晚期糖基化终末产物通过Wnt/β-catenin信号通路调控足细胞自噬和迁移

Modulation of podocyte autophagy and migration by advanced glycationend products through Wnt/β-catenin signaling pathway

目的探讨晚期糖基化终末产物(AGEs)诱导Wnt/β-catenin信号通路活化对足细胞自噬和迁移的影响。方法将小鼠永生化肾足细胞系分为牛血清白蛋白组(100mg/L BSA)、糖基化终末产物组(100 mg/L BSA-AGEs)和糖基化终末产物+DKK-1组(100mg/L BSA-AGEs+100 DKK-1 mg/L),以未加干涉的正常足细胞为对照组。Western blot检测足细胞β-catenin蛋白、自噬膜蛋白微管相关蛋白轻链3-II(LC3-Ⅱ)以及自噬底物p62蛋白表达水平,免疫荧光观察足细胞自噬情况,ELISA检测细胞上清液中Nephrin蛋白含量,划痕试验检测足细胞迁移能力。用AGEs和AGEs+RAGE(糖基化终末产物受体)中和抗体处理足细胞系,Western blot检测足细胞β-catenin蛋白的表达。结果与对照组相比,AGEs组p-β-catenin、p62表达升高(P<0.05),LC3-Ⅱ表达降低(P<0.05)。AGEs+DKK-1组p-β-catenin、p62蛋白表达较AGEs组降低,LC3-Ⅱ表达升高(P<0.05),但与对照组比较差异无显著性(P>0.05)。BSA组各指标与对照组均无显著性差异(P>0.05)。免疫荧光检测发现AGEs组足细胞中LC3荧光强度较对照组明显减弱,AGEs+DKK-1组较AGEs组LC3荧光强度增强,BSA组与对照组比较差异无显著性(P>0.05)。AGEs组细胞培养上清中Nephrin含量较对照组明显降低(P<0.05),AGEs+DKK-1组较AGEs组升高(P<0.05),但仍低于对照组(P<0.05),BSA组与对照组差异无显著性(P>0.05)。与对照组相比,AGEs组足细胞迁移能力降低(P<0.05),AGEs+DKK-1组、BSA组与对照组比较,差异无显著性(P>0.05)。AGEs+RAGE中和抗体处理的足细胞β-catenin蛋白水平较AGEs组显著降低(P<0.05)。结论 AGEs通过RAGE激活Wnt/β-catenin信号通路,抑制足细胞自噬和迁移能力,可能是糖尿病患者足细胞损伤机制之一,在糖尿病肾病的发生和进展中发挥重要调控作用。

Objective To investigate the effect of activated Wnt/β-catenin signaling pathway induced by advanced glycationend products(AGEs)on podocyte autophagy.Methods Mouse immortalized renal cell lines were divided into bovine serum albumin(BSA)group(100 mg/L BSA),AGEs group(100 mg/L BSA-AGEs)and AGEs + DKK-1 group(100 mg/L BSA-AGEs + 100 mg/L DKK-1).Normal cells without intervention were taken as the control group.Western blotting was used to detect the expression of β-catenin,autophagic membrane protein microtubule-associated protein light chain 3-Ⅱ(LC3-Ⅱ)and autophagic substrate p62.Immunofluorescence was used to observe the autophagy of podocytes.The ELISA was used to determine the nephrin level in the cell supernatant.The wound healing assay was used to measure cell migration rate.The cells were treated with AGEs and AGEs+RAGE neutralizing antibody respectively,and the expression of β-catenin protein was detected by Western blotting.Results The expression of p-β-catenin and p62 in AGEs group was increased(P<0.05),while that of LC3-Ⅱ was decreased(P<0.05)as compared with the control group.The expression of p-β-catenin and p62 protein in AGEs + DKK-1 group was lower than that in AGEs group,and that of LC3-Ⅱ was elevated(P<0.05),but there were no significant differences from the control group(P>0.05).There were no significant differences in these indicators between BSA group and control group(P>0.05).Immunofluorescence assay showed that the LC3 fluorescence intensity in AGEs group was significantly lower than that in control group,and the LC3 fluorescence intensity in AGEs + DKK-1 group was higher than that in AGEs group,and there were no significant differences between AGEs group and control group.The content of nephrin in the supernatant of AGEs group was significantly lower than that of the control group(P<0.05),and that in AGEs+DKK-1 group was significantly higher than in AGEs group(P<0.05),but still lower than in control group(P<0.05),there was no significant difference between AGEs group and control group(P>0.05).Compared with control group,the AGEs significantly reduced the migration ability of podocytes(P<0.05),but the AGEs+DKK-1 group and BSA group were not significantly different from control group(P>0.05).The level of β-catenin protein in podocytes treated with AGEs+RAGE neutralizing antibody was significantly lower than that in AGEs group(P<0.05).Conclusions AGEs can inhibit podocyte autophagy and migration by activating Wnt signaling pathway through RAGE,which may be one of the mechanisms in podocyte injury of diabetic patients,and play an important regulatory role in the occurrence and progression of diabetic nephropathy.