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Molecular Basis and Differentiation-Associated Alterations of Anion Secretion in Human Duodenal Enteroid Monolayers.
Cell Mol Gastroenterol Hepatol 2018; 5(4):591-609CM

Abstract

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.

Authors+Show Affiliations

Department of Medicine, Division of Gastroenterology and Hepatology, Johns Hopkins University School of Medicine, Baltimore, Maryland.Department of Medicine, Division of Gastroenterology and Hepatology, Johns Hopkins University School of Medicine, Baltimore, Maryland.Department of Medicine, Division of Gastroenterology and Hepatology, Johns Hopkins University School of Medicine, Baltimore, Maryland.Department of Medicine, Division of Gastroenterology and Hepatology, Johns Hopkins University School of Medicine, Baltimore, Maryland.Department of Medicine, Division of Gastroenterology and Hepatology, Johns Hopkins University School of Medicine, Baltimore, Maryland.Department of Gastroenterology and Hepatology, Erasmus University Medical Center, Rotterdam, The Netherlands.Department of Medicine, Division of Gastroenterology and Hepatology, Johns Hopkins University School of Medicine, Baltimore, Maryland. Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, Maryland.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

29930980

Citation

Yin, Jianyi, et al. "Molecular Basis and Differentiation-Associated Alterations of Anion Secretion in Human Duodenal Enteroid Monolayers." Cellular and Molecular Gastroenterology and Hepatology, vol. 5, no. 4, 2018, pp. 591-609.
Yin J, Tse CM, Avula LR, et al. Molecular Basis and Differentiation-Associated Alterations of Anion Secretion in Human Duodenal Enteroid Monolayers. Cell Mol Gastroenterol Hepatol. 2018;5(4):591-609.
Yin, J., Tse, C. M., Avula, L. R., Singh, V., Foulke-Abel, J., de Jonge, H. R., & Donowitz, M. (2018). Molecular Basis and Differentiation-Associated Alterations of Anion Secretion in Human Duodenal Enteroid Monolayers. Cellular and Molecular Gastroenterology and Hepatology, 5(4), pp. 591-609. doi:10.1016/j.jcmgh.2018.02.002.
Yin J, et al. Molecular Basis and Differentiation-Associated Alterations of Anion Secretion in Human Duodenal Enteroid Monolayers. Cell Mol Gastroenterol Hepatol. 2018;5(4):591-609. PubMed PMID: 29930980.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Molecular Basis and Differentiation-Associated Alterations of Anion Secretion in Human Duodenal Enteroid Monolayers. AU - Yin,Jianyi, AU - Tse,Chung-Ming, AU - Avula,Leela Rani, AU - Singh,Varsha, AU - Foulke-Abel,Jennifer, AU - de Jonge,Hugo R, AU - Donowitz,Mark, Y1 - 2018/02/09/ PY - 2017/07/14/received PY - 2018/02/05/accepted PY - 2018/6/23/entrez PY - 2018/6/23/pubmed PY - 2018/6/23/medline KW - AE2, anion exchanger 2 KW - Bicarbonate Secretion KW - CA, carbonic anhydrase KW - CFTR, cystic fibrosis transmembrane conductance regulator KW - Chloride Secretion KW - DRA KW - DRA, down-regulated-in-adenoma KW - Ion Transport KW - Isc, short-circuit current KW - KRB, Krebs–Ringer bicarbonate KW - NBC, Na+/HCO3- co-transporter KW - NBCe1, electrogenic Na+/HCO3- co-transporter 1 KW - NHE, Na+/H+ exchanger KW - NKCC1, Na+/K+/2Cl- co-transporter 1 KW - SDS, sodium dodecyl sulfate KW - SITS, 4-Acetamido-4′-isothiocyanato-2,2′-stilbenedisulfonic acid disodium salt hydrate KW - TER, transepithelial electrical resistance KW - cAMP, cyclic adenosine monophosphate KW - mRNA, messenger ribonucleic acid KW - qRT-PCR, quantitative real-time polymerase chain reaction KW - ΔIsc, change in short-circuit current SP - 591 EP - 609 JF - Cellular and molecular gastroenterology and hepatology JO - Cell Mol Gastroenterol Hepatol VL - 5 IS - 4 N2 - 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. SN - 2352-345X UR - https://www.unboundmedicine.com/medline/citation/29930980/Molecular_Basis_and_Differentiation_Associated_Alterations_of_Anion_Secretion_in_Human_Duodenal_Enteroid_Monolayers_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S2352-345X(18)30032-8 DB - PRIME DP - Unbound Medicine ER -