Background & Aims

Tight junctions form a barrier to the paracellular passage of luminal antigens. Although most tight junction proteins reside within the apical tight junction complex, claudin-18 localizes mainly to the basolateral membrane where its contribution to paracellular ion transport is undefined. Claudin-18 loss in mice results in gastric neoplasia development and tumorigenesis that may or may not be due to tight junction dysfunction. The aim here was to investigate paracellular permeability defects in stomach mucosa from claudin-18 knockout (Cldn18-KO) mice.

Methods

Stomach tissue from wild-type, heterozygous, or Cldn18-KO mice were stripped of the external muscle layer and mounted in Ussing chambers. Transepithelial resistance, dextran 4 kDa flux, and potential difference (PD) were calculated from the chambered tissues after identifying differences in tissue histopathology that were used to normalize these measurements. Marker expression for claudins and ion transporters were investigated by transcriptomic and immunostaining analysis.

Results

No paracellular permeability defects were evident in stomach mucosa from Cldn18-KO mice. RNAseq identified changes in 4 claudins from Cldn18–KO mice, particularly the up-regulation of claudin-2. Although claudin-2 localized to tight junctions in cells at the base of gastric glands, its presence did not contribute overall to mucosal permeability. Stomach tissue from Cldn18–KO mice also had no PD versus a lumen-negative PD in tissues from wild-type mice. This difference resulted from changes in transcellular Cl^– permeability with the down-regulation of Cl^– loading and Cl^– secreting anion transporters.

Conclusions

Our findings suggest that Cldn18-KO has no effect on tight junction permeability in the stomach from adult mice but rather affects anion permeability. The phenotype in these mice may thus be secondary to transcellular anion transporter expression/function in the absence of claudin-18.

中文翻译：

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Claudin-18 丢失会改变胃上皮细胞中的跨细胞氯离子通量，但不会改变紧密连接离子选择性

背景与目标

紧密连接形成管腔抗原细胞旁通道的屏障。尽管大多数紧密连接蛋白位于顶端紧密连接复合物内，但claudin-18主要定位于基底外侧膜，其对细胞旁离子转运的贡献尚不清楚。小鼠中 Claudin-18 缺失会导致胃瘤形成和肿瘤发生，这可能是或可能不是由于紧密连接功能障碍所致。本次研究的目的是研究claudin-18敲除（Cldn18 -KO）小鼠胃粘膜的细胞旁通透性缺陷。

方法

将来自野生型、杂合子或Cldn18 -KO 小鼠的胃组织剥离外部肌肉层并安装在 Ussing 室中。在确定用于标准化这些测量的组织病理学差异后，从腔室组织计算跨上皮电阻、葡聚糖 4 kDa 通量和电位差 (PD)。通过转录组学和免疫染色分析研究了密蛋白和离子转运蛋白的标记表达。

结果

Cldn18 -KO 小鼠的胃粘膜没有明显的细胞旁通透性缺陷。RNAseq 鉴定了Cldn18 –KO 小鼠中 4 个紧密蛋白的变化，特别是紧密蛋白 2 的上调。尽管claudin-2定位于胃腺底部细胞的紧密连接处，但它的存在总体上对粘膜通透性没有贡献。Cldn18 –KO 小鼠的胃组织也没有出现 PD，而野生型小鼠的组织中则出现管腔阴性的 PD。这种差异是由于跨细胞 Cl ^{-渗透性的变化以及 Cl -}负载和 Cl ^-分泌阴离子转运蛋白的下调所致。

结论

我们的研究结果表明，Cldn18 -KO 对成年小鼠胃中的紧密连接通透性没有影响，但会影响阴离子通透性。因此，这些小鼠的表型可能是在claudin-18不存在的情况下继发于跨细胞阴离子转运蛋白表达/功能的。