FGF23 Is Not Required to Regulate Fetal Phosphorus Metabolism but Exerts Effects Within 12 Hours After Birth.

Loss of fibroblast growth factor-23 (FGF23) causes hyperphosphatemia, extraskeletal calcifications, and early mortality; excess FGF23 causes hypophosphatemia with rickets or osteomalacia. However, FGF23 may not be important during fetal development. FGF23 deficiency (Fgf23 null) and FGF23 excess (Phex null) did not alter fetal phosphorus or skeletal parameters. In this study, we further tested our hypothesis that FGF23 is not essential for fetal phosphorus regulation but becomes important after birth. Although coreceptor Klotho null adults have extremely high FGF23 concentrations, intact FGF23 was normal in Klotho null fetuses, as were fetal phosphorus and skeletal parameters and placental and renal expression of FGF23 target genes. Pth/Fgf23 double mutants had the same elevation in serum phosphorus as Pth null fetuses, as compared with normal serum phosphorus in Fgf23 nulls. We examined the postnatal time courses of Fgf23 null, Klotho null, and Phex null mice. Fgf23 nulls and Klotho nulls were normal at birth, but developed hyperphosphatemia, increased renal expression of NaPi2a and NaPi2c, and reduced renal phosphorus excretion between 5 and 7 days after birth. Parathyroid hormone remained normal. In contrast, excess FGF23 exerted effects in Phex null males within 12 hours after birth, with the development of hypophosphatemia, reduced renal expression of NaPi2a and NaPi2c, and increased renal phosphorus excretion. In conclusion, although FGF23 is present in the fetal circulation at levels that may equal adult values, and there is robust expression of FGF23 target genes in placenta and fetal kidneys, FGF23 itself is not an important regulator of fetal phosphorous metabolism.

CYP24 inhibition as a therapeutic target in FGF23-mediated renal phosphate wasting disorders.

CYP24A1 (hereafter referred to as CYP24) enzymatic activity is pivotal in the inactivation of vitamin D metabolites. Basal renal and extrarenal CYP24 is usually low but is highly induced by its substrate 1,25-dihydroxyvitamin D. Unbalanced high and/or long-lasting CYP24 expression has been proposed to underlie diseases like chronic kidney disease, cancers, and psoriasis that otherwise should favorably respond to supplemental vitamin D. Using genetically modified mice, we have shown that renal phosphate wasting hypophosphatemic states arising from high levels of fibroblast growth factor 23 (FGF23) are also associated with increased renal Cyp24 expression, suggesting that elevated CYP24 activity is pivotal to the pathophysiology of these disorders. We therefore crossed 2 mouse strains, each with distinct etiology for high levels of circulating FGF23, onto a Cyp24-null background. Specifically, we evaluated Cyp24 deficiency in Hyp mice, the murine homolog of X-linked dominant hypophosphatemic rickets, and transgenic mice that overexpress a mutant FGF23 (FGF23R176Q) that is associated with the autosomal dominant form of hypophosphatemic rickets. Loss of Cyp24 in these murine models of human disease resulted in near-complete recovery of rachitic/osteomalacic bony abnormalities in the absence of any improvement in the serum biochemical profile. Moreover, treatment of Hyp and FGF23R1760-transgenic mice with the CYP24 inhibitor CTA102 also ameliorated their rachitic bones. Our results link CYP24 activity to the pathophysiology of FGF23-dependent renal phosphate wasting states and implicate pharmacologic CYP24 inhibition as a therapeutic adjunct for their treatment.

Neither absence nor excess of FGF23 disturbs murine fetal-placental phosphorus homeostasis or prenatal skeletal development and mineralization.

Fibroblast growth factor-23 (FGF23) controls serum phosphorus largely through actions on the kidneys to excrete phosphorus and reduce calcitriol. Although these actions are well established in adults and children, the role that FGF23 plays in regulating fetal phosphorus metabolism has not been previously studied. We used several mouse models to study the effect of endogenous deficiency or excess of FGF23 on fetal phosphorus metabolism. We found that intact FGF23 does not cross the placenta from mother to fetus, but wild-type fetuses normally have intact FGF23 levels that approximately equal the maternal level. Deletion of Fgf23 or 7.8-fold higher serum FGF23 levels did not disturb any parameter of fetal mineral homeostasis, including serum and amniotic fluid phosphorus, skeletal morphology, skeletal mineral content, and placental phosphorus transport. Placentas and fetal kidneys abundantly express FGF23 target genes. Cyp24a1 was significantly reduced in Fgf23 null kidneys and was significantly increased in Phex null placentas and fetal kidneys. Phex null kidneys also showed reduced expression of Klotho. However, these changes in gene expression did not disturb any physiological parameter related to phosphorus. A 50% reduction in FGF23 also failed to affect renal phosphorus excretion into amniotic fluid when either PTH or the vitamin D receptor were absent. In conclusion, FGF23 is not an important regulator of fetal phosphorous metabolism. The active delivery of phosphorus across the placenta does not require FGF23, and that process overrides any effects that absence or excess of FGF23 might otherwise have on phosphate handling by the fetal kidneys.

Upregulation of calcitriol during pregnancy and skeletal recovery after lactation do not require parathyroid hormone.

Pregnancy invokes a doubling of intestinal calcium absorption whereas lactation programs skeletal resorption to provide calcium to milk. Postweaning bone formation restores the skeleton's bone mineral content (BMC), but the factors that regulate this are not established. We used Pth-null mice to test whether parathyroid hormone (PTH) is required for postweaning skeletal recovery. On a normal 1% calcium diet, wild-type (WT) and Pth-null mice each gained BMC during pregnancy, declined 15% to 18% below baseline during lactation, and restored the skeleton above baseline BMC within 14 days postweaning. A 2% calcium diet reduced the lactational decline in BMC without altering the gains achieved during pregnancy and postweaning. The hypocalcemia and hyperphosphatemia of Pth-null mice normalized during lactation and serum calcium remained normal during postweaning. Osteocalcin and propeptide of type 1 collagen (P1NP) each rose significantly after lactation to similar values in WT and Pth-null. Serum calcitriol increased fivefold during pregnancy in both genotypes whereas vitamin D binding protein levels were unchanged. Absence of PTH blocked a normal rise in fibroblast growth factor-23 (FGF23) during pregnancy despite high calcitriol. A 30-fold higher expression of Cyp27b1 in maternal kidneys versus placenta suggests that the pregnancy-related increase in calcitriol comes from the kidneys. Conversely, substantial placental expression of Cyp24a1 may contribute significantly to the metabolism of calcitriol. In conclusion, PTH is not required to upregulate renal expression of Cyp27b1 during pregnancy or to stimulate recovery from loss of BMC caused by lactation. A calcium-rich diet in rodents suppresses skeletal losses during lactation, unlike clinical trials that showed no effect of supplemental calcium on lactational decline in BMC.

The abnormal phenotypes of cartilage and bone in calcium-sensing receptor deficient mice are dependent on the actions of calcium, phosphorus, and PTH.

Patients with neonatal severe hyperparathyroidism (NSHPT) are homozygous for the calcium-sensing receptor (CaR) mutation and have very high circulating PTH, abundant parathyroid hyperplasia, and severe life-threatening hypercalcemia. Mice with homozygous deletion of CaR mimic the syndrome of NSHPT. To determine effects of CaR deficiency on skeletal development and interactions between CaR and 1,25(OH)(2)D(3) or PTH on calcium and skeletal homeostasis, we compared the skeletal phenotypes of homozygous CaR-deficient (CaR(-/-)) mice to those of double homozygous CaR- and 1α(OH)ase-deficient [CaR(-/-)1α(OH)ase(-/-)] mice or those of double homozygous CaR- and PTH-deficient [CaR(-/-)PTH(-/-)] mice at 2 weeks of age. Compared to wild-type littermates, CaR(-/-) mice had hypercalcemia, hypophosphatemia, hyperparathyroidism, and severe skeletal growth retardation. Chondrocyte proliferation and PTHrP expression in growth plates were reduced significantly, whereas trabecular volume, osteoblast number, osteocalcin-positive areas, expression of the ALP, type I collagen, osteocalcin genes, and serum ALP levels were increased significantly. Deletion of 1α(OH)ase in CaR(-/-) mice resulted in a longer lifespan, normocalcemia, lower serum phosphorus, greater elevation in PTH, slight improvement in skeletal growth with increased chondrocyte proliferation and PTHrP expression, and further increases in indices of osteoblastic bone formation. Deletion of PTH in CaR(-/-) mice resulted in rescue of early lethality, normocalcemia, increased serum phosphorus, undetectable serum PTH, normalization in skeletal growth with normal chondrocyte proliferation and enhanced PTHrP expression, and dramatic decreases in indices of osteoblastic bone formation. Our results indicate that reductions in hypercalcemia play a critical role in preventing the early lethality of CaR(-/-) mice and that defects in endochondral bone formation in CaR(-/-) mice result from effects of the marked elevation in serum calcium concentration and the decreases in serum phosphorus concentration and skeletal PTHrP levels, whereas the increased osteoblastic bone formation results from direct effects of PTH.

Endogenous PTH deficiency impairs fracture healing and impedes the fracture-healing efficacy of exogenous PTH(1-34).

BACKGROUND: Although the capacity of exogenous PTH1-34 to enhance the rate of bone repair is well established in animal models, our understanding of the mechanism(s) whereby PTH induces an anabolic response during skeletal repair remains limited. Furthermore it is unknown whether endogenous PTH is required for fracture healing and how the absence of endogenous PTH would influence the fracture-healing capacity of exogenous PTH. METHODOLOGY/PRINCIPAL FINDINGS: Closed mid-diaphyseal femur fractures were created and stabilized with an intramedullary pin in 8-week-old wild-type and Pth null (Pth(-/-)) mice. Mice received daily injections of vehicle or of PTH1-34 (80 µg/kg) for 1-4 weeks post-fracture, and callus tissue properties were analyzed at 1, 2 and 4 weeks post-fracture. Cartilaginous callus areas were reduced at 1 week post-fracture, but were increased at 2 weeks post-fracture in vehicle-treated and PTH-treated Pth(-/-) mice compared to vehicle-treated and PTH-treated wild-type mice respectively. The mineralized callus areas, bony callus areas, osteoblast number and activity, osteoclast number and surface in callus tissues were all reduced in vehicle-treated and PTH-treated Pth(-/-) mice compared to vehicle-treated and PTH-treated wild-type mice, but were increased in PTH-treated wild-type and Pth(-/-) mice compared to vehicle-treated wild-type and Pth(-/-) mice. CONCLUSIONS/SIGNIFICANCE: Absence of endogenous PTH1-84 impedes bone fracture healing. Exogenous PTH1-34 can act in the absence of endogenous PTH but callus formation, including accelerated endochondral bone formation and callus remodeling as well as mechanical strength of the bone are greater when endogenous PTH is present. Results of this study suggest a complementary role for endogenous PTH1-84 and exogenous PTH1-34 in accelerating fracture healing.

The calcium-sensing receptor mediates bone turnover induced by dietary calcium and parathyroid hormone in neonates.

We have investigated, in neonates, whether the calcium-sensing receptor (CaR) mediates the effects of dietary calcium on bone turnover and/or modulates parathyroid hormone (PTH)-induced bone turnover. Wild-type (WT) pups and pups with targeted deletion of the Pth (Pth(-/-)) gene or of both Pth and CaR (Pth(-/-)CaR(-/-)) genes were nursed by dams on a normal or high-calcium diet. Pups nursed by dams on a normal diet received daily injections of vehicle or of PTH(1-34) (80 µg/kg) for 2 weeks starting from 1 week of age. In pups receiving vehicle and fed by dams on a normal diet, trabecular bone volume, osteoblast number, type 1 collagen-positive area, and mineral apposition rate, as well as the expression of bone-formation-related genes, all were reduced significantly in Pth(-/-) pups compared with WT pups and were decreased even more dramatically in Pth(-/-)CaR(-/-) pups. These parameters were increased in WT and Pth(-/-) pups but not in Pth(-/-)CaR(-/-) pups fed by dams on a high-calcium diet compared with pups fed by dams on a normal diet. These parameters also were increased in WT, Pth(-/-), and Pth(-/-)CaR(-/-) pups following exogenous PTH treatment; however, the percentage increase was less in Pth(-/-)CaR(-/-) pups than in WT and Pth(-/-) pups. In vehicle-treated pups fed by dams on either the normal or high-calcium diet and in PTH-treated pups fed by dams on a normal diet, the number and surfaces of osteoclasts and the ratio of RANKL/OPG were reduced significantly in Pth(-/-) pups and less significantly in Pth(-/-)CaR(-/-) pups compared with WT pups. These parameters were further reduced significantly in WT and Pth(-/-) pups from dams fed a high-calcium diet but did not decrease significantly in similarly treated Pth(-/-)CaR(-/-) pups, and they increased significantly in PTH-treated pups compared with vehicle-treated, genotype-matched pups fed by dams on the normal diet. These results indicate that in neonates, the CaR mediates alterations in bone turnover in response to changes in dietary calcium and modulates PTH-stimulated bone turnover.

Vitamin D status and response to daily 400 IU vitamin D3 and weekly alendronate 70 mg in men and women with osteoporosis.

BACKGROUND: Suboptimal vitamin D status is common in elderly individuals. However, the extent of vitamin D inadequacy in men and women being treated for osteoporosis in a family practice setting has not been well characterized. OBJECTIVE: To describe the distribution of serum 25-hydroxyvitamin D (25-[OH] D) in Canadian men and postmenopausal women with osteoporosis taking 400 IU or less of vitamin D daily and to evaluate the safety, tolerability, and impact of vitamin D(3) supplementation 400 IU daily taken concurrently with alendronate sodium 70 mg weekly. METHODS: This was a prospective, single-cohort, open-label, multicenter study. Community-dwelling men and postmenopausal women with osteoporosis were recruited at 197 sites across Canada. Patients received vitamin D(3) 400 IU/day supplementation coadministered with alendronate 70 mg/wk for 16 weeks. The primary outcome was the distribution of serum 25-(OH) D at baseline. Secondary outcome measures included changes from baseline in serum 25-(OH) D levels, adherence to study treatments, and incidence of treatment-related adverse events (AEs). RESULTS: Of the 681 patients included in the analysis, 485 (71.2%) completed the study. Patients were predominantly female (83.1%) with a mean (SD) age of 67.6 (10.7) years. At baseline, mean (SD) serum 25-(OH) D concentration was 25.4 (9.9) ng/mL and 68.0% of the patients had inadequate (less than 30 ng/mL) vitamin D status. At week 16, concentrations increased by 35.1% to 31.2 (9.2) ng/mL (p < 0.001) and the proportion of patients with inadequate 25-(OH) D levels was reduced to 47.0%. Adherence to the treatment regimen was high (greater than 95%). Gastrointestinal disorders were the most frequently reported (6.9%) treatment-related AEs. CONCLUSIONS: About two thirds of patients previously diagnosed with osteoporosis have inadequate vitamin D status. A treatment regimen consisting of alendronate 70 mg/wk administered with daily vitamin D(3) 400 IU supplementation significantly increased patients' serum 25-(OH) D levels, but 47% did not achieve optimal levels. These results support both the National Osteoporosis Foundation and Osteoporosis Canada recommendations for higher vitamin D supplement doses (at least 800 IU daily) in osteoporotic patients receiving pharmacologic therapy for osteoporosis and for monitoring their serum 25-(OH) D response.

Skeletal recovery after weaning does not require PTHrP.

Mice lose 20% to 25% of trabecular bone mineral content (BMC) during lactation and restore it after weaning through unknown mechanisms. We found that tibial Pthrp mRNA expression was upregulated fivefold by 7 days after weaning versus end of lactation in wild-type (WT) mice. To determine whether parathyroid hormone-related protein (PTHrP) stimulates bone formation after weaning, we studied a conditional knockout in which PTHrP is deleted from preosteoblasts and osteoblasts by collagen I promoter-driven Cre (Cre(ColI) ). These mice are osteopenic as adults but have normal serum calcium, calcitriol, and parathyroid hormone (PTH). Pairs of Pthrp(flox/flox) ;Cre(ColI) (null) and WT;Cre(ColI) (WT) females were mated and studied through pregnancy, lactation, and 3 weeks of postweaning recovery. By end of lactation, both genotypes lost lumbar spine BMC: WT declined by 20.6% ± 3.3%, and null decreased by 22.5% ± 3.5% (p < .0001 versus baseline; p = NS between genotypes). During postweaning recovery, both restored BMC to baseline: WT to -3.6% ± 3.7% and null to 0.3% ± 3.7% (p = NS versus baseline or between genotypes). Similar loss and full recovery of BMC were seen at the whole body and hind limb. Histomorphometry confirmed that nulls had lower bone mass at baseline and that this was equal to the value achieved after weaning. Osteocalcin, propeptide of type 1 collagen (P1NP), and deoxypyridinoline increased equally during recovery in WT and null mice; PTH decreased and calcitriol increased equally; serum calcium was unchanged. Urine calcium increased during recovery but remained no different between genotypes. Although osteoblast-derived PTHrP is required to maintain adult bone mass and Pthrp mRNA upregulates in bone after weaning, it is not required for recovery of bone mass after lactation. The factors that stimulate postweaning bone formation remain unknown.

Alterations in phosphorus, calcium and PTHrP contribute to defects in dental and dental alveolar bone formation in calcium-sensing receptor-deficient mice.

To determine whether the calcium-sensing receptor (CaR) participates in tooth formation and dental alveolar bone development in mandibles in vivo, we examined these processes, as well as mineralization, in 2-week-old CaR-knockout (CaR(-/-)) mice. We also attempted to rescue the phenotype of CaR(-/-) mice by genetic means, in mice doubly homozygous for CaR and 25-hydroxyvitamin D 1alpha-hydroxylase [1alpha(OH)ase] or parathyroid hormone (Pth). In CaR(-/-) mice, which exhibited hypercalcemia, hypophosphatemia and increased serum PTH, the volumes of teeth and of dental alveolar bone were decreased dramatically, whereas the ratio of the area of predentin to total dentin and the number and surface of osteoblasts in dental alveolar bone were increased significantly, as compared with wild-type littermates. The normocalcemia present in CaR(-/-);1alpha(OH)ase(-/-) mice only slightly improved the defects in dental and alveolar bone formation observed in the hypercalcemic CaR(-/-) mice. However, these defects were completely rescued by the additional elimination of hypophosphatemia and by an increase in parathyroid hormone-related protein (PTHrP) expression in the apical pulp, Hertwig's epithelial root sheath and mandibular tissue in CaR(-/-); Pth(-/-) mice. Therefore, alterations in calcium, phosphorus and PTHrP contribute to defects in the formation of teeth and alveolar bone in CaR-deficient mice. This study indicates that CaR participates in the formation of teeth and in the development of dental alveolar bone in mandibles in vivo, although it appears to do so largely indirectly.

Klotho ablation converts the biochemical and skeletal alterations in FGF23 (R176Q) transgenic mice to a Klotho-deficient phenotype.

Transgenic mice overexpressing fibroblast growth factor (FGF23) (R176Q) (F(Tg)) exhibit biochemical {hypophosphatemia, phosphaturia, abnormal 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] metabolism} and skeletal (rickets and osteomalacia) abnormalities attributable to FGF23 action. In vitro studies now implicate the aging-related factor Klotho in the signaling mechanism of FGF23. In this study, we used a mouse genetic approach to validate in vivo the pivotal role of Klotho in the metabolic and skeletal derangements associated with FGF23 (R176Q) overexpression. To this end, we crossed mice heterozygous for the hypomorphic Klotho allele (Kl(+/-)) to F(Tg) mice and obtained F(Tg) transgenic mice homozygous for the Kl-hypomorphic allele (F(Tg)/Kl(-/-)). Mice were killed on postnatal day 50, and serum and tissues were procured for analysis and comparison with F(Tg), wild-type, and Kl(-/-) controls. From 4 wk onward, F(Tg)/Kl(-/-) mice were clearly distinguishable from F(Tg) mice and exhibited a striking phenotypic resemblance to the Kl(-/-) controls. Serum analysis for calcium, phosphorus, parathyroid hormone, 1,25(OH)(2)D(3), and alkaline phosphatase activity confirmed the biochemical similarity between the F(Tg)/Kl(-/-) and Kl(-/-) mice and their distinctness from the F(Tg) controls. The characteristic skeletal changes associated with FGF23 (R176Q) overexpression were also dramatically reversed by the absence of Klotho. Hence the wide, unmineralized growth plates and the osteomalacic abnormalities apparent in trabecular and cortical bone were completely reversed in the F(Tg)/Kl(-/-) mice. Nevertheless, independent actions of Klotho on bone were suggested as manifested by alterations in mineralized bone, and in cortical bone volume which were observed in both the Kl(-/-) and F(Tr)/Kl(-/-) mutants. In summary, our findings substantiate in vivo the essential role of Klotho in the mechanism of action of FGF23 in view of the fact that Klotho ablation converts the biochemical and skeletal manifestations resulting from FGF23 overexpression to a phenotype consistent with Klotho deficiency.

Exogenous 1,25-dihydroxyvitamin D3 exerts a skeletal anabolic effect and improves mineral ion homeostasis in mice that are homozygous for both the 1alpha-hydroxylase and parathyroid hormone null alleles.

1,25-Dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] and PTH each modulate calcium and skeletal homeostasis. To identify 1,25(OH)(2)D(3)-mediated skeletal and mineral ion actions independent of PTH, double-knockout mice, which are homozygous for both the 1alpha-hydroxylase and PTH null alleles, were treated with 1,25(OH)(2)D(3), sc, from d 4 to 14 and compared with vehicle-treated animals. Serum calcium rose in 1,25(OH)(2)D(3)-treated double-knockout mice, and messenger RNA and protein levels of the renal calcium transporters TRPV5, calbindin-D(28K), calbindin-D(9K), and Na(+)/Ca(2+) exchanger 1 were up-regulated. Parameters of endochondral bone formation, including long bone length, epiphyseal volume, chondrocyte proliferation and differentiation, and cartilage matrix mineralization, were all increased by 1,25(OH)(2)D(3), Exogenous 1,25(OH)(2)D(3) also increased both trabecular and cortical bone; augmented both osteoblast number and type I collagen deposition in bone matrix; and up-regulated expression levels of the osteoblastic genes alkaline phosphatase, type I collagen, and osteocalcin. Furthermore, in 1,25(OH)(2)D(3)-treated double mutants, osteoclastic bone resorption appeared to decline. The results indicate that administered 1,25(OH)(2)D(3) used intestinal and renal but not skeletal mechanisms to elevate serum calcium and that this sterol can promote endochondral and appositional bone increases independent of endogenous PTH.

The calcium-sensing receptor is required for normal calcium homeostasis independent of parathyroid hormone.

The extracellular calcium-sensing receptor (CaR; alternate gene names, CaR or Casr) is a membrane-spanning G protein-coupled receptor. CaR is highly expressed in the parathyroid gland, and is activated by extracellular calcium (Ca(2+)(o)). Mice homozygous for null mutations in the CaR gene (CaR(-/-)) die shortly after birth because of the effects of severe hyperparathyroidism and hypercalcemia. A wide variety of functions have been attributed to CaR. However, the lethal CaR-deficient phenotype has made it difficult to dissect the direct effect of CaR deficiency from the secondary effects of hyperparathyroidism and hypercalcemia. We therefore generated parathyroid hormone-deficient (PTH-deficient) CaR(-/-) mice (Pth(-/-)CaR(-/-)) by intercrossing mice heterozygous for the null CaR allele with mice heterozygous for a null Pth allele. We show that genetic ablation of PTH is sufficient to rescue the lethal CaR(-/-) phenotype. Pth(-/-)CaR(-/-) mice survive to adulthood with no obvious difference in size or appearance relative to control Pth(-/-) littermates. Histologic examination of most organs did not reveal abnormalities. These Pth(-/-)CaR(-/-) mice exhibit a much wider range of values for serum calcium and renal excretion of calcium than we observe in control littermates, despite the absence of any circulating PTH. Thus, CaR is necessary for the fine regulation of serum calcium levels and renal calcium excretion independent of its effect on PTH secretion.

The autosomal dominant hypophosphatemic rickets R176Q mutation in fibroblast growth factor 23 resists proteolytic cleavage and enhances in vivo biological potency.

Missense mutations in fibroblast growth factor 23 (FGF23) are the cause of autosomal dominant hypophosphatemic rickets (ADHR). The mutations (R176Q, R179W, and R179Q) replace Arg residues within a subtilisin-like proprotein convertase (SPC) cleavage site (RXXR motif), leading to protease resistance of FGF23. The goals of this study were to examine in vivo the biological potency of the R176Q mutant FGF23 form and to characterize alterations in homeostatic mechanisms that give rise to the phenotypic presentation of this disorder. For this, wild type and R176Q mutant FGF23 were overexpressed in the intact animals using a tumor-bearing nude mouse system. At comparable circulating levels, the mutant form was more potent in inducing hypophosphatemia, in decreasing circulating concentrations of 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), and in causing rickets and osteomalacia in these animals compared with wild type FGF23. Parameters of calcium homeostasis were also altered, leading to secondary hyperparathyroidism and parathyroid gland hyperplasia. However, the raised circulating levels of parathyroid hormone were ineffective in normalizing the reduced 1,25(OH)(2)D(3) levels by increasing renal expression of 25(OH)D(3)-1alpha-hydroxylase (Cyp40) to promote its synthesis and by decreasing that of 25(OH)D(3)-24-hydroxylase (Cyp24) to prevent its catabolism. The findings provide direct in vivo evidence that missense mutations from ADHR kindreds are gain-of-function mutations that retain and increase the protein's biological potency. Moreover, for the first time, they define a potential role for FGF23 in dissociating parathyroid hormone actions on mineral fluxes and on vitamin D metabolism at the level of the kidney.