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Relationship: 2440

Title

A descriptive phrase which clearly defines the two KEs being considered and the sequential relationship between them (i.e., which is upstream, and which is downstream). More help

CFTR Function, Decreased leads to ASL Height, Decreased

Upstream event

The causing Key Event (KE) in a Key Event Relationship (KER). More help

CFTR Function, Decreased

Downstream event

The responding Key Event (KE) in a Key Event Relationship (KER). More help

ASL Height, Decreased

Key Event Relationship Overview

The utility of AOPs for regulatory application is defined, to a large extent, by the confidence and precision with which they facilitate extrapolation of data measured at low levels of biological organisation to predicted outcomes at higher levels of organisation and the extent to which they can link biological effect measurements to their specific causes.Within the AOP framework, the predictive relationships that facilitate extrapolation are represented by the KERs. Consequently, the overall WoE for an AOP is a reflection in part, of the level of confidence in the underlying series of KERs it encompasses. Therefore, describing the KERs in an AOP involves assembling and organising the types of information and evidence that defines the scientific basis for inferring the probable change in, or state of, a downstream KE from the known or measured state of an upstream KE. More help

AOPs Referencing Relationship

AOP Name	Adjacency	Weight of Evidence	Quantitative Understanding	Point of Contact	Author Status	OECD Status
Oxidative stress Leading to Decreased Lung Function via CFTR dysfunction	adjacent	High	Moderate	Karsta Luettich (send email)	Open for comment. Do not cite

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) that help to define the biological applicability domain of the KER.In general, this will be dictated by the more restrictive of the two KEs being linked together by the KER. More help

Term	Scientific Term	Evidence	Link
Homo sapiens	Homo sapiens	High	NCBI

Sex Applicability

An indication of the the relevant sex for this KER. More help

Sex	Evidence
Mixed	High

Life Stage Applicability

An indication of the the relevant life stage(s) for this KER. More help

Term	Evidence
All life stages	High

Key Event Relationship Description

Provides a concise overview of the information given below as well as addressing details that aren’t inherent in the description of the KEs themselves. More help

Serous and glandular secretions of the airway epithelium contribute to the ASL, and epithelial ion channel (e.g. CFTR, ENaC, CaCC, BK) function is critical to normal ASL homeostasis. Should the PCL decrease in depth, liquid will be absorbed from the mucus layer until the necessary depth is restored. Conversely, the mucus layer will absorb surplus PCL to reduce any increase in its depth. The regulation of these reabsorption processes is complex and not fully elucidated (Boucher, 2004). Experimental evidence suggests that the balance between Na⁺ absorption and Cl⁻ secretion mediated by ENaC and CFTR plays a major role, with the ion channels affecting each other’s activity (increased CFTR activity leads to decreased ENaC activity and vice versa) (Boucher, 2003; Boucher, 2004; Schmid et al., 2011). Mechanistic studies with selective CFTR and ENaC inhibitors suggest that the sensors for regulating ASL height lie within the ASL itself (Boucher, 2003; Hobbs et al., 2013). Additionally, ATP, adenosine and other purinergic receptor agonists, adenylate cyclase and cyclic adenosine monophosphate (cAMP)-dependent protein kinases acting on CFTR and/or ENaC ensure that the ASL height is adjusted to the appropriate height, resulting in maintenance of PCL depth at approximately the length of cilia (Antunes and Cohen, 2007). If the CFTR-ENaC interaction is perturbed, the airways become “dehydrated” (i.e., the ASL height decreases), resulting in slowing or inhibition of cilia movement and impaired MCC (Munkholm and Mortensen, 2014).

Evidence Collection Strategy

Include a description of the approach for identification and assembly of the evidence base for the KER. For evidence identification, include, for example, a description of the sources and dates of information consulted including expert knowledge, databases searched and associated search terms/strings. Include also a description of study screening criteria and methodology, study quality assessment considerations, the data extraction strategy and links to any repositories/databases of relevant references.Tabular summaries and links to relevant supporting documentation are encouraged, wherever possible. More help

Evidence Supporting this KER

Addresses the scientific evidence supporting KERs in an AOP setting the stage for overall assessment of the AOP. More help

As a major Cl^– channel in the respiratory epithelium, CFTR levels and function are vital for maintenance of ASL homeostasis. In vitro studies on the effects of cigarette smoke exposure on human lung primary cells and cell lines showed a reduction in ASL height, associated with decreased CFTR levels (Hassan et al., 2014; Rasmussen et al., 2014; Xu et al., 2015; Ghosh et al., 2017) and decreased Cl^– current (Lambert et al., 2014; Raju et al., 2016). Moreover, pharmaceutical stimulation and inhibition of CFTR function and expression directly increased and decreased ASL height, respectively (Song et al., 2009; Van Goor et al., 2009; Van Goor et al., 2011; Tuggle et al., 2014).

Biological Plausibility

Addresses the biological rationale for a connection between KEupstream and KEdownstream. This field can also incorporate additional mechanistic details that help inform the relationship between KEs, this is useful when it is not practical/pragmatic to represent these details as separate KEs due to the difficulty or relative infrequency with which it is likely to be measured. More help

Impaired function of the CFTR and ENaC ion channels results in enhanced Na⁺ absorption and reduced Cl⁻ secretion, and as a consequence, reduced ASL height. This phenomenon is well-known from studies in models of cystic fibrosis and acquired CFTR deficiency, even though the exact mechanism of the interaction between these two channels remains to be elucidated (Boucher, 2003; Hassan et al., 2014; Raju et al., 2016a; Rasmussen et al., 2014; Tarran et al., 2001a; Woodworth, 2015; Zhang et al., 2013). Additionally, evidence from studies with pharmacological agents that enhance CFTR expression and/or function or perturb the interaction between CFTR and ENaC provide further support for strong biological plausibility of this KER (Lambert et al., 2014; Van Goor et al., 2009; Van Goor et al., 2011).

Empirical Evidence

Provides specific (citable) evidence that supports the idea of a change in the upstream KE (KEupstream) leading to, or being associated with, a subsequent change in the downstream KE (KEdownstream), assuming the perturbation of KEupstream is sufficient. More help

Addition of 2% cigarette smoke extract (CSE) to differentiated primary human bronchial epithelial cells (HBECs) stimulated with forskolin acutely decreased Cl⁻ transport, which was associated with reduced ASL height and cilia beat frequency (Raju et al., 2016a).

Exposure of differentiated primary HBECs to cigarette smoke for up to 120 h significantly reduced plasma membrane CFTR expression and ASL height (Hassan et al., 2014) in one study, and reduced intracellular cAMP levels, Cl⁻ conductance, ASL volume and cilia beat frequency in another study (Schmid et al., 2015).

Exposure of primary HBECs grown in monolayers to cigarette smoke caused a dose- and time-dependent decrease in forskolin-stimulated Cl⁻ transport, which could be partially restored by treatment with roflumilast via a cAMP-dependent mechanism. Smoke exposure also decreased ASL height, and this effect could also be ameliorated by roflumilast (Lambert et al., 2014).

Treatment of immortalized 16HBE14o- airway epithelial cells with 10% cigarette smoke extract for 48 h resulted in significant reduction in CFTR total and cell surface protein expression as well as in ASL height (Xu et al., 2015).

Repeated exposure of differentiated primary HBECs to smoke from little cigars significantly decreased CFTR protein expression and ASL height (Ghosh et al., 2017).

CFTR protein expression was also reduced following exposure of baby hamster kidney cells expressing human CFTR (BHK^CFTR) or Calu-3 lung cancer cells to cigarette smoke, resulting in cigarette smoke-induced increases in intracellular Ca²⁺, CFTR internalization and subsequent reduction in ASL height (Rasmussen et al., 2014).

In excised pig and human trachea preparations, addition of amiloride and CFTR activators increased ASL height. In pig trachea, CFTR inhibition prevented the increase in ASL height produced by forskolin/3-isobutyl-1-methylxanthine (IBMX). This study did not investigate dose or time responses (Song et al., 2009).

Experiments with pharmacological compounds shown to enhance CFTR cell surface expression or channel open probability in primary human bronchial epithelial cells demonstrated enhanced Cl− currents and increased ASL height (Van Goor et al., 2009; Van Goor et al., 2011).

ASL height in the excised tracheas of rats without functional CFTR expression was approx. half that of wild-type animals (Tuggle et al., 2014).

Exposure of differentiated primary HBECs to supernatant from mucopurulent material (SMM) of cystic fibrosis airways decreased amiloride-sensitive Cl− current in CFTR wild-type but not CFTR mutant HBECs. Forskolin-induced Cl⁻ conductance was absent in treated CFTR mutant HBECs and enhanced in wild-type HBECs, where this effect was both dose- and time-dependent. Enhanced UTP-induced currents were seen in HBECs, independent of CFTR genotype. SMM treatment furthermore induced an increase in ASL height in CFTR wild-type but not mutant HBECs (Abdullah et al., 2018).

Knockdown of SPLUNC1, an allosteric regulator of ENaC, in human bronchial epithelial cells significantly reduced ENaC currents and rapidly and significantly reduced ASL height (Garcia-Caballero et al., 2009). SPLUNC1 is degraded in airway epithelia of cystic fibrosis patients, resulting in failure of ENaC internalization and, consequently, increased ENaC activity. Addition of recombinant SPLUNC1 or sputum supernatants from healthy subjects significantly elevated ASL height in HBECs (Hobbs et al., 2013; Webster et al., 2018). Knock-down of β-ENaC mRNA in HBECs resulted in significantly reduced amiloride-sensitive short-circuit (Na+) currents and significantly increased ASL height (Gianotti et al., 2013). Knockdown of ENaC mRNA in BMI-1 transduced cystic fibrosis BECs resulted in nearly 50% reduction in amiloride short-circuit currents, a ca. 1.5 fold increase in ASL height and increased cilia beat frequency (Tagalakis et al., 2018). Inhibition of channel activating proteases, including prostasin, matriptase, and furin, in ΔF508 cystic fibrosis airway epithelia by QUB-TL1 increased intracellular ENaC, decreased ENaC-mediated Na⁺ transport and increased ASL height (Reihill et al., 2016). In excised tracheas and bronchi from mice overexpressing the β-ENaC subunit in lower airway epithelia, amiloride-sensitive Na⁺ transport was increased ca. 2- to 3-fold, and ASL was significantly depleted (Mall et al., 2004).

Uncertainties and Inconsistencies

Addresses inconsistencies or uncertainties in the relationship including the identification of experimental details that may explain apparent deviations from the expected patterns of concordance. More help

The process of reabsorption of excess liquid to regulate ASL height is also known as “isosmotic volume hypothesis” or “isotonic volume transport/mucus clearance hypothesis” and implies that CFTR assumes a critical role in regulating ASL height by inhibiting ENaC activity (Ganesan et al., 2013; Matsui et al., 1998). However, an alternative, opposing hypothesis exists, the “hypotonic hypothesis” which states “that normal airway epithelia are covered by an ASL with a [NaCl] sufficiently low (≤ 50 mM NaCl) to activate defensins and create an antimicrobial “shield” on airway surfaces”, and there is evidence to both support and refute it (Cowley et al., 1997; Goldman et al., 1997; Jayaraman et al., 2001; Knowles et al., 1997; Landry and Eidelman, 2001; Matsui et al., 1998; Tarran et al., 2001a; Tarran et al., 2001b; Verkman et al., 2003). Other studies suggest the involvement of additional ion channels such as alternative chloride channels (Grasemann et al., 2007) and cyclic nucleotide-gated cation channels, particularly in the alveolar epithelium (Schwiebert et al., 1997; Wilkinson et al., 2011) in the regulation of ASL height. In addition, one study showing that instillation of Pseudomonas aeruginosa-laden agarose beads into excised swine tracheas significantly increased ASL height, and that this increase could be blocked by pre-incubation with the CFTR inhibitor CFTRinh172 (100 μM, 30 minutes) (Luan et al., 2014) presents an inconsistency with the available evidence presented here.

Known modulating factors

This table captures specific information on the MF, its properties, how it affects the KER and respective references.1.) What is the modulating factor? Name the factor for which solid evidence exists that it influences this KER. Examples: age, sex, genotype, diet 2.) Details of this modulating factor. Specify which features of this MF are relevant for this KER. Examples: a specific age range or a specific biological age (defined by...); a specific gene mutation or variant, a specific nutrient (deficit or surplus); a sex-specific homone; a certain threshold value (e.g. serum levels of a chemical above...) 3.) Description of how this modulating factor affects this KER. Describe the provable modification of the KER (also quantitatively, if known). Examples: increase or decrease of the magnitude of effect (by a factor of...); change of the time-course of the effect (onset delay by...); alteration of the probability of the effect; increase or decrease of the sensitivity of the downstream effect (by a factor of...) 4.) Provision of supporting scientific evidence for an effect of this MF on this KER. Give a list of references. More help

Unknown

Quantitative Understanding of the Linkage

Captures information that helps to define how much change in the upstream KE, and/or for how long, is needed to elicit a detectable and defined change in the downstream KE. More help

While there are convincing quantitative data in support of this KER, it becomes clear from the review of the evidence that the downstream KE can only be modulated to a certain extent (e.g., maximal decrease in ASL height did not exceed 50% in the majority of studies), independent of the extent of change in the upstream KE. In addition, the available temporal data indicates that acute exposures predominantly cause a transient, rather than a lasting change in the downstream KE. Since chronic treatment data are not available, we judge our quantitative understanding as being moderate.

Response-response Relationship

Provides sources of data that define the response-response relationships between the KEs. More help

Treatment of fully differentiated primary human bronchial epithelial cells (HBECs) with 2% cigarette smoke extract (CSE; bubbling 10 puffs of smoke from one 3R4F reference into 1 mL DMSO, at 2 s/10 mL puff, 10 puffs over 3 min; defined as 100%) for 20 minutes reduced CFTR channel activity by 50% and ASL by approx. 2-fold (Raju et al., 2016).

Apical treatment of primary HBECs grown in monolayers with 2% CSE (not further described) for 24 h decreased forskolin-induced Cl⁻ currents by ca. 20% and ASL height by approx. 25%, and this could be counteracted by co-treatment with 10 µM ivacaftor, a CFTR potentiator known to significantly augment cAMP-mediated ion transport activity (Sloane et al., 2012).

Exposure of primary HBECs to cigarette smoke (5 min, ca. 12 puffs at 1 puff every 30 s; generated according to ISO standards) resulted in efficient removal of CFTR from the plasma membrane and a ca. 2-fold reduction in ASL height (Xu et al., 2015).

Exposure of primary HBECs, differentiated at the air-liquid interface, to cigarette smoke from 1 cigarette (ten 35-mL puffs, 2R4F reference cigarette) nearly abolished responses of the transepithelial electric potential difference Vt to ADO (i.e., blocking the ADO-A2b-cAMP-CFTR- active ion transport) and significantly decreased ASL volume/height by approx. 2-fold after 30 min (Clunes et al., 2012).

Exposure of fully differentiated primary HBECs to 30 puffs of whole smoke from 2 cigarettes (generated according to ISO standards) every day for 5 days (120 h) resulted in a ca. 40% reduction in CFTR expression and approx. 50% reduction in ASL height (Hassan et al., 2014).

Exposure of primary human airway epithelial cells grown in monolayers to whole smoke (3R4F reference cigarette; inExpose exposure system; 3L/min) resulted in significant reduction of CFTR Cl⁻ currents (ca. 40% for a 30-min exposure) and significantly decreased ASL depth from 11.4± 4.1 to 5.6± 2.0 µm (Lambert et al., 2014).

Exposure of fully differentiated primary HBECs to smoke from 1 cigarette or little cigar every day for 5 days (1 × 35 ml puff per 30 second, up to a butt length of 36 mm) significantly reduced CFTR protein expression by ca. 2- to 4-fold and ASL height by 10 to 20% (Ghosh et al., 2017).

Cell surface CFTR protein expression was reduced by ca. 70% following exposure of baby hamster kidney cells expressing human CFTR (BHK^CFTR) to cigarette smoke for 10 min (3R4F reference cigarette, 1 puff per min according to ISO standards). This was accompanied by a significant reduction in ASL height by approx. 50% (Rasmussen et al., 2014).

Treatment of primary HBECs from a cystic fibrosis patient with the ΔF508 mutation, grown as monolayer at the air-liquid interface, with the CFTR corrector VX-809 for 48 h increased CFTR maturation by ca. 8-fold and enhanced Cl⁻ transport by approx. 4-fold, from 1.9±0.4 to 7.8±1.3 μA/cm². VX-809 treatment for 5 days increased ASL height from 4.5±0.2 to 6.7±0.5 μm. Addition of 3 μM VX-770 further increased the ASL height to 9.2±0.2 μm (Van Goor et al., 2011). Treatment of primary HBECs from a G551D/ΔF508 cystic fibrosis patient, grown as monolayer at the air-liquid interface, with the CFTR potentiator VX-770 (10 µM) for 72 h dose-dependently increased forskolin-mediated Cl⁻ currents by ca. 10-fold to 27±2 μA/cm² and ASL volume to 125% that of controls (Van Goor et al., 2009).

ASL depth in the excised tracheas of rats without functional CFTR expression was approx. half that of wild-type animals (Tuggle et al., 2014).

Knockdown of mRNAs for the α- and β-ENaC subunits resulted in a ca. 70% decrease in amiloride-sensitive currents and a significant increase in ASL height from 6.8±0.5 and 7.4±0.5 µm to 9.8±0.6 and 9.6±0.8 µm in non-CF and CF epithelia, respectively (Gianotti et al., 2013).

Knockdown of α-ENaC mRNA in BMI1-transduced cystic fibrosis bronchial epithelial cells resulted in ca. 50% reduction in protein expression, reduction in amiloride-sensitive short-circuit current from 11.5 (siRNA control) to 6.4 μA/cm² and increase in ASL height from 7.9 (siRNA control) to 12.1 μm (Tagalakis et al., 2018).

Overexpression of the β-ENaC subunit in mouse airways increased basal and amiloride-sensitive short-circuit currents approx. 2-fold (excised tracheas; compared to wild-type) and significantly reduced ASL height in bronchi and tracheas (by approx. 2 µm) (Mall et al., 2004).

Time-scale

Information regarding the approximate time-scale of the changes in KEdownstream relative to changes in KEupstream (i.e., do effects on KEdownstream lag those on KEupstream by seconds, minutes, hours, or days?). More help

Treatment of fully differentiated primary HBECs with 2% CSE (bubbling 10 puffs of smoke from one 3R4F reference into 1 mL DMSO, at 2 s/10 mL puff, 10 puffs over 3 minutes; defined as 100%) for 24 h decreased total CFTR expression and cell surface CFTR expression by approx. 20 and 25%, respectively, but treatment for 20 min did not. A 50% reduction in CFTR channel activity occurred immediately after addition of CSE and lasted for at least 20 minutes. A 2-fold reduction in ASL height was seen after 20 minutes, and ASL height was only partially restored at 1 h after CSE treatment (Raju et al., 2016a).

Following exposure of primary HBECs, differentiated at the air-liquid interface, to cigarette smoke from 1 cigarette (ten 35-mL puffs, 2R4F reference cigarette), ASL volume/height was significantly decreased by approx. 2-fold after 30 min. This decrease lasted for >2.5 h, and ASL height was restored at 4 h post-exposure (Clunes et al., 2012). Exposure of BHK^CFTR cells to cigarette smoke for 10 min (3R4F reference cigarette, 1 puff per min according to ISO standards) resulted in a reduction in ASL height by approx. 50% within 30 min; the decrease lasted for up to 1 h post-exposure (Rasmussen et al., 2014).

Exposure of fully differentiated primary human HBECs to 30 puffs of whole smoke from 2 cigarettes (generated according to ISO standards) was sufficient to decrease ASL height by approx. 50% within 1 h of exposure, and following daily exposure for another 4 days, ASL height remained at around this level (Hassan et al., 2014).

Exposure of fully differentiated primary HBECs to whole smoke from four 3R4F reference cigarettes (generated according to ISO standard 3308; Vitrocell VC10 exposure system) resulted in a small, non-significant increase in ASL volume 4 h post-exposure. ASL volume decreased to baseline levels 7 h post-exposure and continued to drop below baseline levels until 24 h post-exposure (Schmid et al., 2015).

ASL height of primary HBECs dropped within 30 min of exposure to cigarette smoke (5 min, ca. 12 puffs at 1 puff every 30 seconds). ASL height stayed at that reduced level up to until 70 min post-exposure (Xu et al., 2015).

The maximum effect of VX-809 treatment on Cl⁻ currents of primary human bronchial epithelial cells, grown as monolayer at the air-liquid interface, occurred following 24 h, and Cl– transport returned to uncorrected levels within 48 h of compound washout (concurrent ASL data not available) (Van Goor et al., 2011).

Known Feedforward/Feedback loops influencing this KER

Define whether there are known positive or negative feedback mechanisms involved and what is understood about their time-course and homeostatic limits. More help

Unknown

Domain of Applicability

A free-text section of the KER description that the developers can use to explain their rationale for the taxonomic, life stage, or sex applicability structured terms. More help

Phylogenetic analysis of CFTR DNA sequences across multiple species suggests a close evolutionary relationship between human and primate CFTR, followed by rabbit, guinea pig, equine, ovine, and bovine CFTR, whereas rodent CFTR DNA largely diverges from the human DNA (Chen et al., 2001). Of note, CFTR ion permeability differs from species to species (Higgins, 1992). For example, murine CFTR displays reduced channel activity compared with its human counterpart, while ovine CFTR exhibits higher ATP sensitivity, greater single-channel conductance and larger open probability than human CFTR. Moreover, sensitivity to pharmacological agents able to potentiate or block CFTR gating varies greatly from species to species (Bose et al., 2015). Therefore, results from animal studies are not directly transferable to human.

To date, ASL has been investigated in several species including mice, rats, guinea pigs, ferrets, cats, dogs, cows, monkeys, and humans. Although most studies provide data on its composition rather than its height, it is reasonable to assume that regulation of ASL height is equally critical to MCC across these species.

CFTR dysfunction as a consequence of inherited CFTR gene defects is studied in pediatric as well as adult cystic fibrosis patients. Acquired CFTR dysfunction following inhalation exposures (e.g. to cigarette smoke) may also apply to both pediatric and adult populations, depending on the setting and type of exposure, and this also applies to decreased ASL height.

To our knowledge, the role of gender has not been systematically evaluated in acquired CFTR dysfunction and its impact on ASL height. It is thought that the observed suppression of CFTR expression and impairment of CFTR function in cigarette smokers is a contributing factor to the pathogenesis of chronic obstructive pulmonary disease (COPD)(Dransfield et al., 2013; Raju et al., 2016). The main risk factor for COPD is cigarette smoking, and COPD is more common in men than in women, which may be directly related to the higher prevalence of smoking in men, although this gender gap is closing (Hitchman and Fong, 2011; Ntritsos et al., 2018; Syamlal et al., 2014). Nevertheless, the available clinical evidence in support of this AOP suggests that there is no remarkable gender difference.

References

List of the literature that was cited for this KER description. More help

Antunes, M.B., and Cohen, N.A. (2007). Mucociliary clearance–a critical upper airway host defense mechanism and methods of assessment. Curr. Opin. Allergy Clin. Immunol. 7, 5-10.

Bose, S.J., Scott-Ward, T.S., Cai, Z., and Sheppard, D.N. (2015). Exploiting species differences to understand the CFTR Cl− channel. Biochem. Soc. Transact. 43, 975-982.

Boucher, R. (2003). Regulation of airway surface liquid volume by human airway epithelia. Pflügers Arch. 445, 495-498.

Boucher, R.C. (2004). New concepts of the pathogenesis of cystic fibrosis lung disease. Eur. Respir. J. 23, 146-158.

Chen, J.-M., Cutler, C., Jacques, C., Bœuf, G., Denamur, E., Lecointre, G., et al. (2001). A combined analysis of the cystic fibrosis transmembrane conductance regulator: implications for structure and disease models. Mol. Biol. Evol. 18., 1771-1788.

Clunes, L.A., Davies, C.M., Coakley, R.D., Aleksandrov, A.A., Henderson, A.G., Zeman, K.L., et al. (2012). Cigarette smoke exposure induces CFTR internalization and insolubility, leading to airway surface liquid dehydration. FASEB J. 26, 533-545.

Cowley, E., Wang, C., Gosselin, D., Radzioch, D., and Eidelman, D. (1997). Mucociliary clearance in cystic fibrosis knockout mice infected with Pseudomonas aeruginosa. European Respiratory Journal 10, 2312-2318.

Downs, C.A., Kreiner, L.H., Trac, D.Q., and Helms, M.N. (2013). Acute Effects of Cigarette Smoke Extract on Alveolar Epithelial Sodium Channel Activity and Lung Fluid Clearance. Am. J. Respir. Cell Mol. Biol. 49, 251-259.

Dransfield, M.T., Wilhelm, A.M., Flanagan, B., Courville, C., Tidwell, S.L., Raju, S.V., et al. (2013). Acquired cystic fibrosis transmembrane conductance regulator dysfunction in the lower airways in COPD. Chest 144, 498-506.

Ganesan, S., Comstock, A.T., and Sajjan, U.S. (2013). Barrier function of airway tract epithelium. Tissue Barriers 1, e24997.

Garcia-Caballero, A., Rasmussen, J.E., Gaillard, E., Watson, M.J., Olsen, J.C., Donaldson, S.H., et al. (2009). SPLUNC1 regulates airway surface liquid volume by protecting ENaC from proteolytic cleavage. Proc. Natl. Acad. Sci. U. S. A. 106, 11412-11417.

Ghosh, A., Abdelwahab, S.H., Reeber, S.L., Reidel, B., Marklew, A.J., Garrison, A.J., et al. (2017). Little Cigars are More Toxic than Cigarettes and Uniquely Change the Airway Gene and Protein Expression. Sci. Rep. 7, 46239.

Gianotti, A., Melani, R., Caci, E., Sondo, E., Ravazzolo, R., Galietta, L.J., et al. (2013). Epithelial sodium channel silencing as a strategy to correct the airway surface fluid deficit in cystic fibrosis. Am. J. Respir. Cell Mol. Biol. 49, 445-452.

Goldman, M.J., Anderson, G.M., Stolzenberg, E.D., Kari, U.P., Zasloff, M., and Wilson, J.M. (1997). Human β-defensin-1 is a salt-sensitive antibiotic in lung that is inactivated in cystic fibrosis. Cell 88, 553-560.

Grasemann, H., Stehling, F., Brunar, H., Widmann, R., Laliberte, T.W., Molina, L., et al. (2007). Inhalation of Moli1901 in patients with cystic fibrosis. Chest 131, 1461-1466.

Hassan, F., Xu, X., Nuovo, G., Killilea, D.W., Tyrrell, J., Da Tan, C., et al. (2014). Accumulation of metals in GOLD4 COPD lungs is associated with decreased CFTR levels. Respir. Res. 15, 69.

Higgins, C.F. (1992). ABC transporters: from microorganisms to man. Ann. Rev. Cell Biol. 8, 67-113.

Hitchman, S.C., and Fong, G.T. (2011). Gender empowerment and female-to-male smoking prevalence ratios. Bull. World Health Organ. 89, 195-202.

Hobbs, C.A., Blanchard, M.G., Alijevic, O., Tan, C.D., Kellenberger, S., Bencharit, S., et al. (2013). Identification of the SPLUNC1 ENaC-inhibitory domain yields novel strategies to treat sodium hyperabsorption in cystic fibrosis airway epithelial cultures. Am. J. Physiol. Lung Cell. Mol. Physiol. 305(12), L990-L1001.

Jayaraman, S., Song, Y., Vetrivel, L., Shankar, L., and Verkman, A.S. (2001). Noninvasive in vivo fluorescence measurement of airway-surface liquid depth, salt concentration, and pH. J. Clin. Invest. 107, 317-324.

Knowles, M.R., Robinson, J.M., Wood, R.E., Pue, C.A., Mentz, W.M., Wager, G.C., et al. (1997). Ion composition of airway surface liquid of patients with cystic fibrosis as compared with normal and disease-control subjects. J. Clin. Invest. 100, 2588-2595.

Lambert, J.A., Raju, S.V., Tang, L.P., McNicholas, C.M., Li, Y., Courville, C.A., et al. (2014). Cystic fibrosis transmembrane conductance regulator activation by roflumilast contributes to therapeutic benefit in chronic bronchitis. Am. J. Respir. Cell Mol. Biol. 50, 549-558.

Landry, J.S., and Eidelman, D.H. (2001). Airway surface liquid: end of the controversy? J. Gen. Physiol. 117, 419-422.

Luan, X., Campanucci, V.A., Nair, M., Yilmaz, O., Belev, G., Machen, T.E., et al. (2014). Pseudomonas aeruginosa triggers CFTR-mediated airway surface liquid secretion in swine trachea. Proc. Natl. Acad. Sci. U.S.A. 111, 12930-12935.

Mall, M., Grubb, B.R., Harkema, J.R., O'Neal, W.K., and Boucher, R.C. (2004). Increased airway epithelial Na+ absorption produces cystic fibrosis-like lung disease in mice. Nat. Med. 10, 487.

Matsui, H., Grubb, B.R., Tarran, R., Randell, S.H., Gatzy, J.T., Davis, C.W., et al. (1998). Evidence for periciliary liquid layer depletion, not abnormal ion composition, in the pathogenesis of cystic fibrosis airways disease. Cell 95, 1005-1015.

Munkholm, M., and Mortensen, J. (2014). Mucociliary clearance: pathophysiological aspects. Clin. Physiol. Funct. Imaging 34, 171-177.

Ntritsos, G., Franek, J., Belbasis, L., Christou, M.A., Markozannes, G., Altman, P., et al. (2018). Gender-specific estimates of COPD prevalence: a systematic review and meta-analysis. Int. J. Chron. Obstruct. Pulmon. Dis. 13, 1507.

Raju, S.V., Lin, V.Y., Liu, L., Mcnicholas, C.M., Karki, S., Sloane, P.A., et al. (2016). The Cftr Potentiator Ivacaftor Augments Mucociliary Clearance Abrogating Cftr Inhibition by Cigarette Smoke. Am. J. Respir. Cell Mol. Biol. 56, 99-108.

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