肥胖相关肾病表观遗传调控的分子机理

Currently obesity and obesity-associated type 2 diabetes are leading causes of chronic kidney disease (CKD). Obesity-related glomerulopathy (ORG) has increased dramatically in the last decades. Recent studies suggest that ectopic lipid accumulation in the kidney is a critical event leading to ORG and other forms of renal injury. Ectopic accumulation of lipids can cause lipotoxicity by promoting mitochondrial dysfunction, oxidative stress and endoplasmic reticulum stress, which lead to activation of the pro-inflammatory and pro-fibrogenic signaling pathways that eventually cause renal injury. Despite its pathological significance, the molecular mechanism responsible for renal ectopic lipid accumulation in obesity and diabetes remains poorly understood. In this proposal we will test the central hypothesis that ATP-citrate lyase (ACL) is a key metabolic and epigenetic regulator that promotes renal ectopic lipid accumulation and renal injury; as such, it represents a novel therapeutic target for CKD in obesity and type 2 diabetes. ACL is a cytosolic and nuclear enzyme that converts extra-mitochondrial citrate to acetyl-CoA in the nucleocytosolic space. Acetyl-CoA is the common building block for the de novo lipogenesis in eukaryotic cells as well as the required substrate for histone acetylation. Hyperglycemia, hyperlipidemia and inflammation are key features of metabolic syndrome. Our working hypothesis is that hyperglycemia and hyperlipidemia as excess nutrient influx increase cellular metabolism and hence citrate and ATP levels. ATP, via protein kinase A (PKA), and inflammation synergistically stimulate ACL expression, which increases nucleocytosolic acetyl-CoA concentration from citrate transported out of the mitochondria. Acetyl-CoA promotes de novo lipogenesis as well as histone acetylation on the genes that regulate lipogensis (such as ACC, FAS and HMGCR) and fibrogenesis (such as TGF-β). Therefore, ACL senses excess nutrients and links extracellular nutrients to ectopic lipid synthesis and fibrogenesis in the kidney. Thus ACL is a potential therapeutic target for renal injury in obesity and type 2 diabetes. In this proposal we will assess the pro-lipogenic and pro-fibrogenic function of ACL in kidney cells, explore the epigenetic role of ACL in the regulation of renal lipogensis and fibrogenesis under obesity and diabetes, and explore the in vivo role of ACL in renal dysfunction in obesity and type 2 diabetes in animal models as well as assess ACL as a novel therapeutic target for treatment of CKD using ACL inhibitor.

肥胖与二型糖尿病已经成为最主要的慢性肾脏病（CKD）病因之一。近年来肥胖相关肾小球病变（ORG）急剧增加。研究表明肾脏的异位脂质堆积（ELA）是ORG及肾脏纤维化的一个关键因素。ELA通过促使线粒体功能异常、氧化应激和内质应激而引起脂毒性，激活促炎症和促纤维化信号通路而引起肾损伤。尽管ELA在ORG和CKD有重大的病理意义，但它在肥胖和糖尿病状态下形成的分子机理仍然不清。本课题的中心假设是ATP柠檬酸裂解酶（ACL）作为一个关键的代谢和表观遗传调控因子在肥胖和二型糖尿病情况下促进ELA而引发肾脏损伤。ACL把细胞质和细胞核的柠檬酸转化成乙酰辅酶A。在真核细胞中乙酰辅酶A既可以作为底物用于脂合成，也可以用于组蛋白乙酰化。本课题将研究在肥胖和二型糖尿病情况下ACL如何通过改变组蛋白乙酰化来促进肾脏的ELA而造成肾损伤以及探索ACL是否可以作为治疗CKD的新靶点。

本课题基本按照原定计划完成，1. 首次报道了在肥胖相关性肾脏病中针对小鼠ACL蛋白的靶向干预治疗作用，并且通过molecular docking的方式确定了已知的4种ACL抑制剂的亲和力排名；我们发现通过抑制db/db肥胖小鼠的ACL蛋白，能够改善小鼠肾脏异位脂质沉积、肾小球硬化及组织纤维化进程，该发表中科院2区期刊Front Endocrinol (Lausanne)中，同时正在申请国家发明专利一项；2. 研究探讨了ACL蛋白在调控肾脏细胞异位脂质沉积及纤维化中的作用，并且发现了在肥胖和糖尿病状态下ACL促进肾脏异位脂质沉积和纤维化的表观遗传调控机制3. 利用非靶向代谢组学脂质谱技术分析了肥胖小鼠的肾脏组织脂质代谢变化，将差异代谢物富集到KEGG通路中发现了sphingolipid metabolism 和 glycerolipid metabolism为最重要的两条通路。利用生物信息学分析肥胖小鼠肾脏组织RNA-seq数据发现sphingolipid metabolism通路依然具有差异，其中ASAH1作为分解神经酰胺的蛋白酶表达量更是明显下降，以此为基础我们正构建ASAH1（酸性神经酰胺酶1）敲除小鼠及肾小管ASAH1特异性过表达小鼠；4.本课题分析肥胖小鼠肾脏组织RNA-seq数据观察到FOXO通路的富集差异，而且发现FOXO蛋白的乙酰化水平在肥胖小鼠的肾脏组织中上升并且加剧了组织氧化应激损伤，由此我们发现ACL的表达量增加并不仅仅只会影响组蛋白乙酰化水平，乙酰辅酶A作为乙酰化重要的底物来源也会对FOXO的乙酰化水平造成影响。总之，通过该课题的完成，共发表SCI论文7篇，中华医学会肾脏病学分会2021年会壁报2篇，2020年会壁报1篇。