Avaliação do papel da endostatina imobilizada e regulação do NF-kB em células HK-2 submetidas à injúria por estresse oxidativo
Data
2015-08-31
Autores
Rocha Filho, Hugo Nobrega da 
Orientadores
Bellini, Maria Helena
Tipo
Dissertação de mestrado
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Resumo
A lesão renal aguda (LRA) é uma síndrome resultante da queda abrupta na taxa de filtração glomerular, acarretando profunda depleção de ATP intracelular. Tais alterações promovem a geração excessiva de espécies reativas de oxigênio (EROs), ativando mecanismos de estresse oxidativo e morte celular. As EROs participam da ativação do fator de transcrição nuclear NF-kB. A região NC1 do colágeno XVIII pode sofrer clivagem por proteases ou metaloproteinases e liberar endostatina (ES) e suas isoformas. Em alguns trabalhos anteriores nosso grupo demonstrou, in vitro, que uma das isoformas da ES imobilizada promoveu a sobrevivência de células endoteliais por meio do favorecimento da adesão celular. A partir de tais evidências, nosso objetivo foi avaliar o papel da ES imobilizada e a regulação do NF-kB em células HK-2 submetidas a injúria por estresse oxidativo. Desse forma, células HK-2 com (HK-2/ES) e sem ES (HK-2), foram injuriadas pela exposição ao Peróxido de Hidrogênio (H2O2) na concentração de 1mM por 2 horas e após 24 e 48 horas foram analisadas. Células HK-2 que não foram injuriadas foram nomeadas como grupo Controle. Avaliou-se o papel da ES imobilizada e a regulação do NF-kB através dos seguintes ensaios: Avaliação da viabilidade celular (ensaios de MTS e quantificação da enzima Lactato Desidrogenase - LDH), quantificação de EROs (citometria de fluxo com a sonda CM-H2DCFDA) e a expressão gênica do NF-kB através de PCR em tempo real e sua ligação ao DNA pelo ensaio do desvio da mobilidade eletroforética (EMSA). Nossos resultados evidenciaram que as células do grupo HK-2/ES apresentaram taxas de atividade mitocondrial/viabilidade celular de 49% (P < 0,001) após 24horas e de 25% (P < 0,001) após 48horas maiores em relação as HK-2. A quantificação de LDH revelou que após 24hs de injúria as células HK-2/ES apresentaram uma taxa de liberação de LDH 26% menor em relação as células foram injuriadas sem o prévio revestimento com ES. O resultados da quantificação de EROs corroboraram os achados dos ensaios anteriores. As células HK-2/ES apresentaram redução na produção de EROS de 35% em 24hs (HK-2 vs HK-2/ES P<0,005) e de 27% em 48hs (HK-2 vs HK-2/ES P<0,003). As células HK-2/ES apresentaram uma diminuição da transcrição das subunidades p-50 de 3,5 vezes (P < 0,001) e p65 de 3,4 vezes (P < 0,001) da via de sinalização do NF-?B. Os resultados do EMSA mostraram que o revestimento com ES acarretou uma diminuição da ativação do NF-KB. Assim nossos resultados indicam que a ES imobilizada minimiza a ativação do NFK-B após injúria por estresse oxidativo em células tubulares, refletindo no aumento da viabilidade celular.
Acute kidney injury (AKI) is a syndrome denoted by an abrupt decline in glomerular filtration rate. The ischemia is the main cause of AKI, and develops due to a decline in renal blood flow which invariably promotes oxygen restriction, nutrients and accumulation of metabolites in the tissues, leading to a depletion of intracellular ATP. Such changes, lead to injury in the tubular epithelial cells, leading to excessive generation of reactive oxygen species (ROS), mitochondrial dysfunction and the increase of the enzyme lactate dehydrogenase (LDH), thus activating mechanisms of oxidative stress and cell death. The ROS participate in the activation of NF-kB, which is a pleiotropic transcription factor that integrates a complex network of extracellular stimuli and signaling pathways, resulting in the transcriptional regulation of hundreds of genes related to inflammation, immunity, apoptosis, oxidative stress, cell differentiation and proliferation. The collagen XVIII is a non-fibrillar proteoglycan, member of the multiplexin subgroup. Cleavage of the C-terminal region of collagen XVIII, by proteases or metalloproteinases produces endostatin (ES), an inhibitor of angiogenesis. The aim of this study was to evaluate the role of immobilized ES and the regulation of NF-kB in HK-2 subjected to injury by oxidative stress. The first step of this work, cell culture model of oxidative stress was standardized with 1 mM and 2 hours of hydrogen peroxide (H2O2) exposure. It was demonstrated, by means of tests MTS, LDH and exclusion of trypan blue dye, a decrease in cell viability after 24 and 48 hours after exposure to H2O2 (control group vs. HK-2 group; P <0.001). In the second step we evaluated the role of ES immobilized in cellular injury model by oxidative stress. Cells cultured on plates coated with ES had significantly higher viability and lower release of ROS than the groups that were cultured in uncoated plates (HK-2 group vs HK-2 / ES; P <0.001). Subsequently, the role of ES immobilized in the regulation of NF-¿B was verified. By real-time PCR assay, a significant reduction of p-50 and p65 subunits gene expression was found (HK-2 HK group vs group -2 / ES, P <0.001). The presence of p-50 and p-65 protein was also assessed by indirect immunofluorescence assay where positive labeling were observed in all experimental groups. It has also been demonstrated by EMSA gel, decreased NF-kB binding to DNA in the experimental groups coated ES (HK-2 versus Group HK-2 / ES, P <0.001). The supershift gel showed the displacement of the p65 subunit in the control group. Our results indicate that the immobilized ES minimizes the activation of NF-kB after injury by oxidative stress in HK-2 cells, reflecting the decrease in production of ROS and increased cell viability.
Acute kidney injury (AKI) is a syndrome denoted by an abrupt decline in glomerular filtration rate. The ischemia is the main cause of AKI, and develops due to a decline in renal blood flow which invariably promotes oxygen restriction, nutrients and accumulation of metabolites in the tissues, leading to a depletion of intracellular ATP. Such changes, lead to injury in the tubular epithelial cells, leading to excessive generation of reactive oxygen species (ROS), mitochondrial dysfunction and the increase of the enzyme lactate dehydrogenase (LDH), thus activating mechanisms of oxidative stress and cell death. The ROS participate in the activation of NF-kB, which is a pleiotropic transcription factor that integrates a complex network of extracellular stimuli and signaling pathways, resulting in the transcriptional regulation of hundreds of genes related to inflammation, immunity, apoptosis, oxidative stress, cell differentiation and proliferation. The collagen XVIII is a non-fibrillar proteoglycan, member of the multiplexin subgroup. Cleavage of the C-terminal region of collagen XVIII, by proteases or metalloproteinases produces endostatin (ES), an inhibitor of angiogenesis. The aim of this study was to evaluate the role of immobilized ES and the regulation of NF-kB in HK-2 subjected to injury by oxidative stress. The first step of this work, cell culture model of oxidative stress was standardized with 1 mM and 2 hours of hydrogen peroxide (H2O2) exposure. It was demonstrated, by means of tests MTS, LDH and exclusion of trypan blue dye, a decrease in cell viability after 24 and 48 hours after exposure to H2O2 (control group vs. HK-2 group; P <0.001). In the second step we evaluated the role of ES immobilized in cellular injury model by oxidative stress. Cells cultured on plates coated with ES had significantly higher viability and lower release of ROS than the groups that were cultured in uncoated plates (HK-2 group vs HK-2 / ES; P <0.001). Subsequently, the role of ES immobilized in the regulation of NF-¿B was verified. By real-time PCR assay, a significant reduction of p-50 and p65 subunits gene expression was found (HK-2 HK group vs group -2 / ES, P <0.001). The presence of p-50 and p-65 protein was also assessed by indirect immunofluorescence assay where positive labeling were observed in all experimental groups. It has also been demonstrated by EMSA gel, decreased NF-kB binding to DNA in the experimental groups coated ES (HK-2 versus Group HK-2 / ES, P <0.001). The supershift gel showed the displacement of the p65 subunit in the control group. Our results indicate that the immobilized ES minimizes the activation of NF-kB after injury by oxidative stress in HK-2 cells, reflecting the decrease in production of ROS and increased cell viability.
Descrição
Citação
ROCHA FILHO, Hugo Nobrega da. Avaliação do papel da endostatina imobilizada e regulação do NF-kB em células HK-2 submetidas à injúria por estresse oxidativo. 2015. 70 f. Dissertação (Mestrado em Nefrologia) - Escola Paulista de Medicina, Universidade Federal de São Paulo (UNIFESP), São Paulo, 2015.