Single-Nucleus RNA Sequencing Identifies New Classes of Proximal Tubular Epithelial Cells in Kidney Fibrosis

Significance Statement Proximal tubular cells (PTCs) are numerically the predominant constituent of the kidney and are central to regeneration versus organ fibrosis following injury. However, variations in their phenotype are not well characterized. Single-nuclear RNA sequencing revealed phenotypes of PTCs in normal mouse kidney and changes in kidneys undergoing regeneration and fibrosis following aristolochic acid exposure. Five abundant and four rare PTC phenotypes were found, with abundant clusters mapped to different tubular segments and rare phenotypes mapped to proliferative, dedifferentiated, and fibrosis-associated phenotypes. These data identify unrecognized heterogeneity in PTC phenotypes and reveal novel PTCs associated with kidney fibrosis. Background Proximal tubular cells (PTCs) are the most abundant cell type in the kidney. PTCs are central to normal kidney function and to regeneration versus organ fibrosis following injury. This study used single-nucleus RNA sequencing (snRNAseq) to describe the phenotype of PTCs in renal fibrosis. Methods Kidneys were harvested from naïve mice and from mice with renal fibrosis induced by chronic aristolochic acid administration. Nuclei were isolated using Nuclei EZ Lysis buffer. Libraries were prepared on the 10× platform, and snRNAseq was completed using the Illumina NextSeq 550 System. Genome mapping was carried out with high-performance computing. Results A total of 23,885 nuclei were analyzed. PTCs were found in five abundant clusters, mapping to S1, S1–S2, S2, S2-cortical S3, and medullary S3 segments. Additional cell clusters (“new PTC clusters”) were at low abundance in normal kidney and in increased number in kidneys undergoing regeneration/fibrosis following injury. These clusters exhibited clear molecular phenotypes, permitting labeling as proliferating, New-PT1, New-PT2, and (present only following injury) New-PT3. Each cluster exhibited a unique gene expression signature, including multiple genes previously associated with renal injury response and fibrosis progression. Comprehensive pathway analyses revealed metabolic reprogramming, enrichment of cellular communication and cell motility, and various immune activations in new PTC clusters. In ligand-receptor analysis, new PTC clusters promoted fibrotic signaling to fibroblasts and inflammatory activation to macrophages. Conclusions These data identify unrecognized PTC phenotype heterogeneity and reveal novel PTCs associated with kidney fibrosis.