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

Abstract

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 (snRNA-seq) 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 10X platform and snRNA-seq completed using the Illumina NextSeq 550 System. Genome mapping was carried out with high-performance computing. Results: A total of 23,885 nuclei were analysed. PTCs were found in five abundant clusters, mapping to S1, S1-2, 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 labelling 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. Conclusion: These data identify unrecognized PTC phenotype heterogeneity and reveal novel PTCs associated with kidney fibrosis.

In Vivo Inhibition of TRPC6 by SH045 Attenuates Renal Fibrosis in a New Zealand Obese (NZO) Mouse Model of Metabolic Syndrome

“…The histological assessment of deposited collagen and extracellular matrix protein confirmed the expression data of the genes. Of note, renal fibrosis arises after an insult, whereas resident kidney fibroblasts and cells of hematopoietic origin differentiate into myofibroblasts [ 21 , 22 , 23 ]. Myofibroblasts acquire a contractile/proliferative phenotype upon activation by profibrotic factors and become principal kidney collagen-producing cells [ 24 ].…”

New Aspects of Kidney Fibrosis–From Mechanisms of Injury to Modulation of Disease

“…However, injured proximal tubule epithelial cells are still considered important drivers of kidney injury and fibrosis, as e.g., also recently demonstrated by scRNA-seq studies in mouse models of acute kidney injury ( 21 , 22 ). snRNA-seq in a fibrosis mouse model of chronic aristolochic acid administration identified novel classes of proximal tubular cells associated with kidney fibrosis ( 23 ). A scRNA-seq study in kidneys of patients with IgA nephropathy identified a transitional cell type among tubular intercalated cells with fibrosis signatures, suggesting an adverse outcome with interstitial fibrosis ( 24 ).…”

Single-cell transcriptomic profiling of kidney fibrosis identifies a novel specific fibroblast marker and putative disease target

“…Previous studies 30,63,[67][68][69][70] revealed that failed TEC repair elicits pro-fibrotic phenotypes promoting AKIto-CKD progression. Our analysis predicted increased intercellular communications in kidney fibrosis and corroborated the active role of TECs in the fibroblast activation.…”

“…Despite recent advances in understanding the molecular and cellular landscapes of normal and diseased human and murine kidney 30,49,[61][62][63][64][65] , renal quiescent and activated fibroblasts remain an elusive population. Kuppe et al 49 used spatial transcriptomics and genetic fate tracing to examine human CKD and murine UUO induced renal fibrosis.…”

“…Future research will further test Gucy1a3 potential for specific activated renal fibroblast targeting in rodent and human CKD. Previous studies 30,63,[67][68][69][70] revealed that failed TEC repair elicits pro-fibrotic phenotypes promoting AKIto-CKD progression. Our analysis predicted increased intercellular communications in kidney fibrosis and corroborated the active role of TECs in the fibroblast activation.…”