1,25-Dihydroxyvitamin D Alone Improves Skeletal Growth, Microarchitecture, and Strength in a Murine Model of XLH, Despite Enhanced FGF23 Expression

Abstract

X-linked hypophosphatemia (XLH) is characterized by impaired renal tubular reabsorption of phosphate owing to increased circulating FGF23 levels, resulting in rickets in growing children and impaired bone mineralization. Increased FGF23 decreases renal brush border membrane sodium-dependent phosphate transporter IIa (Npt2a) causing renal phosphate wasting, impairs 1-α hydroxylation of 25-hydroxyvitamin D, and induces the vitamin D 24-hydroxylase, leading to inappropriately low circulating levels of 1,25-dihydroxyvitamin D (1,25D). The goal of therapy is prevention of rickets and improvement of growth in children by phosphate and 1,25D supplementation. However, this therapy is often complicated by hypercalcemia and nephrocalcinosis and does not always prevent hyperparathyroidism. To determine if 1,25D or blocking FGF23 action can improve the skeletal phenotype without phosphate supplementation, mice with XLH (Hyp) were treated with daily 1,25D repletion, FGF23 antibodies (FGF23Ab), or biweekly high-dose 1,25D from d2 to d75 without supplemental phosphate. All treatments maintained normocalcemia, increased serum phosphate, and normalized parathyroid hormone levels. They also prevented the loss of Npt2a, α-Klotho, and pERK1/2 immunoreactivity observed in the kidneys of untreated Hyp mice. Daily treatment with 1,25D decreased urine phosphate losses despite a marked increase in bone FGF23 mRNA and in circulating FGF23 levels. Daily 1,25D was more effective than other treatments in normalizing the growth plate and metaphyseal organization. In addition to being the only therapy that normalized lumbar vertebral height and body weight, daily 1,25D therapy normalized bone geometry and was more effective than FGF23Ab in improving trabecular bone structure. Daily 1,25D and FGF23Ab improved cortical microarchitecture and whole-bone biomechanical properties more so than biweekly 1,25D. Thus, monotherapy with 1,25D improves growth, skeletal microarchitecture, and bone strength in the absence of phosphate supplementation despite enhancing FGF23 expression, demonstrating that 1,25D has direct beneficial effects on the skeleton in XLH, independent of its role in phosphate homeostasis. © 2016 American Society for Bone and Mineral Research.

Keywords: BIOMECHANICS; BONE QCT/μCT; GENETIC MOUSE MODELS; GROWTH PLATE; PTH/VIT D/FGF23.

Conflict of interest statement

All authors have declared that no conflict of interest exists.

© 2016 American Society for Bone and Mineral Research.

Figures

Fig 1

1,25D and FGF23Ab treatment increase FGF23 mRNA levels and restore renal Npt2a, α–Klotho and pERK1/2 immunoreactivity in Hyp mice. A. RNA isolated from the humeral diaphysis of d75 mice was subjected to RT-qPCR for evaluation of FGF23 mRNA levels. All treatments dramatically increased long bone FGF23 mRNA expression. Data, normalized for actin in each sample represents RNA isolated from 3 mice per genotype/treatment group. B. Immunohistochemical evaluation of Npt2a, α-Klotho and pERK1/2 was performed on sections of kidneys obtained d75, 24h after the final treatment. All treatments increased Npt2a, α-Klotho and pERK1/2 immunoreactivity relative to untreated Hyp control mice. Data are representative of that obtained from 3 mice per genotype/treatment group. * =p value

Fig. 2

1,25D and FGF23Ab effects on growth plate and metaphyseal morphology and growth in Hyp mice. A. H&E, Col X in situ hybridization, pERK1/2 immunoreactivity and TUNEL labeling of the growth plates of d75 mice. Yellow arrows indicate TUNEL labeled hypertrophic chondrocytes. Data are representative of 3 mice per genotype/treatment group. B. The number of TUNEL labeled nuclei in the last two rows of the hypertrophic chondrocyte layer was quantitated. Data are representative of 3 mice per genotype/treatment group. C. 1,25D increases basal and phosphate-induced mitochondrial pERK1/2. Subcellular fractionation of hypertrophic chondrocytes treated with 10−8 M 1,25D prior to addition of 7mM sodium sulfate (−) or sodium phosphate (Pi). HSP90 is used as a cytosolic control and VDAC as a mitochondrial control. Data are representative of that obtained from 3 independent chondrocyte preparations. D. Weight, lumbar vertebral height, tail length and femur length of d75 mice. Data are representative of that obtained from 3 to 5 mice per genotype/treatment group. E. Radiographs of representative femurs. * =p value <0.05 vs WT, # =p value <0 vs Hyp Con, a =p value <0.05 vs Daily 1,25D, b =p value <0.05 vs FGF23Ab.

Fig. 3

1,25D and FGF23Ab treatment improve microarchitecture. A. MicroCT scans were performed on femurs isolated from d75 mice. Ct. Th, cortical thickness; Ct.Ar/T.Ar, Cortical Area/Total Area; BV/TV, Bone Volume/Tissue Volume. Data represent that obtained from 5 mice per genotype/treatment group. * =p value

Fig. 4

1,25D and FGF23Ab improve bone strength. A. Inferred biomechanical parameters from microCT analyses. pMOI, polar moment of inertia. B. Torsion testing was performed on d75 femurs. Representative torque rotation curves are shown. C. Torsion testing evaluation of whole-bone strength (Max Torque), stiffness and toughness (work to Max Torque). Data represent that obtained from 5 mice per genotype/treatment group. * =p value

Fig. 5

1,25D and FGF23Ab improve mineralization. A. von Kossa and trichrome staining of the distal femur of d75 mice. B. Histomorphometric parameters. BV/TV, Bone Volume/Tissue Volume; Tb.N, Trabecular Number; Tb.Sp., Trabecular Separation; OV/BV, Osteoid Volume/Tissue Volume; MAR, Mineral Apposition Rate; Ct.Th., Cortical Thickness. Data are representative of that obtained from 5–8 mice per treatment/genotype. * =p value

Fig. 6

Osteocyte apoptosis is increased in Hyp cortical bone. A. The number of osteocytes 4 to 5mm proximal and contralateral to the tibiofibular junction in d75 mice was quantitated. Data represent that obtained from three mice per genotype/treatment group. B. Osteocyte apoptosis was quantitated in the tibial cortex of d75 mice. Data represent that obtained from 3 mice per genotype/treatment group. * =p value