Active Vitamin D3 Plays A Protective Role In Podocytes Of Diabetic Nephropathy Rats By Regulating Autophagy Activity

Objective To investigate the protective effect and mechanism of active vitamin D3 on podocyte injury in type 1 diabetic rats.Methods Animals were randomly divided into normal control group(NC group),diabetic nephropathy group(DN group),diabetic nephropathy+active vitamin D3 intervention group(DN+VD group).Body weight,random tail vein blood glucose were measured every 2 weeks,and 24 h urine was collected to observe the dynamic changes of body weight,blood glucose,24 h urine volume and 24 h urine protein.The rats were sacrificed at the end of the 18 th week,and the kidney weight to body weight ratio(KW/BW)was calculated.Serum creatinine(Scr),serum albumin(ALB)and blood urea nitrogen(BUN)levels were measured.Pathological changes of glomeruli were observed by periodic acid-schiff(PAS)staining.Immunohistochemistry and Western blotting were used to observe the slit diaphragm proteins Nephrin and Podocin,podocyte injury marker Desmin and vitamin D receptor protein VDR.Synaptopodin,the slit diaphragm protein was detected by Western blotting.WT1,the podocyte-specific nuclear protein was observed by immunohistochemistry.Immunohistochemistry was used to detecte the expressions of autophagy-related proteins Beclin1 and P62.Western blotting was used to detecte the levels of autophagy-related proteins LC3B/A,Beclin1 and P62.The distribution and expression of Synaptopodin and LC3 B proteins were co-localized by immunofluorescence.The ultrastructure of podocytes and the changes of autophagosomes in podocytes were observed by electron microscopy.Results Compared with normal rats,the weight of diabetic rats was significantly reduced,24 h urine volume,urine protein and blood glucose were significantly increased(P<0.05).24 h urine volume and urine protein were significantly improved after active vitamin D3 treatment(P<0.05),and there were no significant change in body weight and blood glucose(P>0.05).Compared with normal rats,KW/BW,BUN and Scr were significantly increased and ALB was decreased in diabetic rats(P<0.05).KW/BW values were decreased after treatment with active vitamin D3(P<0.05).There were no significant improvement in BUN,Scr and ALB(P>0.05).Compared with normal rats,the glomerular mesangial matrix of diabetic rats was severely hyperplasia(P<0.05),and the glomerular basement membrane(GBM)thickened(P<0.05).the glomeruli of diabetic rats showed foot process fusion or even shedding,the mesangial matrix hyperplasia and basement membrane thickening were improved and the fusion and shedding of the foot processes were partially reversed after treatment with active vitamin D3.Compared with normal rats,the expression of Nephrin,Podocin,Synaptopodin,WT1,VDR protein in diabetic rats were significantly decreased,and the expression of Desmin protein was significantly increased(P<0.05).After treatment with active vitamin D3,the expression of Nephrin,Podocin,Synaptopodin VDR proteins were significantly up-regulated,and Desmin protein was significantly down-regulated(P<0.05).However,there was no significant difference in the expression of WT1 protein(P>0.05).Compared with normal rats,the expression of LC3B/A and Beclin1 proteins in glomeruli of diabetic rats were significantly decreased,and the expression of P62 protein was enhanced(P<0.05).After treatment with active vitamin D3,the expression of LC3B/A and Beclin1 proteins were up-regulated and P62 protein expression was down-regulated(P<0.05).Compared with normal rats,the number of autophagosomes in podocytes of diabetic rats was significantly decreased(P<0.05),after active vitamin D3 intervention,the number of autophagosomes increased(P<0.05).Double immunofluorescence staining showed that in the NC group,LC3 B protein was diffusely distributed throughout the glomerulus and highly coincided with the Synaptopodin protein distributed in the glomerular capillary wall.Different from the NC group,the fluorescence intensity of both proteins in the DN group was significantly weakened,which was enhanced after the intervention of active vitamin D3.Pearson correlation analysis showed that there was a significant positive relationship between VDR and Nephrin(r=0.9002,P<0.05),VDR and Beclin1(r=0.8589,P<0.05).Similarly,Nephrin also showed a significant positive correlation with the expression of Beclin1(r=0.9287,P<0.05).Conclusion Active vitamin D3 inhibits the injury of diabetic nephropathy podocytes by up-regulating VDR expression and enhancing autophagy activity.