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 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.
Hentschel
Dirk M
DM
Renal Division, Department of Medicine, Brigham and Women's Hospital, HIM-550, 4 Blackfan Circle, Boston, MA 02115, USA. dhentschel@partners.org
Mengel
Michael
M
Boehme
Lisa
L
Liebsch
Fabian
F
Albertin
Caroline
C
Bonventre
Joseph V
JV
Haller
Hermann
H
Schiffer
Mario
M
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*
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