Conditional Deletion of Murine Fgf23: Interruption of the Normal Skeletal Responses to Phosphate Challenge and Rescue of Genetic Hypophosphatemia

Abstract

The transgenic and knock out (KO) animals involving Fgf23 have been highly informative in defining novel aspects of mineral metabolism, but are limited by shortened life span, inability of spatial/temporal FGF23 control, and infertility of the global KO. To more finely test the role of systemic and genetic influences in FGF23 production, a mouse was developed that carried a floxed (‘f’)-Fgf23 allele (exon 2 floxed) which demonstrated in vivo recombination when bred to global-Cre transgenic mice (eIIa-cre). Mice homozygous for the recombined allele (‘Δ’) had undetectable serum intact FGF23, elevated serum phosphate (p<0.05), and increased kidney Cyp27b1 mRNA (p<0.05) similar to global Fgf23-KO mice. To isolate cellular FGF23 responses during phosphate challenge Fgf23Δ/f mice were mated with early osteoblast type Iα1 collagen 2.3kb promoter-cre mice (Col2.3-cre) and the late osteoblast/early osteocyte Dentin matrix protein-1-cre (Dmp1-cre). Fgf23Δ/f/Col2.3-cre+ and Fgf23Δ/f/Dmp1-cre+ exhibited reduced baseline serum intact FGF23 versus controls. After challenge with high phosphate diet Cre− mice had 2.1–2.5 fold increased serum FGF23 (p<0.01), but Col2.3-cre+ mice had no significant increase, and Dmp1-cre+ mice had only a 37% increase (p<0.01) despite prevailing hyperphosphatemia in both models. The Fgf23Δ/f/Col2.3-cre was bred onto the Hyp (murine XLH model) genetic background to test the contribution of osteoblasts and osteocytes to elevated FGF23 and Hyp disease phenotypes. Whereas Hyp mice maintained inappropriately elevated FGF23 considering their marked hypophosphatemia, Hyp/Fgf23Δ/f/Col2.3-cre+ mice had serum FGF23 <4% of Hyp (p<0.01), and this targeted restriction normalized serum phosphorus and ricketic bone disease. In summary, deleting FGF23 within early osteoblasts and osteocytes demonstrated that both cell types contribute to baseline circulating FGF23 concentrations, and that targeting osteoblasts/osteocytes for FGF23 production can modify systemic responses to changes in serum phosphate concentrations and rescue the Hyp genetic syndrome.

Heritable and acquired disorders of phosphate metabolism: Etiologies involving FGF23 and current therapeutics

“…The amount of non-mineralized bone was significantly reduced in the treated mice and approached WT levels in the Hyp mice receiving high dose anti-FGF23. These data, in conjunction with bone specific flox- Fgf23 knockout studies in Hyp mice (21), demonstrated the key driver of the hypophosphatemia phenotype is FGF23 and therefore validates the anti-FGF23 antibody treatment regimen.…”

“…These various forms of FGF23 can be detected in the circulation using FGF23 specific enzyme-linked immunosorbent assays (ELISAs) for humans and rodents (Kainos, Inc (20) and Qidel, Inc (21)). Intact FGF23 measurements utilize two antibodies that bind at both the N - and C -terminal portions of the full-length, bioactive form of the hormone (or ‘iFGF23’).…”

FGF23-Klotho signaling axis in the kidney

“…Although other tissues may also contribute to circulating intact FGF23 concentrations [6], bone is probably the major source for circulating FGF23 under physiological circumstances in vivo [5]. There is good evidence that FGF23 is secreted by both osteocytes and osteoblasts in bone [7,8].…”

The metabolic bone disease associated with the Hyp mutation is independent of osteoblastic HIF1α expression

“…Primary cultures of osteoblastic cells from Hyp mice also fail to properly mineralize, thus underscoring an intrinsic cellular defect due to only partially resolved mechanisms (Bai et al, 2004). FGF23 is central to the Hyp metabolic bone disease phenotype, as demonstrated by the fact that global (Sitara et al, 2004) or osteoblast-specific (Clinkenbeard et al, 2016) knockout of Fgf23 in the Hyp genetic background reverses, or rescues, respectively, the low serum phosphate phenotype. However the molecular mechanisms controlling FGF23 in Hyp remain unknown.…”