HNF4A Re-Expression Restores a Proximal Tubular Phenotype in Cultured Primary Cells
- Development, Organoids, Vascularized Kidneys, Nephrons, and More
November 04, 2023 | Location: Exhibit Hall, Pennsylvania Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Development, Stem Cells, and Regenerative Medicine
- 600 Development, Stem Cells, and Regenerative Medicine
- Kha, Michelle, Goteborgs universitet Sahlgrenska Akademin, Goteborg, Sweden
- Altiparmak, Gülay, Goteborgs universitet Sahlgrenska Akademin, Goteborg, Sweden
- Swärd, Karl, Lunds Universitet, Lund, Sweden
- Johansson, Martin E., Goteborgs universitet Sahlgrenska Akademin, Goteborg, Sweden
The tubular part of the nephron is composed of distinct segments. The transcription factor hepatocyte nuclear factor 4α (HNF4A) is a master regulator for the phenotype of the proximal tubules, controlling features such as brush border formation and transport. Culture of primary proximal tubular cells is an essential model system for kidney research, but during culture, the cells display a less differentiated phenotype with expression of markers associated with kidney injury. The aim of this study was to evaluate if HNF4A transduction of cultured primary proximal tubular cells could revert the cells to a more mature phenotype.
Primary proximal tubular cells obtained from human cortical kidney tissue were cultured and harvested at consecutive passages. The change in protein expression at different passages was evaluated using immunohistochemistry. HNF4A adenoviral transduction was performed on primary proximal tubular cells, followed by RNA sequencing and bioinformatic analysis. The effects of HNF4A transduction were furthermore analyzed by qPCR, Western blot, and immuno electron microscopy.
Culture of primary proximal tubular cells resulted in major HNF4A loss. In contrast, the mesenchymal and injury marker vimentin was induced. HNF4A was successfully reintroduced by adenoviral transduction and this caused upregulation of known target genes associated with brush border formation, transport, and metabolism. Gene set enrichment analysis revealed pathways linked to absorption, transport, and digestion as well as microvilli and brush border. Using immuno electron microscopy, morphological features of proximal tubular cells could be visualized in HNF4A-positive cells.
In summary, human primary proximal tubular cells rapidly lose HNF4A expression during culture, but by reintroduction of HNF4A using adenoviral transduction, the cells regain expression of genes essential for brush border formation and transport. This suggests that HNF4A expression may improve the reliability of in vitro models of proximal tubular cells.
- Government Support – Non-U.S.