The Role Of Mitochondrial Fission Factor MFF In Podocyte Injury In Diabetic Nephropathy

Objective:The role of mitochondrial fission factor(MFF)-mediated mitochondrial fission in podocyte injury in diabetic nephropathy is still understudied.Mitochondrial fission is mainly mediated by MFF and fission protein 1,and recent studies have found that the two mediate mitochondrial fission in different ways,but their significance in disease is unclear.This study intends to investigate the molecular mechanism of increased mitochondrial fission in podocytes under the action of high glucose,and to preliminarily study the role of MFF-mediated mitochondrial fission in podocyte injury in diabetic nephropathy.Methods:1.Cell experiment: MPC5 cells,a mouse podocyte line,were selected for in vitro experiments.MPC5 cells were treated with different concentrations of glucose(5.5 mmol/L in the normal control group and 30 mmol/L in the high glucose group)for 72 hours,and the cell survival rates of the two groups were detected by CCK-8 method;Immunofluorescence detection of mitochondrial outer membrane protein TOM20 in cells;JC-10 mitochondrial membrane potential fluorescent probe detected mitochondrial damage;Reverse transcription-quantitative PCR(RT-q PCR)method was used to detect the m RNA expression of MFF at different time points at 6h,12 h,24h,36 h and 48h;Western blotting method was used to detect the protein expression of MFF,DRP1 and TOM20.2.Animal experiments: Male mice with C57BL/6J background were selected for diabetic nephropathy modeling,blood and urine of normal group and model group were collected for related detection and analysis;mouse kidneys were isolated for morphological examination;mouse kidneys were subjected to primary podocyte differentiation.The primary cells were identified by gel electrophoresis detection of podocyte marker proteins Nephrin and Podocin;immunofluorescence detection of TOM20 was used to analyze the number and length of mitochondria;Western blotting was used to detect the expression of TOM20 and MFF protein levels in mouse primary podocytes.3.Action research: MFF si RNA was transfected into MPC5 cells to inhibit the expression of MFF,and the survival rate of cells with RNA interference with MFF or not was analyzed by CCK-8 at 0h,24 h,48h,and 72 h.Results:1.(1)After MPC5 cells were stimulated with high glucose,the cell survival rate was significantly lower than that of the control group(P<0.0001).(2)The results of immunofluorescence showed that the number of mitochondria in MPC5 cells treated with high glucose increased significantly compared with the normal control group(P<0.0001),while the length was significantly shorter than that in the normal control group(P<0.0001).(3)After adding JC-10 fluorescent probe,the two groups were compared,and it was found that the green fluorescence/red fluorescence intensity in MPC5 cells after high glucose stimulation was significantly higher than that in the normal group(P<0.01).(4)According to RT-q PCR results,the m RNA level of MFF in the 12 h high glucose group was significantly higher than that in the normal group,with statistical significance(P<0.0001);but there was no statistical significance at 6h,24 h,36h,and 48 h.significance(P>0.05).(5)Western blotting results also found that the expression of MFF protein was up-regulated at 48 h and 72 h after high glucose stimulation(P<0.01);however,high glucose had no effect on the protein expression level of DRP1,and there was no statistical difference between the two groups(P>0.05).(6)The protein expression level of TOM20 was found to be significantly increased after high glucose treatment(P<0.01),which was statistically significant compared with the control group(P<0.05).2.(1)The levels of urinary microalbumin,blood urea nitrogen,serum creatinine,urinary microalbumin/creatinine,and blood glucose in the diabetic nephropathy model group were significantly higher than normal In the control group,the endogenous creatinine clearance and high-density lipoprotein were lower than those in the normal group,with statistical significance(P<0.01);low-density lipoprotein,triglyceride,total cholesterol and free fatty acid had no significant difference(P>0.05).(2)Kidney HE staining showed that the kidney structure of the normal group was intact,the tissue was not damaged,the nucleus was clearly visible,the structure of the glomerulus and the renal tubule were normal,and the basement membrane was not thickened;while the diabetic nephropathy model group found that the glomerular basement membrane was thickened,the matrix increased,and the contrast between the nucleus and the cytoplasm was not clear enough.Under electron microscope,compared with the normal control group,the glomerular basement membrane of the diabetic nephropathy model group was significantly thickened unevenly,the foot processes were widely fused,and the foot process gap was widened(P<0.01).(3)The expression of Nephrin and Podocin in primary cells can be detected by gel electrophoresis,which proves that primary podocytes are sorted.(4)Immunofluorescence detection of TOM20 showed that the number of mitochondria in podocytes of diabetic nephropathy mice was significantly increased compared with the normal control group(P<0.01),while the length was significantly shorter than that of the normal control group(P<0.001).(5)Western blotting results showed that the expressions of MFF and TOM20 in diabetic nephropathy mice were increased,with statistical significance(P<0.01).3.The survival rate of cells in the high glucose group was significantly lower than that in the control group after RNA interference with MFF(P<0.001).Conclusion:In vitro studies have found that high glucose induces increased mitochondrial fission and podocyte damage.High glucose up-regulated MFF m RNA and protein levels,it is speculated that it may increase its expression by promoting MFF transcription.Consistent with this,mitochondrial fission in podocytes in diabetic nephropathy mice was also significantly increased,while MFF expression was upregulated.Therefore,our study demonstrated that mitochondrial fission was increased in podocytes under diabetic conditions from both in vitro and in vivo levels,and the up-regulation of MFF expression may be one of the reasons for the increased mitochondrial fission.After inhibiting MFF expression,the survival rate of MPC5 cells was significantly decreased under high glucose conditions,suggesting that MFF may play a protective role in podocyte injury in diabetic nephropathy.Our study has certain implications for elucidating the pathogenesis of diabetic nephropathy,and may provide new targets for the treatment of diabetic nephropathy.