背景:足细胞衰老导致足细胞丢失和肾小球病。过量摄入果糖是足细胞损伤的危险因素。然而,高果糖是否促进足细胞衰老尚不清楚。
目的:探讨高果糖驱动足细胞衰老的病理机制,寻找减轻足细胞衰老的天然化合物。
方法:以衰老相关β-半乳糖苷酶(SA-β-gal)染色来表征足细胞衰老,蛋白质印迹,实时定量聚合酶链反应(qRT-PCR),彗星测定和免疫荧光。进行蛋白质组学分析以鉴定高果糖暴露足细胞中差异表达的蛋白质。通过透射电子显微镜观察了足细胞核孔复合物(NPCs)和足过程。通过qRT-PCR检测核孔蛋白155(Nup155)和需要肌醇的突变体80(INO80)的mRNA和蛋白水平,Westernblot和免疫荧光。进行虚拟筛选以发现靶向Nup155的天然化合物。
结果:高果糖增加SA-β-gal活性,p53,p21,p16和磷酸化组蛋白H2AX(γ-H2AX)的蛋白质水平,以及白细胞介素-1β(IL-1β)的mRNA表达,大鼠肾小球和足细胞中IL-6和肿瘤坏死因子α(TNF-α)的表达。蛋白质组学分析揭示了一个关键分子Nup155,该分子在高果糖诱导的足细胞衰老中降低。同时,体内和体外足细胞NPC的数量也减少。始终如一,高果糖抑制了INO80mRNA的核输出,从而下调足细胞衰老中的INO80蛋白表达。Nup155的缺失抑制了INO80mRNA核输出以诱导足细胞衰老,而过表达Nup155或INO80可减轻高果糖诱导的足细胞衰老。发现阿魏酸通过直接结合稳定Nup155蛋白和增强其转录来上调Nup155,促进INO80mRNA核输出缓解高果糖引起的足细胞衰老。
结论:高果糖通过降低Nup155来抑制INO80mRNA核输出,从而诱导足细胞衰老。阿魏酸靶向Nup155可能是预防高果糖诱导的足细胞衰老的潜在策略。
BACKGROUND: Podocyte senescence causes podocyte loss and glomerulopathy. Excessive fructose intake is a risk factor for podocyte injury. However, whether high fructose promotes podocyte senescence remains unknown.
OBJECTIVE: To explore the pathological mechanism by which high fructose drives podocyte senescence and find natural compounds to alleviate podocyte senescence.
METHODS: Podocyte senescence was characterized with senescence-associated beta-galactosidase (SA-β-gal) staining, Western blot, real-time quantitative polymerase chain reaction (qRT-PCR), comet assay and immunofluorescence. Proteomics analysis was performed to identify differentially expressed proteins in high fructose-exposed podocytes. Podocyte nuclear pore complexes (NPCs) and foot processes were observed by transmission electron microscopy. The mRNA and protein levels of nucleoporin 155 (Nup155) and inositol requiring mutant 80 (INO80) were detected by qRT-PCR, Western blot and immunofluorescence. Virtual screening was conducted to find natural compounds that target Nup155.
RESULTS: High fructose increased SA-β-gal activity, protein level of p53, p21, p16 and phosphorylated histone H2AX (γ-H2AX), as well as mRNA expression of interleukin-1β (IL-1β), IL-6 and tumor necrosis factor α (TNF-α) in rat glomeruli and podocytes. Proteomic analysis unraveled a crucial molecule Nup155, which was decreased in high fructose-induced podocyte senescence. Meanwhile, the number of podocyte NPCs was also decreased in vivo and in vitro. Consistently, high fructose suppressed nuclear export of INO80 mRNA, thereby down-regulated INO80 protein expression in podocyte senescence. Deletion of Nup155 inhibited INO80 mRNA nuclear export to induce podocyte senescence, whereas overexpression of Nup155 or INO80 alleviated high fructose-induced podocyte senescence. Ferulic acid was found to up-regulate Nup155 by both direct binding to stabilize Nup155 protein and enhancing its transcription, to promote INO80 mRNA nuclear export in the mitigation of high fructose-caused podocyte senescence.
CONCLUSIONS: High fructose induces podocyte senescence by decreasing Nup155 to inhibit INO80 mRNA nuclear export. Ferulic acid targeting Nup155 may be a potential strategy to prevent high fructose-induced podocyte senescence.