| Background Diabetic nephropathy(DN)is one of the most serious microvascular complications of diabetes and is the leading cause of end-stage kidney disease worldwide.The number of diabetic patients in China is rising,how to prevent the occurrence and development of DN is an urgent problem to be solved.Podocytes are one of the intrinsic cells of the glomeruli,which attach to the lateral side of the glomerular basement membrane(GBM),together with GBM and capillary endothelial cells,forming the final barrier of glomerular hemofiltration.Any change in this barrier can lead to proteinuria.In recent years,it has been found that the change of filtration membrane permeability caused by podocyte injury may be the most important cause of DN and continue to progress to end-stage renal failure,and has become a hot spot in renal disease research.Autophagy and apoptosis are the two most important pathophysiological processes of podocytes.The high level of autophagy of podocytes in physiological state is of great significance to cell homeostasis.Autophagy disorder can lead to podocyte injury,cell loss and ultimately glomerulosclerosis.Diabetic nephropathy is the most common podocyte disease,whether autophagy inhibition leads to the progression of DN has not been studied.Vitamin D,a steroid hormone,is widely known for its role in calcium homeostasis and bone metabolism.Several studies reveal expanding physiological functions of the vitamin D endocrine system far beyond its traditional calcium and phosphorus metabolism that include regulation of renal and cardiovascular functions and immunomodulation.A series of clinical and animal studies have shown that vitamin D and its analogues can reduce proteinuria and delay the progression of diabetic nephropathy.However,the exact mechanism of vitamin D on renal protection has not been fully elucidated.Objective The primary aim of this study was to investigate whether vitamin D can improve podocyte apoptosis induced by high glucose and alleviate podocyte injury by activating autophagy.Methods Part 1: To observe the effect of 1α,25(OH)2D3 on apoptosis of podocytes under high glucose:(1)the cells were incubated with high glucose(30m M)for different times(0,3,6,12,24,36 and 48h).The expression of Bax and Cleaved caspase-3 was examined by Western blotting.Apoptosis was evaluated by flow cytometry.(2)the cells were treated with various concentrations of 1α,25(OH)2D3(1,10,100 n M)and high glucose(30m M)for 48 h.The expression of Bax and Cleaved caspase-3 was examined by Western blotting analysis.Apoptosis was evaluated by flow cytometry.Part 2: To investigate the effects of 1α,25(OH)2D3 on autophagy of podocytes under the high glucose conditions:(1)the cells were incubated with high glucose(30m M)for different times(0,3,6,12,24,36 and 48h).The expression of LC3-Ⅱ、beclin-1、Vps34 was examined by Western blotting.Immunofluorescence staining showed the expression of podocyte marker protein Synaptopodin and endogenous accumulation of LC3.(2)the cells were treated with various concentrations of1α,25(OH)2D3(1,10,100 n M)and high glucose(30m M)for 48 h.Cultured podocytes were divided into six groups according to various treatment: normal glucose(NG,5.6m M glucose),mannitol control group(MA,5.6m M glucose plus24.4m M mannitol),high glucose(HG,30 m M glucose),high glucose plus 1n M1α,25(OH)2D3(HG+1 VD),high glucose plus 10 n M 1α,25(OH)2D3(HG+10 VD),high glucose plus 100 n M 1α,25(OH)2D3(HG+100 VD).Immunofluorescence staining showed the expression of podocyte marker protein Synaptopodin and endogenous accumulation of LC3.Autophagosomes were identified by transmission electron microscopy.The expression of LC3-Ⅱ、beclin-1、Vps34 was examined by Western blotting analysis and RT-PCR,respectively.Part 3: To examine the mechanism of the interaction between autophagy and apoptosis signal on podocyte injury induced by high glucose,immortalized mouse podocytes were treated with NG,HG,high glucose plus 3-MA(HG+3-MA),high glucose plus 100 n M 1α,25(OH)2D3(HG+100 VD),high glucose plus 100 n M1α,25(OH)2D3 and 3-MA(HG+100 VD+3-MA).The expression of nephrin,podocin,LC3-Ⅱ,beclin-1,Vps34,Bax and Cleaved caspase-3 was examined by Western blotting.Results Part 1: The level of Bax and Cleaved caspase-3 increased in a time-dependent manner after high glucose treatment,and the apoptosis rate markedly increased.After adding 1α,25(OH)2D,the expression of Bax and Cleaved caspase-3 decreased significantly,and the apoptosis rate decreased in a concentration-dependent manner.Part 2: The level of LC3-Ⅱ、beclin-1 and Vps34 peaked at 24 hours after high glucose treatment and then decreased gradually,with the most obvious decrease at 48 hours.After adding 1α,25(OH)2D3,the expression of LC3-Ⅱ,Beclin-1 and Vps34 increased in a concentration-dependent manner,the distribution of LC3-Ⅱ increased,and the number of autophagosomes increased.Part 3: Decreases in the expression of nephrin,podocin,LC3 II,Beclin-1 and Vps34,and increases in the level of Bax and Cleaved caspase-3 by co-treatment with 3-MA compared with the group treated with HG or HG+VD alone were observed.Conclusion The activity of autophagy of podocytes increased in the early stage stimulated by high glucose and inhibited by long-term stimulation of autophagy.1 α,25(OH)2D3may be an autophagy activator,which can promote the formation of autophagy lysosomes by regulating the levels of beclin-1 and Vps34,improve the apoptosis induced by high glucose,and alleviate the injury of podocytes. |
PDF Full Text Request