钠钾ATP酶/Src信号复合体在慢性肾损伤中的作用及机制研究

Chronic kidney disease (CKD) is a progressive disease that will finally develop end stage of renal disease. Epithelial-mesenchymal transition (EMT) of renal tubular cells plays key role in the development of chronic renal interstitial fibrosis which is the final common pathological change of CKD patients. The Na/K-ATPase (NKA) is a transmembrane enzyme responsible for pumping Na+ and K+ ions across the plasma membrane by hydrolyzing ATP. Recent studies have revealed that Na/K-ATPase is also involved in tethering and regulating multiple protein and lipid kinases as well as membrane receptors, which means it can also act as a regulator of signal transduction. Reactive oxygen species (ROS) is an important pathogenic factor for CKD and may participate the regulation of EMT process. We had found that the expression of tubular NKA in both CKD patients and animals was decreased; furthermore, we found that ROS could induce the endocytosis and decrease of the amount of NKA in proximal renal tubular cells, with simultaneous activation of intracellular Src/ERK pathway. This stimulation was dependent on NKA/Src receptor complex on plasma membrane (under the support of previous NSF (81041001)). Based on these preliminary data, we want to make studies of the roles and mechanism of NKA/Src receptor complex in ROS-induced EMT of tubular cells by using NKA knockdown/rescued cell lines and NKA/Src interaction mutation cell lines. Furthermore, we want to build CKD animal models with NKA knock-down animals and make interventions with PP2 and pNKtide to observe the involvement of NKA regulated signal transduction pathways in the development of chronic interstitial fibrosis in vivo. The results will help us to make better understand of the potential roles of NKA regulated signal pathways in the development of chronic renal injuries in CKD.

肾小管上皮细胞转分化（EMT）是慢性肾脏病（CKD）肾损伤进展的重要细胞生物学事件，活性氧簇（ROS）可参与其调控。钠钾ATP酶（NKA）是肾小管上皮细胞高表达的跨膜离子转运蛋白，近年来被证实参与细胞内蛋白激酶信号转导通路的调控。我们既往观察到CKD患者和动物中肾小管NKA表达改变；体外细胞研究证实，ROS可引起NKA分布及表达改变，并通过NKA/Src信号复合体调控Src/ERK信号通路活化。本研究拟应用ROS刺激体外培养的正常、NKA敲出/挽救、以及NKA/Src信号复合体结合位点突变的肾小管上皮细胞，探讨NKA/Src信号复合体在ROS诱导的肾小管上皮细胞EMT中的作用及机制；并应用NKA基因敲出小鼠建立慢性肾损伤模型，用PP2及pNKtide进行相关信号干预，观察NKA信号通路与慢性肾脏纤维化病变之间的关系。研究结果将有助于从信号转导调控角度阐明NKA在CKD进展中的作用及机制。

肾间质纤维化是各类CKD发生及进展过程中肾脏的共同病理改变，是CKD肾功能恶化进展的决定因素。肾间质肌成纤维细胞聚集是肾间质纤维化的主要贡献者。许多CKD致病因素均可导致肾脏局部氧化应激增强，ROS产生增加。真核细胞表面的钠钾ATP酶（NKA）除了主动跨膜转运钠钾离子外，还可以固定及调节多个蛋白及脂类激酶以及膜受体，组装成信号转导复合体，在配体的作用下引发细胞内信号转导的级联反应，影响细胞的多种生物学行为。其中，NKA/Src信号复合体是NKA相关信号转导的核心复合体之一，而NKA是该复合体中Src活性的重要调节因子，这个调节功能在上皮细胞内尤为突出。我们在前一项自然基金的研究中（81041001）观察到：ROS可通过NKA/Src信号复合体介导引起肾小管上皮细胞内Src/ERK信号通路的活化。本课题通过在体动物模型及体外细胞实验进一步观察了NKA/Src信号复合体在ROS致肾间质纤维化发生进展中的作用及其相关信号转导通路机制。研究结果显示：NADPH氧化酶家族（NOXs）的活化是UUO肾脏ROS生成增加的主要来源，是导致经典肾间质纤维化动物模型——UUO发生肾间质纤维化的重要原因。抑制NOXs系统及其继发的氧化应激可以有效减轻肾间质纤维化。在上述过程中，可以同时观察到NKA表达的下调及Src活化的增加，可被抑制NOXs系统及继发的氧化应激后部分逆转。进一步的研究显示，给予不同层次阻断干预——夹竹桃麻素抑制NOXs-ROS，PP2抑制Src活化，pNKtide抑制NKA/Src信号复合体后，UUO梗阻肾的Src及ERK活化明显受抑制，肾间质肌成纤维细胞聚集减少，并可见到肾间质纤维化相应指标的减轻，提示NKA/Src信号复合体可以通过ROS-NKA/Src-ERK信号通路诱导肌成纤维细胞聚集介导ROS引起的肾间质纤维化。肾间质纤维化时肌成纤维细胞来源之一为肾小管上皮细胞的转分化（EMT）。我们进一步在体外培养的肾小管上皮细胞-LLC-PK1细胞上通过使用葡萄糖氧化酶刺激模拟慢性氧化应激状态，观察到ROS可通过ROS-NKA/Src信号复合体-MAPK信号通路诱导LLC-PK1细胞发生EMT。上述结果初步证实，NKA/Src信号复合体在氧化应激导致的肾间质纤维化发生中发挥了重要作用，值得进一步研究。