X-linked hypophosphatemic rickets (XLH) is characterized by hypo-mineralization of the bone due to hypophosphatemia. XLH is caused by abnormally high levels of fibroblast growth factor 23, which trigger renal phosphate wasting. Activated fibroblast growth factor receptor 3 (FGFR3) signaling is considered to be involved in XLH pathology. Our previous study revealed that meclozine attenuated FGFR3 signaling and promoted longitudinal bone growth in an achondroplasia mouse model. The present study aimed to examine whether meclozine affected the bone phenotype in a mouse model of XLH [X-linked hypophosphatemic (Hyp) mice]. Meclozine was administered orally to 7-day-old Hyp mice for 10 days, after which the mice were subjected to blood sampling and histological analyses of the first coccygeal vertebra, femur and tibia. Villanueva Goldner staining was used to assess bone mineralization, hematoxylin and eosin staining was used to determine the growth plate structure and tartrate-resistant acid phosphatase staining was used to measure osteoclast activity. The osteoid volume/bone volume of cortical bone was lower in meclozine-treated Hyp mice compared with untreated Hyp mice. Meclozine treatment improved the abnormally thick hypertrophic zone of the growth plate and ameliorated the downregulation of osteoclast surface/bone surface in Hyp mice. However, meclozine had only a marginal effect on mineralization in the trabecular bone and on calcium and phosphate plasma levels. A 10-day-tratment with meclozine partially ameliorated bone mineralization in Hyp mice; hence, meclozine could alleviate XLH symptoms.
X-linked hypophosphatemic rickets (XLH) is the most common hereditary hypophosphatemic disorder. Patients with XLH suffer from short stature, deformed lower extremities, dental abnormalities, bone and joint pain, tinnitus, and hearing loss (
Activating mutations in the
Hyp mice were obtained from the Jackson Laboratory. Hyp mice have a large deletion in the 3' untranslated region of the
Hyp mice were divided into meclozine-treated and untreated (vehicle only) groups; whereas WT mice were treated with vehicle only. The dose-finding study using a mouse model indicated that meclozine at 1 and 2 mg/kg/day attenuated FGFR3 signaling in a dose-dependent manner (
After the 10-day-treatment period, mice were subjected to whole blood collection from the abdominal aorta using a 26-gauge needle and 1-ml syringe under general anesthesia with isoflurane. Mice were initially exposed with 3.5 to 4.0% of isoflurane to fall asleep, then the blood correction was performed with keeping exposure with 2 to 3% of isoflurane. Mice were euthanized caused by this exsanguination procedure. Collected blood samples were left at room temperature for more than 3 h to allow clotting, and were thereafter centrifuged and the obtained serum samples were preserved at -20˚C. Serum calcium and phosphate levels were measured using a DRI-CHEM 7000V biochemical analyzer (Fujifilm). Serum FGF23 levels were measured using the FGF-23 ELISA Kit (RRID: AB_2782966; Kainos Laboratories). Serum samples were excluded from the analysis when the amount of blood drawn was insufficient for measurement, hemolysis was obvious, the blood vessel was damaged, or the body fluid in the abdominal cavity was contaminated.
Following treatment, mice were subjected to histological analysis of the first coccygeal vertebra, femur, and tibia. These samples were collected immediately after euthanasia and fixed with 4% paraformaldehyde at 4˚C. The samples of the first coccygeal vertebra were cut in the sagittal plane, while those of the femur and tibia were cut in the coronal plane; thin sections were stained with Villanueva Goldner (Kureha Special Laboratory). The medial mid-shaft cortical bone of the femur, the central part of the distal metaphyseal cancellous bone (primary spongiosa) of the femur, and the proximal central part of the first coccygeal vertebra were identified in low-magnification images. Osteoid volume (OV) and bone volume (BV) were measured using Image J software, and the OV/BV served as an index of bone mineralization (
Histological and serum parameters were normalized to those of untreated littermate Hyp mice to adjust for environmental differences such as litter skeletal size among kinship. All data are expressed as the mean ± SD. Outliers were removed from analysis using the interquartile range method (
The OV of the femoral and tibial cortical bones, which was higher in untreated Hyp mice than WT mice, decreased after treatment with 2 mg/kg/day of meclozine (
The Hz of the growth plate in the distal femur and proximal tibia was markedly thickened and presented with an increased cell size in untreated Hyp mice (
In the trabecular bone, untreated Hyp mice presented larger OV and smaller BV than WT mice in the distal femur (
Serum calcium and phosphate levels were significantly higher in WT mice than in untreated Hyp mice (
Burosumab, a recombinant human monoclonal antibody that targets FGF23, remains very expensive to administer to all patients with XLH. Additionally, some children fear the burosumab administration via injection, while oral administration of sodium phosphate does not always achieve the correct dosage due to increased renal phosphate wasting (
Histological analyses demonstrated that meclozine decreased OV in both cortical and cancellous bones, and realigned the growth plate structure of Hyp mice. Similar findings have previously been reported after treatment of Hyp mice with other FGFR3 inhibitors, such as NVP-BGJ398 and MAPK inhibitors (
One-week treatment with a MAPK inhibitor was depicted to reduce serum FGF23 levels in Hyp mice (
The current study has several limitations. First, we administered 2 mg/kg/day of meclozine to 7-day-old Hyp mice for 10 days, thus mimicking the protocol used for achondroplasia mice (
In conclusion, repeated administration of meclozine for 10 days partially ameliorated bone quality and growth plate structure in Hyp mice, probably due to inhibition of FGFR3 signaling. Thus, this mechanism successfully suppressed pathological phenotypes in mouse models of both achondroplasia and hypophosphatemic rickets.
The authors would like to thank Mr. Ryusaku Esaki (Department of Orthopaedic Surgery, Nagoya University Graduate School of Medicine, Nagoya, Aichi, Japan) for technical assistance for animal studies and Professor Tamio Ohno (Division of Experimental Animals, Nagoya University Graduate School of Medicine, Nagoya, Aichi, Japan) for technical support.
The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.
YK, MM and HK designed the study. YK and KM acquired the data. YK, BO, TM, SI and KO analyzed and interpreted the data. YK and MM drafted the paper, while TM, SI, KO and HK revised it critically. YK and MM confirm the authenticity of all the raw data. All authors read and approved the final manuscript.
All animal care and experiments conformed to the institutional guidelines of Nagoya University, and all experimental protocols were approved by the Institutional Animal Care and Use Committee of Nagoya University (approval no. M210231; Nagoya, Aichi, Japan).
Not applicable.
The authors declare that they have no competing interests.
Meclozine rescues impaired cortical bone mineralization in Hyp mice. Representative non-decalcified histological images of the (A) femur and (B) tibia stained with Villanueva Goldner. The red and green signals indicate osteoid and bone tissues, respectively. The osteoid signal at the medial mid-shaft cortical bone was lower in meclozine-treated Hyp mice compared with untreated Hyp mice. Scale bars, 100 µm. (C) OV/BV. Dots indicate the OV/BV of each sample, and bars indicate the mean ± SD. Statistical significance was analyzed using one-way ANOVA with or without Welch's correction. *P<0.05, **P<0.01. Hyp mice, X-linked hypophosphatemic mice; OV/BV, osteoid volume/bone volume.
Meclozine improves the structure of the growth plate in Hyp mice. Representative histological images of the (A) distal femoral growth plate and (C) proximal tibial growth plate indicating the width of proliferative and hypertrophic zones in meclozine-treated Hyp mice and untreated Hyp mice. Scale bars, 100 µm. %Hz and %Pz in the (B) distal femur and (D) proximal tibia. Dots indicate the relative %Hz and %Pz of each sample, and bars indicate the mean ± SD. Statistical significance was analyzed using one-way ANOVA with or without Welch's correction. **P<0.01. Hyp mice, X-linked hypophosphatemic mice; %Hz, percentage hypertrophic zone thickness; %Pz, percentage proliferative zone thickness.
Meclozine slightly augments the surface of osteoclasts in Hyp mice. (A) Representative histological images of distal femoral metaphysis stained with tartrate-resistant acid phosphatase in meclozine-treated and untreated Hyp mice. The purple signal indicates osteoclasts. Scale bars, 100 µm. (B) Oc.S and Oc.S/BS. Dots indicate the Oc.S and the Oc.S/BS of each sample, and bars indicate the mean ± SD. Statistical significance was analyzed by one-way ANOVA with post-hoc Tukey's honest significance test. *P<0.05. Hyp mice, X-linked hypophosphatemic mice; Oc.S, osteoclast surface; Oc.S/BS, osteoclast surface/bone surface.
Meclozine partially rescues impaired mineralization of cancellous bone in Hyp mice. Representative non-decalcified histological images of the (A) distal trabecular bone of the femur, (B) proximal trabecular bone of the tibia and (C) proximal trabecular bone of the first tail vertebrae stained with Villanueva Goldner. The red and green signals indicate osteoid and bone tissue, respectively. Scale bars, 100 µm. (D) OV/BV in the tail, femoral and tibial bones. Dots indicate the OV/BV of each sample, and bars indicate the mean ± SD. Statistical significance was analyzed using one-way ANOVA with or without Welch's correction. **P<0.01. Hyp mice, X-linked hypophosphatemic mice; OV/BV, osteoid volume/bone volume.
Meclozine tends to augment serum calcium, phosphate and FGF23 levels in Hyp mice. Relative serum (A) calcium, (B) phosphate and (C) FGF23 levels in meclozine-treated or untreated Hyp mice. Dots indicate the relative serum parameters of each sample, and bars indicate the mean ± SD. Statistical significance was analyzed using one-way ANOVA with post-hoc Tukey's honest significance test. *P<0.05, **P<0.01. Hyp mice, X-linked hypophosphatemic mice; FGF23, fibroblast growth factor 23.