Shen Yue, Yan Yucheng, Lu Liming, Qian Yingying, Guan Xuejing, Zhang Lulu, Qi Yuanyuan, Gu Leyi, Ding Feng
Department of Nephrology, Shanghai Ninth People's Hospital, School of Medicine, Shanghai Jiao Tong University, 639 Zhizaoju Rd., Shanghai, 200011, China.
Department of Nephrology, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, 160 Pujian Rd., Shanghai, 200127, China.
Int Urol Nephrol. 2018 Apr;50(4):787-798. doi: 10.1007/s11255-017-1765-x. Epub 2017 Dec 28.
Defects in Klotho gene expression in mice result in a vulnerability to oxidative injuries. We aimed to identify the expression of Klotho in a mouse tubular epithelial (TCMK-1) cell line, and also to investigate changes in Klotho expression induced by oxidative stress and the potential role of intra- and extracellular Klotho protein.
During exposure to hydrogen peroxide (HO), an overexpression of the Klotho gene was induced and exogenous Klotho protein was added in TCMK-1 cells. The generation of reactive oxidative species (ROS) was examined by flow cytometry, and cell viability was assessed by Cell Counting Kit-8. Cellular apoptosis was determined by flow cytometry and Hoechst 33258 staining followed by Western blotting to evaluate the expression of Klotho, antioxidant enzymes, and apoptosis-associated proteins.
While HO significantly suppressed Klotho expression, cell viability, and the expression of antioxidant enzymes in a concentration-dependent manner, cellular apoptosis was increased and p38/MAPK and JNK/MAPK were activated. Intra- and extracellular Klotho remarkably ameliorated viability inhibition, ROS generation, and cellular apoptosis induced by HO. Intra- and extracellular Klotho also reversed the loss of antioxidant enzymes, the elevation of cleaved Caspase-3 and Bax/Bcl-2, and the phosphorylation of JNK/MAPK and p38/MAPK.
Klotho has posed antioxidant and anti-apoptotic effects on oxidative injuries in TCMK-1 cells, which might be partially related to its inhibition of JNK/MAPK and p38/MAPK phosphorylation and subsequent elevation of antioxidant enzymes. Increasing Klotho expression has played a protective role against oxidative stress in tubular epithelial cells.
小鼠中klotho基因表达缺陷会导致易受氧化损伤。我们旨在确定klotho在小鼠肾小管上皮（TCMK-1）细胞系中的表达，并研究氧化应激诱导的klotho表达变化以及细胞内和细胞外klotho蛋白的潜在作用。
在过氧化氢（HO）暴露期间，诱导TCMK-1细胞中klotho基因过表达并添加外源性klotho蛋白。通过流式细胞术检测活性氧（ROS）的产生，并使用细胞计数试剂盒-8评估细胞活力。通过流式细胞术和Hoechst 33258染色确定细胞凋亡，随后进行蛋白质印迹以评估klotho、抗氧化酶和凋亡相关蛋白的表达。
HO以浓度依赖性方式显著抑制klotho表达、细胞活力和抗氧化酶的表达，同时细胞凋亡增加，p38/丝裂原活化蛋白激酶（MAPK）和c-Jun氨基末端激酶（JNK）/MAPK被激活。细胞内和细胞外klotho显著改善了HO诱导的活力抑制、ROS产生和细胞凋亡。细胞内和细胞外klotho还逆转了抗氧化酶的丧失、裂解的半胱天冬酶-3和Bax/Bcl-2的升高以及JNK/MAPK和p38/MAPK的磷酸化。
klotho对TCMK-1细胞的氧化损伤具有抗氧化和抗凋亡作用，这可能部分与其对JNK/MAPK和p38/MAPK磷酸化的抑制以及随后抗氧化酶的升高有关。增加klotho表达对肾小管上皮细胞的氧化应激起到了保护作用。
