Transepithelial Cl Research Articles

SARS-CoV-2 envelope protein impairs airway epithelial barrier function and exacerbates airway inflammation via increased intracellular Cl− concentration

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection disrupts the epithelial barrier and triggers airway inflammation. The envelope (E) protein, a core virulence structural component of coronaviruses, may play a role in this process. Pathogens could interfere with transepithelial Cl− transport via impairment of the cystic fibrosis transmembrane conductance regulator (CFTR), which modulates nuclear factor κB (NF-κB) signaling. However, the pathological effects of SARS-CoV-2 E protein on airway epithelial barrier function, Cl− transport and the robust inflammatory response remain to be elucidated. Here, we have demonstrated that E protein down-regulated the expression of tight junctional proteins, leading to the disruption of the airway epithelial barrier. In addition, E protein triggered the activation of Toll-like receptor (TLR) 2/4 and downstream c-Jun N-terminal kinase (JNK) signaling, resulting in an increased intracellular Cl− concentration ([Cl−]i) via up-regulating phosphodiesterase 4D (PDE4D) expression in airway epithelial cells. This elevated [Cl−]i contributed to the heightened airway inflammation through promoting the phosphorylation of serum/glucocorticoid regulated kinase 1 (SGK1). Moreover, blockade of SGK1 or PDE4 alleviated the robust inflammatory response induced by E protein. Overall, these findings provide novel insights into the pathogenic role of SARS-CoV-2 E protein in airway epithelial damage and the ongoing airway inflammation during SARS-CoV-2 infection.

Hypoxia-induced cystic fibrosis transmembrane conductance regulator dysfunction is a universal mechanism underlying reduced mucociliary transport in sinusitis.

Hypoxia due to sinus obstruction is a major pathogenic mechanism leading to sinusitis. The objective of the current study is to define the electrophysiologic characteristics of hypoxia in vitro and in vivo. Cystic fibrosis bronchoepithelial cells expressing wild-type cystic fibrosis transmembrane conductance regulator (CFTR) and human sinonasal epithelial cells were exposed to 1% or atmospheric O2 for 24h. Time-dependent production of cytoplasmic free radicals was measured. Cells were subjected to Ussing chamber and patch clamp technique where CFTR currents were recorded in whole-cell and cell-attached mode for single channel studies. Indices of mucociliary transport (MCT) were measured using micro-optical coherence tomography. In a rabbit hypoxic maxillary sinus model, tissue oxygenation, relative mRNA expression of HIF-1α, pH, sinus potential difference (SPD), and MCT were determined. Ussing chamber (p<0.05), whole-cell (p<0.001), and single channel patch-clamp (p<0.0001) showed significant inhibition of Cl- currents in hypoxic cells. Cytoplasmic free radicals showed time-dependent elevation peaking at 4h (p<0.0001). Airway surface liquid (p<0.0001), periciliary liquid (p<0.001), and MCT (p<0.01) were diminished. Co-incubation with the free radical scavenger glutathione negated the impact of hypoxia on single channel currents and MCT markers. In sinusitis rabbits, mucosa exhibited low tissue oxygenation (p<0.0001), increased HIF1α mRNA (p<0.05), reduced pH (p<0.01), and decreased MCT (p<0.001). SPD measurements demonstrated markedly diminished transepithelial Cl- transport (p<0.0001). Hypoxia induces severe CFTR dysfunction via free radical production causing reduced MCT in vitro and in vivo. Improved oxygenation is critical to reducing the impact of persistent mucociliary dysfunction.

Red ginseng aqueous extract improves mucociliary transport dysfunction and histopathology in CF rat airways

BackgroundWe previously discovered that Korean red ginseng aqueous extract (RGAE) potentiates the TMEM16A channel, improved mucociliary transport (MCT) parameters in CF nasal epithelia in vitro, and thus could serve as a therapeutic strategy to rescue the MCT defect in cystic fibrosis (CF) airways. The hypothesis of this study is that RGAE can improve epithelial Cl− secretion, MCT, and histopathology in an in-vivo CF rat model. MethodsSeventeen 4-month old CFTR–/– rats were randomly assigned to receive daily oral control (saline, n = 9) or RGAE (Ginsenosides 0.4mg/kg/daily, n = 8) for 4 weeks. Outcomes included nasal Cl− secretion measured with the nasal potential difference (NPD), functional microanatomy of the trachea using micro-optical coherence tomography, histopathology, and immunohistochemical staining for TMEM16a. ResultsRGAE-treated CF rats had greater mean NPD polarization with UTP (control = -5.48 +/- 2.87 mV, RGAE = -9.49 +/- 2.99 mV, p < 0.05), indicating, at least in part, potentiation of UTP-mediated Cl− secretion through TMEM16A. All measured tracheal MCT parameters (airway surface liquid, periciliary liquid, ciliary beat frequency, MCT) were significantly increased in RGAE-treated CF rats with MCT exhibiting a 3-fold increase (control, 0.45+/-0.31 vs. RGAE, 1.45+/-0.66 mm/min, p < 0.01). Maxillary mucosa histopathology was markedly improved in RGAE-treated cohort (reduced intracellular mucus, goblet cells with no distention, and shorter epithelial height). TMEM16A expression was similar between groups. ConclusionRGAE improves TMEM16A-mediated transepithelial Cl− secretion, functional microanatomy, and histopathology in CF rats. Therapeutic strategies utilizing TMEM16A potentiators to treat CF airway disease are appropriate and provide a new avenue for mutation-independent therapies.

Pentacyclic triterpenes modulate farnesoid X receptor expression in colonic epithelial cells: Implications for colonic secretory function

The nuclear bile acid receptor, farnesoid X receptor (FXR), is an important regulator of intestinal and metabolic function. Previous studies suggest that pentacyclic triterpenes (PCTs), a class of plant-derived bioactive phytochemical, can modulate FXR activity and may therefore offer therapeutic benefits. Here, we investigated the effects of a prototypical PCT, hederagenin (HG), on FXR expression, activity, and antisecretory actions in colonic epithelial cells. T84 cells and murine enteroid-derived monolayers were employed to assess HG effects on FXR expression and activity in colonic epithelia. We measured mRNA levels by qRT-PCR and protein by ELISA and immunoblotting. Transepithelial Cl- secretion was assessed as changes in short circuit current in Ussing chambers. We determined HG treatment (5-10μM) alone did not induce FXR activation but significantly increased expression of the receptor, both in T84cells and murine enteroid-derived monolayers. This effect was accompanied by enhanced FXR activity, as assessed by FGF-15/19 induction in response to the synthetic, GW4064, or natural FXR agonist, chenodeoxycholic acid. Effects of HG on FXR expression and activity were mimicked by another PCT, oleanolic acid. Furthermore, we found FXR-induced downregulation of cystic fibrosis transmembrane conductance regulator Cl- channels and inhibition of transepithelial Cl- secretion were enhanced in HG-treated cells. These data demonstrate that dietary PCTs have the capacity to modulate FXR expression, activity, and antisecretory actions in colonic epithelial cells. Based on these data, we propose that plants rich in PCTs, or extracts thereof, have excellent potential for development as a new class of "FXR-targeted nutraceuticals".

WNK Inhibition Increases Surface Liquid pH and Host Defense in Cystic Fibrosis Airway Epithelia.

In cystic fibrosis (CF), reduced HCO3- secretion acidifies the airway surface liquid (ASL), and the acidic pH disrupts host defenses. Thus, understanding the control of ASL pH (pHASL) in CF may help identify novel targets and facilitate therapeutic development. In diverse epithelia, the WNK (with-no-lysine [K]) kinases coordinate HCO3- and Cl- transport, but their functions in airway epithelia are poorly understood. Here, we tested the hypothesis that WNK kinases regulate CF pHASL. In primary cultures of differentiated human airway epithelia, inhibiting WNK kinases acutely increased both CF and non-CF pHASL. This response was HCO3- dependent and involved downstream SPAK/OSR1 (Ste20/SPS1-related proline-alanine-rich protein kinase/oxidative stress responsive 1 kinase). Importantly, WNK inhibition enhanced key host defenses otherwise impaired in CF. Human airway epithelia expressed two WNK isoforms in secretory cells and ionocytes, and knockdown of either WNK1 or WNK2 increased CF pHASL. WNK inhibition decreased Cl- secretion and the response to bumetanide, an NKCC1 (sodium-potassium-chloride cotransporter 1) inhibitor. Surprisingly, bumetanide alone or basolateral Cl- substitution also alkalinized CF pHASL. These data suggest that WNK kinases influence the balance between transepithelial Cl- versus HCO3- secretion. Moreover, reducing basolateral Cl- entry may increase HCO3- secretion and raise pHASL, thereby improving CF host defenses.

Structural basis for inhibition of the Cation-chloride cotransporter NKCC1 by the diuretic drug bumetanide

Cation-chloride cotransporters (CCCs) NKCC1 and NKCC2 catalyze electroneutral symport of 1 Na+, 1 K+, and 2 Cl− across cell membranes. NKCC1 mediates trans-epithelial Cl− secretion and regulates excitability of some neurons and NKCC2 is critical to renal salt reabsorption. Both transporters are inhibited by the so-called loop diuretics including bumetanide, and these drugs are a mainstay for treating edema and hypertension. Here, our single-particle electron cryo-microscopy structures supported by functional studies reveal an outward-facing conformation of NKCC1, showing bumetanide wedged into a pocket in the extracellular ion translocation pathway. Based on these and the previously published inward-facing structures, we define the translocation pathway and the conformational changes necessary for ion translocation. We also identify an NKCC1 dimer with separated transmembrane domains and extensive transmembrane and C-terminal domain interactions. We further define an N-terminal phosphoregulatory domain that interacts with the C-terminal domain, suggesting a mechanism whereby (de)phosphorylation regulates NKCC1 by tuning the strength of this domain association.

Molecular mechanisms underlying guanylin-induced transcellular Cl− secretion into the intestinal lumen of seawater-acclimated eels

Guanylin (GN) stimulates Cl− secretion into the intestinal lumen of seawater-acclimated eels, but the molecular mechanisms of transepithelial Cl− transport are still unknown. In Ussing chamber experiments, we confirmed that mucosal application of eel GN reversed intestinal serosa-negative potential difference, indicating Cl− secretion. Serosal application of DNDS or mucosal application of DPC inhibited the GN effect, but serosal application of bumetanide had no effect. Removal of HCO3– from the serosal fluid also inhibited the GN effect. In intestinal sac experiments, mucosal GN stimulated luminal secretion of both Cl− and Na+, which was blocked by serosal DNDS. These results suggest that Cl− is taken up at the serosal side by DNDS-sensitive anion exchanger (AE) coupled with Na+–HCO3– cotransporter (NBC) but not by Na+-K+-2Cl− cotransporter 1 (NKCC1), and Cl− is secreted by unknown DPC-sensitive Cl− channel (ClC) at the mucosal side. The transcriptomic analysis combined with qPCR showed low expression of NKCC1 gene and no upregulation of the gene after seawater transfer, while high expression of ClC2 gene and upregulation after seawater transfer. In addition, SO42− transporters (apical Slc26a3/6 and basolateral Slc26a1) are also candidates for transcellular Cl− secretion in exchange of luminal SO42. Na+ secretion could occur through a paracellular route, as Na+-leaky claudin15 was highly expressed and upregulated after seawater transfer. High local Na+ concentration in the lateral interspace produced by Na+/K+-ATPase (NKA) coupled with K+ channels (Kir5.1b) seems to facilitate the paracellular transport. In situ hybridization confirmed the expression of the candidate genes in the epithelial enterocytes. Together with our previous results, we suggest that GN stimulates basolateral NBCela/AE2 and apical ClC2 to increase transcellular Cl− secretion in seawater eel intestine, which differs from the involvement of apical CFTR and basolateral NKCC1 as suggested in mammals and other teleosts.

Correlating genotype with phenotype using CFTR-mediated whole-cell Cl- currents in human nasal epithelial cells.

Dysfunction of the epithelial anion channel cystic fibrosis transmembrane conductance regulator (CFTR) causes a wide spectrum of disease, including cystic fibrosis (CF) and CFTR-related diseases (CFTR-RDs). Here, we investigate genotype-phenotype-CFTR function relationships using human nasal epithelial (hNE) cells from a small cohort of non-CF subjects and individuals with CF and CFTR-RDs and genotypes associated with either residual or minimal CFTR function using electrophysiological techniques. Collected hNE cells were either studied directly with the whole-cell patch-clamp technique or grown as primary cultures at an air-liquid interface after conditional reprogramming. The properties of cAMP-activated whole-cell Cl- currents in freshly isolated hNE cells identified them as CFTR-mediated. Their magnitude varied between hNE cells from individuals within the same genotype and decreased in the rank order: non-CF > CFTR residual function > CFTR minimal function. CFTR-mediated whole-cell Cl- currents in hNE cells isolated from fully differentiated primary cultures were identical to those in freshly isolated hNE cells in both magnitude and behaviour, demonstrating that conditional reprogramming culture is without effect on CFTR expression and function. For the cohort of subjects studied, CFTR-mediated whole-cell Cl- currents in hNE cells correlated well with CFTR-mediated transepithelial Cl- currents measured in vitro with the Ussing chamber technique, but not with those determined in vivo with the nasal potential difference assay. Nevertheless, they did correlate with the sweat Cl- concentration of study subjects. Thus, this study highlights the complexity of genotype-phenotype-CFTR function relationships, but emphasises the value of conditionally reprogrammed hNE cells in CFTR research and therapeutic testing. KEY POINTS: The genetic disease cystic fibrosis is caused by pathogenic variants in the cystic fibrosis transmembrane conductance regulator (CFTR), an ion channel, which controls anion flow across epithelia lining ducts and tubes in the body. This study investigated CFTR function in nasal epithelial cells from people with cystic fibrosis and CFTR variants with a range of disease severity. CFTR function varied widely in nasal epithelial cells depending on the identity of CFTR variants, but was unaffected by conditional reprogramming culture, a cell culture technique used to grow large numbers of patient-derived cells. Assessment of CFTR function in vitro in nasal epithelial cells and epithelia, and in vivo in the nasal epithelium and sweat gland highlights the complexity of genotype-phenotype-CFTR function relationships.

Effects of Bicarbonate Stress on Serum Ions and Gill Transporters in Alkali and Freshwater Forms of Amur Ide (Leuciscus waleckii).

The Amur ide (Leuciscus waleckii) is a fish in the Cyprinidae family. Compared with other Amur ide living in freshwater ecosystems, the Amur ide population in Lake Dali Nor of China is famous for its high tolerance to the alkaline conditions of 54 mM (pH 9.6). Yet, surprisingly, the ionoregulatory mechanism responsible for this remarkable alkaline adaptation remains unclear. Therefore, this study sought to investigate how bicarbonate affects the acid-base balancing and ionoregulatory responses of this animal. Here, using a comparative approach, the alkali form of Amur ide and its ancestral freshwater form living in other freshwater basins were each exposed to 50 mM (pH 9.59 ± 0.09), a level close to the alkalinity of Lake Dali Nor, and their physiological (AE1) adjustment of ions and acid-base regulation were investigated. This study highlighted differences in blood pH and serum ions (e.g., Na+, K+, Cl−, and Ca2+), Na+/K+ ATPase (NKA) activity and its mRNA level, and mRNA expression of gill transporters (Na+/H+ exchanger member 2 and/or 3, Na+/ cotransporter (NBC1), Cl−/ exchanger, Na+/Cl− cotransporter (NCC), Na+/K+/2Cl− (NKCC1), SLC26A5, and SLC26A6) for alkalinity adaptation between the two forms of Amur ide differing in alkalinity tolerance. Specifically, close relationships among the serum Na+ and mRNA levels of NCC, NKCC1, and NHE, and also NKA and NBC1, in addition to serum Cl− and bicarbonate transporters (e.g., SLC26A5 and SLC26A6), characterized the alkali form of Amur ide. We propose that this ecotype can ensure its transepithelial Cl− and Na+ uptake/base secretions are highly functional, by its basolateral NKA with NBC1 and apical ionic transporters, and especially NCC incorporated with other transporters (e.g., SLC26). This suggests an evolved strong ability to maintain an ion osmotic and acid-base balance for more effectively facilitating its adaptability to the high alkaline environment. This study provides new insights into the physiological responses of the alkaline form of the Amur ide fish for adapting to extreme alkaline conditions. This information could be used as a reference to cultivating alkaline-tolerant fish species in abandoned alkaline waters.

LPS decreases CFTR open probability and mucociliary transport through generation of reactive oxygen species

Lipopolysaccharide (LPS) serves as the interface between gram-negative bacteria (GNB) and the innate immune response in respiratory epithelial cells (REC). Herein, we describe a novel biological role of LPS that permits GNB to persist in the respiratory tract through inducing CFTR and mucociliary dysfunction. LPS reduced cystic fibrosis transmembrane conductance regulater (CFTR)-mediated short-circuit current in mammalian REC in Ussing chambers and nearly abrogated CFTR single channel activity (defined as forskolin-activated Cl- currents) in patch clamp studies, effects of which were blocked with toll-like receptor (TLR)-4 inhibitor. Unitary conductance and single-channel amplitude of CFTR were unaffected, but open probability and number of active channels were markedly decreased. LPS increased cytoplasmic and mitochondrial reactive oxygen species resulting in CFTR carbonylation. All effects of exposure were eliminated when reduced glutathione was added in the medium along with LPS. Functional microanatomy parameters, including mucociliary transport, in human sinonasal epithelial cells in vitro were also decreased, but restored with co-incubation with glutathione or TLR-4 inhibitor. In vivo measurements, following application of LPS in the nasal cavities showed significant decreases in transepithelial Cl- secretion as measured by nasal potential difference (NPD) – an effect that was nullified with glutathione and TLR-4 inhibitor. These data provide definitive evidence that LPS-generated reactive intermediates downregulate CFTR function in vitro and in vivo which results in cystic fibrosis-type disease. Findings have implications for therapeutic approaches intent on stimulating Cl- secretion and/or reducing oxidative stress to decrease the sequelae of GNB airway colonization and infection.

Hormonal regulation and functional role of the "renal" tubules in the disease vector, Aedes aegypti.

The Aedes aegypti mosquito is a vector responsible for transmitting various arboviruses including dengue and yellow fever. Their ability to regulate the ionic and water composition of their hemolymph is a major physiological phenomenon, allowing the mosquito to adapt to a range of ecological niches. Hematophagus insects, including the female A. aegypti, face the challenge of excess salt and water intake after a blood meal. Post-prandial diuresis is under rigorous control by neuroendocrine factors, acting on the Malpighian "renal" tubules (MTs), to regulate primary urine production. The MTs are made up of two cell types; mitochondria-rich principal cells, which facilitate active transport of Na+ and K+ cations across the membrane, and thin stellate cells, which allows for transepithelial Cl- secretion. The active driving force responsible for ion transport is the apical V-type H+ ATPase, which creates a proton gradient allowing for Na+ and/or K+ cation exchange through cation/H+ antiporters. Additionally, the basolaterally localized Na+-K+-2Cl- cotransporter (NKCC) is responsible for the transport of these ions from the hemolymph into the principal cells. Numerous studies have examined hormonal regulation of the mosquito MTs and identified several diuretics including serotonin (5HT), a calcitonin-related diuretic hormone 31 (DH31), a corticotropin-related factor like diuretic peptide (DH44), a kinin-related diuretic peptide, as well as anti-diuretic factors including CAPA peptides, all of which are known to regulate fluid and ion transport by the MTs. This review therefore focuses on the control of ionic homeostasis in A. aegypti mosquitoes, emphasizing the importance of the MTs, the channels and transporters involved in maintaining hydromineral balance, and the neuroendocrine regulation of both diuresis and anti-diuresis.

Amphotericin B induces epithelial voltage responses in people with cystic fibrosis

BackgroundApproximately 10% of people with cystic fibrosis (CF) have mutations that result in little to no CFTR production and thus cannot benefit from CFTR modulators. We previously found that Amphotericin B (AmB), a small molecule that forms anion channels, restored HCO3− secretion and increased host defenses in primary cultures of CF airway epithelia. Further, AmB increased ASL pH in CFTR-null pigs, suggesting an alternative CFTR-independent approach to achieve gain-of-function. However, it remains unclear whether this approach can be effective in people. MethodsTo determine whether AmB can impact physiology in people with CF, we first tested whether Fungizone, a clinically approved AmB formulation, could cause electrophysiological effects consistent with anion secretion in primary cultures of CF airway epithelia. We then evaluated the capacity of AmB to change nasal potential difference (NPD), a key clinical biomarker, in people with CF not on CFTR modulators. ResultsAmB increased transepithelial Cl− current and hyperpolarized calculated transepithelial voltage in primary cultures of CF airway epithelia from people with two nonsense mutations. In eight people with CF not on CFTR modulators, intranasal Fungizone treatment caused a statistically significant change in NPD. This change was similar in direction and magnitude to the effect of ivacaftor in people with a G551D mutation. ConclusionsOur results provide the first evidence that AmB can impact a clinical biomarker in people with CF. These results encourage additional clinical studies in people with CF to determine whether small molecule anion channels can provide benefit.

Slc26a3 (DRA) in the Gut: Expression, Function, Regulation, Role in Infectious Diarrhea and Inflammatory Bowel Disease.

The transport of transepithelial Cl- and HCO3- is crucial for the function of the intestinal epithelium and maintains the acid-based homeostasis. Slc26a3 (DRA), as a key chloride-bicarbonate exchanger protein in the intestinal epithelial luminal membrane, participates in the electroneutral NaCl absorption of intestine, together with Na+/H+ exchangers. Increasing recent evidence supports the essential role of decreased DRA function or expression in infectious diarrhea and inflammatory bowel disease (IBD). In this review, we give an overview of the current knowledge of Slc26a3, including its cloning and expression, function, roles in infectious diarrhea and IBD, and mechanisms of actions. A better understanding of the physiological and pathophysiological relevance of Slc26a3 in infectious diarrhea and IBD may reveal novel targets for future therapy. Understanding the physiological function, regulatory interactions, and the potential mechanisms of Slc26a3 in the pathophysiology of infectious diarrhea and IBD will define novel therapeutic approaches in future.

Pendrin stimulates a chloride absorption pathway to increase CFTR-mediated chloride secretion from Cystic Fibrosis airway epithelia.

Cystic Fibrosis (CF), an inherited multi‐system disease, is caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) that disrupt its ability to secrete anions from epithelia. Recovery of functional anion secretion may be curative for CF, so different components of the ion transport machinery have become attractive therapeutic targets. Several members of the SLC26 ion transporter family have been linked to epithelial ion flux, some through putative functional interactions with CFTR. Using a small‐scale qPCR screen, we confirmed that the anion transporter SLC26A4 (pendrin) is downregulated in CF. Upregulation of pendrin using interleukins IL‐4 or IL‐13 increased Cl− secretion through CFTR in human bronchial epithelial cell (HBEC) derived epithelia differentiated in vitro and measured in the Ussing Chamber. Inhibition or knockdown of pendrin prevented this increased secretion. Increased CFTR activity was not driven by increases in CFTR protein or upstream regulatory pathway components. When basolateral Cl− absorption through NKCC1 was inhibited, a pendrin‐dependent Cl− absorption pathway allowing CFTR to continue secreting Cl− from the epithelium was revealed. Although CFTR is often considered the bottleneck in the transepithelial Cl− transport pathway, these studies indicate that basolateral Cl− permeability becomes limiting as CFTR activity increases. Therefore, an increase of epithelial Cl− absorption via pendrin might have additional therapeutic benefit in combination with CFTR modulators.

Differential Chloride Secretory Capacity in Transepithelial Ion Transport Properties in Chronic Rhinosinusitis.

Epithelial ion transport regulates hydration of airway mucosal surfaces, and thus promotes effective mucociliary clearance (MCC). Decreased transepithelial Cl- transport may contribute to epithelial dysfunction by abrogating MCC and increasing mucus viscosity in chronic rhinosinusitis (CRS). The objective of the current study is to evaluate Cl- channel transport properties from cultures of human sinonasal epithelia. Human sinonasal epithelia (HSNE) from patients undergoing sinus surgery were cultured at an air-liquid interface to confluence and full differentiation. The epithelial monolayers were mounted in Ussing Chambers to investigate pharmacological manipulation of ion transport. Epithelial Na+ channel (via Amiloride), CFTR (via forskolin), and Ca2+-activated Cl- channel (CaCC, via UTP) transport were investigated among three different patient groups: Control, CRS and CRS with polyposis. CFTR mRNA levels were evaluated with quantitative RT-PCR. HSNE cultures from 18 patients (Control = 9, CRS = 6, CRS with polyposis = 3) were evaluated in 142 experiments. Summary data from the 18 patients demonstrated that stimulated CFTR-mediated anion transport (Δ ISC) was significantly lower with CRS (7.58+/-2.24 µA/cm2) compared to control (25.86+/-3.44 µA/cm2) and CRS with polyposis (20.16+/-4.0 µA/cm2) (p = 0.004). No statistically significant difference was found for CaCC anion transport between groups (p = 0.39). Significantly decreased mRNA (relative expression) was noted in CRS cultures (CRS = 40.83+/-1.76 vs. control = 116.2+/-24.27, p = 0.03). A substantial decrease in the Cl- secretory capacity of HSNE monolayers was demonstrated in CRS subjects. Data suggest that CFTR may contribute more to abnormal ion transport in CRS than CaCC.

A Novel AAV-mediated Gene Delivery System Corrects CFTR Function in Pigs.

Cystic fibrosis is an autosomal-recessive disease that is caused by a mutant CFTR (cystic fibrosis transmembrane conductance regulator) gene and is characterized by chronic bacterial lung infections and inflammation. Complementation with functional CFTR normalizes anion transport across the airway surface. Adeno-associated virus (AAV) is a useful vector for gene therapy because of its low immunogenicity and ability to persist for months to years. However, because its episomal expression may decrease after cell division, readministration of the AAV vector may be required. To overcome this, we designed an integrating AAV-based CFTR-expressing vector, termed piggyBac (PB)/AAV, carrying CFTR flanked by the terminal repeats of the piggyBac transposon. With codelivery of the piggyBac transposase, PB/AAV can integrate into the host genome. Because of the packaging constraints of AAV, careful consideration was required to ensure that the vector would package and express its CFTR cDNA cargo. In this short-term study, PB/AAV-CFTR was aerosolized to the airways of CF pigs in the absence of the transposase. Two weeks later, transepithelial Cl- current was restored in freshly excised tracheal and bronchial tissue. Additionally, we observed an increase in tracheal airway surface liquid pH and bacterial killing in comparison with untreated CF pigs. Airway surface liquid from primary airway cells cultured from treated CF pigs exhibited increased pH correlating with decreased viscosity. Together, these results show that complementing CFTR in CF pigs with PB/AAV rescues the anion transport defect in a large-animal CF model. Delivery of this integrating viral vector system to airway progenitor cells could lead to persistent, life-long expression in vivo.

Korean Red Ginseng aqueous extract improves markers of mucociliary clearance by stimulating chloride secretion

BackgroundAbnormal chloride (Cl−) transport has a detrimental impact on mucociliary clearance in both cystic fibrosis (CF) and non-CF chronic rhinosinusitis. Ginseng is a medicinal plant noted to have anti-inflammatory and antimicrobial properties. The present study aims to assess the capability of red ginseng aqueous extract (RGAE) to promote transepithelial Cl− secretion in nasal epithelium. MethodsPrimary murine nasal septal epithelial (MNSE) [wild-type (WT) and transgenic CFTR−/−], fisher-rat-thyroid (FRT) cells expressing human WT CFTR, and TMEM16A-expressing human embryonic kidney cultures were utilized for the present experiments. Ciliary beat frequency (CBF) and airway surface liquid (ASL) depth measurements were performed using micro–optical coherence tomography (μOCT). Mechanisms underlying transepithelial Cl− transport were determined using pharmacologic manipulation in Ussing chambers and whole-cell patch clamp analysis. ResultsRGAE (at 30μg/mL of ginsenosides) significantly increased Cl− transport [measured as change in short-circuit current (ΔISC = μA/cm2)] when compared with control in WT and CFTR−/− MNSE (WT vs control = 49.8±2.6 vs 0.1+/−0.2, CFTR−/- = 33.5±1.5 vs 0.2±0.3, p < 0.0001). In FRT cells, the CFTR-mediated ΔISC attributed to RGAE was small (6.8 ± 2.5 vs control, 0.03 ± 0.01, p < 0.05). In patch clamp, TMEM16A-mediated currents were markedly improved with co-administration of RGAE and uridine 5-triphosphate (8406.3 +/− 807.7 pA) over uridine 5-triphosphate (3524.1 +/− 292.4 pA) or RGAE alone (465.2 +/− 90.7 pA) (p < 0.0001). ASL and CBF were significantly greater with RGAE (6.2+/−0.3 μm vs control, 3.9+/−0.09 μm; 10.4+/−0.3 Hz vs control, 7.3 ± 0.2 Hz; p < 0.0001) in MNSE. ConclusionRGAE augments ASL depth and CBF by stimulating Cl− secretion through CaCC, which suggests therapeutic potential in both CF and non-CF chronic rhinosinusitis.

Prostaglandin E2‐Stimulated Cl− Secretion in Airway Epithelium Involves Protein Kinase A and Calcium Crosstalk

BackgroundMucociliary clearance is an integral defense mechanism against pulmonary infections and is impaired in Cystic Fibrosis (CF) airways. Prostaglandin E2 (PGE2) is abundant in infected airways and stimulates transepithelial Cl− secretion and mucociliary clearance, both of which are impaired in CF. Previously, we found that PGE2‐stimulated Cl− secretion was cystic fibrosis transmembrane conductance regulator (CFTR)‐dependent in bronchial epithelial cells, but in submucosal gland cells utilized both CFTR and Ca2+‐activated Cl− channels (CaCC).AimTo determine the signaling mechanism(s) whereby PGE2 stimulates airway Cl− secretion in order to identify ways to circumvent the mucociliary defect in CF.MethodsHuman bronchial epithelial cell cultures (CFBE41o‐ with wildtype CFTR) were used for all experiments. Clsecretion was measured by short‐circuit current (Isc) with cell monolayers mounted in Ussing chambers and exposed to a serosal to mucosal Cl− gradient. These conditions minimize contribution from basolateral K+ channels and Isc reflects primarily Cl− transport. EP receptor expression was determined by qPCR.ResultsIn bronchial epithelial cell cultures, qPCR showed that EP4 receptor expression was 148% greater than EP3 receptor expression. CAY10598 (1 μM), a selective EP4 agonist, stimulated Cl− secretion to a similar magnitude as PGE2 (Cay: 85.5 +/− 8.6 μA/cm2 (n=4) vs. PGE2: 81.6 +/− 10.2 μA/cm2 (n=17); mean +/− SE). In contrast, sulprostone, an EP3 agonist, was less effective at 1 or 10 μM (5.6+/− 2.0 μA/cm2 (n=6) and 14.5+/− 7.3 μA/cm2 (n=2), respectively). Pre‐treatment with H‐89 (10 μM), a protein kinase A (PKA) inhibitor, decreased PGE2‐stimulated Cl− secretion by 74% (n=9). Similarly, pretreatment with BAPTA‐AM (100 μM), a Ca2+ chelator, inhibited PGE2‐stimulated Cl− secretion by 71% (n=7). Simultaneous pre‐treatment with H‐89 and BAPTA‐AM was additive and completely abolished PGE2‐stimulated Cl− secretion (94% inhibition, n=3). Additionally, wortmannin (1μM), a phosphoinositide 3‐kinase (PI3K) inhibitor, decreased PGE2‐stimulated Clsecretion by 56% (n=4).Summary and ConclusionsPGE2‐stimulated Cl− secretion in human bronchial epithelial cells predominantly occurs via EP4 receptors. While utilizing classic EP4 receptor signaling (PKA and PI3K), there is also substantial crosstalk with intracellular Ca2+. Interestingly, the latter does not activate CaCC, but rather CFTR. Ongoing experiments are aimed at further delineating the intracellular signaling mechanisms involved in PGE2‐stimulated Cl− secretion in bronchial epithelial cells, as well as comparing these mechanisms to PGE2‐stimulated Cl− secretion in submucosal gland cells. Through this we hope to identify potential ways to augment PGE2‐stimulated mucociliary clearance in CF, resulting in improved clearance of pulmonary infections.Support or Funding InformationCystic Fibrosis Foundation (Z.M.S), Elizabeth Nash Foundation (BI)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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.

Probing Structure, Topology and Dynamics of KCNE3 in Proteoliposomes using Site-Direct Spin Labeling and EPR Spectroscopy

KCNE3 (E3) is a β-subunit of voltage gated potassium ion channels (Kv) that modulates the function of Kv by interacting with its a subunits including KCNQ1 and KCNQ4 (known as Q1 and Q4). Compared to other Kv channels, E3-Q1 interactions have been well studied in intestinal and tracheal epithelial cells where the complex maintains ion homeostasis. E3 interaction with Q1 allows Ky to act as a voltage independent and constitutively active channel that facilitates transepithelial Cl- ion secretion. Mutations in KCNE3 are responsible for diseases related to salt and fluid disorders including cystic fibrosis. Hence, it is important to elucidate KCNE3 structural and dynamic information with functional studies. Previously, the structure of E3 has been studied in a bicelle environment. However, in physiological conditions, lipids are organized into bilayers instead of single sheets of molecules. This research has been focused on protein structure, dynamic and topology of KCNE3 membrane protein in POPC/POPG vesicle usingSite-Directed Spin Labeling (SDSL) and Electron Paramagnetic Resonance Spectroscopy (EPR)techniques. EPR coupled with SDSL provides structural and dynamic information about proteins. In this study we have successfully introduced S-(2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl) methyl methanesulfonothioate spin label (MTSL) on several site-specific position of E3 by site-directed mutagenesis to obtain EPR spectra. CW-EPR (Continuous Wave Electron Paramagnetic Resonance)line shape analysis has been performed to investigate the dynamic of the protein in a lipid bilayer, while EPR power saturation has been used to study the topology of KCNE3 in a vesicle. Our data are consistent with the model of KCNE3 in a bicelle and revealed more promising structure and dynamic information for the protein in a native membrane environment.