Knockout mice reveal key roles for claudin-18 in alveolar fluid homeostasis

Claudin proteins are major constituents of epithelial and endothelial tight junctions (TJ), where they serve as regulators of paracellular permeability to ions and solutes. Claudin-18, a member of the large claudin family, is highly expressed in lung epithelium. To elucidate the role of claudin-18 in alveolar epithelial barrier function and fluid homeostasis, we generated claudin-18 knockout (C18 KO) mice. Increased alveolar fluid clearance (AFC) observed in C18 KO mice may have accounted for absence of lung edema despite increased alveolar solute permeability compared to wild type (WT) controls. Higher AFC in C18 KO mice was associated with higher Na-K-ATPase activity and increased expression of the Na-K-ATPase beta1 subunit compared to WT controls. Consistent with in vivo findings, alveolar epithelial cell (AEC) monolayers derived from C18 KO mice exhibited lower transepithelial electrical resistance (RT) accompanied by increased solute and ion permeability without changes in ion selectivity. Expression of claudin-3 and claudin-4 was markedly increased in whole lung and in freshly isolated AEC from C18 KO mice, while claudin-5 was unchanged. In contrast, occludin, another major component of the TJ complex, was significantly decreased in C18 KO lung. Further analysis revealed rearrangements in the F-actin cytoskeleton in C18 KO MAECM. These findings demonstrate a crucial non-redundant role for claudin-18 in regulation of alveolar epithelial tight junction composition and permeability to ions and solutes. Importantly, increased AFC in C18 KO mice identifies additional roles for claudin-18 in alveolar fluid homeostasis beyond its direct contributions to barrier properties of the alveolar epithelium. Animals with a ubiquitous knockout (C18 KO) were obtained by crossing mice harboring a conditional (floxed) allele of claudin-18 (Cldn18F/F) with CMV-cre deleter mice to delete exons 2 and 3 by Cre/loxP recombination.紧密连接蛋白（Claudin proteins）是上皮细胞与内皮细胞紧密连接（tight junctions, TJ）的核心组成成分，可通过该结构调控离子与溶质的细胞旁通透性。作为庞大的紧密连接蛋白家族成员之一，紧密连接蛋白18（Claudin-18）在肺上皮细胞中呈高表达。为阐明紧密连接蛋白18在肺泡上皮屏障功能与液体稳态中的调控作用，本研究构建了紧密连接蛋白18基因敲除（Claudin-18 knockout, C18 KO）小鼠模型。与野生型（wild type, WT）对照小鼠相比，C18 KO小鼠的肺泡溶质通透性升高，但未出现肺水肿，这一表型可能与其观测到的肺泡液体清除率（alveolar fluid clearance, AFC）升高有关。相较于WT对照小鼠，C18 KO小鼠的AFC升高与钠钾ATP酶（Na-K-ATPase）活性提升及该酶β1亚基的表达上调显著相关。与体内实验结果一致，从C18 KO小鼠分离得到的肺泡上皮细胞（alveolar epithelial cell, AEC）单层培养物的跨上皮电阻（transepithelial electrical resistance, RT）降低，同时溶质与离子通透性升高，但离子选择性未发生改变。相较于WT对照小鼠，C18 KO小鼠的全肺组织与新鲜分离的AEC中，紧密连接蛋白3（Claudin-3）与紧密连接蛋白4（Claudin-4）的表达显著上调，而紧密连接蛋白5（Claudin-5）的表达无明显变化。与之相反，紧密连接复合物的另一核心组分闭合蛋白（occludin）在C18 KO小鼠肺组织中的表达水平显著降低。进一步分析显示，C18 KO小鼠的MAECM中存在丝状肌动蛋白（F-actin）细胞骨架的重排。本研究结果证实，紧密连接蛋白18在调控肺泡上皮紧密连接的组成成分及离子、溶质通透性方面发挥着不可或缺的关键作用。尤为重要的是，C18 KO小鼠体内升高的AFC表明，紧密连接蛋白18除了直接影响肺泡上皮的屏障特性外，还在肺泡液体稳态中承担着额外的调控功能。本研究通过将携带紧密连接蛋白18条件性（floxed）等位基因（Cldn18F/F）的小鼠与CMV-cre敲除小鼠杂交，经Cre/loxP重组删除第2、3号外显子，成功获得了全身性基因敲除（C18 KO）小鼠。