RENAL SINGLE-NUCLEAR TRANSCRIPTOMICS IDENTIFIES NOVEL THERAPEUTIC TARGETS IN A PRECLINICAL MODEL OF CHRONIC KIDNEY DISEASE.

Background and hypothesis: The prevalence of chronic kidney disease (CKD) is on the rise, and precision strategies to offset the progression to end-stage kidney disease are needed. We used a well-established pre-clinical translational model of CKD in swine and single nucleus RNA sequencing (snRNA-seq) to identify potential therapeutic targets via characterization of the renal cell-specific transcriptomic landscape.

Methods: Normal and CKD pigs were studied in vivo and ex vivo after 14 weeks (n=6/group). In randomly selected pigs (n=3/group), kidneys were harvested, nuclei isolated, libraries prepared, and snRNA-seq performed. Protein expression of candidate differentially expressed genes (DEGs, log2FC>0.25, adjusted p-value<0.05) was determined by immunohistochemistry, and their expression in primary normal and CKD renal vascular endothelial cells (RECs) was modulated (siRNA) in vitro.

Results: A total of 52,213 nuclei were analyzed. Thirty clusters were identified and filtered by canonical gene markers, revealing 16 unique renal cell types. Endothelial cells were the top cell type exhibiting the highest number of DEGs; which were subsequently filtered by angiogenesis-, inflammation-, and fibrosis (major injurious pathways altered in CKD kidneys). Venn diagram analysis identified 5 unique overlapping DEGs in endothelial cells: VWF, LAMA3, and KDR upregulated, and PTGIS and ICAM1 downregulated in CKD versus normal kidneys. Venn diagram analysis indicates that VWF, LAMA3, and ICAM1 participate in inflammatory and fibrotic signaling. Renal protein expression of these DEGs matched snRNA-seq findings. Furthermore, in vitro silencing of VWF and LAMA3 ameliorated endothelial cell inflammatory and fibrotic signaling.

Conclusions: Our work characterized the single-nuclear renal cell transcriptomic landscape of a translational model of CKD and singled out genes implicated in major renal injury pathways. These genes could serve as potential targets to pave the way for new therapeutic strategies in patients with CKD.