PCSK9对足细胞脂质稳态和细胞损伤的作用及机制  

Effect and mechanism of PCSK9 on lipid homeostasis and cell damage of podocytes

作　　者：吴美延[1] 陈志[1] 臧崇森[1] 马福哲[1] 侯洁[1] 许钟镐[1] Wu Meiyan;Chen Zhi;Zang Chongsen;Ma Fuzhe;Hou Jie;Xu Zhonggao(Department of Nephrology,the First Hospital of Jilin University,Changchun 130021,China)

机构地区：[1]吉林大学第一医院肾病科,长春130021

出　　处：《中华肾脏病杂志》2021年第7期583-590,共8页Chinese Journal of Nephrology

基　　金：国家自然科学基金青年科学基金项目(82000689);国家自然科学基金面上项目(81974094);吉林省教育厅"十三五"科学研究规划项目(JJKH20201066K);中华医学会临床医学科研专项资金-施维雅肾脏病研究与发展项目(20010090801);吉林省医疗卫生人才专项项目(JLSCZD2019-066)。

摘　　要：目的:探讨前蛋白转化酶枯草杆菌蛋白酶9(proprotein convertase subtilisin/kexin type 9,PCSK9)对足细胞脂质稳态和细胞损伤的影响及其作用机制。方法:选取12周龄C57BL/6野生型小鼠(对照组,n=10)和系统性敲除PCSK9基因型小鼠(PCSK9 KO组,n=10)为动物模型,经心脏全身灌注后取肾组织。体外培养小鼠足细胞,用干扰小RNA(siRNA)敲低足细胞PCSK9的表达。用荧光染料BODIPY 493/503染色法观察小鼠肾组织肾小球及体外培养足细胞内的脂质蓄积程度;透射电子显微镜观察足细胞的足突、线粒体结构和脂滴大小及其分布;TUNEL染色法评估肾小球内的细胞凋亡;实时荧光定量PCR(qPCR)和Western印迹法检测线粒体功能相关关键酶过氧化物酶体增殖物激活受体C辅激活因子1α(peroxisome proliferator-activated receptor-C coactivator 1α,PGC-1α)、肉毒碱棕榈酰转移酶1(carnitine palmitoyltransferase 1,CPT-1)、乙酰辅酶A氧化酶1(acetyl CoA oxidase 1,Acox-1),以及凋亡相关指标的mRNA和蛋白表达水平。结果:与对照组相比,PCSK9 KO组小鼠肾小球内脂质蓄积更显著;肾组织内线粒体功能相关关键酶PGC-1α蛋白和mRNA相对表达量均显著降低,CPT-1和Acox-1的mRNA相对表达量也均显著降低(均P<0.05);肾小球足细胞内线粒体肿胀、嵴消失,足细胞的足突部分融合、消失;肾小球内细胞凋亡指数增加(P<0.05)。与对照组比较,体外培养的PCSK9 siRNA组足细胞内脂质蓄积显著,线粒体功能相关关键酶PGC-1α、CPT-1和Acox-1的mRNA相对表达量下降,线粒体结构受损,细胞凋亡指数增加(均P<0.05)。结论:PCSK9参与足细胞的脂代谢平衡,PCSK9表达减少增加足细胞内脂质沉积,诱导线粒体结构及功能受损,导致细胞凋亡。Objective To evaluate the effect of proprotein convertase subtilisin/kexin type 9(PCSK9)on lipid homeostasis and cellular injury of podocytes,and to clarify its mechanism.Methods Twelve-week old C57BL/6 wild-type mice(n=10)and PCSK9 knockout(PCSK9 KO)mice(n=10)were selected as the animal models.The renal tissues were taken after perfusion through heart.Mouse podocytes were transfected with PCSK9 siRNA to downregulate PCSK9 expression.BODIPY 493/503 staining was performed for evaluating lipid accumulation,and standard transmission electron microscopy(TEM)was used to observe the foot process of podocytes,the shape of mitochondria and lipid droplet in podocytes.TUNEL staining was carried out to evaluate cell apoptosis in glomerulus.The parameters about mitochondria function(key enzymes such as PGC-1α,CPT-1 and Acox-1)and apoptosis were quantified through qPCR and western blotting.Results The lipid accumulation in glomerulus of PCSK9 KO mice were more serious than controls.The expression of PGC-1αprotein and PGC-1α,CPT-1 and Acox-1 mRNA in PCSK9 KO mouse kidney tissues were decreased than controls(all P<0.05),and mitochondria swelling and cristae disappearance in podocytes of PCSK9 KO mice were observed.In PCSK9 KO group,the foot process of podocytes partially fused and disappeared,and the apoptosis index increased compared with the control group(P<0.05).In vitro,compared with the control group,the lipid accumulation was more significant,transcription level of key enzymes related to mitochondrial function was decreased,mitochondrial structure was damaged and the apoptosis index was increased in cultured podocyte PCSK9 siRNA group(all P<0.05).Conclusions PCSK9 is involved in the lipid homeostasis of podocytes.The decrease of PCSK9 results in the increase of intracellular lipid accumulation,accompanied by the mitochondrial structure damage and disfunction of podocytes,and leads to cell apoptosis.

关 键 词：足细胞 线粒体 脂代谢障碍 脂质沉积 前蛋白转化酶枯草杆菌蛋白酶9 凋亡 

分 类 号：R692[医药卫生—泌尿科学]