title: Establishment of Nephrin Reporter Mice and Use for Chemical Screening.
abstract: Nephrin is a critical component of glomerular filtration barrier, which is important to maintain glomerular structure and avoid proteinuria. Downregulation of nephrin expression is commonly observed at early stage of glomerular disorders, suggesting that methods to increase nephrin expression in podocytes may have therapeutic utility. Here, we generated a knockin mouse line carrying single copy of 5.5 kb nephrin promoter controlling expression of enhanced green fluorescent protein (EGFP) at Rosa26 genomic locus (Nephrin-EGFP mouse). In these mice, EGFP was specifically expressed in podocytes. Next, we isolated and cultivated glomeruli from these mice, and developed a protocol to automatically quantitate EGFP expression in cultured glomeruli. EGFP signal was markedly reduced after 5 days of culture but reduction was inhibited by vitamin D treatment. We confirmed that vitamin D increased mRNA and protein expression of endogenous nephrin in cultivated glomeruli. Thus, we generated a mouse line converting nephrin promoter activity into fluorescence, which can be used to screen compounds having activity to enhance nephrin gene expression.
authors.affiliation: TMK Project, Medical Innovation Center, Kyoto University Graduate School of Medicine, Kyoto, Japan.|Research Unit/Nephrological & Endocrinological Science, Sohyaku, Innovative Research Division, Mitsubishi Tanabe Pharma Corporation, Toda, Saitama, Japan.
authors.forename: Junichi
authors.initials: J
authors.lastname: Tsuchida
authors.affiliation: Institute of Medical Science, Tokai University, Isehara, Kanagawa, Japan.|Department of Molecular Life Science, Division of Basic Medical Science and Molecular Medicine, Tokai University School of Medicine, Isehara, Kanagawa, Japan.
authors.forename: Taiji
authors.initials: T
authors.lastname: Matsusaka
authors.affiliation: Institute of Medical Science, Tokai University, Isehara, Kanagawa, Japan.|Department of Molecular Life Science, Division of Basic Medical Science and Molecular Medicine, Tokai University School of Medicine, Isehara, Kanagawa, Japan.
authors.forename: Masato
authors.initials: M
authors.lastname: Ohtsuka
authors.affiliation: Institute of Medical Science, Tokai University, Isehara, Kanagawa, Japan.|Department of Molecular Life Science, Division of Basic Medical Science and Molecular Medicine, Tokai University School of Medicine, Isehara, Kanagawa, Japan.
authors.forename: Hiromi
authors.initials: H
authors.lastname: Miura
authors.affiliation: Medical Research Support Center, Graduate School of Medicine, Kyoto University, Kyoto, Japan.
authors.forename: Yukiko
authors.initials: Y
authors.lastname: Okuno
authors.affiliation: TMK Project, Medical Innovation Center, Kyoto University Graduate School of Medicine, Kyoto, Japan.
authors.forename: Katsuhiko
authors.initials: K
authors.lastname: Asanuma
authors.affiliation: TMK Project, Medical Innovation Center, Kyoto University Graduate School of Medicine, Kyoto, Japan.
authors.forename: Takahiko
authors.initials: T
authors.lastname: Nakagawa
authors.affiliation: TMK Project, Medical Innovation Center, Kyoto University Graduate School of Medicine, Kyoto, Japan.|Department of Nephrology, Kyoto University Graduate School of Medicine, Kyoto, Japan.
authors.forename: Motoko
authors.initials: M
authors.lastname: Yanagita
authors.affiliation: TMK Project, Medical Innovation Center, Kyoto University Graduate School of Medicine, Kyoto, Japan.|School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka, Japan.|Department of Nephrology and Kidney Research, Shizuoka General Hospital, Shizuoka, Japan.
authors.forename: Kiyoshi
authors.identifier: http://orcid.org/0000-0002-7285-8351
authors.initials: K
authors.lastname: Mori
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D007678:Kidney Glomerulus / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008822:Mice, Transgenic; D050199:Podocytes / Q000378:metabolism*; D011401:Promoter Regions, Genetic
chemical_list: D008565:Membrane Proteins; C485184:enhanced green fluorescent protein; C113267:nephrin; D049452:Green Fluorescent Proteins