钠钾ATP酶DR区特异性抗体在肾脏缺血再灌注损伤中的保护作用及其机制的研究

Renal ischemia reperfusion injury is an important injury part of ischemic acute renal failure, and it's also the main factors to affect the early functional recovery and the long-term survival of transplanted kidney in renal transplantation. Recently, It was reported that activated PI3K/AKT, ERK and PKCε could increase the tolerance of tissue to ischemia/reperfusion. It was also found that NKA (Na+/K+-ATPase) DR region specific antibody could induce cardioprotection against ischemic injury via Src/PI3K/Akt/ERK signal pathway. Based on these mechanism, we proposed that if DR region specific antibody (DRSAb) could also induce protective effect against ischemia/reperfusion injury on kidney. This proposal is therefore designed to: ..1).Comfirm if DRSAb could induce protective effect against ischemia on HK-2 cell line. Specifically, we will make and purify the DRSAb firstly. After that, HK-2 cells will be incubated with DRSAb or control. Cells were then subjected to ischemia. The viability of HK-2 cell will be measured 10 min after reperfusion by MTT assay...2).Investigate the signaling mechanisms underlying DRSAb induced protective effect.Specifically, we will examine the involvement of protein kinase C ε (PKCε), Extracellular signal-regulated kinases (ERK), and phosphoinositide-3 kinase (PI3K/Akt) pathways with western blot...3).Investigate the protect effect of DRSAb in kidney ischemia-reperfusion injury animal models. We will establish kidney ischemia/reperfusion injury animal models and examine the protective effect of DRSAb by detecting the inflammation factors and biochemistry index of serum after ischemia/reperfusion. We will also discuss the relationship among the activated protein kinase including PKCε, ERK, and PI3K/Akt pathways with western blot，the level of inflammation factors，such as TNF-a、IL-6、IL-18 and IL-17, the extent of neutrophile granulocyte infiltration and ROS level in tissue...This study focuses on the main factors of ischemia/reperfusion injury for functional recovery after ischemic acute renal failure and renal transplantation. Based on the point of cell survival kinase activation could improve the cell tolerance to ischemia/reperfusion injury, this project is advanced on theoretically, and has important theoretical and practical significance for improving the initial graft organ recovery efficiency and long-term survival of renal transplant patients.

肾缺血再灌注损伤是缺血性急性肾功能衰竭的重要损伤环节,也是肾移植中影响移植肾早期功能恢复及长期存活的主要因素。PI3K/Akt激酶，ERK激酶及PKCε激酶等，在被激活的情况下可以显著提高组织细胞对缺血再灌注损伤的耐受作用。本课题基于钠钾ATP酶DR区特异性抗体通过诱导钠钾ATP酶活化而激活与细胞存活相关的细胞信号传导通路，深入研究PI3K/Akt，PKC ε及ERKs等蛋白激酶在肾缺血再灌注损伤中的保护作用，以及与组织中ROS水平，中性粒细胞浸润程度，各种血清炎症因子的表达水平的关系。本研究关注缺血性急性肾功能衰竭和肾移植后功能恢复的主要影响因素缺血再灌注损伤，从激活与细胞存活相关激酶的角度出发,提高组织细胞自身对缺血再灌注损伤的耐受，在理论上具有先进性，而且对于提高移植肾早期器官功能恢复效率及肾移植患者长期存活率具有重要理论价值和现实意义。

项目背景:肾缺血再灌注损伤是缺血性急性肾功能衰竭的重要损伤环节,也是肾移植中影响移植肾早期功能恢复及长期存活的主要因素。PI3K/Akt激酶，ERK激酶及PKCε激酶等，在被激活的情况下可以显著提高组织细胞对缺血再灌注损伤的耐受作用。.主要研究内容:本课题基于钠钾ATP酶DR区特异性抗体通过诱导钠钾ATP酶活化而激活与细胞存活相关的细胞信号传导通路，深入研究PI3K/Akt，PKC ε及ERKs等蛋白激酶在肾缺血再灌注损伤中的保护作用。.重要结果及关键数据: 自本年度项目启动以来，我们按照申请书的年度安排计划制备和纯化了DRSAb,并对其免疫学特性进行了检测。我们还另外构建了NKAβ的胞外区，其具有与NKA α DR 区结合的功能，同时我们对DRSAb 及tNKAβ的胞外区对缺氧HK-2细胞及大鼠肾脏缺血再灌注损伤模型的保护作用及机制进行了研究。实验证实DRSAb和tNKAβ对缺氧HK-2细胞具有保护作用，并发现保护机制与PI3K/Akt, ERKs 和PKC ε通路活化相关。检测模型鼠缺血再灌注后各项生化指标组织病理形态学改变证明了DRSAb和tNKAβ对SD大鼠肾脏缺血再灌注模型的保护作用， 证明了DRSAb及tNKAβ对肾脏缺血再灌注损伤保护作用机制与PI3K/Akt, ERKs 和PKC ε通路活化相关。.科学意义:本研究关注缺血性急性肾功能衰竭和肾移植后功能恢复的主要影响因素缺血再灌注损伤，从激活与细胞存活相关激酶的角度出发,提高组织细胞自身对缺血再灌注损伤的耐受，在理论上具有先进性，而且对于提高移植肾早期器官功能恢复效率及肾移植患者长期存活率具有重要理论价值和现实意义。