Fibroblast growth factor 23 regulates renal 1,25-dihydroxyvitamin D and phosphate metabolism via the MAP kinase signaling pathway in Hyp mice

Abstract

In X-linked hypophosphatemia (XLH) and in its murine homologue, the Hyp mouse, increased circulating concentrations of fibroblast growth factor 23 (FGF-23) are critical to the pathogenesis of disordered metabolism of phosphate (P(i)) and 1,25-dihydroxyvitamin D [1,25(OH)(2)D]. In this study, we hypothesized that in Hyp mice, FGF-23-mediated suppression of renal 1,25(OH)(2)D production and P(i) reabsorption depends on activation of mitogen-activated protein kinase (MAPK) signaling. Wild-type and Hyp mice were administered either vehicle or the MEK inhibitor PD0325901 (12.5 mg/kg) orally daily for 4 days. At baseline, the renal abundance of early growth response 1 (egr1) mRNA was approximately 2-fold greater in Hyp mice than in wild-type mice. Treatment with PD0325901 greatly suppressed egr1 mRNA abundance in both wild-type and Hyp mice. In Hyp mice, PD0325901 induced an 8-fold increase in renal 1α-hydroxylase mRNA expression and a 4-fold increase in serum 1,25(OH)(2)D concentrations compared with vehicle-treated Hyp mice. Serum P(i) levels in Hyp mice increased significantly after treatment with PD0325901, and the increase was associated with increased renal Npt2a mRNA abundance and brush-border membrane Npt2a protein expression. These findings provide evidence that in Hyp mice, MAPK signaling is constitutively activated in the kidney and support the hypothesis that the FGF-23-mediated suppression of renal 1,25(OH)(2)D production and P(i) reabsorption depends on activation of MAPK signaling via MEK/ERK1/2. These findings demonstrate the physiologic importance of MAPK signaling in the actions of FGF-23 in regulating renal 1,25(OH)(2)D and P(i) metabolism.

Fig. 1

Effect of FGF-23 on renal MEK/ERK1/2 signaling in wild-type mice. Mice were injected with FGF-23 or vehicle and euthanized 10 and 60 minutes later. (A) Whole-kidney tissue protein extracts were probed with rabbit anti-phospho-ERK1/2 antibodies (top panel). Protein loading was determined using total ERK2 protein (bottom panel). (B). Egr1 mRNA abundance was quantitated by real-time PCR, normalized to that of Gus mRNA, and expressed as a percent relative to vehicle. Bars depict mean ± SEM (n = 4 to 6 mice/group). ^*p <.05 compared with the vehicle group.

Fig. 2

Effect of PD0325901 on renal MEK/ERK1/2 signaling in wild-type and Hyp mice. Mice were administered PD0325901 or vehicle for 4 days. (A) Whole-kidney tissue protein extracts were probed with rabbit anti-phospho-ERK1/2 antibodies. (B) Egr1 mRNA abundance, normalized to that of Gus mRNA, is expressed as a percent relative to vehicle-treated wild-type mice. Bars depict mean ± SEM (n =7 to 8 mice/group). ^#p <.05 compared with wild-type mice treated with vehicle. ^*p <.05 compared with vehicle-treated mice within each mouse strain.

Fig. 3

Effects of MEK inhibition by PD0325901 on 1,25(OH)₂D metabolism in wild-type and Hyp mice. Mice were administered PD0325901 or vehicle for 4 days. (A) Renal 1α-hydroxylase mRNA abundance was quantitated by real-time PCR, normalized to that of Gus mRNA, and expressed as a percent relative to vehicle-treated wild-type mice. (B) Renal mitochondrial 1α-hydroxylase protein abundance in Hyp mice determined by a Western blot analysis. Protein loading was determined using β-actin. The immunoblot represents data from 2 mice/group. (C) Renal 24-hydroxylase mRNA abundance was quantitated as above. Bars depict mean ± SEM (n =7 to 12 mice/group). ^#p <.05 compared with wild-type mice within each treatment group. ^*p <.05 compared with vehicle-treated mice within each mouse strain.

Fig. 4

Effects of MEK inhibition by PD0325901 on serum 1,25(OH)₂D concentrations in wild-type and Hyp mice. Mice were administered PD0325901 or vehicle for 4 days. ^#p <.05 compared with wild-type mice within each treatment group. ^*p <.05 compared with vehicle-treated mice within each mouse strain.

Fig. 5

Effects of MEK inhibition by PD0325901 on phosphate metabolism in wild-type and Hyp mice. Mice were administered PD0325901 or vehicle for 4 days. (A) Serum P_i concentrations. (B) Renal Npt2a mRNA abundance was quantitated by real-time PCR, normalized to that of Gus mRNA, and expressed as a percent relative to vehicle-treated wild-type mice. (C) Renal brush-border Npt2a protein abundance. Protein loading was determined using β-actin. Bars depict mean ± SEM (n =7 to 12 mice/group) ^#p <.05 compared with wild-type mice within each treatment group. ^*p <.05 compared with vehicle-treated mice within each mouse strain.

Fig. 6

Effects of MEK inhibition by PD0325901 on serum calcium and iPTH concentrations in wild-type and Hyp mice. Mice were administered PD0325901 or vehicle for 4 days. (A) Serum calcium concentrations. (B) Serum iPTH concentrations. Bars depict mean ± SEM (n =7 to 12 mice/ group) ^#p <.05 compared with wild-type mice within each treatment group. ^*p <.05 compared with vehicle-treated mice within each mouse strain.

Fig. 7

Effects of MEK inhibition by PD0325901 on fgf23 mRNA in bone in wild-type and Hyp mice. Mice were administered PD0325901 or vehicle for 4 days. Femoral bone fgf23 mRNA abundance was quantitated by real-time PCR, normalized to that of Gapdh mRNA, and expressed as a percent relative to vehicle-treated mice. Bars depict mean ± SEM (n =7 to 12 mice/group) ^#p <.05 compared with wild-type mice within each treatment group. ^*p <.05 compared with vehicle-treated mice within each mouse strain.