Single-Cell and Spatial Transcriptomics Identify Immediate Early Genes as Central Transcriptomic Factors during Renal Endothelial Regeneration In Vivo

Background: Immediate early genes play a central role in the regulation of cell stress, cell proliferation and differentiation. Individual studies indicate that immediate early genes significantly influence vascular repair, of aortic and lung endothelial cells. Less is known about the regenerative mechanisms in renal endothelial cells. To test the relevance of mediated early genes in the kidney, renal endothelial injury was induced. Transcriptomic patterns of the endothelium were evaluated on a single-cell level to uncover potential mechanisms of renal vascular regeneration. Methods: For single-cell transcriptomics (scRNA), endothelial-specific injury was induced in Tie2 eGFP reporter mice (n=48) by renal arterial perfusion with Concanavalin A (ConA) /anti-ConA. Nineteen mice served as sham-operated controls. Kidneys were harvested 24 hours, 48 hours, 4 days, and 7 days after injury induction. For scRNA transcriptomics, cells were isolated from glomerular and extraglomerular renal tissue. Biopsies from 15 mice were used to perform 10x Xenium spatial transcriptomics. Endothelial cell damage was evaluated using periodic acid–Schiff and endomucin stainings. Results: Both endothelial cell injury (24h) and remarkable regeneration (d4-d7) were observed using glomerular injury evaluation. Following quality control measurements, 10 glomerular and 7 peritubular endothelial cell subclusters were identified. Based on scRNA transcriptomics, we identified cells which increased significantly with proportions of 6% (d2), 17% (d4), and 20% (d7), along with the detected healing process. Immediate early genes like Atf3, Erg1, Fos, and Junb were differentially expressed in these cell population. A similar pattern was observed in peritubular endothelial cells. With the help of spatial transcriptomics, findings were successfully confirmed. Conclusion: In summary, state-of-the-art transcriptomics allowed us to initially identify endothelial renal subpopulations expressing the immediate early genes, which potentially regulate the subsequent endothelial cell repair mediated by cell differentiation and proliferation. Next, gene expression has to be modified by depletion or overactivation to evaluate the participation of immediate early response genes in renal regenerative processes.