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Keywords:

  • FIBROBLAST GROWTH FACTOR 23;
  • X-LINKED HYPOPHOSPHATEMIA;
  • PHOSPHATONIN;
  • NEUTRALIZING ANTIBODY

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References

Fibroblast growth factor 23 (FGF-23) plays causative roles in the development of several hypophosphatemic rickets/osteomalacia such as X-linked hypophosphatemic rickets/osteomalacia (XLH) and tumor-induced rickets/osteomalacia. Patients with hypophosphatemic rickets/osteomalacia often complain of muscle weakness and bone pain that severely affect daily activities of these patients. The purpose of this study was to examine whether anti-FGF-23 antibodies, which have been shown to improve hypophosphatemia and rachitic changes of juvenile Hyp mice in a murine model of XLH, also ameliorate hypophosphatemic osteomalacia and affect muscle force and spontaneous motor activity in adult Hyp mice. Repeated injections of anti-FGF-23 antibodies increased serum phosphate and 1,25-dihydroxyvitmain D levels and enhanced mineralization of osteoid in adult Hyp mice, whereas bone length did not change. We found that grip strength was weaker and that spontaneous movement was less in adult Hyp mice than in wild-type mice. In addition, FGF-23 antibodies increased grip strength and spontaneous movement. These results suggest that the inhibition of excess FGF-23 action not only ameliorates hypophosphatemia and impaired mineralization of bone but also improves muscle weakness and daily activities of patients with FGF-23-related hypophosphatemic rickets/osteomalacia. © 2011 American Society for Bone and Mineral Research.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References

Rickets and osteomalacia are diseases characterized by impaired mineralization of bone matrix. While there are many causes of rickets and osteomalacia, chronic hypophosphatemia is present in most cases of rickets/osteomalacia. Patients with hypophosphatemic rickets/osteomalacia often complain of muscle weakness and bone pain that deeply affect daily activities of the affected subjects.1–4 Fibroblast growth factor 23 (FGF-23) has been shown not only to be a physiologic regulator of serum phosphate and 1,25-dihydroxyvitmain D [1,25(OH)2D] levels but also to play important pathophysiologic roles in the development of several disorders of phosphate and vitamin D metabolism.5, 6 FGF-23 works to reduce serum phosphate and 1,25(OH)2D levels on renal proximal tubules. Since it is a physiologic hormone, appropriate action of FGF-23 is necessary to keep serum phosphate and 1,25(OH)2D within reference ranges because Fgf23 knockout mice showed hyperphosphatemia and high 1,25(OH)2D levels.7 However, excess actions of FGF-23 cause hypophosphatemic rickets/osteomalacia, including X-linked hypophosphatemic rickets/osteomalacia (XLH),8, 9 autosomal dominant and recessive hypophosphatemic rickets/osteomalacia (ADHR and ARHR),10–12 tumor-induced rickets/osteomalacia (TIO),8, 9 hypophosphatemic rickets/osteomalacia associated with McCune-Albright syndrome/fibrous dysplasia,13, 14 and hypophosphatemic diseases caused by intravenous administration of iron polymaltose.15, 16 Hypophosphatemic rickets/osteomalacia usually has been treated by oral phosphate and active vitamin D3.1 However, these medications sometimes cause adverse events, including diarrhea and secondary or tertiary hyperparathyroidism.17, 18

After the cloning of Fgf23, we have obtained several kinds of anti-FGF-23 antibodies. Using these antibodies, we have developed a sandwich enzyme-linked immunosorbent assay (ELISA) for FGF-23.8 In addition, some of these antibodies were found to antagonize the endogenous actions of FGF-23. Actually, injections of some anti-FGF-23 antibodies increased serum phosphate and 1,25(OH)2D levels in wild-type mice, confirming that FGF-23 is a physiologic humoral factor.19 Furthermore, we have reported recently that anti-FGF-23 neutralizing antibodies increase serum phosphate and 1,25(OH)2D levels and ameliorate rachitic bone of juvenile Hyp mice, murine homologues of XLH.20 These results suggested that the inhibition of circulating FGF-23 activity is promising as a new therapeutic tool for diseases caused by excess actions of this hormone. However, it is unknown whether the muscle weakness and bone pain observed in patients with XLH also improve with inhibition of FGF-23 activity. Furthermore, it is not clear whether the suppression of FGF-23 activity ameliorates impaired mineralization in adults either. In this study, the effects of anti-FGF-23 antibodies were examined in adult Hyp mice to address these questions.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References

Antibodies

Mouse monoclonal antibodies (IgG1) FN1 and FC1 were prepared as described previously.19, 20 In this study, we used a 1:1 mixture of FN1 and FC1 denoted as FGF-23Ab. As an isotype-matched control antibody (control Ab), we used anti-human thrombopoietin (TPO) mouse monoclonal antibody (IgG1), which has no cross-reactivity to murine TPO.19, 20

Mice

We used male Hyp/Y and age-matched normal littermates, which were obtained by breeding male C57BL/6J mice and female Hyp/X mice originally purchased from Jackson Laboratory (Bar Harbor, ME, USA). The Hyp mouse is a murine homologue of XLH with a large deletion in the 3' region of Phosphate-regulating gene with homologies to endopeptidases on the X chromosome.21 All mice were maintained under standardized conditions with an artificial 12 hours dark/light cycle, fed with standard rodent chow CE-2 (Clea, Tokyo, Japan), and tap water ad libitum. All animal studies were reviewed and approved by the institutional animal care and use committee at the Fuji Research Park, Kyowa Hakko Kirin Co., Ltd.

Study design

In the study of single injections, FGF-23Ab was administered subcutaneously to male Hyp mice (16 to 18 weeks of age) at 4 mg/kg (2 mg/kg of FN1 and 2 mg/kg of FC1) or 16 mg/kg (8 mg/kg of FN1 and 8 mg/kg of FC1) as a bolus injection. As a control, 16 mg/kg of control Ab was injected into wild-type or Hyp mice. To monitor the time course of changes in serum phosphate and 1,25(OH)2D after the single injection of the antibodies, mice were euthanized on days 1, 4, 7, and 14 after dosing to collect blood samples from the hearts. In the study of repeated injections, FGF-23Ab (4 or 16 mg/kg) or control Ab (16 mg/kg) was administered subcutaneously into Hyp or normal littermates at 21 weeks of age (day 0), followed by an additional seven injections on days 7, 14, 21, 28, 35, 42, and 49. Mice were euthanized on day 56 to collect blood samples from the hearts as well as bones and quadriceps muscles.

Serum and urine parameters

Serum samples were prepared in MicroTainer (BD Biosciences, San Jose, CA, USA). Serum and urinary concentrations of phosphate and creatinine for the single-injection study were determined by an autoanalyzer (Hitachi, Tokyo, Japan). Serum phosphate levels for the repeated-injection study were measured manually using Phosphor C Test Wako Kit (Wako, Tokyo, Japan). Serum calcium was assayed by Calcium C Test Wako Kit (Wako). 1,25(OH)2D and FGF-23 levels were measured by 1,25(OH)2D RIA Kit (TFB Inc., Tokyo, Japan) and FGF-23 ELISA (Kainos Laboratories Inc., Tokyo, Japan), respectively. Intact parathyroid hormone (PTH) levels were determined by Mouse Intact-PTH ELISA Kit (Immutopics Inc., Burlington, Ontario, Canada).

Histologic analyses of bone

Soft X-ray radiographs of femurs were obtained by µFX-1000 (Fujifilm, Tokyo, Japan). The tibias were dried at 100 °C for 48 hours and ashed at 600 °C for 24 hours to determine the bone mineral content. Bone ash content was calculated as a percentage of ash weight to dry tissue weight. Femurs were fixed in 70% ethanol and stained with Villanueva bone stain. Then the tissues were embedded in methyl methacrylate, sectioned at 5 µm, and stained with Villanueva Goldner stain.

Measurement of grip strength and muscle weight

Grip strength of forelimbs and hind limbs was determined using a Grip Strength Meter MK-380 (Muromachi Kikai, Inc., Tokyo, Japan).22, 23 The mice were placed on a metal mesh (mesh dimensions 180 mm wide × 120 mm deep, 6-mm intervals) and allowed to grasp the wire gauzes by the forelimbs and hind limbs. Then the tail was pulled backward horizontally until the mice released the gauzes so that the maximal force was recorded. Three such trials were undertaken for each mouse, and these trials were repeated four times (on days 0, 14, 35, and 54). The grip strength was normalized by body weight. Right quadriceps weight also was evaluated after adjustment by body weight.

Histologic analyses of quadriceps

Removed left quadriceps muscles were embedded in Tissue-TEK O.T.C. compound (Sakura Finetek, Tokyo, Japan), rapidly frozen in liquid nitrogen, and then cross-sectioned by 5 µm and fixed in acetone. Quadriceps tissue sections were subjected to hematoxylin and eosin staining for calculations of the cross-sectional area of myofiber and the number of myofibers per given area.

Measurement of spontaneous movement

Spontaneous movement was measured by Supermex (Muromachi Kikai, Tokyo, Japan).24 The Supermex has a thermosensor that was mounted above the cage to detect changes in heat across multiple zones of the cage through an array of Fresnel lenses. The body heat radiated by the animal was detected with paired infrared pyroelectric detectors on the sensor head of the monitor. In this way, the system could monitor and count every spontaneous movement, including locomotion, rearing, movement of the head, and so on. Each animal was prehoused individually in the experimental cage with freely available food and water for 24 hours to be accustomed to this experimental condition, and spontaneous activity then was measured during the nocturnal period for 12 hours. All counts were totaled and recorded automatically every 10 minutes.

Statistical analysis

Statistical significance compared with wild-type mice was determined by one-way ANOVA followed by Dunnett's test. Dose-related statistical significance was evaluated by one-way ANOVA followed by William's test. All statistical analyses of data were performed using SAS software (Release 9.1.3, SAS Institute, Inc., Cary, NC, USA). Significance for all tests was set at p < .05. Data are presented as means ± SEM.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References

Effects of anti-FGF-23 antibodies in adult Hyp mice

We have shown previously that a single injection of FGF-23Ab increased serum phosphate and 1,25(OH)2D in juvenile Hyp mice.20 Circulatory FGF-23 level was high in Hyp mice, and there were no age-dependent changes of FGF-23 levels (96 ± 20 pg/mL in wild-type mice and 1578 ± 798 pg/mL in Hyp mice at 4 weeks of age and 76 ± 22 pg/mL in wild-type mice and 1665 ± 356 pg/mL in Hyp mice at 20 weeks of age, mean ± SE, n = 20, 101, 22, and 113, respectively). These results suggested that FGF-23Ab is effective not only in juvenile but also in adult Hyp mice. To confirm the effectiveness of FGF-23Ab in adult Hyp mice, we first conducted a study of single injections. Similarly, serum phosphate and 1,25(OH)2D increased in a dose-dependent manner in adult Hyp mice when FGF-23Ab was injected (Fig. 1). Since fractional excretion of phosphate, which is known to be significantly elevated in Hyp mice, was completely normalized by an injection of FGF-23Ab (wild type 36.8% ± 8.3%; Hyp + control Ab 89.5% ± 20.6%; Hyp + FGF-23Ab 37.1% ± 6.2%, n = 9 each), the increase in serum phosphate was thought to be derived at least in part from enhanced renal phosphate reabsorption. These results indicate that the basic abnormality of high FGF-23 is persistent throughout life in Hyp mice and that FGF-23Ab effectively inhibited the activity of endogenous FGF-23 in Hyp mice.

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Figure 1. Time course of changes in serum phosphate (A) and 1,25(OH)2D (B) levels after a single injection of FGF-23Ab. The numbers in parentheses indicate the doses of FGF-23Ab given (in mg/kg). Each datum represents mean ± SE (n = 6). Statistical analysis was conducted for the results at each time point by Dunnett's method, ap < .05 and bp < .01 versus wild-type control Ab, and Williams' method, cp < .01 versus Hyp control Ab.

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In juvenile Hyp mice, once-weekly injections of FGF-23Ab enhanced longitudinal growth of long bones, increased bone mineral density (BMD), and corrected impaired mineralization of bone.20 In order to examine whether similar changes are observed in adult Hyp mice, FGF-23Ab was administered once weekly for 8 weeks to adult Hyp mice. Again, serum phosphate level was increased by FGF-23Ab administration at 24 hours after the eighth injection (day 50; Table 1). Serum calcium and 1,25(OH)2D levels also were increased by FGF-23Ab administration (Table 1). Repeated injections of FGF-23Ab did not change the intact PTH levels in Hyp mice (Table 1). In contrast to juvenile Hyp mice, the lengths of long bones in Hyp mice treated with FGF-23Ab did not differ from those of Hyp mice that had received control Ab (Fig. 2B). In addition, FGF-23Ab did not change the contour of femoral bones in Hyp mice (Fig. 2A). The body weights of Hyp mice injected with control Ab clearly were less than those of wild-type mice with the same antibody (Table 1). Repeated injections of FGF-23Ab increased the body weights of adult Hyp mice. However, the body weights of Hyp mice treated with FGF-23Ab were significantly less than those of wild-type mice throughout the experiment. Tibial ash content also increased as a result of the injections of FGF-23Ab, although it did not reach the level of wild-type mice (Fig. 2C). Histologically, increased osteoid in untreated Hyp mice decreased by FGF-23Ab. Osteoid volume of the treated mice was almost similar to that of control mice (Fig. 3). FGF-23Ab also increased calvarial bone ash content (% dry weight) in FGF-23Ab-treated Hyp mice, indicating that treatment with FGF-23Ab also had an ameliorative effect on intramembranous ossification (66.3% ± 3.2% in control Ab-treated wild-type mice, 52.2% ± 0.5% in control Ab-treated Hyp mice, 60.2% ± 1.1% in 4 mg/kg of FGF-23Ab-treated Hyp mice, and 64.7% ± 0.5% in 16 mg/kg of FGF-23Ab-treated Hyp mice, mean ± SE, n = 9, 10, 10, and 10, respectively). Therefore, while FGF-23Ab did not correct short bones of adult Hyp mice, it improved impaired mineralization, as in juvenile mice.

Table 1. Serum Parameters and Growth After Repeated Injections of FGF-23Ab in Adult Mice
 Wild typeHyp
 Control Ab (16) (n = 9)Control Ab (16) (n = 10)FGF-23Ab (4) (n = 10)FGF-23Ab (16) (n = 10)
  • Note: The serum samples were collected 1 and 7* days after the eighth dosing (days 50 and 56*). The numbers in parentheses indicate the doses of FGF-23Ab (in mg/kg). Results are mean ± SE (n = 9 to 10). Δ Body weight represents body weight gain from day 0 to day 56.

  • a, a,d

    p < .05 and

  • b, b,d

    p < .01 versus wild-type mice treated with control Ab (Dunnett's test).

  • c

    p < .05 and

  • d

    p < .01 versus Hyp mice treated with control Ab (Williams' test).

Serum P (mg/dL)5.4 ± 0.32.3 ± 0.1b5.6 ± 0.2d7.3 ± 0.2b,d
Serum Ca (mg/dL)9.4 ± 0.18.9 ± 0.1a9.6 ± 0.1d9.9 ± 0.2a,d
Serum 1,25(OH)2D (pg/mL)172 ± 6177 ± 10660 ± 27b,d474 ± 29b,d
Serum PTH (pg/mL)*20.4 ± 3.537.8 ± 8.850.8 ± 7.325.8 ± 7.7
Body weight (g)35.5 ± 1.324.4 ± 0.7b26.8 ± 0.4b,c27.9 ± 0.7b,d
Δ Body weight (g)1.0 ± 0.50.3 ± 0.33.2 ± 0.4b,d4.1 ± 0.2b,d
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Figure 2. Soft X-ray radiograph (A), longitudinal length of femoral bone (B), and tibial ash weight (C) isolated on day 56. The results represent mean ± SE (n = 9 to 10). ap < .01 versus wild-type mice treated with control Ab (Dunnett's test). bp < .01 versus Hyp mice treated with control Ab (Williams' test).

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Figure 3. Histologic analysis of femoral bone on day 56. Undecalcified sections visualized by Villanueva-Goldner stain (green = mineralized bone; red = unmineralized osteoid) with counterstain by hematoxylin (violet = growth plate cartilage). (A) Distal metaphysis (magnification × 40). (B) Cancellous bone (× 400). (C) Cortical bone (×400).

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Effects of FGF-23Ab on grip strength

Muscle weakness is one of symptoms associated with hypophosphatemic rickets/osteomalacia and is very important clinically in the management of hypophosphatemic patients.4 However, muscle weakness has not been well addressed in the murine model of hypophosphatemia. Many studies have revealed that a measurement of grip strength is a convincing method for assessing muscle strength in vivo.22, 23, 25, 26 Therefore, in this study we measured grip strength as described under “Materials and Methods.” Because there is positive correlation between body weight and grip strength, the data on maximum grip strength divided by body weight were evaluated.27 Grip strength of Hyp mice clearly was lower than that of wild-type control mice (Fig. 4A). However, treatment with FGF-23Ab gradually increased grip strength in Hyp mice. On day 35, grip strength of treated Hyp mice was not different from that of wild-type control mice. In order to clarify whether this improvement in grip strength derived from the increase in muscle mass, we measured quadriceps weight. However, FGF-23Ab did not increase muscle weight in Hyp mice (Fig. 4B). In contrast, repeated injections of FGF-23Ab did not affect grip strength in wild-type mice (data not shown). There was no statistical difference in the thickness and number of myofibers of the quadriceps between wild-type and Hyp mice (Table 2). In addition, FGF-23Ab did not change the number and thickness of myofibers in Hyp mice (Table 2).

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Figure 4. Maximal grip strength and quadriceps muscle weight. (A) Time course of changes in maximal grip strength per body weight during treatment with FGF-23Ab. All results represent means ± SE (n = 9 to 10). Statistical analysis was conducted for the results at each time point by Dunnett's method, ap < .05 and bp < .01 versus wild-type mice treated with control Ab, and Williams' method, cp < .01 versus Hyp mice treated with control Ab. (B) Weight (mg) of quadriceps femoris isolated on day 56 adjusted for body weight (g). Quadriceps weight of Hyp mice treated with either FGF-23Ab or control Ab was statistically lower than that of wild-type mice (ap < .01, Dunnett's test).

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Table 2. Histologic Analysis of Quadriceps Muscle
 Wild typeHyp
Control Ab (16) (n = 9)Control Ab (16) (n = 10)FGF-23Ab (4)FGF-23Ab (16)
  1. Note: The numbers in parentheses indicate the doses of FGF-23Ab (in mg/kg). Results are means ± SE (n = 9 to 10).

Cross-sectional area of myofiber (µm2)614 ± 45450 ± 39503 ± 53498 ± 33
Number of myofibers per given area45.8 ± 2.359.3 ± 3.254.7 ± 3.954.3 ± 2.6

Effects of FGF-23Ab on spontaneous movement

Muscle weakness and bone pain impair the daily activities of patients with hypophosphatemic rickets/osteomalacia. While it is difficult to analyze daily activity in murine models, we hypothesized that muscle weakness and pain might affect spontaneous movements. From preclinical studies, pain is known to depress spontaneous behaviors such as locomotion, exploration, and rearing and enhance noxious stimulus–related withdrawal responses such as writhing and flinching.28–32 Moreover, many analgesic treatments restore suppressed spontaneous behaviors along with providing pain relief.29–31 Therefore, we recorded spontaneous movement in Hyp and wild-type mice during the nighttime. As shown in Fig. 5, the frequency of spontaneous movement was higher during the first several hours of the dark period and decreased thereafter in all groups (Fig. 5A). Figure 5B shows the average spontaneous movement of each group. The frequency of spontaneous movement of Hyp mice was significantly less than that of wild-type mice. However, treatment with FGF-23Ab for 8 weeks increased the frequency of spontaneous movement in a dose-dependent manner. In contrast, a single injection of FGF-23Ab did not affect spontaneous movement in Hyp mice, whereas this treatment resulted in higher serum phosphate and 1,25(OH)2D levels than in wild-type mice (Fig. 1; data not shown). In addition, repeated injections of FGF-23Ab did not affect spontaneous movement in wild-type mice either (data not shown).

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Figure 5. Time course of changes in spontaneous motor activity (count) recorded at 10-minute intervals (A) and total counts of spontaneous movement (B) during 12 hours in the dark phase counted on day 52. The results represent mean ± SE (n = 9 to 10). ap < .01 versus wild-type mice treated with control Ab (Dunnett's test). bp < .05 versus Hyp mice treated with control Ab (Williams' test).

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References

Several results indicate that hypophosphatemic rickets/osteomalacia in Hyp mice is caused by overproduction of FGF-23 in bone.33 Actually, we have reported that the inhibition of excess activity of FGF-23 by FGF-23Ab corrects hypophosphatemia and impaired renal tubular phosphate reabsorption and mineralization and enhances longitudinal growth of long bones of 4-week-old Hyp mice.20 These results indicated that inhibition of FGF-23 activity is promising as a novel therapy for hypophosphatemia in children caused by excess action of FGF-23. In contrast, there is no consensus about therapy for adult patients with XLH. While phosphate and active vitamin D3 were reported to improve symptoms and histologic changes in adult XLH patients,1 it is not clear whether these treatments are necessary for all patients. In this study, we used Hyp and wild-type mice that were 21 weeks of age. These mice showed only marginal body weight gain during the experimental period of 8 weeks (Table 1), indicating that these animals were not in a growth stage. Indeed, morphologic abnormalities in the growth plates of Hyp mice were not completely reversed, and elongation of the long bones, as seen in juvenile Hyp mice, was not achieved by treatment with FGF-23Ab. These results may be explained either by the late start of the treatment to prevent irreversible changes or the short duration of treatment. In contrast, we have shown that FGF-23Ab corrected hypophosphatemia and improved impaired mineralization in adult Hyp mice. These observations may provide important insights in considering a clinical use of FGF-23Ab in adult XLH patients; that is, while the therapeutic effect of FGF-23Ab on bone growth may be limited, it would have a significant impact on improvement of the bone mineralization defect.

Patients with hypophosphatemic rickets/osteomalacia often complain of muscle weakness and bone pain that severely impair their quality of life.1–4 Several mechanisms, such as depletion of ATP34, 35 and phosphodiesters,36 have been proposed for this muscle weakness associated with chronic hypophosphatemia. While FGF-23Ab improves hypophosphatemia and rachitic changes of bone, it has been unclear whether inhibition of FGF-23 activity results in recovery from muscle weakness. In this study, we used grip strength as a marker of muscle power. Previous studies indicated that grip power of the forelimb reflects muscle strength of isolated muscle, thus validating the use of grip power.25, 26 Grip strength in Hyp mice clearly was weaker than that in wild-type mice even after correction by body weight. In addition, FGF-23Ab gradually improved this reduced grip strength. However, muscle weight did not change with FGF-23Ab administration. While muscle weight of only quadriceps was measured, it is unlikely that FGF-23Ab affected muscle weight in other regions. Therefore, we assume that the effects of FGF-23Ab seen in the quadriceps properly represents the effects in other muscles as well. Furthermore, histologic analysis indicated that FGF-23Ab did not increase the number and thickness of myofibers in Hyp mice (Table 2). These results indicate that muscle weakness is caused by chronic hypophosphatemia and that inhibition of FGF-23 activity increases contractile power by each myofiber without causing muscular hypertrophy.

It is reported that the frequency of spontaneous movement is reduced in animal models of pain,28–31 motor disability,37 and several neurogenic and mental disorders.38, 39 In this study, we discovered that Hyp mice move less than wild-type mice. It is not clear what this reduction in the frequency of spontaneous movement actually represents. However, repeated injections, but not a single injection, of FGF-23Ab increased the frequency of spontaneous movement. Therefore, we speculate that muscle weakness and pain in Hyp mice caused by the actions of chronically excess FGF-23 may have affected spontaneous movement.

This study did not directly address the underlying mechanism by which FGF-23Ab ameliorated muscle weakness and decreased spontaneous activity in Hyp mice. We observed a reduction in grip strength and spontaneous movement in Fgf23 transgenic mice as well (data not shown), indicating that these defects are not specific to Hyp mice but can be seen in mice with FGF-23-related hypophosphatemia. However, patients with hypophosphatemic rickets/osteomalacia as a result of Fanconi syndrome and vitamin D deficiency also complain of muscle weakness and bone pain. In contrast to patients with XLH, FGF-23 levels in these hypophosphatemic diseases are rather low. Furthermore, Klotho, which is an essential component of the FGF-23 receptor complex, is not expressed in muscle or bone.40 These results indicate that it is unlikely that FGF-23 has a direct role in these organs. While it is possible that increased 1,25(OH)2D in treated Hyp mice may have affected muscle strength and movement in addition to the increase in serum phosphate and calcium, muscle weakness is reported in patients with hereditary hypophosphatemic rickets with hypercalciuria who have high 1,25(OH)2D levels.3

Therefore, it is more likely that FGF-23Ab improved muscle power and spontaneous movement at least in part by increasing the serum phosphate level. This is also compatible with our preliminary results in hypophosphatemic mice induced by a low-phosphate diet. In this study, we fed normal adult mice (male C57BL/6J, 20 weeks of age) with a normal (0.9%) or low-phosphate (0.1%) diet for 3 weeks (n = 10 each). The low-phosphate diet caused significant hypophosphatemia (2.9 ± 0.1 mg/dL versus 4.8 ± 0.1 mg/dL by Phosphor C Test Wako, p < .001) accompanied by a 20% reduction in grip strength/body weight (7.6 ± 0.2 g/g versus 9.4 ± 0.4 g/g, p < .01). In addition, a recent report showed that normalization of serum phosphate levels improved muscle weakness in vitamin D–deficient rats.41 However, there was no significant correlation between grip strength and serum phosphate or FGF-23 level in untreated Hyp mice (data not shown). In addition, there is no report showing the correlation between serum phosphate level and muscle power in various hypophosphatemic diseases caused by excess FGF-23 action. It is possible that there is a threshold in serum phosphate level to prevent the development of muscle weakness. Otherwise, serum phosphate level may not correctly reflect intracellular phosphate level in muscle. Nonetheless, these findings provide an additional insight into the relationship between phosphate metabolism and muscle power that would be more important in patients with hypophosphatemic conditions.

In summary, FGF-23Ab improved the hypophosphatemia and impaired mineralization of adult Hyp mice. In addition, FGF-23Ab also increased muscle strength and frequency of spontaneous movement. While further study is necessary to determine whether these findings in mice can be applied to human, these results suggest that the inhibition of excess FGF-23 activity is useful for the improvement of not only biochemical, morphologic, and histologic changes but also of symptoms and quality of life in patients with FGF-23-related hypophosphatemia.

Disclosures

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References

YA, HH, YY, TS, and TY are employees of Kyowa Hakko Kirin Company, Ltd. SF serves as a consultant for Kyowa Hakko Kirin Company, Ltd.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References

We thank K Ono, J Murakami, and N Yoshii for excellent technical assistance and Drs M Wada, K Yao, T Sakai, I Urakawa, T Kawata, J Yasutake, T Kawakami, and T Sudo for fruitful discussion. This work was supported in part by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References