ABCA1在血管紧张素Ⅱ诱导足细胞胆固醇蓄积中的作用

Role of ABCA1 in angiotensin Ⅱ -induced cholesterol accumulation in podocytes

目的研究血管紧张素Ⅱ（AngII）作用下大鼠肾小球及条件永生性人足细胞三磷酸腺苷结合盒转运子A1（ATP—bindingcassettetransporterA1，ABCAl）的表达变化，探讨ABCAl在Ang11诱导足细胞胆固醇蓄积中的作用。方法12只无特定病原（SPF）级雄性Wistar大鼠皮下埋置微量渗透泵，随机数字表法分为Angll组（AngII400ng·kg-1·min-1，n=6）和正常对照组（生理盐水替代Angll，n=6），造模8周后处死大鼠取肾。实时荧光定量PCR及Western印迹检测肾小球ABCAlmRNA及蛋白水平。体外培养条件永生性人足细胞，分为正常对照组、AngⅡ刺激组（浓度10-mol／L，刺激时间24h）、AngII＋无功能小干扰RNA（scrambledsiRNA）组和AngⅡ＋ABCAIsiRNA组，免疫荧光法观察足细胞ABCAl表达，实时荧光定量PCR及Western印迹法检测足细胞ABCAlmRNA和蛋白表达，油红O染色观察足细胞内脂滴沉积情况，胆固醇外流试剂盒检测胆固醇外流率，异硫氰酸荧光素（FITC）-鬼笔环肽（phalloidin）染色观察足细胞骨架改变。结果体内实验中，与正常对照组相比，Angll灌注8周组大鼠肾小球ABCAlmRNA及蛋白表达均降低（P〈0．05）；体外实验中免疫荧光结果显示ABCAl沿足细胞膜表达，与正常对照组相比，AngⅡ刺激后ABCAImRNA及蛋白表达下降（均P〈0．05），足细胞内脂滴沉积增加，胆固醇外流减少（P〈0．05），足细胞出现骨架重构；ABCAlsiRNA可在AngⅡ刺激基础上进一步降低ABCAl表达，与AngⅡ组比较，足细胞脂滴沉积程度更重，胆固醇外流更少（P〈0．05）。结论AngII可通过诱导ABCAl表达下调导致足细胞胆固醇蓄积。

Objective To investigate the effects of angiotensin II （Ang Ⅱ ） on the expression of ATP- binding cassette transporter AI（ABCA1） in Ang Ⅱ -infused rat model and cultured human podocytes, and to explore the role of ABCA1 in Ang Ⅱ -induced cholesterol accumulation of podocytes. Methods Twelve Wistar rats were randomly subjected to normal saline infusion, or Ang Ⅱinfusion at 400 ng·kg-1·min-1（via subcutaneous osmotic minipumps） for 8 weeks. The expression of glomerular ABCA1 was analyzed by Western blotting and real- time fluorescent quantitative PCR. In vitro, conditionally immortalized human podocytes were divided into normal group, Ang Ⅱ group, Ang Ⅱ＋ scrambled siRNA group, Aug Ⅱ＋ ABCA1 siRNA group. The expression of podocyte ABCA1 was assessed by immunofluorescence, Western blotting and real-time fluorescent quantitative PCR. Oil Red O staining was used to observe the lipid droplets in podocytes and cholesterol efflux assay kit was usedto measure the cholesterol efflux rate of podocytes. Fluorescein isothiocyanate （F1TC）- conjugated phalloidin was used to observe the podocyte cytoskeleton. Results In vivo, compared with normal group, the protein and mRNA expression of glomerular ABCA1 in Ang Ⅱ -infused rats were decreased （P 〈 0.05）. In vitro, ABCA1 was distributed in the cytomembrane of podocytes, and compared with normal group, Ang II treatment down- regulated the expression of ABCA1 （P 〈 0.05）. Increased lipid droplets, decreased cholesterol efflux and cytoskeletal rearrangement were observed in Ang Ⅱ -treated podocytes. When compared to Ang II group, podocytes stimulated by Ang II and then transfected with ABCAI siRNA had lower expression level of ABCA1 mRNA and protein （all P 〈 0.05）. More lipid droplets and lower cholesterol efflux rate could be observed in Ang Ⅱ＋ ABCA1 siRNA group （P 〈 0.05）. Conclusion The reduced expression of ABCA1 may be involved in Ang Ⅱ -induced cholesterol accumulation in podocytes.