Department of Surgery/Division of General Surgery, Beth Israel Deaconess Medical Center, Boston, Massachusetts; Division of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, Massachusetts; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts.
Department of Surgery/Division of General Surgery, Beth Israel Deaconess Medical Center, Boston, Massachusetts.
Cell Mol Gastroenterol Hepatol. 2021;11(3):783-801. doi: 10.1016/j.jcmgh.2020.10.005. Epub 2020 Oct 16.
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.
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.
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.
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.
紧密连接形成了管腔抗原旁细胞通过的屏障。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，其对旁细胞离子转运的贡献尚不清楚。Claudin-18 在小鼠中的缺失导致胃肿瘤的发展和肿瘤的形成，这可能是也可能不是由于紧密连接功能障碍。本研究旨在研究 Claudin-18 敲除（Cldn18-KO）小鼠胃黏膜的旁细胞通透性缺陷。
从野生型、杂合型或 Cldn18-KO 小鼠中取出胃组织，去除外肌层，置于 Ussing 室中。用 chambered 组织计算跨上皮电阻、4 kDa 葡聚糖通量和跨膜电位差（PD），并根据组织病理学差异对这些测量值进行归一化。通过转录组和免疫染色分析研究 Claudin 和离子转运体的表达。
Cldn18-KO 小鼠胃黏膜无明显的旁细胞通透性缺陷。RNAseq 鉴定出 Cldn18-KO 小鼠 Claudin-2 等 4 种 Claudin 的表达发生变化，尤其是 Claudin-2 的上调。虽然 Claudin-2 定位于胃腺底部细胞的紧密连接，但它的存在并不能增加黏膜通透性。与野生型小鼠的组织相比，Cldn18-KO 小鼠的胃组织没有 PD 差异，而是表现为负 PD。这种差异是由于 Cl 通透性的变化导致 Cl 加载和 Cl 分泌阴离子转运体下调所致。
我们的研究结果表明，Cldn18-KO 对成年小鼠胃的紧密连接通透性没有影响，但影响阴离子通透性。因此，这些小鼠的表型可能是 Claudin-18 缺失导致阴离子转运体表达/功能改变的结果。
