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

肺功能依赖于肺泡上皮细胞，肺泡上皮细胞负责表面活性物质的产生和跨上皮离子/液体的运输。事实上，为了保持肺泡腔几乎没有液体，液体清除是必需的，而液体是气体交换所必需的。已经证实，液体吸收主要是由通过Na+通道ENaC的活性离子传输驱动的。然而，ENaC和Na+/流体转运的复杂调控机制仍然不完全清楚。 在我们之前的NSERC资助条款中，我们在了解ENaC通道和通过初级肺泡II型(ATII)细胞的Na+/液体运输的机制方面取得了重大进展。我们的工作还揭示了另一类通道，即K+通道(KCH，即KATP和KvQT1通道)，它通过改变启动子活性来调节ENaC的表达，并参与Na+/液体吸收的控制。最后，我们实现了一个综合的小鼠模型来研究液体的清除和肺功能参数。我们的初步体内数据表明，ENaC和KCH与KCH对肺泡液清除、表面活性物质表达和肺顺应性的控制有关。我们现在建议继续我们的研究计划，通过细胞和动物模型分析ENaC和KCH在控制液体吸收和肺功能方面的互补作用。 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ENaC启动子区域参与了KCH对ENaC的调节；2)ENaC和KCH在基础条件下以及随后的肾上腺素能激动剂或皮质类固醇刺激下通过ATII细胞的液体吸收方面的互补功能和相对贡献。表面活性蛋白的表达也将被评估。我们的第二个目标将是在离子通道沉默后，或相反地，在注入刺激Na+和K+转运的药物化合物后，在小鼠身上进行我们的体内研究。基于我们的体外观察显示KCH功能与ENaC表达的关系，我们将检测参与肺泡原位清除的离子通道和转运体的表达水平。最后，我们将确定ENaC和KCH对液体清除率、表面活性物质产生、肺功能参数以及血气的调节作用；在基础条件下以及在肾上腺素能激动剂或皮质类固醇刺激后。 我们的研究计划将使我们能够更好地了解在非病理条件下，肺泡上皮细胞在肺上皮生理学中控制主要功能的调节机制。我们的NSERC项目结合了分子和细胞方面以及一个研究小鼠肺生理学的综合模型，还在我们的实验室为HQP提供了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.