The Role Of COP1 In Apoptosis Of Glomerular Podocytes With High Glucose-Induced

Objective To investigate the effect of COP1 on apoptosis in podocytes by high glucose exposure in vitro and the mechanism.Methods Podocytes were incubated in experimental medium. Podocytes were stimulated with 15, 30, 45mmol/L glucose for 12, 24, 48h, respectively. Then apoptosis index and mortality index were detected by Flow Cytometry. Then podocytes were divided into 3 groups: the control group, the high glucose group and the mannitol group. Podocytes apoptosis were evaluated by Flow Cytometry and TUNEL.The recombinant plasmid pEGFP-COP1 and pEGFP-C1 were digested by two restrict endonuclease respectively. Then the pEGFP-COP1 was transiently transfected into podocytes. The rate of the podocytes transfected with the recombinant plasmid were evaluated by FCM, and the expression of COP1 gene were detected by RT-PCR,Western blot and florescence observation.Podocytes were stimulated with 30mmol/L glucose for 48h. Cells were divided into control group, podocytes+mannitol group, podocytes+ glucose group, pEGFP-COP1+glucose group, pEGFP-N1+glucose group, respectively. Podocytes apoptosis were evaluated by Flow Cytometry and TUNEL. The expression of COP1 were detected by RT-PCR and Western blot.Results The apoptosis of podocytes induced by glucose in a dose and time way. Podocytes were stimulated for 48h with 30mmol/L glucose, the expression of COP1 mRNA and protein were decreased, accompanicied with increased apoptosis in podocytes(P<0.05). There was no significant difference between the control group and the mannitol group(P>0.05).The accurate eukaryotic recombinant plasmid pEGFP-COP1 was identified by restrict endonuclease. Green fluorescent signal in podocytes was detected. The rate of the podocytes transfected with the recombinant plasmid was 30%-40% by FCM. The COP1 gene expression was significantly higher in pEGFP-COP1 transfected cells than in the control group by RT-PCR and Western blot(P<0.05).Incubating pEGFP-COP1 transfected podocytes with 30mmol/L gluose for 48h, the apoptosis was remarkably reduced than the glucose group(P<0.05),it was no different with the control group and the mannitol group(P>0.05). The apoptosis of the pEGFP-N1 group was no different with the glucose group(P>0.05),it was remarkably increased than the control group and the mannitol group(P<0.05). The expression of COP1 mRNA in the pEGFP-COP1 group was remarkably increased than the glucose group(P<0.05),it was no different with the control group and the mannitol group(P>0.05). The expression of COP1 mRNA in the pEGFP-N1 group was no different with the glucose group(P>0.05),it was remarkably reduced than the control group and the mannitol group(P<0.05). The expression of COP1 protein was in agreement with COP1 mRNA.Conclusion Glucose can increase the apoptosis of the glomerular podocytes and reduce the expression of COP1. Over expression of COP1 can reduce the apoptosis of the glomerular podocytes induced by glucose. These results suggest that the increased expression of COP1 plays an important role in the mechanisms of high glucose induced podocyte apoptosis.