ABSTRACT
There is increasing evidence that the study of normal human enteroids duplicates many known aspects of human intestinal physiology. However, this epithelial cell-only model lacks the many nonepithelial intestinal cells present in the gastrointestinal tract and exposure to the mechanical forces to which the intestine is exposed. We tested the hypothesis that physical shear forces produced by luminal and blood flow would provide an intestinal model more closely resembling normal human jejunum. Jejunal enteroid monolayers were studied in the Emulate, Inc. Intestine-Chip under conditions of constant luminal and basolateral flow that was designed to mimic normal intestinal fluid flow, with human umbilical vein endothelial cells (HUVECs) on the basolateral surface and with Wnt3A, R-spondin, and Noggin only on the luminal surface. The jejunal enteroids formed monolayers that remained confluent for 6-8 days, began differentiating at least as early as day 2 post plating, and demonstrated continuing differentiation over the entire time of the study, as shown by quantitative real-time polymerase chain reaction and Western blot analysis. Differentiation impacted villus genes and proteins differently with early expression of regenerating family member 1α (REG1A), early reduction to a low but constant level of expression of Na+-K+-2Cl- cotransporter 1 (NKCC1), and increasing expression of sucrase-isomaltase (SI) and downregulated in adenoma (DRA). These results were consistent with continual differentiation, as was shown to occur in mouse villus enterocytes. Compared with differentiated enteroid monolayers grown on Transwell inserts, enteroids exposed to flow were more differentiated but exhibited increased apoptosis and reduced carbohydrate metabolism, as shown by proteomic analysis. This study of human jejunal enteroids-on-chip suggests that luminal and basolateral flow produce a model of continual differentiation over time and NaCl absorption that mimics normal intestine and should provide new insights in intestinal physiology.NEW & NOTEWORTHY This study showed that polarized enteroid models in which there is no basolateral Wnt3a, are differentiated, regardless of the Wnt3a status of the apical media. The study supports the concept that in the human intestine villus differentiation is not an all or none phenomenon, demonstrating that at different days after lack of basolateral Wnt exposure, clusters of genes and proteins exist geographically along the villus with different domains having different functions.
Subject(s)
Cell Differentiation , Jejunum/cytology , Microfluidics/methods , Primary Cell Culture/methods , Stress, Mechanical , Adult , Apoptosis , Carrier Proteins/metabolism , Cells, Cultured , Enterocytes/cytology , Enterocytes/metabolism , Female , Human Umbilical Vein Endothelial Cells/metabolism , Humans , Jejunum/metabolism , Lithostathine/metabolism , Solute Carrier Family 12, Member 2/metabolism , Thrombospondins/metabolism , Wnt3A Protein/metabolismABSTRACT
Acetylcholine induces robust electrogenic anion secretion in mammalian intestine and it has long been hypothesized that it mediates the epithelial response through the M3 and, to a lesser extent, the M1 muscarinic receptors in the mouse. However, nicotinic receptors have recently been identified in intestinal enterocytes by quantitative real-time (qRT)-PCR/RNAseq, although any direct influence on intestinal transport has not been identified. We tested the hypothesis that cholinergic-induced anion secretion in the intestine is a result of both muscarinic and nicotinic pathways that are intrinsic to the intestinal epithelia. We developed a method to generate mouse jejunal enteroid monolayers which were used to measure active electrogenic anion secretion by the Ussing chamber/voltage-clamp technique. Here, we show that the cholinergic agonist carbachol (CCh) and the muscarinic agonist bethanechol (BCh) stimulate short-lived, concentration-dependent anion secretion in the epithelial cell-only enteroid monolayers. The muscarinic antagonist atropine completely inhibited CCh- and BCh-induced secretion, while the nicotinic antagonist hexamethonium reduced the CCh response by ~45%. While nicotine alone did not alter anion secretion, it increased the BCh-induced increase in short-circuit current in a concentration-dependent manner; this synergy was prevented by pretreatment with hexamethonium. In addition to being sensitive to hexamethonium, monolayers express both classes of cholinergic receptor by qRT-PCR, including 13 of 16 nicotinic receptor subunits. Our findings indicate that an interaction between muscarinic and nicotinic agonists synergistically stimulates anion secretion in mouse jejunal epithelial cells and identify a role for epithelial nicotinic receptors in anion secretion.
Subject(s)
Muscarinic Agonists/pharmacology , Non-Neuronal Cholinergic System/genetics , Receptors, Muscarinic/genetics , Receptors, Nicotinic/genetics , Acetylcholine/pharmacology , Animals , Anions/metabolism , Atropine/pharmacology , Cholinergic Agonists/pharmacology , Enterocytes/drug effects , Enterocytes/metabolism , Hexamethonium/pharmacology , Humans , Intestinal Mucosa/drug effects , Intestinal Mucosa/metabolism , Intestines/drug effects , Mice , Non-Neuronal Cholinergic System/drug effects , Receptors, Muscarinic/metabolism , Receptors, Nicotinic/metabolismABSTRACT
Modeling host-pathogen interactions with human intestinal epithelia using enteroid monolayers on permeable supports (such as Transwells) represents an alternative to animal studies or use of colon cancer-derived cell lines. However, the static monolayer model does not expose epithelial cells to mechanical forces normally present in the intestine, including luminal flow and serosal blood flow (shear force) or peristaltic forces. To determine the contribution of mechanical forces in the functional response of human small intestine to a virulence factor of a pathogenic intestinal bacterium, human jejunal enteroids were cultured as monolayers in microengineered fluidic-based Organ-Chips (Intestine-Chips) exposed to enterotoxigenic Escherichia coli heat-stable enterotoxin A (ST) and evaluated under conditions of static fluid, apical and basolateral flow, and flow plus repetitive stretch. Application of flow increased epithelial cell height and apical and basolateral secretion of cyclic GMP (cGMP) under baseline, unstimulated conditions. Addition of ST under flow conditions increased apical and basolateral secretion of cGMP relative to the level under static conditions but did not enhance intracellular cGMP accumulation. Cyclic stretch did not have any significant effect beyond that contributed by flow. This study demonstrates that fluid flow application initiates changes in intestinal epithelial cell characteristics relative to those of static culture conditions under both baseline conditions and with exposure to ST enterotoxin and suggests that further investigations of the application of these mechanical forces will provide insights into physiology and pathophysiology that more closely resemble intact intestine than study under static conditions.
Subject(s)
Cyclic GMP/physiology , Enterotoxigenic Escherichia coli/physiology , Enterotoxins/physiology , Escherichia coli Infections/physiopathology , Escherichia coli Proteins/physiology , Intestine, Small/physiology , Signal Transduction/physiology , Stress, Mechanical , Bacterial Toxins , Humans , Jejunum/cytology , Virulence Factors/physiologyABSTRACT
Cholera toxin (CT) causes severe diarrhea by increasing intracellular cAMP leading to a PKA-dependent increase in Cl- secretion through CFTR and decreased Na+ absorption through inhibition of Na+/H+ exchanger 3 (NHE3; also known as SLC9A3). The mechanism(s) by which CT inhibits NHE3 is partially understood, although no drug therapy has been successful at reversing this inhibition. We now describe that CT phosphorylates an amino acid in the PDZ domain of SNX27, which inhibits SNX27-mediated trafficking of NHE3 from the early endosomes to the plasma membrane (PM), and contributes to reduced basal NHE3 activity through a mechanism that involves reduced PM expression and reduced endocytic recycling. Importantly, mutagenesis studies (Ser to Asp) showed that the effect of this phosphorylation of SNX27 phenocopies the effects seen upon loss of SNX27 function, affecting PM trafficking of cargo proteins that bind SNX27-retromer. Additionally, CT destabilizes retromer function by decreasing the amount of core retromer proteins. These effects of CT can be partially rescued by enhancing retromer stability by using 'pharmacological chaperones'. Moreover, pharmacological chaperones can be used to increase basal and cholera toxin-inhibited NHE3 activity and fluid absorption by intestinal epithelial cells.This article has an associated First Person interview with the first author of the paper.
Subject(s)
Cell Membrane/metabolism , Cholera Toxin/pharmacology , Endosomes/drug effects , Endosomes/metabolism , Sorting Nexins/metabolism , Caco-2 Cells , Cell Membrane/drug effects , Cells, Cultured , Down-Regulation/drug effects , Endocytosis/drug effects , HEK293 Cells , HeLa Cells , Humans , Molecular Chaperones/genetics , Molecular Chaperones/metabolism , Protein Transport/drug effects , Sorting Nexins/genetics , Vesicular Transport Proteins/genetics , Vesicular Transport Proteins/metabolismABSTRACT
The investigation of orphan GPCRs (GPRs) has the potential to uncover novel insights into whole animal physiology. In this study, our goal was to determine the renal localization of Gprc5c, a receptor that we previously reported to be highly expressed in murine whole kidney, and to examine physiologic parameters in Gprc5c knockout (KO) mice to gain insight into function. Gprc5c localized to the apical membrane of renal proximal tubules (PTs) in mice, rats, and humans. With the comparison of Gprc5c wild-type (WT) and KO mice, we found that Gprc5c KO mice have altered acid-base homeostasis. Specifically, Gprc5c KO mice have lower blood pH and higher urine pH compared with WT mice, with a reduced level of titratable acids in their urine. In an in vitro GPCR internalization assay, we observed that Gprc5c internalization (an index of activation) was triggered by alkaline extracellular pH. Furthermore, with the use of an in vitro BCECF assay, we observed that Gprc5c increases Na+/H+ exchanger 3 (NHE3) activity at alkaline pH. We also find that the NHE3 activity is reduced in Gprc5c KO mice by 2 photon imaging in seminaphthorhodafluors (SNARF)-4F-loaded kidney sections. NHE3 is a primary contributor to apical transport of H+ in the renal PT. Together, these data imply that Gprc5c modulates the renal contribution to systemic pH homeostasis, at least in part, by taking part in the regulation of NHE3.-Rajkumar, P., Cha, B., Yin, J., Arend, L. J., Paunescu, T. G., Hirabayashi, Y., Donowitz, M., Pluznick, J. L. Identifying the localization and exploring a functional role for Gprc5c in the kidney.
Subject(s)
Kidney Tubules, Proximal/metabolism , Receptors, G-Protein-Coupled/metabolism , Acids/blood , Acids/urine , Alkalies/blood , Alkalies/urine , Animals , HEK293 Cells , Humans , Kidney Tubules, Proximal/physiology , Mice , Mice, Inbred C57BL , Protein Transport , Receptors, G-Protein-Coupled/genetics , Sodium-Hydrogen Exchanger 3/metabolism , Water-Electrolyte BalanceABSTRACT
Secretory diarrheas caused by bacterial enterotoxins, including cholera and traveler's diarrhea, remain a major global health problem. Inappropriate activation of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel occurs in these diarrheas. We previously reported that the benzopyrimido-pyrrolo-oxazinedione (R)-BPO-27 inhibits CFTR chloride conductance with low-nanomolar potency. Here, we demonstrate using experimental mouse models and human enterocyte cultures the potential utility of (R)-BPO-27 for treatment of secretory diarrheas caused by cholera and Escherichia coli enterotoxins. (R)-BPO-27 fully blocked CFTR chloride conductance in epithelial cell cultures and intestine after cAMP agonists, cholera toxin, or heat-stable enterotoxin of E. coli (STa toxin), with IC50 down to â¼5 nM. (R)-BPO-27 prevented cholera toxin and STa toxin-induced fluid accumulation in small intestinal loops, with IC50 down to 0.1 mg/kg. (R)-BPO-27 did not impair intestinal fluid absorption or inhibit other major intestinal transporters. Pharmacokinetics in mice showed >90% oral bioavailability with sustained therapeutic serum levels for >4 h without the significant toxicity seen with 7-d administration at 5 mg/kg/d. As evidence to support efficacy in human diarrheas, (R)-BPO-27 blocked fluid secretion in primary cultures of enteroids from human small intestine and anion current in enteroid monolayers. These studies support the potential utility of (R)-BPO-27 for therapy of CFTR-mediated secretory diarrheas.-Cil, O., Phuan, P.-W., Gillespie, A. M., Lee, S., Tradtrantip, L., Yin, J., Tse, M., Zachos, N. C., Lin, R., Donowitz, M., Verkman, A. S. Benzopyrimido-pyrrolo-oxazine-dione CFTR inhibitor (R)-BPO-27 for antisecretory therapy of diarrheas caused by bacterial enterotoxins.
Subject(s)
Cystic Fibrosis Transmembrane Conductance Regulator/antagonists & inhibitors , Diarrhea/chemically induced , Diarrhea/drug therapy , Oxazines/pharmacology , Pyrimidinones/pharmacology , Pyrroles/pharmacology , Animals , Dose-Response Relationship, Drug , Female , Humans , Intestines/drug effects , Mice , Molecular Structure , Oxazines/chemical synthesis , Pyrimidinones/chemical synthesis , Pyrroles/chemical synthesisABSTRACT
Congenital sodium diarrhea (CSD) refers to an intractable diarrhea of intrauterine onset with high fecal sodium loss. CSD is clinically and genetically heterogeneous. Syndromic CSD is caused by SPINT2 mutations. While we recently described four cases of the non-syndromic form of CSD that were caused by dominant activating mutations in intestinal receptor guanylate cyclase C (GC-C), the genetic cause for the majority of CSD is still unknown. Therefore, we aimed to determine the genetic cause for non-GC-C non-syndromic CSD in 18 patients from 16 unrelated families applying whole-exome sequencing and/or chromosomal microarray analyses and/or direct Sanger sequencing. SLC9A3 missense, splicing and truncation mutations, including an instance of uniparental disomy, and whole-gene deletion were identified in nine patients from eight families with CSD. Two of these nine patients developed inflammatory bowel disease (IBD) at 4 and 16 years of age. SLC9A3 encodes Na(+)/H(+) antiporter 3 (NHE3), which is the major intestinal brush-border Na(+)/H(+) exchanger. All mutations were in the NHE3 N-terminal transport domain, and all missense mutations were in the putative membrane-spanning domains. Identified SLC9A3 missense mutations were functionally characterized in plasma membrane NHE null fibroblasts. SLC9A3 missense mutations compromised NHE3 activity by reducing basal surface expression and/or loss of basal transport function of NHE3 molecules, whereas acute regulation was normal. This study identifies recessive mutations in NHE3, a downstream target of GC-C, as a cause of CSD and implies primary basal NHE3 malfunction as a predisposition for IBD in a subset of patients.
Subject(s)
Abnormalities, Multiple/genetics , Diarrhea/congenital , Metabolism, Inborn Errors/genetics , Mutation , Sodium-Hydrogen Exchangers/genetics , Abnormalities, Multiple/metabolism , Abnormalities, Multiple/physiopathology , Adolescent , Adult , Child , Child, Preschool , DNA Mutational Analysis , Diarrhea/genetics , Diarrhea/metabolism , Diarrhea/physiopathology , Female , Genes, Recessive , Humans , Infant , Infant, Newborn , Inflammatory Bowel Diseases/genetics , Inflammatory Bowel Diseases/metabolism , Inflammatory Bowel Diseases/physiopathology , Intestinal Mucosa/metabolism , Intestines/physiopathology , Male , Metabolism, Inborn Errors/metabolism , Metabolism, Inborn Errors/physiopathology , Microvilli/metabolism , Oligonucleotide Array Sequence Analysis , Sodium-Hydrogen Exchanger 3 , Young AdultABSTRACT
Na+/H+ exchanger NHE3 mediates the majority of intestinal and renal electroneutral sodium absorption. Dysfunction of NHE3 is associated with a variety of diarrheal diseases. We previously reported that the NHE3 gene (SLC9A3) has more than 400 single-nucleotide polymorphisms (SNPs) but few nonsynonymous polymorphisms. Among the latter, one polymorphism (rs2247114-G>A), which causes a substitution from arginine to cysteine at amino acid position 799 (p.R799C), is common in Asian populations. To improve our understanding of the population distribution and potential clinical significance of the NHE3-799C variant, we investigated the frequency of this polymorphism in different ethnic groups using bioinformatics analyses and in a cohort of Japanese patients with cardiovascular or renal disease. We also characterized the function of human NHE3-799C and its sensitivity to regulatory ligands in an in vitro model. NHE3-799C had an allele frequency of 29.5-57.6% in Asian populations, 11.1-23.6% in European populations, and 10.2-22.7% in African populations. PS120/FLAG-NHERF2 fibroblasts stably expressing NHE3-799C had lower total protein expression but a higher percentage of surface expression than those expressing NHE3-799R. NHE3-799C had similar basal activity to NHE3-799R and was similarly stimulated or inhibited, by serum or forskolin, respectively. Tenapanor, a small-molecule NHE3 inhibitor, dose-dependently inhibited NHE3-799R and NHE3-799C activities. The IC50 values of tenapanor for NHE3-799C and NHE3-799R were significantly different, but both were in the nanomolar range. These results suggest that NHE3-799C is a common variant enriched in Asian populations, is not associated with compromised function or abnormal regulation, and is unlikely to contribute to clinical disease.NEW & NOTEWORTHY This study reports results on the functional significance of human NHE3-799C under basal conditions and in response to regulatory ligands, including a novel NHE3 inhibitor called tenapanor. We demonstrate that NHE3-799C is a common variant of NHE3 that is enriched in Asian populations; however, in contrast to our previous studies using rabbit NHE3, its presence seems to have limited clinical significance in humans and is not associated with compromised function or abnormal transport regulation.
Subject(s)
Alleles , Gene Frequency , Polymorphism, Single Nucleotide , Sodium-Hydrogen Exchangers/genetics , Asian People/genetics , Cardiovascular Diseases/genetics , Computational Biology , Genotype , Humans , Kidney Diseases/genetics , Mutation , Sodium-Hydrogen Exchanger 3 , White People/geneticsABSTRACT
BACKGROUND & AIMS: Human intestinal crypt-derived enteroids are a model of intestinal ion transport that require validation by comparison with cell culture and animal models. We used human small intestinal enteroids to study neutral Na(+) absorption and stimulated fluid and anion secretion under basal and regulated conditions in undifferentiated and differentiated cultures to show their functional relevance to ion transport physiology and pathophysiology. METHODS: Human intestinal tissue specimens were obtained from an endoscopic biopsy or surgical resections performed at Johns Hopkins Hospital. Crypts were isolated, enteroids were propagated in culture, induced to undergo differentiation, and transduced with lentiviral vectors. Crypt markers, surface cell enzymes, and membrane ion transporters were characterized using quantitative reverse-transcription polymerase chain reaction, immunoblot, or immunofluorescence analyses. We used multiphoton and time-lapse confocal microscopy to monitor intracellular pH and luminal dilatation in enteroids under basal and regulated conditions. RESULTS: Enteroids differentiated upon withdrawal of WNT3A, yielding decreased crypt markers and increased villus-like characteristics. Na(+)/H(+) exchanger 3 activity was similar in undifferentiated and differentiated enteroids, and was affected by known inhibitors, second messengers, and bacterial enterotoxins. Forskolin-induced swelling was completely dependent on cystic fibrosis transmembrane conductance regulator and partially dependent on Na(+)/H(+) exchanger 3 and Na(+)/K(+)/2Cl(-) cotransporter 1 inhibition in undifferentiated and differentiated enteroids. Increases in cyclic adenosine monophosphate with forskolin caused enteroid intracellular acidification in HCO3(-)-free buffer. Cyclic adenosine monophosphate-induced enteroid intracellular pH acidification as part of duodenal HCO3(-) secretion appears to require cystic fibrosis transmembrane conductance regulator and electrogenic Na(+)/HCO3(-) cotransporter 1. CONCLUSIONS: Undifferentiated or crypt-like, and differentiated or villus-like, human enteroids represent distinct points along the crypt-villus axis; they can be used to characterize electrolyte transport processes along the vertical axis of the small intestine. The duodenal enteroid model showed that electrogenic Na(+)/HCO3(-) cotransporter 1 might be a target in the intestinal mucosa for treatment of secretory diarrheas.
Subject(s)
Cell Differentiation , Intestine, Small/metabolism , Membrane Transport Proteins/metabolism , Sodium/metabolism , Gene Expression Regulation , Humans , Hydrogen-Ion Concentration , Intestinal Secretions/metabolism , Intestine, Small/pathology , Intestine, Small/physiopathology , Ion Transport , Kinetics , Membrane Transport Proteins/genetics , Microscopy, Confocal , Microscopy, Fluorescence, Multiphoton , Microscopy, Video , Time-Lapse Imaging , Tissue Culture Techniques , Transduction, Genetic , TransfectionABSTRACT
In the brush border of intestinal and kidney epithelial cells, scaffolding proteins ezrin, Na(+)-H(+) exchanger regulatory factor (NHERF)1 and NHERF2 play important roles in linking transmembrane proteins to the cytoskeleton and assembling signalling regulatory complexes. The last 30 carboxyl residues of NHERF1 and NHERF2 form the EBDs [ezrin, radixin and moesin (ERM)-binding domain]. The current study found that NHERF1/2 contain an ERM-binding regulatory sequence (EBRS), which facilitates the interaction between the EBD and ezrin. The EBRSs are located within 24 and 19 residues immediately upstream of EBDs for NHERF1 and NHERF2 respectively. In OK (opossum kidney) epithelial cells, EBRSs are necessary along with the EBD to distribute NHERF1 and NHERF2 exclusively to the apical domain. Furthermore, phosphorylation of Ser(303) located in the EBRS of NHERF2, decreases the binding affinity for ezrin, dislocates apical NHERF2 into the cytosol and increases the NHERF2 microvillar mobility rate. Moreover, increased phosphorylation of Ser(303) was functionally significant preventing acute stimulation of NHE3 (Na(+)-H(+) exchanger 3) activity by dexamethasone.
Subject(s)
DNA-Binding Proteins/metabolism , Dexamethasone/pharmacology , Phosphoproteins/metabolism , Sodium-Hydrogen Exchangers/metabolism , Transcription Factors/metabolism , Amino Acid Sequence , Animals , Binding Sites/drug effects , Binding Sites/physiology , Caco-2 Cells , DNA-Binding Proteins/genetics , HEK293 Cells , Humans , Molecular Sequence Data , Opossums , Phosphoproteins/genetics , Sodium-Hydrogen Exchanger 3 , Sodium-Hydrogen Exchangers/genetics , Transcription Factors/geneticsABSTRACT
Th17 cells that produce Interleukin IL-17 are pathogenic in many human diseases, including inflammatory bowel disease, but are, paradoxically, essential for maintaining the integrity of the intestinal barrier in a non-inflammatory state. However, the intracellular mechanisms that regulate distinct transcriptional profiles and functional diversity of Th17 cells remain unclear. Here we show Raftlin1, a lipid raft protein, specifically upregulates and forms a complex with RORγt in pathogenic Th17 cells. Disruption of the RORγt-Raftlin1 complex results in the reduction of pathogenic Th17 cells in response to Citrobacter rodentium; however, there is no effect on nonpathogenic Th17 cells in response to commensal segmented filamentous bacteria. Mechanistically, we show that Raftlin1 recruits distinct phospholipids to RORγt and promotes the pathogenicity of Th17 cells. Thus, we have identified a mechanism that drives the pathogenic function of Th17 cells, which could provide a platform for advanced therapeutic strategies to dampen Th17-mediated inflammatory diseases.
Subject(s)
Nuclear Receptor Subfamily 1, Group F, Member 3 , Th17 Cells , Humans , Nuclear Receptor Subfamily 1, Group F, Member 3/genetics , Virulence , Inflammation , ColonABSTRACT
Eosinophilic gastrointestinal diseases are rare disorders characterized by infiltration of eosinophils in one or multiple segments of the gastrointestinal tract. Hypersensitivity to food or environmental allergens is believed to play an important role in the pathogenesis. In this case report, we describe a 61-year-old man who developed eosinophilic gastroenteritis and colitis with severe peripheral eosinophilia after intragastric balloon (IGB) placement for weight loss. His symptoms and peripheral eosinophilia improved rapidly after removal of the IGB without the need for immunomodulatory therapies or diet modifications. This case suggests a possible association between IGB and eosinophilic gastrointestinal diseases, which warrants clinicians' awareness and further studies.
ABSTRACT
INTRODUCTION: Immune checkpoint inhibitors (ICIs) have become a standard of care in metastatic renal cell carcinoma (mRCC) but are associated with immune-related adverse events (irAEs) including colitis. Growing evidence suggests proton pump inhibitors (PPIs) increase the risk of inflammatory bowel disease (IBD). Given the pathophysiological overlap between IBD and ICI colitis, we sought to evaluate the relationship between PPI use and ICI colitis in mRCC patients. PATIENTS AND METHODS: We performed a retrospective study of adult patients who received ICI therapy for mRCC between 2015 and 2018 at University of Texas Southwestern Medical Center affiliated hospitals. Clinical characteristics, oncological outcomes, ICI colitis details, and PPI use details were collected by manual chart review. The diagnosis of ICI colitis was made via biopsy when available, or by clinical criteria (symptoms and response to immunosuppressive therapy) when biopsy specimens were unavailable or inconclusive. Univariable and multivariable logistic regression analyses were conducted to assess the potential contribution of PPIs to ICI colitis. RESULTS: A total of 176 patients received ICI therapy for mRCC, of which 16 (9.1%) were diagnosed with ICI colitis. Patients with ICI colitis presented with elevated stool lactoferritin and calprotectin and a wide range of endoscopic and histologic findings. There were no significant differences between patients with and without ICI colitis in age, gender, medical comorbidities, RCC history, and overall survival. However, exposure to ipilimumab and PPI use were more frequently observed in patients with ICI colitis than those without. In univariable and multivariable logistic regression analyses, exposure to ipilimumab and chronic use of PPIs > 8 weeks were significantly associated with ICI colitis. CONCLUSION: In addition to ipilimumab use, chronic use of PPIs may be associated with ICI colitis in patients with mRCC.
Subject(s)
Carcinoma, Renal Cell , Colitis , Inflammatory Bowel Diseases , Kidney Neoplasms , Adult , Carcinoma, Renal Cell/drug therapy , Colitis/chemically induced , Colitis/drug therapy , Humans , Immune Checkpoint Inhibitors/adverse effects , Inflammatory Bowel Diseases/drug therapy , Ipilimumab/therapeutic use , Kidney Neoplasms/drug therapy , Proton Pump Inhibitors/adverse effects , Retrospective StudiesABSTRACT
BACKGROUND & AIMS: One of the features of ulcerative colitis (UC) is a defect in the protective mucus layer. This has been attributed to a reduced number of goblet cells (GCs). However, it is not known whether abnormal GC mucus secretion also contributes to the reduced mucus layer. Our aims were to investigate whether GC secretion was abnormal in UC and exists as a long-term effect of chronic inflammation. METHODS: Colonoids were established from intestinal stem cells of healthy subjects (HS) and patients with UC. Colonoids were maintained as undifferentiated (UD) or induced to differentiate (DF) and studied as three-dimensional or monolayers on Transwell filters. Total RNA was extracted for quantitative real-time polymerase chain reaction analysis. Carbachol and prostaglandin E2 mediated mucin stimulation was examined by MUC2 IF/confocal microscopy and transmission electron microscopy. RESULTS: Colonoids from UC patients can be propagated over many passages; however, they exhibit a reduced rate of growth and transepithelial electrical resistance compared with HS. Differentiated UC colonoid monolayers form a thin and non-continuous mucus layer. UC colonoids have increased expression of secretory lineage markers ATOH1 and SPDEF, along with MUC2 positive GCs, but failed to secrete mucin in response to the cholinergic agonist carbachol and prostaglandin E2, which caused increased secretion in HS. Exposure to tumor necrosis factor α (5 days) reduced the number of GCs, with a greater percentage decrease in UC colonoids compared with HS. CONCLUSIONS: Chronic inflammation in UC causes long-term changes in GCs, leading to abnormal mucus secretion. This continued defect in GC mucus secretion may contribute to the recurrence in UC.
Subject(s)
Colitis, Ulcerative , Colitis, Ulcerative/pathology , Goblet Cells/pathology , Humans , Inflammation/pathology , Intestinal Mucosa/metabolism , Mucins/metabolismABSTRACT
OBJECTIVE: The mechanisms behind the efficacy of bariatric surgery (BS) for treating obesity and type 2 diabetes, particularly with respect to the influence of the small bowel, remain poorly understood. In vitro and animal models are suboptimal with respect to their ability to replicate the human intestinal epithelium under conditions induced by obesity. Human enteroids have the potential to accelerate the development of less invasive anti-obesity therapeutics if they can recapitulate the pathophysiology of obesity. Our aim was to determine whether adult stem cell-derived enteroids preserve obesity-characteristic patient-specific abnormalities in carbohydrate absorption and metabolism. METHODS: We established 24 enteroid lines representing 19 lean, overweight, or morbidly obese patients, including post-BS cases. Dietary glucose absorption and gluconeogenesis in enteroids were measured. The expression of carbohydrate transporters and gluconeogenic enzymes was assessed and a pharmacological approach was used to dissect the specific contribution of each transporter or enzyme to carbohydrate absorption and metabolism, respectively. RESULTS: Four phenotypes representing the relationship between patients' BMI and intestinal dietary sugar absorption were found, suggesting that human enteroids retain obese patient phenotype heterogeneity. Intestinal glucose absorption and gluconeogenesis were significantly elevated in enteroids from a cohort of obese patients. Elevated glucose absorption was associated with increased expression of SGLT1 and GLUT2, whereas elevated gluconeogenesis was related to increased expression of GLUT5, PEPCK1, and G6Pase. CONCLUSIONS: Obesity phenotypes preserved in human enteroids provide a mechanistic link to aberrant dietary carbohydrate absorption and metabolism. Enteroids can be used as a preclinical platform to understand the pathophysiology of obesity, study the heterogeneity of obesity mechanisms, and identify novel therapeutics.
Subject(s)
Gluconeogenesis/physiology , Glucose/metabolism , Intestine, Small/metabolism , Obesity, Morbid/metabolism , Phenotype , Stem Cells/metabolism , Animals , Bariatric Surgery , Diabetes Mellitus, Type 2/metabolism , Dietary Carbohydrates/metabolism , Glucose Transporter Type 2/metabolism , Glucose Transporter Type 5/metabolism , Humans , Intestinal Absorption , Intestinal Mucosa/metabolism , Sodium-Glucose Transporter 1/metabolismABSTRACT
[This corrects the article DOI: 10.1093/ofid/ofz492.].
ABSTRACT
Induction of intestinal drug metabolizing enzymes can complicate the development of new drugs, owing to the potential to cause drug-drug interactions (DDIs) leading to changes in pharmacokinetics, safety and efficacy. The development of a human-relevant model of the adult intestine that accurately predicts CYP450 induction could help address this challenge as species differences preclude extrapolation from animals. Here, we combined organoids and Organs-on-Chips technology to create a human Duodenum Intestine-Chip that emulates intestinal tissue architecture and functions, that are relevant for the study of drug transport, metabolism, and DDI. Duodenum Intestine-Chip demonstrates the polarized cell architecture, intestinal barrier function, presence of specialized cell subpopulations, and in vivo relevant expression, localization, and function of major intestinal drug transporters. Notably, in comparison to Caco-2, it displays improved CYP3A4 expression and induction capability. This model could enable improved in vitro to in vivo extrapolation for better predictions of human pharmacokinetics and risk of DDIs.
Subject(s)
Drug Evaluation, Preclinical/instrumentation , Drug Interactions , Duodenum/metabolism , ATP Binding Cassette Transporter, Subfamily B/metabolism , Animals , Caco-2 Cells , Computational Biology , Cytochrome P-450 CYP3A/metabolism , Gene Expression Profiling , Gene Expression Regulation , Humans , Microvilli , Organ Culture Techniques , Organoids/metabolism , Permeability , TranscriptomeABSTRACT
Intestinal regeneration and crypt hyperplasia after radiation or pathogen injury relies on Wnt signaling to stimulate stem cell proliferation. Mesenchymal Wnts are essential for homeostasis and regeneration in mice, but the role of epithelial Wnts remains largely uncharacterized. Using the enterohemorrhagic E. coli-secreted cytotoxin EspP to induce injury to human colonoids, we evaluated a simplified, epithelial regeneration model that lacks mesenchymal Wnts. Here, we demonstrate that epithelial-produced WNT2B is upregulated following injury and essential for regeneration. Hedgehog signaling, specifically activation via the ligand Desert Hedgehog (DHH), but not Indian or Sonic Hedgehog, is another driver of regeneration and modulates WNT2B expression. These findings highlight the importance of epithelial WNT2B and DHH in regulating human colonic regeneration after injury.
ABSTRACT
BACKGROUND & AIMS: SLC26A3 (DRA) is an electroneutral Cl-/HCO3- exchanger that is present in the apical domain of multiple intestinal segments. An area that has continued to be poorly understood is related to DRA regulation in acute adenosine 3',5'-cyclic monophosphate (cAMP)-related diarrheas, in which DRA appears to be both inhibited as part of NaCl absorption and stimulated to contribute to increased HCO3- secretion. Different cell models expressing DRA have shown that cAMP inhibits, stimulates, or does not affect its activity. METHODS: This study re-evaluated cAMP regulation of DRA using new tools, including a successful knockout cell model, a specific DRA inhibitor (DRAinh-A250), specific antibodies, and a transport assay that did not rely on nonspecific inhibitors. The studies compared DRA regulation in colonoids made from normal human colon with regulation in the colon cancer cell line, Caco-2. RESULTS: DRA is an apical protein in human proximal colon, differentiated colonoid monolayers, and Caco-2 cells. It is glycosylated and appears as 2 bands. cAMP (forskolin) acutely stimulated DRA activity in human colonoids and Caco-2 cells. In these cells, DRA is the predominant apical Cl-/HCO3- exchanger and is inhibited by DRAinh-A250 with a median inhibitory concentration of 0.5 and 0.2 µmol/L, respectively. However, there was no effect of cAMP in HEK293/DRA cells that lacked a cystic fibrosis transmembrane conductance regulator (CFTR). When CFTR was expressed in HEK293/DRA cells, cAMP also stimulated DRA activity. In all cases, cAMP stimulation of DRA was not inhibited by CFTRinh-172. CONCLUSIONS: DRA is acutely stimulated by cAMP by a process that is CFTR-dependent, but appears to be one of multiple regulatory effects of CFTR that does not require CFTR activity.