Liu Changxuan, Lou Meiyu, Ding Yu, Wang Yin, Huang Yunfang, Shao Danni, Chen Wenli
Division of Nephrology, The Central Hospital of Wuhan, Tongji Medical College, Huazhong University of Science and Technology Wuhan 430014, China.
Key Laboratory for Molecular Diagnosis of Hubei Province, The Central Hospital of Wuhan, Tongji Medical College, Huazhong University of Science and Technology Wuhan 430014, China.
Int J Clin Exp Pathol. 2018 May 1;11(5):2305-2313. eCollection 2018.
This study aimed to investigate the underlying molecular mechanism of ouabain-induced apoptosis and inhibited viability of tubulointerstitial cells in lupus nephritis (LN). Serum ouabain expression in 21 LN patients and 20 healthy controls (HCs) was detected by ELISA. Na-K-ATPase α1 (NKA) expression in kidney tissue from 21 LN patients and 6 controls who underwent nephrectomy was determined by immunohistochemistry assay. HK-2 cells were treated by ouabain with concentration 0 nM (control), 0.1 nM, 1 nM, 10 nM, 100 nM and 1000 nM for 72 h; MTT assay was performed at 24 h, 48 h and 72 h to detect cells viability, AV/PI assay was conducted at 24 h to detect the cell apoptosis. NKA expression was detected by immunofluorescence assay and western blot (WB) while pSrc, pERK, pAkt, pS6K and caspase 3 expression was detected by WB after 100 nM ouabain treatment. Serum ouabain was elevated in LN patients compared to HCs, while kidney NKA was reduced in LN patients compared with controls. HK-2 cell viability by MTT assay was decreased while cell apoptosis by AV/PI was increased at each time point after ouabain 0.1 nM, 1 nM, 10 nM, 100 nM and 1000 nM treatment compared to control group. Both immunofluorescence and WB disclosed that NKA expression was decreased at 12 h and 24 h compared to 0 h after 100 nM ouabain treatment. Moreover, pSrc, pERK, pAkt, pS6K and caspase 3 expressions were elevated after 100 nM ouabain treatment. In conclusion, ouabain may contribute to LN etiology by inhibiting human proximal tubular cell viability and promoting cells apoptosis through regulating NKA, pSrc, pERK, pAkt, pS6K and caspase 3.
本研究旨在探讨哇巴因诱导狼疮性肾炎（LN）肾小管间质细胞凋亡及抑制其活力的潜在分子机制。采用酶联免疫吸附测定（ELISA）检测21例LN患者和20例健康对照者（HCs）血清中哇巴因的表达。采用免疫组织化学法检测21例LN患者及6例接受肾切除术的对照者肾组织中钠钾ATP酶α1（NKA）的表达。将人肾小管上皮细胞系（HK-2细胞）分别用浓度为0 nM（对照）、0.1 nM、1 nM、10 nM、100 nM和1000 nM的哇巴因处理72小时；分别于24小时、48小时和72小时进行MTT试验检测细胞活力，于24小时进行Annexin V/碘化丙啶（AV/PI）试验检测细胞凋亡。采用免疫荧光法和蛋白质免疫印迹法（WB）检测NKA表达，用蛋白质免疫印迹法检测100 nM哇巴因处理后磷酸化Src（pSrc）、磷酸化细胞外信号调节激酶（pERK）、磷酸化蛋白激酶B（pAkt）、磷酸化核糖体蛋白S6激酶（pS6K）和半胱天冬酶3的表达。与健康对照者相比，LN患者血清中哇巴因升高；与对照者相比，LN患者肾组织中NKA降低。与对照组相比，经0.1 nM、1 nM、10 nM、100 nM和1000 nM哇巴因处理后的各时间点，MTT法检测显示HK-2细胞活力降低，AV/PI法检测显示细胞凋亡增加。免疫荧光法和蛋白质免疫印迹法均显示，100 nM哇巴因处理后12小时和24小时，NKA表达较0小时降低。此外，100 nM哇巴因处理后，pSrc、pERK、pAkt、pS6K和半胱天冬酶3的表达升高。综上所述，哇巴因可能通过调节NKA、pSrc、pERK、pAkt、pS6K和半胱天冬酶3，抑制人近端肾小管细胞活力并促进细胞凋亡，从而参与LN的发病机制。
