Rapid screening of glomerular slit diaphragm integrity in larval zebrafish

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 knock-out mice for each candidate. Here we present a functional screening assay based on the clearance of a 70kDa fluorescent dextran in another vertebrate system that is rapid and low in cost. In the pronephric glomerulus of larval zebrafish we demonstrate quantifiable loss of slit diaphragm integrity in a zebrafish model of puromycin aminonucleoside (PA) toxicity. In addition, after knock-down of CD2AP and podocin, two in mammals well characterized genetic contributors to podocyte differentiation, we observe 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. Key words: podocyte, nephrotic syndrome, puromycin aminonucleoside, CD2AP, podocin.