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Kidney Week

Abstract: PO0656

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

Session Information

  • CKD Mechanisms - 2
    October 22, 2020 | Location: On-Demand
    Abstract Time: 10:00 AM - 12:00 PM

Category: CKD (Non-Dialysis)

  • 2103 CKD (Non-Dialysis): Mechanisms

Authors

  • Lu, Yuehan, Cardiff University, Cardiff, United Kingdom
  • Liao, Chia-te, Cardiff University, Cardiff, South Glamorgan, United Kingdom
  • Raybould, Rachel, Cardiff University, Cardiff, South Glamorgan, United Kingdom
  • Talabani, Bnar, Cardiff University, Cardiff, South Glamorgan, United Kingdom
  • Szomolay, Barbara, Cardiff University, Cardiff, South Glamorgan, United Kingdom
  • Bowen, Timothy, Cardiff University, Cardiff, South Glamorgan, United Kingdom
  • Andrews, Robert, Cardiff University, Cardiff, South Glamorgan, United Kingdom
  • Taylor, Philip R., Cardiff University, Cardiff, South Glamorgan, United Kingdom
  • Fraser, Donald, Cardiff University, Cardiff, South Glamorgan, United Kingdom

Group or Team Name

  • Wales Kidney Research Unit
Background

Proximal tubular cells (PTC) play a central role in nephron recovery versus fibrosis following renal injury. PTC heterogeneity is well-documented but poorly-characterized in extant single-cell sequencing data. Here we have determined PTC phenotype in renal fibrosis by single-nucleus RNA sequencing (snRNA-seq).

Methods

Kidneys were harvested from naïve mice and 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 Illumina NextSeq 550. Downstream bioinformatics analyses used Seurat.

Results

A total of 23,885 nuclei were analyzed. PTC’s were found in five abundant clusters, mapping to S1, S1-2, S2-cortical S3, and medullary S3 segments. Additional cell clusters were present (“new PTC clusters”) 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, dedifferentiated-intermediate, dedifferentiated-regenerating, and (present only following injury) dedifferentiated-senescence. Each of these clusters exhibited a unique gene expression signature, including multiple genes 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

SnRNA-seq permits the dissection of cell-type and cell-subtype-specific responses. We identified previously unknown, injury-associated PTC clusters. These exhibit highly specific and restricted gene signatures, including canonical PTC injury genes previously assumed to be expressed at low level throughout injured PTC, on the basis of bulk expression analyses.

Funding

  • Government Support - Non-U.S.