医药学论文：不同时期应用氯沙坦治疗糖尿病肾病的比较研究

【摘要】 目的 观察在微量白蛋白尿发生前后应用氯沙坦对糖尿病肾病的治疗作用及其机制。方法 单侧肾切除的STZ糖尿病大鼠，随机分为正常对照组（NC）、糖尿病肾病组（DN）、氯沙坦早期治疗组（DL1）和氯沙坦晚期治疗组（DL2）。DL1和DL2组分别于糖尿病模型建立后即刻或第9周起开始给予氯沙坦20mg/（kg·d)灌胃，疗程均为8周。第16周时，观测肾脏形态学及肾功能变化，并采用荧光实时定量RTPCR检测肾脏TGF-β1、CTGF、PAI-1等细胞因子的表达情况。结果 DN对照组大鼠尿微量白蛋白（UAER）持续上升；血肌酐（SCr）、肾脏肥大指数（KW/BW）、肾小球平均体积（MGV）、系膜面积比（FMA）以及肾脏TGF-β1、CTGF、PAI-1和FN的mRNA表达均明显升高，而肌酐清除率（CCr）已开始下降。DL1和DL2组以上指标均明显改善，但两组之间差异无显著性。结论 氯沙坦可通过下调肾脏细胞因子基因表达，起到肾脏保护作用；但在微量白蛋白尿出现之前进行干预并不比在微量白蛋白尿出现之后进行治疗的效果更好。
【关键词】 糖尿病肾病；转化生长因子；结缔组织生长因子；纤溶酶原激活物抑制剂-1；氯沙坦
【Abstract】 Objective To estimate the renoproctive effect of angiotensin receptor antagonists on DN by applying losartan before or after microalbuminuria.Methods 40 uninephrectomized male Wistar rats are randomatically divided into 4 groups：normal control (NC)，diabetic nephropathy control (DN)，Losartan early treatment group (DL1) and Losartan late treatment group (DL2).Rats were made diabetic by single intraperitoneal injection of streptozotocin (STZ，60mg/kg body weight).DL1 group was treated with Losartan ［20mg/(kg·d)，ig］ for 8 weeks immediately after the diabetic mould was made，while DL2 group began their treatment 8 weeks later.At the end of the 16th week，all rats were sacrificed，the left kidney was removed and weighed.Mean glomerular volume (MGV) and fractional mesangial area (FMA) were calculated by medical image analysis system.The mRNA expression of TGFβ1，CTGF，PAI1 and FN were detected by quantitative real time RTPCR.Urinary albumin excretion rate (UAER)，as well as serum creatinine (SCr) and creatinine clearance rate (CCr)，was detected.Results Compared with NC group，UAER increased 12.68 folds at the end of the 8thweek and 21.92 folds at the end of the 16thweek in group DN.At the same time，Scr in rats of group DN elevated significantly (136%)，KW/BW，MGV and FMA in DN elevated significantly，which were 129%，98% and 183% higher separately，and the mRNA expression of TGFβ1，CTGF，PAI1 and FN in DN group increased 22.92，13.42，28.61 and 5.96 folds separately. All these indices decreased in rats of group DL1 and DL2 (P< 0.05). And there was no significant differences between group DL1 and DL2(P＞0.05). However，Ccr in rats of group DN began to decline already at the end of the 16th week (14.8%，P< 0.05)，which in rats of all treated diabetic groups was still higher than that in group NC (P< 0.05).③FMA is negatively correlated with Ccr in rats of group DN at the 16th week.And the mRNA expression level of TGFβ1，CTGF，PAI1 and FN is positively correlated with UAER，KW/BW，MGV and FMA.Conclusion The mRNA expression level of TGF-β1，CTGF，PAI1 and FN is higher in kidneys of diabetic rats，which was responsible for renal hypertrophy，excess ECM deposition and increased UAER.Losartan could downregulate the mRNA expression of such renal cytokines，therefore prevent the progress of DN.The therapeutic effect is similar no matter we began treatment before or after microalbuminuria，which suggested that microalbuminuria be the appropriate time to begin clinical treatment of DN.
【Key words】 diabetic nephropathy；transforming growth factor；connective tissue growth factor；plasminogen activator inhibitor-1；losartan
在西方国家，糖尿病肾病（diabetic nephropathy，DN）已成为终末期肾病（end stage renal disease，ESRD）最主要的病因。由此带来的透析、肾移植等治疗费用在整个医疗支出中所占比例正在逐年提高，给病人和社会造成极大的经济负担［1］。近年来ARB类药物的肾脏保护作用日益受到关注。不少学者提出，糖尿病患者即使血压正常也应尽早应用ACEI和ARBs类药物，以达到早期保护肾脏的作用。但此类药物的应用时机究竟应"早"到何时？至今缺乏循证医学证据。本研究将就在微量白蛋白尿出现前后的不同时期应用氯沙坦对DN的治疗作用进行探讨，为临床防治DN的策略选择和时机把握提供实验依据。
1 材料与方法
1.1 动物分组及模型建立 40只清洁级雄性Wistar大鼠，体重180～200g（山东大学实验动物中心提供），随机分为正常对照组(NC)、糖尿病肾病组(DN)、氯沙坦早期治疗组（DL1）和氯沙坦晚期治疗组（DL2）。所有大鼠均经背部切口行右肾切除术。2周后，一次性腹腔内注射链尿佐菌素(STZ，Sigma公司，60mg/kg，溶于pH 4.5、0.1mmol/L枸橼酸缓冲液中)诱导糖尿病模型，NC组仅给予等量缓冲液。72h后，尾静脉采血测随机血糖＞16.7mmol/L，作为糖尿病动物模型建立标准。
1.2 实验方法 DL1和DL2组分别于糖尿病模型建立后即刻或第9周起开始治疗，给予氯沙坦（科素亚，杭州默沙东制药有限公司生产）20mg/（kg·d）灌胃，疗程均为8周。正常对照组（NC）和糖尿病肾病对照组（DN）不予治疗。实验期间，每4周测体重、血糖。第8周和16周末，代谢笼收集24h尿，2000r/min离心10min，去沉淀，-20℃保存待测尿肌酐、尿白蛋白排泄率。第16周末，戊巴比妥钠(30mg/kg)腹腔麻醉，腹部正中切口，腹主动脉取血，2000r/min离心10min，取上清，-20℃保存待测血肌酐。取残余肾，剥离被膜，称重后，经肾门纵向剖开，部分以10%中性福尔马林固定，石蜡切片，PAS染色；部分以液氮保存，用于荧光实时定量RTPCR检测。
1.3 生化指标检测 尿白蛋白检测采用放免法（试剂盒购自中国原子能科学研究所）。血、尿肌酐用HITACH-7150自动生化分析仪检测，CCr按公式尿肌酐浓度×每分钟尿量(ml)/血肌酐浓度计算，并以体重校正。
1.4 肾组织形态学定量分析 每张PAS染色切片在光镜下（×400）随机取30个肾小球，用HPIAS-1000生物医学图像分析系统测定肾小球平均截面积(MGA)、系膜平均面积（MMA），并根据公式［2］：肾小球平均体积(MGV)=1.25·(MGA)3/2计算MGV；根据公式：系膜面积比（FMA）＝MMA/MGA计算FMA。
1.5 荧光实时定量RTPCR检测肾脏TGF-β1、CTGF、PAI1和FN mRNA表达 Trizol(上海生工)提取肾皮质总RNA。电泳鉴定RNA完整性。取总RNA 2μg逆转录合成cDNA。逆转录反应体系包括随机引物2μl、5×Reaction Buffer 4μl、10mM dNTP 2μl、ribonuclease inhibitor 1μl、MMLV逆转录酶1μl，总体积20μl。采用ABI PRISM 7000 SDS进行realtime PCR。TGFβ1、CTGF、PAI-1、FN与GAPDH引物由上海生工合成(见表1)。PCR反应体系：5×SYBR Green PCR Buffer 2μl，dNTP 0.5μl，MgCl28μl，AmpliTaq Glod 0.25μl，目的基因上、下游引物各2μl，逆转录产物2μl，总体积25μl。PCR参数：预变性94℃2min；94℃15s、55℃30s、72℃45s，共50个循环。结果以目的基因与内参照GADPH的比值表示，以平衡样本中细胞含量的差异。