Endothelial Cells of Afferent Arteriole: Novel Potential Regenerative Niche of Glomerular Endothelial Regeneration?

Background: Recent research has demonstrated that endothelial cells (EC) exhibit significant heterogeneity, displaying remarkable plasticity in both normal and pathophysiological conditions. However, the precise cellular responses of renal EC during injury and regeneration remain poorly understood. Therefore, we characterize the process of regeneration of renal EC using single-cell RNA sequencing (SC RNA-seq). This approach enables the identification of differentially expressed genes (DEG) at the single-cell level, which may facilitate the identification of potential regenerative gene patterns or niches within the renal endothelium. Methods: EC-specific injury was induced in Tie2-eGFP reporter mice (n=48) by renal arterial perfusion with ConcanavalinA (ConA)/anti-ConA serum. Ten mice served as sham-operated controls. Kidneys were harvested at 24h, 48h, 4 days, and 7 days post-injury induction. For SC RNA-seq more than 40,000 glomerular (gEC) and peritubular (pEC) endothelial cells were isolated and further separated using fluorescence-activated cell sorting (FACS). EC damage was evaluated by endomucin staining. Bioinformatic analyses of the SC RNAseq data were conducted using the Seurat, clusterProfiler, and venn.diagram packages for R. Results: EC injury (24 h) and remarkable regeneration (d4-d7) were observed in histology and SC RNA-seq data. In addition to known glomerular and peritubular endothelial subclusters, Gene Set Enrichment Analyses (GSEA) revealed several clusters with DEG associated with injury, a transition from injury to regeneration, remodeling, regeneration and proliferation. A strong resemblance between the regeneration cluster and the already known afferent arteriole-associated cluster was evident. 29% of the DEG of the regeneration cluster can also be found in the afferent arteriole associated cluster. A lot of these DEG are associated with regenerative processes. In contrast, only 1% of the regenerative cluster DEG can be found in the cluster linked with the EC of the efferent arteriole. Conclusion: In conclusion, our SC RNA-seq data illuminates the transcriptional changes that occur during injury and regeneration of renal EC. The analyses strongly suggest a role of the EC of afferent arteriole in initiating or transducing regeneration processes in the renal endothelium.