Hentschel Dirk M, Mengel Michael, Boehme Lisa, Liebsch Fabian, Albertin Caroline, Bonventre Joseph V, Haller Hermann, Schiffer Mario
Renal Division, Department of Medicine, Brigham and Women's Hospital, HIM-550, 4 Blackfan Circle, Boston, MA 02115, USA.
Am J Physiol Renal Physiol. 2007 Nov;293(5):F1746-50. doi: 10.1152/ajprenal.00009.2007. Epub 2007 Aug 15.
Gene array-type experiments have identified large numbers of genes thought to be important for the integrity of the glomerular slit diaphragm. Confirmation of individual proteins has been limited by the expenses and time involved in generating transgenic or knockout mice for each candidate. We present a functional screening assay based on the clearance of a 70-kDa fluorescent dextran in another vertebrate system that is rapid and low in cost. In the pronephric glomerulus of larval zebrafish, we have demonstrated quantifiable loss of slit diaphragm integrity in a zebrafish model of puromycin aminonucleoside (PA) toxicity. In addition, after knockdown of CD2-associated protein (CD2AP) and podocin, two well-characterized genetic contributors to podocyte differentiation in mammals, we observed glomerular loss of serum macromolecules similar to that seen in mammalian kidneys with inborn mutations in these genes. Increased filtration of 70-kDa FITC-labeled dextran correlates with effacement of podocyte foot processes in ultrastructural analysis. These findings document the value of the zebrafish model in genomics and pharmacological screening applications.
基因阵列类型的实验已经鉴定出大量被认为对肾小球裂孔隔膜完整性很重要的基因。由于为每个候选基因生成转基因或基因敲除小鼠涉及费用和时间问题，对单个蛋白质的确认受到了限制。我们基于另一种脊椎动物系统中70 kDa荧光葡聚糖的清除率，提出了一种快速且低成本的功能筛选测定法。在斑马鱼幼体的前肾肾小球中，我们已经在嘌呤霉素氨基核苷（PA）毒性的斑马鱼模型中证明了裂孔隔膜完整性的可量化损失。此外，在敲低CD2相关蛋白（CD2AP）和足突蛋白后，这两种在哺乳动物足细胞分化中已得到充分表征的基因贡献者，我们观察到肾小球血清大分子的丢失，类似于在这些基因存在先天性突变的哺乳动物肾脏中所见到的情况。在超微结构分析中，70 kDa异硫氰酸荧光素（FITC）标记的葡聚糖过滤增加与足细胞足突消失相关。这些发现证明了斑马鱼模型在基因组学和药理学筛选应用中的价值。
