自发性2型糖尿病KKAy小鼠肾小球microRNA表达谱和氯沙坦治疗效应

The glomerular microRNA expression profile in spontaneous type 2 diabetes KKAy mice and the effect of losartan treatment

目的分析糖尿病肾病（DN）发病过程中肾小球miRNA表达谱的变化，观察血管紧张素受体拮抗剂（ARB）氯沙坦对DN肾小球miRNA表达谱的影响，确认在DN发病过程中发挥关键作用的miRNA。方法8周龄KKAy小鼠随机分为氯沙坦治疗组（10mg kg-1·d-1）和非治疗组，C57BL／6小鼠作为正常对照组。于20周龄检测体质量、随机血糖、尿微量白蛋白、尿肌酐，观察肾脏形态改变。应用磁珠灌注法分离肾小球，提取总RNA，应用Affymetri。GeneChipmiRNA芯片，分析KKAy小鼠肾小球microRNA表达谱的变化，以及氯沙坦对microRNA表达谱的影响。结果KKAy小鼠的体质量和血糖较正常对照C57BL／6组小鼠显著升高（均P〈0．05），氯沙坦治疗显著改善2型糖尿病KKAy小鼠的尿白蛋白／肌酐比值[（539．71±100．23）mg／g比（728．00±177．19）mg／g，P〈0．051和肾脏病理损害，而对血糖无影响。miRNA芯片分析结果发现，与正常对照C57BL／6小鼠相比，20周龄KKAy小鼠肾小球内10个miRNA的表达上调；12个miRNA的表达下调。与KKAy非治疗组小鼠相比，20周龄氯沙坦治疗组KKAy小鼠肾小球内共有4个miRNA表达下调，其中miR-503和miR-181d在KKAy非治疗组小鼠肾小球内的表达显著上调，氯沙坦治疗可抑制其过表达。结论miR-503和miR-181d在糖尿病KKAy小鼠肾小球内的表达显著上调，氯沙坦治疗可抑制其在糖尿病状态下的异常表达，可能为糖尿病肾病新的治疗靶点。

Objective To identify susceptible miRNAs for the pathogenesis of diabetic nephropathy （DN） and the molecular targets of losartan treatment. Methods The 8-week age KKAy mice were divided into losartan treatment group （10 mg kg-1 d-1） and non-treatment group, C57BL/6 mice were used as the control group. At age of 20 weeks, body weight, random blood glucose, urinary albumin and urinary creatinine were tested, and kidney morphology was observed. Glomeruli were separated by magnetic beads perfusion, and total RNA were extracted. MiRNAs expression profiles were analyzed by the Affymetrix GeneChip miRNAs arrays. Results At age of 20 weeks, KKAy mice developed higher body weight, higher blood glucose and higher urinary microalbumin creatinine ratio than C57BL/6 mice, and the glomerular basement membrane thickened, mesangial matrix widened. Losartan treatment markedly improved the level of urinary albumin creatinine ratio [（539.71±100.23） mg/g vs （728±177.19） mg/g, P〈0.05）] and pathological lesion of KKAy mice. The miRNA array analysis showed that there were 22 miRNAs differentially expressed between KKAy non-treatment mice and C57BL/6 mice glomeruli at age of 20 weeks. Among them, 10 miRNAs were up-regulated, and 12 miRNAs were down-regulated. The expression of 4 miRNAs was down-regulated in glumeruli of KKAy mice treated by losartan compared with that of non-treatment mice. The expressions of miRNA-503 and miRNA-181d were significantly up-regulated in the glumeruli of KKAy mice and inhibited by losartan treatment. Conclusion The expressions of miRNA-503 and miRNA-181d are significantly up-regulated in the glumeruli of KKAy mice and inhibited by losartan treatment, which may be new therapeutic targets of DN.