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Paracellular bicarbonate flux across human cystic fibrosis airway epithelia tempers changes in airway surface liquid pH.

ABSTRACT:

Key points

Cl^- and HCO₃ ^- had similar paracellular permeabilities in human airway epithelia. P_Cl /P_Na of airway epithelia was unaltered by pH 7.4 vs. pH 6.0 solutions. Under basal conditions, calculated paracellular HCO₃ ^- flux was secretory. Cytokines that increased airway surface liquid pH decreased or reversed paracellular HCO₃ ^- flux. HCO₃ ^- flux through the paracellular pathway may counterbalance effects of cellular H⁺ and HCO₃ ^- secretion.

Abstract

Airway epithelia control the pH of airway surface liquid (ASL), thereby optimizing respiratory defences. Active H⁺ and HCO₃ ^- secretion by airway epithelial cells produce an ASL that is acidic compared with the interstitial space. The paracellular pathway could provide a route for passive HCO₃ ^- flux that also modifies ASL pH. However, there is limited information about paracellular HCO₃ ^- flux, and it remains uncertain whether an acidic pH produced by loss of cystic fibrosis transmembrane conductance regulator anion channels or proinflammatory cytokines might alter the paracellular pathway function. To investigate paracellular HCO₃ ^- transport, we studied differentiated primary cultures of human cystic fibrosis (CF) and non-CF airway epithelia. The paracellular pathway was pH-insensitive at pH 6.0 vs. pH 7.4 and was equally permeable to Cl^- and HCO₃ ^- . Under basal conditions at pH ∼6.6, calculated paracellular HCO₃ ^- flux was weakly secretory. Treating epithelia with IL-17 plus TNFα alkalinized ASL pH to ∼7.0, increased paracellular HCO₃ ^- permeability, and paracellular HCO₃ ^- flux was negligible. Applying IL-13 increased ASL pH to ∼7.4 without altering paracellular HCO₃ ^- permeability, and calculated paracellular HCO₃ ^- flux was absorptive. These results suggest that HCO₃ ^- flux through the paracellular pathway counterbalances, in part, changes in the ASL pH produced via cellular mechanisms. As the pH of ASL increases towards that of basolateral liquid, paracellular HCO₃ ^- flux becomes absorptive, tempering the alkaline pH generated by transcellular HCO₃ ^- secretion.

SUBMITTER: Thornell IM

PROVIDER: S-EPMC7589346 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Respiratory failure, driven by airways mucus obstruction, chronic inflammation and bacterial infections, is the main cause of mortality and morbidity in people with cystic fibrosis (CF) due to defects in the Cl- and HCO3- transport activity of the CF Transmembrane conductance Regulator (CFTR). Most recent pre-clinical and clinical studies have focused on restoring CFTR function by enhancing its trafficking or transport activity and show promising results. However, there are a significant number of patients that will not benefit from these CFTR-targeted therapies and it is therefore important to identify new non-CFTR targets that will restore lung function, by-passing CFTR dysfunction. The H+/K+-ATPase, ATP12A, has recently been identified as a potential novel target for CF therapies, since its acute inhibition by ouabain was shown to help restore mucus viscosity, mucociliary transport, and antimicrobial activity using in vitro CF airway models, and this effect was linked to an increase in the pH of the airway surface liquid (ASL). Here, we have evaluated the potential therapeutic use of ouabain by investigating the effect of chronically treating fully differentiated CF primary human airway epithelial cells (hAECs) with ouabain, under thin film conditions, resembling the in vivo situation. Our results show that although chronic treatment increased ASL pH, this correlated with a deleterious effect on epithelial integrity as assessed by LDH release, transepithelial electrical resistance, fluorescein flux, and ion transport. Since ATP12A shares approximately 65% identity with the gastric H+/K+-ATPase (ATP4A), we investigated the potential of using clinically approved ATP4A proton pump inhibitors (PPIs) for their ability to restore ASL pH in CF hAECs. We show that, despite not expressing ATP4A transcripts, acute exposure to the PPI esomeprezole, produced changes in intracellular pH that were consistent with the inhibition of H+ secretion, but this response was independent of ATP12A. More importantly, chronic exposure of CF hAECs to esomeprazole alkalinized the ASL without disrupting the epithelial barrier integrity, but this increase in ASL pH was consistent with a decrease in mRNA expression of ATP12A. We conclude that PPIs may offer a new approach to restore ASL pH in CF airways, which is independent of CFTR.

Project description:Cystic fibrosis (CF) is a genetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) protein, a plasma membrane protein expressed on the apical surface of secretory epithelia of the airways. In the airways, defective or absent function of the CFTR protein determines abnormalities of chloride and bicarbonate secretion and, in general, of the transepithelial homeostasis that lead to alterations of airway surface liquid (ASL) composition and properties. The reduction of ASL volume impairs ciliary beating with the consequent accumulation of a sticky mucus. This situation prevents normal mucociliary clearance, favoring the survival and proliferation of bacteria and contributing to the genesis of the CF pulmonary disease. We explored the potential of some CFTR modulators, namely ivacaftor, tezacaftor, elexacaftor and their combination KaftrioTM, capable of partially recovering the basic defects of the CFTR protein, to ameliorate the transepithelial fluid transport and the viscoelastic properties of the mucus when used singly or in combination. Primary human bronchial epithelial cells obtained from CF and non-CF patients were differentiated into a mucociliated epithelia in order to assess the effects of correctors tezacaftor, elexacaftor and their combination with potentiator ivacaftor on the key properties of ASL, such as fluid reabsorption, viscosity, protein content and pH. The treatment of airway epithelia bearing the deletion of a phenylalanine at position 508 (F508del) in the CFTR gene with tezacaftor and elexacaftor significantly improved the pericilial fluid composition, reducing the fluid reabsorption, correcting the ASL pH and reducing the viscosity of the mucus. KaftrioTM was more effective than single modulators in improving all the evaluated parameters, demonstrating once more that this combination recently approved for patients 6 years and older with cystic fibrosis who have at least one F508del mutation in the CFTR gene represents a valuable tool to defeat CF.

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Paracellular bicarbonate flux across human cystic fibrosis airway epithelia tempers changes in airway surface liquid pH.

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