The WNK-SPAK/OSR1 kinases and NKCC1 control epithelial barrier function by regulating claudin-2 expression

Conditions that cause the loss of epithelial barrier integrity 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 deficits, including chronic gastrointestinal inflammation, gastrointestinal hemorrhage, intestinal obstruction, and constipation. Although the intestinal inflammation observed in patients with loss of NKCC1 function may or may not be due to tight junction dysfunction, we investigated whether the loss of NKCC1 function affects paracellular ion transport and epithelial barrier function. Wild-type HT29-MTX-E12 and CRISPR/Cas9-mediated NKCC1 knockout HT29 clones were tested for tight junction protein expression and localization. Tightness of epithelial cell monolayer was assessed by measurement of transepithelial resistance and permeability of molecular tracers in transwell filters. Tight junction protein localization was assessed by immunofluorescence. Loss of NKCC1 expression strongly increases the expression of claudin-2. Genetic deletion of WNK1, WNK4, SPAK and OSR1 resulted in a substantial increase in claudin-2 expression, confirming an essential role of WNK-SPAK/OSR1 pathway in tight junction regulation. Loss of NKCC1 significantly reduces the transepithelial electrical resistance (TER) indicating an increase in paracellular ions flux, consistent with upregulation of the cation-selective and channel-forming claudin-2. In addition, NKCC1-KO monolayers showed a significant increase in the paracellular flux of small molecules like fluorescein (0.33 kDa), while the permeability of higher molecular weight TRITC-Dextran (4 kDa and 70 kDa) remained unchanged. Thus, NKCC1 regulates tight junction protein expression and loss of NKCC1 function affects epithelial barrier integrity. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.