Abstract: TH-OR15
The Spatial-Temporal Heterogeneity Dictating Kidney FGF23 Bioactivity as Identified by Single-Cell RNA Sequencing
Session Information
- CKD-MBD Potpourri: Emerging Clinical and Basic Science in Mineral and Bone Disease
November 04, 2021 | Location: Simulive, Virtual Only
Abstract Time: 04:30 PM - 06:00 PM
Category: Bone and Mineral Metabolism
- 401 Bone and Mineral Metabolism: Basic
Authors
- Agoro, Rafiou, Indiana University School of Medicine Department of Medical and Molecular Genetics, Indianapolis, Indiana, United States
- Myslinski, Jered, Indiana University School of Medicine, Department of Medicine, Indianapolis, Indiana, United States
- Ni, Pu, Indiana University School of Medicine, Department of Medicine, Indianapolis, Indiana, United States
- Janosevic, Danielle, Indiana University School of Medicine, Department of Medicine, Indianapolis, Indiana, United States
- Marambio, Yamil, Indiana University School of Medicine Department of Medical and Molecular Genetics, Indianapolis, Indiana, United States
- Dagher, Pierre C., Indiana University School of Medicine, Department of Medicine, Indianapolis, Indiana, United States
- Hato, Takashi, Indiana University School of Medicine, Department of Medicine, Indianapolis, Indiana, United States
- White, Kenneth E., Indiana University School of Medicine Department of Medical and Molecular Genetics, Indianapolis, Indiana, United States
Background
FGF23 is critical for maintaining phosphate balance via interactions with renal FGFRs and Klotho (KL), and its effects on gene expression have been described at tissue levels. However, KL is expressed in multiple nephron cell types thus the full spectrum and spatial-temporal mechanisms dictating FGF23 bioactivity remain undefined.
Methods
A single cell RNA-seq approach was used to identify the dynamics of FGF23-mediated bioactivity. Kidneys were isolated from FGF23 (400ng/g)-injected C57BL/6 mice at 1, 4 and 12h, and single cell transcriptomics were analyzed.
Results
From libraries of 10,000 male/female kidney cells, 21 UMAP cluster enriched markers distinctly identified epithelial, endothelial, and immune cells. At baseline, KL mRNA had diffuse expression in proximal tubule (PT) S1-S3 cells, overlap in loop of Henle, and was more concentrated in distal/connecting tubule (DT/CNT). In response to FGF23, at 1h Egr1, other MAPK genes, and eIF2 signaling were increased, tracking with 80% of KL-positive PT and DT cells. The vitamin D 24-OHase (Cyp24a1) was the most up-regulated gene in PT-S1/S2 at 4h, whereas KL was reduced, and Egr1 and vitamin D 1-alpha-OHase (Cyp27b1) were completely suppressed out to 12h. Pathway Analysis showed that most expressional changes were not cell type unique, including PT and DT up-regulation (8-20 fold) of TNF pathway member Tnfrs12a, supporting that FGF23 initially signals via common mechanisms but its function is defined by nephron-site specific gene expression. To segregate KL-dependent FGF23 responses in PT-S1, a critical FGF23 target, KL+ and KL- cells were subsetted. Of note, in response to FGF23, KL+ cells showed distinct clustering at each time point, whereas KL- cells remained indistinguishable, highlighting that temporal FGF23 responses drive unique and transient cellular identity. At later time points, PXR signaling was specific to PT, and Epithelial Remodeling and Actin-based Motility signaling to DT.
Conclusion
Kidney KL distribution was pinpointed at the single cell level, and we demonstrated that FGF23 bioactivity controls nephron segment-unique and -general transcriptional events that regulate mineral metabolism. Identification of these pathways is critical for the isolation of novel disease targets.
Funding
- NIDDK Support