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Abstract: TH-OR32

Spatial Genomics Localized Nephron Segment-Specific FGF23 and CKD Klotho-Dependent and -Independent Transcriptional Reprogramming

Session Information

Category: Bone and Mineral Metabolism

  • 501 Bone and Mineral Metabolism: Basic

Authors

  • Hibbard, Lainey M., Indiana University School of Medicine, Indianapolis, Indiana, United States
  • Liu, Sheng, Indiana University School of Medicine, Indianapolis, Indiana, United States
  • Agoro, Rafiou, Indiana University School of Medicine, Indianapolis, Indiana, United States
  • Marambio, Yamil, Indiana University School of Medicine, Indianapolis, Indiana, United States
  • Jennings, Kayleigh Nicole, Indiana University School of Medicine, Indianapolis, Indiana, United States
  • Welc, Steven S., 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

FGF23 acts in the kidney via Klotho (KL) to control phosphate metabolism. However, in CKD, KL is lost and FGF23 is pathologically increased, disrupting mineral homeostasis. KL is expressed in multiple nephron segments, thus localization of FGF23 actions in the nephron remain unclear. Herein, we tested for novel FGF23 bioactivity localized with KL, as well as hypothesized that CKD alters FGF23/KL signaling and causes spatially unique transcriptional reprogramming.

Methods

Visium spatial transcriptomics (ST) was performed on kidney sections from normal mice injected with FGF23 (250 ng/kg) for 1 or 4 h, and male mice with adenine diet induced CKD (0.2% for 4 weeks).

Results

The FGF23-injected and CKD kidney sections had >6900 genes/sequencing spot and formed 12 and 10 UMAP cell clusters, respectively. Mapping nephron segment markers showed clear demarcation of cortical and medullary gene expression. PT S1/S2 marker Slc5a2 was highly expressed in the cortex and S3 marker Eci3 localized to the outer stripe of the medulla (OSOM). FGF23 increased MAPK-dependent transcription factor Egr1 more at 1 h than 4 h. The vitamin D metabolic enzymes Cyp24a1 and Cyp27b1 mRNAs were increased or decreased, respectively, at 4 h and overlapped with KL. ST data was validated by qPCR and independent scRNAseq data from FGF23-injected mice. We also identified novel changes in response to FGF23, including increased cortical Cyp4b1 at 4 h, which CKD decreased. Mice with CKD had reduced KL mRNA and increased Cyp27b1. Consistent with CKD fibrosis, Col1a1, -1a2, -3a1, and -4a1 were ubiquitously increased, although Col3a1 was more focused to the inner stripe of the medulla (ISOM). Further, pro-fibrotic Tgfb1 broadly increased, whereas its target Mmp7 increased in the ISOM. In contrast, damage and injury markers C3 and Havcr1 were restricted to the cortex and OSOM, respectively. Finally, evidence of wider immune infiltration was present in CKD with elevated neutrophil marker Lcn2, and macrophage markers Cd68 and Ptprc.

Conclusion

Using ST, we identified unbiased, spatially identifiable effects of FGF23 bioactivity in kidney, including new potential FGF23 targets. We also localized pathologic KL-dependent and -independent CKD gene alterations that differentially occur in distinct cell populations.

Funding

  • NIDDK Support