酸性环境对慢性肾衰竭大鼠血管钙化的影响及其机制  

Effect and mechanism of acidification on vascular calcification in chronic renal failure rats

作　　者：张胜雷[1] 徐金升[1] 冯雨[1] 张俊霞[1] 崔立文[1] 何雷[1] 俞啟遥[1] 白亚玲[1] 

机构地区：[1]河北医科大学第四医院肾内科,石家庄050011

出　　处：《中华肾脏病杂志》2015年第12期924-931,共8页Chinese Journal of Nephrology

基　　金：河北省自然科学基金（H2012206157）

摘　　要：目的 探讨酸性环境对慢性肾衰竭大鼠血管钙化的影响及可能机制.方法 将22只雄性SD大鼠随机分为4组：假手术组（甲基纤维素灌胃）、慢性肾衰竭组（5/6肾切除术构建慢性肾衰竭大鼠模型,甲基纤维素灌胃）、慢性肾衰竭血管钙化组（甲基纤维素加骨化三醇灌胃）、酸干预组[甲基纤维素及骨化三醇灌胃加氯化铵（0.28 mmol/L）饮水].采用von Kossa染色及邻甲酚酞络合酮比色法检测胸主动脉钙化情况;免疫组织化学方法检测胸主动脉runt相关转录因子2 （runt-related transcription factor 2,runx2）和L型钙通道（calcium channels,Ltype,LTCC） β3亚基表达.体外原代培养大鼠胸主动脉血管平滑肌细胞（VSMCs）,用β-甘油磷酸盐（β-glycerophosphate,β-GP）诱导钙化.将细胞随机分为4组：正常＋pH7.4组、高磷＋pH7.4组（10 mmol/L β-GP）、高磷＋pH7.1组（高磷培养基基础上添加盐酸调整pH值为7.1）和维拉帕米干预组[高磷培养基基础上添加维拉帕米（调整浓度为20 mmol/L）].采用茜素红染色及邻甲酚酞络合酮比色法检测细胞钙化情况;酶联免疫吸附法检测细胞碱性磷酸酶（ALP）活性;钙离子探针Fluo-3/AM检测血管平滑肌细胞胞外钙离子内流的效应变化;RT-PCR和Western印迹法检测LTCCβ3亚基、runx2的表达.结果 体内实验中,成功制备了慢性肾衰竭大鼠的血管钙化模型,与慢性肾衰竭血管钙化组相比,给予酸干预后,大鼠血管钙盐沉积明显减轻（P＜0.05）,LTCCβ3亚基和runx2表达降低（P＜0.05）.体外实验证明,酸性环境减少了VSMCs钙盐沉积,降低了ALP活性（P＜0.05）,抑制了LTCCβ3亚基表达（P＜0.05）,阻滞细胞外钙离子内流（P＜0.05）,降低runx2的表达（P＜0.05）.维拉帕米干预组VSMCs钙盐沉积和runx2表达均降低（P＜0.05）.结论 酸性环境可以抑制血管钙化,其可能机制之一是通过抑制VSMCsLTCCβ3亚基表达,阻滞钙离子内流,抑Objective To explore the effect and mechanism of acidification on calcification in chronic renal failure rats.Methods In vivo, male SD rats （n=22） were randomly divided into sham operated group （n=6）, 5/6 nephrectomy （Nx, n=5）, 5/6 Nx＋calcitriol group （n=5）, 5/6 Nx＋calcitriol ＋ acidosis group（n=6）.Vascular calcification was determined by yon Kossa stain and quantification of calcium.L-type calcium channels（LTCC）β3 subunit and runt-related transcription factor 2 （runx2） in aortic were measured by immunohistochemistry.In vitro, vascular smooth muscle cells （VSMCs） were primarily cultured and calcification was induced by β-glycerophosphate（β-GP）.They were then randomly divided into control ＋pH7.4 group, high phosphorus ＋pH7.4 group （10 mmol/L β-GP）, high phosphorus ＋pH7.1 group （10 mmol/L β-GP） and verapamil intervention group （10 mmol/L β-GP＋20 mmol/L verapamil）.Calcium deposition was measured by Alizarin red staining and quantification of calcium;and alkaline phosphatase （ALP） activity was measured by enzyme linked immunosorbent assay.RT-PCR and Western blotting were used to observe the expression of VSMCs LTCCβ3 subunit and runx2 mRNA and protein respectively.Results In vivo, compared to that in 5/6 Nx＋calcitriol group, calcification was significantly reduced in 5/6Nx ＋ calcitriol ＋ acidosis group （P 〈 0.05）;the expressions of LTCCβ3 subunit and runx2 were obviously down-regulated by acidification （P 〈 0.05）.In vitro, compared to that in high phosphorus ＋pH7.4 group, calcification was significantly reduced in high phosphorus＋pH7.1 group （P 〈 0.05）, as well as ALP activity （P 〈 0.05）;the expressions of runx2 and LTCCβ3 subunit were down-regulated in high phosphorus ＋pH7.1 group （P 〈 0.05）.Calcium influx in VSMCs was partially blocked during acidification （P 〈 0.05）.The calcification level and expression of runx2 in verapamil intervention group were lower than that in high phosphorus ＋ pH7.4

关 键 词：肾功能衰竭 慢性 钙质沉着症 肌细胞 平滑肌 

分 类 号：R692.5[医药卫生—泌尿科学]