RESUMEN
Pancreatic secretions become viscous and acidic in Cystic fibrosis (CF), highlighting the role of CFTR in pancreatic fluid and bicarbonate secretion. Forskolin-induced swelling (FIS) assay developed in intestinal organoids measures residual CFTR function. It is not known whether FIS reflects bicarbonate secretion in pancreas, an organ that secretes near-isotonic NaHCO3 levels. To investigate this, we generated pancreatic duct organoids from CF and non-CF pigs. Epithelial and ductal origin was confirmed with epithelial markers, ion transporters and lack of acinar, islet cell markers. CF organoids were small with no identifiable lumen; CFTR was expressed only in non-CF organoids. Utilizing FIS, organoid size increased only in response to chloride, not bicarbonate. This report highlights pancreatic duct organoids isolated for the first time from CF pigs and evidence for chloride and not bicarbonate driving pancreatic organoid swelling. These organoids would be useful to test chloride permeability of CFTR mutations that cause CF pancreatic disease.
Asunto(s)
Fibrosis Quística , Animales , Porcinos , Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Cloruros/metabolismo , Bicarbonatos/metabolismo , Conductos Pancreáticos/metabolismo , Colforsina/farmacología , Organoides/metabolismoRESUMEN
Fluid and anion secretion are important functions of the biliary tract. It has been established that cAMP regulates Na+ absorption through NHE3. However, mechanisms of gallbladder anion transport are less defined. We created organoids and organoid-derived monolayers from human gallbladder tissue to measure organoid swelling and transepithelial electrophysiology. In our in vitro models, forskolin-stimulation caused organoid swelling and increased transepithelial anion transport. Full organoid swelling required Cl-while changes in short-circuit current were HCO3 --dependent. Organoids and monolayers from an individual homozygous for the cystic fibrosis-causing ΔF508 CFTR mutation had no apical expression of CFTR and minimal changes in transepithelial current and conductance with forskolin treatment. However, organoid swelling remained intact. Dilution potential studies revealed that forskolin treatment increased the paracellular permeability to anions relative to cations. These data suggest a novel paracellular contribution to forskolin-stimulated fluid transport across the gallbladder epithelium.
RESUMEN
Submucosal glands (SMGs) protect lungs but can also contribute to disease. For example, in cystic fibrosis (CF), SMGs produce abnormal mucus that disrupts mucociliary transport. CF is an ion transport disease, yet knowledge of the ion transporters expressed by SMG acini, which produce mucus, and SMG ducts that carry it to the airway lumen is limited. Therefore, we isolated SMGs from newborn pigs and used single-cell messenger RNA sequencing, immunohistochemistry, and in situ hybridization to identify cell types, gene expression, and spatial distribution. Cell types and transcript levels were the same in non-CF and CF SMGs, suggesting that loss of epithelial anion secretion rather than an intrinsic cell defect causes CF mucus abnormalities. Gene signatures of acinar mucous and acinar serous cells revealed specialized functions in producing mucins and antimicrobials, respectively. However, surprisingly, these two cell types expressed the same ion transporters and neurohumoral receptors, suggesting the importance of balancing mucin and liquid secretion to produce optimal mucus properties. SMG duct cell transcripts suggest that they secrete HCO3- and Cl-, and thus have some similarity to pancreatic ducts that are also defective in CF. These and additional findings suggest the functions of the SMG acinus and duct and provide a baseline for understanding how environmental and genetic challenges impact their contribution to lung disease.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Fibrosis Quística/genética , Fibrosis Quística/metabolismo , Mutación , Mucosa Respiratoria/metabolismo , Células Acinares/metabolismo , Animales , Biomarcadores , Fibrosis Quística/etiología , Fibrosis Quística/patología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Modelos Animales de Enfermedad , Técnica del Anticuerpo Fluorescente , Expresión Génica , Técnicas de Silenciamiento del Gen , Predisposición Genética a la Enfermedad , Mucinas/metabolismo , Depuración Mucociliar , Moco/metabolismo , Mucosa Respiratoria/patología , PorcinosRESUMEN
[This retracts the article DOI: 10.7759/cureus.17850.].
RESUMEN
Light chain deposition disease (LCDD) is a rare cause of kidney failure. It is associated with multiple myeloma and is diagnosed by the evaluation of serum and urine free light chains. Patients who are diagnosed with this disease often develop rapidly progressive renal failure. To our knowledge, it does not have any association with other malignancies aside from multiple myeloma. We present a case that highlights a novel association between renal cell carcinoma and light chain-mediated renal disease. Our patient was admitted due to acute renal failure and underwent a comprehensive diagnostic evaluation with an eventual diagnosis of light chain glomerulopathy in the setting of metastatic renal cell carcinoma.
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Hepatobiliary disease causes significant morbidity and mortality in people with cystic fibrosis (CF), yet this problem remains understudied. Previous studies in the newborn CF pig demonstrated decreased bile flow into the small intestine and a microgallbladder with increased luminal mucus and fluid secretion defects. In this study, we examined the intrahepatic bile ducts of the newborn CF pig. We assessed whether our findings from the gallbladder are present elsewhere in the porcine biliary tract and if CF pig cholangiocytes have fluid secretion defects. Immunohistochemistry demonstrated apical CFTR expression in non-CF pig intrahepatic bile ducts of a variety of sizes; CF pig intrahepatic bile ducts lacked CFTR expression. Assessment of serum markers did not reveal significant signs of hepatobiliary disease except for an elevation in direct bilirubin. Quantitative histology demonstrated that CF pigs had smaller bile ducts that more frequently contained luminal mucus. CF intrahepatic cholangiocyte organoids were smaller and lacked cAMP-mediated fluid secretion. Together these data suggest that cholangiocyte fluid secretion is decreased in the CF pig, contributing to structural changes in bile ducts and decreased biliary flow.
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Conductos Biliares Intrahepáticos/metabolismo , Conductos Biliares Intrahepáticos/patología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/deficiencia , Fibrosis Quística/metabolismo , Fibrosis Quística/patología , Modelos Animales de Enfermedad , Animales , Animales Modificados Genéticamente , Animales Recién Nacidos , Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , PorcinosRESUMEN
Submucosal glands (SMGs) are a prominent structure that lines human cartilaginous airways. Although it has been assumed that SMGs contribute to respiratory defense, that hypothesis has gone without a direct test. Therefore, we studied pigs, which have lungs like humans, and disrupted the gene for ectodysplasin (EDA-KO), which initiates SMG development. EDA-KO pigs lacked SMGs throughout the airways. Their airway surface liquid had a reduced ability to kill bacteria, consistent with SMG production of antimicrobials. In wild-type pigs, SMGs secrete mucus that emerges onto the airway surface as strands. Lack of SMGs and mucus strands disrupted mucociliary transport in EDA-KO pigs. Consequently, EDA-KO pigs failed to eradicate a bacterial challenge in lung regions normally populated by SMGs. These in vivo and ex vivo results indicate that SMGs are required for normal antimicrobial activity and mucociliary transport, two key host defenses that protect the lung.
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Ectodisplasinas/genética , Glándulas Exocrinas/inmunología , Mucosa Respiratoria/inmunología , Staphylococcus aureus/fisiología , Sus scrofa/inmunología , Animales , Ectodisplasinas/inmunología , Femenino , Técnicas de Inactivación de Genes , Masculino , Sus scrofa/genéticaRESUMEN
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.
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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
Mucus produced by submucosal glands is a key component of respiratory mucociliary transport (MCT). When it emerges from submucosal gland ducts, mucus forms long strands on the airway surface. However, the function of those strands is uncertain. To test the hypothesis that mucus strands facilitate transport of large particles, we studied newborn pigs. In ex vivo experiments, interconnected mucus strands moved over the airway surface, attached to immobile spheres, and initiated their movement by pulling them. Stimulating submucosal gland secretion with methacholine increased the percentage of spheres that moved and shortened the delay until mucus strands began moving spheres. To disrupt mucus strands, we applied reducing agents tris-(2-carboxyethyl)phosphine and dithiothreitol. They decreased the fraction of moving spheres and delayed initiation of movement for spheres that did move. We obtained similar in vivo results with CT-based tracking of microdisks in spontaneously breathing pigs. Methacholine increased the percentage of microdisks moving and reduced the delay until they were propelled up airways. Aerosolized tris-(2-carboxyethyl)phosphine prevented those effects. Once particles started moving, reducing agents did not alter their speed either ex vivo or in vivo. These findings indicate that submucosal glands produce mucus in the form of strands and that the strands initiate movement of large particles, facilitating their removal from airways.
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Abnormal airway smooth muscle function can contribute to cystic fibrosis (CF) airway disease. We previously found that airway smooth muscle from newborn CF pigs had increased basal tone, an increased bronchodilator response, and abnormal calcium handling. Since CF pigs lack airway infection and inflammation at birth, these findings suggest intrinsic airway smooth muscle dysfunction in CF. In this study, we tested the hypothesis that CFTR loss in airway smooth muscle would produce a distinct set of changes in the airway smooth muscle transcriptome that we could use to develop novel therapeutic targets. Total RNA sequencing of newborn wild-type and CF airway smooth muscle revealed changes in muscle contraction-related genes, ontologies, and pathways. Using connectivity mapping, we identified several small molecules that elicit transcriptional signatures opposite of CF airway smooth muscle, including NVP-TAE684, an inhibitor of proline-rich tyrosine kinase 2 (PYK2). In CF airway smooth muscle tissue, PYK2 phosphorylation was increased and PYK2 inhibition decreased smooth muscle contraction. In vivo NVP-TAE684 treatment of wild-type mice reduced methacholine-induced airway smooth muscle contraction. These findings suggest that studies in the newborn CF pig may provide an important approach to enhance our understanding of airway smooth muscle biology and for discovery of novel airway smooth muscle therapeutics for CF and other diseases of airway hyperreactivity.
Asunto(s)
Bronquios/metabolismo , Fibrosis Quística/genética , Quinasa 2 de Adhesión Focal/metabolismo , Músculo Liso/metabolismo , Transcriptoma , Animales , Animales Recién Nacidos , Bronquios/enzimología , Bronquios/fisiopatología , Fibrosis Quística/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Expresión Génica , Músculo Liso/enzimología , Músculo Liso/fisiopatología , Cadenas Ligeras de Miosina/metabolismo , Fosforilación , ARN Mensajero/genética , Hipersensibilidad Respiratoria , PorcinosRESUMEN
Ankyrin-B is a multifunctional adapter protein responsible for localization and stabilization of select ion channels, transporters, and signaling molecules in excitable cells including cardiomyocytes. Ankyrin-B dysfunction has been linked with highly penetrant sinoatrial node (SAN) dysfunction and increased susceptibility to atrial fibrillation. While previous studies have identified a role for abnormal ion homeostasis in ventricular arrhythmias, the molecular mechanisms responsible for atrial arrhythmias and SAN dysfunction in human patients with ankyrin-B syndrome are unclear. Here, we develop a computational model of ankyrin-B dysfunction in atrial and SAN cells and tissue to determine the mechanism for increased susceptibility to atrial fibrillation and SAN dysfunction in human patients with ankyrin-B syndrome. Our simulations predict that defective membrane targeting of the voltage-gated L-type Ca(2+) channel Cav1.3 leads to action potential shortening that reduces the critical atrial tissue mass needed to sustain reentrant activation. In parallel, increased fibrosis results in conduction slowing that further increases the susceptibility to sustained reentry in the setting of ankyrin-B dysfunction. In SAN cells, loss of Cav1.3 slows spontaneous pacemaking activity, whereas defects in Na(+)/Ca(2+) exchanger and Na(+)/K(+) ATPase increase variability in SAN cell firing. Finally, simulations of the intact SAN reveal a shift in primary pacemaker site, SAN exit block, and even SAN failure in ankyrin-B-deficient tissue. These studies identify the mechanism for increased susceptibility to atrial fibrillation and SAN dysfunction in human disease. Importantly, ankyrin-B dysfunction involves changes at both the cell and tissue levels that favor the common manifestation of atrial arrhythmias and SAN dysfunction.