title: Cellular chloride and bicarbonate retention alters intracellular pH regulation in Cftr KO crypt epithelium.
abstract: Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR), an anion channel providing a major pathway for Cl(-) and HCO3 (-) efflux across the apical membrane of the epithelium. In the intestine, CF manifests as obstructive syndromes, dysbiosis, inflammation, and an increased risk for gastrointestinal cancer. Cftr knockout (KO) mice recapitulate CF intestinal disease, including intestinal hyperproliferation. Previous studies using Cftr KO intestinal organoids (enteroids) indicate that crypt epithelium maintains an alkaline intracellular pH (pHi). We hypothesized that Cftr has a cell-autonomous role in downregulating pHi that is incompletely compensated by acid-base regulation in its absence. Here, 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein microfluorimetry of enteroids showed that Cftr KO crypt epithelium sustains an alkaline pHi and resistance to cell acidification relative to wild-type. Quantitative real-time PCR revealed that Cftr KO enteroids exhibit downregulated transcription of base (HCO3 (-))-loading proteins and upregulation of the basolateral membrane HCO3 (-)-unloader anion exchanger 2 (Ae2). Although Cftr KO crypt epithelium had increased Ae2 expression and Ae2-mediated Cl(-)/HCO3 (-) exchange with maximized gradients, it also had increased intracellular Cl(-) concentration relative to wild-type. Pharmacological reduction of intracellular Cl(-) concentration in Cftr KO crypt epithelium normalized pHi, which was largely Ae2-dependent. We conclude that Cftr KO crypt epithelium maintains an alkaline pHi as a consequence of losing both Cl(-) and HCO3 (-) efflux, which impairs pHi regulation by Ae2. Retention of Cl(-) and an alkaline pHi in crypt epithelium may alter several cellular processes in the proliferative compartment of Cftr KO intestine.
authors.affiliation: Dalton Cardiovascular Research Center, University of Missouri, Columbia, Missouri; and.
authors.forename: Nancy M
authors.initials: NM
authors.lastname: Walker
authors.affiliation: Dalton Cardiovascular Research Center, University of Missouri, Columbia, Missouri; and.
authors.forename: Jinghua
authors.initials: J
authors.lastname: Liu
authors.affiliation: Dalton Cardiovascular Research Center, University of Missouri, Columbia, Missouri; and.
authors.forename: Sydney R
authors.initials: SR
authors.lastname: Stein
authors.affiliation: Dalton Cardiovascular Research Center, University of Missouri, Columbia, Missouri; and Department of Biomedical Sciences, University of Missouri, Columbia, Missouri.
authors.forename: Casey D
authors.initials: CD
authors.lastname: Stefanski
authors.affiliation: Dalton Cardiovascular Research Center, University of Missouri, Columbia, Missouri; and Department of Biomedical Sciences, University of Missouri, Columbia, Missouri.
authors.forename: Ashlee M
authors.initials: AM
authors.lastname: Strubberg
authors.affiliation: Dalton Cardiovascular Research Center, University of Missouri, Columbia, Missouri; and Department of Biomedical Sciences, University of Missouri, Columbia, Missouri clarkel@missouri.edu.
authors.forename: Lane L
authors.initials: LL
authors.lastname: Clarke
mesh_terms: D000818:Animals; D001639:Bicarbonates / Q000378:metabolism*; D027963:Chloride-Bicarbonate Antiporters / Q000235:genetics / Q000378:metabolism*; D002712:Chlorides / Q000378:metabolism*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007413:Intestinal Mucosa / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout
chemical_list: D001639:Bicarbonates; D027963:Chloride-Bicarbonate Antiporters; D002712:Chlorides; D019005:Cystic Fibrosis Transmembrane Conductance Regulator
keywords: anion exchanger 2; cystic fibrosis; enteroid; intestine; organoid