title: Simultaneous assessment of glomerular filtration and barrier function in live zebrafish.
abstract: The zebrafish pronephros is a well-established model to study glomerular development, structure, and function. A few methods have been described to evaluate glomerular barrier function in zebrafish larvae so far. However, there is a need to assess glomerular filtration as well. In the present study, we extended the available methods by simultaneously measuring the intravascular clearances of Alexa fluor 647-conjugated 10-kDa dextran and FITC-conjugated 500-kDa dextran as indicators of glomerular filtration and barrier function, respectively. After intravascular injection of the dextrans, mean fluorescence intensities of both dextrans were measured in the cardinal vein of living zebrafish (4 days postfertilization) by confocal microscopy over time. We demonstrated that injected 10-kDa dextran was rapidly cleared from the circulation, became visible in the lumen of the pronephric tubule, quickly accumulated in tubular cells, and was detectably excreted at the cloaca. In contrast, 500-kDa dextran could not be visualized in the tubule at any time point. To check whether alterations in glomerular function can be quantified by our method, we injected morpholino oligonucleotides (MOs) against zebrafish nonmuscle myosin heavy chain IIA (zMyh9) or apolipoprotein L1 (zApol1). While glomerular filtration was reduced in zebrafish nonmuscle myosin heavy chain IIA MO-injected larvae, glomerular barrier function remained intact. In contrast, in zebrafish apolipoprotein L1 MO-injected larvae, glomerular barrier function was compromised as 500-kDa dextran disappeared from the circulation and became visible in tubular cells. In summary, we present a novel method that allows to simultaneously assess glomerular filtration and barrier function in live zebrafish.
authors.affiliation: Department of Anatomy and Cell Biology, University Medicine Greifswald, Greifswald, Germany; and.
authors.forename: Ahmed M
authors.initials: AM
authors.lastname: Kotb
authors.affiliation: Department of Anatomy and Cell Biology, University Medicine Greifswald, Greifswald, Germany; and.
authors.forename: Tobias
authors.initials: T
authors.lastname: Müller
authors.affiliation: Department of Ophthalmic Research, Cole Eye Institute, Cleveland Clinic, Cleveland, Ohio.
authors.forename: Jing
authors.initials: J
authors.lastname: Xie
authors.affiliation: Department of Ophthalmic Research, Cole Eye Institute, Cleveland Clinic, Cleveland, Ohio.
authors.forename: Bela
authors.initials: B
authors.lastname: Anand-Apte
authors.affiliation: Department of Anatomy and Cell Biology, University Medicine Greifswald, Greifswald, Germany; and nicole.endlich@uni-greifswald.de.
authors.forename: Karlhans
authors.initials: K
authors.lastname: Endlich
authors.affiliation: Department of Anatomy and Cell Biology, University Medicine Greifswald, Greifswald, Germany; and.
authors.forename: Nicole
authors.initials: N
authors.lastname: Endlich
mesh_terms: D000818:Animals; D001053:Apolipoproteins / Q000235:genetics / Q000378:metabolism; D002232:Carbocyanines / Q000378:metabolism; D003911:Dextrans / Q000097:blood; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives; D005456:Fluorescent Dyes / Q000378:metabolism; D060730:Glomerular Filtration Barrier / Q000378:metabolism*; D005919:Glomerular Filtration Rate; D007814:Larva / Q000378:metabolism; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D060172:Morpholinos / Q000008:administration & dosage; D018995:Myosin Heavy Chains / Q000235:genetics / Q000378:metabolism; D065667:Renal Elimination; D013997:Time Factors; D015027:Zebrafish / Q000097:blood / Q000196:embryology / Q000235:genetics / Q000378:metabolism*; D029961:Zebrafish Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: C569686:Alexa Fluor 647; D001053:Apolipoproteins; D002232:Carbocyanines; D003911:Dextrans; D005456:Fluorescent Dyes; D060172:Morpholinos; D029961:Zebrafish Proteins; C015219:fluorescein isothiocyanate dextran; D018995:Myosin Heavy Chains; D016650:Fluorescein-5-isothiocyanate
keywords: glomerular filtration; in vivo observations; kidney; pronephros; zebrafish