NKA/Src信号复合体氧化应激放大作用发生机制及在肾间质纤维化中的作用研究

Oxidative stress is an important cause of renal interstitial fibrosis, with the NADPH oxidase (NOXs) as the major contributor to the generation of reactive oxygen species (ROS). The Na/K-ATPase (NKA)/Src receptor complex is the key receptor complex among NKA-related receptor complexes, with NKA acts as an important regulator of Src activation and subsequent signal transduction. In our previous study, we found that exogenous ROS could induce the activation of the NKA/Src complex with subsequent activation of Src/ERK pathway in LLCPK1 cells. We also found that inhibition of NOXs activation and subsequent ROS production could partly reversed the decreased expression of NKA and increased activation of Src in UUO animals. Furthermore, inhibition of NOXs or Src activation could alleviate renal interstitial fibrosis in UUO. Interestingly, it was reported that activation of the NKA/Src complex per ce could induce intracellular ROS production in previous studies. Based on these data, we hypothesized that NKA/Src complex might contribute to the renal fibrosis via its oxidative stress amplification effect in the present project. We will explore the potential role of NOXs in the generation of oxidative stress amplification effect of NKA/Src complex in in vitro studies. The contribution of the oxidative stress amplification effect to the development of interstitial fibrosis will be further explored in UUO animals. We hope the results would help to elucidate the role of NKA/Src complex in the development of renal interstitial fibrosis from the point of view of oxidative stress regulation.

氧化应激是肾间质纤维化的重要致病因素。NOXs系统活化是肾脏活性氧簇（ROS）产成的主要来源。我们既往研究证实：钠钾ATP酶（NKA）/Src信号复合体可被外源性ROS激活；抑制NOXs活化可部分逆转UUO鼠肾小管NKA表达的下调及Src的活化；抑制NOXs活化或抑制Src活化均可显著减轻UUO肾间质纤维化。基于NKA/Src信号复合体活化本身又可使细胞内ROS生成增加的文献背景，本课题拟通过细胞实验，观察NOXs系统活化与NKA/Src信号复合体这种氧化应激放大作用之间的关系及调控机制；并通过给予UUO模型不同干预，在在体水平观察NKA/Src信号复合体氧化应激放大作用在肾间质纤维化中的病理生理作用。研究结果有助于从氧化应激调控角度阐明NKA/Src信号复合体在慢性肾间质纤维化进展中的作用。

肾间质纤维化的发生/进展机制及干预一直是肾脏病研究领域中的重点。在多种肾脏间质纤维化致病因素的作用过程中，都可观察到肾脏局部的氧化应激增强，表现为活性氧簇(ROS)生成增加。NOXs是重要的促氧化系统之一。Src激酶可调控多条细胞内信号转导通路。钠钾ATP酶(NKA)通过与Src激酶组装成NKA/Src信号复合体，可以调控Src激酶的活化。当NKA/Src信号复合体激活后可以观察到细胞内ROS生成增加，但机制尚不清楚。近年来研究揭示，ROS亦可像NKA经典配体一样激活NKA/Src信号复合体，从而提出了NKA/Src/ROS放大环路学说。基于上述背景，我们提出本课题假说：在肾间质纤维化时，NKA/Src/ROS氧化应激放大环路通过正性反向回馈作用，使肾脏局部氧化应激持续，导致Src及其下游相关信号通路持续活化，进而推进肾间质纤维化进展。而NOXs系统则作为重要的促氧化系统参与NKA/Src/ROS放大环路的发生/持续。我们分别采用NAK/Src信号复合体的特异阻滞剂-pNaKtide以及夹竹桃麻素干预处理UUO小鼠，观察到抑制NKA/Src信号复合体及其下游信号通路的活化后以及阻断NOXs系统后均可减轻小鼠肾间质纤维化。在给予pNaKtide处理后，UUO肾脏NKA表达下降未受影响，但NOXs系统活化降低伴ROS生成减少；而在夹竹桃麻素干预模型中，除NOXs系统受抑外，UUO小鼠Src激酶及其下游ERK/p38/AKT活化受到抑制。这些结果提示：肾间质纤维化时，NOXs既可以从源头上通过ROS生成激活NKA/Src信号复合体，又可以参与NKA/Src信号复合体活化后氧化应激的发生及相关信号通路活化的调控，通过NKA/Src/ROS氧化应激放大环路的正性反向回馈效应，推动肾间质纤维化进程。我们进一步应用LLC-PK1细胞系，采用NKA的经典配体-哇巴因为刺激物，观察到哇巴因刺激后，NKA/Src信号复合体及其下游信号通路活化，同时伴有细胞内ROS增加及NOX2表达增加，NOX4无明显改变。应用PP2和pNaKtide预处理细胞后，哇巴因诱导的NOX2表达升高受抑。应用NAC阻断氧化应激后，哇巴因对Src下游信号ERK/p38的活化受抑，而Src及NOX2的活化未受影响。上述研究结果初步证实了NOX2可能作为NOXs系统的主要亚型，参与NKA/Src/ROS氧化应激放大