RESUMO
Diabetic nephropathy (DN) is the main cause of morbidity and mortality in diabetes and is characterized by mesangial matrix deposition and podocytopathy, including podocyte loss. The risk factors and mechanisms involved in the pathogenesis of DN are still not completely defined. In the present study, we aimed to understand the cellular mechanisms through which activation of B2 kinin receptors contribute to the initiation and progression of DN. Stimulation of cultured rat podocytes with bradykinin (BK) resulted in a significant increase in ROS generation, and this was associated with a significant increase in NADPH oxidase (NOX)1 and NOX4 protein and mRNA levels. BK stimulation also resulted in a signicant increase in the phosphorylation of ERK1/2 and Akt, and this effect was inhibited in the presence of NOX1 and Nox4 small interfering (si)RNA. Furthermore, podocytes stimulated with BK resulted in a significant increase in protein and mRNA levels of connective tissue growth factor (CTGF) and, at the same time, a significant decrease in protein and mRNA levels of nephrin. siRNA targeted against NOX1 and NOX4 significantly inhibited the BK-induced increase in CTGF. Nephrin expression was increased in response to BK in the presence of NOX1 and NOX4 siRNA, thus implicating a role for NOXs in modulating the BK response in podocytes. Moreover, nephrin expression in response to BK was also significantly increased in the presence of siRNA targeted against CTGF. These findings provide novel aspects of BK signal transduction pathways in pathogenesis of DN and identify novel targets for interventional strategies.
Assuntos
Bradicinina/farmacologia , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Proteínas de Membrana/metabolismo , Podócitos/efeitos dos fármacos , Animais , Células Cultivadas , Fator de Crescimento do Tecido Conjuntivo/genética , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteínas de Membrana/genética , NADH NADPH Oxirredutases/genética , NADH NADPH Oxirredutases/metabolismo , NADPH Oxidase 1 , NADPH Oxidase 4 , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Fosforilação , Podócitos/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , RNA Mensageiro/metabolismo , Ratos , Receptor B2 da Bradicinina/agonistas , Receptor B2 da Bradicinina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Transfecção , Regulação para CimaRESUMO
The contribution of plasma prekallikrein (PK) to vascular remodeling is becoming increasingly recognized. Plasma PK is activated when the zymogen PK is digested to an active enzyme by activated factor XII (FXII). Here, we present our findings that vascular smooth muscle cells (VSMC) activate plasma PK in the absence of FXII. Extracted plasma membrane and cytosolic fractions of VSMCs activate PK, but the rate of PK activation was greater by the membrane fraction. FXII neutralizing antibody did not affect PK activation by extracted proteins of VSMCs. VSMC PKA was inhibited by the serine protease inhibitors such as aprotinin, phenylmethylsulfonyl fluoride, leupeptin and CTI with CI50 of 0.78 µM, 1 mM, 3.13 µM and 40 nM on the cultured cells, respectively. No inhibition of PK activation by cysteine, aspartic acid, and metalloprotease inhibitors was observed. This is the first report of the presence of an intrinsic PKA in VSMC. Considering that VSMCs are normally separated from the circulating blood by endothelial cells, direct PK activation by VSMCs may play a role in disease states like diabetes, hyperlipidemia or hypertension where the endothelial layer is damaged.