Transcription profiling of mouse glomeruli from a model of Denys-Drash Syndrome

The Wilms tumor-suppressor gene WT1, a key player in renal development, also has a crucial role in maintenance of the glomerulus in the mature kidney. However, molecular pathways orchestrated by WT1 in podocytes, where it is highly expressed, remain unknown. Their defects are thought to modify the cross-talk between podocytes and other glomerular cells and ultimately lead to glomerular sclerosis, as observed in diffuse mesangial sclerosis (DMS) a nephropathy associated with WT1 mutations. To identify podocyte WT1 targets, we generated a novel DMS mouse line, performed gene expression profiling in isolated glomeruli, and identified excellent candidates that may modify podocyte differentiation and growth factor signalling in glomeruli. Scel, encoding sciellin, a protein of the cornified envelope in the skin, and sulf1, encoding a 6-O endosulfatase, are shown to be expressed in wild type podocytes and to be strongly down-regulated in mutants. Co-expression of Wt1, Scel and Sulf1 was also found in a mesonephric cell line, and siRNA-mediated knockdown of WT1 decreased Scel and Sulf1 mRNAs and proteins. By ChIP we show that Scel and Sulf1 are direct WT1 targets. Cyp26a1, encoding an enzyme involved in the degradation of retinoic acid, is shown to be up-regulated in mutant podocytes. Cyp26a1 may play a role in the development of glomerular lesions but does not seem to be regulated by WT1. These results provide novel clues in our understanding of normal glomerular function and early events involved in glomerulosclerosis. Experiment Overall Design: Isolation of glomeruli from mutant (FVB-N4 Wt1+/R394W) and wild-type (FVB-N4 Wt1+/+) was performed after cardiac Dynabead perfusion. GeneChip analysis of glomeruli from 5 Wt1+/R394W mice and 5 Wt1+/+ littermates (N4-FVB) were performed independently. Animals were unweaned 27-day-old males. The Wt1+/R394W mice used were showing little albuminuria (<3 ug/ul on Coomassie blue stained SDS-PAGE gel) and no evidence of mesangial lesions by light microscopy.威尔姆斯肿瘤抑制基因WT1（Wilms tumor-suppressor gene WT1）是肾脏发育中的关键调控因子，同时在成熟肾脏的肾小球（glomerulus）维持过程中发挥至关重要的作用。不过，在高表达WT1的足细胞（podocytes）中，由WT1介导的分子调控通路仍未明确。研究认为，足细胞的功能缺陷会改变足细胞与其他肾小球细胞间的细胞串扰，最终引发肾小球硬化，这与弥漫性系膜硬化症（diffuse mesangial sclerosis, DMS）的病理表现一致——DMS是一种与WT1突变相关的肾病。 为鉴定足细胞中WT1的靶基因，本研究构建了一株新型DMS小鼠模型，对分离得到的肾小球开展基因表达谱分析，并筛选出可调控肾小球内足细胞分化与生长因子信号通路的优质候选基因。编码皮肤角质被膜蛋白外皮蛋白（sciellin）的Scel基因，以及编码6-O-内切硫酸酯酶的Sulf1基因，在野生型足细胞中均有表达，且在突变体足细胞中显著下调。 我们还在中肾细胞系中检测到Wt1、Scel与Sulf1的共表达，且通过小干扰RNA（siRNA）介导的WT1敲低实验证实，Scel和Sulf1的mRNA与蛋白水平均出现显著下降。通过染色质免疫沉淀（ChIP）实验，我们确认Scel与Sulf1是WT1的直接靶基因。编码视黄酸降解酶的Cyp26a1基因在突变体足细胞中呈现上调表达，其可能参与肾小球病变的发生进程，但似乎并不受WT1调控。 上述研究结果为我们理解正常肾小球功能及肾小球硬化的早期发病机制提供了全新线索。 实验整体设计：通过心脏磁珠灌注法（cardiac Dynabead perfusion），从突变型（FVB-N4 Wt1+/R394W）与野生型（FVB-N4 Wt1+/+）小鼠中分离肾小球。对5只Wt1+/R394W突变小鼠与5只同窝野生型小鼠（N4-FVB）的肾小球开展独立的基因芯片（GeneChip）分析。实验所用动物为未断奶的27日龄雄性小鼠，所用Wt1+/R394W小鼠仅表现出轻度蛋白尿（考马斯亮蓝染色的SDS-聚丙烯酰胺凝胶电泳检测结果<3 ug/ul），且光学显微镜下未观察到系膜病变。