Department of Ophthalmology and Eye Hospital, University of Leipzig, Leipzig, Germany.
Institute of Anatomy, Medical Faculty, University of Leipzig, Germany.
Mol Vis. 2020 Dec 30;26:797-817. eCollection 2020.
The expression of aquaporin-8 (), which plays a crucial role in the maintenance of the cellular fluid and electrolyte balance, was shown to be increased in RPE cells under hyperosmotic conditions. The aim of the present study was to investigate the mechanisms of hyperosmotic gene expression and the localization of AQP8 in cultured human RPE cells.
Hyperosmolarity was produced with the addition of 100 mM NaCl or 200 mM sucrose. Hypoxia was induced by cell culture in a 0.2% O atmosphere or the addition of the hypoxia mimetic CoCl. Oxidative stress was induced by the addition of HO. Gene expression was determined with real-time RT-PCR analysis. AQP8 protein localization and secretion of VEGF were evaluated with immunocytochemistry, western blotting, and enzyme-linked immunosorbent assay (ELISA).
Immunocytochemical and western blot data suggest that the AQP8 protein is mainly located in the mitochondria. Extracellular hyperosmolarity, hypoxia, and oxidative stress induced increases in gene expression. Hyperosmotic gene expression was reduced by inhibitors of the p38 MAPK and PI3K signal transduction pathways, and by JAK2 and PLA inhibitors, and was in part mediated by the transcriptional activity of CREB. Hyperosmotic gene expression was also reduced by autocrine/paracrine interleukin-1 signaling, the sulfonylureas glibenclamide and glipizide, which are known inhibitors of K channel activation, and a pannexin-blocking peptide. The K channel opener pinacidil increased the expression of under control conditions. The cells contained and gene transcripts and displayed Kir6.1 immunoreactivity. siRNA-mediated knockdown of caused increases in hypoxic VEGF gene expression and secretion and decreased cell viability under control, hyperosmotic, and hypoxic conditions.
The data indicate that hyperosmotic expression of in RPE cells is dependent on the activation of K channels. The data suggest that AQP8 activity decreases the hypoxic VEGF expression and improves the viability of RPE cells which may have impact for ischemic retinal diseases like diabetic retinopathy and age-related macular degeneration.
水通道蛋白-8()的表达在维持细胞液和电解质平衡中起着至关重要的作用，在高渗条件下 RPE 细胞中的表达增加。本研究旨在探讨高渗条件下基因表达的机制和 AQP8 在培养的人 RPE 细胞中的定位。
通过添加 100mM NaCl 或 200mM 蔗糖产生高渗。通过在 0.2%O 气氛下培养细胞或添加缺氧模拟物 CoCl 诱导缺氧。通过添加 H2O2 诱导氧化应激。通过实时 RT-PCR 分析确定基因表达。通过免疫细胞化学、Western blot 和酶联免疫吸附试验(ELISA)评估 AQP8 蛋白定位和 VEGF 的分泌。
免疫细胞化学和 Western blot 数据表明，AQP8 蛋白主要位于线粒体。细胞外高渗、缺氧和氧化应激诱导基因表达增加。高渗基因表达被 p38 MAPK 和 PI3K 信号转导通路抑制剂、JAK2 和 PLA 抑制剂以及部分由 CREB 转录活性介导的抑制剂所降低。高渗基因表达也被自分泌/旁分泌白细胞介素-1 信号、已知的 K 通道激活抑制剂磺酰脲类格列本脲和格列吡嗪以及泛肽阻断肽所降低。K 通道开放剂吡那地尔在对照条件下增加基因的表达。细胞含有和基因转录本，并显示 Kir6.1 免疫反应性。siRNA 介导的敲低导致缺氧条件下 VEGF 基因表达和分泌增加，对照、高渗和缺氧条件下细胞活力降低。
数据表明，RPE 细胞中基因的高渗表达依赖于 K 通道的激活。数据表明，AQP8 活性降低了缺氧条件下 VEGF 的表达，提高了 RPE 细胞的活力，这可能对糖尿病性视网膜病变和年龄相关性黄斑变性等缺血性视网膜疾病产生影响。
