The Regulation Of Glucose-6-phosphate Dehydrogenase By The Ubiquitin Proteasome Pathwaymediates The Damage Of Podocyte In Diabetic Nephropathy

PART 1 High glucose decreases the expression of G6PD in podocyte through the ubiquitin proteasome pathwayObjective:To observe the effects of high glucose and high palmitic acid on the proliferation, apoptosis, cytoskeleton and redox of potocytes.Whether overexpression of G6PD can improve these effects. And to observe the ubiquitination of glucose-6-phosphate dehydrogenase (G6PD).Methods:MTT was used to detect the effects of the high glucoseon podocytes proliferation; detection the expression of cleaved caspase-3 underhighpalmitic acid and high glucose; detection the vivo oxidation reduction balance of potocytes cultured under high glucose; using phalloidin ring peptide fluorescence staining to detectF-actin arrangement of potocytes; using plasmid technology to build pCDNA3.0-Flag-G6PD and pRK5-His-Ub plasmid.. Podocytes in accordance with the purpose of the experiment were divided into different groups:normal glucose (NG,5.6 mmol/L) and high concentration glucose (HG,25 mmol/L) and high concentration glucose plus overexpression of G6PD adenovirus (HG/Ad2) and high concentration glucose aplusempty adenovirus (HG/Laz); BSA,500 u mol/1 palmitic acid,500 u mol/L palmitic acid plusoverexpression of G6PD adenovirus,500 u mol/L palmitic acid and empty adenovirus expression. HEK293T cells according to the experimental group were divided into differenr groups:His-Ub plasmid transfection group; His-Ub and Flag-G6PD plasmid cotransfection group; His-Ub plasmid transfection +MG132 (20uM) group; His-Ub and Flag-G6PD plasmid cotransfection plus MG132 (20uM) group. After 48 hours plasmid were transfected, Ripa lysis buffer and Anti-flag beads were used to enrich protein of cell lysate, and His antibody was used to verify the ubiquitylated G6PD. We use corresponding antibody by immunoblotting to detect the corresponding protein of Input.Results:1.The apoptosis of podocytes induced by high glucose and high palmitic acid could be improved by G6PD.2.High palmitic acid could lead to the disarrangement of F-actin in podocyte and overexpression of G6PD could partly reverse this situation.3.High glucose coulddecrease NADPH and GSH/GSSG, increase ROSand cause the imbalance of redox, and overexpression of G6PD can improve such redox indicators.4.We successfully constructed thepCDNA3.0-Flag-G6PD plasmid. 5.Compared with other groups, the cotransfectionof His-Ub and Flag-G6PD plasmid plus MG132 group could significantly increase theubiquitylated G6PD.Conclusion:Overexpression of G6PD can partially reverse the effects of high glucose and high palmitic acid on the proliferation, apoptosis, morphology and redox of podocytes,and high glucose decreases the expression of G6PD in podocyte through the ubiquitin proteasome pathway.PART2VHL participates in the degradation of G6PD in ubiquitin proteasome pathwayObjective:To find and verify the E3 ubiquitin ligase involved in the ubiquitination of G6PD.Methods:The yeast two hybrid technique was used to screen the E3 enzyme involved in G6PD. Co-immunoprecipitation experiment:HEK293T cells were divided into two groups:HA-VHL plasmid transfected group, Flag-G6PD plasmid and HA-VHL plasmid cotransfected group. In the last 6 hours both groups were added the proteasome inhibitor MG132 (20um). Co-immunoprecipitation lysis buffer and Anti-flag beads were used to enrich the protein of cell lysate, and HA antibody was used to verify to detect HA-VHL protein. In the vivo ubiquitin experiment, HEK293Tcells were divided into threegroups: His-Ub plasmid transfected group; Flag-G6PD and His-Ub plasmid cotransfected group; Flag-G6PD, His-Ub and HA-VHL plasmid cotransfected group. In the last 6 hours all groups were added the proteasome inhibitor MG132 (20um), Ripa lysis buffer and Anti-Flag beads were used to enrich in the protein of cell lysate, His antibody was used to detect the ubiquitylated G6PD. We use corresponding antibody by immunoblotting to detect the corresponding protein of input.Results:1. We successfully construted pCDNA3.0-HA-VHL plasmid.2.VHL (Hippel-Lindau tumor suppressor Von) was screened by the yeast two hybrid experiment.3. We proved that G6PD and VHL hang together in HEK293T cells by the method of Co-immunoprecipitation.4. VHL could increase the ubiquitylated G6PD protein which was verified by in vivo ubiquitin experiment.Conclusion:VHL as an E3 ubiquitin ligase is involved in the ubiquitination of G6PD in ubiquitin proteasome pathway.