Vera Manel, Torramade-Moix Sergi, Martin-Rodriguez Susana, Cases Aleix, Cruzado Josep M, Rivera Jose, Escolar Gines, Palomo Marta, Diaz-Ricart Maribel
Nephrology Department, Hospital Clinic of Barcelona, Barcelona, Spain.
Institute of Biomedical Research August Pi i Sunyer (IDIBAPS), University of Barcelona, Barcelona, Spain.
Cell Physiol Biochem. 2018;51(3):1287-1300. doi: 10.1159/000495540. Epub 2018 Nov 27.
BACKGROUND/AIMS: Accelerated atherosclerosis in chronic kidney disease (CKD) is preceded by endothelial dysfunction (ED), which exhibits a proinflammatory and prothrombotic phenotype and enhanced oxidative stress. In this study, the effect of several compounds with anti-inflammatory and/or antioxidant properties on uremia-induced endothelial dysfunction has been evaluated in an in vitro model.
Endothelial cells (ECs) were exposed to sera from uremic patients in the absence and presence of the flavonoids apigenin, genistein and quercetin, the antioxidant enzyme mimetics (AEM) ebselen (glutathione peroxidase mimetic), EUK-134 and EUK-118 (both superoxide dismutase mimetics), and the pharmacological drug N-acetylcysteine (NAC). We explored changes in the expression of adhesion receptors on the cell surface, by immunofluorescence, the production of radical oxygen species (ROS), by fluorescence detection, and the activation of signaling proteins related to inflammation, by both a phosphospecific antibody cell-based ELISA and immunoblotting techniques.
Uremic media induced a significantly increased expression of ICAM-1, overproduction of radical oxygen species (ROS) and activation of p38 mitogen activated protein kinase (p38MAPK) and Nuclear Factor kB (NFkB) in ECs. Quercetin, the AEM and NAC showed a significant inhibitory effect on both ICAM-1 expression and ROS generation (p<0.05). All the compounds reduced p38MAPK activation, but only the AEM, especially ebselen, and NAC, both potentiating the glutathione peroxidase pathway, also inhibited NFkB activation. These two compounds were capable of increasing endothelial glutathione levels, especially in response to uremia.
Our results indicate that the potentiation of the antioxidant pathways can be an effective strategy to improve endothelial dysfunction in uremia and a potential target to reduce the cardiovascular risk in this population.
背景/目的：慢性肾脏病（CKD）患者动脉粥样硬化加速之前存在内皮功能障碍（ED），其表现为促炎和促血栓形成表型以及氧化应激增强。在本研究中，已在体外模型中评估了几种具有抗炎和/或抗氧化特性的化合物对尿毒症诱导的内皮功能障碍的影响。
在内皮细胞（ECs）不存在和存在黄酮类化合物芹菜素、染料木黄酮和槲皮素、抗氧化酶模拟物（AEM）依布硒仑（谷胱甘肽过氧化物酶模拟物）、EUK-134和EUK-118（均为超氧化物歧化酶模拟物）以及药物N-乙酰半胱氨酸（NAC）的情况下，将其暴露于尿毒症患者的血清中。我们通过免疫荧光探索细胞表面黏附受体表达的变化，通过荧光检测探索活性氧（ROS）的产生，并通过基于磷酸特异性抗体的细胞酶联免疫吸附测定和免疫印迹技术探索与炎症相关的信号蛋白的激活。
尿毒症培养基诱导ECs中ICAM-1表达显著增加、活性氧（ROS）过量产生以及p38丝裂原活化蛋白激酶（p38MAPK）和核因子κB（NFkB）激活。槲皮素、AEM和NAC对ICAM-1表达和ROS生成均显示出显著抑制作用（p<0.05）。所有化合物均降低了p38MAPK激活，但只有AEM，尤其是依布硒仑，以及NAC，二者均增强了谷胱甘肽过氧化物酶途径，也抑制了NFkB激活。这两种化合物能够增加内皮谷胱甘肽水平，尤其是对尿毒症的反应。
我们的结果表明，增强抗氧化途径可能是改善尿毒症患者内皮功能障碍的有效策略，也是降低该人群心血管风险的潜在靶点。
