缺氧诱导因子-2α调控树突状细胞功能参与肾脏缺血再灌注损伤的机制

Dendritic cell (DC) is among the key immune cells that influence renal ischemia-reperfusion injury (IRI). It has been reported that hypoxia and hypoxia-inducible factor-1 (HIF-1) play important roles in the maturation and function of DCs. However, the role of HIF-2 has not been documented before. In the preliminary studies employing Cd11cCre+HIF-2α-/- mice, we found that HIF-2α knockout led to much more severe renal IRI, which couldn’t be mitigated by CGS21680, an activator of adenosine A2a receptor (adora2a). We speculate that there is a regulatory relationship between HIF-2 and adora2a in DCs, which affect the function of specific DC subpopulations as well as the inflammatory process in renal IRI. We plan to produce DC-specific HIF-2α knockout and overexpression mice by Cre-Loxp system and based on these mice we can define the role of HIF-2α on the maturation, phenotype and function of DCs. The regulatory relationship between HIF-2 and adora2a in DCs will be determined by promoter studies. Mice genetically modified to overexpress HIF-2α but lack adora2a will help to further define the relationship between HIF-2 and adora2a in vivo. We also plan to employ HIF-2α and Rag1 double knockout mice to define the role of T/NKT cells in HIF-2α knockout-induced aggravated IRI. Adoptive transfer of WT or HIF-2α-deficient DC subpopulations into mice that have no DCs will help to find out the specific DC subpopulation that determines the outcome of renal IRI. This study may further disclose how hypoxia regulates the function of DCs, and also, may lead to new therapeutic protocols to alleviate renal IRI.

树突状细胞（DC）是影响肾缺血再灌注损伤（IRI）的关键性免疫细胞之一。缺氧和缺氧诱导因子（HIF）-1α已被证实对DC功能有影响，HIF-2α则未见相关研究报道。我们在预实验中发现HIF-2α敲除导致DC自发性成熟，肾IRI明显加重，且不能被腺苷A2a受体（adora2a）激活剂缓解。我们推测DC中存在着HIF-2α对Adora2a的表达调控，HIF-2α因此影响特定DC亚群的功能进而改变肾IRI炎症反应的转归。我们设想获得DC特异性HIF-2α敲除或高表达小鼠，观察其DC表型和功能变化。对DC中HIF-2α/Adora2a调控关系进行研究确认并观察这种调控关系对肾IRI的影响。观察T/NKT细胞在上述研究体系中的作用。通过磁珠分选不同DC亚群过继输注无DC小鼠从而确定起关键作用的DC亚群。本研究将进一步揭示缺氧对DC功能的影响及其调控机制，并可能为肾IRI提供新的干预靶点。

肾移植是终末期肾病最有效的治疗手段，然而缺血再灌注损伤（Ischemia-reperfusion injury, IRI）是临床肾移植面临的一个相对棘手的问题。开展肾脏IRI机制研究，为肾脏IRI的干预提供新的靶点和理论基础，对移植医学和重症医学都有着十分重要的意义。树突状细胞（Dendritic cell，DC）是影响肾IRI的关键性免疫细胞之一。在IRI中，缺氧和缺氧诱导因子（HIF）是影响DC功能、调控炎症反应的关键因素之一。本项目中我们通过条件性基因敲除构建了DC特异性HIF-2α敲除小鼠模型。通过肾IRI建模我们发现HIF-2α敲除导致小鼠肾IRI明显加重，且肾组织铁死亡明显升高。DC中HIF-2α缺失一方面增强了DC的糖酵解水平，促进DC细胞激活和共刺激分子表达增加；另一方面，释放外泌体激活巨噬细胞向M1型极化，促进足细胞铁死亡，加重肾IRI。研究过程中我们还发现 HIF-2α通过调控鞘氨醇激酶1（Sphingosine kinase 1，SK1）的表达，促进生成1-磷酸-鞘氨醇（sphingosine 1-phosphate，S1P）保护肾IRI。进一步深入研究发现人多能干细胞（hiPSC）分化的间充质干细胞（MSC）分泌的胞外囊泡（hiPSC-MSCs-EVs）可以促进SK1的表达，进而导致S1P合成增加，抑制肾小管细胞的程序性坏死，同时抑制DC细胞的激活，减少炎症因子的释放，显著减轻肾IRI。我们在RNA表观调控方面也进行了探索，发现N6-甲基腺嘌呤（N6-methyladenosine，m6A）mRNA甲基化酶METTL14通过抑制YAP1加重肾IRI。综上，本项目中我们初步阐明了DCs中HIF-2α的缺失通过调控DC的代谢重编程和M1型巨噬细胞活化，促进肾IRI的作用机制，另一方面在肾IRI分子调控机制方面进行了探索，为肾IRI的干预提供了新的思路。