Ubiquitin COOH-terminal hydrolase L1 deletion is associated with urinary α-klotho deficiency and perturbed phosphate homeostasis.

Loss of ubiquitin COOH-terminal hydrolase L1 (UCHL1), a deubiquitinating enzyme required for neuronal function, led to hyperphosphatemia accompanied by phosphaturia in mice, while calcium homeostasis remained intact. We therefore investigated the mechanisms underlying the phosphate imbalance in Uchl1-/- mice. Interestingly, phosphaturia was not a result of lower renal brush border membrane sodium-phosphate cotransporter expression as sodium-phosphate cotransporter 2a and 2c expression levels was similar to wild-type levels. Plasma parathyroid hormone and fibroblast growth factor 23 levels were not different; however, fibroblast growth factor 23 mRNA levels were significantly increased in femur homogenates from Uchl1-/- mice. Full-length and soluble α-klotho levels were comparable in kidneys from wild-type and Uchl1-/- mice; however, soluble α-klotho was reduced in Uchl1-/- mice urine. Consistent with unchanged components of 1,25(OH)2D3 metabolism (i.e., CYP27B1 and CYP24A1), sodium-phosphate cotransporter 2b protein levels were not different in ileum brush borders from Uchl1-/- mice, suggesting that the intestine is not the source of hyperphosphatemia. Nonetheless, when Uchl1-/- mice were fed a low-phosphate diet, plasma phosphate, urinary phosphate, and fractional excretion of phosphate were significantly attenuated and comparable to levels of low-phosphate diet-fed wild-type mice. Our findings demonstrate that Uchl1-deleted mice exhibit perturbed phosphate homeostasis, likely consequent to decreased urinary soluble α-klotho, which can be rescued with a low-phosphate diet. Uchl1-/- mice may provide a useful mouse model to study mild perturbations in phosphate homeostasis.

Prenatal diagnosis of familial lethal hypophosphatasia using imaging, blood enzyme levels, chorionic villus sampling and archived fetal tissue.

Hypophosphatasia is an inheritable disorder characterized by defective bone mineralization and a deficiency of tissue-nonspecific alkaline phosphatase (TNSALP) activity. Screening for mutations in the TNSALP gene allows genetic counseling and prenatal diagnosis of the disease in families with severe forms of hypophosphatasia. A 33-year-old, gravida 4, para 3 Japanese woman was referred to Nagoya City University Hospital for prenatal genetic counseling because of two previous occurrences of fetal bone anomalies. The molecular examination showed that the fetus was homozygous for the TNSALP gene mutation c.1559delT, each parent being heterozygous. Genetic counseling was offered and at the next pregnancy, chorionic villus sampling was performed, whereupon genetic analysis confirmed that the fetus did not carry the familial mutation c.1559delT. Postnatal molecular genetic analysis using the cord tissue can provide a diagnosis of lethal hypophosphatasia and prenatal genetic diagnosis of the TNSALP gene allows time for parental counseling and delivery planning.

Normal FGF23 levels in adult idiopathic phosphate diabetes.

Fibroblast growth factor 23 (FGF23), a recently discovered phosphaturic substance playing a key role in genetic and oncogenic phosphate diabetes, is involved in the physiological regulation of phosphate metabolism. Moderate idiopathic phosphate diabetes (IPD) leading to male osteoporosis and diffuse pain resembling fibromyalgia has been described. The aim of our study was to define the role of FGF23 in the mechanism of IPD. The study concerned 29 patients with IPD, mean age 53 +/- 11 years, of whom 72% were men. Fifteen subjects without bone disease and with normal serum phosphate and calcium levels were used as controls. Phosphate diabetes was confirmed by phosphate reabsorption level <85% and phosphate reabsorption threshold (TmPO4/GFR) <0.83. Known causes of phosphate diabetes were excluded. Fasting level of FGF23, serum phosphate, 1-25(OH)2D3, and parathyroid hormone were measured in patients and compared with FGF23 and serum phosphate in healthy controls. Spinal and hip bone mineral density (BMD) were measured by osteodensitometry. Sixteen of 29 patients had diffuse pain, 10 had osteoporosis according to the World Health Organization criteria, and 11 had osteopenia. Serum phosphate was significantly lower in patients than in controls, but FGF23 levels did not differ. Compared to patients with normal bone status, patients with osteopenia and osteoporosis had significantly decreased FGF23 levels, whereas serum phosphate was identical in the two groups. In all patients, serum phosphate and FGF23 were positively correlated and FGF23 and 1-25(OH)2D3 were negatively correlated. FGF23 seems not be a cause of IPD, and the FGF23/phosphate/1-25(OH)2D3 axis appeared to be functional.

Serum parathyroid hormone in X-linked hypophosphatemia.

Serum immunoreactive parathyroid hormone(IPTH) is normal in patients with X-linked hypophosphatemic rickets who are not treated with phosphate salts. Phosphate raises IPTH in these patients. Endogenous IPTH does not influence the existing defect in tubular reabsorption of phosphate in male patients.

Phosphaturic mesenchymal tumor and related wound problem.

INTRODUCTION: Phosphaturic mesenchymal tumor mixed connective tissue type (PMT/MCT) is the most common type (up to 90%) of phosphaturic mesenchymal tumor (PMT), a rare clinicopathologic entity. Besides overproduction of fibroblast growth factor 23 (FGF23), there is a big variation of immunohistochemical characteristic across types of PMT, which makes it difficult to obtain an early diagnosis of PMT/MCT. As a benign tumor, PMT/MCT usually happens in subcutaneous tissues and leads to nonhealing of wound. A complete excision of PMT/MCT facilitates wound healing. CONCLUSIONS: Review of the existing evidence indicates that early diagnosis of PMT/MCT is critically important when treating PMT/MCT wound. Hence standardization of early diagnosis for PMT/MCT is mandated.

Metabolism of vitamin D3-3H in vitamin D-resistant rickets and familial hypophosphatemia.

The fate of an intravenous dose of tritiated vitamin D(3) was studied in seven normal subjects, four children with vitamin D-resistant rickets, and four adults with a familial history of vitamin D-resistant rickets and persistent hypophosphatemia. An abnormal metabolism of vitamin D in vitamin D-resistant rickets was defined and characterized by a decrease in the plasma fractional turnover rate, a marked increase in plasma water-soluble metabolites, and a relative decrease in the conversion of vitamin D to a polar, biologically active metabolite. Alterations in vitamin D metabolism in the adults with persistent hypophosphatemia were similar but less severe than those of affected children with vitamin D-resistant rickets. It is tentatively concluded that the abnormalities in vitamin D metabolism documented in patients with vitamin D-resistant rickets and familial hypophosphatemia may account for the observed osseous and biochemical changes.

Long-term nocturnal calcium infusions can cure rickets and promote normal mineralization in hereditary resistance to 1,25-dihydroxyvitamin D.

We report the beneficial effects of calcium infusions in a child with hereditary resistance to 1,25(OH)2D and alopecia. This patient after transient responsiveness to vitamin D derivatives became unresponsive to all therapy despite serum 1,25(OH)2D concentrations maintained at levels approximately 100-fold normal. A 7-mo trial with calcium infusions led to correction of biochemical abnormalities and healing of rickets. Bone biopsies (n = 3) showed a normal mineralization and the disappearance of the osteomalacia. Cultures of bone-derived cells demonstrated a lack of activation of 25-hydroxyvitamin D 24-hydroxylase and osteocalcin synthesis by 1,25(OH)2D3 (10(-9) and 10(-6) M). These results demonstrate that even in the absence of a normal 1,25(OH)2D3 receptor-effector system in bone cells, normal mineralization can be achieved in humans if adequate serum calcium and phosphorus concentrations are maintained; and calcium infusions may be an efficient alternative for the management of patients with this condition who are unresponsive to large doses of vitamin D derivatives.

Orthophosphate transport in the erythrocyte of normal subjects and of patients with X-linked hypophosphatemia.

We have examined the mechanism of TCA-soluble orthophosphate (Pi) transfer across the membrane of mature human erythrocytes in normal subjects and in patients with X-linked hypophosphatemia (X-LH). The studies were carried out largely at pH 7.4 and 37 degrees C, in partial stimulation of conditions in vivo. (a) At physiological concentrations (1-2 mM) Pi enters the intact normal erythrocyte down its chemical gradient and under no conditions could we identify a steady-state trans-membrane gradient for Pi greater than 0.6. Calculations of the phosphate anion distribution ratio using the Nernst equation yield theoretical values that closely approximate observed values. (b) Glycolytic inhibitors have little effect on total entry of 32Pi inti erythrocytes but they do affect the intracellular distribution of Pi. In the presence of iodoacetamide, label accumulates almost exclusively in the orthophosphate pool and less than 1% enters the organic phosphate pool. (c) Specific activity measurements in unblocked cells indicate that Pi anion equilibrates first with its intracellular Pi pool. These initial findings imply that neither group translocation, nor energy coupling, influence Pi permeation into the human erythrocytes. (d) The relationship between 32P entry and extracellular Pi concentration is parabolic in the presence of chloride, and linear in the presence of sulfate. The kinetics of concentration dependent entrance cannot be examined and saturability of Pi entry cannot be identified under these conditions. (e) The competitive inhibitor arsenate partially inhibits the initial rate and steady-state flux of orthophosphate in erythrocytes treated with iodoacetamide to inhibit glycolysis. However, a significant portion of Pi transport escapes arsenate inhibition. (f) Activation energies for Pi entry, in nonglycolizing erythrocytes are much higher than those required by simple diffusion in an aqueous system. (g) Neither the inward or outward movement of Pi is modulated by trans-phosphate. These latter findings suggest that transport of phosphate across the human erythrocyte is compatible with slow facilitated diffusion with symmetry for influex and efflux. The transmembrane chemical distribution ratio, and the equilibrium flux of Pi were not different from normal in the X-LH erythrocyte. Nor did the extracellular Pi concentration, arsenate, or temperature affect Pi entry differently in the two types of cells. We dedjce that different gene products serve the diffusional type of Pi transport in the erythrocyte membrane and the saturable component of transepithelial absorption in the gut and kidney. Only the latter is affected by the X-LH mutation. The former is apparently present not only in erythrocytes but also in epithelial tissue, where it can serve the absorption of pharmacologic amounts of Pi in the therapeutic repair of the depleted phosphate pools in X-LH.

Fibroblast growth factor 23 in oncogenic osteomalacia and X-linked hypophosphatemia.

BACKGROUND: Mutations in fibroblast growth factor 23 (FGF-23) cause autosomal dominant hypophosphatemic rickets. Clinical and laboratory findings in this disorder are similar to those in oncogenic osteomalacia, in which tumors abundantly express FGF-23 messenger RNA, and to those in X-linked hypophosphatemia, which is caused by inactivating mutations in a phosphate-regulating endopeptidase called PHEX. Recombinant FGF-23 induces phosphaturia and hypophosphatemia in vivo, suggesting that it has a role in phosphate regulation. To determine whether FGF-23 circulates in healthy persons and whether it is elevated in those with oncogenic osteomalacia or X-linked hypophosphatemia, an immunometric assay was developed to measure it. METHODS: Using affinity-purified, polyclonal antibodies against [Tyr223]FGF-23(206-222)amide and [Tyr224]FGF-23(225-244)amide, we developed a two-site enzyme-linked immunosorbent assay that detects equivalently recombinant human FGF-23, the mutant form in which glutamine is substituted for arginine at position 179 (R179Q), and synthetic human FGF-23(207-244)amide. Plasma or serum samples from 147 healthy adults (mean [+/-SD] age, 48.4+/-19.6 years) and 26 healthy children (mean age, 10.9+/-5.5 years) and from 17 patients with oncogenic osteomalacia (mean age, 43.0+/-13.3 years) and 21 patients with X-linked hypophosphatemia (mean age, 34.9+/-17.2 years) were studied. RESULTS: Mean FGF-23 concentrations in the healthy adults and children were 55+/-50 and 69+/-36 reference units (RU) per milliliter, respectively. Four patients with oncogenic osteomalacia had concentrations ranging from 426 to 7970 RU per milliliter, which normalized after tumor resection. FGF-23 concentrations were 481+/-528 RU per milliliter in those with suspected oncogenic osteomalacia and 353+/-510 RU per milliliter (range, 31 to 2335) in those with X-linked hypophosphatemia. CONCLUSIONS: FGF-23 is readily detectable in the plasma or serum of healthy persons and can be markedly elevated in those with oncogenic osteomalacia or X-linked hypophosphatemia, suggesting that this growth factor has a role in phosphate homeostasis. FGF-23 measurements might improve the management of phosphate-wasting disorders.

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.

Serum insulin-like growth factor binding protein-3 in the hypophosphatemic mouse: decreased activity and abnormal modulation by dietary phosphate.

The hypophosphatemic mouse, the murine homologue of X-linked hypophosphatemia, is characterized by renal defects in phosphate reabsorption and 1,25-dihydroxy vitamin D3 (1,25(OH)2D3) production and by an osteoblast dysfunction. In view of the potential importance of insulin-like growth factors (IGFs) in the regulation of these processes and the role of IGF-binding proteins (IGFBPs) as modulators of IGF action, we asked whether Hyp mice have alterations in IGFs or IGFBPs. Using specific radioimmunoassays and Western ligand blot analysis, we evaluated serum levels of IGFs (IGF-1 and IGF-II) and IGFBPs, respectively, in normal and Hyp mice. We also examined the effect of dietary phosphatase on these parameters. Serum levels of IGF-1 and IGF-II in Hyp mice were not significantly different from those in normal mice, but IGFBP-3 levels were significantly lower (70% of normal, p < 0.05) in the mutant strain. The other IGFBP species appear unchanged. Phosphate supplementation normalized serum phosphate levels in Hyp mice and elicited a significant decrease in serum IGF-I levels (23%, p < 0.05) and a further deduction in IGFBP-3 (22%, p < 0.02). Phosphate deprivation induced hypophosphatemia IGF-II. The present results indicate that the low serum IGFBP-3 activity in Hyp mice is not related to hypophosphatemia per se. Based on the documented effects of parathyroid hormone (PTH) on IGF-I and IGFBP-3, we propose that the secondary hyperparathyroidism displayed by Hyp mice and its exacerbation by phosphate supplementation may contribute to low IGFBP-3 levels in control Hyp mice and to the decreases in serum IGF-I and IGFBP-3 in phosphate-supplemented Hyp mice.

Serum FGF23 levels in normal and disordered phosphorus homeostasis.

UNLABELLED: We investigated if the circulating levels of the phosphaturic factor FGF23 are elevated in subjects with XLH. Although we failed to find a statistically significant increase, FGF23 levels were significantly correlated with the degree of hypophosphatemia in XLH. In contrast, FGF23 levels were markedly increased in subjects with ESRD and correlated inversely with the degree of hyperphosphatemia. INTRODUCTION: Inactivating mutations of PHEX cause renal phosphate wasting in X-linked hypophosphatemic rickets (XLH) because of the accumulation of a phosphaturic hormone called phosphatonin. The recent discovery that FGF23 is the circulating phosphaturic factor in autosomal dominant hypophosphatemia raises the possibility that FGF23 is phosphatonin. METHODS: Fasting serum FGF23 levels and serum biochemical parameters were measured using a human FGF23 (C-terminal) ELISA assay in 11 subjects with XLH and 42 age-matched controls, 5 subjects with hypophosphatemia of unknown cause, and 14 hyperphosphatemic subjects with end stage renal disease (ESRD). Associations between variables were examined using the Spearman's correlation coefficient and linear regression analysis. RESULTS AND CONCLUSIONS: FGF23 (RU/ml) concentrations were not different (p = 0.11) between control and hypophosphatemic XLH subjects, but were significantly increased in hyperphosphatemic subjects with ESRD (p < 0.001). Western blot analysis found the presence of both full-length and C-terminal FGF23 fragments in serum from ESRD subjects. There was a strong inverse correlation between FGF23 and serum phosphorus (r = -0.60) and calcium and phosphorus (Ca x P) product (r = -0.65) in XLH, and a strong positive relationship between FGF23 and Pi (r = 0.50) and Ca x P product (r = 0.62) in ESRD. FGF23 levels were variably elevated in subjects with hypophosphatemia of unknown cause, one of which had tumor-induced osteomalacia (TIO). Removal of the tumor resulted in rapid reduction in serum FGF23 levels. These findings suggest that FGF23 has a possible role in mediating hypophosphatemia in XLH and TIO, but the overlapping levels of FGF23 in hypophosphatemic disorders and normal subjects indicate that serum phosphorus and FGF23 can also be independently regulated.

Evidence that low plasma 1,25-dihydroxyvitamin D causes intestinal malabsorption of calcium and phosphate in juvenile X-linked hypophosphatemic mice.

X-linked hypophosphatemic (Hyp) mice are a model for human sex-linked vitamin D-resistant rickets. We have reported intestinal malabsorption of calcium in young Hyp mice, and in this report we have explored the mechanism for it. To test for resistance of the intestine to 1,25(OH)2 vitamin D3, this hormone was continually infused via osmotic minipumps into 4-week-old normal and Hyp mice at 0, 17, 50 or 150 ng/kg/day. After 3 days, 45Ca and inorganic 32P were administered by gavage, and the mice were sacrificed on the fifth day. The Hyp mice showed responses to the hormone equivalent to the normal mice in terms of increased intestinal absorption of both 45Ca and 32P, increased plasma isotope levels, increased femoral isotope content, and increased duodenal and renal 9 kD vitamin D-dependent calcium-binding protein (calbindin-D9K; CaBP). Plasma 1,25(OH)2D was measured in these mice. There were significant correlations of plasma 1,25(OH)2D to the intestinal absorption of 45Ca and 32P and to duodenal and renal CaBP. Plasma 1,25(OH)2D was also measured in stock normal and Hyp mice and was found to be lower in 4-week-old Hyp mice than in 4-week-old normal mice (113 +/- 10 pM (n = 18) vs. 67 +/- 10 (n = 20), normal vs. Hyp, p less than .01), but unchanged at 13 weeks of age (77 +/- 13 (n = 13) vs. 70 +/- 15 (n = 15), NS). This observed difference in plasma 1,25(OH)2D between normal and Hyp mice at 4 weeks of age was sufficient to explain the observed normal-to-Hyp differences in intestinal absorption of 45Ca and duodenal and renal CaBP. It also explained 72 +/- 18% of the observed difference in 32P absorption. We conclude that Hyp mouse intestine is not resistant to 1,25(OH)2D and that the lower plasma 1,25(OH)2D of 4-week-old Hyp mice causes intestinal malabsorption of calcium and phosphate.

Plasma 24,25-dihydroxyvitamin D3 concentrations in X-linked hypophosphatemic mice: studies using mass fragmentographic and radioreceptor assays.

Previous studies have suggested that both plasma 24,25-dihydroxyvitamin D [24,25-(OH)2D] concentrations and renal 25-hydroxyvitamin D-24-hydroxylase activity are increased in mice with X-linked hypophosphatemia (Hyp mice). However, because the plasma levels of 24,25-(OH)2D seemed surprisingly high, we repeated these assays using two different techniques. Mass fragmentographic and radioreceptor assays were employed to compare the plasma concentrations of 25-hydroxyvitamin D (25-OHD) and 24,25-(OH)2D in normal mice with those in Hyp mice. These assays yielded 24,25-(OH)2D concentrations much lower than previously reported in mice (both normal and Hyp). The concentrations of 25-OHD3 and 24,25-(OH)2D3, determined by mass fragmentography, were lower in Hyp mice than in controls [25-OHD3, 9.7 +/- 0.4 versus 14.6 +/- 0.6 ng/ml, p less than 0.01; 24,25-(OH)2D3, 7.1 +/- 0.3 versus 10.4 +/- 0.4 ng/ml, p less than 0.01]. Plasma 25-OHD concentration was the main determinant of plasma 24,25-(OH)2D, and the ratio of 25-OHD3 to 24,25-(OH)2D3 obtained from mass fragmentographic measurements did not differ between the two groups (1.40 +/- 0.05 versus 1.36 +/- 0.03 ng/ml, NS in normal and Hyp groups, respectively). Separate measurement of plasma 25-OHD, 24,25-(OH)2D, and 25-OHD3-26,23-lactone by radioreceptor assay showed no difference between either plasma 24,25-(OH)2D, or the ratio of 25-OHD concentration to 24,25-(OH)2D concentration among Hyp and control animals. In neither study was plasma phosphate concentration related to the 25-OHD3:24,25-(OH)2D3 ratio.(ABSTRACT TRUNCATED AT 250 WORDS)

X-linked hypophosphatemic rickets: a study (with literature review) of linear growth response to calcitriol and phosphate therapy.

Not all children with X-linked hypophosphatemia (XLH) have demonstrated improved linear growth with calcitriol [1,25-(OH)2D3] and inorganic phosphate (Pi) therapy. To assess which factors are associated with a favorable growth response during this treatment, we retrospectively compared demographics and biochemical parameters of bone metabolism to the linear growth patterns of 20 children with XLH who were prepubertal and had not required osteotomy. A total of 15 patients had family histories consistent with XLH; 5 appeared to be sporadic cases. During 3 years of therapy, the growth velocities of 12 patients had been at or above the mean for age (good growers) and those of 8 patients had been below the mean (poor growers). Data from the two groups were contrasted. We found no difference between the good growers and poor growers before or after the 3 year period of therapy in mean age, dietary calcium, calcitriol dose or compliance, or Pi dose or compliance. Both groups increased their mean fasting serum Pi levels with treatment. The TmP/GFR (mean +/- SEM) of the good growers improved with therapy (1.9 +/- 0.2 to 2.6 +/- 0.2 mg/dl, p = 0.01), and their posttreatment value was higher compared to that of the poor growers (2.6 +/- 0.1 versus 2.2 +/- 0.1 mg/dl, p = 0.02). However, their enhanced TmP/GFR was not associated with a reduction in serum iPTH levels (before, 693 +/- 50; after, 688 +/- 76 pg/ml; p = 0.9). The Z test for binomial proportions showed that the group that grew well contained a disproportionate number of girls (10 of 12, p = 0.04). Our findings suggest that calcitriol may exert a direct effect on the renal tubule to improve Pi reclamation in XLH. The observation that heterozygous girls appear to respond better than hemizygous boys to calcitriol and Pi therapy provides evidence for a gene dosage effect in the expression of this X-linked dominant disorder.

Development and validation of a radioimmunoassay for mouse osteocalcin: paradoxical response in the Hyp mouse.

The hypophosphatemic (Hyp) mouse is a model for human familial hypophosphatemic rickets. To test the hypothesis that there is an osteoblastic defect in these animals, serum osteocalcin levels were measured in Hyp mice and their normal littermates. Furthermore, the effects of phosphorus deprivation, phosphorus loading, and 1,25-dihydroxyvitamin D3 administration on serum osteocalcin levels were examined. Osteocalcin was purified from mouse hindlimbs, and a polyclonal antibody to this material was produced in a goat. The antibody recognized native and decarboxylated mouse osteocalcin, but could not recognize osteocalcin from several other species. A RIA was developed which had a minimal detection limit of 0.4 nmol/liter (2.2 micrograms/liter) and half-maximal displacement at 2.7-3.3 nmol/liter (14.8-18.2 micrograms/liter). The intraassay coefficient of variation was 6.4%, while the interassay coefficient of variation was 12%. Dilutions of mouse serum samples varied by less than 15%. Analytical recovery was typically greater than 90%. Serum osteocalcin concentrations in Hyp and normal mice were shown to decrease with age. However, circulating osteocalcin levels in Hyp mice were higher than those in their normal littermates regardless of the age of the animal (P less than 0.001). One week of a high phosphorus diet resulted in an increase in serum phosphate in normal and Hyp mice, but serum osteocalcin concentrations were unaffected. On the other hand, dietary phosphorus deprivation for 4 weeks resulted in comparable hypophosphatemia in both Hyp and normal mice, and serum osteocalcin increased in both groups of animals. Intraperitoneal injection of 30 ng/day 1,25-dihydroxyvitamin D3 for 7 days resulted in a 215 +/- 33% increase in serum osteocalcin in normal animals, while the same regimen produced a 250 +/- 29% decrease in the Hyp mouse. Our results are consistent with the hypothesis that abnormal osteoblastic activity is present in Hyp mice. Furthermore, hypophosphatemia may be a general regulator of osteocalcin synthesis or secretion in the mouse.

Abnormal vitamin D metabolism in the X-linked hypophosphatemic mouse.

The vitamin D metabolites, 25-hydroxyvitamin D (250HD) and 1,25-dihydroxyvitamin D [1,25-(OH)2D], were measured in samples of plasma from normal and X-linked hypophosphatemic (Hyp) mice, which are a model for this common form a human vitamin D-resistant rickets. Each sample was the pooled plasma from the exsanguination of 10-15 mice. On stock diets plasma 1,25-(OH)2D was the same in Hyp mice as in normal mice [normal vs. Hyp, 70 +/- 4 vs. 66 +/- 4 pM; (mean +/- SE) n = 8; P = NS]. However, plasma 25OHD was lower in Hyp mice (59 +/- 4 vs. 37 +/- 4 nM; n = 6; P < 0.01). Since hypophosphatemia usually elevates plasms 1,25-(OH)2D in rats, we suspected that Hyp mice were unresponsive to hypophosphatemia. To test this, mice were challenged with a low phosphorus (P) diet for 6 days . A low P diet lowered plasma P and raised plasma calcium levels in both normal and Hyp mice. In both genotypes, the low P diet also increased magnesium levels in urine and transiently in plasma. The low P diet raised plasma 1,25-(OH)2D in normal mice, but lowered it in Hyp mice to nondetectable levels. There was no significant effect of the low P diet on plasma levels of 25OHD. We conclude that Hyp mice have a defective control system for plasma levels of 1,25-(OH)2D that does not respond to a low P stimulus with elevated plasma levels of the hormone.

Increased plasma 1,25-dihydroxyvitamin D after low calcium challenge in X-linked hypophosphatemic mice.

Hyp mice are a model for human X-linked hypophosphatemic (vitamin D-resistant) rickets. We have reported reduced plasma 25-hydroxyvitamin D (25OHD) and normal plasma 1,25-dihydroxyvitamin D [1,25-(OH)2D] levels and (2) failure of low phosphate diets to increase plasma 1,25-(OH)2D in Hyp mice. In this study, we tested the other principal regulatory system of plasma 1,25-(OH)2D by challenging with a low calcium diet. Normal and Hy p mice were fed a control or a low calcium diet for 3 or 7 days. Both normal and Hyp mice showed unchanged plasma phosphate levels and slightly but significantly reduced plasma calcium levels on the low calcium diet. The Hyp mice had significantly elevated plasma parathyroid hormone levels while on the low calcium diet. Plasma 25OHD and 1,25-(OH)2D were measured in the same 4-ml plasma sample by modifications of the Eisman method for 1,25-(OH)2D and the competitive binding assay of Haddad for 25OHD. Each sample was the pooled plasma of 10-14 mice at 13 weeks of age. On the control diet, plasma 1,25-(OH)2D levels were not significantly different in Hyp and normal mice. Plasma 1,25-(OH)2D was elevated in both genotypes on the low calcium diet, but the level in Hyp animals was significantly (P less than 0.05) higher than the level in normal mice. Plasma 25OHD was lower in Hyp than normal and was unaffected by the low calcium diet in either genotype. In summary, whereas Hyp mice do not respond to low phosphate challenge with elevated plasma 1,25-(OH)2D levels, they do respond to low calcium challenge.

Abnormal 24-hydroxylation of 25-hydroxyvitamin D in the X-linked hypophosphatemic mouse.

The effect of extracellular phosphate on the control of 25-hydroxyvitamin D3 24-hydroxylase was studied in normal mice and littermates with X-linked hypophosphatemic rickets (Hyp). 24-Hydroxylase activity and plasma concentrations of 24,25-dihydroxyvitamin D3 were significantly higher in Hyp mice than in normal mice when both groups were fed a normal diet containing 1.22% calcium (Ca) and 0.8% phosphorus (Pi). The differential in 24-hydroxylase activity was exaggerated when serum phosphate was reduced in normal mice by means of a low Pi diet or increased in Hyp mice by means of a high Pi diet. Differences in 24-hydroxylase activity between the two groups of mice were also demonstrated in the presence of varying Pi concentrations in vitro. Thus, in both Hyp and normal mice, 24-hydroxylase activity is influenced in a qualitatively similar manner by serum Pi. Plasma concentrations of 1,25-dihydroxyvitamin D3 were the same in normal and Hyp mice. The data are consistent with the hypothesis that control the renal metabolism of 25-hydroxyvitamin D3 in Hyp mice is reset such tht 24-hydroxylase activity is inappropriate high for the prevailing serum phosphate over a wide range of concentrations.

Transgenic mice overexpressing human fibroblast growth factor 23 (R176Q) delineate a putative role for parathyroid hormone in renal phosphate wasting disorders.

Fibroblast growth factor 23 (FGF23) is a recently characterized protein likely involved in the regulation of serum phosphate homeostasis. Increased circulating levels of FGF23 have been reported in patients with renal phosphate-wasting disorders, but it is unclear whether FGF23 is the direct mediator responsible for the decreased phosphate transport at the proximal renal tubules and the altered vitamin D metabolism associated with these states. To examine this question, we generated transgenic mice expressing and secreting from the liver human FGF23 (R176Q), a mutant form that fails to be degraded by furin proteases. At 1 and 2 months of age, mice carrying the transgene recapitulated the biochemical (decreased urinary phosphate reabsorption, hypophosphatemia, low serum 1,25-dihydroxyvitamin D(3)) and skeletal (rickets and osteomalacia) alterations associated with these disorders. Unexpectantly, marked changes in parameters of calcium homeostasis were also observed, consistent with secondary hyperparathyroidism. Moreover, in the kidney the anticipated alterations in the expression of hydroxylases associated with vitamin D metabolism were not observed despite the profound hypophosphatemia and increased circulating levels of PTH, both major physiological stimuli for 1,25-dihydroxyvitamin D(3) production. Our findings strongly support the novel concept that high circulating levels of FGF23 are associated with profound disturbances in the regulation of phosphate and vitamin D metabolism as well as calcium homeostasis and that elevated PTH levels likely also contribute to the renal phosphate wasting associated with these disorders.