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A critical shortage of donors exists for liver transplantation, which non-heart-beating cadaver donors could help ease. This study evaluated ischemic preconditioning to improve graft viability after non-heart-beating liver donation in rats. Ischemic preconditioning was performed by clamping the portal vein and hepatic artery for 10 min followed by unclamping for 5 min. Subsequently, the aorta was cross-clamped for up to 120 min. After 2 h of storage, livers were either transplanted or perfused with warm buffer containing trypan blue. Aortic clamping for 60 and 120 min prior to liver harvest markedly decreased 30-day graft survival from 100% without aortic clamping to 50% and 0%, respectively, which ischemic preconditioning restored to 100 and 50%. After 60 min of aortic clamping, loss of viability of parenchymal and nonparenchymal cells was 22.6 and 5.6%, respectively, which preconditioning decreased to 3.0 and 1.5%. Cold storage after aortic clamping further increased parenchymal and non-parenchymal cell killing to 40.4 and 10.1%, respectively, which ischemic preconditioning decreased to 12.4 and 1.8%. In conclusion, ischemic preconditioning markedly decreased cell killing after subsequent sustained warm ischemia. Most importantly, ischemic preconditioning restored 100% graft survival of livers harvested from non-heart-beating donors after 60 min of aortic clamping.
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Background. Transplantation of ethanol-induced steatotic livers causes increased graft injury. We hypothesized that upregulation of hepatic ICAM-1 after ethanol produces increased leukocyte adherence, resulting in increased generation of reactive oxygen species (ROS) and injury after liver transplantation (LT). Methods. C57BL/6 wildtype (WT) and ICAM-1 knockout (KO) mice were gavaged with ethanol (6 g/kg) or water. LT was then performed into WT recipients. Necrosis and apoptosis, 4-hydroxynonenal (4-HNE) immunostaining, and sinusoidal leukocyte movement by intravital microscopy were assessed. Results. Ethanol gavage of WT mice increased hepatic triglycerides 10-fold compared to water treatment (P < 0.05). ICAM-1 also increased, but ALT was normal. At 8 h after LT of WT grafts, ALT increased 2-fold more with ethanol than water treatment (P < 0.05). Compared to ethanol-treated WT grafts, ALT from ethanol-treated KO grafts was 78% less (P < 0.05). Apoptosis also decreased by 75% (P < 0.05), and 4-HNE staining after LT was also decreased in ethanol-treated KO grafts compared to WT. Intravital microscopy demonstrated a 2-fold decrease in leukocyte adhesion in KO grafts compared to WT grafts. Conclusions. Increased ICAM-1 expression in ethanol-treated fatty livers predisposes to leukocyte adherence after LT, which leads to a disturbed microcirculation, oxidative stress and graft injury.
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BACKGROUND: Massive hepatectomy (MHX) leads to failure of remnant livers. Excessive metabolic burden in remnant livers may cause mitochondrial dysfunction. This study investigated whether blockade of the mitochondrial permeability transition (MPT) with N-methyl-4-isoleucine cyclosporine (NIM811) improves the outcome of MHX. METHODS: Mice were gavaged with NIM811 (10 mg/kg before surgery and 5 mg/kg daily afterward) and underwent sham-operation or approximately 90% partial hepatectomy. RESULTS: Serum alanine aminotransferase, necrosis, and apoptosis increased, respectively, to approximately 1200 U/L, 6.1%, and 7% after MHX. NIM811 decreased peak alanine aminotransferase release, necrosis, and apoptosis by 70%, 100%, and 42%, respectively. 5-Bromo-2'-deoxyuridine incorporation, proliferating cell nuclear antigen expression, and the remnant liver weights were all increased significantly by NIM811 treatment, indicating improved liver regeneration. NIM811 also blunted hyperbilirubinemia by 54%, increased serum albumin by 51%, and improved survival from 6% to 40% after MHX. Hepatic mitochondrial depolarization, cell death, and MPT were detected by intravital confocal/multiphoton microscopy of rhodamine 123, propidium iodide, and calcein. Mitochondrial depolarization occurred in many viable hepatocytes (13 cells/high-power field), and nonviable hepatocytes increased slightly to approximately 1 cell/high-power field at 3 hr after MHX. Entry of calcein into mitochondria after MHX indicated MPT onset. Importantly, NIM811 decreased mitochondria depolarization by more than 60%, blocked MPT onset, and prevented cell death. Decreases of hepatic ATP, mitochondrial cytochrome c release, and caspase-3 activation after MHX were also partially blocked by NIM811. CONCLUSIONS: NIM811 minimized liver injury and improved liver regeneration after MHX, at least in part, by preventing MPT onset and subsequent compromised energy supply and proapoptotic cytochrome c release.
Assuntos
Ciclosporina/uso terapêutico , Hepatectomia/efeitos adversos , Regeneração Hepática/efeitos dos fármacos , Doenças Mitocondriais/prevenção & controle , Trifosfato de Adenosina/metabolismo , Alanina Transaminase/sangue , Animais , Caspase 3/metabolismo , Morte Celular/efeitos dos fármacos , Citocromos c/metabolismo , Fluoresceínas/metabolismo , Fígado/efeitos dos fármacos , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Tamanho do Órgão/efeitos dos fármacos , Antígeno Nuclear de Célula em Proliferação/metabolismoRESUMO
In primary culture, hepatocytes dedifferentiate, and their cytoplasm undergoes remodeling. Here, our aim was to characterize changes of mitochondria during remodeling. Hepatocytes were cultured one to five days in complete serumcontaining Waymouth's medium. In rat hepatocytes loaded with MitoTracker Green (MTG), tetramethylrhodamine methylester (TMRM), and/or LysoTracker Red (LTR), confocal microscopy revealed that mitochondria number and mass decreased by approximately 50% between Day 1 and Day 3 of culture. As mitochondria disappeared, lysosomes/autophagosomes proliferated five-fold. Decreased mitochondrial content correlated with (a) decreased cytochrome c oxidase activity and mitochondrial number observed by electron microscopy and (b) a profound decrease of PGC-1alpha mRNA expression. By contrast, mtDNA content per cell remained constant from the first to the third day of culture, although ethidium bromide (de novo mtDNA synthesis inhibitor) caused mtDNA to decrease by half from the first to the third culture day. As mitochondria disappeared, their MTG label moved into LTR-labeled lysosomes, which was indicative of autophagic degradation. A multiwell fluorescence assay revealed a 2.5-fold increase of autophagy on Day 3 of culture, which was decreased by 3-methyladenine, an inhibitor of autophagy, and also by cyclosporin A and NIM811, both selective inhibitors of the mitochondrial permeability transition (MPT). These findings indicate that mitochondrial autophagy (mitophagy) and the MPT underlie mitochondrial remodeling in cultured hepatocytes.
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Autofagia , Hepatócitos/citologia , Hepatócitos/metabolismo , Mitocôndrias Hepáticas/metabolismo , Adenina/análogos & derivados , Adenina/farmacologia , Aminas/metabolismo , Animais , Autofagia/efeitos dos fármacos , Transporte Biológico/efeitos dos fármacos , Compartimento Celular/efeitos dos fármacos , Células Cultivadas , Ciclosporina/farmacologia , Citocromos c/metabolismo , DNA Mitocondrial/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Hepatócitos/enzimologia , Hepatócitos/ultraestrutura , Lisossomos/efeitos dos fármacos , Lisossomos/ultraestrutura , Masculino , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/ultraestrutura , Permeabilidade/efeitos dos fármacos , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Fagossomos/efeitos dos fármacos , Fagossomos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ratos , Ratos Sprague-Dawley , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The mitochondrial permeability transition (MPT) plays an important role in hepatocyte death caused by ischemia-reperfusion (IR). This study investigated whether activation of the cellular oxygen-sensing signal cascade by prolyl hydroxylase inhibitors (PHI) protects against the MPT after hepatic IR. Ethyl 3,4-dihyroxybenzoate (EDHB, 100 mg/kg ip), a PHI, increased mouse hepatic hypoxia-inducible factor-1alpha and heme oxygenase-1 (HO-1). EDHB-treated and untreated mice were subjected to 1 h of warm ischemia to approximately 70% of the liver followed by reperfusion. Mitochondrial polarization, cell death, and the MPT were assessed by intravital confocal/multiphoton microscopy of rhodamine 123, propidium iodide, and calcein. EDHB largely blunted alanine aminotransferase (ALT) release and necrosis after reperfusion. In vehicle-treated mice at 2 h after reperfusion, viable cells with depolarized mitochondria were 72%, and dead cells were 2%, indicating that depolarization preceded necrosis. Mitochondrial voids excluding calcein disappeared, indicating MPT onset in vivo. NIM811, a specific inhibitor of the MPT, blocked mitochondrial depolarization after IR, further confirming that mitochondrial depolarization was due to MPT onset. EDHB decreased mitochondrial depolarization to 16% and prevented the MPT. Tin protoporphyrin (10 micromol/kg sc), an HO-1 inhibitor, partially abrogated protection by EDHB against ALT release, necrosis, and mitochondrial depolarization. In conclusion, IR causes the MPT and mitochondrial dysfunction, leading to hepatocellular death. PHI prevents MPT onset and liver damage through an effect mediated partially by HO-1.
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Mitocôndrias Hepáticas/fisiologia , Proteínas de Transporte da Membrana Mitocondrial/fisiologia , Traumatismo por Reperfusão/metabolismo , Animais , Heme Oxigenase-1/metabolismo , Hidroxibenzoatos/antagonistas & inibidores , Hidroxibenzoatos/farmacologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/biossíntese , Masculino , Metaloporfirinas/farmacologia , Camundongos , Mitocôndrias Hepáticas/efeitos dos fármacos , Poro de Transição de Permeabilidade Mitocondrial , Oxigênio/metabolismo , Pró-Colágeno-Prolina Dioxigenase/antagonistas & inibidores , Protoporfirinas/farmacologia , Transdução de Sinais/fisiologiaRESUMO
Cholestasis causes hepatocyte death, possibly because of mitochondrial injury. This study investigated whether NIM811 (N-methyl-4-isoleucine cyclosporine), an inhibitor of the mitochondrial permeability transition (MPT), attenuates cholestatic liver injury in vivo. Cholestasis was induced in mice by bile duct ligation (BDL). NIM811 was gavaged (20 mg/kg) before BDL and daily (10 mg/kg) afterward. Mitochondrial depolarization, cell death, and MPT onset were assessed by intravital confocal/multiphoton microscopy of rhodamine 123, propidium iodide, and calcein. After BDL, serum alanine aminotransferase (ALT), hepatic necrosis, and apoptosis all increased. NIM811 decreased ALT, necrosis, and apoptosis by 60 to 86%. In vehicle-treated mice at 6 h after BDL, viable hepatocytes with depolarized mitochondria were 18/high-power field (hpf), and nonviable cells were approximately 1/hpf, showing that depolarization preceded necrosis. Calcein entered mitochondria after BDL, indicating MPT onset in vivo. NIM811 decreased depolarization by 72%, prevented calcein entry into mitochondria, and blocked release of cytochrome c. Hepatic tumor necrosis factor alpha, transforming growth factor-beta1, procollagen alpha1(I) mRNA, alpha-smooth muscle actin, and Sirius red staining for collagen increased after BDL but were not different in vehicle- and NIM811-treated mice. Taken together, NIM811 decreased cholestatic necrosis and apoptosis but did not block fibrosis, indicating that the MPT plays an important role in cholestatic cell death in vivo.
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Colestase/tratamento farmacológico , Ciclosporina/farmacologia , Membranas Mitocondriais/fisiologia , Animais , Morte Celular/efeitos dos fármacos , Colágeno/análise , Fibrose , Hepatócitos/metabolismo , Hepatócitos/ultraestrutura , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Permeabilidade/efeitos dos fármacosRESUMO
UNLABELLED: Graft failure after liver transplantation may involve mitochondrial dysfunction. We examined whether prevention of mitochondrial injury would improve graft function. Orthotopic rat liver transplantation was performed after 18 hours' cold storage in University of Wisconsin solution and treatment with vehicle, minocycline, tetracycline, or N-methyl-4-isoleucine cyclosporin (NIM811) of explants and recipients. Serum alanine aminotransferase (ALT), necrosis, and apoptosis were assessed 6 hours after implantation. Mitochondrial polarization and cell viability were assessed by intravital microscopy. Respiration and the mitochondrial permeability transition (MPT) were assessed in isolated rat liver mitochondria. After transplantation with vehicle or tetracycline, ALT increased to 5242 U/L and 4373 U/L, respectively. Minocycline and NIM811 treatment decreased ALT to 2374 U/L and 2159 U/L, respectively (P < 0.01). Necrosis and terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) also decreased from 21.4% and 21 cells/field, respectively, after vehicle to 10.1% and 6 cells/field after minocycline and to 8.7% and 5.2 cells/field after NIM811 (P < 0.05). Additionally, minocycline decreased caspase-3 activity in graft homogenates (P < 0.05). Long-term graft survival was 27% and 33%, respectively, after vehicle and tetracycline treatment, which increased to 60% and 70% after minocycline and NIM811 (P < 0.05). In isolated mitochondria, minocycline and NIM811 but not tetracycline blocked the MPT. Minocycline blocked the MPT by decreasing mitochondrial Ca(2+) uptake, whereas NIM811 blocks by interaction with cyclophilin D. Intravital microscopy showed that minocycline and NIM811 preserved mitochondrial polarization and cell viability after transplantation (P < 0.05). CONCLUSION: Minocycline and NIM811 attenuated graft injury after rat liver transplantation and improved graft survival. Minocycline and/or NIM811 might be useful clinically in hepatic surgery and transplantation.
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Antibacterianos/uso terapêutico , Ciclosporina/uso terapêutico , Transplante de Fígado/efeitos adversos , Minociclina/uso terapêutico , Proteínas de Transporte da Membrana Mitocondrial/antagonistas & inibidores , Traumatismo por Reperfusão/prevenção & controle , Difosfato de Adenosina/metabolismo , Alanina Transaminase/sangue , Animais , Antibacterianos/farmacologia , Apoptose/efeitos dos fármacos , Cálcio/metabolismo , Ciclosporina/farmacologia , Sobrevivência de Enxerto/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/patologia , Masculino , Minociclina/farmacologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Doenças Mitocondriais/prevenção & controle , Poro de Transição de Permeabilidade Mitocondrial , Necrose/prevenção & controle , Ratos , Ratos Endogâmicos Lew , Traumatismo por Reperfusão/etiologia , Tetraciclina/farmacologiaRESUMO
Mitochondria become targets for autophagic degradation after nutrient deprivation, a process also termed mitophagy. In this study, we used LysoTracker Red (LTR) and MitoTracker Green to characterize the kinetics of autophagosomal proliferation and mitophagy in cultured rat hepatocytes. Autophagy induced by nutrient deprivation plus glucagon increased LTR uptake assessed with a fluorescence plate reader and the number of LTR-labeled acidic organelles assessed with confocal microscopy in individual hepatocytes both by 4- to 6-fold. Serial imaging of hepatocytes coloaded with MitoTracker Green (MTG) revealed an average mitochondrial digestion time of 7.5 min after autophagic induction. In the presence of protease inhibitors, digestion time more than doubled, and the total number of LTR-labeled organelles increased about 40%, but the proportion of the LTR-labeled acidic organelles containing MTG fluorescence remained constant at about 75%. Autophagy inhibitors, 3-methyladenine, wortmannin and LY204002, suppressed the increase of LTR uptake after nutrient deprivation by up to 85%, confirming that increased LTR uptake reflected autophagy induction. Cyclosporin A and NIM811, specific inhibitors of the mitochondrial permeability transition (MPT), also decreased LTR uptake, whereas tacrolimus, an immunosuppressive reagent that does not inhibit the MPT, was without effect. In addition, the c-Jun N-terminal kinase (JNK) inhibitors, SCP25041 and SP600125, blocked LTR uptake by 47% and 61%, respectively, but ERK1, p38 and caspase inhibitors had no effect. The results show that mitochondria once selected for mitophagy are rapidly digested and support the concept that mitochondrial autophagy involves the MPT and signaling through PI3 kinase and possibly JNK.
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Aminas/análise , Autofagia , Corantes Fluorescentes/análise , Hepatócitos/química , Microscopia Confocal/métodos , Mitocôndrias Hepáticas/química , Adenina/análogos & derivados , Adenina/farmacologia , Aldeídos/análise , Aminas/metabolismo , Androstadienos/farmacologia , Animais , Autofagia/efeitos dos fármacos , Células Cultivadas , Corantes Fluorescentes/metabolismo , Glucagon/farmacologia , Hepatócitos/metabolismo , Hepatócitos/ultraestrutura , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Mitocôndrias Hepáticas/metabolismo , Mitocôndrias Hepáticas/ultraestrutura , Fagossomos/química , Fagossomos/efeitos dos fármacos , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteases/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Ratos , WortmaninaRESUMO
BACKGROUND/AIMS: The purpose of the present study was to characterize the role of Na+, pH and cellular swelling in the pathogenesis of hypoxic injury to rat livers. METHODS AND RESULTS: When livers were perfused with hypoxic Krebs-Henseleit bicarbonate buffer (KHB) containing 143 mM Na+, release of LDH began after 30 min and was maximal after 60 min. In livers perfused with choline-substituted low-Na+ KHB (25 mM Na+), LDH release began after 60 min and peaked after 120 min or longer. Supplementation of KHB with mannitol, a permeant sugar with antioxidant properties, suppressed LDH release, whereas sucrose, an impermeant disaccharide, did not afford protection. At the end of hypoxic perfusions with KHB and low-Na+ KHB, liver weight was not different, whereas mannitol but not sucrose increased liver weight after hypoxia. At pH 7.4, monensin, a Na+-H+ ionophore, reversed protection against hypoxia by low-Na+ KHB (10 mM Na+) but had no effect at pH 6.8. As measured directly by confocal microscopy of biscarboxyethylcarboxyfluorescein fluorescence, pH was lower during perfusion with low-Na+ KHB than KHB. CONCLUSIONS: Cytoprotection by low Na+ was not mediated by prevention of Na+-dependent tissue swelling. Rather, promotion of intracellular acidification likely mediates cytoprotection in low-Na+ buffer.
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Concentração de Íons de Hidrogênio/efeitos dos fármacos , Hipóxia/patologia , Fígado/patologia , Preservação de Órgãos/métodos , Sódio/farmacologia , Animais , Sobrevivência Celular , Diuréticos Osmóticos/farmacologia , Edema/patologia , Edema/prevenção & controle , Glucose/farmacologia , Hepatócitos/patologia , Técnicas In Vitro , Ionóforos/farmacologia , Masculino , Manitol/farmacologia , Monensin/farmacologia , Soluções para Preservação de Órgãos/farmacologia , Tamanho do Órgão/efeitos dos fármacos , Perfusão , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Sacarose/farmacologia , Trometamina/farmacologiaRESUMO
Kidneys preserved for transplantation surgery sustain injuries caused by cold ischemia during storage. Additionally, kidneys harvested from non-heart-beating donors encounter the stress of warm ischemia. The aim of this study was to determine the specific cell types losing viability after warm and cold ischemia. In warm ischemia studies, the pedicles of left kidneys of Lewis rats were cross-clamped for up to 90 min. In cold ischemia studies, kidneys were flushed with cold University of Wisconsin solution and stored up to 48h at 0-1 degrees C. After warm or cold ischemia, kidneys were perfused via the renal arteries with Krebs-Henseleit bicarbonate (KHB) buffer at 37 degrees C, followed by trypan blue to label the nuclei of nonviable cells. Warm ischemia for 90 min caused renal failure and led to injury of proximal tubular cells, e.g., loss of brush borders, cast formation and trypan blue labeling. Cold ischemia for 48 h also caused renal failure but, unlike warm ischemia, caused trypan blue labeling of glomerular podocytes and peritubular endothelial cells. In warm ischemia-induced injury, electron microscopy showed shedding of microvilli and marked swelling of proximal tubular cells, microvilli and mitochondria. In cold ischemia-induced injury, podocytes were blebbed and swollen, and their pedicels were detached from the basement membrane, but disruption in proximal tubules was milder. In conclusion, warm ischemia triggers injury primarily to proximal tubular cells, whereas cold ischemia damages glomerular podocytes and peritubular endothelial cells in addition to proximal tubules.
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Temperatura Baixa/efeitos adversos , Temperatura Alta/efeitos adversos , Isquemia/complicações , Isquemia/patologia , Transplante de Rim/efeitos adversos , Rim/lesões , Rim/patologia , Insuficiência Renal/etiologia , Insuficiência Renal/patologia , Animais , Modelos Animais de Doenças , Rim/irrigação sanguínea , Transplante de Rim/patologia , Masculino , Necrose , Néfrons/irrigação sanguínea , Néfrons/lesões , Néfrons/patologia , Ratos , Ratos Endogâmicos LewRESUMO
BACKGROUND: Kidney damage caused by cold ischemia-reperfusion injury promotes adverse outcomes after renal transplantation. The purpose of this study was to determine whether Carolina rinse solution (CRS) used at the end of cold ischemic storage decreases kidney injury and improves graft function and survival. METHODS: Inbred male Lewis rats were used as donors and recipients. Left kidneys were removed from donor rats, infused with cold University of Wisconsin solution, and stored for 24, 30, or 48 hr at 0-1 degrees C. Just before implantation, kidneys were flushed with either Ringer's solution or CRS at 35-37 degrees C or were not treated. Kidneys were then transplanted into recipient rats with removal of both native kidneys. RESULTS: Survival and renal function were analyzed over a 14-day postoperative period. Among rats receiving kidneys after 24-hr cold storage, creatinine clearance was 75% greater in rats transplanted with kidneys flushed with CRS compared with Ringer's solution or nontreatment. In animals receiving kidneys after 30-hr cold storage, recipient survival after CRS was significantly higher than with Ringer's solution or without flushing (80% vs. 25% and 17%, respectively). However, CRS failed to prevent renal graft failure after 48 hr of cold storage (14% survival with CRS vs. 0% with Ringer's solution). In separate ex vivo studies, nonviable cell nuclei were labeled by trypan blue after cold preservation and brief warm reperfusion. CRS decreased podocyte and peritubular endothelial cell killing associated with cold ischemia-reperfusion injury. CONCLUSION: Flushing renal explants with warm CRS before implantation diminishes cold ischemia-reperfusion injury and improves the function and survival of transplanted kidneys.