琥珀酸/GPR91/cAMP 信号在肾间质纤维化中的作用及机制研究

Succinate has become a new pathogenic factor from the bystanders of mitochondrial metabolism. Succinate acts an extracellular ligand by binding to G-protein coupled receptor (GPR91) and regulating cyclic adenosine monophosphate (cAMP) signaling, leading to a wide array of physiological and pathological effects.GPR91 is highly expressed in kidney. Local levels of succinate in the kidney together with GPR91 activates the renin- angiotensin system may play a key role in the development of hypertension and the complications of diabetes mellitus, metabolic disease. Succinic acid is mainly absorbed in the proximal renal tubule, and the receptor GPR91 is also expressed in proximal tubules. But the role of succinate/GPR91 remains to be studied in detail. cAMP level in proximal tubular epithelial cells is correlated with tubular injury and renal fibrosis by modulating mitochondrial biogenesis. Therefore, the hypothesis is that through manipulating GPR91/cAMP signaling, succinate plays a critical role in renal interstitial fibrosis..This research project will be set up as follows. First of all, in vitro and in vivo models of renal interstitial fibrosis will be established. Then we will analysis the metabolites changes in the progress of renal interstitial fibrosis, and find the mechanism of succinate metabolism. Nest, we will compare cAMP level and the degree of fibrosis in the fibrotic kidneys between proximal tubular epithelial cells- GPR91 specific knockout mice and control mice to explore the mechanism and the role of succinate/GPR91/cAMP in renal interstitial fibrosis. .This study will not only help us to understand the pathogenesis of renal interstitial fibrosis from the new perspective of energy metabolism, but also provide a new clue for the prevention and treatment of renal interstitial fibrosis.

三羧酸循环中间代谢产物琥珀酸已成为新型致病分子，其可通过琥珀酸受体（GPR91）调节胞内环磷酸腺苷(cAMP)水平参与组织的损伤，如高血压肾病、糖尿病肾病的发生发展。GPR91高表达于肾近端肾小管，琥珀酸/GPR91/cAMP信号对近端肾小管的直接作用尚未有深入研究。本项目以近端肾小管上皮细胞为研究对象，应用模式动物和细胞/分子生物学等技术与方法，分析肾脏病变过程中三羧酸循环中间代谢产物琥珀酸的变化特点、调节机制以及与肾间质纤维化的关系；同时利用近端肾小管上皮细胞GPR91特异性敲除小鼠，重点研究琥珀酸通过GPR91对cAMP的调控作用，探索琥珀酸/GPR91/cAMP在肾间质纤维化发生发展中的作用机制，确立细胞代谢异常在慢性肾脏病中的作用。本项目以期通过能量代谢新的角度阐明肾间质纤维化的病理机制，为深入认识慢性肾间质纤维化的病理机制及未来设计全新的防治肾间质纤维化的治疗方案提供理论基础。

琥珀酸作为三羧酸循环的核心代谢产物，被认为是表观遗传的调节者、致癌的代谢物、缺氧反应的中介体、活性氧代谢的重要传输者以及炎症反应调节。琥珀酸受到肾脏排泄和重吸收的调节，其特异性G蛋白受体GPR91在肾脏表达。本课题以近端肾小管上皮细胞为研究对象，重点探讨琥珀酸/GRP91在肾间质纤维化过程中的作用以及相关调控机制。主要内容是分析纤维化进程中肾小管上皮细胞琥珀酸水平的变化及增加的原因；从琥珀酸结合琥珀酸受体GPR91方面，分析在肾间质纤维化中的作用及机制；从琥珀酰化修饰的角度分析了肾间质纤维化进程中琥珀酸和琥珀酰辅酶A调控的作用及调控机制。我们研究结果发现肾间质纤维化进程中肾小管上皮细胞（PTCs）分泌琥珀酸增多，琥珀酸脱氢酶逆向催化激活是小管上皮细胞分泌琥珀酸的主要来源。体外TGF-b1刺激原代PTCs，体外UUO和I/R模型都观察到肾小管上皮细胞GPR91的增加。GPR91敲除可以抑制UUO造成的肾间质纤维化和TGF-b1的作用；而过表达GPR91则具有相反的作用。肾间质纤维化进程中cAMP-Epac通路受到抑制。给予特异性Epac激动剂可以阻断TGF-b1的作用。过表达GPR91可以进一步减少cAMP的含量和Epac的表达；而敲除GPR91则可以恢复cAMP水平和Epac的表达。证实琥珀酸通过GPR91抑制cAMP/Epac通路是促进肾间质纤维化的重要机制。我们发现琥珀酸可以刺激PTCs琥珀酰化修饰的增强。琥珀酰化修饰增强的现象同样在小鼠肾纤维化的肾小管和慢性肾脏病（CKD）患者的肾小管中出现。SIRT5为重要的去琥珀酰化酶。其表达在CKD患者肾小管上皮细胞下降。我们通过构建肾小管上皮细胞SIRT5敲除小鼠和SIRT5过表达小鼠证实SIRT5调节去琥珀酰功能在肾间质纤维化中具有保护作用。琥珀酰化组学发现HSP60是重要的靶蛋白，K249位点是重要的修饰位点。HSP60琥珀酰化修饰增加后其折叠功能下降，导致未折叠蛋白在线粒体内的沉积，抑制线粒体功能，并进一步激活mtUPR反应。该研究证实肾间质纤维化早期琥珀酰化修饰导致线粒体蛋白未折叠是引起线粒体功能障碍的重要机制。