The transport of bicarbonate across the enterocyte cell membrane regulates the intracellular as well as the luminal pH and is an essential part of directional fluid movement in the gut. Since the ...first description of “active” transport of HCO
3
−
ions against a concentration gradient in the 1970s, the fundamental role of HCO
3
−
transport for multiple intestinal functions has been recognized. The ion transport proteins have been identified and molecularly characterized, and knockout mouse models have given insight into their individual role in a variety of functions. This review describes the progress made in the last decade regarding novel techniques and new findings in the molecular regulation of intestinal HCO
3
−
transport in the different segments of the gut. We discuss human diseases with defects in intestinal HCO
3
−
secretion and potential treatment strategies to increase luminal alkalinity. In the last part of the review, the cellular and organismal mechanisms for acid/base sensing in the intestinal tract are highlighted.
Highlights • Fluorometic imaging and microelectrodes allow dynamic assessment of pH microenvironments. • Transgenic and knockout mice allow delineation of new regulatory networks that govern ...gastrointestinal HCO3− transport. • A multitude of luminal sensing mechanisms regulate the luminal pH microenvironment. • This knowledge can be employed in the design of new mucosal protective drug development strategies.
NBCn1 (SLC4A7) is one of the two major Na
-HCO
cotransporters in the human colonic epithelium, expressed predominantly in the highly proliferating colonocytes at the cryptal base. Increased NBCn1 ...expression levels are reported in tumors, including colorectal cancer. The study explores its importance for maintenance of the intracellular pH (pH
), as well as the proliferative, adhesive, and migratory behavior of the self-differentiating Caco2BBe colonic tumor cell line. In the self-differentiating Caco2BBe cells,
mRNA was highly expressed from the proliferative stage until full differentiation. The downregulation of NBCn1 expression by RNA interference affected proliferation and differentiation and decreased intracellular pH (pH
) of the cells in correlation with the degree of knockdown. In addition, a disturbed cell adhesion and reduced migratory speed were associated with NBCn1 knockdown. Murine colonic
enteroids also displayed reduced proliferative activity. In the migrating Caco2BBe cells, NBCn1 was found at the leading edge and in colocalization with the focal adhesion markers vinculin and paxillin, which suggests that NBCn1 is involved in the establishment of cell-matrix adhesion. Our data highlight the physiological significance of NBCn1 in modulating epithelial pH homeostasis and cell-matrix interactions in the proliferative region of the colonic epithelium and unravel the molecular mechanism behind pathological overexpression of this transporter in human colorectal cancers.
The transporter NBCn1 plays a central role in maintaining homeostasis within Caco2BBe colonic epithelial cells through its regulation of intracellular pH, matrix adhesion, migration, and proliferation. These observations yield valuable insights into the molecular mechanism of the aberrant upregulation of this transporter in human colorectal cancers.
NBCn1 (SLC4A7) is one of the two major Na+-HCO3− cotransporters in the human colonic epithelium, expressed predominantly in the highly proliferating colonocytes at the cryptal base. Increased NBCn1 ...expression levels are reported in tumors, including colorectal cancer. The study explores its importance for maintenance of the intracellular pH (pHi), as well as the proliferative, adhesive, and migratory behavior of the self-differentiating Caco2BBe colonic tumor cell line. In the self-differentiating Caco2BBe cells, NBCn1 mRNA was highly expressed from the proliferative stage until full differentiation. The downregulation of NBCn1 expression by RNA interference affected proliferation and differentiation and decreased intracellular pH (pHi) of the cells in correlation with the degree of knockdown. In addition, a disturbed cell adhesion and reduced migratory speed were associated with NBCn1 knockdown. Murine colonic Nbcn1–/– enteroids also displayed reduced proliferative activity. In the migrating Caco2BBe cells, NBCn1 was found at the leading edge and in colocalization with the focal adhesion markers vinculin and paxillin, which suggests that NBCn1 is involved in the establishment of cell-matrix adhesion. Our data highlight the physiological significance of NBCn1 in modulating epithelial pH homeostasis and cell-matrix interactions in the proliferative region of the colonic epithelium and unravel the molecular mechanism behind pathological overexpression of this transporter in human colorectal cancers.
Loss of function mutations in the
gene cause chloride-losing diarrhea in mice and humans. Although systemic adaptive changes have been documented in these patients and in the corresponding knockout ...mice, how colonic enterocytes adapt to loss of this highly expressed and highly regulated luminal membrane anion exchanger remains unclear. To address this question,
was deleted in the self-differentiating Caco2BBe colonic cell line by the CRISPR/Cas9 technique. We selected a clone with loss of SLC26A3 protein expression and morphological features indistinguishable from those of the native cell line. Neither growth curves nor development of transepithelial electrical resistance (TEER) differed between wild-type (WT) and SLC26A3 knockout (KO) cells. Real-time qPCR and Western analysis in SLC26A3-KO cells revealed an increase in AE2 expression without significant change in NHE3 expression or localization. Steady-state pH
and apical and basolateral Cl
/HCO
exchange activities were assessed fluorometrically in a dual perfusion chamber with independent perfusion of luminal and serosal baths. Apical Cl
/HCO
exchange rates were strongly reduced in SLC26A3-KO cells, accompanied by a surface pH more acidic than that of WT cells. Steady-state pH
was not significantly different from that of WT cells, but basolateral Cl
/HCO
exchange rates were higher in SLC26A3-KO than in WT cells. The data show that CRISPR/Cas9-mediated
deletion strongly reduced apical Cl
/HCO
exchange rate and apical surface pH, but sustained a normal steady-state pH
due to increased expression and function of basolateral AE2. The low apical surface pH resulted in functional inhibition of NHE-mediated fluid absorption despite normal expression of NHE3 polypeptide.
SLC26A3 gene mutations cause chloride-losing diarrhea. To understand how colonic enterocytes adapt, SLC26A3 was deleted in Caco2BBe cells using CRISPR/Cas9. In comparison to the wild-type cells, SLC26A3 knockout cells showed similar growth and transepithelial resistance but substantially reduced apical Cl
/HCO
exchange rates, and an acidic surface pH. Steady-state intracellular pH was comparable between the WT and KO cells due to increased basolateral AE2 expression and function.
Summary Background The interleukin-23 pathway is implicated genetically and biologically in the pathogenesis of Crohn's disease. We aimed to assess the efficacy and safety of risankizumab (BI 655066, ...Boehringer Ingelheim, Ingelheim, Germany), a humanised monoclonal antibody targeting the p19 subunit of interleukin-23, in patients with moderately-to-severely active Crohn's disease. Methods In this randomised, double-blind, placebo-controlled phase 2 study, we enrolled patients at 36 referral sites in North America, Europe, and southeast Asia. Eligible patients were aged 18–75 years, with a diagnosis of Crohn's disease for at least 3 months, assessed as moderate-to-severe Crohn's disease at screening, defined as a Crohn's Disease Activity Index (CDAI) of 220–450, with mucosal ulcers in the ileum or colon, or both, and a Crohn's Disease Endoscopic Index of Severity (CDEIS) of at least 7 (≥4 for patients with isolated ileitis) on ileocolonoscopy scored by a masked central reader. Patients were randomised 1:1:1 using an interactive response system to a double-blind investigational product, and stratified by previous exposure to TNF antagonists (yes vs no). Patients received intravenous 200 mg risankizumab, 600 mg risankizumab, or placebo, at weeks 0, 4, and 8. The primary outcome was clinical remission (CDAI <150) at week 12 (intention-to-treat population). Safety was assessed in patients who received at least one dose of study drug. This study is registered with ClinicalTrials.gov , number NCT02031276. Findings Between March, 2014, and September, 2015, 213 patients were screened, and 121 patients randomised. At baseline, 113 patients (93%) had been previously treated with at least one tumour necrosis factor (TNF) antagonist (which had failed in 96 79%). At week 12, 25 (31%) of 82 risankizumab patients (pooled 41 patients in 200 mg and 41 patients in 600 mg arms) had clinical remission versus six (15%) of 39 placebo patients (difference vs placebo 15·0%, 95% CI 0·1 to 30·1; p=0·0489). Ten (24%) of 41 patients who received 200 mg risankizumab had clinical remission (9·0%, −8·3 to 26·2; p=0·31) and 15 (37%) of 41 who received the 600 mg dose (20·9%, 2·6 to 39·2; p=0·0252). 95 (79%) patients had adverse events (32 in the placebo group, 32 randomised to 200 mg risankizumab, 31 randomised to 600 mg risankizumab); 18 had severe adverse events (nine, six, three); 12 discontinued (six, five, one); 24 had serious adverse events (12, nine, three). The most common adverse event was nausea and most common serious adverse event was worsening of underlying Crohn's disease. No deaths occurred. Interpretation In this short-term study, risankizumab was more effective than placebo for inducing clinical remission in patients with active Crohn's disease. Therefore, selective blockade of interleukin-23 via inhibition of p19 might be a viable therapeutic approach in Crohn's disease. Funding Boehringer Ingelheim.
The five plasma membrane Na
/H
exchanger (NHE) isoforms in the gastrointestinal tract are characterized by distinct cellular localization, tissue distribution, inhibitor sensitivities, and ...physiological regulation. NHE1 (Slc9a1) is ubiquitously expressed along the gastrointestinal tract in the basolateral membrane of enterocytes, but so far, an exclusive role for NHE1 in enterocyte physiology has remained elusive. NHE2 (Slc9a2) and NHE8 (Slc9a8) are apically expressed isoforms with ubiquitous distribution along the colonic crypt axis. They are involved in pH
regulation of intestinal epithelial cells. Combined use of a knockout mouse model, intestinal organoid technology, and specific inhibitors revealed previously unrecognized actions of NHE2 and NHE8 in enterocyte proliferation and differentiation. NHE3 (Slc9a3), expressed in the apical membrane of differentiated intestinal epithelial cells, functions as the predominant nutrient-independent Na
absorptive mechanism in the gut. The new selective NHE3 inhibitor (Tenapanor) allowed discovery of novel pathophysiological and drug-targetable NHE3 functions in cystic-fibrosis associated intestinal obstructions. NHE4, expressed in the basolateral membrane of parietal cells, is essential for parietal cell integrity and acid secretory function, through its role in cell volume regulation. This review focuses on the expression, regulation and activity of the five plasma membrane Na
/H
exchangers in the gastrointestinal tract, emphasizing their role in maintaining intestinal homeostasis, or their impact on disease pathogenesis. We point to major open questions in identifying NHE interacting partners in central cellular pathways and processes and the necessity of determining their physiological role in a system where their endogenous expression/activity is maintained, such as organoids derived from different parts of the gastrointestinal tract.
Cation and anion transport in the colonocyte apical membrane is highly spatially organized along the cryptal axis. Because of lack of experimental accessibility, information about the functionality ...of ion transporters in the colonocyte apical membrane in the lower part of the crypt is scarce. The aim of this study was to establish an in vitro model of the colonic lower crypt compartment, which expresses the transit amplifying/progenitor (TA/PE) cells, with accessibility of the apical membrane for functional study of lower crypt-expressed Na
/H
exchangers (NHEs). Colonic crypts and myofibroblasts were isolated from human transverse colonic biopsies, expanded as three-dimensional (3D) colonoids and myofibroblast monolayers, and characterized. Filter-grown colonic myofibroblast-colonic epithelial cell (CM-CE) cocultures (myofibroblasts on the bottom of the transwell and colonocytes on the filter) were established. The expression pattern for ion transport/junctional/stem cell markers of the CM-CE monolayers was compared with that of nondifferentiated (EM) and differentiated (DM) colonoid monolayers. Fluorometric pH
measurements were performed to characterize apical NHEs. CM-CE cocultures displayed a rapid increase in transepithelial electrical resistance (TEER), paralleled by downregulation of claudin-2. They maintained proliferative activity and an expression pattern resembling TA/PE cells. The CM-CE monolayers displayed high apical Na
/H
exchange activity, mediated to >80% by NHE2. Human colonoid-myofibroblast cocultures allow the study of ion transporters that are expressed in the apical membrane of the nondifferentiated colonocytes of the cryptal neck region. The NHE2 isoform is the predominant apical Na
/H
exchanger in this epithelial compartment.
Mutations in the CFTR chloride channel result in intestinal obstructive episodes in cystic fibrosis (CF) patients and in CF animal models. In this study, we explored the possibility of reducing the ...frequency of obstructive episodes in cftr−/− mice through the oral application of a gut-selective NHE3 inhibitor tenapanor and searched for the underlying mechanisms involved. Sex- and age-matched cftr+/+ and cftr−/− mice were orally gavaged twice daily with 30 mg kg−1 tenapanor or vehicle for a period of 21 days. Body weight and stool water content was assessed daily and gastrointestinal transit time (GTT) once weekly. The mice were sacrificed when an intestinal obstruction was suspected or after 21 days, and stool and tissues were collected for further analysis. Twenty-one day tenapanor application resulted in a significant increase in stool water content and stool alkalinity and a significant decrease in GTT in cftr+/+ and cftr−/− mice. Tenapanor significantly reduced obstructive episodes to 8% compared to 46% in vehicle-treated cftr−/− mice and prevented mucosal inflammation. A decrease in cryptal hyperproliferation, mucus accumulation, and mucosal mast cell number was also observed in tenapanor- compared to vehicle-treated, unobstructed cftr−/− mice. Overall, oral tenapanor application prevented obstructive episodes in CFTR-deficient mice and was safe in cftr+/+ and cftr−/− mice. These results suggest that tenapanor may be a safe and affordable adjunctive therapy in cystic fibrosis patients to alleviate constipation and prevent recurrent DIOS.
Haploinsufficiency A20 (HA20) is a newly described monogenic disease characterized by a wide spectrum of manifestations and caused by heterozygous mutations in
which encodes A20 protein.
mutation ...leads to disruption of the A20 ovarian tumor (OTU) domain and/or the zinc finger (ZnF) domain. This study aims at exploring the association between the various manifestations of HA20 and different domains disruption of A20.
We reviewed the HA20 cases in previous literature and summarized the clinical features,
mutation loci and the disrupted domains caused by different sites and patterns of mutations. Patients were classified into three groups according to the A20 domains disruption.
A total of 89 patients from 39 families with a genetic diagnosis of HA20 were included. Overall, the age at onset of HA20 was early (median:5.92, IQR:1-10). Patients in the ZnF group showed the earliest onset (median:2.5, IQR:0.6-5), followed by patients in the OTU+ZnF group (median:6, IQR:1-10) and patients in the OTU group (median:10, IQR:8-14). The main manifestations of HA20 patients were recurrent oral ulcers (70%), recurrent fever (42%), gastrointestinal ulcers (40%), skin lesion (38%), genital ulcers (36%), and musculoskeletal disorders (34%). The percentage of patients with musculoskeletal disorders was significantly different among the three groups (
= 0.005). Patients in the OTU+ZnF group and ZnF group were more likely to develop musculoskeletal disorders than patients in the OTU group (
= 0.002 and
= 0.035, respectively). Besides, forty-three percent of HA20 patients were initially diagnosed as Behcet's disease (BD). Compared to the ZnF group, the OTU+ZnF group and OTU group had a higher percentage of patients initially diagnosed as BD (
= 0.006 and
< 0.001, respectively).
HA20 is characterized by early-onset and the most common symptoms of HA20 are recurrent oral ulcers, fever and gastrointestinal ulcers. The onset of HA20 in patients with the ZnF domain disruption is earlier than patients with the OTU domain disruption. Compared to the OTU domain, the ZnF domain may be more closely related to musculoskeletal disorders.
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