High Glucose And High Homocysteine Can Induce Podocyte Apoptosis And Down Regulation Of Podocin MRNA Expression Through The Activation Of PKC

Objective To explore the effect of the high glucose、high homocysteineonapoptosis and the expression of PKC、podocin in mouse podocyte，and to analysis theeffect of PKC、podocin in diabetic nephropathy（DN）.Methods：1.Cultivate conditions immortalization mice podocyte cell lines, takeundifferentiated cell with RPMI1640containing10%FBS,10U/ml mice IFN-γ,100U/ml of penicillin and100U/ml of streptomycin,use5%、330C、CO2incubator topassage. After passage induce podocyte differentiate in2weeks with37℃、non-IFN-γ culture. Observe cell morphous of undifferentiated and already differentiatedunder invertion microscope.2.Put the mature podocytes in1640without FBS to synchronous culture for24hours，The podocytes were randomly divided into①control group: normal controlgroup （5mmol/L glucose）, hypertonic control group （5mmol/L glucose+20mmol/Lmannitol）; injured group: high Hcy group （1mmol/L Hcy）, high glucose group（25mmol/L glucose）, combination group （1mmol/L Hcy+25mmol/L glucose）; PKCgroup: high Hcy+chelerythrine (10^-5/L), high glucose+chelerythrine(10^-5/L), high Hcy+high+glucose+chelerythrine (10^-5/L); the cells wereincubated for24,48, to72h, to observe the growth state of cells by invertedmicroscope; cell apoptosis was measured by flow cytometry at24,48and72hour, theactivity of PKC was demonstrated by ELISA and the mRNA expression of podocinwas examined by RT–PCR. Results：1. Compared to normal group，the apoptosis rate, PKC activity and expression ofpodocin mRNA of hypertonic control group showed no significant differences.;72h,the cell apoptosis of three groups was more obvious than48h （P <0.05）, apoptosisrate was （20.27±1.80）%,（21.49±1.20）%,（27.77±1.81）%; the high Hcy group,high glucose group and mixed group of cells incubated with24h, PKC activity washigher than the normal group （P <0.05）, expression of Podocin mRNA decreasedcompared with normal control group （P <0.05）, mixed group was more obvious（P <0.01）, with the passage of time,48,72h, the differences were more obvious （P <0.05）.2. There was no significant difference between the inhibitor group to it’scorresponding group when podocytes were incubated with24h, at48h, cell injurygroup （P>0.05）, apoptosis rate of high Hcy, high glucose, mixed group were （10.10±1.57）%,（10.33±1.98）%,（16.99±2.44）%compared with the injury group wasdecreased, the difference was statistically significant （P <0.05）,there was samedifference at72h;24h, the expression of podocin mRNA than corresponding injurygroup increased （P <0.05）, there was same difference at72h.Conclusion:1. High Hcy、high glucose can induce mouse podocyte apoptosis in atime-dependent manner, the two have superposition effect.2. High Hcy、high glucose can down-regulate the expression of mRNA in atime-dependent manner, the two have superposition effect.3. High Hcy and high glucose on podocyte apoptosis and down-regulation ofpodocin mRNAexpression through the activation of PKC.