Airway Surface Liquid Dehydration Research Articles

Evaluation of chronic cigarette smoke exposure in human bronchial epithelial cultures.

Cigarette smoke (CS) exposure induces both cytotoxicity and inflammation, and often causes COPD, a growing cause of morbidity and mortality. CS also inhibits the CFTR Cl- channel, leading to airway surface liquid dehydration, which is predicated to impair clearance of inhaled pathogens and toxicants. Numerous in vitro studies have been performed that utilize acute (≤24 h) CS exposures. However, CS exposure is typically chronic. We evaluated the feasibility of using British-American Tobacco (BAT)-designed CS exposure chambers for chronically exposing human bronchial epithelial cultures (HBECs) to CS. HBECs are polarized and contain mucosal and serosal sides. In vivo, inhaled CS interacts with mucosal membranes, and BAT chambers are designed to direct CS to HBEC mucosal surfaces while keeping CS away from serosal surfaces via a perfusion system. We found that serosal perfusion was absolutely required to maintain HBEC viability over time following chronic CS exposure. Indeed, with this system, we found that CS increased inflammation and mucin levels, while decreasing CFTR function. Without this serosal perfusion, CS was extremely toxic within 24 h. We therefore propose that 5- and 10-day CS exposures with serosal perfusion are suitable for measuring chronic CS exposure and can be used for monitoring new and emerging tobacco products.

Allicin Facilitates Airway Surface Liquid Hydration by Activation of CFTR.

Airway epithelium plays critical roles in regulating airway surface liquid (ASL), the alteration of which causes mucus stasis symptoms. Allicin is a compound released from garlic and harbors the capacity of lung-protection. However, the potential regulatory effects of allicin on airway epithelium remain elusive. This study aimed to investigate the effects of allicin on ion transport across airway epithelium and evaluate its potential as an expectorant. Application of allicin induced Cl− secretion across airway epithelium in a concentration-dependent manner. Blockade of cystic fibrosis transmembrane conductance regulator (CFTR) or inhibition of adenylate cyclase-cAMP signaling pathway attenuated allicin-induced Cl− secretion in airway epithelial cells. The in vivo study showed that inhaled allicin significantly increased the ASL secretion in mice. These results suggest that allicin induces Cl− and fluid secretion across airway epithelium via activation of CFTR, which might provide therapeutic strategies for the treatment of chronic pulmonary diseases associated with ASL dehydration.

Hyperinflammation and airway surface liquid dehydration in cystic fibrosis: purinergic system as therapeutic target.

The exacerbate inflammatory response contributes to the progressive loss of lung function in cystic fibrosis (CF), a genetic disease that affects the osmotic balance of mucus and mucociliary clearance, resulting in a microenvironment that favors infection and inflammation. The purinergic system, an extracellular signaling pathway characterized by nucleotides, enzymes and receptors, may have a protective role in the disease, through its action in airway surface liquid (ASL) and anti-inflammatory response. To make up this review, studies covering topics of CF, inflammation, ASL and purinergic system were selected from the main medical databases, such as Pubmed and ScienceDirect. We propose several ways to modulate the purinergic system as a potential therapy for CF, like inhibition of P2X7, activation of P2Y2, A2A and A2B receptors and blocking of adenosine deaminase. Among them, we postulate that the most suitable strategy is to block the action of adenosine deaminase, which culminates in the increase of Ado levels that presents anti-inflammatory actions and improves mucociliary clearance. Furthermore, it is possible to maintain the physiological levels of ATP to control the hydration of ASL. These therapies could correct the main mechanisms that contribute to the progression of CF.

Dysfunctional Inflammation in Cystic Fibrosis Airways: From Mechanisms to Novel Therapeutic Approaches.

Cystic fibrosis (CF) is an inherited disorder caused by mutations in the gene encoding for the cystic fibrosis transmembrane conductance regulator (CFTR) protein, an ATP-gated chloride channel expressed on the apical surface of airway epithelial cells. CFTR absence/dysfunction results in defective ion transport and subsequent airway surface liquid dehydration that severely compromise the airway microenvironment. Noxious agents and pathogens are entrapped inside the abnormally thick mucus layer and establish a highly inflammatory environment, ultimately leading to lung damage. Since chronic airway inflammation plays a crucial role in CF pathophysiology, several studies have investigated the mechanisms responsible for the altered inflammatory/immune response that, in turn, exacerbates the epithelial dysfunction and infection susceptibility in CF patients. In this review, we address the evidence for a critical role of dysfunctional inflammation in lung damage in CF and discuss current therapeutic approaches targeting this condition, as well as potential new treatments that have been developed recently. Traditional therapeutic strategies have shown several limitations and limited clinical benefits. Therefore, many efforts have been made to develop alternative treatments and novel therapeutic approaches, and recent findings have identified new molecules as potential anti-inflammatory agents that may exert beneficial effects in CF patients. Furthermore, the potential anti-inflammatory properties of CFTR modulators, a class of drugs that directly target the molecular defect of CF, also will be critically reviewed. Finally, we also will discuss the possible impact of SARS-CoV-2 infection on CF patients, with a major focus on the consequences that the viral infection could have on the persistent inflammation in these patients.

Engineered mutant α-ENaC subunit mRNA delivered by lipid nanoparticles reduces amiloride currents in cystic fibrosis-based cell and mice models.

Cystic fibrosis (CF) results from mutations in the chloride-conducting CF transmembrane conductance regulator (CFTR) gene. Airway dehydration and impaired mucociliary clearance in CF is proposed to result in tonic epithelial sodium channel (ENaC) activity, which drives amiloride-sensitive electrogenic sodium absorption. Decreasing sodium absorption by inhibiting ENaC can reverse airway surface liquid dehydration. Here, we inhibit endogenous heterotrimeric ENaC channels by introducing inactivating mutant ENaC α mRNA (αmutENaC). Lipid nanoparticles carrying αmutENaC were transfected in CF-based airway cells in vitro and in vivo. We observed a significant decrease in macroscopic as well as amiloride-sensitive ENaC currents and an increase in airway surface liquid height in CF airway cells. Similarly, intranasal transfection of αmutENaC mRNA decreased amiloride-sensitive nasal potential difference in CFTRKO mice. These data suggest that mRNA-based ENaC inhibition is a powerful strategy for reducing mucus dehydration and has therapeutic potential for treating CF in all patients, independent of genotype.

High-mobility group box-1 increases epithelial sodium channel activity and inflammation via the receptor for advanced glycation end products.

Cystic fibrosis (CF) lung disease persists and remains life-limiting for many patients. Elevated high-mobility group box-1 protein (HMGB-1) levels and epithelial sodium channel hyperactivity (ENaC) are hallmark features of the CF lung. The objective of this study was to better understand the pathogenic role of HMGB-1 signaling and ENaC in CF airway cells. We hypothesize that HMGB-1 links airway inflammation [via signaling to the receptor for advanced glycation end products (RAGE)] and airway surface liquid dehydration (via upregulation of ENaC) in the CF lung. We calculated equivalent short-current (Isc) and single-channel ENaC open probability (Po) in normal and CF human small airway epithelial cells (SAEC) in the presence and absence of human HMGB-1 peptide (0.5 μg/mL). In normal SAECs, HMGB-1 increased amiloride-sensitive Isc and elevated ENaC Po from 0.15 ± 0.03 to 0.28 ± 0.04 (P < 0.01). In CF SAECs, ENaC Po increased from 0.45 ± 0.06 to 0.73 ± 0.04 (P < 0.01). Pretreatment with 1 μM FPS-ZM1 (a RAGE inhibitor) attenuated all HMGB-1 effects on ENaC current in normal and CF SAECs. Confocal analysis of SAECs indicates that nuclear size and HMBG-1 localization can be impacted by ENaC dysfunction. Masson's trichrome labeling of mouse lung showed that intraperitoneally injected HMGB-1 significantly increased pulmonary fibrosis. Bronchoalveolar lavage fluid from HMGB-1-treated mice showed significant increases in IL-1β, IL-10, IL-6, IL-27, IL-17A, IFN-β, and granulocyte-macrophage colony-stimulating factor compared with vehicle-injected mice (P < 0.05). These studies put forth a new model in which HMGB-1 signaling to RAGE plays an important role in perpetuating ENaC dysfunction and inflammation in the CF lung.

Immunopathology of Airway Surface Liquid Dehydration Disease.

The primary purpose of pulmonary ventilation is to supply oxygen (O2) for sustained aerobic respiration in multicellular organisms. However, a plethora of abiotic insults and airborne pathogens present in the environment are occasionally introduced into the airspaces during inhalation, which could be detrimental to the structural integrity and functioning of the respiratory system. Multiple layers of host defense act in concert to eliminate unwanted constituents from the airspaces. In particular, the mucociliary escalator provides an effective mechanism for the continuous removal of inhaled insults including pathogens. Defects in the functioning of the mucociliary escalator compromise the mucociliary clearance (MCC) of inhaled pathogens, which favors microbial lung infection. Defective MCC is often associated with airway mucoobstruction, increased occurrence of respiratory infections, and progressive decrease in lung function in mucoobstructive lung diseases including cystic fibrosis (CF). In this disease, a mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene results in dehydration of the airway surface liquid (ASL) layer. Several mice models of Cftr mutation have been developed; however, none of these models recapitulate human CF-like mucoobstructive lung disease. As an alternative, the Scnn1b transgenic (Scnn1b-Tg+) mouse model overexpressing a transgene encoding sodium channel nonvoltage-gated 1, beta subunit (Scnn1b) in airway club cells is available. The Scnn1b-Tg+ mouse model exhibits airway surface liquid (ASL) dehydration, impaired MCC, increased mucus production, and early spontaneous pulmonary bacterial infections. High morbidity and mortality among mucoobstructive disease patients, high economic and health burden, and lack of scientific understanding of the progression of mucoobstruction warrants in-depth investigation of the cause of mucoobstruction in mucoobstructive disease models. In this review, we will summarize published literature on the Scnn1b-Tg+ mouse and analyze various unanswered questions on the initiation and progression of mucobstruction and bacterial infections.

High mobility group box‐1 increases epithelial sodium channel activity and inflammation in small airway epithelial cells

In Cystic Fibrosis (CF) patients, inherited defects in anion transport leads to hyperactive epithelial Na+ channel (ENaC) activity. ENaC dysfunction in CF lung is known to cause airway surface liquid dehydration. Thick and dry mucus, in turn, leads to an inflammatory lung phenotype that often occurs without detection of infection in CF patients. Many investigative groups have reported that airway tissue and fluid from murine models exhibiting the CF lung phenotype (Scnn1b‐Tr mice), as well as CF patients, have increased levels of high mobility group box‐1 (HMGB‐1). HMGB‐1 is a cytokine mediator of inflammation and is a biomarker for severity of CF lung disorder. Herein, we hypothesize that HMGB‐1 plays an important role in the pathogenic regulation of ENaC, inflammation, and fibrosis in sterile airways. To begin testing this hypothesis, we measured transepithelial sodium current (Isc) and single channel ENaC open probability (Po) in human small airway epithelial cells (SAEC) in the presence or absence of 1 mg/mL human HMGB‐1 peptide. Electrophysiological measurements show that HMGB‐1 significantly increased amiloride‐sensitive ENaC Isc from −9.7±1.4 μA/cm2 to −18.4±2.9 μA/cm2 in confluent monolayers of SAEC (n=12; p=0.01) within 30 min. Subsequent amiloride treatment of the same SEAC monolayers decreased Isc 71%. Single channel measurements were conducted in primary SAEC obtained from 4 separate donor lungs in the cell‐attached configuration; HMGB‐1 increased ENaC Po from 0.18±0.02 to 0.35±0.04 (n=17; p&lt;0.01) in single channel analysis. Mean absolute total cell counts were significantly higher in bronchoalveolar lavage fluid (BALF) from mice intraperitoneally (IP) injected with HMBG‐1 (10 mg in 1 mL saline/kg) vs vehicle injected mice. Total immune cell counts were 27.4×103± 8.8×103 (in HMGB‐1) vs. 5.2×103±1.3X103 (in vehicle) injected mice (n=8 counts from 2 independent studies; p&lt;0.05). Masson's Trichrome labeling of IP injected mice show that HMGB‐1 significantly increases pulmonary fibrosis (see Fig 1). Flow cytometric analysis of HMGB‐1 and vehicle injected mice showed significant increase in IL‐1β, IL‐10, IL‐6, IL‐27, IL‐17A, IFN‐β, and GM‐CSF (n=3; p&lt;0.05; see Fig 2). Together, our data suggests that HMGB‐1 plays an important role in the CF lung phenotype; additional studies are underway to strengthen these observations. Impact: Successful completion of our work can lead to novel therapies needed to counteract ENaC dysfunction and inflammation in the CF lung.Support or Funding InformationThis work was supported by NIH 1 R01 HL137033‐01A1 awarded to MH.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

SPLUNC1 degradation by the cystic fibrosis mucosal environment drives airway surface liquid dehydration.

The multi-organ disease cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane regulator gene (CFTR) that lead to diminished transepithelial anion transport. CF lungs are characterised by airway surface liquid (ASL) dehydration, chronic infection/inflammation and neutrophilia. Dysfunctional CFTR may upregulate the epithelial Na+ channel (ENaC), further exacerbating dehydration. We previously demonstrated that short palate lung and nasal epithelial clone 1 (SPLUNC1) negatively regulates ENaC in normal airway epithelia.Here, we used pulmonary tissue samples, sputum and human bronchial epithelial cells (HBECs) to determine whether SPLUNC1 could regulate ENaC in a CF-like environment.We found reduced endogenous SPLUNC1 in CF secretions, and rapid degradation of recombinant SPLUNC1 (rSPLUNC1) by CF secretions. Normal sputum, containing SPLUNC1 and SPLUNC1-derived peptides, inhibited ENaC in both normal and CF HBECs. Conversely, CF sputum activated ENaC, and rSPLUNC1 could not reverse this phenomenon. Additionally, we observed upregulation of ENaC protein levels in human CF bronchi. Unlike SPLUNC1, the novel SPLUNC1-derived peptide SPX-101 resisted protease degradation, bound apically to HBECs, inhibited ENaC and prevented ASL dehydration following extended pre-incubation with CF sputum.Our data indicate that CF mucosal secretions drive ASL hyperabsorption and that protease-resistant peptides, e.g. SPX-101, can reverse this effect to rehydrate CF ASL.

Cigarette smoke modifies and inactivates SPLUNC1, leading to airway dehydration.

Chronic obstructive pulmonary disease (COPD) is a growing cause of morbidity and mortality worldwide. Cigarette smoke (CS) exposure, a major cause of COPD, dysregulates airway epithelial ion transport and diminishes airway surface liquid (ASL) volume. Short palate lung and nasal epithelial clone 1 (SPLUNC1) is secreted into the airway lumen where it maintains airway hydration via interactions with the epithelial Na+ channel (ENaC). Although ASL hydration is dysregulated in CS-exposed/COPD airways, effects of CS on SPLUNC1 have not been elucidated. We hypothesized that CS alters SPLUNC1 activity, therefore contributing to ASL dehydration. CS exposure caused irreversible SPLUNC1 aggregation and prevented SPLUNC1 from internalizing ENaC and maintaining ASL hydration. Proteomic analysis revealed αβ-unsaturated aldehyde modifications to SPLUNC1's cysteine residues. Removal of these cysteines prevented SPLUNC1 from regulating ENaC/ASL volume. In contrast, SPX-101, a peptide mimetic of natural SPLUNC1, that internalizes ENaC, but does not contain cysteines was unaffected by CS. SPX-101 increased ASL hydration and attenuated ENaC activity in airway cultures after CS exposure and prolonged survival in a chronic airway disease model. These findings suggest that the CS-induced defects in SPLUNC1 can be circumvented, thus making SPX-101 a novel candidate for the treatment of mucus dehydration in COPD. -Moore, P. J., Reidel, B., Ghosh, A., Sesma, J., Kesimer, M., Tarran, R. Cigarette smoke modifies and inactivates SPLUNC1, leading to airway dehydration.

SPLUNC1 degradation by the cystic fibrosis mucosal environment drives airway surface liquid dehydration

SPLUNC1 degradation by the cystic fibrosis mucosal environment drives airway surface liquid dehydration

Assessment of acquired mucociliary clearance defects using micro‐optical coherence tomography

Dehydration of airway surface liquid (ASL) disrupts normal mucociliary clearance (MCC) in sinonasal epithelium, which may lead to chronic rhinosinusitis (CRS). Abnormal chloride (Cl- ) transport is one such mechanism that contributes to this disorder and can be acquired secondary to environmental perturbations, such as hypoxia at the tissue surface. The objective of this study was to assess the technological feasibility of the novel micro-optical coherence tomography (μOCT) imaging technique for investigating acquired MCC defects in cultured human sinonasal epithelial (HSNE) cells. Primary HSNE cell cultures were subjected to a 1% oxygen environment for 12 hours to induce acquired cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction. Ion transport characteristics were assessed with pharmacologic manipulation in Ussing chambers. ASL, periciliary fluid (PCL), and ciliary beat frequency (CBF) were evaluated using μOCT. Amiloride-sensitive transport (ΔISC ) was greater in cultures exposed to hypoxia (hypoxia: -13.2 ± 0.6μA/cm2 ; control: -6.5 ± 0.1 μA/cm2 ; p < 0.01), whereas CFTR-mediated anion transport was significantly diminished (hypoxia: 28.6 ± 0.3 μA/cm2 ; control: 36.2 ± 1.6μA/cm2 ; p < 0.01), consistent with acquired CFTR dysfunction and sodium hyperabsorption. Hypoxia diminished all markers of airway surface function microanatomy as observed with μOCT, including ASL (hypoxia: 5.0 ± 0.4 μm; control: 9.0 ± 0.9 μm; p < 0.01) and PCL depth (hypoxia: 2.5 ± 0.1 μm; control: 4.8 ± 0.3 μm; p < 0.01), and CBF (hypoxia: 8.7 ± 0.3 Hz; control: 10.2 ± 0.3 Hz; p < 0.01). Hypoxia-induced defects in epithelial anion transport in HSNE led to predictable effects on markers of MCC measured with novel μOCT imaging. This imaging method represents a technological leap forward and is feasible for assessing acquired defects impacting the airway surface.

Glandular Proteome Identifies Antiprotease Cystatin C as a Critical Modulator of Airway Hydration and Clearance.

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel lead to viscous secretions from submucosal glands that cannot be properly hydrated and cleared by beating cilia in cystic fibrosis (CF) airways. The mechanisms by which CFTR, and the predominant epithelial sodium channel (ENaC), control the hydration and clearance of glandular secretions remain unclear. We used a proteomics approach to characterize the proteins contained in CF and non-CF submucosal gland fluid droplets and found that differentially regulated proteases (cathepsin S and H) and their antiprotease (cystatin C) influenced the equilibration of fluid on the airway surface and tracheal mucociliary clearance (MCC). Contrary to prevailing models of airway hydration and clearance, cystatin C, or raising the airway surface liquid (ASL) pH, inhibited cathepsin-dependent ENaC-mediated fluid absorption and raised the height of ASL, and yet decreased MCC velocity. Importantly, coupling of both CFTR and ENaC activities were required for effective MCC and for effective ASL height equilibration after volume challenge. Cystatin C-inhibitable cathepsins controlled initial phases of ENaC-mediated fluid absorption, whereas CFTR activity was required to prevent ASL dehydration. Interestingly, CF airway epithelia absorbed fluid more slowly owing to reduced cysteine protease activity in the ASL but became abnormally dehydrated with time. Our findings demonstrate that, after volume challenge, pH-dependent protease-mediated coupling of CFTR and ENaC activities are required for rapid fluid equilibration at the airway surface and for effective MCC. These findings provide new insights into how glandular fluid secretions may be equilibrated at the airway surface and how this process may be impaired in CF.

Epithelial Sodium and Chloride Channels and Asthma.

Objective:To focus on the asthmatic pathogenesis and clinical manifestations related to epithelial sodium channel (ENaC)/chlorine ion channel.Data Sources:The data analyzed in this review were the English articles from 1980 to 2015 from journal databases, primarily PubMed and Google Scholar. The terms used in the literature search were: (1) ENaCs; cystic fibrosis (CF) transmembrane conductance regulator (CFTR); asthma/asthmatic, (2) ENaC/sodium salt; CF; asthma/asthmatic, (3) CFTR/chlorine ion channels; asthma/asthmatic, (4) ENaC/sodium channel/scnn1a/scnn1b/scnn1g/scnn1d/amiloride-sensitive/amiloride-inhibtable sodium channels/sodium salt; asthma/asthmatic, lung/pulmonary/respiratory/tracheal/alveolar, and (5) CFTR; CF; asthma/asthmatic (ti).Study Selection:These studies included randomized controlled trials or studies covering asthma pathogenesis and clinical manifestations related to ENaC/chlorine ion channels within the last 25 years (from 1990 to 2015). The data involving chronic obstructive pulmonary disease and CF obtained from individual studies were also reviewed by the authors.Results:Airway surface liquid dehydration can cause airway inflammation and obstruction. ENaC and CFTR are closely related to the airway mucociliary clearance. Ion transporters may play a critical role in pathogenesis of asthmatic exacerbations.Conclusions:Ion channels have been the center of many studies aiming to understand asthmatic pathophysiological mechanisms or to identify therapeutic targets for better control of the disease.

Model demonstrates functional purpose of the nasal cycle.

BackgroundDespite the occurrence of the nasal cycle being well documented, the functional purpose of this phenomenon is not well understood. This investigation seeks to better understand the physiological objective of the nasal cycle in terms of airway health through the use of a computational nasal air-conditioning model.MethodA new state-variable heat and water mass transfer model is developed to predict airway surface liquid (ASL) hydration status within each nasal airway. Nasal geometry, based on in-vivo magnetic resonance imaging (MRI) data is used to apportion inter-nasal air flow.ResultsThe results demonstrate that the airway conducting the majority of the airflow also experiences a degree of ASL dehydration, as a consequence of undertaking the bulk of the heat and water mass transfer duties. In contrast, the reduced air conditioning demand within the other airway allows its ASL layer to remain sufficiently hydrated so as to support continuous mucociliary clearance.ConclusionsIt is quantitatively demonstrated in this work how the nasal cycle enables the upper airway to accommodate the contrasting roles of air conditioning and the removal of entrapped contaminants through fluctuation in airflow partitioning between each airway.

Model of mucociliary clearance in cystic fibrosis lungs.

Mucus clearance is a primary innate defense mechanism in the human airways. Cystic fibrosis (CF) is a genetic disease caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein. CF is characterized by dehydration of airway surface liquid and impaired mucociliary clearance. As a result, microorganisms are not efficiently removed from the airways, and patients experience chronic pulmonary infections and inflammation. We propose a new physiologically based mathematical model of muco-ciliary transport consisting of the two major components of the mucociliary clearance system: (i) periciliary liquid layer (PCL) and (ii) mucus layer. We study mucus clearance under normal conditions and in CF patients. Restoring impaired clearance of airway secretions in one of the major goals of therapy in patients with CF. We consider the action of the aerosolized and inhaled medication dornase alfa, which reduces the viscosity of cystic fibrosis mucus, by selectively cleaving the long DNA strands it contains. The results of the model simulations stress the potential relevance of the location of the drug deposition in the central or peripheral airways. Mucus clearance was increased in case the drug was primarily deposited peripherally, i.e. in the small airways.

Linking increased airway hydration, ciliary beating, and mucociliary clearance through ENaC inhibition.

Airway dehydration causes mucus stasis and bacterial overgrowth in cystic fibrosis and chronic bronchitis (CB). Rehydration by hypertonic saline is efficacious but suffers from a short duration of action. We tested whether epithelial sodium channel (ENaC) inhibition would rehydrate normal and dehydrated airways to increase mucociliary clearance (MCC) over a significant time frame. For this, we used a tool compound (Compound A), which displays nanomolar ENaC affinity and retention in the airway surface liquid (ASL). Using normal human bronchial epithelial cultures (HBECs) grown at an air-liquid interface, we evaluated in vitro potency and efficacy using short-circuit current (I(sc)) and ASL height measurements where it inhibited I(sc) and increased ASL height by ∼ 50% (0.052 μM at 6 h), respectively. The in vivo efficacy was investigated in a modified guinea pig tracheal potential difference model, where we observed an effective dose (ED50) of 5 μg/kg (i.t.), and by MCC measures in rats and sheep, where we demonstrated max clearance rates at 100 μg/kg (i.t.) and 75 μg/kg (i.t.), respectively. Acute cigarette smoke-induced ASL height depletion in HBECs was used to mimic the situation in patients with CB, and pretreatment prevented both cigarette smoke-induced ASL dehydration and lessened the decrease in ciliary beat frequency. Furthermore, when added after cigarette smoke exposure, Compound A increased the rate of ASL rehydration. In conclusion, Compound A demonstrated significant effects and a link between increased airway hydration, ciliary function, and MCC. These data support the hypothesis that ENaC inhibition may be efficacious in the restoration of mucus hydration and transport in patients with CB.

Cigarette Smoke-induced Ca2+ Release Leads to Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Dysfunction

Chronic obstructive pulmonary disease affects 64 million people and is currently the fourth leading cause of death worldwide. Chronic obstructive pulmonary disease includes both emphysema and chronic bronchitis, and in the case of chronic bronchitis represents an inflammatory response of the airways that is associated with mucus hypersecretion and obstruction of small airways. Recently, it has emerged that exposure to cigarette smoke (CS) leads to an inhibition of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel, causing airway surface liquid dehydration, which may play a role in the development of chronic bronchitis. CS rapidly clears CFTR from the plasma membrane and causes it to be deposited into aggresome-like compartments. However, little is known about the mechanism(s) responsible for the internalization of CFTR following CS exposure. Our studies revealed that CS triggered a rise in cytoplasmic Ca(2+) that may have emanated from lysosomes. Furthermore, chelation of cytoplasmic Ca(2+), but not inhibition of protein kinases/phosphatases, prevented CS-induced CFTR internalization. The macrolide antibiotic bafilomycin A1 inhibited CS-induced Ca(2+) release and prevented CFTR clearance from the plasma membrane, further linking cytoplasmic Ca(2+) and CFTR internalization. We hypothesize that CS-induced Ca(2+) release prevents normal sorting/degradation of CFTR and causes internalized CFTR to reroute to aggresomes. Our data provide mechanistic insight into the potentially deleterious effects of CS on airway epithelia and outline a hitherto unrecognized signaling event triggered by CS that may affect the long term transition of the lung into a hyper-inflammatory/dehydrated environment.

Hypertonic Saline and Acute Wheezing in Preschool Children

Most acute wheezing episodes in preschool children are associated with rhinovirus. Rhinovirus decreases extracellular adenosine triphosphate levels, leading to airway surface liquid dehydration. This, along with submucosal edema, mucus plaques, and inflammation, causes failure of mucus clearance. These preschool children do not respond well to available treatments, even oral steroids. This calls for pro-mucus clearance and prohydration treatments such as hypertonic saline in wheezing preschool children. Randomized, controlled, double-blind study. Forty-one children (mean age 31.9 ± 17.4 months, range 1-6 years) presented with wheezing to the emergency department were randomized after 1 albuterol inhalation to receive either 4 mL of hypertonic saline 5% (HS) (n = 16) or 4 mL of normal saline (NS) (n = 25), both with 0.5 mL albuterol, twice every 20 minutes in the emergency department and 4 times a day thereafter if hospitalized. The primary outcome measured was length of stay (LOS) and the secondary outcomes were admission rate (AR) and clinical severity score. The LOS was significantly shorter in the HS than in the NS group: median 2 days (range 0-6) versus 3 days (range 0-5) days (P = .027). The AR was significantly lower in the HS than the NS group: 62.2% versus 92%. Clinical severity score improved significantly in both groups but did not reach significance between them. Using HS inhalations significantly shortens LOS and lowers AR in preschool children presenting with an acute wheezing episode to the emergency department.

Hypertonic Saline and Acute Wheezing in Preschool Children

Background: Most acute wheezing episodes in preschool children are associated with viral respiratory tract infections, most commonly rhinovirus. Rhinovirus, like RSV, decreases extra-cellular ATP, leading to airway surface liquid dehydration. This, along with sub-mucosal edema, mucus plaques and inflammation cause failure of mucus clearance (MC). Such preschool children do not respond well to available treatments, including oral steroids. This calls for pro-MC and pro-hydration treatment like hypertonic saline. Methods: Randomized, controlled, double-blind study. Forty one children (mean age 31.9±17.4 months, range 1-6 years) presented with wheezing to the emergency department (ED) were randomized after one albuterol inhalation to receive either 4 ml of Hypertonic Saline 5% (HS), (n=16) or 4 ml of Normal saline (NS), (n=25) both with 0.5 ml albuterol, twice every 20 minutes in the ED and four times a day thereafter if hospitalized. Primary outcome measured was length of stay (LOS) and secondary outcomes were admission rate (AR) and clinical severity score (CS). Results: LOS was significantly shorter in the HS than in the NS group: median (range) 2 (0-6) days, versus 3 (0-5) days (P=0.027). AR was significantly lower in the HS than the NS group: 62.2% versus 92%. CS improved significantly in both groups but did not reach significance between them. View this table: Hospitalization rate Conclusions: Using HS inhalations significantly shortens LOS and lowers AR in preschool children presenting with acute wheezing episode to the ED.