肌源性外泌体特异性携带miRNA-29c调控肾间质纤维化的机制研究

Despite the renal interstitial fibrosis (RIF) is regarded as the final common pathway for most forms of progressive renal disease, little is known about the exact pathogenic mechanisms and the therapeutic effect is unsatisfied. The international studied has found that over expression of miRNA-29c can relieve RIF and its accompanying skeletal muscle atrophy. Our study also verified the expression level was declined in kidneys and skeletal muscle. The essential question is how to present miRNA-29c to the damaged areas of kidney accurately and effectively. Exosomes play a key role in the communication of cell-to-cell and using exosomes to transmit microRNA-29c is a kind of new treatment. We have successfully extracted exosomes in the preliminary experiments. We also verified the expression level was increased in blood exosomes. Thus, this research is designed to establish the mice model of renal interstitial fibrosis injecting with muscle source exosomes and establish the mice model of renal interstitial fibrosis which are knocked out of miRNA-29c injecting with miRNA-29c in kidneys. Molecular biology, immunohistochemistry and immunofluorescence tracer techniques will be applied to investigate the degree of RIF and the recovery degree from RIF. This research is to clarify the mechanism that muscle source exosomes specifically present miRNA-29c to relieve the degree of RIF. Findings will contribute to provide theoretical foundation in therapy of RIF with miRNA-29c.

肾间质纤维化目前发病机制不明，治疗效果不理想。国际上最新研究发现过表达miRNA-29 能有效缓解肾间质纤维化及其伴随的骨骼肌萎缩，我们前期实验也证实miRNA-29c在肾脏、肌肉中表达下降，提示miRNA-29c是改善肾间质纤维化的重要靶点，但如何递呈至肾脏受损部位目前尚无有效解决方案。外泌体是重要的细胞间信息传递分子，使用外泌体传送miRNA-29c是一种崭新的治疗方法。本课题组前期已成功提取外泌体，并发现miRNA-29c在血液外泌体中表达升高。因此，本项目通过构建肾间质纤维化小鼠模型，注射肌源性外泌体；构建miRNA-29c 敲除鼠肾间质纤维化模型，肾脏原位注射miRNA-29c，比较肾间质纤维化程度及恢复程度，旨在阐明肌源性外泌体特异性携带 miRNA-29c 到达肾脏受损部位缓解肾间质纤维化的机制。研究结果将阐释miRNAs-29c递呈途径，为临床治疗肾间质纤维化提供崭新方法。

肾纤维化导致终末期肾病，但抗纤维化药物难以开发。慢性肾脏疾病常常导致肌肉萎缩，从而增加发病率和死亡率。我们的研究发现MIRNA-29a在UUO小鼠肾脏和骨骼肌中表达下调。腺相关病毒（AAV）介导的miRNA-29a过表达可以对抗单侧输尿管梗阻（UUO）小鼠的肾脏纤维化和肌肉萎缩。我们使用了一个工程化的外泌体载体，它含有一个外膜蛋白基因Lamp2b，它与靶向多肽RVG（狂犬病病毒糖蛋白多肽）融合在一起。RVG将外切体导向表达乙酰胆碱受体的器官，如肾脏。我们在体内的研究发现，Exo/miRNA29干预增加了肌肉横截面积，降低了UUO诱导的TRIM63/MuRF1和FBXO32/ atrogin-1的上调。有趣的是，肌肉注射Exo/miRNA29的UUO小鼠的肾脏纤维化得到部分抑制。UUO小鼠肾脏中TGF-β、α-平滑肌肌动蛋白、纤维连接蛋白和胶原1A1的减少证实了这一点。当我们使用荧光标记的Exo/miRNA29在体内追踪Exo/ miRNA途径时，发现在未注射的肌肉和肾脏中都可以看到荧光。我们发现miRNA-29可以直接抑制YY1和TGF-β3的表达，这为Exo/miRNA29抑制肌肉萎缩和肾脏纤维化提供了可能的机制。除此外，我们的研究发现UUO模型中MMP9通过增加肾脏梗阻的炎症水平，巨噬细胞蓄积和肾纤维化来促进中性粒细胞浸润，从而引起肾脏纤维化。CoCl2模拟的缺氧环境可以激活CNA/NFATc1和myostatin信号通路，增加MyHCⅠ型肌纤维的比例。结论：Exo/miRNA29通过下调YY1和TGF-β途径蛋白的表达，改善骨骼肌萎缩，减轻肾脏纤维化。