基于WNK4/SPAK信号通路探讨调节ENaC的分子机制及在急性肺损伤中的保护作用

Alveolar fluid clearance (AFC) is an effective way to reduce the mortality rate of ALI/ARDS.The epithelial sodium channel (ENaC) is a critical procedure in AFC. In the previously doctoral research project, PI3K/Akt pathway was an important signaling chain in AFC, but its downstream signaling interlinkage has not been completely clarified.As an inevitable connection"bridge" in the downstream of PI3K/Akt signaling chain, WNK4-SPAK pathway was a central way which maintained sodium and water transport and was a powerful regulator in AFC with ideal intervention,but the relationship with ENaC has not been clear so far .The primary alveolar epithelial cell and ALI animal model are served as subjects, whether the regulation of ENaC is by WNK4-SPAK pathway as a corn and the methods including gene overexpression and RNAi interference are used to investigate the function of WNK4-SPAK pathway in the regulation of ENaC expression and activity in vivo and in vitro, which explore the effect of the pathway in AFC and ultimately reveal the molecular interlinkage foundation between WNK4-SPAK pathway and AFC.The results of the study might further improve the role of PI3K/Akt signaling chain played in AFC and provide an effective intervention target for the prevention and treatment of ALI/ARDS.

肺泡水肿液清除（AFC）是降低ALI/ARDS病死率的有效途径。上皮钠通道（ENaC）是AFC的关键环节。申请人在前期博士课题研究中发现PI3K/Akt通路是参与AFC的重要信号链，但其下游的信号链接仍未完全阐明。作为PI3K/Akt信号链下游必经的连接“桥梁”，WNK4-SPAK通路是维持钠水转运的一条中心通路，是AFC强有力的调节器，具有理想可干预性，但与ENaC的关系目前尚不明确。本项目以原代肺泡上皮细胞和ALI动物模型为研究对象，以WNK4-SPAK通路是否调控ENaC为研究核心，采用基因过表达、RNAi干扰等方法，从体内和体外两个层面研究WNK4-SPAK通路调节ENaC表达和活性的功能，探讨其在AFC中的作用，最终揭示WNK4-SPAK通路与AFC的分子链接基础。研究结果将进一步完善PI3K/Akt信号链在AFC中扮演的角色,并为ALI/ARDS防治提供有效的干预靶点。

一、研究背景：急性肺损伤(acute lung injury, ALI)/急性呼吸窘迫综合征（acute respiratorydistress syndrome, ARDS）是临床常见的呼吸危重症，近10年病死率达40%~50%。肺泡上皮钠通道（epithelial sodium channel, ENaC）在钠水转运过程中能清除肺泡腔内聚集的水肿液，是能降低ARDS患者病死率的有效途径。近年研究显示WNK4-SPAK通路在AFC 中起到“调节器”作用，并与介导肺水肿液清除的ENaC 密切相关，可能成为干预肺水肿的理想靶点。.二、研究内容：1. 通过构建WNK4、SPAK 过表达载体及siRNA-WNK4 和siRNA-SPAK 干扰质粒，构建动物及体外基因过表达模型或基因沉默模型。2. 以ALI/ARDS 小鼠模型为基础证实WNK4-SPAK 通路对肺泡液体清除（alveolar fluid clearance, AFC）的影响和在肺泡上皮细胞中的保护性。3.研究WNK4-SPAK 通路调控ENaC 的机制，揭示PI3K/Akt 信号链下游的分子与ENaC 的信号链接机制。.三、重要结果. 1. ARDS小鼠模型在转染pCMV-Myc-WNK4、pCMV-Myc-SPAK后AFC显著增加，肺水含量减少，肺损伤减轻，α-ENaC、β-ENaC、γ-ENaC的表达显著增加；ARDS小鼠模型在转染siRNA-WNK4和siRNA-SPAK后，AFC显著减少，肺水含量增加，肺损伤加重，α-ENaC、β-ENaC、γ-ENaC的表达显著减少。. 2.通过肺泡上皮细胞成功转染pCMV-Myc-WNK4 和pCMV-Myc-SPAK后，α-ENaC、β-ENaC、γ-ENaC的表达显著增加，SPAK磷酸化水平增加；成功转染siRNA-WNK4和siRNA-SPAK后，α-ENaC、β-ENaC、γ-ENaC的表达显著降低，SPAK磷酸化水平降低。.四、科学意义 . 本研究显示WNK4-SPAK 通路是调控ENaC 的门控“通道”，从而参与肺泡腔内的钠水转运，在ALI/ARDS 肺水肿清除中起到保护作用。对临床治疗ALI/ARDS奠定了一定的理论基础，在国内外同类研究中具有一定的创新性，发表SCI1篇，中文核心期刊3篇。