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Kidney Week

Abstract: FR-OR15

Spatial Mapping of Murine Diabetic Kidney Disease (DKD) Transcriptomics at Single-Cell Resolution

Session Information

Category: Diabetic Kidney Disease

  • 601 Diabetic Kidney Disease: Basic

Authors

  • Myshkin, Eugene, Janssen Research and Development LLC, Boston, Massachusetts, United States
  • Wu, Haojia, Washington University in St Louis, St Louis, Missouri, United States
  • Kota, Savithri B., Janssen Research and Development LLC, Boston, Massachusetts, United States
  • Dixon, Eryn E., Washington University in St Louis, St Louis, Missouri, United States
  • Gonzalez-Villalobos, Romer Andres, Janssen Research and Development LLC, Boston, Massachusetts, United States
  • Natoli, Thomas A., Janssen Research and Development LLC, Boston, Massachusetts, United States
  • Reilly, Dermot F., Janssen Research and Development LLC, Boston, Massachusetts, United States
  • Humphreys, Benjamin D., Washington University in St Louis, St Louis, Missouri, United States
  • Magnone, Maria chiara, Janssen Research and Development LLC, Boston, Massachusetts, United States
  • Breyer, Matthew Douglas, Janssen Research and Development LLC, Boston, Massachusetts, United States
Background

DKD is the major cause of kidney failure in the USA, yet the molecular pathogenesis of DKD and the spatial distribution of the transcriptomic response to injury is poorly characterized. Single cell RNA sequencing and cell clustering has been used on a limited basis in DKD to explore distinct cell-type transcriptomic responses. Here we applied Multiplexed error-robust fluorescence in situ hybridization (MERFISH) to anatomically validate snRNAseq cell clustering in diabetic mouse kidney.

Methods

MERFISH was used to localize a panel of 260 cell selective markers derived from single nuclear RNAseq clustering in frozen kidney sections from 3 murine models: C57BLKS db/m, db/db LacZ and db/db Renin-AAV.

Results

Each section contained ∼100,000 cells. Single-cell gene expression profiling and cell identification by MERFISH allowed us to map the spatial organization of 11 major cell types: PTS1, PTS3, EC, DCT, Podo, DTL, injPT, PC, mTAL, cTAL, and fibroblasts. Podocyte cluster transcripts Cdkn1c, Dendrin, Sema3g,and EphA6 were specifically expressed in glomeruli and EphA6 and Sema3g were significantly increased in diabetes. Top PTS1 markers including Ppara, Slc7a9 and Slc5a2 exhibited superficial cortical localization whereas PTS3 markers Slc22a19, Acox2 and Kcnc3 were in the cortical/medullary region. Myh11 selectively labled JGA while Nos1 marked the macula densa. Endothelial (EC) markers including Egfl7, Cdh5, Ehd3, Plxnd1, Pi16, EphB4, exhibited distinct anatomic expression, with Ehd3 most highly expressed in glomeruli (figure), while Cdh5 and Plvap were low to absent in glomeruli and rather predominated in peri-tubular interstitium.

Conclusion

This application of MERFISH single cell spatial transcriptomics to murine diabetic kidney identified nephron specific cell clusters and confirmed anatomically separate gene expression patterns in PT and EC subpopulations.

mRNA expression in Glomeruli

Funding

  • Commercial Support