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Abstract: TH-PO151

FGF23 Drives Transcriptional and Genomic Accessibility Reprogramming as Identified in the KL-KO Mouse at the Single Cell Level

Session Information

  • CKD-MBD: Targets and Outcomes
    November 03, 2022 | Location: Exhibit Hall, Orange County Convention Center‚ West Building
    Abstract Time: 10:00 AM - 12:00 PM

Category: Bone and Mineral Metabolism

  • 401 Bone and Mineral Metabolism: Basic

Authors

  • Solis, Emmanuel, Indiana University School of Medicine, Indianapolis, Indiana, United States
  • Agoro, Rafiou, Indiana University School of Medicine, Indianapolis, Indiana, United States
  • Liu, Sheng, Indiana University School of Medicine, Indianapolis, Indiana, United States
  • Wan, Jun, Indiana University School of Medicine, Indianapolis, Indiana, United States
  • White, Kenneth E., Indiana University School of Medicine, Indianapolis, Indiana, United States
Background

The high affinity FGF23/Klotho(KL)/FGFR receptor complex is critical for controlling 1,25D and phosphate utilization. However, KL is expressed in multiple nephron cell types thus the full spectrum and mechanisms dictating the spatial modes of FGF23-mediated actions remain undefined.

Methods

10X Multiome (single cell RNAseq (scRNAseq) and the Assay for Transposase-Accessible Chromatin (scATACseq)) was used to test KL-dependent FGF23 activity. Kidneys from male KL-KO and wild-type (WT) littermates underwent nuclei isolation/library preparation. Nephron segment-specific RNA expression and chromatin accessibility clustering analysis was performed and alterations of key signaling pathways tested using Ingenuity Pathway Analysis (IPA).

Results

High quality sequencing was obtained from libraries of ~10,000 cells, and integrative scRNAseq and scATACseq analysis identified 26 UMAP clusters distinctly comprised of epithelial, endothelial, and immune cells. With the deletion of KL, Cyp24a1 was reduced in proximal tubule (PT) cells, consistent with lack of FGF23 activity in this model, validating our approach. Using cluster enriched markers, the PT was sub-segmented into S1, S2, S3 cells. IPA analyses on these populations demonstrated that in the KL-null vitamin D receptor (VDR) and PTEN signaling were upregulated in PT S1-S3, whereas phagosome formation, involved in protein maturation was down-regulated. Further, with loss of FGF23-mediated activity, genes associated with VDR and PPAR signaling were upregulated in PT S1-S2. However, transcripts associated with xenobiotic metabolism were increased specifically in PT-S3, suggesting a novel role of FGF23 in this segment. Distal convoluted tubule and connecting tubule exhibited decreases in mRNAs controlling Autophagy and CLEAR pathways, but increased AMPK-mediated signaling. Finally, scATACseq revealed increased accessibility of both the VDR and Osteopontin (Spp1) genes in the PT-S1.

Conclusion

Our studies pinpoint transcriptional and genomic accessibility reprogramming for specific kidney cell types due to loss of KL-mediated FGF23 bioactivity and show that FGF23/KL interactions control nephron segment-unique and -general mechanisms that regulate mineral metabolism. Identification of these pathways is critical for isolating novel FGF23-related disease targets.

Funding

  • NIDDK Support