Ion Transport In Cystic Fibrosis Research Articles

Potential of Intestinal Current Measurement for Personalized Treatment of Patients with Cystic Fibrosis.

Refinement of personalized treatment of cystic fibrosis (CF) with emerging medicines targeting the CF basic defect will likely benefit from biomarkers sensitive to detect improvement of cystic fibrosis transmembrane conductance regulator (CFTR) function in individual patients. Intestinal current measurement (ICM) is a technique that enables quantitative assessment of CFTR chloride channel function in rectal tissues or other intestinal epithelia. ICM was originally developed to study the CF ion transport defect in the intestine and has been established as a sensitive biomarker of CFTR function and diagnostic test for CF. With the emergence of CFTR-directed therapeutics, ICM has become an important tool to estimate the level of rescue of CFTR function achieved by approved CFTR modulators, both at the level of CFTR genotype groups, as well as individual patients with CF. In combination with preclinical patient-derived cell culture models, ICM may aid the development of targeted therapies for patients with rare CFTR mutations. Here, we review the principles of ICM and examine how this CFTR biomarker may be used to support diagnostic testing and enhance personalized medicine for individual patients with common as well as rare CFTR mutations in the new era of medicines targeting the underlying cause of CF.

Epithelial vectorial ion transport in cystic fibrosis: Dysfunction, measurement, and pharmacotherapy to target the primary deficit.

Cystic fibrosis patients display multi-organ system dysfunction (e.g. pancreas, gastrointestinal tract, and lung) with pathogenesis linked to a failure of Cl− secretion from the epithelial surfaces of these organs. If unmanaged, organ dysfunction starts early and patients experience chronic respiratory infection with reduced lung function and a failure to thrive due to gastrointestinal malabsorption. Early mortality is typically caused by respiratory failure. In the past 40 years of newborn screening and improved disease management have driven the median survival up from the mid-teens to 43–53, with most of that improvement coming from earlier and more aggressive management of the symptoms. In the last decade, promising pharmacotherapies have been developed for the correction of the underlying epithelial dysfunction, namely, Cl− secretion. A new generation of systemic drugs target the mutated Cl− channels in cystic fibrosis patients and allow trafficking of the immature mutated protein to the cell membrane (correctors), restore function to the channel once in situ (potentiators), or increase protein levels in the cells (amplifiers). Restoration of channel function prior to symptom development has the potential to significantly change the trajectory of disease progression and their evidence suggests that a modest restoration of Cl− secretion may delay disease progression by decades. In this article, we review epithelial vectorial ion and fluid transport, its quantification and measurement as a marker for cystic fibrosis ion transport dysfunction, and highlight some of the recent therapies targeted at the dysfunctional ion transport of cystic fibrosis.

Influence of perfusate temperature on nasal potential difference

Nasal potential difference (NPD) quantifies abnormal ion transport in cystic fibrosis. It has gained acceptance as an outcome measure for the investigation of new therapies. To quantify the effect of solution temperature on NPD, we first examined the effect of switching from room temperature (20-25°C) to warmed (32-37°C) solutions and vice versa during each perfusion step. Secondly, standard protocols were repeated at both temperatures in the same subjects. Changing solution temperature did not alter NPD during perfusion with Ringer's solution (<1 mV) (p>0.1). During perfusion with zero chloride solution, changing from room temperature to warmed solutions tended to decrease absolute NPD (i.e. it became less negative) by 0.9 mV (p>0.1); changing from warmed to room temperature increased NPD by 2.1 mV (p<0.05). During isoprenaline perfusion, changing from room temperature to warmed solutions increased NPD by 1.5 mV (p<0.01) and from warmed to room temperature decreased NPD by 1.4 mV (p<0.05). For full protocols at room temperature or warmed in the same subjects, mean values were similar (n = 24). During warmed perfusion, group results for total chloride response had a larger standard deviation. As this increased variability will probably decrease the power of trials, this study suggests that solutions at room temperature should be recommended for the measurement of NPD.

The ENaC-overexpressing mouse as a model of cystic fibrosis lung disease

Chronic lung disease remains the major cause of morbidity and mortality of cystic fibrosis (CF) patients. Cftr mutant mice developed severe intestinal obstruction, but did not exhibit the characteristic CF ion transport defects (i.e. deficient cAMP-dependent Cl − secretion and increased Na + absorption) in the lower airways, and failed to develop CF-like lung disease. These observations led to the generation of transgenic mice with airway-specific overexpression of the epithelial Na + channel (ENaC) as an alternative approach to mimic CF ion transport pathophysiology in the lung. Studies of the phenotype of βENaC-transgenic mice demonstrated that increased airway Na + absorption causes airway surface liquid (ASL) depletion, reduced mucus transport and a spontaneous CF-like lung disease with airway mucus obstruction and chronic airway inflammation. Here, we summarize approaches that can be applied for studies of the complex in vivo pathogenesis and preclinical evaluation of novel therapeutic strategies in this model of CF lung disease.

Airway Delivery of Low-Dose Miglustat Normalizes Nasal Potential Difference in F508del Cystic Fibrosis Mice

N-butyldeoxynojyrimicin (NB-DNJ, miglustat [Zavesca]) an approved drug for treating Gaucher disease, was reported to be able to correct the defective trafficking of the F508del-CFTR protein. To evaluate the efficacy of in vivo airway delivery of miglustat for restoring ion transport in cystic fibrosis (CF). We used nasal transepithelial potential difference (PD) as a measure of sodium and chloride transport. The effect of nasal instillation of a single dose of miglustat was investigated in F508del, cftr knockout and normal homozygous mice. The galactose iminosugar analog N-butyldeoxygalactonojirimycin (NB-DGJ) was used as a placebo. In F508del mice, sodium conductance (evaluated by basal hyperpolarization) and chloride conductance (evaluated by perfusing the nasal mucosa with chloride-free solution in the presence of amiloride and forskolin) were normalized 1 hour after an intranasal dose of 50 picomoles of miglustat. Chloride conductance in the presence of 200 microM 4-4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS), an inhibitor of alternative chloride channels, was much higher after miglustat than after placebo. In cftr knockout mice, a normalizing effect was observed on sodium but not on chloride conductance. Our results provide clear evidence that nasal delivery of miglustat, at picomolar doses, normalizes sodium and Cftr-dependent chloride transport in F508del transgenic mice; they highlight the potential of topical miglustat as a therapy for CF.

Normal and Cystic Fibrosis Airway Surface Liquid Homeostasis: THE EFFECTS OF PHASIC SHEAR STRESS AND VIRAL INFECTIONS

Mammalian airways normally regulate the volume of a thin liquid layer, the periciliary liquid (PCL), to facilitate the mucus clearance component of lung defense. Studies under standard (static) culture conditions revealed that normal airway epithelia possess an adenosine-regulated pathway that blends Na+ absorption and Cl- secretion to optimize PCL volume. In cystic fibrosis (CF), the absence of CF transmembrane conductance regulator results in a failure of adenosine regulation of PCL volume, which is predicted to initiate mucus stasis and infection. However, under conditions that mimic the phasic motion of the lung in vivo, ATP release into PCL was increased, CF ion transport was rebalanced, and PCL volume was restored to levels adequate for lung defense. This ATP signaling system was vulnerable, however, to insults that trigger CF bacterial infections, such as viral (respiratory syncytial virus) infections, which up-regulated extracellular ATPase activity and abolished motion-dependent ATP regulation of CF PCL height. These studies demonstrate (i) how the normal coordination of opposing ion transport pathways to maintain PCL volume is disrupted in CF, (ii) the hitherto unknown role of phasic motion in regulating key aspects of normal and CF innate airways defense, and (iii) that maneuvers directed at increasing motion-induced nucleotide release may be therapeutic in CF patients.

Culture of murine nasal epithelia: model for cystic fibrosis

The ion transport defects reported for human cystic fibrosis (CF) airways are reproduced in nasal epithelia of the CF mouse. Although this tissue has been studied in vivo using the nasal potential difference technique and as a native tissue mounted in the Ussing chamber, little information is available on cultured murine nasal epithelia. We have developed a polarized cell culture model of primary murine nasal epithelia in which the CF tissue exhibits not only a defect in cAMP-mediated Cl- secretion but also the Na+ hyperabsorption and upregulation of the Ca2+-activated Cl- conductance observed in human airways. Both the wild-type and CF cultures were constituted predominantly of undifferentiated cuboidal columnar cells, with most cultures exhibiting a small number of ciliated cells. Although no goblet cells were observed, RT-PCR demonstrated the expression of Muc5ac RNA after approximately 22 days in culture. The CF tissue exhibited an adherent layer of mucus similar to the mucus plaques reported in the distal airways of human CF patients. Furthermore, we found that treatment of CF preparations with a Na+ channel blocker for 7 days prevented formation of mucus adherent to epithelial surfaces. The cultured murine nasal epithelial preparation should be an excellent model tissue for gene transfer studies and pharmacological studies of Na+ channel blockers and mucolytic agents as well as for further characterization of CF ion transport defects. Culture of nasal epithelia from DeltaF508 mice will be particularly useful in testing drugs that allow DeltaF508 CFTR to traffic to the membrane.

Blue Native/SDS-PAGE Analysis Reveals Reduced Expression of the mClCA3 Protein in Cystic Fibrosis Knock-out Mice

Cystic fibrosis (CF) is a frequent autosomal recessive disorder caused by mutation of a gene encoding a multifunctional transmembrane protein, the cystic fibrosis transmembrane conductance regulator (CFTR), located in the apical membrane of epithelial cells lining exocrine glands. In an attempt to get a more complete picture of the pleiotropic effects of the CFTR defect on epithelial cells and particularly on the membrane compartment, a bidimensional blue native (BN)/SDS-PAGE-based proteomic approach was used on colonic crypt samples from control and CFTR knock-out mice (cftr-/-). This approach overcomes the difficulties of membrane protein analysis by conventional two-dimensional PAGE and is able to resolve multiprotein complexes. Used here for the first time on crude membrane proteins that were extracted from murine colonic crypts, BN/SDS-PAGE allows effective separation of protein species and complexes of various origins, including mitochondria, plasma membrane, and intracellular compartments. The major statistically significant difference in protein maps obtained with samples from control and cftr-/- mice was unambiguously identified as mClCA3, a member of a family of calcium-activated chloride channels considered to be key molecules in mucus secretion by goblet cells. On the basis of this finding, we evaluated the overall expression and localization of mClCA3 in the colonic epithelium and in the lung of mice by immunoblot analysis and immunohistochemistry. We found that mClCA3 expression was significantly decreased in the colon and lung of the cftr-/- mice. In an ex vivo assay, we found that the Ca2+-dependent (carbachol-stimulated) glycoprotein secretion strongly inhibited by the calcium-activated chloride channel blocker niflumic acid (100 microm) was impaired in the distal colon of cftr-/- mice. These results support the conclusion that a ClCA-related function in the CF colon depends on CFTR expression and may be correlated with the impaired expression of mClCA3.

350. Pre-Clinical Assays for Cystic Fibrosis Gene Therapy

Cystic fibrosis (CF) has long been a model disease to study lung gene therapy and we and others have carried out a number of phase I/II clinical trials using viral or non-viral vectors carrying the cystic fibrosis transmembrane conductance regulator (CFTR) gene. In addition to safety, measurements of potential difference (PD) across the airway epithelium, which is altered in CF due to the altered ion transport, have often been included as a primary endpoint. However, although important, the information obtained from PD measurements in pre-clinical and clinical gene therapy research has to be interpreted carefully, because changes in PD after transfection with the CFTR gene do not provide information about the number of cells transfected. In addition, it is unclear, how a change in PD after gene therapy correlates with more clinically relevant endpoints. The UK CF Gene Therapy Consortium has, therefore, devoted a significant amount of time and resources to refine further existing assays and set-up and validate a panel of new pre-clinical and clinical assays to screen non-viral gene transfer agents.The CF knockout mouse is the only available CF animal model and although the CF mouse does not develop traditional CF lung disease, it has long been demonstrated that the CF ion transport abnormalities are recapitulated in the murine nasal epithelium. Importantly, the cell composition of the murine nasal epithelium is also similar to the human lower airways. We have chosen the FABp-CF knockout mouse (Zhou 1994) as the standard model in our core facility. Fatty acid binding promoter (FABp)-mediated expression of CFTR in the gut, but not the airways, prevents the intestinal disease of the CF knockout mouse, thus easing work in a core facility setting. When choosing assays we aimed to cover several aspects of defective CFTR level and function [1. CFTR protein (vector- and epithelial cell-specific detection of CFTR mRNA and protein). 2. Ion transport (PD and short circuit current measurements). 3. Airway surface liquid (ASL) (ASL height), 4. Host bacterial interaction (Bacterial adherence to airway epithelial cells). During assay validation we compared FABP-CF mice and wild-type littermates and used the results to carry out power calculations that informed cohort size when assessing efficiency of gene transfer agents. Importantly, we learnt that n numbers have to be large to detect small, but potentially clinically relevant, changes in these assays and that care has to be taken not to underpower assays for assumed treatment effects.

The Effect of Varying Tonicity on Nasal Epithelial Ion Transport in Cystic Fibrosis

There is reasonable evidence that the fluid layer of the airway epithelium is exposed to changes in tonicity. The inspiration of cool, dry air causes an increased tonicity, whereas this tonicity may be decreased by glandular secretions. We hypothesized that the cystic fibrosis transmembrane conductance regulator (CFTR) is involved in the responses to changes in tonicity and that these may be altered in cystic fibrosis (CF). Using nasal potential difference (PD) protocols in 8 subjects with CF and 10 subjects without CF, we investigated the effects of hyper- and hypotonicity on ion transport processes. We found significant differences between the two groups. In response to a hypertonic challenge (mannitol 500 mM), there was a decreased PD in both groups, suggesting decreased sodium absorption. However, after the prior inhibition of sodium transport using amiloride, there was an increased PD in the non-CF group alone, suggesting CFTR-mediated chloride secretion in response to luminal hypertonicity. For the hypotonic solution, we found that hypotonicity inhibited CFTR-mediated chloride secretion in the non-CF group. These data suggest that CFTR plays a role in the recognition and regulation of airway fluid tonicity.

Macromolecular interactions and ion transport in cystic fibrosis.

Cystic fibrosis (CF) is a genetic disease caused by autosomal recessive mutations of the CF transmembrane regulator, CFTR. CFTR functions in the plasma membrane of epithelial cells lining the lung, pancreas, liver, intestines, sweat duct, and the epididymis. The primary problem in CF is that mutations in CFTR affect its ability to be made, processed, and trafficked to the plasma membrane and/or its function as a Cl(-) channel and conductance regulator. Many proteins and processes normally interact with normal CFTR throughout its life cycle and mutant CFTR during the disease process. Understanding the function of these proteins and processes is expected to provide a clearer understanding of how normal CFTR is involved in salt movement and how mutant CFTR is handled by the cell and leads to the pathophysiology of CF. Recently, efforts to find therapies that correct defective CFTR have been intensifying. To facilitate our understanding of normal and mutant CFTR and the identification of new drug targets for developing novel therapies, a panel of experts was convened by the National Heart, Lung, and Blood Institute to explore the critical questions, challenges, and current opportunities to highlight new areas of research that would facilitate a integrated understanding of the processes and proteins that impact CFTR. The meeting highlighted the multiple pathways and interacting proteins involved in CFTR folding and biosynthesis, processing, and trafficking. A number of critical areas for future study were identified. Although these therapies are promising, a big question remains as to whether simply correcting defective CFTR will lead to significant improvement in patient health or whether the symptoms manifested in CF will require therapies in addition to those that target defective CFTR specifically.

Optimisation of real-time quantitative RT-PCR for the evaluation of non-viral mediated gene transfer to the airways.

Naked plasmid DNA and DNA/liposome complexes are currently being considered as gene therapy treatments for cystic fibrosis (CF) pulmonary disease. Current methods of gene delivery to the airways result only in transient correction of the CF ion transport defect, and disease treatment is likely to require repeated administrations of vector. However, it is unclear if repeat administration will be tolerated by CF individuals. Technologies including TaqMan (Applied Biosystems) real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) can be used to determine the efficacy of gene transfer formulations. TaqMan RT-PCR assays were designed and optimised to detect plasmid vector-derived and endogenous gene expression. Subsequently, these assays were used to quantify vector-derived mRNA after delivery of naked DNA and DNA/liposome formulations expressing human and murine cystic fibrosis transmembrane conductance regulator (CFTR) to the mouse airways. Vector-derived mRNA was detected in samples following the delivery of naked DNA or DNA/liposomes to the mouse airways, and no reduction in vector-derived mRNA was observed upon repeat administration, a finding that is consistent with the murine and human CFTR being tolerated by the mouse. Although it remains to be seen if CF patients can tolerate long-term expression of wild-type CFTR, these data demonstrate that TaqMan RT-PCR is an effective tool to accurately quantify transgene expression in the airways.

The CF mouse: an important tool for studying cystic fibrosis

Cystic fibrosis (CF) is a common and fatal recessive disease, which is caused by dysfunction of a chloride ion channel, termed the CF transmembrane conductance regulator (CFTR). The CF gene was cloned in 1989; subsequently, several mouse models have been created using gene targeting within embryonic stem cells. This report describes how such animal models provide the opportunity to elucidate disease pathogenesis, correlate genotype with phenotype and develop novel therapies. The current models encompass mice with a complete knockout of CFTR function, with residual CFTR function, and with precise mutations corresponding to those in humans that precipitate CF. All the CF mice demonstrate the characteristic CF ion-transport defect and show some evidence of intestinal disease, but they have a variable level of survival. Genetic background has also been shown to affect the intestinal phenotype of CF mice and this has allowed identification of a genetic modifier locus of CF in humans. Lung disease in human CF is the major cause of death in early adulthood. This is not entirely reproduced in CF mice, but repeat exposure of the lung to clinical pathogens does reveal a significantly abnormal pathogen-related response in the residual-function mice. CF mice have been successfully used to investigate the safety and efficacy of various pharmacological and gene-therapy protocols. As new cloning techniques become available, the models can be refined to ensure that in vivo models continue to be an essential tool for studying CF.

Outpatient care of the cystic fibrosis patient after lung transplantation.

Lung transplantation has become an accepted treatment for respiratory failure due to cystic fibrosis (CF). Effective means of patient selection, surgical technique, immunosuppression, and post-transplant management permit survival as good as that of transplant patients with other diseases. The new lungs do not acquire the CF ion transport abnormalities but are subject to the usual post-transplant complications. CF problems in other organ systems persist and may be worsened by some of the immunosuppressive regimens. Prolonged survival increases the risk of age-related CF and other complications. Effective medical management requires expert knowledge of CF and lung transplantation and of how their problems interact, and good communications among the participating care teams.

A new device for in vivo measurement of nasal transepithelial potential difference in cystic fibrosis patients and normal subjects.

Measurement of transepithelial potential difference (PD) on the nasal mucosa has been proposed to test for defective ion transport in cystic fibrosis (CF), and its possible correction after gene therapy or other treatments. The "classical" method records nasal PD under the inferior turbinate, with the disadvantage that the tip of the electrode is not seen by the operator. We have developed a purpose-designed perfusion electrode for PD recording on the visible, medial/posterior aspect of the turbinate. We wanted to determine whether such PD recordings adequately discriminate between CF patients and normal subjects. Measurements of baseline PD and response to a standardized perfusion protocol were performed in 20 normal subjects and 12 CF patients. Solutions of amiloride, with or without low chloride buffer were applied for 3 min. Increased baseline PD and depolarization after amiloride discriminated CF patients from normal subjects. Only one CF patient overlapped with the normal range. Superfusion of low chloride buffer with amiloride and terbutaline caused repolarization in 18 out of 20 normal subjects (90%), consistent with physiological Cl- secretion process, but in none of the CF patients. We conclude that measurements of potential difference on the medial/posterior aspect of the turbinate can discriminate between cystic fibrosis patients and normal subjects. At this site, visual control of the measurement is possible, and the mucosa is easily accessible for subsequent cytological sampling or biopsy.

X-ray microanalysis of cAMP-induced ion transport in cystic fibrosis fibroblasts

cAMP-induced ion transport in normal and cystic fibrosis (CF) fibroblasts was investigated by X-ray microanalysis. Stimulation with cAMP causes an increase in cellular Na content and a decrease in cellular Cl and K content. No significant difference in response between CF and normal cells was noted. In this respect, fibroblasts differ from epithelial cells, where cAMP-induced Cl − efflux is blocked in CF patients. Isoproterenol produced similar changes in Na and K content as cAMP, but did not effect Cl content.

Defective epithelial ion transport in cystic fibrosis.

In America, cystic fibrosis (CF) is the most common fatal inherited disease. In the past few years new concepts have evolved regarding the etiology of CF, changing from an almost universal acceptance of the abnormality as a disease of abnormal mucus production to that of a disease of abnormal fluid and electrolyte transport. Although mucus production is increased, this probably occurs as a secondary defense or protection against more primary disturbances in electrolyte transport brought on by a defect in Cl- permeability that affects both fluid absorption and fluid secretion. Here I review the progress that has been made and some of the puzzles uncovered in recent investigations of electrolyte transport in secretory and absorptive processes in cystic fibrosis.

Kinetic Parameters of Rubidium Transport Pathways Are Normal in Cystic Fibrosis Red Cells

The abnormalities in ion transport in cystic fibrosis (CF) respiratory and sweat duct epithelia have prompted studies of ion permeability in CF red blood cells (RBC) although previous reports have been contradictory. In this study, the kinetic characteristics of the three major cation transport systems in RBC were evaluated by measuring rubidium (Rb) uptake at various external Rb concentrations. The maximal velocity and affinity for external Rb (K1/2) of the NaK pump were normal in CF RBC, as were the maximal velocity and Km for Rb of the NaK cotransport system. Residual (ouabain and bumetanide insensitive) Rb uptake, and steady state RBC Na and K contents were also normal. These data indicate the NaK pump and cotransport system do not exhibit primary or secondary perturbations in CF RBC, and suggest that the noncarrier-mediated membrane permeability to cations is also normal in these cells.

Abnormal Respiratory Epithelial Ion Transport in Cystic Fibrosis

The respiratory epithelium of cystic fibrosis patients exhibits excessive sodium (and volume) absorption and an absence of chloride (and volume) secretion in response to usual stimuli. These abnormalities likely contribute to the pathophysiology of cystic fibrosis lung disease, and provide a rationale for a novel therapeutic approach to this problem.