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.