The Effect Of Active Vitamin D On The Expression Of Nestin In High Glucose-induced Mouse Podocytes

Objectives: 1. To investigate the effect on the expression of nestin in mouse podocytes induced by the gradient glucose concentrations and different time points. 2. To investigate whether active vitamin D(1,25(OH)2D3, calcitriol) ameliorating mouse podocytes impairment is associated with regulation of the expression of nestin in high glucose-induced mouse podocytes, in order to provide the theoretical basis for the treatment of diabetic kidney disease.Methods:(1). In this study, we cultured immortalized mouse podocytes in vitro, the logarithmic growth phase podocytes were divided into experiment group(glucose concentration gradient 15,25,35mmol/l groups) 、normal glucose group(glucose 5mmol/l group) and mannitol contrast group(mannitol 30mmol/l+glucose 5mmol/l group) were further divided in to 24,48,72 h group depend on the induce time. The vitality of podocytes was detected by Cell Counting Kit-8(CCK-8) technique.(2). Detected the expression of nestin in the differentiated podocytes by Immunocytochemistry;(3). The differentiated podocytes were divided into different glucose concentration groups and different time groups. The protein and mRNA expression levels of nestin were detected by western blot and RT-PCR.(4). The differentiated podocytes were divided into 6 groups: NG group(normal glucose), HG group(high glucose), MN group(mannitol+normal glucose), intervention groups(high glucose+vitamin D 10-7, 10-8, 10-9mol/l groups). The protein and mRNA expression levels of nestin were detected by western blot and RT-PCR.Results:(1). Podocytes were maintained in an undifferentiated state at the permissive temperature(33℃) in RPMI 1640 media supplemented with 10% FBS and 10U/ml γ- interferon in complete medium. Podocytes were gradually differentiated and matured in growth-restricted conditions(37℃) without γ- interferon.(2). As the glucose concentration increased, the viability of podocytes was decreased(p<0.05);(3). According to immunocytochemistry, we knew the expression of nestin in cytoplasm of MPC.(4). With the increased of concentration and time, the results of Western Blot and RT-PCR were shown that the protein level and the mRNA level of nestin were decreased(p<0.05).(5). The results of Western Blot and RT-PCR were shown that the HG + active vitamin D groups were significantly higher than HG group of the protein level and the mRNA level of nestin(p<0.05).Conclusion:(1). The viability of podocytes is decreased in High glucose.(2). The expression of nestin was decreased in a concentration and time dependent manner in HG-induced MPC.(3). Active vitamin D could significantly up-regulate the expressions of nestin mRNA and protein which might protect podocyte in HG-induced MPC.