School of Medicine, State Key Laboratory of Medicinal Chemical Biology, Key Laboratory of Bioactive Materials Ministry of Education, Nankai University, Tianjin 300071, PR China; Tianjin Medical University Cancer Institute and Hospital, Tianjin 300200, PR China.
College of Life Sciences, Nankai University, Tianjin 300071, PR China; School of Mathematical Sciences, Nankai University, Tianjin 300071, PR China.
Chem Biol Interact. 2017 Nov 1;277:110-118. doi: 10.1016/j.cbi.2017.09.010. Epub 2017 Sep 12.
As well known, abnormalities of Angiotensin II (Ang II) is closely related with glomerular damage. This study was to investigate whether Ang II could affect autophagy in podocytes via oxidative stress, and whether autophagy had a positive role in protecting podocytes impaired by Ang II. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that Ang II induced podocyte death. The measurements of malondialdehyde (MDA) and HO levels, and flow cytometry assay revealed that Ang II considerably increased reactive oxygen species (ROS) generation in podocytes. Meaningfully, treatment with ROS scavenger N-(mercaptopropionyl)-glycine (N-MPG) could inhibit podocyte death and attenuate accumulation of ROS induced by Ang II. The patch-clamp experiments indicated that Ang II increased the current of transient receptor potential canonical 6 (TRPC6). Moreover, measurement of Fluo-3 image showed that Ang II increased intracellular Ca level, as N-MPG and La impeded Ang II induced Ca influx. Acridine orange staining indicated that Ang II induced accumulation of acidic vacuoles. Beclin-1 and LC3 are essential for autophagosome formation. Furthermore, as one of the selective substrates for autophagy, P62 plays a key role in the formation of cytoplasmic proteinaceous inclusion. Western blot assay presented that Ang II obviously elevated LC3-II/LC3-I ratio and expression of beclin-1, and reduced expression of P62. Meanwhile, N-MPG expectedly down-regulated autophagy in Ang II-treated podocytes. Rapamycin can enhance the level of autophagy by inhibiting mTOR, and 3-methyladenine (3-MA) can inhibit autophagosome formation through blocking class III phosphatidylinositol 3-kinase. MTT assay exhibited that rapamycin significantly enhanced the cell viability, while 3-MA considerably reduced it in Ang II-treated podocytes. Consequently, this study demonstrated that Ang II could increase TRPC6 induced Ca influx and enhance autophagy through increasing ROS levels in podocytes, and autophagy could protect Ang II-treated podocytes. Improving TRPC6 channels and autophagy may become a new targeted therapy to relieve glomerular damage induced by Ang II.
众所周知，血管紧张素 II（Ang II）的异常与肾小球损伤密切相关。本研究旨在探讨 Ang II 是否可以通过氧化应激影响足细胞的自噬，以及自噬是否对 Ang II 损伤的足细胞具有保护作用。3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐（MTT）测定表明 Ang II 诱导足细胞死亡。丙二醛（MDA）和 HO 水平的测定以及流式细胞术分析表明，Ang II 可显著增加足细胞中活性氧（ROS）的产生。有意义的是，用 ROS 清除剂 N-（巯基丙酰基）-甘氨酸（N-MPG）处理可抑制 Ang II 诱导的足细胞死亡和 ROS 积累。膜片钳实验表明，Ang II 增加了瞬时受体电位经典型 6（TRPC6）的电流。此外，Fluo-3 图像的测量表明，Ang II 增加了细胞内 Ca 水平，而 N-MPG 和 La 则阻碍了 Ang II 诱导的 Ca 内流。吖啶橙染色表明 Ang II 诱导酸性空泡的积累。Beclin-1 和 LC3 是自噬体形成的必需物质。此外，作为自噬的选择性底物之一，P62 在细胞质蛋白包涵体的形成中起关键作用。Western blot 分析表明，Ang II 明显增加了 LC3-II/LC3-I 比值和 beclin-1 的表达，并降低了 P62 的表达。同时，N-MPG 预期下调了 Ang II 处理的足细胞中的自噬。雷帕霉素通过抑制 mTOR 可以增强自噬水平，而 3-甲基腺嘌呤（3-MA）可以通过阻断 III 类磷酸肌醇 3-激酶来抑制自噬体的形成。MTT 测定表明，雷帕霉素显著增强了 Ang II 处理的足细胞的细胞活力，而 3-MA 则显著降低了细胞活力。因此，本研究表明，Ang II 可以通过增加足细胞中 ROS 水平来增加 TRPC6 诱导的 Ca 内流并增强自噬，并且自噬可以保护 Ang II 处理的足细胞。改善 TRPC6 通道和自噬可能成为缓解 Ang II 诱导的肾小球损伤的新靶向治疗方法。
