Effect Of High Glucose High Ang-Ⅱ On PKC Activity Expression Of Nephrin And CD2AP In Podocyte Of Mice

Objective1.To cultivate mice podocyte with high glucose and high Ang-Ⅱ, observe themorphocytology of podocyte.2.Observe changes of PKC activation with injuriescultivations.3.Detect the expression of nephrin and CD2AP in podocyte,measure theapoptosis of podocyte with different cultivations in different time.Methods Cultivate conditions immortalization mice podocyte cell lines, takeundifferentiated cell with RPMI1640containing10%FBS,10U/ml mice IFN-γ,100U/mlof penicillin and100U/ml of streptomycin,use5%330C CO₂incubator to passage. Afterpassage induce podocyte differentiate in2weeks with37non-IFN-γ culture. Observecell morphous of undifferentiated and already differentiated under invertion microscope.Plante cells in6pore plate by2104/cm2.After24h randomly divided into five groups,5mmol/L glucose,25mmol/L high glucose,10-6mol/L Ang-sole and joint high glucose,mannitol.After72hours observe cell morphology change, detect cell apoptosis rate usingflow cytometer;and detect PKC activity （ELISA） in24hours,48hours, detecte nephrin,CD2AP mRNA expression with RT-PCR.Results When high glucose, high Ang-role24hours, PKC is activated clearly（P<0.05）,both damage factors to activate more obvious degree（P<0.01）, and in a time dependence.High glucose, high Ang-respectively cut down nephrin and CD2AP expression,compared with controls significantly, and nephrin expression is reduced in early24hours（P<0.05）, the joint action of two damage factors are significant thanseparated（P<0.01）.48hours after apoptosis rate rise compared with the controlpresent（P<0.05）, and in a time dependence, damage factors stacked rose apoptosis rate moreobvious（P<0.01）.Compared with Ang-Ⅱ group,Valsartan group PKC activity was significantly reduced （P<0.05） in48hours; nephrin mRNA expression of Valsartan group were higher than those ofAng-II group （P <0.05） in48,72hours,carrying CD2AP mRNA level in no significantchange in24hours; the apoptosis rate of valsartan and Ang-II group were not statisticallydifferent; and elevated compared with control group （P <0.05）.Conclusion1. High glucose, Ang-can activate podocyte PKC activity, and may inducecell apoptosis through PKC pathways.2. Under the action of high glucose the podocytelnephrin expression is reduced, Ang-either as damage factors alone cut podocyte nephrin expression, but also as a superposition in high sugar nephrin aggravating factors.3. Thechanged expression of CD2AP also participate in the process of cell damage, but have nodirect correlation with nephrin expression changes in the degree and the speed.4. Highsugar, Ang-Ⅱ can induce podocyte apoptosis in vitro, and apoptosis is in a timedependence pattern.5.2"10^-5mol/L valsartan can intervent the activation of PKCpathway couse of Ang-II-induced in vitro mouse podocytes.6. Valsartan can upregulatenephrin,CD2AP mRNA expression level couse of Ang-II-induced in vitro murinepodocytes, but it can not completely antagonistic the apoptosis of podocytes in mice couseof Ang-Ⅱ-induced.