ENaC-α mediates lung fluid clearance and capillary barrier function in pneumonia

ENaC-α 介导肺炎中的肺液清除和毛细血管屏障功能

• 批准号: 9976342
• 负责人: Rudolf Lucas
• 金额: $ 53.16万
• 依托单位: AUGUSTA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-07-01 至 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9976342
• 关键词: AGTR2 gene; ASIC channel; Actin-Binding Protein; Actins; Acute Lung Injury; Alveolar; Bacteria; Bacterial Pneumonia; Binding; Blood Vessels; Blood capillaries; Capillary Endothelial Cell; Cations; Cell Line; Cells; Data; Development; Edema; Endothelial Cells; Endothelium; Epithelial; Epithelial Cells; Epithelium; Evaluation; Female; Fluid Balance; Fostering; Functional disorder; Genetic; Goals; Human; Hybrids; Impairment; In Vitro; Individual; Infection; Ion Channel; Knockout Mice; Life; Liquid substance; Lung; Mediating; Molecular; Mus; Outcome; Pathway interactions; Peptides; Permeability; Pharmacological Treatment; Pharmacology; Phosphorylation; Pneumococcal Pneumonia; Pneumonia; Preparation; Probability; Proteins; Publishing; Recycling; Regulation; Resolution; Role; Signal Transduction; Signaling Molecule; Slice; Sodium; Sodium Channel; Streptococcus pneumoniae; Streptococcus pneumoniae plY protein; Stress Fibers; Surface; TNF gene; Testing; Toxin; Vascular Permeabilities; alveolar epithelium; epithelial Na+ channel; filamin; improved; in vivo; lung injury; male; mutant; novel; novel therapeutic intervention; overexpression; prevent; therapeutic target; uptake; vector

PROJECT SUMMARY. Pulmonary permeability edema (PPE) associated with pneumococcal pneumonia is a life-threatening condition, resulting from capillary barrier dysfunction in conjunction with impaired alveolar liquid clearance (ALC), with no proven treatment. Vectorial Na+ uptake mediates ALC. The identification of novel therapeutic approaches to simultaneously restore both ALC and barrier function represents a critical unmet need for pneumonia- associated PPE. Our main hypothesis is that the a subunit of the epithelial sodium channel (ENaC-α) represents a promising therapeutic target in PPE since it is a component of the highly-selective cation channel (HSC), which consists of ENaC-α, β and γ subunits, and of the non-selective cation channel (NSC), which contains ASIC1a and ENaC-α. Both HSC and NSC channels mediate Na+ uptake. We also propose ENaC-α as a signaling molecule that strengthens barrier function in lung capillaries in the presence of pneumococci or their pore-forming toxin pneumolysin (PLY). Our main hypothesis is that ENaC-α exerts these actions mainly by blunting phosphorylation of the actin-binding protein filamin-A. In its non-phosphorylated form, filamin-A on the one hand promotes Na+ uptake capacity in sodium channels in alveolar epithelial cells and on the other hand it prevents stress fiber formation in capillary endothelial cells. Our novel hypothesis centers on the concept that ENaC-α functions as both an ion channel component and a signaling molecule, whose specific pharmacological activation restores ALC and barrier function during pneumococcal pneumonia. We will study the effect of specific activators of NSC (MitTx), of HSC (S3969 which binds to ENaC-β, not present in NSC) or of both (TIP peptide, which binds to ENaC-α), as well as genetic depletion or overexpression of ENaC-α on Na+ uptake capacity in pneumococci- or PLY-treated alveolar epithelial cells in vitro. We will moreover investigate whether ENaC-α activation or overexpression corrects S. pneumoniae- or PLY-induced barrier dysfunction in MVEC in vitro, through inhibition of Ca2+-dependent pathways that mediate filamin-A phosphorylation. Finally, we will test our hypothesis that direct ENaC-α activation is sufficient to mediate ALC and capillary barrier function during pneumococcal pneumonia in mice and in isolated perfused human lungs. Our expected outcomes include a better characterization of the unique role of ENaC-α in ALC during pneumococcal pneumonia, demonstration of a hitherto unknown role for ENaC-α in capillary barrier regulation and evaluation of the relative role of NSC versus HSC in protection from bacterial pneumonia-induced acute lung injury. Unraveling the unique mechanisms by which ENaC-α mediates ALC and barrier function during bacterial pneumonia can foster development of a novel breakthrough treatment for pulmonary permeability edema.

项目总结。 肺炎球菌肺炎相关的肺通透性水肿(PPE)是一种危及生命的疾病， 由毛细血管屏障功能障碍和肺泡液体清除障碍(ALC)所致，无 久经考验的治疗方法。载体Na+摄取介导ALC。新的治疗方法的确定 同时恢复ALC和屏障功能代表着肺炎的严重需求尚未得到满足- 关联的PPE。我们的主要假设是上皮钠通道的α亚单位(ENaC-α) 由于它是高选择性阳离子通道的一部分，因此在PPE中是一个很有前途的治疗靶点 (Hsc)，由ENaC-α，β和γ亚基组成，以及非选择性阳离子通道(Nsc)，它 含有ASIC1a和ENaC-α。HSC和NSC通道均介导Na+摄取。我们还提出了ENaC-α 作为一种信号分子，在肺炎球菌或肺炎球菌存在的情况下加强肺毛细血管屏障功能 它们的致孔毒素--溶气素(PLE)。我们的主要假设是ENaC-α主要发挥这些作用 通过钝化肌动蛋白结合蛋白细丝-A的磷酸化。以其非磷酸化形式，丝胺-A 一方面促进肺泡上皮细胞钠离子通道的钠摄取能力，另一方面促进肺泡上皮细胞钠离子的摄取 另一方面，它可以防止毛细血管内皮细胞中应力纤维的形成。我们的新假设集中在 ENaC-α既是离子通道成分，又是信号分子，其特有的 在肺炎球菌肺炎期间，药物激活可恢复ALC和屏障功能。我们会研究 神经干细胞(MitTx)、肝星状细胞(与ENaC-β结合的S3969，在神经干细胞中不存在)或 两者(与ENaC-α结合的TIP肽)以及ENaC-α的遗传缺失或过表达 肺炎球菌或PLY处理的肺泡上皮细胞对Na+的摄取能力。此外，我们还会 研究ENaC-α激活或过表达是否纠正肺炎链球菌或PLE诱导的屏障 通过抑制介导细丝蛋白A的钙依赖通路，体外培养的微血管内皮细胞功能障碍 磷酸化。最后，我们将测试我们的假设，即直接ENaC-α激活足以调节ALC 在小鼠肺炎球菌肺炎期间和在隔离灌流的人肺中的毛细血管屏障功能。 我们的预期结果包括更好地表征ENaC-α在ALC中的独特作用 肺炎球菌肺炎：ENaC-α在毛细血管屏障调节中的未知作用 并评价NSC和HSC在细菌性肺炎所致急性肺炎保护中的相对作用 肺损伤。ENaC-α介导碱性磷酸酶和屏障功能的独特机制 细菌性肺炎可以促进一种新的突破性治疗方法的开发 浮肿。