Intestinal Epithelial Cell Brush Border Membrane Cl:HCO3 Exchanger Regulation by Mast Cells in Chronic Ileitis.

Malabsorption of NaCl is the primary cause of diarrhea in inflammatory bowel disease (IBD). Coupled NaCl absorption occurs via the dual operation of Na:H and Cl:HCO3 exchange in the brush border membrane (BBM) of villus cells. Cl:HCO3 exchange is mediated by BBM transporters DRA (downregulated in adenoma) and PAT1 (putative anion transporter 1) in the mammalian small intestine. DRA/PAT1-mediated Cl:HCO3 exchange was significantly downregulated in the BBM of villus cells in a rabbit model of chronic ileitis, while Na:H exchange was unaffected. The inhibition of Cl:HCO3 exchange was restored in the rabbits when treated with a broad-spectrum immunomodulator, i.e. a glucocorticoid, indicating that the downregulation of DRA/PAT1 is likely to be immune-mediated during chronic enteritis. Mucosal mast cells are one type of key immune cells that are known to proliferate and release immune inflammatory mediators, thus playing a significant role in the pathogenesis of IBD. However, how mast cells may regulate DRA- and PAT1-mediated Cl:HCO3 exchange in a rabbit model of chronic ileitis is unknown. In this study, treatment of rabbits with chronic intestinal inflammation with the mast cell stabilizer ketotifen did not affect the mucosal architecture of the inflamed intestine. However, ketotifen treatment reversed the inhibition of Cl:HCO3 activity in the BBM of villus cells. This restoration of Cl:HCO3 activity to normal levels by ketotifen was found to be secondary to restoring the affinity of the exchangers for its substrate chloride. This observation was consistent with molecular studies, where the mRNA and BBM protein expressions of DRA and PAT1 remained unaffected in the villus cells under all experimental conditions. Thus, this study indicates that mast cells mediated the inhibition of coupled NaCl absorption by inhibiting Cl:HCO3 exchange in a rabbit model of chronic enteritis.

Decreased expression of DRA (SLC26A3) by a p38-driven IL-1α response contributes to diarrheal disease following in vivo challenge with Brachyspira spp.

In this study, we uncovered the novel mechanism of IL-1α-mediated downregulated in adenoma (DRA) (SLC26A3) downregulation in the context of Brachyspira spp.-induced malabsorptive diarrhea. Experimentally infected pigs with Brachyspira spp. had significantly reduced DRA expression in the colon accompanied by IL-1α upregulation. This response was recapitulated in vitro by exposing Caco-2 cells to either Brachyspira lysate or IL-1α. Both p38 and MAPK-activated protein kinase 2 (MAPKAPK-2 also referred as MK-2) showed an increased phosphorylation after exposure to either. SB203580 application, a p38 inhibitor blocked the MK-2 phosphorylation and attenuated the DRA and IL-1α response to both lysate and IL-1α. Exposure to IL-1 receptor antagonist (IL-1RA) produced a similar response. In addition, exposure of cells to either of these blockers without IL-1α or lysate results in increased DRA and decreased IL-1α expression, revealing that DRA needs IL-1α signaling for basal physiological expression. Dual inhibition with both blockers completely inhibited the effect from IL-1α while significantly attenuating the response from Brachyspira lysate, suggesting a minor contribution from another pathway. Together this demonstrates that Brachyspira activates p38 MAPK signaling driving IL-1α expression, which activates IL-1R1 causing DRA downregulation while also driving upregulation of IL-1α through p38 in a positive feedback mechanism. In conclusion, we elucidated a major pathway involved in DRA downregulation and its role in Brachyspira-induced diarrhea. In addition, these observations will aid in our understanding of other inflammatory and infectious diarrhea conditions.NEW & NOTEWORTHY The diarrheal disease caused by the two infectious spirochete spp. B. hyodysenteriae and B. hampsonii reduced the expression of DRA (SLC26A3), a major Cl-/HCO-3 exchanger involved in Cl- absorption. This is attributed to the upregulation of IL-1α driven by p38 MAPK. This work also describes a potential new mechanism in inflammatory diseases while showing the importance of IL-1α in maintaining DRA levels.

DRA involvement in linaclotide-stimulated bicarbonate secretion during loss of CFTR function.

Duodenal bicarbonate secretion is critical to epithelial protection, as well as nutrient digestion and absorption, and is impaired in cystic fibrosis (CF). We examined if linaclotide, typically used to treat constipation, may also stimulate duodenal bicarbonate secretion. Bicarbonate secretion was measured in vivo and in vitro using mouse and human duodenum (biopsies and enteroids). Ion transporter localization was identified with confocal microscopy, and de novo analysis of human duodenal single-cell RNA sequencing (scRNA-Seq) data sets was performed. Linaclotide increased bicarbonate secretion in mouse and human duodenum in the absence of cystic fibrosis transmembrane conductance regulator (CFTR) expression (Cftr-knockout mice) or function (CFTRinh-172). Na+/H+ exchanger 3 inhibition contributed to a portion of this response. Linaclotide-stimulated bicarbonate secretion was eliminated by down-regulated in adenoma (DRA, SLC26A3) inhibition during loss of CFTR activity. ScRNA-Seq identified that 70% of villus cells expressed SLC26A3, but not CFTR, mRNA. Loss of CFTR activity and linaclotide increased apical brush border expression of DRA in non-CF and CF differentiated enteroids. These data provide further insights into the action of linaclotide and how DRA may compensate for loss of CFTR in regulating luminal pH. Linaclotide may be a useful therapy for CF individuals with impaired bicarbonate secretion.

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.

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.

Acid-adapted cancer cells alkalinize their cytoplasm by degrading the acid-loading membrane transporter anion exchanger 2, SLC4A2.

Acidic environments reduce the intracellular pH (pHi) of most cells to levels that are sub-optimal for growth and cellular functions. Yet, cancers maintain an alkaline cytoplasm despite low extracellular pH (pHe). Raised pHi is thought to be beneficial for tumor progression and invasiveness. However, the transport mechanisms underpinning this adaptation have not been studied systematically. Here, we characterize the pHe-pHi relationship in 66 colorectal cancer cell lines and identify the acid-loading anion exchanger 2 (AE2, SLC4A2) as a regulator of resting pHi. Cells adapt to chronic extracellular acidosis by degrading AE2 protein, which raises pHi and reduces acid sensitivity of growth. Acidity inhibits mTOR signaling, which stimulates lysosomal function and AE2 degradation, a process reversed by bafilomycin A1. We identify AE2 degradation as a mechanism for maintaining a conducive pHi in tumors. As an adaptive mechanism, inhibiting lysosomal degradation of AE2 is a potential therapeutic target.

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 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.

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.

Vitamin D receptor involves in the protection of intestinal epithelial barrier function via up-regulating SLC26A3.

BACKGROUND: Vitamin D receptor (VDR) and SLC26A3 (DRA) have been identified as pivotal protective factors in maintaining gut homeostasis in IBD patients. However, the specific mechanism underlying the increased intestinal susceptibility to inflammation induced by the loss of VDR and whether DRA participates in the role of VDR regulating intestinal epithelial barrier function are undefined. AIM: The current study is undertaken to elucidate the regulatory effects of VDR on DRA and VDR prevents intestinal epithelial barrier dysfunction via up-regulating the expression of DRA. METHODS: WT and VDR-/- mice are used as models for intestinal epithelial response. Paracellular permeability is measured by TEER and FD-4 assays. Immunohistochemistry, immunofluorescence, qPCR and immunoblotting are performed to determine the effects of VDR and DRA on gut epithelial barrier function. RESULTS: VDR-/- mice exhibits significant hyperpermeability of intestine with greatly decreased levels of ZO-1 and Claudin1 proteins. DRA is located on the intestinal epithelial apical membrane and is tightly modulated by VDR in vivo and in vitro via activating ERK1/2 MAPK signaling pathway. Notably, the current study for the first time demonstrates that VDR maintains intestinal epithelial barrier integrity via up-regulating DRA expression and the lack of DRA induced by VDR knockdown leads to a more susceptive condition for intestine to DSS-induced colitis. CONCLUSION: Our study provides evidence and deep comprehension regarding the role of VDR in modulating DRA expression in gut homeostasis and makes novel contributions to better generally understanding the links between VDR, DRA and intestinal epithelial barrier function.

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.

PRDX6: A protein bridging S-palmitoylation and diabetic neuropathy.

Diabetic neuropathy is regarded as one of the most debilitating outcomes of diabetes. It can affect both the peripheral and central nervous systems, leading to pain, decreased motility, cognitive decline, and dementia. S-palmitoylation is a reversible posttranslational lipid modification, and its dysregulation has been implicated in metabolic syndrome, cancers, neurological disorders, and infections. However, the role of S-palmitoylation in diabetic neuropathy remains unclear. Here we demonstrate a potential association between activating protein palmitoylation and diabetic neuropathy. We compared the proteomic data of lumbar dorsal root ganglia (DRG) of diabetes mice and palmitoylome profiling data of the HUVEC cell line. The mapping results identified peroxiredoxin-6 (PRDX6) as a novel target in diabetic neuropathy, whose biological mechanism was associated with S-palmitoylation. Bioinformatic prediction revealed that PRDX6 had two palmitoylation sites, Cys47 and Cys91. Immunofluorescence results indicated PRDX6 translocating between the cytoplasm and cell membrane. Protein function analysis proposed that increased palmitoylation could competitively inhibit the formation of disulfide-bond between Cys47 and Cys91 and change the spatial topology of PRDX6 protein. Cl-HCO3- anion exchanger 3 (AE3) was one of the AE family members, which was proved to express in DRG. AE3 activity evoked Cl- influx in neurons which was generally associated with increased excitability and susceptibility to pain. We demonstrated that the S-palmitoylation status of Cys47 could affect the interaction between PRDX6 and the C-terminal domain of AE3, thereby regulating the activity of AE3 anion exchanger enzyme in the nervous system. The results highlight a central role for PRDX6 palmitoylation in protection against diabetic neuropathy.

Population-based meta-analysis and gene-set enrichment identifies FXR/RXR pathway as common to fatty liver disease and serum lipids.

Nonalcoholic fatty liver disease (NAFLD) is prevalent worldwide. NAFLD is associated with elevated serum triglycerides (TG), low-density lipoprotein cholesterol (LDL), and reduced high-density lipoprotein cholesterol (HDL). Both NAFLD and blood lipid levels are genetically influenced and may share a common genetic etiology. We used genome-wide association studies (GWAS)-ranked genes and gene-set enrichment analysis to identify pathways that affect serum lipids and NAFLD. We identified credible genes in these pathways and characterized missense variants in these for effects on serum traits. We used MAGENTA to identify 58 enriched pathways from publicly available TG, LDL, and HDL GWAS (n = 99,000). Three of these pathways were also enriched for associations with European-ancestry NAFLD GWAS (n = 7176). One pathway, farnesoid X receptor (FXR)/retinoid X receptor (RXR) activation, was replicated for association in an African-ancestry NAFLD GWAS (n = 3214) and plays a role in serum lipids and NAFLD. Credible genes (proteins) in FXR/RXR activation include those associated with cholesterol/bile/bilirubin transport/absorption (ABCC2 (MRP2) [ATP binding cassette subfamily C member (multidrug resistance-associated protein 2)], ABCG5, ABCG8 [ATP-binding cassette (ABC) transporters G5 and G8], APOB (APOB) [apolipoprotein B], FABP6 (ILBP) [fatty acid binding protein 6 (ileal lipid-binding protein)], MTTP (MTP) [microsomal triglyceride transfer protein], SLC4A2 (AE2) [solute carrier family 4 member 2 (anion exchange protein 2)]), nuclear hormone-mediated control of metabolism (NR0B2 (SHP) [nuclear receptor subfamily 0 group B member 2 (small heterodimer partner)], NR1H4 (FXR) [nuclear receptor subfamily 1 group H member 4 (FXR)], PPARA (PPAR) [peroxisome proliferator activated receptor alpha], FOXO1 (FOXO1A) [forkhead box O1]), or other pathways (FETUB (FETUB) [fetuin B]). Missense variants in ABCC2 (MRP2), ABCG5 (ABCG5), ABCG8 (ABCG8), APOB (APOB), MTTP (MTP), NR0B2 (SHP), NR1H4 (FXR), and PPARA (PPAR) that associate with serum LDL levels also associate with serum liver function tests in UK Biobank. Conclusion: Genetic variants in NR1H4 (FXR) that protect against liver steatosis increase serum LDL cholesterol while variants in other members of the family have congruent effects on these traits. Human genetic pathway enrichment analysis can help guide therapeutic development by identifying effective targets for NAFLD/serum lipid manipulation while minimizing side effects. In addition, missense variants could be used in companion diagnostics to determine their influence on drug effectiveness.

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.

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.

Rescue of chloride and bicarbonate transport by elexacaftor-ivacaftor-tezacaftor in organoid-derived CF intestinal and cholangiocyte monolayers.

BACKGROUND: In cystic fibrosis (CF), loss of CF transmembrane conductance regulator (CFTR)-dependent bicarbonate secretion precipitates the accumulation of viscous mucus in the lumen of respiratory and gastrointestinal epithelial tissues. We investigated whether the combination of elexacaftor (ELX), ivacaftor (IVA) and tezacaftor (TEZ), apart from its well-documented effect on chloride transport, also restores Phe508del-CFTR-mediated bicarbonate transport. METHODS: Epithelial monolayers were cultured from intestinal and biliary (cholangiocyte) organoids of homozygous Phe508del-CFTR patients and controls. Transcriptome sequencing was performed, and bicarbonate and chloride transport were assessed in the presence or absence of ELX/IVA/TEZ, using the intestinal current measurement technique. RESULTS: ELX/IVA/TEZ markedly enhanced bicarbonate and chloride transport across intestinal epithelium. In biliary epithelium, it failed to enhance CFTR-mediated bicarbonate transport but effectively rescued CFTR-mediated chloride transport, known to be requisite for bicarbonate secretion through the chloride-bicarbonate exchanger AE2 (SLC4A2), which was highly expressed by cholangiocytes. Biliary but not intestinal epithelial cells expressed an alternative anion channel, anoctamin-1/TMEM16A (ANO1), and secreted bicarbonate and chloride upon purinergic receptor stimulation. CONCLUSIONS: ELX/IVA/TEZ has the potential to restore both chloride and bicarbonate secretion across CF intestinal and biliary epithelia and may counter luminal hyper-acidification in these tissues.

Alterations in SLC4A2, SLC26A7 and SLC26A9 Drive Acid-Base Imbalance in Gastric Neuroendocrine Tumors and Uncover a Novel Mechanism for a Co-Occurring Polyautoimmune Scenario.

Autoimmune polyendocrine syndrome (APS) is assumed to involve an immune system malfunction and entails several autoimmune diseases co-occurring in different tissues of the same patient; however, they are orphans of its accurate diagnosis, as its genetic basis and pathogenic mechanism are not understood. Our previous studies uncovered alterations in the ATPase H+/K+ Transporting Subunit Alpha (ATP4A) proton pump that triggered an internal cell acid-base imbalance, offering an autoimmune scenario for atrophic gastritis and gastric neuroendocrine tumors with secondary autoimmune pathologies. Here, we propose the genetic exploration of APS involving gastric disease to understand the underlying pathogenic mechanism of the polyautoimmune scenario. The whole exome sequencing (WES) study of five autoimmune thyrogastric families uncovered different pathogenic variants in SLC4A2, SLC26A7 and SLC26A9, which cotransport together with ATP4A. Exploratory in vitro studies suggested that the uncovered genes were involved in a pathogenic mechanism based on the alteration of the acid-base balance. Thus, we built a custom gene panel with 12 genes based on the suggested mechanism to evaluate a new series of 69 APS patients. In total, 64 filtered putatively damaging variants in the 12 genes of the panel were found in 54.17% of the studied patients and none of the healthy controls. Our studies reveal a constellation of solute carriers that co-express in the tissues affected with different autoimmune diseases, proposing a unique genetic origin for co-occurring pathologies. These results settle a new-fangled genetics-based mechanism for polyautoimmunity that explains not only gastric disease, but also thyrogastric pathology and disease co-occurrence in APS that are different from clinical incidental findings. This opens a new window leading to the prediction and diagnosis of co-occurring autoimmune diseases and clinical management of patients.

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.

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.