Department of Pediatrics, Second Hospital, Jilin University, Changchun, China.
The Key Laboratory of Pathobiology, Ministry of Education, Norman Bethune College of Medicine, Jilin University, Changchun, China.
PLoS One. 2020 Mar 2;15(3):e0229747. doi: 10.1371/journal.pone.0229747. eCollection 2020.
Angiotensin II (Ang II) is a key contributor to glomerular disease by predominantly resulting in podocyte injury, whereas the underlying molecular mechanisms has not been fully understood. This study aimed to investigate if and how ADP-ribosylation factor 6 (Arf6), a small GTP-binding protein, involves Ang II-induced cellular injury in cultured human podocytes. Cellular injury was evaluated with caspase 3 activity, reactive oxygen species (ROS) level and TUNEL assay. Arf6 activity was measured using an Arf6-GTP Pull-Down Assay. Ang II significantly enhanced Arf6 expressions accompanied by increase of Arf6-GTP. The TUNEL-positive cells as well as activated caspase 3, NADPH oxidase 4 protein (Nox4) and ROS levels were dramatically increased in Ang II-treated podocytes, which was prevented by secinH3, an Arf6 activity inhibitor. Induction of ROS by Ang II was inhibited in podocytes with Nox4 knockdown. Ang II-induced elevation of Nox4 and ROS was prevented by Arf6 knockdown. Phpspho-Erk1/2Thr202/Tyr204 levels were upregulated remarkably following Ang II treatment, and Erk inhibitor LY3214996 significantly downregulated Nox4 expression. In addition, Ang II decreased CD2AP expression. Overexpression of CD2AP prevented Ang II-induced upregulation of Arf6-GTP. Our data demonstrated that Ang II promotes ROS production and podocytes injury through activation of Arf6-Erk1/2-Nox4 signaling. We also provided evidence that Ang II activates Arf6 by degradation of CD2AP.
血管紧张素 II（Ang II）主要通过导致足细胞损伤而成为肾小球疾病的重要致病因素，但其潜在的分子机制尚未完全阐明。本研究旨在探讨小 G 蛋白 ADP-ribosylation factor 6（Arf6）是否以及如何参与培养的人足细胞中 Ang II 诱导的细胞损伤。用 caspase 3 活性、活性氧（ROS）水平和 TUNEL 测定评估细胞损伤。使用 Arf6-GTP 下拉测定法测量 Arf6 活性。Ang II 显著增强了 Arf6 的表达，同时增加了 Arf6-GTP。Ang II 处理的足细胞中 TUNEL 阳性细胞以及活化的 caspase 3、NADPH 氧化酶 4 蛋白（Nox4）和 ROS 水平显著增加，而 Arf6 活性抑制剂 secinH3 可预防这种增加。用 Nox4 敲低抑制 Ang II 诱导的 ROS 生成。Arf6 敲低可预防 Ang II 诱导的 Nox4 和 ROS 升高。Ang II 处理后，磷酸化-Erk1/2Thr202/Tyr204 水平显著上调，Erk 抑制剂 LY3214996 显著下调 Nox4 表达。此外，Ang II 降低了 CD2AP 的表达。CD2AP 的过表达可防止 Ang II 诱导的 Arf6-GTP 上调。我们的数据表明，Ang II 通过激活 Arf6-Erk1/2-Nox4 信号通路促进 ROS 产生和足细胞损伤。我们还提供了证据表明，Ang II 通过降解 CD2AP 激活 Arf6。
