Epithelial Fluid Secretion Research Articles

Combining different ion-selective channelrhodopsins to control water flux by light

Water transport through water channels, aquaporins (AQPs), is vital for many physiological processes including epithelial fluid secretion, cell migration and adipocyte metabolism. Water flux through AQPs is driven by the osmotic gradient that results from concentration differences of solutes including ions. Here, we developed a novel optogenetic toolkit that combines the light-gated anion channel GtACR1 either with the light-gated K+ channel HcKCR1 or the new Na+ channelrhodopsin HcNCR1 with high Na+ permeability, to manipulate water transport in Xenopus oocytes non-invasively. Water efflux through AQP was achieved by light-activating K+ and Cl- efflux through HcKCR1 and GtACR1. Contrarily, when GtACR1 was co-expressed with HcNCR1, inward movement of Na+ and Cl- was light-triggered, and the resulting osmotic gradient led to water influx through AQP1. In sum, we demonstrate a novel optogenetic strategy to manipulate water movement into or out of Xenopus oocytes non-invasively. This approach provides a new avenue to interfere with water homeostasis as a means to study related biological phenomena across cell types and organisms.

Human pancreatic ductal organoids with controlled polarity provide a novel ex vivo tool to study epithelial cell physiology

Epithelial ion and fluid secretion determine the physiological functions of a broad range of organs, such as the lung, liver, or pancreas. The molecular mechanism of pancreatic ion secretion is challenging to investigate due to the limited access to functional human ductal epithelia. Patient-derived organoids may overcome these limitations, however direct accessibility of the apical membrane is not solved. In addition, due to the vectorial transport of ions and fluid the intraluminal pressure in the organoids is elevated, which may hinder the study of physiological processes. To overcome these, we developed an advanced culturing method for human pancreatic organoids based on the removal of the extracellular matrix that induced an apical-to-basal polarity switch also leading to reversed localization of proteins with polarized expression. The cells in the apical-out organoids had a cuboidal shape, whereas their resting intracellular Ca2+ concentration was more consistent compared to the cells in the apical-in organoids. Using this advanced model, we demonstrated the expression and function of two novel ion channels, the Ca2+ activated Cl− channel Anoctamin 1 (ANO1) and the epithelial Na+ channel (ENaC), which were not considered in ductal cells yet. Finally, we showed that the available functional assays, such as forskolin-induced swelling, or intracellular Cl− measurement have improved dynamic range when performed with apical-out organoids. Taken together our data suggest that polarity-switched human pancreatic ductal organoids are suitable models to expand our toolset in basic and translational research.Graphical abstract

#3679 ANOCTAMIN 1 INHIBITION SUPPRESSES CYSTOGENESIS BY ENHANCING CILIOGENESIS AND THE CILIARY DOSAGE OF POLYCYSTINS

Abstract Background and Aims Autosomal dominant polycystic kidney disease (ADPKD) is a ciliopathy which is characterized by abnormal tubular epithelial proliferation and fluid secretion. Anoctamin 1 (ANO1) is a calcium-dependent chloride channel, which has recently been shown to be involved in ADPKD progression. However, how ANO1 contributes to ADPKD is largely unexplored. Method 1. Based on mRNA gene chips, the expression of ANO1 abnormally increased in human ADPKD kidneys. 2. Immunohistochemical staining showed ANO1 location. 3. Real-time PCR and Western blot showed mRNAs and protein of ANO1 in ADPKD patients and animal model-PKD1RC/RC mice. 4. Confocal immunofluorescence showed ANO1 and PC2 location in the cilia. 5. 3D gel culture to verify the function of ANO1 in cysts formation. 6. ANO1 inhibitors and siRNA to discover STAT3 and ERK pathway in ADPKD cell by WB. Results We found that ANO1 is consistently upregulated inhuman and mouse PKD kidneys. Intriguingly, ANO1 located in a vesicle-like patten at the ciliary base but not on the ciliary surface. ANO1 deficiency promotes both ciliogenesis and the ciliary dosage of polycystin-2 in human Pkd1-/- cells, and reduced cyst formation in 3D culture models. Moreover, inhibition of ANO1 abolished the activation of STAT3 and ERK pathways in PKD cells. Conclusion Our data indicate ANO1 may aggravate ADPKD by regulating the cilia length and PC2 expression. Moreover, ANO1 could support cyst growth by activating STAT3 and ERK pathways. Thus ANO1 is a negative regulator for both cilia formation and cilia trafficking of polycystins and provide mechanistic insights regarding the therapeutic potential of ANO1 pathway in ADPKD treatment.

Conjunctival Fluid Secretion Impairment via CaCC-CFTR Dysfunction Is the Key Mechanism in Environmental Dry Eye.

Dry eye disease (DED) is a multifactorial disease with an incidence of approximately 50% worldwide. DED seriously affects quality of life and work. The prevalence of environmental DED (eDED) ranges from 35 to 48%. Conjunctival fluid secretion dysfunction may be one of the major causes of DED. Notably, the Cl- flux corresponds to the conjunctival fluid secretion and could be affected by ATP. Both the cystic fibrosis transmembrane conductance regulator (CFTR) and the Ca2+-activated Cl- channel (CaCC) are Cl- channels involved in epithelial fluid secretion. Conjunctival fluid secretion could be increased by activating P2Y2R (an ATP receptor) in DED. However, the role of the CaCC and CFTR channels regulated by P2Y2R in eDED remains unclear. In this study, we established a rabbit eDED model using a controlled drying system. A Ussing chamber was used to perform a conjunctival short-circuit current induced by ATP to evaluate the reactivity of the ion channels to the ATP. Our results revealed that eDED accompanied by conjunctival fluid secretion impairment was caused by a P2Y2R dysfunction, which is related to CaCC-CFTR signaling in the conjunctiva epithelium. Notably, the coupling effect of the ATP-induced CaCC-CFTR activation and intracellular Ca2+ may represent a promising therapeutic target for treating eDED.

Drug Repurposing for Cystic Fibrosis: Identification of Drugs That Induce CFTR-Independent Fluid Secretion in Nasal Organoids.

Individuals with cystic fibrosis (CF) suffer from severe respiratory disease due to a genetic defect in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which impairs airway epithelial ion and fluid secretion. New CFTR modulators that restore mutant CFTR function have been recently approved for a large group of people with CF (pwCF), but ~19% of pwCF cannot benefit from CFTR modulators Restoration of epithelial fluid secretion through non-CFTR pathways might be an effective treatment for all pwCF. Here, we developed a medium-throughput 384-well screening assay using nasal CF airway epithelial organoids, with the aim to repurpose FDA-approved drugs as modulators of non-CFTR-dependent epithelial fluid secretion. From a ~1400 FDA-approved drug library, we identified and validated 12 FDA-approved drugs that induced CFTR-independent fluid secretion. Among the hits were several cAMP-mediating drugs, including β2-adrenergic agonists. The hits displayed no effects on chloride conductance measured in the Ussing chamber, and fluid secretion was not affected by TMEM16A, as demonstrated by knockout (KO) experiments in primary nasal epithelial cells. Altogether, our results demonstrate the use of primary nasal airway cells for medium-scale drug screening, target validation with a highly efficient protocol for generating CRISPR-Cas9 KO cells and identification of compounds which induce fluid secretion in a CFTR- and TMEM16A-indepent manner.

Anoctamin 1 Inhibition Suppresses Cystogenesis by Enhancing Ciliogenesis and the Ciliary Dosage of Polycystins.

Autosomal dominant polycystic kidney disease (ADPKD) is a ciliopathy characterized by abnormal tubular epithelial proliferation and fluid secretion. Anoctamin 1 (ANO1) is a calcium-dependent chloride channel. However, how ANO1 contributes to ADPKD is largely unexplored. Kidney tissues from ADPKD patients, Pkd1RC/RC mice model, WT9-7 human PKD1+⁣/- cells, and 3D culture models in vitro were used. Localization of ANO1 and cilium length were investigated by confocal immunofluorescence. We found that ANO1 was consistently upregulated in human and mouse PKD kidneys. Intriguingly, ANO1 located in a vesicle-like pattern at the ciliary base but not on the ciliary surface. ANO1 deficiency enhanced ciliogenesis and the ciliary dosage of polycystin-2 in human PKD cells, and reduced cyst formation in 3D culture models. Moreover, inhibition of ANO1 abolished the activation of STAT3 and ERK pathways in PKD cells. Our data indicate ANO1 is a negative regulator for both cilia length and cilia trafficking of polycystin-2 and provide mechanistic insights regarding the therapeutic potential of ANO1 pathway in ADPKD treatment.

A PI3Kγ mimetic peptide triggers CFTR gating, bronchodilation, and reduced inflammation in obstructive airway diseases.

Cyclic adenosine 3',5'-monophosphate (cAMP)-elevating agents, such as β2-adrenergic receptor (β2-AR) agonists and phosphodiesterase (PDE) inhibitors, remain a mainstay in the treatment of obstructive respiratory diseases, conditions characterized by airway constriction, inflammation, and mucus hypersecretion. However, their clinical use is limited by unwanted side effects because of unrestricted cAMP elevation in the airways and in distant organs. Here, we identified the A-kinase anchoring protein phosphoinositide 3-kinase γ (PI3Kγ) as a critical regulator of a discrete cAMP signaling microdomain activated by β2-ARs in airway structural and inflammatory cells. Displacement of the PI3Kγ-anchored pool of protein kinase A (PKA) by an inhaled, cell-permeable, PI3Kγ mimetic peptide (PI3Kγ MP) inhibited a pool of subcortical PDE4B and PDE4D and safely increased cAMP in the lungs, leading to airway smooth muscle relaxation and reduced neutrophil infiltration in a murine model of asthma. In human bronchial epithelial cells, PI3Kγ MP induced unexpected cAMP and PKA elevations restricted to the vicinity of the cystic fibrosis transmembrane conductance regulator (CFTR), the ion channel controlling mucus hydration that is mutated in cystic fibrosis (CF). PI3Kγ MP promoted the phosphorylation of wild-type CFTR on serine-737, triggering channel gating, and rescued the function of F508del-CFTR, the most prevalent CF mutant, by enhancing the effects of existing CFTR modulators. These results unveil PI3Kγ as the regulator of a β2-AR/cAMP microdomain central to smooth muscle contraction, immune cell activation, and epithelial fluid secretion in the airways, suggesting the use of a PI3Kγ MP for compartment-restricted, therapeutic cAMP elevation in chronic obstructive respiratory diseases.

The Distribution and Role of the CFTR Protein in the Intracellular Compartments.

Cystic fibrosis is a hereditary disease that mainly affects secretory organs in humans. It is caused by mutations in the gene encoding CFTR with the most common phenylalanine deletion at position 508. CFTR is an anion channel mainly conducting Cl− across the apical membranes of many different epithelial cells, the impairment of which causes dysregulation of epithelial fluid secretion and thickening of the mucus. This, in turn, leads to the dysfunction of organs such as the lungs, pancreas, kidney and liver. The CFTR protein is mainly localized in the plasma membrane; however, there is a growing body of evidence that it is also present in the intracellular organelles such as the endosomes, lysosomes, phagosomes and mitochondria. Dysfunction of the CFTR protein affects not only the ion transport across the epithelial tissues, but also has an impact on the proper functioning of the intracellular compartments. The review aims to provide a summary of the present state of knowledge regarding CFTR localization and function in intracellular compartments, the physiological role of this localization and the consequences of protein dysfunction at cellular, epithelial and organ levels. An in-depth understanding of intracellular processes involved in CFTR impairment may reveal novel opportunities in pharmacological agents of cystic fibrosis.

NBD2 Is Required for the Rescue of Mutant F508del CFTR by a Thiazole-Based Molecule: A Class II Corrector for the Multi-Drug Therapy of Cystic Fibrosis.

Cystic fibrosis (CF) is caused by loss-of-function mutations in the CF transmembrane conductance regulator (CFTR) protein, an anion channel that regulates epithelial surface fluid secretion. The deletion of phenylalanine at position 508 (F508del) is the most common CFTR mutation. F508del CFTR is characterized by folding and trafficking defects, resulting in decreased functional expression of the protein on the plasma membrane. Several classes of small molecules, named correctors, have been developed to rescue defective F508del CFTR. Although individual correctors failed to improve the clinical status of CF patients carrying the F508del mutation, better results were obtained using correctors combinations. These results were obtained according to the premise that the administration of correctors having different sites of action should enhance F508del CFTR rescue. We investigated the putative site of action of an aminoarylthiazole 4-(3-chlorophenyl)-N-(3-(methylthio)phenyl)thiazol-2-amine, named FCG, with proven CFTR corrector activity, and its synergistic effect with the corrector VX809. We found that neither the total expression nor the maturation of WT CFTR transiently expressed in human embryonic kidney 293 cells was influenced by FCG, administrated alone or in combination with VX809. On the contrary, FCG was able to enhance F508del CFTR total expression, and its combination with VX809 provided a further effect, being able to increase not only the total expression but also the maturation of the mutant protein. Analyses on different CFTR domains and groups of domains, heterologously expressed in HEK293 cells, show that NBD2 is necessary for FCG corrector activity. Molecular modelling analyses suggest that FCG interacts with a putative region located into the NBD2, ascribing this molecule to class II correctors. Our study indicates that the continuous development and testing of combinations of correctors targeting different structural and functional defects of mutant CFTR is the best strategy to ensure a valuable therapeutic perspective to a larger cohort of CF patients.

A chalcone derivative retards renal cyst enlargement by inhibiting fluid secretion and cell proliferation in an in vitro model of polycystic kidney disease.

Renal bilateral fluid filled-cyst in polycystic kidney disease (PKD) is associated with abnormal epithelial cell proliferation and transepithelial fluid secretion which leads to end-stage renal disease (ESRD). A chalcone derivative, isoliquiritigenin (ISLQ), has been shown to have various pharmacological properties. Since several studies have shown that ISLQ could inhibit CFTR channel activity, it is interesting to see whether it can inhibit renal cyst enlargement. The present study was aimed to determine an inhibitory effect and the mechanism of chalcone derivatives on MDCK cyst progression and Pkd1 mutant cells. MDCK cyst growth and cyst formation experiments, MTT assay, Ussing chamber experiment, BrdU cell proliferation assay and western blot analysis were performed in this study. Among four compounds of chalcone derivatives tested, CHAL-005 (100µM) was found to inhibit MDCK cyst growth in a dose-dependent manner without cytotoxicity. It inhibited short-circuit current of chloride secretion as well as CFTR protein expression in MDCK cells. CHAL-005 significantly suppressed cell proliferation. In addition, CHAL-005 strongly reduced phosphorylation ERK1/2 and phosphorylation S6 kinase in MDCK and Pkd1 mutant cells. Interestingly, CHAL-005 activated phosphorylation of AMP kinase protein expression in MDCK and Pkd1 mutant cells. CHAL-005 slowed MDCK cyst progression by inhibiting CFTR expression and reducing ERK1/2 and mTOR/S6K signaling pathways as well as activating AMPK expression. Therefore, a chalcone derivative could represent as a promising drug candidate for polycystic kidney disease intervention.

Emerging roles of calcium-sensing receptor in the local regulation of intestinal transport of ions and calcium.

Whether the intestinal mucosal cells are capable of sensing calcium concentration in the lumen and pericellular interstitium remains enigmatic for decades. Most calcium-regulating organs, such as parathyroid gland, kidney, and bone, are capable of using calcium-sensing receptor (CaSR) to detect plasma calcium and trigger appropriate feedback responses to maintain calcium homeostasis. Although both CaSR transcripts and proteins are abundantly expressed in the crypt and villous enterocytes of the small intestine as well as the surface epithelial cells of the large intestine, the studies of CaSR functions have been limited to amino acid sensing and regulation of epithelial fluid secretion. Interestingly, several lines of recent evidence have indicated that the enterocytes use CaSR to monitor luminal and extracellular calcium levels, thereby reducing the activity of transient receptor potential channel, subfamily V, member 6, and inducing paracrine and endocrine feedback responses to restrict calcium absorption. Recent investigations in zebra fish and rodents have also suggested the role of fibroblast growth factor (FGF)-23 as an endocrine and/or paracrine factor participating in the negative control of intestinal calcium transport. In this review article, besides the CaSR-modulated ion transport, we elaborate the possible roles of CaSR and FGF-23 as well as their crosstalk as parts of a negative feedback loop for counterbalancing the seemingly unopposed calciotropic effect of 1,25-dihydroxyvitamin D3 on the intestinal calcium absorption.

IMPACT OF TRIKAFTA ON LUNG FUNCTION IN A CYSTIC FIBROSIS TRANSPLANT PATIENT

SESSION TITLE: Fellows Transplantation Posters SESSION TYPE: Fellow Case Report Posters PRESENTED ON: October 18-21, 2020 INTRODUCTION: Cystic fibrosis (CF) patients with at least one copy of F508del are eligible for Tezacaftor/Ivacaftor/Elexacaftor (Trikafta), a novel CFTR corrector/potentiator drug associated with increased lung function1 and likely improved sinus clearance. Trikata effects in lung transplant patients are unknown. We report a CF transplant patient, who developed reversible profound leukopenia, severely decreased lung function, and CT findings of peribronchial thickening and gas trapping after starting Trikafta for recurrent severe sinus disease. CASE PRESENTATION: A 30 year old CF female (homozygous F508del), underwent bilateral orthoptic lung transplant for end-stage lung disease and was treated with tacrolimus, azathioprine, prednisone valganciclovir, and sulfamethoxazole/trimethoprim for immunosuppression and infection prophylaxis. FEV1/FVC 3 months post-op were 3.36L/3.58L. Complaints of severe chronic sinusitis persisted and Trikafta was initiated with symptomatic benefit. 4 weeks later, she developed profound neutropenia, despite no antecedent changes in myelosuppressive drug therapy. Despite G-CSF, she was admitted with worsening respiratory symptoms and declining PFTs. She was afebrile with normal vital signs. Initial testing for bacterial and viral infections was negative. Azathioprine, Bactrim and Valcyte were held. Eravacycline was started empirically based on prior cultures. FEV1/ FVC declined to 2.62L/2.80L. Initial CT chest was essentially normal. Leucocyte counts slowly recovered with G-CSF. During this time, she began expectorating copious greenish ’sea-foam’ liquid. Repeat expiratory CT chest showed diffuse bronchial wall thickening and extensive air trapping. Bronchoscopy showed diffuse frothy secretions. Bronchoalveolar lavage had >80% return. Cell counts showed 100 nucleated cells/ml with benign differential. Following a negative cardiac and other etiologic workup, antibiotics were stopped and Trikata was discontinued as causation was suspected. Respiratory symptoms resolved and FEV1/FVC improved to supra-normal baseline over 2 weeks. DISCUSSION: Trikafta’s effect on transplanted lungs (where CFTR function is intrinsically normal) is unknown. Inflammatory cell-derived mediators are known to augment native CFTR function and increase airway epithelial cell fluid secretion in vitro2. We hypothesize that our patient’s symptoms and findings were due to exaggerated airway fluid secretion associated with repopulation of inflammatory cells. We propose that this led to airway edema, and bronchial wall thickening + gas trapping observed on imaging. Improvement in symptoms/PFTs after drug discontinuation support this. Etiology of profound leukopenia is less clear, though a previously undescribed direct drug-effect or altered GI absorption of other myelosuppressive medications are possible. CONCLUSIONS: Trikafta may have unexpected detrimental effects on a lung transplant physiology in CF patients Reference #1: Heijerman HGM, McKone EF, Downey DG, et al. Efficacy and safety of the elexacaftor plus tezacaftor plus ivacaftor combination regimen in people with cystic fibrosis homozygous for the F508del mutation: a double-blind, randomised, phase 3 trial. Lancet. 2019. Reference #2: Abdullah LH, Coakley R, Webster MJ, et al. Mucin Production and Hydration Responses to Mucopurulent Materials in Normal versus Cystic Fibrosis Airway Epithelia. Am J Respir Crit Care Med. 2018; 197(4):481–491. doi:10.1164/rccm.201706-1139OC DISCLOSURES: No relevant relationships by Raymond Coakley, source=Admin input No relevant relationships by Christina Doligalski, source=Web Response No relevant relationships by Kunal Jakharia, source=Web Response No relevant relationships by leonard lobo, source=Web Response

TMEM16A Potentiation: A Novel Therapeutic Approach for the Treatment of Cystic Fibrosis.

Rationale: Enhancing non–CFTR (cystic fibrosis transmembrane conductance regulator)-mediated anion secretion is an attractive therapeutic approach for the treatment of cystic fibrosis (CF) and other mucoobstructive diseases.Objectives: To determine the effects of TMEM16A potentiation on epithelial fluid secretion and mucociliary clearance.Methods: The effects of a novel low-molecular-weight TMEM16A potentiator (ETX001) were evaluated in human cell and animal models of airway epithelial function and mucus transport.Measurements and Main Results: Potentiating the activity of TMEM16A with ETX001 increased the Ca2+-activated Cl− channel activity and anion secretion in human bronchial epithelial (HBE) cells from patients with CF without impacting calcium signaling. ETX001 rapidly increased fluid secretion and airway surface liquid height in CF-HBE cells under both static conditions and conditions designed to mimic the shear stress associated with tidal breathing. In ovine models of mucus clearance (tracheal mucus velocity and mucociliary clearance), inhaled ETX001 was able to accelerate clearance both when CFTR function was reduced by administration of a pharmacological blocker and when CFTR was fully functional.Conclusions: Enhancing the activity of TMEM16A increases epithelial fluid secretion and enhances mucus clearance independent of CFTR function. TMEM16A potentiation is a novel approach for the treatment of patients with CF and non-CF mucoobstructive diseases.

TMEM16A: An Alternative Approach to Restoring Airway Anion Secretion in Cystic Fibrosis?

The concept that increasing airway hydration leads to improvements in mucus clearance and lung function in cystic fibrosis has been clinically validated with osmotic agents such as hypertonic saline and more convincingly with cystic fibrosis transmembrane conductance regulator (CFTR) repair therapies. Although rapidly becoming the standard of care in cystic fibrosis (CF), current CFTR modulators do not treat all patients nor do they restore the rate of decline in lung function to normal levels. As such, novel approaches are still required to ensure all with CF have effective therapies. Although CFTR plays a fundamental role in the regulation of fluid secretion across the airway mucosa, there are other ion channels and transporters that represent viable targets for future therapeutics. In this review article we will summarise the current progress with CFTR-independent approaches to restoring mucosal hydration, including epithelial sodium channel (ENaC) blockade and modulators of SLC26A9. A particular emphasis is given to modulation of the airway epithelial calcium-activated chloride channel (CaCC), TMEM16A, as there is controversy regarding whether it should be positively or negatively modulated. This is discussed in light of a recent report describing for the first time bona fide TMEM16A potentiators and their positive effects upon epithelial fluid secretion and mucus clearance.

Ca2+ Signaling in Exocrine Cells.

Calcium (Ca2+) and cyclic AMP (cAMP) signaling cross talk and synergize to stimulate the cardinal functions of exocrine cells, regulated exocytosis, and fluid and electrolyte secretion. This physiological process requires the organization of the two signaling pathways into complexes at defined cellular domains and close placement. Such domains are formed by membrane contact sites (MCS). This review discusses the basic properties of Ca2+ signaling in exocrine cells, the role of MCS in the organization of cell signaling and in cross talk and synergism between the Ca2+ and cAMP signaling pathways and, finally, the mechanism by which the Ca2+ and cAMP pathways synergize to stimulate epithelial fluid and electrolyte secretion.

Physiological and pathological impact of AQP1 knockout in mice.

Aquaporin 1 (AQP1) is a glycoprotein responsible for water passive transport quickly across biological membrane. Here, we reviewed the structural and functional impacts of AQP1 knockout (AQP1-KO) in animal or cell culture models. AQP1 gene deletion can cause a large number of abnormalities including the disturbance in epithelial fluid secretion, polyhydramnios, deficiency of urinary concentrating function, and impairment of pain perception. AQP1-KO mice also displayed aberrations of cardiovascular, gastrointestinal and hepatobiliary, and kidney functions as well as placenta and embryo development. Moreover, AQP1-KO perturbed tumor angiogenesis and led to reduced brain injury upon trauma. On the cellular level, AQP1-KO caused neuroinflammation, aberrant cell proliferation and migration, and macrophages infiltration. Mechanistic studies confirmed that AQP1 gene products regulate the secretory function and participated in balancing the osmotic water flux across the peritoneal membrane. The available data indicated that AQP1 might serve as a potential target for developing novel therapeutic approaches against diverse human diseases.

125Iodide as a Surrogate for 36Chloride in Tracing Transepithelial Intestinal Chloride Transport

Chloride (Cl−) transport by the large intestine is critically important, driving epithelial fluid secretion and absorption. Dysregulation of Cl− transport by the intestine results in diarrhea‐associated pathologies. Measurement of epithelial Cl− transport has long been achieved using the radioisotope 36Cl−. However, the price of 36Cl has sky‐rocketed, now costing over $100 per μCi compared to just $1.50 per μCi 15 years ago, making it prohibitively expensive and a barrier to future research. 125Iodide (125I−) has been used as an alternative to 36Cl− in some transport assays, including Cl− efflux and influx by cultured cells, but has never before been validated in parallel with 36Cl as an alternative for tracing transepithelial Cl− transport. The goal of this study was to examine whether 125I− can serve as a reliable surrogate for measuring Cl− transport by the intestinal epithelium. Under symmetrical short‐circuited conditions in the Ussing chamber, we simultaneously measured 36Cl− and 125I− fluxes across the cecum and distal colon from mice lacking the main Cl−/HCO3− exchanger, DRA (Slc26a3), relative to wild‐type controls. In addition, we used a cocktail of forskolin and 3‐isobutyl‐1‐methylxanthine (IBMX) to stimulate cAMP production and net Cl− secretion by these tissues. We found the unidirectional fluxes of 125I− correlated well with 36Cl− in all circumstances, even in the absence of DRA, with a significant R2 value of 0.86 across all fluxes, or higher (up to 0.93) when data was separated by genotype and intestinal segment. Unexpectedly, cAMP induced net Cl− secretion predominantly through a reduction in the absorptive, mucosal‐to‐serosal, Cl− flux rather than stimulating the secretory serosal‐to‐mucosal Cl− flux, which is noteworthy considering antidiarrheal drugs often target secretory processes. Also, rates of Cl− and I− absorption in DRA‐knockout mice were decreased by 79% and 50%, respectively, in the ceca, and 65% and 34% in the distal colon, suggesting this Cl− transporter may have a previously unrecognized role in I− uptake. In summary, we demonstrated 125I− is an adequate, cost‐effective alternative for tracing unidirectional Clfluxes across the intestinal epithelium. For example, the amount of 36Cl− used in this study cost $536 compared to a mere $4 worth of 125I−, thus making future research into epithelial Cltransport accessible. In addition, this study also provides insight into mechanisms of net Clsecretion and suggests that DRA may have a role in intestinal I− absorption.Support or Funding InformationThis study was supported by grants DK088892 and DK108755 to M. Hatch from the National Institutes of Health.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A truncation mutation in human SLC12A2 leads to abnormal goblet cell mucus secretion

The basolateral Na+‐K+‐2Cl− cotransporter (NKCC1) plays a critical role in maintaining the intracellular concentrations of Na+, K+, and Cl− ions, and cell volume. The cotransporter also plays a key role in mediating epithelial fluid secretion. In previous studies, we have described the first known human SLC12A2 (NKCC1) mutation (Cold Spring Harbor Mol. Case Stud. 2(6): a001289, 2016). The now 16‐year‐old patient carries a carboxyl‐terminal truncation mutation (NKCC1‐DFX) in the transporter that leads to complex clinical features including complete gastrointestinal and bladder failure, endocrine insufficiencies, small intestinal dysmotility, seizure‐like episodes, and multi‐organ failure. The 11 bp deletion leads to truncation of the cytosolic COOH‐terminal domain of the cotransporter, making it non‐functional. In polarized epithelial MDCK cells and a mouse model that recapitulates the patient mutation we demonstrated that expression of the truncated Na‐K‐2Cl cotransporter causes mistargeting of the mutant and wild‐type cotransporter to the apical membrane of Cl− secreting epithelia and affects proper lumen formation in MDCK cysts (Am. J. Physiol. Cell Physiol. 315: C258–C276, 2018). Recently due to chronic infections, the patient's colon was surgically resected and lately, the patient has been battling chronic yeast infections in the small intestine. To investigate whether NKCC1‐DFX affect the integrity and function of the gut epithelia, we used MDCK cells expressing the mutant transporter and a mouse model recapitulating the patient mutation. We show that expression of the NKCC1‐DFX mutant impairs tight junction integrity, as indicated by a decrease in trans‐epithelial electrical resistance and inability of MDCK‐I cells expressing NKCC1‐DFX to re‐establish barrier function after a calcium‐switch challenge. Compared to wild‐type mice, NKCC1‐DFX have increased intestinal transit time and displayed an increase in unhealthy mitochondria characterize by dense inclusion bodies. Relative to wild‐type mice, NKCC1‐DFX mice showed decreased expression of Claudin‐2, a tight junction protein involved in paracellular Na+ and water transport. NKCC1‐DFX expression causes defective goblet cells mucus granules exocytosis leading to secretion of intact granules in the lumen of the large intestine. In addition, NKCC1‐DFX colon submucosal glands secrete mucus that remained attached to the epithelium. Taken together, these data indicate that NKCC1‐DFX likely impairs gut barrier function by affecting epithelia tight junction integrity and mucus secretion.Support or Funding InformationSupported by NIH grant GM118944.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Importance of Aquaporin 1 in Pancreatitis and Its Relation to the CFTR Cl- Channel.

Aquaporins (AQPs) facilitate the transepithelial water flow involved in epithelial fluid secretion in numerous tissues; however, their function in the pancreas is less characterized. Acute pancreatitis (AP) is a serious disorder in which specific treatment is still not possible. Accumulating evidence indicate that decreased pancreatic ductal fluid secretion plays an essential role in AP; therefore, the aim of this study was to investigate the physiological and pathophysiological role of AQPs in the pancreas. Expression and localization of AQPs were investigated by real-time PCR and immunocytochemistry, whereas osmotic transmembrane water permeability was estimated by the dye dilution technique, in Capan-1 cells. The presence of AQP1 and CFTR in the mice and human pancreas were investigated by immunohistochemistry. Pancreatic ductal HCO3- and fluid secretion were studied on pancreatic ducts isolated from wild-type (WT) and AQP1 knock out (KO) mice using microfluorometry and videomicroscopy, respectively. In vivo pancreatic fluid secretion was estimated by magnetic resonance imaging. AP was induced by intraperitoneal injection of cerulein and disease severity was assessed by measuring biochemical and histological parameters. In the mice, the presence of AQP1 was detected throughout the whole plasma membrane of the ductal cells and its expression highly depends on the presence of CFTR Cl- channel. In contrast, the expression of AQP1 is mainly localized to the apical membrane of ductal cells in the human pancreas. Bile acid treatment dose- and time-dependently decreased mRNA and protein expression of AQP1 and reduced expression of this channel was also demonstrated in patients suffering from acute and chronic pancreatitis. HCO3- and fluid secretion significantly decreased in AQP1 KO versus WT mice and the absence of AQP1 also worsened the severity of pancreatitis. Our results suggest that AQP1 plays an essential role in pancreatic ductal fluid and HCO3- secretion and decreased expression of the channel alters fluid secretion which probably contribute to increased susceptibility of the pancreas to inflammation.

Ductal Mucus Obstruction and Reduced Fluid Secretion Are Early Defects in Chronic Pancreatitis.

Objective: Defective mucus production in the pancreas may be an important factor in the initiation and progression of chronic pancreatitis (CP), therefore we aimed to (i) investigate the qualitative and quantitative changes of mucus both in human CP and in an experimental pancreatitis model and (ii) to correlate the mucus phenotype with epithelial ion transport function.Design: Utilizing human tissue samples and a murine model of cerulein induced CP we measured pancreatic ductal mucus content by morphometric analysis and the relative expression of different mucins in health and disease. Pancreatic fluid secretion in CP model was measured in vivo by magnetic resonance cholangiopancreatography (MRCP) and in vitro on cultured pancreatic ducts. Time-changes of ductal secretory function were correlated to those of the mucin production.Results: We demonstrate increased mucus content in the small pancreatic ducts in CP. Secretory mucins MUC6 and MUC5B were upregulated in human, Muc6 in mouse CP. In vivo and in vitro fluid secretion was decreased in cerulein-induced CP. Analysis of time-course changes showed that impaired ductal ion transport is paralleled by increased Muc6 expression.Conclusion: Mucus accumulation in the small ducts is a combined effect of mucus hypersecretion and epithelial fluid secretion defect, which may lead to ductal obstruction. These results suggest that imbalance of mucus homeostasis may have an important role in the early-phase development of CP, which may have novel diagnostic and therapeutic implications.