CysRS促进ATP利用抑制肾小管上皮细胞焦亡在早期肾缺血再灌注损伤中的作用与机制

Renal ischemia reperfusion injury (IRI) is one of the major causes of acute renal injury. The main pathological feature is the death of renal tubular epithelium, and its molecular mechanism is complex. Current studies have confirmed pyroptosis is closely related to the death of renal tubular epithelium in renal IRI. In the previous study, we confirmed in vivo and in vitro that renal tubular epithelium pyroptosis occurred in early stage of renal IRI, and was accompanied by impaired ATP utility. We have found cysteinyl-tRNA synthetase (CysRS) which is directly related to ATP metabolism was significantly downregulated in human renal tubular epithelium IRI model using quantitative differential proteomic method, and further confirmed CysRS's downregulation in pyroptotic renal tubular epithelium in early stage of renal IRI. Therefore, we proposed the hypothesis that ‘CysRS inhibits ATP induced renal tubular epithelium pyroptosis by improving ATP utility in early stage of renal IRI; upregulating CysRS helps to alleviate IRI induced renal injury and delay renal function loss’. Based on ATP utilization, the project reveals a new mechanism of CysRS against renal tubular epithelium injury and death in early stage of IRI, providing a new insight and target for optimizing clinical prevention and treatment of IRI induced AKI.

肾缺血再灌注损伤(IRI)是急性肾损伤的重要诱因之一，主要病理特征为肾小管上皮细胞死亡，其分子机制复杂。现有研究证实焦亡与肾IRI时肾小管上皮细胞死亡密切相关。本课题组前期研究通过体内及体外实验证实IRI早期肾小管上皮细胞内发生了伴随ATP利用障碍的细胞焦亡，利用定量差异蛋白质组学方法从人肾小管上皮细胞IRI模型中筛选出与ATP代谢直接相关的半胱氨酰tRNA合成酶（CysRS），并进一步证实IRI肾小管上皮细胞焦亡时CysRS表达显著下调。据此，本课题组提出“肾IRI早期CysRS促进ATP利用，继而抑制ATP诱导肾小管上皮细胞焦亡；上调CysRS表达可减轻IRI诱导的肾组织损害并延缓肾功能丢失”的科学假说。该研究项目以ATP利用为切入点揭示CysRS对抗IRI早期肾小管上皮细胞损伤和死亡的新机制，为优化IRI引起AKI的临床防治提供新的思路和作用靶点。

本研究成功构建、鉴过表达载体pRP[Exp]-EGFP-EF1A>FLAG/hCARS（以下简称Flag/hCARS）和重组腺病毒载体pAV[shRNA]-EGFP-U6-mCars（以下简称mCars-shRNA）；体外培养人HK-2细胞并成功建立体外HK-2 H/R细胞模型，观察Flag/hCARS和mCars-shRNA转染后H/R条件下HK-2细胞的焦亡状态，确认H/R条件下HK-2细胞的CysRS与焦亡相关蛋白表达有明显改变，改变CARS表达对焦亡相关蛋白表达有影响。这种影响在焦亡激动剂7-DG和抑制剂MCC950作用下也存在，提示CARS有潜在对抗焦亡的作用。同时，本研究通过建立小鼠IRI模型，用重组腺病毒载体mCars-shRNA对小鼠IRI动物模型进行CARS敲减，观察小鼠模型肾脏组织的焦亡蛋白表达情况。结果提示CARS敲减后小鼠IRI模型的焦亡相关蛋白表达增加，与体外试验结果一致，共同证实CARS有对抗焦亡的潜在作用。但CARS通过影响焦亡参与调控肾IRI时小管上皮细胞损伤的具体通路和机制有待进一步研究。