Department of Physiology and Anatomy, University of North Texas Health Science Center, Fort Worth, Texas, USA.
The North Texas Eye Research Institute and Department of Pharmacology and Neuroscience, University of North Texas Health Science Center, Fort Worth, Texas, USA.
J Biol Chem. 2022 Jun;298(6):101990. doi: 10.1016/j.jbc.2022.101990. Epub 2022 Apr 29.
Podocyte injury induced by hyperglycemia is the main cause of kidney dysfunction in diabetic nephropathy. However, the underlying mechanism is unclear. Store-operated Ca entry (SOCE) regulates a diversity of cellular processes in a variety of cell types. Calpain, a Ca-dependent cysteine protease, was recently shown to be involved in podocyte injury. In the present study, we sought to determine whether increased SOCE contributed to high glucose (HG)-induced podocyte injury through activation of the calpain pathway. In cultured human podocytes, whole-cell patch clamp indicated the presence of functional store-operated Ca channels, which are composed of Orai1 proteins and mediate SOCE. Western blots showed that HG treatment increased the protein abundance of Orai1 in a dose-dependent manner. Consistently, calcium imaging experiments revealed that SOCE was significantly enhanced in podocytes following HG treatment. Furthermore, HG treatment caused overt podocyte F-actin disorganization as well as a significant decrease in nephrin protein abundance, both of which are indications of podocyte injury. These podocyte injury responses were significantly blunted by both pharmacological inhibition of Orai1 using the small molecule inhibitor BTP2 or by genetic deletion of Orai1 using CRISPR-Cas9 lentivirus. Moreover, activation of SOCE by thapsigargin, an inhibitor of Ca pump on the endoplasmic/sarcoplasmic reticulum membrane, significantly increased the activity of calpain, which was inhibited by BTP2. Finally, the calpain-1/calpain-2 inhibitor calpeptin significantly blunted the nephrin protein reduction induced by HG treatment. Taken together, our results suggest that enhanced signaling via an Orai1/SOCE/Calpain axis contributes to HG-induced podocyte injury.
高血糖引起的足细胞损伤是糖尿病肾病肾功能障碍的主要原因。然而，其潜在机制尚不清楚。储存操纵的钙内流（SOCE）调节多种细胞类型中的多种细胞过程。钙依赖性半胱氨酸蛋白酶 calpain 最近被证明参与了足细胞损伤。在本研究中，我们试图确定增加的 SOCE 是否通过激活 calpain 途径导致高葡萄糖（HG）诱导的足细胞损伤。在培养的人足细胞中，全细胞膜片钳表明存在功能储存操纵的钙通道，该通道由 Orai1 蛋白组成，介导 SOCE。Western blot 显示 HG 处理以剂量依赖性方式增加 Orai1 蛋白的丰度。一致地，钙成像实验表明，HG 处理后 SOCE 在足细胞中显著增强。此外，HG 处理导致足细胞 F-肌动蛋白明显解聚以及nephrin 蛋白丰度显著降低，这两者都是足细胞损伤的迹象。这些足细胞损伤反应均被小分子抑制剂 BTP2 抑制 Orai1 的药理学抑制或通过 CRISPR-Cas9 慢病毒基因敲除 Orai1 显著减弱。此外，内质网/肌浆网上钙泵抑制剂 thapsigargin 激活 SOCE 可显著增加 calpain 的活性，BTP2 可抑制 calpain 的活性。最后，calpain-1/calpain-2 抑制剂 calpeptin 显著减弱了 HG 处理引起的 nephrin 蛋白减少。总之，我们的结果表明，Orai1/SOCE/Calpain 轴的增强信号传导导致 HG 诱导的足细胞损伤。
