Effect Of Active Vitamin D₃ On Podocyte Injury In Rats With Adriamycin-induced Nephropathy Via Nephrin-PI3K/Akt Signaling Pathway

Objective:The case of human chronic kidney disease?CKD?originates from the glomerulus,which is a filter that forms primary urine.The selective glomerular filtration process depends largely on the structural integrity and functional capacity of the podocytes.The podocyte surface core protein nephrin is its central signaling platform and plays a role in signal transmission.Increasing evidence suggests that vitamin D and its analogs exert a renal protective effect independent of calcium and phosphorus metabolism,there are fewer reports on its effects on podocytes,and the mechanism of podocyte injury has not yet been determined.Therefore,we explored the effect of active vitamin D₃?AVD₃?on podocyte injury and the PI3K/Akt pathway,and explored the underlying mechanism of its role.Methods:1.Healthy 6-week-old male SD rats were randomly divided into control group?NC group?,adriamycin nephropathy model group?AN group?,and adriamycin nephropathy active vitamin D₃?0.25?g/kg/d?intervention group?AN+AVD₃ group?,36 in each group.Model rats?AN,AN+AVD₃ group?received a single tail vein injection of adriamycin?adriamycin,ADR,7.5mg/kg?.A large amount of proteinuria,renal dysfunction and renal pathological damage were used as experimental standards.10 weeks.Every 2 weeks,various indicators of rats in each group were measured in different periods,including:weight,renal coefficient,renal function indicators,renal pathological damage;immunofluorescence observation of nephrin,desmin,PI3K?p85??and p-Akt in the glomerulus Localization,semi-quantitative immunohistochemistry and Western Blot quantitative analysis of nephrin,desmin,PI3K?p85??,AKT and p-Akt protein expression levels,real-time fluorescence quantitative PCR?qPCR?detection of nephrin,desmin,PI3K?p85??and AKT mRNA Level.2.The podocytes cultured in vitro were divided into:normal control group?NC group?,pure 1,25-dihydroxyvitamin D₃ group?10nmol/L,AVD₃ group?,ADR podocyte injury model group(1.5?mol/L,ADR grou-dihydroxyvitamin D₃ intervention group?1.5?mol/L ADR+10nmol/L AVD₃,ADR+AVD₃ group?;rat morphology,free of fluorescent identification of rat podocytes;MTT assay was used to detect the inhibitory effect of ADR on podocyte cell viability and the protective effect of AVD₃ on podocyte cell viability;Western Blot was used to detect the effects of different concentrations of ADR and AVD₃ on nephrin protein expression;Western Blot was used to detect AVD₃ on podocyte desmin under ADR,Nephrin,PI3K?p85??,AKT and p-Akt protein expression.Results:Part 1:Effects of active vitamin D₃ on podocyte injury and PI3K/Akt pathway in AN rats1.1 Evaluation of AN rat modelCompared with the NC group,rats in the model group?AN,AN+AVD₃ group?showed general signs and mental abnormalities:fluffy and dark yellow hair,polyuria and sparse stools,dystrophy,and irritability;rats produced a large amount of proteinuria?>30mg/100g?and abnormal renal phenotype:renal capsule widens,thickened basement membrane,cell shedding in renal tubules,and renal interstitial inflammatory infiltration.1.2 Renal protective effect of AVD₃ on AN ratsCompared with the NC group in the same period,the body weight of the rats in the AN group was significantly reduced?P<0.05?,renal index,24h urine protein?24h UP?,Significantly increased,renal function indicators:total serum protein?TP?and albumin?albumin,Alb?decreased?both P<0.05?,creatinine?crein?and total cholesterol?TC?increased High?all P<0.05?,renal pathological staining showed significantly increased mesangial matrix index and percentage of fibrotic area?all P<0.05?.Rats in the AN+AVD3group had increased body weight,kidney coefficient,and proteinuria in varying degrees.,TP and Alb levels increased,Cre and TC levels decreased,mesangial matrix index and fibrosis area percentage decreased?all P<0.05?.1.3 Effects of AVD₃ on nephrin,desmin,PI3K?p85??and p-Akt in AN ratsImmunofluorescence showed that nephrin,desmin,PI3K?p85??and p-Akt proteins were localized in the glomerulus.Immunohistochemical staining and Western Blot detection showed that nephrin protein expression level in the renal cortex of AN group rats was decreased?P<0.05?,and desmin,PI3K?p85??and p-Akt protein levels were significantly increased?all P<0.05?,Further qPCR results were consistent with protein results;active vitamin D₃ treatment promoted the expression of nephrin protein and mRNA levels?P<0.05?,reduced desmin,PI3K?p85??protein and mRNA levels?both P<0.05?,and total AKT in protein and There was no difference between the groups in mRNA level?all P<0.05?.1.4 Podocyte injury is closely related to the production of proteinuriaSpearman correlation analysis showed that nephrin expression and podocyte injury in AN rats Negative correlation?r=-0.4938,P<0.05?,and negative correlation with 24h UP?r=-0.4588,P<0.05?;desmin protein expression was positively correlated with 24h UP?r=0.8165,P<0.01?.Part 2:Active vitamin D₃ may reduce podocyte damage through the nephrin-PI3K/Akt pathway2.1 Identification of rat podocytes in vitroUndifferentiated podocytes have a cobblestone-like appearance,no obvious foot processes are seen,and mature and mature podocytes are fibroblast-like.The middle is raised and the two ends are slender.Immunofluorescence staining shows that the cell line expresses podocyte WT-1 and nephrin proteins.It can be seen that the cell line is derived from podocytes,which lays the foundation for subsequent experiments.2.2 Effect of different ADR and AVD₃ interventions on cell viabilityDifferent concentrations and different times were used to stimulate podocytes.MTT assay of cell viability showed that ADR had Viability inhibition decreased with concentration and stimulation time.1.5?mol/L ADR stimulated podocytes had a significant inhibitory effect on cell viability?P<0.05?,which was 64.21%of the control group;under these conditions,podocyte injury was induced.With the intervention of active vitamin D₃,the cell viability increased in a concentration-dependent manner,and the 10 nmol/L intervention concentration had a significant protective effect on cell viability?P<0.05?.Nephrin protein expression detection showed consistent results.2.3 Effect of AVD₃ on the expression of nephrin,desmin,PI3K?p85??,AKT and p-Akt proteins in podocytesCompared with the control group,the expression of nephrin in podocytes was reduced by ADR stimulation?P<0.05?,and the expression of desmin,PI3K?p85??and p-Akt were increased?all P<0.05?.Inhibited the expression of desmin,PI3K?p85??and p-Akt?all P<0.05?,there was no significant difference in the expression of total AKT protein in each group?all P>0.05?.2.4 Correlation analysis of nephrin and PI3K/Akt signal pathwaySpearman correlation analysis showed that in the ADR-induced podocyte injury model,nephrin protein expression was significantly negatively correlated with the PI3K/Akt pathway protein?r=-0.6082,-0.3671;all P<0.05?.There is co-expression of nephrin protein and PI3K/Akt pathway protein in cortical glomeruli.Conclusions:1.In adriamycin nephropathy podocyte injury model,nephrin expression is down-regulated,and there is a correlation with 24h urinary protein.Shows that podocyte injury is an important cause of proteinuria produced by CKD.2.Animal kidney disease model and podocyte injury model experiments show that active vitamin D₃ promotes nephrin expression and improves kidney function,suggesting that podocytes are the target organs for active vitamin D₃ to exert renal protective effects.3.Active vitamin D₃ inhibits ADR-induced activation of the PI3K/Akt signaling pathway,and nephrin expression is related to PI3K/Akt There is a correlation between signaling pathway activation,suggesting that the nephrin-PI3K/Akt signaling pathway is a potential molecular mechanism for active vitamin D₃ to exert renal protection.