Sodium-coupled neutral amino acid transporter SNAT2 – a regulatory hub of alveolar homeostasis and its critical role in acute lung injury/ARDS

急性呼吸窘迫综合征(ARDS)是重症监护医学中最常见的死亡原因，其特征是炎症过度、上皮细胞凋亡、肺泡-毛细血管屏障功能丧失，并连续形成蛋白性肺水肿，最终导致呼吸衰竭。尽管进行了大量的临床试验，但仍缺乏有效的药物治疗来提高ARDS患者的总体存活率。这一未得到满足的医疗需求强调了更好地了解ARDS潜在的病理机制以及肺泡内稳态和修复的调节的必要性。在我们的项目提案中，我们假设Na+偶联的中性氨基酸转运体SNAT2在ARDS的肺水肿形成和肺泡上皮细胞凋亡中起关键作用。到目前为止，SNAT2还没有与任何肺部病理有关。然而，由于SNAT2介导中性氨基酸与Na+的共转运，它可能会影响Na+驱动的肺泡液清除(AFC)和氨基酸调节的细胞信号转导。在初步的概念验证数据中，我们表明：1)在ARDS特有的炎症刺激下，SNAT2的表达下调；2)SNAT2的缺失抑制了AFC，从而促进了水肿的形成；3)SNAT2的缺失通过细胞内氨基酸剥夺加剧了内质网应激、ROS的形成和自噬介导的细胞凋亡。因此，SNAT2可能演变为一种新的肺泡动态平衡的主调节因子，SNAT2的缺失促进了ARDS的经典特征。在拟议的工作中，我们将首先在分离的灌流小鼠肺中、在体内的急性肺损伤小鼠模型中以及在体外的肺上皮细胞培养系统中，巩固SNAT2在AFC中与水肿消退的相关性。接下来，我们将分析SNAT2在完整和损伤的肺泡上皮中的调控。为此，我们将重点研究SNAT2在体外和原位响应炎症刺激、细菌毒素或活菌感染时表达和功能的变化，并剖析潜在的调控机制。最后，我们将深入探讨SNAT2在上皮损伤和修复过程中的调节作用。具体地说，我们提出，在缺乏SNAT2功能的细胞中，由于内质网应激标志物的损伤表达，凋亡和自噬蛋白以及ROS的产生将增加。我们期待我们的研究结果产生基本的新知识，因为它们将关键地推动我们对肺泡液运输和水肿形成的理解，并对氨基酸及其辅助转运体在与肺泡上皮损伤相关的促凋亡和抗凋亡信号通路中的作用产生重要的见解。我们的发现有望通过确定SNAT2作为一个潜在的靶点来改善ARDS患者的水肿消退和维持或恢复上皮屏障功能，从而提供显著的翻译益处。

Acute respiratory distress syndrome (ARDS), the most frequent cause of mortality in critical care medicine, is characterized by hyperinflammation, epithelial apoptosis, and a loss of alveolar-capillary barrier function with consecutive formation of a proteinaceous lung edema that ultimately results in respiratory failure. Despite numerous clinical trials, effective pharmacological treatments to improve overall survival in ARDS patients are still lacking. This unmet medical need stresses the necessity for a better understanding of the pathomechanisms underlying ARDS and the regulation of alveolar homeostasis and repair. In our project proposal, we postulate a key role for Na+-coupled neutral amino acid transporter SNAT2 in lung edema formation and alveolar epithelial apoptosis in ARDS. So far SNAT2 has not been implicated in any pulmonary pathologies. Yet, as SNAT2 mediates cellular uptake of neutral amino acids in cotransport with Na+, it may impact both Na+-driven alveolar fluid clearance (AFC) and amino acid-regulated cell signaling. In preliminary proof-of-concept data we show i) that SNAT2 expression is downregulated in response to inflammatory stimuli characteristic for ARDS, ii) that loss of SNAT2 inhibits AFC thus promoting edema formation, and iii) that SNAT2 loss aggravates ER-stress, ROS formation, and autophagy-mediated apoptosis through intracellular amino acid deprivation. SNAT2 may thus evolve as a novel master regulator of alveolar homeostasis, with SNAT2 loss promoting the classic hallmarks of ARDS.In the proposed work, we will first consolidate the relevance of SNAT2 in AFC and edema resolution in situ in isolated perfused mouse lungs, in vivo in a murine model of acute lung injury, and in vitro in a pulmonary epithelial cell culture system. Next, we will analyze SNAT2 regulation in the intact and injured alveolar epithelium. To this end, we will focus on changes in SNAT2 expression and function in response to inflammatory stimuli, bacterial toxins, or infection with live bacteria both in vitro and in situ, and dissect underlying regulatory mechanisms. Lastly, we will probe in depth for the regulatory role of SNAT2 on epithelial injury and repair processes. Specifically, we propose that in response to injury expression of ER stress markers, apoptotic and autophagic proteins, and ROS production will be increased in cells lacking functional SNAT2.We anticipate the results of our research to generate fundamental new knowledge, in that they will critically propel our understanding of dysregulated alveolar fluid transport and edema formation and generate important insights into the role of amino acids and their co-transporters in pro- and anti-apoptotic signaling pathways pertinent to alveolar epithelial injury. Our findings are expected to provide significant translational benefits by identifying SNAT2 as a potential target to improve edema resolution and maintain or restore epithelial barrier function in ARDS patients.