Efeitos do lipopolissacarídeo de escherichia coli (ecoli) sobre as catecolaminas nas células mesangiais humanas imortalizadas e em situação de hipóxia
Data
2013-02-27
Autores
Peralta, Dayane Cabo Grosso 
Orientadores
Casarini, Dulce Elena
Tipo
Dissertação de mestrado
Título da Revista
ISSN da Revista
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Resumo
As células mesangiais (CM) possuem toda a maquinaria para a produção das catecolaminas (CA): dopamina (DA), adrenalina (AD) e noradrenalina (NOR), bem como expressam o mCD14, um dos componentes do receptor de lipopolissacarídeo (LPS) que está associado ao Toll Like receptor tipo 4 na superfície celular. O LPS e a hipóxia celular presentes na sepse são fatores que podem desencadear a insuficiência renal aguda. O objetivo deste estudo foi avaliar possíveis alterações na produção/liberação de catecolaminas (CAs) pelas células mesangiais humanas imortalizadas (CMHI) frente ao estímulo com LPS/hipóxia. As CMHI foram cultivadas em meio DMEM (Dulbecco’s Modified Eagle’s Medium) com 10% de soro bovino fetal até a confluência. No dia do experimento estas células foram cultivadas com meio DMEM sem soro fetal bovino, e posteriormente submetidas à incubação com LPS e à situação de hipóxia. As CAs e o cofator tetrahidrobiopterina (BH4) foram quantificados utilizando-se cromatografia líquida de alta performance (HPLC). A expressão das enzimas que sintetizam CA foi avaliada por Western Blotting. As CMHI apresentaram-se viáveis frente ao processo de LPS/hipóxia. LPS 100 μg/mL elevou no compartimento intracelular os níveis de NOR e AD discretamente, sendo significante o aumento de L-DOPA e DA. Em paralelo, reduziu a concentração de NOR, AD, DA e L-DOPA no meio extracelular. Houve um aumento na expressão da enzima tirosina hidroxilase (TH) nas CMHI incubadas com LPS 100 μg/mL, similar aos níveis de CA observados nestas células frente ao mesmo estímulo. Os níveis de BH4 no compartimento extracelular estão diretamente relacionados aos níveis de elevados L-DOPA no grupo LPS/hipóxia. Os resultados sugerem que o LPS/hipóxia pode influenciar a atividade da enzima passo limitante da cascata de produção de catecolaminas a TH e seu cofator, ambos aumentados no meio intracelular, bem como a produção/liberação das CA nas CMHI, um modelo sem interferência neuronal. Esses resultados sugerem que as CAs produzidas nas CMHI podem agir de forma autócrina e parácrina no rim, ora vasodilatando ora vasoconstritando as arteríolas, contraindo as CMs, participando desta forma na fisiopatologia da insuficiência renal aguda, podendo levar a perda da função renal.
Mesangial cells (MC) have the entire machinery for the production of catecholamines (CA), dopamine (DA), adrenaline (AD) and noradrenaline (NOR), are able to express mCD14, a component of the receptor lipopolysaccharide (LPS) that is associated with type Toll like 4 receptor on the cell surface. The LPS in sepsis can trigger acute renal failure. We aimed to evaluate production/release of CA by immortalized human mesangial cell (IHMC) treated with LPS/hypoxia. The IHMC were cultured in DMEM with 10% fetal bovine serum. For the experiments in presence of LPS/hypoxia the cells were incubated with DMEM without FBS. The CA and cofactor tetrahydrobiopterin (BH4) were quantified using HPLC. The enzymes as tyrosine hydroxylase (TH), dopa decarboxylase (DDC) and dopamine - hydroxylase (DH) were detected by Western blotting. Incubation of HMC with LPS 100 g/ml increased intracellular levels of L-DOPA and DA, and at the same time reduced the concentration of AD and L-DOPA in the extracellular. There was an increase in expression of TH in IHMC incubated with LPS 100 g/ml that was correlated with the levels of CA. BH4, the enzymatic cofactor of tyrosine hydroxylase levels, in extracellular compartment are directly related to increased levels of CA in the cells treated with LPS. CAs are involved in various physiological processes in the kidney, being factors that impact on renal hemodynamics. The results suggested that LPS/hypoxia can influenciate the activity of TH and their cofactor, both increased intracellularly, as well as, production/release of CA in IHMC, a model without neuronal interferences. These results suggest that the IHMC through the CAs production would be able to act as a vasodilator or vasoconstrictor paracrine hormone upon afferent and efferent arterioles and to contract the MC participating in the pathophysiology of acute renal failure.
Mesangial cells (MC) have the entire machinery for the production of catecholamines (CA), dopamine (DA), adrenaline (AD) and noradrenaline (NOR), are able to express mCD14, a component of the receptor lipopolysaccharide (LPS) that is associated with type Toll like 4 receptor on the cell surface. The LPS in sepsis can trigger acute renal failure. We aimed to evaluate production/release of CA by immortalized human mesangial cell (IHMC) treated with LPS/hypoxia. The IHMC were cultured in DMEM with 10% fetal bovine serum. For the experiments in presence of LPS/hypoxia the cells were incubated with DMEM without FBS. The CA and cofactor tetrahydrobiopterin (BH4) were quantified using HPLC. The enzymes as tyrosine hydroxylase (TH), dopa decarboxylase (DDC) and dopamine - hydroxylase (DH) were detected by Western blotting. Incubation of HMC with LPS 100 g/ml increased intracellular levels of L-DOPA and DA, and at the same time reduced the concentration of AD and L-DOPA in the extracellular. There was an increase in expression of TH in IHMC incubated with LPS 100 g/ml that was correlated with the levels of CA. BH4, the enzymatic cofactor of tyrosine hydroxylase levels, in extracellular compartment are directly related to increased levels of CA in the cells treated with LPS. CAs are involved in various physiological processes in the kidney, being factors that impact on renal hemodynamics. The results suggested that LPS/hypoxia can influenciate the activity of TH and their cofactor, both increased intracellularly, as well as, production/release of CA in IHMC, a model without neuronal interferences. These results suggest that the IHMC through the CAs production would be able to act as a vasodilator or vasoconstrictor paracrine hormone upon afferent and efferent arterioles and to contract the MC participating in the pathophysiology of acute renal failure.
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Citação
PERALTA, Dayane Cabo Grosso. Efeitos do lipopolissacarídeo de escherichia coli (ecoli) sobre as catecolaminas nas células mesangiais humanas imortalizadas e em situação de hipóxia. 2013. Dissertação (Mestrado) - Escola Paulista de Medicina, Universidade Federal de São Paulo (UNIFESP), São Paulo, 2013.