Regulation of alveolar epithelial barrier function by claudins

描述(申请人提供)：急性呼吸窘迫综合征(ARDS)是最严重的急性肺损伤形式，是导致呼吸衰竭的常见原因，总体死亡率接近40%；然而，目前还没有专门的药物疗法。ARDS的一个特点是肺泡屏障通透性增加，液体从空气中清除减少，导致肺水肿。尽管我们和其他人的工作已经开始阐明跨细胞上皮离子转运的调控，但对于急性肺损伤中细胞旁通透性和离子转运是如何调控的知之甚少。对细胞旁通透性的调节和通过紧密连接的选择性转运的新研究对于了解ARDS的上皮功能具有重要意义。在初步研究中，我们发现紧密连接蛋白在急性肺损伤中有不同的表达。Claudin蛋白家族的不同调节可能代表了上皮细胞改变细胞旁途径的物理性质以控制通透性和离子移动的中心机制。我们的假设是，肺泡上皮细胞控制着特定的紧密连接蛋白在环境刺激下的表达。我们发现，在急性肺损伤过程中，claudin 4的表达显著增加。这可能代表了一种适应性反应，以限制空间水肿的形成，并允许更高的水肿率清除，因为claudin 4降低了细胞旁对大分子的通透性，并有利于排除钠但允许氯运输的细胞旁途径。这些特性将促进空域流体的清除。在目标1中，我们将使用一种肽抑制剂和RNAi在原代大鼠和人肺泡II型上皮细胞中确定claudin 4对紧密连接的功能贡献。我们还将确定claudin 4和其他紧密连接蛋白表达的调节机制。目的2研究Claudin 4在呼吸机诱导的小鼠肺损伤模型中的作用和调控。在目标3中，我们将首次检测排斥移植的人肺中几个Claudins的表达，并使用我们的灌流人肺模型，将表达水平与体外培养的上皮功能指标相关联。这些研究将对肺损伤过程中肺泡上皮屏障的调节机制提供更全面的了解，并有助于开发ARDS患者的新治疗策略。

DESCRIPTION (provided by applicant): Acute Respiratory Distress Syndrome (ARDS), the most severe form of acute lung injury, is a common cause of respiratory failure with an overall mortality rate of nearly 40%; however, there are currently no specific pharmacological therapies. A hallmark of ARDS is increased alveolar barrier permeability and decreased clearance of fluid from the airspaces resulting in pulmonary edema. Although work by our group and others has begun to shed light on the regulation of transcellular epithelial ion transport, much less is known about how paracellular permeability and ion transport are regulated in acute lung injury. New research into the regulation of paracellular permeability and selective transport through tight junctions is of fundamental importance to understanding epithelial function in ARDS. In preliminary studies, we have found that tight junction claudins are differentially expressed in acute lung injury. Differential regulation of the claudin family of proteins may represent a central mechanism by which epithelial cells alter the physical properties of the paracellular pathway to control permeability and ion movement. Our hypothesis is that alveolar epithelial cells control the particular tight junction claudins expressed in response to environmental stimuli. We found a significant increase in claudin 4 expression during acute lung injury. This may represent an adaptive response to limit airspace edema formation and allow higher rates of edema clearance because claudin 4 decreases paracellular permeability to large molecules and favors a paracellular pathway that excludes sodium but allows chloride transport. These properties would promote airspace fluid clearance. In Aim 1, we will determine the functional contribution of claudin 4 to tight junctions using a peptide inhibitor and RNAi in primary rat and human alveolar epithelial type II cells. We will also determine the mechanisms by which claudin 4 and other tight junction protein expression is regulated. In Aim 2, we will study the function and regulation of claudin 4 in our mouse model of ventilator-induced lung injury. In Aim 3, we will for the first time examine expression of several claudins in human lungs rejected for transplantation and, using our perfused human lung model, correlate expression levels with measures of epithelial function ex vivo. These studies will provide a more complete understanding of the mechanisms of alveolar epithelial barrier regulation during lung injury and facilitate the development of new therapeutic strategies for ARDS patients.