Metabolic acidosis increases fibroblast growth factor 23 in neonatal mouse bone

Abstract

Fibroblast growth factor 23 (FGF23) significantly increases with declining renal function, leading to reduced renal tubular phosphate reabsorption, decreased 1,25-dihydroxyvitamin D, and increased left ventricular hypertrophy. Elevated FGF23 is associated with increased mortality. FGF23 is synthesized in osteoblasts and osteocytes; however, the mechanisms by which it is regulated are not clear. Patients with chronic kidney disease have decreased renal acid excretion leading to metabolic acidosis, which has a direct effect on bone cell activity. We hypothesized that metabolic acidosis would directly increase bone cell FGF23 production. Using cultured neonatal mouse calvariae, we found that metabolic acidosis increased medium FGF23 protein levels as well as FGF23 RNA expression at 24 h and 48 h compared with incubation in neutral pH medium. To exclude that the increased FGF23 was secondary to metabolic acidosis-induced release of bone mineral phosphate, we cultured primary calvarial osteoblasts. In these cells, metabolic acidosis increased FGF23 RNA expression at 6 h compared with incubation in neutral pH medium. Thus metabolic acidosis directly increases FGF23 mRNA and protein in mouse bone. If these results are confirmed in humans with chronic kidney disease, therapeutic interventions to mitigate acidosis, such as bicarbonate administration, may also lower levels of FGF23, decrease left ventricular hypertrophy, and perhaps even decrease mortality.

Fig. 1.

Medium fibroblast growth factor 23 (FGF23) concentration after incubation of calvariae in metabolic acidosis (MET) or parathyroid hormone (PTH). Calvariae were incubated in medium at neutral physiological pH (NTL), at reduced pH simulating metabolic acidosis (MET), reduced pH simulating respiratory acidosis (RESP), or with 10⁻⁸ M parathyroid hormone (PTH) in NTL for 24 h (0–24 h, top) or continued for an additional 24 h in fresh preequilibrated medium (24–48 h, bottom). Initial medium pH, Pco₂, and [HCO₃⁻] for ELISA experiments are shown in Table 1. Medium FGF23 concentration was assayed by ELISA at the end of the indicated incubation time (n = 4–29 pairs of calvariae/group). Values are means ± SE. *P < 0.05 vs. NTL; +P < 0.05 vs. MET.

Fig. 2.

FGF23 mRNA expression after incubation of calvariae in MET or PTH. Calvariae were incubated in medium at neutral physiological pH (NTL), at reduced pH simulating metabolic acidosis (MET), or with 10⁻⁸ M parathyroid hormone (PTH) in NTL for 24 h (0–24 h, top) or continued for an additional 24 h in fresh preequilibrated medium (24–48 h, bottom). Initial medium pH, Pco₂, and [HCO₃⁻] were not different from those shown in Table 1 for ELISA experiments. FGF23 mRNA expression in calvariae was assayed by real-time PCR at the end of the indicated incubation time (n = 8–13 pairs of calvariae/group). Rel, relative. Values are means ± SE. *P < 0.05 vs. NTL; +P < 0.05 vs. MET, same time period.

Fig. 3.

Net calcium (Ca) efflux from calvariae in response to MET or PTH. Calvariae were incubated in medium at neutral physiological pH (NTL), at reduced pH simulating metabolic acidosis (MET), or with 10⁻⁸ M parathyroid hormone (PTH) in NTL for 24 h (0–24 h, top) or continued for an additional 24 h in fresh preequilibrated medium (24–48 h, bottom). Initial medium pH, Pco₂, and [HCO₃⁻] were not different from Table 1. The Ca fluxes for 6–13 pairs of calvariae/group are means ± SE. *P < 0.05 vs. NTL.

Fig. 4.

Net inorganic phosphate (P_i) efflux from calvariae in response to MET or PTH. Calvariae were incubated in medium at neutral physiological pH (NTL), at reduced pH simulating metabolic acidosis (MET), or with 10⁻⁸ M parathyroid hormone (PTH) in NTL for 24 h (0–24 h, top) or continued for an additional 24 h in fresh preequilibrated medium (24–48 h, bottom). Initial medium pH, Pco₂, and [HCO₃⁻] were not different from Table 1. The P_i fluxes for 10–15 pairs of calvariae/group are means ± SE. *P < 0.05 vs. NTL.

Fig. 5.

FGF23 expression in primary osteoblasts after incubation in MET. Primary osteoblasts were isolated from neonatal mouse calvariae. At confluence, some plates were collected for a baseline measurement of FGF23 expression (0 h), and the remaining cells were incubated at neutral physiological pH (NTL; white bars) or at reduced pH simulating metabolic acidosis (MET; black bars) for 6 or 24 h. Initial medium pH, Pco₂, and [HCO₃⁻] were not different from Table 1. At the end of the incubation period, cells were collected for RNA isolation and FGF23 expression was assayed by real-time PCR. Data are means ± SE for 10–20 samples/group. *P < 0.05 vs. 0; +P < 0.05 vs. NTL, same time period.