Bicarbonate Secretion Research Articles

Solute carrier family 4 member 4 (SLC4A4) is associated with cell proliferation, migration and immune cell infiltration in colon cancer

BackgroundSolute Carrier Family 4 Member 4 (SLC4A4) is a membrane protein‐coding gene for a Na+/HCO3− cotransporter and plays a crucial role in regulating pH, bicarbonate secretion and homeostasis. However, the prognostic and immunological role of SLC4A4 in colon cancer remains unknown.MethodIn this study, expression profiles of SLC4A4 were retrieved from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases, to which a variety of bioinformatic analyses were performed. Sangerbox, Xiantao, ESTIMATE and TIMER online tools were used to delve into the relationship between SLC4A4 expression and immune cell infiltration. The role of SLC4A4 in the proliferation and migration of colon cancer cells was verified by CCK8, EdU and wound healing assays. The related molecules and pathways that SLC4A4 may affect were validated by bioinformatic prediction and western blotting analysis.ResultsThe expression levels of SLC4A4 were significantly lower in colon cancer tissues than in normal tissues and its low expression was positively correlated with poor prognosis. TIMER and ESTIMATE showed that SLC4A4 broadly influenced immune cell infiltration. Experiments in vitro demonstrated that SLC4A4 inhibited partial epithelial-mesenchymal transition (EMT) phenotypes.ConclusionsTo conclude, our study revealed that SLC4A4 is lowly expressed in colon cancer tissues, and SLC4A4 may inhibit the progression of colon cancer via regulating partial EMT phenotypes and immune cell infiltration, which may provide new perspectives for the development of more precise and personalized immune anti-tumor therapies.

Bile Acids-Based Therapies for Primary Sclerosing Cholangitis: Current Landscape and Future Developments.

Primary sclerosing cholangitis (PSC) is a rare, chronic liver disease with no approved therapies. The ursodeoxycholic acid (UDCA) has been widely used, although there is no evidence that the use of UDCA delays the time to liver transplant or increases survival. Several candidate drugs are currently being developed. The largest group of these new agents is represented by FXR agonists, including obeticholic acid, cilofexor, and tropifexor. Other agents that target bile acid metabolism are ASTB/IBAP inhibitors and fibroblasts growth factor (FGF)19 analogues. Cholangiocytes, the epithelial bile duct cells, play a role in PSC development. Recent studies have revealed that these cells undergo a downregulation of GPBAR1 (TGR5), a bile acid receptor involved in bicarbonate secretion and immune regulation. Additional agents under evaluation are PPARs (elafibranor and seladelpar), anti-itching agents such as MAS-related G-protein-coupled receptors antagonists, and anti-fibrotic and immunosuppressive agents. Drugs targeting gut bacteria and bile acid pathways are also under investigation, given the strong link between PSC and gut microbiota.

Low prevalence of peptic ulcer disease in hospitalized patients with cystic fibrosis: A national database study

BackgroundAlthough cystic fibrosis (CF) is widely considered a lung disease, the prevalence of CF-specific gastrointestinal symptoms and diseases has continued to rise. Peptic ulcer disease (PUD) has not been well-studied among people with CF (PwCF) and may be a common cause of abdominal symptoms. In PwCF, impaired bicarbonate secretion and unbuffered gastric acid production have been attributed to the development of ulcers, although ulcers remain uncommon. The objective of this study was to evaluate the prevalence of PUD in PwCF and assess for possible contributing factors. MethodsThis study utilized the National Inpatient Sample (NIS) database. All patients 18 years or older with CF were identified from 2014 to 2019. Relevant patient characteristics and procedures were identified using ICD-9 and ICD-10 codes. Linear trend, bivariate analyses, and multiple regression analysis were performed. The outcomes of interest were peptic ulcer disease, pancreatic insufficiency, and nonalcoholic steatohepatitis or NASH. All analyses accounted for complex sampling scheme of the NIS. ResultsThe total prevalence of PwCF in the National Inpatient Sample (NIS) database was 0.08 %, and the number was stable year to year from 2014 to 2019. Hispanic patients were more likely to be diagnosed with PUD than other white (aOR 1.802 [1.311,2.476]). Multiple regression analysis indicated that PUD in PwCF was strongly associated with a diagnosis of NASH (aOR 2.421[1.197, 4.898]). PUD patients were less likely to have pancreatic insufficiency compared to the non-PUD group (aOR 0.583 [0.455, 0.745]). ConclusionAlthough cystic fibrosis has been historically known as a disease of childhood, advancements in therapy have led to prolonged life expectancy and higher prevalence for cystic fibrosis-related digestive diseases. This study revealed a low prevalence of PUD in PwCF. Hispanics and those with NASH are more likely to develop peptic ulcers.

THE INTERRELATION BETWEEN ESOPHAGITIS AND HYDROGEN SULFIDE: A LITERATURE REVIEW

Esophagitis is characterized by irritation and inflammation of the esophagus, caused by several factors, such as gastroesophageal reflux, radiation, infections, medications and eosinophilic esophagitis. Its symptoms include chest pain, odynophagia, dysphagia, nausea, vomiting, throat clearing, heartburn, dry cough and manifestations related to gastroesophageal reflux. And its pathophysiology involves lesions in the esophageal mucosa due to the abnormal and frequent increase in gastric acid. With regard to hydrogen sulfide (H2S), it is a toxic gaseous compound with an odor similar to rotten eggs, it performs essential physiological functions, stabilizing mucus, preventing injuries and strengthening gastrointestinal defense mechanisms by stimulating the secretion of bicarbonate in the duodenum. . The purpose of this article is to analyze the relationship between hydrogen sulfide and esophagitis. Therefore, this integrative review covered original articles in Portuguese and English, published from 2003 to 2022, in the VHL, SciELO and PUBMED databases. Using descriptors such as “esophagitis,” “gastrointestinal,” and “hydrogen sulfide,” the research followed strict inclusion criteria to provide a comprehensive analysis of the relationship between esophagitis, gastrointestinal health, and hydrogen sulfide. As for the results and discussions, they highlight the relationship between H2S and esophagitis, exploring its antioxidant and anti-inflammatory function by protecting cells from oxidative stress by increasing glutathione (GSH) and contributing to quenching reactive oxygen species and promoting the resolution of inflammation. mainly how it interferes with the production of pro-inflammatory cytokines, such as IL-1, IL-2, IL-8 and TNFα, in addition to maintaining or increasing the production of IL-10. Through the results obtained, it can be concluded that esophagitis has an intimate relationship with the modulation of hydrogen sulfide levels in the human body, particularly in the context of the H2S/CSE/HIF-2α axis and that they should be better studied in order to promote the development of H2S-based drugs to treat inflammatory processes, including esophagitis, which have the potential to significantly improve the well-being and quality of life of the population.

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.

Heavy metals in cigarette smoke strongly inhibit pancreatic ductal function and promote development of chronic pancreatitis.

Smoking is recognised as an independent risk factor in the development of chronic pancreatitis (CP). Cystic fibrosis transmembrane conductance regulator (CFTR) function and ductal fluid and bicarbonate secretion are also known to be impaired in CP, so it is crucial to understand the relationships between smoking, pancreatic ductal function and the development of CP. We measured sweat chloride (Cl-) concentrations in patients with and without CP, both smokers and non-smokers, to assess CFTR activity. Serum heavy metal levels and tissue cadmium concentrations were determined by mass spectrometry in smoking and non-smoking patients. Guinea pigs were exposed to cigarette smoke, and cigarette smoke extract (CSE) was prepared to characterise its effects on pancreatic HCO3 - and fluid secretion and CFTR function. We administered cerulein to both the smoking and non-smoking groups of mice to induce pancreatitis. Sweat samples from smokers, both with and without CP, exhibited elevated Cl- concentrations compared to those from non-smokers, indicating a decrease in CFTR activity due to smoking. Pancreatic tissues from smokers, regardless of CP status, displayed lower CFTR expression than those from non-smokers. Serum levels of cadmium and mercury, as well as pancreatic tissue cadmium, were increased in smokers. Smoking, CSE, cadmium, mercury and nicotine all hindered fluid and HCO3 - secretion and CFTR activity in pancreatic ductal cells. These effects were mediated by sustained increases in intracellular calcium ([Ca2+]i), depletion of intracellular ATP (ATPi) and mitochondrial membrane depolarisation. Smoking impairs pancreatic ductal function and contributes to the development of CP. Heavy metals, notably cadmium, play a significant role in the harmful effects of smoking. Smoking and cigarette smoke extract diminish pancreatic ductal fluid and HCO3 - secretion as well as the expression and function of CFTR Cd and Hg concentrations are significantly higher in the serum samples of smokers Cd accumulates in the pancreatic tissue of smokers.

Selective Inhibition of Hepatocyte Claudin-3 Ameliorates Fibrotic and Cholestatic Liver Damage by Enhancing Bicarbonate Secretion and Bile Dynamics.

Abstract Background Cholestatic-fibrotic liver diseases have a high unmet medical need. Tight junctions, which maintain the blood-biliary-barrier in the liver, have important biological functions that impact cholestatic diseases. A main component of the hepatic tight junction is claudin-3. Aims In this study, we explored the function of claudin-3 and demonstrate its potential as a new drug target in cholestatic-fibrotic liver diseases. Methods Adult C57BL/6 N background Cldn3+/+ or Cldn3-/- mice were subjected to extra- and intrahepatic cholestasis via surgical ligation of the common bile duct (BDL) or chemically, by gavage or feeding alpha-naphthyl isothiocyanate (ANIT). Hepatic necrosis and serum liver injury markers were determined. Bile flow and bicarbonate levels were quantified. Bile acid levels were measured by LC-MS/MS in serum and liver tissue. Claudin-3 was knocked down using liver specific claudin-3 GalNAc-siRNAs. Results Claudin-3 is expressed in normal- and cholestatic liver tissues. Cldn3-/- mice have a higher bile-flow rate, more bicarbonate secretion, and the bile is less concentrated with bile acids and bilirubin. Following bile duct ligation or ANIT, Cldn3-/- mice showed macroscopically less signs of bile acid retention and a different bile color. Hepatic bile acid levels were significantly lower in normal- or cholestasis challenged Cldn3-/- mice. Following BDL, ANIT gavage- or ANIT feeding, liver injury in was remarkably ameliorated in Cldn3-/- mice. Liver specific knockdown of claudin-3 using GalNAc-siRNAs ameliorated cholestasis at a lower success rate compared to the complete knockout. Conclusion Knockout or GalNAc-siRNA mediated inhibition of claudin-3 protected the liver from injury in models of extra- or intrahepatic cholestasis. Further pre-clinical and clinical studies need to be conducted to show whether claudin-3 inhibition is an efficient and safe therapeutic approach for the treatment of cholestatic liver diseases. As next step towards translation of our results, we are currently screening and optimizing human GalNAc conjugated claudin-3 inhibitors.

A Direct Link Implicating Loss of SLC26A6 to Gut Microbial Dysbiosis, Compromised Barrier Integrity, and Inflammation

BackgroundPutative 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 diseases (IBD) 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. MethodsAge-matched PKO and wild-type (WT) littermates were administered 3.5%-DSS in drinking water for 6 days. Ileum and colon of control and treated mice were harvested. mRNA and protein expression of tight junction (TJ) proteins were determined by RT-PCR and western blotting. Severity of inflammation was assessed by measuring diarrheal phenotype, cytokine expression and H&E staining. Gut microbiome and associated metabolome were analyzed by 16S rRNA sequencing and mass spectrometry, respectively. ResultsPKO mice exhibited significantly higher loss of body weight, gut permeability, colonic inflammation, and diarrhea in response to DSS treatment. Additionally, PKO mice showed microbial dysbiosis and significantly reduced levels of butyrate and butyrate-producing microbes compared to controls. Cohousing WT and PKO mice for 4 weeks resulted in PKO-like signatures on the expression of TJ proteins in the colon of WT mice. ConclusionOur 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 IBD.

The digestive Enzyme-Based nasal dysbiosis in Empty nose Syndrome: A theory

Empty nose syndrome (ENS) is described as an empty space in the region of the inferior and middle turbinates of patients who underwent inferior and middle turbinectomy. Patients commonly complained of dry nose, paradoxical nasal obstruction, and crust. Symptoms significantly worsen the quality of life of patients. The pathophysiology of ENS is poorly understood. Several abnormalities in the nasal airflow dynamics, air humidification and warming, mucociliary clearance, and trigeminal-related sensory function were reported but it is misunderstood why some patients with anatomical turbinate defects developed ENS, while others do not experience symptoms with similar anatomy. This paper proposes a theory associating the development of ENS with the presence of laryngopharyngeal reflux disease (LPRD). LPRD is associated with the deposit of gastroduodenal enzymes into the upper aerodigestive tract mucosa, which lead to a decrease of the defense mechanisms of mucosa (mucus hydration, bicarbonate secretion, growth factor production) and an increase of mucosa injuries. In ENS patients, the deposit of digestive enzymes into the nasal mucosa may lead to concurrent injuries of the nasal cells involved in air humidification, warming, or sensory function, and modifications of the nasal microbiome that cannot heal the injured mucosa.

SLC26A6 Deficiency Differentially Alters the Expression of MicroRNAs and RNA Binding Proteins in the Intestine

BACKGROUND: Putative anion transporter 1 (PAT1, SLC26A6), expressed at the luminal membrane of the intestinal epithelial cells, plays a key role in oxalate homeostasis and bicarbonate secretion. Additionally, we have recently shown that PAT1 knockout mice (PKO) exhibit compromised barrier integrity, gut dysbiosis and enhanced susceptibility to colitis. As defective PAT1 function and/or expression alters intestinal vulnerability to inflammation and miRNAs and RNA binding proteins are implicated in pathophysiology of gut inflammation, the focus of the current study was to investigate if loss of PAT1 modulates the expression of these post transcriptional regulators. Methods: microRNA was extracted from the colonic mucosa of age-matched PKO and wild type (WT) littermates (Qiagen miRNeasy mini kit) and subjected to miRNA expression profiling by Nanostring nCounter Mouse miRNA panel. Acquired raw data was further analyzed using the nSolver Analysis Software and ROSALIND online software platform to identify the differential gene expression. RT-PCR and western blotting was used to analyze the mRNA and protein expression of RNA binding proteins. Results: Genetic deficiency of PAT-1 resulted in differential expression of miRNAs compared to WT mice, where 60 miRNAs were notably upregulated, and 5 miRNAs were significantly downregulated in the colon. miR-150, known to be downregulated in ulcerative colitis patients, showed the maximum decrease (~6.9-fold change, P< 0.02) in PKO mice. Of note, miR-767 (known to promote tumor progression) was the top upregulated miRNA (~5.2-fold change, p<0.01). Interestingly, in silico analysis using TargetScan revealed that most of the upregulated miRNAs have been associated with the decrease in gut barrier function as previously shown in PKO mice. Further, a remarkable decrease in the protein expression of RNA binding proteins, HUR (0.82 ± 0.07 WT vs 0.49 + 0.03 PKO, P< 0.5) and CUGBP-1(0.37 ± 0.08 WT vs 0. 0.12 ± 0.03 PKO, P< 0.5) was also noted in PKO compared to WT mice. CONCLUSION: Our data provide evidence that PAT-1 deficiency in mice results in differential expression of several posttranscriptional regulators including miRNAs and RNA binding proteins with gene targets implicated in maintenance of epithelial barrier integrity. We speculate that altered expression of posttranscriptional regulators may potentially contribute to altered gut homeostasis observed in diseases associated with PAT1 deficiency. Supported by VA/NIH. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Secretin Inhibits Small Intestinal Motility via Interstitial Cells of Cajal and cAMP Mechanisms

Background: Secretin is a member of the secretin-glucagon-vasoactive intestinal peptide hormone superfamily and is a multifunctional gastrointestinal- (GI) and neuro- peptide hormone. Secretin is primarily secreted postprandially from the crypts of Lieberkühn of duodenal enteroendocrine S cells into circulation where it reaches secretin receptor targets in the central nervous system and periphery. This dynamic hormone has been shown to act as a key signaling molecule in the regulation of digestion, metabolism and energy expenditure, water retention, reproduction, thermogenesis in adipose tissue, and in gastric and intestinal motility. Secretin’s canonical role in the GI tract is to stimulate the secretion of bicarbonate and bile from pancreatic ducts and bile ducts to neutralize acidic chyme exiting the stomach. Secretin has also been shown to slow intestinal motility, but its targets and mechanism of action is poorly understood. Aims: Several studies have proposed that secretin acts to slow intestinal motility in the intestines primarily through the many secretin receptors present on vagal afferents in the GI tract, here we discuss new data that suggests an alternate and complementary signaling pathway via interstitial cells of Cajal (ICC). ICC act as a liaison to facilitate a reduction in force of GI smooth muscle contraction through the activation of the secretin receptor (SCTR) and subsequent stimulation of the second messenger, cyclic adenosine monophosphate (cAMP). Here we provide evidence to show how ICC mediates changes in myogenic activity and motility in the small intestine. Methods: Spinning-disk confocal microscopy was used to monitor Ca2+ signaling in ICC from small intestinal muscles of GCaMP6f x KitiCre mice. Additionally, cAMP levels were evaluated using CAMPER mice. Intestinal muscle contractility was assessed using muscle strip myography experiments. Results: Secretin reduced small intestinal force of contraction in the presence of tetrodotoxin (TTX) and dampened the effect of cholinergic transmission. The secretin receptor (SCTR) is expressed primarily on ICC, specifically ICC within the deep muscular plexus (ICC-DMP) in the small intestine and Ca2+ imaging confirmed the effects are primarily localized within ICC-DMP. Secretin reduced carbachol-induced contractions and Ca2+ transients in ICC-DMP in response to electrical field stimulation (EFS) in the presence of LNNA (NO synthase inhibitor) and MRS2500 (P2Y1 antagonist). Secretin caused an increase in cAMP levels in ICC-DMP in muscles from Kit-iCre-CAMPER mice. PKA inhibitors rescued some of the effects of secretin on ICC-DMP Ca2+ signaling. Measurements of diameter change in large, intact, intestinal segments (4-5 cm) showed a significant decrease in response to Secretin. Conclusions: Secretin can inhibit small intestinal motility through the activation SCTRs on ICC-DMP via cAMP-mediated mechanisms. These results show how novel secretin targets on ICC influence GI muscles and reduce propulsive and segmental motility to facilitate nutrient absorption and digestion after a meal. Funding: This project was supported by R01 DK-120759 from the National Institute of Diabetes and digestive and Kidney (NIDDK). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Comparative assessment of anti-ulcer effects: Hydro-ethanolic extract from air-dried ray and disc florets vs essential oil of traditional Tagetes erecta (Asteraceae) in Swiss Albino rats

The Tagetes erecta Linn (family: Asteraceae) herb possesses medicinal and antioxidants value. The herb is traditionally used to treat various ailments, including ulcers. In order to investigate antiulcer activity of T. erecta herb, experimental study was performed between ray and disc florets extracted with 30 parts of water and 70 parts of ethanol solvents using Soxhlet apparatus and leaf's essential oil extracted in Clevenger apparatus by steam distillation method. Two doses of the hydroethanolic extract (400 and 800 mg/kg, Per os) and single dose of essential oil (10 ml/Kg, Per os) were administered for five consecutive days. Rat's pyloric ligation and aspirin (200 mg/kg, Per os) were used to induce acute and chronic gastric ulcers respectively. Antiulcer activity of both the herb's parts was compared on gastric juice parameters (pH, free acidity, total acidity) ulcer-index and ulcer percent protection. The p value of less than 0.05 was considered significant in statistical analysis. Elevation in pH, reduction in free acidity, total acidity and ulcer-index in both the ulcer models confirm that the test drug showed the ulcer-protective activity in rats. Dose-dependent effects were observed in the antiulcer activity of the hydroethanolic extract. However, the leaf's essential oil showed 94 % and 96.93 % ulcer protection in the pyloric ligation and aspirin-induced gastric ulcer models respectively. The hydroethanolic extract and the essential oil may be capable in part, regulating gastric acid secretion pathways or stimulating the cytoprotective secretion of bicarbonate, mucus and prostaglandins. Histological studies supported the observed antiulcer activity of the herb. The essential oil has more potent and significant anti-ulcer activity than hydro-ethanolic extract and respective standard drugs: ranitidine (50 mg/kg, Per os) & pantoprazole (20 mg/kg, Per os). The herb exhibits antiulcer activity, which could be due to the presence of lead phytochemicals: Quercetin-3-methyl ether, quercetin-7-methyl-ether & kaempferol flavonoids in the extract and limonene & terpinolene in leaf's essential oil and, thus, confirmed the traditional uses of T. erecta in the treatment of ulcers. The herb may cure acid pepsin disorders.

Gastroprotective Effect of Quercus infectoria Olivier Galls on Ethanol-Induced Gastritis in Rats.

One of the common inflammatory disorders that substantially affects the stomach and its mucosa is gastritis. It can be induced by non-steroidal anti-inflammatory drugs (NSAIDs), antibiotics, alcohol, Helicobacter pylori infection, and stress. These factors affect cellular regeneration, mucus production, and bicarbonate secretion, resulting finally in inflammation and ulceration. Ethanol-induced gastritis is one of the commonly used models for studying the pathology of gastritis and investigating the effect of drugs in managing the disease. Several drugs, such as proton pump inhibitors (PPIs), are available to control and correct the pathological signs of gastritis; however, the side effects of such drugs represent an obstacle to their applications in many cases. Quercus infectoria(QI) Olivier galls are formed as a pathological response to wasp insults to the tree. They are rich in several bioactive molecules, e.g., gallotannins that have been shown to be effective in several inflammatory conditions due to their antioxidant and anti-inflammatory potentials. In this study, we aimed to evaluate the therapeutic potential of QI gall extract (QIGE) in treating ethanol-induced gastritis in rats. To test this, 20 adult male Swiss rats were divided into four groups: healthy control, ethanol-treated (80% in water, 5 ml/kg, per oral gavage), ethanol + omeprazole (20 mg/kg, per oral gavage), and ethanol + QIGE (300 mg/kg, per oral gavage). QIGE was administered for seven days before ethanol administration, which took place three hours after the last QIGE dose. Three hours after ethanol intake, animals were euthanized, gastric content was collected, and stomach tissue was examined for macroscopic changes and then fixed to be further utilized for histological assessment by hematoxylin and eosin (H&E), periodic acid-Schiff (PAS), and Masson's trichrome staining. Ethanol treatment significantly decreased gastric pH and increased gastric acidity compared to healthy control. It also induced clear morphological and histological damage and ulceration, depleted mucus on the gastric epithelium, and induced edema and collagen deposition in gastric submucosa. The QIGE treatment ameliorated the changes in gastric pH and total acidity. It also protected stomach tissue from ethanol-induced ulceration, histopathological changes, edema, and collagen deposition. The protective effects of QIGE were comparable to those of omeprazole. In conclusion, QI gall extract possesses a promising gastroprotective effect against ethanol-induced gastritis.

CFTR function is impaired in a subset of patients with pancreatitis carrying rare CFTR variants

BackgroundMany affected by pancreatitis harbor rare variants of the cystic fibrosis (CF) gene, CFTR, which encodes an epithelial chloride/bicarbonate channel. We investigated CFTR function and the effect of CFTR modulator drugs in pancreatitis patients carrying CFTR variants. MethodsNext-generation sequencing was performed to identify CFTR variants. Sweat tests and nasal potential difference (NPD) assays were performed to assess CFTR function in vivo. Intestinal current measurement (ICM) was performed on rectal biopsies. Patient-derived intestinal epithelial monolayers were used to evaluate chloride and bicarbonate transport and the effects of a CFTR modulator combination: elexacaftor, tezacaftor and ivacaftor (ETI). ResultsOf 32 pancreatitis patients carrying CFTR variants, three had CF-causing mutations on both alleles and yielded CF-typical sweat test, NPD and ICM results. Fourteen subjects showed a more modest elevation in sweat chloride levels, including three that were provisionally diagnosed with CF. ICM indicated impaired CFTR function in nine out of 17 non-CF subjects tested. This group of nine included five carrying a wild type CFTR allele. In epithelial monolayers, a reduction in CFTR-dependent chloride transport was found in six out of 14 subjects tested, whereas bicarbonate secretion was reduced in only one individual. In epithelial monolayers of four of these six subjects, ETI improved CFTR function. ConclusionsCFTR function is impaired in a subset of pancreatitis patients carrying CFTR variants. Mutations outside the CFTR locus may contribute to the anion transport defect. Bioassays on patient-derived intestinal tissue and organoids can be used to detect such defects and to assess the effect of CFTR modulators.

Pro-inflammatory cytokines stimulate CFTR-dependent anion secretion in pancreatic ductal epithelium.

Loss of CFTR-dependent anion and fluid secretion in the ducts of the exocrine pancreas is thought to contribute to the development of pancreatitis, but little is known about the impact of inflammation on ductal CFTR function. Here we used adult stem cell-derived cell cultures (organoids) obtained from porcine pancreas to evaluate the effects of pro-inflammatory cytokines on CFTR function. Organoids were cultured from porcine pancreas and used to prepare ductal epithelial monolayers. Monolayers were characterized by immunocytochemistry. Epithelial bicarbonate and chloride secretion, and the effect of IL-1β, IL-6, IFN-γ, and TNF-α on CFTR function was assessed by electrophysiology. Immunolocalization of ductal markers, including CFTR, keratin 7, and zonula occludens 1, demonstrated that organoid-derived cells formed a highly polarized epithelium. Stimulation by secretin or VIP triggered CFTR-dependent anion secretion across epithelial monolayers, whereas purinergic receptor stimulation by UTP, elicited CFTR-independent anion secretion. Most of the anion secretory response was attributable to bicarbonate transport. The combination of IL-1β, IL-6, IFN-γ, and TNF-α markedly enhanced CFTR expression and anion secretion across ductal epithelial monolayers, whereas these cytokines had little effect when tested separately. Although TNF-α triggered apoptotic signaling, epithelial barrier function was not significantly affected by cytokine exposure. Pro-inflammatory cytokines enhance CFTR-dependent anion secretion across pancreatic ductal epithelium. We propose that up-regulation of CFTR in the early stages of the inflammatory response, may serve to promote the removal of pathogenic stimuli from the ductal tree, and limit tissue injury.

Laminin 511-E8, an autoantigen in IgG4-related cholangitis, contributes to cholangiocyte protection

IgG4-related cholangitis (IRC) is the hepatobiliary manifestation of IgG4-related disease. Anti-laminin 511-E8 autoantibodies have been identified in its pancreatic manifestation. Laminin 511-E8 promotes endothelial barrier function, lymphocyte recruitment, and cholangiocyte differentiation. Here, we investigate anti-laminin 511-E8 autoantibody presence in IRC, and mechanisms via which laminin 511 may contribute to cholangiocyte protection. Anti-laminin 511-E8 serum autoantibody positivity was assessed by ELISA. RNA sequencing and RT-qPCR were performed on human H69 cholangiocytes treated with recombinant laminin 511-E8. H69 cholangiocytes were subjected to shRNA knockdown targeting genes encoding laminin 511 (LAMA5, LAMB1, LAMC1) or treated with recombinant laminin 511-E8. Cholangiocellular bile acid influx was quantified radiochemically using 22,23-3H-glycochenodeoxycholic acid (GCDC). GCDC-induced apoptosis was determined by Caspase-3/7 assays. Cholangiocellular barrier function was assessed by FITC-Dextran permeability assays. Immunofluorescent staining of laminin 511 and claudin 1 was performed on extrahepatic bile duct tissue of control and anti-laminin 511-E8 positive individuals with IRC. Seven out of 52 individuals with IRC had autoantibodies against laminin 511-E8. Recombinant laminin 511-E8 led to differential expression of genes involved in secretion, barrier function, and inflammation. Knockdown of laminin 511 constituents increased toxic bile acid permeation and GCDC-induced apoptosis. Laminin 511-E8 treatment decreased toxic bile acid permeation and dose-dependently alleviated GCDC-induced apoptosis. LAMA5 and LAMC1 knockdown increased transepithelial permeability. Laminin 511-E8 treatment reduced transepithelial permeability and prevented T lymphocyte-induced barrier dysfunction. Laminin 511 and claudin 1 staining patterns appeared altered in anti-laminin 511-E8 positive individuals with IRC. Laminin 511-E8 is an autoantigen in subsets of individuals with IRC. Laminin 511 enhances cholangiocellular barrier function and protects cholangiocytes against T lymphocyte-induced barrier dysfunction, toxic bile acid permeation and bile acid-induced apoptosis. A subset of patients with IgG4-related cholangitis (IRC) has autoantibodies against laminin 511-E8. In human cholangiocytes, laminin 511 protects against (T lymphocyte-induced) epithelial barrier dysfunction and hydrophobic bile acids. Laminin 511 and claudin 1 staining may be altered in extrahepatic bile ducts of patients with IRC who are anti-laminin 511-E8 positive. This makes it tempting to speculate that a decreased epithelial barrier function with attraction of immune cells and impaired bicarbonate secretion as a result of dysfunction of laminin 511 by autoantibody binding could potentially be a common systemic pathogenic mechanism in a subset of patients with IgG4-RD.

A Comparative Kidney Transcriptome Analysis of Bicarbonate-Loaded insrr-Null Mice.

The maintenance of plasma pH is critical for life in all organisms. The kidney plays a critical role in acid-base regulation in vertebrates by controlling the plasma concentration of bicarbonate. The receptor tyrosine kinase IRR (insulin receptor-related receptor) is expressed in renal β-intercalated cells and is involved in alkali sensing due to its ability to autophosphorylate under alkalization of extracellular medium (pH > 7.9). In mice with a knockout of the insrr gene, which encodes for IRR, urinary bicarbonate secretion in response to alkali loading is impaired. The specific regulatory mechanisms in the kidney that are under the control of IRR remain unknown. To address this issue, we analyzed and compared the kidney transcriptomes of wild-type and insrr knockout mice under basal or bicarbonate-loaded conditions. Transcriptomic analyses revealed a differential regulation of a number of genes in the kidney. Using TaqMan real-time PCR, we confirmed different expressions of the slc26a4, rps7, slc5a2, aqp6, plcd1, gapdh, rny3, kcnk5, slc6a6 and atp6v1g3 genes in IRR knockout mice. Also, we found that the expression of the kcnk5 gene is increased in wild-type mice after bicarbonate loading but not in knockout mice. Gene set enrichment analysis between the IRR knockout and wild-type samples identified that insrr knockout causes alterations in expression of genes related mostly to the ATP metabolic and electron transport chain processes.

B-067 Performance Assessment of a Newly Developed Lateral Flow Assay for the Quantification of Faecal Pancreatic Elastase

Abstract Background Pancreatic exocrine insufficiency (PEI) can be defined as an insufficient secretion of pancreatic enzymes and bicarbonate to maintain a normal digestion (1). PEI is associated to fat malabsorption, weight loss and malnutrition-related complications. Due to its important role in the digestive process, as a proteolytic enzyme and the high stability of the enzyme during intestinal transit, fecal pancreatic elastase (fPELA) is an ideal biomarker for the assessment of pancreatic function in patients suffering from PEI. The aim of this work was to develop and optimize a new rapid test for the quantification of fPELA. Methods A quantitative lateral flow assay was designed for the selective measurement of elastase antigen concentrations from stool extracts. The BÜHLMANN Quantum Blue® fPELA is a sandwich immunoassay. A monoclonal detection antibody is conjugated to gold nanoparticles. Elastase present in the sample binds to this gold conjugate. A polyclonal capture antibody, highly specific for elastase, is immobilized on the analytical membrane and captures the complex of elastase bound to the anti-elastase gold conjugate, resulting in a coloring of the test line. The remaining free anti-elastase gold conjugate binds to the control line. The signal intensities of the test line and the control line are measured quantitatively by the Quantum Blue® Reader. Pancreatic elastase is quantified in extracts of human stool samples collected with the BÜHLMANN CALEX® Cap device. Several analytical studies, such as sensitivity, linearity, High Dose Hook Effect (HDHE), were performed during optimization phase to assess the performance of the assay. Moreover, a method comparison with the commercially available high throughput turbidimetric BÜHLMANN fPELA® turbo assay was also performed. Results Both analytical and clinical performance were evaluated. Preliminary results of the assay performance were obtained during the optimization phase. Analytical sensitivity was determined as 12.8 µg/g LoB, 16.6 µg/g LoD and 17.2 µg/g LoQ. The linearity evaluation revealed a linear range of 10 to 472 µg/g. No HDHE was observed in native samples up to 4000 µg/g. Method comparison of the new lateral flow assay with BÜHLMANN fPELA® turbo assay, with 80 clinical samples, revealed good analytical agreement with a slope of 1.04 (Passing-Bablok) and a bias at cutoff (200 µg/g) of 5.6%. Relative bias (Bland-Altman) of the two assays was 6.9%. Considering the turbidimetric assay as the reference method, the sensitivity and specificity were 92% and 83%, respectively. Conclusion The newly developed quantitative lateral flow assay, BÜHLMANN Quantum Blue® fPELA, is an accurate, non-invasive and rapid test to determine fPELA levels in fecal extracts. The lateral flow assay is an attractive alternative to ELISA with a turn-around time of only 15 minutes enabling patients’ management in a more patient near clinical environment.

ATP12A Proton Pump as an Emerging Therapeutic Target in Cystic Fibrosis and Other Respiratory Diseases.

ATP12A encodes the catalytic subunit of the non-gastric proton pump, which is expressed in many epithelial tissues and mediates the secretion of protons in exchange for potassium ions. In the airways, ATP12A-dependent proton secretion contributes to complex mechanisms regulating the composition and properties of the fluid and mucus lining the respiratory epithelia, which are essential to maintain the airway host defense and the respiratory health. Increased expression and activity of ATP12A in combination with the loss of other balancing activities, such as the bicarbonate secretion mediated by CFTR, leads to excessive acidification of the airway surface liquid and mucus dysfunction, processes that play relevant roles in the pathogenesis of cystic fibrosis and other chronic inflammatory respiratory disorders. In this review, we summarize the findings dealing with ATP12A expression, function, and modulation in the airways, which led to the consideration of ATP12A as a potential therapeutic target for the treatment of cystic fibrosis and other airway diseases; we also highlight the current advances and gaps regarding the development of therapeutic strategies aimed at ATP12A inhibition.

The Impact of CFTR Modulating Therapy on Chronic Lung Infection in Patients with Cystic Fibrosis

Cystic fibrosis is the most common lethal genetic disease in the white population, affecting approximately 80 000 people worldwide. It is an autosomal recessive, monogenic, and multisystemic disease, with over 2000 mutations described in the CFTR protein gene. The dysfunction of this protein leads to a decrease in the secretion of chlorine and bicarbonate, sodium hyperabsorption, and consequent water absorption, resulting in the thickening of secretions and accumulation of pathogens. These changes culminate in inflammation, chronic pulmonary infection, and recurrent exacerbations, with lung disease being the main cause of morbidity and mortality. In the early stages of the disease, Staphylococcus aureus is generally the agent responsible for chronic infection. Over time, Pseudomonas aeruginosa becomes more prevalent, being the most frequent bacteria in adults. However, in up to 70% of patients, colonization is polymicrobial, with frequent isolation of S. aureus and P. aeruginosa, associated with Haemophilus influenzae or Streptococcus pneumoniae, as well as isolation of other bacterial agents, viruses, or fungi. In recent years, drugs modulating CFTR have been developed which have shown a positive effect on lung function, body mass index, exacerbation rate, chlorine concentration, and quality of life. Currently, four drugs are approved that act by improving the function or increasing the amount of protein produced and consequently the ion transport. [...].