Sidahmed, M. | Search bioRxiv

bioRxiv2023

Claudin-2 limits pancreatitis development through regulating tight junction-controlled pancreatic ductal transport

Pancreatitis is an inflammatory disease of the pancreas that can arise due to various factors, including environmental risks such as diet, alcohol, and smoking, as well as genetic predispositions. In some cases, pancreatitis may progress and become chronic, leading to irreversible damage and impaired pancreatic function. Genome-wide association studies (GWAS) have identified polymorphisms at the X-linked CLDN2 locus as risk factors for both sporadic and alcohol-related chronic pancreatitis. CLDN2 encodes claudin-2 (CLDN2), a paracellular cation-selective channel localized at tight junctions and expressed in the pancreas and other secretory organs. However, whether and how CLDN2 may modify pancreatitis susceptibility remains poorly understood. We aimed to clarify the potential role of CLDN2 in the onset and progression of pancreatitis. We employed multiple methodologies to examine the role of CLDN2 in human pancreatic tissue, caerulein-induced experimental pancreatitis mouse model, and pancreatic ductal epithelial organoids. In both human chronic pancreatitis tissues and caerulein-induced experimental pancreatitis, CLDN2 protein was significantly upregulated in pancreatic ductal epithelial cells. Our studies using pancreatic ductal epithelial organoids and mice demonstrated the inflammatory cytokine IFN{gamma} upregulates claudin-2 expression at both RNA and protein levels. Following caerulein treatment, Ifng KO mice had diminished upregulation of CLDN2 relative to WT mice, indicating that caerulein-induced claudin-2 expression is partially driven by IFN{gamma}. Functionally, Cldn2 knockout mice developed more severe caerulein-induced experimental pancreatitis, indicating CLDN2 plays a protective role in pancreatitis development. Pancreatic ductal epithelial organoid-based studies demonstrated that CLDN2 is critical for sodium-dependent water transport and necessary for cAMP-driven, CFTR-dependent fluid secretion. These findings suggest that functional crosstalk between CLDN2 and CFTR is essential for fluid transport in pancreatic ductal epithelium, which may protect against pancreatitis by adjusting pancreatic ductal secretion to prevent worsening autodigestion and inflammation. In conclusion, our studies suggest CLDN2 upregulation during pancreatitis may play a protective role in limiting disease development, and decreased CLDN2 function may increase pancreatitis severity. These results point to the possibility of modulating pancreatic ductal CLDN2 function as an approach for therapeutic intervention of pancreatitis.

pathology↗

bioRxiv2023

Molecular dynamics analyses of CLDN15 pore size and charge selectivity

Claudin-15 (CLDN15) molecules form channels that directly regulate cation and water transport. In the gastrointestinal tract, this transport indirectly impacts nutrient absorption. However, the mechanisms governing ion transport through these channels remain poorly understood. We addressed this question by building on our previous cell culture studies and all atom molecular dynamic simulation model of CLDN15. By mutating D55 to a bulkier glutamic acid (E) or neutral amino acid asparagine (N), our in vitro measurements showed that the D55E mutation decreased charge selectivity and favored small ion permeability, while the D55N mutation led to reduced charge selectivity without markedly altering size selectivity. By establishing a simplified (reduced) CLDN15 molecular dynamics model that excludes non-essential transmembrane regions, we were able to probe how D55 modified cation dehydration, charge interaction, and permeability. These results provide novel insight into organization of the CLDN15 selectivity filter and suggests that D55 plays a dual role in shaping both electrostatic and steric properties of the pore, but its electrostatic role is more prominent in determining CLDN15 cation permeability. This knowledge can be used toward the development of effective strategies to modulate CLDN15 function. The experimental approach established can be further extended to study the function of other claudin channels. Together, these advancements will help us to modulate tight junctions to promote human health. SUMMARYCell culture and molecular dynamics simulations reveal the role of the CLDN15-D55 residue in ion size and charge selectivity. A reduced CLDN15 model offers novel insights into ion conductance, providing a valuable tool for therapeutic modulation of tight junctions.

physiology↗