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Putative anion transporter-1 (PAT1, SLC26A6) plays a key role in intestinal oxalate and bicarbonate secretion. PAT1 knockout (PKO) mice exhibit hyperoxaluria and nephrolithiasis. Notably, diseases such as inflammatory bowel disease are also associated with higher risk of hyperoxaluria and nephrolithiasis. However, the potential role of PAT1 deficiency in gut-barrier integrity and susceptibility to colitis is currently elusive. Age-matched PKO and wild-type littermates were administered 3.5% dextran sulfate sodium in drinking water for 6 days. Ileum and colon of control and treated mice were harvested. Messenger RNA and protein expression of tight junction proteins were determined by reverse transcription polymerase chain reaction and western blotting. Severity of inflammation was assessed by measuring diarrheal phenotype, cytokine expression, and hematoxylin and eosin staining. Gut microbiome and associated metabolome were analyzed by 16S ribosomal RNA sequencing and mass spectrometry, respectively. PKO mice exhibited significantly higher loss of body weight, gut permeability, colonic inflammation, and diarrhea in response to dextran sulfate sodium treatment. In addition, PKO mice showed microbial dysbiosis and significantly reduced levels of butyrate and butyrate-producing microbes compared with controls. Co-housing wild-type and PKO mice for 4 weeks resulted in PKO-like signatures on the expression of tight junction proteins in the colons of wild-type mice. Our data demonstrate that loss of PAT1 disrupts gut microbiome and related metabolites, decreases gut-barrier integrity, and increases host susceptibility to intestinal inflammation. These findings, thus, highlight a novel role of the oxalate transporter PAT1 in promoting gut-barrier integrity, and its deficiency appears to contribute to the pathogenesis of inflammatory bowel diseases. [Display omitted] This study demonstrates that deficiency of intestinal oxalate transporter putative anion transporter 1 results in compromised gut-barrier integrity, altered composition of gut microbes/metabolites leading to enhanced susceptibility to inflammatory bowel diseases. [ABSTRACT FROM AUTHOR]

The gut hormone, glucagon like peptide-1 (GLP-1) exerts anti-inflammatory effects. However, its clinical use is limited by its short half-life. Previously, we have shown that GLP-1 as a nanomedicine (GLP-1 in sterically stabilized phospholipid micelles, GLP-1-SSM) has increased in vivo stability. The current study was aimed at testing the efficacy of this GLP-1 nanomedicine in alleviating colonic inflammation and associated diarrhea in dextran sodium sulfate (DSS) induced mouse colitis model. Our results show that GLP-1-SSM treatment markedly alleviated the colitis phenotype by reducing the expression of pro-inflammatory cytokine IL-1β, increasing goblet cells and preserving intestinal epithelial architecture in colitis model. Further, GLP-1-SSM alleviated diarrhea (as assessed by luminal fluid) by increasing protein expression of intestinal chloride transporter DRA (down regulated in adenoma). Our results indicate that GLP-1 nanomedicine may act as a novel therapeutic tool in alleviating gut inflammation and associated diarrhea in inflammatory bowel disease (IBD). [ABSTRACT FROM AUTHOR]

Tumor necrosis factor (TNF)-α, a homotrimeric, pleiotropic cytokine, is secreted in response to inflammatory stimuli in diseases such as rheumatoid arthritis and inflammatory bowel disease. TNA-α mediates both apoptosis and inflammation, stimulating an inflammatory cascade through the non-canonical pathway of NF-κB activation, leading to increased nuclear RelB and p52. In contrast, the common food additive carrageenan (CGN) stimulates inflammation through both the canonical and non-canonical pathways of NF-κB activationand utilizes the adaptor molecule BCL10 (B-cell leukemia/lymphoma 10). In a series of experiments, colonic epithelial cells and mouse embryonic fibroblasts were treated with TNA-α and carrageenan in order to simulate the possible effects of exposure to dietary CGN in the setting of a TNA-α-mediated inflammatory disease process. A marked increase in secretion of IL-8 occurred, attributable to synergistic effects on phosphorylated NF-κB-inducing kinase (NIK) in the non-canonical pathway. TNA-α induced the ubiquitination of TRAF2 (TNF receptor-associated factor 2), which interacts with NIK, and CGN induced phosphorylation of BCL10, leading to increased NIK phosphorylation. These results suggest thit TNF-α and CGN in combination act to increase NIK phosphorylation, thereby increasing activation of the non-canonical pathway of NF-κB activation. In contrast, the apoptotic effects of TNA-α, including activation of caspase-8 and PARP-1 (poly(ADP-ribose) polymerase 1) fragmentation, were markedly reduced in the presence of CGN, and CGN caused reduced expression of Fas. These findings demonstrate that exposure to CGN drives TNA-α-stimulated cells toward inflammation rather than toward apoptotic cell death and suggest that CGN exposure may compromise the effectiveness of anti-TNA-α therapy. [ABSTRACT FROM AUTHOR]

Background & Aims: The current model of duodenal HCO3 − secretion proposes that basal secretion results from Cl−/HCO3 − exchange, whereas cyclic adenosine monophosphate (cAMP)-stimulated secretion depends on a cystic fibrosis transmembrane conductance regulator channel (Cftr)-mediated HCO3 − conductance. However, discrepancies in applying the model suggest that Cl−/HCO3 − exchange also contributes to cAMP-stimulated secretion. Of 2 candidate Cl−/HCO3 − exchangers, studies of putative anion transporter-1 knockout (KO) mice find little contribution of putative anion transporter-1 to basal or cAMP-stimulated secretion. Therefore, the role of down-regulated in adenoma (Dra) in duodenal HCO3 − secretion was investigated using DraKO mice. Methods: Duodenal HCO3 − secretion was measured by pH stat in Ussing chambers. Apical membrane Cl−/HCO3 − exchange was measured by microfluorometry of intracellular pH in intact villous epithelium. Dra expression was assessed by immunofluorescence. Results: Basal HCO3 − secretion was reduced ∼55%–60% in the DraKO duodenum. cAMP-stimulated HCO3 − secretion was reduced ∼50%, but short-circuit current was unchanged, indicating normal Cftr activity. Microfluorimetry of villi demonstrated that Dra is the dominant Cl−/HCO3 − exchanger in the lower villous epithelium. Dra expression increased from villous tip to crypt. DraKO and wild-type villi also demonstrated regulation of apical Na+/H+ exchange by Cftr-dependent cell shrinkage during luminal Cl− substitution. Conclusions: In murine duodenum, Dra Cl−/HCO3 − exchange is concentrated in the lower crypt-villus axis where it is subject to Cftr regulation. Dra activity contributes most basal HCO3 − secretion and ∼50% of cAMP-stimulated HCO3 − secretion. Dra Cl−/HCO3 − exchange should be considered in efforts to normalize HCO3 − secretion in duodenal disorders such as ulcer disease and cystic fibrosis. [Copyright &y& Elsevier]

Background & Aims: Electroneutral NaCl absorption across small intestine contributes importantly to systemic fluid balance. Disturbances in this process occur in both obstructive and diarrheal diseases, eg, cystic fibrosis, secretory diarrhea. NaCl absorption involves coupling of Cl−/HCO3 − exchanger(s) primarily with Na+/H+ exchanger 3 (Nhe3) at the apical membrane of intestinal epithelia. Identity of the coupling Cl−/HCO3 − exchanger(s) was investigated using mice with gene-targeted knockout (KO) of Cl−/HCO3 − exchangers: Slc26a3, down-regulated in adenoma (Dra) or Slc26a6, putative anion transporter-1 (Pat-1). Methods: Intracellular pH (pHi) of intact jejunal villous epithelium was measured by ratiometric microfluoroscopy. Ussing chambers were used to measure transepithelial 22Na36Cl flux across murine jejunum, a site of electroneutral NaCl absorption. Expression was estimated using immunofluorescence and quantitative polymerase chain reaction. Results: Basal pHi of DraKO epithelium, but not Pat-1KO epithelium, was alkaline, whereas pHi in the Nhe3KO was acidic relative to wild-type. Altered pHi was associated with robust Na+/H+ and Cl−/HCO3 − exchange activity in the DraKO and Nhe3KO villous epithelium, respectively. Contrary to genetic ablation, pharmacologic inhibition of Nhe3 in wild-type did not alter pHi but coordinately inhibited Dra. Flux studies revealed that Cl− absorption was essentially abolished (>80%) in the DraKO and little changed (<20%) in the Pat-1KO jejunum. Net Na+ absorption was unaffected. Immunofluorescence demonstrated modest Dra expression in the jejunum relative to large intestine. Functional and expression studies did not indicate compensatory changes in relevant transporters. Conclusions: These studies provide functional evidence that Dra is the major Cl−/HCO3 − exchanger coupled with Nhe3 for electroneutral NaCl absorption across mammalian small intestine. [Copyright &y& Elsevier]

Probiotics are viable nonpathogenic microorganisms that are considered to confer health benefits to the host. Recent studies indicated that some Lactobacillus species function as probiotics and have been used as alternative treatments for diarrhea, which occurs due to increased secretion, decreased absorption, or both. However, the direct effects of probiotics on intestinal electrolyte absorption are not known. Therefore, we examined the effects of Lactobacillus on luminal chloride/hydroxyl (CI-/OH-) exchange activity in human intestinal epithelial cells. Postconfluent Caco-2 cells were treated with the Lactobacillus species Lactobacillus acidophilus ILA), Lactobacillus casei, Lactobacillus plantarum, or Lactobacillus rhamnosus ILR) for 3 h at a multiplicity of infection of 50. Cl-/OH- exchange activity was measured as 4,4′- diisothiocyanostilbene-2, 2′-disulfonic acid-sensitive 36C1 uptake in base-loaded cells. Treatment with live, but not heat- killed, LA and LR significantly increased Cl-/OH- exchange activity (~50%), whereas other species were ineffective. Similarly, the conditioned medium (supernatant) of live LA increased Cl-/OH- exchange. The ability of LA or its conditioned culture medium to enhance Cl-/OH- exchange activity was blocked by Pl-3 kinase inhibition but was unaffected by inhibition of mitogen-activated protein kinases. Corresponding to the increased Cl-/OH- exchange activity, LA treatment increased the surface expression of the apical anion exchanger, SLC26A3 [Down Regulated in Adenoma (DRA)]. The increased DRA membrane localization might contribute to the increased C1- absorption by LA. Our results suggest that LA secretes soluble effector molecule(s) into the culture medium that stimulate apical Cl-/OH- exchange activity via phosphatidylinositol-3 kinase mediated mechanism. [ABSTRACT FROM AUTHOR]

Multiple studies in animal models have shown that the commonly used food additive carrageenan (CGN) induces inflammation and intestinal neoplasia. We performed the first studies to determine the effects of CGN exposure on human intestinal epithelial cells (IEC) in tissue culture and tested the effect of very low concentrations (1-10 mg/L) of undegraded, high-molecular weight CGN. These concentrations of CGN are less than the anticipated exposure of the human colon to CGN from the average Western diet. In the human colonic epithelial cell line NCM460 and in primary human colonic epithelial cells that were exposed to CGN for 1-8 d, we found increased cell death, reduced cell proliferation, and cell cycle arrest compared with unexposed control cells. After 6-8 d of CGN exposure, the percentage of cells reentering G0-G1 significantly decreased and the percentages of cells in S and G2-M phases significantly increased. Increases in activated p53, p21, and p15 followed CGN exposure, consistent with CGN-induced cell cycle arrest. Additional data, including DNA ladder, poly ADP ribose polymerase Western blot, nuclear DNA staining, and activities of caspases 3 and 7, indicated no evidence of increased apoptosis following CGN exposure and were consistent with CGN-induced necrotic cell death. These data document for the first time, to our knowledge, marked adverse effects of low concentrations of CGN on survival of normal human IEC and suggest that CGN exposure may have a role in development of human intestinal pathology. [ABSTRACT FROM AUTHOR]

Background & Aims: : Increased serotonin levels have been implicated in the pathophysiology of diarrhea associated with celiac and inflammatory diseases. However, the effects of serotonin on Na+/H+ exchange (NHE) activity in the human intestine have not been investigated fully. The present studies examined the acute effects of 5-hydroxytryptamine (5-HT) on NHE activity using Caco-2 cells as an in vitro model. Methods: : Caco-2 cells were treated with 5-HT (.1 μmol/L, 1 h) and NHE activity was measured as ethyl-isopropyl-amiloride (EIPA)-sensitive 22Na uptake. The effect of 5-HT receptor-specific agonists and antagonists was examined. The role of signaling intermediates in 5-HT-mediated effects on NHE activity was elucidated using pharmacologic inhibitors and immunoblotting. Results: : NHE activity was inhibited significantly (∼50%–75%, P < .05) by .1 μmol/L 5-HT via inhibition of maximal velocity (Vmax) without any changes in apparent affinity (Km) for the substrate Na+. NHE inhibition involved a decrease of both NHE2 and NHE3 activities. Studies using specific inhibitors and agonists showed that the effects of 5-HT were mediated by 5-HT4 receptors. 5-HT-mediated inhibition of NHE activity was dependent on phosphorylation of phospholipase C γ 1 (PLCγ1) via activation of src-kinases. Signaling pathways downstream of PLCγ1 involved increase of intracellular Ca2+ levels and subsequent activation of protein kinase C α (PKCα). The effects of 5-HT on NHE activity were not cell-line specific because T84 cells also showed NHE inhibition. Conclusions: : A better understanding of the regulation of Na+ absorption by 5-HT offers the potential for providing insights into molecular and cellular mechanisms involved in various diarrheal and inflammatory disorders. [Copyright &y& Elsevier]

The down-regulated in adenoma (DRA) protein, encoded by SLC26A3 , a key intestinal chloride anion exchanger, has recently been identified as a novel susceptibility gene for inflammatory bowel disease (IBD). However, the mechanisms underlying the increased susceptibility to inflammation induced by the loss of DRA remain elusive. Compromised barrier is a key event in IBD pathogenesis. The current studies were undertaken to elucidate the impact of DRA deficiency on epithelial barrier integrity and to define underlying mechanisms. Wild-type and DRA-knockout (KO) mice and crypt-derived colonoids were used as models for intestinal epithelial response. Paracellular permeability was measured by using fluorescein isothiocyanate–dextran flux. Immunoblotting, immunofluorescence, immunohistochemistry, and ribonucleoprotein immunoprecipitation assays were performed. Gut microbiome analysis was conducted to investigate the impact of DRA deficiency on gut microbial communities. DRA-KO mice exhibited an increased colonic paracellular permeability with significantly decreased levels of tight junction/adherens junction proteins, including ZO-1, occludin, and E-cadherin. A similar expression pattern of occludin and E-cadherin was observed in colonoids derived from DRA-KO mice and short hairpin RNA–mediated DRA knockdown in Caco-2 cells. Microbial analysis showed gut dysbiosis in DRA-KO mice. However, cohousing studies showed that dysbiosis played only a partial role in maintaining tight junction protein expression. Furthermore, our results showed increased binding of RNA-binding protein CUGBP1 with occludin and E-cadherin genes in DRA-KO mouse colon, suggesting that posttranscriptional mechanisms play a key role in gut barrier dysfunction. To our knowledge, our studies demonstrate a novel role of DRA in maintaining the intestinal epithelial barrier function and potential implications of its dysregulation in IBD pathogenesis. [Display omitted] [ABSTRACT FROM AUTHOR]

Background & Aims Inactivating mutations in MYO5B cause microvillus inclusion disease (MVID), but the physiological cause of the diarrhea associated with this disease is unclear. We investigated whether loss of MYO5B results in aberrant expression of apical enterocyte transporters. Methods We studied alterations in apical membrane transporters in MYO5B-knockout mice, as well as mice with tamoxifen-inducible, intestine-specific disruption of Myo5b (VilCre ERT2; Myo5b flox/flox mice) or those not given tamoxifen (controls). Intestinal tissues were collected from mice and analyzed by immunostaining, immunoelectron microscopy, or cultured enteroids were derived. Functions of brush border transporters in intestinal mucosa were measured in Ussing chambers. We obtained duodenal biopsy specimens from individuals with MVID and individuals without MVID (controls) and compared transporter distribution by immunocytochemistry. Results Compared to intestinal tissues from littermate controls, intestinal tissues from MYO5B-knockout mice had decreased apical localization of SLC9A3 (also called NHE3), SLC5A1 (also called SGLT1), aquaporin (AQP) 7, and sucrase isomaltase, and subapical localization of intestinal alkaline phosphatase and CDC42. However, CFTR was present on apical membranes of enterocytes from MYO5B knockout and control mice. Intestinal biopsies from patients with MVID had subapical localization of NHE3, SGLT1, and AQP7, but maintained apical CFTR. After tamoxifen administration, VilCre ERT2; Myo5b flox/flox mice lost apical NHE3, SGLT1, DRA, and AQP7, similar to germline MYO5B knockout mice. Intestinal tissues from VilCre ERT2; Myo5b flox/flox mice had increased CFTR in crypts and CFTR localized to the apical membranes of enterocytes. Intestinal mucosa from VilCre ERT2; Myo5b flox/flox mice given tamoxifen did not have an intestinal barrier defect, based on Ussing chamber analysis, but did have decreased SGLT1 activity and increased CFTR activity. Conclusions Although trafficking of many apical transporters is regulated by MYO5B, trafficking of CFTR is largely independent of MYO5B. Decreased apical localization of NHE3, SGLT1, DRA, and AQP7 might be responsible for dysfunctional water absorption in enterocytes of patients with MVID. Maintenance of apical CFTR might exacerbate water loss by active secretion of chloride into the intestinal lumen. [ABSTRACT FROM AUTHOR]

Background & Aims Diarrhea associated with inflammatory bowel diseases has been associated with increased levels of inflammatory cytokines, including tumor necrosis factor (TNF). The intestinal mucosa of patients with inflammatory bowel diseases has reduced expression of solute carrier family 26 member 3 (SLC26A3, also called DRA). We investigated whether TNF directly affects expression of DRA in human intestinal epithelial cells (IECs) and in the intestines of mice, and studied the mechanisms of these effects. Methods We performed quantitative reverse transcription polymerase chain reaction, immunofluorescence, and immunoblot analyses in Caco-2, HT-29, and T-84 cells human IECs cultured in 2 or 3 dimensions with or without TNF (50 ng/mL for 6–24 hours). We purified nuclear extracts and quantified nuclear factor−κB (NF-κB) activation and DNA binding. We isolated intestinal crypts from C57BL/6 mice, cultured enteroids, incubated these with TNF (50 ng/mL, 24 hours), and quantified messenger RNAs. DRA-mediated exchange of Cl − for HCO 3 − was measured by uptake of 125 I. Expression of the NF-κB inhibitor α (IkBa) was knocked down in Caco-2 cells with small interfering RNAs. Activation of NF-κB in response to TNF was measured by luciferase reporter assays; binding of the NF-κB subunit p65 in cells was analyzed in chromatin immunoprecipitation assays. DRA promoter activity was measured in a luciferase reporter assay. C57BL/6 mice were injected with TNF (5 μg/mouse for 3–6 hours) or vehicle (control); intestines were collected and analyzed by immunofluorescence, or RNA and protein were collected from the mucosa. Results Incubation of IECs with TNF reduced expression of DRA. Knockdown of NF-κB inhibitor α in IECs led to nuclear translocation of the NF-κB subunit p65 and reduced levels of DRA messenger RNA and protein. Expression of a transgene encoding p65 or p50 in IECs led to significant reductions in the promoter activity of DRA and its expression. In chromatin immunoprecipitation assays, p65 bound directly to the promoter of DRA , at the regions of −935 to −629 and −375 to −84. Injection of mice with TNF or incubation of crypt-derived enteroids with TNF reduced their expression of DRA messenger RNA and protein. Conclusions In human IECs and intestinal tissues from mice, we found TNF to activate NF-κB, which reduced expression of the Cl − / HCO 3 − exchanger DRA (SLC26A3), via direct binding to the promoter of DRA. This pathway is an important therapeutic target for inflammatory bowel disease−associated diarrhea. [ABSTRACT FROM AUTHOR]