RESUMEN
AIMS: Estradiol 17ß-d-glucuronide (E217G) induces cholestasis by triggering endocytosis and further intracellular retention of the canalicular transporters Bsep and Mrp2, in a cPKC- and PI3K-dependent manner, respectively. Pregnancy-induced cholestasis has been associated with E217G cholestatic effect, and is routinely treated with ursodeoxycholic acid (UDCA). Since protective mechanisms of UDCA in E217G-induced cholestasis are still unknown, we ascertained here whether its main metabolite, tauroursodeoxycholate (TUDC), can prevent endocytosis of canalicular transporters by counteracting cPKC and PI3K/Akt activation. MAIN METHODS: Activation of cPKC and PI3K/Akt was evaluated in isolated rat hepatocytes by immunoblotting (assessment of membrane-bound and phosphorylated forms, respectively). Bsep/Mrp2 function was quantified in isolated rat hepatocyte couplets (IRHCs) by assessing the apical accumulation of their fluorescent substrates, CLF and GS-MF, respectively. We also studied, in isolated, perfused rat livers (IPRLs), the status of Bsep and Mrp2 transport function, assessed by the biliary excretion of TC and DNP-SG, respectively, and Bsep/Mrp2 localization by immunofluorescence. KEY FINDINGS: E217G activated both cPKC- and PI3K/Akt-dependent signaling, and pretreatment with TUDC significantly attenuated these activations. In IRHCs, TUDC prevented the E217G-induced decrease in apical accumulation of CLF and GS-MF, and inhibitors of protein phosphatases failed to counteract this protection. In IPRLs, E217G induced an acute decrease in bile flow and in the biliary excretion of TC and DNP-SG, and this was prevented by TUDC. Immunofluorescence studies revealed that TUDC prevented E217G-induced Bsep/Mrp2 endocytosis. SIGNIFICANCE: TUDC restores function and localization of Bsep/Mrp2 impaired by E217G, by preventing both cPKC and PI3K/Akt activation in a protein-phosphatase-independent manner.
Asunto(s)
Colestasis , Endocitosis , Estradiol , Hepatocitos , Fosfatidilinositol 3-Quinasas , Transducción de Señal , Ácido Tauroquenodesoxicólico , Animales , Colestasis/metabolismo , Colestasis/inducido químicamente , Colestasis/prevención & control , Ratas , Transducción de Señal/efectos de los fármacos , Estradiol/metabolismo , Estradiol/farmacología , Estradiol/análogos & derivados , Hepatocitos/metabolismo , Hepatocitos/efectos de los fármacos , Endocitosis/efectos de los fármacos , Ácido Tauroquenodesoxicólico/farmacología , Ácido Tauroquenodesoxicólico/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Miembro 11 de la Subfamilia B de Transportador de Casetes de Unión al ATP/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas Wistar , Femenino , Masculino , Proteína Quinasa C/metabolismo , Transportadoras de Casetes de Unión a ATP/metabolismoRESUMEN
The endogenous metabolite of estradiol, estradiol 17ß-D-glucuronide (E17G), is considered the main responsible of the intrahepatic cholestasis of pregnancy. E17G alters the activity of canalicular transporters through a signaling pathway-dependent cellular internalization, phenomenon that was attributed to oxidative stress in different cholestatic conditions. However, there are no reports involving oxidative stress in E17G-induced cholestasis, representing this the aim of our work. Using polarized hepatocyte cultures, we showed that antioxidant compounds prevented E17G-induced Mrp2 activity alteration, being this alteration equally prevented by the NADPH oxidase (NOX) inhibitor apocynin. The model antioxidant N-acetyl-cysteine prevented, in isolated and perfused rat livers, E17G-induced impairment of bile flow and Mrp2 activity, thus confirming the participation of reactive oxygen species (ROS) in this cholestasis. In primary cultured hepatocytes, pretreatment with specific inhibitors of ERK1/2 and p38MAPK impeded E17G-induced ROS production; contrarily, NOX inhibition did not affect ERK1/2 and p38MAPK phosphorylation. Both, knockdown of p47phox by siRNA and preincubation with apocynin in sandwich-cultured rat hepatocytes significantly prevented E17G-induced internalization of Mrp2, suggesting a crucial role for NOX in this phenomenon. Concluding, E17G-induced cholestasis is partially mediated by NOX-generated ROS through internalization of canalicular transporters like Mrp2, being ERK1/2 and p38MAPK necessary for NOX activation.
Asunto(s)
Estradiol , Hepatocitos , NADPH Oxidasas , Especies Reactivas de Oxígeno , Animales , NADPH Oxidasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Ratas , Hepatocitos/metabolismo , Hepatocitos/efectos de los fármacos , Estradiol/farmacología , Estradiol/metabolismo , Estradiol/análogos & derivados , Femenino , Colestasis/inducido químicamente , Colestasis/metabolismo , Colestasis/patología , Ratas Wistar , Acetofenonas/farmacología , Estrés Oxidativo/efectos de los fármacos , Acetilcisteína/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Células Cultivadas , Antioxidantes/farmacología , Antioxidantes/metabolismo , Colestasis Intrahepática , Complicaciones del Embarazo , Transportadoras de Casetes de Unión a ATPRESUMEN
Ursodeoxycholic acid (UDCA) is a natural substance physiologically produced in the liver. Initially used to dissolve gallstones, it is now successfully used in treating primary biliary cirrhosis and as adjuvant therapy for various hepatobiliary cholestatic diseases. However, the mechanisms underlying its beneficial effects still need to be clarified. Evidence suggests three mechanisms of action for UDCA that could benefit humans with cholestatic liver disease (CLD): protection of cholangiocytes against hydrophobic bile acid (BA) cytotoxicity, stimulation of hepatobiliary excretion, and protection of hepatocytes against BA-induced apoptosis. These mechanisms may act individually or together to potentiate them. At the molecular level, it has been observed that UDCA can generate modifications in the transcription and translation of proteins essential in the transport of BA, correcting the deficit in BA secretion in CLD, in addition to activating signaling pathways to translocate these transporters to the sites where they should fulfill their function. Inhibition of BA-induced hepatocyte apoptosis may play a role in CLD, characterized by BA retention in the hepatocyte. Thus, different mechanisms of action contribute to the improvement after UDCA administration in CLD. On the other hand, the effects of UDCA on tissues that possess receptors that may interact with BAs in pathological contexts, such as skeletal muscle, are still unclear. This work aims to describe the main molecular mechanisms by which UDCA acts in the human body, emphasizing the interaction in tissues other than the liver.
Asunto(s)
Colestasis , Hepatopatías , Humanos , Ácido Ursodesoxicólico/farmacología , Ácido Ursodesoxicólico/uso terapéutico , Ácido Ursodesoxicólico/metabolismo , Ácidos y Sales Biliares , Colestasis/tratamiento farmacológico , Colestasis/metabolismo , Hepatopatías/tratamiento farmacológicoRESUMEN
BACKGROUND: Skeletal muscle is sensitive to bile acids (BA) because it expresses the TGR5 receptor for BA. Cholic (CA) and deoxycholic (DCA) acids induce a sarcopenia-like phenotype through TGR5-dependent mechanisms. Besides, a mouse model of cholestasis-induced sarcopenia was characterised by increased levels of serum BA and muscle weakness, alterations that are dependent on TGR5 expression. Mitochondrial alterations, such as decreased mitochondrial potential and oxygen consumption rate (OCR), increased mitochondrial reactive oxygen species (mtROS) and unbalanced biogenesis and mitophagy, have not been studied in BA-induced sarcopenia. METHODS: We evaluated the effects of DCA and CA on mitochondrial alterations in C2C12 myotubes and a mouse model of cholestasis-induced sarcopenia. We measured mitochondrial mass by TOM20 levels and mitochondrial DNA; ultrastructural alterations by transmission electronic microscopy; mitochondrial biogenesis by PGC-1α plasmid reporter activity and protein levels by western blot analysis; mitophagy by the co-localisation of the MitoTracker and LysoTracker fluorescent probes; mitochondrial potential by detecting the TMRE probe signal; protein levels of OXPHOS complexes and LC3B by western blot analysis; OCR by Seahorse measures; and mtROS by MitoSOX probe signals. RESULTS: DCA and CA caused a reduction in mitochondrial mass and decreased mitochondrial biogenesis. Interestingly, DCA and CA increased LC3II/LC3I ratio and decreased autophagic flux concordant with raised mitophagosome-like structures. In addition, DCA and CA decreased mitochondrial potential and reduced protein levels in OXPHOS complexes I and II. The results also demonstrated that DCA and CA decreased basal, ATP-linked, FCCP-induced maximal respiration and spare OCR. DCA and CA also reduced the number of cristae. In addition, DCA and CA increased the mtROS. In mice with cholestasis-induced sarcopenia, TOM20, OXPHOS complexes I, II and III, and OCR were diminished. Interestingly, the OCR and OXPHOS complexes were correlated with muscle strength and bile acid levels. CONCLUSION: Our results showed that DCA and CA decreased mitochondrial mass, possibly by reducing mitochondrial biogenesis, which affects mitochondrial function, thereby altering potential OCR and mtROS generation. Some mitochondrial alterations were also observed in a mouse model of cholestasis-induced sarcopenia characterised by increased levels of BA, such as DCA and CA.
Asunto(s)
Colestasis , Sarcopenia , Animales , Ratones , Sarcopenia/metabolismo , Sarcopenia/patología , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Mitocondrias , Modelos Animales de Enfermedad , Colestasis/metabolismo , Colestasis/patologíaRESUMEN
Chronic liver diseases are a group of pathologies affecting the liver with high prevalence worldwide. Among them, cholestatic chronic liver diseases (CCLD) are characterized by alterations in liver function and increased plasma bile acids. Secondary to liver disease, under cholestasis, is developed sarcopenia, a skeletal muscle dysfunction with decreased muscle mass, strength, and physical function. CCL5/RANTES is a chemokine involved in the immune and inflammatory response. Indeed, CCL5 is a myokine because it is produced by skeletal muscle. Several studies show that bile acids induce CCL5/RANTES expression in liver cells. However, it is unknown if the expression of CCL5/RANTES is changed in the skeletal muscle of mice with cholestatic liver disease. We used a murine model of cholestasis-induced sarcopenia by intake of hepatotoxin 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC diet), in which we detected the mRNA levels for ccl5. We determined that mice fed the DDC diet presented high levels of serum bile acids and developed typical features of sarcopenia. Under these conditions, we detected the ccl5 gene expression in diaphragm muscle showing elevated mRNA levels compared to mice fed with a standard diet (chow diet). Our results collectively suggest an increased ccl5 gene expression in the diaphragm muscle concomitantly with elevated serum bile acids and the development of sarcopenia.
Asunto(s)
Colestasis , Hepatopatías , Sarcopenia , Ratones , Animales , Sarcopenia/patología , Diafragma/metabolismo , Diafragma/patología , Regulación hacia Arriba , Quimiocina CCL5/metabolismo , Colestasis/complicaciones , Colestasis/metabolismo , Colestasis/patología , Hígado/metabolismo , Ácidos y Sales Biliares , Hepatopatías/metabolismo , Expresión Génica , Ratones Endogámicos C57BLRESUMEN
BACKGROUND: Skeletal muscle is sensitive to bile acids (BA) because it expresses the TGR5 receptor for BA. Cholic (CA) and deoxycholic (DCA) acids induce a sarcopenia-like phenotype through TGR5-dependent mechanisms. Besides, a mouse model of cholestasis-induced sarcopenia was characterised by increased levels of serum BA and muscle weakness, alterations that are dependent on TGR5 expression. Mitochondrial alterations, such as decreased mitochondrial potential and oxygen consumption rate (OCR), increased mitochondrial reactive oxygen species (mtROS) and unbalanced biogenesis and mitophagy, have not been studied in BA-induced sarcopenia.METHODS: We evaluated the effects of DCA and CA on mitochondrial alterations in C2C12 myotubes and a mouse model of cholestasis-induced sarcopenia. We measured mitochondrial mass by TOM20 levels and mitochondrial DNA; ultrastructural alterations by transmission electronic microscopy; mitochondrial biogenesis by PGC-1α plasmid reporter activity and protein levels by western blot analysis; mitophagy by the co-localisation of the MitoTracker and LysoTracker fluorescent probes; mitochondrial potential by detecting the TMRE probe signal; protein levels of OXPHOS complexes and LC3B by western blot analysis; OCR by Seahorse measures; and mtROS by MitoSOX probe signals. RESULTS: DCA and CA caused a reduction in mitochondrial mass and decreased mitochondrial biogenesis. Interestingly, DCA and CA increased LC3II/LC3I ratio and decreased autophagic flux concordant with raised mitophagosome-like structures. In addition, DCA and CA decreased mitochondrial potential and reduced protein levels in OXPHOS complexes I and II. The results also demonstrated that DCA and CA decreased basal, ATP-linked, FCCP-induced maximal respiration and spare OCR. DCA and CA also reduced the number of cristae. In addition, DCA and CA increased the mtROS. In mice with cholestasis-induced sarcopenia, TOM20, OXPHOS complexes I, II and III, and OCR were diminished. Interestingly, the OCR and OXPHOS complexes were correlated with muscle strength and bile acid levels. CONCLUSION: Our results showed that DCA and CA decreased mitochondrial mass, possibly by reducing mitochondrial biogenesis, which affects mitochondrial function, thereby altering potential OCR and mtROS generation. Some mitochondrial alterations were also observed in a mouse model of cholestasis-induced sarcopenia characterised by increased levels of BA, such as DCA and CA.
Asunto(s)
Animales , Ratones , Colestasis/metabolismo , Colestasis/patología , Sarcopenia/metabolismo , Sarcopenia/patología , Músculo Esquelético/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Modelos Animales de Enfermedad , MitocondriasRESUMEN
BACKGROUND: Platelet α-granule biogenesis in precursor megakaryocytes is critically dependent on VPS33B and VPS16B, as demonstrated by the platelet α-granule deficiency seen in the rare multisystem disorder arthrogryposis, renal dysfunction, and cholestasis (ARC) syndrome associated with biallelic pathogenic variants in VPS33B and VIPAS39 (encoding VPS16B). VPS33B and VPS16B are ubiquitously expressed proteins that are known to interact and play key roles in protein sorting and trafficking between subcellular locations. However, there remain significant gaps in our knowledge of the nature of these interactions in primary cells from patients with ARC syndrome. OBJECTIVES: To use primary cells from patients with ARC syndrome to better understand the interactions and roles of VPS33B and VPS16B in platelets and precursor megakaryocytes. PATIENTS/METHODS: The proband and his male sibling were clinically suspected to have ARC syndrome. Confirmatory genetic testing and platelet phenotyping, including electron microscopy and protein expression analysis, was performed with consent in a research setting. RESULTS: We describe the first case of ARC syndrome identified in Costa Rica, associated with a novel homozygous nonsense VPS33B variant that is linked with loss of expression of both VPS33B and VPS16B in platelets. CONCLUSION: These results indicate that stable expression of VPS16B in platelets, their precursor megakaryocytes, and other cells is dependent on VPS33B. We suggest that systematic evaluation of primary cells from patients with a range of VPS33B and VIPAS39 variants would help to elucidate the interactions and functions of these proteins.
Asunto(s)
Artrogriposis , Colestasis , Artrogriposis/diagnóstico , Artrogriposis/genética , Artrogriposis/metabolismo , Plaquetas/metabolismo , Colestasis/diagnóstico , Colestasis/genética , Colestasis/metabolismo , Humanos , Masculino , Insuficiencia Renal , Hermanos , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoRESUMEN
Hepatocyte growth factor (HGF) has been proved to protect the liver against α-naphthylisothiocyanate (ANIT)-induced cholestasis by acting as an antioxidant agent and redirecting toxic biliary solutes towards blood for urinary excretion. However, this may represent an additional potential risk for kidney integrity, which is already compromised by the cholestatic process itself (cholemic nephropathy). Therefore, in the present work, we studied the renal damage caused by ANIT-induced cholestasis and whether it is aggravated or, on the contrary, counteracted by HGF; if the latter holds, the involvement of its antioxidant properties will be ascertained. ANIT-induced cholestatic deleterious renal effects were corroborated by the presence of urine bile salts, impairment of renal function, and the alterations of renal damage markers, such as HSP72, creatinine clearance, and albuminuria. HGF fully reverted all these, and the cast formation in the tubules was significantly decreased. These findings were associated with the control of renal oxidative stress. In summary, despite HGF enhancing the overload of potentially harmful biliary constituents that the kidney should remove from the bloodstream as an alternative depuration organ in cholestasis, it simultaneously protects the kidney from this damage by counteracting the prooxidant effects resulting from this harmful exposure.
Asunto(s)
Colestasis/tratamiento farmacológico , Factor de Crecimiento de Hepatocito/farmacología , Enfermedades Renales/fisiopatología , 1-Naftilisotiocianato/efectos adversos , 1-Naftilisotiocianato/farmacología , Animales , Antioxidantes/farmacología , Ácidos y Sales Biliares/metabolismo , Conductos Biliares/fisiopatología , Colestasis/sangre , Colestasis/metabolismo , Modelos Animales de Enfermedad , Factor de Crecimiento de Hepatocito/metabolismo , Riñón/metabolismo , Enfermedades Renales/metabolismo , Hígado/metabolismo , Masculino , Ratones , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Bile acids (BA) are recognized by their role in nutrient absorption. However, there is growing evidence that BA also have endocrine and metabolic functions. Besides, the steroidal-derived structure gives BA a toxic potential over the biological membrane. Thus, cholestatic disorders, characterized by elevated BA on the liver and serum, are a significant cause of liver transplant and extrahepatic complications, such as skeletal muscle, central nervous system (CNS), heart, and placenta. Further, the BA have an essential role in cellular damage, mediating processes such as membrane disruption, mitochondrial dysfunction, and the generation of reactive oxygen species (ROS) and oxidative stress. The purpose of this review is to describe the BA and their role on hepatic and extrahepatic complications in cholestatic diseases, focusing on the association between BA and the generation of oxidative stress that mediates tissue damage.
Asunto(s)
Ácidos y Sales Biliares/metabolismo , Colestasis/patología , Animales , Ácidos y Sales Biliares/química , Ácidos y Sales Biliares/farmacología , Colestasis/metabolismo , Hígado/metabolismo , Músculo Esquelético/metabolismo , Oxidación-Reducción , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/química , Especies Reactivas de Oxígeno/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismoRESUMEN
The mechanism of damage of the biliary epithelium remains partially unexplored. However, recently many works have offered new evidence regarding the cholangiocytes' damage process, which is the main target in a broad spectrum of pathologies ranging from acute cholestasis, cholangiopathies to cholangiocarcinoma. This is encouraging since some works addressed this epithelium's relevance in health and disease until a few years ago. The biliary tree in the liver, comprised of cholangiocytes, is a pipeline for bile flow and regulates key hepatic processes such as proliferation, regeneration, immune response, and signaling. This review aimed to compile the most recent advances on the mechanisms of cholangiocellular damage during cholestasis, which, although it is present in many cholangiopathies, is not necessarily a common or conserved process in all of them, having a relevant role cAMP and PKA during obstructive cholestasis, as well as Ca2+-dependent PKC in functional cholestasis. Cholangiocellular damage could vary according to the type of cholestasis, the aggressor, or the bile ducts' location where it develops and what kind of damage can favor cholangiocellular carcinoma development.
Asunto(s)
Procedimientos Quirúrgicos del Sistema Biliar/efectos adversos , Sistema Biliar/patología , Colestasis/patología , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismo , Animales , Sistema Biliar/metabolismo , Proliferación Celular , Colestasis/metabolismo , Colestasis/cirugía , Humanos , Ligadura , Transducción de SeñalRESUMEN
Significance: Most hepatopathies are primarily or secondarily cholestatic in nature. Oxidative stress (OS) is a frequent trait among them, and impairs the machinery to generate bile by triggering endocytic internalization of hepatocellular transporters, thus causing cholestasis. This is critical, since it leads to accelerated transporter degradation, which could explain the common post-transcriptional downregulation of transporter expression in human cholestatic diseases. Recent Advances: The mechanisms involved in OS-induced hepatocellular transporter internalization are being revealed. Filamentous actin (F-actin) cytoskeleton disorganization and/or detachment of crosslinking actin proteins that afford transporter stability have been characterized as causal factors. Activation of redox-sensitive signaling pathways leading to changes in phosphorylation status of these structures is involved, including Ca2+-mediated activation of "classical" and "novel" protein kinase C (PKC) isoforms or redox-signaling cascades downstream of NADPH oxidase. Critical Issues: Despite the well-known occurrence of hepatocellular transporter internalization in human hepatopathies, the cholestatic implications of this phenomenon have been overlooked. Accordingly, no specific treatment has been established in the clinical practice for its prevention/reversion. Future Directions: We need to improve our knowledge on the pro-oxidant triggering factors and the multiple signaling pathways that mediate this oxidative injury in each cholestatic hepatopathy, so as to envisage tailor-made therapeutic strategies for each case. Meanwhile, administration of antioxidants or heme oxygenase-1 induction to elevate the hepatocellular levels of the endogenous scavenger bilirubin are promising alternatives that need to be re-evaluated and implemented. They may complement current treatments in cholestasis aimed to enhance transcriptional carrier expression, by providing membrane stability to the newly synthesized carriers. Antioxid. Redox Signal. 35, 808-831.
Asunto(s)
Bilis/metabolismo , Colestasis/metabolismo , Hepatocitos/metabolismo , Factores de Transcripción/metabolismo , Animales , Humanos , Estrés Oxidativo , Transducción de SeñalRESUMEN
BACKGROUND AND AIMS: Chronically administered parenteral nutrition (PN) in patients with intestinal failure carries the risk for developing PN-associated cholestasis (PNAC). We have demonstrated that farnesoid X receptor (FXR) and liver X receptor (LXR), proinflammatory interleukin-1 beta (IL-1ß), and infused phytosterols are important in murine PNAC pathogenesis. In this study we examined the role of nuclear receptor liver receptor homolog 1 (LRH-1) and phytosterols in PNAC. APPROACH AND RESULTS: In a C57BL/6 PNAC mouse model (dextran sulfate sodium [DSS] pretreatment followed by 14 days of PN; DSS-PN), hepatic nuclear receptor subfamily 5, group A, member 2/LRH-1 mRNA, LRH-1 protein expression, and binding of LRH-1 at the Abcg5/8 and Cyp7a1 promoter was reduced. Interleukin-1 receptor-deficient mice (Il-1r-/- /DSS-PN) were protected from PNAC and had significantly increased hepatic mRNA and protein expression of LRH-1. NF-κB activation and binding to the LRH-1 promoter were increased in DSS-PN PNAC mice and normalized in Il-1r-/- /DSS-PN mice. Knockdown of NF-κB in IL-1ß-exposed HepG2 cells increased expression of LRH-1 and ABCG5. Treatment of HepG2 cells and primary mouse hepatocytes with an LRH-1 inverse agonist, ML179, significantly reduced mRNA expression of FXR targets ATP binding cassette subfamily C member 2/multidrug resistance associated protein 2 (ABCC2/MRP2), nuclear receptor subfamily 0, groupB, member 2/small heterodimer partner (NR0B2/SHP), and ATP binding cassette subfamily B member 11/bile salt export pump (ABCB11/BSEP). Co-incubation with phytosterols further reduced expression of these genes. Similar results were obtained by suppressing the LRH-1 targets ABCG5/8 by treatment with small interfering RNA, IL-1ß, or LXR antagonist GSK2033. Liquid chromatography-mass spectrometry and chromatin immunoprecipitation experiments in HepG2 cells showed that ATP binding cassette subfamily G member 5/8 (ABCG5/8) suppression by GSK2033 increased the accumulation of phytosterols and reduced binding of FXR to the SHP promoter. Finally, treatment with LRH-1 agonist, dilauroyl phosphatidylcholine (DLPC) protected DSS-PN mice from PNAC. CONCLUSIONS: This study suggests that NF-κB regulation of LRH-1 and downstream genes may affect phytosterol-mediated antagonism of FXR signaling in the pathogenesis of PNAC. LRH-1 could be a potential therapeutic target for PNAC.
Asunto(s)
Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 5/metabolismo , Transportador de Casete de Unión a ATP, Subfamilia G, Miembro 8/metabolismo , Colestasis/etiología , Lipoproteínas/metabolismo , FN-kappa B/metabolismo , Nutrición Parenteral/efectos adversos , Fitosteroles/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Animales , Colestasis/metabolismo , Inmunoprecipitación de Cromatina , Modelos Animales de Enfermedad , Cromatografía de Gases y Espectrometría de Masas , Técnicas de Silenciamiento del Gen , Células Hep G2 , Humanos , Ratones , Ratones Endogámicos C57BLRESUMEN
INTRODUCTION AND OBJECTIVES: Free and conjugated bile acids (BA's) cannot cross cell membranes; therefore, a particular transport system is required by the cell. Members of the family of ABC (ATP-binding proteins) transporters transfer bile acids in and out of the cell, preventing their accumulation. High intracellular concentrations of bile acids, such as those observed in cholestasis, have been related to oxidative stress and apoptosis, which in many cases are the leading causes of hepatocyte damage. MRP3 and MRP4 (multidrug resistance-associated protein 3 and 4) proteins belong to the ABC subfamily C, and are transporters of the hepatocyte's basolateral membrane with a compensatory role. Both transporters' increased expression constitutes an essential role in the protective and adaptive responses of bile acid overload, such as cholestasis. This work aimed to analyze both transporters' mRNA and protein expression in an in vitro model of cholestasis using HepG2 cell line treated with main bile acids. METHODS: The expression of transporters was investigated through confocal microscopy immunofluorescence, Western Blot, and RT-qPCR after the main bile acids in HepG2 line cells. RESULTS: The results showed the relation between confluence and expression of both transporters in the plasma membrane. MRP3 showed atypical and heterogeneous distribution in this cell line. CDCA (chenodeoxycholic acid) at low concentrations induced the expression of mRNA of both transporters. In contrast, protein expression was induced by CA (cholic acid) at high concentrations. CONCLUSION: Primary bile acids (CDCA and CA) induce overexpression of the MRP4 and MRP3 transporters in the HepG2 cell line.
Asunto(s)
Ácidos y Sales Biliares/farmacología , Colestasis/genética , Colestasis/patología , Fármacos Gastrointestinales/farmacología , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Técnicas de Cultivo de Célula , Colestasis/metabolismo , Células Hep G2 , Humanos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , ARN Mensajero/metabolismoRESUMEN
AIMS: Lipopolysaccharide (LPS) induces inflammatory cholestasis by impairing expression, localization, and function of carriers involved in bile formation, e.g. bile salt export pump (Bsep) and multidrug resistance-associated protein 2 (Mrp2). A specific therapy against this disease is still lacking. Therefore, we evaluated the anticholestatic effects of spironolactone (SL), a PXR ligand that regulates bile salt homeostasis, up-regulates Mrp2, and bears anti-inflammatory properties. MAIN METHODS: Male Wistar rats were divided into four groups: Control, SL (83.3 mg/kg/day of SL, i.p., for 3 days), LPS (2.5 mg/kg/day, i.p., at 8 am of the last 2 days, and 1.5 mg/kg/day at 8 pm of the last day), and SL + LPS. Biliary and plasma parameters and the expression, function, and localization of Mrp2 and Bsep were evaluated. KEY FINDINGS: SL partially prevented LPS-induced drop of basal bile flow by normalizing the bile salt-independent fraction of bile flow (BSIBF), via improvement of glutathione output. This was due to a recovery in Mrp2 transport function, the major canalicular glutathione transporter, estimated by monitoring the output of its exogenously administered substrate dibromosulfophthalein. SL counteracted the LPS-induced downregulation of Mrp2, but not that of Bsep, at both mRNA and protein levels. LPS induced endocytic internalization of both transporters, visualized by immunofluorescence followed by confocal microscopy, and SL partially prevented this relocalization. SL did not prevent the increase in IL-1ß, IL-6, and TNF-α plasma levels. SIGNIFICANCE: SL prevents the impairment in Mrp2 expression and localization, and the resulting recovery of Mrp2 function normalizes the BSIBF by improving glutathione excretion.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Colestasis/tratamiento farmacológico , Espironolactona/uso terapéutico , Miembro 11 de la Subfamilia B de Transportador de Casetes de Unión al ATP/metabolismo , Animales , Bilis/metabolismo , Colestasis/sangre , Colestasis/metabolismo , Citocinas/sangre , Regulación de la Expresión Génica/efectos de los fármacos , Lipopolisacáridos/efectos adversos , Masculino , Ratas , Ratas Wistar , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
BACKGROUND AND AIMS: The Wnt/ß-catenin signaling pathway has a well-described role in liver pathobiology. Its suppression was recently shown to decrease bile acid (BA) synthesis, thus preventing the development of cholestatic liver injury and fibrosis after bile duct ligation (BDL). APPROACH AND RESULTS: To generalize these observations, we suppressed ß-catenin in Mdr2 knockout (KO) mice, which develop sclerosing cholangitis due to regurgitation of BA from leaky ducts. When ß-catenin was knocked down (KD) in KO for 2 weeks, hepatic and biliary injury were exacerbated in comparison to KO given placebo, as shown by serum biochemistry, ductular reaction, inflammation, and fibrosis. Simultaneously, KO/KD livers displayed increased oxidative stress and senescence and an impaired regenerative response. Although the total liver BA levels were similar between KO/KD and KO, there was significant dysregulation of BA transporters and BA detoxification/synthesis enzymes in KO/KD compared with KO alone. Multiphoton intravital microscopy revealed a mixing of blood and bile in the sinusoids, and validated the presence of increased serum BA in KO/KD mice. Although hepatocyte junctions were intact, KO/KD livers had significant canalicular defects, which resulted from loss of hepatocyte polarity. Thus, in contrast to the protective effect of ß-catenin KD in BDL model, ß-catenin KD in Mdr2 KO aggravated rather than alleviated injury by interfering with expression of BA transporters, hepatocyte polarity, canalicular structure, and the regenerative response. CONCLUSIONS: The resulting imbalance between ongoing injury and restitution led to worsening of the Mdr2 KO phenotype, suggesting caution in targeting ß-catenin globally for all cholestatic conditions.
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Colangitis Esclerosante/complicaciones , Colangitis Esclerosante/genética , Colestasis/etiología , Colestasis/metabolismo , Vía de Señalización Wnt , beta Catenina/metabolismo , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Animales , Ácidos y Sales Biliares/metabolismo , Conductos Biliares/patología , Colestasis/genética , Modelos Animales de Enfermedad , Hepatocitos/metabolismo , Hepatocitos/patología , Uniones Intercelulares/metabolismo , Uniones Intercelulares/patología , Ratones Noqueados , Miembro 4 de la Subfamilia B de Casete de Unión a ATPRESUMEN
Multidrug resistance-associated protein 2 (MRP2/ABCC2), a hepatocyte canalicular transporter involved in bile secretion, is downregulated in cholestasis triggered by lipopolysaccharide. The human aquaporin-1 (hAQP1) adenovirus-mediated gene transfer to liver improves cholestasis by incompletely defined mechanisms. Here we found that hAQP1 did not affect MRP2/ABCC2 expression, but significantly increased its transport activity assessed in situ with endogenous and exogenous substrates, likely by a hAQP1-induced increase in canalicular membrane cholesterol amount. Our results suggest that hAQP1-induced MRP2/ABCC2 activation contributes to the cholestasis improvement.
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Transportadoras de Casetes de Unión a ATP/metabolismo , Acuaporina 1/fisiología , Bilis/metabolismo , Colestasis/metabolismo , Hepatocitos/metabolismo , Animales , Acuaporina 1/genética , Colestasis/terapia , Técnicas de Transferencia de Gen , Hepatocitos/citología , Masculino , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Ratas WistarRESUMEN
In obstructive cholestasis, there is an integral adaptive response aimed to diminish the bile flow and minimize the injury of bile ducts caused by increased intraluminal pressure and harmful levels of bile salts and bilirrubin. Canalicular bicarbonate secretion, driven by the anion exchanger 2 (AE2), is an influential determinant of the canalicular bile salt-independent bile flow. In this work, we ascertained whether AE2 expression and/or activity is reduced in hepatocytes from rats with common bile duct ligation (BDL), as part of the adaptive response to cholestasis. After 4 days of BDL, we found that neither AE2 mRNA expression (measured by quantitative real-time PCR) nor total levels of AE2 protein (assessed by western blot) were modified in freshly isolated hepatocytes. However, BDL led to a decrease in the expression of AE2 protein in plasma membrane fraction as compared with SHAM control. Additionally, AE2 activity (JOH-, mmol/L/min), measured in primary cultured hepatocytes from BDL and SHAM rats, was decreased in the BDL group versus the control group (1.9 ± 0.3 vs. 3.1 ± 0.2, p<0.005). cAMP-stimulated AE2 activity, however, was not different between SHAM and BDL groups (3.7 ± 0.3 vs. 3.5 ± 0.3), suggesting that cAMP stimulated insertion into the canalicular membrane of AE2-containing intracellular vesicles, that had remained abnormally internalized after BDL. In conclusion, our results point to the existence of a novel adaptive mechanism in cholestasis aimed to reduce biliary pressure, in which AE2 internalization in hepatocytes might result in decreased canalicular HCO3- output and decreased bile flow.
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Bicarbonatos/metabolismo , Antiportadores de Cloruro-Bicarbonato/biosíntesis , Cloruros/metabolismo , Colestasis/metabolismo , Regulación hacia Abajo , Hepatocitos/metabolismo , Animales , Colestasis/patología , Modelos Animales de Enfermedad , Hepatocitos/patología , Transporte Iónico , Masculino , Ratas , Ratas WistarRESUMEN
Bile flow generation is driven by the vectorial transfer of osmotically active compounds from sinusoidal blood into a confined space, the bile canaliculus. Hence, localization of hepatocellular transporters relevant to bile formation is crucial for bile secretion. Hepatocellular transporters are localized either in the plasma membrane or in recycling endosomes, from where they can be relocated to the plasma membrane on demand, or endocytosed when the demand decreases. The balance between endocytic internalization/ exocytic targeting to/from this recycling compartment is therefore the main determinant of the hepatic capability to generate bile, and to dispose endo- and xenobiotics. Furthermore, the exacerbated endocytic internalization is a common pathomechanisms in both experimental and human cholestasis; this results in bile secretory failure and, eventually, posttranslational transporter downregulation by increased degradation. This review summarizes the proposed structural mechanisms accounting for this pathological condition (e.g., alteration of function, localization or expression of F-actin or F-actin/transporter cross-linking proteins, and switch to membrane microdomains where they can be readily endocytosed), and the mediators implicated (e.g., triggering of "cholestatic" signaling transduction pathways). Lastly, we discussed the efficacy to counteract the cholestatic failure induced by transporter internalization of a number of therapeutic experimental approaches based upon the use of compounds that trigger exocytic targetting of canalicular transporters (e.g., cAMP, tauroursodeoxycholate). This therapeutics may complement treatments aimed to transcriptionally improve transporter expression, by affording proper localization and membrane stability to the de novo synthesized transporters.
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Sistema Biliar/metabolismo , Colestasis/metabolismo , Hígado/metabolismo , Regulación hacia Abajo , Endocitosis , Humanos , Transducción de SeñalRESUMEN
Clinical conditions that result in endotoxemia, such as sepsis and alcoholic hepatitis (AH), often are accompanied by cholestasis. Although hepatocellular changes in response to lipopolysaccharide (LPS) have been well characterized, less is known about whether and how cholangiocytes contribute to this form of cholestasis. We examined effects of endotoxin on expression and function of the type 3 inositol trisphosphate receptor (ITPR3), because this is the main intracellular Ca2+ release channel in cholangiocytes, and loss of it impairs ductular bicarbonate secretion. Bile duct cells expressed the LPS receptor, Toll-like receptor 4 (TLR4), which links to activation of nuclear factor-κB (NF-κB). Analysis of the human ITPR3 promoter revealed five putative response elements to NF-κB, and promoter activity was inhibited by p65/p50. Nested 0.5- and 1.0-kilobase (kb) deletion fragments of the ITPR3 promoter were inhibited by NF-κB subunits. Chromatin immunoprecipitation (ChIP) assay showed that NF-κB interacts with the ITPR3 promoter, with an associated increase in H3K9 methylation. LPS decreased ITPR3 mRNA and protein expression and also decreased sensitivity of bile duct cells to calcium agonist stimuli. This reduction was reversed by inhibition of TLR4. ITPR3 expression was decreased or absent in cholangiocytes from patients with cholestasis of sepsis and from those with severe AH. Conclusion: Stimulation of TLR4 by LPS activates NF-κB to down-regulate ITPR3 expression in human cholangiocytes. This may contribute to the cholestasis that can be observed in conditions such as sepsis or AH.
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Conductos Biliares/efectos de los fármacos , Conductos Biliares/metabolismo , Endotoxemia/metabolismo , Endotoxinas/toxicidad , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Adulto , Señalización del Calcio/efectos de los fármacos , Colestasis/etiología , Colestasis/metabolismo , Endotoxemia/complicaciones , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Hepatitis Alcohólica/metabolismo , Humanos , Receptores de Inositol 1,4,5-Trifosfato/genética , Masculino , Persona de Mediana Edad , FN-kappa B/metabolismoRESUMEN
Background and rationale for the study. Bacterial translocation is an important triggering factor of infection and mortality in cirrhosis. In a rat model using bile duct ligation (BDL), bacterial translocation appears within 24 h after ligation. The dynamic between TH1/TH2/TH17 cytokines and the integrity of the colonic mucosa in the context of cirrhosis is little known. This study aims to determine the link between bacterial translocation and intestinal inflammation in a cholestasis model. Additionally, alterations of the colonic mucus layer and the bacterial load were also addressed. RESULTS: Bacterial translocation detected by microbiological cultures and MALDI-TOF showed that Escherichia coli predominates in mesenteric lymph nodes of BDL rats. Intestinal bacterial load analyzed by qPCR indicates a dramatic Escherichia/Shigella overgrowth at 8 and 30 days post-BDL. IFN-γ, IL-4, and IL-17 evaluated by Western blotting were increased at 8 and 30 days in the small intestine. In the colon, in contrast, only IFN-γ was significantly increased. The colonic mucus layer and mucin-2 expression determined by Alcian blue staining and immunohistochemistry surprisingly showed an increase in the mucus layer thickness related to increased mucin-2 expression during the entire process of liver damage. Hepatic enzymes, as well as collagen I, collagen III, TNF-α, and IL-6 liver gene expression were increased. In conclusion, bacterial overgrowth associated with bacterial translocation is linked to the over-expression of IFN-γ, IL-4, IL-17 and mucin-2. These molecules might facilitate the intestinal permeability through exacerbating the inflammatory process and disturbing tight junctions, leading to the perpetuation of the liver damage.