Regulatory mechanisms of fluid clearance and lung function by alveolar ion channels

肺功能依赖于负责表面活性剂产生和跨上皮离子/液体转运的肺泡上皮细胞。实际上，需要流体清除来维持肺泡空间几乎没有液体，这是气体交换所必需的。已经确定，液体吸收主要由通过Na+通道ENaC的活性离子转运驱动。然而，ENaC和Na+/液体转运的复杂调节机制仍不完全清楚。 在我们以前的NSERC赠款的条款，我们已经取得了重大进展的机制，通过初级肺泡II型（ATII）细胞调节ENaC通道和Na+/液体运输的理解。我们的工作还揭示了另一类通道，即K+通道（KCh，即KATP和KvQT 1通道），其通过改变启动子活性来调节ENaC表达并参与Na+/液体吸收的控制。最后，我们已经实施了一个综合小鼠模型，以研究液体的清除率和肺功能参数。我们的初步体内数据表明ENaC和KCh之间的关系，并通过KCh控制肺泡液体清除率、表面活性物质表达和肺顺应性。我们现在建议继续我们的研究计划，使用细胞和动物模型来剖析ENaC和KCh在控制液体吸收和肺功能中的互补作用。 我们的第一个目标是在细胞水平上研究：1）控制ENaC表达的信号和参与KCh调节ENaC的ENaC启动子区域，和2）ENaC和KCh在基础条件下以及在用β-肾上腺素能激动剂或皮质类固醇刺激后对通过ATII细胞的液体吸收的互补功能和相对贡献。还将评价表面活性蛋白的表达。我们的第二个目标是在离子通道沉默后，或相反地在滴注刺激Na+和K+转运的药理学化合物后，在小鼠中进行我们的体内研究。基于我们的体外观察显示KCh功能和ENaC表达之间的关系，我们将测量原位肺泡清除中涉及的离子通道和转运蛋白的表达水平。最后，我们将定义ENaC和KCh调节对液体清除率、表面活性剂产生、肺功能参数以及血气的生理作用;在基础条件下和用β-肾上腺素能激动剂或皮质类固醇刺激后。 我们的研究计划将允许更好地了解负责控制肺泡细胞在肺上皮生理学中的主要功能的调节机制，在非病理条件下。结合分子和细胞方面以及一个综合模型来研究小鼠的肺生理学，我们的NSERC计划还为我们实验室的HQP提供高水平的多学科培训。

Lung function is dependent on alveolar epithelial cells that are responsible for surfactant production and transepithelial ion/liquid transport. Indeed, fluid clearance is required to maintain alveolar spaces virtually free of liquid, which is necessary for gas exchange. It has been established that liquid absorption is mainly driven by active ion transport through the Na+ channel ENaC. However, the complex regulatory mechanisms of ENaC and Na+/fluid transport remain incompletely understood. Over the terms of our previous NSERC grants, we have made significant progress in the understanding of the mechanisms regulating ENaC channels and Na+/liquid transport through primary alveolar type II (ATII) cells. Our work also unveiled another class of channels, i.e. K+ channels (KCh, namely KATP and KvQT1 channels) that regulates ENaC expression via changes in promoter activity and participate in the control of Na+/liquid absorption. Finally, we have implemented an integrative murine model to study the clearance of fluids and lung function parameters. Our preliminary in vivo data indicated a relationship between ENaC and KCh and a control of alveolar fluid clearance, surfactant expression and lung compliance by KCh. We now propose to pursue our research program to dissect the complementary roles of ENaC and KCh in the control of fluid absorption and lung function using both cellular and animal models. Our first goal will be to investigate, at the cellular level: 1) the signals controlling ENaC expression and the region of the ENaC promoter involved in ENaC regulation by KCh and 2) the complementary function, and relative contribution, of ENaC and KCh, on liquid absorption through ATII cells under basal conditions as well as following stimulation with ß-adrenergic agonists or corticosteroids. Expression of surfactant proteins will also be evaluated. Our second aim will be to pursue our in vivo studies in mice after ion channel silencing, or conversely after instillation of pharmacological compounds stimulating Na+ and K+ transport. Based on our in vitro observation showing a relationship between KCh function and ENaC expression, we will measure the level of expression of ion channels and transporters involved in alveolar clearance in situ. Finally, we will define the physiological effects of ENaC and KCh modulation on liquid clearance, surfactant production, lung function parameters as well as blood gases; in basal conditions and after stimulation with ß-adrenergic agonists or corticosteroids. Our research program will allow a better understanding of the regulatory mechanisms responsible for the control of major functions of alveolar cells in lung epithelial physiology, in non-pathological conditions. Combining molecular and cellular aspects as well as an integrative model to study lung physiology in mice, our NSERC program also provides high level, multidisciplinary training to HQP in our lab.