Establishment of Nephrin Reporter Mice and Use for Chemical Screening

PLoS One. 2016 Jun 30;11(6):e0157497. doi: 10.1371/journal.pone.0157497. eCollection 2016.

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.

MeSH terms

  • Animals
  • Cells, Cultured
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • Kidney Glomerulus / metabolism*
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism*
  • Mice
  • Mice, Transgenic
  • Podocytes / metabolism*
  • Promoter Regions, Genetic

Substances

  • Membrane Proteins
  • enhanced green fluorescent protein
  • nephrin
  • Green Fluorescent Proteins

Grant support

This work was supported in part by a research fund from Mitsubishi Tanabe Pharma Corporation (MTPC, KM), by Grant-in-Aid for Scientific Research from the Japanese Ministry of Education, Culture, Sports, Science and Technology (KM, TM), by Grant-in-Aid for Young Scientists (B) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT, MO, 23700514), by Takeda Science Foundation (MO), and by 2011–2012 Tokai University School of Medicine, Project Research to M.O. MTPC provided support in the form of salaries for authors JT, KA, TN, and KM, but MTPC and other funders did not have any additional role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.