RESUMO
The effects of each type of machine perfusion preservation (MP) of liver grafts donated after cardiac death on the bile canaliculi of hepatocytes remain unclear. We analyzed the intracellular three-dimensional ultrastructure of the bile canaliculi and hepatocyte endomembrane systems in porcine liver grafts after warm ischemia followed by successive MP with modified University of Wisconsin gluconate solution. Transmission and osmium-maceration scanning electron microscopy revealed that lumen volume of the bile canaliculi decreased after warm ischemia. In liver grafts preserved by hypothermic MP condition, bile canaliculi tended to recover in terms of lumen volume, while their microvilli regressed. In contrast, midthermic MP condition preserved the functional form of the microvilli of the bile canaliculi. Machine perfusion preservation potentially restored the bile canaliculus lumen and alleviated the cessation of cellular endocrine processes due to warm ischemia. In addition, midthermic MP condition prevented the retraction of the microvilli of bile canaliculi, suggesting further mitigation of the damage of the bile canaliculi.
Assuntos
Canalículos Biliares/ultraestrutura , Morte , Fígado/ultraestrutura , Preservação de Órgãos , Animais , Feminino , Hepatócitos/ultraestrutura , Transplante de Fígado , Perfusão , Suínos , Temperatura , Isquemia QuenteRESUMO
Background: Neonates on long-term parenteral nutrition (PN) may develop parenteral nutrition-associated liver disease (PNALD). Aluminum (Al) is a known contaminant of infant PN, and we hypothesize that it substantially contributes to PNALD. In this study, we aim to assess the impact of Al on hepatocytes in a piglet model. Methods: We conducted a randomized control trial using a Yucatan piglet PN model. Piglets, aged 3â»6 days, were placed into two groups. The high Al group (n = 8) received PN with 63 µg/kg/day of Al, while the low Al group (n = 7) received PN with 24 µg/kg/day of Al. Serum samples for total bile acids (TBA) were collected over two weeks, and liver tissue was obtained at the end of the experiment. Bile canaliculus morphometry were studied by transmission electron microscopy (TEM) and ImageJ software analysis. Results: The canalicular space was smaller and the microvilli were shorter in the high Al group than in the low Al group. There was no difference in the TBA between the groups. Conclusions: Al causes structural changes in the hepatocytes despite unaltered serum bile acids. High Al in PN is associated with short microvilli, which could decrease the functional excretion area of the hepatocytes and impair bile flow.
Assuntos
Alumínio/toxicidade , Canalículos Biliares/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Hepatopatias/etiologia , Soluções de Nutrição Parenteral/toxicidade , Nutrição Parenteral/efeitos adversos , Animais , Animais Recém-Nascidos , Ácidos e Sais Biliares/metabolismo , Canalículos Biliares/metabolismo , Canalículos Biliares/ultraestrutura , Hepatócitos/ultraestrutura , Hepatopatias/metabolismo , Hepatopatias/patologia , Microscopia Eletrônica de Transmissão , Microvilosidades/efeitos dos fármacos , Microvilosidades/ultraestrutura , Suínos , Porco Miniatura , Fatores de TempoRESUMO
Spermine oxidase (SMOX) catalyzes oxidation of spermine to generate spermidine, hydrogen peroxide (H2O2) and 3-aminopropanal, which is spontaneously converted to acrolein. SMOX is induced by a variety of stimuli including bacterial infection, polyamine analogues and acetaldehyde exposure. However, the physiological functions of SMOX are not yet fully understood. We investigated the physiological role of SMOX in liver cells using human hepatocellular carcinoma cell line HepG2. SMOX localized to the bile canalicular lumen, as determined by F-actin staining. Knockdown of SMOX reduced the formation of bile canalicular lumen. We also found that phospho-Akt (phosphorylated protein kinase B) was localized to canalicular lumen. Treatment with Akt inhibitor significantly reduced the formation of bile canalicular lumen. Acrolein scavenger also inhibited the formation of bile canalicular lumen. PTEN, phosphatase and tensin homolog and an inhibitor of Akt, was alkylated in a SMOX-dependent manner. Our results suggest that SMOX plays a central role in the formation of bile canalicular lumen in liver cells by activating Akt pathway through acrolein production.
Assuntos
Acroleína/metabolismo , Canalículos Biliares/ultraestrutura , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/fisiologia , Actinas/metabolismo , Aldeídos/metabolismo , Alquilação , Canalículos Biliares/química , Células Hep G2 , Humanos , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/análise , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , PTEN Fosfo-Hidrolase/metabolismo , Fosforilação , Propilaminas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Poliamina OxidaseRESUMO
A primary amino-functionalized methyl methacrylate-based statistical copolymer is covalently coupled with retinoic acid (RA) and a fluorescent dye (DY590) in order to investigate the feasibility of the RA containing polymeric nanoparticles for Raman imaging studies and to study the possible selectivity of RA for hepatic stellate cells via intravital microscopy. Cationic nanoparticles are prepared by utilizing the nanoprecipitation method using modified polymers. Raman studies show that RA functional nanoparticles can be detectable in all tested cells without any need of additional label. Moreover, intravital microscopy indicates that DY590 is eliminated through the hepatobiliary route but not if used as covalently attached tracing molecule for nanoparticles. However, it is a suitable probe for sensitive detection of polymeric nanoparticles.
Assuntos
Canalículos Biliares/metabolismo , Células Estreladas do Fígado/metabolismo , Fígado/metabolismo , Nanopartículas/química , Polimetil Metacrilato/química , Tretinoína/química , Animais , Canalículos Biliares/ultraestrutura , Transporte Biológico , Portadores de Fármacos , Corantes Fluorescentes/química , Células Estreladas do Fígado/ultraestrutura , Humanos , Microscopia Intravital/métodos , Fígado/ultraestrutura , Camundongos , Nanopartículas/administração & dosagem , Microscopia Óptica não Linear/métodosRESUMO
BACKGROUND: We studied histologic changes of bile canalicular-ductule networks in the future liver remnant (FLR) while associating liver partition and portal vein occlusion for staged hepatectomy (ALPPS), since little is known about regeneration of these networks during the relatively short interval between procedures in ALPPS. METHODS: Bile canalicular-ductule networks were examined in specimens from eight patients treated with ALPPS and six patients undergoing hepatectomy following portal vein embolization (PVE). Expression of multidrug resistance-1 (MDR1), a membrane transporter in bile canaliculi (BC), was analyzed immunohistochemistcally. Morphologic changes of BC and tight junctions (TJs) adjoining BC were also assessed electron microscopically. RESULTS: Extrapolated kinetic growth of the FLR was greater during ALPPS (17.2 ± 6.8 mL/day) than after PVE (6.3 ± 3.4 mL/day; p = 0.005), and continuity of the MDR1-positive bile canalicular networks was less evident in ALPPS than PVE (p < 0.001). Electron microscopically, no significant difference was evident in numbers of BC or BC lumen size between the two groups; however, development of microvilli in BC was poorer in the ALPPS group than in the PVE group (p < 0.001). TJ/desmosome complexes were shorter in the ALPPS group (0.69 ± 0.52 µm) than in the PVE group (1.09 ± 0.50 µm; p < 0.001), and leaky TJs were seen more frequently in the ALPPS group (64.9 vs. 23.6%; p = 0.001). CONCLUSIONS: Regeneration of bile canalicular-ductule networks in the FLR was poorer in ALPPS than PVE, which may be associated with prolonged cholestasis following final hepatectomy in ALPPS.
Assuntos
Canalículos Biliares/patologia , Canalículos Biliares/ultraestrutura , Embolização Terapêutica , Hepatectomia/métodos , Neoplasias Hepáticas/terapia , Veia Porta , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Canalículos Biliares/metabolismo , Desmossomos/ultraestrutura , Feminino , Humanos , Imuno-Histoquímica , Fígado/crescimento & desenvolvimento , Masculino , Microscopia Eletrônica de Transmissão , Microvilosidades/ultraestrutura , Pessoa de Meia-Idade , Junções Íntimas/ultraestruturaRESUMO
Rift Valley fever (RVF) is a zoonotic disease that primarily affects ruminant animals and can also cause fatal disease in humans. In the current report, we present the ultrastructural changes in the liver of a man aged 60 years who died from RVF in the Aseer Central Hospital, Abha, Saudi Arabia. The main hepatic changes by transmission electron microscopy included the presence of 95-115â nm electron-dense particles consistent with RVF virions, nuclear condensation, vacuolar degeneration, lipid droplet accumulation and mitochondrial damage and dilation. There were also viral inclusion bodies with electron-dense aggregates, dilation of intercellular spaces, damage of sinusoidal microvilli with widening of space of Disse, dilation of bile canaliculi and increasing number of phagolysosomes.
Assuntos
Fígado/ultraestrutura , Febre do Vale de Rift/patologia , Vírus da Febre do Vale do Rift , Canalículos Biliares/patologia , Canalículos Biliares/ultraestrutura , Evolução Fatal , Humanos , Corpos de Inclusão Viral , Masculino , Microvilosidades , Pessoa de Meia-Idade , Fagossomos , Febre do Vale de Rift/virologia , Arábia Saudita , VírionRESUMO
AIM: To study whether transfer of blood between the right gastroepiploic artery and gastroduodenal artery could lessens the damage to bile canaliculi. METHODS: Forty male Bama miniature pigs were divided into four groups as follows: a control group, two hepatic artery ischemia groups (1 h and 2 h), and a hepatic artery bridging group. The hemodynamics of the hepatic artery in the hepatic artery bridging group was measured using color Doppler ultrasound. Morphological changes in the bile canaliculus were observed by transmission electron microscopy. Cofilin, heat shock protein 27 and F-actin expression was detected by immunohistochemistry, Western blot, and real-time polymerase chain reaction. Terminal deoxynucleotidyl transferase-mediated nick end-labeling method was used to evaluate liver injury. RESULTS: The hemodynamics was not changed in the hepatic artery bridging group. The microvilli in the bile canaliculus were impaired in the two hepatic artery ischemia groups. The down-regulation of cofilin and F-actin and up-regulation of heat shock protein 27 were observed in the two hepatic artery ischemia groups, while there were no significant differences between the control group and hepatic artery bridging group. CONCLUSION: Hepatic artery ischemia aggravates damage to bile canaliculi, and this damage can be diminished by a hepatic artery bridging duct.
Assuntos
Canalículos Biliares/ultraestrutura , Artéria Gastroepiploica/cirurgia , Artéria Hepática/fisiopatologia , Isquemia/prevenção & controle , Circulação Hepática , Fatores de Despolimerização de Actina/genética , Fatores de Despolimerização de Actina/metabolismo , Actinas/genética , Actinas/metabolismo , Animais , Canalículos Biliares/irrigação sanguínea , Canalículos Biliares/metabolismo , Biomarcadores/sangue , Velocidade do Fluxo Sanguíneo , Modelos Animais de Doenças , Artéria Gastroepiploica/fisiopatologia , Regulação da Expressão Gênica , Proteínas de Choque Térmico HSP27/genética , Proteínas de Choque Térmico HSP27/metabolismo , Artéria Hepática/diagnóstico por imagem , Isquemia/sangue , Isquemia/diagnóstico por imagem , Isquemia/genética , Isquemia/patologia , Isquemia/fisiopatologia , Masculino , Suínos , Porco Miniatura , Fatores de Tempo , Ultrassonografia Doppler em CoresRESUMO
The liver is unique in its capacity to regenerate after injury, during which hepatocytes actively divide and establish cell-cell contacts through cell adhesion complexes. Here, we demonstrate that the loss of α-catenin, a well-established adhesion component, dramatically disrupts liver regeneration. Using a partial hepatectomy model, we show that regenerated livers from α-catenin knockdown mice are grossly larger than control regenerated livers, with an increase in cell size and proliferation. This increased proliferation correlated with increased YAP activation, implicating α-catenin in the Hippo/YAP pathway. Additionally, α-catenin knockdown mice exhibited a phenotype reminiscent of clinical cholestasis, with drastically altered bile canaliculi, elevated levels of bile components and signs of jaundice and inflammation. The disrupted regenerative capacity is a result of actin cytoskeletal disorganisation, leading to a loss of apical microvilli, dilated lumens in the bile canaliculi, and leaky tight junctions. This study illuminates a novel, essential role for α-catenin in liver regeneration.
Assuntos
Colestase/genética , Regeneração Hepática/fisiologia , alfa Catenina/genética , Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Canalículos Biliares/patologia , Canalículos Biliares/ultraestrutura , Proteínas de Ciclo Celular , Proliferação de Células , Colestase/sangue , Feminino , Hepatócitos/fisiologia , Camundongos , Camundongos Knockout , Microvilosidades/ultraestrutura , Modelos Animais , Fosfoproteínas/metabolismo , Proteínas de Sinalização YAP , alfa Catenina/deficiênciaRESUMO
BACKGROUND: The bile canaliculus is the smallest and first biliary channel and is formed by two or three adjacent hepatocytes. Previous studies of chronic cholangiopathies such as primary sclerosing cholangitis have focused on the bile ductules. However, little is known about the pathological alterations in bile canaliculi in the early phase of cholangiopathies. AIM: To characterize the bile canalicular morphology in the early phase of sclerosing cholangitis we used 3,5-diethoxycarbonyl-1,4-dihydrocollidine-induced mouse model of sclerosing cholangitis. METHODS: Mice were fed a diet with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (0.1%). Serum biochemical, histological, immunohistochemical, and electron microscopic analyses were performed 1, 2, 4, and 7 days after feeding. RESULTS: All experimental groups showed significantly increased serum aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase levels. From day 1, bile canalicular abnormalities such as dilatation and meandering and loss of microvilli were observed. After bile canalicular abnormalities had appeared, substantial infiltration of inflammatory cells was observed amongst the necrotic cells and periductal region. After these inflammatory changes, cholangiocytes proliferated in the portal area and formed ductular reactions. Finally, periductal fibrosis appeared. CONCLUSION: This study provides novel evidence of the occurrence of bile canalicular abnormalities during the early phase of sclerosing cholangitis.
Assuntos
Canalículos Biliares/patologia , Colangite Esclerosante/patologia , Fígado/patologia , Animais , Canalículos Biliares/ultraestrutura , Ductos Biliares Intra-Hepáticos/patologia , Colangite Esclerosante/induzido quimicamente , Modelos Animais de Doenças , Progressão da Doença , Fígado/ultraestrutura , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Eletrônica , PiridinasRESUMO
Opisthorchis viverrini infection causes inflammation and liver injury leading to periductal fibrosis. Little is known about the pathological alterations in bile canaliculi in opisthorchiasis. This study aimed to investigate bile canalicular alterations in O. viverrini-infected hamsters and to examine the chemopreventive effects of curcumin on such changes. Hamsters were infected with O. viverrini and one group of animals was fed with 1% dietary curcumin supplement. Animals were examined during the acute infection phase, days 21 and 30 post-infection (PI) and chronic infection phase (day 90 PI). Scanning electron microscopy revealed that in the infected group fed with a normal diet, bile canaliculi became slightly tortuous by 30 day PI and more tortuous at day 90 PI. Transmission electron microscopy showed a reduction in microvilli density of canaliculi starting at day 30 PI, with a marked loss of microvilli at day 90 PI. These ultrastructral changes were slightly seen at day 21 PI, which was similar to that found in infected animals fed with 1% curcumin-supplemented diet. Notably, curcumin treatment prevented the reduction of microvilli density, reduced the dilation of bile canaliculi, and decreased the tortuosity of the bile canaliculi relative to non-infected animals on a normal diet at days 30 and 90 PI. These results suggest that curcumin reduces alteration of bile canaliculi and may be a promising agent to prevent the onset of bile duct abnormalities induced by O. viverrini infection.
Assuntos
Anti-Helmínticos/administração & dosagem , Canalículos Biliares/patologia , Curcumina/administração & dosagem , Opistorquíase/patologia , Opistorquíase/prevenção & controle , Opisthorchis/crescimento & desenvolvimento , Animais , Canalículos Biliares/ultraestrutura , Quimioprevenção/métodos , Cricetinae , Modelos Animais de Doenças , Elétrons , Fígado/patologia , Fígado/ultraestrutura , Masculino , Mesocricetus , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Opistorquíase/parasitologiaRESUMO
Hepatocytes are highly polarized cells where intercellular junctions, including tight junctions (TJs), determine the polarity. Recently, the TJ-associated proteins claudin-1 and occludin have been implicated in hepatitis C virus (HCV) entry and spread. Nevertheless, cell line-based experimental systems that exhibit hepatocyte-like polarity and permit robust infection and virion production are not currently available. Thus, we sought to determine whether cell line-based, Matrigel-embedded cultures could be used to study hepatitis C virus (HCV) infection and virion production in a context of hepatocyte-like polarized cells. In contrast to standard bidimensional cultures, Matrigel-cultured Huh-7 cells adopted hepatocyte polarization features forming a continuous network of functional proto-bile canaliculi structures. These 3D cultures supported HCV infection by JFH-1 virus and produced infective viral particles which shifted towards lower densities with higher associated specific infectivity. In conclusion, our findings describe a novel use of Matrigel to study the entire HCV cycle in a more relevant context.
Assuntos
Materiais Biocompatíveis/química , Técnicas de Cultura de Células/métodos , Colágeno/química , Hepacivirus/patogenicidade , Laminina/química , Proteoglicanas/química , Inclusão do Tecido/métodos , Vírion/metabolismo , Canalículos Biliares/ultraestrutura , Linhagem Celular Tumoral , Polaridade Celular , Combinação de Medicamentos , Hepacivirus/genética , Hepacivirus/metabolismo , Hepacivirus/ultraestrutura , Hepatite C/virologia , Hepatócitos/metabolismo , Hepatócitos/virologia , Humanos , Microscopia Confocal , Junções Íntimas/ultraestrutura , Vírion/ultraestruturaRESUMO
Hepatocytes are epithelial cells whose apical poles constitute the bile canaliculi. The establishment and maintenance of canalicular poles is a finely regulated process that dictates the efficiency of primary bile secretion. Protein kinase A (PKA) modulates this process at different levels. AKAP350 is an A-kinase anchoring protein that scaffolds protein complexes involved in modulating the dynamic structures of the Golgi apparatus and microtubule cytoskeleton, facilitating microtubule nucleation at this organelle. In this study, we evaluated whether AKAP350 is involved in the development of bile canaliculi-like structures in hepatocyte derived HepG2 cells. We found that AKAP350 recruits PKA to the centrosomes and Golgi apparatus in HepG2 cells. De-localization of AKAP350 from these organelles led to reduced apical cell polarization. A decrease in AKAP350 expression inhibited the formation of canalicular structures and impaired F-actin organization at canalicular poles. Furthermore, loss of AKAP350 expression led to diminished polarized expression of the p-glycoprotein (MDR1/ABCB1) at the apical "canalicular" membrane. AKAP350 knock down effects on canalicular structures formation and actin organization could be mimicked by inhibition of Golgi microtubule nucleation by depletion of CLIP associated proteins (CLASPs). Our data reveal that AKAP350 participates in mechanisms which determine the development of canalicular structures as well as accurate canalicular expression of distinct proteins and actin organization, and provide evidence on the involvement of Golgi microtubule nucleation in hepatocyte apical polarization.
Assuntos
Proteínas de Ancoragem à Quinase A/metabolismo , Canalículos Biliares/metabolismo , Canalículos Biliares/ultraestrutura , Polaridade Celular/fisiologia , Proteínas do Citoesqueleto/metabolismo , Hepatócitos/metabolismo , Hepatócitos/ultraestrutura , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestrutura , Centrossomo/metabolismo , Centrossomo/ultraestrutura , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Imunofluorescência , Complexo de Golgi/metabolismo , Complexo de Golgi/ultraestrutura , Células Hep G2 , Humanos , Immunoblotting , Microscopia Confocal , Microscopia de FluorescênciaRESUMO
PURPOSE: Parenteral nutrition-associated cholestasis remains a significant problem, especially for the surgical neonate. Aluminum is a toxic element known to contaminate parenteral nutrition. We hypothesize that parenterally administered aluminum causes liver injury similar to that seen in parenteral nutrition-associated cholestasis. METHODS: Twenty 3- to 6-day-old domestic pigs were divided into 5 equal groups. A control group received daily intravenous 0.9% sodium chloride. Each subject in experimental groups received intravenous aluminum chloride at 1500 µg/kg per day for 1, 2, 3, or 4 weeks. At the end of the study, blood was sampled for direct bilirubin and total bile acid levels. Liver, bile, and urine were sampled for aluminum content. Liver tissue was imaged by transmission electron microscopy for ultrastructural changes. RESULTS: Transmission electron microscopy revealed marked blunting of bile canaliculi microvilli in all experimental subjects but not the controls. Serum total bile acids correlated with the duration of aluminum exposure. The hepatic aluminum concentration correlated with the duration of aluminum exposure. CONCLUSIONS: Parenterally infused aluminum resulted in liver injury as demonstrated by elevated bile acids and by blunting of the bile canaliculi microvilli. These findings are similar to those reported in early parenteral nutrition-associated liver disease.
Assuntos
Compostos de Alumínio/toxicidade , Alumínio/análise , Canalículos Biliares/efeitos dos fármacos , Cloretos/toxicidade , Colestase/induzido quimicamente , Modelos Animais de Doenças , Fígado/efeitos dos fármacos , Nutrição Parenteral/efeitos adversos , Sus scrofa , Alumínio/sangue , Alumínio/urina , Cloreto de Alumínio , Compostos de Alumínio/administração & dosagem , Animais , Animais Recém-Nascidos , Bile/química , Ácidos e Sais Biliares/sangue , Canalículos Biliares/ultraestrutura , Bilirrubina/sangue , Cloretos/administração & dosagem , Colestase/patologia , Relação Dose-Resposta a Droga , Contaminação de Medicamentos , Microanálise por Sonda Eletrônica , Injeções Intravenosas , Fígado/química , Fígado/ultraestrutura , Microscopia Eletrônica , Microvilosidades/efeitos dos fármacos , Microvilosidades/ultraestrutura , Soluções de Nutrição Parenteral/efeitos adversos , SuínosRESUMO
There are different types of membranes used for hepatocyte cultivation. In our studies, spongy polyethersulfone (PES) membranes were examined as a support for hepatic cell cultivation in vitro. The extended surface of the membranes allows to introduce a high cell number especially in three-dimensional gel structure. Scanning electron microscopy analysis indicated that C3A cells used in our experiments grew well on PES membranes forming microvilli characteristic for normal hepatocytes. Analysis of cell viability proved that spongy PES membrane is well tolerated by J774 macrophages and did not stimulate nitric oxide synthesis. Bile canalicular structures were observed in fluorescence microscopy after F-actin staining with tetramethyl rhodamine iso-thiocyanate (TRITC)-phalloidin. The C3A cells showed high affinity to the PES membranes and adhered to almost 90% during the initial 24 h of incubation. Albumin production increased during static culture from the value of 805.2 +/- 284.4 (ng/24 h/initial 10(6) cells) during the first days, to 2017.6 +/- 505.9 (ng/24 h/initial 10(6) cells) after 10 days of culture. In conclusion, the spongy PES membranes can be used as scaffold for hepatocyte cultivation, especially for the creation of three-dimensional environments.
Assuntos
Técnicas de Cultura de Células , Hepatócitos/citologia , Hepatócitos/metabolismo , Membranas Artificiais , Polímeros/química , Sulfonas/química , Animais , Canalículos Biliares/ultraestrutura , Carcinoma Hepatocelular/metabolismo , Adesão Celular , Linhagem Celular Tumoral , Sobrevivência Celular , Células Cultivadas/metabolismo , Humanos , Macrófagos/metabolismo , CamundongosRESUMO
Cholestasis, induced by liver ischemia-reperfusion injury (IRI), is characterized by dilatation of bile canaliculi and loss of microvilli. Tauroursodeoxycholic acid (TUDCA) is an anti-cholestatic agent, modulating protein kinase C (PKC) alpha pathway. PKC reduces ischemic damage in several organs, its isoform alpha modulates ezrin, a key protein in the maintenance of cell lamellipoidal extensions. We evaluated the effects of TUDCA on cholestasis, canalicular changes and PKCalpha-ezrin expression in a rat model of liver IRI. Livers flushed and stored with Belzer solution or Belzer + 10 mm TUDCA (4 degrees C for 6 h) were reperfused (37 degrees C with O(2)) with Krebs-Ringer bicarbonate + 2.5 micromol/min of Taurocholate or TUDCA. Bile was harvested for bile flow assessment. Liver tissue was employed for Electron Microscopy (EM) and for PKCalpha and ezrin immunoblot and immunofluorescence. The same experiments were conducted with the PKCalpha inhibitor Go-6976. TUDCA-treated livers showed increased bile flow (0.25+/-0.17 vs. 0.042+/-0.02 microl/min/g liver, P<0.05) and better preservation of microvilli and bile canalicular area at EM. These effects were associated with increased PKCalpha and ezrin expression (P=0.03 and P=0.04 vs. control respectively), as also confirmed by immunofluorescence data. PKCalpha inhibition abolished these TUDCA effects. TUDCA administration during IRI reduces cholestasis and canalicular damage in the liver modulating PKCalpha-ezrin pathway.
Assuntos
Canalículos Biliares/patologia , Colagogos e Coleréticos/uso terapêutico , Colestase/prevenção & controle , Proteínas do Citoesqueleto/metabolismo , Fígado/irrigação sanguínea , Proteína Quinase C-alfa/metabolismo , Traumatismo por Reperfusão/prevenção & controle , Ácido Tauroquenodesoxicólico/uso terapêutico , Animais , Bile , Canalículos Biliares/ultraestrutura , Carbazóis/farmacologia , Colestase/etiologia , Colestase/patologia , Colestase/fisiopatologia , Proteínas do Citoesqueleto/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , L-Lactato Desidrogenase/metabolismo , Fígado/metabolismo , Masculino , Microscopia Eletrônica de Varredura , Proteína Quinase C-alfa/antagonistas & inibidores , Proteína Quinase C-alfa/efeitos dos fármacos , Ratos , Ratos Wistar , Traumatismo por Reperfusão/complicações , Traumatismo por Reperfusão/patologia , Ácido Tauroquenodesoxicólico/farmacologiaRESUMO
Calcium-mobilizing hormones and neurotransmitters are known to affect cell morphology and function including cell differentiation or division. In this study, we examined vasopressin (AVP)-induced morphological changes in a polarized system of rat hepatocytes. Light and electron microscope observations showed that AVP induced microvilli formation and a remodeling of the isolated hepatocyte F-actin submembrane cytoskeleton, these two events being correlated. We showed that these effects were rapid, reversible, observed at nanomolar AVP concentration and mediated by the V(1a) receptor. On polarized multicellular systems of hepatocytes, we observed a rapid reduction of the bile canaliculi lumen at the apical pole and micovilli formation at the basolateral domain with an enlarged F-actin cytoskeleton. Neither activation of protein kinase C nor A via phorbol ester or dibutyryl cAMP induced such rapid morphological changes, at variance with ionomycin, suggesting that AVP-induced intracellular calcium rise plays a crucial role in those effects. By using spectrofluorimetry and cytochemistry, we showed that calcium release from intracellular stores was involved in bile canaliculus contraction, while calcium entry from the extracellular space controlled microvilli formation. Taken together, AVP and calcium-mobilizing agonists differentially regulate physiological hepatocyte plasma membrane events at the basal and the apical domains via topographically specialized calcium-dependent mechanisms.
Assuntos
Arginina Vasopressina/farmacologia , Cálcio/metabolismo , Hepatócitos/ultraestrutura , Citoesqueleto de Actina/ultraestrutura , Actinas/análise , Animais , Canalículos Biliares/efeitos dos fármacos , Canalículos Biliares/ultraestrutura , Polaridade Celular , Células Cultivadas , Feminino , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Microvilosidades/efeitos dos fármacos , Microvilosidades/ultraestrutura , Ratos , Ratos WistarRESUMO
BACKGROUND AND AIM: The precise mechanism of bile regurgitation from the biliary system to the blood stream still remains to be elucidated. The aim of this study was to examine the initial site of bile regurgitation in vivo after common bile duct (CBD) obstruction by digitally enhanced fluorescence microscopy. METHODS: The fluorescence excreted into bile canaliculi after the administration of sodium fluorescein was continuously observed in CBD obstruction, using video-enhanced contrast (VEC) microscopy equipped with a silicon intensified target (SIT) camera. The liver histology and the localization of Mg(2+)-ATPase were examined by light and electron microscopy. RESULTS: By the continuous recording of canalicular fluorescence, the sequential regurgitation of the fluorescence from the canaliculi to the hepatocyte cytoplasm to the sinusoids was distinctively recognized after CBD obstruction. Bile canalicular fluorescence was enhanced, and then the fluorescence of the hepatocyte cytoplasm increased in intensity, followed by regurgitation of the fluorescence to the sinusoids. These in vivo sequences closely correlated with changes in CBD pressure. In zone 1, canalicular fluorescence focally burst into hepatocyte cytoplasm, thus resulting in the formation of fluorescent cells. By light and electron microscopy, the fluorescent cells were found to correspond to the liver cell injury. The reaction products of Mg(2+)-ATPase were incorporated into vesicles with a decreased canalicular activity, and then were transported to the sinusoidal surface after CBD obstruction. CONCLUSIONS: The initial site of bile regurgitation may be transcellular, and partly involves liver cell injury in zone 1 in extrahepatic biliary obstruction, associated with increased pressure of the biliary system.
Assuntos
Canalículos Biliares/metabolismo , Bile/metabolismo , Colestase Extra-Hepática/metabolismo , Fígado/metabolismo , Microscopia de Fluorescência , Microscopia de Vídeo/métodos , Animais , Canalículos Biliares/enzimologia , Canalículos Biliares/ultraestrutura , Vasos Sanguíneos/metabolismo , ATPase de Ca(2+) e Mg(2+)/metabolismo , Colestase Extra-Hepática/sangue , Colestase Extra-Hepática/patologia , Citoplasma/metabolismo , Modelos Animais de Doenças , Feminino , Fluoresceína , Corantes Fluorescentes , Hepatócitos/metabolismo , Fígado/ultraestrutura , Microscopia Eletrônica , Ratos , Ratos Wistar , Processamento de Sinais Assistido por Computador , Fatores de TempoRESUMO
Arsenic is a double-edged sword to human health. The excretion of various organic anions into bile is mediated by an adenosine triphosphate-dependent conjugate export pump, which has been identified as the canalicular isoform of the multidrug resistance protein 2 (Mrp2). It has been proved that Mrp2 can transport arsenite in vitro, but its effects in vivo are not clear. The aim of this study was to investigate whether Mrp2 plays a role in exportation of arsenic in vivo and its protective effects on liver function. Mrp2 protein level in rat liver was determined by Western blot analysis. Total arsenic concentrations in whole blood and bile were measured using hydride generation atomic absorption spectrometry. Alanine aminotransferase (ALT) activity, aspartate aminotransferase activity (AST), glutathione peroxidase (GSH-PX) activity, malon dialdehyde (MDA) and total bilirubin were measured by biochemical assays. The morphological changes were observed by electron microscopy. Total arsenic levels in blood and bile of arsenite-treated rats were significantly higher than those of control rats (P<0.05) at all three different time points. The overexpression of Mrp2 was 36.61%, 32.36% and 12.73% at 2, 4 and 6 weeks, respectively (percentage of controls, P<0.05), which was significantly higher than controls. A positive correlation between Mrp2 expression level and total arsenic concentration in bile indicated that Mrp2 accelerated the transport of arsenic. Electron microscopy showed that microvilli of bile canaliculi became swollen and sparse. ALT and AST activities in serum were markedly raised at 6 weeks. MDA level in serum increased (P<0.05) and GSH-PX activity in serum decreased except for 2 weeks. Damage of liver function became worse following decreased expression of Mrp2. In conclusion, overexpression of Mrp2 may explain increased biliary excretion of arsenic and it may protect liver function.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Intoxicação por Arsênico/sangue , Arsenitos/toxicidade , Doença Hepática Induzida por Substâncias e Drogas/sangue , Inibidores Enzimáticos/toxicidade , Fígado/efeitos dos fármacos , Compostos de Sódio/toxicidade , Alanina Transaminase/sangue , Animais , Arsênio/sangue , Intoxicação por Arsênico/patologia , Arsenitos/metabolismo , Aspartato Aminotransferases/sangue , Bile/química , Bile/metabolismo , Canalículos Biliares/efeitos dos fármacos , Canalículos Biliares/ultraestrutura , Bilirrubina/sangue , Western Blotting , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Doença Hepática Induzida por Substâncias e Drogas/patologia , Inibidores Enzimáticos/metabolismo , Feminino , Glutationa Peroxidase/sangue , Fígado/metabolismo , Masculino , Malondialdeído/sangue , Ratos , Ratos Wistar , Compostos de Sódio/metabolismo , Espectrofotometria AtômicaRESUMO
In rodent livers, integral tight junction (TJ) proteins claudin-1, -2, -3, -5 and -14 are detected and play crucial roles in the barrier to keep bile in bile canaculi away from the blood circulation. Claudin-2 shows a lobular gradient increasing from periportal to pericentral hepatocytes, whereas claudin-1 and -3 are expressed in the whole liver lobule. Although claudin-2 expression induces cation-selective channels in tight junctions of epithelial cells, the physiological functions and regulation of claudin-2 in hepatocytes remain unclear. Oncostatin M (OSM) is a multifunctional cytokine implicated in the differentiation of hepatocytes that induces formation of E-cadherin-based adherens junctions in fetal hepatocytes. In this study, we examined whether OSM could induce expression and function of claudin-2 in rodent hepatocytes, immortalized mouse and primary cultured proliferative rat hepatocytes. In the immortalized mouse and primary cultured proliferative rat hepatocytes, treatment with OSM markedly increased mRNA and protein of claudin-2 together with formation of developed networks of TJ strands. The increase of claudin-2 enhanced the paracellular barrier function which depended on molecular size. The increase of claudin-2 expression induced by OSM in rodent hepatocytes was regulated through distinct signaling pathways including PKC. These results suggest that expression of claudin-2 in rodent hepatocytes may play a specific role as controlling the size of paracellular permeability in the barrier to keep bile in bile canaculi.