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Forward genetic screen in human podocytes identifies diphthamide biosynthesis genes as regulators of adhesion.
Cinà, Davide P; Ketela, Troy; Brown, Kevin R; Chandrashekhar, Megha; Mero, Patricia; Li, Chengjin; Onay, Tuncer; Fu, Yulong; Han, Zhe; Saleem, Moin; Moffat, Jason; Quaggin, Susan E.
Afiliação
  • Cinà DP; Feinberg Cardiovascular and Renal Research Institute, Northwestern University Feinberg School of Medicine, Chicago, Illinois.
  • Ketela T; Division of Nephrology and Hypertension, Department of Medicine, Northwestern University Feinberg School of Medicine, Chicago, Illinois.
  • Brown KR; Donnelly Centre, Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario, Canada.
  • Chandrashekhar M; Donnelly Centre, Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario, Canada.
  • Mero P; Donnelly Centre, Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario, Canada.
  • Li C; Donnelly Centre, Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario, Canada.
  • Onay T; Tanenbaum-Lunenfeld Research Institute, University of Toronto, Toronto, Ontario, Canada.
  • Fu Y; Feinberg Cardiovascular and Renal Research Institute, Northwestern University Feinberg School of Medicine, Chicago, Illinois.
  • Han Z; Division of Nephrology and Hypertension, Department of Medicine, Northwestern University Feinberg School of Medicine, Chicago, Illinois.
  • Saleem M; Center for Genetic Medicine Research, Children's National Health System, Washington, District of Columbia.
  • Moffat J; Center for Genetic Medicine Research, Children's National Health System, Washington, District of Columbia.
  • Quaggin SE; School of Clinical Sciences, Children's Renal Unit and Academic Renal Unit, University of Bristol, Bristol, United Kingdom.
Am J Physiol Renal Physiol ; 317(6): F1593-F1604, 2019 12 01.
Article em En | MEDLINE | ID: mdl-31566424
ABSTRACT
Podocyte function is tightly linked to the complex organization of its cytoskeleton and adhesion to the underlying glomerular basement membrane. Adhesion of cultured podocytes to a variety of substrates is reported to correlate with podocyte health. To identify novel genes that are important for podocyte function, we designed an in vitro genetic screen based on podocyte adhesion to plates coated with either fibronectin or soluble Fms-like tyrosine kinase-1 (sFLT1)/Fc. A genome-scale pooled RNA interference screen on immortalized human podocytes identified 77 genes that increased adhesion to fibronectin, 101 genes that increased adhesion to sFLT1/Fc, and 44 genes that increased adhesion to both substrates when knocked down. Multiple shRNAs against diphthamide biosynthesis protein 1-4 (DPH1-DPH4) were top hits for increased adhesion. Immortalized human podocyte cells stably expressing these hairpins displayed increased adhesion to both substrates. We then used CRISPR-Cas9 to generate podocyte knockout cells for DPH1, DPH2, or DPH3, which also displayed increased adhesion to both fibronectin and sFLT1/Fc, as well as a spreading defect. Finally, we showed that Drosophila nephrocyte-specific knockdown of Dph1, Dph2, and Dph4 resulted in altered nephrocyte function. In summary, we report here a novel high-throughput method to identify genes important for podocyte function. Given the central role of podocyte adhesion as a marker of podocyte health, these data are a rich source of candidate regulators of glomerular disease.
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Texto completo: 1 Base de dados: MEDLINE Assunto principal: Adesão Celular / Podócitos / Histidina / Rim Tipo de estudo: Prognostic_studies Limite: Animals / Humans Idioma: En Ano de publicação: 2019 Tipo de documento: Article

Texto completo: 1 Base de dados: MEDLINE Assunto principal: Adesão Celular / Podócitos / Histidina / Rim Tipo de estudo: Prognostic_studies Limite: Animals / Humans Idioma: En Ano de publicação: 2019 Tipo de documento: Article