Abstract: TH-OR13
HIF-PHI Have Direct Actions in Osteocytes: Implications for Anemia Treatment in CKD
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
- Noonan, Megan L., Indiana University School of Medicine, Indianapolis, Indiana, United States
- Ni, Pu, Indiana University School of Medicine, Indianapolis, Indiana, United States
- 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
- Clinkenbeard, Erica, Indiana University School of Medicine, Indianapolis, Indiana, United States
- Thompson, William R., 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
Patients with CKD manifest overlapping oxygen sensing/endocrine dysfunction as osteocyte-produced FGF23 is highly elevated under prevailing anemia, however the cellular mechanisms driving FGF23 production are not understood. Our goal was to test the molecular context of osteocyte oxygen sensing, and the roles of these systems in FGF23 induction which can have severe effects on CKD bone disease.
Methods
A hypoxia-inducible factor prolyl hydroxylase inhibitor (HIF-PHI; FG-4592, ‘Roxadustat’) was used to treat parent undifferentiated MSCs (‘MPC2’, mesenchymal progenitor cell clone 2) and 3-week differentiated osteocytes (Ocy), to mimic changes in cellular oxygen status in vitro, followed by ATACseq and RNAseq. Conditional Fgf23-KO mice were treated with FG-4592 in vivo.
Results
Following FG-4592 (50µM) treatment of MSCs and Ocy, unbiased RNAseq and Gene Ontology analysis validated Ocy enrichment for bone ossification/mineralization processes as well as revealed unforeseen pathways critical for oxygen and iron utilization. ATACseq showed that FG-4592 acutely (48 h) increased genome-wide chromatin accessibility, with HOMER motif analysis identifying highly significant enrichment in Ocy HIF-1α/β and -2α transcription factor binding motif accessibility (p<1e-33). In contrast, HIF motif accessibility in FG-treated MSC was unchanged, revealing a predisposition of Ocy to mediate oxygen responses. RNAseq (confirmed by qPCR) also showed significant upregulation of Fgf23 in FG-4592-treated Ocy cultures (logFC 5.8; FDR<0.001) but not in MSCs (logFC 0.08; FDR NS), and HIF1α inhibition completely suppressed Ocy Fgf23. Further, the iron chelator DFO increased Fgf23 (80-fold), which was dose-dependently reduced by holo-transferrin (p<0.001), underscoring direct effects of oxygen/iron on Ocy. In normal mice, FG-4592 injections induced plasma iFGF23 (450-900 pg/mL, p<0.001). In contrast, conditional Fgf23 deletion from Ocy (flox-Fgf23/Dmp1-cre+) completely abolished this response despite similarly elevated plasma EPO (8,000-77,000 pg/mL, p<0.001) in both genotypes.
Conclusion
These data show Ocy are poised to respond to oxygen/iron via rapid genomic accessibility and transcriptional mechanisms, which together may drive Ocy biomineralization potential through FGF23 and thus have important implications for CKD-MBD.
Funding
- NIDDK Support