Mechanism Of Glucose-mediated Induction Of Endothelial Nitric Oxide Synthase Expression In Rat Mesangial Cells

Background and Aim:Diabetic nephropathy is one of the major complications of diabetes with 30% of type 1 and 5-30% of type 2 diabetic patients developing diabetic nephropathy and end stage renal disease. The initial phase of diabetic nephropathy is characterized by an increase of glomerular filtration rate (GFR) or glomerular hyperfiltration, which may gradually progress to end stage renal failure. Increased nitric oxide (NO) production in the kidney, especially NO generation from endothelial/neuronal NO synthase (eNOS/nNOS), has been shown to play critical roles in the development of diabetic hyperfiltration. The expression of eNOS as well as NO generation in the kidney endothelial and epithelial cells is regulated by glucose.In the current work, we showed that both eNOS expression and NO generation is induced by high glucose in rat mesangial cells. The mechanism of glucose-mediated eNOS expression in rat mesangial cells was further characterized as well.Methods:Rat mesangial cells (RMC) were cultured in RPMI 1640 medium containing 1,000 mg/l (5.5 mM) glucose unless otherwise specified, supplemented with 14% fetal bovine serum, and penicillin (50 U/ml)/streptomycin (50μg/ml), at 37°C in a humidified 5% CO₂ atmosphere incubator. Mesangial cells were harvested with trypsin (0.05%)-EDTA (0.02%) when reached confluence (usually 72h culture following passage). [Ca²⁺]_i and NO production were simultaneously measured using the multi-dye function of the QM-6 fluorometer. The mRNA and protein levels of eNOS were analysed by RT/Real Time-PCR method and immunoblot analysis, respectively. Sequence of rat eNOS promotor and pDs Red Express-1 Vector were used to construct plasmid pDsRed eNOS promotor. Reagent Lipofectamine RNAiMAX was used to transfect plasmid into RMC.Results:The steady-state mRNA and protein levels of eNOS were increased significantly in RMC cultured in high concentrations of glucose (11 and 30mM, 72h) compared with cells grown in control concentration of glucose (5.5mM, 72h) (p<0.05). The parallel increases in eNOS mRNA and protein by high glucose (11mM) were time dependent and at least 24h treatment was required for the induction of eNOS mRNA and protein. Both cyclohexamide, an inhibitor of protein translation, inhibited high glucose (11mM)-induced eNOS protein expression. 2-deoxyglucose inhibited high glucose (11mM)-induced eNOS protein expression. Phosphoralytion of eNOS and PKC/ERK inhibitor have no effects on eNOS expression. High glucose did not affect the stability of eNOS mRNA. Furthermore a plasmid named pDsRed-eNOS promotor was constructed and transfected into RMC. The expression of eNOS protein was enhanced significantly in transfected RMC cultured in high concentrations of glucose (11 and 30mM, 72h) compared to cells grown in control concentration of glucose (5.5mM, 72h) (p<0.05). Conclusion:High glucose enhanced the expression of eNOS protein and NO generation in RMC. The up-regulation of eNOS protein by high glucose required novel protein synthesis. The expression of eNOS protein under high glucose was regulated through eNOS promoter. The enhanced eNOS expression and NO generation in RMC under hyperglycemic condition might be responsible for glomerular hyperfiltration in diabetes. Background and Aim:Angiogenesis plays an important role in heart coronary disease. The central role of vascular endothelial cell growth factor (VEGF) in vessel growth makes this protein a tantalizing target for either stimulation or inhibition of angiogenesis. The extracts of radix angelicae sinensis (angelica) and chuanxiong rhizoma (chuan-xiong) have been used to cure ischemic heart disease in China. But the angiogenic effects of angelicais and chuan-xiong were currently unknown.Methods:Rats were relegated randomly into either precautionary or therapeutic group. Three rats were fed a regular diet for 2 weeks as normal control. The precautionary group (15 rats) was fed regular diet and the herbal extracts for 2 weeks. After 2 weeks they were led to acute blood-lacking in myocardium for 2 h. The therapeutic group (15 rats) were made myocardial infarction model and then fed regular diet and the herbal extracts for 2 weeks. All rats were sacrificed at the end of their treatments. A portion of the myocardial specimens was fixed in 4 % paraformaldehyde and embedded in paraffin for histological analysis. The rest of the myocardium were snap frozen in liquid nitrogen and stored at -80℃until use for Western blot analysis. Results:The extract of Angelica increased vascular endothelial growth factor (VEGF) protein expression significantly in precautionary and therapeutic groups (p<0.05). The extract of ChuanXiong increased VEGF protein expression significantly in precautionary groups (p<0.05), yet it significantly decreased the expression of VEGF protein in therapeutic groups (p<0.05). Meanwhile, the angiogenic activity of the extracts of Angelica and ChuanXiong was used to quantify vessels on chick embryo chorioallantoic membrane (CAM), to measure cell proliferation of cultured rat cardiac microvascular endothelial cells (CMECs) and human umbilical vein endothelial cells (HUVECs).Conclusion:The extracts of Angelica and ChuanXiong increased the expression of VEGF protein, enhanced cell proliferation (p<0.05) and stimulated revascularity (p<0.05) in CAM, CMECs and HUVECs. Our data might provide some insight into the mechanism for the treatment of myocardial infaction and peripheral ischemia.