摘要
目的:研究Pleckstrin同原序列富亮氨酸重复片段蛋白磷酸酶(PHLPP2)在急性肾损伤(AKI)发生发展中的作用及机制;方法:将20只野生型C57BL/6(WT)小鼠与20只敲除PHLPP2(PHLPP2^-/-)表达的转基因小鼠按是否使用顺铂(CDDP)处理进行分组,即WT对照组、WT CDDP处理组、PHLPP2^-/-对照组和PHLPP2^-/-CDDP处理组(每组10只)。3d后处死小鼠,RT-PCR及Western Blot检测各组小鼠中PHLPP2 mRNA及蛋白水平;免疫组织化学染色检测肾组织PHLPP2表达及定位;HE与TUNEL染色检测肾组织损害和细胞的凋亡情况;分离并鉴定小鼠肾小管上皮细胞(RTECs),透射电子显微镜观察RTECs中线粒体结构;JC-1法检测RTECs中线粒体膜电位变化;Western Blot检测RTECs中促凋亡蛋白Bax、Caspase-3,及凋亡抑制蛋白Bcl-2和Akt的Thr308与Ser473位点磷酸化水平。结果:CDDP成功诱导AKI模型,肾脏组织中PHLPP2表达显著增加;与WT CDDP组相比,PHLPP2^-/-CDDP组小鼠的血尿素氮、血清肌酐明显降低,肾组织结构损害较轻,凋亡细胞减少,线粒体结构损伤减轻,线粒体膜电位下降减少;敲除PHLPP2基因表达可显著促进Bcl-2蛋白表达,而抑制Bax、Caspase-3蛋白表达,且PHLPP2基因通过对Akt的Thr308与Ser473位点去磷酸化而调节上述蛋白表达。结论:敲除PHLPP2表达能够通过减轻线粒体膜电位的下降,保护线粒体的正常结构及功能,从而通过线粒体途径的凋亡改善AKI损伤。
Objective:To study the role and mechanism of protein phosphatase PHLPP2 in development of acute kidney injury(AKI);Methodology:Wild type C57BL/6(WT)and knockout PHLPP2(PHLPP2^-/-)mice were divided into WT control group,WT cisplatin(CDDP)treatment group,PHLPP2^-/- control group and PHLPP2^-/- CDDP treatment group(10 mice in each group)to establish AKI model.Three days later,the mice were killed.The mRNA and protein levels of PHLPP2 were detected by RT-PCR and Western Blot;HE and TUNEL staining were used to detect renal tissue damage and cell apoptosis,renal tubular epithelial cells(RTECS)were isolated and identified;JC-1 method was used to detect the changes of mitochondrial membrane potential;The protein levels of Bax,Caspase-3,and Bcl-2 and the phosphorylation levels of Thr308 and Ser473 in AKt Kinase were detected by Western Blot assay.Results:In AKI model induced by CDDP,expression of PHLPP2 in kidney tissue was significantly increased;compared with WT CDDP group,renal tissue structure damage and apoptosis were alleviated.Mitochondrial structure damage was reduced,and mitochondrial membrane potential was decreased;Knockout of PHLPP2 gene could significantly promote expression of Bcl-2 protein,but inhibit expression of Bax and caspase-3.PHLPP2 gene regulated expression of these proteins through dephosphorylation of thr308 and ser473 of Akt.Conclusion:Knockout of PHLPP2 can protect normal structure and function of mitochondria by alleviating the decline of mitochondrial membrane potential,thus improving AKI injury through mitochondrial apoptosis.
作者
陈丽
朱美娟
陈娟
应海玲
唐文庄
CHEN Li;ZHU Meijuan;CHEN Juan;YING Hailing;TANG Wenzhuang(Department of Blood Purification,The First Affiliated Hospital of Hainan Medical College,Haikou 570102,China;School of Basic Medicine and Life Science,Hainan Medical College,Haikou 571199,China)
出处
《肾脏病与透析肾移植杂志》
CAS
CSCD
北大核心
2020年第5期443-449,共7页
Chinese Journal of Nephrology,Dialysis & Transplantation
基金
海南省卫生计生行业科研项目(1801320712A2060)。
