促红素调节内质网应激改善肾脏缺血再灌注损伤的机制研究

Ischemia and reperfusion injury (IRI) plays an important role in renal tissue damage and dysfunction, but so far no effective treatment is available. Endoplasmic reticulum stress (ER stress) occurs in early stage of renal ischemia and reperfusion injury, and then leads to tissue damage through activating apoptosis and inflammation pathway. Our previous studies showed that erythropoietin (EPO) significantly alleviated renal IRI and improved renal function via inhibiting cell apoptosis and tissue inflammation. In this project, we try to investigate the effects of EPO on renal ER stress, the common upstream pathway of apoptosis and inflammation, in the IRI and then elucidate its underlying mechanisms. In preliminary experiments, we discovered that EPO significantly reduced ER stress protein Grp78 expression; however, it is not clear how EPO may modulate the three branches of ER stress (IRE-1, PERK and ATF-6). Using animal and cell models of renal IRI, we try to clarify the detailed regulating mechanisms of EPO on ER stress signaling pathways in renal IRI, and identify the key molecules and then regulate these molecules for the treatment of renal IRI. Here, we hope to clarify the novel renal protection mechanism of EPO in renal IRI and provide a new potential target for treatment of renal IRI.

缺血再灌注是造成肾组织损伤和功能不全的重要因素,目前尚无有效治疗手段。以往研究发现：肾脏缺血再灌注早期发生内质网应激（ER stress），进而通过促进细胞凋亡及炎症反应导致组织损伤。我们前期研究发现促红细胞生成红素(EPO)可通过抑制缺血再灌注损伤中凋亡和炎症反应，显著减轻大鼠肾损伤和改善肾功能。本课题重点研究EPO对肾脏缺血再灌注损伤中ER stress的调节及其分子机制。预实验发现经EPO可以显著下调小鼠肾ER stress标志性蛋白Grp78表达，但对ER stress三条通路IRE-1,PERK和ATF-6的调节机制仍不清楚。拟在肾脏缺血再灌注小鼠模型及小鼠肾小管上皮细胞模型中，探讨EPO对ER stress的影响及其信号通路，筛选关键分子，通过上调和下调该分子以治疗肾脏缺血再灌注损伤。通过本研究有望阐明EPO发挥肾脏保护作用的新机制，也有望找到治疗肾脏缺血再灌注损伤的新靶点。

缺血再灌注损伤（ishemia and reperfusion injury，IRI）是血液灌注不足和血流恢复后所致的组织、器官损伤。在肾移植过程中，移植肾不可避免经受不同程度的IRI，且影响术后移植肾功能的早期恢复和长期存活。目前IRI缺乏有效的治疗措施，研究IRI的病理生理机制，发现新的治疗措施是该领域研究的重要方向。本课题组建立小鼠肾脏缺血再灌注损伤模型，使用具有肾保护作用的EPO作为干预手段，研究ER stress信号通路中相关分子，探索其发挥肾脏保护作用的分子机制。通过研究ERstress下游炎症信号通路，发现抑炎因子IL-35在EPO减轻缺血再灌注ER stress下游炎症应答中表达异常升高。进而通过重组IL-35质粒预处理后行肾脏IRI模型，发现IL-35预处理能够明显改善IRI小鼠生存率和肾损伤程度，其机制主要是通过抑制促炎因子TNF-α、IL-6、IL-1β的产生，减少IRI肾脏局部炎症细胞浸润，降低肾小管上皮细胞凋亡。基于动物实验的结果，我们将其转化至IRI临床研究中，研究了肾移植术后急性肾损伤炎症应答特征，发现术后两周内，移植肾功能延迟恢复患者血清及尿液IL-35的含量显著降低，而尿液中促炎细胞因子IL-6、IL-1β、TNF-α的含量显著升高。本课题研究阐明了EPO调节ER stress下游炎症应答发挥肾脏缺血再灌注损伤保护作用的机制是通过促进IL-35的表达，进而抑制肾脏IRI过程中过强的炎症应答，减少肾小管上皮细胞凋亡；临床转化研究中，发现肾移植患者术后早期患者血清及尿液中IL-35含量降低是移植肾功能延迟恢复的危险因素。本研究结果有望成为早期预警缺血再灌注肾损伤及其预后的分子标志，也为临床急性肾损伤的干预提供理论依据和实验基础，为指导临床实践具有一定现实意义。