To date, the spatial difference in ion transport along the crypt-villus axis of the small intestine remains not fully understood. It is widely believed that absorption and secretion are 2 distinct functions occurring at different sites of the intestinal epithelium, with the former occurring in villi and the latter in crypts. However, a number of studies have suggested that the 2 transport processes may not be completely spatially separated along the crypt-villus axis, and reasons why this separation is unlikely to be so distinct have been described.
1Intestinal ion transport and the pathophysiology of diarrhea.
, 4The response of small intestinal villous and crypt epithelium to choleratoxin in rat and guinea pig. Evidence against a specific role of the crypt cells in choleragen-induced secretion.
, 5- Jakab R.L.
- Collaco A.M.
- Ameen N.A.
Physiological relevance of cell-specific distribution patterns of CFTR, NKCC1, NBCe1, and NHE3 along the crypt-villus axis in the intestine.
, 6Origin of cAMP-dependent Cl- secretion from both crypts and surface epithelia of rat intestine.
, 7- McNicholas C.M.
- Brown C.D.
- Turnberg L.A.
Na-K-Cl cotransport in villus and crypt cells from rat duodenum.
In addition, a previous study from our group showed the expression and function of Na
+/H
+ exchanger 3 (NHE3), which is responsible for the majority of electroneutral sodium absorption in the small intestine and a potential drug target for treating diarrhea/constipation,
8- Yin J.
- Tse C.M.
- Cha B.
- Sarker R.
- Zhu X.C.
- Walentinsson A.
- Greasley P.J.
- Donowitz M.
A common NHE3 single nucleotide polymorphism has normal function and sensitivity to regulatory ligands.
in both crypt-like–undifferentiated enteroids and villus-like–differentiated enteroids, suggesting sodium absorption is not confined to villi.
9- Foulke-Abel J.
- In J.
- Yin J.
- Zachos N.C.
- Kovbasnjuk O.
- Estes M.K.
- de Jonge H.
- Donowitz M.
Human enteroids as a model of upper small intestinal ion transport physiology and pathophysiology.
Also, several ion transporters that are thought to contribute to anion secretion, including cystic fibrosis transmembrane conductance regulator (CFTR), Na
+/K
+/2Cl
- co-transporter 1 (NKCC1), and electrogenic Na
+/HCO
3- co-transporter 1 (NBCe1), were detectable by immunofluorescence in rat villus enterocytes and co-localized with NHE3.
5- Jakab R.L.
- Collaco A.M.
- Ameen N.A.
Physiological relevance of cell-specific distribution patterns of CFTR, NKCC1, NBCe1, and NHE3 along the crypt-villus axis in the intestine.
CFTR, NBCe1, and, to a lesser extent NKCC1, also were documented as being expressed in human villus enterocytes (Turner JR, unpublished data). Moreover, a chloride-dependent depolarization of apical membrane potential difference was observed upon administration of secretagogues in villi as well as in crypts of rat small intestine.
10- Stewart C.P.
- Turnberg L.A.
A microelectrode study of responses to secretagogues by epithelial cells on villus and crypt of rat small intestine.
Hence, it is reasonable to speculate that anion secretion also may occur in villi, although the extent may not be as large as it is in crypts.
1Intestinal ion transport and the pathophysiology of diarrhea.
However, this speculation lacks strong evidence from human studies, largely owing to the lack of a reliable method to functionally and physically separate crypt cells and villus cells of human small intestinal epithelium.
In this study, we used the model of human duodenal enteroid monolayers to investigate cyclic adenosine monophosphate (cAMP)-stimulated anion secretion. The aim was to determine the molecular basis of anion secretion and the relevant changes upon enteroid differentiation. We herein report that cAMP-stimulated anion secretion in human duodenal enteroids is composed primarily of Cl- secretion with a smaller component of HCO3- secretion, and is highly dependent on CFTR, NKCC1, cAMP-activated K+ channel(s), Na+/K+–adenosine triphosphatase (ATPase), and carbonic anhydrase(s). Many of these ion transporters and carbonic anhydrase isoforms are subject to regulation by differentiation at both the messenger RNA (mRNA) and protein levels, contributing to a quantitatively reduced but preserved secretory phenotype of differentiated enteroids.
Discussion
The present study documents the changes in cAMP-stimulated anion secretion and expression of relevant ion transporters during differentiation of normal human duodenal enteroid monolayers, showing the following: (1) similarities with the previous characterization of 3-dimensional human duodenal enteroids, although the changes in expansion medium seem to cause changes in some aspects of the secretory processes
9- Foulke-Abel J.
- In J.
- Yin J.
- Zachos N.C.
- Kovbasnjuk O.
- Estes M.K.
- de Jonge H.
- Donowitz M.
Human enteroids as a model of upper small intestinal ion transport physiology and pathophysiology.
, 22- Fujii M.
- Matano M.
- Nanki K.
- Sato T.
Efficient genetic engineering of human intestinal organoids using electroporation.
; (2) that cAMP-stimulated anion secretion occurs in both undifferentiated and differentiated enteroid monolayers, further supporting that ion transport processes in human undifferentiated and differentiated enteroids are more quantitatively than qualitatively different
9- Foulke-Abel J.
- In J.
- Yin J.
- Zachos N.C.
- Kovbasnjuk O.
- Estes M.K.
- de Jonge H.
- Donowitz M.
Human enteroids as a model of upper small intestinal ion transport physiology and pathophysiology.
, 13- Yu H.
- Hasan N.M.
- In J.G.
- Estes M.K.
- Kovbasnjuk O.
- Zachos N.C.
- Donowitz M.
The contributions of human mini-intestines to the study of intestinal physiology and pathophysiology.
; (3) that both undifferentiated and differentiated enteroids perform what appears to be cAMP-stimulated Cl
- secretion and HCO
3- secretion, with Cl
- secretion being predominant; and (4) that although total anion secretion is much less in differentiated enteroids, the extent of HCO
3- secretion is similar in differentiated and undifferentiated enteroids.
Initially, we showed that the removal of Wnt3A/R-spondin1/SB202190 induced differentiation in duodenal enteroid monolayers, as supported by several phenotypic changes characterized at day 5. Comparison between undifferentiated and differentiated enteroid monolayers also showed several differentiation-related alterations in the expression of multiple ion transporters and carbonic anhydrase isoforms, consistent with the results based on studies of intact human (Turner JR, unpublished data) and animal small intestine.
5- Jakab R.L.
- Collaco A.M.
- Ameen N.A.
Physiological relevance of cell-specific distribution patterns of CFTR, NKCC1, NBCe1, and NHE3 along the crypt-villus axis in the intestine.
These data support our ability to separately study intestinal epithelial cells that represent crypts and villi using undifferentiated and differentiated enteroids. However, where along the villus our differentiation protocol is modeling is difficult to state given that not all villus cells are differentiated to the same extent, presumably continuing to differentiate as they move from the base of villus to the tip. Moreover, data are lacking that show the differentiation state and expression of transport proteins in epithelial cells at multiple positions along the human intestinal villus. Similarly, although the crypt base has LGR5-positive stem cells, Paneth cells, and transit-amplifying cells that proliferate, it is likely that cells of multiple states of differentiation are present, particularly at the upper crypt. Importantly, it is not yet known where in the crypt the anion secretory cells appear.
A major contribution of this study is the identification of the transport processes and cell populations that contribute to small intestinal anion secretion stimulated by increased intracellular cAMP. Although the functional relevance of enteroids to understanding human intestinal ion transport physiology has been shown by several previous studies,
9- Foulke-Abel J.
- In J.
- Yin J.
- Zachos N.C.
- Kovbasnjuk O.
- Estes M.K.
- de Jonge H.
- Donowitz M.
Human enteroids as a model of upper small intestinal ion transport physiology and pathophysiology.
, 24- 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.
the characterization of human intestinal anion secretory processes remains incomplete. In the present study, we defined active electrogenic anion secretion stimulated by forskolin in human duodenal enteroid monolayers by the Ussing chamber/voltage-current clamp technique. Specifically, this includes cAMP-stimulated Cl
- secretion (HCO
3--independent and HCO
3--dependent) and HCO
3- secretion (Cl
--independent and Cl
--dependent). It is important to point out that only electrogenic ion transport is defined by the approach of measuring I
sc; this did not allow us to specifically quantitate the electroneutral Cl
--dependent HCO
3- secretion that is provided by an apical anion exchanger, such as DRA, which exchanges Cl
- that is secreted by CFTR for intracellular HCO
3-, and that together with the linked CFTR-related Cl
- secretion is an overall electrogenic process.
28- Singh A.K.
- Riederer B.
- Chen M.
- Xiao F.
- Krabbenhöft A.
- Engelhardt R.
- Nylander O.
- Soleimani M.
- Seidler U.
The switch of intestinal Slc26 exchangers from anion absorptive to HCO3- secretory mode is dependent on CFTR anion channel function.
Similarly, the specific contribution of HCO
3--dependent Cl
- secretion, which includes the putative activated DRA stimulation of CFTR activity, could not be determined,
29- Shan J.
- Liao J.
- Huang J.
- Robert R.
- Palmer M.L.
- Fahrenkrug S.C.
- O'Grady S.M.
- Hanrahan J.W.
Bicarbonate-dependent chloride transport drives fluid secretion by the human airway epithelial cell line Calu-3.
, 30- Hong J.H.
- Yang D.
- Shcheynikov N.
- Ohana E.
- Shin D.M.
- Muallem S.
Convergence of IRBIT, phosphatidylinositol (4,5) bisphosphate, and WNK/SPAK kinases in regulation of the Na+-HCO3- cotransporters family.
nor is it known if it occurs in intact mammalian small intestine. In addition, the current study was not able to simultaneously quantitate the amount of active HCO
3- transport, given that our available equipment could not accomplish simultaneous pH titration and short-circuiting. In addition, although we have found many similarities in cAMP-stimulated secretion between enteroids derived from duodenum and jejunum, the findings of the current study in duodenal enteroids may not be completely extrapolated to other small intestinal segments given the special functions of each segment.
cAMP-stimulated anion secretion was present in both the crypt-like–undifferentiated enteroids and the villus-like–differentiated enteroids with the following characteristics. First, the magnitude of forskolin-stimulated anion secretion in differentiated enteroids was approximately 33% of that in undifferentiated enteroids. Second, a reduction in the magnitude of forskolin-induced ΔI
sc was observed in both undifferentiated and differentiated enteroids after the removal of extracellular Cl
-, presumably owing to loss of Cl
- secretion and also Cl
--dependent HCO
3- secretion. Similarly, the removal of extracellular HCO
3-/CO
2, which causes loss of HCO
3- secretion and HCO
3--dependent Cl
- secretion, resulted in reduced magnitude of forskolin-induced ΔI
sc, but this was modest compared with that with extracellular Cl
- removal. Similar effects of extracellular anion removal also were found in mouse duodenum.
31- Clarke L.L.
- Stien X.
- Walker N.M.
Intestinal bicarbonate secretion in cystic fibrosis mice.
, 32- Seidler U.
- Blumenstein I.
- Kretz A.
- Viellard-Baron D.
- Rossmann H.
- Colledge W.H.
- Evans M.
- Ratcliff R.
- Gregor M.
A functional CFTR protein is required for mouse intestinal cAMP-, cGMP- and Ca2+-dependent HCO3- secretion.
Although the cAMP-stimulated anion secretion in the absence of extracellular Cl
- or HCO
3-/CO
2 does not represent the entire HCO
3- or Cl
- secretion, this method of extracellular anion removal at least allowed us to confirm the dependency of cAMP-stimulated anion secretion on extracellular anions. Third, Cl
- secretion, primarily defined as the bumetanide-sensitive component of forskolin-induced ΔI
sc, makes up the bulk of anion secretion in undifferentiated enteroids and a significant part in differentiated enteroids. Specifically, 75% of the forskolin-induced ΔI
sc was inhibited by bumetanide in undifferentiated enteroids and 44% in differentiated enteroids. This is consistent with the in vivo observation that bumetanide inhibited a large part of forskolin-induced ΔI
sc without affecting bicarbonate secretion in mouse duodenum.
33- Walker N.M.
- Flagella M.
- Gawenis L.R.
- Shull G.E.
- Clarke L.L.
An alternate pathway of cAMP-stimulated Cl- secretion across the NKCC1-null murine duodenum.
Fourth, a bumetanide-insensitive component of forskolin-induced ΔI
sc was observed in both undifferentiated and differentiated enteroids. This residual component is either Cl
- secretion dependent on another bumetanide-insensitive basolateral Cl
- uptake pathway or HCO
3- secretion. The former has not been identified in the small intestine. Also, although the basolateral anion exchanger AE2, which is present similarly in undifferentiated and differentiated enteroids, could provide such a transport pathway,
25- Gawenis L.R.
- Bradford E.M.
- Alper S.L.
- Prasad V.
- Shull G.E.
AE2 Cl-/HCO3- exchanger is required for normal cAMP-stimulated anion secretion in murine proximal colon.
, 33- Walker N.M.
- Flagella M.
- Gawenis L.R.
- Shull G.E.
- Clarke L.L.
An alternate pathway of cAMP-stimulated Cl- secretion across the NKCC1-null murine duodenum.
the lack of effect on forskolin-induced ΔI
sc by blocking AE2 with SITS suggests it unlikely to be involved. Of note, this bumetanide-insensitive component was seen only in KRB buffer that contains HCO
3-/CO
2; it was not observed in HCO
3--free buffer, suggesting that it involves HCO
3- secretion. In fact, a similar bumetanide-insensitive component also was found in mouse duodenum only when extracellular HCO
3- was present.
31- Clarke L.L.
- Stien X.
- Walker N.M.
Intestinal bicarbonate secretion in cystic fibrosis mice.
That this was likely to be HCO
3- secretion was supported further by its sensitivity to acetazolamide, which has been shown to inhibit basal and stimulated HCO
3- secretion in intact human/animal duodenum.
34- Knutson T.W.
- Koss M.A.
- Hogan D.L.
- Isenberg J.I.
- Knutson L.
Acetazolamide inhibits basal and stimulated HCO3- secretion in the human proximal duodenum.
, 35- Muallem R.
- Reimer R.
- Odes H.S.
- Schwenk M.
- Beil W.
- Sewing K.F.
Role of carbonic anhydrase in basal and stimulated bicarbonate secretion by the guinea pig duodenum.
, 36- Jacob P.
- Christiani S.
- Rossmann H.
- Lamprecht G.
- Vieillard-Baron D.
- Müller R.
- Gregor M.
- Seidler U.
Role of Na+HCO3- cotransporter NBC1, Na+/H+ exchanger NHE1, and carbonic anhydrase in rabbit duodenal bicarbonate secretion.
Taken together, these results show the similarity of human duodenal enteroid monolayers and intact human/mouse duodenum in many aspects of cAMP-stimulated Cl
- and HCO
3- secretion.
As we proposed previously,
13- Yu H.
- Hasan N.M.
- In J.G.
- Estes M.K.
- Kovbasnjuk O.
- Zachos N.C.
- Donowitz M.
The contributions of human mini-intestines to the study of intestinal physiology and pathophysiology.
that forskolin-induced ΔI
sc is detectable in villus-like–differentiated enteroids suggests anion secretion is not strictly confined to crypts, although a significant quantitative difference exists between differentiated and undifferentiated enteroids. The lesser anion secretion in differentiated enteroids seems to be caused largely by the reduction in Cl
- secretion, which is related to the reduced expression of CFTR, NKCC1 and KCNE3, all of which are known to be necessary for cAMP-stimulated Cl
- secretion. Interestingly, several ion transporters and carbonic anhydrase isoforms that are involved in HCO
3- secretion, including DRA, NBCe1, CA2, and CA4, were increased in differentiated enteroids. These changes may explain that the absolute amount of forskolin-induced ΔI
sc that was inhibited by acetazolamide, which represents a component of HCO
3- secretion, was similar between undifferentiated and differentiated enteroids despite a significant reduction in CFTR expression in the latter. Further studies are needed to determine whether these changes in expression lead to increased DRA-related Cl
--dependent HCO
3- secretion in differentiated enteroids, a process that was not determined by the Ussing chamber/voltage-current clamp technique used in this study.
The present study allowed further dissection of the contribution of specific transporters to cAMP-stimulated anion secretion in duodenal enteroids. On the apical surface, CFTR was found to be necessary for both Cl
- and HCO
3- secretion because CFTR
inh-172 abolished the entire forskolin-induced ΔI
sc in the presence and absence of extracellular Cl
- and HCO
3-/CO
2 in differentiated as well as undifferentiated enteroids. Thus, CFTR is the major apical transporter for all duodenal cAMP-stimulated anion secretion and no other Cl
- channel seems to contribute significantly, unless that contribution is indirect and involves CFTR. Similarly, there appears no role for NHE3 in cAMP-stimulated anion secretion because forskolin-induced ΔI
sc was not altered by tenapanor. However, NHE3 still could have a role in electroneutral HCO
3- secretion,
28- Singh A.K.
- Riederer B.
- Chen M.
- Xiao F.
- Krabbenhöft A.
- Engelhardt R.
- Nylander O.
- Soleimani M.
- Seidler U.
The switch of intestinal Slc26 exchangers from anion absorptive to HCO3- secretory mode is dependent on CFTR anion channel function.
, 31- Clarke L.L.
- Stien X.
- Walker N.M.
Intestinal bicarbonate secretion in cystic fibrosis mice.
which could not be quantitated in the current study. Any specific role for DRA and PAT-1 could not be evaluated in this study, although they are likely to contribute to cAMP-stimulated anion secretion through their interaction with CFTR.
28- Singh A.K.
- Riederer B.
- Chen M.
- Xiao F.
- Krabbenhöft A.
- Engelhardt R.
- Nylander O.
- Soleimani M.
- Seidler U.
The switch of intestinal Slc26 exchangers from anion absorptive to HCO3- secretory mode is dependent on CFTR anion channel function.
, 37- Walker N.M.
- Simpson J.E.
- Brazill J.M.
- Gill R.K.
- Dudeja P.K.
- Schweinfest C.W.
- Clarke L.L.
Role of down-regulated in adenoma anion exchanger in HCO3- secretion across murine duodenum.
In terms of the basolateral transporters participating in cAMP-stimulated Cl
- secretion, our results confirmed NKCC1 as the essential basolateral Cl
- loader and the necessary contributions of cAMP-activated K
+ channel(s) and Na
+/K
+-ATPase.
32- Seidler U.
- Blumenstein I.
- Kretz A.
- Viellard-Baron D.
- Rossmann H.
- Colledge W.H.
- Evans M.
- Ratcliff R.
- Gregor M.
A functional CFTR protein is required for mouse intestinal cAMP-, cGMP- and Ca2+-dependent HCO3- secretion.
, 38- Preston P.
- Wartosch L.
- Günzel D.
- Fromm M.
- Kongsuphol P.
- Ousingsawat J.
- Kunzelmann K.
- Barhanin J.
- Warth R.
- Jentsch T.J.
Disruption of the K+ channel beta-subunit KCNE3 reveals an important role in intestinal and tracheal Cl- transport.
, 39- Seidler U.
- Bachmann O.
- Jacob P.
- Christiani S.
- Blumenstein I.
- Rossmann H.
Na+/HCO3- cotransport in normal and cystic fibrosis intestine.
As for cAMP-stimulated HCO
3- secretion, there are 2 potential pathways that could supply HCO
3- for apical extrusion. One is a Na
+/HCO
3- co-transporter that interacts with NHE1 to mediate HCO
3- uptake across the basolateral membrane.
36- Jacob P.
- Christiani S.
- Rossmann H.
- Lamprecht G.
- Vieillard-Baron D.
- Müller R.
- Gregor M.
- Seidler U.
Role of Na+HCO3- cotransporter NBC1, Na+/H+ exchanger NHE1, and carbonic anhydrase in rabbit duodenal bicarbonate secretion.
Our study did not provide any evidence for the involvement of NBCs because a specific inhibitor of the NBC family (S0859) had no significant inhibitory effect but rather a small stimulatory effect on forskolin-induced ΔI
sc. A similar transient stimulatory effect of S0859 on forskolin-induced ΔI
sc also was seen in bronchial epithelial cells, which could be inhibited by calcium-activated chloride channel (CaCC) inhibitor.
40- Gorrieri G.
- Scudieri P.
- Caci E.
- Schiavon M.
- Tomati V.
- Sirci F.
- Napolitano F.
- Carrella D.
- Gianotti A.
- Musante I.
- Favia M.
- Casavola V.
- Guerra L.
- Rea F.
- Ravazzolo R.
- Di Bernardo D.
- Galietta L.J.
Goblet cell hyperplasia requires high bicarbonate transport to support mucin release.
The second source of HCO
3- is the production of intracellular HCO
3- by carbonic anhydrase(s), the contribution of which is supported by the inhibitory effect of acetazolamide on HCO
3- secretion, as shown by multiple studies.
34- Knutson T.W.
- Koss M.A.
- Hogan D.L.
- Isenberg J.I.
- Knutson L.
Acetazolamide inhibits basal and stimulated HCO3- secretion in the human proximal duodenum.
, 35- Muallem R.
- Reimer R.
- Odes H.S.
- Schwenk M.
- Beil W.
- Sewing K.F.
Role of carbonic anhydrase in basal and stimulated bicarbonate secretion by the guinea pig duodenum.
, 36- Jacob P.
- Christiani S.
- Rossmann H.
- Lamprecht G.
- Vieillard-Baron D.
- Müller R.
- Gregor M.
- Seidler U.
Role of Na+HCO3- cotransporter NBC1, Na+/H+ exchanger NHE1, and carbonic anhydrase in rabbit duodenal bicarbonate secretion.
In the current study, an acetazolamide-sensitive component was present in cAMP-stimulated anion secretion, indicating the requirement for carbonic anhydrase(s) in HCO
3- secretion. However, the specific carbonic anhydrase isoform was not identified. Two isoforms were up-regulated significantly with differentiation in duodenal enteroids; CA2 is thought to be the major enzyme involved in intracellular HCO
3- production, and CA4 is expressed only in differentiated enteroids and is reported to interact with CFTR and facilitate CO
2 influx.
41- Fanjul M.
- Salvador C.
- Alvarez L.
- Cantet S.
- Hollande E.
Targeting of carbonic anhydrase IV to plasma membranes is altered in cultured human pancreatic duct cells expressing a mutated (ΔF508) CFTR.
, 42- Musa-Aziz R.
- Occhipinti R.
- Boron W.F.
Evidence from simultaneous intracellular- and surface-pH transients that carbonic anhydrase IV enhances CO2 fluxes across Xenopus oocyte plasma membranes.
In conclusion, this study describes some of the molecular basis of cAMP-stimulated anion secretion in normal human duodenal enteroid monolayers and compares the ion transport processes taking part in anion secretion in crypt-like–undifferentiated enteroids and villus-like–differentiated enteroids. We suggest that human enteroid monolayers are a useful tool to study multiple physiological and pathophysiological models of intestinal anion secretion, including the chloride and bicarbonate components, as well as those that are caused separately by crypt-like and villus-like epithelial cells of human small intestine.
Article Info
Publication History
Published online: February 09, 2018
Accepted:
February 5,
2018
Received:
July 14,
2017
Footnotes
Author contributions Jianyi Yin, Chung-Ming Tse, and Mark Donowitz designed the experiments; Jianyi Yin, Chung-Ming Tse, Leela Rani Avula, Varsha Singh, and Jennifer Foulke-Abel performed experiments and analyzed data; Jianyi Yin drafted the manuscript; and Chung-Ming Tse, Hugo R. de Jonge, and Mark Donowitz revised the manuscript.
Conflicts of interest The authors disclose no conflicts.
Funding This study was supported in part by National Institutes of Health grants RO1 DK26523 , RO1 DK61765 , P01 DK072084 , P30 DK089502 , UO1AI125181 , UH3TR00003 , U18TR000552 , and R24 DK99803 .
Copyright
© 2018 The Authors. Published by Elsevier Inc. on behalf of the AGA Institute.