Search

Background: Intestinal epithelial barrier dysfunction is intricately linked to the pathogenesis of ulcerative colitis (UC). Dietary interventions that bolster intestinal epithelial barrier function can effectively thwart UC onset. Our prior research revealed that p-Hydroxy benzaldehyde (HD), a phenolic compound from Nostoc commune (an edible cyanobacterium), markedly upregulated the expression of E-cadherin, a pivotal protein in intestinal mucosa, thereby mitigating mucosal damage in mice afflicted with dextran sulfate sodium (DSS)-induced colitis. Nevertheless, the precise molecular mechanisms underpinning HD's ameliorative effects on intestinal epithelial barrier dysfunction remain elusive. Methods: Dextran sodium sulfate (DSS)-induced colitis mouse model was established, and the successful establishment of the model was determined by evaluating the changes in body weight, disease activity index (DAI), colonic histopathology, and white blood cell count. Transmission electron microscopy (TEM) observed the ultrastructural changes of intestinal villi. The levels of inflammatory factors (IFN-γ IL-13) and intestinal permeability indicators (FITC-Dextran, DAO, ET, and D-LA) were detected by Enzyme-linked immunosorbent assay (ELISA). Western blotting (WB) and immunohistochemistry (IHC) were used to detect the expression of intestinal barrier integrity-related factors such as tight junction protein TJs (ZO-1, occludin) and adhesion junction protein AJs (E-cadherin). Furthermore, WB, Pull-down assay, drug affinity reaction target stability (DARTS) assay, molecular docking and molecular dynamics (MD) simulation were used to determine the potential target and molecular mechanism of HD. Results: HD intervention significantly alleviated the symptoms of colitis mice, inhibited the weight loss and colon shortening, reduced DAI score and colon pathological score, maintained the ultrastructure of intestinal villi in colon tissue, and significantly reduced the inflammatory factors IFN-γ, IL-13 and the number of white blood cells in colon tissue of colitis mice. HD could also reduce the levels of FITC-Dextran, DAO, ET, and D-LA and increase the expression of ZO-1, occludin, and E-cadherin in the colonic tissues of colitis mice, thereby maintaining the impaired intestinal barrier function caused by colitis. Mechanically, HD augmented the expression of hepatocyte nuclear factor 1β (HNF-1β) and DRA. Adeno-associated virus (AAV)-HNF-1β shRNA or Lentivirus-mediated HNF-1β knockdown effectively abolished HD-induced intestinal barrier protection, as well as the promotion of solute carrier family 26 member 3 (SLC26A3) expression levels. SLC26A3 siRNA effectively reversed the inhibition of intestinal permeability by HD. Pull-down assay, DARTS analysis, molecular docking, and MD results showed high binding strength, interaction efficiency and remarkable stability between HNF-1β and HD. Conclusion: This study elucidates HD's role in forestalling intestinal epithelial barrier disruption under colitis conditions. Mechanistic investigations revealed that HD fortifies TJs and AJs expression via the HNF-1β/SLC26A3 pathway, thus preserving the lower intestinal epithelial barrier's integrity in UC. [ABSTRACT FROM AUTHOR]

Summary: The intestinal epithelium is covered by mucus that protects the tissue from the luminal content. Studies have shown that anion secretion via the cystic fibrosis conductance regulator (Cftr) regulates mucus formation in the small intestine. However, mechanisms regulating mucus formation in the colon are less understood. The aim of this study was to explore the role of anion transport in the regulation of mucus formation during steady state and in response to carbamylcholine (CCh) and prostaglandin E2 (PGE2). The broad-spectrum anion transport inhibitor 4,4′-diisothiocyanatostilbene-2,2′-disulfonate (DIDS), CftrdF508 (CF) mice, and the slc26a3 inhibitor SLC26A3-IN-2 were used to inhibit anion transport. In the distal colon, steady-state mucus expansion was reduced by SLC26A3-IN-2 and normal in CF mice. PGE2 stimulated mucus expansion without de novo mucus release in wild type (WT) and CF colon via slc26a3 sensitive mechanisms, while CCh induced de novo mucus secretion in WT but not in CF colon. However, when added simultaneously, CCh and PGE2 stimulated de novo mucus secretion in the CF colon via DIDS-sensitive pathways. A similar response was observed in CF ileum that responded to CCh and PGE2 with DIDS-sensitive de novo mucus secretion. In conclusion, this study suggests that slc26a3 regulates colonic mucus expansion, while Cftr regulates CCh-induced de novo mucus secretion from ileal and distal colon crypts. Furthermore, these findings demonstrate that in the absence of a functional Cftr channel, parallel stimulation with CCh and PGE2 activates additional anion transport processes that help release mucus from intestinal goblet cells. [ABSTRACT FROM AUTHOR]

BackgroundIntestinal epithelial barrier dysfunction is intricately linked to the pathogenesis of ulcerative colitis (UC). Dietary interventions that bolster intestinal epithelial barrier function can effectively thwart UC onset. Our prior research revealed that p-Hydroxy benzaldehyde (HD), a phenolic compound from Nostoc commune (an edible cyanobacterium), markedly upregulated the expression of E-cadherin, a pivotal protein in intestinal mucosa, thereby mitigating mucosal damage in mice afflicted with dextran sulfate sodium (DSS)-induced colitis. Nevertheless, the precise molecular mechanisms underpinning HD’s ameliorative effects on intestinal epithelial barrier dysfunction remain elusive.MethodsDextran sodium sulfate (DSS)-induced colitis mouse model was established, and the successful establishment of the model was determined by evaluating the changes in body weight, disease activity index (DAI), colonic histopathology, and white blood cell count. Transmission electron microscopy (TEM) observed the ultrastructural changes of intestinal villi. The levels of inflammatory factors ( IFN-γ IL-13 ) and intestinal permeability indicators (FITC-Dextran, DAO, ET, and D-LA ) were detected by Enzyme-linked immunosorbent assay (ELISA). Western blotting (WB) and immunohistochemistry (IHC) were used to detect the expression of intestinal barrier integrity-related factors such as tight junction protein TJs (ZO-1, occludin) and adhesion junction protein AJs (E-cadherin). Furthermore, WB, Pull-down assay, drug affinity reaction target stability (DARTS) assay, molecular docking and molecular dynamics (MD) simulation were used to determine the potential target and molecular mechanism of HD.ResultsHD intervention significantly alleviated the symptoms of colitis mice, inhibited the weight loss and colon shortening, reduced DAI score and colon pathological score, maintained the ultrastructure of intestinal villi in colon tissue, and significantly reduced the inflammatory factors IFN-γ, IL-13 and the number of white blood cells in colon tissue of colitis mice. HD could also reduce the levels of FITC-Dextran, DAO, ET, and D-LA and increase the expression of ZO-1, occludin, and E-cadherin in the colonic tissues of colitis mice, thereby maintaining the impaired intestinal barrier function caused by colitis. Mechanically, HD augmented the expression of hepatocyte nuclear factor 1β (HNF-1β) and DRA. Adeno-associated virus (AAV)-HNF-1β shRNA or Lentivirus-mediated HNF-1β knockdown effectively abolished HD-induced intestinal barrier protection, as well as the promotion of solute carrier family 26 member 3 (SLC26A3) expression levels. SLC26A3 siRNA effectively reversed the inhibition of intestinal permeability by HD. Pull-down assay, DARTS analysis, molecular docking, and MD results showed high binding strength, interaction efficiency and remarkable stability between HNF-1β and HD.ConclusionThis study elucidates HD’s role in forestalling intestinal epithelial barrier disruption under colitis conditions. Mechanistic investigations revealed that HD fortifies TJs and AJs expression via the HNF-1β/SLC26A3 pathway, thus preserving the lower intestinal epithelial barrier’s integrity in UC.

Congenital chloride diarrhea (CLD) is a rare autosomal recessive disease caused by mutations in the solute carrier family 26 member 3 (SLC26A3) gene on chromosome 7q31. Affected neonates are vulnerable to dehydration, electrolyte imbalance in the form of hyponatremia, metabolic alkalosis, failure to thrive, or even death if left untreated. Genetic testing for mutations should be considered if the clinical diagnosis remains uncertain because early diagnosis and appropriate management are critical to the disease course in CLD. Several mutations have been reported in Korean patients with CLD, with the most common being the c.2063-1G>T mutation. Here, we report the case of a neonate with prenatally suspected CLD with confirmed novel mutations in the SLC26A3 gene (c.2147C>G; p.Ala716Gly).

Congenital chloride diarrhea (CLD) is a rare autosomal recessive disease caused by mutations in the solute carrier family 26 member 3 (SLC26A3) gene on chromosome 7q31. Affected neonates are vulnerable to dehydration, electrolyte imbalance in the form of hyponatremia, metabolic alkalosis, failure to thrive, or even death if left untreated. Genetic testing for mutations should be considered if the clinical diagnosis remains uncertain because early diagnosis and appropriate management are critical to the disease course in CLD. Several mutations have been reported in Korean patients with CLD, with the most common being the c.2063-1G>T mutation. Here, we report the case of a neonate with prenatally suspected CLD with confirmed novel mutations in the SLC26A3 gene (c.2147C>G; p.Ala716Gly). [ABSTRACT FROM AUTHOR]

Cholesterol-rich membrane domains, also called lipid rafts (LRs), are specialized membrane domains that provide a platform for intracellular signal transduction. Membrane proteins often cluster in LRs that further aggregate into larger platform-like structures that are enriched in ceramides and are called ceramide-rich platforms (CRPs). The role of CRPs in the regulation of intestinal epithelial functions remains unknown. Down-regulated in adenoma (DRA) is an intestinal Cl−/HCO3− antiporter that is enriched in LRs. However, little is known regarding the mechanisms involved in the regulation of DRA activity. The air–liquid interface (ALI) was created by removing apical media for a specified number of days; from 12–14 days post-confluency, Caco-2/BBe cells or a colonoid monolayer were grown as submerged cultures. Confocal imaging was used to examine the dimensions of membrane microdomains that contained DRA. DRA expression and activity were enhanced in Caco-2/BBe cells and human colonoids using an ALI culture method. ALI causes an increase in acid sphingomyelinase (ASMase) activity, an enzyme responsible for enhancing ceramide content in the plasma membrane. ALI cultures expressed a larger number of DRA-containing platforms with dimensions >2 µm compared to cells grown as submerged cultures. ASMase inhibitor, desipramine, disrupted CRPs and reduced the ALI-induced increase in DRA expression in the apical membrane. Exposing normal human colonoid monolayers to ALI increased the ASMase activity and enhanced the differentiation of colonoids along with basal and forskolin-stimulated DRA activities. ALI increases DRA activity and expression by increasing ASMase activity and platform formation in Caco-2/BBe cells and by enhancing the differentiation of colonoids. [ABSTRACT FROM AUTHOR]

Intestinal disorders are common in metabolic syndrome. However, their pathogenesis is still not fully understood. Pig and human intestines are highly similar in terms of associated metabolic processes. Here, we successfully constructed a metabolic disease-susceptible transgenic (TG) Bama pig model by knocking in three humanized disease risk genes with the CRISPR / Cas9 technique to assess its potential as a model for human intestinal diseases and explore the possible pathological mechanisms involved. We found that jejunal barrier integrity was disrupted and that the infiltration of inflammatory cells increased in TG pigs after high-fat and high-sucrose diet (HFHSD) treatment. We revealed significant differences in the transcriptome, associated microbiome profiles and microbial metabolite short-chain fatty acid (SCFA) content of the jejunum of TG pigs. Notably, we found that SLC26A3 was significantly downregulated in TG pigs. Knockdown or overexpression of the SLC26A3 gene in IPEC-J2 cells significantly affected the expression of MUC2 , MUC13 and occludin. Furthermore, in vitro experiments further verified that CDX2 directly regulated the expression of SLC26A3. Mechanistically, CDX2 mediated intestinal barrier function by enhancing the expression of SLC26A3 by binding to its promoter region between −1120 and − 1070 bp. TG pigs represent a promising model that provides new insights into preclinical research on human intestinal metabolic diseases associated with metabolic disorders and revealed that SLC26A3 may be a potential therapeutic target for intestinal metabolic diseases. [ABSTRACT FROM AUTHOR]

ObjectivesOral salt substitutive therapy is pivotal for the survival of patients with congenital chloride diarrhea (CLD), however this therapy is unable to influence the symptoms severity. Butyrate has been proposed to limit diarrhea severity in CLD. Unfortunately, the optimal dose schedule is still largely undefined. In addition, butyrate seems not to be well-tolerated by all patients, with some subjects reporting diarrhea worsening. We investigated the efficacy of a step-up therapeutic approach with sodium butyrate in patients who experienced a diarrhea worsening or an absent improvement after the direct administration of 100 mg/kg/day of sodium butyrate.MethodsThe efficacy of a step-up therapeutic approach starting from 50 mg/Kg/day with a subsequent 25 mg/kg/day weekly increase up to 100 mg/kg/day of oral sodium butyrate was investigated in previously three unresponsive CLD children.ResultsThe step-up therapeutic approach resulted effective in limiting diarrhea severity in all our three previously unresponsive CLD patients.ConclusionsOur results suggest the efficacy of the step-up therapeutic approach in CLD children.

Background SATB2 is a diagnostic biomarker and a favourable prognostic marker for colorectal cancer [CRC], but its role in colitis and colitis-associated colorectal cancer [CAC] is unknown. Methods Colitis was induced in intestinal epithelial-specific Satb2 knockout [Satb2 IEC-KO] and control mice using dextran sulphate sodium [DSS]. RNA-seq analysis was performed on colonic tissues, and 16S rDNA-Seq on faecal bacterial DNA from Satb2 IEC-KO and control mice. Immunohistochemistry and flow cytometry were performed to reveal the proportions of different immune cells. Chromatin immunoprecipitation [ChIP] and luciferase reporter were applied to show the regulatory role of SATB2 on SLC26A3, of which the Cl-/HCO3- exchange activity was measured fluorometrically by the pHi-sensitive dye. Bacteroides were detected by fluorescence in situ hybridisation [FISH] on colonic tissue. Results Satb2 IEC-KO mice suffered from intestinal epithelial damage spontaneously, and developed more severe colitis and CAC. The expression of SLC26A3 correlated well with SATB2 revealed by RNA-seq and The Cancer Genome Atlas [TCGA] data, and was governed by SATB2 confirmed by ChIP and luciferase reporter experiments. Decreased intestinal flora diversity was seen in Satb2 IEC-KO mice. Bacteroides were more abundant and could colonise into the inner layer of colonic mucosa in Satb2 IEC-KO mice. Faecal microbiome transplantation from Satb2 IEC-KO mice aggravated colitis and M1 macrophages infiltration. Conclusions SATB2 plays a vital role in maintaining intestinal homeostasis, and its deficiency promotes the development of colitis and CAC by influencing the intestinal luminal environment and gut flora. [ABSTRACT FROM AUTHOR]

Background Congenital chloride diarrhoea [CLD] is a rare autosomal recessive disease caused by mutations in the solute family carrier 26 member 3 [ SLC26A3 ] gene. Patients suffer from life-long watery diarrhoea and chloride loss. Inflammatory bowel disease [IBD] has been reported in individual patients with CLD and in scl26a3 -deficient mice. Methods We performed an international multicentre analysis to build a CLD cohort and to identify cases with IBD. We assessed clinical and genetic characteristics of subjects and studied the cumulative incidence of CLD-associated IBD. Results In a cohort of 72 patients with CLD caused by 17 different SLC26A3 mutations, we identified 12 patients [17%] diagnosed with IBD. Nine patients had Crohn's disease, two ulcerative colitis and one IBD-unclassified [IBD-U]. The prevalence of IBD in our cohort of CLD was higher than the highest prevalence of IBD in Europe [ p < 0.0001]. The age of onset was variable [13.5 years, interquartile range: 8.5–23.5 years]. Patients with CLD and IBD had lower z-score for height than those without IBD. Four of 12 patients had required surgery [ileostomy formation n = 2, ileocaecal resection due to ileocaecal valve stenosis n = 1 and colectomy due to stage II transverse colon cancer n = 1]. At last follow-up, 5/12 were on biologics [adalimumab, infliximab or vedolizumab], 5/12 on immunosuppressants [azathioprine or mercaptopurine], one on 5-ASA and one off-treatment. Conclusions A substantial proportion of patients with CLD develop IBD. This suggests the potential involvement of SL26A3-mediated anion transport in IBD pathogenesis. Patients with CLD-associated IBD may require surgery for treatment failure or colon cancer. [ABSTRACT FROM AUTHOR]

Background: Anti-Cluster of differentiation (CD)-40-induced colitis, driven by innate inflammatory responses in the intestine, is a potent animal model exhibiting IBD pathophysiology including diarrhea. However, the ion transport basis of diarrhea and some key mucosal pathways (Paneth cells, stem cell niche, and mechanosensory) in this model have not been investigated., Methods: Mucosal scrapings and intestinal tissue from control and CD40 antibody (150 µg) treated Rag2-/- mice were examined for gut inflammation, Paneth cell numbers, expression of key transporters, tight/adherens junction proteins, stem cell niche, and mechanosensory pathway via hematoxylin and eosin staining, quantitative polymerase chain reaction, and western blotting., Results: Compared with control, anti-CD40 antibody treatment resulted in a significant loss of body weight (P < .05) and diarrhea at day 3 postinjection. Distal colonic tissues of anti-CD40 mice exhibited increased inflammatory infiltrates, higher claudin-2 expression, and appearance of Paneth cell-like structures indicative of Paneth cell metaplasia. Significantly reduced expression (P < .005) of downregulated in adenoma (key Cl- transporter), P-glycoprotein/multidrug resistantance-1 (MDR1, xenobiotic transporter), and adherens junction protein E-cadherin (~2-fold P < .05) was also observed in the colon of anti-CD40 colitis mice. Interestingly, there were also marked alterations in the stem cell markers and upregulation of the mechanosensory YAP-TAZ pathway, suggesting the activation of alternate regeneration pathway post-tissue injury in this model., Conclusion: Our data demonstrate that the anti-CD40 colitis model shows key features of IBD observed in the human disease, hence making it a suitable model to investigate the pathophysiology of ulcerative colitis (UC)., (Published by Oxford University Press on behalf of Crohn’s & Colitis Foundation 2024.)

Aim: To evaluate the efficacy of locally delivered nanomedicine, vasoactive intestinal peptide in sterically stabilized micelles (VIP-SSM) to the colon and conduct in vitro release studies of a potential oral formulation. Materials & methods: Intracolonic instillation of VIP-SSM was tested in a mouse model of dextran sulfate sodium-induced colitis. Based on the effective mouse dose, human equivalent dose containing nanomedicine powder was filled into enteric coated capsules for in vitro release testing. Results: Colonic delivery of VIP-SSM significantly alleviated colitis. VIP-SSM containing capsules completely dissolved at colonic pH allowing micelles to reform with active VIP. Capsule formulations exhibited reproducible release profiles when stored up to 6 weeks demonstrating stability. Conclusion: VIP-SSM is an effective nanomedicine formulation which appears to have potential for oral treatment of colitis in humans. [ABSTRACT FROM AUTHOR]

DRA (downregulated in adenoma, SLC26A3) and NHE3 (Na+/H+ exchanger 3, SLC9A3) together mediate intestinal electroneutral NaCl absorption. Both transporters contain PDZ (postsynaptic density 95, disc large, zonula occludens 1) binding motifs and interact with PDZ adaptor proteins regulating their activity and recycling. SNX27 (sorting nexin 27) contains a PDZ domain and is involved in the recycling of cargo proteins including NHE3. The interaction of SNX27 with DRA and its potential role for the activity and recycling of DRA have been evaluated in this study. SNX27 specifically interacts with DRA via its PDZ domain. The knockdown (KD) of SNX27 reduced DRA activity by 50% but was not accompanied by a decrease of DRA surface expression. This indicates that DRA is trafficked to specific functional domains in the plasma membrane in which DRA is particularly active. Consistently, the disruption of lipid raft integrity by methyl-β-cyclodextrin has an inhibitory effect on DRA activity that was strongly reduced after SNX27 KD. In differentiated intestinal Caco2 cells, superresolution microscopy and a novel quantitative axial approach revealed that DRA and SNX27 colocalize in rab5-positive early endosomes at the apical pole. SNX27 regulates the activity of DRA in the apical plasma membrane through binding with its PDZ domain. This interaction occurs in rab5-positive early endosomes at the apical pole of differentiated intestinal Caco2 cells. SNX27 is involved in the direct recycling of DRA to the plasma membrane where it is inserted into lipid rafts facilitating increased activity. [ABSTRACT FROM AUTHOR]

ClopHensor, a fluorescent fusion protein, is a dual function biosensor that has been utilized as a tool for the simultaneous measurement of intracellular chloride and pH in cells. ClopHensor has traditionally been used in conjunction with fluorescence microscopy for single cell measurements. Here, we present a promising multi-well format advancement for the use of ClopHensor as a potential high-throughput method capable of measuring fluorescence signal intensity across a well of confluent cells with highly reproducible results. Using this system, we gained mechanistic insight into an endogenous oxalate transporter in Chinese hamster ovary (CHO) cells expressing ClopHensor and the human chloride transporter, SLC26A3. SLC26A3, a known anion exchanger, has been proposed to play a role in colonic oxalate absorption in humans. Our attempt to study the role of SLC26A3 in oxalate transport revealed the presence of an endogenous oxalate transporter in CHO cells. This transporter was strongly inhibited by niflumate, and exhibited clear saturability. Use of ClopHensor in a multi-well cell assay allowed us to quickly demonstrate that the endogenous oxalate transporter was unable to exchange chloride for bicarbonate, unlike SLC26A3.

The protozoan parasite Cryptosporidium parvum (CP) causes cryptosporidiosis, a diarrheal disease worldwide. Infection in immunocompetent hosts typically results in acute, self-limiting, or recurrent diarrhea. However, in immunocompromised individuals infection can cause fulminant diarrhea, extraintestinal manifestations, and death. To date, the mechanisms underlying CP-induced diarrheal pathogenesis are poorly understood. Diarrheal diseases most commonly involve increased secretion and/or decreased absorption of fluid and electrolytes. We and others have previously shown impaired chloride absorption in infectious diarrhea due to dysregulation of SLC26A3 [downregulated in adenoma (DRA)], the human intestinal apical membrane Cl–/HCO3– exchanger protein. However, there are no studies on the effects of CP infection on DRA activity. Therefore, we examined the expression and function of DRA in intestinal epithelial cells in response to CP infection in vitro and in vivo. CP infection (0.5 X 106 oocysts/well in 24-well plates, 24 h) of Caco-2 cell monolayers significantly decreased Cl–/HCO3– exchange activity (measured as DIDS-sensitive 125I uptake) as well as DRA mRNA and protein levels. Substantial downregulation of DRA mRNA and protein was also observed following CP infection ex vivo in mouse enteroid-derived monolayers and in vivo in the ileal and jejunal mucosa of C57BL/6 mice for 24 h. However, at 48 h after infection in vivo, the effects on DRA mRNA and protein were attenuated and at 5 days after infection DRA returned to normal levels. Our results suggest that impaired chloride absorption due to downregulation of DRA could be one of the contributing factors to CP-induced acute, self-limiting diarrhea in immunocompetent hosts. [ABSTRACT FROM AUTHOR]

New Findings: What is the central question of this study?The tracer 36Cl−, currently used to measure transepithelial Cl− fluxes, has become prohibitively expensive, threatening its future use. 125Iodide, previously validated alongside 36Cl− as a tracer of Cl− efflux by cells, has not been tested as a surrogate for 36Cl− across epithelia.What is the main finding and its importance?We demonstrate that 125I− can serve as an inexpensive replacement for measuring Cl− transport across mouse large intestine, tracking Cl− transport in response to cAMP stimulation (inducing Cl− secretion) in the presence and absence of the main gastrointestinal Cl−–HCO3− exchanger, DRA. Chloride transport is important for driving fluid secretion and absorption by the large intestine, with dysregulation resulting in diarrhoea‐associated pathologies. The radioisotope 36Cl− has long been used as a tracer to measure epithelial Cl− transport but is prohibitively expensive. 125Iodide has been used as an alternative to 36Cl− in some transport assays but has never been validated as an alternative for tracing bidirectional transepithelial Cl− fluxes. The goal of this study was to validate 125I− as an alternative to 36Cl− for measurement of Cl− transport by the intestine. Simultaneous fluxes of 36Cl− and 125I− were measured across the mouse caecum and distal colon. Net Cl− secretion was induced by the stimulation of cAMP with a cocktail of forskolin (FSK) and 3‐isobutyl‐1‐methylxanthine (IBMX). Unidirectional fluxes of 125I− correlated well with 36Cl− fluxes after cAMP‐induced net Cl− secretion, occurring predominantly through a reduction in the absorptive mucosal‐to‐serosal Cl− flux rather than by stimulation of the secretory serosal‐to‐mucosal Cl− flux. Correlations between 125I− fluxes and 36Cl− fluxes were maintained in epithelia from mice lacking DRA (Slc26a3), the main Cl−–HCO3− exchanger responsible for Cl− absorption by the large intestine. Lower rates of Cl− and I− absorption in the DRA knockout intestine suggest that DRA might have a previously unrecognized role in iodide uptake. This study validates that 125I− traces transepithelial Cl− fluxes across the mouse large intestine, provides insights into the mechanism of net Cl− secretion and suggests that DRA might be involved in intestinal iodide absorption. [ABSTRACT FROM AUTHOR]

Purpose: Abnormalities of the solute-linked carrier family 26 member A3 (SLC26A3) are implicated in the pathogenesis of several diseases including ulcerative colitis (UC). The short communication aimed at investigating the associations of UC with SLC26A3 (rs17154444, rs7810937, rs7785539, rs2108225 and rs6951457) polymorphisms and its expression in colonic tissues.Methods: The techniques of SNaPshot method, quantitative real-time PCR and immunohistochemical analysis were conducted.Results and conclusion: We found that the rs2108225 variation in SLC26A3 might increase the risk of UC and affect its expression at both the mRNA and protein levels in colonic tissues of patients with UC. Moreover, the rs17154444 variation might influence the severity of UC. [ABSTRACT FROM AUTHOR]

SLC26A3 [downregulated in adenoma (DRA)] plays a key role in mammalian intestinal NaCl absorption, in that it mediates apical membrane Cl-/HCO3- exchange. DRA function and expression are significantly decreased in diarrhea associated with inflammatory bowel disease. DRA is also considered to be a marker of cellular differentiation and is predominantly expressed in differentiated epithelial cells. Caudal-type homeobox protein-2 (CDX2) is known to regulate genes involved in intestinal epithelial differentiation and proliferation. Reduced expression of both DRA and CDX2 in intestinal inflammation prompted us to study whether the DRA gene is directly regulated by CDX2. Our initial studies utilizing CDX2 knockout (CDX2fV/fV;Cre+) mice showed a marked reduction in DRA mRNA and protein levels in proximal and distal colon. In silico analysis of the DRA promoter showed two consensus sites for CDX2 binding. Therefore, we utilized Caco-2 cells as an in vitro model to examine if DRA is a direct target of CDX2 regulation. siRNAmediated silencing of CDX2 in Caco-2 cells resulted in a marked (~50%) decrease in DRA mRNA and protein levels, whereas ectopic overexpression of CDX2 upregulated DRA expression and also stimulated DRA promoter activity, suggesting transcriptional regulation. Electrophoretic mobility shift and chromatin immunoprecipitation assays demonstrated direct binding of CDX2 to one of the two putative CDX2 binding sites in the DRA promoter (+645/+663). In summary, our studies, for the first time, demonstrate transcriptional regulation of DRA expression by CDX2, implying that reduced expression of DRA in inflammatory bowel disease-associated diarrhea may, in part, be due to downregulation of CDX2 in the inflamed mucosa. [ABSTRACT FROM AUTHOR]

In polarized intestinal epithelial cells, downregulated in adenoma (DRA) is an apical Cl - /[Formula: see text] exchanger that is part of neutral NaCl absorption under baseline conditions, but in cyclic adenosine monophosphate (cAMP)-driven diarrheas, it is stimulated and contributes to increased anion secretion. To further understand the regulation of DRA in conditions mimicking some diarrheal diseases, Caco-2/BBE cells were exposed to forskolin (FSK) and adenosine 5'-triphosphate (ATP). FSK and ATP stimulated DRA in a concentration-dependent manner, with ATP acting via P2Y1 receptors. FSK at 1 µM and ATP at 0.25 µM had minimal to no effect on DRA given individually; however, together, they stimulated DRA to levels seen with maximum concentrations of FSK and ATP alone. In Caco-2/BBE cells expressing the Ca 2+ indicator GCaMP6s, ATP increased intracellular Ca 2+ (Ca 2+ i ) in a concentration-dependent manner, whereas FSK (1 µM), which by itself did not significantly alter Ca 2+ i , followed by 0.25 µM ATP produced a large increase in Ca 2+ that was approximately equal to the elevation caused by 1 µM ATP. 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-AM) pretreatment prevented the ATP and FSK/ATP synergistically increased the DRA activity and the increase in Ca 2+ i caused by FSK/ATP. FSK/ATP synergistic stimulation of DRA was similarly observed in human colonoids. In Caco-2/BBE cells, subthreshold concentrations of FSK (cAMP) and ATP (Ca 2+ ) synergistically increased Ca 2+ i and stimulated DRA activity with both being blocked by BAPTA-AM pretreatment. Diarrheal diseases, such as bile acid diarrhea, in which both cAMP and Ca 2+ are elevated, are likely to be associated with stimulated DRA activity contributing to increased anion secretion, whereas separation of DRA from Na + /H + exchanger isoform-3 (NHE3) contributes to reduced NaCl absorption. NEW & NOTEWORTHY The BB Cl - /[Formula: see text] exchanger DRA takes part in both neutral NaCl absorption and stimulated anion secretion. Using intestinal cell line, Caco-2/BBE high concentrations of cAMP and Ca 2+ individually stimulated DRA activity, whereas low concentrations, which had no/minimal effect, synergistically stimulated DRA activity that required a synergistic increase in intracellular Ca 2+ . This study increases understanding of diarrheal diseases, such as bile salt diarrhea, in which both cAMP and elevated Ca 2+ are involved.

Congenital chloride diarrhea (CCD) is a rare autosomal recessive disease, which is characterized by electrolyte absorption defect due to impaired function of the Cl-/ HCO3- exchanger in the ileum and the colon. Its main features are profuse watery diarrhea, high fecal chloride concentration, and failure to thrive. Profuse watery diarrhea characterized by a high concentration of chloride in stools results in hypochloremia, hyponatremia, and dehydration with metabolic alkalosis. Early detection and therapeutic intervention can prevent life-threatening symptoms of CCD and growth failure. Recently, several therapies, such as proton pump inhibitors and butyrate, have been suggested for amelioration of diarrhea. Here, we report a case of CCD in a preterm male infant who was successfully treated with an oral proton pump inhibitor. [ABSTRACT FROM AUTHOR]

The solute carrier 26 (SLC26) family emerges as a distinct class of anion transporters with its members SLC26A3 (Slc26a3) and SLC26A6 (Slc26a6) reported to be electrogenic Cl−/HCO3− exchangers. While it is known that uterine fluid has high HCO3− content and that HCO3− is essential for sperm capacitation, the molecular mechanisms underlying the transport of HCO3− across uterine epithelial cells and sperm have not been fully investigated. The present review re-examines the results from early reports studying anion transport, finding clues for the involvement of Cl−/HCO3− anion exchangers in electrogenic HCO3− transport across endometrial epithelium. We also summarise recent work on Slc26a3 and Slc26a6 in uterine epithelial cells and sperm, revealing their functional role in working closely with the cystic fibrosis transmembrane conductance regulator (CFTR) for HCO3− transport in these cells. The possible involvement of these anion exchangers in other HCO3− dependent reproductive processes and their implications for infertility are also discussed. [ABSTRACT FROM AUTHOR]

Congenital chloride diarrhea (CLD, OMIM#214700) is an autosomal recessive disorder caused by mutations in the solute carrier family 26 member 3 ( SLC26A3) gene, which encodes an intestinal chloride/bicarbonate exchanger. While more than 50 mutations have been identified throughout the world, there have been no data on the genetic characteristics of the patients of East Asian ethnic origin. In this study, we performed genetic analysis by direct sequencing of the 20 exons and parts of exon-intron boundaries of the SLC26A3 gene in eight patients of Korean origin with non-consanguineous parents. We identified three novel mutations, including two splice-site mutations (c.2063-1G>T in intron 18, c.1047+3 A>C in intron 12) and one missense mutation (p.Ser134Asn in exon 5). One previously identified mutation was also found (p.Pro131Leu in exon 5). The most common mutation was c.2063-1G>T, which was found in at least one allele of all patients. Conclusion: This is the first report to demonstrate the genetic background of CLD in a single ethnic group of East Asian descent. The c.2063-1G>T mutation could be suggested as a founder mutation in Korean population so that the targeting sequencing for the mutation would be a cost-efficient screening method to confirm a diagnosis of CLD in patients of Korean descent. [ABSTRACT FROM AUTHOR]

Background: Congenital chloride diarrhea (CLD) is an autosomal recessive disorder characterized by life-long, severe diarrhea with intestinal Cl- malabsorption. It results from a reduced activity of the down regulated in adenoma exchanger (DRA), due to mutations in the solute carrier family 26, member 3 (SLC26A3) gene. Currently available therapies are not able to limit the severity of diarrhea in CLD. Conflicting results have been reported on the therapeutic efficacy of oral butyrate.Methods: We investigated the effect of oral butyrate (100 mg/kg/day) in seven CLD children with different SLC26A3 genotypes. Nasal epithelial cells were obtained to assess the effect of butyrate on the expression of the two main Cl- transporters: DRA and putative anion transporter-1 (PAT-1).Results: A variable clinical response to butyrate was observed regarding the stool pattern and fecal ion loss. The best response was observed in subjects with missense and deletion mutations. Variable response to butyrate was also observed on SLC26A3 (DRA) and SLC26A6 (PAT1) gene expression in nasal epithelial cells of CLD patients.Conclusions: We demonstrate a genotype-dependency for butyrate therapeutic efficacy in CLD. The effect of butyrate is related in part on a different modulation of the expression of the two main apical membrane Cl- exchangers of epithelial cells, members of the SLC26 anion family.Trial Registration: Australian New Zealand Clinical trial Registry ACTRN12613000450718. [ABSTRACT FROM AUTHOR]