CLDN18 Loss Affects Transcellular Cl‐Permeability Rather than Dysregulating Tight Junctions
Authors: Caron Tyler J , Sinha Nishita , Scott Kathleen E , Ang Lay‐Hong , Fox James G , Hagen Susan J 
Published: November 2023

Loss of tight junction integrity is an important risk factor for H. pylori‐mediated gastric cancer (GC) development. The loss of claudin (CLDN)18 in particular, a membrane spanning protein of the tight junction complex in stomach, is associated with an aggressive phenotype and poor patient outcome. The loss of CLDN18 occurs early, after the onset of intestinal metaplasia, and is thought to promote GC development. To model this outcome in mice, we recently demonstrated that CLDN18‐deficient (KO) mice have severe atrophy, glandular dysplasia, and a rapid progression to intraepithelial neoplasia. CLDN18 KO mice also upregulate CLDN2, which is a leaky CLDN that increases paracellular permeability. Because the loss of CLDN18 may affect tight junction function to drive GC development, the AIM of this work was to examine the permeability of stomachs from CLDN18 KO mice using Ussing chambers.

Methods

Frozen embryos from CLDN18 KO mice (B6:129S5‐Cldn18tm1Lex/Mmucd) were purchased from the MMRRC and re‐derived. Isolated gastric mucosa from 7 week old wild‐type (WT), heterozygous (HET), or knockout (KO) mice were stripped of the muscularis externa and then mounted between two Lucite halves of an Ussing‐type chamber. Tissues were maintained at 37° C with a balanced nutrient buffer on the luminal and basolateral sides and were gassed with 95% O2/5% CO2. Gastric acid secretion was blocked with an H2 receptor antagonist. Transepithelial resistance (TER)/conductance, potential difference (PD), the luminal to basolateral flux of FITC‐labeled dextran (FD‐4), and dilution potentials were measured. Confocal microscopy was used to analyze CLDN2 expression and RNAseq was used to assess transcriptional differences between WT and KO mucosa at 7 weeks. All data were analyzed using Systat software.

Results

In WT and HET mice, the PD was highly lumen negative, due to constitutive transcellular chloride flux from the basolateral to luminal surface. In contrast, the PD of tissues from KO mice was nearly zero, concomitant with a significant reduction in chloride permeability. This result occurred with no significant change in TER/conductance and no change in paracellular flux between groups. Although RNAseq indicated a large‐fold increase in CLDN2 expression, confocal microscopy analysis showed substantially lower protein expression. Gene expression for the majority of known chloride channels and transporters in glandular epithelial cells was significantly down‐regulated in KO mice.

Conclusions

Our results suggest that there is no dysregulation of tight junction function in CLDN18 KO mice but rather, a decrease in transcellular chloride permeability. This is likely due to the lack of chloride transporters in the pre‐neoplastic mucosa. We conclude that the loss of CLDN18 in GC development does not affect tight junction integrity or alter paracellular permeability per se, but instead drives neoplastic change by other mechanisms.

Support or Funding Information

Funding was from the Department of Surgery, BIDMC and NIH P30 DK034854 (SJH), R01 CA093405 and OD011141, P30 ES002109, and P01 CA028842 (JGF). Dr. Caron was supported by T32 OD0109978 (JGF).