Klotho converts canonical FGF receptor into a specific receptor for FGF23.

FGF23 is a unique member of the fibroblast growth factor (FGF) family because it acts as a hormone that derives from bone and regulates kidney functions, whereas most other family members are thought to regulate various cell functions at a local level. The renotropic activity of circulating FGF23 indicates the possible presence of an FGF23-specific receptor in the kidney. Here we show that a previously undescribed receptor conversion by Klotho, a senescence-related molecule, generates the FGF23 receptor. Using a renal homogenate, we found that Klotho binds to FGF23. Forced expression of Klotho enabled the high-affinity binding of FGF23 to the cell surface and restored the ability of a renal cell line to respond to FGF23 treatment. Moreover, FGF23 incompetence was induced by injecting wild-type mice with an anti-Klotho monoclonal antibody. Thus, Klotho is essential for endogenous FGF23 function. Because Klotho alone seemed to be incapable of intracellular signalling, we searched for other components of the FGF23 receptor and found FGFR1(IIIc), which was directly converted by Klotho into the FGF23 receptor. Thus, the concerted action of Klotho and FGFR1(IIIc) reconstitutes the FGF23 receptor. These findings provide insights into the diversity and specificity of interactions between FGF and FGF receptors.

Direct evidence for a causative role of FGF23 in the abnormal renal phosphate handling and vitamin D metabolism in rats with early-stage chronic kidney disease.

Circulating levels of fibroblast growth factor 23 (FGF23) are elevated in patients with early chronic kidney disease (CKD) and are postulated to cause low blood levels of 1,25-dihydroxyvitamin D, as well as normal phosphate levels. In order to provide more direct evidence for the pathophysiological role of FGF23 in the settings of mineral ion homeostasis typically seen in early CKD, we studied rats with progressive CKD treated with anti-FGF23 neutralizing antibody. Without antibody treatment, rats with CKD exhibited high circulating levels of FGF23 and parathyroid hormone, low 1,25-dihydroxyvitamin D, and normal serum phosphate levels, accompanied by increased fractional excretion of phosphate. Antibody treatment, however, lessened fractional excretion of phosphate, thus increasing serum phosphate levels, and normalized serum 1,25-dihydroxyvitamin D by increased 1α-OHase and decreased 24-OHase expressions in the kidney. These antibody-induced changes were followed by increased serum calcium levels, leading to decreased serum parathyroid hormone. Hence, our study shows that FGF23 normalizes serum phosphate and decreases 1,25-dihydroxyvitamin D levels in early-stage CKD, and suggests a pathological sequence of events for the development of secondary hyperparathyroidism triggered by increased FGF23, followed by a reduction of 1,25-dihydroxyvitamin D and calcium levels, thereby increasing parathyroid hormone secretion.

Establishment of sandwich ELISA for soluble alpha-Klotho measurement: Age-dependent change of soluble alpha-Klotho levels in healthy subjects.

BACKGROUND: Alpha-Klotho (alphaKl) regulates mineral metabolism such as calcium ion (Ca(2+)) and inorganic phosphate (Pi) in circulation. Defects in mice result in clinical features resembling disorders found in human aging. Although the importance of transmembrane-type alphaKl has been demonstrated, less is known regarding the physiological importance of soluble-type alphaKl (salphaKl) in circulation. OBJECTIVES: The aims of this study were: (1) to establish a sandwich ELISA system enabling detection of circulating serum salphaKl, and (2) to determine reference values for salphaKl serum levels and relationship to indices of renal function, mineral metabolism, age and sex in healthy subjects. RESULTS: We successively developed an ELISA to measure serum salphaKl in healthy volunteers (n=142, males 66) of ages (61.1+/-18.5year). The levels (mean+/-SD) in these healthy control adults were as follows: total calcium (Ca; 9.46+/-0.41mg/dL), Pi (3.63+/-0.51mg/dL), blood urea nitrogen (BUN; 15.7+/-4.3mg/dL), creatinine (Cre; 0.69+/-0.14mg/dL), 1,25 dihydroxyvitamin D (1,25(OH)(2)D; 54.8+/-17.7pg/mL), intact parathyroid hormone (iPTH; 49.2+/-20.6pg/mL), calcitonin (26.0+/-12.3pg/mL) and intact fibroblast growth factor (FGF23; 43.8+/-17.6pg/mL). Serum levels of salphaKl ranged from 239 to 1266pg/mL (mean+/-SD; 562+/-146pg/mL) in normal adults. Although salphaKl levels were not modified by gender or indices of mineral metabolism, salphaKl levels were inversely related to Cre and age. However, salphaKl levels in normal children (n=39, males 23, mean+/-SD; 7.1+/-4.8years) were significantly higher (mean+/-SD; 952+/-282pg/mL) than those in adults (mean+/-SD; 562+/-146, P<0.001). A multivariate linear regression analysis including children and adults in this study demonstrated that salphaKl correlated negatively with age and Ca, and positively with Pi. Finally, we measured a serum salphaKl from a patient with severe tumoral calcinosis derived from a homozygous missense mutation of alpha-klotho gene. In this patient, salphaKl level was notably lower than those of age-matched controls. CONCLUSION: We established a detection system to measure human serum salphaKl for the first time. Age, Ca and Pi seem to influence serum salphaKl levels in a normal population. This detection system should be an excellent tool for investigating salphaKl functions in mineral metabolism.

Association between fibroblast growth factor 23 and left ventricular hypertrophy in maintenance hemodialysis patients. Comparison with B-type natriuretic peptide and cardiac troponin T.

BACKGROUND: Fibroblast growth factor 23 (FGF-23) is a novel bone-derived phosphate-regulating hormone, and serum FGF-23 levels are associated with mortality among hemodialysis (HD) patients. However, the pathophysiological role of FGF-23 in those patients remains unclear, so the association between serum FGF-23 levels and known cardiac biomarkers or echocardiographic measurements were investigated in long-term HD patients without cardiac symptoms. METHODS AND RESULTS: The 87 consecutive patients treated in a single HD center (51 males, 36 females; mean age 64 years, mean HD duration 5.8 years) were included in this study. Comprehensive echocardiography was performed after HD. Blood samples were obtained before HD. Serum FGF-23 levels in dialysis patients were 1,171±553pg/ml. In univariate analysis, serum phosphate (r=0.443, P<0.001) and calcium levels (r=0.256, P=0.04), left ventricular mass index (LVMI) (r=0.268, P=0.039) were significantly associated with FGF-23 levels. Neither the B-type natriuretic peptide (BNP) nor the cardiac troponin T level was correlated with FGF-23. In multivariate regression analysis, only LVMI (ß=0.287, P=0.031, confidence interval (CI) 0.390-8.040) and phosphate levels (ß=0.419, P=0.001, CI 57.12-207.7) and calcium levels (ß=0.277, P=0.025, CI 24.95-360.1) remained significantly correlated with FGF-23. CONCLUSIONS: Beside BNP, FGF-23 was identified as a factor that is significantly associated with LVMI. FGF-23 could be a novel biomarker of left ventricular overload, which is closely associated with the increased risk of death in HD patients.

Targeted deletion of the tybe IIb Na(+)-dependent Pi-co-transporter, NaPi-IIb, results in early embryonic lethality.

NaPi-IIb encodes a Na(+)-dependent Pi co-transporter, which is expressed in various adult tissues and mediates transport of extracellular Pi ions coupling with Na(+) ion. To define the role of NaPi-IIbin vivo, NaPi-IIb gene deficient mice were generated utilizing targeted mutagenesis, yielding viable, heterozygous NaPi-IIb mice. In contrast, homozygous NaPi-IIb mice died in utero soon after implantation, indicating that NaPi-IIb was essential for early embryonic development. In situ hybridization revealed NaPi-IIb mRNA expression in the parietal endoderm, followed by the visceral endoderm, at a time point prior to establishment of a functioning chorio-allantoic placenta. At the time point of functional placenta development, the main site of NaPi-IIb production resided in the labyrinthine zone, where embryonic and maternal circulations were in closest contact. Expression patterns of NaPi-IIb suggest that NaPi-IIb plays an important role in Pi absorption from maternal circulation.

Hyperostosis-hyperphosphatemia syndrome: a congenital disorder of O-glycosylation associated with augmented processing of fibroblast growth factor 23.

UNLABELLED: Two hyperphosphatemic patients with mutations in GALNT3 showed low intact FGF23 levels with marked increase of processed C-terminal fragments. FGF23 protein has three O-linked glycans and FGF23 with incomplete glycosylation is susceptible to processing. Silencing GALNT3 resulted in enhanced processing of FGF23. Decreased function of FGF23 by enhanced processing is the cause of hyperphosphatemia in patients with GALNT3 mutation. INTRODUCTION: Hyperostosis-hyperphosphatemia syndrome (HHS) is an autosomal recessive entity manifesting as severe hyperphosphatemia associated with episodic bone pain and radiological findings of cortical hyperostosis and periosteal reaction. Persistent hyperphosphatemia is not counterbalanced by PTH or 1,25-dihydroxyvitamin D, posing a mirror image of hypophosphatemic states attributed to increased fibroblast growth factor (FGF)23 activity. MATERIALS AND METHODS: We describe two children with HHS who were found to be homozygous for a mutation in GALNT3 encoding a peptide involved in mucin-type O-glycosylation (ppGaNTase-T3). FGF23 levels were evaluated by two ELISAs and Western blotting. FGF23 protein was analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Effect of silencing GALNT3 was evaluated using siRNA in cells transfected with expression vector for FGF23. RESULTS: Both patients had low levels of the full-length FGF23 with markedly augmented amounts of the inactive fragments. Biologically active FGF23 has three O-linked glycans. FGF23 with only one or two O-linked glycans is processed into inactive fragments. Decreasing the expression of the GALNT3 gene by RNA interference resulted in enhanced processing of FGF23. CONCLUSIONS: The primary defect in HHS is impairment of glycosylation of FGF23 resulting from mutations in GALNT3 and leading to augmented processing of FGF23. These changes in FGF23 abolish its phosphaturic effect and lead to severe persistent hyperphosphatemia. This study provides the pathogenetic mechanism of the first mucin-type O-glycosylation defect identified.

Targeted ablation of Fgf23 demonstrates an essential physiological role of FGF23 in phosphate and vitamin D metabolism.

Inorganic phosphate is essential for ECM mineralization and also as a constituent of important molecules in cellular metabolism. Investigations of several hypophosphatemic diseases indicated that a hormone-like molecule probably regulates serum phosphate concentration. FGF23 has recently been recognized as playing important pathophysiological roles in several hypophosphatemic diseases. We present here the evidence that FGF23 is a physiological regulator of serum phosphate and 1,25-dihydroxyvitamin D (1,25[OH]2D) by generating FGF23-null mice. Disruption of the Fgf23 gene did not result in embryonic lethality, although homozygous mice showed severe growth retardation with abnormal bone phenotype and markedly short life span. The Fgf23(-/-) mice displayed significantly high serum phosphate with increased renal phosphate reabsorption. They also showed an elevation in serum 1,25(OH)2D that was due to the enhanced expression of renal 25-hydroxyvitamin D-1alpha-hydroxylase (1alpha-OHase) from 10 days of age. These phenotypes could not be explained by currently known regulators of mineral homeostasis, indicating that FGF23 is essential for normal phosphate and vitamin D metabolism.

FGF23 is a hormone-regulating phosphate metabolism--unique biological characteristics of FGF23.

FGF23 was identified as the last member of FGF23 family. Recent investigations indicate that excess actions of FGF23 cause several hypophosphatemic diseases whereas deficient FGF23 activity results in hyperphosphatemic tumoral calcinosis. These results indicate that FGF23 is a hormone that regulates serum phosphate level in contrast to other FGF family members that work as local factors. Furthermore, FGF23 requires Klotho for its signaling in addition to a canonical FGF receptor. These unique characteristics of FGF23 expanded our knowledge about the diversity of FGF family members and specificity of FGF23.

Dietary phosphorus regulates serum fibroblast growth factor-23 concentrations in healthy men.

CONTEXT: Fibroblast growth factor 23 (FGF-23) is important in the regulation of phosphorus and vitamin D metabolism. States of excess circulating FGF-23 are associated with renal phosphate wasting and inappropriately low serum 1,25-dihydroxyvitamin D [1,25(OH)(2)D] concentrations. Conversely, states of absent or biologically inactive circulating FGF-23 are associated with increased serum phosphorus and 1,25(OH)(2)D concentrations. Restriction of the dietary intake of phosphorus increases renal phosphate reabsorption and 1,25(OH)(2)D production, whereas the opposite occurs when dietary phosphorus is supplemented. OBJECTIVE: We sought to determine whether serum FGF-23 concentration is regulated by dietary phosphorus and thereby mediates the physiological response of serum 1,25(OH)(2)D to changes in dietary phosphorus. DESIGN, SETTING, AND PARTICIPANTS: We studied 13 healthy men as inpatients during a 4-wk dietary phosphorus intervention study. INTERVENTION: Subjects consumed a constant diet that provided 500 mg of phosphorus per day, which was supplemented to achieve three phosphorus intakes, each of 9 d: 1) control = 1500 mg/d; 2) supplemented = 2300 mg/d; 3) restricted = 625 mg/d. Intakes of calcium, sodium, potassium, magnesium, and energy were constant. MAIN OUTCOME MEASURE: Serum FGF-23, 1,25(OH)(2)D, phosphorus, and calcium concentrations were measured. RESULTS: Serum FGF-23 concentrations decreased significantly from 30.7 +/- 8.7 pg/ml during phosphorus supplementation to 19.6 +/- 7.0 pg/ml during phosphorus restriction. Serum 1,25(OH)(2)D concentrations increased significantly from 29 +/- 10 pg/ml (75 +/- 26 pmol/liter) during phosphorus supplementation to 40 +/- 16 pg/ml (104 +/- 42 pmol/liter) during phosphorus restriction (P < 0.001). Serum 1,25(OH)(2)D concentrations varied inversely with those of serum FGF-23 (r = -0.67, P < 0.001). CONCLUSIONS: We conclude that in healthy men, changes in dietary phosphorus within the physiological range of intakes regulate serum FGF-23 concentrations and suggest that dietary phosphorus regulation of 1,25(OH)(2)D production is mediated, at least in part, by changes in circulating FGF-23.

Dietary and serum phosphorus regulate fibroblast growth factor 23 expression and 1,25-dihydroxyvitamin D metabolism in mice.

Fibroblast growth factor-23 (FGF-23) is a novel circulating peptide that regulates phosphorus (Pi) and vitamin D metabolism, but the mechanisms by which circulating FGF-23 itself is regulated are unknown. To determine whether the serum FGF-23 concentration is regulated by dietary intake of Pi, we fed wild-type (WT), Npt2a gene-ablated (Npt2a(-/-)), and Hyp mice diets containing varying Pi contents (0.02-1.65%). In WT mice, increases in dietary Pi intake from 0.02-1.65% induced a 7-fold increase in serum FGF-23 and a 3-fold increase in serum Pi concentrations. Across the range of dietary Pi, serum FGF-23 concentrations varied directly with serum Pi concentrations (r(2) = 0.72; P < 0.001). In Npt2a(-/-) mice, serum FGF-23 concentrations were significantly lower than in WT mice, and these differences could be accounted for by the lower serum Pi levels in Npt2a(-/-) mice. The serum concentrations of FGF-23 in Hyp mice were 5- to 25-fold higher than values in WT mice, and the values varied with dietary Pi intake. Fgf-23 mRNA abundance in calvaria was significantly higher in Hyp mice than in WT mice on the 1% Pi diet; in both groups of mice, fgf-23 mRNA abundance in calvarial bone was suppressed by 85% on the low (0.02%) Pi diet. In WT mice fed the low (0.02%) Pi diet, renal mitochondrial 1alpha-hydroxylase activity and renal 1alpha-hydroxylase (P450c1alpha) mRNA abundance were significantly higher than in mice fed the higher Pi diets and varied inversely with serum FGF-23 concentrations (r(2) = 0.86 and r(2) = 0.64; P < 0.001, respectively). The present data demonstrate that dietary Pi regulates the serum FGF-23 concentration in mice, and such regulation is independent of phex function. The data suggest that genotype-dependent and dietary Pi-induced changes in the serum FGF-23 concentration reflect changes in fgf-23 gene expression in bone.

Fibroblast growth factor-23 in patients with Graves' disease before and after antithyroid therapy: its important role in serum phosphate regulation.

OBJECTIVE: Hyperthyroidism is a well-described cause of hyperphosphatemia. We aimed to clarify the physiological role of fibroblast growth factor (FGF)-23 in serum phosphate homeostasis in patients with Graves' disease during the course of treatment for hyperthyroidism. CONTEXT: The study group comprised 56 patients (45 for a cross-sectional study and 11 for a longitudinal study) with Graves' disease. For the cross-sectional study, patients were assigned, on the basis of their serum phosphate level, to a hypophosphatemia group (n = 14), a normophosphatemia group (n = 16), or a hyperphosphatemia group (n = 15). Serum FGF-23, calcium, phosphate, PTH, and 1,25-dihydroxyvitamin D [1,25(OH)(2)D] levels were compared between the three groups. For the longitudinal study, we assessed changes in these biochemical indices before and after antithyroid treatment. RESULTS: In the cross-sectional study, the serum FGF-23 level was significantly higher (P < 0.05) in the hyperphosphatemia group than in the other groups (61 +/- 36 ng/liter vs. 31 +/- 22 ng/liter and 30 +/- 9 ng/liter). In the longitudinal study, serum levels of FGF-23 decreased significantly (P < 0.05) from a high of 54 +/- 12 ng/liter before treatment to 29 +/- 14 ng/liter after treatment. In contrast, the serum 1,25(OH)(2)D level increased significantly (P < 0.005) from 55 +/- 22 pmol/liter before treatment to 185 +/- 76 pmol/liter 3 months after treatment. Serum FGF-23 levels were positively correlated with serum phosphate levels (P < 0.0001) and negatively correlated with serum 1,25(OH)(2)D levels (P < 0.0001). CONCLUSIONS: The significant positive correlation between serum levels of phosphate and FGF-23 indicates that FGF-23 may play an important role in serum phosphate homeostasis by its up-regulation in the hyperphosphatemic condition.

A novel mutation in fibroblast growth factor 23 gene as a cause of tumoral calcinosis.

CONTEXT: Tumoral calcinosis is a disease characterized by ectopic calcification and hyperphosphatemia due to enhanced renal tubular phosphate reabsorption. Fibroblast growth factor (FGF)23 was identified as a responsible factor in hypophosphatemic diseases caused by renal phosphate leak. OBJECTIVE: The objective of the study was to analyze the involvement of FGF23 in the development of tumoral calcinosis. DESIGN: Serum FGF23 level was evaluated in a patient with tumoral calcinosis by two kinds of ELISA: full-length assay that detects only full-length FGF23 with phosphate-lowering activity and C-terminal assay that measures full-length as well as C-terminal fragment of FGF23. FGF23 gene was analyzed by direct sequencing of PCR products, and mutant FGF23 was analyzed by Western blotting after expression in mammalian cells. PATIENTS: A family of tumoral calcinosis patients were studied. RESULTS: Serum FGF23 was extremely high when measured by C-terminal assay. In contrast, it was low normal by full-length assay. Analysis of FGF23 gene detected a serine to phenylalanine mutation in codon 129. No wild-type allele of this codon was found in the patient. The brother of the proband showed the same base change. When this mutant FGF23 was expressed in vitro, full-length and N-terminal fragments were barely detectable by Western blotting, whereas C-terminal fragment with the same molecular weight as that from wild-type FGF23 could be detected. CONCLUSION: The production and serum level of C-terminal fragment of FGF23 are increased in this patient with tumoral calcinosis. Together with the recent similar report of FGF23 mutation, impaired action of full-length FGF23 seems to result in tumoral calcinosis.

Intravenous calcitriol therapy increases serum concentrations of fibroblast growth factor-23 in dialysis patients with secondary hyperparathyroidism.

BACKGROUND/AIMS: Fibroblast growth factor-23 (FGF-23) is a recently discovered phosphaturic factor. Although increased levels of serum FGF-23 have been reported in dialysis patients, the role of high FGF-23 levels remains unclear. Since FGF-23 is associated also with vitamin D metabolism, we examined the changes of serum FGF-23 levels in chronic dialysis patients treated with intravenous calcitriol therapy. METHODS: Thirty patients with severe secondary hyperparathyroidism were treated with intravenous calcitriol (0.5-1.0 microg) two or three times per week for 6 months. The changes of serum levels of calcium, phosphate, intact PTH, and FGF-23 were evaluated. RESULTS: Baseline serum FGF-23 levels were markedly high. By intravenous calcitriol therapy, intact PTH levels decreased effectively in the first month (p < 0.001). In contrast, FGF-23 levels increased gradually during the study period (p = 0.027). The Delta serum FGF-23 level was significantly correlated with the total doses of calcitriol injected intravenously in 6 months in patients with refractory secondary hyperparathyroidism (R2 = 0.147; p = 0.036). CONCLUSIONS: Intravenous calcitriol decreased serum intact PTH level and increased serum FGF-23 levels significantly. Extremely high levels of serum FGF-23 in these patients may be attributed, at least in part, to the cumulative dose of vitamin D.

Structural and biochemical properties of fibroblast growth factor 23.

Fibroblast growth factor (FGF) 23 shares a fundamentally common structure with the members of the FGF family and has a unique sequence extension at the C-terminus. The molecular behavior of FGF23 as a systemic factor can be justified by the altered conformation of the beta-trefoil structure similar to that suspected in FGF19. On the other hand, the biological activity of FGF23 is quite distinct from those of other FGFs and requires the C-terminal unique extended structure. Two types of enzyme-linked immunosorbent assays (ELISA) have been developed to detect the intact mature form of FGF23 and its C-terminal portion. The former ELISA method enables the detection of rodent FGF23 and human FGF23. Studies on experimental animal models and laboratory examinations of physiologic and disordered conditions using these assays are contributing toward elucidating the physiology and pathophysiology of FGF23.

[Fibroblast growth factor (FGF)-23 in patients with hypoparathyroidism].

Hypoparathyroidism is a well-described cause of hyperphosphatemia. We aimed to clarify the physiological role of FGF-23 in serum phosphate homeostasis in hypoparathyroidism after thyroidectomy. Increased serum FGF-23 levels were found in patients with hyperphosphatemia and hypocalcemia, caused by hypoparathyroidism after thyroidectomy. After the recovery of parathyroid function, the serum level of calcium, phosphate, and FGF-23 was normalized. Serum FGF-23 levels were significantly higher in patients with permanent hypoparathyroidism than in healthy controls. These results indicate that FGF-23 may play an important role in serum phosphate homeostasis by its up-regulation in the hyperphosphatemic condition.

FGF-23 is a potent regulator of vitamin D metabolism and phosphate homeostasis.

UNLABELLED: We analyzed the effects of an FGF-23 injection in vivo. FGF-23 caused a reduction in serum 1,25-dihydroxyvitamin D by altering the expressions of key enzymes for the vitamin D metabolism followed by hypophosphatemia. This study indicates that FGF-23 is a potent regulator of the vitamin D and phosphate metabolism. INTRODUCTION: The pathophysiological contribution of FGF-23 in hypophosphatemic diseases was supported by animal studies in which the long-term administration of recombinant fibroblast growth factor-23 reproduced hypophosphatemic rickets with a low serum 1,25-dihydroxyvitamin D [1,25(OH)2D] level. However, there is no clear understanding of how FGF-23 causes these changes. MATERIALS AND METHODS: To elucidate the molecular mechanisms of the FGF-23 function, we investigated the short-term effects of a single administration of recombinant FGF-23 in normal and parathyroidectmized animals. RESULTS: An injection of recombinant FGF-23 caused a reduction in serum phosphate and 1,25(OH)2D levels. A decrease in serum phosphate was first observed 9 h after the injection and was accompanied with a reduction in renal mRNA and protein levels for the type IIa sodium-phosphate cotransporter (NaPi-2a). There was no increase in the parathyroid hormone (PTH) level throughout the experiment, and hypophosphatemia was reproduced by FGF-23 in parathyroidectomized rats. Before this hypophosphatemic effect, the serum 1,25(OH)2D level had already descended at 3 h and reached the nadir 9 h after the administration. FGF-23 reduced renal mRNA for 25-hydroxyvitamin D-1alpha-hydroxylase and increased that for 25-hydroxyvitamin D-24-hydroxylase starting at 1 h. In addition, an injection of calcitriol into normal mice increased the serum FGF-23 level within 4 h. CONCLUSIONS: FGF-23 regulates NaPi-2a independently of PTH and the serum 1,25(OH)2D level by controlling renal expressions of key enzymes of the vitamin D metabolism. In conclusion, FGF-23 is a potent regulator of phosphate and vitamin D homeostasis.

Mutant FGF-23 responsible for autosomal dominant hypophosphatemic rickets is resistant to proteolytic cleavage and causes hypophosphatemia in vivo.

FGF-23 is involved in the pathogenesis of two similar hypophosphatemic diseases, autosomal dominant hypophosphatemic rickets/osteomalacia (ADHR) and tumor-induced osteomalacia (TIO). We have shown that the overproduction of FGF-23 by tumors causes TIO. In contrast, ADHR derives from missense mutations in FGF-23 gene. However, it has been unclear how those mutations affect phosphate metabolism. Therefore, we produced mutant as well as wild-type FGF-23 proteins and examined their biological activity. Western blot analysis using site-specific antibodies showed that wild-type FGF-23 secreted into conditioned media was partially cleaved between Arg(179) and Ser(180). In addition, further processing of the cleaved N-terminal portion was observed. In constrast, mutant FGF-23 proteins found in ADHR were resistant to the cleavage. In order to clarify which molecule has the biological activity to induce hypophosphatemia, we separated full-length protein, the N-terminal and C-terminal fragments of wild-type FGF-23. When the activity of each fraction was examined in vivo, only the full-length FGF-23 decreased serum phosphate. Mutant FGF-23 protein that was resistant to the cleavage also retained the activity to induce hypophosphatemia. The extent of hypophosphatemia induced by the single administration of either wild-type or the mutant full-length FGF-23 protein was similar. In addition, implantation of CHO cells expressing the mutant FGF-23 protein caused hypophosphatemia and the decrease of bone mineral content. We conclude that ADHR is caused by hypophosphatemic action of mutant full-length FGF-23 proteins that are resistant to the cleavage between Arg(179) and Ser(180).

Venous sampling for fibroblast growth factor-23 confirms preoperative diagnosis of tumor-induced osteomalacia.

Tumor-induced osteomalacia (TIO) is a paraneoplastic disorder characterized by hypophosphatemia, phosphaturia, inappropriately low serum levels of 1,25-dihydroxyvitamin D for hypophosphatemia, and skeletal undermineralization. Patients with TIO suffer from severe muscle weakness and pain. Because surgical removal of the responsible tumors is the only satisfactory treatment for TIO, identification of the tumors is clinically essential. However, because they are predominantly slow-growing neoplasms of benign mesenchymal origin, localization of the responsible tumors is often very difficult. Moreover, even if a tumor is found in a patient with hypophosphatemic osteomalacia, we have had no way to know that the tumor is actually causing the disease. Fibroblast growth factor-23 (FGF-23) was recently identified as a causative factor for TIO and was shown to induce renal phosphate wasting. We have recently shown that the circulatory FGF-23 level was high in a patient with TIO and rapidly decreased after removal of the responsible tumor. For the first time, we describe a patient with adult-onset hypophosphatemic osteomalacia in whom a clinical diagnosis of TIO was confirmed before surgical removal of the tumor by localizing the responsible tumor using venous sampling for FGF-23 together with magnetic resonance imaging. This combinatorial procedure would be clinically useful for sporadic cases of hypophosphatemic rickets/osteomalacia.

Increased circulatory level of biologically active full-length FGF-23 in patients with hypophosphatemic rickets/osteomalacia.

Hypophosphatemic rickets/osteomalacia with inappropriately low serum 1,25-dihidroxyvitamin D level is commonly observed in X-linked hypophosphatemic rickets/osteomalacia, autosomal dominant hypophosphatemic rickets/osteomalacia and tumor-induced osteomalacia. Although the involvement of a newly identified factor, FGF-23, in the pathogenesis of ADHR and TIO has been suggested, clinical evidence indicating the role of FGF-23 has been lacking. We have previously shown that FGF-23 is cleaved between Arg(179) and Ser(180), and this processing abolished biological activity of FGF-23 to induce hypophosphatemia. Therefore, sandwich ELISA for biologically active intact human FGF-23 was developed using two kinds of monoclonal antibodies that requires the simultaneous presence of both the N-terminal and C-terminal portion of FGF-23. The serum levels of FGF-23 in healthy adults were measurable and ranged from 8.2 to 54.3 ng/L. In contrast, those in a patient with TIO were over 200 ng/L. After the resection of the responsible tumor, the elevated FGF-23 level returned to normal level within 1 h. The increase of serum concentrations of 1,25-dihidroxyvitamin D and phosphate, and the decrease of serum 24,25-dihydroxyvitamin D followed the change of FGF-23. In addition, the elevated serum FGF-23 levels were demonstrated in most patients with XLH. It is likely that increased serum levels of FGF-23 contributes to the development of hypophosphatemia not only in TIO but also in XLH.

Possible involvement of circulating fibroblast growth factor 23 in the development of secondary hyperparathyroidism associated with renal insufficiency.

BACKGROUND: Fibroblast growth factor 23 (FGF-23) is a recently identified polypeptide that promotes renal phosphate excretion and decreases serum 1,25-dihydroxyvitamin D3 (1,25D) levels. Serum FGF-23 levels are extraordinarily elevated in patients with end-stage renal failure. METHODS: Blood and urine samples were obtained from 62 predialysis patients (age, 51.3 +/- 14.0 years; range, approximately 18 to 76 years; 32 men, 30 women). Serum FGF-23 levels were determined by means of a sandwich enzyme-linked immunosorbent assay system using 2 kinds of monoclonal antibodies that does not detect biologically inactive N-terminal and C-terminal fragments derived from an identified internal cleavage site to date. RESULTS: Serum FGF-23 levels increased with the decrease in creatinine clearance (Ccr). Both intact parathyroid hormone (PTH) and 1-84 PTH levels correlated closely with FGF-23 levels (r2 = 0.857; r2 = 0.860). A negative correlation between serum concentrations of FGF-23 and 1,25D (r2 = 0.255) was found. The maximum tubular reabsorptive rate of phosphate correlated negatively with serum FGF-23 concentrations (r2 = 0.460). However, the amount of daily urinary phosphate excretion was significantly less in patients with a Ccr less than 30 mL/min (<0.50 mL/s; P < 0.01), whereas their circulating FGF-23 levels were significantly greater (P < 0.001). CONCLUSION: Circulating FGF-23 levels increase with the decrease in renal function. FGF-23 is a likely candidate to lead the reduction in serum 1,25D levels. FGF-23 becomes a potential uremic toxin to decrease 1,25D levels when it loses its hypophosphatemic action because of a decreased number of viable nephrons in patients with advanced renal failure. As such, FGF-23 may be an important determinant in the regulation of mineral metabolism with renal insufficiency.