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[摘要] 目的：观察并探讨血管紧张素受体拮抗剂（ARB，氯沙坦）对进展性肾炎大鼠肾小管间质低氧诱导因子-1α（HIF-1α）的影响及肾小管间质损害的抑制作用。方法：将大鼠随机分为假手术组、模型组和不同剂量氯沙坦治疗组，除假手术组外，其余各组行右肾切除并两次注射IgG（OX-7）mouse monoclonal anti-Thy1.1 antibody 建立进展性肾炎大鼠模型，各组分别予饮用水或不同剂量氯沙坦灌胃4周，观察各组大鼠肾功能、尿蛋白、收缩压、HIF-1α表达及肾组织病理的改变。结果：与模型组比较，氯沙坦组大鼠尿蛋白减少，肾功能改善，肾小管间质HIF-1α mRNA含量显著减少，肾组织病理改变明显减轻，尤其在大剂量氯沙坦组。结论：氯沙坦抑制了进展性肾炎大鼠肾小管间质的损害可能与改变HIF-1α的表达有关。
[关键词] 血管紧张素受体拮抗剂；慢性低氧；进展性肾炎；低氧诱导因子-1α
[中图分类号] R-332 [文献标识码]A[文章编号]1674-4721（2011）08（a）-０15－０4
Chronic hypoxia inducing tubulointerstitial injury is inhibited by Angiotensin II receptor blocker in a progressive model of rat glomerulonephritis
ZHA Yan1, YU Fangfang1, SHEN Yan1, ZHA Yong2*
1.Department of Nephrology, Guizhou Province People′s Hospital, Guizhou Province, Guiyang 550004, China; 2. Guizhou Province Department of Public Health, Guizhou Province, Guiyang550004, China
[Abstract] Objective: To observe and probe into the effects of angiotensin Ⅱ receptor blocker (ARB, losertan) on hypoxia inducible factor-1α (HIF-1α) and the inhibition of tubulointerstitial injury in a progressive model of rat glomerulonephritis. Methods: The rats were randomly divided into sham operation, model and different doses losartan therapy groups. Excepted the sham operation group, the rest groups underwent right nephrectomy and injected IgG (OX-7) mouse monoclonal anti-Thy1.1 antibody twice, were established as progressive nephritis model. Either drink water or different doses losartan were irrigated into the stomachs of the rats in all groups for 4 weeks. And the effects of losartan on renal function, proteinuria, SBP, HIF-1α mRNA, and the tubulointerstitial pathological changes were examined. Results: In losartan therapy groups, proteinuria was decreased, the renal function was improved, and these expression of HIF-1α mRNA was suppressed. Tubulointers titial injury was ameliorated. Regression of histological damage was observed in losartan therapy groups, especially in high dose losartan group. Conclusion: Losartan may inhibit the tubulointerstitial injury by decreasing the high level of HIF-1α in the progressive model of rat glomerulonephritis.
[Key words] Angiotensin Ⅱ receptor blocker; Chronic hypoxia; Progressive nephritis; Hypoxia inducible factor-1α
“慢性低氧学说”是目前肾脏病学界的研究热点，该学说提出：肾小管间质部分的慢性氧缺失是促进肾脏疾病进展和纤维化的重要原因[1]。体外实验表明低氧可以刺激细胞分泌低氧诱导因子1（hypoxia inducible factor-1α, HIF-1α） 的表达[2]。本课题旨在通过动物实验探讨肾小管间质细胞HIF-1α mRNA的高表达在慢性肾间质纤维化中的作用，及氯沙坦对其表达的影响。
1 材料与方法
1.1 试剂
氯沙坦购自杭州默沙东制药公司，小鼠抗人HIF-1α单克隆抗体购于美国 Neo Markers 公司，IgG（OX-7）mouse monoclonal anti-Thy1.1 antibody购于美国Santa Cruz Biotechnology 公司。
1.2 仪器
全自动多功能生化分析仪（日本Hitachi公司）；SN-628放射免疫γ计数仪（中国科学院上海原子核研究所）；光学显微镜（日本Olympus公司）。
1.3 动物模型及分组
雄性Wistar大鼠30只（由重庆腾鑫生物技术公司提供，二级），体重（126.7±10.0）g。将大鼠随机分为4组：假手术组（10只，只做肾包膜剥离，不做肾脏切除）；模型组（20只，选用进展性肾炎模型[2]），行右肾摘除术，分别于术后第1、2周予鼠尾静脉注射IgG（OX-7）mouse monoclonal anti-Thy1.1 antibody 1.2 mg/kg 2次，于第4周时在麻醉下处死大鼠（n=5）；剩余模型组大鼠随机分为未给药组（n=5，每日饮用水灌胃，连续4周）、小剂量氯沙坦组[n=5，20 mg/（kg・d）]氯沙坦加入饮用水中灌胃，连续4周）、大剂量氯沙坦组[n=5，80 mg/（kg・d）氯沙坦加入饮用水中灌胃，连续4周]，第8周在麻药下处死以上各组大鼠。大鼠处死前留取24 h尿液，麻醉后即心脏采血及处死后即取左肾标本，于-70℃冰箱保存，用作生化、组织病理和分子生物学检查。
1.4 观察指标与方法
1.4.1 24 h尿蛋白定量分别于第4、8周收集24 h尿液，用双缩脲法测定尿蛋白含量。
1.4.2 血生化检测 取大鼠心脏血，测定血肌酐（Scr）。
1.4.3 肾组织病理学检查5％中性甲醛固定肾组织，石蜡包埋，常规HE、PAS、MASSON染色，显微镜下观察肾脏病理改变。
1.4.4 原位杂交法检测取3 μm厚石蜡切片，常规脱蜡至水，灭活内原性酶，热修复抗原，抗原抗体反应，DAB显色、脱水、透明、封片、显微镜观察。HIF-1α mRNA原位杂交所用探针为经地高辛标记的针对HIF-1α基因的多相寡核苷酸探针，操作步骤按HIF-1α原位杂交试剂盒（Boster）说明书进行。阳性产物呈深紫色，不加探针为阴性对照。
1.4.5 肾脏病理、原位杂交分析①肾间质面积测量：在400倍光镜下用PAS染色标本，每个标本选取10个肾小管间质视野（避开肾小球和大血管），每个视野分别测量肾间质面积与统计场面积的比值。②原位杂交分析：采用显微图象分析系统（Olympus C3040-ADU）对各组染色结果进行积分光密度值（integral optical density，IOD）测定，每张切片随机选取10个高倍视野，每个视野代表0.13 mm2的区域面积，计算其IOD，取平均值。
1.5 统计学处理
数据用平均值±标准差（x±s）表示。用SPSS 11.5统计软件分析，组间差异比较采用t检验，相关性检验采用Pearson分析。
2 结果
2.1 收缩压
第4周时模型组和假手术组间无明显改变，第8周大剂量氯沙坦组较8周未给药组（P [参考文献]
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（收稿日期：2011-06-10）
