肾脏5-羟色胺合成和降解在高血糖诱导肾损伤时的作用研究

The role of renal 5-hydroxytryptamine synthesis and degradation in hyperglycemia-induced kidney injury

糖尿病患者常出现高血糖性肾损伤(hyperglycemic kidney injury,HKI)并发症。本文研究了高血糖致肾脏5-羟色胺(5-hydroxytryptamine,5-HT)系统表达异常与HKI的关系。动物实验用高脂饲料喂养结合腹腔注射链脲佐菌素建立小鼠2型糖尿病(type 2 diabetes mellitus,T2DM)模型,用5-HT_(2A)受体(5-HT_(2A)receptor,5-HT_(2A)R)拮抗剂盐酸沙格雷酯(sarpogrelate hydrochloride,SH)及5-HT合成抑制剂卡比多巴(carbidopa,CDP)分别或联合给药治疗。细胞实验用D-葡萄糖(D-glucose,D-Glu)刺激人肾小球系膜细胞(human glomerular mesangial cells,HMC),并用SH、CDP或单胺氧化酶A(monoamine oxidase A,MAO-A)抑制剂氯吉兰分别抑制5-HT_(2A)R、5-HT合成及5-HT降解。实验用PAS染色法(periodic acid-Schiff stain)和Masson染色、免疫组化和Western blot、荧光探针、酶联免疫吸附剂测定(enzyme linked immunosorbent assay,ELISA)和酶试剂分别检测组织病理学、蛋白表达、细胞内活性氧和生化指标。本文中,动物福利和实验过程均遵循中国药科大学动物伦理委员会的规定。结果表明,小鼠肾脏的肾小球基底膜、肾小管上皮细胞和HMC细胞均存在5-HT_(2A)R、5-HT合成酶及MAO-A表达,且在T2DM小鼠及高浓度D-Glu刺激HMC细胞时被上调;以肾功能异常、肾小球肿胀及基底膜增厚和纤维化为主要表现的HKI与肾脏5-HT_(2A)R表达上调、5-HT合成及5-HT降解的增加密切相关。其中,5-HT_(2A)R可介导细胞的5-HT合成酶及MAO-A的表达;而MAO-A通过催化5-HT降解、使线粒体活性氧(reactive oxygen species,ROS)产生增多,导致核因子κB(nuclear factor kappa B,NF-κB)的磷酸化、炎症因子产生及基质金属蛋白酶-2(matrix metalloproteinases-2,MMP-2)、α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)表达上调及胶原产生。用SH和CDP可有效治疗HKI,且联合用药有协同效应。

Hyperglycemic kidney injury(HKI)is a common complication of diabetic patients.We examined the relationship between HKI and the abnormal expression of 5-hydroxytryptamine(5-HT)system induced by hyperglycemia in type 2 diabetes mellitus(T2 DM).In animal experiments,a T2 DM model was established in mice by feeding a high-fat diet with intraperitoneal injection of streptozotocin.The mice were treated with the 5-HT2A receptor(5-HT2AR)antagonist sarpogrelate hydrochloride(SH)and 5-HT synthesis inhibitor carbidopa(CDP)(respectively or in combination).In cell culture experiments,human glomerular mesangial cells(HMC)were stimulated with D-glucose(D-Glu),and 5-HT2AR,5-HT synthesis,and 5-HT degradation were inhibited by SH,CDP,or monoamine oxidase A(MAO-A)inhibitor clorgyline.Periodic acid-Schiff(PAS)staining and Masson staining,immunohistochemistry and Western blot,fluorescent probe,and enzyme linked immunosorbent assay(ELISA)and enzyme reagent were respectively used to detect histopathology,protein expression,intracellular reactive oxygen species(ROS),and biochemical indexes.The animal experiments were in accordance with the regulations of the Animal Ethics Committee of China Pharmaceutical University.The results showed that 5-HT2AR,5-HT synthases,and MAO-A were expressed in glomerular basement membrane and kidney tubular epithelial cells of mouse kidney and HMC.The expression of these proteins was significantly up-regulated in T2 DM mice or when HMC cells were exposed to high concentration of D-Glu.HKI,characterized by abnormal renal function,glomerular swelling,and glomerular basement membrane thickening and fibrosis,is closely associated with an increase in kidney 5-HT_(2A)R,5-HT synthesis,and 5-HT degradation.Among them,5-HT_(2A)R can mediate the expression of 5-HT synthases and MAO-A;MAO-A can catalyze the degradation of 5-HT to increase the production of mitochondrial ROS,leading to the phosphorylation of nuclear factor kappa B(NF-κB)with the production of inflammatory cytokines,and the up-regulation of matrix metalloproteinase-2(MMP-2)andα-smooth muscle actin(α-SMA)with the production of collagens.SH and CDP can effectively treat HKI,and the combination of SH and CDP has a clear synergistic effect.