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Abstract: SA-PO015

Rapid Loss of Proximal-Tubule-Specific Gene Expression in Primary Culture Associates with HNF4a Downregulation

Session Information

Category: Bioengineering

  • 400 Bioengineering


  • Telang, Asha Claire, University of Michigan, Ann Arbor, Michigan, United States
  • McElliott, Madison Chelise, University of Michigan, Ann Arbor, Michigan, United States
  • Ference-Salo, Jenna T., University of Michigan, Ann Arbor, Michigan, United States
  • Beamish, Jeffrey A., University of Michigan, Ann Arbor, Michigan, United States

Cell culture models are used widely to study the functions of proximal tubule epithelial cells on the implicit assumption that in vitro cell behavior represents in vivo cell function. The advent of tools for transcriptome-wide analysis in culture models has revealed that immortalized cell lines poorly mimic the native proximal tubule. While the dedifferentiation of proximal tubules induced by cell culture has long been appreciated, the dynamics of genome-wide transcription changes accompanying this process have not been described in detail yet are critical to interpret findings in bioengineered and standard cell culture models.


We isolated proximal tubules from 6-week-old female C57B6 mice by enzymatic and mechanical dissociation followed by gradient centrifugation yielding > 90% proximal tubules. We cultured cells on standard non-porous tissue culture plastic in a standard incubator (37 °C, 5% CO2) in DMEM/F-12 with standard glucose (3.8 g/L) supplemented with low serum (0.5%) and epidermal growth factor (EGF) as representative conditions among the myriad of reported techniques. At various timepoints, RNA was harvested and subjected to bulk RNA sequencing. Cells also were characterized by immunofluorescence staining.


Proximal-tubule-specific genes with more than 15 transcripts per million detected decreased from 77% to 18% after only 4 d. This fraction did not increase with culture duration despite the formation of morphologically mature epithelial structures including typical cuboidal morphology, apical Zo-1 staining, and basolateral sodium potassium ATPase identified by confocal microscopy. Gene set enrichment against the Molecular Signatures Database Hallmark gene sets indicated cultured cells permanently downregulate genes associated with oxidative and fatty acid metabolism within the first 24 h of culture. The transcriptional networks inferred by these changes using ChEA3 point to a central role of decreased HNF4a activity, which itself is silenced by 1 week of culture.


Our results indicate that cultured proximal tubules irreversibly silence most proximal tubule specific genes within just a few days of culture. These findings highlight the need for optimized culture conditions from the moment of isolation.


  • NIDDK Support