Abstract
Large-conductance, Ca2+-activated, voltage-dependent K+(BK) channel function is critical for adequate airway hydration and mucociliary function. In airway epithelia, BK function is regulated by its γ-subunit, leucine-rich repeat-containing protein 26 (LRRC26). Since patients with cystic fibrosis (CF)-related diabetes mellitus (CFRD) have worse lung function outcomes, this study determined the effects of hyperglycaemia on BK function in CF bronchial epithelial (CFBE) cellsin vitroand evaluated the correlation between glycaemic excursions and mRNA expression ofLRRC26in the upper airways of CF and CFRD patients.
CFBE cells were redifferentiated at the air–liquid interface (ALI) in media containing either 5.5 mM or 12.5 mM glucose. BK activity was measured in an Ussing chamber. Airway surface liquid (ASL) volume was estimated by meniscus scanning and inflammatory marker expression was measured by quantitative real-time PCR and enzyme-linked immunosorbent assay (ELISA). CF patients were assessed by 7 days of continuous glucose monitoring (CGM).LRRC26mRNA expression was measured by quantitative real-time PCR from nasal cells obtained at the end of glucose monitoring.
BK currents were significantly decreased in CFBE cells cultured under high glucose. These cells revealed significantly lower ASL volumes and increased inflammation, including the receptor for advanced glycation endproducts (RAGE), compared to cells cultured in normal glucose.In vivo, nasal cell expression ofLRRC26mRNA was inversely correlated with hyperglycaemic excursions, consistent with thein vitroresults.
Our findings demonstrate that hyperglycaemia induces inflammation and impairs BK channel function in CFBE cellsin vitro. These data suggest that declining lung function in CFRD patients may be related to BK channel dysfunction.
Abstract
In CF patients, hyperglycaemia downregulates airway epithelial BK channels that are critical for mucociliary clearance. Mechanistically, hyperglycaemia suppresses expression of the BK γ-subunitLRRC26, which is required for its function in the airway.https://bit.ly/315FCTV
Introduction
Cystic fibrosis (CF)-related diabetes mellitus (CFRD) is a major predictor of worse lung function and affects ∼20% of adolescents and >40% of adults with CF [1–3]. However, little is known about the mechanisms by which elevated glucose levels lead to worse lung function outcomes.
Advances in care and therapeutic treatments for CF patients have led to a dramatic increase in life expectancy. However, the consequence of prolonged life is an increase in the prevalence of age-related and CF-related comorbidities that can worsen pulmonary function. In fact, CFRD is the most common CF comorbidity and is associated with worse lung function, increased number and severity of pulmonary exacerbations, increased risk of infection byPseudomonas aeruginosaand poorer prognosis compared to CF patients without diabetes [4]. Thus, it is imperative to initiate mechanistic studies that could be translated into lung function preservation trials in CFRD beyond glycaemic control.
Hyperglycaemia initiates pro-inflammatory signalling pathways [5], including the formation of advanced glycation endproducts (AGE). These bind to the receptor for advanced glycation endproducts (RAGE or AGER), a member of the immunoglobulin superfamily of cell-surface receptors, which functions as a pattern-recognition receptor and engages pro-inflammatory signalling pathways when activated [6]. RAGE is highly expressed in the lung and is also important for cell adhesion and epithelial repair [7,8]. RAGE signalling has been implicated in the pathogenesis of a number of respiratory disorders, including chronic obstructive pulmonary disease (COPD), asthma and pulmonary fibrosis [9]. In CF patients, even more so in CFRD patients, levels of RAGE ligands in the lung inversely correlate with lung function [10,11].
It is known that inflammation and hyperglycaemia impair airway epithelial ion channel function [12] and thereby possibly contribute to respiratory decline in CFRD patients, but little is known about mechanism. Here, we specifically studied the impact of hyperglycaemia on apically expressed large-conductance, Ca2+-activated, voltage-dependent K+(BK) channels because they are critical to maintaining mucus clearance in CF bronchial epithelial (CFBE) cells [13].
Our data reveal that airway surface liquid (ASL) volume reduction under hyperglycaemic conditions, in CFBE cellsin vitro, correlated with reduced BK channel function and increased expression of RAGE, which could initiate pro-inflammatory signalling cascades under conditions of persistent hyperglycaemia [14]. BK channel dysfunction was likely due to a decrease in expression of leucine-rich repeat-containing protein 26 (LRRC26), the BK γ-subunit needed for its function in non-excitable cells [15,16]. Furthermore, continuous monitoring of glucose levels in CF patients with or without CFRD over a 1-week period revealed that hyperglycaemic excursions inversely correlated with mRNA expression ofLRRC26.
Methods
Lungs
所有non-CF人类支气管上皮细胞(NHBE)were from nonsmoking donors with no known pre-existing airway diseases or lung diseases, whose lungs were not used for transplant by the Life Alliance Organ Recovery Agency (University of Miami, Miami, FL, USA), the LifeCenter Northwest (Seattle, WA, USA), or the Midwest Transplant Network (Kansas City, KS, USA). Organ donation was performed on donors who had consented appropriately with the organ procurement agencies. Thus, cell and tissue use are not considered human subjects research (deceased individuals). Non-CF lung donor information is presented insupplementary table S1. Most CFBE cells stemmed from explanted lungs from patients undergoing lung transplantation, who gave with appropriate consent and which was approved by the University of Miami's Institutional Review Board (IRB). Lungs from one deceased CF donor were procured through LifeCenter Northwest. CF lung donor information is presented insupplementary table S2.
Cell culture
Culturing of NHBE and CFBE cells at the air–liquid interface (ALI) was performed as described (supplementary material) [17–19]. Cells were re-differentiated at an ALI in media containing 5.5 mM or 12.5 mM glucose for a minimum of 3 weeks before experiments were performed. Glucose levels in media were measured with a calibrated OneTouch Verio meter (LifeScan, Malvern, PA, USA). For high mobility group box 1 (HMGB1) experiments, 100 ng·mL−1recombinant human HMGB1 (H4652, MilliporeSigma, Burlington, MA, USA) was added to the basolateral media 24 h before Ussing chamber and ASL volume measurements.
Quantitative PCR
NHBE and CFBE cells were lysed and total RNA was isolated using the E.Z.N.A. Total RNA Kit (Omega Bio-tek, Norcross, GA, USA). Quantitative real-time PCR was performed as described [20,21] using TaqMan Gene Expression Assays (ThermoFisher Scientific, Waltham, MA, USA) forAGER(Hs00153957_m1),IL-1β(Hs01555410_m1),IL-6(Hs00985639_m1),IL-8(Hs00174103),KCNMA1(Hs00266938_m1),KCNN4(Hs00158470_m1),LRRC26(Hs02385555_g1),MMP-9(Hs00234579_m1),COX-2(Hs00153133_m1) andTGF-β1(Hs00998133_m1), and normalised to reference geneGAPDH(GAPDH: glyceraldehyde 3-phosphate dehydrogenase).
Enzyme-linked immunosorbent assay
Basolateral media from CFBE cells cultured in ALI media with starting glucose concentrations of 5.5 mM or 12.5 mM were collected 48 h after the previous media change. Media samples were analysed using the Ella Automated Immunoassay System and Simple Plex cartridge-based immunoassay for interleukin (IL)-1β, IL-6 and Il-8 (Bio-Techne Corp., Minneapolis, MN, USA).
Ussing chamber
CF transmembrane conductance regulator (CFTR) and large conductance, Ca2+-activated and voltage-dependent K+channel (BK) activities were recorded in Ussing chambers as previously described (supplementary material) [16,22]. Ussing chamber experiments were performed 24 h after apical wash and basolateral media change unless otherwise noted. CFTR and BK measurements in NHBE cells on Snapwell inserts (Corning Inc, Corning, NY, USA) were conducted using 1.12 cm2aperture sliders (P2302, Physiologic Instruments, San Diego, CA, USA). BK measurements in CFBE cells on Transwell inserts (Corning Inc) were conducted using 0.10 cm2aperture sliders (P2303, Physiologic Instruments). The smaller aperture size likely allows for greater epithelial uniformity, which may account for the larger BK currents observed in CFBE cells.
Airway surface liquid volume
ASL volume estimation was performed by meniscus scanning as previously published [21,23]. Briefly, the apical surface of the cells was washed with Dulbecco's phosphate-buffered saline (Corning Inc) and ASL volumes were measured after 24 h. Basolateral media was replenished with ALI media containing either 5.5 mM or 12.5 mM glucose and ASL volumes were measured again after another 24 h. Change in ASL (ΔASL) represents the difference between these two measurements.
Human Study approval
The study was approved by the University of Kansas Medical Center IRB and informed consent was obtained from each participant.
Human subjects
Patients aged 18 years or older in the University of Kansas Health System, with known CF (with or without CFRD), were enroled in an observational cohort study (seesupplementary materialfor additional enrolment criteria). CFRD was defined as fasting blood glucose >126 mg·dL−1or 2 h blood glucose >200 mg·dL−1during an oral glucose tolerance test (OGTT). Those with a normal OGTT (<126 mg·dL−1at baseline or <140 mg·dL−1at 2 h) or an impaired OGTT (<126 mg·dL−1at baseline or 140–200 mg·dL−1at 2 h) were defined as CF only. Demographics and baseline clinical variables were collected at the initial study visit and haemoglobin A1C was measured from capillary blood using A1CNow+ (PTS Diagnostics, Whitestown, IN, USA). Study participants wore blinded continuous glucose monitors (Dexcom G6, Dexcom Inc., San Diego, CA, USA) for 5–10 days. Nasal cells were collected from study participants at the time of monitor removal using sterile cytology brushes (Medical Packaging Corp., Camarillo, CA, USA) as described in thesupplementary material.
Continuous glucose monitoring analyses
Continuous glucose monitoring (CGM) variables were calculated using the R statistical package “cgmanalysis” (The R Project for Statistical Computing,www.r-project.org) [24]. Imputation was used to fill missing intervals <20 min in length and, for those intervals >20 min in length, the corresponding 24 h period was censored from the final analysis. CGM variables of interest were selected due to being previously associated with outcomes in CFRD (mean amplitude of glycaemic excursion (MAGE) and percentage time glucose >140 mg·dL−1) or due to being reflective of hyperglycaemic excursions (glucose management indicator (GMI), percentage time glucose >200 mg·dL−1and area under the curve (AUC) of glucose >180 mg·dL−1) [25,26].
Statistical analyses
Forin vitrostudies, data are shown as mean±standard error of the mean (sem). Data that followed normal distribution by Shapiro–Wilk normality testing were analysed by parametric tests. Otherwise, nonparametric statistical analyses were performed. Multiple groups were analysed by one-way ANOVA followed by the appropriatepost hoc测试。两组比较配对或联合国paired t-test as appropriate. Results were considered statistically significant at p<0.05. Clinical study data are shown as dot plot/bar graph combinations with mean±sem或趋势线95%置信区间(CIs). Differences between two groups were compared by parametric or nonparametric tests as indicated in the figure captions depending on whether the data passed Shapiro–Wilk testing. Pearson's correlation coefficient was used to assess correlation between continuous variables. Results were considered statistically significant at p<0.05. All analyses were performed using Prism 8 (GraphPad Software, San Diego, CA, USA).
Results
Hyperglycaemia differentially affects ion channel function in NHBE cellsin vitro
NHBE cells cultured at the ALI were exposed to high levels of glucose in the basolateral media (12.5 mM or 225 mg·dL−1). Glucose levels decreased over time, reaching normal blood levels 48 h after media change (figure 1a). BK channel conductance was measured at 24 h and 72 h after media change, corresponding to time points with high glucose conditions (24 h range: 7.2–12.5 mM) and normal glucose conditions (72 h range: 3.1–5.7 mM), respectively. BK channel currents were reduced by 50% in NHBE cells under high glucose conditions (at 24 h) compared to normal glucose conditions (at 72 h) (figures 1band 1c).
BK channels comprise the pore-forming α-subunit, encoded by theKCNMA1gene and the auxiliary β- and γ-subunits [28]. BK channel function in airway epithelial cells requires the γ-regulatory subunit LRRC26 and is directly related toLRRC26expression [16]. We found that NHBE cells under high glucose conditions expressed lower levels ofLRRC26mRNA compared to cells under normal glucose conditions (figure 1d), consistent with the functional BK channel data. On the other hand,KCNMA1mRNA expression levels remained unchanged (figure 1e). NHBE cells under high glucose conditions showed a significantly greater response to mallotoxin, a BK channel opener that is more effective in the absence of LRRC26 (figures 1fand 1g), providing further evidence that decreases in BK channel conductance induced by high glucose conditions are likely due to reducedLRRC26expression.
To determine the extent by which chronic elevation in glucose induces changes in ion channel function, we cultured NHBE cells in ALI media supplemented with either a 5.5 mM (normal) or 12.5 mM (high) glucose level throughout redifferentiation at the ALI. High glucose level did not significantly impact the transepithelial electrical resistance (TEER) of cultures (supplementary figure S1). However, BK channel activity was significantly reduced in NHBE cells cultured under high glucose levels compared to normal glucose (figures 2aand 2b). On the other hand, NHBE cells cultured under high glucose conditions showed a significant increase in CFTR conductance (figures 2d–2f), with a concomitant decrease in the activity of calcium-activated chloride channels (CaCCs) (supplementary figure S1). Expression ofCFTR信使rna是glucos与水平升高相关e (supplementary figure S1). Despite this increase, the change in ASL volume over a 24-h period was not significantly different between NHBE cells cultured under high glucose compared to those cultured under normal glucose (figure 2c), likely due to the balance of changes in ion channel activities.
Hyperglycaemia induces the expression of inflammatory markers in NHBE cellsin vitro
Hyperglycaemia can activate a pronounced inflammatory response [5], but whether it increases expression of inflammatory markers in the airway epithelium remains largely unknown. Compared to NHBE cells cultured under normal glucose conditions, NHBE cells cultured under high glucose conditions showed significant increases in the mRNA expression levels of a number of proinflammatory cytokines, includingIL-1β,IL-6andIL-8(figures 3a–3c), as well as the inflammatory markers matrix metalloproteinase-9 (MMP-9) and transforming growth factor-β1 (TGF-β1) (figures 3dand 3e).
Hyperglycaemia induces BK channel dysfunction, loss of ASL volume and inflammation in CFBE cellsin vitro
We next sought to determine the impact of high glucose conditions on BK channel function and ASL volume in primary CFBE cellsin vitro(CFTR mutations of donor lungs are listed insupplementary table S2). CFBE cells cultured under high glucose conditions (24 h range: 10.1–12.5 mM) showed a significant reduction in BK channel function compared to CFBE cells cultured under normal glucose conditions (24 h range: 2.5–5.5 mM) (figures 4aand 4b andsupplementary figure S2). The decrease in BK channel activity in CFBE cells also correlated with reducedLRRC26mRNA expression (figure 4c). Furthermore, CFBE cells under high glucose conditions showed significantly greater rates of ASL absorption over a 24 h period (figure 4d).KCNMA1mRNA expression was not affected by glucose levels (figure 4e), while ATP-stimulated K+currents in CFBE cells cultured under both normal and high glucose conditions were completely inhibited by the BK channel blocker paxilline (PAX) (figures 4gand 4h).
KCa3.1 potassium channels, which are encoded by theKCNN4gene, are also expressed at the apical membrane of bronchial epithelial cells and can be blocked by 1-((2-chlorophenyl)diphenylmethyl)-1H-pyrazole (TRAM-34) [29]. Glucose levels did not impactKCNN4mRNA expression in CFBE cells (figure 4f). Furthermore, while TRAM-34 had no effect on ATP-stimulated K+currents in CFBE cells cultured under normal glucose conditions (figure 4g), such currents were reduced by TRAM-34 in CFBE cells cultured under high glucose conditions, although the decrease was not significant (figure 4h).
High glucose conditions did not lead to a significant increase in the expression ofIL-1β,IL-8, orTGF-β1mRNA in CFBE cells (supplementary figure S3). On the other hand, CFBE cells cultured under high glucose conditions did show significant increases in the mRNA expression ofMMP-9and cyclooxygenase-2 (COX-2) compared to CFBE cells cultured under normal glucose media (figures 5aand 5b). AGE are formed during hyperglycaemia and bind to the pro-inflammatory RAGE [14]. Interestingly, CFBE cells under high glucose conditions demonstrated a significant increase in the expression ofRAGEmRNA compared to CFBE cells under normal glucose conditions (figure 5c). A similar increase inRAGEmRNA expression was not observed in NHBE cells in response to high glucose conditions (figure 3f). We also detected a significant increase in secreted IL-1β and IL-8 protein in the basolateral media of CFBE cells cultured under high glucose conditions compared to CFBE cells cultured under normal glucose conditions (figures 5dand 5e). Levels of IL-6 in the basolateral media remained unchanged (figure 5e).
The RAGE agonist HMGB1 induces BK channel dysfunction and ASL volume loss in CFBE cellsin vitro
It is possible that the increase inRAGEexpression in CFBE cells is caused by BK channel dysfunction induced by hyperglycaemia. To address this possibility, we treated CFBE cells cultured under normal (5.5 mM) glucose conditions with recombinant HMGB1 protein and measured BK channel function. HMGB1 is a RAGE ligand and marker of airway epithelial injury that induces a strong pro-inflammatory response [30–32]. CFBE cells treated with HMGB1 (100 ng·mL−1) in the basolateral media for 24 h showed a significant reduction in BK channel function compared to controls (figures 6aand 6b). The reduction in BK channel activity correlated with increased ASL absorption (figure 6c). These data suggest that RAGE activation is upstream of BK channel dysfunction.
CGM variables for hyperglycaemia correlate with decreasedLRRC26expression in those with CF and CFRD
Based on ourin vitrodata, we initiated an observational study to determine whether there was a correlation between glycaemic excursions (as measured by CGM) andLRRC26expression in the upper airways of CF patients. A total of 21 participants (CF (n=6) and CFRD (n=15)) were enrolled in the study (seesupplementary table S3for baseline participant characteristics). Four subjects (CF (n=1) and CFRD (n=3)) were excluded from analysis due to monitor malfunction, monitor use less than 5 days, or inadequate mRNA in nasal brush samples. The CFRD group was older (mean age: 32 yearsversus22 years; 95% CI of difference between groups 5.3–14.7) and had lower baseline lung function (mean % predicted forced expiratory volume in 1 s (FEV1): 64%versus98%; 95% CI of difference between groups −50.77 to −17.23) as compared to the CF group without diabetes. There were no differences inCFTRmutation group (F508del homozygous or heterozygote/minimum function) or CFTR modulator use between groups. CGM variables of interest were uniformly higher in the CFRD group as compared to the CF group (supplementary table S4).
A significant, inverse correlation was found betweenLRRC26expression in nasal cells and percentage time of glucose >140 mg·dL−1(r= −0.51, p=0.03), percentage time glucose >200 mg·dL−1(r= −0.54, p=0.02) and AUC for glucose >180 mg·dL−1(r= −0.48, p=0.04) (figures 7a–7c). Other CGM variables of interest, including GMI (r= −0.46, p=0.06) and MAGE (r= −0.35, p=0.17), demonstrated a negative trend withLRRC26expression but did not achieve significance (figures 7dand 7e). There was no significant association between age andLRRC26expression (or a difference inLRRC26expression) between female and male sex or CF and CFRD (supplementary figure S4). A significant correlation was found in the CFRD group betweenRAGEexpression and CGM variables including percentage time glucose >140 mg·dL−1and MAGE (p<0.05) (figures 8aand 8b). Other CGM variables of interest also demonstrated positive trends but failed to reach significance. As this small sample size is sensitive to outliers, excluding the participant with the greatestRAGEexpression resulted in a significant association betweenRAGEexpression and AUC for glucose >180 mg·dL−1, percentage time glucose >200 mg·dL−1and GMI (p<0.05) (figures 7c–7e).
Discussion
CFRD is a common extrapulmonary comorbidity in adults with CF and is associated with increased lung function decline [1,2]. However, the mechanisms underlying this association are unknown. In this study, we demonstrated in anin vitromodel of the CF airway epithelia that hyperglycaemia leads to impaired BK channel function, decreased ASL volume and elevation in the expression of inflammatory markers. This finding was confirmedin vivowhereLRRC26expression, which predicts BK channel function in airway epithelia [13,27,33,34], was inversely correlated with CGM variables for hyperglycaemia in study participants with CF and CFRD.
BK channels are likely to be the primary apically expressed K+channels in the airway epithelium [13,16]. BK channel function is critical for ASL hydration since inhibition, either by knockdown of the pore-forming α-subunit KCNMA1 or of LRRC26, causes a significant reduction in ASL volume, even in the presence of fully functional CFTR [16]. Interestingly, although high glucose conditions correlated with reduced BK channel function in NHBE cells, ASL volumes were largely unaffected. This was likely due to the unexpected increase in CFTR conductance in these cells, suggesting that the airway epithelium employs mechanisms that differentially affect ion channel function to maintain ASL homeostasis during hyperglycaemic excursions. Although a previous study found that hyperglycaemia leads to a reduction in CFTR conductance, this study was conducted at higher glucose concentration (25 mM) in CFBE cell monolayers transduced with wild-type CFTR and may not necessarily reflect endogenous CFTR function in NHBE cells [12]. When CFTR function is limited or absent, as in CF, the airway epithelium is unlikely to maintain ASL homeostasis when glucose levels become elevated. Indeed, we found increased rates of ASL absorption in CFBE cells challenged with high glucose levels that correlated with reduced BK channel function andLRRC26expression.
It is likely that hyperglycaemia also affects other ion channels that are important for ASL hydration. For example, activation of KCa3.1 channels, which are also expressed at the apical membrane, is thought to increase chloride secretion and improve airway hydration [28,35]. Although high glucose levels did not have a significant impact on the expression or function of KCa3.1 channels in our experiments, we cannot completely rule out the possibility that they contribute to hyperglycaemia-induced changes in mucociliary clearance. Furthermore, our data in NHBE cells show that uridine-5'-triphosphate (UTP)-stimulated Cl−currents are impacted by high glucose levels. However, these studies provide some of the firstin vitroevidence that high glucose conditions can directly impact the expression and function of an ion channel known to be critical for mucociliary clearance in the CF airways.
The underlying link between BK channel dysfunction and hyperglycaemia is unclear, but we hypothesise that it is related to inflammation. In hyperglycaemia the AGE–RAGE signalling axis is upregulated and this contributes to excess inflammation [14]. We found that CFBE cells under high glucose conditions have significantly elevated levels ofRAGEmRNA expression. Interestingly, we did not see a similar increase inRAGEexpression in NHBE cells under high glucose conditions. This is consistent with previous reports that showed significantly higher levels ofRAGEexpression in the sputum of CF and CFRD subjects compared to non-CF diabetic subjects [11]. Indeed, levels of RAGE and RAGE ligands have been found to be increased in CF airways and levels of AGE have been shown to negatively correlate with % predicted FEV1[10,11]. Furthermore, levels of HMGB1 are significantly elevated in the sputum and serum of CF patients and particularly at the onset of CFRD [33]. The increase in IL-1β secretion in CFBE cells in response to high glucose levels is also particularly interesting in light of a recent study demonstrating the importance of IL-1β in promoting mucus hypersecretion and increasing the expression of proinflammatory mediators in CF airways in the absence of infection [36]. Levels ofLRRC26expression are sensitive to inflammation in CFBE cells [33]. Thus, future studies will focus on how RAGE activation and possibly Il-1β may potentially regulate BK channel function.
Based on thesein vitrodata, we initiated a clinical study to determine whetherLRRC26expression is impacted by variations in glucose in CF patients. Interestingly, there was no significant difference inLRRC26expression between CF and CFRD patients. Instead, the inverse correlation withLRRC26表达发生在一系列的血糖control in both groups. This suggests glycaemic control is an important factor in both CF and CFRD. Also, a correlation betweenRAGEexpression and hyperglycaemia was observed in participants with CFRD, mirroring thein vitroresults. However, there were several limitations to our study. First, we relied on historical OGTT to define CF and CFRD, which could have led to incorrect categorisation of participants. However, as our results suggest that level of glycaemic control was more strongly associated withLRRC26expression than CFRD status, this was not likely to affect our findings. Secondly, a nonsignificant but noticeable discrepancy in CGM sampling periods between the two groups was observed. While the CGM variables are means and proportions, which are less likely to be affected by differences in sampling length, this difference could lead to misestimation of true glycaemic control. Thirdly, while CFTR modulator use was not found to be associated withLRRC26expression, we were not able to compare differences between specific CFTR modulators given our relatively small number of subjects. It should be noted that our aims were exploratory and no corrections were made for multiple comparisons given the small sample size.
Importantly, we believe these findings remain relevant in the context of highly effective CFTR modulator therapy. Previous observational studies have demonstrated that there are improvements in insulin secretion after initiation of highly effective CFTR modulator therapy. However, their potential effects on hyperglycaemic excursions are less well known [37,38]. A study of subjects on ivacaftor showed that, while there was an increase in insulin after 16 weeks of ivacaftor use, there was no significant improvement in peak glucose or AUC for glucose during a mixed meal tolerance test [39]. While this study was small in size and subjects had mostly normal OGTTs prior to ivacaftor use, it highlights that glycaemic excursions may persist in those receiving highly effective modulators. Furthermore, impaired pancreatic function and glycaemic abnormalities are present at birth in the CF ferret model and are prevalent in infants and young children with CF, suggesting earlier interventions are needed to preserve lung function in CFRD [40,41]. Thus, there is a need for continued investigation into the effects of hyperglycaemia on pulmonary function decline in CF.
In conclusion and summarised infigure 9, we have demonstrated that hyperglycaemia increases inflammation with a detrimental effect onLRRC26expression and therefore impairment of BK channel function. To our knowledge this is the first study to provide a connection between hyperglycaemic excursions and worse parameters important for mucociliary function in CF. Further mechanistic and interventional studies are needed to define therapies that will improve the disparity in outcomes between CF and CFRD.
Supplementary material
Supplementary Material
Please note:supplementary material is not edited by the Editorial Office, and is uploaded as it has been supplied by the author.
Supplementary materialERJ-00509-2020.SUPPLEMENT
Shareable PDF
Supplementary Material
This one-page PDF can be shared freely online.
Shareable PDFERJ-00509-2020.Shareable
Acknowledgements
We would like to acknowledge Michael Myerburg (University of Pittsburgh School of Medicine, PA, USA) for providing the ASL meniscus scanning software. We also thank the Life Alliance Organ Recovery Agency (University of Miami, FL, USA), LifeCenter Northwest (Seattle, WA, USA) and the Midwest Transplant Network (Kansas City, KS, USA) for providing the lungs. Finally, we owe a great deal of thanks to all of the CF patients in the University of Kansas Health System who participated in the study.
Footnotes
This article has supplementary material available fromwww.qdcxjkg.com
Author contributions: Conceived and designed the study: C.D. Bengtson, A. Anabtawi, N. Baumlin, M.D. Kim and M. Salathe. Executed experiments and analysed the data: all authors. Clinical study recruitment, data collection and data interpretation: C.D. Bengtson, A. Anabtawi, M.D. Kim and M. Salathe. Wrote the manuscript: C.D. Bengtson, M.D. Kim and M. Salathe. Discussed the results and commented on the manuscript: all authors.
Conflict of interest: C.D. Bengtson reports grants from the Cystic Fibrosis Foundation and NCATS, during the conduct of the study.
Conflict of interest: M.D. Kim reports grants from the NIH and the Cystic Fibrosis Foundation, during the conduct of the study; and grants from the Flight Attendant Medical Research Institute and the James and Esther King Florida Biomedical Research Program, outside the submitted work.
Conflict of interest: A. Anabtawi reports grants from the Cystic Fibrosis Foundation and University of Kansas Pilot Support, during the conduct of the study.
Conflict of interest: J. He reports grants from the Cystic Fibrosis Foundation, during the conduct of the study.
Conflict of interest: J.S. Dennis has nothing to disclose.
Conflict of interest: S. Miller has nothing to disclose.
Conflict of interest: M. Yoshida has nothing to disclose.
Conflict of interest: N. Baumlin reports grants from the NIH and the Cystic Fibrosis Foundation, during the conduct of the study; and grants from the Flight Attendant Medical Research Institute and the James and Esther King Florida Biomedical Research Program, outside the submitted work.
Conflict of interest: M. Salathe reports grants and personal fees from the NIH and the Cystic Fibrosis Foundation, during the conduct of the study; grants and personal fees from Arrowhead Pharmaceuticals and the Flight Attendant Medical Research Institute, outside the submitted work; and grants from the COPD Foundation and the James and Esther King Florida Biomedical Research Program, outside the submitted work.
支持声明:本研究在b部分资助y the Cystic Fibrosis Foundation (SALATH18I0, BENGTS19AC0), the National Heart, Lung, and Blood Institute (NHLBI) (R01 HL-133240) and a CTSA TL-1 training grant under UL1-TR002366 (NCATS) awarded to the University of Kansas for Frontiers: University of Kansas Clinical and Translational Science Institute (TL1TR002368). The contents are solely the responsibility of the authors and do not necessarily represent the official views of the National Institutes of Health (NIH) or NCATS. Funding information for this article has been deposited with theCrossref Funder Registry.
- ReceivedMarch 1, 2020.
- AcceptedJuly 20, 2020.
- Copyright ©ERS 2021