ABCA1介导胆固醇外流障碍致细胞胆固醇超负荷在糖尿病肾病肾小球内皮细胞免疫炎症损伤中的作用机制研究

Cholesterol efflux from glomerular endothelial cell in diabetic nephropathy is a new research field in the pathogenesis of diabetic nephropathy. Our previous study had found that: cholesterol metabolites and regulators were increased in diabetic nephropathy compared with normal control; intracellular lipid droplets and cholesterol accumulation were found in glomerular endothelial cells detected by electron microscope and specific stainings. Our further studies had shown that: ABCA1, one of the most important regulators of cholesterol efflux, was downregulated in isolated glomeruli and glomerular endothelial cells in diabetic nephropathy; high glucose could upregulate the expression of ABCA1in human glomerular endothelial cells in vitro which could be downregulated by cholesterol. It may imply that cholesterol efflux mediated by ABCA1 may be involved in the pathogenesis of diabetic nephropathy and may be the interventional target. Some other studies have also shown that cholesterol accumulation can promote inflammatory responses by activating Toll-like receptor (TLR) /MyD88-NF-κB signalling pathway. All these revealed that cholesterol efflux impairmment mediated by downregulation of ABCA1 may lead to intracellular cholesterol overload in glomerular endothelial cell in diabetic nephropathy, in whichTLRs/MyD88-NF-κB signaling pathway may be involved and plays an important role in the cholesterol-mediated inflammatory injury. Specifically glomerular endothelial cell ABCA1 gene knockout and transgenic mice and glomerular endothelial cells will be used in this study to investigate the role and related mechanisms of ABCA1 in two models of diabetic nephropathy.

胆固醇外流障碍致糖尿病肾病肾小球内皮细胞免疫炎症损伤是糖尿病肾病发病机制研究的新领域。我们前期研究发现：糖尿病肾病中，肾脏胆固醇代谢物及调节物较正常对照升高；电镜及肾小球脂质、胆固醇染色显示肾小球内皮中大量脂滴及胆固醇的积聚；进一步研究显示调控细胞胆固醇外流的膜蛋白ABCA1在糖尿病肾病肾小球及肾小球内皮中表达下调；高糖可诱导体外培养人肾小球内皮细胞ABCA1表达下调，而胆固醇可诱导其上调。有研究显示，细胞内胆固醇积聚可通过激活天然免疫系统及炎症反应TLRs/MyD88-NF-κB信号通路致细胞炎症损伤。由此推测：糖尿病肾病中，ABCA1下调所介导的胆固醇外流障碍致肾小球内皮细胞内胆固醇超负荷可能通过激活TLRs/MyD88-NF-κB信号通路导致肾脏免疫炎症损伤。本研究将在ABCA1特异性肾小球内皮敲除及转基因糖尿病肾病小鼠模型及肾小球内皮细胞中研究ABCA1作用及机制。

随着糖尿病发病率和患病率全球剧增，糖尿病肾病已经成为全球终末期肾脏病和慢性肾脏病的首位病因。高血糖及血脂异常是糖尿病的两大特征。持续的高血糖导致脂肪酸的合成增加、甘油三酯的堆积及胆固醇的糖基化与氧化修饰，在肾脏等非脂肪组织的异位脂质沉积可导致肾脏等脏器损伤和功能障碍。细胞内胆固醇稳态对细胞发挥正常生理作用至关重要，其中任何一个环节异常都可能导致胞内胆固醇稳态失衡、胆固醇异常积聚而引发一系列病理生理过程。ATP结合盒转运子A1（ABCA1）在调节胆固醇外流，维持细胞内胆固醇稳态中起着重要作用。细胞内胆固醇蓄积带来的细胞损伤中，细胞凋亡是细胞损伤的主要表现之一，而内质网应激是近年来新发现的介导细胞凋亡的途径之一。因此，本研究在体内部分通过构建肾小球内皮细胞特异性敲除ABCA1基因小鼠，并建立糖尿病肾病模型开展研究，体外在人肾小球内皮细胞中通过敲低ABCA1开展研究。研究结果发现：1、ABCA1缺陷致胆固醇外流障碍促使脂质大量沉积于肾小球内皮细胞，其脂质毒性介导了促炎反应及细胞凋亡损伤，破坏了内皮细胞表面的糖萼层，损害肾小球滤过屏障，加重了糖尿病肾病小鼠的肾脏损害；2、ABCA1缺陷介导的胆固醇外流障碍可以显著增加糖尿病肾病小鼠肾组织的内质网应激，进而激活NFκB及caspase 3信号通路引起炎症及细胞凋亡；3、体外实验证实敲低肾小球内皮细胞ABCA1介导的胆固醇外流障碍致胆固醇在细胞内蓄积，可加重内质网应激反应而激活NFκB及caspase3信号通路，从而加重肾小球内皮细胞炎症损伤及凋亡，而过表达ABCA1增加胆固醇外流及抑制内质网应激反应可以缓解肾小球内皮细胞损伤及凋亡。本研究初步证实肾小球内皮细胞胆固醇聚集在糖尿病肾病发生发展中的重要作用；初步证实ABCA1 通过调节肾小球内皮细胞胆固醇外流平衡而可能成为未来糖尿病肾病治疗的干预靶点，值得未来开展深入研究并向临床转化。