肾小管上皮细胞释放携带HE4的外泌体参与缺氧诱导的肾间质纤维化

Hypoxia is an important initial factor of renal interstitial fibrosis (RIF). Previous studies and our results demonstrated that human epididymis protein-4 (HE4) plays an important role in RIF. However, the molecular mechanism of HE4 for RIF has not been well elucidated. Exosome participates in many biological processes. We found that hypoxia upregulated the expression of p53 and HE4 and induced renal epithelial cells to release HE4 exosomes. Several studies proved that p53 can medicate exocytosis. And, our bioinformatics analysis showed that HIF-1α could bind to the 3‘UTR of p53 and HE4. And, in vitro experiments suggested that the HE4 exosome can promote the activation and proliferation of fibroblasts and activated the NF-κB pathway. Therefore, the purposes of our present study are following: i) to confirm the role of HE4 exosome from renal epithelial cells on the hypoxia induced RIF, ii) to figure out whether hypoxia induces renal epithelial cells to release HE4 exosomes through the HIF-1α/p53 and HIF-1α/HE4 pathways, and iii) to clarify if the HE4 exosome promotes RIF through the NF-κB pathway. Our present study will clarify the molecular pathways of the hypoxia-induced HE4 exosome release and the HE4 exosome induced RIF. Most likely, these results will be associated with important theoretical significance.

缺氧是肾间质纤维化（RIF）的始动因素。文献及我们前期研究表明，人附睾蛋白4（HE4）在RIF过程中发挥重要作用，但机制尚未阐明。外泌体可参与多种生物学过程。我们发现，缺氧可上调肾小管上皮细胞内HE4和p53表达，并促使其释放携带HE4的外泌体。文献报道，p53可调控多种细胞胞吐。进一步生物信息学分析发现，p53和HE4启动子区均存在HIF-1α结合位点。同时证实，携带HE4的外泌体可刺激成纤维细胞增殖活化并激活NF-κB通路。本课题拟在此基础上，深入研究缺氧诱导肾小管上皮细胞释放携带HE4的外泌体在RIF中的作用；揭示HIF-1α、p53和HE4之间的关系在缺氧诱导肾小管上皮细胞释放携带HE4的外泌体中的意义；阐明NF-κB通路在携带HE4的外泌体调控RIF中的作用和机制。这些研究可以明确缺氧通过调控肾小管上皮细胞释放携带HE4的外泌体参与RIF的分子机制，具有重要理论意义。

慢性肾脏病以其高达10.8%的患病率已成为威胁人类健康的主要疾病之一。肾间质纤维化是各种慢性肾脏病进展至终末期肾病的共同病理表现，缺氧参与了肾间质纤维化的整个过程。缺氧相关的肾间质纤维化机制的研究是创立新的抗纤维化策略的基础。本项目通过外泌体抽提、细胞共培养技术明确缺氧诱导肾小管上皮细胞外泌体释放增加，且抽提到的外泌体中HE4蛋白表达量也增加，携带HE4的外泌体能促进成纤维细胞的活化增殖；进一步通过质粒转染、ChIP、荧光素酶报告基因实验证实缺氧可以通过HIF-1α调控p53，进而引起肾小管上皮细胞G2/M周期阻滞，促进肾间质纤维化，通过过表达质粒和p53抑制剂通路上调和下调p53通路后，肾小管上皮细胞出现相应的细胞周期和纤维化蛋白分泌的变化；另外，通过质粒转染、ChIP、荧光素酶报告基因证实缺氧可通过HIF-1α通路调控HE4的表达且HE4在缺氧相关的肾间质纤维化中发挥重要作用，进一步通过慢病毒转染、抑制剂等途径，明确NF-κB通路在HIF-1α/HE4介导的缺氧相关肾间质纤维化过程中发挥重要作用。研究阐明了缺氧诱导肾纤维化新分子机制，为通过HIF-1α、p53、HE4、NF-κB通路开发肾间质纤维化的治疗和预防策略提供了新的靶点和思路，具有重要的理论意义。