Inactivation of the basolateral Na‐K‐2Cl cotransporter, NKCC1, increases intestinal barrier permeability

Abstract

Background Epithelial tight junctions are defective in gastrointestinal diseases. Conditions that causes epithelial barrier loss are often accompanied by dysregulation of tight junction protein expression and/or localization. Recently we have reported that patients with mutations in SLC12A2; the gene encoding the basolateral Na+-K+-2Cl- cotransporter (NKCC1), suffer from severe gastrointestinal deficit; including chronic gastrointestinal inflammation, gastrointestinal hemorrhage, intestinal obstruction, and constipation. NKCC1-KO mice develop spontaneous gastrointestinal inflammation with bleeding in the intestine. We therefore ask whether loss of NKCC1 function affect intestinal epithelial barrier function. Methods Wild-type HT29-MTX-E12 and CRISPR/Cas9 NKCC1-deleted HT29 clones were tested for tight junction protein expression and localization. Epithelial cell monolayers permeability to molecular tracers was assessed in transwell filters. Tight junction protein localization was assessed by immunofluorescence. Results Loss of NKCC1 expression strongly increases the expression of claudin-2, occluding, and ZO-1 in epithelial cell monolayers. Loss of NKCC1 significantly reduces the trans-epithelial electrical resistance (TER) indicating an increase in paracellular ions flux, which is consistent with upregulation of the cation-selective and channel-forming claudin-2. In addition, NKCC1-KO monolayers showed a significant increase in paracellular flux of small molecules, as the 0.33 kDa fluorescein permeability increases in the NKCC1-KO monolayers compared to wild-type, while the high molecular weight 4 kDa TRITC-Dextran and 70 kDa TRITC-Dextran permeability remained unchanged. In addition, Loss of NKCC1 causes mis-localization of occludin from the tight junction complex. Conclusions NKCC1 regulate tight junction protein expression and localization. Loss of NKCC1 function is a previously unknown mechanism that causes intestinal epithelial barrier loss.