Entry of cystic fibrosis transmembrane conductance potentiator ivacaftor into the developing brain and lung

BackgroundThe potential effects of ivacaftor during pregnancy and breastfeeding on the offspring are still unknown. This study aimed to investigate pre-/postnatal age-related entry into the brain and lungs and transfer of maternally administered drug by the placental and via the milk.MethodsIn acute experiments Sprague Dawley rats at embryonic day (E) 19, postnatal days (P) 4, 9, 16, and adult were administered an intraperitoneal injection of ivacaftor (40 mg/kg) traced with [3H] ivacaftor. To determine tissue entry, plasma, cerebrospinal fluid (CSF), lungs and brains were collected, and radioactivity measured using liquid scintillation counting. For long term experiments pregnant dams were orally treated at 25 mg/kg/day for 7 days and pups collected at E19. For postnatal pups, dams received treatment for 7 or 14 days and pups were collected at P6, 9, 13 and 16. To estimate placental and milk transfer concentration of ivacaftor in pup & maternal plasma was determined by liquid chromatography–mass spectrometry.ResultsAt all ages, entry of ivacaftor into lungs, following either acute or prolonged exposure, was much higher than into brain & CSF. Brain entry appeared higher at earlier ages. Transfer across the placenta and breast milk. was estimated to be around ~40% of maternal plasma.ConclusionsFetal and postnatal rats were exposed to maternally administered ivacaftor via placental and milk transfer. Preferential entry in the lungs at all ages suggests the possibility that exposing CF babies to maternally administered ivacaftor could be beneficial for limiting progression of CF pathology in early development.     

Defects in processing and trafficking of cystic fibrosis transmembrane conductance regulator

In most epithelial tissues Cl(-) transport relies on the cystic fibrosis transmembrane conductance regulator (CFTR) which has dual function as a Cl(-) channel and as a regulator of other ion channels. More than 900 different mutations in the CFTR gene are the cause for defective transport of Cl(-) and Na(+) and impaired secretion or absorption of electrolytes in cystic fibrosis. However, the CFTR mutation delta F508 is the most common reason for the frequently inherited disease among the Caucasian population. Maturation and processing of delta F508-CFTR is defective which leads to expression of only very little but functional CFTR in the cell membrane. Understanding the processing and trafficking of CFTR may give a clue to the question as to how the expression and residual function of delta F508-CFTR can be enhanced, and may lead to the development of new pharmacological tools for the treatment of cystic fibrosis.     

Defects in processing and trafficking of the cystic fibrosis transmembrane conductance regulator

Cystic fibrosis (CF) is caused by inherited mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-regulated chloride channel expressed in epithelial tissues. Most mutations in CF patients result in rapid intracellular degradation of the CFTR protein. While this defect is thought to result from abnormal protein folding, it is unclear how mutant and wild-type (WT) proteins differ in structure, how the cell is able to distinguish these differences, and how the fate of the mutant protein is determined. By examining the initial steps of CFTR assembly into the endoplasmic reticulum (ER) membrane, it has recently been shown that CFTR utilizes two redundant translocation pathways to direct N-terminus folding events. Mutations that block one pathway therefore do not alter transmembrane topology, but rather appear to disrupt intracellular trafficking through perturbations in higher order tertiary structure. These studies suggest that cellular quality control machinery acts at least in part, by monitoring proper interactions between CFTR subdomains. The end result of this process is the conversion of misfolded CFTR into a membrane-bound, polyubiquitinated complex. This complex recruits cytosolic degradation machinery to the endoplasmic reticulum membrane where CFTR is degraded as it is extracted from the lipid bilayer. Understanding how cellular machinery mediates this process will be an important step in designing strategies to modify protein folding and degradation in CF and related ion channelopathies.     

Up-regulated cystic fibrosis transmembrane conductance regulator after anal stenosis in rats

BackgroundThe pathophysiology of fecal incontinence from fecal impaction and rectal distension is poorly understood. We hypothesize that fecal impaction elicits up-regulation of cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated mucosal chloride channel.MethodsThe anus was ligated to produce 75% stenosis in rats. Controls received ligation without inducing stenosis. 24 to 48 hours after ligation the colon was removed. Mucosal short-circuit current was measured by Ussing chamber. Western blot analysis was used to detect CFTR expression in the colonic mucosa. Ligated rats failed to defecate, whereas control rats stooled normally.ResultsLigated colons were markedly stool filled and dilated. Water content of feces was significantly increased to 66.5% ± 1.1% (P < .01, n = 12) 24 hours after ligation, vs controls (49.5 ± 5.2%, n = 12). Baseline short-circuit current was significantly increased in the distal (78.8 ± 7.4 μA/cm², n = 8, P < .01) and mid colon (24.5 ± 2.5 μA/cm², n = 8, P < .05) 24 hours after ligation, compared to control rats (12.5 ± 3.2 μA/cm2, n = 8). CFTR expression was significantly increased 24 hours after ligation in the mid and distal colon.ConclusionWe observe that fecal impaction from anal ligation induces early compensatory up-regulation of CFTR, altering function from net absorption to net secretion in the mid and distal colon.     

The cystic fibrosis transmembrane regulator (CFTR) in the kidney

The cystic fibrosis transmembrane regulator (CFTR) is a Cl - channel. Mutations of this transporter lead to a defect of chloride secretion by epithelial cells causing the Cystic Fibrosis disease (CF). In spite of the high expression of CFTR in the kidney, patients with CF do not show major renal dysfunction, but it is known that both the urinary excretion of drugs and the renal capacity to concentrate and dilute urine is deficient. CFTR mRNA is expressed in all nephron segments and its protein is involved with chloride secretion in the distal tubule, and the principal cells of the cortical (CCD) and medullary (IMCD) collecting ducts. Several studies have demonstrated that CFTR does not only transport Cl - but also secretes ATP and, thus, controls other conductances such as Na+ (ENaC) and K+ (ROMK2) channels, especially in CCD. In the polycystic kidney the secretion of chloride through CFTR contributes to the cyst enlargement. This review is focused on the role of CFTR in the kidney and the implications of extracellular volume regulators, such as hormones, on its function and expression.     

Immunolocalization and regulation of cystic fibrosis transmembrane conductance regulator in the adult rat epididymis

Cystic fibrosis is the most common serious autosomal recessive condition in whites, and more than 95% of men with cystic fibrosis are infertile. The cystic fibrosis transmembrane conductance regulator (CFTR), a cyclic adenosine monophosphate (cAMP)-regulated chloride channel, has been localized in the efferent ducts; however, to our knowledge, its expression and regulation in the epididymis by testicular factors have not been examined. In the present study, these parameters were examined immunocytochemically by the light microscope with an anti-CFTR antibody in Bouin-fixed, paraffin-embedded control adult rat epididymides and both orchidectomized adult rats with or without testosterone supplementation and efferent duct-ligated rats sacrificed at different time points. In control animals, a thick dense band of immunoperoxidase reaction product was visualized over the apical plasma membrane of the principal cells but not their microvilli. The apical band was prominent only in the corpus and cauda regions. While there was no CFTR expression in basal cells, clear cells of the corpus and cauda regions showed a weak-to-moderate band of apical plasma membrane staining. An examination of orchidectomized, orchidectomized and testosterone, and efferent duct-ligated rats revealed that CFTR was no longer expressed as an intense band on the apical plasma membrane of the principal cells of the corpus and cauda regions. However, under these conditions, an intense apical/supranuclear reaction was noted in the form of small vesicular structures. Clear cells were unaffected by the different experimental treatments. Together, these data indicate that CFTR is expressed in a cell- and region-specific manner and that, while its synthesis in principal cells is not under the control of testicular factors, targeting to the apical plasma membrane is regulated by a testicular luminal factor.     

AR-13 reduces antibiotic-resistant bacterial burden in cystic fibrosis phagocytes and improves cystic fibrosis transmembrane conductance regulator function

BackgroundThere are no effective treatments for Burkholderia cenocepacia in patients with cystic fibrosis (CF) due to bacterial multi-drug resistance and defective host killing. We demonstrated that decreased bacterial killing in CF is caused by reduced macrophage autophagy due to defective cystic fibrosis transmembrane conductance regulator (CFTR) function. AR-12 is a small molecule autophagy inducer that kills intracellular pathogens such as Francisella. We evaluated the efficacy of AR-12 and a new analogue AR-13 in reducing bacterial burden in CF phagocytes.MethodsHuman CF and non-CF peripheral blood monocyte-derived macrophages, neutrophils, and nasal epithelial cells were exposed to CF bacterial strains in conjunction with treatment with antibiotics and/or AR compounds.ResultsAR-13 and not AR-12 had growth inhibition on B. cenocepacia and methicillin-resistantStaphylococcus aureus (MRSA) in media alone. There was a 99% reduction in MRSA in CF macrophages, 71% reduction in Pseudomonas aeruginosa in CF neutrophils, and 70% reduction in non-CF neutrophils using AR-13. Conversely, there was no reduction in B. cenocepacia in infected CF and non-CF macrophages using AR-13 alone, but AR-13 and antibiotics synergistically reduced B. cenocepacia in CF macrophages. AR-13 improved autophagy in CF macrophages and CF patient-derived epithelial cells, and increased CFTR protein expression and channel function in CF epithelial cells.ConclusionsThe novel AR-12 analogue AR-13, in combination with antibiotics, reduced antibiotic-resistant bacterial burden in CF phagocytes, which correlated with increased autophagy and CFTR expression. AR-13 is a novel therapeutic for patients infected with B. cenocepacia and other resistant organisms that lack effective therapies.     

囊性纤维化跨膜转导调节器在培养人子宫内膜上皮细胞的表达

目的　探讨雌、孕激素对体外培养人子宫内膜腺上皮细胞囊性纤维化跨膜转导调节器(CFTR)基因和蛋白质表达的影响。　方法　采用逆转录 聚合酶链反应(RT PCR)、原位杂交、免疫组织化学方法检测CFTR mRNA和蛋白质表达量的变化。　结果　RT PCR结果显示单纯雌二醇、雌二醇与孕酮合用、对照组均扩增出特异的 CFTR mRNA条带,孕酮组则未见到;原位杂交结果也证实雌二醇刺激CFTR mRNA的表达(P<0.05),孕酮则抑制CFTR mRNA的表达。免疫组织化学检测子宫内膜腺上皮细胞中CFTR蛋白质含量表明雌二醇显著刺激其生成。　结论　卵巢性激素雌二醇上调、孕酮下调体外培养人子宫内膜腺上皮细胞CFTR mRNA及蛋白质的表达,从而调节月经周期不同时相宫腔液微环境中水与电解质含量,适应不同的生殖阶段。     

Cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations in pancreatitis

BackgroundThe pancreas is one of the primary organs affected by dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) protein. While exocrine pancreatic insufficiency is a well-recognized complication of cystic fibrosis (CF), symptomatic pancreatitis is often under-recognized.ResultsThe aim of this review is to provide a general overview of CFTR mutation-associated pancreatitis, which affects patients with pancreatic sufficient CF, CFTR-related pancreatitis, and idiopathic pancreatitis. The current hypothesis regarding the role of CFTR dysfunction in the pathogenesis of pancreatitis, and concepts on genotype–phenotype correlations between CFTR and symptomatic pancreatitis will be reviewed.Symptomatic pancreatitis occurs in 20% of pancreatic sufficient CF patients. In order to evaluate genotype–phenotype correlations, the Pancreatic Insufficiency Prevalence (PIP) score was developed and validated to determine severity in a large number of CFTR mutations. Specific CFTR genotypes are significantly associated with pancreatitis. Patients who carry genotypes with mild phenotypic effects have a greater risk of developing pancreatitis than patients carrying genotypes with moderate–severe phenotypic consequences at any given time.ConclusionsThe genotype–phenotype correlation in pancreatitis is unique compared to other organ manifestations but still consistent with the complex monogenic nature of CF. Paradoxically, genotypes associated with otherwise mild phenotypic effects have a greater risk for causing pancreatitis; compared with genotypes associated with moderate to severe disease phenotypes. Greater understanding into the underlying mechanisms of disease is much needed. The emergence of CFTR-assist therapies may potentially play a future role in the treatment of CFTR-mutation associated pancreatitis.     

The cystic fibrosis transmembrane conductance regulator: an intriguing protein with pleiotropic functions.

Cystic fibrosis is a frequent autosomal recessive disorder that is caused by the malfunctioning of a small chloride channel, the cystic fibrosis transmembrane conductance regulator. The protein is found in the apical membrane of epithelial cells lining exocrine glands. Absence of this channel results in imbalance of ion concentrations across the cell membrane. As a result, fluids secreted through these glands become more viscous and, in the end, ducts become plugged and atrophic. Little is known about the pathways that link the malfunctioning of the CFTR protein with the observed clinical phenotype. Moreover, there is no strict correlation between specific CFTR mutations and the CF phenotype. This might be explained by the fact that environmental and additional genetic factors may influence the phenotype. The CFTR protein itself is regulated at the maturational level by chaperones and SNARE proteins and at the functional level by several protein kinases. Moreover, CFTR functions also as a regulator of other ion channels and of intracellular membrane transport processes. In order to be able to function as a protein with pleiotropic actions, CFTR seems to be linked with other proteins and with the cytoskeleton through interaction with PDZ-domain-containing proteins at the apical pole of the cell. Progress in cystic fibrosis research is substantial, but still leaves many questions unanswered.     

CF gene and cystic fibrosis transmembrane conductance regulator expression in autosomal dominant polycystic kidney disease

Disease-modifying genes might participate in the significant intrafamilial variability of the renal phenotype in autosomal dominant polycystic kidney disease (ADPKD). Cystic fibrosis (CF) transmembrane conductance regulator (CFTR) is a chloride channel that promotes intracystic fluid secretion, and thus cyst progression, in ADPKD. The hypothesis that mutations of the CF gene, which encodes CFTR, might be associated with a milder renal phenotype in ADPKD was tested. A series of 117 unrelated ADPKD probands and 136 unaffected control subjects were screened for the 12 most common mutations and the frequency of the alleles of the intron 8 polymorphic TN: locus of CF. The prevalence of CF mutations was not significantly different in the ADPKD (1.7%, n = 2) and control (3.7%, n = 5) groups. The CF mutation was DeltaF508 in all cases, except for one control subject (1717-1G A). The frequencies of the 5T, 7T, and 9T intron 8 alleles were also similar in the ADPKD and control groups. Two additional patients with ADPKD and the DeltaF508 mutation were detected in the families of the two probands with CF mutations. Kidney volumes and renal function levels were similar for these four patients with ADPKD and DeltaF508 CFTR (heterozygous for three and homozygous for one) and for control patients with ADPKD collected in the University of Colorado Health Sciences Center database. The absence of a renal protective effect of the homozygous DeltaF508 mutation might be related to the lack of a renal phenotype in CF and the variable, tissue-specific expression of DeltaF508 CFTR. Immunohistochemical analysis of a kidney from the patient with ADPKD who was homozygous for the DeltaF508 mutation substantiated that hypothesis, because CFTR expression was detected in 75% of cysts (compared with <50% in control ADPKD kidneys) and at least partly in the apical membrane area of cyst-lining cells. These data do not exclude a potential protective role of some CFTR mutations in ADPKD but suggest that it might be related to the nature of the mutation and renal expression of the mutated CFTR.     

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) in Chinese patients with congenital bilateral absence of vas deferens

BackgroundGenetic testing of the cystic fibrosis transmembrane conductance (CFTR) gene is currently performed in patients with congenital bilateral absence of vas deferens (CBAVD). This study was conducted to investigate the role of mutations in the CFTR gene in CBAVD-dependent male infertility.Methods73 Chinese patients diagnosed with CBAVD were studied. The entire coding regions and splice sites of 27 exons of the CFTR gene were sequenced in 146 chromosomes from the 73 CBAVD patients. Screening was carried out using PCR, gel electrophoresis and DNA sequencing to identify novel variants of the entire coding regions and boundaries of the 27 exons.ResultsFive novel nonsynonymous mutations, three novel splice site mutations and one deletion were identified by sequencing. Apart from the novel variants, we also found 19 previously reported mutations and polymorphism sites. Thirty-four patients (46.57%) had the 5T variant (6 homozygous and 28 heterozygous) and in two of them it was not associated with any detectable mutation of the CFTR gene. All potential pathogenic mutations are not contained in the 1000 Genome Project database. In total, the present study identified 30 potential pathogenic variations in the CFTR gene, 9 of which had not previously been described.ConclusionsMost patients with CBAVD have mutations in the CFTR gene. A mild genotype with one or two mild or variable mutations was observed in all the patients. These findings improve our understanding of the distribution of CFTR alleles in CBAVD patients and will facilitate the development of more sensitive CFTR mutation screening.     

Sperm cystic fibrosis transmembrane conductance regulator expression level is relevant to fecundity of healthy couples

Cystic fibrosis transmembrane conductance regulator (CFTR) is relevant to sperm quality, sperm capacitation and male fertility. However, it is still unknown whether CFTR can be a potential parameter for fecundity prediction in healthy couples. In this study, 135 healthy couples were divided into groups according to their fertility. We demonstrated that the sperm CFTR expression level of healthy males who never impregnated their partners (49 cases, 38.68 ± 2.71%) was significantly lower than that of fertile men (86 cases, 46.35 ± 2.32%). Sperm CFTR expression level accurately corresponded with fertility through the logistic regression model. Receiver operating characteristic (ROC) curve analysis showed that the cut‐off value of sperm CFTR expression level for fecundity prediction was 43.75%. Furthermore, cumulative pregnancy rates (CPRs) of CFTR > 43.75% group and CFTR ≤ 43.75% group during the follow‐up periods were 80.6% and 49.3% respectively. Meanwhile, the mean time to pregnancy (TTP) of CFTR ≤ 43.75% group (26.79 ± 2.35) was significantly longer than that of CFTR > 43.75% group (16.46 ± 2.42). Therefore, sperm CFTR expression level is relevant to fecundity of healthy couples and shows potential predictive capacity of fecundity.     

Airway hyperresponsiveness in FVB/N delta F508 cystic fibrosis transmembrane conductance regulator mice

BackgroundAirway hyperresponsiveness is a feature of clinical CF lung disease. In this study, we investigated whether the FVB/N ΔF508 CFTR mouse model has altered airway mechanics.MethodsMechanics were measured in 12–14 week old FVB/N Cftr^tm1Eur (ΔF508) mice and wildtype littermates using the FlexiVent small animal ventilator. Lung disease was assayed by immunohistochemistry, histology and bronchoalveolar lavage analysis.ResultsCftr^tm1Eur mice presented with increased airway resistance, compared to wildtype littermates, in response to methacholine challenge. No differences in bronchoalveolar cell number or differential, or in tissue lymphocyte, goblet cell or smooth muscle actin levels were evident in mice grouped by Cftr genotype. The bronchoalveolar lavage of Cftr^tm1Eur mice included significantly increased levels of interleukin 12(p40) and CXCL1 compared to controls.ConclusionWe conclude that the pulmonary phenotype of Cftr^tm1Eur mice includes airway hyperresponsiveness in the absence of overt lung inflammation or airway remodeling.     

The study of cystic fibrosis transmembrane conductance regulator gene mutations in a group of patients from Romania

BackgroundCystic fibrosis (CF) is produced by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator Gene (CFTR) gene.MethodsOne hundred twenty eight patients with CF were analysed for mutations in the CFTR gene in order to establish the frequency of CF mutations in the Romanian population. The chief methods of analysis were polymerase chain reaction (PCR) of DNA extracted from blood and electrophoresis of PCR products.ResultsThe frequency of F508del in CF chromosomes from Romania is approximately 56.3%. Other frequent mutations noted are: G542X (3.9%), W1282X (2.3%), and CFTRdele2,3(21 kb)(1.6%); the remaining mutations have frequencies below 1%.ConclusionsWe consider that the frequency of F508del in CF patients from Romania is higher than in previous reports, reaching 56.3%, probably owing to more rigorous selection of patients for genetic testing, allowing improved calculation of mutation frequencies.     

Cystic fibrosis transmembrane conductance regulator (CFTR) gene mutation associated with a congenital bilateral absence of vas deferens

Abstract:   Cystic fibrosis transmembrane conductance regulator ( CFTR ) gene mutations associated with cystic fibrosis have been reported to be rare in Japanese patients with congenital bilateral absence of vas deferens (CBAVD).
A 28-year-old Japanese male was referred for infertility. Vas deferens and epididymis were not palpable bilaterally. Semen analyses showed azoospermia with volumes below 2.0 ml. Serum follicle-stimulating hormone value was slightly elevated. Seminal fructose concentration was also very low. Scrotal ultrasonography showed absence of the bodies and tails of the right and left epididymides. Imaging studies showed cystic dysplasia of the right seminal vesicle and agenesis of the left seminal vesicle. A CFTR gene mutation of I556V was found.
Recent studies show that prevalence of CFTR gene mutation in Japanese CBAVD patients may be approximately equal to that of the Caucasian population. Genetic counselling may be recommended for any couple attempting assisted reproduction technology when the man has CBAVD.