Sclerostin antibody treatment increases bone mass and normalizes circulating phosphate levels in growing Hyp mice.

Abstract

X-linked hypophosphatemia (XLH), caused by a loss-of-function mutation in the phosphate regulating gene with homology to endopeptidase located on the X chromosome (PHEX), is the most common form of vitamin D-resistant rickets. Loss of functional PHEX results in elevated fibroblast growth factor 23 (FGF23) levels, impaired phosphate reabsorption, and inhibited skeletal mineralization. Sclerostin, a protein produced primarily in osteocytes, suppresses bone formation by antagonizing Wnt-signaling and is reported to be elevated in XLH patients. This study used the Hyp mouse model to investigate sclerostin’s role in the pathophysiology of XLH by evaluating the use of a monoclonal antibody to sclerostin in a mouse model of XLH, the Hyp mouse. Male and female wild-type and Hyp littermates were injected with 25mg/kg of vehicle or sclerostin-antibody (Scl-Ab) twice weekly, beginning at 4-weeks of age and sacrificed at 8-weeks of age. Scl-Ab treatment increased serum phosphate levels and suppressed circulating levels of intact FGF23 in treated wild-type and Hyp mice of both sexes. Cortical area, trabecular bone volume fraction (BV/TV), metaphyseal apparent density, and the peak load increased with Scl-Ab treatment in both sexes. This short-term treatment study suggests that Scl-Ab treatment can effectively improve some of the pathologies associated with XLH, including normalization of phosphate, and that sclerostin may play a role in regulating FGF23 and phosphate metabolism in XLH.

Introduction

X-linked hypophosphatemia (XLH) is the most common cause of vitamin D-resistant rickets, affecting 1 in 20,000 people⁽¹⁾. The condition results from a loss-of-function mutation in the phosphate regulating gene with homology to endopeptidase located on the X chromosome (PHEX)⁽²⁾. This mutation leads to elevated fibroblast growth factor 23 (FGF23) levels, which subsequently impairs phosphate reabsorption in the kidney and inhibits skeletal mineralization. Clinical manifestations of XLH include hypophosphatemia, lower limb deformities, and a stunted growth rate⁽³⁾, which are first evident in children and will persist until adulthood if left untreated⁽⁴⁾. XLH patients of all ages have decreased bone mass and are at an increased risk for fracture⁽⁵⁾.

Sclerostin (encoded by the SOST gene) is a protein produced primarily by osteocytes that suppresses bone formation by antagonizing Wnt-signaling⁽⁶⁾. Neutralizing antibodies to sclerostin (Scl-Ab) have been used both in animal studies and clinically to effectively promote bone formation in osteoporosis^(7,8). In addition to its clear positive effects on bone mass, sclerostin antibody treatment may also increase mineralization kinetics⁽⁹⁾ and reduce mineralization lag time⁽⁷⁾.

Interestingly, previous literature has linked sclerostin with FGF23 in various disease models. Circulating levels of sclerostin and FGF23 are positively correlated in patients with sclerosteosis and in those with chronic kidney disease^(10–12) and FGF23 levels are suppressed in the sclerostin null mouse⁽¹³⁾. Further, circulating sclerostin levels are higher in humans with XLH⁽¹⁴⁾. This evidence of an association between sclerostin and FGF23, led us to hypothesize that suppressing sclerostin with Scl-Ab treatment could effectively improve both skeletal and endocrine pathologies in XLH. To test this idea, we investigated the effects of Scl-Ab treatment on bone mass and phosphate metabolism in growing 4-week old Hyp mouse model of XLH. Our results show that Scl-Ab treatment significantly decreased the circulating levels of intact FGF23 and improved phosphate levels, while also significantly improving bone mass and strength in Hyp mice.

Materials and Methods

Animals

Female heterozygous (+/Hyp) [000528] and male wild-type (WT; +/y) mice were purchased from Jackson Laboratory [Bar Harbor, ME, USA]. The breeding strategy generated heterozygous (+/Hyp) and WT females and hemizygous (Hyp/y) and WT males. Mice were caged in groups of 3 to 5, maintained on a 12-hour dark/light cycle, and were provided standard Teklad Global 18% protein rodent chow (2018, Teklad; 1% Ca, 0.7% Phosphorus) and water ad libitum. Sixty (23 female, 37 male) Hyp mice and wild type littermates in total were used for this study. All mice were randomly assigned to twice weekly subcutaneous injections of either 25 mg/kg Scl-Ab (Amgen Inc, Thousand Oaks, CA and UCB, Brussels, Belgium) or vehicle (saline). The Scl-Ab dosing strategy was chosen based on previous publications testing the efficacy of Scl-Ab in preclinical rodent models ^(7,15–17). Treatment was initiated at weaning (4 weeks of age) and continued for 4 weeks until sacrifice at 8 weeks of age. All animal studies were approved by the Rush University Institutional Animal Care and Use Committee.

Animals were sacrificed 24-hours after the last treatment injection. Body mass was measured immediately after sacrifice. Blood was collected via cardiac puncture and allowed to clot at room temperature for 30 minutes before being centrifuged at 3,400 rpm for 15 min at 4°C to separate serum. Right femurs were collected, stored in 70% ethanol and refrigerated. Left femurs were collected, wrapped in phosphate-buffered saline soaked gauze and frozen for future 3-point bending mechanical testing. Left and right tibias were collected and cleaned of all soft tissue and the proximal and distal epiphyses were removed. The resulting tibia diaphyseal tissue was centrifuged at 5,000 rpm for 10 min at 4°C to remove medullary contents to ensure enrichment of osteocytes. Tibia diaphyses and kidneys were then stored in RNAlater (Ambion) and frozen at −20°C. The sample size for each of the 8 experimental groups ranged between 5–12 animals.

Serum parameters

Serum phosphate levels were measured using a colorimetric assay (BioVision). Serum FGF23 levels were measured using immunoassays for mouse/rat intact FGF23 and mouse/rat C-terminal FGF23 (Immutopics). Serum sclerostin levels were measured using a mouse/rat SOST/sclerostin quantikine immunoassay (R&D Systems). The sample size for circulating protein levels ranged between n = 3 to 11 mice.

Gene expression

Tibia diaphyses and kidneys were removed from RNAlater, snap frozen using liquid nitrogen, and subsequently crushed with a mortar and pestle. The resulting tissue was then submerged in Trizol (Ambion) and homogenized with a Polytron PT 10–35 Homogenizer (Brinkmann). RNA was extracted using the manufacturer’s protocol (Trizol) before being reverse transcribed using an Applied Biosystems High capacity cDNA Reverse Transcription Kit. qPCR (QuantStudio™ 7 Flex System using SYBR Green reagents) was completed to detect gene expression of sclerostin (Sost), Polypeptide N-Acetylgalactosaminyltransferase 3 (Galnt3), FAM20C Golgi Associated Secretory Pathway Kinase (Fam20c), dentin matrix acidic phosphoprotein (Dmp1), osteopontin (Opn), matrix extracellular phosphoglycoprotein (Mepe), sodium-dependent phosphate transport protein 2A and 2C (Npt2a and Npt2c). Gapdh was used as the internal control. Primer sequences are presented in Supplemental Table 1. All groups had a sample size of n = 5 for skeletal gene expression.

Micro-computed tomography (μCT)

Right femurs were μCT scanned while submerged in 70% ethanol, perpendicular to the bone’s long-axis. Scanning parameters were 55 kVp and 145 μA, with a 500 ms integration time and a 6μm isotropic voxel size (μCT50, Scanco Medical). Cortical geometry was measured in the middle 100 slices of the femoral diaphysis. Primary cortical parameters included cortical area (Ct.Ar), total area (Tt.Ar), medullary area (Ma.Ar), cortical thickness (Ct.Th), cortical porosity (Ct.Po), the polar moment of inertia (pMOI), and cortical tissue mineral density (Ct.TMD). Trabecular bone architecture was measured from the distal 30% slice of the total femoral length to the distal growth plate. Primary trabecular parameters included bone volume per total volume (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular spacing (Tb.Sp). Analysis of the metaphyseal region included trabecular and cortical bone measured from the distal 30% slice of the total femoral length to the distal growth plate (Figure 1). The primary metaphyseal parameter was apparent density (Ap.Dens) and metaphyseal tissue mineral density (M.TMD). All parameters are reported using conventional nomenclature⁽¹⁸⁾. Group sample sizes for all μCT analyses ranged from n = 5 to 12 mice.

Figure 1.

μCT image of a WT femur showing the region of interest for cortical (green), trabecular (yellow), and metaphyseal (red) analyses.

Mechanical testing

Left femurs were thawed in phosphate-buffered saline prior to 3-point bending. Femurs were loaded to failure in the anterior-posterior direction. A lower support span length of 7mm was used for WT mice and 5mm for Hyp mice. A load rate of 0.1 mm/s, and a data acquisition rate of 100 Hz (MTS CriterionTM). A preload of ~0.5 N was applied to each bone to prevent shifting during testing. Load-displacement curves were used to determine the peak load and bending stiffness. Group sizes for mechanical testing ranged from n = 6 to 8 mice.

Histological mineralization analysis

Following μCT scanning, the femoral head was removed from the right femurs to improve infiltration and the remaining bone was dehydrated through a series of graded alcohol solutions. Femurs were then embedded in polymethyl methacrylate (PMMA). Longitudinal sections were cut to at a 5μm thickness and mounted onto glass slides. Slides were stained with Goldner’s trichrome bone stain. Cortical and trabecular osteoid surface was evaluated using Osteomeasure (Osteometrics). Cortical bone osteoid was measured on the endocortical surface in a region of interest that began 1.5mm proximal to the growth plate and continued 1.5mm up the medial and lateral sides of the femur. Trabecular bone osteoid was measured in the medullary cavity just proximal to the growth plate and continued 1.5mm proximally. Primary outcome measures included osteoid width (O.Wi) and osteoid surface/bone surface (OS/BS). Group sample sizes for histological analyses ranged from n = 4 to 6.

Statistical analyses

All variables were compared separately for males and females using a two-way analysis of variance (ANOVA) with genotype and treatment as the independent factors. Outcomes from the ANOVA included genotype and treatment effects and the genotype by treatment interaction. When main effects were significant, post-hoc analysis (independent student’s T-test) was performed to compare the effects of the Scl-Ab treatment. A p-value of <0.05 was considered statistically significant.

Results

Scl-Ab treatment did not affect body weight or femoral length

Hyp mice had a significantly lower body weight at sacrifice (p < 0.001, both sexes). Scl-Ab treatment did not increase body weight in either WT or Hyp mice. Similarly, Hyp mice had significantly shorter femurs (p < 0.001, both sexes), and Scl-Ab treatment did not improve this phenotype in either WT or Hyp mice (Figure 2).

Figure 2.

Body mass (A) and femur length (B) in male (left) and female (right) mice. Data are presented as the mean ± standard deviation. Results from the two-way ANOVA are presented in the figure legends. Significant post-hoc differences between animals of the same genotype are reported above the data with horizontal bars and corresponding p-values. Letters highlight significant post-hoc differences between Hyp vehicle and WT vehicle (a); Hyp vehicle and WT Scl-Ab (b); Hyp Scl-Ab and WT vehicle (c); Hyp Scl-Ab and WT Scl-Ab (d).

Scl-Ab treatment significantly increased circulating phosphate and decreased intact FGF23 levels

Circulating phosphate levels were significantly lower in Hyp mice compared to WT littermates (Genotype effect: p < 0.001 both sexes) (Figure 3). Scl-Ab treatment significantly increased circulating phosphate concentrations in male and female mice of both genotypes (Treatment effect: p < 0.001 both sexes), with no significant genotype*treatment interaction.

Figure 3.

Circulating phosphate (A), intact FGF23 (B), and c-term FGF23 (C) protein levels in male (left) and female (right) mice. Data are presented as the mean ± standard deviation. Results from the two-way ANOVA are presented in the figure legends. Significant post-hoc differences between animals of the same genotype are reported above the data with horizontal bars and corresponding p-values. Letters highlight significant post-hoc differences between Hyp vehicle and WT vehicle (a); Hyp vehicle and WT Scl-Ab (b); Hyp Scl-Ab and WT vehicle (c); Hyp Scl-Ab and WT Scl-Ab (d).

Hyp mice had significantly elevated circulating intact FGF23 levels compared to WT littermates (Genotype effect: p < 0.001, both sexes) (Figure 3). Scl-Ab treatment significantly decreased circulating intact FGF23 concentrations (Treatment effect: p < 0.001, both sexes). Although Scl-Ab reduced intact FGF23 by ~30% in both genotypes, the magnitude was larger in the Hyp mice, resulting in a significant genotype*treatment interaction (p = 0.002 males; p = 0.001 females). Hyp mice had elevated circulating C-term FGF23 levels compared to WT littermates (Genotype effect: p < 0.001, both sexes). Scl-Ab treatment had no effect on the circulating C-term FGF23 levels in male or female mice.

Scl-Ab treatment significantly increased bone mass

Cortical area was significantly lower in Hyp mice compared to WT littermates in both male and female mice (Genotype effect: p < 0.001, both sexes, Figure 4). Scl-Ab treatment significantly increased cortical area in both male and female mice (Treatment effect: p < 0.001). Post-hoc analysis revealed that Scl-Ab significantly increased cortical area in WT and Hyp mice of both sexes. Representative μCT images are presented as Supplemental Figures 1 and 2.

Figure 4.

μCT results of cortical area (A), trabecular BV/TV (B), and metaphyseal apparent density (C) in male (left) and females (right) mice. Data are presented as the mean ± standard deviation. Results from the two-way ANOVA are presented in the figure legends. Significant post-hoc differences between animals of the same genotype are reported above the data with horizontal bars and corresponding p-values. Letters highlight significant post-hoc differences between Hyp vehicle and WT vehicle (a); Hyp vehicle and WT Scl-Ab (b); Hyp Scl-Ab and WT vehicle (c); Hyp Scl-Ab and WT Scl-Ab (d).

Hyp mice also had significantly decreased cortical thickness, cortical tissue mineral density, and polar moment of inertia (pMOI), and increased medullary area and cortical porosity compared to WT mice (Tables 1 & 2). There were no significant genotype effects for the total area. Scl-Ab treatment significantly increased total area, cortical thickness, and pMOI in both sexes.

Table 1.

μCT results for males of each genotype and treatment group. Data are presented as mean values ± standard deviation. P-values are from the two-way ANOVA results.

μCT variable	Male WT vehicle	Male WT Scl-Ab	Male Hyp vehicle	Male Hyp Scl-Ab	Genotype effect	Treatment effect	Interaction term
Tt.Ar (mm2)	1.85 ± 0.14	2.13 ± 0.33a	1.76 ± 0.26	2.10 ± 0.48	0.577	0.005	0.731
Ma.Ar (mm2)	1.04 ± 0.11	1.05 ± 0.24	1.32 ± 0.23b	1.52 ± 0.37c	<0.001	0.200	0.248
Ct.Th (mm)	0.17 ± 0.02	0.19 ± 0.03a	0.07 ± 0.01b	0.08 ± 0.01a,c	<0.001	0.004	0.193
Ct.Po (%)	1.24 ± 1.19	1.89 ± 1.22	21.45 ± 5.94a,b	19.96 ± 5.90c	<0.001	0.736	0.391
Ct.TMD (mgHA/mm3)	930.55 ± 29.72	936.97 ± 47.30	902.84 ± 24.61	881.47 ± 20.66c	0.001	0.509	0.219
pMOI	0.40 ± 0.06	0.58 ± 0.16a	0.24 ± 0.04b	0.37 ± 0.15c	<0.001	0.001	0.455
Tb.N (1/mm)	5.26 ± 0.34	5.90 ± 0.74a	2.19 ± 0.44b	2.46 ± 0.32c	<0.001	0.012	0.289
Tb.Th (μm)	45.63 ± 3.91	57.68 ± 4.18a	38.90 ± 10.91	44.27 ± 4.79c	<0.001	<0.001	0.090
Tb.Sp (mm)	0.19 ± 0.01	0.17 ± 0.02a	0.48 ± 0.12b	0.43 ± 0.05c	<0.001	0.856	0.664
M.TMD (mgHA/mm3)	865.82 ± 18.59	878.53 ± 26.75	790.22 ± 22.33b	774.66 ± 26.73c	<0.001	0.853	0.083

^aindicates significant difference between vehicle and Scl-Ab treated mice of the same genotype.

^bindicates significant differences from vehicle treated WT mice.

^cindicates significant differences from Scl-Ab treated WT mice.

Table 2.

μCT results for females of each genotype and treatment group. Data are presented as mean values ± standard deviation. P-values are from the two-way ANOVA results.

μCT variable	Female WT vehicle	Female WT Scl-Ab	Female Hyp vehicle	Female Hyp Scl-Ab	Genotype effect	Treatment effect	Interaction term
Tt.Ar (mm2)	1.49 ± 0.06	1.71 ± 0.07a	1.49 ± 0.11	1.56 ± 0.06c	0.046	0.001	0.034
Ma.Ar (mm2)	0.89 ± 0.08	0.85 ± 0.10	1.03 ± 0.11b	1.04 ± 0.06c	<0.001	0.597	0.555
Ct.Th (mm)	0.14 ± 0.01	0.19 ± 0.01a	0.08 ± 0.01b	0.09 ± 0.01a,c	<0.001	<0.001	<0.001
Ct.Po (%)	2.12 ± 1.45	1.09 ± 0.34	13.88 ± 8.99b	13.12 ± 5.07c	0.001	0.701	0.953
Ct.TMD (mgHA/mm3)	940.37 ± 48.96	972.07 ± 17.83	929.14 ± 21.98	920.20 ± 17.12c	0.019	0.366	0.115
pMOI	0.24 ± 0.05	0.37 ± 0.03a	0.21 ± 0.07	0.22 ± 0.03c	0.001	0.005	0.015
Tb.N (1/mm)	3.87 ± 0.33	4.20 ± 0.26	1.79 ± 0.27b	1.78 ± 0.29c	<0.001	0.102	0.316
Tb.Th (μm)	32.72 ± 2.43	47.85 ± 2.40a	37.73 ± 8.12	46.70 ± 6.65	0.514	<0.001	0.275
Tb.Sp (mm)	0.26 ± 0.01	0.24 ± 0.01	0.58 ± 0.08b	0.59 ± 0.08c	<0.001	0.308	0.873
M.TMD (mgHA/mm3)	864.87 ± 12.78	880.32 ± 11.93	832.38 ± 12.39b	811.38 ± 17.07a,c	<0.001	0.631	0.005

^aindicates significant difference between vehicle and Scl-Ab treated mice of the same genotype.

^bindicates significant differences from vehicle treated WT mice.

^cindicates significant differences from Scl-Ab treated WT mice.

At the distal femoral metaphysis, Hyp mice had significantly decreased trabecular BV/TV in both males and females compared to WT littermates (Genotype effect: p < 0.001, both sexes, Figure 4). Scl-Ab treatment significantly increased trabecular BV/TV in both males and females (Treatment effect: p < 0.001 and p = 0.001, respectively). Post-hoc analysis revealed that Scl-Ab significantly increased BV/TV in WT and Hyp of both sexes. The genotype*treatment interaction for BV/TV was significant for both sexes (p = 0.010 and 0.005, for males and females, respectively), owing to a larger treatment response in WT animals.

Hyp male mice also had significantly decreased trabecular thickness, while Hyp mice of both sexes had significantly fewer trabecular number and significantly increased trabecular spacing (Genotype effect: p < 0.001 for all variables). Scl-Ab treatment significantly increased trabecular thickness in both sexes (Treatment effect: p < 0.001) and the trabecular number (Treatment effect: p = 0.012) in male mice (Tables 1 & 2).

Distal femoral metaphyseal apparent density (Ap.Dens) was significantly decreased in Hyp mice compared to WT mice (Genotype effect: p < 0.001, both sexes, Fig. 4). Scl-Ab treatment significantly increased Ap.Dens in males and females (Treatment affect: p < 0.001, both sexes). Post-hoc analyses revealed that Scl-Ab significantly increased Ap.Dens in WT and Hyp mice of both sexes.

Hyp mice had significantly decreased metaphyseal tissue mineral density (M.TMD) compared to their WT littermates (Genotype effect: p < 0.001, both sexes). There was no treatment effect for M.TMD for either sex. Female mice had a significant genotype*treatment effect (p = 0.008) for M.TMD, attributed to an increase in M.TMD in WT animals following treatment (Tables 1 & 2).

Scl-Ab treatment significantly increased bone strength

Hyp mice had significantly reduced peak load compared to WT littermates (Genotype effect: p < 0.001, both sexes, Figure 5). Scl-Ab treatment significantly increased peak load in both sexes (Treatment effect: p < 0.001). Post-hoc analysis revealed that Scl-Ab increased peak load in male and female WTs and male Hyp mice. There was also a significant interaction for both males and females (p = 0.002 and 0.001, respectively), which is attributed to larger gains in the peak load in WT mice.

Figure 5.

Peak load (A), and stiffness (B) in male (left) and females (right) mice. Data are presented as the mean ± standard deviation. Results from the two-way ANOVA are presented in the figure legends. Significant post-hoc differences between animals of the same genotype are reported above the data with horizontal bars and corresponding p-values. Letters highlight significant post-hoc differences between Hyp vehicle and WT vehicle (a); Hyp vehicle and WT Scl-Ab (b); Hyp Scl-Ab and WT vehicle (c); Hyp Scl-Ab and WT Scl-Ab (d).

Hyp similarly had significantly reduced bending stiffness compared to WT mice (Genotype effect: p < 0.001, both sexes). Scl-Ab significantly increased the bending stiffness in both sexes (Treatment effect: p = 0.009 and 0.025, for male and females, respectively), although the post-hoc analysis found that Scl-Ab significantly increased bending stiffness only in the male WT mice.

Scl-Ab treatment had little effect on the amount of unmineralized osteoid

Hyp mice did not differ significantly from WT mice in cortical osteoid width (O.Wi) (Tables 3 & 4). In both sexes, there was a small non-significant reduction in O.Wi following Scl-Ab treatment, which only approached significance in female mice (Treatment effect: p=0.077). Cortical osteoid surface/bone surface (OS/BS) was similarly not affected by genotype, although vehicle treated Hyp mice of both sexes had the highest OS/BS. Scl-Ab treated Hyp mice showed a non-significant reduction in the OS/BS parameter in both sexes. Representative histology images from male mice are presented in Figure 6 and female mice are presented as Supplemental Figure 3.

Table 3.

Histological osteoid results for males of each genotype and treatment group. Data are presented as mean ± standard deviation. P-values are from the two-way ANOVA.

	Variable	Male WT vehicle	Male WT Scl-Ab	Male Hyp vehicle	Male Hyp Scl-Ab	Genotype effect	Treatment effect	Interaction term
Cortical	O.Wi (μm)	4.97 ± 1.95	3.81 ± 0.58	5.17 ± 1.78	4.51 ± 1.12	0.557	0.240	0.735
	OS/BS (%)	7.84 ± 6.09	6.94 ± 5.93	15.32 ± 14.37	5.69 ± 7.42	0.487	0.248	0.335
Trabecular	O.Wi (μm)	2.47 ± 6.05	0.00 ± 0.00	7.36 ± 6.37	8.43 ± 9.76	0.032	0.808	0.542
	OS/BS (%)	0.03 ± 0.08	0.00 ± 0.00	15.22 ± 14.78	17.23 ± 15.76	0.003	0.831	0.825

Table 4.

Histological osteoid results for females of each genotype and treatment group. Values are presented as mean ± standard deviation. P-values are from the two-way ANOVA.

	variable	Female WT vehicle	Female WT Scl-Ab	Female Hyp vehicle	Female Hyp Scl-Ab	Genotype effect	Treatment effect	Interaction term
Cortical	O.Wi (μm)	4.06 ± 0.45	3.43 ± 1.13	4.77 ± 0.64	3.84± 1.09	0.195	0.077	0.720
	OS/BS (%)	5.86± 4.24	6.43 ± 8.09	12.01 ± 5.34	3.68 ± 2.76	0.511	0.145	0.098
Trabecular	O.Wi (μm)	1.13 ± 2.52	0.79 ± 1.76	4.54 ± 4.24	4.65 ± 4.78	0.036	0.943	0.888
	OS/BS (%)	0.16 ± 0.35	0.07 ± 0.15	12.30 ± 10.04	14.70 ± 10.09	0.001	0.718	0.697

Figure 6.

Representative histology demonstrating the extent of osteoid present in WT and Hyp male mice treated with either vehicle or Scl-Ab. Images on the right are magnified cortical regions from the same sample presented on the left.

Trabecular osteoid width (O.Wi) was higher in Hyp mice compared to WTs of both sexes (Genotype effect: p = 0.032 males; p = 0.036 females). Scl-Ab treatment did not affect the trabecular O.Wi in either sex. Similarly, Hyp mice had significantly elevated trabecular osteoid surface/bone surface (OS/BS) compared to WT mice (Genotype effect: p = 0.003 males; p = 0.001 females). There was no treatment effect on trabecular OS/BS in males or females (Tables 3 & 4).

Scl-Ab treatment affected skeletal gene expression

Skeletal gene expression was assessed in osteocyte-enriched tibiae samples. As expected, Hyp mice had increased Fgf23 expression (Genotype effect: p = 0.002 and 0.003 for males and females, respectively), which increased further following Scl-Ab treatment (Figure 7). Hyp mice also had reduced skeletal Sost levels, although these differences were not significant, due to a variable treatment response in both WT and Hyp mice. Skeletal expression of Sost trended back toward WT levels in Hyp mice following Scl-Ab treatment. Despite the low skeletal gene levels of Sost, circulating levels of sclerostin protein were similar between WT and Hyp mice; 398.2 ± 178.2 vs 273.0 ± 142.7 pg/mL in male WT compared to Hyp mice and 320.1 ± 119.0 vs 248.35 ± 107.2 ng/mL in females. Circulating sclerostin levels were above the detection limit of the assay (1000 pg/mL) for all sexes and genotype following Scl-Ab treatment.

Figure 7.

Skeletal gene expression of Fgf23 (A), and Sost (B) in male (left) and females (right) mice. Data are presented as the mean ± standard deviation. Results from the two-way ANOVA are presented in the figure legends. Significant post-hoc differences between animals of the same genotype are reported above the data with horizontal bars and corresponding p-values. Letters highlight significant post-hoc differences between Hyp vehicle and WT vehicle (a); Hyp vehicle and WT Scl-Ab (b); Hyp Scl-Ab and WT vehicle (c); Hyp Scl-Ab and WT Scl-Ab (d).

Additional analysis of genes thought to be involved in the regulation of FGF23 failed to show any treatment effects. There was a significant genotype effect for Mepe in male mice, with Hyp mice having increased Mepe gene levels (Supplemental Figure 4). No effects were detected for Dmp1 or Opn. Similarly, no treatment or genotype effects were noted for either Galnt3 or Fam20c (Supplemental Figure 5).

Analysis of the gene expression of sodium-phosphate transporters, Npt2a and Npt2c did not show any significant effects (Supplemental Figure 6). Although, there was evidence of a non-significant increased expression of both transporters in Scl-Ab treated female Hyp mice, no similar trend was noted in males.

Discussion

The current study used the growing Hyp mouse model to evaluate the use of Scl-Ab as a treatment option for XLH-related pathologies. Short term treatment with Scl-Ab significantly improved phosphate levels in Hyp mice to near WT levels. Additionally, consistent with the effects of Scl-Ab in other diseases models, we find a significant increase in bone mass in both trabecular and cortical bone compartments of the Hyp mice. More specifically, changes were primarily noted in the periosteal surface where cortical area was restored to WT levels and resulted in improved mechanical properties. These results suggest that Scl-Ab is capable of partially rescuing the disease pathologies associated with XLH and provide evidence for a novel role of sclerostin in the regulation of phosphate metabolism.

XLH is characterized by significant growth inhibition and treatment of children heavily focuses on improving longitudinal growth. Despite an increase in bone mass, Scl-Ab treatment did not improve femoral length or body weight in Hyp animals. However, other studies have found that calcitriol is capable of normalizing body weight and femur length in Hyp mice^(19,20). Therefore, it is possible that combining calcitriol with Scl-Ab treatment, as has been done in Scl-Ab clinical trials for osteoporosis ⁽⁸⁾, could potentially improve growth in XLH. Additionally, Burosumab, a monoclonal antibody to FGF23 protein, has been reported to increase bone length in Hyp mice⁽²¹⁾ and may present another potential option for combinatory therapy with Scl-Ab.

The primary goal of current clinical XLH treatment strategies is to increase circulating phosphate levels⁽⁵⁾. Conventional treatment of XLH includes a combination of calcitriol and phosphate, which is effective at partially alleviating symptoms. However, if not closely monitored, hypercalcemia, hypercalciuria, hyperparathyroidism, or nephrocalcinosis can develop as a direct result of this treatment⁽⁵⁾. One novel alternative treatment strategy that has emerged is anti-FGF23 antibody (Burosumab), which successfully inhibits the action of FGF23 to prevent renal phosphate wasting^(21–24). Herein, we show that Scl-Ab is another potential treatment strategy that increases phosphate. However, future clinical use of Scl-Ab for XLH would need to be balanced against the risk of adverse cardiac events, which has been reported in postmenopausal osteoporotic women ⁽²⁵⁾.

Short-term treatment with Scl-Ab decreased circulating intact (i.e., hormonally active) FGF23 levels, suggesting that sclerostin acts as an upstream regulator of FGF23. This finding is consistent with the finding that circulating intact FGF23 is reduced in sclerostin-null mice⁽¹³⁾. Interestingly, Scl-Ab treatment decreased FGF23 protein levels nearly 30% in both WT and Hyp mice, suggesting that the connection between sclerostin and FGF23 is likely independent of PHEX, which is mutated in the Hyp mice. The regulation of FGF23 within the skeleton is complex and not fully understood, although several mineralization-related genes have been implicated in its regulation ^(26–28). In the current study we investigated the skeletal gene expression of several of these genes, including Dmp1, Opn, and Mepe, but were unable to detect any significant Scl-Ab treatment effects. Future unbiased genomics approaches are likely needed to determine the mechanism by which sclerostin regulates FGF23.

While the circulating intact FGF23 levels were reduced by Scl-Ab treatment, the C-terminal levels were unchanged. As the C-terminal FGF23 assay measures both the intact and cleaved C-terminal form of FGF23, these data point to an increase in the amount of circulating C-terminal FGF23 following Scl-Ab treatment. The cleavage of intact FGF23 is regulated by Galnt3 and Fam20c, which either inhibits or promotes the cleavage of intact FGF23, respectively⁽²⁹⁾. Despite the increased C-terminal FGF23, we did not detect a Scl-Ab treatment induced change in the expression of Galnt3 or Fam20c. It is possible that the protein levels are not reflected in the gene expression levels and future studies will concentrate on investigating the concentration of both proteins following Scl-Ab treatment.

Although Scl-Ab treatment reduced circulating intact FGF23 levels in both WT and Hyp mice, the absolute level of FGF23 protein remained elevated in treated Hyp mice compared to WTs. Despite the persistently high FGF23 levels, circulating phosphate in Scl-Ab treated Hyp mice were near WT levels. It is possible that the increase in C-terminal FGF23 may be serving to antagonize the action of the hormonally active intact FGF23⁽³⁰⁾. FGF23 suppresses renal phosphate reabsorption by reducing the levels of the sodium-phosphate transporters, Npt2a and Npt2c ^(27,31). In the current study, we investigated the renal expression of these two genes, but found no significant treatment effects on gene expression. While the renal expression of sodium-phosphate co-transporter genes have been reported to be responsive to FGF23 ⁽³²⁾, current data points to FGF23-induced destruction the proteins ⁽³³⁾, which may explain why we did not detect a difference at the gene level. Future experiments will aim to investigate the protein level of Npt2a and Npt2c to determine whether the reduction in FGF23 protects these two proteins from degradation. Finally, it is also possible that a concomitant increase in active vitamin D levels increases the amount of intestinal phosphate absorption. Although the current study did not evaluate vitamin D due to limited sera, increased 1,25 dihydroxyvitamin D has been noted in the sclerostin knockout mouse⁽¹³⁾.

Scl-Ab treatment increases bone mass and subsequently, bone strength in several preclinical animal models, including postmenopausal osteoporosis⁽³⁴⁾, osteogenesis imperfecta^(16,35), and autosomal recessive hypophosphatemic rickets⁽¹⁷⁾. Consistent with this, we find that Scl-Ab induces gains in bone mass in Hyp animals with the largest gains in the cortical compartment. Previous studies have demonstrated that Scl-Ab activates bone lining cells on existing bone surfaces, and sustains osteoprogenitor cell recruitment to increase bone mass ^(15,36). It was observed in this study that Hyp mice began at a BV/TV deficit compared to WT mice. Taken together, this may explain the relatively small increase in this parameter with Scl-Ab treatment in the Hyp model.

Poor skeletal mineralization and the accumulation of osteoid (osteomalacia) is a hallmark of XLH. In the current study, suppressing sclerostin via Scl-Ab treatment showed moderate but non-significant improvements in the cortical OS/BS of Hyp mice, without affecting the trabecular surfaces. Interestingly, the osteoid parameters failed to improve despite a restoration of circulating phosphate levels. FGF23 itself has been suggested to directly inhibit bone matrix mineralization^(37–39). Circulating FGF23 levels remained elevated in treated Hyp mice, which may explain why Scl-Ab did not completely restore the mineralization defects in Hyp mice to WT levels.

In the current study we found reduced skeletal Sost expression in Hyp mice, which is consistent with several other publications ^(19,40). However, there have also been reports of increased Sost expression in the bone of Hyp mice^(41,42). The source of this discrepancy is unclear. In addition, we find that despite reduced gene expression, circulating sclerostin levels in Hyp mice are unchanged compared to WT mice. This is inconsistent with the only published study of XLH in humans, in which sclerostin levels were found to be elevated in XLH patients⁽¹⁴⁾. The discrepancy may be a consequence of the relatively young age of the mice used in our study. It is worth noting that other studies have also found that serum levels of sclerostin do not always correlate with Sost skeletal gene expression⁽⁴³⁾, which may reflect distinct paracrine and endocrine functions of sclerostin⁽⁴⁴⁾. After treatment there is an increase in both the gene expression and circulating levels of sclerostin, demonstrating positive feedback in response to the antibody. Despite the relatively modest increase in SOST expression, serum sclerostin levels spiked beyond the detection limit upon Scl-Ab treatment, which may be due to the assay detecting both unbound and antibody-bound sclerostin and the stabilization of bound sclerostin by Scl-Ab ⁽⁴⁵⁾.

Limitations of this study include the use of relatively young, growing animals, where sclerostin levels are expected to be lowest⁽⁴⁴⁾. Despite the low circulating sclerostin levels, Scl-Ab treatment improved several XLH-related pathologies in young Hyp mice. However, it is worth noting that not all of the XLH-related pathologies were improved with treatment, which may indicate that the Scl-Ab dosing used in the current study was not optimal for the treatment of XLH. We based our dosing strategy on previously published studies using various mouse models of metabolic bone diseases, but dosing experiments are warranted to determine whether increasing Scl-Ab treatment can further improve phenotypes in the Hyp mouse model. Although we noted improvements in the circulating phosphate levels following Scl-Ab treatment, we are not able to fully investigate the renal phosphate reabsorption as we did not collect urine samples from these mice. Finally, we assessed intact FGF23 levels in serum samples using the Immutopics assay, which likely resulted in reduced values compared to those measured in plasma samples⁽⁴⁶⁾, however, as all samples were treated the same way, we believe that the comparison between groups represent a true treatment response.

Conclusion

Short term Scl-Ab treatment of Hyp mice corrected some of the pathophysiological changes associated with XLH, including dysregulated circulating phosphate and intact FGF23 levels, and abnormally low bone mass. These findings suggest that Scl-Ab may be a viable treatment option for XLH, and provide additional evidence for a potential role of sclerostin in the regulation of FGF23.

Supplementary Material

Supplemental Figure 2

Supplemental Figure 2. Representative two-dimensional μCT images of female WT and Hyp mice.

Supplemental Figure 3

Supplemental Figure 3. Representative histology demonstrating the extent of osteoid present in WT and Hyp female mice treated with either vehicle or Scl-Ab. Images on the right are magnified cortical regions from the same sample presented on the left.

Supplemental Figure 1

Supplemental Figure 1. Representative two-dimensional μCT images of male WT and Hyp mice.

Supplemental Figure 5

Supplemental Figure 5. Skeletal gene expression of Galnt3 (A), and Fam20C (B) in male (left) and females (right) mice. Data are presented as the mean ± standard deviation. Results from the two-way ANOVA are presented in the figure legends.

Supplemental Figure 6

Supplemental Figure 6. Skeletal gene expression of Npt2a (A), and Npt2c (B) in male (left) and females (right) mice. Data are presented as the mean ± standard deviation. Results from the two-way ANOVA are presented in the figure legends.

Supplemental Figure 4

Supplemental Figure 4. Skeletal gene expression of Dmp1 (A), Opn (B), and Mepe (C) in male (left) and females (right) mice. Data are presented as the mean ± standard deviation. Results from the two-way ANOVA are presented in the figure legends. Significant post-hoc differences between animals of the same genotype are reported above the data with horizontal bars and corresponding p-values. Letters highlight significant post-hoc differences between Hyp vehicle and WT vehicle (a); Hyp vehicle and WT Scl-Ab (b); Hyp Scl-Ab and WT vehicle (c); Hyp Scl-Ab and WT Scl-Ab (d).