摘要
目的:探讨钙/钙调素依赖性蛋白激酶Ⅱ(CaMKⅡ)和Yes相关蛋白(YAP)是否参与急性肾损伤(AKI)中肾小管上皮细胞(RTEC)凋亡及其机制。方法:15只6~8周雌性BALB/c小鼠随机分为正常对照组、阿霉素(ADR)干预组、ADR+ CaMKⅡ抑制剂(K)干预组( n=5)。ADR干预小鼠建立AKI模型,HE染色观察小鼠肾小管细胞损伤,酶联免疫吸附检测小鼠血肌酐值。ADR干预体外培养HK-2细胞建立RTEC凋亡模型,流式细胞仪检测ADR、ADR+K、YAP-siRNA、CaMKⅡ活化剂(C)、C +oYAP(过表达YAP)对细胞凋亡率的影响。免疫印迹检测小鼠肾组织和HK-2细胞总蛋白Bax和Bcl-2、胞质蛋白pCaMKⅡ和CaMKⅡ、核YAP蛋白的表达。 结果:与正常对照组比较,ADR干预小鼠肾小管细胞损伤增加和血肌酐值升高,予K后肾小管细胞损伤减少、血肌酐值降低(均 P<0.05)。ADR干预的RTEC凋亡率明显高于对照组( P<0.05),予K后细胞凋亡减轻( P<0.05)。与正常对照组和siRNA空白对照组比较,YAP-siRNA和C干预的RTEC中细胞凋亡率明显增加(均 P<0.05),而C干预RTEC的同时予oYAP后细胞凋亡又减轻( P<0.05)。与正常对照组比较,ADR干预的RTEC和小鼠肾组织细胞中,pCaMKⅡ/CaMKⅡ、Bax蛋白的表达增加而核YAP蛋白、Bcl-2蛋白的表达减少(均 P<0.05),予K后这些作用减轻(均 P<0.05)。 结论:CaMKⅡ可能通过YAP调节ADR诱导AKI小鼠的RTEC凋亡。
Objective To investigate whether calcium/calmodulin dependent protein kinaseⅡ(CaMKⅡ)and Yes-associated protein(YAP)are involved in apoptosis of renal tubular epithelial cells(RTEC)in acute renal injury(AKI)and the underlying mechanisms.Methods Fifteen female BALB/c mice aged 6-8 weeks were randomly divided into the normal control group,adriamycin(ADR)intervention group and ADR+CaMKⅡinhibitor(K)intervention group(n=5 each).AKI was modeled in mice by ADR intervention.HE staining was used to observe the renal tubular cell injury.Enzyme-linked immunosorbent assay was used to measure the serum creatinine level in the mice.The RTEC apoptosis model was established in vitro by ADR intervention of HK-2 cells.Flow cytometry was used to examine the effects of ADR,ADR+K,YAP siRNA,CaMKⅡactivator(C),and C+YAP overexpression(oYAP)on the apoptosis rates.Western blotting was used to measure the expression of total Bax and Bcl-2 proteins,cytoplasmic(pCaMKⅡ)and total proteins of CaMKⅡ,and nuclear YAP protein in mouse renal tissues and the HK-2 cells.Results Compared with the normal control group,ADR intervention increased the RTEC injury and serum creatinine level in mice,and the RTEC injury and the serum creatinine level were lowered after administration of K(all P<0.05).The ADR intervention group had significantly higher rate of RTEC apoptosis than that in the control group(P<0.05),and the apoptosis was attenuated after administration of K(P<0.05).Compared with the normal control group and the siRNA blank control group,the apoptosis rate of RTEC treated with YAP siRNA and C was significantly increased(both P<0.05).The apoptosis rate of C-treated RTEC was reduced in the presence of oYAP(P<0.05).Compared with the normal control group,the RTEC and mouse renal tissue cells intervened by ADR showed higher protein expression of pCaMKⅡ/CaMKⅡand Bax,and lower protein expression of nuclear YAP and total Bcl-2(all P<0.05).These effects were alleviated after administration of K(all P<0.05).Conclusion CaMKⅡmay regulate ADR-induced apoptosis of RTEC in AKI mice via YAP.
作者
黄宗顺
肖洁
陈亚琦
Huang Zongshun;Xiao Jie;Chen Yaqi(Department of Nephrology,First Affiliated Hospital of Guangzhou Medical University,Guangzhou 510120,China)
出处
《中华生物医学工程杂志》
CAS
2022年第5期522-527,共6页
Chinese Journal of Biomedical Engineering
基金
国家自然科学基金青年项目(81700594)
广东省自然科学基金博士启动纵向协同单位管理项目(2017A030310075)
广州市科技计划基础与应用基础研究项目(202201020403)。
