MDM2膜转位在AKI供肾诱发移植后T细胞介导排斥反应中的作用与机制

Allogenic kidney transplantation is the optimal replacement treatment for the patients with End Stage Renal Disease. The application of Expanded Criteria Donor alleviates the shortage of donor kidney. However, Acute Kidney Injury (AKI), the commonest feature of Expanded Criteria Donor, tightly associates with the T cell mediated rejection (TCMR) and early graft function loss. And the proximal tubular epithelium (PTE) plays the central role in AKI related TCMR by increased immunogenicity. To resolve this challenge is pivotal for the improvement of survival rate of graft and patient...Our preliminary data indicated that under AKI in PTE MDM2, a well-established E3 ubiquitin ligase, translocates to cell membrane which along with the downregulation of PD-L1, a T cell co-inhibitory molecule. Meanwhile, the expression of Cdk5 is enhanced and positive correlates with the serum creatinine of donor at organ procurement. Also, in vitro study revealed overexpression of Cdk5 augments the membrane translocation of MDM2 in a dose-dependent manner. Therefore, we hypothesize that during AKI Cdk5 induces membrane translocation of MDM2 which subsequently ubiquitinates and degrades PD-L1. As a result, the immunogenicity of PTE is elevated along with the diminished inhibitory signaling for T cell activation which consequently leads to the TCMR. Blocking Cdk5 is a promising strategy to manage donor AKI related TCMR. ..To this end, we are going to employ the mice transplant model under life support as well as the in vitro co-culture of PTE and T cell as research subjects. By genetic or pharmacologic interference to analyze the roles and mechanisms of MDM2 membrane translocation in donor AKI initiated TCMR. And finally reconfirm above mechanisms in clinic by kidney biopsy samples from donor and transplant allograft...By this study, we wish to elucidate the mechanisms of donor AKI related TCMR and provide a promising strategy for further application of AKI donor and the improvement of graft survival.

急性肾损伤（AKI）供体肾脏的应用缓解了器官短缺的困境，但其增加移植后T细胞介导排斥反应（TCMR）仍是待解难题。肾小管上皮细胞（PTE）损伤后免疫原性增强是启动TCMR的关键事件，但其机制尚不明了。我们前期工作证实：AKI状态下PTE泛素连接酶MDM2的表达分布发生膜转位并伴随膜蛋白PD-L1（T细胞协同共抑制分子）表达下调；此外，AKI供肾中Cdk5表达增强，而体外过表达Cdk5促进MDM2膜转位。据此推测Cdk5介导的MDM2膜转位通过增加PD-L1泛素化修饰与降解，导致PTE免疫原性增强，促发T细胞活化与TCMR。为证实上述假说，本项目拟以小鼠肾移植模型与PTE/T细胞共培养体系为研究对象，通过基因与药物干预，阐明MDM2膜转位在AKI诱发TCMR中的作用与机制，并通过临床资料进一步验证之。通过本研究，期望为AKI供肾的推广与TCMR的防控提供理论依据与临床策略。

扩展标准供肾（Expanded Criteria Donor, ECD）的采用缓解了临床移植供肾短缺的困境，但其相关的排斥反应，尤其是T 细胞介导的排斥反应（T Cell Mediated Rejection, TCMR）的增加仍是移植领域亟待解决的难题。相当一部分ECD来源于缺血再灌注（Ischemia/Reperfusion, I/R）所致的AKI供肾，肾小管上皮细胞（Tubular Epithelium Cells, TECs）是AKI病理损伤中主要靶细胞。与此同时，TECs具有免疫原性，在同种异体免疫过程中，特别是在以小管炎为主要病理形态学表现的TCMR中扮演重要角色。本研究中，我们旨在探索I/R诱导的AKI供肾TECs免疫原性的变化及其可能的病理机制。我们既往研究发现鼠双微体2（Murine Double Minute 2, MDM2）参与了顺铂诱导的AKI及AKI向CKD进展，但MDM2是否参与了I/R诱导的AKI还尚未阐明。鉴于T细胞协同共抑制因子PD-L1不仅是调控T细胞活化的重要分子，其亦在TECs中有丰富表达，故在本研究中我们通过患者肾活检标本、小鼠缺血再灌注损伤模型（IRI）与大鼠肾小管上皮细胞（NRK-52E），探索与揭示了MDM2在AKI损伤中TECs免疫原性异常中的作用及其对PD-L1的调控机制。我们的研究结果显示：①IRI小鼠肾皮质区域以及缺氧/复氧刺激下的TECs中，MDM2在胞膜上的表达增加，且其主要富集于TECs胞膜基底部；与此同时，PD-L1在肾皮质区域中与TECs胞膜上的表达下调。②通过质粒干预MDM2在胞膜和胞核的表达分布，我们证实胞膜MDM2通过泛素化修饰PD-L1减少其在TECs中的表达，随之导致TECs免疫原性增强。③通过TECs与CD4+T细胞共培养模拟移植免疫微环境，进一步证实缺氧/复氧状态下TECs的免疫原性与其活化T细胞的能力增强。本研究提示干预MDM2-PD-L1通路是防治AKI供肾相关TCMR的重要途径之一，这一研究成果为临床缓解供肾短缺、减少移植术后TCMR、提高移植肾存活率提供可行的干预策略。