytokine-induced NO synthesis was assayed by chemiluminescence using an NO analyzer. High media glucose (25 mM) inhibited NO synthesis in a time-dependent fashion. This inhibition was posttranslational as revealed by analysis of inducible nitric oxide synthase (iNOS) gene and protein expression. L-Arginine supplementation partially reversed the inhibition whereas addition of tetrahydrobiopterin (BH4), a cofactor for NOS, restored the inducibility of NO synthesis. The in vitro [H-3]citrulline assay for iNOS activity indicated that high glucose decreased BH4 availability whereas examination of the BH4 synthetic pathway suggested decreased BH4 stability rather than synthesis, a defect that was corrected by ascorbic acid. We conclude that hyperglycemia inhibits NO synthesis in mesangial cells by a posttranslational defect that might involve the stability and hence availability of BH4.