Jianyi Yin

Human Enteroids as a Model of Upper Small Intestinal Ion Transport Physiology and Pathophysiology.

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.

Reduced sodium/proton exchanger NHE3 activity causes congenital sodium diarrhea

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.

Benzopyrimido-pyrrolo-oxazine-dione CFTR inhibitor (R)-BPO-27 for antisecretory therapy of diarrheas caused by bacterial enterotoxins

Secretory diarrheas caused by bacterial enterotoxins, including cholera and travelers 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‐pyrrolooxazinedione (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 ~5 nM. (R)‐BPO‐27 prevented cholera toxin and STa toxin‐induced fluid accumulation in small intestinal loops, with IC50 down to 0.1 μg/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 μg/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. FASEB J. 31, 751–760 (2017). http://www.fasebj.org

Sorting nexin 27 regulates basal and stimulated brush border trafficking of NHE3.

In polarized epithelial cells, SNX27 regulates PDZ domain–directed trafficking of NHE3 from endosomes to the plasma membrane and increases the stability of brush border NHE3. This establishes SNX27 as an important regulator of polarized sorting in epithelial cells.

The NHERF2 sequence adjacent and upstream of the ERM-binding domain affects NHERF2-ezrin binding and dexamethasone stimulated NHE3 activity.

The microvillar localization of Na+-H+ exchanger regulatory factor (NHERF)1/2 requires not only ezrin, radixin and moesin (ERM)-binding domain (EBD) but also a newly defined ERM-binding regulatory sequence (EBRS) that modulates NHERF1/2–ezrin binding. NHERF2 EBRS is also regulated by phosphorylation, which affects NHE3 (Na+-H+ exchanger 3) stimulation by dexamethasone.

A Common NHE3 Single Nucleotide Polymorphism Has Normal Function and Sensitivity to Regulatory Ligands

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.

Molecular Basis and Differentiation-Associated Alterations of Anion Secretion in Human Duodenal Enteroid Monolayers

Background & Aims Human enteroids present a novel tool to study human intestinal ion transport physiology and pathophysiology. The present study describes the contributions of Cl- and HCO3- secretion to total cyclic adenosine monophosphate (cAMP)-stimulated electrogenic anion secretion in human duodenal enteroid monolayers and the relevant changes after differentiation. Methods Human duodenal enteroids derived from 4 donors were grown as monolayers and differentiated by a protocol that includes the removal of Wnt3A, R-spondin1, and SB202190 for 5 days. The messenger RNA level and protein expression of selected ion transporters and carbonic anhydrase isoforms were determined by quantitative real-time polymerase chain reaction and immunoblotting, respectively. Undifferentiated and differentiated enteroid monolayers were mounted in the Ussing chamber/voltage-current clamp apparatus, using solutions that contained as well as lacked Cl- and HCO3-/CO2, to determine the magnitude of forskolin-induced short-circuit current change and its sensitivity to specific inhibitors that target selected ion transporters and carbonic anhydrase(s). Results Differentiation resulted in a significant reduction in the messenger RNA level and protein expression of cystic fibrosis transmembrane conductance regulator, (CFTR) Na+/K+/2Cl- co-transporter 1 (NKCC1), and potassium channel, voltage gated, subfamily E, regulatory subunit 3 (KCNE3); and, conversely, increase of down-regulated-in-adenoma (DRA), electrogenic Na+/HCO3- co-transporter 1 (NBCe1), carbonic anhydrase 2 (CA2), and carbonic anhydrase 4 (CA4). Both undifferentiated and differentiated enteroids showed active cAMP-stimulated anion secretion that included both Cl- and HCO3- secretion as the magnitude of total active anion secretion was reduced after the removal of extracellular Cl- or HCO3-/CO2. The magnitude of total anion secretion in differentiated enteroids was approximately 33% of that in undifferentiated enteroids, primarily owing to the reduction in Cl- secretion with no significant change in HCO3- secretion. Anion secretion was consistently lower but detectable in differentiated enteroids compared with undifferentiated enteroids in the absence of extracellular Cl- or HCO3-/CO2. Inhibiting CFTR, NKCC1, carbonic anhydrase(s), cAMP-activated K+ channel(s), and Na+/K+-adenosine triphosphatase reduced cAMP-stimulated anion secretion in both undifferentiated and differentiated enteroids. Conclusions Human enteroids recapitulate anion secretion physiology of small intestinal epithelium. Enteroid differentiation is associated with significant alterations in the expression of several ion transporters and carbonic anhydrase isoforms, leading to a reduced but preserved anion secretory phenotype owing to markedly reduced Cl- secretion but no significant change in HCO3- secretion.

110 Missense Mutations of SLC9A3 in Patients With Congenital Sodium Diarrhea Are Associated With Reduced Na+/H+ Exchanger 3 (NHE3) Activity: Identification of the Cause of the Phenotype

Myo5b null mice display microvillus inclusions. Ezrin and p120 immunostaining were assessed in Myo5b null and wild type littermate mice. In a wildtype littermate, ezrin (left panel and red-right panel) was only expressed on the apical surface of the enterocytes. In all Myo5b null mice examined to date, ezrin was found in inclusions within the enterocytes. p120 localization (green-right panel) was unchanged in null mice. Heterozygote mice were identical to wild type mice and did not show microvillus inclusions. Scale bars = 50 μm.

Epithelial WNT2B and Desert Hedgehog are necessary for human colonoid regeneration after bacterial cytotoxin injury

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.

cAMP stimulates SLC26A3 activity in human colon by a CFTR-dependent mechanism that does not require CFTR activity

Background & Aims DRA (SLC26A3) 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 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 reevaluated 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 non-specific 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 two 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 IC50 of 0.5 μmol/L and 0.2 µmol/L, respectively. However, there was no effect of cAMP in HEK293/DRA cells that lacked 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.