RESUMO
Three-dimensional (3D) geometrical models are potent tools for quantifying complex tissue features and exploring structure-function relationships. However, these models are generally incomplete due to experimental limitations in acquiring multiple (> 4) fluorescent channels in thick tissue sections simultaneously. Indeed, predictive geometrical and functional models of the liver have been restricted to few tissue and cellular components, excluding important cellular populations such as hepatic stellate cells (HSCs) and Kupffer cells (KCs). Here, we combined deep-tissue immunostaining, multiphoton microscopy, deep-learning techniques, and 3D image processing to computationally expand the number of simultaneously reconstructed tissue structures. We then generated a spatial single-cell atlas of hepatic architecture (Hep3D), including all main tissue and cellular components at different stages of post-natal development in mice. We used Hep3D to quantitatively study 1) hepatic morphodynamics from early post-natal development to adulthood, and 2) the effect on the liver's overall structure when changing the hepatic environment after removing KCs. In addition to a complete description of bile canaliculi and sinusoidal network remodeling, our analysis uncovered unexpected spatiotemporal patterns of non-parenchymal cells and hepatocytes differing in size, number of nuclei, and DNA content. Surprisingly, we found that the specific depletion of KCs results in morphological changes in hepatocytes and HSCs. These findings reveal novel characteristics of liver heterogeneity and have important implications for both the structural organization of liver tissue and its function. Our next-gen 3D single-cell atlas is a powerful tool to understand liver tissue architecture, opening up avenues for in-depth investigations into tissue structure across both normal and pathological conditions.
Assuntos
Hepatócitos , Fígado , Camundongos , Animais , Fígado/patologia , Células de Kupffer/patologia , Células Estreladas do Fígado/patologia , Canalículos BiliaresRESUMO
Gaucher disease (GD) is an autosomal recessive lysosomal disorder caused by a disturbance in the metabolism of glucocerebroside in the macrophages. Most of its manifestations - hepatosplenomegaly, anemia, thrombocytopenia, and bone pain - are amenable to a macrophage-target therapy such as enzyme replacement. However, there is increasing evidence that abnormalities of the liver persist despite the specific GD treatment. In this work, we adapted histomorphometry techniques to the study of hepatocytes in GD using liver tissue of treated patients, developing the first morphometrical method for canalicular quantification in immunohistochemistry-stained liver biopsies, and exploring histomorphometric characteristics of GD. This is the first histomorphometric technique developed for canalicular analysis on histological liver biopsy samples.
Assuntos
Humanos , Citometria por Imagem/métodos , Doença de Gaucher/terapia , Canalículos Biliares , Hepatócitos , Biópsia com Agulha de Grande CalibreRESUMO
In previous studies, we showed that the pro-oxidant model agent tert-butyl hydroperoxide (tBuOOH) induces alterations in hepatocanalicular secretory function by activating Ca2+-dependent protein kinase C isoforms (cPKC), via F-actin disorganization followed by endocytic internalization of canalicular transporters relevant to bile formation (Mrp2, Bsep). Since mitogen-activated protein kinases (MAPKs) may be downstream effectors of cPKC, we investigated here the involvement of the MAPKs of the ERK1/2, JNK1/2, and p38MAPK types in these deleterious effects. tBuOOH (100 µM, 15 min) increased the proportion of the active, phosphorylated forms of ERK1/2, JNK1/2, and p38MAPK, and panspecific PKC inhibition with bisindolylmaleimide-1 (100 nM) or selective cPKC inhibition with Gö6976 (1 µM) prevented the latter two events. In isolated rat hepatocyte couplets, tBuOOH (100 µM, 15 min) decreased the canalicular vacuolar accumulation of the fluorescent Bsep and Mrp2 substrates, cholylglycylamido fluorescein, and glutathione-methylfluorescein, respectively, and selective inhibitors of ERK1/2 (PD098059), JNK1/2 (SP600125), and p38MAPK (SB203580) partially prevented these alterations. In in situ perfused rat livers, these three MAPK inhibitors prevented tBuOOH (75 µM)-induced impairment of bile flow and the decrease in the biliary output of the Bsep and Mrp2 substrates, taurocholate, and dinitrophenyl-S-glutathione, respectively. The changes in Bsep/Mrp2 and F-actin localization induced by tBuOOH, as assessed by (immuno)fluorescence staining followed by analysis of confocal images, were prevented total or partially by the MAPK inhibitors. We concluded that MAPKs of the ERK1/2, JNK1/2, and p38MAPK types are all involved in cholestasis induced by oxidative stress, by promoting F-actin rearrangement and further endocytic internalization of canalicular transporters critical for bile formation.
Assuntos
Canalículos Biliares/efeitos dos fármacos , Colestase/induzido quimicamente , Fígado/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Estresse Oxidativo/efeitos dos fármacos , terc-Butil Hidroperóxido/toxicidade , Animais , Canalículos Biliares/metabolismo , Canalículos Biliares/fisiopatologia , Colestase/metabolismo , Fígado/metabolismo , Fígado/fisiopatologia , Masculino , Proteína Quinase C/metabolismo , Ratos WistarRESUMO
UNLABELLED: The adenoviral gene transfer of human aquaporin-1 (hAQP1) water channels to the liver of 17α-ethinylestradiol-induced cholestatic rats improves bile flow, in part by enhancing canalicular hAQP1-mediated osmotic water secretion. To gain insight into the mechanisms of 17α-ethinylestradiol cholestasis improvement, we studied the biliary output of bile salts (BS) and the functional expression of the canalicular BS export pump (BSEP; ABCB11). Adenovector encoding hAQP1 (AdhAQP1) or control vector was administered by retrograde intrabiliary infusion. AdhAQP1-transduced cholestatic rats increased the biliary output of major endogenous BS (50%-80%, P < 0.05) as well as that of taurocholate administered in choleretic or trace radiolabel amounts (around 60%, P < 0.05). Moreover, liver transduction with AdhAQP1 normalized serum BS levels, otherwise markedly elevated in cholestatic animals. AdhAQP1 treatment was unable to improve BSEP protein expression in cholestasis; however, its transport activity, assessed by adenosine triphosphate-dependent taurocholate transport in canalicular membrane vesicles, was induced by 90% (P < 0.05). AdhAQP1 administration in noncholestatic rats induced no significant changes in either biliary BS output or BSEP activity. Canalicular BSEP, mostly present in raft (high cholesterol) microdomains in control rats, was largely found in nonraft (low cholesterol) microdomains in cholestasis. Considering that BSEP activity directly depends on canalicular membrane cholesterol content, decreased BSEP presence in rafts may contribute to BSEP activity decline in 17α-ethinylestradiol cholestasis. In AdhAQP1-transduced cholestatic rats, BSEP showed a canalicular microdomain distribution similar to that of control rats, which provides an explanation for the improved BSEP activity. CONCLUSION: Hepatocyte canalicular expression of hAQP1 through adenoviral gene transfer promotes biliary BS output by modulating BSEP activity in estrogen-induced cholestasis, a novel finding that might help us to better understand and treat cholestatic disorders. (Hepatology 2016;64:535-548).
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Aquaporina 1/genética , Ácidos e Sais Biliares/metabolismo , Colestase/terapia , Terapia Genética , Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Animais , Aquaporina 1/metabolismo , Ácidos e Sais Biliares/sangue , Canalículos Biliares/metabolismo , Colestase/induzido quimicamente , Etinilestradiol , Hepatócitos/metabolismo , Humanos , Masculino , Ratos WistarRESUMO
Estradiol-17ß-D-glucuronide (E17G) induces acute endocytic internalization of canalicular transporters, including multidrug resistance-associated protein 2 (Abcc2) in rat, generating cholestasis. Several proteins organized in at least two different signaling pathways are involved in E17G cholestasis: one pathway involves estrogen receptor alpha (ERα), Ca(2+)-dependent protein kinase C and p38-mitogen activated protein kinase, and the other pathway involves GPR30, PKA, phosphoinositide 3-kinase/AKT and extracellular signal-related kinase 1/2. EGF receptor (EGFR) can potentially participate in both pathways since it interacts with GPR30 and ERα. Hence, the aim of this study was to analyze the potential role of this receptor and its downstream effectors, members of the Src family kinases in E17G-induced cholestasis. In vitro, EGFR inhibition by Tyrphostin (Tyr), Cl-387785 or its knockdown with siRNA strongly prevented E17G-induced impairment of Abcc2 function and localization. Activation of EGFR was necessary but not sufficient to impair the canalicular transporter function, whereas the simultaneous activation of EGFR and GPR30 could impair Abcc2 transport. The protection of Tyr was not additive to that produced by the ERα inhibitor ICI neither with that produced by Src kinase inhibitors, suggesting that EGFR shared the signaling pathway of ERα and Src. Further analysis of ERα, EGFR and Src activations induced by E17G, demonstrated that ERα activation precedes that of EGFR and EGFR activation precedes that of Src. In conclusion, activation of EGFR is a key factor in the alteration of canalicular transporter function and localization induced by E17G and it occurs before that of Src and after that of ERα.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Receptores ErbB/metabolismo , Estradiol/análogos & derivados , Receptor alfa de Estrogênio/metabolismo , Hepatócitos/metabolismo , Animais , Canalículos Biliares/efeitos dos fármacos , Canalículos Biliares/metabolismo , Canalículos Biliares/fisiopatologia , Células Cultivadas , Colestase/induzido quimicamente , Colestase/metabolismo , Receptores ErbB/genética , Estradiol/metabolismo , Estradiol/farmacologia , Antagonistas do Receptor de Estrogênio/farmacologia , Feminino , Fulvestranto , Técnicas de Silenciamento de Genes , Hepatócitos/efeitos dos fármacos , Quinazolinas/farmacologia , Ratos , Ratos Wistar , Tirfostinas/farmacologia , Quinases da Família src/metabolismoRESUMO
OBJETIVO: Avaliar a prevalência da baixa densidade mineral óssea (DMO) em mulheres na pós-menopausa tratadas de câncer de mama. MÉTODOS: Estudo de corte transversal que incluiu 115 mulheres tratadas de câncer de mama atendidas em Hospital Universitário do Sudeste do Brasil. Foram incluídas mulheres com amenorreia há 12 meses ou mais e 45 anos ou mais de idade, tratadas de câncer de mama e livres de doença há pelo menos 5 anos. A DMO foi mensurada pelos raios-X de dupla energia em coluna lombar (L1 a L4) e colo de fêmur. Considerou-se baixa DMO quando valores de T-score de coluna total e/ou colo de fêmur <-1,0 Score de Delphi (DP) (osteopenia e osteoporose). Por meio de entrevista, foram avaliados fatores de risco para baixa DMO. Na análise estatística, empregaram-se os testes do χ2 ou Exato de Fisher. RESULTADOS: A média de idade das pacientes foi 61,6±10,1 anos e o tempo de menopausa, 14,2±5,6 anos, com tempo médio de seguimento de 10,1±3,9 anos. Considerando coluna e colo de fêmur, 60% das mulheres tratadas de câncer de mama apresentavam baixa DMO. Avaliando os fatores de risco para baixa DMO, foi encontrada diferença significativa na distribuição percentual quanto à idade (maior porcentagem de mulheres com mais de 50 anos e baixa DMO), história pessoal de fratura prévia (11,6% com baixa DMO e nenhuma com DMO normal) e índice de massa corpórea. Maior frequência de obesidade foi observada entre mulheres com DMO normal (63%) quando comparadas àquelas com baixa DMO (26,1%; p<0,05). CONCLUSÃO: Mulheres na pós-menopausa tratadas de câncer de mama apresentaram elevada prevalência de baixa DMO (osteopenia e/ou osteoporose). .
PURPOSE: To evaluate the prevalence of low bone mineral density (BMD) in postmenopausal breast cancer survivors. METHODS: In this cross-sectional study, 115 breast cancer survivors, seeking healthcare at a University Hospital in Brazil, were evaluated. Eligibility criteria included women with amenorrhea ≥12 months and age ≥45 years, treated for breast cancer and metastasis-free for at least five years. BMD was measured by DEXA at the lumbar spine (L1-L4) and femoral neck. Low BMD was considered when total-spine and/or femoral-neck T-score values were <-1.0 Delphi Score (DP) (osteopenia and osteoporosis). The risk factors for low BMD were assessed by interview. Data were analyzed statistically by the χ2 test and Fisher's exact test. RESULTS: The mean age of breast cancer survivors was 61.6±10.1 years and time since menopause was 14.2±5.6 years, with a mean follow-up of 10.1±3.9 years. Considering spine and femoral neck, 60% of breast cancer survivors had low BMD. By evaluating the risk factors for low BMD, a significant difference was found in the percent distribution for age (higher % of women >50 years with low BMD), personal history of previous fracture (11.6% with low BMD versus 0% with normal BMD) and BMI. A higher frequency of obesity was observed among women with normal BMD (63%) compared to those with low BMD (26.1%) (p<0.05). CONCLUSION: Postmenopausal breast cancer survivors had a high prevalence of osteopenia and osteoporosis. .
Assuntos
Animais , Ratos , Ácidos e Sais Biliares/metabolismo , Canalículos Biliares/metabolismo , Proteínas de Transporte/metabolismo , Hidroxiesteroide Desidrogenases , Glicoproteínas de Membrana , Adenosina Trifosfatases/metabolismo , Transporte Biológico , Células COS , Antígeno Carcinoembrionário/biossíntese , Proteínas de Transporte/biossíntese , Primers do DNA , DNA Complementar , Íleo/metabolismo , Cinética , Mutagênese Sítio-Dirigida , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/metabolismo , Transfecção , Ácido Taurocólico/metabolismoRESUMO
At present, it has not been systematically evaluated whether the functional alterations induced by cholestatic compounds in canalicular transporters involved in bile formation can be reproduced in sandwich-cultured rat hepatocytes (SCRHs). Here, we focused on two clinically relevant cholestatic agents, such as estradiol 17ß-D-glucuronide (E17G) and taurolithocholate (TLC), also testing the ability of dibutyryl cyclic AMP (DBcAMP) to prevent their effects. SCRHs were incubated with E17G (200 µM) or TLC (2.5 µM) for 30 min, with or without pre-incubation with DBcAMP (10 µM) for 15 min. Then, the increase in glutathione methyl fluorescein (GS-MF)-associated fluorescence inside the canaliculi was monitored by quantitative time-lapse imaging, and Mrp2 transport activity was calculated by measuring the slope of the time-course fluorescence curves during the initial linear phase, which was considered to be the Mrp2-mediated initial transport rate (ITR). E17G and TLC impaired canalicular bile formation, as evidenced by a decrease in both the bile canaliculus volume and the bile canaliculus width, estimated from 3D and 2D confocal images, respectively. These compounds decreased ITR and induced retrieval of Mrp2, a main pathomechanism involved in their cholestatic effects. Finally, DBcAMP prevented these effects, and its well-known choleretic effect was evident from the increase in the canalicular volume/width values; this choleretic effect is associated in part with its capability to increase Mrp2 activity, evidenced here by the increase in ITR of GS-MF. Our study supports the use of SCRHs as an in vitro model useful to quantify canalicular transport function under conditions of cholestasis and choleresis.
Assuntos
Canalículos Biliares/metabolismo , Bile/metabolismo , Transporte Biológico , Colestase/metabolismo , Hepatócitos/metabolismo , Modelos Biológicos , Animais , Canalículos Biliares/efeitos dos fármacos , Bucladesina/farmacologia , Técnicas de Cultura de Células , Células Cultivadas , Colestase/induzido quimicamente , Estradiol/análogos & derivados , Estradiol/farmacologia , Hepatócitos/efeitos dos fármacos , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Ratos , Ácido Taurolitocólico/farmacologiaRESUMO
OBJECTIVES: Partial external bile diversion (PEBD) is an established therapy for low-γ-glutamyl transferase (GGT) progressive familial intrahepatic cholestasis (PFIC). This study sought to determine whether the dynamics of the cholic acid (CA) and chenodeoxycholic acid (CDCA) pools in subjects with low-GGT-PFIC with successful PEBD were equivalent to those achieved with successful liver transplantation (LTX). METHODS: The kinetics of CA and CDCA metabolism were measured by stable isotope dilution in plasma samples in 5 subjects with PEBD, all with intact canalicular bile salt export pump expression and compared with subjects with low-GGT-PFIC with successful LTX. Stomal loss of bile acids was measured in subjects with PEBD. RESULTS: The fractional turnover rate for CA in the PEBD group ranged from 0.5 to 4.2/day (LTX group, range 0.2-0.9/day, P = 0.076) and for CDCA from 0.7 to 4.5/day (LTX group 0.3-0.4/day, P = 0.009). The CA and CDCA pool sizes were equivalent between groups; however, pool composition in PEBD was somewhat more hydrophilic. The CA/CDCA ratio in PEBD ranged from 0.9 to 19.5, whereas in LTX it ranged from 0.5 to 2.6. Synthesis rates computed from isotope dilution correlated well with timed output for both CA (r2 = 0.760, P = 0.024) and CDCA (r2 = 0.690, P = 0.021). CONCLUSIONS: PEBD results in bile acid fractional turnover rates greater than LTX, pool sizes equivalent to LTX, and pool composition that is at least as hydrophilic as produced by LTX.
Assuntos
Ácidos e Sais Biliares/metabolismo , Coledocostomia/efeitos adversos , Colestase Intra-Hepática/cirurgia , Fígado/metabolismo , Adolescente , Adulto , Ácidos e Sais Biliares/sangue , Canalículos Biliares/metabolismo , Canalículos Biliares/patologia , Ductos Biliares Intra-Hepáticos/cirurgia , Criança , Pré-Escolar , Colestase Intra-Hepática/sangue , Colestase Intra-Hepática/metabolismo , Colestase Intra-Hepática/patologia , Deutério , Feminino , Humanos , Interações Hidrofóbicas e Hidrofílicas , Lactente , Jejuno/cirurgia , Cinética , Fígado/patologia , Transplante de Fígado/efeitos adversos , Masculino , Técnica de Diluição de Radioisótopos , Adulto JovemRESUMO
UNLABELLED: Estradiol-17ß-D-glucuronide (E17G) activates different signaling pathways (e.g., Ca(2+) -dependent protein kinase C, phosphoinositide 3-kinase/protein kinase B, mitogen-activated protein kinases [MAPKs] p38 and extracellular signal-related kinase 1/2, and estrogen receptor alpha) that lead to acute cholestasis in rat liver with retrieval of the canalicular transporters, bile salt export pump (Abcb11) and multidrug resistance-associated protein 2 (Abcc2). E17G shares with nonconjugated estradiol the capacity to activate these pathways. G-protein-coupled receptor 30 (GPR30) is a receptor implicated in nongenomic effects of estradiol, and the aim of this study was to analyze the potential role of this receptor and its downstream effectors in E17G-induced cholestasis. In vitro, GPR30 inhibition by G15 or its knockdown with small interfering RNA strongly prevented E17G-induced impairment of canalicular transporter function and localization. E17G increased cyclic adenosine monophosphate (cAMP) levels, and this increase was blocked by G15, linking GPR30 to adenylyl cyclase (AC). Moreover, AC inhibition totally prevented E17G insult. E17G also increased protein kinase A (PKA) activity, which was blocked by G15 and AC inhibitors, connecting the links of the pathway, GPR30-AC-PKA. PKA inhibition prevented E17G-induced cholestasis, whereas exchange protein activated directly by cyclic nucleotide/MAPK kinase, another cAMP downstream effector, was not implicated in cAMP cholestatic action. In the perfused rat liver model, inhibition of the GPR30-AC-PKA pathway totally prevented E17G-induced alteration in Abcb11 and Abcc2 function and localization. CONCLUSION: Activation of GPR30-AC-PKA is a key factor in the alteration of canalicular transporter function and localization induced by E17G. Interaction of E17G with GPR30 may be the first event in the cascade of signaling activation.
Assuntos
Adenilil Ciclases/metabolismo , Colestase/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Estradiol/análogos & derivados , Receptores Acoplados a Proteínas G/metabolismo , Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Canalículos Biliares/metabolismo , Células Cultivadas , Colestase/induzido quimicamente , Estradiol/toxicidade , Técnicas de Silenciamento de Genes , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/enzimologia , Ratos , Receptores Acoplados a Proteínas G/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
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
UDCA (ursodeoxycholic acid) is the therapeutic agent most widely used for the treatment of cholestatic hepatopathies. Its use has expanded to other kinds of hepatic diseases, and even to extrahepatic ones. Such versatility is the result of its multiple mechanisms of action. UDCA stabilizes plasma membranes against cytolysis by tensioactive bile acids accumulated in cholestasis. UDCA also halts apoptosis by preventing the formation of mitochondrial pores, membrane recruitment of death receptors and endoplasmic-reticulum stress. In addition, UDCA induces changes in the expression of metabolizing enzymes and transporters that reduce bile acid cytotoxicity and improve renal excretion. Its capability to positively modulate ductular bile flow helps to preserve the integrity of bile ducts. UDCA also prevents the endocytic internalization of canalicular transporters, a common feature in cholestasis. Finally, UDCA has immunomodulatory properties that limit the exacerbated immunological response occurring in autoimmune cholestatic diseases by counteracting the overexpression of MHC antigens and perhaps by limiting the production of cytokines by immunocompetent cells. Owing to this multi-functionality, it is difficult to envisage a substitute for UDCA that combines as many hepatoprotective effects with such efficacy. We predict a long-lasting use of UDCA as the therapeutic agent of choice in cholestasis.
Assuntos
Colagogos e Coleréticos/farmacologia , Colestase/tratamento farmacológico , Ácido Ursodesoxicólico/farmacologia , Animais , Apoptose/efeitos dos fármacos , Ácidos e Sais Biliares/fisiologia , Canalículos Biliares/efeitos dos fármacos , Colagogos e Coleréticos/uso terapêutico , Colestase/patologia , Colestase/fisiopatologia , Humanos , Fatores Imunológicos/farmacologia , Fatores Imunológicos/uso terapêutico , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ácido Ursodesoxicólico/uso terapêuticoRESUMO
In estradiol 17ß-d-glucuronide (E17G)-induced cholestasis, the canalicular hepatocellular transporters bile salt export pump (Abcb11) and multidrug-resistance associated protein 2 (Abcc2) undergo endocytic internalization. cAMP stimulates the trafficking of transporter-containing vesicles to the apical membrane and is able to prevent internalization of these transporters in estrogen-induced cholestasis. Hepatocyte levels of cAMP are regulated by hormones such as glucagon and adrenaline (via the ß2 receptor). We analyzed the effects of glucagon and salbutamol (a ß2 adrenergic agonist) on function and localization of Abcb11 and Abcc2 in isolated rat hepatocyte couplets exposed to E17G and compared the mechanistic bases of their effects. Glucagon and salbutamol partially prevented the impairment in Abcb11 and Abcc2 transport capacity. E17G also induced endocytic internalization of Abcb11 and Abcc2, which partially colocalized with the endosomal marker Rab11a. This effect was completely prevented by salbutamol, whereas some transporter-containing vesicles remained internalized and mainly colocalizing with Rab11a in the perinuclear region after incubation with glucagon. Glucagon prevention was dependent on cAMP-dependent protein kinase (PKA) and independent of exchange proteins activated directly by cAMP (Epac) and microtubules. In contrast, salbutamol prevention was PKA independent and Epac/MEK and microtubule dependent. Anticholestatic effects of glucagon and salbutamol were additive in nature. Our results show that increases in cAMP could activate different anticholestatic signaling pathways, depending on the hormonal mediator involved.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Albuterol/farmacologia , Canalículos Biliares/efeitos dos fármacos , AMP Cíclico/metabolismo , Estradiol/análogos & derivados , Glucagon/farmacologia , Hepatócitos/efeitos dos fármacos , Transdução de Sinais , Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Agonistas de Receptores Adrenérgicos beta 2/farmacologia , Animais , Canalículos Biliares/metabolismo , Colestase/tratamento farmacológico , Colestase/metabolismo , Colestase/fisiopatologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Endocitose/efeitos dos fármacos , Epinefrina/farmacologia , Estradiol/efeitos adversos , Estradiol/farmacologia , Feminino , Hepatócitos/metabolismo , Transporte Proteico/efeitos dos fármacos , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Vesículas Transportadoras/efeitos dos fármacos , Vesículas Transportadoras/metabolismo , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
UNLABELLED: Estradiol 17ß-D-glucuronide (E(2)17G) is an endogenous, cholestatic metabolite that induces endocytic internalization of the canalicular transporters relevant to bile secretion: bile salt export pump (Bsep) and multidrug resistance-associated protein 2 (Mrp2). We assessed whether phosphoinositide 3-kinase (PI3K) is involved in E(2)17G-induced cholestasis. E(2)17G activated PI3K according to an assessment of the phosphorylation of the final PI3K effector, protein kinase B (Akt). When the PI3K inhibitor wortmannin (WM) was preadministered to isolated rat hepatocyte couplets (IRHCs), it partially prevented the reduction induced by E(2)17G in the proportion of IRHCs secreting fluorescent Bsep and Mrp2 substrates (cholyl lysyl fluorescein and glutathione methylfluorescein, respectively). 2-Morpholin-4-yl-8-phenylchromen-4-one, another PI3K inhibitor, and an Akt inhibitor (Calbiochem 124005) showed similar protective effects. IRHC immunostaining and confocal microscopy analysis revealed that endocytic internalization of Bsep and Mrp2 induced by E(2)17G was extensively prevented by WM; this effect was fully blocked by the microtubule-disrupting agent colchicine. The protection of WM was additive to that afforded by the classical protein kinase C (cPKC) inhibitor 5,6,7,13-tetrahydro-13-methyl-5-oxo-12H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-12-propanenitrile (Gö6976); this suggested differential and complementary involvement of the PI3K and cPKC signaling pathways in E(2)17G-induced cholestasis. In isolated perfused rat liver, an intraportal injection of E(2)17G triggered endocytosis of Bsep and Mrp2, and this was accompanied by a sustained decrease in the bile flow and the biliary excretion of the Bsep and Mrp2 substrates [(3)H]taurocholate and glutathione until the end of the perfusion period. Unlike Gö6976, WM did not prevent the initial decay, but it greatly accelerated the recovery to normality of these parameters and the reinsertion of Bsep and Mrp2 into the canalicular membrane in a microtubule-dependent manner. CONCLUSION: The PI3K/Akt signaling pathway is involved in the biliary secretory failure induced by E(2)17G through sustained internalization of canalicular transporters endocytosed via cPKC.
Assuntos
1-Fosfatidilinositol 4-Quinase/fisiologia , Colestase/induzido quimicamente , Proteína Quinase C/fisiologia , Proteínas Proto-Oncogênicas c-akt/fisiologia , Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/metabolismo , Androstadienos/farmacologia , Animais , Canalículos Biliares/efeitos dos fármacos , Canalículos Biliares/fisiologia , Sistema Biliar/metabolismo , Carbazóis/farmacologia , Colchicina/farmacologia , Endocitose/efeitos dos fármacos , Estradiol/análogos & derivados , Glutationa/metabolismo , Técnicas In Vitro , Masculino , Microtúbulos/efeitos dos fármacos , Microtúbulos/fisiologia , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Perfusão , Proteína Quinase C/antagonistas & inibidores , Ratos , Ratos Wistar , Transdução de Sinais , Ácido Taurocólico/metabolismo , WortmaninaRESUMO
The chronic exposure to Aluminum (Al) may compromise different liver functions, mainly during the hepatic regeneration. The aim of this study is to investigate the interactions between the chronic i.p. exposure to Al and hepatic regeneration (HR) on bile flow and organic anion transport in experimental animals. For this purpose, we studied bile flow and fractional transfer rates for the transport of hepatic organic anions (hepatic uptake, sinusoidal efflux, and canalicular excretion), as well as parameters related with the oxidative stress (OS), on rats chronically treated with Al at 0 and 2 days of HR. The Al treatment and time of HR caused a decrease in the biliary flow and in the hepatic uptake and canalicular excretion constants. In addition, Al and HR increased the lipoperoxidation associated with a reduction of the glutathione content and glutathione peroxidase and catalase enzyme's activities. Since the effects of Al and HR on biliary flow and transport systems were additive, but not on the oxidative status, different mechanisms might be involved on these alterations. Even though the OS may play a key role on the hepatic deleterious effects, there is no unique cause-effect relationship between OS and liver dysfunction in this experimental animal model.
Assuntos
Alumínio/toxicidade , Bile/fisiologia , Transporte de Íons , Regeneração Hepática/fisiologia , Fígado/fisiologia , Alumínio/análise , Alumínio/sangue , Análise de Variância , Animais , Bile/química , Ácidos e Sais Biliares/metabolismo , Canalículos Biliares/metabolismo , Catalase/metabolismo , Glutationa/metabolismo , Glutationa Peroxidase/metabolismo , Peróxidos Lipídicos/fisiologia , Fígado/enzimologia , Masculino , Estresse Oxidativo/fisiologia , Ratos , Ratos Wistar , ATPase Trocadora de Sódio-Potássio/metabolismo , Espectrofotometria AtômicaRESUMO
Aquaporin-8 (AQP8) water channels, which are expressed in rat hepatocyte bile canalicular membranes, are involved in water transport during bile formation. Nevertheless, there is no conclusive evidence that AQP8 mediates water secretion into the bile canaliculus. In this study, we directly evaluated whether AQP8 gene silencing by RNA interference inhibits canalicular water secretion in the human hepatocyte-derived cell line, HepG2. By RT-PCR and immunoblotting we found that HepG2 cells express AQP8 and by confocal immunofluorescence microscopy that it is localized intracellularly and on the canalicular membrane, as described in rat hepatocytes. We also verified the expression of AQP8 in normal human liver. Forty-eight hours after transfection of HepG2 cells with RNA duplexes targeting two different regions of human AQP8 molecule, the levels of AQP8 protein specifically decreased by 60-70%. We found that AQP8 knockdown cells showed a significant decline in the canalicular volume of approximately 70% (P < 0.01), suggesting an impairment in the basal (nonstimulated) canalicular water movement. We also found that the decreased AQP8 expression inhibited the canalicular water transport in response either to an inward osmotic gradient (-65%, P < 0.05) or to the bile secretory agonist dibutyryl cAMP (-80%, P < 0.05). Our data suggest that AQP8 plays a major role in water transport across canalicular membrane of HepG2 cells and support the notion that defective expression of AQP8 causes bile secretory dysfunction in human hepatocytes.
Assuntos
Aquaporinas/metabolismo , Canalículos Biliares/metabolismo , Técnicas de Silenciamento de Genes , Hepatócitos/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Água/metabolismo , Aquaporinas/genética , Canalículos Biliares/efeitos dos fármacos , Linhagem Celular Tumoral , CMP Cíclico/análogos & derivados , CMP Cíclico/farmacologia , Hepatócitos/efeitos dos fármacos , Humanos , Microscopia Confocal , Osmose , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , TransfecçãoRESUMO
BACKGROUND: Recent data suggest that canalicular bile secretion involves selective expression and coordinated regulation of aquaporins (AQPs), a family of water channels proteins. In order to further characterize the role of AQPs in this process, an in vitro cell system with retained polarity and expression of AQPs and relevant solute transporters involved in bile formation is highly desirable. The WIF-B cell line is a highly differentiated and polarized rat hepatoma/human fibroblast hybrid, which forms abundant bile canalicular structures. This cell line has been reported to be a good in vitro model for studying hepatocyte polarity. RESULTS: Using RT-PCR, immunoblotting and confocal immunofluorescence, we showed that WIF-B cells express the aquaporin water channels that facilitate the osmotically driven water movements in the liver, i.e. AQP8, AQP9, and AQP0; as well as the key solute transporters involved in the generation of canalicular osmotic gradients, i.e., the bile salt export pump Bsep, the organic anion transporter Mrp2 and the chloride bicarbonate exchanger AE2. The subcellular localization of the AQPs and the solute transporters in WIF-B cells was similar to that in freshly isolated rat hepatocytes and in intact liver. Immunofluorescent costaining studies showed intracellular colocalization of AQP8 and AE2, suggesting the possibility that these transporters are expressed in the same population of pericanalicular vesicles. CONCLUSION: The hepatocyte cell line WIF-B retains the expression and subcellular localization of aquaporin water channels as well as key solute transporters for canalicular bile secretion. Thus, these cells can work as a valuable tool for regulatory and mechanistic studies of the biology of bile formation.
Assuntos
Aquaporinas/metabolismo , Polaridade Celular , Hepatócitos/fisiologia , Frações Subcelulares/metabolismo , Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Proteínas de Transporte de Ânions/metabolismo , Antiporters/metabolismo , Canalículos Biliares/metabolismo , Proteínas de Transporte/metabolismo , Linhagem Celular , Proteínas do Olho/metabolismo , Imunofluorescência , Hepatócitos/metabolismo , Humanos , Células Híbridas , Canais Iônicos/metabolismo , Glicoproteínas de Membrana/metabolismo , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas SLC4A , Distribuição TecidualRESUMO
BACKGROUND: Obese Zucker rats (ZR) have been used as an experimental model for non-alcoholic fatty liver disease and are particularly susceptible to various types of liver injury. Bile secretory function has not been assessed in ZR. AIM: To study bile secretion and expression of the main hepatobiliary transporters in ZR. METHODS: Bile flow and biliary secretion of lipids and glutathione were determined in eight and 14 week old obese ZR and their lean controls. Protein mass and mRNA of the Na(+)/taurocholate cotransporting polypeptide (Ntcp), the bile salt export pump (Bsep), and the multidrug resistant associated protein 2 (Mrp2) were assessed by western and northern blot, respectively. The effects of administration of a tumour necrosis factor alpha inactivator (etanercept) and an insulin sensitiser (rosiglitazone) were assessed in obese ZR while leptin was given to non-obese rats to study its effect on Mrp2 expression. RESULTS: ZR exhibited increased body weight and hyperlipidaemia. Only 14 week old obese ZR has fatty liver. Decreased bile flow and biliary lipid and glutathione secretion as well as reduced hepatic transport of both taurocholate and bromosulphthalein were found in obese ZR. Hepatic Mrp2 protein mass was markedly reduced (-70%) in obese rats while Ntcp and Bsep protein levels were similar to lean rats. Downregulation of Mrp2 seems to involve both transcriptional and post-transcriptional mechanisms probably related to insulin and leptin resistance. CONCLUSIONS: Obese ZR exhibit an impaired bile secretory function with significant functional and molecular alterations consistent with mild cholestasis. A defective hepatobiliary transport capacity may be a contributory factor in rendering the obese ZR more susceptible to liver injury.
Assuntos
Canalículos Biliares/metabolismo , Bile/metabolismo , Colestase/metabolismo , Obesidade/metabolismo , Animais , Transporte Biológico Ativo , Peso Corporal , Colestase/etiologia , Colestase/patologia , Regulação para Baixo , Fígado/patologia , Masculino , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Obesidade/complicações , Obesidade/patologia , Tamanho do Órgão , RNA Mensageiro/genética , Ratos , Ratos ZuckerRESUMO
The effects of a chronic aluminum (Al) exposure on biliary secretory function, with special emphasis on hepatic handling of non-bile salt organic anions, was investigated. Male Wistar rats received, intraperitoneally, either 27 mg/kg body weight of Al, as Al hydroxide [Al (+) rats], or the vehicle saline [Al (-) rats] three times a week for 3 months. Serum and hepatic Al levels were increased by the treatment (approximately 9- and 4-fold, respectively). This was associated with enhanced malondialdehyde formation (+110%) and a reduction in GSH content (-17%) and in the activity of the antioxidant enzymes catalase (-84%) and GSH peroxidase (-46%). Bile flow (-23%) and the biliary output of bile salts (-39%), cholesterol (-43%), and proteins (-38%) also decreased. Compartmental analysis of the plasma decay of the model organic anion bromosulphophthalein revealed that sinusoidal uptake and canalicular excretion of the dye were significantly decreased in Al (+) rats (-53 and -43%, respectively). Expression of multidrug resistance-associated protein 2 (Mrp2), the main, multispecific transporter involved in the canalicular excretion of organic anions, was also decreased (-40%), which was associated with a significant decrease in the cumulative biliary excretion of the Mrp2 substrate, dinitrophenyl-S-glutathione (-50%). These results show that chronic Al exposure leads to oxidative stress, cholestasis, and impairment of the hepatic handling of organic anions by decreasing both sinusoidal uptake and canalicular excretion. The alteration of the latter process seems to be causally related to impairment of Mrp2 expression. We have addressed some possible mechanisms involved in these deleterious effects.
Assuntos
Alumínio/intoxicação , Canalículos Biliares/efeitos dos fármacos , Canalículos Biliares/metabolismo , Bile/metabolismo , Glutationa/análogos & derivados , Hidróxido de Alumínio/administração & dosagem , Hidróxido de Alumínio/sangue , Hidróxido de Alumínio/intoxicação , Animais , Bile/efeitos dos fármacos , Ácidos e Sais Biliares/antagonistas & inibidores , Ácidos e Sais Biliares/metabolismo , Catalase/antagonistas & inibidores , Catalase/metabolismo , Colestase/induzido quimicamente , Colesterol/metabolismo , Doença Crônica , Esquema de Medicação , Avaliação Pré-Clínica de Medicamentos/métodos , Expressão Gênica , Glutationa/antagonistas & inibidores , Glutationa/química , Glutationa/efeitos dos fármacos , Glutationa/metabolismo , Glutationa Peroxidase/antagonistas & inibidores , Glutationa Peroxidase/metabolismo , Glutationa Transferase/química , Glutationa Transferase/metabolismo , Injeções Intraperitoneais , Fígado/química , Fígado/efeitos dos fármacos , Masculino , Proteínas Mitocondriais/antagonistas & inibidores , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Proteínas/antagonistas & inibidores , Proteínas/metabolismo , Ratos , Ratos Wistar , Proteínas Ribossômicas/antagonistas & inibidores , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Sulfobromoftaleína/metabolismo , Sulfobromoftaleína/farmacocinética , Substâncias Reativas com Ácido Tiobarbitúrico/química , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismoRESUMO
Abstract Bile is primarily secreted in hepatocytes (i.e. the canalicular bile) and subsequently delivered to the intrahepatic bile ducts, where is modified by cholangiocytes (i.e. the ductal bile). Bile formation is the result of the coordinated interactions of membrane-transport systems that generate the vectorial movement of solutes and osmotically driven water molecules. Hepatocytes and cholangiocytes express aquaporins, specialized membrane channel proteins that facilitate the osmotic transport of water. In this review, we provide a summary of what is known on liver AQPs and their significance in canalicular and ductal bile formation under normal and pathological conditions.