Loss of luminal carbonic anhydrase XIV results in decreased biliary bicarbonate output, liver fibrosis, and cholangiocyte proliferation in mice.

Carbonic anhydrase XIV (Car14) is highly expressed in the hepatocyte, with predominance in the canalicular membrane and its active site in the extracellular milieu. The aim of this study is to determine the physiological relevance of Car14 for biliary fluid and acid/base output, as well as its role in the maintenance of hepatocellular and cholangiocyte integrity. The common bile duct of anesthetized car14-/- and car14+/+ mice was cannulated and hepatic HCO3- output was measured by microtitration and bile flow gravimetrically before and during stimulation with intravenously applied tauroursodeoxycholic acid (TUDCA). Morphological alterations and hepatic damage were assessed histologically and immunohistochemically in liver tissue from 3- to 52-week-old car14-/- and car14+/+ mice, and gene and/or protein expression was measured for pro-inflammatory cytokines, fibrosis, and cholangiocyte markers. Biliary basal and more so TUDCA-stimulated HCO3- output were significantly reduced in car14-/- mice of all age groups, whereas bile flow and hepatic and ductular morphology were normal at young age. Car14-/- mice developed fibrotic and proliferative changes in the small bile ducts at advanced age, which was accompanied by a reduction in bile flow, and an upregulation of hepatic cytokeratin 19 mRNA and protein expression. Membrane-bound Car14 is essential for biliary HCO3- output, and its loss results in gradual development of small bile duct disease and hepatic fibrosis. Bile flow is not compromised in young adulthood, suggesting that Car14-deficient mice may be a model to study the protective role of biliary canalicular HCO3- against luminal noxi to the cholangiocyte.

The NHE3 Inhibitor Tenapanor Prevents Intestinal Obstructions in CFTR-Deleted Mice.

Mutations in the CFTR chloride channel result in intestinal obstructive episodes in cystic fibrosis (CF) patients and in CF animal models. In this study, we explored the possibility of reducing the frequency of obstructive episodes in cftr-/- mice through the oral application of a gut-selective NHE3 inhibitor tenapanor and searched for the underlying mechanisms involved. Sex- and age-matched cftr+/+ and cftr-/- mice were orally gavaged twice daily with 30 mg kg-1 tenapanor or vehicle for a period of 21 days. Body weight and stool water content was assessed daily and gastrointestinal transit time (GTT) once weekly. The mice were sacrificed when an intestinal obstruction was suspected or after 21 days, and stool and tissues were collected for further analysis. Twenty-one day tenapanor application resulted in a significant increase in stool water content and stool alkalinity and a significant decrease in GTT in cftr+/+ and cftr-/- mice. Tenapanor significantly reduced obstructive episodes to 8% compared to 46% in vehicle-treated cftr-/- mice and prevented mucosal inflammation. A decrease in cryptal hyperproliferation, mucus accumulation, and mucosal mast cell number was also observed in tenapanor- compared to vehicle-treated, unobstructed cftr-/- mice. Overall, oral tenapanor application prevented obstructive episodes in CFTR-deficient mice and was safe in cftr+/+ and cftr-/- mice. These results suggest that tenapanor may be a safe and affordable adjunctive therapy in cystic fibrosis patients to alleviate constipation and prevent recurrent DIOS.

Cleistanthus collinus poisoning affects mitochondrial respiration and induces oxidative stress in the rat kidney.

Cleistanthus collinus is a poisonous shrub used for deliberate self-harm in rural areas of South India and intake of boiled decoction of leaves is a common method of self-harm. Distal renal tubular acidosis (dRTA) is an important clinical symptom observed in C. collinus poisoning, and renal V-ATPases may be potential targets of damage. However, a lack of understanding of molecular mediators involved hampers medical management, which is mainly supportive. We hypothesized that C. collinus poisoning induces renal oxidative stress; probably by inducing mitochondrial uncoupling, which compromises V-ATPase activity to ultimately produce dRTA. This was tested by exposing renal BBMV, kidney cells in culture, and Wistar rats to C. collinus poisoning. Exposure to C. collinus aqueous extract resulted in significant elevations in the lipid peroxidation marker, conjugated dienes, in cell culture and in vivo. A significant decrease in mitochondrial respiratory control ratio was observed in kidneys from C. collinus-treated animals suggesting that mitochondrial oxidative phosphorylation is uncoupled. This was accompanied by significant increase in ADP levels and a decrease in proton pump activity. Thus, these results demonstrate that C. collinus poisoning induces oxidative stress which influences proton pump activity, probably due to feedback inhibition by elevated ADP levels because of mitochondrial dysfunction in the rat kidney.

Slc26a3 deficiency is associated with epididymis dysplasia and impaired sperm fertilization potential in the mouse.

Members of the solute carrier 26 (SLC26) family have emerged as important players in mediating anions fluxes across the plasma membrane of epithelial cells, in cooperation with the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. Among them, SLC26A3 acts as a chloride/bicarbonate exchanger, highly expressed in the gastrointestinal, pancreatic and renal tissues. In humans, mutations in the SLC26A3 gene were shown to induce congenital chloride-losing diarrhea (CLD), a rare autosomal recessive disorder characterized by life-long secretory diarrhea. In view of some reports indicating subfertility in some male CLD patients together with SLC26-A3 and -A6 expression in the male genital tract and sperm cells, we analyzed the male reproductive parameters and functions of SLC26A3 deficient mice, which were previously reported to display CLD gastro-intestinal features. We show that in contrast to Slc26a6, deletion of Slc26a3 is associated with severe lesions and abnormal cytoarchitecture of the epididymis, together with sperm quantitative, morphological and functional defects, which altogether compromised male fertility. Overall, our work provides new insight into the pathophysiological mechanisms that may alter the reproductive functions and lead to male subfertility in CLD patients, with a phenotype reminiscent of that induced by CFTR deficiency in the male genital tract.

Coexistence of aberrant hematopoietic and stromal elements in myelodysplastic syndromes.

Myelodysplastic syndromes (MDS) are a group of clonal hematopoietic disorders related to hematopoietic stem and progenitor cell dysfunction. Several studies have shown the role of the bone marrow microenvironment in regulating hematopoietic stem, and progenitor function and their individual abnormalities have been associated with disease pathogenesis. In this study, we simultaneously evaluated hematopoietic stem cells (HSC), hematopoietic stem progenitor cells (HSPCs) and different stromal elements in a cohort of patients with MDS-refractory cytopenia with multilineage dysplasia (RCMD). Karyotyping of these patients revealed variable chromosomal abnormalities in 73.33% of patients. Long-term HSC and lineage-negative CD34+CD38- cells were reduced while among the HPCs, there was an expansion of common myeloid progenitor and loss of granulocyte-monocyte progenitors. Interestingly, loss of HSCs was accompanied by aberrant frequencies of endothelial (ECs) (CD31+CD45-CD71-) and mesenchymal stem cells (MSCs) (CD31-CD45-71-) and its subsets associated with HSC niche. We further demonstrate down-regulation of HSC maintenance genes such as Cxcl12, VEGF in mesenchymal cells and a parallel upregulation in endothelial cells. Altogether we report for the first time quantitative and qualitative de novo changes in hematopoietic stem and its associated niche in a cohort of MDS-RCMD patients. These findings further reinforce the role of different components of the bone marrow microenvironment in MDS pathogenesis and emphasize the need for comprehensive simultaneous evaluation of all niche elements in such studies.

Enteral glutamine differentially regulates Nrf 2 along the villus-crypt axis of the intestine to enhance glutathione levels.

BACKGROUND AND AIM: Glutamine is an important energy source for the intestinal epithelium, and its supplementation protects intestinal epithelial cells by induction of glutathione. However, mechanisms of glutathione induction in cells at various stages of differentiation along the crypt to villus axis are not well understood. This study examined induction of glutathione in response to glutamine along the intestinal villus-crypt axis and evaluated regulatory mediators involved in the process. METHODS: Animals were administered 4% glutamine in feed for 7 days, following which enterocytes at various stages of differentiation were isolated and glutathione levels and signaling mediators involved in its regulation were studied. RESULTS: In control animals, glutathione levels were higher in the intestinal crypt than in the villus or middle region. This was accompanied by elevated expression of the modifier subunit of glutathione synthetase (GCLM) and the transcription factor Nrf2 when compared with cells from the villus and middle regions. These levels were further enhanced by glutamine throughout the intestine, although the effects were more dramatic in the crypt. In parallel to glutathione induction, glutamine supplementation also altered actin dynamics and proliferation in cells of the crypt. CONCLUSIONS: These results suggest that the variation of glutathione levels along the villus-crypt axis in the intestine is due to gradients in expression of mediators such as glutamate cysteine ligase modifier subunit and Nrf2. The protective effects of glutamine supplementation seem to be most pronounced in the crypt, where it upregulates proliferation, glutathione levels and alters actin dynamics.

An empirical examination of customer advocacy influenced by engagement behaviour and predispositions of FinTech customers in India.

Background: The extensive adoption and usage of emerging technologies furthered by the global coronavirus disease 2019 (COVID-19) pandemic, has reduced direct face to face communications. New FinTech (financial technology) apps and technologies are flooding the Indian digital payments market and competitors are striving hard to attract and retain their customers. Especially when customer engagement behaviours (CEBs) are digital in nature, it is essential to gauge the intrinsically driven customer motivations which drive a positive CEB. The objective of this paper was to empirically test the influence of customer-based antecedents such as emotions, moral identity, self-concept, communal focus, perceived cost and perceived benefits on non-transactional experiential customer engagement behaviours (CEBs) and CEB's effect on customer advocacy in the FinTech industry. Methods: Data from 380 financial app users in south India were gathered by administering a survey that captured customer predispositions, CEBs, and customer advocacy. Structural equation modelling (SEM) using smart PLS (partial least squares) 3.0 was applied to test the theoretical model. Results: Results indicate that CEB fully mediates the relationship between self-concept and customer advocacy. The positive CEBs get formed through customer predispositions leading to referral/advocacy behaviours. Conclusions: This paper provides directions for FinTech practitioners, marketers, technologists, and academicians to devise marketing strategies customized to customer needs and factors. This is one of the first research studies to demonstrate and empirically validate the CEB model for the FinTech industry during the COVID-19 pandemic.

Upregulation of antimicrobial peptide expression in slc26a3-/- mice with colonic dysbiosis and barrier defect.

Genetic defects in SLC26A3 (DRA), an intestinal Cl-/HCO3- exchanger, result in congenital chloride diarrhea (CLD), marked by lifelong acidic diarrhea and a high risk of inflammatory bowel disease. Slc26a3-/- mice serve as a model to understand the pathophysiology of CLD and search for treatment options. This study investigates the microbiota changes in slc26a3-/- colon, the genotype-related causes for the observed microbiota alterations, its inflammatory potential, as well as the corresponding host responses. The luminal and the mucosa-adherent cecal and colonic microbiota of cohoused slc26a3-/- and wt littermates were analyzed by 16S rRNA gene sequencing. Fecal microbiota transfer from cohoused slc26a3-/- and wt littermates to germ-free wt mice was performed to analyze the stability and the inflammatory potential of the communities.The cecal and colonic luminal and mucosa-adherent microbiota of slc26a3-/- mice was abnormal from an early age, with a loss of diversity, of short-chain fatty acid producers, and an increase of pathobionts. The transfer of slc26a3-/- microbiota did not result in intestinal inflammation and the microbial diversity in the recipient mice normalized over time. A strong increase in the expression of Il22, Reg3ß/γ, Relmß, and other proteins with antimicrobial functions was observed in slc26a3-/- colon from juvenile age, while the mucosal and systemic inflammatory signature was surprisingly mild. The dysbiotic microbiota, low mucosal pH, and mucus barrier defect in slc26a3-/- colon are accompanied by a stark upregulation of the expression of a panel of antimicrobial proteins. This may explain the low inflammatory burden in the gut of these mice.

Slc26a3 deletion alters pH-microclimate, mucin biosynthesis, microbiome composition and increases the TNFα expression in murine colon.

AIM: SLC26A3 (DRA) mediates the absorption of luminal Cl- in exchange for HCO3- in the distal intestine. Its expression is lost in congenital chloride diarrhoea (CLD) and strongly decreased in the presence of intestinal inflammation. To characterize the consequences of a loss of Slc26a3 beyond disturbed electrolyte transport, colonic mucus synthesis, surface accumulation and composition, pH microclimate, microbiome composition and development of inflammation was studied in slc26a3-/- mice. METHODS: The epithelial surface pH microclimate and the surface mucus accumulation in vivo was assessed by two photon microscopy in exteriorized mid colon of anaesthetized slc26a3-/- and wt littermates. Mucus synthesis, composition and inflammatory markers were studied by qPCR and immunohistochemistry and microbiome composition by 16S rRNA sequencing. RESULTS: Colonic pH microclimate was significantly more acidic in slc26a3-/- and to a lesser extent in cftr-/- than in wt mice. Goblet cell thecae per crypt were decreased in slc26a3-/- and increased in cftr-/- colon. Mucus accumulation in vivo was reduced, but much less so than in cftr-/- colon, which is possibly related to the different colonic fluid balance. Slc26a3-/- colonic luminal microbiome displayed strong decrease in diversity. These alterations preceded and maybe causally related to increased mucosal TNFα mRNA expression levels and leucocyte infiltration in the mid-distal colon of slc26a3-/- but not of cftr-/- mice. CONCLUSIONS: These findings may explain the strong increase in the susceptibility of slc26a3-/- mice to DSS damage, and offer insight into the mechanisms leading to an increased incidence of intestinal inflammation in CLD patients.

Aberrant Niche Signaling in the Etiopathogenesis of Ulcerative Colitis.

BACKGROUND: Primary colonic epithelial defects leading to inflammatory responses are considered central to the development of ulcerative colitis (UC). However, a systematic analysis of various colonic subcompartments in the pathogenesis of UC before inflammation remains elusive. Here, we explored changes in colonic subcompartments and their associated niche signals in patient mucosal biopsies and in an animal model of colitis. METHODS: Analysis of mucosal biopsies obtained from uninvolved and involved regions of patients with UC and Crohn's disease was performed and compared with normal subjects. Temporal analysis of colonic subcompartments was performed in mice administered with 5% dextran sodium sulphate. Phenotypic enumeration of the crypt subcompartment was complemented with flow cytometric analysis. Members of Notch and Wnt signaling pathways were analyzed by molecular, biochemical, and colocalization studies. RESULTS: Phenotypic enumeration of colonocytes' subcompartments from patients revealed significant alterations of the lower crypt, enriched in stem cell and progenitors, independent of inflammation. These changes, unique to UC, were confirmed by immunohistochemistry and molecular analysis. In parallel, a defect in proliferation and Muc2 synthesis was observed. Animal data before inflammation recapitulated human studies. Mechanistic studies revealed that changes in signaling through Wnt primarily affected colonic stem cells, whereas Notch affected progenitor function. CONCLUSIONS: Our results thus provide new insights into the development of inflammation and relapse in UC and suggest that the stem cell niche in the colon may influence pathogenesis of the disease.

Acute liver injury induced by low dose dimethylnitrosamine alters mediators of hepatic vascular flow.

Alterations in liver vascular tone play an important role in chronic liver disease. The hepatic stellate cell (HSC) and mediators such as nitric oxide (NO) and hydrogen sulfide (H2S) have been implicated in regulation of vascular tone and intra-hepatic pressure. Though these have been studied in chronic liver damage, changes in response to acute liver injury induced by hepatotoxins such as dimethyl nitrosamine are not well understood. Liver injury was induced in mice by a single intra-peritoneal injection of dimethylnitrosamine (DMN), following which animals were sacrificed at 24, 48 and 72 h. Changes in vascular mediators such as NO and H2S as well as stellate cell activation was then examined. It was found that a single low dose of DMN in mice is sufficient to induce activation of hepatic stellate cells within 24 h, accompanied by oxidative stress, compromised metabolism of H2S and decreased levels of the von Willebrand factor (vWF) cleaving protease; a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13), which functions in intravascular thrombosis. A suppression of hepatic NO levels is also initiated at this time point, which progresses further and is sustained up to 72 h, at which point the HSC activation is still present. Compromised levels of ADAMTS13 and H2S metabolism however, begin to recover by 48 h and are almost similar to control by 72 h. In conclusion, these data suggest that even moderate acute insults in the liver can have far reaching consequences on a number of mediators of vascular flow in the liver.