Mechanism of glucose-mediated induction of endothelial nitric oxide synthase expression in rat glomerular mesangial cells

AIM: To explore the high glucose-induced changes of eNOS expression and NO generation in rat glomerular mesangial cells (GMCs) and the mechanism of glucose-mediated eNOS expression in GMCs. METHODS: Rat glomerular mesangial cells were cultured in RPMI1640 medium containing 1000 mg/L (5.5 mmol/L) glucose unless otherwise specified and supplemented with 14% fetal bovine serum and penicillin (50 U/ml)/streptomycin (50 mg/L) at 37℃ in a humidified 5% CO2 atmosphere incubator. They were harvested with trypsin (0.05%)-EDTA (0.02%) when culture reached confluence (usually 72 h culture following passage). NO production was measured using the QM-6 fluorometer. mRNA and protein levels of eNOS were analyzed by RT-real time-PCR method and immunoblot analysis, respectively. RESULTS: Steady-state mRNA and protein levels of eNOS increased significantly in GMCs cultured in high concentrations of glucose (11 and 30mmol/L, 72h) compared with glucose (5.5mmol/L, 72h) (P<0.05). The parallel increases in eNOS mRNA and protein by high glucose (11 mmol/L) were cells grown in control concentration of glucose (5.5 mmol/L, 72 h) (P<0.05). The parallel increases in eNOS mRNA and protein by high glucose (11 mmol/L) were time dependent and at least 24 h treatment was required for the induction of eNOS mRNA and protein. Cyclohexamide, an inhibitor of protein translation, inhibited high glucose (11 mmol/L)-induced eNOS protein expression and high glucose did not affect the stability of eNOS mRNA. CONCLUSION: High glucose enhances the expression of eNOS protein and NO generation in GMCs. Upregulation of eNOS protein by high glucose requires novel protein synthesis and the expression of eNOS protein under high glucose is regulated through protein translation. The enhanced eNOS expression and NO generation in GMCs under hyperglycemic condition may be responsible for the glomerular hyperfiltration in diabetes.