Oxidative stress and reactive oxygen species (ROS) generation can be influenced by G-protein coupled receptor (GPCR)-mediated regulation of intracellular Ca2+ ([Ca2+]i) signaling. ROS production are much higher in proximal tubular (PT) cells; in addition, the lack of antioxidants enhances the vulnerability to oxidative damage. Despite such predispositions, PT cells show resiliency, and therefore must possess some inherent mechanism to protect from oxidative damage. While the mechanism in unknown, we tested the effect of l-ornithine, since it is abundantly present in PT luminal fluid and can activate Ca2+-sensing receptor (CaSR), a GPCR, expressed in the PT luminal membrane. We used human kidney 2 (HK2) cells, a PT cell line, and performed Ca2+ imaging and electrophysiological experiments to show that l-ornithine has a concentration-dependent effect on CaSR activation. We further demonstrate that the operation of CaSR activated Ca2+ signaling in HK-2 cells mediated by the transient receptor potential canonical (TRPC) dependent receptor-operated Ca2+ entry (ROCE) using pharmacological and siRNA inhibitors. Since PT cells are vulnerable to ROS, we simulated such deleterious effects using genetically encoded peroxide-induced ROS production (HyperRed indicator) to show that the l-ornithine-induced ROCE mediated [Ca2+]i signaling protects from ROS production. Furthermore, we performed cell viability, necrosis and apoptosis assays, and mitochondrial oxidative gene expression to establish that presence of l-ornithine rescued the ROS-induced damage in HK-2 cells. Moreover, l-ornithine-activation of CaSR can reverse ROS production and apoptosis via mitogen-activated protein kinase p38 activation. Such nephroprotective role of l-ornithine can be useful as the translational option for reversing kidney diseases involving PT cell damage due to oxidative stress or crystal nephropathies.

中文翻译：

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l-鸟氨酸激活 Ca2+ 信号传导，对人近端肾小管细胞发挥保护作用。


氧化应激和活性氧 (ROS) 的产生可能受到 G 蛋白偶联受体 (GPCR) 介导的细胞内 Ca2+ ([Ca2+]i) 信号传导调节的影响。近端肾小管 (PT) 细胞中 ROS 的产生量要高得多；此外，缺乏抗氧化剂会加剧氧化损伤的脆弱性。尽管有这样的倾向，PT 细胞仍表现出弹性，因此必须具有一些固有的机制来防止氧化损伤。虽然机制尚不清楚，但我们测试了 L-鸟氨酸的作用，因为它大量存在于 PT 管腔液中，并且可以激活 Ca2+ 感应受体 (CaSR)，一种在 PT 管腔膜中表达的 GPCR。我们使用人肾 2 (HK2) 细胞（一种 PT 细胞系）进行 Ca2+ 成像和电生理学实验，表明 L-鸟氨酸对 CaSR 激活具有浓度依赖性作用。我们进一步证明，使用药理学和 siRNA 抑制剂，CaSR 的操作激活了 HK-2 细胞中由瞬时受体电位规范 (TRPC) 依赖性受体操作的 Ca2+ 进入 (ROCE) 介导的 Ca2+ 信号传导。由于 PT 细胞容易受到 ROS 的影响，我们使用基因编码的过氧化物诱导的 ROS 产生（HyperRed 指示剂）模拟了此类有害影响，以表明 L-鸟氨酸诱导的 ROCE 介导的 [Ca2+]i 信号传导可防止 ROS 产生。此外，我们进行了细胞活力、坏死和凋亡测定以及线粒体氧化基因表达，以确定 L-鸟氨酸的存在可以挽救 HK-2 细胞中 ROS 诱导的损伤。此外，CaSR 的 L-鸟氨酸激活可以通过丝裂原激活蛋白激酶 p38 激活来逆转 ROS 产生和细胞凋亡。 L-鸟氨酸的这种肾保护作用可作为逆转肾脏疾病的转化选择，这些疾病涉及氧化应激或晶体肾病导致的 PT 细胞损伤。