Abstract: FR-PO295
Merged In Vitro/In Vivo RNAseq/ATACseq Pinpointed FGF23 Target Genes Dysregulated with Klotho Deletion in Kidney Single-Cell Subpopulations
Session Information
- Bone and Mineral Metabolism: Basic
November 03, 2023 | Location: Exhibit Hall, Pennsylvania Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Bone and Mineral Metabolism
- 501 Bone and Mineral Metabolism: Basic
Authors
- Solis, Emmanuel, Indiana University School of Medicine, Indianapolis, Indiana, United States
- Jennings, Kayleigh Nicole, Indiana University School of Medicine, Indianapolis, Indiana, United States
- Noonan, Megan L., 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
- 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
FGF23 controls phosphate and vitamin D synthesis in the kidney via its co-receptor αKlotho (KL), however, the cellular responses to FGF23 during normal and disease states are not fully understood. We hypothesize that FGF23 induces unique and generalized changes in transcription and genomic accessibility within specific nephron cell populations.
Methods
HEK293 cell line stably expressing membrane Klotho (HEK-mKL cells) was treated with FGF23 (50 ng/mL) for 4 and 16 hours, then processed for ATACseq and RNAseq libraries. Differentially expressed genes were validated by qPCR as well as in an independent single-cell 10X Multiomics dataset from KL-KO mice. Dimensionality reduction via Uniform Manifold Approximation and Projection (UMAP) was used to identify distinct nephron cell types.
Results
HEK-mKL cell groups treated with FGF23 displayed clear segregation for both RNAseq and ATACseq following principal component analysis (PCA), with 9 and 7-fold increases in MAPK target genes EGR1 and FOS. Vitamin D 24-hydroxylase CYP24A1 (2-fold) and VDR (1.5-fold) increased at 4 and 16 hours, supporting this model. ATACseq showed FGF23 rapidly influenced genomic regions to control MAPK signaling as demonstrated by opening chromatin accessibility of an EGR1 distal enhancer by 4h. In confirmation, HOMER motif discovery predicted enrichment in transcription factor binding for MAPK targets FOS, JUN, AP1, and EGR1 across the genome (P<0.05). At both 4 and 16h, FGF23 bioactivity was associated with novel induction of ETV transcription factor family mRNAs (ETV1 (3.7-fold), ETV4 (8-fold), and ETV5 (7.5-fold). Furthermore, FGF23 bioactivity increased the expression of ETV1/4/5 target genes MMP1, PTGS2, and VEGF. Conversely, in vivo, 10X Multiome analysis of KL-KO mouse kidney proximal tubule-S1/S2 cells showed a 27% decrease in Etv5 mRNA. Further, Etv1 expression and chromatin accessibility decreased by 70% and 92%, respectively, contrary to the increase of these key factors with FGF23 treatment.
Conclusion
A unique combination of unbiased in vitro ATACseq/RNAseq pinpointed novel FGF23-induced transcriptional and genomic reprogramming. Translation to in vivo kidney cell subpopulations demonstrated these changes may influence cell-specific outcomes in FGF23-related diseases.
Funding
- NIDDK Support