SHP-1调控肾小管上皮细胞TLR信号通路的分子机制及其在肾缺血再灌注损伤中的作用

Ischemia reperfusion (IR) injury is an inevitable consequence occurring in organ procurement and transplantation. Reactive oxygen species(ROS) played a critical role in the induction of IR injury. Studys identified that renal-associated Toll like receptor (TLR) in Tubular epithelial cells(TEC) as an important initiator of inflammatory responses leading to renal injury and dysfunction after IR insult. In our previous study, we found that IR significantly decreased the mRNA as well as the protein content of SHP-1 both in the kidney and in the primary cultured cells. The SHP-1 phosphatase activity in kidney homogenate also decreased after IR. ROS generated from renal IR further decreased the expression and phosphotase activity of SHP-1 in kidney. We also found that the SHP-1 deficient mice are more susceptible to renal IR than the WT controls. In this study, we intend to study the precise role of SHP-1 in the regulation of TLRs pathway in tubular epithelial cells during renal ischemia-reperfusion injury. Using transgenes techniques and Methylmion Specific PCR, we will further investigate the mechanism that oxidative stress downregulating SHP-1 expression. This study will provide theraputic targets for the future clinical treatment for renal IR injury.

移植肾缺血再灌注（IR）损伤主要发生在肾小管，氧化应激反应是重要的致伤因素。肾小管上皮细胞（TEC）的Toll样受体（TLR）信号是介导IR损伤的关键通路，课题组在前期研究中发现蛋白酪氨酸磷酸酶SHP-1可负相调控TLR信号通路，但其在IR背景下的表达和作用机制尚不明确。在本课题的预探索研究中，我们首次发现IR后的氧化应激可显著下调TEC的SHP-1表达，SHP-1缺陷的mev+/-小鼠在肾脏IR后损伤明显加重。在此基础上，本项目拟采用SHP-1缺陷小鼠和腺病毒转染过表达，在体外细胞模拟IR、小鼠肾蒂阻断热缺血和肾移植冷缺血模型上，研究SHP-1对IR后TEC TLR通路的调节机制及其在移植肾IR损伤中的作用；并通过基因启动子区域甲基化分析和信号蛋白磷酸化检测等方法探讨IR后氧化应激下调SHP-1表达的分子机制。本课题可为临床减轻移植物IR损伤，促进修复、增加移植物存活时间提供新的治疗靶点

移植肾缺血再灌注（ischemia reperfusion, IR）损伤是一种常见的急性肾损伤，主要发生在肾小管。氧化应激是IR损伤的关键环节，尤其是IR激活了炎症信号通路和损伤了线粒体的结构和功能。SHP-1是机体重要的酪氨酸磷酸酶，通过抑制激酶酪氨酸残基磷酸化发挥负调节作用。本课题研究了肾脏IR后SHP-1的表达、天然免疫分子的活化，使SHP-1体内和体外缺失后天然免疫分子的表达、信号通路的改变、最终表型的改变，重新体内外使SHP-1过表达后相应分子和表型的恢复。最终明确了SHP-1在移植肾缺血再灌注损伤中通过抑制天然免疫中保守的模式识别受体TLR4（Toll-like receptor 4）的表达，阻断了MAPK信号通路的活化，从而减轻了移植肾缺血再灌注损伤。在探讨氧化应激在急性肾损伤中的作用时，本课题着重研究了沉默配型信息调节2同聚物3 (silent mating type information regulation 2 homolog 3，SIRT3)通过抗氧化损伤减轻急性肾损伤的作用机制。通过SIRT3的基因敲除小鼠模型结合急性肾损伤的手术模型，以及在体外H2O2 刺激肾小管上皮细胞的细胞模型的基础上过表达和敲减SIRT3，检测下游分子和相关信号通路的分子表达，明确了SIRT3是保护线粒体功能的关键调节分子，通过抑制活性氧簇（reactive oxygen species，ROS）的生成，稳定线粒体的结构和维持其在氧化呼吸链中的作用，最终减轻急性肾损伤。