RESUMO
Anterior mesoderm (AM) and definitive endoderm (DE) progenitors represent the earliest embryonic cell types that are specified during germ layer formation at the primitive streak (PS) of the mouse embryo. Genetic experiments indicate that both lineages segregate from Eomes-expressing progenitors in response to different Nodal signaling levels. However, the precise spatiotemporal pattern of the emergence of these cell types and molecular details of lineage segregation remain unexplored. We combined genetic fate labeling and imaging approaches with single-cell RNA sequencing (scRNA-seq) to follow the transcriptional identities and define lineage trajectories of Eomes-dependent cell types. Accordingly, all cells moving through the PS during the first day of gastrulation express Eomes AM and DE specification occurs before cells leave the PS from Eomes-positive progenitors in a distinct spatiotemporal pattern. ScRNA-seq analysis further suggested the immediate and complete separation of AM and DE lineages from Eomes-expressing cells as last common bipotential progenitor.
Assuntos
Linhagem da Célula , Endoderma/citologia , Endoderma/metabolismo , Gastrulação , Mesoderma/citologia , Mesoderma/metabolismo , Alelos , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Diferenciação Celular , Células Epiteliais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Camadas Germinativas/citologia , Camundongos , Modelos Biológicos , Linha Primitiva/embriologia , Linha Primitiva/metabolismo , Células-Tronco/metabolismo , Proteínas com Domínio T/metabolismo , Fatores de Tempo , Transcrição GênicaRESUMO
OBJECTIVE: The aim of the study was to determine whether magnetic resonance imaging (MRI) can be used in assessment of biologic activity of intraluminal thrombus (ILT) and proteolytic processes of the abdominal aortic aneurysm wall. METHODS: Using MRI, 50 patients with asymptomatic infrarenal abdominal aortic aneurysm were analyzed at the maximum aneurysm diameter on T1-weighted images in the arterial phase after administration of contrast material. Relative ILT signal intensity (SI) was determined as the ratio between ILT SI and psoas muscle SI. During surgery, the full thickness of the ILT and the adjacent part of the aneurysm wall were harvested at the maximal diameter for biochemical analysis. The concentrations of matrix metalloproteinase 9 and neutrophil elastase (NE/ELA) were analyzed in harvested thrombi, and the concentrations of collagen type III, elastin, and proteoglycans were analyzed in harvested aneurysm walls. RESULTS: A significant positive correlation was found between the NE/ELA concentration of the ILT and the relative SI (ρ = 0.309; P = .029). Furthermore, a negative correlation was observed between the elastin content of the aneurysm wall and the relative SI (ρ = -0.300; P = .034). No correlations were found between relative SI and concentration of matrix metalloproteinase 9, NE/ELA, collagen type III, or proteoglycan 4 in the aneurysm wall. CONCLUSIONS: These findings indicate a potential novel use of MRI in prediction of thrombus proteolytic enzyme concentrations and the extracellular matrix content of the aneurysm wall, thus providing additional information for the risk of potential aneurysm rupture.
Assuntos
Aorta Abdominal/diagnóstico por imagem , Aneurisma da Aorta Abdominal/diagnóstico por imagem , Elastase de Leucócito/análise , Imageamento por Ressonância Magnética , Metaloproteinase 9 da Matriz/análise , Trombose/diagnóstico por imagem , Idoso , Aorta Abdominal/enzimologia , Aorta Abdominal/cirurgia , Aneurisma da Aorta Abdominal/enzimologia , Aneurisma da Aorta Abdominal/cirurgia , Colágeno Tipo III/análise , Estudos Transversais , Elastina/análise , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Proteoglicanas/análise , Proteólise , Trombose/enzimologia , Trombose/cirurgiaRESUMO
The immunoglobulin superfamily adhesion molecule close homolog of L1 (CHL1) plays important roles during nervous system development. Here, we identified the hedgehog receptor patched-1 (PTCH1) as a novel CHL1-binding protein and showed that CHL1 interacts with the first extracellular loop of PTCH1 via its extracellular domain. Colocalization and co-immunoprecipitation of CHL1 with PTCH1 suggest an association of CHL1 with this major component of the hedgehog signaling pathway. The trans-interaction of CHL1 with PTCH1 promotes neuronal survival in cultures of dissociated cerebellar granule cells and of organotypic cerebellar slices. An inhibitor of the PTCH1-regulated hedgehog signal transducer, smoothened (SMO), and inhibitors of RhoA and Rho-associated kinase (ROCK) 1 and 2 prevent CHL1-dependent survival of cultured cerebellar granule cells and survival of cerebellar granule and Purkinje cells in organotypic cultures. In histological sections from 10- and 14-day-old CHL1-deficient mice, enhanced apoptosis of granule, but not Purkinje, cells was observed. The results of the present study indicate that CHL1 triggers PTCH1-, SMO-, RhoA- and ROCK-dependent signal transduction pathways to promote neuronal survival after cessation of the major morphogenetic events during mouse cerebellar development.
Assuntos
Apoptose , Moléculas de Adesão Celular/metabolismo , Receptor Patched-1/metabolismo , Células de Purkinje/metabolismo , Transdução de Sinais , Animais , Moléculas de Adesão Celular/genética , Camundongos , Camundongos Knockout , Receptor Patched-1/genéticaRESUMO
Menopause is characterized by deep metabolic disturbances, including decreased insulin sensitivity, adiposity, and changes in lipid profiles. Estrogen replacement therapy can partially reverse these changes, and while it is safe in most healthy postmenopausal women, there are still existing concerns regarding an increased risk for breast and endometrial cancer as well as a risk for cardiovascular and thromboembolic disease. Therefore, certain natural compounds with positive metabolic effects may be considered as a possible alternative or adjunctive treatment in patients not willing to take estrogens or patients with contraindications for estrogens. The aim of this study was to investigate the influence of Sideritis scardica (mountain tea) extract on metabolic disturbances induced by ovariectomy in rats. The study included 24 rats divided into three groups: ovariectomized rats treated with 200 mg/kg S. scardica extract for 24 weeks (n = 8), ovariectomized non-treated (n = 8), and Sham-operated (n = 8) rats. Food intake, weight gain, body composition, fasting glucose levels, response to oral glucose challenge, liver glycogen content, catalase activity, thiol groups, and malondialdehyde concentrations as well as AMP-activated protein kinase activity in liver cells were studied. Ovariectomized rats treated with S. scardica extract had lower blood triglycerides, reduced fasting glucose levels, as well lower glucose peaks after oral glucose challenge, increased liver glycogen content, and significantly higher catalase activity and thiol group concentration than non-treated ovariectomized rats. The ability of S. scardica extract to attenuate metabolic disturbances associated with ovariectomy was associated with the activation of AMP-activated protein kinase in liver cells.
Assuntos
Glicemia/efeitos dos fármacos , Ovariectomia , Estresse Oxidativo/efeitos dos fármacos , Extratos Vegetais/farmacologia , Sideritis , Triglicerídeos/sangue , Animais , Glicemia/análise , Cromatografia Líquida de Alta Pressão , Teste de Tolerância a Glucose , Glicogênio Hepático/análise , Ovariectomia/efeitos adversos , Ratos , Ratos Wistar , Sideritis/químicaRESUMO
Growing knowledge of how cell identity is determined at the molecular level has enabled the generation of diverse tissue types, including renal cells from pluripotent or somatic cells. Recently, several in vitro protocols involving either directed differentiation or transcription-factor-based reprogramming to kidney cells have been established. Embryonic stem cells or induced pluripotent stem cells can be guided towards a kidney fate by exposing them to combinations of growth factors or small molecules. Here, renal development is recapitulated in vitro resulting in kidney cells or organoids that show striking similarities to mammalian embryonic nephrons. In addition, culture conditions are also defined that allow the expansion of renal progenitor cells in vitro. Another route towards the generation of kidney cells is direct reprogramming. Key transcription factors are used to directly impose renal cell identity on somatic cells, thus circumventing the pluripotent stage. This complementary approach to stem-cell-based differentiation has been demonstrated to generate renal tubule cells and nephron progenitors. In-vitro-generated renal cells offer new opportunities for modelling inherited and acquired renal diseases on a patient-specific genetic background. These cells represent a potential source for developing novel models for kidney diseases, drug screening and nephrotoxicity testing and might represent the first steps towards kidney cell replacement therapies. In this review, we summarize current approaches for the generation of renal cells in vitro and discuss the advantages of each approach and their potential applications.
Assuntos
Técnicas de Reprogramação Celular/métodos , Reprogramação Celular , Regulação da Expressão Gênica , Rim , Engenharia Tecidual/métodos , Fatores de Transcrição , Animais , Humanos , Rim/citologia , Rim/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Arylpiperazine-based dopaminergic/serotonergic ligands exert neuroprotective activity. We examined the effect of arylpiperazine D2 /5-HT1A ligands, N-{4-[2-(4-phenyl-piperazin-1-yl)-ethyl}-phenyl]-picolinamide (6a) and N-{3-[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}-picolinamide (6b), in experimental autoimmune encephalomyelitis (EAE), a model of neuroinflammation. Both compounds (10 mg/kg i.p.) reduced EAE clinical signs in spinal cord homogenate-immunized Dark Agouti rats. Compound 6b was more efficient in delaying the disease onset and reducing the maximal clinical score, which correlated with its higher affinity for D2 and 5-HT1A receptors. The protection was retained if treatment was limited to the effector (from day 8 onwards), but not the induction phase (day 0-7) of EAE. Compound 6b reduced CNS immune infiltration and expression of mRNA encoding the proinflammatory cytokines tumor necrosis factor, IL-6, IL-1, and GM-CSF, TH 1 cytokine IFN-γ, TH 17 cytokine IL-17, as well as the signature transcription factors of TH 1 (T-bet) and TH 17 (RORγt) cells. Arylpiperazine treatment reduced apoptosis and increased the activation of anti-apoptotic mediators Akt and p70S6 kinase in the CNS of EAE animals. The in vitro treatment with 6b protected oligodendrocyte cell line OLN-93 and neuronal cell line PC12 from mitogen-activated normal T cells or myelin basic protein-activated encephalitogenic T cells. In conclusion, arylpiperazine dopaminergic/serotonergic ligands suppress EAE through a direct neuroprotective action and decrease in CNS inflammation. Arylpiperazine dopaminergic/serotonergic ligands reduce neurological symptoms of acute autoimmune encephalomyelitis in rats without affecting the activation of autoreactive immune response, through mechanisms involving a decrease in CNS immune infiltration, as well as direct protection of CNS from immune-mediated damage. These data indicate potential usefulness of arylpiperazine-based compounds in the treatment of neuroinflammatory disorders such as multiple sclerosis.
Assuntos
Dopaminérgicos/farmacologia , Encefalomielite Autoimune Experimental/tratamento farmacológico , Esclerose Múltipla/tratamento farmacológico , Fármacos Neuroprotetores/farmacologia , Animais , Dopamina/metabolismo , Encefalomielite Autoimune Experimental/imunologia , Feminino , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Interleucina-6/metabolismo , Ligantes , Esclerose Múltipla/imunologia , Células PC12 , Ratos , Serotonina/metabolismo , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologiaRESUMO
The altered expression of the SOX2 transcription factor is associated with oncogenic or tumor suppressor functions in human cancers. This factor regulates the migration and invasion of different cancer cells. In this study we investigated the effect of constitutive SOX2 overexpression on the migration and adhesion capacity of embryonal teratocarcinoma NT2/D1 cells derived from a metastasis of a human testicular germ cell tumor. We detected that increased SOX2 expression changed the speed, mode and path of cell migration, but not the adhesion ability of NT2/D1 cells. Additionally, we demonstrated that SOX2 overexpression increased the expression of the tumor suppressor protein p53 and the HDM2 oncogene. Our results contribute to the better understanding of the effect of SOX2 on the behavior of tumor cells originating from a human testicular germ cell tumor. Considering that NT2/D1 cells resemble cancer stem cells in many features, our results could contribute to the elucidation of the role of SOX2 in cancer stem cells behavior and the process of metastasis.
Assuntos
Movimento Celular/fisiologia , Fatores de Transcrição SOXB1/metabolismo , Teratocarcinoma/metabolismo , Adesão Celular/fisiologia , Linhagem Celular Tumoral , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Masculino , Teratocarcinoma/patologia , Análise Serial de TecidosRESUMO
The protective activity of dry olive leaf extract (DOLE) in carbon tetrachloride (CCl4)-induced liver damage and possible mechanisms involved in this protection were investigated in rats. Acute CCl4 intoxication resulted in a massive hepatic necrosis, in increased serum transaminases, and in a perturbation of oxidative stress parameters in liver tissue [malondyaldehide, glutathione (GSH), catalase]. CCl4 did not affect the expression of caspase-3 and cytochrome c as markers of apoptosis; however, CCl4 increased the AMP-activated protein kinase (AMPK) activity and the expression of autophagy-related protein LC3II and decreased the expression of p62 protein. The pre-treatment with DOLE significantly improved serum markers of liver damage, liver catalase activity, and GSH concentration, suggesting that antioxidative mechanism is responsible for hepatoprotection. Oral administration of DOLE did not influence LC3II conversion and p62 degradation in liver, but AMPK activity was significantly decreased, suggesting the energy balance perturbation as an additional potential mechanism of DOLE hepatoprotective effect.
Assuntos
Antioxidantes/farmacologia , Tetracloreto de Carbono/toxicidade , Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Olea/química , Extratos Vegetais/farmacologia , Substâncias Protetoras/farmacologia , Animais , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos WistarRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
For adequate hemodialysis, functional vascular access is obligatory. Neointimal hyperplasia (NIH) has a central role in stenosis and thrombosis development, which represent the most frequent causes of vascular access failure. Polymorphism of different genes that have a significant role in endothelial function may have an impact on NIH development. Therefore, the aim of our study is to determine the effect of angiotensin-converting enzyme (ACE) I/D and matrix metalloproteinase-3 (MMP3) 5A/6A polymorphism on risk for developing vascular access failure in hemodialysis patients. The study included 200 patients on regular hemodialysis at Nephrology Department, University Medical Center Zvezdara. Retrospective analysis included a collection of general and vascular access data from medical records. Genetic analysis was performed by using polymerase chain reaction-restriction fragment length polymorphism method (PCR-RFLP). Patients were divided into two groups: Group 1-patients who have never experienced vascular access failure and Group 2-patients who have at least one spontaneous vascular access failure. There was no difference in age, gender, hemodialysis vintage, main diagnosis, presence of hypertension, and diabetes mellitus between the two groups. There were no statistically significant differences in the frequencies of ACE and MMP3 genotypes between the two groups. Without statistical significance, it was found that homozygotes for I allele had two times higher risk for developing vascular access failure than homozygotes for D allele (OR 2.00; 95%CI: 0.727-5.503; P = 0.180). In addition, patients with 5A allele have 1.7 times higher risk for developing vascular access failure compared with patients without this allele (OR 1.745; 95% CI: 0.868-3.507; P = 0.118). Patients with vascular access failure do not have different genotype distribution regarding ACE gene and MMP3 gene polymorphism as compared with patients without vascular access failure. Still, homozygotes for I allele and homozygotes for 5A allele have higher risk for developing vascular access failure compared with other patients.
RESUMO
We examined the effects of betaine, an endogenous and dietary methyl donor essential for the methionine-homocysteine cycle, on oxidative stress, inflammation, apoptosis, and autophagy in methionine-choline deficient diet (MCD)-induced non-alcoholic fatty liver disease (NAFLD). Male C57BL/6 mice received standard chow (control), standard chow and betaine (1.5% w/v in drinking water), MCD, or MCD and betaine. After six weeks, serum and liver samples were collected for analysis. Betaine reduced MCD-induced increase in liver transaminases and inflammatory infiltration, as well as hepatosteatosis and serum levels of low-density lipoprotein, while it increased that of high-density lipoprotein. MCD-induced hepatic production of reactive oxygen and nitrogen species was significantly reduced by betaine, which also improved liver antioxidative defense by increasing glutathione content and superoxide-dismutase, catalase, glutathione peroxidase, and paraoxonase activity. Betaine reduced the liver expression of proinflammatory cytokines tumor necrosis factor and interleukin-6, as well as that of proapoptotic mediator Bax, while increasing the levels of anti-inflammatory cytokine interleukin-10 and antiapoptotic Bcl-2 in MCD-fed mice. In addition, betaine increased the expression of autophagy activators beclin 1, autophagy-related (Atg)4 and Atg5, as well as the presence of autophagic vesicles and degradation of autophagic target sequestosome 1/p62 in the liver of NAFLD mice. The observed effects of betaine coincided with the increase in the hepatic phosphorylation of mammalian target of rapamycin (mTOR) and its activator Akt. In conclusion, the beneficial effect of betaine in MCD-induced NAFLD is associated with the reduction of liver oxidative stress, inflammation, and apoptosis, and the increase in cytoprotective Akt/mTOR signaling and autophagy.
Assuntos
Betaína/uso terapêutico , Deficiência de Colina/metabolismo , Metionina/deficiência , Hepatopatia Gordurosa não Alcoólica/metabolismo , Estresse Oxidativo/fisiologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Animais , Autofagia/efeitos dos fármacos , Autofagia/fisiologia , Betaína/farmacologia , Deficiência de Colina/complicações , Fármacos Gastrointestinais/farmacologia , Fármacos Gastrointestinais/uso terapêutico , Inflamação/tratamento farmacológico , Inflamação/etiologia , Inflamação/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Hepatopatia Gordurosa não Alcoólica/etiologia , Estresse Oxidativo/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
The first lineage specification of pluripotent mouse epiblast segregates neuroectoderm (NE) from mesoderm and definitive endoderm (ME) by mechanisms that are not well understood. Here we demonstrate that the induction of ME gene programs critically relies on the T-box transcription factors Eomesodermin (also known as Eomes) and Brachyury, which concomitantly repress pluripotency and NE gene programs. Cells deficient in these T-box transcription factors retain pluripotency and differentiate to NE lineages despite the presence of ME-inducing signals transforming growth factor ß (TGF-ß)/Nodal and Wnt. Pluripotency and NE gene networks are additionally repressed by ME factors downstream of T-box factor induction, demonstrating a redundancy in program regulation to safeguard mutually exclusive lineage specification. Analyses of chromatin revealed that accessibility of ME enhancers depends on T-box factor binding, whereas NE enhancers are accessible and already activation primed at pluripotency. This asymmetry of the chromatin landscape thus explains the default differentiation of pluripotent cells to NE in the absence of ME induction that depends on activating and repressive functions of Eomes and Brachyury.
Assuntos
Cromatina/genética , Proteínas Fetais/genética , Camadas Germinativas/fisiologia , Células-Tronco Pluripotentes/fisiologia , Proteínas com Domínio T/genética , Animais , Diferenciação Celular/genética , Linhagem Celular , Separação Celular/métodos , Endoderma/fisiologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Masculino , Camundongos , Placa Neural/fisiologia , Fator de Crescimento Transformador beta/genéticaRESUMO
We investigated the therapeutic capacity of nano-sized graphene sheets, called graphene quantum dots (GQD), in experimental autoimmune encephalomyelitis (EAE), an animal model of immune-mediated central nervous system (CNS) damage. Intraperitoneally administered GQD (10â¯mg/kg/day) accumulated in the lymph node and CNS cells of Dark Agouti rats in which EAE was induced by immunization with spinal cord homogenate in complete Freund's adjuvant. GQD significantly reduced clinical signs of EAE when applied throughout the course of the disease (day 0-32), while the protection was less pronounced if the treatment was limited to the induction (day 0-7 post-immunization) or effector (from day 8 onwards) phase of the disease. GQD treatment diminished immune infiltration, demyelination, axonal damage, and apoptotic death in the CNS of EAE animals. GQD also reduced the numbers of interferon-γ-expressing T helper (Th)1â¯cells, as well as the expression of Th1 transcription factor T-bet and proinflammatory cytokines tumor necrosis factor, interleukin-1, and granulocyte-macrophage colony-stimulating factor in the lymph nodes and CNS immune infitrates. The protective effect of GQD in EAE was associated with the activation of p38 and p42/44 mitogen-activated protein kinases (MAPK) and Akt in the lymph nodes and/or CNS. Finally, GQD protected oligodendrocytes and neurons from T cell-mediated damage in the in vitro conditions. Collectively, these data demonstrate the ability of GQD to gain access to both immune and CNS cells during neuroinflammation, and to alleviate immune-mediated CNS damage by modulating MAPK/Akt signaling and encephalitogenic Th1 immune response.
Assuntos
Encefalomielite/imunologia , Encefalomielite/terapia , Grafite/uso terapêutico , Pontos Quânticos/uso terapêutico , Animais , Sistema Nervoso Central/imunologia , Sistema Nervoso Central/metabolismo , Citocinas/biossíntese , Citocinas/efeitos dos fármacos , Doenças Desmielinizantes , Encefalomielite Autoimune Experimental , Inflamação , Injeções Intraperitoneais , Linfonodos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Medula EspinalRESUMO
Pluripotency can be induced in vitro from adult somatic mammalian cells by enforced expression of defined transcription factors regulating and initiating the pluripotency network. Despite the substantial advances over the last decade to improve the efficiency of direct reprogramming, exact mechanisms underlying the conversion into the pluripotent stem cell state are still vaguely understood. Several studies suggested that induced pluripotency follows reversed embryonic development. For somatic cells of mesodermal and endodermal origin that would require the transition through a Primitive streak-like state, which would necessarily require an Eomesodermin (Eomes) expressing intermediate. We analyzed reprogramming in human and mouse cells of mesodermal as well as ectodermal origin by thorough marker gene analyses in combination with genetic reporters, conditional loss of function and stable fate-labeling for the broad primitive streak marker Eomes. We unambiguously demonstrate that induced pluripotency is not dependent on a transient primitive streak-like stage and thus does not represent reversal of mesendodermal development in vivo.
Assuntos
Reprogramação Celular/genética , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Linha Primitiva/citologia , Linha Primitiva/metabolismo , Proteínas com Domínio T/metabolismo , Animais , Reprogramação Celular/fisiologia , Ectoderma/citologia , Ectoderma/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Mesoderma/citologia , Mesoderma/metabolismo , Camundongos , Proteínas com Domínio T/genéticaRESUMO
We investigated the role of the intracellular energy-sensing AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway in the in vitro antiglioma effect of the cyclooxygenase (COX) inhibitor indomethacin. Indomethacin was more potent than COX inhibitors diclofenac, naproxen, and ketoprofen in reducing the viability of U251 human glioma cells. Antiglioma effect of the drug was associated with p21 increase and G2M cell cycle arrest, as well as with oxidative stress, mitochondrial depolarization, caspase activation, and the induction of apoptosis. Indomethacin increased the phosphorylation of AMPK and its targets Raptor and acetyl-CoA carboxylase (ACC), and reduced the phosphorylation of mTOR and mTOR complex 1 (mTORC1) substrates p70S6 kinase and PRAS40 (Ser183). AMPK knockdown by RNA interference, as well as the treatment with the mTORC1 activator leucine, prevented indomethacin-mediated mTORC1 inhibition and cytotoxic action, while AMPK activators metformin and AICAR mimicked the effects of the drug. AMPK activation by indomethacin correlated with intracellular ATP depletion and increase in AMP/ATP ratio, and was apparently independent of COX inhibition or the increase in intracellular calcium. Finally, the toxicity of indomethacin towards primary human glioma cells was associated with the activation of AMPK/Raptor/ACC and subsequent suppression of mTORC1/S6K. By demonstrating the involvement of AMPK/mTORC1 pathway in the antiglioma action of indomethacin, our results support its further exploration in glioma therapy.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Glioma/tratamento farmacológico , Glioma/metabolismo , Indometacina/farmacologia , Complexos Multiproteicos/antagonistas & inibidores , Serina-Treonina Quinases TOR/antagonistas & inibidores , Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Proteínas Quinases Ativadas por AMP/genética , Monofosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Apoptose/efeitos dos fármacos , Cálcio/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Inibidores de Ciclo-Oxigenase/farmacologia , Glioblastoma/tratamento farmacológico , Glioblastoma/metabolismo , Glioma/patologia , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina , Modelos Biológicos , Complexos Multiproteicos/metabolismo , RNA Interferente Pequeno/genética , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismoRESUMO
Direct reprogramming by forced expression of transcription factors can convert one cell type into another. Thus, desired cell types can be generated bypassing pluripotency. However, direct reprogramming towards renal cells remains an unmet challenge. Here, we identify renal cell fate-inducing factors on the basis of their tissue specificity and evolutionarily conserved expression, and demonstrate that combined expression of Emx2, Hnf1b, Hnf4a and Pax8 converts mouse and human fibroblasts into induced renal tubular epithelial cells (iRECs). iRECs exhibit epithelial features, a global gene expression profile resembling their native counterparts, functional properties of differentiated renal tubule cells and sensitivity to nephrotoxic substances. Furthermore, iRECs integrate into kidney organoids and form tubules in decellularized kidneys. Our approach demonstrates that reprogramming factors can be identified by targeted in silico analysis. Renal tubular epithelial cells generated ex vivo by forced expression of transcription factors may facilitate disease modelling, drug and nephrotoxicity testing, and regenerative approaches.
Assuntos
Reprogramação Celular , Células Epiteliais/citologia , Fibroblastos/citologia , Túbulos Renais/citologia , Fatores de Transcrição/metabolismo , Animais , Agregação Celular , Linhagem da Célula , Proliferação de Células , Forma Celular , Células Cultivadas , Análise por Conglomerados , Embrião de Mamíferos/citologia , Células Epiteliais/ultraestrutura , Imunofluorescência , Perfilação da Expressão Gênica , Humanos , Camundongos , Néfrons/citologia , Néfrons/metabolismo , Organoides/citologia , XenopusRESUMO
INTRODUCTION: Retrospective studies showed that hemodiafiltration was associated with a reduced risk of mortality compared with standard hemodialysis in the patients with end-stage renal disease. Recently, a few prospective randomized clinical trials found no advantage in survival with hemodiafiltration as compared with high-flux hemodialysis and low-flux hemodialysis. The aim of this study was to compare the parameters of hemodialysis adequacy and two-year survival of patients depending on the modality of hemodialysis. MATERIAL AND METHODS: A total of 159 hemodialysis patients were divided into 3 groups according to the type of hemodialysis treatment: group A - low-flux hemodialysis, group B - high-flux hemodialysis, and group C - hemodiafiltration. All patients had the same duration of hemodialysis sessions. The analysis included average one-year biochemical parameters, and two-year survival of patients. RESULTS: The patients on hemodiafiltration were significantly younger, they had longer dialysis vintage and higher index of dialysis adequancy as compared with the patients on low-flux hemodialysis and high-flux hemodialysis, but without a difference between the two latter groups. Compared to the patients on low-flux hemodialysis, the patients on hemodiafiltration and high-flux hemodialysis had significantly higher hemoglobin value with less frequent erythropoietin stimulating agent use. According to Kaplan-Meier survival analysis, the patients on hemodiafiltration and high-flux hemodialysis had significantly better two-year survival than the patients on low-flux hemodialysis. Cox proportional hazards model confirmed that high-flux hemodialysis caused a significantly lower relative risk of mortality (56% reduction) compared to low-flux hemodialysis (hazard ratio 0.44; P=0.026), and hemodiafiltration caused a 58% reduction in the relative risk of mortality compared to low-flux dialysis (hazard ratio 0.42; P=0.105), but without a statistical significance. CONCLUSION: This study has demonstrated two-year survival benefit with high-flux hemodialysis and hemodiafiltration compared with low-flux hemodialysis. There was no difference in survival between high-flux hemodialysis and hemodiafiltration groups.
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Hemodiafiltração/métodos , Falência Renal Crônica/terapia , Diálise Renal/métodos , Idoso , Anemia/complicações , Anemia/tratamento farmacológico , Eritropoetina/uso terapêutico , Feminino , Hematínicos/uso terapêutico , Humanos , Estimativa de Kaplan-Meier , Falência Renal Crônica/complicações , Falência Renal Crônica/mortalidade , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Estudos Retrospectivos , Taxa de Sobrevida , Resultado do TratamentoRESUMO
INTRODUCTION: The use of non-steroidal anti-inflammatory drugs may lead to stricture of the small intestine and less frequently of the colon. Colonic strictures have not been described in patients on dialysis and the aim of this report is to show the case of dialysis patient who was followed for recurrent and prolonged diarrhea. CASE REPORT: We present the patient on chronic dialysis for 15 years who used non-steroidal anti-inflammatory drugs due to chronic pain and who developed recurrent diarrhea. Diagnosis was made by endoscopy and confirmed by histology. Specific therapy was applied with a good response. CONCLUSION: Although not described in the literature, non-steroidal anti-inflammatory drug-induced colitis and/or diaphragm disease could be a potential reason for recurrent or prolonged diarrhea in dialysis patients.
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Anti-Inflamatórios não Esteroides/efeitos adversos , Colite/induzido quimicamente , Colite/diagnóstico , Diálise Renal , Insuficiência Renal/terapia , Colite/terapia , Diafragma , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
INTRODUCTION: The optimal length of dialysis is still under debate and current regimen of 12 hours a week is medically acceptable. The aim of this observational study was to confirm the relationship between different length of dialysis per week and the parameters of dialysis adequacy and cardiovascular morbidity. MATERIAL AND METHODS: The study included 206 patients (128 man and 78 females) who were on maintenance hemodialysis for more than 6 months. They were classified into three groups according to the length of dialysis per week: group I (12 hours), group II (15 hours) and group III (≥17.5 hours). RESULTS: Index of dyalysis adequacy values did not differ among the groups (group I, II, III = 1.32 vs. 1.51 vs. 1.42; p>0.05); however, the patients from group III had the best bicarbonate level (group I, II and III = 22.7; 21.4; 17.6 mmol/L; p<0.001). In comparison with group I (12 hours), longer dialysis duration was associated with significantly higher hemoglobin values (12.2 vs. 11.4 vs. 10.5 g/dL), less frequent use of erythropoietin stimulating agents (26.9% vs. 65% vs. 86.3%), lower stimulating agents weekly dose (median in group I, II, III = 2000 vs. 5000 vs. 4000 I.J.), lower stimulating agents resistance index (4.9 vs. 7.8 vs. 8.8), significantly higher level of serum albumin (42.3 vs. 40.7 vs. 38.2 g/dL), total cholesterol (5.1 vs. 4.7 vs. 4.5 mmol/L) and serum calcium level (2.38 vs. 2.42 vs. 2.28 mmol/L), less frequent use of phosphate binders (53.8% vs. 85% vs. 84.4%) and calcitriol (19.2% vs. 65% vs. 50.6%) and lower intact parathyroid hormone level (336 vs. 363 vs. 446 pg/ml). In addition, longer dialysis duration was associated with lower cardiovascular morbidity score (0.52 vs. 1.05 vs. 1.26). CONCLUSION: Duration of dialysis per week above the current standard positively correlates with parameters of hemodialysis adequacy.
Assuntos
Doenças Cardiovasculares/epidemiologia , Diálise Renal/métodos , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estado Nutricional , Estudos Retrospectivos , Fatores de Tempo , Resultado do TratamentoRESUMO
INTRODUCTION: Cardiovascular morbidity and mortality are the major concern in dialysis patients and many risk factors are thought to be involved in its pathogenesis. Apart from traditional and non-traditional risk factors, the genetic susceptibility may be of importance, including renin-angiotensin system gene polymorphism. The aim of this study was to analyse renin-angiotensin system polymorphism in our group of hemodialysis patients and to correlate the findings with cardiovascular morbidity. MATERIAL AND METHODS: The study included 196 patients on regular hemodialysis on polysulphone membrane three times per week for more than six months. Genetic analysis was performed by using polymerase chain reaction-restriction fragment length polymorphism method. RESULTS: Out of 196 patients, 55% had I/D genotype, 35% had D/D and 10% had I/I, including angiotensin-converting enzyme polymorphism. It was shown that the patients with D allele genotype developed a significantly higher incidence of left ventricular hypertrophy and peripheral vascular disease. The angiotensin-converting enzyme polymorphism showed a significant association with the incidence of cerebrovascular accident and hyperlipoproteinemia in our group of hemodialysis patients. CONCLUSION: The angiotensin-converting enzyme gene polymorphism is associated with the development of cerebrovascular accidents and hyperlipoproteinemia. Allele D of this gene increases the risk for the development of left ventricular hypertrophy and peripheral vascular disease significantly in hemodialysis patients. A longer follow-up is needed to make the definitive conclusion about the influence of angiotensin-converting enzyme polymorphism on cardiovascular morbidity and its importance in everyday clinical practice.