Drp1介导线粒体分裂调控成纤维细胞活化增殖和肾脏纤维化的研究

Activation and proliferation of interstitial fibroblasts is the predominant cellular event indicating the development and progression of renal fibrosis. Our recent preliminary data showed that mitochondrial fragmentation in kidney of patient with chronic kidney disease negatively correlated with the estimated glomerular filtration rate. Inhibition of mitochondrial fission attenuated renal fibrosis in unilateral ureteral obstruction mice. Knockdown of dynamic related protein 1 (Drp1) by siRNA blocked TGF-β1-induced the expression levels of H3K27 acetylation (H3K27ac) and α-SMA in NRK-49F cells. Moreover, TGF-β1 treatment resulted in the increased H3K27ac in the promoter of α-SMA gene by chromatin immunoprecipitation. Our findings suggest that mitochondria fission regulates fibroblast activation and renal fibrosis. However, the mechanism(s) were unclear. In this proposal, mice models of chronic kidney nephropathy were used to further study the relationship among mitochondrial fission, activation and proliferation, and renal fibrosis. The direct effects of mitochondrial fission on activation and proliferation of fibroblasts were investigated by upregulating/downregulating/mutation Drp1 expression in NRK-49F cells. We also explore the mechanisms that suppression of Drp1-mediated mitochondrial fission inhibits acetylation of H3K27 and its related targets of gene. Our study will provide significant insight into the mechanism by which mitochondrial fission promotes renal fibrosis. Manipulation of mitochondrial fission might be a novel approach for the prevention and treatment of renal fibrosis.

成纤维细胞活化增殖是肾脏纤维化发生发展的关键事件。我们新近发现慢性肾脏病患者肾组织成纤维细胞的线粒体呈分裂样改变，其程度与肾小球滤过率呈负相关；抑制小鼠慢性肾病模型Drp1介导的线粒体分裂可延缓肾脏纤维化；下调成纤维细胞株Drp1表达可降低TGF-β1诱导的H3K27乙酰化和α-SMA蛋白水平；乙酰化H3K27可与α-SMA基因启动子结合。提示线粒体分裂可调控成纤维细胞活化和肾脏纤维化，但其机制不清。本项目拟利用小鼠慢性肾病模型，阐明成纤维细胞线粒体分裂、活化增殖与纤维化的关系；应用Drp1特异性抑制剂、上调/沉默/变异线粒体Drp1水平/活性，探讨线粒体分裂直接调控成纤维细胞活化增殖的作用，研究抑制Drp1介导的线粒体分裂靶向阻止H3K27乙酰化调控细胞活化的机制。本项目有助于揭示成纤维细胞线粒体分裂促进肾脏纤维化的作用和机制，为探索和开发线粒体靶向分子防治肾脏纤维化提供可靠的实验依据。

成纤维细胞活化增殖是肾脏纤维化发生发展的关键事件，过度的线粒体分裂与肿瘤、肺纤维化等多种疾病的发生有关。然而，线粒体分裂如何调控成纤维细胞活化和肾脏纤维化尚不清楚。本项目研究的主要内容包括：①建立小鼠梗阻性肾病模型，利用Drp1特异性抑制剂Mdivi-1，明确成纤维细胞线粒体分裂、增殖活化与纤维化的关系；②上调/沉默/变异NRK-49F细胞线粒体Drp1水平/活性，探讨线粒体分裂直接调控成纤维细胞活化增殖的作用；③利用上述细胞模型检测组蛋白乙酰化和下游靶基因的情况，探索线粒体分裂介导组蛋白乙酰化和调控靶基因转录的信号通路。.我们的研究结果发现：慢性肾脏病患者和梗阻性肾病（UUO）小鼠肾组织中成纤维细胞的线粒体呈分裂样改变；UUO小鼠肾组织中第616位丝氨酸磷酸化的Drp1和乙酰化的H3K27表达增多；药物Mdivi-1抑制线粒体分裂可延缓UUO小鼠的H3K27乙酰化和肾脏纤维化；Mdivi-1抑制或下调成纤维细胞株Drp1表达可降低α-SMA和胶原蛋白1的表达水平，进而抑制TGF-β1引发的细胞活化和增殖；此外，Drp1的缺失增强了TGF-β1诱导的细胞凋亡并降低了mtROS的产生和糖酵解代谢水平。相反，在Drp1缺失的成纤维细胞中过表达野生型Drp1质粒，而不是突变型Drp1S616A质粒可恢复TGF-β1诱导的Drp1磷酸化、H3K27乙酰化和细胞活化。在TGF-β1刺激下，ac-H3K27在α-SMA和PCNA基因启动子区域结合增加，沉默Drp1可下调α-SMA和PCNA的转录活性，过表达野生型Drp1质粒而不是突变型Drp1S616A质粒可逆转抑制Drp1介导的α-SMA和PCNA的转录活性下调。.本研究阐明了抑制Drp1介导的线粒体分裂通过对纤维化相关基因转录的表观遗传学调控来抑制成纤维细胞的活化和增殖，从而减轻肾脏纤维化。