宫内发育迟缓大鼠肾脏Wilms瘤1基因DNA甲基化与蛋白尿关系

目的:观察宫内环境对肾脏Wilms瘤1（WT1）基因甲基化状态的影响及其与肾脏功能的关系，探讨宫内环境引发肾脏疾病的可能分子机制。方法:采用孕期全程低蛋白饮食法建立宫内发育迟缓（IUGR）大鼠模型，至自然分娩。对照组以孕期常规饲料饲养至自然分娩。12周龄时，比色法测定24 h尿蛋白定量，光镜下计数肾小球数目，实时PCR方法检测肾脏WT1基因mRNA水平及甲基转移酶DNMT1、DNMT3a和DNMT3b mRNA水平，MassARRAY定量分析检测WT1基因启动子区DNA甲基化状态。结果:①IUGR组新生鼠出生体重显著低于对照组（P<0.000 1），直至12周龄时体重仍低于对照组（P=0.043）。②与对照组相比，12周龄时IUGR组大鼠24 h尿蛋白定量显著升高（P=0.016）；血清胱抑素C水平显著升高（P =0.036），肾小球数目显著下降（P=0.001）。③与对照组相比，12周龄时IUGR组大鼠肾组织WT1基因mRNA的表达显著增高（P=0.047），WT1基因启动子区甲基化水平显著降低（P=0.029），并且其M1段甲基化水平与WT1基因mRNA的表达呈负相关（r=-0.939，P=0.000 1），DNMT1和DNMT3b mRNA表达水平也显著下降（P值分别为0.003和0.010）。结论:不良的宫内环境可以影响大鼠肾脏WT1基因的甲基化状态，继而导致其异常表达，可能参与了IUGR大鼠成年期蛋白尿的发生。

Study on the relationship between WT1 DNA methylation and proteinuria in intrauterine growth retardation rat

Objective To investigate the influence of intrauterine environment on DNA methylation status of Wilms＇ tumor gene 1 （ WT1 ） in the kidney and renal function, and to explore the potential molecular mechanism by which the fetal environment leads to postnatal kidney diseases. Methods A rat model of intrauterine growth retardation （IUGR） was established by maternal low-protein diet throughout pregnancy. At 12 weeks of age, 24-hour urine protein was detected, glomerular number was counted with the light microscope, real-time PCR was used to test the mRNA levels of WT1 and the methyhransferases, such as DNMT1, DNMT3a and DNMT3b. MassARRAY was used to analyse the methylation of WT1 gene promoter region quantitatively. Results The body weights of IUGR newborn rats were significantly lower than normal rats （ P 〈 0. 000 1 ） , and the glomernlar number was lower. The total number of glomeruli per kidney was also decreased （ P = 0. 001 ）. At week 12, they had mi]d proteinuria （P =0. 016）, with increased serum cystatin C （P = 0. 036）. Quantitative methylation results showed that the WT1 promoter methylation levels significantly increased （ P = 0. 029 ） at this time, and was strikingly associated with its mRNA expression （r = - 0. 939, P = 0. 000 1 ）. It was also found that the mRNA expression of DNMT1 and DNMT3a significantly increased respectively （P was 0. 003 and 0. 010, respectively ）. Conclusions Poor intrauterine environment can affect the methylation state of WT1 and lead to its abnormal expression, which may contribute to the proteinuria of IUGR rats in adulthood.