Forward genetic screen in human podocytes identifies diphthamide biosynthesis genes as regulators of adhesion

Davide P Cinà, Troy Ketela, Kevin R Brown, Megha Chandrashekhar, Patricia Mero, Chengjin Li, Tuncer Onay, Yulong Fu, Zhe Han, Moin Saleem, Jason Moffat, Susan E Quaggin

Research output: Contribution to journalArticle (Academic Journal)

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.

Original languageEnglish
Pages (from-to)F1593-F1604
JournalAmerican journal of physiology. Renal physiology
Volume317
Issue number6
DOIs
Publication statusPublished - 30 Sep 2019

Keywords

  • Animals
  • Cell Adhesion/genetics
  • Cell Line
  • Drosophila
  • Fibronectins/metabolism
  • HSP40 Heat-Shock Proteins/genetics
  • High-Throughput Screening Assays
  • Histidine/analogs & derivatives
  • Humans
  • Intracellular Signaling Peptides and Proteins/genetics
  • Kidney/metabolism
  • Minor Histocompatibility Antigens/genetics
  • Podocytes/metabolism
  • Proteins/genetics
  • RNA, Small Interfering
  • Tumor Suppressor Proteins/genetics
  • Vascular Endothelial Growth Factor Receptor-1/genetics

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