RESUMEN
The lungs and kidneys are pivotal organs in the regulation of body acid-base homeostasis. In cystic fibrosis (CF), the impaired renal ability to excrete an excess amount of HCO3- into the urine leads to metabolic alkalosis [P. Berg et al., J. Am. Soc. Nephrol. 31, 1711-1727 (2020); F. Al-Ghimlas, M. E. Faughnan, E. Tullis, Open Respir. Med. J. 6, 59-62 (2012)]. This is caused by defective HCO3- secretion in the ß-intercalated cells of the collecting duct that requires both the cystic fibrosis transmembrane conductance regulator (CFTR) and pendrin for normal function [P. Berg et al., J. Am. Soc. Nephrol. 31, 1711-1727 (2020)]. We studied the ventilatory consequences of acute oral base loading in normal, pendrin knockout (KO), and CFTR KO mice. In wild-type mice, oral base loading induced a dose-dependent metabolic alkalosis, fast urinary removal of base, and a moderate base load did not perturb ventilation. In contrast, CFTR and pendrin KO mice, which are unable to rapidly excrete excess base into the urine, developed a marked and transient depression of ventilation when subjected to the same base load. Therefore, swift renal base elimination in response to an acute oral base load is a necessary physiological function to avoid ventilatory depression. The transient urinary alkalization in the postprandial state is suggested to have evolved for proactive avoidance of hypoventilation. In CF, metabolic alkalosis may contribute to the commonly reduced lung function via a suppression of ventilatory drive.
Asunto(s)
Alcalosis/fisiopatología , Fibrosis Quística/fisiopatología , Hipoventilación/fisiopatología , Equilibrio Ácido-Base/fisiología , Alcalosis/metabolismo , Animales , Bicarbonatos/metabolismo , Antiportadores de Cloruro-Bicarbonato , Fibrosis Quística/complicaciones , Fibrosis Quística/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/fisiología , Modelos Animales de Enfermedad , Femenino , Hipoventilación/etiología , Hipoventilación/metabolismo , Transporte Iónico , Riñón/metabolismo , Riñón/patología , Pulmón/metabolismo , Pulmón/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Eliminación Renal , Reabsorción Renal/fisiologíaRESUMEN
Cystic fibrosis (CF) is a life-limiting autosomal recessive genetic disease caused by variants in the CFTR gene, most commonly by the [F508del] variant. Although CF is a classical Mendelian disease, genetic variants in several modifier genes have been associated with variation of the clinical phenotype for pulmonary and gastrointestinal function and urogenital development. We hypothesized that whole genome sequencing of a well-phenotyped CF populations might identify novel variants in known, or hitherto unknown, modifier genes. Whole genome sequencing was performed on the Illumina HiSeq X platform for 98 clinically diagnosed cystic fibrosis patient samples from the Adult CF Clinic at the University of California San Diego (UCSD). We compared protein-coding, non-silent variants genome wide between CFTR [F508del] homozygotes vs CFTR compound heterozygotes. Based on a single variant score test, we found 3 SNPs in common variants (MAF >5%) that occurred at significantly different rates between homozygous [F508del]CFTR and compound heterozygous [F508del]CFTR patients. The 3 SNPs were all located in one gene on chromosome 2: Tensin 1 (TNS1: rs3796028; rs2571445: and rs918949). We observed significantly lower BMIs in homozygous [F508del]CFTR patients who were also homozygous for Tensin 1 rs918949 (T/T) (p = 0.023) or rs2571445 (G/G) (p = 0.02) variants. The Tensin 1 gene is thus a potential modifier gene for low BMI in CF patients homozygous for the [F508del]CFTR variant.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Tensinas/fisiología , Delgadez/genética , Adulto , Índice de Masa Corporal , Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/fisiología , Femenino , Heterocigoto , Homocigoto , Humanos , Masculino , Polimorfismo de Nucleótido Simple/genética , Tensinas/genética , Secuenciación Completa del GenomaRESUMEN
The ongoing severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic has been a formidable global challenge. As yet, there are very few drugs to treat this infection and no vaccine is currently available. It has gradually become apparant that coronavirus disease 2019 (COVID-19) is not a simple disease involving a single organ; rather, many vital organs and systems are affected. The endothelium is one target of SARS-CoV-2. Damaged endothelial cells, which break away from organs and enter the bloodstream to form circulating endothelial cells, were recently reported as putative biomarkers for COVID-19. Modulation of the expression level of sphingosine-1 phosphate via sphingosine kinase activation can control endothelial cell proliferation and apoptosis. As such, it may be possible to obtain a sensitive and specific diagnosis of the severity of COVID-19 by assessing the absolute number and the viable/apoptotic ratio of circulating endothelial cells. Furthermore, a focus on the endothelium could help to develop a strategy for COVID-19 treatment from the perspective of endothelial protection and repair.
Asunto(s)
COVID-19/diagnóstico , Células Endoteliales/patología , SARS-CoV-2 , Biomarcadores , COVID-19/terapia , Regulador de Conductancia de Transmembrana de Fibrosis Quística/fisiología , Humanos , Lisofosfolípidos/análisis , Esfingosina/análogos & derivados , Esfingosina/análisisRESUMEN
Optical measurement of CFTR-dependent sweat secretion stimulated by a beta-adrenergic cocktail (C-phase) vs. CFTR-independent sweat secretion induced by methacholine (M-phase) can discriminate cystic fibrosis (CF) patientts from controls and healthy carriers by the ratio of sweat rate in the C-phase vs. the M-phase (C/M ratio). However, image analysis is experimentally demanding and time-consuming. Here, sweat droplet number (SDN) in the C-phase, corresponding to the number of sweat-secreting glands, was a statistically significant predictor for detecting the effects of CFTR-targeted therapy. We show that in 44 non-CF subjects and 110 CF patients, SDN in the C-phase provides a linear readout of CFTR function that is more sensitive than that using the C/M ratio. In CF patients, increased SDN in the C-phase during treatment with (LUMA/IVA) was associated with a trend toward improved lung function (FEV1). Our method is suitable for multicenter monitoring of the effects of CFTR modulators.
Asunto(s)
Aminofenoles/uso terapéutico , Aminopiridinas/uso terapéutico , Benzodioxoles/uso terapéutico , Regulador de Conductancia de Transmembrana de Fibrosis Quística/fisiología , Fibrosis Quística/diagnóstico , Fibrosis Quística/tratamiento farmacológico , Quinolonas/uso terapéutico , Glándulas Sudoríparas/metabolismo , Sudor/metabolismo , Combinación de Medicamentos , Humanos , Óptica y Fotónica , Sudor/efectos de los fármacos , Glándulas Sudoríparas/efectos de los fármacosRESUMEN
The transmembrane Hsp40 DNAJB12 and cytosolic Hsp70 cooperate on the endoplasmic reticulum's (ER) cytoplasmic face to facilitate the triage of nascent polytopic membrane proteins for folding versus degradation. N1303K is a common mutation that causes misfolding of the ion channel CFTR, but unlike F508del-CFTR, biogenic and functional defects in N1303K-CFTR are resistant to correction by folding modulators. N1303K is reported to arrest CFTR folding at a late stage after partial assembly of its N-terminal domains. N1303K-CFTR intermediates are clients of JB12-Hsp70 complexes, maintained in a detergent-soluble state, and have a relatively long 3-h half-life. ER-associated degradation (ERAD)-resistant pools of N1303K-CFTR are concentrated in ER tubules that associate with autophagy initiation sites containing WIPI1, FlP200, and LC3. Destabilization of N1303K-CFTR or depletion of JB12 prevents entry of N1303K-CFTR into the membranes of ER-connected phagophores and traffic to autolysosomes. In contrast, the stabilization of intermediates with the modulator VX-809 promotes the association of N1303K-CFTR with autophagy initiation machinery. N1303K-CFTR is excluded from the ER-exit sites, and its passage from the ER to autolysosomes does not require ER-phagy receptors. DNAJB12 operates in biosynthetically active ER microdomains to triage membrane protein intermediates in a conformation-specific manner for secretion versus degradation via ERAD or selective-ER-associated autophagy.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Degradación Asociada con el Retículo Endoplásmico/fisiología , Proteínas del Choque Térmico HSP40/metabolismo , Animales , Autofagosomas , Autofagia/fisiología , Células COS , Línea Celular , Chlorocebus aethiops , Cricetinae , Regulador de Conductancia de Transmembrana de Fibrosis Quística/fisiología , Retículo Endoplásmico/metabolismo , Células HEK293 , Proteínas del Choque Térmico HSP40/genética , Proteínas del Choque Térmico HSP40/fisiología , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas de Choque Térmico/metabolismo , Humanos , Pliegue de ProteínaRESUMEN
The redox states of NAD and NADP are linked to each other in the mitochondria thanks to the enzyme nicotinamide nucleotide transhydrogenase (NNT) which, by utilizing the mitochondrial membrane potential (mΔΨ), catalyzes the transfer of redox potential between these two coenzymes, reducing one at the expense of the oxidation of the other. In order to define NNT reaction direction in CF cells, NNT activity under different redox states of cell has been investigated. Using spectrophotometric and western blotting techniques, the presence, abundance and activity level of NNT were determined. In parallel, the levels of NADPH and NADH as well as of mitochondrial and cellular ROS were also quantified. CF cells showed a 70% increase in protein expression compared to the Wt sample; however, regarding NNT activity, it was surprisingly lower in CF cells than healthy cells (about 30%). The cellular redox state, together with the low mΔΨ, pushes to drive NNT reverse reaction, at the expense of its antioxidant potential, thus consuming NADPH to support NADH production. At the same time, the reduced NNT activity prevents the NADH, produced by the reaction, from causing an explosion of ROS by the damaged respiratory chain, in accordance with the reduced level of mitochondrial ROS in NNT-loss cells. This new information on cellular bioenergetics represents an important building block for further understanding the molecular mechanisms responsible for cellular dysfunction in cystic fibrosis.
Asunto(s)
Fibrosis Quística/metabolismo , NADP Transhidrogenasas/metabolismo , Catálisis , Células Cultivadas , Fibrosis Quística/genética , Fibrosis Quística/patología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/fisiología , Metabolismo Energético/genética , Humanos , Potencial de la Membrana Mitocondrial/fisiología , Redes y Vías Metabólicas/genética , Mitocondrias/metabolismo , NAD/metabolismo , NADP/metabolismo , Oxidación-Reducción , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Since the discovery of the gene responsible for cystic fibrosis (CF) in 1989, hopes have been pinned on a future with novel therapies tackling the basis of the disease rather than its symptoms. These have become a reality over the last decade with the development through to the clinic of CF transmembrane conductance regulator (CFTR) modulators. These are oral drugs which improve CFTR protein function through either increasing the time the channel pore is open (potentiators) or facilitating its trafficking through the cell to its location on the cell membrane (correctors). The first potentiator, ivacaftor, is now licensed and available clinically in many parts of the world. It is highly effective with impressive clinical impact in the lungs and gastrointestinal tract; longer-term data from patient registries show fewer exacerbations, a slower rate of lung function loss and reduced need for transplantation in patients receiving ivacaftor. However, as a single drug, it is suitable for only a small minority of patients. The commonest CFTR mutation, F508del, requires both correction and potentiation for clinical efficacy. Two dual-agent drugs (lumacaftor/ivacaftor and tezacaftor/ivacaftor) have progressed through to licensing, although their short term impact is more modest than that of ivacaftor; this is likely due to only partial correction of protein misfolding and trafficking. Most recently, triple compounds have been developed: two different corrector molecules (elexacaftor and tezacaftor) which, by addressing different regions in the misfolded F508del protein, more effectively improve trafficking. In addition to large improvements in clinical outcomes in people with two copies of F508del, the combination is sufficiently effective that it works in patients with only one copy of F508del and a second, nonmodulator responsive mutation. For the first time, we thus have a drug suitable for around 85% of people with CF. Even more gains are likely to be possible when these drugs can be used in younger children, although more sensitive outcome measures are needed for this age group. Special consideration is needed for people with very rare mutations; those with nonmodulatable mutation combinations will likely require gene or messenger RNA-based therapeutic approaches, many of which are being explored. Although this progress is hugely to be celebrated, we still have more work to do. The international collaboration between trials networks, pharma, patient organizations, registries, and people with CF is something we are all rightly proud of, but innovative trial design and implementation will be needed if we are to continue to build on this progress and further develop drugs for people with CF.
Asunto(s)
Aminofenoles/uso terapéutico , Agonistas de los Canales de Cloruro/uso terapéutico , Regulador de Conductancia de Transmembrana de Fibrosis Quística/efectos de los fármacos , Fibrosis Quística/tratamiento farmacológico , Quinolonas/uso terapéutico , Aminofenoles/administración & dosificación , Aminofenoles/farmacología , Aminopiridinas/administración & dosificación , Benzodioxoles/administración & dosificación , Niño , Agonistas de los Canales de Cloruro/administración & dosificación , Agonistas de los Canales de Cloruro/farmacología , Ensayos Clínicos como Asunto , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/fisiología , Quimioterapia Combinada , Humanos , Indoles/administración & dosificación , Mutación , Pirazoles/administración & dosificación , Piridinas/administración & dosificación , Pirrolidinas/administración & dosificación , Quinolonas/administración & dosificación , Quinolonas/farmacologíaRESUMEN
Although effective cystic fibrosis transmembrane conductance regulator (CFTR) modulator therapy has the potential to change the lives of many patients with cystic fibrosis (CF), it is unlikely that these drugs will be a game changing therapy for all. There are about 10% of patients with CF who don't produce a mutant protein tomodulate, potentiate, or optimize and for these patients such therapies are unlikely to be of significant benefit. There is a need to develop new therapeutic approaches that can work for this patient population and can advance CF therapies. These new therapies will be genetic-based therapies and each approach will result in functional CFTR protein inpreviously affected CF cells. In this review we will examine the potential of RNA therapies, gene transfer therapies, and gene editing therapies for the treatment of CF as well as the challenges that will need to be facedas we harness the power of these emerging therapies towards a one-time cure.
Asunto(s)
Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Regulador de Conductancia de Transmembrana de Fibrosis Quística/fisiología , Fibrosis Quística/terapia , Terapia Genética/métodos , Fibrosis Quística/fisiopatología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Humanos , Mutación , ARN Mensajero/genéticaRESUMEN
BACKGROUND: Increased (abnormal) ventilation inhomogeneity in individuals with mild Cystic Fibrosis (CF) lung disease may become a treatable trait for small-molecule therapeutics improving Cystic Fibrosis Transmembrane Regulator (CFTR) function. The relationship between CFTR function and ventilation inhomogeneity is unknown. We aimed to identify and quantify increased ventilation inhomogeneity in relation to CFTR function. METHODS: This was an international, multi-center, cross-sectional study. We collated data from individuals aged 3-25 years with minimal (CFTR-MF) or residual (CFTR-RF) function of a variety of CFTR genotypes and FEV1 ≥ 70% predicted. We measured lung function using nitrogen multiple-breath washout and spirometry. We compared lung clearance index (LCI) and FEV1 between individuals with CFTR-MF vs CFTR-RF using a mixed effects multi-variable linear regression model to account for study differences and a logistic model based on propensity-score matching to adjust for possible confounding. RESULTS: We included 141 with CFTR-MF and 35 with CFTR-RF. LCI (> 1.96 z-score) was elevated in 71.6% individuals with CFTR-MF and in 40.0% with CFTR-RF. FEV1 (< -1.96 z-score) was reduced in 11.3% individuals with CFTR-MF and in 5.7% with CFTR-RF. The mean difference (95% CI) of LCI and FEV1 between CFTR-MF and CFTR-RF was 3.71 (1.63 to 5.79) and -0.40 (-0.83 to 0.02) z-score. The LCI differences were similar after adjustment for confounders and in individuals with normal FEV1. CONCLUSION: Increased ventilation inhomogeneity is associated with less CFTR function. In individuals with mild CF lung disease, LCI can identify and quantify increased ventilation inhomogeneity, a candidate treatable trait.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/fisiología , Fibrosis Quística/fisiopatología , Adolescente , Niño , Preescolar , Estudios Transversales , Femenino , Humanos , Masculino , Ventilación Pulmonar , Pruebas de Función Respiratoria , Estudios RetrospectivosRESUMEN
BACKGROUND: The pathophysiological roles of cystic fibrosis transmembrane-conductance regulator (CFTR) Cl- channels in the regulation of blood pressure (BP) remain controversial. Here we studied the function of CFTR Cl- channels in regulation of BP and in the high-fructose-salt-diet (HFSD) induced hypertension in mice. METHODS: The systolic, diastolic and mean BP (SBP, DBP and MBP, respectively) were continuously monitored from unrestricted conscious wild-type (cftr+/+) FVB and CFTR-knockout (cftr-/-) mice (8-week old, male). HFSD (64.7% fructose, 2% NaCl water) or control normal starch diet (CNSD, 58.9% corn starch, 0 NaCl water) was given for 8 weeks and vascular Doppler were performed. Real-time PCR and Western blot were used to examine mRNA and protein expression, respectively. RESULTS: The aortic stiffness, daytime and nighttime SBP, DBP, and MBP of the cftr-/- mice were significantly higher than those in the age- and gender-matched cftr+/+ mice, which is consistent with the findings of increased vascular resistance in cystic fibrosis patients. The aortic stiffness, daytime and nighttime SBP, DBP, and MBP of cftr+/+ mice fed with HFSD were all significantly higher than those fed with CNSD. Importantly, HFSD caused a significant decrease in mRNA and protein expression of WINK1, WINK4 and CFTR in aorta and mesenteric arteries, but not in the kidney, corroborating that HSFD-induced downregulation of WINKs and loss of CFTR function specifically in the arteries may mediate the increased BP. CONCLUSIONS: CFTR regulates peripheral arterial resistance and BP in vivo. HFSD-induced CFTR downregulation specifically in the arteries may be a novel mechanism for hypertension.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/fisiología , Fibrosis Quística/fisiopatología , Resistencia Vascular/fisiología , Animales , Presión Sanguínea/fisiología , Dieta Alta en Grasa , Carbohidratos de la Dieta/administración & dosificación , Regulación hacia Abajo , Fructosa/administración & dosificación , Masculino , Ratones , Ultrasonografía DopplerRESUMEN
Cystic fibrosis (CF) is a lethal autosomal recessive genetic disease, caused by a mutation in the cystic fibrosis transmembrane conductance regulator gene (CFTR), which primarily affects the lungs and digestive system. This gene encodes the CFTR protein, a distinctive membrane transporter of the ATP-binding cassette (ABC) superfamily. It functions as a chloride channel, allowing the balance and transport of chloride through the apical membrane of epithelial cells. Due to its ubiquitous location, mutations in the CFTR gene trigger multiple changes in ion transport and metabolic pathways, affecting various organs, as it will be herein explained. Pulmonary impairment is the most characteristic comorbidity of CF and respiratory failure is the main cause of death. This review presents the importance of an early diagnosis of CF to establish, as soon as possible, a primary therapy for symptomatic prevention and relief. It also mentions new therapeutic approaches that include CFTR modulators. They are correctors and/or potentiators of the deficient CFTR channel. In an attempt to overcome the disadvantages of CFTR modulators, the application of biotechnology techniques is addressed, such as gene therapy, gene editing, RNA therapy and therapeutic microRNAs. The potential of the intranasal administration route is another presented aspect.
Asunto(s)
Fibrosis Quística , Animales , Biotecnología , Fibrosis Quística/diagnóstico , Fibrosis Quística/tratamiento farmacológico , Fibrosis Quística/genética , Fibrosis Quística/fisiopatología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/fisiología , HumanosRESUMEN
Spermatozoa released from the testis are unable to fertilize an egg without a coordinated process of maturation in the lumen of the epididymis. Relatively little is known about the molecular events that integrate this critical progression along the male genital ducts in man. Here, we use single cell RNA-sequencing to construct an atlas of the human proximal epididymis. We find that the CFTR, which is pivotal in normal epididymis fluid transport, is most abundant in surface epithelial cells in the efferent ducts and in rare clear cells in the caput epididymis, suggesting region-specific functional properties. We reveal transcriptional signatures for multiple cell clusters, which identify the individual roles of principal, apical, narrow, basal, clear, halo, and stromal cells in the epididymis. A marked cell type-specific distribution of function is seen along the duct with local specialization of individual cell types integrating processes of sperm maturation.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Epidídimo/citología , Adulto , Regulador de Conductancia de Transmembrana de Fibrosis Quística/fisiología , Epidídimo/metabolismo , Epidídimo/fisiología , Células Epiteliales/metabolismo , Epitelio/metabolismo , Genitales Masculinos , Humanos , Masculino , Persona de Mediana Edad , Análisis de Secuencia de ARN/métodos , Análisis de la Célula Individual/métodos , Maduración del Esperma , Espermatozoides/citología , Espermatozoides/fisiología , TestículoRESUMEN
The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) anion channel is essential for epithelial salt-water balance. CFTR mutations cause cystic fibrosis, a lethal incurable disease. In cells CFTR is activated through the cAMP signaling pathway, overstimulation of which during cholera leads to CFTR-mediated intestinal salt-water loss. Channel activation is achieved by phosphorylation of its regulatory (R) domain by cAMP-dependent protein kinase catalytic subunit (PKA). Here we show using two independent approaches--an ATP analog that can drive CFTR channel gating but is unsuitable for phosphotransfer by PKA, and CFTR mutants lacking phosphorylatable serines--that PKA efficiently opens CFTR channels through simple binding, under conditions that preclude phosphorylation. Unlike when phosphorylation happens, CFTR activation by PKA binding is completely reversible. Thus, PKA binding promotes release of the unphosphorylated R domain from its inhibitory position, causing full channel activation, whereas phosphorylation serves only to maintain channel activity beyond termination of the PKA signal. The results suggest two levels of CFTR regulation in cells: irreversible through phosphorylation, and reversible through R-domain binding to PKA--and possibly also to other members of a large network of proteins known to interact with the channel.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Canales Aniónicos Dependientes del Voltaje/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Aniones/metabolismo , Fenómenos Biofísicos , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/fisiología , Activación del Canal Iónico/fisiología , Mutagénesis Sitio-Dirigida , Nucleótidos/metabolismo , Oocitos/metabolismo , Técnicas de Placa-Clamp/métodos , Fosforilación , Unión Proteica/fisiología , Serina/metabolismo , Canales Aniónicos Dependientes del Voltaje/fisiología , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismoRESUMEN
Cystic fibrosis (CF) is an inherited recessive autosomal disorder that affects the lungs, the digestive system, and secretory glands. It is a lethal condition caused by a mutation in the gene cystic-fibrosis-transmembrane-conductance- regulator (CFTR), which leads to defects in ion channels and results in obstruction of mucus in airway channels. Unbalanced ion exchange causes impaired water transport and accumulation of viscous mucus in the air way leads to bacterial colonization, for example, with Staphylococcus aureus. The most common mutation is the deletion of nucleotides in epithelial membrane; hence, it is a multiple-organ-defective disease that mostly effects the lungs. Researchers are working on gene therapy that aims to introduce a normal CFTR gene copy into the epithelial cells of lungs. Several approaches have been designed to improve transepithelial ion transport in CF patients. Normal CFTR gene delivery has been performed using viral and nonviral vectors, but these approaches are not more efficient against the cell barriers. Enzymes may be used that inhibit the sphingolipid to provide proper microenvironment for the CFTR gene product. Thymosin alpha-1 has also been reported as a potential corrector in treatment of CF.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/fisiología , Fibrosis Quística/terapia , Quimioterapia , Terapia Genética , Fibrosis Quística/complicaciones , Epitelio/fisiopatología , Glucosilceramidasa/antagonistas & inhibidores , Humanos , Transporte Iónico , Liposomas/metabolismo , Proteínas de la Membrana/fisiología , Mutación , Infecciones Estafilocócicas/complicaciones , Infecciones Estafilocócicas/tratamiento farmacológico , Staphylococcus aureus/efectos de los fármacos , Timalfasina/uso terapéuticoRESUMEN
Cystic fibrosis (CF) is a genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, resulting in chronic bacterial lung infections and tissue damage. CF macrophages exhibit reduced bacterial killing and increased inflammatory signaling. Iron is elevated in the CF lung and is a critical nutrient for bacteria, including the common CF pathogen Pseudomonas aeruginosa (Pa). While macrophages are a key regulatory component of extracellular iron, iron metabolism has yet to be characterized in human CF macrophages. Secreted and total protein levels were analyzed in non-CF and F508del/F508del CF monocyte derived macrophages (MDMs) with and without clinically approved CFTR modulators ivacaftor/lumacaftor. CF macrophage transferrin receptor 1 (TfR1) was reduced with ivacaftor/lumacaftor treatment. When activated with LPS, CF macrophage expressed reduced ferroportin (Fpn). After the addition of exogenous iron, total iron was elevated in conditioned media from CF MDMs and reduced in conditioned media from ivacaftor/lumacaftor treated CF MDMs. Pa biofilm formation and viability were elevated in conditioned media from CF MDMs and biofilm formation was reduced in the presence of conditioned media from ivacaftor/lumacaftor treated CF MDMs. Defects in iron metabolism observed in this study may inform host-pathogen interactions between CF macrophages and Pa.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/efectos de los fármacos , Fibrosis Quística/metabolismo , Hierro/metabolismo , Macrófagos/metabolismo , Pseudomonas aeruginosa/fisiología , Adolescente , Adulto , Aminofenoles/farmacología , Aminopiridinas/farmacología , Benzodioxoles/farmacología , Biopelículas/efectos de los fármacos , Niño , Medios de Cultivo Condicionados/farmacología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/fisiología , Combinación de Medicamentos , Femenino , Perfilación de la Expresión Génica , Interacciones Huésped-Patógeno , Humanos , Proteínas Reguladoras del Hierro/biosíntesis , Proteínas Reguladoras del Hierro/genética , Masculino , Persona de Mediana Edad , Quinolonas/farmacología , Esputo/microbiologíaRESUMEN
Autosomal dominant polycystic kidney disease (ADPKD) is the most frequent inherited kidney disease. Transepithelial fluid secretion is one of the key factors of cystogenesis in ADPKD. Multiple studies have suggested that fluid secretion across ADPKD cyst-lining cells is driven by the secretion of chloride, essentially mediated by the CFTR channel and stimulated by increased intracellular levels of 3',5'-cyclic adenosine monophosphate. This review focuses on the pathophysiology of fluid secretion in ADPKD based on the pioneering studies of Jared Grantham and colleagues, and on the follow-up investigations from the molecular level to the potential applications in ADPKD patients. Altogether, the studies of fluid and chloride transport in ADPKD paved the way for innovative therapeutic targets to prevent cyst volume expansion and thus, kidney disease progression.
Asunto(s)
AMP Cíclico/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/fisiología , Células Epiteliales/metabolismo , Riñón Poliquístico Autosómico Dominante/metabolismo , Animales , Células Cultivadas , Cloruros/metabolismo , Células Epiteliales/patología , Humanos , Transporte IónicoRESUMEN
BACKGROUND: Patients with cystic fibrosis (CF) do not respond with increased urinary HCO3- excretion after stimulation with secretin and often present with metabolic alkalosis. METHODS: By combining RT-PCR, immunohistochemistry, isolated tubule perfusion, in vitro cell studies, and in vivo studies in different mouse models, we elucidated the mechanism of secretin-induced urinary HCO3- excretion. For CF patients and CF mice, we developed a HCO3- drinking test to assess the role of the cystic fibrosis transmembrane conductance regulator (CFTR) in urinary HCO3-excretion and applied it in the patients before and after treatment with the novel CFTR modulator drug, lumacaftor-ivacaftor. RESULTS: ß-Intercalated cells express basolateral secretin receptors and apical CFTR and pendrin. In vivo application of secretin induced a marked urinary alkalization, an effect absent in mice lacking pendrin or CFTR. In perfused cortical collecting ducts, secretin stimulated pendrin-dependent Cl-/HCO3- exchange. In collecting ducts in CFTR knockout mice, baseline pendrin activity was significantly lower and not responsive to secretin. Notably, patients with CF (F508del/F508del) and CF mice showed a greatly attenuated or absent urinary HCO3--excreting ability. In patients, treatment with the CFTR modulator drug lumacaftor-ivacaftor increased the renal ability to excrete HCO3-. CONCLUSIONS: These results define the mechanism of secretin-induced urinary HCO3- excretion, explain metabolic alkalosis in patients with CF, and suggest feasibility of an in vivo human CF urine test to validate drug efficacy.
Asunto(s)
Bicarbonatos/metabolismo , Fibrosis Quística/metabolismo , Riñón/metabolismo , Animales , AMP Cíclico/fisiología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/fisiología , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratas Endogámicas F344 , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/fisiología , Receptores de la Hormona Gastrointestinal/genética , Receptores de la Hormona Gastrointestinal/fisiología , Secretina/farmacologíaRESUMEN
Hepatobiliary disease causes significant morbidity in people with cystic fibrosis (CF), yet this problem remains understudied. We previously found that newborn CF pigs have microgallbladders with significant luminal obstruction in the absence of infection and consistent inflammation. In this study, we sought to better understand the early pathogenesis of CF pig gallbladder disease. We hypothesized that loss of CFTR would impair gallbladder epithelium anion/liquid secretion and increase mucin production. CFTR was expressed apically in non-CF pig gallbladder epithelium but was absent in CF. CF pig gallbladders lacked cAMP-stimulated anion transport. Using a novel gallbladder epithelial organoid model, we found that Cl- or HCO3- was sufficient for non-CF organoid swelling. This response was absent for non-CF organoids in Cl-/HCO3--free conditions and in CF. Single-cell RNA-sequencing revealed a single epithelial cell type in non-CF gallbladders that coexpressed CFTR, MUC5AC, and MUC5B. Despite CF gallbladders having increased luminal MUC5AC and MUC5B accumulation, there was no significant difference in the epithelial expression of gel-forming mucins between non-CF and CF pig gallbladders. In conclusion, these data suggest that loss of CFTR-mediated anion transport and fluid secretion contribute to microgallbladder development and luminal mucus accumulation in CF.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/fisiología , Fibrosis Quística/complicaciones , Enfermedades de la Vesícula Biliar/etiología , Vesícula Biliar/metabolismo , Animales , Animales Recién Nacidos , Fibrosis Quística/metabolismo , Fibrosis Quística/fisiopatología , Modelos Animales de Enfermedad , Células Epiteliales/metabolismo , Vesícula Biliar/fisiopatología , Enfermedades de la Vesícula Biliar/metabolismo , Mucina 5AC/metabolismo , Mucina 5B/metabolismo , Porcinos , TranscriptomaRESUMEN
Organoid cultures derived from primary human tissues facilitate the study of disease processes and the development of new therapeutics. Most men with cystic fibrosis (CF) are infertile due to defects in the epididymis and vas deferens; however, the causative mechanisms are still unclear. We used human epididymis epithelial cell (HEE) organoids and polarized HEE cell cultures to assay the CF transmembrane conductance regulator (CFTR) in the human epididymis. 3D HEE organoids and polarized 2D HEE cell cultures on membrane inserts were established from human caput epididymis. Single-cell RNA sequencing (scRNA-seq) was performed to map cell type-specific gene expression in the organoids. Using forskolin (FSK) to activate CFTR and inhibitor CFTRinh172 to block its activity, we assessed how CFTR contributes to organoid swelling and epithelial barrier function. The scRNA-seq data showed key caput epididymis cell types present in HEE organoid cultures. FSK at 10 µM induced HEE organoid swelling by 20% at 16 h, while 5 and 10 µM CFTRinh172 treatment significantly reduced HEE organoid size. In transepithelial resistance (TER) measurements, FSK reduced TER, while inhibition of CFTR increased TER; also, depletion of CFTR with specific siRNAs significantly increased TER. FSK treatment significantly increased the flux of 4-kDa but not 70-kDa dextran, suggesting activation of CFTR mainly enhances transcellular diffusion. We have demonstrated that CFTR contributes to the maintenance of HEE cell TER and that cultured HEE organoids are a useful model to investigate human epididymis function. These results facilitate progress in elucidating how CFTR-dependent cellular processes impair fertility in CF.