外泌体调控肾小管上皮细胞损伤引起的肾脏纤维化的机理研究

Chronic kidney disease (CKD) is a seriously public health problem in China. Kidney fibrosis is the final outcome of the development of CKD, but its occurrence and development mechanism is unclear. Exosome is one kind of membrane vesicles which consist of a limiting lipid bilayer, transmembrane proteins and a hydrophilic core containing proteins, mRNAs and microRNAs. They are transported in tissue fluid and protected by lipid bilayers and act as excellular vehicles through the delivery of their functional cargo to recipient cells, with many important biological, physiological and pathological functions. In fact, exosomes and microvesicles are difficult to distinguish and so we all called them as exosomes. There are many studies about exosomes explored in kidney in which exosomes can be early diagnosis criteria and in another aspect they can contribute to antigen presentation, pathogen transfer and communication in kidney diseases. Our previous study found that the secretion of exosomes were dramatically increased in renal tubulointerstitial injury model. In vitro, we also confirmed that exosomes derived from TGF-β1-treated tubular epithelial cell line HKC-8 could promote proliferation and fibrosis in fibroblast cell line 49F, and fibroblast activation was dependent on exosomes delivering SHH/Gli1 signaling pathway, but the mechanism has not elucidated yet. This study intends to explain the molecular mechanism of exosomes regulating renal fibrosis induced by tubulointerstitial injury. On the one hand, we need to continuely expose the physiological function of exosomes and regulation of SHH/Gli1 signal pathways in renal fibrosis; on the other hand, by using the methods of RNA-seq, lncRNA-seq accurately analyze substances of exosomes, reveals the key factors and important signaling pathways regulating renal fibrosis.

慢性肾病在我国是一个重要的公共卫生问题，肾脏纤维化是慢性肾病发展的最终结果，然而其发生和发展机制尚不清晰。外泌体是一种细胞自身分泌的囊泡，因其能够携带多种物质在体内进行远距离运输而成为细胞间信息传递的优良载体并发挥着重要的作用。我们前期研究发现在肾小管间质损伤模型的小鼠肾脏内外泌体的分泌急剧升高，体外细胞实验也证实TGF-β刺激的肾小管上皮细胞系HKC-8分泌的外泌体能够显著促进成纤维细胞系49F的纤维化和增殖，而SHH/Gli1信号通路在其中发挥了重要的调控作用，但具体机制尚无人阐明。本研究拟深入阐释外泌体调控肾小管间质损伤引起的肾脏纤维化的分子作用机制。一方面，在已有基础上进一步确认外泌体在肾脏纤维化过程中的生理功能和SHH/Gli1信号通路的调控作用；另一方面，利用RNA-seq、lncRNA-seq等方法精确解析外泌体内的物质组成，揭示其调控肾脏纤维化的关键因子和重要信号通路。

慢性肾病在我国是一个重要的公共卫生问题，肾脏纤维化是慢性肾病发展的最终结果，然而其发生和发展机制尚不清晰。外泌体是一种细胞自身分泌的囊泡，因其能够携带多种物质在体内进行远距离运输而成为细胞间信息传递的优良载体并发挥着重要的作用。我们在前期研究中发现肾小管间质损伤模型的小鼠肾脏内外泌体的分泌急剧升高，体外细胞实验也证实TGF-β1刺激的肾小管上皮细胞系HKC-8分泌的外泌体能够显著促进成纤维细胞系49F的纤维化和增殖，但具体机制尚无人阐明。为深入阐释外泌体调控肾小管间质损伤引起的肾脏纤维化的分子作用机制，我们在多种CKD动物模型中发现外泌体的产生和分泌随着肾脏纤维化的进程不断增加，这种变化主要发生在肾小管上皮细胞中，并且伴随着Shh的表达上升。体外实验中，由于TGF-β1处理造成的损伤，人源肾小管上皮细胞系HKC-8产生和分泌的外泌体急剧增加，并且能够诱导大鼠源肾脏间质成纤维细胞系NRK-49F的增殖和纤维化。利用药物处理抑制HKC-8细胞中外泌体的分泌后，缺乏外泌体的条件培养上清抑制了NRK-49F细胞活性。同时，干扰HKC-8细胞中Shh的表达同样能够抑制条件培养上清对NRK-49F细胞活性的诱导。体内实验中，注射损伤的肾小管细胞来源的外泌体明显加剧了肾脏损伤和纤维化程度，而该效应能够被Shh信号通路的抑制剂阻断。我们进一步解析了外泌体内的物质组成变化，发现Shh/Gli1信号通路在其中发挥了重要的调控作用，抑制外泌体途径的Shh/Gli1信号传递可以显著改善肾脏功能，缓解肾脏纤维化的进程。这一系列结果表明肾小管细胞分泌的外泌体通过携带和转运Shh信号在调控肾脏纤维化的过程中发挥着重要作用。本项目为肾小管间质病变的发生和发展提供新的理论依据，同时也为以外泌体为靶标治疗肾脏纤维化提供新的研究思路。