The cellular mechanisms of neuronal swelling underlying cytotoxic edema.

Cytotoxic brain edema triggered by neuronal swelling is the chief cause of mortality following brain trauma and cerebral infarct. Using fluorescence lifetime imaging to analyze contributions of intracellular ionic changes in brain slices, we find that intense Na(+) entry triggers a secondary increase in intracellular Cl(-) that is required for neuronal swelling and death. Pharmacological and siRNA-mediated knockdown screening identified the ion exchanger SLC26A11 unexpectedly acting as a voltage-gated Cl(-) channel that is activated upon neuronal depolarization to membrane potentials lower than -20 mV. Blockade of SLC26A11 activity attenuates both neuronal swelling and cell death. Therefore cytotoxic neuronal edema occurs when sufficient Na(+) influx and depolarization is followed by Cl(-) entry via SLC26A11. The resultant NaCl accumulation causes subsequent neuronal swelling leading to neuronal death. These findings shed light on unique elements of volume control in excitable cells and lay the ground for the development of specific treatments for brain edema.

Alkalosis-induced hypoventilation in cystic fibrosis: The importance of efficient renal adaptation.

The lungs and kidneys are pivotal organs in the regulation of body acid-base homeostasis. In cystic fibrosis (CF), the impaired renal ability to excrete an excess amount of HCO3- into the urine leads to metabolic alkalosis [P. Berg et al., J. Am. Soc. Nephrol. 31, 1711-1727 (2020); F. Al-Ghimlas, M. E. Faughnan, E. Tullis, Open Respir. Med. J. 6, 59-62 (2012)]. This is caused by defective HCO3- secretion in the ß-intercalated cells of the collecting duct that requires both the cystic fibrosis transmembrane conductance regulator (CFTR) and pendrin for normal function [P. Berg et al., J. Am. Soc. Nephrol. 31, 1711-1727 (2020)]. We studied the ventilatory consequences of acute oral base loading in normal, pendrin knockout (KO), and CFTR KO mice. In wild-type mice, oral base loading induced a dose-dependent metabolic alkalosis, fast urinary removal of base, and a moderate base load did not perturb ventilation. In contrast, CFTR and pendrin KO mice, which are unable to rapidly excrete excess base into the urine, developed a marked and transient depression of ventilation when subjected to the same base load. Therefore, swift renal base elimination in response to an acute oral base load is a necessary physiological function to avoid ventilatory depression. The transient urinary alkalization in the postprandial state is suggested to have evolved for proactive avoidance of hypoventilation. In CF, metabolic alkalosis may contribute to the commonly reduced lung function via a suppression of ventilatory drive.

Reduced surface pH and upregulated AE2 anion exchange in SLC26A3-deleted polarized intestinal epithelial cells.

Loss of function mutations in the SLC26A3 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, SLC26A3 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 pHi and apical and basolateral Cl-/HCO3- exchange activities were assessed fluorometrically in a dual perfusion chamber with independent perfusion of luminal and serosal baths. Apical Cl-/HCO3- exchange rates were strongly reduced in SLC26A3-KO cells, accompanied by a surface pH more acidic than that of WT cells. Steady-state pHi was not significantly different from that of WT cells, but basolateral Cl-/HCO3- exchange rates were higher in SLC26A3-KO than in WT cells. The data show that CRISPR/Cas9-mediated SLC26A3 deletion strongly reduced apical Cl-/HCO3- exchange rate and apical surface pH, but sustained a normal steady-state pHi 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.NEW & NOTEWORTHY 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-/HCO3- 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.

Novel functions of the anion exchanger AE4 (SLC4A9).

The kidney plays a crucial role in acid-base homeostasis. In the distal nephron, α-intercalated cells contribute to urinary acid (H+) secretion and ß-intercalated cells accomplish urinary base (HCO3-) secretion. ß-intercalated cells regulate the acid base status through modulation of the apical Cl-/HCO3- exchanger pendrin (SLC26A4) activity. In this review, we summarize and discuss our current knowledge of the physiological role of the renal transporter AE4 (SLC4A9). The AE4, as cation-dependent Cl-/HCO3- exchanger, is exclusively expressed in the basolateral membrane of ß-intercalated cells and is essential for the sensing of metabolic acid-base disturbances in mice, but not for renal sodium reabsorption and plasma volume control. Potential intracellular signaling pathways are discussed that might link basolateral acid-base sensing through the AE4 to apical pendrin activity.

Biallelic variants in SLC4A10 encoding a sodium-dependent bicarbonate transporter lead to a neurodevelopmental disorder.

PURPOSE: SLC4A10 encodes a plasma membrane-bound transporter, which mediates Na+-dependent HCO3- import, thus mediating net acid extrusion. Slc4a10 knockout mice show collapsed brain ventricles, an increased seizure threshold, mild behavioral abnormalities, impaired vision, and deafness. METHODS: Utilizing exome/genome sequencing in families with undiagnosed neurodevelopmental disorders and international data sharing, 11 patients from 6 independent families with biallelic variants in SLC4A10 were identified. Clinico-radiological and dysmorphology assessments were conducted. A minigene assay, localization studies, intracellular pH recordings, and protein modeling were performed to study the possible functional consequences of the variant alleles. RESULTS: The families harbor 8 segregating ultra-rare biallelic SLC4A10 variants (7 missense and 1 splicing). Phenotypically, patients present with global developmental delay/intellectual disability and central hypotonia, accompanied by variable speech delay, microcephaly, cerebellar ataxia, facial dysmorphism, and infrequently, epilepsy. Neuroimaging features range from some non-specific to distinct neuroradiological findings, including slit ventricles and a peculiar form of bilateral curvilinear nodular heterotopia. In silico analyses showed 6 of 7 missense variants affect evolutionarily conserved residues. Functional analyses supported the pathogenicity of 4 of 7 missense variants. CONCLUSION: We provide evidence that pathogenic biallelic SLC4A10 variants can lead to neurodevelopmental disorders characterized by variable abnormalities of the central nervous system, including altered brain ventricles, thus resembling several features observed in knockout mice.

SLC4A10 mutation causes a neurological disorder associated with impaired GABAergic transmission.

SLC4A10 is a plasma-membrane bound transporter that utilizes the Na+ gradient to drive cellular HCO3- uptake, thus mediating acid extrusion. In the mammalian brain, SLC4A10 is expressed in principal neurons and interneurons, as well as in epithelial cells of the choroid plexus, the organ regulating the production of CSF. Using next generation sequencing on samples from five unrelated families encompassing nine affected individuals, we show that biallelic SLC4A10 loss-of-function variants cause a clinically recognizable neurodevelopmental disorder in humans. The cardinal clinical features of the condition include hypotonia in infancy, delayed psychomotor development across all domains and intellectual impairment. Affected individuals commonly display traits associated with autistic spectrum disorder including anxiety, hyperactivity and stereotyped movements. In two cases isolated episodes of seizures were reported in the first few years of life, and a further affected child displayed bitemporal epileptogenic discharges on EEG without overt clinical seizures. While occipitofrontal circumference was reported to be normal at birth, progressive postnatal microcephaly evolved in 7 out of 10 affected individuals. Neuroradiological features included a relative preservation of brain volume compared to occipitofrontal circumference, characteristic narrow sometimes 'slit-like' lateral ventricles and corpus callosum abnormalities. Slc4a10 -/- mice, deficient for SLC4A10, also display small lateral brain ventricles and mild behavioural abnormalities including delayed habituation and alterations in the two-object novel object recognition task. Collapsed brain ventricles in both Slc4a10-/- mice and affected individuals suggest an important role of SLC4A10 in the production of the CSF. However, it is notable that despite diverse roles of the CSF in the developing and adult brain, the cortex of Slc4a10-/- mice appears grossly intact. Co-staining with synaptic markers revealed that in neurons, SLC4A10 localizes to inhibitory, but not excitatory, presynapses. These findings are supported by our functional studies, which show the release of the inhibitory neurotransmitter GABA is compromised in Slc4a10-/- mice, while the release of the excitatory neurotransmitter glutamate is preserved. Manipulation of intracellular pH partially rescues GABA release. Together our studies define a novel neurodevelopmental disorder associated with biallelic pathogenic variants in SLC4A10 and highlight the importance of further analyses of the consequences of SLC4A10 loss-of-function for brain development, synaptic transmission and network properties.

The Oscillating Stimulus Transporter Assay, OSTA: Quantitative Functional Imaging of Transporter Protein Activity in Time and Frequency Domains.

Transmembrane transporter proteins allow the passage of essentially all biologically important molecules across the lipid membranes of cells and organelles and are therefore of central importance to all forms of life. Current methods of transporter measurement, however, are lacking in several dimensions. Herein, a method is presented in which oscillating stimuli are presented to transporter-expressing cells, and activity is measured through imaging the corresponding oscillating responses of intracellular fluorescent sensors. This approach yields continuous temporal readouts of transporter activity and can therefore be used to measure time-dependent responses to drugs and other stimuli. Because of the periodic nature of the response, temporal Fourier transforms can be used to identify and quantify regions of interest in the xy plane and to overcome noise. This technique, called the Oscillating Stimulus Transporter Assay (OSTA), should greatly facilitate both functional characterization of transporters as well as high-throughput screening of drugs for transporters of particular pathophysiological interest.

Congenital chloride diarrhoea in a Chinese infant with a compound heterozygous SLC26A3 mutation.

INTRODUCTION: Congenital chloride diarrhoea (CCD) is an autosomal recessive condition that causes secretory diarrhoea and potentially deadly electrolyte imbalances in infants because of solute carrier family 26 member 3 (SLC26A3) gene mutations. CASE PRESENTATION: A 7-month-old Chinese infant with a history of maternal polyhydramnios presented with frequent watery diarrhoea, severe dehydration, hypokalaemia, hyponatraemia, failure to thrive, metabolic alkalosis, hyperreninaemia, and hyperaldosteronaemia. Genetic testing revealed a compound heterozygous SLC26A3 gene mutation in this patient (c.269_270dup and c.2006 C > A). Therapy was administered in the form of oral sodium and potassium chloride supplements, which decreased stool frequency. CONCLUSIONS: CCD should be considered when an infant presents with prolonged diarrhoea during infancy, particularly in the context of maternal polyhydramnios and dilated foetal bowel loops.

Chloride/Multiple Anion Exchanger SLC26A Family: Systemic Roles of SLC26A4 in Various Organs.

Solute carrier family 26 member 4 (SLC26A4) is a member of the SLC26A transporter family and is expressed in various tissues, including the airway epithelium, kidney, thyroid, and tumors. It transports various ions, including bicarbonate, chloride, iodine, and oxalate. As a multiple-ion transporter, SLC26A4 is involved in the maintenance of hearing function, renal function, blood pressure, and hormone and pH regulation. In this review, we have summarized the various functions of SLC26A4 in multiple tissues and organs. Moreover, the relationships between SLC26A4 and other channels, such as cystic fibrosis transmembrane conductance regulator, epithelial sodium channel, and sodium chloride cotransporter, are highlighted. Although the modulation of SLC26A4 is critical for recovery from malfunctions of various organs, development of specific inducers or agonists of SLC26A4 remains challenging. This review contributes to providing a better understanding of the role of SLC26A4 and development of therapeutic approaches for the SLC26A4-associated hearing loss and SLC26A4-related dysfunction of various organs.

K+-Driven Cl-/HCO3- Exchange Mediated by Slc4a8 and Slc4a10.

Slc4a genes encode various types of transporters, including Na+-HCO3- cotransporters, Cl-/HCO3- exchangers, or Na+-driven Cl-/HCO3- exchangers. Previous research has revealed that Slc4a9 (Ae4) functions as a Cl-/HCO3- exchanger, which can be driven by either Na+ or K+, prompting investigation into whether other Slc4a members facilitate cation-dependent anion transport. In the present study, we show that either Na+ or K+ drive Cl-/HCO3- exchanger activity in cells overexpressing Slc4a8 or Slc4a10. Further characterization of cation-driven Cl-/HCO3- exchange demonstrated that Slc4a8 and Slc4a10 also mediate Cl- and HCO3--dependent K+ transport. Full-atom molecular dynamics simulation on the recently solved structure of Slc4a8 supports the coordination of K+ at the Na+ binding site in S1. Sequence analysis shows that the critical residues coordinating monovalent cations are conserved among mouse Slc4a8 and Slc4a10 proteins. Together, our results suggest that Slc4a8 and Slc4a10 might transport K+ in the same direction as HCO3- ions in a similar fashion to that described for Na+ transport in the rat Slc4a8 structure.

SLC26A3 (DRA) is stimulated in a synergistic, intracellular Ca2+-dependent manner by cAMP and ATP in intestinal epithelial cells.

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 Ca2+ indicator GCaMP6s, ATP increased intracellular Ca2+ (Ca2+i) in a concentration-dependent manner, whereas FSK (1 µM), which by itself did not significantly alter Ca2+i, followed by 0.25 µM ATP produced a large increase in Ca2+ 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 Ca2+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 (Ca2+) synergistically increased Ca2+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 Ca2+ 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 Ca2+ individually stimulated DRA activity, whereas low concentrations, which had no/minimal effect, synergistically stimulated DRA activity that required a synergistic increase in intracellular Ca2+. This study increases understanding of diarrheal diseases, such as bile salt diarrhea, in which both cAMP and elevated Ca2+ are involved.

The transcription factor Foxi1 promotes expression of V-ATPase and Gpr116 in M-1 cells.

The diverse functions of each nephron segment rely on the coordinated action of specialized cell populations that are uniquely defined by their transcriptional profile. In the collecting duct, there are two critical and distinct cell populations: principal cells and intercalated cells. Principal cells play key roles in the regulation of water, Na+, and K+, whereas intercalated cells are best known for their role in acid-base homeostasis. Currently, there are no in vitro systems that recapitulate the heterogeneity of the collecting ducts, which limits high-throughput and replicate investigations of genetic and physiological phenomena. Here, we demonstrated that the transcription factor Foxi1 is sufficient to alter the transcriptional identity of M-1 cells, a murine cortical collecting duct cell line. Specifically, overexpression of Foxi1 induces the expression of intercalated cell transcripts including Gpr116, Atp6v1b1, Atp6v1g3, Atp6v0d2, Slc4a9, and Slc26a4. These data indicate that overexpression of Foxi1 differentiates M-1 cells toward a non-A, non-B type intercalated cell phenotype and may provide a novel in vitro tool to study transcriptional regulation and physiological function of the renal collecting duct.NEW & NOTEWORTHY Transfection of M-1 cells with the transcription factor Foxi1 generates cells that express V-ATPase and Gpr116 as well as other genes associated with renal intercalated cells. This straightforward and novel in vitro system could be used to study processes including transcriptional regulation and cell specification and differentiation in renal intercalated cells.

Secretin alleviates biliary and liver injury during late-stage primary biliary cholangitis via restoration of secretory processes.

BACKGROUND & AIMS: Primary biliary cholangitis (PBC) is characterised by ductopenia, ductular reaction, impairment of anion exchanger 2 (AE2) and the 'bicarbonate umbrella'. Ductulo-canalicular junction (DCJ) derangement is hypothesised to promote PBC progression. The secretin (Sct)/secretin receptor (SR) axis regulates cystic fibrosis transmembrane receptor (CFTR) and AE2, thus promoting choleresis. We evaluated the role of Sct/SR signalling on biliary secretory processes and subsequent injury in a late-stage PBC mouse model and human samples. METHODS: At 32 weeks of age, female and male wild-type and dominant-negative transforming growth factor beta receptor II (late-stage PBC model) mice were treated with Sct for 1 or 8 weeks. Bulk RNA-sequencing was performed in isolated cholangiocytes from mouse models. RESULTS: Biliary Sct/SR/CFTR/AE2 expression and bile bicarbonate levels were reduced in late-stage PBC mouse models and human samples. Sct treatment decreased bile duct loss, ductular reaction, inflammation, and fibrosis in late-stage PBC models. Sct reduced hepatic bile acid levels, modified bile acid composition, and restored the DCJ and 'bicarbonate umbrella'. RNA-sequencing identified that Sct promoted mature epithelial marker expression, specifically anterior grade protein 2 (Agr2). Late-stage PBC models and human samples exhibited reduced biliary mucin 1 levels, which were enhanced by Sct treatment. CONCLUSION: Loss of Sct/SR signalling in late-stage PBC results in a faulty 'bicarbonate umbrella' and reduced Agr2-mediated mucin production. Sct restores cholangiocyte secretory processes and DCJ formation through enhanced mature cholangiocyte phenotypes and bile duct growth. Sct treatment may be beneficial for individuals with late-stage PBC. IMPACT AND IMPLICATIONS: Secretin (Sct) regulates biliary proliferation and bicarbonate secretion in cholangiocytes via its receptor, SR, and in mouse models and human samples of late-stage primary biliary cholangitis (PBC), the Sct/SR axis is blunted along with loss of the protective 'bicarbonate umbrella'. We found that both short- and long-term Sct treatment ameliorated ductular reaction, immune cell influx, and liver fibrosis in late-stage PBC mouse models. Importantly, Sct treatment promoted bicarbonate and mucin secretion and hepatic bile acid efflux, thus reducing cholestatic and toxic bile acid-associated injury in late-stage PBC mouse models. Our work perpetuates the hypothesis that PBC pathogenesis hinges on secretory defects, and restoration of secretory processes that promote the 'bicarbonate umbrella' may be important for amelioration of PBC-associated damage.

Key Risk Genes Identified From the Postmortem Brain of Patients With Major Depressive Disorder and Their Potential Clinical Applications.

BACKGROUND: Major depressive disorder (MDD) is a type of emotional dysfunction, and its pathogenesis has not been fully elucidated. Specifically, the key molecules in depression-related brain regions involved in this disease and their contributions to this disease are currently unclear. METHODS: GSE53987 and GSE54568 were selected from the Gene Expression Omnibus database. The data were standardized to identify the common differentially expressed genes (DEGs) in the cortex of MDD patients in the 2 datasets. The DEGs were subjected to Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses. The STRING database was used to build protein-protein interaction networks, and the cytoHubba plugin was used to identify hub genes. Furthermore, we selected another blood transcriptome dataset that included 161 MDD and 169 control samples to explore the changes in the screened hub genes. Mice were subjected to 4 weeks of chronic unpredictable mild stress to establish an animal model of depression, and the expression of these hub genes in tissues of the prefrontal cortex was then detected by quantitative real time polymerase chain reaction (qRT-PCR). We subsequently predicted the possible posttranscriptional regulatory networks and traditional Chinese medicine according to the hub genes using a few online databases. RESULTS: The analysis identified 147 upregulated genes and 402 downregulated genes were identified in the cortex of MDD patients compared with that of the controls. Enrichment analyses revealed that DEGs were predominantly enriched in synapse-related cell functions, linoleic acid metabolism, and other pathways. Protein-protein interaction analysis identified 20 hub genes based on the total score. The changes in KDM6B, CUX2, NAAA, PHKB, NFYA, GTF2H1, CRK, CCNG2, ACER3, and SLC4A2 in the peripheral blood of MDD patients were consistent with those in the brain. Furthermore, the prefrontal cortex of mice with depressive-like behaviors showed significantly increased Kdm6b, Aridb1, Scaf11, and Thoc2 expression and decreased Ccng2 expression compared with that of normal mice, which was consistent with the results found for the human brain. Potential therapeutic candidates, such as citron, fructus citri, leaves of Panax Notoginseng, sanchi flower, pseudoginseng, and dan-shen root, were selected via traditional Chinese medicine screening. CONCLUSIONS: This study identified several novel hub genes in specific brain regions involved in the pathogenesis of MDD, which may not only deepen our understanding of depression but may also provide new ideas for its diagnosis and treatment.

The Air-Liquid Interface Reorganizes Membrane Lipids and Enhances the Recruitment of Slc26a3 to Lipid-Rich Domains in Human Colonoid Monolayers.

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.

Diarrhoeal pathogenesis in Salmonella infection may result from an imbalance in intestinal epithelial differentiation through reduced Notch signalling.

Infections with non-typhoidal Salmonella spp. represent the most burdensome foodborne illnesses worldwide, yet despite their prevalence, the mechanism through which Salmonella elicits diarrhoea is not entirely known. Intestinal ion transporters play important roles in fluid and electrolyte homeostasis in the intestine. We have previously shown that infection with Salmonella caused decreased colonic expression of the chloride/bicarbonate exchanger SLC26A3 (down-regulated in adenoma; DRA) in a mouse model. In this study, we focused on the mechanism of DRA downregulation during Salmonella infection, by using murine epithelial enteroid-derived monolayers (EDMs). The decrease in DRA expression caused by infection was recapitulated in EDMs and accompanied by increased expression of Atonal Homolog 1 (ATOH1), the goblet cell marker Muc2 and the enteroendocrine cell marker ChgA. This suggested biased epithelial differentiation towards the secretory, rather than absorptive phenotype. In addition, the downstream Notch effector, Notch intracellular domain (NICD) and Hes1 were decreased following Salmonella infection. The relevance of Notch signalling was further investigated using a γ-secretase inhibitor, which recapitulated the downregulation in Hes1 and DRA as well as upregulation in ATOH1 and Muc2 seen following infection. Our findings suggest that Salmonella infection may result in a shift from absorptive to secretory cell types through Notch inhibition, which explains why there is a decreased capacity for absorption and ultimately the accumulation of diarrhoeal fluid. Our work also shows the value of EDMs as a model to investigate mechanisms that might be targeted for therapy of diarrhoea caused by Salmonella infection. KEY POINTS: Salmonella is a leading foodborne pathogen known to cause high-chloride-content diarrhoea. Salmonella infection of murine enteroid-derived monolayers decreased DRA expression. Salmonella infection resulted in upregulation of the secretory epithelial marker ATOH1, the goblet cell marker Muc2 and the enteroendocrine cell marker ChgA. Downregulation of DRA may result from infection-induced Notch inhibition, as reflected by decreased expression of Notch intracellular domain and Hes1, as well as from decreased HNF1α signalling. The imbalance in intestinal epithelial differentiation favouring secretory over absorptive cell types is a possible mechanism by which Salmonella elicits diarrhoea and may be relevant therapeutically.

A Novel Role of SLC26A3 in the Maintenance of Intestinal Epithelial Barrier Integrity.

BACKGROUND & AIMS: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

The rs12532734 Polymorphism Near the Solute Carrier 26A3 Gene Locus Is Associated With Gallstone Disease in Children.

Gallstones are increasingly frequent in children. In this candidate gene study, we genotyped 5 gene variants ( ANO1 , SPTLC3 , TMEM147 , TNRC6B , rs12532734) from a recent gallstone genome-wide association study (GWAS) in a cohort of 214 children with gallstones and 172 gallstone-free adult controls. In total, 138 genotyped children presented with symptomatic gallstone disease, 47 underwent cholecystectomy, and 126 received ursodeoxycholic acid (UDCA) as therapy for stones. Among 5 tested variants, the rs12532734 polymorphism modulated the gallstone risk in the studied cohort. Its genotype distribution significantly ( P = 0.025) departed from the Hardy-Weinberg equilibrium among cases, and the common allele was associated with increased odds of developing gallstones at young age (OR = 1.69, P = 0.014). SLC26A3 is the nearest gene to rs12532734 and is involved in the transepithelial bicarbonate and chloride transport. The association of rs12532734 with pediatric gallstones is a novel finding warranting further investigations also with regard to biliary bicarbonate flux and bile composition.

SLC4A2 anion exchanger promotes tumour cell malignancy via enhancing net acid efflux across golgi membranes.

Proper functioning of each secretory and endocytic compartment relies on its unique pH micro-environment that is known to be dictated by the rates of V-ATPase-mediated H+ pumping and its leakage back to the cytoplasm via an elusive "H+ leak" pathway. Here, we show that this proton leak across Golgi membranes is mediated by the AE2a (SLC4A2a)-mediated bicarbonate-chloride exchange, as it is strictly dependent on bicarbonate import (in exchange for chloride export) and the expression level of the Golgi-localized AE2a anion exchanger. In the acidic Golgi lumen, imported bicarbonate anions and protons then facilitate a common buffering reaction that yields carbon dioxide and water before their egress back to the cytoplasm via diffusion or water channels. The flattened morphology of the Golgi cisternae helps this process, as their high surface-volume ratio is optimal for water and gas exchange. Interestingly, this net acid efflux pathway is often upregulated in cancers and established cancer cell lines, and responsible for their markedly elevated Golgi resting pH and attenuated glycosylation potential. Accordingly, AE2 knockdown in SW-48 colorectal cancer cells was able to restore these two phenomena, and at the same time, reverse their invasive and anchorage-independent growth phenotype. These findings suggest a possibility to return malignant cells to a benign state by restoring Golgi resting pH.

Mouse organoid culture is a suitable model to study esophageal ion transport mechanisms.

Altered esophageal ion transport mechanisms play a key role in inflammatory and cancerous diseases of the esophagus, but epithelial ion processes have been less studied in the esophagus because of the lack of a suitable experimental model. In this study, we generated three-dimensional (3D) esophageal organoids (EOs) from two different mouse strains and characterized the ion transport processes of the EOs. EOs form a cell-filled structure with a diameter of 250-300 µm and were generated from epithelial stem cells as shown by FACS analysis. Using conventional PCR and immunostaining, the presence of Slc26a6 Cl-/HCO3- anion exchanger (AE), Na+/H+ exchanger (NHE), Na+/HCO3- cotransporter (NBC), cystic fibrosis transmembrane conductance regulator (CFTR), and anoctamin 1 Cl- channels was detected in EOs. Microfluorimetric techniques revealed high NHE, AE, and NBC activities, whereas that of CFTR was relatively low. In addition, inhibition of CFTR led to functional interactions between the major acid-base transporters and CFTR. We conclude that EOs provide a relevant and suitable model system for studying the ion transport mechanisms of esophageal epithelial cells, and they can be also used as preclinical tools to assess the effectiveness of novel therapeutic compounds in esophageal diseases associated with altered ion transport processes.