Advertisement

Hepatobiliary acid-base homeostasis: Insights from analogous secretory epithelia

  • David C. Trampert
    Affiliations
    Amsterdam UMC, University of Amsterdam, Department of Gastroenterology and Hepatology, Tytgat Institute for Liver and Intestinal Research, Amsterdam Gastroenterology Endocrinology Metabolism (AGEM), Meibergdreef 9, Amsterdam, the Netherlands
    Search for articles by this author
  • Stan F.J. van de Graaf
    Affiliations
    Amsterdam UMC, University of Amsterdam, Department of Gastroenterology and Hepatology, Tytgat Institute for Liver and Intestinal Research, Amsterdam Gastroenterology Endocrinology Metabolism (AGEM), Meibergdreef 9, Amsterdam, the Netherlands
    Search for articles by this author
  • Aldo Jongejan
    Affiliations
    Amsterdam UMC, University of Amsterdam, Department of Clinical Epidemiology, Biostatistics and Bioinformatics, Meibergdreef 9, Amsterdam, the Netherlands
    Search for articles by this author
  • Ronald P.J. Oude Elferink
    Affiliations
    Amsterdam UMC, University of Amsterdam, Department of Gastroenterology and Hepatology, Tytgat Institute for Liver and Intestinal Research, Amsterdam Gastroenterology Endocrinology Metabolism (AGEM), Meibergdreef 9, Amsterdam, the Netherlands
    Search for articles by this author
  • Ulrich Beuers
    Correspondence
    Corresponding author. Address: Department of Gastroenterology and Hepatology, Tytgat Institute for Liver and Intestinal Research, Amsterdam UMC, University of Amsterdam, location AMC (C2-327), Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. Tel.: +31 20 5662422; fax: +31 20 566 9701.
    Affiliations
    Amsterdam UMC, University of Amsterdam, Department of Gastroenterology and Hepatology, Tytgat Institute for Liver and Intestinal Research, Amsterdam Gastroenterology Endocrinology Metabolism (AGEM), Meibergdreef 9, Amsterdam, the Netherlands
    Search for articles by this author
Open AccessPublished:October 23, 2020DOI:https://doi.org/10.1016/j.jhep.2020.10.010

      Summary

      Many epithelia secrete bicarbonate-rich fluid to generate flow, alter viscosity, control pH and potentially protect luminal and intracellular structures from chemical stress. Bicarbonate is a key component of human bile and impaired biliary bicarbonate secretion is associated with liver damage. Major efforts have been undertaken to gain insight into acid-base homeostasis in cholangiocytes and more can be learned from analogous secretory epithelia. Extrahepatic examples include salivary and pancreatic duct cells, duodenocytes, airway and renal epithelial cells. The cellular machinery involved in acid-base homeostasis includes carbonic anhydrase enzymes, transporters of the solute carrier family, and intra- and extracellular pH sensors. This pH-regulatory system is orchestrated by protein-protein interactions, the establishment of an electrochemical gradient across the plasma membrane and bicarbonate sensing of the intra- and extracellular compartment. In this review, we discuss conserved principles identified in analogous secretory epithelia in the light of current knowledge on cholangiocyte physiology. We present a framework for cholangiocellular acid-base homeostasis supported by expression analysis of publicly available single-cell RNA sequencing datasets from human cholangiocytes, which provide insights into the molecular basis of pH homeostasis and dysregulation in the biliary system.

      Keywords

      Linked Article

      Introduction

      Many epithelia are specialised in bicarbonate (HCO3-)-rich fluid secretion, which generates flow, alters viscosity, controls pH and potentially protects luminal and intracellular structures from chemical stress. The cellular machinery involved is largely preserved across epithelial cells, allowing for comparison. Chronic cholestatic liver diseases affect bile formation and secretion, causing accumulation of cytotoxic cholephiles, biliary fibrosis, cirrhosis and end-stage liver disease if left untreated. As models of cholestatic liver diseases, primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC) affect small intrahepatic and larger intra- and extrahepatic bile ducts, respectively.
      • Terziroli Beretta-Piccoli B.
      • Mieli-Vergani G.
      • Vergani D.
      • Vierling J.M.
      • Adams D.
      • Alpini G.
      • et al.
      The challenges of primary biliary cholangitis: what is new and what needs to be done.
      ,
      • Karlsen T.H.
      • Folseraas T.
      • Thorburn D.
      • Vesterhus M.
      Primary sclerosing cholangitis – a comprehensive review.
      Notably, impaired HCO3- secretion was described in PBC 20 years ago and is putatively caused by microRNA-506-dependent defective expression of the Cl-/HCO3- exchanger anion exchanger 2 (AE2).
      • Prieto J.
      • García N.
      • Martí-Climent J.
      • Peñuelas I.
      • Richter J.
      • Medina J.
      Assessment of biliary bicarbonate secretion in humans by positron emission tomography.
      ,
      • Banales J.
      • Sáez E.
      • Uriz M.
      • Sarvide S.
      • Urribarri A.
      • Splinter P.
      • et al.
      Upregulation of mir-506 leads to decreased AE2 expression in biliary epithelium of patients with primary biliary cirrhosis.
      Meanwhile, the widely accepted concept of a biliary HCO3- umbrella specifies that bile duct integrity depends on a protective HCO3- layer facilitating deprotonation of hydrophobic membrane-permeable apolar bile acids, thereby preventing cholangiocellular cytotoxicity.
      • Beuers U.
      • Hohenester S.
      • de Buy Wenniger L.J.M.
      • Kremer A.E.
      • Jansen P.L.M.
      • Oude Elferink R.P.J.
      The biliary HCO3- umbrella: a unifying hypothesis on pathogenetic and therapeutic aspects of fibrosing cholangiopathies.
      ,
      • Hohenester S.
      • Maillette de Buy Wenniger L.
      • Paulusma C.C.
      • van Vliet S.J.
      • Jefferson D.M.
      • Oude Elferink R.P.
      • et al.
      A biliary HCO3- umbrella constitutes a protective mechanism against bile acid-induced injury in human cholangiocytes.
      First-line treatment for patients with PBC consists of ursodeoxycholic acid (UDCA), which enhances the secretory capacity of hepatocytes and cholangiocytes, thereby improving biliary HCO3- secretion and underlining the clinical relevance of this concept.
      • Beuers U.
      Drug insight: mechanisms and sites of action of ursodeoxycholic acid in cholestasis.
      Major progress in research over the past decade has led to the development of second-line treatment options, which notably also directly or indirectly stabilise the biliary HCO3- umbrella.
      • Beuers U.
      • Trauner M.
      • Jansen P.
      • Poupon R.
      New paradigms in the treatment of hepatic cholestasis: from UDCA to FXR, PXR and beyond.
      Growing evidence also suggests a role for pH dysregulation in cystic fibrosis-associated liver disease (CFLD) and IgG4-related cholangitis (IRC). In patients with cystic fibrosis (CF) and associated pancreatic insufficiency, genetic mutations result in impaired HCO3- secretion with maintenance of substantial Cl- secretion, supporting the view that impaired HCO3- secretion increases disease severity.
      • Choi J.Y.
      • Muallem D.
      • Kiselyov K.
      • Lee M.G.
      • Thomas P.J.
      • Muallem S.
      Aberrant CFTR-dependent HCO3- transport in mutations associated with cystic fibrosis.
      In systemic IgG4-related disease (IgG4-RD), target organs such as the pancreas, bile ducts, salivary glands, kidney or airways all deal with HCO3- fluxes. The importance of adequate biliary acid-base homeostasis is further demonstrated in the assessment of liver grafts for transplantation. In ex situ normothermic machine perfusion of liver grafts a biliary pH above 7.48 and biliary [HCO3-] above 18 mmol/L are accurate protective predictors of bile duct injury and may serve as biomarkers for pretransplant graft assessment.
      • Matton A.P.M.
      • Vries Y de
      • Burlage L.C.
      • Rijn R van
      • Fujiyoshi M.
      • de Meijer V.E.
      • et al.
      Biliary bicarbonate, pH, and glucose are suitable biomarkers of biliary viability during ex situ normothermic machine perfusion of human donor livers.
      In this review, we aim to identify conserved principles of acid-base homeostasis in analogous secretory epithelia, which have often been unravelled in equal or more detail than those in cholangiocytes. We present a framework for cholangiocellular acid-base homeostasis supported by expression analysis of publicly available single-cell RNA sequencing datasets from human cholangiocytes, which provide insights into the molecular basis of pH homeostasis and dysregulation in the biliary system (Fig. 1) .
      Carbonic anhydrase enzymes, transporters of the solute carrier family, and intra- and extracellular pH sensors are conserved across secretory epithelia.
      Figure thumbnail gr1
      Fig. 1Heatmaps depicting expression levels of cellular acid-base machinery in human cholangiocyte clusters.
      Cholangiocyte clusters (positive for EPCAM, KRT19, CFTR and negative for hepatocyte markers ALB, ASGR1) were obtained from public single cell RNA sequencing datasets. (A) Expression values provided by Aizarani et al. Nature. 2019,
      • Aizarani N.
      • Saviano A.
      • Sagar
      • Mailly L.
      • Durand S.
      • Herman J.S.
      • et al.
      A human liver cell atlas reveals heterogeneity and epithelial progenitors.
      GEO accession GSE124395, were aggregated using the provided cluster IDs, centred and scaled per gene. (B) Expression values provided by Hu et al. Cell. 2018,
      • Hu H.
      • Gehart H.
      • Artegiani B.
      • LÖpez-Iglesias C.
      • Dekkers F.
      • Basak O.
      • et al.
      Long-term expansion of functional mouse and human hepatocytes as 3D organoids.
      GEO accession GSE111301, from both available human single cell RNA sequencing samples (GSM3490459 and GSM3490460) were combined and filtered according to the original publication (i.e. minimal number of transcripts per cell > 4000) using the RaceID3 algorithm. Using the t-distributed stochastic neighbour embedding approach, clustering and dimensionality reduction were performed. Clusters positive for EPCAM and KRT19 were identified as containing cholangiocytes and expression values centred and scaled per gene. Genes not present in the dataset are depicted with grey bars. ALB, albumin; EPCAM, epithelial cell adhesion molecule; ASGR1, asialoglycoprotein receptor 1; Chol, cholangiocytes; Chol Orgs, cholangiocyte organoids; Cl, cluster; KRT19, cytokeratin 19. Further details on the genes of interest are in the list of abbreviations.

      Acid-base production

      Carbonic anhydrases (CAs) catalyse the reversible hydration of CO2, generating HCO3- and H+. The equilibrium equation consists of 2 reactions: CO2 + H2O ⇌ H2CO3 ⇌ HCO3- + H+. The initial formation of carbonic acid (H2CO3) is slow in the absence of a catalyst. Hence, ubiquitously expressed CAs facilitate cellular HCO3- supply and regulate pH by enhancing buffering capacity. The CA α-family is present in humans and consists of 15 isoforms, which can be characterised by their subcellular localisation, protein architecture, catalytic activity and efficiency (Table 1). For instance, membrane-bound CA IV is attached via the lipid anchor glycosylphosphatidylinositol and can be released by phospholipase treatment, whilst CA IX, XII and XIV are transmembrane isoforms. Unique to CA IX is the extracellular proteoglycan-like region, which represents an N-terminal extension of the catalytic domain, with evidence suggesting it has both extra- and intracellular catalytic activity.
      • Klier M.
      • Jamali S.
      • Ames S.
      • Schneider H.P.
      • Becker H.M.
      • Deitmer J.W.
      Catalytic activity of human carbonic anhydrase isoform IX is displayed both extra- and intracellularly.
      CA VIII, X and XI lack an active site and are accordingly referred to as carbonic anhydrase-related proteins (CARPs). CA isoforms exhibit a tailored expression pattern across tissues and are particularly concentrated in epithelia specialised in HCO3--rich fluid secretion. The heterogeneous characteristics and spatial organisation of CAs across a luminal structure may potentiate functionality at sites dealing with a harsh microenvironment comprising relatively acidic fluids, digestive enzymes or lipophilic molecules.
      Table 1Characteristics of proteins containing an α-CA domain.
      Subcellular localizationCytogenic locationProtein domainsCA activityCA efficiency

      (M-1 s-1)
      CA ICyt8q21.2α-CAModerate5.0 × 107
      CA IICyt8q21.2α-CAHigh1.5 × 108
      CA IIICyt8q21.2α-CALow4.0 × 105
      CA IVTeth, EDs?17q23.1α-CA, SP, GPI, PPHigh5.1 × 107
      CA VAMito16q24.2α-CA, mTPModerate2.9 × 107
      CA VBMitoXp22.2α-CA, mTPHigh9.8 × 107
      CA VISecr1p36.23α-CA, SPModerate4.9 × 107
      CA VIICyt16q22.1α-CAHigh8.3 × 107
      CA(RP) VIIICyt8q12.1α-CAAcatalyticAcatalytic
      CA IXMemb, EDs9p13.3α-CA, SP, PG, TM, ICHigh1.6 × 108
      CA(RP) XCyt17q21.33-q22α-CAAcatalyticAcatalytic
      CA(RP) XICyt, Secr?19q13.33α-CA, SPAcatalyticAcatalytic
      CA XIIMemb15q22.2α-CA, SP, TM, ICModerate3.5 × 107
      CA XIIICyt8q21.2α-CAModerate1.1 × 107
      CA XIVMemb1q21.2α-CA, SP, TM, ICModerate3.9 × 107
      PTPRGMemb, EDs3p14.2α-CA, FNIII, PTPaseAcatalyticAcatalytic
      PTPRZ1Memb, EDs7q31.32α-CA, FNIII, PTPaseAcatalyticAcatalytic
      Refs
      • Purkerson J.M.
      • Schwartz G.J.
      The role of carbonic anhydrases in renal physiology.
      ,
      • Zatovicova M.
      • Sedlakova O.
      • Svastova E.
      • Ohradanova A.
      • Ciampor F.
      • Arribas J.
      • et al.
      Ectodomain shedding of the hypoxia-induced carbonic anhydrase IX is a metalloprotease-dependent process regulated by TACE/ADAM17.
      ,
      • Moratti E.
      • Vezzalini M.
      • Tomasello L.
      • Giavarina D.
      • Sorio C.
      Identification of protein tyrosine phosphatase receptor gamma extracellular domain (sPTPRG) as a natural soluble protein in plasma.
      ,
      • Maurel P.
      • Rauch U.
      • Flad M.
      • Margolis R.K.
      • Margolis R.U.
      Phosphacan, a chondroitin sulfate proteoglycan of brain that interacts with neurons and neural cell-adhesion molecules, is an extracellular variant of a receptor-type protein tyrosine phosphatase.
      NCBI Genome Data ViewerEMBL-EBI InterPro, Swiss-Prot,
      • Lee H.
      • Yi J.S.
      • Lawan A.
      • Min K.
      • Bennett A.M.
      Mining the function of protein tyrosine phosphatases in health and disease.
      • Parkkila S.
      Significance of pH regulation and carbonic anhydrases in tumour progression and implications for diagnostic and therapeutic approaches.
      ,
      • Mboge M.Y.
      • Mahon B.P.
      • McKenna R.
      • Frost S.C.
      Carbonic anhydrases: role in pH control and cancer.
      • Parkkila S.
      Significance of pH regulation and carbonic anhydrases in tumour progression and implications for diagnostic and therapeutic approaches.
      ,
      • Mboge M.Y.
      • Mahon B.P.
      • McKenna R.
      • Frost S.C.
      Carbonic anhydrases: role in pH control and cancer.
      Note the differing subcellular localization, cytogenic location, enzymatic activity and efficiency. The enzymatic activity reflects Kcat (s-1), whilst enzymatic efficiency is defined as Kcat/Km (M-1 s-1). NCBI Genome Data Viewer was used to retrieve data on cytogenic location. EMBL-EBI InterPro and Swiss-Prot were used to retrieve protein domain details. Cyt, cytosolic; EDs, ectodomain shedding; FNIII, fibronectin type III; GPI, glycosylphosphatidylinositol anchor; IC, intracellular tail; Memb, membrane-bound; Mito, mitochondrial; mTP, mitochondrial transit peptide; PG, proteoglycan-like region; PP, propeptide; PTPase, Protein tyrosine phosphatase; SP, signal peptide; Teth, tethered; TM, transmembrane.

      Carbonic anhydrases in analogous secretory epithelia

      Urogenital tract

      Systemic pH homeostasis is regulated by the urogenital and respiratory tract and possibly the liver according to the Henderson-Hasselbalch equation: pH = pK + log ([HCO3-]/[CO2]). A high [HCO3-] is present in the glomerular ultrafiltrate, for which CAs facilitate luminal HCO3- reclamation to the peritubular capillaries of the nephron. CA activity and expression is concentrated in the renal proximal convoluted tubule (PCT) where 80% of the nephronal HCO3- reclamation takes place.
      • Hamm L.L.
      • Nakhoul N.
      • Hering-Smith K.S.
      Acid-base homeostasis.
      This is exploited in the management of metabolic alkalosis with oedema by pharmacologically inhibiting CAs, which results in nephronal HCO3- and fluid loss, consequently lowering systemic pH and promoting diuresis. Specifically, CA II, IV and XII are expressed along the human nephron, whilst CA XIV is additionally present in rodents.
      • Purkerson J.M.
      • Schwartz G.J.
      The role of carbonic anhydrases in renal physiology.
      CA IV is generally localised to the apical membrane, but CA IV has also been detected at the basolateral membrane in stained kidney sections and by cell-surface biotinylation of confluent monolayers.
      • Purkerson J.M.
      • Kittelberger A.M.
      • Schwartz G.J.
      Basolateral carbonic anhydrase IV in the proximal tubule is a glycosylphosphatidylinositol-anchored protein.
      This indicates that trafficking of CA isoforms may be an adaptive process, similar to the adaptive trafficking of the vacuolar H+-ATPase (V-ATPase) in renal intercalated cells,
      • Wall S.
      Recent advances in our understanding of intercalated cells.
      making manipulation possible. In the epididymis, HCO3- is absorbed from testicular fluid to keep spermatozoa in a quiescent state and promote maturation.
      • Pastor-soler N.
      • Piétrement C.
      • Breton S.
      Role of acid / base transporters in the male reproductive tract and potential consequences of their malfunction.
      To this end, CA II, IV and the V-ATPase are expressed in epididymal clear cells. In line with this, CA II and IV knockout (KO) mice show reduced sperm motility, swimming speed and flagellar beating frequency, processes that are regulated by HCO3-.
      • Wandernoth P.M.
      • Mannowetz N.
      • Szczyrba J.
      • Grannemann L.
      • Wolf A.
      • Becker H.M.
      • et al.
      Normal fertility requires the expression of carbonic anhydrases II and IV in sperm.
      Additionally, CA IX and XII can be detected throughout the male excurrent duct system; the latter is co-expressed with aquaporin 1 (AQP1), indicating a role in compartmental fluid regulation.
      • Karhumaa P.
      • Kaunisto K.
      • Parkkila S.
      • Waheed A.
      • Pastoreková S.
      • Pastorek J.
      • et al.
      Expression of the transmembrane carbonic anhydrases, CA IX and CA XII, in the human male excurrent ducts.

      Respiratory tract

      CO2 produced by cellular respiration in peripheral tissues diffuses to the capillaries and is hydrated in erythrocytes by CA I and II, after which HCO3- is shuttled out. Upon arrival in the pulmonary capillaries, CO2 is regenerated in erythrocytes, from which it diffuses into the alveolar space along its partial pressure gradient, allowing for alveolar O2 uptake. CAs in the respiratory tract appear to be involved in inflammation, as treatment with the pan-CA inhibitor acetazolamide suppressed the pro-inflammatory cytokines IL-1, IL-6, IL-17 and tumour necrosis factor-α and decreased neutrophil sequestering in a model of pulmonary ischemia-reperfusion injury.
      • Lan C.C.
      • Peng C.K.
      • Tang S.E.
      • Huang K.L.
      • Wu C.P.
      Carbonic anhydrase inhibitor attenuates ischemia-reperfusion induced acute lung injury.
      The mechanism behind this is currently unknown but may be relevant for immune-mediated diseases where HCO3- secretion appears defective.

      Gastrointestinal tract and pancreas

      Salivary duct cells express CA II, IX and XII, and secrete high concentrations of CA VI, protecting against gastric acid and potentially gastroduodenal reflux of bile acids.
      • Parkkila S.
      • Parkkila A.K.
      • Lehtola J.
      • Reinilä A.
      • Södervik H.J.
      • Rannisto M.
      • et al.
      Salivary carbonic anhydrase protects gastroesophageal mucosa from acid injury.
      HCl-secreting gastric parietal cells express basolateral CA IX and XII, facilitating H+ supply.
      • Pastorekova S.
      • Parkkila S.
      • Parkkila A.K.
      • Opavsky R.
      • Zelnik V.
      • Saarnio J.
      • et al.
      Carbonic anhydrase IX, MN/CA IX: analysis of stomach complementary DNA sequence and expression in human and rat alimentary tracts.
      To neutralise gastric acid, duodenocytes secrete a HCO3--rich fluid and accordingly have the highest CA activity in the gastrointestinal tract, expressing CA II, IV and IX to facilitate HCO3- supply.
      • Kivelä A.J.
      • Kivelä J.
      • Saarnio J.
      • Parkkila S.
      Carbonic anhydrases in normal gastrointestinal tract and gastrointestinal tumours.
      Colonocytes have the primary function of absorbing fluid. Notably, colonocytes from cystic fibrosis transmembrane conductance regulator (Cftr) KO mice show a defect in peroxisome proliferator-activated receptor (PPAR) signalling; pharmacological correction of this defect leads to upregulation of CA II and IV expression resulting in increased HCO3- secretion and reduced disease severity.
      • Harmon G.S.
      • Dumlao D.S.
      • Ng D.T.
      • Barrett K.E.
      • Dennis E.A.
      • Dong H.
      • et al.
      Pharmacological correction of a defect in PPAR-γ signaling ameliorates disease severity in Cftr-deficient mice.
      This indicates that CFTR, CAs and PPAR signalling are connectively involved in HCO3- secretion.
      Pancreatic acini secrete NaCl, whilst the ductular system secretes a HCO3--rich fluid to generate flow, control pH and potentially protect luminal and intracellular structures from chemical stress.
      • Lee M.G.
      • Ohana E.
      • Park H.W.
      • Yang D.
      • Muallem S.
      Molecular mechanism of pancreatic and salivary gland fluid and HCO3- secretion.
      Pancreatic duct cells and cholangiocytes have a common embryonic ancestor, with the biliary system conforming to a similar architecture of closed-end canaliculi draining into consecutively larger ducts. Moreover, there appears to be functional overlap as administration of luminal chenodeoxycholic acid in a polarised human pancreatic duct cell line increases intracellular [Ca2+] and apical Cl-/HCO3- exchange.
      • Ignáth I.
      • Hegyi P.
      • Venglovecz V.
      • Székely C.A.
      • Carr G.
      • Hasegawa M.
      • et al.
      CFTR expression but not Cl- transport is involved in the stimulatory effect of bile acids on apical Cl-/HCO3- exchange activity in human pancreatic duct cells.
      In pancreatic duct cells, CA II, IV, IX and XII are detected.
      • Nishimori I.
      • Onishi S.
      Carbonic anhydrase isozymes in the human pancreas.
      ΔF508 CFTR mutated human pancreatic duct cells show a marked decrease in apical CA IV protein levels due to accumulation in the Golgi apparatus.
      • Fanjul M.
      • Salvador C.
      • Alvarez L.
      • Cantet S.
      • Hollande E.
      Targeting of carbonic anhydrase IV to plasma membrane is altered in cultured human pancreatic duct cells expressing a mutated (ΔF508) CFTR.
      This indicates that the ΔF508 CFTR mutation, which causes defective transporter targeting to the plasma membrane, also disrupts apical trafficking of CA IV.

      Carbonic anhydrases in the hepatobiliary tract

      In various immune-mediated diseases, including cholestatic liver diseases such as PBC and IgG4-RD, anti-CA II IgG antibodies are found in patient sera regardless of anti-mitochondrial antibody (AMA) status.
      • Ueno Y.
      • Ishii M.
      • Igarashi T.
      • Mano Y.
      • Yahagi K.
      • Kisara N.
      • et al.
      Primary biliary cirrhosis with antibody against carbonic anhydrase II associates with distinct immunological backgrounds.
      Despite CA IX being a transmembrane protein, fragments can be detected in body fluids of patients with cancer due to ectodomain shedding; thus, it has justly received considerable attention in the field of oncology.
      • Zatovicova M.
      • Sedlakova O.
      • Svastova E.
      • Ohradanova A.
      • Ciampor F.
      • Arribas J.
      • et al.
      Ectodomain shedding of the hypoxia-induced carbonic anhydrase IX is a metalloprotease-dependent process regulated by TACE/ADAM17.
      CA IX is upregulated in hepatocellular carcinoma (HCC) and many other malignancies by the transcription factor hypoxia inducible factor 1, whilst healthy cholangiocytes endogenously express high levels of CA IX (Fig. 1), which is actually downregulated in cholangiocarcinoma (CCA).
      • Saarnio J.
      • Parkkila S.
      • Parkkila A.K.
      • Pastoreková S.
      • Haukipuro K.
      • Pastorek J.
      • et al.
      Transmembrane carbonic anhydrase, MN/CA IX, is a potential biomarker for biliary tumours.
      In line with this, CA IX is an unfavourable prognostic marker in primary resectable HCC but not in CCA.
      • Huang W.
      • Jeng Y.
      • Lai H.
      • Fong I.
      Expression of hypoxic marker carbonic anhydrase IX Predicts poor prognosis in resectable hepatocellular carcinoma.
      ,
      • Bi C.
      • Liu M.
      • Rong W.
      • Wu F.
      • Zhang Y.
      • Lin S.
      • et al.
      High Beclin-1 and ARID1A expression corelates with poor survival and high recurrence in intrahepatic cholangiocarcinoma: a histopathological retrospective study.
      Notably, bile acids are classified as endogenous CA inhibitors and incorporation of bile acid tail moieties increases the potency of classical CA inhibitors.
      • Scozzafava A.
      • Supuran C.T.
      Carbonic anhydrase inhibitors. Preparation of potent sulfonamides inhibitors incorporating bile acid tails.
      The primary unconjugated bile acid cholic acid promotes indirect binding of CA II through reconfiguration of the enzyme's active site.
      • Boone C.D.
      • Tu C.
      • Mckenna R.
      Structural elucidation of the hormonal inhibition mechanism of the bile acid cholate on human carbonic anhydrase II.
      The glycine conjugated primary bile acid glycocholic acid (GCA) and secondary bile acid deoxycholic acid (DCA) appear to be potent CA II inhibitors.
      • Milov D.E.
      • Jou W.S.
      • Shireman R.B.
      • Chun P.W.
      The effect of bile salts on carbonic anhydrase.
      Possibly bile acid-induced inhibition of luminal CAs may reduce the deprotonation efficiency of hydrophobic membrane-permeable apolar bile acids. Although a potential correlation between bile acid hydrophobicity indexes and the magnitude of CA inhibition is yet to be investigated, one must note that GCA and DCA themselves are on the hydrophobic side of the bile acid spectrum. Secretory CA VI has been detected in animal bile and immunohistochemical analysis shows positivity for CA VI in the gallbladder epithelium besides the previously reported presence of CA IV.
      • Nishita T.
      • Itoh S.
      • Arai S.
      • Ichihara N.
      • Arishima K.
      Measurement of carbonic anhydrase isozyme VI (CA-VI) in swine sera, colostrums, saliva, bile, seminal plasma and tissues.
      ,
      • Parkkila S.
      • Parkkila A.K.
      • Juvonen T.
      • Waheed A.
      • Sly W.S.
      • Saarnio J.
      • et al.
      Membrane-bound carbonic anhydrase IV is expressed in the luminal plasma membrane of the human gallbladder epithelium.
      Functionally, CA inhibition decreases secretin-induced biliary HCO3- secretion,
      • Nilsson B.
      • Valantinas J.
      • Hedin L.
      • Friman S.
      • Svanvik J.
      Acetazolamide inhibits stimulated feline liver and gallbladder bicarbonate secretion.
      UDCA-induced biliary HCO3- secretion and bile flow.
      • Garcia-Marin J.J.
      • Dumont M.
      • Corbic M.
      • de Couet G.
      • Erlinger S.
      Effect of acid-base balance and acetazolamide on ursodeoxycholate-induced biliary bicarbonate secretion.
      This indicates that the enzymatic production of HCO3- can become a rate-limiting factor in ductular secretion.

      Carbonic anhydrases: rate-limiting or redundant?

      CA II deficiency in humans is an autosomal recessive disorder characterised by renal tubular acidosis (RTA) type III, osteopetrosis and cerebral calcifications.
      • Sly W.
      • Hewett-Emmett D.
      • Whyte M.
      • Yu Y.
      • Tashian R.
      Carbonic anhydrase II deficiency identified as the primary defect in the autosomal recessive syndrome of osteopetrosis with renal tubular acidosis and cerebral calcification.
      RTA is detected in approximately 60% of patients with PBC and to a lesser extent in patients with PSC showing normalisation of urinary acidification after liver transplantation.
      • Golding P.L.
      • Mason A.S.
      Renal tubular acidosis and autoimmune liver disease.
      • Parés A.
      • Rimola A.
      • Bruguera M.
      • Mas E.
      • Rodés J.
      Renal tubular acidosis in primary biliary cirrhosis.
      • Goutaudier V.
      • Szwarc I.
      • Serre J.E.
      • Pageaux G.P.
      • Argilés À.
      • Ribstein J.
      Primary sclerosing cholangitis: a new cause of distal renal tubular acidosis.
      The aetiology of RTA in these patients remains unclear but note the common occurrence of pH dysregulation in bile ducts and nephrons. In cholestatic liver diseases, RTA type I caused by inadequate H+ secretion into the renal collecting duct (CD) is most frequently observed.
      • Scheiner B.
      • Lindner G.
      • Reiberger T.
      • Schneeweiss B.
      • Trauner M.
      • Zauner C.
      • et al.
      Acid-base disorders in liver disease.
      Notably, CAs not only appear essential in the PCT to reclaim HCO3- but also in the CD to determine cellular composition and to regulate acid-base secretion.
      • Bagnis C.
      • Marshansky V.
      • Breton S.
      • Brown D.
      Remodeling the cellular profile of collecting ducts by chronic carbonic anhydrase inhibition.
      Besides CA II KO mice having RTA, absence of duodenal HCO3- secretion in response to prostaglandins and H+ is observed, suggesting involvement in luminal acid sensing.
      • Leppilampi M.
      • Parkkila S.
      • Karttunen T.
      • Gut M.O.
      • Gros G.
      • Sjoblom M.
      Carbonic anhydrase isozyme-II-deficient mice lack the duodenal bicarbonate secretory response to prostaglandin E2.
      ,
      • Sjöblom M.
      • Singh A.K.
      • Zheng W.
      • Wang J.
      • Tuo B.G.
      • Krabbenhöft A.
      • et al.
      Duodenal acidity “sensing” but not epithelial HCO3- supply is critically dependent on carbonic anhydrase II expression.
      Compensatory upregulation of other isoforms occurs in CA II KO mice but is limited in CA IX KO mice.
      • Pan P.
      • Leppilampi M.
      • Pastorekova S.
      • Pastorek J.
      • Waheed A.
      • Sly W.S.
      • et al.
      Carbonic anhydrase gene expression in CA II-deficient (Car2-/-) and CAIX-deficient (Car9-/-) mice.
      CA XII is present in many secretory epithelia including airway epithelium, pancreatic and salivary duct cells (Table 2). A subset of patients expressing a CF-like phenotype consisting of lung disease, elevated sweat Cl- levels and failure to thrive, appear to have adequate Cl- secretion through CFTR but largely reduced CA XII function.
      • Lee M.
      • Vecchio-Pagán B.
      • Sharma N.
      • Waheed A.
      • Li X.
      • Raraigh K.S.
      • et al.
      Loss of carbonic anhydrase XII function in individuals with elevated sweat chloride concentration and pulmonary airway disease.
      Moreover, homozygous mutations of CA XII cause a dry mouth and tongue with further investigation revealing a key role in salivary and pancreatic ductular HCO3- secretion.
      • Hong J.H.
      • Muhammad E.
      • Zheng C.
      • Hershkovitz E.
      • Alkrinawi S.
      • Loewenthal N.
      • et al.
      Essential role of carbonic anhydrase XII in secretory gland fluid and HCO3- secretion revealed by disease causing human mutation.
      Mitochondrial CAs consist of CA VA, which is predominant in hepatocytes, and the more uniformly distributed CA VB. Sequencing revealed homozygous mutations in CA VA that cause defective hepatic HCO3- production in a paediatric patient cohort suffering from a metabolic crisis comprising hyperammonaemia, elevated lactate and ketonuria.
      • Diez-Fernandez C.
      • Rüfenacht V.
      • Santra S.
      • Lund A.M.
      • Santer R.
      • Lindner M.
      • et al.
      Defective hepatic bicarbonate production due to carbonic anhydrase VA deficiency leads to early-onset life-threatening metabolic crisis.
      Furthermore, membrane-bound CA XIV is upregulated in hepatocytes by INT-767, a dual nuclear farnesoid X receptor (FXR) and G protein-coupled bile acid receptor (GPBAR1/TGR5) agonist, which reduces liver damage in Mdr2 KO mice by promoting bile flow with a concomitant increase in biliary HCO3- secretion.
      • Baghdasaryan A.
      • Claudel T.
      • Gumhold J.
      • Silbert D.
      • Adorini L.
      • Roda A.
      • et al.
      Dual farnesoid X receptor/TGR5 agonist INT-767 reduces liver injury in the Mdr2−/− (Abcb4−/−) mouse Cholangiopathy model by promoting biliary HCO3- Output.
      This collectively suggests that CAs can become rate-limiting in humans and rodents if their levels become sufficiently low, making them interesting candidates for further research into the molecular basis of pH homeostasis and dysregulation in the biliary system. Note these findings do not imply that increased CA expression results in enhanced HCO3- secretion.
      Table 2Overview of acid-base machinery expressed in secretory epithelia.
      CholangiocyteDuodenocytePancreatic duct cellSalivary duct cellAirway epitheliumPCT epitheliumα-intercalated cell
      Carbonic anhydraseIIC, IVA, IXBL, XIUNDETIIC, IVA, IXBLIIC, IVA, IXBL, XIIBLIIC, VIS, IXBL, XIIBLIIC, IVA, VIS, XIIBLIIC, IVA, XIIBLIIC, IVA, XIIBL
      H+ transporterNHE1BL

      NHE2A

      NHE3A
      NHE1BL

      NHE2A

      NHE3A
      NHE1BL

      NHE2A

      NHE3A
      NHE1BL

      NHE2A

      NHE3A
      NHE1BL

      V-ATPaseA
      NHE3A

      V-ATPaseA
      V-ATPaseA
      Na+ indep. HCO3- transporterAE2A

      CFTRA
      AE2BL

      CFTRA

      SLC26A3A

      SLC26A6A
      AE2BL

      CFTRA

      SLC26A3A

      SLC26A6A
      AE2BL

      CFTRA

      SLC26A4A

      SLC26A6A
      AE2BL

      CFTRA

      SLC26A4A

      SLC26A9A
      AE2BL

      SLC26A6A
      AE1BL

      SLC26A7BL
      Na+ dep. HCO3- transporterNBCe1UNDET (1:2)

      NDCBEBL
      NBCe1BL (1:2)

      NBCn1BL
      NBCe1BL (1:2)

      NBCn1A
      NBCe1BL (1:2)

      NBCn1A
      NBCe1BL (1:n)NBCe1BL (1:3)

      NBCe2A

      NBCn2A
      NBCn1A
      pH sensorsACC

      ALPS

      BASICA

      PTPRG/Z1UNDET
      ALPS

      ASICA
      Undet.Undet.Undet.PTPRGBLsACC
      Luminal pH~ 7.5–8.0~ 5.5–7.5~ 7.5–8.0~ 6.5–7.5~ 6.5–7.0~ 6.8–7.0~ 4.5–5.5
      Refs
      • Kivelä A.J.
      • Kivelä J.
      • Saarnio J.
      • Parkkila S.
      Carbonic anhydrases in normal gastrointestinal tract and gastrointestinal tumours.
      ,
      • Banales J.M.
      • Prieto J.
      • Medina J.F.
      Cholangiocyte anion exchange and biliary bicarbonate excretion.
      ,
      • Tabibian J.H.
      • Masyuk A.I.
      • Masyuk T.V.
      • Hara S.P.O.
      • Larusso N.F.
      Physiology of cholangiocytes.
      ,
      • Chang J.C.
      • Go S.
      • de Waart D.R.
      • Munoz-Garrido P.
      • Beuers U.
      • Paulusma C.C.
      • et al.
      Soluble adenylyl cyclase regulates bile Salt-induced apoptosis in human cholangiocytes.
      ,
      • Michelotti G.A.
      • Tucker A.
      • Swiderska-Syn M.
      • Machado M.V.
      • Choi S.S.
      • Kruger L.
      • et al.
      Pleiotrophin regulates the ductular reaction by controlling the migration of cells in liver progenitor niches.
      ,
      • Wiemuth D.
      • Sahin H.
      • Falkenburger B.H.
      • Lefèvre C.M.T.
      • Wasmuth H.E.
      • Gründer S.
      Basic - a bile acid-sensitive ion channel highly expressed in bile ducts.
      • Kivelä A.J.
      • Kivelä J.
      • Saarnio J.
      • Parkkila S.
      Carbonic anhydrases in normal gastrointestinal tract and gastrointestinal tumours.
      ,
      • Akiba Y.
      • Kaunitz J.D.
      Duodenal chemosensing and mucosal defenses.
      ,
      • Dong X.
      • Ko K.H.
      • Chow J.
      • Tuo B.
      • Barrett K.E.
      • Dong H.
      Expression of acid-sensing ion channels in intestinal epithelial cells and their role in the regulation of duodenal mucosal bicarbonate secretion.
      ,
      • Seidler U.E.
      Gastrointestinal HCO3- transport and epithelial protection in the gut: new techniques, transport pathways and regulatory pathways.
      • Lee M.G.
      • Ohana E.
      • Park H.W.
      • Yang D.
      • Muallem S.
      Molecular mechanism of pancreatic and salivary gland fluid and HCO3- secretion.
      ,
      • Park H.W.
      • Lee M.G.
      Transepithelial bicarbonate secretion: lessons from the pancreas.
      ,
      • Inada A.
      • Nienaber C.
      • Katsuta H.
      • Fujitani Y.
      • Levine J.
      • Morita R.
      • et al.
      Carbonic anhydrase II-positive pancreatic cells are progenitors for both endocrine and exocrine pancreas after birth.
      ,
      • Fanjul M.
      • Alvarez L.
      • Salvador C.
      • Gmyr V.
      • Kerr-Conte J.
      • Pattou F.
      • et al.
      Evidence for a membrane carbonic anhydrase IV anchored by its C-terminal peptide in normal human pancreatic ductal cells.
      ,
      • Kivelä A.J.
      • Parkkila S.
      • Saarnio J.
      • Karttunen T.J.
      • Kivelä J.
      • Parkkila A.K.
      • et al.
      Expression of transmembrane carbonic anhydrase isoenzymes IX and XII in normal human pancreas and pancreatic tumours.
      • Kivelä A.J.
      • Kivelä J.
      • Saarnio J.
      • Parkkila S.
      Carbonic anhydrases in normal gastrointestinal tract and gastrointestinal tumours.
      ,
      • Lee M.G.
      • Ohana E.
      • Park H.W.
      • Yang D.
      • Muallem S.
      Molecular mechanism of pancreatic and salivary gland fluid and HCO3- secretion.
      ,
      • Parkkila S.
      • Parkkila A.K.
      • Juvonen T.
      • Rajaniemi H.
      Distribution of the carbonic anhydrase isoenzymes I, II, and VI in the human alimentary tract.
      ,
      • Shcheynikov N.
      • Yang D.
      • Wang Y.
      • Zeng W.
      • Karniski L.P.
      • So I.
      • et al.
      The Slc26a4 transporter functions as an electroneutral Cl-/I-/HCO3- exchanger: role of Slc26a4 and Slc26a6 in I- and HCO3- secretion and in regulation of CFTR in the parotid duct.
      • Lee M.
      • Vecchio-Pagán B.
      • Sharma N.
      • Waheed A.
      • Li X.
      • Raraigh K.S.
      • et al.
      Loss of carbonic anhydrase XII function in individuals with elevated sweat chloride concentration and pulmonary airway disease.
      ,
      • Shan J.
      • Liao J.
      • Huang J.
      • Robert R.
      • Palmer M.L.
      • Fahrenkrug S.C.
      • et al.
      Bicarbonate-dependent chloride transport drives fluid secretion by the human airway epithelial cell line Calu-3.
      ,
      • Fischer H.
      • Widdicombe J.H.
      Mechanisms of acid and base secretion by the airway epithelium.
      ,
      • Esbaugh A.J.
      • Tufts B.L.
      The structure and function of carbonic anhydrase isozymes in the respiratory system of vertebrates.
      ,
      • Leinonen J.S.
      • Saari K.A.
      • Seppänen J.M.
      • Myllylä H.M.
      • Rajaniemi H.J.
      Immunohistochemical demonstration of carbonic anhydrase isoenzyme VI (CA VI) expression in rat lower airways and lung.
      ,
      • Saint-Criq V.
      • Gray M.A.
      Role of CFTR in epithelial physiology.
      • Purkerson J.M.
      • Schwartz G.J.
      The role of carbonic anhydrases in renal physiology.
      ,
      • Felder R.A.
      • Jose P.A.
      • Xu P.
      • Gildea J.J.
      The renal sodium bicarbonate cotransporter NBCe2: is it a major contributor to sodium and pH homeostasis?.
      ,
      • Guo Y.
      • Liu Y.
      • Liu M.
      • Wang J.
      • Xie Z.
      • Chen K.
      • et al.
      Na+/HCO3- cotransporter NBCn2 mediates HCO3- reclamation in the apical membrane of renal proximal tubules.
      ,
      • Zhou Y.
      • Skelton L.A.
      • Xu L.
      • Chandler M.P.
      • Berthiaume J.M.
      • Boron W.F.
      Role of receptor protein tyrosine phosphatase γ in sensing extracellular CO2 and HCO3−.
      ,
      • Skelton L.A.
      • Boron W.F.
      • Zhou Y.
      Acid-base transport by the renal proximal tubule.
      ,
      • Soleimani M.
      SLC26 Cl-/HCO3- exchangers in the kidney: roles in health and disease.
      • Purkerson J.M.
      • Schwartz G.J.
      The role of carbonic anhydrases in renal physiology.
      ,
      • Wall S.
      Recent advances in our understanding of intercalated cells.
      ,
      • Pǎunescu T.G.
      • Da Silva N.
      • Russo L.M.
      • McKee M.
      • Lu H.A.J.
      • Breton S.
      • et al.
      Association of soluble adenylyl cyclase with the V-ATPase in renal epithelial cells.
      ,
      • Soleimani M.
      SLC26 Cl-/HCO3- exchangers in the kidney: roles in health and disease.
      A similar CA isoform expression pattern exists across secretory epithelia. Due to the current lack of compelling evidence, mitochondrial CAs have been excluded. NHE1 is expressed on the basolateral membrane, whilst NHE2-3 are expressed on the apical membrane of secretory epithelia. In all secretory epithelia discussed, AE2 is expressed on the basolateral membrane, whilst in cholangiocytes it appears apical. Secretory epithelia dealing with high HCO3- fluxes express electrogenic HCO3- transporters. Luminal pH of the respective secretory epithelia is given. A, apical; BL, basolateral; C, cytosolic; M, Mitochondrial; S, secretory; UNDET, undetermined. Details on the protein names are in the list of abbreviations.

      Acid-base transport

      Expression of acid-base transporters is also concentrated in secretory epithelia with cross-tissue co-expression network analysis showing a strong connection based on solute carrier (SLC) transporter clustering between the liver, intestine and kidney.
      • Rosenthal S.B.
      • Bush K.T.
      • Nigam S.K.
      A network of SLC and ABC transporter and DME genes involved in Remote sensing and signaling in the gut-liver-kidney Axis.
      In brief, SLC9 transporters encode Na+/H+ exchangers (NHE), whilst SLC4 transporters are functionally divided into Na+-independent Cl-/HCO3- exchangers (AE), electrogenic Na+/HCO3- cotransporters (NBCe) and electroneutral Na+-coupled HCO3- transporters (NBCn and NDCBE). The SLC26 subfamily encodes multifunctional HCO3- transporters, which appear absent in the hepatobiliary tract but have a prominent role in analogous secretory epithelia. Electrogenic SLC26A3 (DRA) and SLC26A6 (PAT1) show specific Cl-:HCO3- stoichiometries of 2:1 and 1:2, respectively, share oxalate as an additional substrate and appear able to uncouple substrate exchange.
      • Shcheynikov N.
      • Wang Y.
      • Park M.
      • Ko S.B.H.
      • Dorwart M.
      • Naruse S.
      • et al.
      Coupling modes and stoichiometry of Cl-/HCO3- exchange by slc26a3 and slc26a6.

      SLC transporters in analogous secretory epithelia

      Urogenital tract

      To reclaim 80% of all HCO3- from the glomerular ultrafiltrate in the renal PCT, basolateral NBCe1 is required to work with a Na+:HCO3- stoichiometry of 1:3 (Fig. 2).
      • Kurtz I.
      • Zhu Q.
      Structure, function, and regulation of the SLC4 NBCe1 transporter and its role in causing proximal renal tubular acidosis.
      Under basal conditions NBCe2 is concentrated near the Golgi apparatus, but it is trafficked towards the apical membrane when extracellular [Na+] increases.
      • Felder R.A.
      • Jose P.A.
      • Xu P.
      • Gildea J.J.
      The renal sodium bicarbonate cotransporter NBCe2: is it a major contributor to sodium and pH homeostasis?.
      For electroneutral transport in the kidney, mathematical modelling estimates that CAs facilitate 65% and apical NBCn2 15% of luminal HCO3- reclamation.
      • Guo Y.
      • Liu Y.
      • Liu M.
      • Wang J.
      • Xie Z.
      • Chen K.
      • et al.
      Na+/HCO3- cotransporter NBCn2 mediates HCO3- reclamation in the apical membrane of renal proximal tubules.
      To this end, CAs work in conjunction with apical NHE3.
      • Wang T.
      • Hropot M.
      • Aronson P.S.
      • Giebisch G.
      Role of NHE isoforms in mediating bicarbonate reabsorption along the nephron.
      Similarly, luminal HCO3- uptake in the epididymis is facilitated by NBCn1 and CAs working in conjunction with apical NHEs to acidify the lumen and facilitate the movement of HCO3- towards the basolateral membrane for NBCe1 or AE2 to reclaim.
      • Liu Y.
      • Wang D.-K.
      • Chen L.-M.
      The physiology of bicarbonate transporters in mammalian reproduction.
      Figure thumbnail gr2
      Fig. 2Cellular acid-base machinery expressed in epithelial cells reclaiming HCO3- compared to epithelial cells secreting HCO3-.
      In both cell types cellular acid-base machinery is highly similar and HCO3- flux is facilitated by CAs working in conjunction with NHEs and Na+-coupled HCO3- transporters. (A) Renal epithelial cells from the proximal convoluted tubule reclaim 80% of all HCO3- from the glomerular ultrafiltrate. NBCe1 working in a 1:3 mode allows for basolateral HCO3- reclamation in proximal convoluted tubule cells as the electrical gradient exceeds the opposing Na+ and HCO3- gradients. (B) Pancreatic duct cells secrete large amounts of HCO3--rich fluid into the ductular system. NBCe1 working in a 1:2 mode allows for basolateral HCO3- uptake in pancreatic duct cells as the Na+ and HCO3- gradients exceed the opposing electrical gradient. Details on the protein names are in the list of abbreviations.

      Respiratory tract

      Ciliated and goblet epithelial cells in the airways maintain the airway surface liquid (ASL) which captures and transports inhaled particles. Increased ASL viscosity and host defence abnormalities are common occurrences in patients with CF, which are attributed to ASL acidification and increased [Ca2+].
      • Tang X.X.
      • Ostedgaard L.S.
      • Hoegger M.J.
      • Moninger T.O.
      • Karp P.H.
      • Mcmenimen J.D.
      • et al.
      Acidic pH increases airway surface liquid viscosity in cystic fibrosis.
      ,
      • Shah V.S.
      • Meyerholz D.K.
      • Tang X.X.
      • Reznikov L.
      • Alaiwa M.A.
      • Ernst S.E.
      • et al.
      Airway acidification initiates host defense abnormalities in cystic fibrosis mice.
      As HCO3- alkalinises the ASL and decreases mucin viscosity by sequestering Ca2+, the importance of defective HCO3- secretion in the pathogenesis of CF is becoming increasingly apparent.
      • Chen E.Y.T.
      • Yang N.
      • Quinton P.M.
      • Chin W.C.
      A new role for bicarbonate in mucus formation.
      The mucociliary transport rate upon stimulation is dependent upon HCO3- secretion, whilst the basal rate is dependent upon Cl- secretion.
      • Cooper J.L.
      • Quinton P.M.
      • Ballard S.T.
      Mucociliary transport in porcine trachea: differential effects of inhibiting chloride and bicarbonate secretion.
      Besides apical CFTR, HCO3- secretion takes place via SLC26A4 (pendrin), which is upregulated by IL-4 and is particularly responsible for HCO3- secretion during inflammation.
      • Shan J.
      • Liao J.
      • Huang J.
      • Robert R.
      • Palmer M.L.
      • Fahrenkrug S.C.
      • et al.
      Bicarbonate-dependent chloride transport drives fluid secretion by the human airway epithelial cell line Calu-3.
      ,
      • Kim D.
      • Huang J.
      • Billet A.
      • Abu-Arish A.
      • Goepp J.
      • Matthes E.
      • et al.
      Pendrin mediates bicarbonate secretion and enhances cystic fibrosis transmembrane conductance regulator function in airway surface epithelia.
      Basolateral AE2 is generally responsible for Cl- uptake, whilst HCO3- supply is facilitated by NBCe1 and CAs working in conjunction with NHE1.
      • Fischer H.
      • Widdicombe J.H.
      Mechanisms of acid and base secretion by the airway epithelium.

      Gastrointestinal tract and pancreas

      The relative contribution of acid-base transporters to secretory fluid composition may differ depending on the endogenous cellular function and microenvironment. In gastric parietal cells, basolateral AE2 appears to have the prevailing role over SLC26A7 (SUT2) as HCl secretion is disrupted in Ae2 KO mice.
      • Gawenis L.R.
      • Ledoussal C.
      • Judd L.M.
      • Prasad V.
      • Alper S.L.
      • Stuart-Tilley A.
      • et al.
      Mice with a targeted disruption of the AE2 Cl-/HCO3- exchanger are achlorhydric.
      • Recalde S.
      • Muruzábal F.
      • Looije N.
      • Kunne C.
      • Burrell M.A.
      • Sáez E.
      • et al.
      Inefficient chronic activation of parietal cells in Ae2a,b-/- mice.
      • Petrovic S.
      • Ju X.
      • Barone S.
      • Seidler U.
      • Alper S.L.
      • Lohi H.
      • et al.
      Identification of a basolateral Cl-/HCO3- exchanger specific to gastric parietal cells.
      In duodenocytes, SLC26A3 (DRA) and SLC26A6 (PAT1) are presumed to have roughly equal roles, as basal and cAMP-induced HCO3- secretion are decreased by approximately 50% in Dra KO mice.
      • Walker N.M.
      • Simpson J.E.
      • Brazill J.M.
      • Gill R.K.
      • Dudeja P.K.
      • Schweinfest C.W.
      • et al.
      Role of down-regulated in adenoma anion exchanger in HCO3- secretion across Murine Duodenum.
      ,
      • Wang Z.
      • Wang T.
      • Petrovic S.
      • Tuo B.
      • Riederer B.
      • Barone S.
      • et al.
      Renal and intestinal transport defects in Slc26a6-null mice.
      As for cellular HCO3- supply in duodenocytes, NBCe1 and CA inhibition individually resulted in an approximately 50% reduction of HCO3- flux and simultaneous inhibition had a cumulative effect, indicating parallel activity of electrogenic HCO3- transporters and CAs.
      • Jacob P.
      • Christiani S.
      • Rossmann H.
      • Lamprecht G.
      • Vleillard-Baron D.
      • Müller R.
      • et al.
      Role of Na+ HCO3− cotransporter NBC1, Na+/H+ exchanger NHE1, and carbonic anhydrase in rabbit duodenal bicarbonate secretion.
      Pancreatic fluid has a particularly high [HCO3-] in humans and secretion is stimulated by secretin, acetylcholine, ATP and Ca2+ with expression of inositol 1,4,5-trisphosphate receptors, Ca2+-activated Cl- channels and CFTR.
      • Lee M.G.
      • Ohana E.
      • Park H.W.
      • Yang D.
      • Muallem S.
      Molecular mechanism of pancreatic and salivary gland fluid and HCO3- secretion.
      Despite the cellular machinery being highly similar to that of cholangiocytes, a notable difference is that AE2 is located basolaterally in pancreatic duct cells, whilst SLC26 transporters are responsible for luminal HCO3- secretion (Fig. 2). Furthermore, basolateral NBCe1 functioning with a stoichiometry of 1:2 has been identified as the main source of HCO3- supply alongside CAs working in conjunction with NHE1. Despite competing with an electrogenic transporter, the latter pathway appears physiologically relevant as pancreatic duct cells treated with the pan-CA inhibitor acetazolamide exhibit decreased HCO3- secretion.
      • Pak B.
      • Hong S.
      • Pak H.
      • Hong S.
      Effects of acetazolamide and acid-base changes on biliary and pancreatic secretion.
      ,
      • Dyck W.P.
      • Hightower N.C.
      • Janowitz H.D.
      Effect of acetazolamide on human pancreatic secretion.
      At the distal end of the pancreatic duct system, apical NBCn1, NHE2 and NHE3 are expressed, facilitating HCO3- salvage and fluid uptake when HCO3--rich secretion is deemed unnecessary.
      • Lee M.G.
      • Ohana E.
      • Park H.W.
      • Yang D.
      • Muallem S.
      Molecular mechanism of pancreatic and salivary gland fluid and HCO3- secretion.

      SLC transporters in the hepatobiliary tract

      Epithelial cells secrete HCO3- through CFTR and Cl-/HCO3- exchangers with AE2 localizing apically in the hepatobiliary tract but basolaterally in analogous secretory epithelia.
      To date, CFTR and ubiquitous AE2 are the only base extruders identified in human cholangiocytes.
      • Banales J.M.
      • Prieto J.
      • Medina J.F.
      Cholangiocyte anion exchange and biliary bicarbonate excretion.
      ,
      • Concepcion A.R.
      • Lopez M.
      • Ardura-Fabregat A.
      • Medina J.F.
      Role of AE2 for pHi regulation in biliary epithelial cells.
      It is striking that in all of the secretory epithelia discussed, AE2 is expressed on the basolateral membrane, whilst in cholangiocytes it appears apical (Table 2).
      • Martínez-Ansó E.
      • Castillo J.
      • Díez J.
      • Medina J.
      • Prieto J.
      Immunohistochemical detection of chloride/bicarbonate anion exchangers in human liver.
      ,
      • Spirli C.
      • Granato A.
      • Zsembery A.
      • Anglani F.
      • Okolicsanyi L.
      • LaRusso N.F.
      • et al.
      Functional polarity of Na+/H+ and Cl-/HCO3- exchangers in a rat cholangiocyte cell line.
      AE2 activity is regulated by intra- and extracellular pH, which is attributed to the NH2-terminal domain whose deletion results in reduced responsiveness.
      • Stewart A.K.
      • Chernova M.N.
      • Shmukler B.E.
      • Wilhelm S.
      • Alper S.L.
      Regulation of AE2-mediated Cl- transport by intracellular or by extracellular pH requires highly conserved amino acid residues of the AE2 NH2-terminal cytoplasmic domain.
      ,
      • Stewart A.K.
      • Chernova M.N.
      • Kunes Y.Z.
      • Alper S.L.
      Regulation of AE2 anion exchanger by intracellular pH: critical regions of the NH2-terminalcytoplasmic domain.
      Cholangiocellular HCO3- supply is achieved by CAs working in conjunction with NHEs and/or by Na+-coupled HCO3- transport.
      • Strazzabosco M.
      New insights into cholangiocyte physiology.
      It appears that secretory epithelia dealing with high HCO3- fluxes express electrogenic HCO3- transporters (Table 2). NBCe1 is present in mouse cholangiocytes and is upregulated in cultured Ae2 KO mouse cholangiocytes, accounting for a separate route through which HCO3- may be transported.
      • Uriarte I.
      • Banales J.M.
      • Śaez E.
      • Arenas F.
      • Elferink R.P.J.O.
      • Prieto J.
      • et al.
      Bicarbonate secretion of mouse cholangiocytes involves Na+ -HCO3- cotransport in addition to Na+ -independent Cl-/HCO3- exchange.
      NBCe1 activity is greater in Ae2 KO mouse cholangiocytes but appears to be downregulated by cAMP and ATP. In rat liver, NBCe2 localises apically in cholangiocytes but basolaterally in hepatocytes.
      • Abuladze N.
      • Pushkin A.
      • Tatishchev S.
      • Newman D.
      • Sassani P.
      • Kurtz I.
      Expression and localization of rat NBC4c in liver and renal uroepithelium.
      Human cholangiocytes may also express electrogenic HCO3- transporters (Fig. 1), but when comparing ductular secretion to renal HCO3- reclamation, HCO3- flux is estimated to be at least a factor of 40 higher in the latter.
      • Al-Atabi M.
      • Ooi R.C.
      • Luo X.Y.
      • Chin S.B.
      • Bird N.C.
      Computational analysis of the flow of bile in human cystic duct.
      ,
      • Boyer J.L.
      Bile formation and secretion.
      Electroneutral NDCBE has been identified at the basolateral membrane of human cholangiocytes but appears inactive at physiological intracellular pH ranges.
      • Tabibian J.H.
      • Masyuk A.I.
      • Masyuk T.V.
      • Hara S.P.O.
      • Larusso N.F.
      Physiology of cholangiocytes.
      ,
      • Strazzabosco M.
      • Joplin R.
      • Zsembery À.
      • Wallace L.
      • Spirlì C.
      • Fabris L.
      • et al.
      Na+ -dependent and -independent Cl-/HCO3- exchange mediate cellular HCO3- transport in cultured human intrahepatic bile duct cells.
      Notably, despite its electroneutrality, NDCBE transports 2 HCO3- ions. The importance of CAs working in conjunction with NHEs for cholangiocellular HCO3- supply is underlined by the dependence of secretin-induced ductular secretion and choleresis on basolateral NHE1 activity.
      • Hübner C.
      • Stremmel W.
      • Elsing C.
      Sodium, hydrogen exchange type 1 and bile ductular secretory activity in the Guinea pig.
      Apical Nhe3 KO mice and intrahepatic bile duct units from mice treated with the Na+ channel blocker amiloride fail to absorb fluid.
      • Mennone A.
      • Biemesderfer D.
      • Negoianu D.
      • Yang C.L.
      • Abbiati T.
      • Schultheis P.J.
      • et al.
      Role of sodium/hydrogen exchanger isoform NHE3 in fluid secretion and absorption in mouse and rat cholangiocytes.
      This indicates that apical NHEs alter the viscosity of bile via Na+-dependent fluid absorption and concomitant luminal acidification, the suppression of which may be beneficial in chronic cholestatic liver diseases. In the gallbladder, HCO3- salvaging is facilitated by NHEs which acidify bile, leading to a linear increase in partial CO2 pressure.
      • Marteau C.
      • Sastre B.
      • Iconomidis N.
      • Portugal H.
      • Pauli A.-M.
      • Gérolami A.
      pH regulation in human gallbladder bile: study in patients with and without gallstones.
      Secretory epithelia dealing with high HCO3- fluxes, e.g. cholangiocytes, express electrogenic HCO3- transporters and may utilize transport metabolons.

      Metabolons and electrochemical gradients

      CAs may interact with acid-base transporters to enhance their transportation rate, however controversies exist. Evidence against such a physical interaction, i.e. a transport metabolon, includes colocalisation not being reproducible across cell lines and mathematical modelling indicating that CO2/HCO3- transport occurs more rapidly when cytosolic CAs are homogeneously distributed as opposed to being concentrated at the interior portion of the plasma membrane.
      • Piermarini P.M.
      • Kim E.Y.
      • Boron W.F.
      Evidence against a direct interaction between intracellular carbonic anhydrase II and pure C-terminal domains of SLC4 bicarbonate transporters.
      ,
      • Al-Samir S.
      • Papadopoulos S.
      • Scheibe R.J.
      • Meißner J.D.
      • Cartron J.P.
      • Sly W.S.
      • et al.
      Activity and distribution of intracellular carbonic anhydrase II and their effects on the transport activity of anion exchanger AE1/SLC4A1.
      Evidence supporting such a physical interaction includes NHE1 and AE1 both having a CA II amino acid binding sequence, the latter being identified in the C-terminal domain.
      • Li X.
      • Liu Y.
      • Alvarez B.V.
      • Casey J.R.
      • Fliegel L.
      A novel carbonic anhydrase II binding site regulates NHE1 activity.
      ,
      • Vince J.W.
      • Reithmeier R.A.F.
      Identification of the carbonic anhydrase II binding site in the Cl-/HCO3- anion exchanger AE1.
      AE1 polypeptides with missense mutations in the C-terminal domain show markedly reduced HCO3- transport.
      • Dahl N.K.
      • Jiang L.
      • Chernova M.N.
      • Stuart-Tilley A.K.
      • Shmukler B.E.
      • Alper S.L.
      Deficient HCO3- transport in an AE1 mutant with normal Cl- transport can be Rescued by carbonic anhydrase II presented on an adjacent AE1 Protomer.
      Moreover, the transport activity of AEs is reduced by up to 60% in HEK293 cells transfected with functionally inactive CA II.
      • Sterling D.
      • Reithmeier R.A.F.
      • Casey J.R.
      A transport metabolon: functional interaction of carbonic anhydrase II and chloride/bicarbonate exchangers.
      Glutathione-S-transferase pull-down assays with domain deleted forms of CA IX revealed that the catalytic domain physically interacts with AE2.
      • Morgan P.E.
      • Pastoreková S.
      • Stuart-Tilley A.K.
      • Alper S.L.
      • Casey J.R.
      Interactions of transmembrane carbonic anhydrase, CAIX, with bicarbonate transporters.
      Co-expression of CA IX and AE2 increases transporter activity by approximately 30% and migration assays demonstrate a spatial cooperation.
      • Svastova E.
      • Witarski W.
      • Csaderova L.
      • Kosik I.
      • Skvarkova L.
      • Hulikova A.
      • et al.
      Carbonic anhydrase IX interacts with bicarbonate transporters in lamellipodia and increases cell migration via its catalytic domain.
      CAs are also capable of enhancing electrogenic HCO3- transport, indicating synergistic functioning as opposed to separate routes of HCO3- movement.
      • Schueler C.
      • Becker H.M.
      • McKenna R.
      • Deitmer J.W.
      Transport activity of the sodium bicarbonate cotransporter NBCe1 is enhanced by different isoforms of carbonic anhydrase.
      NBCe1 changes stoichiometry upon phosphorylation, with CA inhibition drastically reducing the short-circuit current of NBCe1 working in a 1:3 mode while having no effect on the 1:2 mode.
      • Gross E.
      • Pushkin A.
      • Abuladze N.
      • Fedotoff O.
      • Kurtz I.
      Regulation of the sodium bicarbonate cotransporter kNBC1 function: role of Asp986, Asp988 and kNBC1-carbonic anhydrase II binding.
      This suggests an added level of complexity, namely that the enhancement of HCO3- flux by CAs depends on transporter phosphorylation status. Taken together, the functional significance of transport metabolons may only be relevant in secretory epithelia that deal with high acid-base fluxes.
      Basolateral Na+/K+ ATPase sets a potential difference over the plasma membrane with a low intracellular [Na+] facilitating NHE-mediated antiport of H+. Membrane-bound CAs generate CO2 and H2O, which diffuse or move through AQPs to the cytosol, where CA II regenerates H+ and HCO3-.
      • Wang Y.
      • Cohen J.
      • Boron W.F.
      • Schulten K.
      • Tajkhorshid E.
      Exploring gas permeability of cellular membranes and membrane channels with molecular dynamics.
      H+ and HCO3- are extruded over opposing membranes creating a flux. Generally, in the renal PCT, H+ is extruded into the lumen and HCO3- is reclaimed basolaterally, whilst in secretory epithelia, i.e. pancreatic duct cells and cholangiocytes, H+ is extruded basolaterally and HCO3- is secreted into the lumen to generate flow. In addition, the low intracellular [Na+] facilitates Na+-coupled HCO3- transport. In pancreatic duct cells, NBCe1-mediated HCO3- import occurs when the inward facing chemical gradients exceed the outward facing electrical gradient. Notably, plasma membrane depolarisation promotes electrogenic HCO3- import. When hyperpolarisation of the basolateral membrane exceeds the reversal potential of NBCe1, basolateral HCO3- import is blocked or reversed. Thus, NBCe1 working in a 1:2 mode allows for basolateral HCO3- uptake in pancreatic duct cells and possibly cholangiocytes, whilst NBCe1 working in a 1:3 mode allows for basolateral HCO3- reclamation in the renal PCT (Fig. 2). The electrochemical gradient also determines the ability of CFTR to switch between Cl- and HCO3- transport.
      • Bridges R.J.
      Mechanisms of bicarbonate secretion: lessons from the airways.
      ,
      • Park H.W.
      • Lee M.G.
      Transepithelial bicarbonate secretion: lessons from the pancreas.
      CFTR has been calculated to conduct HCO3- and Cl- at a ratio of 0.26:1,
      • Poulsen J.H.
      • Fischer H.
      • Illek B.
      • Machen T.E.
      Bicarbonate conductance and pH regulatory capability of cystic fibrosis transmembrane conductance regulator.
      however it is increased by low intracellular [Cl-].
      • Kim Y.
      • Jun I.
      • Shin D.H.
      • Yoon J.G.
      • Piao H.
      • Jung J.
      • et al.
      Regulation of CFTR bicarbonate channel activity by WNK1: implications for pancreatitis and CFTR-related disorders.
      Thus, the membrane potential may function as an “on/off switch” for electrogenic HCO3- transporters and thereby partially determine the substrate of CFTR alongside Cl- availability. In addition, intracellular [Cl-] functions as a regulator of electrogenic Na+-coupled HCO3- transport.
      • Shcheynikov N.
      • Son A.
      • Hong J.H.
      • Yamazaki O.
      • Ohana E.
      • Kurtz I.
      • et al.
      Intracellular Cl- as a signaling ion that potently regulates Na+/HCO3- transporters.
      In human cholangiocytes, the regulation of HCO3- uptake by basolateral de- and hyperpolarisation and intracellular [Cl-] is of importance if NBCe1 is indeed functionally present alongside electroneutral NDCBE. Notably NDCBE takes up 2 HCO3- and 1 Na+ to extrude 1 Cl-, thereby not excluding the possibility of CFTR secreting HCO3- in cholangiocytes.
      Membrane potential and intracellular [Cl-] may distinguish between electrogenic transporters and carbonic anhydrases as the preferred source of HCO3- supply.

      Acid-base sensing

      Acid-base sensors exist in a variety of forms including receptors, enzymes, ion channels and signalling molecules. Soluble adenylyl cyclase (sAC) is an intracellular HCO3- sensor, whilst extracellular acid-base sensors include G protein-coupled receptors; tyrosine kinase and phosphatase receptors, such as the type 5 protein tyrosine phosphatase receptors (PTPRG and PTPRZ1); and cation channels, such as acid sensing ion channels (ASICs). Besides these sensory proteins, biliary and intestinal alkaline phosphatase (ALP) activity is pH-dependent: preserving ATP at low pH values but degrading it when luminal pH increases.
      • Akiba Y.
      • Kaunitz J.D.
      Duodenal chemosensing and mucosal defenses.
      Thus, ALP regulates ATP-mediated purinergic HCO3- secretion along the enterohepatic circulation.

      Acid-base sensors in analogous secretory epithelia

      Intracellular compartment

      sAC is responsive to [HCO3-] and [Ca2+], catalyses the conversion of ATP to cAMP and works together with CAs and the V-ATPase as an enclosed sensory system able to modulate extracellular pH in secretory epithelia.
      • Pastor-Soler N.
      • Beaulieu V.
      • Litvin T.N.
      • Da Silva N.
      • Chen Y.
      • Brown D.
      • et al.
      Bicarbonate-regulated adenylyl cyclase (sAC) is a sensor that regulates pH-dependent V-ATPase recycling.
      The subcellular localisation of sAC and the V-ATPase differ in renal intercalated cells and expression appears dynamic as metabolic acidosis decreases the number of HCO3--secreting β-intercalated cells, whilst the V-ATPase is inserted in the apical membrane with sAC in close proximity in H+-secreting α-intercalated cells.
      • Pǎunescu T.G.
      • Da Silva N.
      • Russo L.M.
      • McKee M.
      • Lu H.A.J.
      • Breton S.
      • et al.
      Association of soluble adenylyl cyclase with the V-ATPase in renal epithelial cells.
      sAC additionally appears to regulate other acid-base machinery as inhibition reduces CA II and CFTR expression.
      • Mardones P.
      • Chang J.C.
      • Oude Elferink R.P.J.
      Cyclic AMP and alkaline pH downregulate carbonic anhydrase 2 in mouse fibroblasts.
      ,
      • Wang Y.
      • Lam C.S.
      • Wu F.
      • Wang W.
      • Duan Y.
      • Huang P.
      Regulation of CFTR channels by HCO3- -sensitive soluble adenylyl cyclase in human airway epithelial cells.

      Extracellular compartment

      PTPRG and PTPRZ1 contain an extracellular domain of 266 amino acids with striking homology to CA.
      • Barnea G.
      • Silvennoinen O.
      • Shaanan B.
      • Honegger A.M.
      • Canoll P.D.
      • D'Eustachio P.
      • et al.
      Identification of a carbonic anhydrase-like domain in the extracellular region of RPTP gamma defines a new subfamily of receptor tyrosine phosphatases.
      Based on the crystal structure of PTPRG, 11 out of 19 residues forming the CA active site and 1 out of 3 residues responsible for CAs' catalytic activity are conserved, suggesting the type 5 PTPRs have additional functions besides dephosphorylation. Indeed, ubiquitous PTPRG has been identified as a novel HCO3- sensor.
      • Zhou Y.
      • Skelton L.A.
      • Xu L.
      • Chandler M.P.
      • Berthiaume J.M.
      • Boron W.F.
      Role of receptor protein tyrosine phosphatase γ in sensing extracellular CO2 and HCO3−.
      Basolateral HCO3- flux in the renal PCTs of Ptprg KO mice was irresponsive to alterations in basolateral [HCO3-] and [CO2], rendering these mice unable to defend against chronic metabolic acidosis. Notably, a downregulation of CA II and IV was observed. The vasomotor effects of HCO3- on arteries are also abolished in Ptprg KO mice.
      • Boedtkjer E.
      • Hansen K.B.
      • Boedtkjer D.M.B.
      • Aalkjaer C.
      • Boron W.F.
      Extracellular HCO3- is sensed by mouse cerebral arteries: regulation of tone by receptor protein tyrosine phosphatase γ.
      As PTPRG functions as a HCO3- sensor in various tissues and has an extracellular CA-like domain, it raises the question whether PTPRZ1 and the CARPs are also conserved HCO3- sensors.
      • Occhipinti R.
      • Boron W.F.
      Role of carbonic anhydrases and inhibitors in acid–base physiology: insights from mathematical modeling.
      In addition, type 5 PTPRs are implicated in inflammation, underlined by their expression in the spleen and specialised macrophages; they are a functional receptor for IL-34 and Ptprg KO mice have altered B-cell levels.
      • Lissandrini D.
      • Vermi W.
      • Vezzalini M.
      • Sozzani S.
      • Facchetti F.
      • Bellone G.
      • et al.
      Receptor-type protein tyrosine phosphatase gamma (PTPgamma), a new identifier for myeloid dendritic cells and specialized macrophages.
      • Nandi S.
      • Cioce M.
      • Yeung Y.G.
      • Nieves E.
      • Tesfa L.
      • Lin H.
      • et al.
      Receptor-type protein-tyrosine phosphatase ζ is a functional receptor for interleukin-34.
      • Cohen S.
      • Shoshana O.
      • Zelman-Toister E.
      • Maharshak N.
      • Binsky-Ehrenreich I.
      • Gordin M.
      • et al.
      The cytokine midkine and its receptor RPTPζ regulate B cell survival in a pathway induced by CD74.
      Thus, proteins possessing an acatalytic CA domain are potential candidates for HCO3- sensing in bile ducts and may even be involved in local immunological processes.
      ASICs are phylogenetically related to epithelial Na+ channels and primarily transport Na+, but also Ca2+ and K+.
      • Waldmann R.
      • Champigny G.
      • Bassilana F.
      • Heurteaux C.
      • Lazdunski M.
      A proton-gated cation channel involved in acid-sensing.
      ASICs are H+-gated trimeric protein complexes that have been shown to arise from 5 different genes excluding splice variants. ASIC isoforms are functionally expressed in intestinal epithelial cells and are involved in H+-stimulated duodenal HCO3- secretion through a Ca2+ signalling pathway.
      • Dong X.
      • Ko K.H.
      • Chow J.
      • Tuo B.
      • Barrett K.E.
      • Dong H.
      Expression of acid-sensing ion channels in intestinal epithelial cells and their role in the regulation of duodenal mucosal bicarbonate secretion.
      ASIC expression is also upregulated during inflammation, thereby associating inflammation with an acidic microenvironment.
      • Voilley N.
      • de Weille J.
      • Mamet J.
      • Lazdunski M.
      Nonsteroid anti-inflammatory drugs inhibit both the activity and the inflammation-induced expression of acid-sensing ion channels in nociceptors.
      Moreover, there appears to be an interplay between ASICs and CFTR. This is indicated physically by apical colocalisation in human airway epithelial cells and functionally by whole cell patch clamp studies demonstrating that cAMP-induced CFTR activation reduces the H+-gated Na+ current and, oppositely, ASIC activation inhibits cAMP-induced CFTR activation.
      • Su X.
      • Li Q.
      • Shrestha K.
      • Cormet-Boyaka E.
      • Chen L.
      • Smith P.R.
      • et al.
      Interregulation of proton-gated Na+ channel 3 and cystic fibrosis transmembrane conductance regulator.

      Acid-base sensors in the hepatobiliary tract

      Intracellular compartment

      In cholangiocytes, AE2 knockdown causes intracellular build-up of HCO3- and consequently sAC upregulation and activation.
      • Chang J.C.
      • Go S.
      • de Waart D.R.
      • Munoz-Garrido P.
      • Beuers U.
      • Paulusma C.C.
      • et al.
      Soluble adenylyl cyclase regulates bile Salt-induced apoptosis in human cholangiocytes.
      Bile acid exposure activates sAC causing downstream cytochrome c release and apoptosis, which can be prevented by sAC inhibition. These findings provide insight into the promising role of sAC in the pathogenesis of PBC.
      • Chang J.C.
      • Beuers U.
      • Elferink R.P.J.O.
      The emerging role of soluble adenylyl cyclase in primary biliary cholangitis.

      Extracellular compartment

      PTPRZ1-positive bile ducts appear prominent in liver biopsies of patients with PBC and PSC.
      • Michelotti G.A.
      • Tucker A.
      • Swiderska-Syn M.
      • Machado M.V.
      • Choi S.S.
      • Kruger L.
      • et al.
      Pleiotrophin regulates the ductular reaction by controlling the migration of cells in liver progenitor niches.
      Furthermore, PTPRZ1 is expressed in hepatic stellate cells and is increased in liver sections of bile duct ligated (BDL) mice. Ductular reaction appeared to be inhibited in Ptprz1 KO mice undergoing BDL. This suggests that PTPRZ1 signalling regulates ductular reaction in cholestatic liver diseases, such as PBC and PSC. Similar to CA IX, PTPRZ1 and PTPRG can shed their ectodomains, the latter having plasma levels correlating with liver damage in humans.
      • Moratti E.
      • Vezzalini M.
      • Tomasello L.
      • Giavarina D.
      • Sorio C.
      Identification of protein tyrosine phosphatase receptor gamma extracellular domain (sPTPRG) as a natural soluble protein in plasma.
      Hepatic PTPRG expression is increased during inflammation, but this requires NF-kB to bind to its promotor region.
      • Brenachot X.
      • Ramadori G.
      • Ioris R.M.
      • Veyrat-Durebex C.
      • Altirriba J.
      • Aras E.
      • et al.
      Hepatic protein tyrosine phosphatase receptor gamma links obesity-induced inflammation to insulin resistance.
      A bile acid sensitive cation channel phylogenetically related to ASICs has been identified in cholangiocytes and named ASIC5 or bile acid sensitive ion channel (BASIC).
      • Wiemuth D.
      • Sahin H.
      • Falkenburger B.H.
      • Lefèvre C.M.T.
      • Wasmuth H.E.
      • Gründer S.
      Basic - a bile acid-sensitive ion channel highly expressed in bile ducts.
      This cation channel localises to the apical membrane in close proximity to CFTR and is kept in a resting state by extracellular divalent cations; removal of these cations and exposure to bile acids such as UDCA, lead to activation of BASIC.
      • Wiemuth D.
      • Sahin H.
      • Lefèvre C.M.T.
      • Wasmuth H.E.
      • Gründer S.
      Strong activation of bile acid-sensitive ion channel (BASIC) by ursodeoxycholic acid.
      Evidence suggests that bile acids do not exert their agonistic effect on BASIC through direct interaction but by altering the properties of the environment surrounding the membrane.
      • Schmidt A.
      • Lenzig P.
      • Oslender-Bujotzek A.
      • Kusch J.
      • Lucas S.D.
      • Gründer S.
      • et al.
      The bile acid-sensitive ion channel (BASIC) is activated by alterations of its membrane environment.
      As opposed to ASICs, BASIC is not activated by protons, but stepwise reduction of extracellular pH reduces channel conductance, suggesting that H+ has an inhibitory effect. Notably, BASIC itself is permeable for H+. This novel cation channel that is sensitive to bile acids and closely related to ASICs may be a potential extracellular pH sensor in bile ducts.
      Type 5 protein tyrosine phosphatase receptors (PTPRG, PTPRZ1) and the bile acid sensitive ion channel (BASIC) are candidate luminal pH sensors in cholangiocytes.

      Framework for cholangiocellular acid-base homeostasis

      Many epithelia are specialised in HCO3--rich fluid secretion, which generates flow, alters viscosity, controls pH and potentially protects luminal and intracellular structures from chemical stress. Based on these conserved principles identified in secretory epithelia, and the biliary system conforming to an exocrine gland architecture of acini, i.e. canaliculi, draining into consecutively larger ducts, a framework is proposed for cholangiocellular acid-base homeostasis (Fig. 3): Basolateral Na+/K+ ATPase alongside K+ channels set a potential difference over the plasma membrane, with a low intracellular [Na+] used by basolateral NHE1 to extrude H+. In conjunction with basolateral CA IX, CO2 and H2O are formed and enter the cytosol where CA II regenerates HCO3- and H+ allowing the latter to be recycled by NHE1 creating a closed loop. Intracellular HCO3- diffuses to the apical region and is primarily extruded via AE2 coupled to CFTR to form a biliary HCO3- umbrella and generate flow. If an electrogenic HCO3- transporter, such as NBCe1, is indeed functionally present in human cholangiocytes, the regulation of HCO3- uptake by basolateral de- and hyperpolarisation and intracellular [Cl-] is of importance. In addition, the Na+ gradient drives electroneutral NDCBE, thereby possibly depleting intracellular Cl- stores causing CFTR to favour HCO3- transport. Bile acids can endogenously inhibit luminal CAs. In turn, the deprotonation of hydrophobic apolar bile acids by luminal HCO3- may be enhanced via tethered CA IV and secretory CA VI in bile. sAC is responsible for intracellular HCO3- sensing, whilst extracellular pH sensors in cholangiocytes remain elusive. Potential candidates are the type 5 PTPRs and BASIC. This framework provides insight into the molecular basis of pH homeostasis and dysregulation in the biliary system.
      Figure thumbnail gr3
      Fig. 3Framework for cholangiocellular acid-base homeostasis.
      Basolateral CA IX and cytosolic CA II work in conjunction with NHE1 to facilitate cellular HCO3- supply and extrude H+ creating a closed loop. A parallel source of HCO3- supply is via Na+-coupled HCO3- transporters, such as electrogenic NBCe1 and electroneutral NDCBE. Intracellular HCO3- diffuses to the apical region and is primarily extruded via AE2 coupled to CFTR to form a biliary HCO3- umbrella and generate flow. Bile acids can endogenously inhibit luminal CAs. In turn, the deprotonation of hydrophobic apolar bile acids by luminal HCO3- may be enhanced via luminal CAs. sAC is responsible for intracellular HCO3- sensing, whilst extracellular pH sensors in cholangiocytes remain elusive. Potential candidates are the type 5 PTPRs and BASIC. CALD, carbonic anhydrase-like domain; ER, endoplasmic reticulum; GCDCA, glycochenodeoxycholic acid; InsP3R, inositol 1,4,5-trisphosphate receptor (type III); KC, K+ channel. Further details on the protein names are in the list of abbreviations.

      Therapeutic considerations and future directions

      Dysregulation of the acid-base balance may be a common occurrence in cholestatic liver diseases, such as PBC, PSC, cystic fibrosis-related liver disease (CFLD) and IgG4-related cholangitis (IRC).
      Dysregulation of the acid-base balance may be a common occurrence in cholestatic liver diseases, such as PBC, PSC, CFLD and IRC. Defective biliary HCO3- secretion is thought to be pathogenic in PBC
      • Prieto J.
      • García N.
      • Martí-Climent J.
      • Peñuelas I.
      • Richter J.
      • Medina J.
      Assessment of biliary bicarbonate secretion in humans by positron emission tomography.
      • Banales J.
      • Sáez E.
      • Uriz M.
      • Sarvide S.
      • Urribarri A.
      • Splinter P.
      • et al.
      Upregulation of mir-506 leads to decreased AE2 expression in biliary epithelium of patients with primary biliary cirrhosis.
      • Beuers U.
      • Hohenester S.
      • de Buy Wenniger L.J.M.
      • Kremer A.E.
      • Jansen P.L.M.
      • Oude Elferink R.P.J.
      The biliary HCO3- umbrella: a unifying hypothesis on pathogenetic and therapeutic aspects of fibrosing cholangiopathies.
      • Hohenester S.
      • Maillette de Buy Wenniger L.
      • Paulusma C.C.
      • van Vliet S.J.
      • Jefferson D.M.
      • Oude Elferink R.P.
      • et al.
      A biliary HCO3- umbrella constitutes a protective mechanism against bile acid-induced injury in human cholangiocytes.
      but remains enigmatic in PSC. A common occurrence in these chronic cholestatic liver diseases is that patients suffer from RTA, indicating involvement beyond the biliary system. The concept of pH dysregulation may be expanded to CFLD and IRC due to the emerging role of defective HCO3- secretion in CF and the HCO3--rich secretion by target organs in IgG4-RD. Anticholestatic treatment options for patients with PBC can alter the acid-base balance by directly or indirectly stabilising the biliary HCO3- umbrella,
      • Beuers U.
      Drug insight: mechanisms and sites of action of ursodeoxycholic acid in cholestasis.
      ,
      • Beuers U.
      • Trauner M.
      • Jansen P.
      • Poupon R.
      New paradigms in the treatment of hepatic cholestasis: from UDCA to FXR, PXR and beyond.
      thereby improving biochemical markers of cholestasis and surrogate markers of prognosis, as well as slowing histological disease progression.
      • Rudic J.S.
      • Poropat G.
      • Krstic M.N.
      • Bjelakovic G.
      • Gluud C.
      Ursodeoxycholic acid for primary biliary cirrhosis.
      Currently no registered therapeutics are available for PSC despite major research efforts. UDCA improves biochemical markers of cholestasis and surrogate markers of prognosis,
      • Poropat G.
      • Giljaca V.
      • Stimac D.
      • Gluud C.
      Bile acids for primary sclerosing cholangitis.
      however, no survival benefit has been revealed to date. An often neglected fact is that no methodologically sound trials have assessed the effect of moderate dose UDCA on long-term prognosis in patients with PSC. Very high dose UDCA resulted in more complications and should be avoided.
      • Lindor K.D.
      • Kowdley K.V.
      • Luketic V.A.C.
      • Harrison M.E.
      • McCashland T.
      • Befeler A.S.
      • et al.
      High-dose ursodeoxycholic acid for the treatment of primary sclerosing cholangitis.
      The effect of UDCA on long-term prognosis is also unclear in patients suffering from CFLD, again due to a lack of adequately sized, controlled prospective studies. Besides UDCA, FXR and PPAR agonists also appear to modulate hepatobiliary acid-base balance, potentially leading to anticholestatic effects.
      • Beuers U.
      • Trauner M.
      • Jansen P.
      • Poupon R.
      New paradigms in the treatment of hepatic cholestasis: from UDCA to FXR, PXR and beyond.
      Collectively this underlines the clinical relevance of hepatobiliary acid-base homeostasis and the necessity for additional therapeutics to treat these cholestatic liver diseases. Detailed insight into the regulation of (and interplay between) CA enzymes, transporters of the solute carrier family, and intra- and extracellular pH sensors in cholangiocytes may reveal additional targets for repairing defective biliary HCO3- secretion. The molecular processes and targets we think are most promising for this purpose are: 1) The interactions between CA isoforms and acid-base transporters, including their potential to become rate-limiting in cholangiocellular HCO3- flux; 2) The contribution of Na+-coupled (electrogenic) HCO3- transporters to cholangiocellular HCO3- supply; 3) Identification of extracellular pH sensors in cholangiocytes, for which we propose the type 5 PTPRs possessing a CA-like domain and BASIC.
      Modulation of hepatobiliary acid-base machinery may be a therapeutic strategy for cholestatic liver diseases, supported by the molecular mechanisms of action of current anticholestatics.

      Abbreviations

      AE1 (SLC4A1), anion exchanger 1; AE2 (SLC4A2), anion exchanger 2; ALP, alkaline phosphatase; AMA, anti-mitochondrial antibodies; AQP, aquaporin; ASIC, acid sensing ion channel; ASL, airway surface liquid; BASIC, bile acid sensing ion channel; BDL, bile duct ligation; CA, carbonic anhydrase; CARP, carbonic anhydrase-related protein; CCA, cholangiocarcinoma; CD, collecting duct; CF, cystic fibrosis; CFLD, cystic fibrosis-associated liver disease; CFTR, cystic fibrosis transmembrane conductance regulator; DCA, deoxycholic acid; DRA (SLC26A3), down-regulated in adenoma; FXR, farnesoid X receptor; GCA, glycocholic acid; GPBAR1, G protein-coupled bile acid receptor; HCC, hepatocellular carcinoma; IgG4-RD, IgG4-related disease; IRC, IgG4-related cholangitis; KO, knockout; Mdr2, multidrug resistance protein 2; NBCe1 (SLC4A4), electrogenic Na+/HCO3- cotransporter 1; NBCe2 (SLC4A5), electrogenic Na+/HCO3- cotransporter 2; NBCn1 (SLC4A7), electroneutral Na+/HCO3- cotransporter 1; NBCn2 (SLC4A10), electroneutral Na+/HCO3- cotransporter 2; NDCBE, Na+-driven Cl-/HCO3- exchanger; NHE1 (SLC9A1), Na+/H+ exchanger 1; NHE2 (SLC9A2), Na+/H+ exchanger 2; NHE3 (SLC9A3), Na+/H+ exchanger 3; PAT1 (SLC26A6), putative anion transporter 1; PBC, primary biliary cholangitis; PCT, proximal convoluted tubule; PPAR, peroxisome proliferator-activated receptor; PSC, primary sclerosing cholangitis; PTPRG, protein tyrosine phosphatase receptor gamma; PTPRZ1, protein tyrosine phosphatase receptor zeta; RTA, renal tubular acidosis; sAC (ADCY10), soluble adenylyl cyclase; SLC, solute carrier; UDCA, ursodeoxycholic acid; V-ATPase, vacuolar H+-ATPase.

      Financial support

      This work was supported by an Amsterdam UMC , AMC PhD Scholarship (to DT) and a South-African PSC Patient Foundation (to UB). DT, SvdG, AJ and ROE have nothing to disclose. UB received grant support via Amsterdam UMC for investigator-initiated studies from Dr. Falk GmbH (Freiburg) and Intercept (San Diego), and lecture fees from Abbvie, Falk Foundation, Gilead and Intercept.

      Authors' contributions

      All authors contributed to the concept and design of the manuscript. The original draft of the manuscript was written by DT, after which reviewing and editing was done by SvdG, ROE and UB. The publicly available single cell RNA sequencing datasets were analysed and graphically displayed by AJ. The final manuscript was approved by all authors. Funding was facilitated by DT and UB.

      Conflict of interest

      The authors declare no conflicts of interest that pertain to this work.
      Please refer to the accompanying ICMJE disclosure forms for further details.

      Acknowledgements

      Publicly available single cell RNA sequencing datasets were analysed and graphically displayed in collaboration with the Department of Clinical Epidemiology, Biostatistics and Bioinformatics of Amsterdam UMC, Academic Medical Center. Specifically, we wish to express our gratitude towards Professor Antoine H.C. van Kampen for his recommendations on analysis of the datasets obtained from Gene Expression Omnibus (GEO). In addition, we would like to thank N. Aizarani et al. (GEO accession GSE124395) and H. Hu et al. (GEO accession GSE111301) for depositing their single cell RNA sequencing datasets in GEO available to the public. Finally, we would also like to thank Remco Kersten for the fruitful dialogue on protein tyrosine phosphatase receptor gamma (PTPRG).

      Supplementary data

      References

        • Terziroli Beretta-Piccoli B.
        • Mieli-Vergani G.
        • Vergani D.
        • Vierling J.M.
        • Adams D.
        • Alpini G.
        • et al.
        The challenges of primary biliary cholangitis: what is new and what needs to be done.
        J Autoimmun. 2019; 105: 102328
        • Karlsen T.H.
        • Folseraas T.
        • Thorburn D.
        • Vesterhus M.
        Primary sclerosing cholangitis – a comprehensive review.
        J Hepatol. 2017; 67: 1298-1323
        • Prieto J.
        • García N.
        • Martí-Climent J.
        • Peñuelas I.
        • Richter J.
        • Medina J.
        Assessment of biliary bicarbonate secretion in humans by positron emission tomography.
        Gastroenterology. 1999; 117: 167-172
        • Banales J.
        • Sáez E.
        • Uriz M.
        • Sarvide S.
        • Urribarri A.
        • Splinter P.
        • et al.
        Upregulation of mir-506 leads to decreased AE2 expression in biliary epithelium of patients with primary biliary cirrhosis.
        Hepatology. 2012; 56: 687-697
        • Beuers U.
        • Hohenester S.
        • de Buy Wenniger L.J.M.
        • Kremer A.E.
        • Jansen P.L.M.
        • Oude Elferink R.P.J.
        The biliary HCO3- umbrella: a unifying hypothesis on pathogenetic and therapeutic aspects of fibrosing cholangiopathies.
        Hepatology. 2010; 52: 1489-1496
        • Hohenester S.
        • Maillette de Buy Wenniger L.
        • Paulusma C.C.
        • van Vliet S.J.
        • Jefferson D.M.
        • Oude Elferink R.P.
        • et al.
        A biliary HCO3- umbrella constitutes a protective mechanism against bile acid-induced injury in human cholangiocytes.
        Hepatology. 2012; 55: 173-183
        • Beuers U.
        Drug insight: mechanisms and sites of action of ursodeoxycholic acid in cholestasis.
        Nat Clin Pract Gastroenterol Hepatol. 2006; 3: 318-328
        • Beuers U.
        • Trauner M.
        • Jansen P.
        • Poupon R.
        New paradigms in the treatment of hepatic cholestasis: from UDCA to FXR, PXR and beyond.
        J Hepatol. 2015; 62: 25-37
        • Choi J.Y.
        • Muallem D.
        • Kiselyov K.
        • Lee M.G.
        • Thomas P.J.
        • Muallem S.
        Aberrant CFTR-dependent HCO3- transport in mutations associated with cystic fibrosis.
        Nature. 2001; 410: 94-97
        • Matton A.P.M.
        • Vries Y de
        • Burlage L.C.
        • Rijn R van
        • Fujiyoshi M.
        • de Meijer V.E.
        • et al.
        Biliary bicarbonate, pH, and glucose are suitable biomarkers of biliary viability during ex situ normothermic machine perfusion of human donor livers.
        Transplantation. 2019; 103: 1405-1413
        • Klier M.
        • Jamali S.
        • Ames S.
        • Schneider H.P.
        • Becker H.M.
        • Deitmer J.W.
        Catalytic activity of human carbonic anhydrase isoform IX is displayed both extra- and intracellularly.
        FEBS J. 2016; 283: 191-200
        • Hamm L.L.
        • Nakhoul N.
        • Hering-Smith K.S.
        Acid-base homeostasis.
        Clin J Am Soc Nephrol. 2015; 10: 2232-2242
        • Purkerson J.M.
        • Schwartz G.J.
        The role of carbonic anhydrases in renal physiology.
        Kidney Int. 2007; 71: 103-115
        • Purkerson J.M.
        • Kittelberger A.M.
        • Schwartz G.J.
        Basolateral carbonic anhydrase IV in the proximal tubule is a glycosylphosphatidylinositol-anchored protein.
        Kidney Int. 2007; 71: 407-416
        • Wall S.
        Recent advances in our understanding of intercalated cells.
        Curr Opin Nephrol Hypertens. 2005; 14: 480-484
        • Pastor-soler N.
        • Piétrement C.
        • Breton S.
        Role of acid / base transporters in the male reproductive tract and potential consequences of their malfunction.
        Physiology. 2005; 20: 417-428
        • Wandernoth P.M.
        • Mannowetz N.
        • Szczyrba J.
        • Grannemann L.
        • Wolf A.
        • Becker H.M.
        • et al.
        Normal fertility requires the expression of carbonic anhydrases II and IV in sperm.
        J Biol Chem. 2015; 290: 29202-29216
        • Karhumaa P.
        • Kaunisto K.
        • Parkkila S.
        • Waheed A.
        • Pastoreková S.
        • Pastorek J.
        • et al.
        Expression of the transmembrane carbonic anhydrases, CA IX and CA XII, in the human male excurrent ducts.
        Mol Hum Reprod. 2001; 7: 611-616
        • Lan C.C.
        • Peng C.K.
        • Tang S.E.
        • Huang K.L.
        • Wu C.P.
        Carbonic anhydrase inhibitor attenuates ischemia-reperfusion induced acute lung injury.
        PLoS One. 2017; 12: 1-20
        • Parkkila S.
        • Parkkila A.K.
        • Lehtola J.
        • Reinilä A.
        • Södervik H.J.
        • Rannisto M.
        • et al.
        Salivary carbonic anhydrase protects gastroesophageal mucosa from acid injury.
        Dig Dis Sci. 1997; 42: 1013-1019
        • Pastorekova S.
        • Parkkila S.
        • Parkkila A.K.
        • Opavsky R.
        • Zelnik V.
        • Saarnio J.
        • et al.
        Carbonic anhydrase IX, MN/CA IX: analysis of stomach complementary DNA sequence and expression in human and rat alimentary tracts.
        Gastroenterology. 1997; 112: 398-408
        • Kivelä A.J.
        • Kivelä J.
        • Saarnio J.
        • Parkkila S.
        Carbonic anhydrases in normal gastrointestinal tract and gastrointestinal tumours.
        World J Gastroenterol. 2005; 11: 155-163
        • Harmon G.S.
        • Dumlao D.S.
        • Ng D.T.
        • Barrett K.E.
        • Dennis E.A.
        • Dong H.
        • et al.
        Pharmacological correction of a defect in PPAR-γ signaling ameliorates disease severity in Cftr-deficient mice.
        Nat Med. 2010; 16: 313-318
        • Lee M.G.
        • Ohana E.
        • Park H.W.
        • Yang D.
        • Muallem S.
        Molecular mechanism of pancreatic and salivary gland fluid and HCO3- secretion.
        Physiol Rev. 2012; 92: 39-74
        • Ignáth I.
        • Hegyi P.
        • Venglovecz V.
        • Székely C.A.
        • Carr G.
        • Hasegawa M.
        • et al.
        CFTR expression but not Cl- transport is involved in the stimulatory effect of bile acids on apical Cl-/HCO3- exchange activity in human pancreatic duct cells.
        Pancreas. 2009; 38: 921-929
        • Nishimori I.
        • Onishi S.
        Carbonic anhydrase isozymes in the human pancreas.
        Dig Liver Dis. 2001; 33: 68-74
        • Fanjul M.
        • Salvador C.
        • Alvarez L.
        • Cantet S.
        • Hollande E.
        Targeting of carbonic anhydrase IV to plasma membrane is altered in cultured human pancreatic duct cells expressing a mutated (ΔF508) CFTR.
        Eur J Cell Biol. 2002; 81: 437-447
        • Ueno Y.
        • Ishii M.
        • Igarashi T.
        • Mano Y.
        • Yahagi K.
        • Kisara N.
        • et al.
        Primary biliary cirrhosis with antibody against carbonic anhydrase II associates with distinct immunological backgrounds.
        Hepatol Res. 2001; 20: 18-27
        • Zatovicova M.
        • Sedlakova O.
        • Svastova E.
        • Ohradanova A.
        • Ciampor F.
        • Arribas J.
        • et al.
        Ectodomain shedding of the hypoxia-induced carbonic anhydrase IX is a metalloprotease-dependent process regulated by TACE/ADAM17.
        Br J Cancer. 2005; 93: 1267-1276
        • Saarnio J.
        • Parkkila S.
        • Parkkila A.K.
        • Pastoreková S.
        • Haukipuro K.
        • Pastorek J.
        • et al.
        Transmembrane carbonic anhydrase, MN/CA IX, is a potential biomarker for biliary tumours.
        J Hepatol. 2001; 35: 643-649
        • Huang W.
        • Jeng Y.
        • Lai H.
        • Fong I.
        Expression of hypoxic marker carbonic anhydrase IX Predicts poor prognosis in resectable hepatocellular carcinoma.
        PLoS One. 2015; 10: 1-14
        • Bi C.
        • Liu M.
        • Rong W.
        • Wu F.
        • Zhang Y.
        • Lin S.
        • et al.
        High Beclin-1 and ARID1A expression corelates with poor survival and high recurrence in intrahepatic cholangiocarcinoma: a histopathological retrospective study.
        BMC Cancer. 2019; 19: 213
        • Scozzafava A.
        • Supuran C.T.
        Carbonic anhydrase inhibitors. Preparation of potent sulfonamides inhibitors incorporating bile acid tails.
        Bioorg Med Chem Lett. 2002; 12: 1551-1557
        • Boone C.D.
        • Tu C.
        • Mckenna R.
        Structural elucidation of the hormonal inhibition mechanism of the bile acid cholate on human carbonic anhydrase II.
        Acta Crystallogr Sect D Biol Crystallogr. 2014; 70: 1758-1763
        • Milov D.E.
        • Jou W.S.
        • Shireman R.B.
        • Chun P.W.
        The effect of bile salts on carbonic anhydrase.
        Hepatology. 1992; 15: 288-296
        • Nishita T.
        • Itoh S.
        • Arai S.
        • Ichihara N.
        • Arishima K.
        Measurement of carbonic anhydrase isozyme VI (CA-VI) in swine sera, colostrums, saliva, bile, seminal plasma and tissues.
        Anim Sci J. 2011; 82: 673-678
        • Parkkila S.
        • Parkkila A.K.
        • Juvonen T.
        • Waheed A.
        • Sly W.S.
        • Saarnio J.
        • et al.
        Membrane-bound carbonic anhydrase IV is expressed in the luminal plasma membrane of the human gallbladder epithelium.
        Hepatology. 1996; 24: 1104-1108
        • Nilsson B.
        • Valantinas J.
        • Hedin L.
        • Friman S.
        • Svanvik J.
        Acetazolamide inhibits stimulated feline liver and gallbladder bicarbonate secretion.
        Acta Physiol Scand. 2002; 174: 117-123
        • Garcia-Marin J.J.
        • Dumont M.
        • Corbic M.
        • de Couet G.
        • Erlinger S.
        Effect of acid-base balance and acetazolamide on ursodeoxycholate-induced biliary bicarbonate secretion.
        Am J Physiol. 1985; 248: G20-G27
        • Sly W.
        • Hewett-Emmett D.
        • Whyte M.
        • Yu Y.
        • Tashian R.
        Carbonic anhydrase II deficiency identified as the primary defect in the autosomal recessive syndrome of osteopetrosis with renal tubular acidosis and cerebral calcification.
        Proc Natl Acad Sci U S A. 1983; 80: 2752-2756
        • Golding P.L.
        • Mason A.S.
        Renal tubular acidosis and autoimmune liver disease.
        Gut. 1971; 12: 153-157
        • Parés A.
        • Rimola A.
        • Bruguera M.
        • Mas E.
        • Rodés J.
        Renal tubular acidosis in primary biliary cirrhosis.
        Gastroenterology. 1981; 80: 681-686
        • Goutaudier V.
        • Szwarc I.
        • Serre J.E.
        • Pageaux G.P.
        • Argilés À.
        • Ribstein J.
        Primary sclerosing cholangitis: a new cause of distal renal tubular acidosis.
        Clin Kidney J. 2016; 9: 811-813
        • Scheiner B.
        • Lindner G.
        • Reiberger T.
        • Schneeweiss B.
        • Trauner M.
        • Zauner C.
        • et al.
        Acid-base disorders in liver disease.
        J Hepatol. 2017; 67: 1062-1073
        • Bagnis C.
        • Marshansky V.
        • Breton S.
        • Brown D.
        Remodeling the cellular profile of collecting ducts by chronic carbonic anhydrase inhibition.
        Am J Physiol Ren Physiol. 2001; 280: 437-448
        • Leppilampi M.
        • Parkkila S.
        • Karttunen T.
        • Gut M.O.
        • Gros G.
        • Sjoblom M.
        Carbonic anhydrase isozyme-II-deficient mice lack the duodenal bicarbonate secretory response to prostaglandin E2.
        Proc Natl Acad Sci U S A. 2005; 102: 15247-15252
        • Sjöblom M.
        • Singh A.K.
        • Zheng W.
        • Wang J.
        • Tuo B.G.
        • Krabbenhöft A.
        • et al.
        Duodenal acidity “sensing” but not epithelial HCO3- supply is critically dependent on carbonic anhydrase II expression.
        Proc Natl Acad Sci U S A. 2009; 106: 13094-13099
        • Pan P.
        • Leppilampi M.
        • Pastorekova S.
        • Pastorek J.
        • Waheed A.
        • Sly W.S.
        • et al.
        Carbonic anhydrase gene expression in CA II-deficient (Car2-/-) and CAIX-deficient (Car9-/-) mice.
        J Physiol. 2006; 571: 319-327
        • Lee M.
        • Vecchio-Pagán B.
        • Sharma N.
        • Waheed A.
        • Li X.
        • Raraigh K.S.
        • et al.
        Loss of carbonic anhydrase XII function in individuals with elevated sweat chloride concentration and pulmonary airway disease.
        Hum Mol Genet. 2016; 25: 1923-1933
        • Hong J.H.
        • Muhammad E.
        • Zheng C.
        • Hershkovitz E.
        • Alkrinawi S.
        • Loewenthal N.
        • et al.
        Essential role of carbonic anhydrase XII in secretory gland fluid and HCO3- secretion revealed by disease causing human mutation.
        J Physiol. 2015; 593: 5299-5312
        • Diez-Fernandez C.
        • Rüfenacht V.
        • Santra S.
        • Lund A.M.
        • Santer R.
        • Lindner M.
        • et al.
        Defective hepatic bicarbonate production due to carbonic anhydrase VA deficiency leads to early-onset life-threatening metabolic crisis.
        Genet Med. 2016; 18: 991-1000
        • Baghdasaryan A.
        • Claudel T.
        • Gumhold J.
        • Silbert D.
        • Adorini L.
        • Roda A.
        • et al.
        Dual farnesoid X receptor/TGR5 agonist INT-767 reduces liver injury in the Mdr2−/− (Abcb4−/−) mouse Cholangiopathy model by promoting biliary HCO3- Output.
        Hepatology. 2011; 54: 1303-1312
        • Rosenthal S.B.
        • Bush K.T.
        • Nigam S.K.
        A network of SLC and ABC transporter and DME genes involved in Remote sensing and signaling in the gut-liver-kidney Axis.
        Sci Rep. 2019; 9: 1-19
        • Shcheynikov N.
        • Wang Y.
        • Park M.
        • Ko S.B.H.
        • Dorwart M.
        • Naruse S.
        • et al.
        Coupling modes and stoichiometry of Cl-/HCO3- exchange by slc26a3 and slc26a6.
        J Gen Physiol. 2006; 127: 511-524
        • Kurtz I.
        • Zhu Q.
        Structure, function, and regulation of the SLC4 NBCe1 transporter and its role in causing proximal renal tubular acidosis.
        Curr Opin Nephrol Hypertens. 2013; 22: 572-583
        • Felder R.A.
        • Jose P.A.
        • Xu P.
        • Gildea J.J.
        The renal sodium bicarbonate cotransporter NBCe2: is it a major contributor to sodium and pH homeostasis?.
        Curr Hypertens Rep. 2016; 18: 1-9
        • Guo Y.
        • Liu Y.
        • Liu M.
        • Wang J.
        • Xie Z.
        • Chen K.
        • et al.
        Na+/HCO3- cotransporter NBCn2 mediates HCO3- reclamation in the apical membrane of renal proximal tubules.
        J Am Soc Nephrol. 2017; 28: 2409-2417
        • Wang T.
        • Hropot M.
        • Aronson P.S.
        • Giebisch G.
        Role of NHE isoforms in mediating bicarbonate reabsorption along the nephron.
        Am J Physiol Ren Physiol. 2001; 281: 1117-1122
        • Liu Y.
        • Wang D.-K.
        • Chen L.-M.
        The physiology of bicarbonate transporters in mammalian reproduction.
        Biol Reprod. 2012; 86: 1-13
        • Tang X.X.
        • Ostedgaard L.S.
        • Hoegger M.J.
        • Moninger T.O.
        • Karp P.H.
        • Mcmenimen J.D.
        • et al.
        Acidic pH increases airway surface liquid viscosity in cystic fibrosis.
        J Clin Invest. 2016; 126: 879-891
        • Shah V.S.
        • Meyerholz D.K.
        • Tang X.X.
        • Reznikov L.
        • Alaiwa M.A.
        • Ernst S.E.
        • et al.
        Airway acidification initiates host defense abnormalities in cystic fibrosis mice.
        Science. 2016; 351: 503-507
        • Chen E.Y.T.
        • Yang N.
        • Quinton P.M.
        • Chin W.C.
        A new role for bicarbonate in mucus formation.
        Am J Physiol Lung Cell Mol Physiol. 2010; 299: 542-549
        • Cooper J.L.
        • Quinton P.M.
        • Ballard S.T.
        Mucociliary transport in porcine trachea: differential effects of inhibiting chloride and bicarbonate secretion.
        Am J Physiol Lung Cell Mol Physiol. 2013; 304: 184-190
        • Shan J.
        • Liao J.
        • Huang J.
        • Robert R.
        • Palmer M.L.
        • Fahrenkrug S.C.
        • et al.
        Bicarbonate-dependent chloride transport drives fluid secretion by the human airway epithelial cell line Calu-3.
        J Physiol. 2012; 590: 5273-5297
        • Kim D.
        • Huang J.
        • Billet A.
        • Abu-Arish A.
        • Goepp J.
        • Matthes E.
        • et al.
        Pendrin mediates bicarbonate secretion and enhances cystic fibrosis transmembrane conductance regulator function in airway surface epithelia.
        Am J Respir Cell Mol Biol. 2019; 60: 705-716
        • Fischer H.
        • Widdicombe J.H.
        Mechanisms of acid and base secretion by the airway epithelium.
        J Membr Biol. 2006; 211: 139-150
        • Gawenis L.R.
        • Ledoussal C.
        • Judd L.M.
        • Prasad V.
        • Alper S.L.
        • Stuart-Tilley A.
        • et al.
        Mice with a targeted disruption of the AE2 Cl-/HCO3- exchanger are achlorhydric.
        J Biol Chem. 2004; 279: 30531-30539
        • Recalde S.
        • Muruzábal F.
        • Looije N.
        • Kunne C.
        • Burrell M.A.
        • Sáez E.
        • et al.
        Inefficient chronic activation of parietal cells in Ae2a,b-/- mice.
        Am J Pathol. 2006; 169: 165-176
        • Petrovic S.
        • Ju X.
        • Barone S.
        • Seidler U.
        • Alper S.L.
        • Lohi H.
        • et al.
        Identification of a basolateral Cl-/HCO3- exchanger specific to gastric parietal cells.
        Am J Physiol Gastrointest Liver Physiol. 2003; 284: 1093-1103
        • Walker N.M.
        • Simpson J.E.
        • Brazill J.M.
        • Gill R.K.
        • Dudeja P.K.
        • Schweinfest C.W.
        • et al.
        Role of down-regulated in adenoma anion exchanger in HCO3- secretion across Murine Duodenum.
        Gastroenterology. 2009; 136: 893-901
        • Wang Z.
        • Wang T.
        • Petrovic S.
        • Tuo B.
        • Riederer B.
        • Barone S.
        • et al.
        Renal and intestinal transport defects in Slc26a6-null mice.
        Am J Physiol Cell Physiol. 2005; 288: 957-965
        • Jacob P.
        • Christiani S.
        • Rossmann H.
        • Lamprecht G.
        • Vleillard-Baron D.
        • Müller R.
        • et al.
        Role of Na+ HCO3− cotransporter NBC1, Na+/H+ exchanger NHE1, and carbonic anhydrase in rabbit duodenal bicarbonate secretion.
        Gastroenterology. 2000; 119: 406-419
        • Pak B.
        • Hong S.
        • Pak H.
        • Hong S.
        Effects of acetazolamide and acid-base changes on biliary and pancreatic secretion.
        Am J Physiol. 1966; 210: 624-628
        • Dyck W.P.
        • Hightower N.C.
        • Janowitz H.D.
        Effect of acetazolamide on human pancreatic secretion.
        Gastroenterology. 1972; 62: 547-552
        • Banales J.M.
        • Prieto J.
        • Medina J.F.
        Cholangiocyte anion exchange and biliary bicarbonate excretion.
        World J Gastroenterol. 2006; 12: 3496-3511
        • Concepcion A.R.
        • Lopez M.
        • Ardura-Fabregat A.
        • Medina J.F.
        Role of AE2 for pHi regulation in biliary epithelial cells.
        Front Physiol. 2014; 4: 1-7
        • Martínez-Ansó E.
        • Castillo J.
        • Díez J.
        • Medina J.
        • Prieto J.
        Immunohistochemical detection of chloride/bicarbonate anion exchangers in human liver.
        Hepatology. 1994; 19: 1400-1406
        • Spirli C.
        • Granato A.
        • Zsembery A.
        • Anglani F.
        • Okolicsanyi L.
        • LaRusso N.F.
        • et al.
        Functional polarity of Na+/H+ and Cl-/HCO3- exchangers in a rat cholangiocyte cell line.
        Am J Physiol Gastrointest Liver Physiol. 1998; 275: G1236-1245
        • Stewart A.K.
        • Chernova M.N.
        • Shmukler B.E.
        • Wilhelm S.
        • Alper S.L.
        Regulation of AE2-mediated Cl- transport by intracellular or by extracellular pH requires highly conserved amino acid residues of the AE2 NH2-terminal cytoplasmic domain.
        J Gen Physiol. 2002; 120: 707-722
        • Stewart A.K.
        • Chernova M.N.
        • Kunes Y.Z.
        • Alper S.L.
        Regulation of AE2 anion exchanger by intracellular pH: critical regions of the NH2-terminalcytoplasmic domain.
        Am J Physiol Cell Physiol. 2001; 281: 1344-1354
        • Strazzabosco M.
        New insights into cholangiocyte physiology.
        J Hepatol. 1997; 27: 945-952
        • Uriarte I.
        • Banales J.M.
        • Śaez E.
        • Arenas F.
        • Elferink R.P.J.O.
        • Prieto J.
        • et al.
        Bicarbonate secretion of mouse cholangiocytes involves Na+ -HCO3- cotransport in addition to Na+ -independent Cl-/HCO3- exchange.
        Hepatology. 2010; 51: 891-902
        • Abuladze N.
        • Pushkin A.
        • Tatishchev S.
        • Newman D.
        • Sassani P.
        • Kurtz I.
        Expression and localization of rat NBC4c in liver and renal uroepithelium.
        Am J Physiol Cell Physiol. 2004; 287: 781-789
        • Al-Atabi M.
        • Ooi R.C.
        • Luo X.Y.
        • Chin S.B.
        • Bird N.C.
        Computational analysis of the flow of bile in human cystic duct.
        Med Eng Phys. 2012; 34: 1177-1183
        • Boyer J.L.
        Bile formation and secretion.
        Compr Physiol. 2013; 3: 1035-1078
        • Tabibian J.H.
        • Masyuk A.I.
        • Masyuk T.V.
        • Hara S.P.O.
        • Larusso N.F.
        Physiology of cholangiocytes.
        Compr Physiol. 2013; 3: 1-49
        • Strazzabosco M.
        • Joplin R.
        • Zsembery À.
        • Wallace L.
        • Spirlì C.
        • Fabris L.
        • et al.
        Na+ -dependent and -independent Cl-/HCO3- exchange mediate cellular HCO3- transport in cultured human intrahepatic bile duct cells.
        Hepatology. 1997; 25: 976-985
        • Hübner C.
        • Stremmel W.
        • Elsing C.
        Sodium, hydrogen exchange type 1 and bile ductular secretory activity in the Guinea pig.
        Hepatology. 2000; 31: 562-571
        • Mennone A.
        • Biemesderfer D.
        • Negoianu D.
        • Yang C.L.
        • Abbiati T.
        • Schultheis P.J.
        • et al.
        Role of sodium/hydrogen exchanger isoform NHE3 in fluid secretion and absorption in mouse and rat cholangiocytes.
        Am J Physiol Gastrointest Liver Physiol. 2001; 280: 247-254
        • Marteau C.
        • Sastre B.
        • Iconomidis N.
        • Portugal H.
        • Pauli A.-M.
        • Gérolami A.
        pH regulation in human gallbladder bile: study in patients with and without gallstones.
        Hepatology. 1990; 11: 997-1002
        • Piermarini P.M.
        • Kim E.Y.
        • Boron W.F.
        Evidence against a direct interaction between intracellular carbonic anhydrase II and pure C-terminal domains of SLC4 bicarbonate transporters.
        J Biol Chem. 2007; 282: 1409-1421
        • Al-Samir S.
        • Papadopoulos S.
        • Scheibe R.J.
        • Meißner J.D.
        • Cartron J.P.
        • Sly W.S.
        • et al.
        Activity and distribution of intracellular carbonic anhydrase II and their effects on the transport activity of anion exchanger AE1/SLC4A1.
        J Physiol. 2013; 591: 4963-4982
        • Li X.
        • Liu Y.
        • Alvarez B.V.
        • Casey J.R.
        • Fliegel L.
        A novel carbonic anhydrase II binding site regulates NHE1 activity.
        Biochemistry. 2006; 45: 2414-2424
        • Vince J.W.
        • Reithmeier R.A.F.
        Identification of the carbonic anhydrase II binding site in the Cl-/HCO3- anion exchanger AE1.
        Biochemistry. 2000; 39: 5527-5533
        • Dahl N.K.
        • Jiang L.
        • Chernova M.N.
        • Stuart-Tilley A.K.
        • Shmukler B.E.
        • Alper S.L.
        Deficient HCO3- transport in an AE1 mutant with normal Cl- transport can be Rescued by carbonic anhydrase II presented on an adjacent AE1 Protomer.
        J Biol Chem. 2003; 278: 44949-44958
        • Sterling D.
        • Reithmeier R.A.F.
        • Casey J.R.
        A transport metabolon: functional interaction of carbonic anhydrase II and chloride/bicarbonate exchangers.
        J Biol Chem. 2001; 276: 47886-47894
        • Morgan P.E.
        • Pastoreková S.
        • Stuart-Tilley A.K.
        • Alper S.L.
        • Casey J.R.
        Interactions of transmembrane carbonic anhydrase, CAIX, with bicarbonate transporters.
        Am J Physiol Cell Physiol. 2007; 293: C738-C748
        • Svastova E.
        • Witarski W.
        • Csaderova L.
        • Kosik I.
        • Skvarkova L.
        • Hulikova A.
        • et al.
        Carbonic anhydrase IX interacts with bicarbonate transporters in lamellipodia and increases cell migration via its catalytic domain.
        J Biol Chem. 2012; 287: 3392-3402
        • Schueler C.
        • Becker H.M.
        • McKenna R.
        • Deitmer J.W.
        Transport activity of the sodium bicarbonate cotransporter NBCe1 is enhanced by different isoforms of carbonic anhydrase.
        PLoS One. 2011; 6: e27167
        • Gross E.
        • Pushkin A.
        • Abuladze N.
        • Fedotoff O.
        • Kurtz I.
        Regulation of the sodium bicarbonate cotransporter kNBC1 function: role of Asp986, Asp988 and kNBC1-carbonic anhydrase II binding.
        J Physiol. 2002; 544: 679-685
        • Wang Y.
        • Cohen J.
        • Boron W.F.
        • Schulten K.
        • Tajkhorshid E.
        Exploring gas permeability of cellular membranes and membrane channels with molecular dynamics.
        J Struct Biol. 2007; 157: 534-544
        • Bridges R.J.
        Mechanisms of bicarbonate secretion: lessons from the airways.
        Cold Spring Harb Perspect Med. 2012; 2: 1-11
        • Park H.W.
        • Lee M.G.
        Transepithelial bicarbonate secretion: lessons from the pancreas.
        Cold Spring Harb Perspect Med. 2012; 2: a009571
        • Poulsen J.H.
        • Fischer H.
        • Illek B.
        • Machen T.E.
        Bicarbonate conductance and pH regulatory capability of cystic fibrosis transmembrane conductance regulator.
        Proc Natl Acad Sci U S A. 1994; 91: 5340-5344
        • Kim Y.
        • Jun I.
        • Shin D.H.
        • Yoon J.G.
        • Piao H.
        • Jung J.
        • et al.
        Regulation of CFTR bicarbonate channel activity by WNK1: implications for pancreatitis and CFTR-related disorders.
        CMGH. 2020; 9: 79-103
        • Shcheynikov N.
        • Son A.
        • Hong J.H.
        • Yamazaki O.
        • Ohana E.
        • Kurtz I.
        • et al.
        Intracellular Cl- as a signaling ion that potently regulates Na+/HCO3- transporters.
        Proc Natl Acad Sci U S A. 2015; 112: E329-E337
        • Akiba Y.
        • Kaunitz J.D.
        Duodenal chemosensing and mucosal defenses.
        Digestion. 2011; 83: 25-31
        • Pastor-Soler N.
        • Beaulieu V.
        • Litvin T.N.
        • Da Silva N.
        • Chen Y.
        • Brown D.
        • et al.
        Bicarbonate-regulated adenylyl cyclase (sAC) is a sensor that regulates pH-dependent V-ATPase recycling.
        J Biol Chem. 2003; 278: 49523-49529
        • Pǎunescu T.G.
        • Da Silva N.
        • Russo L.M.
        • McKee M.
        • Lu H.A.J.
        • Breton S.
        • et al.
        Association of soluble adenylyl cyclase with the V-ATPase in renal epithelial cells.
        Am J Physiol Ren Physiol. 2008; 294: 130-138
        • Mardones P.
        • Chang J.C.
        • Oude Elferink R.P.J.
        Cyclic AMP and alkaline pH downregulate carbonic anhydrase 2 in mouse fibroblasts.
        Biochim Biophys Acta. 2014; 1840: 1765-1770
        • Wang Y.
        • Lam C.S.
        • Wu F.
        • Wang W.
        • Duan Y.
        • Huang P.
        Regulation of CFTR channels by HCO3- -sensitive soluble adenylyl cyclase in human airway epithelial cells.
        Am J Physiol Cell Physiol. 2005; 289: 1145-1151
        • Barnea G.
        • Silvennoinen O.
        • Shaanan B.
        • Honegger A.M.
        • Canoll P.D.
        • D'Eustachio P.
        • et al.
        Identification of a carbonic anhydrase-like domain in the extracellular region of RPTP gamma defines a new subfamily of receptor tyrosine phosphatases.
        Mol Cell Biol. 1993; 13: 1497-1506
        • Zhou Y.
        • Skelton L.A.
        • Xu L.
        • Chandler M.P.
        • Berthiaume J.M.
        • Boron W.F.
        Role of receptor protein tyrosine phosphatase γ in sensing extracellular CO2 and HCO3−.
        J Am Soc Nephrol. 2016; 27: 2616-2621
        • Boedtkjer E.
        • Hansen K.B.
        • Boedtkjer D.M.B.
        • Aalkjaer C.
        • Boron W.F.
        Extracellular HCO3- is sensed by mouse cerebral arteries: regulation of tone by receptor protein tyrosine phosphatase γ.
        J Cereb Blood Flow Metab. 2015; 36: 965-980
        • Occhipinti R.
        • Boron W.F.
        Role of carbonic anhydrases and inhibitors in acid–base physiology: insights from mathematical modeling.
        Int J Mol Sci. 2019; 20: 3841
        • Lissandrini D.
        • Vermi W.
        • Vezzalini M.
        • Sozzani S.
        • Facchetti F.
        • Bellone G.
        • et al.
        Receptor-type protein tyrosine phosphatase gamma (PTPgamma), a new identifier for myeloid dendritic cells and specialized macrophages.
        Blood. 2006; 108: 4223-4231
        • Nandi S.
        • Cioce M.
        • Yeung Y.G.
        • Nieves E.
        • Tesfa L.
        • Lin H.
        • et al.
        Receptor-type protein-tyrosine phosphatase ζ is a functional receptor for interleukin-34.
        J Biol Chem. 2013; 288: 21972-21986
        • Cohen S.
        • Shoshana O.
        • Zelman-Toister E.
        • Maharshak N.
        • Binsky-Ehrenreich I.
        • Gordin M.
        • et al.
        The cytokine midkine and its receptor RPTPζ regulate B cell survival in a pathway induced by CD74.
        J Immunol. 2012; 188: 259-269
        • Waldmann R.
        • Champigny G.
        • Bassilana F.
        • Heurteaux C.
        • Lazdunski M.
        A proton-gated cation channel involved in acid-sensing.
        Nature. 1997; 386: 173-177
        • Dong X.
        • Ko K.H.
        • Chow J.
        • Tuo B.
        • Barrett K.E.
        • Dong H.
        Expression of acid-sensing ion channels in intestinal epithelial cells and their role in the regulation of duodenal mucosal bicarbonate secretion.
        Acta Physiol. 2011; 201: 97-107
        • Voilley N.
        • de Weille J.
        • Mamet J.
        • Lazdunski M.
        Nonsteroid anti-inflammatory drugs inhibit both the activity and the inflammation-induced expression of acid-sensing ion channels in nociceptors.
        J Neurosci. 2001; 21: 8026-8033
        • Su X.
        • Li Q.
        • Shrestha K.
        • Cormet-Boyaka E.
        • Chen L.
        • Smith P.R.
        • et al.
        Interregulation of proton-gated Na+ channel 3 and cystic fibrosis transmembrane conductance regulator.
        J Biol Chem. 2006; 281: 36960-36968
        • Chang J.C.
        • Go S.
        • de Waart D.R.
        • Munoz-Garrido P.
        • Beuers U.
        • Paulusma C.C.
        • et al.
        Soluble adenylyl cyclase regulates bile Salt-induced apoptosis in human cholangiocytes.
        Hepatology. 2016; 64: 522-534
        • Chang J.C.
        • Beuers U.
        • Elferink R.P.J.O.
        The emerging role of soluble adenylyl cyclase in primary biliary cholangitis.
        Dig Dis. 2017; 35: 217-223
        • Michelotti G.A.
        • Tucker A.
        • Swiderska-Syn M.
        • Machado M.V.
        • Choi S.S.
        • Kruger L.
        • et al.
        Pleiotrophin regulates the ductular reaction by controlling the migration of cells in liver progenitor niches.
        Gut. 2016; 65: 683-692
        • Moratti E.
        • Vezzalini M.
        • Tomasello L.
        • Giavarina D.
        • Sorio C.
        Identification of protein tyrosine phosphatase receptor gamma extracellular domain (sPTPRG) as a natural soluble protein in plasma.
        PLoS One. 2015; 10: e0119110
        • Brenachot X.
        • Ramadori G.
        • Ioris R.M.
        • Veyrat-Durebex C.
        • Altirriba J.
        • Aras E.
        • et al.
        Hepatic protein tyrosine phosphatase receptor gamma links obesity-induced inflammation to insulin resistance.
        Nat Commun. 2017; 8: 1820
        • Wiemuth D.
        • Sahin H.
        • Falkenburger B.H.
        • Lefèvre C.M.T.
        • Wasmuth H.E.
        • Gründer S.
        Basic - a bile acid-sensitive ion channel highly expressed in bile ducts.
        FASEB J. 2012; 26: 4122-4130
        • Wiemuth D.
        • Sahin H.
        • Lefèvre C.M.T.
        • Wasmuth H.E.
        • Gründer S.
        Strong activation of bile acid-sensitive ion channel (BASIC) by ursodeoxycholic acid.
        Channels. 2013; 7: 38-42
        • Schmidt A.
        • Lenzig P.
        • Oslender-Bujotzek A.
        • Kusch J.
        • Lucas S.D.
        • Gründer S.
        • et al.
        The bile acid-sensitive ion channel (BASIC) is activated by alterations of its membrane environment.
        PLoS One. 2014; 9: e111549
        • Rudic J.S.
        • Poropat G.
        • Krstic M.N.
        • Bjelakovic G.
        • Gluud C.
        Ursodeoxycholic acid for primary biliary cirrhosis.
        Cochrane Database Syst Rev. 2012; 12: CD000551
        • Poropat G.
        • Giljaca V.
        • Stimac D.
        • Gluud C.
        Bile acids for primary sclerosing cholangitis.
        Cochrane Database Syst Rev. 2011; 2011: CD003626
        • Lindor K.D.
        • Kowdley K.V.
        • Luketic V.A.C.
        • Harrison M.E.
        • McCashland T.
        • Befeler A.S.
        • et al.
        High-dose ursodeoxycholic acid for the treatment of primary sclerosing cholangitis.
        Hepatology. 2009; 50: 808-814
        • Maurel P.
        • Rauch U.
        • Flad M.
        • Margolis R.K.
        • Margolis R.U.
        Phosphacan, a chondroitin sulfate proteoglycan of brain that interacts with neurons and neural cell-adhesion molecules, is an extracellular variant of a receptor-type protein tyrosine phosphatase.
        Proc Natl Acad Sci U S A. 1994; 91: 2512-2516
        • Lee H.
        • Yi J.S.
        • Lawan A.
        • Min K.
        • Bennett A.M.
        Mining the function of protein tyrosine phosphatases in health and disease.
        Semin Cell Dev Biol. 2015; 37: 66-72
        • Parkkila S.
        Significance of pH regulation and carbonic anhydrases in tumour progression and implications for diagnostic and therapeutic approaches.
        BJU Int. 2008; 101: 16-21
        • Mboge M.Y.
        • Mahon B.P.
        • McKenna R.
        • Frost S.C.
        Carbonic anhydrases: role in pH control and cancer.
        Metabolites. 2018; 8: 19
        • Seidler U.E.
        Gastrointestinal HCO3- transport and epithelial protection in the gut: new techniques, transport pathways and regulatory pathways.
        Curr Opin Pharmacol. 2013; 13: 900-908
        • Inada A.
        • Nienaber C.
        • Katsuta H.
        • Fujitani Y.
        • Levine J.
        • Morita R.
        • et al.
        Carbonic anhydrase II-positive pancreatic cells are progenitors for both endocrine and exocrine pancreas after birth.
        Proc Natl Acad Sci U S A. 2008; 105: 19915-19919
        • Fanjul M.
        • Alvarez L.
        • Salvador C.
        • Gmyr V.
        • Kerr-Conte J.
        • Pattou F.
        • et al.
        Evidence for a membrane carbonic anhydrase IV anchored by its C-terminal peptide in normal human pancreatic ductal cells.
        Histochem Cell Biol. 2004; 121: 91-99
        • Kivelä A.J.
        • Parkkila S.
        • Saarnio J.
        • Karttunen T.J.
        • Kivelä J.
        • Parkkila A.K.
        • et al.
        Expression of transmembrane carbonic anhydrase isoenzymes IX and XII in normal human pancreas and pancreatic tumours.
        Histochem Cell Biol. 2000; 114: 197-204
        • Parkkila S.
        • Parkkila A.K.
        • Juvonen T.
        • Rajaniemi H.
        Distribution of the carbonic anhydrase isoenzymes I, II, and VI in the human alimentary tract.
        Gut. 1994; 35: 646-650
        • Shcheynikov N.
        • Yang D.
        • Wang Y.
        • Zeng W.
        • Karniski L.P.
        • So I.
        • et al.
        The Slc26a4 transporter functions as an electroneutral Cl-/I-/HCO3- exchanger: role of Slc26a4 and Slc26a6 in I- and HCO3- secretion and in regulation of CFTR in the parotid duct.
        J Physiol. 2008; 586: 3813-3824
        • Esbaugh A.J.
        • Tufts B.L.
        The structure and function of carbonic anhydrase isozymes in the respiratory system of vertebrates.
        Respir Physiol Neurobiol. 2006; 154: 185-198
        • Leinonen J.S.
        • Saari K.A.
        • Seppänen J.M.
        • Myllylä H.M.
        • Rajaniemi H.J.
        Immunohistochemical demonstration of carbonic anhydrase isoenzyme VI (CA VI) expression in rat lower airways and lung.
        J Histochem Cytochem. 2004; 52: 1107-1112
        • Saint-Criq V.
        • Gray M.A.
        Role of CFTR in epithelial physiology.
        Cell Mol Life Sci. 2017; 74: 93-115
        • Skelton L.A.
        • Boron W.F.
        • Zhou Y.
        Acid-base transport by the renal proximal tubule.
        J Nephrol. 2010; 23: 1-25
        • Soleimani M.
        SLC26 Cl-/HCO3- exchangers in the kidney: roles in health and disease.
        Kidney Int. 2013; 84: 657-666
        • Aizarani N.
        • Saviano A.
        • Sagar
        • Mailly L.
        • Durand S.
        • Herman J.S.
        • et al.
        A human liver cell atlas reveals heterogeneity and epithelial progenitors.
        Nature. 2019; 572: 199-204
        • Hu H.
        • Gehart H.
        • Artegiani B.
        • LÖpez-Iglesias C.
        • Dekkers F.
        • Basak O.
        • et al.
        Long-term expansion of functional mouse and human hepatocytes as 3D organoids.
        Cell. 2018; 175: 1591-1606