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Abstract: FR-PO372

Single-Cell RNA Sequencing Reveals Renal Endothelium Heterogeny During Injury and Regeneration in a Murine Model of Renal Thrombotic Microangiopathy

Session Information

Category: Development‚ Stem Cells‚ and Regenerative Medicine

  • 500 Development‚ Stem Cells‚ and Regenerative Medicine

Authors

  • Sradnick, Jan, Division of Nephrology, Department of Internal Medicine III, University Hospital CGC, Dresden, Germany
  • Wirth, Anika, Division of Nephrology, Department of Internal Medicine III, University Hospital CGC, Dresden, Germany
  • Kroeger, Hannah, Division of Nephrology, Department of Internal Medicine III, University Hospital CGC, Dresden, Germany
  • Arndt, Patrick, Division of Nephrology, Department of Internal Medicine III, University Hospital CGC, Dresden, Germany
  • Todorov, Vladimir T., Division of Nephrology, Department of Internal Medicine III, University Hospital CGC, Dresden, Germany
  • Hugo, Christian, Division of Nephrology, Department of Internal Medicine III, University Hospital CGC, Dresden, Germany
Background

Analyses of the marker genes, pathways, and biological functions revealed that endothelial cells (EC) are highly heterogeneous showing plasticity both in normal and pathophysiological conditions. However, the heterogeneity and cellular responses of renal endothelial cells during injury and regeneration have not been well characterized.

Methods

Endothelial specific injury was induced in Tie2 eGFP mice (n=48) by renal arterial perfusion with ConcanavalinA (ConA) /anti-ConA. Nineteen mice served as sham operated controls. Kidneys were harvested 24h, 48h, 3 days 4 days and 7 days after injury induction. For 10x single-cell RNA sequencing, cells were isolated from glomerular and extraglomerular renal tissue. Tie2 EGFP+CD102+CD105+CD45- endothelial cells were further separated using FACS cell sorting. After 10x single cell sequencing, Seurat clustering analysis was performed to identify EC cell clusters. Endothelial cell damage was evaluated using periodic acid–Schiff staining and histology in zinc fixed paraffin embedded slices.

Results

Endothelial cell loss was observed 24h following injury, while the EC number already markedly increased on day 3 and was back to baseline levels after 4–7 days. 10x single-cell RNA sequencing was performed on 42,000 sorted renal cells. Following quality control measurements almost all sequenced cells expressed endothelial specific genes. In glomerular and peritubular ECs, different cluster (17 and 7, respectively) could be identified following t-distributed stochastic neighbour embedding (t-SNE). In both glomerular and peritubular EC transcriptomics, cell clusters demonstrating predominant regulation of cellular injury genes was seen 24h but also 48h after model induction. Gene clusters consistent with cell remodeling and differentiation were mostly identified on days 4-7.

Conclusion

In summary, our study provides a high-resolution atlas of the renal endothelium during EC specific injury and regeneration. Our data highlight the phenotypic heterogeny and changes of endothelial subtypes which were potentially involved during EC injury and regeneration after selective renal endothelial cell injury.