PCSK9对C57BL/6小鼠肾组织脂质平衡及损伤的作用

摘要

目的 评估PCSK9(proprotein convertase subtilisin kexin type 9)对C57BL/6小鼠肾脏组织脂质平衡的影响以及损伤作用.方法 12周龄的C57BL/6野生型小鼠和系统性敲除PCSK9基因小鼠通过代谢笼收集24 h尿,经心全身灌注,取出肾脏.采用ELISA方法检测尿微量白蛋白、血尿肌酐、血总胆固醇、肾组织内总胆固醇和甘油三酯水平,BODIPY 493/503染色,透射电镜(TEM)观察肾组织内脂质蓄积;PAS染色、TUNNEL染色以及qPCR和Western印迹法评估肾组织损伤及细胞凋亡.结果 与C57BL/6野生型对照组相比,PCSK9 KO组肾组织内总胆固醇及甘油三酯水平升高(均P<0.05),BODIPY 493/503染色显示肾小球及肾小管细胞内脂质染色明显加深,TEM提示肾小管细胞内脂滴数目显著增多.系统性敲除PCSK9基因小鼠较C57BL/6野生型对照组尿微量白蛋白/尿肌酐升高(P<0.05),Podocin和Nephrin的转录水平下降(均P<0.05),TEM可见肾小球内足细胞足突变短、不同程度的融合.与野生型对照组相比,PCSK9 KO组小鼠肾组织内Bax和Cleaved Caspase 3表达升高,Bcl?2表达下降(均P<0.05).结论 PCSK9表达下降增加肾组织细胞内脂质蓄积,继而诱导肾固有细胞的损伤和凋亡.%Objectives To evaluate the role of PCSK9 (proprotein convertase subtilisin kexin type 9) on the lipid accumulation and kidney injury of C57BL/6 mice. Methods The 24 h urine of 12 weeks old wide type C57BL/6 mice and PCSK9 knockout (KO) mice were collected through a metabolic cage, followed by perfusion and sacrifice. Urinary microalbumin?to?creatinine ratio (UACr), total cholesterol and triglyceride in kidney tissues were measured by ELISA. BODIPY 493/503 staining and standard transmission electron microscopy (TEM) of kidney tissues was performed for evaluating lipid accumulation and podocyte foot effacement in the kidney. Kidney tissues were also evaluated by PAS stain and TUNNEL stain. PCSK9, podocin and nephrin were quantified through real?time PCR, and the Bcl?2, Bax and cleaved caspase 3 were evaluated by Western blotting. Results Total cholesterol and triglyceride contents were higher in the kidneys of PCSK9 KO mice than controls (P<0.05). The level of lipid accumulation in glomeruli and tubules through BODIPY 493/503 stain, and the amount of lipid drop in TEM were more serious in PCSK9 KO mice. UACr and podocyte foot process effacement were increased, and the transcription of podocin and nephrin were decreased in the kidneys of PCSK9 KO mice (all P<0.05). The expression of Bcl?2 was decreased, and Bax and cleavedcaspase 3 were increased in the kidney samples of PCSK9 KO mice. Conclusion PCSK9 might be reversely involved in lipid homeostasis and accumulation, resulting in injury and apoptosis in the kidneys of C57BL/6 mice.