Regulation of alveolar epithelial barrier function by claudins

描述（由申请人提供）：急性呼吸窘迫综合征（ARDS）是最严重的急性肺损伤形式，是呼吸衰竭的常见原因，总死亡率接近40%；然而，目前还没有专门的药物治疗方法。急性呼吸窘迫综合征的一个标志是肺泡屏障通透性增加和空气间隙液体清除减少，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节，但对于急性肺损伤中细胞旁通透性和离子转运是如何调节的，我们知之甚少。对细胞旁通透性调控和紧密连接选择性转运的新研究对理解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存在差异表达。claudin家族蛋白的差异调控可能是上皮细胞通过改变细胞旁通路的物理特性来控制通透性和离子运动的主要机制。我们的假设是，肺泡上皮细胞控制特定的紧密连接蛋白表达，以应对环境刺激。我们发现急性肺损伤时claudin 4的表达明显增加。这可能代表了一种限制空域水肿形成的适应性反应，并允许更高的水肿清除率，因为claudin 4降低了对大分子的细胞旁通透性，有利于排除钠但允许氯化物运输的细胞旁通路。这些特性将促进空气空间流体的清除。在Aim 1中，我们将使用肽抑制剂和RNAi在原代大鼠和人肺泡上皮II型细胞中确定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受到调节的机制。在Aim 2中，我们将研究claudin 4在呼吸机肺损伤小鼠模型中的功能和调控。在Aim 3中，我们将首次检测几种claudins在因移植而被排斥的人肺中的表达，并使用我们的灌注人肺模型，将表达水平与体外上皮功能的测量相关联。这些研究将提供对肺损伤过程中肺泡上皮屏障调节机制的更完整的理解，并促进ARDS患者新的治疗策略的发展。

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.