Sun Yan, Kang Juening, Guan Xiaofeng, Xu Hua, Wang Xiang, Deng Yaoliang
Department of Urology, The Langdong Hospital of Guangxi Medical University, Nanning, 530028, China.
Department of Urology, The First Affiliated Hospital of Guangxi Medical University, Nanning, China.
Urolithiasis. 2021 Aug;49(4):291-299. doi: 10.1007/s00240-021-01261-7. Epub 2021 Mar 31.
This study aimed to observe whether calcium oxalate (CaOx) crystals can induce the activation of endoplasmic reticulum (ER) stress in human renal cortex proximal tubule epithelial (HK-2) cells and to explore the regulatory of ER stress on the damage and apoptosis of HK-2 cells induced by CaOx crystals. We detected the optimal CaOx crystal concentration and intervention time by Western blot. ER stress modifiers tunicamycin (TM) and 4-phenylbutyric acid (4-PBA) were used to regulate the ER stress of HK-2 cells. The activities of ER stress marker proteins GRP78 and CHOP were evaluated by Western blot and immunohistochemistry. Western blot and TUNEL staining were used to detect cell apoptosis. We observed cell-crystal adhesion with an optical microscope. Lactate dehydrogenase (LDH) test kit and IL-1β enzyme-linked immunosorbent assay kit were used to detect and evaluate HK-2 cell damage. We found that the expression of ER stress marker proteins GRP78 and CHOP gradually increased with the increase in CaOx crystal concentration and intervention time and reached the maximum at 2.0 mmol/L and 24 h. The use of ER stress modifiers TM and 4-PBA can effectively regulate the ER stress level induced by CaOx crystals, and the level of apoptosis is positively correlated with the level of ER stress. 4-PBA pretreatment remarkably reduced cell-crystal adhesion and the secretions of IL-1β and LDH, whereas the results of TM pretreatment were the opposite. In summary, the damage and apoptosis of HK-2 cells induced by CaOx crystals are closely related to the level of ER stress. Inhibiting the ER stress of HK-2 cells can substantially reduce the cell damage and apoptosis induced by CaOx crystals.
本研究旨在观察草酸钙（CaOx）晶体是否能诱导人肾皮质近端小管上皮（HK-2）细胞内质网（ER）应激的激活，并探讨ER应激对CaOx晶体诱导的HK-2细胞损伤和凋亡的调控作用。我们通过蛋白质免疫印迹法检测了最佳的CaOx晶体浓度和干预时间。使用ER应激调节剂衣霉素（TM）和4-苯基丁酸（4-PBA）来调节HK-2细胞的ER应激。通过蛋白质免疫印迹法和免疫组织化学法评估ER应激标记蛋白GRP78和CHOP的活性。采用蛋白质免疫印迹法和TUNEL染色检测细胞凋亡。我们用光学显微镜观察细胞与晶体的黏附情况。使用乳酸脱氢酶（LDH）检测试剂盒和IL-1β酶联免疫吸附测定试剂盒检测并评估HK-2细胞损伤。我们发现，随着CaOx晶体浓度和干预时间的增加，ER应激标记蛋白GRP７８和CHOP的表达逐渐增加，并在2.0 mmol/L和24小时时达到最大值。使用ER应激调节剂TM和4-PBA可以有效调节CaOx晶体诱导的ER应激水平，且凋亡水平与ER应激水平呈正相关。4-PBA预处理显著降低了细胞与晶体的黏附以及IL-1β和LDH的分泌，而TM预处理的结果则相反。综上所述，CaOx晶体诱导的HK-2细胞损伤和凋亡与ER应激水平密切相关。抑制HK-2细胞的ER应激可显著减少CaOx晶体诱导的细胞损伤和凋亡。
