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Abstract: FR-PO393

The Scattered Tubular Cell Phenotype Is Induced in Cultured Primary Human Tubular Cells and Correlates With Loss of HNF4A Expression

Session Information

Category: Development‚ Stem Cells‚ and Regenerative Medicine

  • 500 Development‚ Stem Cells‚ and Regenerative Medicine

Authors

  • 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
Background

Scattered tubular cells (STCs) are found in the proximal tubules (PTs) of healthy human kidneys. The STCs differ from the PT cells by having less cytoplasm, fewer mitochondria and reduced brush border. Furthermore, several markers have been identified for the STCs. Shown to be involved in injury and regeneration, their phenotype is believed to be induced by injury or more controversially to represent a putative progenitor cell population. HNF4A is a transcription factor controlling the mature PT phenotype, including brush border formation and regulation of genes associated with transport and metabolism. We aimed to study if primary tubular cell cultures model induction of the STC phenotype by loss of HNF4A.

Methods

Primary PT cells were obtained from dissociated human kidney tissue. Histological material was collected from tissue and primary cells at consecutive passages. In parallel, we developed a method for correlative light and electron microscopy (CLEM) to seamlessly detect the STCs in light microscopy and study the ultrastructure of these cells in electron microscopy using the same tissue sample. Finally, HNF4A adenoviral transduction was used to reintroduce the PT phenotype in primary cells, followed by RNA sequencing.

Results

Colocalization of HNF4A and the STC marker VIM in human kidney tissue revealed two STC populations. A minority of VIM+ cells had complete loss of HNF4A expression. Most of the STCs were VIM+/HNF4A+, indicating an intermediate form between the PT and STC phenotype. CLEM showed reduced brush border and fewer mitochondria in most VIM+ cells, while a subset had complete loss of brush border and considerably less cytoplasm corresponding to the VIM+/HNF4A- population. Primary cells showed rapid reciprocal HNF4A loss and VIM induction following tissue dissociation. By reexpressing HNF4A using viral transduction, the PT phenotype was regained, including expression of HNF4A target genes associated with metabolism and brush border formation.

Conclusion

Collectively, this study suggests that HNF4A loss is a major event during development of the STC phenotype. In vitro culture of PT cells represents an induced STC model rather than a PT model, a process reversed by reintroduction of HNF4A. Loss of HNF4A is a gradual process possibly triggered by injurious events.

Funding

  • Government Support – Non-U.S.