Kidney Week

Abstract: SA-OR21

Single-Cell Transcriptomic Analysis to Define Cellular Heterogeneity in Human ADPKD

Session Information

• Kidneyomics: From Cysts to Populations
  October 24, 2020 | Location: Simulive
  Abstract Time: 05:00 PM - 07:00 PM

Category: Genetic Diseases of the Kidneys

• 1001 Genetic Diseases of the Kidneys: Cystic

Authors

• Muto, Yoshiharu, Washington University School of Medicine, St. Louis, Missouri, United States

Yoshiharu Muto, MD, PhD



Dr. Yoshiharu Muto is a postdoctoral fellow at the Humphreys Lab at Washington University in St. Louis. His research focuses on single cell analysis of kidney diseases to find new therapeutic targets.

• Dixon, Eryn E., University of Maryland School of Medicine, Baltimore, Maryland, United States

Eryn E. Dixon,

• Ramachandran, Chidambaram, Chinook Therapeutics, Inc., Vancouver, British Columbia, Canada

Chidambaram Ramachandran,

• King, Andrew J., Chinook Therapeutics, Inc., Vancouver, British Columbia, Canada

Andrew J. King,

• Seliger, Stephen L., University of Maryland School of Medicine, Baltimore, Maryland, United States

Stephen L. Seliger,

• Woodward, Owen M., University of Maryland School of Medicine, Baltimore, Maryland, United States

Owen M. Woodward,

• Welling, Paul A., Johns Hopkins School of Medicine, Baltimore, Maryland, United States

Paul A. Welling,

• Watnick, Terry J., University of Maryland School of Medicine, Baltimore, Maryland, United States

Terry J. Watnick,

• Humphreys, Benjamin D., Washington University School of Medicine, St. Louis, Missouri, United States

Benjamin D. Humphreys,

Background

Deciphering the molecular pathogenesis of human autosomal dominant polycystic kidney disease (ADPKD) requires a detailed understanding of the distinct cell types and cell states driving cyst growth. Unlike single cell RNA-seq, single nucleus RNA-seq can be performed on cryopreserved samples, and we hypothesized that it might reveal unique cell states from biobanked human ADPKD samples.

Methods

We performed snRNA-seq on 8 ADPKD kidney samples (4 males and 4 females, mean = 50 +/- 8.5 years old, all had ESRD). Cystic kidneys weighed 1631 +/- 728 g. Nuclear preparations were processed using 10x Genomics Chromium 3’ kit and sequenced by NovaSeq. Reads were counted with CellRanger 3.1.0 and analyzed with Seurat v3. Gene expression was validated by fluorescence in situ hybridization.

Results

Samples were stored at -80^oC prior to processing (median, 20 mo; range 7-40 mo). All samples yielded good libraries (Avg. genes/nucleus = 1560 +/- 526). 63,289 nuclei originating from all 8 ADPKD kidneys passed quality control filters. Large clusters of cystic epithelial cells could be distinctly identified as originating from PT, TAL and CD. Compared to non-cystic epithelia, TAL-derived cystic epithelia showed strong induction of ERBB4 and gene ontology terms including fibroblasts/ECM and secretion of TGFb. CDH6 ^high /LRP2^high cystic epithelia expressed the PT injury markers HAVCR1 and VCAM1, suggesting stress-induced dedifferentiation. Cyst-specific marker gene expression was validated by RNAscope on human ADPKD sections. Analysis also revealed four separate fibroblast subtypes, most expressing markers of activation and inflammation, as well as endothelial clusters, podocytes and four separate macrophage sub-clusters.

Conclusion

To our knowledge this is the first single cell transcriptomic atlas of human ADPKD. We demonstrate the utility of this approach by revealing (1) excellent gene expression from all samples including those stored > 3 yrs, (2) segment specific cystic epithelial expression profiles, (3) activated interstitial fibroblast subsets and (4) pro-inflammatory macrophage cell types and states.

Funding

• NIDDK Support – Chinook Therapeutics