title: Rapid screening of glomerular slit diaphragm integrity in larval zebrafish.
abstract: 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.
authors: Renal Division, Department of Medicine, Brigham and Women's Hospital, HIM-550, 4 Blackfan Circle, Boston, MA 02115, USA. dhentschel@partners.org
authors: Dirk M
authors: DM
authors: Hentschel
authors: Michael
authors: M
authors: Mengel
authors: Lisa
authors: L
authors: Boehme
authors: Fabian
authors: F
authors: Liebsch
authors: Caroline
authors: C
authors: Albertin
authors: Joseph V
authors: JV
authors: Bonventre
authors: Hermann
authors: H
authors: Haller
authors: Mario
authors: M
authors: Schiffer
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics; D000818:Animals; D003598:Cytoskeletal Proteins / Q000235:genetics; D003911:Dextrans / Q000493:pharmacokinetics; D003950:Diagnostic Techniques, Urological; D004487:Edema / Q000139:chemically induced; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives / Q000493:pharmacokinetics; D017353:Gene Deletion; D007267:Injections; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics; D007674:Kidney Diseases / Q000175:diagnosis* / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D007678:Kidney Glomerulus / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D007814:Larva / Q000033:anatomy & histology; D046911:Macromolecular Substances / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics; D008854:Microscopy, Electron; D050199:Podocytes / Q000473:pathology; D011692:Puromycin Aminonucleoside / Q000008:administration & dosage / Q000506:poisoning; D013997:Time Factors; D015027:Zebrafish / Q000254:growth & development*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C114545:CD2-associated protein; D003598:Cytoskeletal Proteins; D003911:Dextrans; D047908:Intracellular Signaling Peptides and Proteins; D046911:Macromolecular Substances; D008565:Membrane Proteins; C407223:NPHS2 protein; C015219:fluorescein isothiocyanate dextran; D011692:Puromycin Aminonucleoside; D016650:Fluorescein-5-isothiocyanate