IGFBP6-Setdb2-FH轴调控成纤维细胞有氧糖酵解诱导肾纤维化的作用及机制研究

It is still an urgent issue for urologists that how to prevent persistent progression of kidney fibrosis caused by hydronephrosis. A switch of metabolism to aerobic glycolysis for renal fibroblasts facilitate their activation into myofibroblasts, leading to kidney fibrosis, but the underline mechanism is obscure. In our preliminary investigation, metabolism modulatory protein IGFBP6 increased in human and mice severe hydronephrotic kidney tissues. Silencing IGFBP6 in rat kidney fibroblast(NRK)-49F lead to decreased expression of histone lysine methylation transferase-2(Setdb2) but increased expression of fumarate hydratase(FH); and then inhibited the related enzymes of aerobic glycolysis and prevent their activation into myofibroblasts. We speculate that in the process of kidney fibrosis, high expression of IGFBP6 may promote the expression of Setdb2 and histone lysine methylation so as to inhibit FH transcription, which consequently enhance aerobic glycolysis of fibroblasts and finally facilitate kidney fibrosis. Our project will respectively employ IGFBP6-/- mice model and primary mice kidney fibroblast, and then silencing FH in vivo and in vitro to investigate the role and mechanism of IGFBP6-Setdb2-FH axis in promoting aerobic glycolysis of fibroblast leading to kidney fibrosis. Our study will provide important theoretical and experimental basis for elucidating the mechanisms of kidney fibrosis and investigating the potential treatment strategy of hydronephrosis.

如何缓解肾积水所致的肾纤维化持续进展，是泌尿外科临床上亟待解决的问题。肾成纤维细胞有氧糖酵解增强促进其活化为肌成纤维细胞，引起肾纤维化，但具体的调控机制所知甚少。我们预实验发现：代谢调节蛋白IGFBP6在人和小鼠重度积水肾组织中高表达，沉默鼠肾成纤维细胞IGFBP6后，组蛋白赖氨酸甲基转移酶2（Setdb2）表达减少、延胡索酸酶（FH）表达升高，并抑制有氧糖酵解相关酶，阻止其向肌成纤维细胞活化。我们推测：肾纤维化过程中，IGFBP6高表达促进Setdb2表达升高，使组蛋白赖氨酸甲基化进而抑制FH转录，致成纤维细胞有氧糖酵解增强，诱导肾纤维化。本项目拟使用IGFBP6-/-小鼠模型及原代肾成纤维细胞，在整体和细胞水平沉默FH，探讨IGFBP6-Setdb2-FH轴调控成纤维细胞有氧糖酵解介导肾纤维化的作用及机制；本研究为解析肾纤维化的发生机制，探寻肾积水的防治策略，提供重要的理论依据。

肾纤维化是所有慢性肾脏病的最终病理通路，尽管进行了大量研究但仍机制不清，临床上缺少有效的治疗靶点。研究报道，IGFBP6在慢性肾病患者血清中表达升高，但其在肾纤维化中的作用目前尚不清楚。本研究从分子、细胞、动物模型三个层面，采用基因修饰等方法，以IGFBP6-HK2-有氧糖酵解为主线，研究IGFBP6在肾纤维化中的功能及机制。我们创新性发现：1).IGFBP6在人、小鼠纤维化肾脏组织中表达显著升高；2).我们构建了IGFBP6全身敲除或肾小管上皮细胞条件性敲除小鼠，并行单侧输尿管梗阻造模，发现IGFBP6敲除可显著改善肾纤维化。3）我们进行了IGFBP6的功能回复实验：进一步通过IGFBP6敲除小鼠尾静脉注射IGFBP6-AAV9后可逆转IGBFP敲除后肾纤维化的缓解。4) 在体外实验中，我们通过在细胞上过表达或沉默IGFBP6，发现其在肾小管上皮细胞或肾成纤维细胞中均具有促纤维化的功能。5）进一步研究发现，IGFBP6升高可通过促进肾小管上皮细胞的有氧糖酵解促进肾纤维化，且依赖于已糖激酶（HK-2）的表达上调。这些研究揭示了IGFBP6-HK2—有氧糖酵解轴在肾纤维化发生中的全新机制，并发现IGFBP6可能作为肾纤维化治疗的新靶点提供了实验依据，具有深远的科学意义和临床价值。