Background: Podocyte injury, characterized by podocyte hypertrophy, apoptosis, and epithelial-mesenchymal transition (EMT), is the major causative factor of diabetic nephropathy (DN). Autophagy dysfunction is regarded as the major risk factor for podocyte injury including EMT and apoptosis. High mobility group box 1 (HMGB1) is involved in the progression of DN through the induction of autophagy. However, the underlying mechanism remains unknown.
Methods: Plasma HMGB1 concentrations were determined in DN patients using ELISA. Apoptosis of DN serum-treated podocytes was evaluated by flow cytometry. Podocyte autophagy flux was measured using immunofluorescence. Western blotting analysis was used to investigate HMGB1 expression, EMT, and autophagy-related signaling pathways.
Results: Upregulation of HMGB1 was found in DN patients and DN serum-treated podocytes. Removal of HMGB1 inhibited DN serum-mediated podocyte apoptosis by inhibiting autophagy and activating AKT/mammalian target of rapamycin (mTOR) signaling. In addition, HMGB1 depletion repressed the progression of podocyte EMT by inhibiting transforming growth factor (TGF)-β/smad1 signaling in vitro and in vivo. The combination of HMGB1 short interference (si) RNA and the autophagy activator rapamycin protected against podocyte apoptosis and EMT progression by inhibiting the AKT/mTOR and TGF-β/smad signaling pathway, respectively.
Conclusions: Although HMGB1 siRNA and rapamycin treatment had opposite effects on autophagy and AKT/mTOR signaling, there was no contradiction about the role of HMGB1 siRNA and rapamycin on AKT/mTOR pathway because autophagy and AKT/mTOR signaling play dual roles in intracellular biological processes. Based on the findings of this study, we may assume that HMGB1-initiated autophagy is harmful, whereas rapamycin is beneficial to podocyte survival. Possibly combined treatment with HMGB1 siRNA and rapamycin improved podocyte damage and EMT by regulating autophagy homeostasis.
摘要: 背景 以肥大、凋亡、上皮-间充质转化(EMT)为特征的足细胞损伤是糖尿病肾病(DN)的主要病因。自噬功能障碍被认为是包括EMT和凋亡在内的足细胞损伤的主要危险因素。HMGB1 (High mobility group box 1) 也通过自噬参与DN的发展进程，但具体机制尚未明确。 方法:采用ELISA法测定DN患者血浆HMGB1浓度。采用流式细胞术检测经DN患者血清处理后的足细胞的凋亡情况。用免疫荧光法测定足细胞自噬流。采用Western blotting分析HMGB1表达、EMT和自噬相关信号通路。 结果 在DN患者和经过DN患者血清处理的足细胞中均发现HMGB1表达量上升。通过抑制HMGB1可抑制自噬和激活AKT/mTOR信号通路，进而抑制DN患者血清介导的足细胞凋亡。此外，HMGB1的损耗可通过抑制体内、外的转化生长因子(TGF) -β/ smad1信号，抑制足细胞的EMT进程。HMGB1 siRNA和自噬激活剂雷帕霉素的结合可分别通过抑制 AKT/mTOR和TGF-β/smad信号通路而起到抑制足细胞凋亡和EMT进程的作用。 结论 虽然HMGB1 siRNA和雷帕霉素处理对自噬和AKT/mTOR信号通路有相反的作用，但因自噬和AKT/mTOR信号通路在细胞内的生物进程中起双重作用，所以二者对AKT/mTOR通路的作用并不矛盾。基于此项研究发现，我们可以推测HMGB1启动的自噬是有害的，而雷帕霉素对足细胞的存活有益。HMGB1 siRNA联合雷帕霉素可能通过调节自噬稳态而改善足细胞损伤和EMT。.
Keywords: AKT/mTOR信号通路; AKT/mammalian target of (mTOR) signaling; HMGB1; epithelial-mesenchymal transdifferentiation; high mobility group box 1 (HMGB1); podocyte apoptosis; rapamycin; 上皮-间充质转分化; 足细胞凋亡; 雷帕霉素.
© 2019 Ruijin Hospital, Shanghai Jiaotong University School of Medicine and John Wiley & Sons Australia, Ltd.