Single-nucleus RNA sequencing of proximal tubular cells in progressive renal fibrosis

Proximal tubular cells (PTCs) are the most abundant cell type in the kidney. PTCs are central to normal kidney function, and to kidney regeneration versus organ fibrosis following kidney injury. This study determined PTC phenotype in healthy and fibrotic kidneys by single-nucleus RNA sequencing (snRNA-Seq), aiming to improve understanding of the character of PTCs in chronic kidney disease and fibrosis. The nuclear isolation protocol was optimised to achieve the best yield and nuclear RNA quality. SnRNA-Seq using healthy kidneys and fibrotic kidneys induced by aristolochic acid injection from adult male mice was performed. PTCs mapped to five abundant clusters, corresponding to tubular segments S1, S1-2, S2-cortical S3, and medullary S3. Novel clusters that were present at low abundance in normal kidneys and in increased number in kidneys undergoing regeneration and fibrosis following injury were identified. These clusters exhibited clear molecular phenotypes and were categorised as, proliferating, dedifferentiated-intermediate, dedifferentiated regenerating, and a dedifferentiated-senescent category that was present only after injury. Using trajectory and RNA velocity analysis, two major processes of PTC transition and differentiation were described, the path toward cellular senescence and the path toward tubular regeneration. Comprehensive pathway analyses revealed metabolic reprogramming and various immune activations in new PTC clusters. In ligand-receptor analysis, new PTC clusters promoted fibrotic signalling to fibroblasts and inflammatory activation to macrophages. The new identified PTC clusters were validated using confocal microscope. SnRNA-Seq using growing mouse kidneys from 2 and 4-week-old mice were than carried out to investigate PTC proliferation and differentiation. The 2-week-old mouse kidneys had a larger proportion of proliferative cells and the male to female difference of PTCs became significant in the 4-week-old mouse kidneys. These data identified unappreciated heterogeneity in PTC phenotypes, revealed novel PTCs associated with fibrosis and regeneration and inferred the possible PTC differentiation pathways after kidney injury