Primary biliary cholangitis: pathogenic mechanisms.

PURPOSE OF REVIEW: Primary biliary cholangitis (PBC) is characterized by autoimmune damage of intrahepatic bile ducts associated with a loss of tolerance to mitochondrial antigens. PBC etiopathogenesis is intriguing because of different perplexing features, namely: a) although mitochondria are present in all cell types and tissues, the damage is mainly restricted to biliary epithelial cells (BECs); b) despite being an autoimmune disorder, it does not respond to immunosuppressive drugs but rather to ursodeoxycholic acid, a bile salt that induces HCO3- rich choleresis; c) the overwhelming female preponderance of the disease remains unexplained. Here we present an etiopathogenic view of PBC which sheds light on these puzzling facts of the disease. RECENT FINDINGS: PBC develops in patients with genetic predisposition to autoimmunity in whom epigenetic mechanisms silence the Cl-/HCO3- exchanger AE2 in both cholangiocytes and lymphoid cells. Defective AE2 function can produce BECs damage as a result of decreased biliary HCO3- secretion with disruption of the protective alkaline umbrella that normally prevents the penetration of toxic apolar bile salts into cholangiocytes. AE2 dysfunction also causes increased intracellular pH (pHi) in cholangiocytes, leading to the activation of soluble adenylyl cyclase, which sensitizes BECs to bile salt-induced apoptosis. Recently, mitophagy was found to be inhibited by cytosolic alkalization and stimulated by acidification. Accordingly, we propose that AE2 deficiency may disturb mitophagy in BECs, thus, promoting the accumulation of defective mitochondria, oxidative stress and presentation of mitochondrial antigens to the immune cells. As women possess a more acidic endolysosomal milieu than men, mitophagy might be more affected in women in an AE2-defective background. Apart from affecting BECs function, AE2 downregulation in lymphocytes may also contribute to alter immunoregulation facilitating autoreactive T-cell responses. SUMMARY: PBC can be considered as a disorder of Cl-/HCO3- exchange in individuals with genetic predisposition to autoimmunity.

Evaluation of the Integrated Tuberculosis Research Program Sponsored by the Spanish Society of Pulmonology and Thoracic Surgery: 11 Years on. / Evaluación del Programa Integrado de Investigación en Tuberculosis promovido por la sociedad española de Neumología y Cirugía Torácica tras 11 años de funcionamiento.

OBJECTIVE: The objective of the study was to determine the trend of variables related to tuberculosis (TB) from the Integrated Tuberculosis Research Program (PII-TB) registry of the Spanish Society of Pulmonology and Thoracic Surgery (SEPAR), and to evaluate the PII-TB according to indicators related to its scientific objectives. METHOD: Cross-sectional, population-based, multicenter study of new TB cases prospectively registered in the PII-TB between 2006 and 2016. The time trend of quantitative variables was calculated using a lineal regression model, and qualitative variables using the χy test for lineal trend. RESULTS: A total of 6,892 cases with an annual median of 531 were analyzed. Overall, a significant downward trend was observed in women, immigrants, prisoners, and patients initially treated with 3 drugs. Significant upward trends were observed in patients aged 40-50 and > 50 years, first visit conducted by a specialist, hospitalization, diagnostic delay, disseminated disease and single extrapulmonary location, culture(+), sensitivity testing performed, drug resistance, directly observed treatment, prolonged treatment, and death from another cause. The scientific objectives of the PII-TB that showed a significant upward trend were publications, which reached a maximum of 8 in 2016 with a total impact factor of 49,664, numbers of projects initiated annually, presentations at conferences, and theses. CONCLUSIONS: PII-TB provides relevant information on TB and its associated factors in Spain. A large team of researchers has been created; some scientific aspects of the registry were positive, while others could have been improved.

Targeting the anion exchanger 2 with specific peptides as a new therapeutic approach in B lymphoid neoplasms.

Regulatory T (Treg) cells can weaken antitumor immune responses, and inhibition of their function appears to be a promising therapeutic approach in cancer patients. Mice with targeted deletion of the gene encoding the Cl-/HCO3- anion exchanger AE2 (also termed SLC4A2), a membrane-bound carrier involved in intracellular pH regulation, showed a progressive decrease in the number of Treg cells. We therefore challenged AE2 as a potential target for tumor therapy, and generated linear peptides designed to bind the third extracellular loop of AE2, which is crucial for its exchange activity. Peptide p17AE2 exhibited optimal interaction ability and indeed promoted apoptosis in mouse and human Treg cells, while activating effector T-cell function. Interestingly, this linear peptide also induced apoptosis in different types of human leukemia, lymphoma and multiple myeloma cell lines and primary malignant samples, while it showed only moderate effects on normal B lymphocytes. Finally, a macrocyclic AE2 targeting peptide exhibiting increased stability in vivo was effective in mice xenografted with B-cell lymphoma. These data suggest that targeting the anion exchanger AE2 with specific peptides may represent an effective therapeutic approach in B-cell malignancies.

Composition of mineralizing incisor enamel in cystic fibrosis transmembrane conductance regulator-deficient mice.

Formation of crystals in the enamel space releases protons that need to be buffered to sustain mineral accretion. We hypothesized that apical cystic fibrosis transmembrane conductance regulator (CFTR) in maturation ameloblasts transduces chloride into forming enamel as a critical step to secrete bicarbonates. We tested this by determining the calcium, chloride, and fluoride levels in developing enamel of Cftr-null mice by quantitative electron probe microanalysis. Maturation-stage enamel from Cftr-null mice contained less chloride and calcium than did wild-type enamel, was more acidic when stained with pH dyes ex vivo, and formed no fluorescent modulation bands after in vivo injection of the mice with calcein. To acidify the enamel further we exposed Cftr-null mice to fluoride in drinking water to stimulate proton release during formation of hypermineralized lines. In Cftr-deficient mice, fluoride further lowered enamel calcium without further reducing chloride levels. The data support the view that apical CFTR in maturation ameloblasts tranduces chloride into developing enamel as part of the machinery to buffer protons released during mineral accretion.

Functional crosstalk between the adenosine transporter CNT3 and purinergic receptors in the biliary epithelia.

BACKGROUND & AIMS: Both hepatocytes and cholangiocytes release ATP into the bile, where it acts as a potent autocrine/paracrine stimulus that activates biliary secretory mechanisms. ATP is known to be metabolized into multiple breakdown products, ultimately yielding adenosine. However, the elements implicated in the adenosine-dependent purinergic regulation of cholangiocytes are not known. METHODS: Normal rat cholangiocytes (NRCs) were used to study the expression of adenosine receptors and transporters and their functional interactions at the apical and basolateral membrane domains of polarized cholangiocytes. RESULTS: We found that: (1) cholangiocytes exclusively express two concentrative nucleoside transporters (CNT) known to be efficient adenosine carriers: CNT3, located at the apical membrane, and CNT2, located at apical and basolateral membrane domains; (2) in both domains, NRCs also express the high affinity adenosine receptor A2A, which modulated the activity of apical CNT3 in a domain-specific manner; (3) the regulation exerted by A2A on CNT3 was dependent upon the cAMP/PKA/ERK/CREB axis, intracellular trafficking mechanisms and AMPK phosphorylation; (4) secretin increased the activity of the apically-located CNT3, and promoted additional basolateral CNT3-related activity; and (5) extracellular ATP (a precursor of adenosine) was able to exert an inhibitory effect on the apical activity of both CNT3 and CNT2. CONCLUSIONS: This study uncovered the functional expression of nucleoside transporters in cholangiocytes and provides evidence for direct crosstalks between adenosine transporters and receptors for adenosine and its natural extracellular precursor, ATP. Our data anticipate the possibility of adenosine playing a major role in the physiopathology of the biliary epithelia.

Identification of fibroblast growth factor 15 as a novel mediator of liver regeneration and its application in the prevention of post-resection liver failure in mice.

OBJECTIVE: Cholestasis is associated with increased liver injury and morbidity after partial hepatectomy (PH), yet bile acids (BAs) are emerging as important mediators of liver regeneration. Fibroblast growth factor 15 (Fgf15, human FGF19) is a BA-induced ileum-derived enterokine that governs BA metabolism. We evaluated the relevance of Fgf15 in the preservation of BA homeostasis after PH and its potential role in the regenerative process. DESIGN: Liver regeneration after PH was studied in Fgf15 (-/-) and Fgf15 (+/+) mice. The effects of the BA sequestrant cholestyramine and adenovirally delivered Fgf15 were examined in this model. The role of Fgf15 in BA-induced liver growth was tested in Fgf15 (-/-) mice upon cholic acid (CA) feeding. The direct mitogenic effect of Fgf15 was evaluated in cultured mouse hepatocytes and cholangiocytes. RESULTS: Fgf15 (-/-) mice showed marked liver injury and mortality after PH accompanied by persistently elevated intrahepatic BA levels. Cholestyramine feeding and adenovirally delivered Fgf15 reduced BA levels and significantly prevented this lethal outcome. Fgf15 also reduced mortality after extensive hepatectomy in Fgf15(+/+) animals. Liver growth elicited by CA feeding was significantly diminished in Fgf15 (-/-) mice. Proliferation of hepatocytes and cholangiocytes was also noticeably reduced in CA-fed Fgf15 (-/-) mice. Fgf15 induced intracellular signalling and proliferation of cultured hepatocytes and cholangiocytes. CONCLUSIONS: Fgf15 is necessary to maintain BA homeostasis and prevent liver injury during liver regeneration. Moreover, Fgf15 is an essential mediator of the liver growth-promoting effects of BA. Preoperative administration of this enterokine to patients undergoing liver resection might be useful to reduce damage and foster regeneration.

Up-regulation of microRNA 506 leads to decreased Cl-/HCO3- anion exchanger 2 expression in biliary epithelium of patients with primary biliary cirrhosis.

UNLABELLED: Cl(-) /HCO3- anion exchanger 2 (AE2) participates in intracellular pH homeostasis and secretin-stimulated biliary bicarbonate secretion. AE2/SLC4A2 gene expression is reduced in liver and blood mononuclear cells from patients with primary biliary cirrhosis (PBC). Our previous findings of hepatic and immunological features mimicking PBC in Ae2-deficient mice strongly suggest that decreased AE2 expression might be involved in the pathogenesis of PBC. Here, we tested the potential role of microRNA 506 (miR-506) - predicted as candidate to target AE2 mRNA - for the decreased expression of AE2 in PBC. Real-time quantitative polymerase chain reaction showed that miR-506 expression is increased in PBC livers versus normal liver specimens. In situ hybridization in liver sections confirmed that miR-506 is up-regulated in the intrahepatic bile ducts of PBC livers, compared with normal and primary sclerosing cholangitis livers. Precursor-mediated overexpression of miR-506 in SV40-immortalized normal human cholangiocytes (H69 cells) led to decreased AE2 protein expression and activity, as indicated by immunoblotting and microfluorimetry, respectively. Moreover, miR-506 overexpression in three-dimensional (3D)-cultured H69 cholangiocytes blocked the secretin-stimulated expansion of cystic structures developed under the 3D conditions. Luciferase assays and site-directed mutagenesis demonstrated that miR-506 specifically may bind the 3'untranslated region (3'UTR) of AE2 messenger RNA (mRNA) and prevent protein translation. Finally, cultured PBC cholangiocytes showed decreased AE2 activity, together with miR-506 overexpression, compared to normal human cholangiocytes, and transfection of PBC cholangiocytes with anti-miR-506 was able to improve their AE2 activity. CONCLUSION: miR-506 is up-regulated in cholangiocytes from PBC patients, binds the 3'UTR region of AE2 mRNA, and prevents protein translation, leading to diminished AE2 activity and impaired biliary secretory functions. In view of the putative pathogenic role of decreased AE2 in PBC, miR-506 may constitute a potential therapeutic target for this disease.

Omalizumab therapy in severe asthma: experience from the Spanish registry--some new approaches.

OBJECTIVE: The efficacy of omalizumab in severe asthma has been widely demonstrated. The main objective of this study was to evaluate the efficacy and tolerability of omalizumab in a real-life setting in Spain, particularly in those patients with immunoglobulin E (IgE) levels out of range. METHODS: Totally 266 uncontrolled severe asthma patients receiving high-dose inhaled corticosteroids (ICSs) plus long-acting ß2-agonist (LABA) were recruited. Main efficacy outcomes were asthma exacerbation rate (AER), asthma control test (ACT), and global evaluation of treatment effectiveness (GETE). RESULTS: AER was reduced from 3.6 (3.6) in previous year to 0.67 (1.2) at 4 months (p < .05) and to 1.04 (1.8) at 2 years (p < .05). ACT increased significantly from 14.3 (4.7) at baseline to 18.4 (4.4) at 4 months (p < .05) and to 20.3 (4.0) (p < .05) at 2 years. After 4 months, 74.6% of patients had reached a good or excellent rate on the GETE scale (p < .05). This rate continued increasing up to 81.6% at 2 years. These efficacy results were similar for patients with "off-label" IgE > 700 IU/ml. At follow-up, maintenance treatment with oral steroids was discontinued in a considerable number of patients: from 89 to 19 (p < .05). Omalizumab was discontinued because of lack of efficacy only in 28/266 (10.5%) patients. Overall, 30 patients (11.4%) reported adverse events. Severe adverse events were not observed. CONCLUSION: This real-life study confirms that omalizumab is very efficacious and very well tolerated in patients with uncontrolled severe asthma. Results did not vary in the subgroup of patients with IgE levels >700 IU/ml.

Ursodeoxycholic acid is conjugated with taurine to promote secretin-stimulated biliary hydrocholeresis in the normal rat.

BACKGROUND & AIMS: Secretin induces bicarbonate-rich hydrocholeresis in healthy individuals, but not in untreated patients with primary biliary cirrhosis (PBC). Ursodeoxycholic acid (UDCA)--the first choice treatment for PBC--restores the secretin response. Compared with humans, secretin has poor effect in experimental normal-rat models with biliary drainage, although it may elicit hydrocholeresis when the bile-acid pool is maintained. In view of the benefits of UDCA in PBC, we used normal-rat models to unravel the acute contribution of UDCA (and/or taurine-conjugated TUDCA) for eliciting the biliary secretin response. METHODS: Intravascular and/or intrabiliary administration of agonists and inhibitors was performed in normal rats with biliary monitoring. Secretin/bile-acid interplay was analyzed in 3D cultured rat cholangiocytes that formed expansive cystic structures with intralumenal hydroionic secretion. RESULTS: In vivo, secretin stimulates hydrocholeresis upon UDCA/TUDCA infusion, but does not modify the intrinsic hypercholeretic effect of dehydrocholic acid (DHCA). The former effect is dependent on microtubule polymerization, and involves PKCα, PI3K and MEK pathways, as shown by colchicine (i.p.) and retrograde biliary inhibitors. In vitro, while secretin alone accelerates the spontaneous expansion of 3D-cystic structures, this effect is enhanced in the presence of TUDCA, but not UDCA or DHCA. Experiments with inhibitors and Ca(2+)-chelator confirmed that the synergistic effect of secretin plus TUDCA involves microtubules, intracellular Ca(2+), PKCα, PI3K, PKA and MEK pathways. Gene silencing also demonstrated the involvement of the bicarbonate extruder Ae2. CONCLUSIONS: UDCA is conjugated in order to promote secretin-stimulated hydrocholeresis in rats through Ae2, microtubules, intracellular Ca(2+), PKCα, PI3K, PKA, and MEK.

Biliary secretion of S-nitrosoglutathione is involved in the hypercholeresis induced by ursodeoxycholic acid in the normal rat.

UNLABELLED: Ursodeoxycholic acid (UDCA) induces bicarbonate-rich hypercholeresis by incompletely defined mechanisms that involve the stimulation of adenosine triphosphate (ATP) release from cholangiocytes. As nitric oxide (NO) at a low concentration can stimulate a variety of secretory processes, we investigated whether this mediator could be implicated in the choleretic response to UDCA. Our in vivo experiments with the in situ perfused rat liver model in anesthetized rats, showed that UDCA infusion increased the biliary secretion of NO derivatives, hepatic inducible NO synthase expression, and NO synthase activity in liver tissue. UDCA also stimulated NO release by isolated rat hepatocytes. In contrast to UDCA, cholic acid was a poor inducer of NO secretion, and tauroursodeoxycholic acid showed no effect on NO secretion. Upon UDCA administration, NO was found in bile as low-molecular-weight nitrosothiols, of which S-nitrosoglutathione (GSNO) was the predominant species. UDCA-stimulated biliary NO secretion was abolished by the inhibition of inducible NO synthase with N(omega)-nitro-L-arginine methyl ester in isolated perfused livers and also in rats whose livers were depleted of glutathione with buthionine sulfoximine. Moreover, the biliary secretion of NO species was significantly diminished in UDCA-infused transport mutant [ATP-binding cassette C2 (ABCC2)/multidrug resistance-associated protein 2 (Mrp2)-deficient] rats, and this finding was consistent with the involvement of the glutathione carrier ABCC2/Mrp2 in the canalicular transport of GSNO. It was particularly noteworthy that in cultured normal rat cholangiocytes, GSNO activated protein kinase B, protected against apoptosis, and enhanced UDCA-induced ATP release to the medium; this effect was blocked by phosphoinositide 3-kinase inhibition. Finally, retrograde GSNO infusion into the common bile duct increased bile flow and biliary bicarbonate secretion. CONCLUSION: UDCA induces biliary secretion of GSNO, which contributes to stimulating ductal secretion.

Bicarbonate secretion of mouse cholangiocytes involves Na(+)-HCO(3)(-) cotransport in addition to Na(+)-independent Cl(-)/HCO(3)(-) exchange.

UNLABELLED: Bicarbonate secretion from cholangiocytes is required for appropriate adjustment of primary canalicular bile along the biliary tract. In human and rat cholangiocytes, bicarbonate secretion is mediated by anion exchanger (AE) 2, an electroneutral Na(+)-independent Cl(-)/HCO(3) (-) AE also involved in intracellular pH (pH(i)) regulation. In Ae2(a,b)-deficient mice, pH(i) is increased in lymphocytes and fibroblasts, whereas it is surprisingly normal in cholangiocytes. Here, we analyze the mechanisms for HCO(3) (-) secretion in cultured Ae2(a,b) (+/+) and Ae2(a,b) (-/-) mouse cholangiocytes by microfluorimetric measurement of pH(i) changes upon established perfusion maneuvers. Cl(-) withdrawal by isethionate-based perfusions showed that Ae2(a,b) (+/+) but not Ae2(a,b) (-/-) mouse cholangiocytes can display Cl(-)/HCO(3) (-) exchange, which is therefore entirely mediated by Ae2. Nevertheless, simultaneous withdrawal of Cl(-) and Na(+) revealed that mouse cholangiocytes possess an additional transport activity for HCO(3) (-) secretion not observed in control rat cholangiocytes. Propionate-based maneuvers indicated that this supplemental Na(+)-driven HCO(3) (-)-secreting activity is Cl(-)-independent, consistent with a Na(+)-HCO(3) (-) cotransport (NBC). NBC activity is greater in Ae2(a,b) (-/-) than Ae2(a,b) (+/+) mouse cholangiocytes, and membrane-depolarization experiments showed that it is electrogenic. Consistent with the potential role of Slc4a4/Nbc1 as the involved transporter, Ae2(a,b) (-/-) mouse cholangiocytes exhibit up-regulated expression of this electrogenic NBC carrier. Whereas Ae2-mediated Cl(-)/HCO(3) (-) exchange in Ae2(a,b) (+/+) mouse cholangiocytes is stimulated by cyclic adenosine monophosphate (cAMP) and acetylcholine, the NBC activity is down-regulated by cAMP and adenosine triphosphate (ATP) in Ae2(a,b) (-/-) mouse cholangiocytes. Polarized Ae2(a,b) (-/-) mouse cholangiocytes placed in Ussing chambers show decreased (but not abolished) cAMP-dependent Cl(-) current and increased ATP-dependent/Ca(2+)-activated Cl(-) secretion, which run in parallel with decreased cystic fibrosis transmembrane conductance regulator messenger RNA expression and increased intracellular Ca(2+) levels. CONCLUSION: Bicarbonate secretion in mouse cholangiocytes involves two differentially regulated activities: Ae2-mediated Cl(-)/HCO(3) (-) exchange and Na(+)-HCO(3) (-) cotransport.

The cAMP effectors Epac and protein kinase a (PKA) are involved in the hepatic cystogenesis of an animal model of autosomal recessive polycystic kidney disease (ARPKD).

UNLABELLED: PCK rats, an animal model of autosomal recessive polycystic kidney disease (ARPKD), develop cholangiocyte-derived liver cysts associated with increased intracellular adenosine 3',5'-cyclic adenosine monophosphate (cAMP), the inhibition of which suppresses cyst growth. We hypothesized that elevated cAMP stimulates cholangiocyte proliferation via two downstream effectors, exchange proteins activated by cAMP (Epac1 and Epac2 isoforms) and protein kinase A (PKA), and that intracellular calcium is also involved in this process. Assessment of Epac isoforms and PKA regulatory subunits at the messenger RNA and protein level showed that cultured normal rat cholangiocytes express Epac1, Epac2, and all regulatory PKA subunits. Epac isoforms and the PKA RIbeta subunit were overexpressed in cultured PCK cholangiocytes. Proliferation analysis in response to Epac and PKA activation indicated that both normal and PCK cholangiocytes increase their growth upon Epac-specific stimulation, while PKA-specific stimulation results in differential effects, suppressing proliferation in normal cholangiocytes but accelerating this process in PCK cholangiocytes. On the other hand, both PKA and Epac activation of cystic structures generated by normal and PCK cholangiocytes when cultured under three-dimensional conditions resulted in increased cyst growth, particularly in PCK-cholangiocyte derived cysts. Pharmacological inhibitors and small interfering RNA-mediated gene silencing demonstrated the specificity of each effector activation, as well as the involvement of MEK-ERK1/2 signaling in all the observed effector-associated proliferation changes. Hyperproliferation of PCK cholangiocytes in response to PKA stimulation, but not to Epac stimulation, was found to be associated with decreased intracellular calcium, and restoration of calcium levels blocked the PKA-dependent proliferation via the PI3K/AKT pathway. CONCLUSION: Our data provide strong evidence that both cAMP effectors, Epac and PKA, and the levels of intracellular calcium are involved in the hepatic cystogenesis of ARPKD.

Hepatic cystogenesis is associated with abnormal expression and location of ion transporters and water channels in an animal model of autosomal recessive polycystic kidney disease.

Polycystic kidney (PCK) rats are a spontaneous model of autosomal recessive polycystic kidney disease that exhibit cholangiocyte-derived liver cysts. We have previously reported that in normal cholangiocytes a subset of vesicles contain three proteins (ie, the water channel AQP1, the chloride channel CFTR, and the anion exchanger AE2) that account for ion-driven water transport. Thus, we hypothesized that altered expression and location of these functionally related proteins contribute to hepatic cystogenesis. We show here that under basal conditions and in response to secretin and hypotonicity, cysts from PCK rats expanded to a greater degree than cysts formed by normal bile ducts. Quantitative reverse transcriptase-polymerase chain reaction, immunoblot analysis, and confocal and immunoelectron microscopy all indicated increased expression of these three proteins in PCK cholangiocytes versus normal cholangiocytes. AQP1, CFTR, and AE2 were localized preferentially to the apical membrane in normal rats while overexpressed at the basolateral membrane in PCK rats. Exposure of the cholangiocyte basolateral membrane to CFTR inhibitors [5-nitro-2-(3-phenylpropylamino)-benzoic acid and CFTRinh172], or Cl(-)/HCO(3)(-) exchange inhibitors (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid disodium salt hydrate and 4-acetamido-4'-isothiocyanato-2,2'-stilbenedisulfonic acid disodium salt hydrate) blocked secretin-stimulated fluid accumulation in PCK but not in normal cysts. Our data suggest that hepatic cystogenesis in autosomal recessive polycystic kidney disease may involve increased fluid accumulation because of overexpression and abnormal location of AQP1, CFTR, and AE2 in cystic cholangiocytes. Therapeutic interventions that block the activation of these proteins might inhibit cyst expansion in polycystic liver disease.

Ae2a,b-deficient mice develop antimitochondrial antibodies and other features resembling primary biliary cirrhosis.

BACKGROUND & AIMS: Cl(-)/HCO(3)(-) anion exchanger 2 (AE2) is involved in intracellular pH (pH(i)) regulation and transepithelial acid-base transport, including secretin-stimulated biliary bicarbonate excretion. AE2 gene expression was found to be reduced in liver biopsy specimens and blood mononuclear cells from patients with primary biliary cirrhosis (PBC), a disease characterized by chronic nonsuppurative cholangitis associated with antimitochondrial antibodies (AMA) and other autoimmune phenomena. In mice with widespread Ae2 gene disruption, we previously reported altered spermiogenesis and reduced gastric acid secretion. We now describe the hepatobiliary and immunologic changes observed in these Ae2(a.b)-deficient mice. METHODS: In this murine model, splenocyte pH(i) and T-cell populations were studied by flow cytometry. CD3-stimulated cytokine secretion was estimated using cytokine arrays. AMA were evaluated by immunoblotting and proteomics. Hepatobiliary changes were assessed by immunohistopathology, flow cytometry, and serum biochemistry. Cholangiocyte gene expression was analyzed by real-time polymerase chain reaction. RESULTS: Ae2(a,b)(-/-) mice exhibit splenomegaly, elevated pH(i) in splenocytes, increased production of interleukin-12p70 and interferon gamma, expanded CD8(+) T-cell population, and under represented CD4(+)FoxP3(+)/regulatory T cells. Most Ae2(a,b)(-/-) mice tested positively for AMA, showing increased serum levels of immunoglobulin M and G, and liver-specific alkaline phosphatase. About one third of Ae2(a,b)(-/-) mice had extensive portal inflammation with CD8(+) and CD4(+) T lymphocytes surrounding damaged bile ducts. Cholangiocytes isolated from Ae2(a,b)(-/-) mice showed gene expression changes compatible with oxidative stress and increased antigen presentation. CONCLUSIONS: Ae2 deficiency alters pH(i) homeostasis in immunocytes and gene expression profile in cholangiocytes, leading to immunologic and hepatobiliary changes that resemble PBC.

Activation of cyclic AMP Signaling in Ae2-deficient mouse fibroblasts.

Anion exchanger 2 (AE2, SLC4A2) is a ubiquitously expressed membrane solute carrier that regulates intracellular pH (pH(i)) by exchanging cytosolic bicarbonate for extracellular chloride. We used fibroblasts from Ae2-deficient (Ae2(a,b)(-/-)) mice to study the effects of an alkaline shift in resting intracellular pH (pH(i)) on the activation of cAMP signaling and gene expression. Ae2(a,b)(-/-) fibroblasts show increased pH(i) (by 0.22 +/- 0.03 unit) compared with wild type cells at extracellular pH (pH(o)) 7.4 and 37 degrees C. This shift in resting pH(i) is associated with an up-regulation of bicarbonate-activated soluble adenylyl cyclase expression, increased cAMP production, Creb phosphorylation, inducible cAMP early repressor 1 mRNA expression, and impaired activation of c-Fos transcription by forskolin. These results highlight the importance of bicarbonate transport via Ae2 in maintaining pH(i) homeostasis in cultured mouse fibroblasts and unveil the role of cAMP in the cellular response to chronic alkalization, which putatively includes an inducible cAMP early repressor 1-mediated attenuation of phosphorylated Creb activity.

Efecto de la biofertilización en vivero del cacao (Theobroma cacao L) con Azospirillum brasilense tarrand, Krieg et Dõbereiner y Glomus intraradices Schenk et Smith / Effect of cocoa (theobroma cacao L) biofertilitation in nursely whit Azospirillum brasilense Tarrand, Krieg et D”bereiner and Glomus intraradices Schenk et Smith

Theobroma cacao L. es una especie originaria de América y ha estado ligada al desarrollo de diversas culturas indígenas en las regiones tropicales húmedas. Su semilla se utiliza para la elaboración de alimentos, bebidas y golosinas y su demanda se ha incrementado cuando se cultiva sin agroquímicos. La nutrición de la planta mediante biofertilizantes microbianos es una alternativa para incrementar la oferta de cacao orgánico. En este trabajo se identificó el aporte de dos microsimbiontes en el desarrollo vegetal y nutrimental del cacao en dos condiciones de suelo del Soconusco, Chiapas, México, uno de ellos tratado con bromuro de metilo y otro sin tratar. Las semillas de cacao se inocularon con Azospirillum brasilense y Glomus intraradices, solos o combinados al momento de la siembra. Se registraron variables morfológicas y fisiológicas del rendimiento y el contenido de N2, P y Ca²+ en el tejido vegetal cada 30 días durante seis meses. Los resultados indicaron una respuesta diferencial entre condiciones de suelo y microsimbiontes en la asignación de materia seca. Los órganos de la planta más modificados fueron la raíz y la lámina foliar. Las plantas inoculadas mostraron mayor concentración de N2 en suelo no tratado. G. intraradices fue más efectivo en promover la incorporación de P en suelo no tratado y de Ca²+ en ambas condiciones del suelo.

Cholangiocyte anion exchange and biliary bicarbonate excretion.

Primary canalicular bile undergoes a process of fluidization and alkalinization along the biliary tract that is influenced by several factors including hormones, innervation/neuropeptides, and biliary constituents. The excretion of bicarbonate at both the canaliculi and the bile ducts is an important contributor to the generation of the so-called bile-salt independent flow. Bicarbonate is secreted from hepatocytes and cholangiocytes through parallel mechanisms which involve chloride efflux through activation of Cl- channels, and further bicarbonate secretion via AE2/SLC4A2-mediated Cl-/HCO3- exchange. Glucagon and secretin are two relevant hormones which seem to act very similarly in their target cells (hepatocytes for the former and cholangiocytes for the latter). These hormones interact with their specific G protein-coupled receptors, causing increases in intracellular levels of cAMP and activation of cAMP-dependent Cl- and HCO3- secretory mechanisms. Both hepatocytes and cholangiocytes appear to have cAMP-responsive intracellular vesicles in which AE2/SLC4A2 colocalizes with cell specific Cl- channels (CFTR in cholangiocytes and not yet determined in hepatocytes) and aquaporins (AQP8 in hepatocytes and AQP1 in cholangiocytes). cAMP-induced coordinated trafficking of these vesicles to either canalicular or cholangiocyte lumenal membranes and further exocytosis results in increased osmotic forces and passive movement of water with net bicarbonate-rich hydrocholeresis.

Bicarbonate-rich choleresis induced by secretin in normal rat is taurocholate-dependent and involves AE2 anion exchanger.

Canalicular bile is modified along bile ducts through reabsorptive and secretory processes regulated by nerves, bile salts, and hormones such as secretin. Secretin stimulates ductular cystic fibrosis transmembrane conductance regulator (CFTR)-dependent Cl- efflux and subsequent biliary HCO3- secretion, possibly via Cl-/HCO3- anion exchange (AE). However, the contribution of secretin to bile regulation in the normal rat, the significance of choleretic bile salts in secretin effects, and the role of Cl-/HCO3- exchange in secretin-stimulated HCO3- secretion all remain unclear. Here, secretin was administered to normal rats with maintained bile acid pool via continuous taurocholate infusion. Bile flow and biliary HCO3- and Cl- excretion were monitored following intrabiliary retrograde fluxes of saline solutions with and without the Cl- channel inhibitor 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) or the Cl-/HCO3- exchange inhibitor 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS). Secretin increased bile flow and biliary excretion of HCO3- and Cl-. Interestingly, secretin effects were not observed in the absence of taurocholate. Whereas secretin effects were all blocked by intrabiliary NPPB, DIDS only inhibited secretin-induced increases in bile flow and HCO3- excretion but not the increased Cl- excretion, revealing a role of biliary Cl-/HCO3- exchange in secretin-induced, bicarbonate-rich choleresis in normal rats. Finally, small hairpin RNA adenoviral constructs were used to demonstrate the involvement of the Na+-independent anion exchanger 2 (AE2) through gene silencing in normal rat cholangiocytes. AE2 gene silencing caused a marked inhibition of unstimulated and secretin-stimulated Cl-/HCO3- exchange. In conclusion, maintenance of the bile acid pool is crucial for secretin to induce bicarbonate-rich choleresis in the normal rat and that this occurs via a chloride-bicarbonate exchange process consistent with AE2 function.

Anion exchanger 2 is essential for spermiogenesis in mice.

Na+-independent anion exchangers (AE) mediate electroneutral exchange of Cl- for HCO3- ions across cell membranes, being involved in intracellular pH and cell volume regulation and in transepithelial hydroionic fluxes. Bicarbonate activation of adenylyl cyclase is known to be necessary for sperm motility and sperm capacitation, and a few studies have suggested a possible role of AE carriers in reproduction. Among the four AE genes identified in mammals thus far, only Ae2 (Slc4a2) has been determined to be expressed in the male reproductive system, especially in developing spermatozoa and in epididymal epithelium. Most AE genes drive alternative transcription, which in mouse Ae2 results in several Ae2 isoforms. Here, we generated mice carrying a targeted disruption of Ae2 that prevents the expression of the three AE2 isoforms (Ae2a, Ae2b1, and Ae2b2) normally found in mouse testes. Male Ae2-/- mice (but not female Ae2-/- mice) are infertile. Histopathological analysis of Ae2-/- testes shows an interruption of spermiogenesis, with only a few late spermatids and a complete absence of spermatozoa in the seminiferous tubules. The number of apoptotic bodies is increased in the seminiferous tubules and in the epididymis, which also shows squamous metaplasia of the epididymal epithelium. Our findings reveal an essential role of Ae2 in mouse spermiogenesis and stress the recently postulated involvement of bicarbonate in germ-cell differentiation through the bicarbonate-sensitive soluble-adenylyl-cyclase pathway.

HNF1alpha upregulates the human AE2 anion exchanger gene (SLC4A2) from an alternate promoter.

The human AE2 gene (SLC4A2) is transcribed in a widespread fashion from the upstream promoter, the resultant full-length transcript AE2a being encountered in most tissues. Moreover, alternate promoter sequences within intron 2 may drive tissue-restricted expression of variants AE2b(1) and AE2b(2), mainly in liver and kidney. AE2b(2) proximal promoter sequences are highly active in transfected liver-derived HepG2 cells and contain an HNF1 motif. Mutation-disruption of this motif dramatically decreased alternate promoter activity in HepG2 cells but not in prostate-derived PC-3 cells. Electromobility shift and supershift assays indicated that HNF1alpha from HepG2 nuclear extracts binds the HNF1 sequence. Transactivation studies in PC-3 cells showed enhanced activity of the wild-type construct upon cotransfection with an HNF1alpha expression plasmid, while activity of the HNF1-mutated construct remained unaffected. Since liver AE2 is putatively involved in the biliary secretion of bicarbonate, HNF1alpha may have a role in increasing bicarbonate secretion in response to certain stimuli.