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Abstract: PO0232

Proximal Tubular S3 Cells Expressing Angiotensinogen Localize to the Outer Stripe of the Outer Medulla

Session Information

  • AKI Mechanisms - 3
    October 22, 2020 | Location: On-Demand
    Abstract Time: 10:00 AM - 12:00 PM

Category: Acute Kidney Injury

  • 103 AKI: Mechanisms

Authors

  • Janosevic, Danielle, Indiana University School of Medicine, Indianapolis, Indiana, United States
  • Melo ferreira, Ricardo, Indiana University School of Medicine, Indianapolis, Indiana, United States
  • Eadon, Michael T., Indiana University School of Medicine, Indianapolis, Indiana, United States
  • Syed, Farooq, Indiana University School of Medicine, Indianapolis, Indiana, United States
  • Hato, Takashi, Indiana University School of Medicine, Indianapolis, Indiana, United States
  • Dagher, Pierre C., Indiana University School of Medicine, Indianapolis, Indiana, United States
Background

The S3 segments of proximal tubules (PT) are observed in the cortex as well as the outer stripe of the outer medulla (OS-OM). Using single cell RNA sequencing (scRNAseq), we and others have identified a unique S3 population, referred to as S3-Type 2 (S3T2, Figure a). These S3T2 cells express Rnf24 and Slc22a7 as defining genes, in addition to other classical S3 markers. In this work, we combined spatial transcriptomics (SpT) and scRNAseq to further localize this novel S3T2 cell population in the murine kidney.

Methods

We performed SpT on OCT frozen murine kidney sections stained with hematoxylin & eosin. Images were collected with Keyence BZ-810 microscope. RNA was isolated from tissue, sequenced, and clustering performed with Seurat R package. This yielded seven clusters that could be overlaid over the tissue section. To increase resolution, we combined scRNAseq data with SpT. Gene expression was visualized with the Loupe Browser. Expression of specific genes was also confirmed with single molecule FISH (smFISH).

Results

The combination of scRNAseq with SpT increased the spatial resolution and resulted in 15 unique cell clusters that could be easily overlaid over the tissue section. While regular S3 cells localized to the cortex, S3T2 cells localized specifically to the OS-OM (Fig. b). In addition to Rnf24 and Slc22a7, S3T2 cells showed strong expression of angiotensinogen (Agt, Fig. c). smFISH of Agt and Aqp1 confirmed these results (Fig. d).

Conclusion

The combination of scRNAseq and SpT allows for a greater resolution in the spatial layout of renal cell populations. This approach localized S3T2 cells specifically to the OS-OM. These cells also exhibited strong expression of Agt, possibly indicating an important role in the renin-angiotensin system. These transcriptomic differences between classical S3 and S3T2 are likely dictated by the unique microenvironments of the cortex and the OS-OM.

Figure a-d: a scRNAseq UMAP highlights S3 and S3T2 b SpT highlighting S3 and S3T2. c-d Feature plot of Agt and smFISH of Agt (green) localizes to S3T2.

Funding

  • NIDDK Support