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.     

Variable phosphate-mediated regulation of vitamin D metabolism in the murine hypophosphatemic rachitic/osteomalacic disorders

The familial hypophosphatemic (vitamin D-resistant) disorders are a variety of genetic and acquired syndromes that exhibit unexpected biochemical heterogeneity, manifest as variably abnormal or apparently normal regulation of 1,25-dihydroxyvitamin D [1,25-(OH)2D] production. Recently, we observed that Hyp and Gy mice, murine homologs of X-linked hypophosphatemic rickets, exhibit similarly disparate regulation of vitamin D metabolism. While Gy mice under basal conditions maintain an appropriate elevation (relative to hypophosphatemia) of renal 25-hydroxyvitamin D (25OHD)-1 alpha-hydroxylase, Hyp mice manifest only normal, not increased, enzyme activity. Whether such diversity results from maintenance of phosphate (P)-regulated 1,25-(OH)2D production in Gy mice and loss of this function in Hyp mice or from other variations remains unknown. Therefore, we examined the integrity of P-regulated enzyme activity in the Gy and Hyp mice by testing the effects of enzyme inhibition and alteration of the serum phosphorus concentration on 1,25-(OH)2D production. Initially, we discovered that inhibition of renal 25OHD-1 alpha-hydroxylase suppressed enzyme function in Hyp mice, but did not prevent expression of P-mediated activity in Gy mice. In this regard, while administration of a high calcium diet or 1,25-(OH)2D (0.4 ng/h, sc, for 48 h) resulted in a comparable inhibition of enzyme activity in Hyp (5.9 +/- 0.5 vs. 2.8 +/- 0.7 fmol/mg.min) and normal mice (4.4 +/- 0.6 vs. 2.0 +/- 0.2 fmol/mg.min), similar treatment did not effect complete inhibition of 25OHD-1 alpha-hydroxylase in Gy (10.3 +/- 0.6 vs. 4.9 +/- 0.3 fmol/mg.min) or P-depleted mice (10.2 +/- 0.5 vs. 5.1 +/- 0.4 fmol/mg.min). In accord with the apparent persistence of P-mediated stimulation of enzyme function in Gy mice, dietary P repletion in this mutant resulted in a serum phosphorus concentration similar to that of normal mice and decreased enzyme activity (4.0 +/- 0.8 fmol/mg.min) to a level no different from that expressed in controls (3.4 +/- 0.3 fmol/mg.min). However, in the absence of apparent P-mediated stimulation of enzyme activity, identical treatment of Hyp mice increased the serum phosphorus level comparably, but paradoxically enhanced 25OHD-1 alpha-hydroxylase (3.1 +/- 0.4 vs. 11.7 +/- 2.0 fmol/mg.min). Collectively, these data indicate that enhanced renal 25OHD-1 alpha-hydroxylase expressed in Gy mice and probably in related human diseases results from normally maintained P regulation of enzyme activity, an action absent or mutated in the genetically distinct Hyp mouse.     

Chronic inhibition of ERK1/2 signaling improves disordered bone and mineral metabolism in hypophosphatemic (Hyp) mice

The X-linked hypophosphatemic (Hyp) mouse carries a loss-of-function mutation in the phex gene and is characterized by hypophosphatemia due to renal phosphate (Pi) wasting, inappropriately suppressed 1,25-dihydroxyvitamin D [1,25(OH)?D] production, and rachitic bone disease. Increased serum fibroblast growth factor-23 concentration is responsible for the disordered metabolism of Pi and 1,25(OH)?D. In the present study, we tested the hypothesis that chronic inhibition of fibroblast growth factor-23-induced activation of MAPK signaling in Hyp mice can reverse their metabolic derangements and rachitic bone disease. Hyp mice were administered the MAPK inhibitor, PD0325901 orally for 4 wk. PD0325901 induced a 15-fold and 2-fold increase in renal 1α-hydroxylase mRNA and protein abundance, respectively, and thereby higher serum 1,25(OH)?D concentrations (115 ± 13 vs. 70 ± 16 pg/ml, P < 0.05), compared with values in vehicle-treated Hyp mice. With PD0325901, serum Pi levels were higher (5.1 ± 0.5 vs. 3 ± 0.2 mg/dl, P < 0.05), and the protein abundance of sodium-dependent phosphate cotransporter Npt2a, was greater than in vehicle-treated mice. The rachitic bone disease in Hyp mice is characterized by abundant unmineralized osteoid bone volume, widened epiphyses, and disorganized growth plates. In PD0325901-treated Hyp mice, mineralization of cortical and trabecular bone increased significantly, accompanied by a decrease in unmineralized osteoid volume and thickness, as determined by histomorphometric analysis. The improvement in mineralization in PD0325901-treated Hyp mice was confirmed by microcomputed tomography analysis, which showed an increase in cortical bone volume and thickness. These findings provide evidence that in Hyp mice, chronic MAPK inhibition improves disordered Pi and 1,25(OH)?D metabolism and bone mineralization.     

The mobilization of bone mineral by 1,25-dihydroxyvitamin D3 in hypophosphatemic rats.

Administration of 1,25-dihydroxyvitamin D3 to thyroparathyroidectomized, hypophosphatemic rats on a low-phosphorus diet increases serum inorganic phosphorus and calcium concentrations. Experiments utilizing such rats treated with 45Ca and 32P ten days before use revealed that the rise in serum phosphorus in response in 1,25-dihydroxy-vitamin D3 is derived at least in part from bone mineral. This suggests that under conditions of hypophosphatemia, 1,25-dihydroxyvitamin D3 can mobilize bone in the absence of parathyroid hormone.     

Investigation of the mechanism for abnormal renal 25-hydroxyvitamin D3-1-hydroxylase activity in the X-linked Hyp mouse

Renal mitochondria from mutant hypophosphatemic male mice (Hyp/Y) fed a vitamin D-deficient, low calcium diet synthesize significantly less 1,25-dihydroxyvitamin D3 than mitochondria from normal male (+/Y) littermates on the same diet. Kinetic studies reveal that maximum velocity (Vmax) for 25-hydroxyvitamin D3-1-hydroxylase (1-hydroxylase) is lower in Hyp/Y relative to +/Y mice (0.21 +/- 0.02 vs. 1.06 +/- 0.12 pmol/mg protein X min) whereas the apparent Michaelis-Menten constant (Km) for the reaction is not different in both genotypes (0.55 +/- 0.05 vs. 0.50 +/- 0.08 microM). The presence of an inhibitor for 1-hydroxylase activity in renal mitochondria of Hyp/Y mice was ruled out by estimating enzyme activity in mixtures of renal mitochondria from +/Y and Hyp/Y mice. Phosphate in the incubation medium stimulated 1-hydroxylase activity in +/Y mitochondria. In Hyp/Y mice, the stimulation achieved was smaller in magnitude and the added phosphate did not restore mutant 1-hydroxylase activity to normal. The vitamin D-deficient, low calcium diet led to a significant and comparable increase in serum PTH and urinary excretion of cAMP in +/Y and Hyp/Y, suggesting that the mutant strain had an appropriate PTH response to the diet-induced fall in serum calcium. Furthermore, the fractional excretion index of phosphate which is significantly greater in Hyp/Y than +/Y mice fed the control diet increased 3-fold in both genotypes fed the vitamin D-deficient, low calcium diet. These results suggest that the abnormal renal 1-hydroxylase response in Hyp mice is not the result of generalized renal resistance to PTH in the mutant strain and suggest that the defect in Hyp/Y mice may reside at a regulatory step subsequent to cAMP production.     

Disordered regulation of renal 25-hydroxyvitamin D-1alpha-hydroxylase gene expression by phosphorus in X-linked hypophosphatemic (hyp) mice

X-linked hypophosphatemic (Hyp) mice exhibit hypophosphatemia, impaired renal phosphate reabsorption, defective skeletal mineralization, and disordered regulation of vitamin D metabolism: In Hyp mice, restriction of dietary phosphorus induces a decrease in serum concentration of 1,25-dihydroxyvitamin D and renal activity of 25-hydroxyvitamin D-1alpha-hydroxylase (1alpha-hydroxylase), and induces an increase in renal activity of 25-hydroxyvitamin D-24-hydroxylase (24-hydroxylase). In contrast, in wild-type mice, phosphorus restriction stimulates renal 1alpha-hydroxylase gene expression and suppresses that of 24-hydroxylase. To determine the molecular basis for the disordered regulation of vitamin D metabolism in Hyp mice, we determined renal mitochondrial 1alpha-hydroxylase activity and the renal abundance of p450c1alpha and p450c24 mRNA in wild-type and Hyp mice fed either control, low-, or high-phosphorus diets for 5 d. In wild-type mice, phosphorus restriction increased 1alpha-hydroxylase activity and p450c1alpha mRNA expression by 6-fold and 3-fold, respectively, whereas in the Hyp strain the same diet induced changes of similar magnitude but opposite in direction. Phosphorus supplementation was without effect in wild-type mice, whereas in Hyp mice the same diet induced 3-fold and 2-fold increases, respectively, in enzyme activity and p450c1alpha mRNA abundance. In wild-type mice, both renal 1alpha-hydroxylase activity and p450c1alpha mRNA abundance varied inversely and significantly with serum phosphorus concentrations, whereas in Hyp mice the relationship between both renal parameters and serum phosphorus concentration was direct. In Hyp mice, phosphorus restriction induced a significant increase in renal p450c24 mRNA abundance, in contrast to the lack of effect observed in wild-type mice. The present findings demonstrate that regulation of both the p450c1alpha and p45024 genes by phosphorus is disordered in Hyp mice at the level of renal 1alpha-hydroxylase activity and renal p450c1alpha and p450c24 mRNA expression.     

Effect of the X-linked Hyp mutation and vitamin D status on induction of renal 25-hydroxyvitamin D3-24-hydroxylase

The present study was undertaken to evaluate the response of Hyp mice to regulators known to inhibit renal 25-hydroxyvitamin D3-1-hydroxylase (1-hydroxylase) and stimulate renal 25-hydroxyvitamin D-24-hydroxylase (24-hydroxylase). Renal mitochondrial metabolism of 25-hydroxyvitamin D3 (25OHD3) was initially examined in vitamin D- and calcium-deprived normal and mutant mice (with no detectable 24-hydroxylase) treated with either calcium, 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], or both calcium + 1,25-(OH)2D3. In normal mice, 1,25-(OH)2D3 treatment was more effective than calcium in turning off 1-hydroxylase and turning on 24-hydroxylase activity; serum calcium, however, was similarly increased by both treatments. Although calcium + 1,25-(OH)2D3 did not result in a further change in 25OHD3 metabolism in normal mice, a further elevation in serum calcium was apparent. In Hyp mice, treatment with calcium + 1,25-(OH)2D3 resulted in a greater decrease in 1-hydroxylase and a greater increase in 24-hydroxylase and in serum calcium than treatment with either agent alone. In spite of similar serum calcium levels in both genotypes, 24-hydroxylase was 20-fold, 3-fold, and 8-fold greater in Hyp mice relative to normals treated with calcium, 1,25-(OH)2D3, and calcium + 1,25-(OH)2D3, respectively. Kinetic studies revealed that the maximum velocity (Vmax) for induced 24-hydroxylase was 6-fold greater than normal in Hyp mice whereas the apparent Michaelis-Menten constant (Km) was not different in the two groups of calcium + 1,25-(OH)2D3-treated mice. The effect of 1,25-(OH)2D3 treatment on the above serum and renal parameters was also examined in vitamin D replete normal and Hyp mice. A sharp rise in serum phosphate was observed in 1,25-(OH)2D3-treated Hyp mice whereas normal littermates experienced marked hypercalcemia in response to treatment. Renal 24-hydroxylase was significantly stimulated by 1,25-(OH)2D3 treatment in both normal and Hyp mice and genotype differences were not apparent. The present study demonstrates that vitamin D- and calcium-deprived Hyp mice are more responsive to signals which induce 24-hydroxylase than normal littermates; Vmax for induced 24-hydroxylase is 6-fold greater in Hyp mice than in normal littermates whereas apparent Km is unchanged; the inhibitory control of 1-hydroxylase appears to be intact in the mutant strain; induced 24-hydroxylase is similar in vitamin D replete normal and Hyp mice; and vitamin D status can thus modify the response of both genotypes to treatment with 1,25-(OH)2D3.(ABSTRACT TRUNCATED AT 400 WORDS)     

Decreased nuclear uptake of 1,25-dihydroxyvitamin D3 by duodenal mucosal cells in the X-linked hypophosphatemic mouse

We have shown that there is a significant decrease in the nuclear uptake of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] by duodenal mucosal cells in the X-linked hypophosphatemic (Hyp) mouse. Duodenal mucosal cells prepared from control and Hyp mice were incubated with 1,25(OH)2[26,27-methyl-3H]D3 ([3H]-1,25(OH)2D3) for 30 min. to evaluate the time-course and perform saturation analysis. The results of time-course studies showed that saturation was attained in 30 min., reaching an average nuclear uptake of 10.4 fmol/tube in the control mice and 6.1 fmol/tube in the Hyp mice. The results of Scatchard analyses were as follows: dissociation constant (Kd) 5.71 X 10(-10) M and maximal binding sites 7.31 X 10(4) sites/cell in the control mice, and Kd 2.92 X 10(-10) M and maximal binding sites 4.88 X 10(4) sites/cell in the Hyp mice, the maximal binding sites of the latter showed a significant decrease (P less than 0.05) by Student's t test. In addition, there was no significant difference in the binding of [3H]-1,25(OH)2D3 to its residual cytosol receptors between the control and Hyp mice. On the basis of these data, we speculate that the reported resistance of Hyp mice to vitamin D may be due to decreased nuclear uptake of 1,25(OH)2D3 by their duodenal mucosal cells.     

Defective bone formation by transplanted Hyp mouse bone cells into normal mice

The hypophosphatemic (Hyp) mouse is a model for human X-linked hypophosphatemia (XLH). To test the hypothesis of an abnormal osteoblast function in XLH, periostea and osteoblasts isolated from normal and Hyp mice were transplanted im into normal and mutant mice. The thickness of the osteoid seams at the periphery of the bone nodules and the osteoid volume were measured in transplants as an index of bone formation. Impaired mineralization was evidenced in transplants of Hyp cells into Hyp mice by excessive osteoid thickness and volume compared with transplants of normal cells into normal mice. When normal cells were transplanted into mutant mice, the osteoid thickness and volume were markedly increased, demonstrating that the extracellular environment is critical for bone formation. In contrast, when Hyp cells were transplanted into normal mice, reduction, but not normalization, of the osteoid thickness and volume was observed. This abnormal bone formation supports the hypothesis of an osteoblast defect in the Hyp mouse.     

I alpha, 25-Dihydroxyvitamin D3 receptor in the X-linked hypophosphatemic mouse

We have attempted to determine if the resistance of the X-linked hypophosphatemic mouse to the actions of 1,25(OH)2D3 is due to abnormal cytosolic receptors for this hormone. Cytoplasmic 1,25-dihydroxy-vitamin D3 [1,25(OH)3D3] receptors were demonstrated in hypophosphatemic (HYP) mouse tissues including intestine, kidney and testis. Cytosol preparations from 14 murine tissues were prepared using hypertonic buffer and incubated for three hours at 0 C with 1,25(OH)2 [23,24(n)-(-3)H]D3 ([3H]-1,25(OH)2D3). The results of studies using 5-20% sucrose density gradients revealed th at cytosol preparations from intestine, kidney and testis exhibited a 3.2 S peak which ws specific for 1,25(OH)2D3. Scatchard analysis of intestinal, renal and testicular cytosol preparations of Hyp mice revealed a single class of noninteracting binding sites with a range of equilibrium dissociation constants (KD) of 0.9-3.5 X 10(-10) M, and a range of specific binding sites of 15-317 fmol/mg protein. There were no significant differences in values of KD, and numbers of specific binding sites among Hyp and control mice. We conclude the reported resistance of Hyp mice to vitamin D is not due to abnormal 1,24(OH)2D3 receptor in Hyp mouse.     

Transgenic mice expressing fibroblast growth factor 23 under the control of the alpha1(I) collagen promoter exhibit growth retardation, osteomalacia, and disturbed phosphate homeostasis

Mutations in the fibroblast growth factor 23 gene, FGF23, cause autosomal dominant hypophosphatemic rickets (ADHR). The gene product, FGF-23, is produced by tumors from patients with oncogenic osteomalacia (OOM), circulates at increased levels in most patients with X-linked hypophosphatemia (XLH) and is phosphaturic when injected into rats or mice, suggesting involvement in the regulation of phosphate (Pi) homeostasis. To better define the precise role of FGF-23 in maintaining Pi balance and bone mineralization, we generated transgenic mice that express wild-type human FGF-23, under the control of the alpha1(I) collagen promoter, in cells of the osteoblastic lineage. At 8 wk of age, transgenic mice were smaller (body weight = 17.5 +/- 0.57 vs. 24.3 +/- 0.37 g), exhibited decreased serum Pi concentrations (1.91 +/- 0.27 vs. 2.75 +/- 0.22 mmol/liter) and increased urinary Pi excretion when compared with wild-type littermates. The serum concentrations of human FGF-23 (undetectable in wild-type mice) was markedly elevated in transgenic mice (>7800 reference units/ml). Serum PTH levels were increased in transgenic mice (231 +/- 62 vs. 139 +/- 44 pg/ml), whereas differences in calcium and 1,25-dihydroxyvitamin D were not apparent. Expression of Npt2a, the major renal Na(+)/Pi cotransporter, as well as Npt1 and Npt2c mRNAs, was significantly decreased in the kidneys of transgenic mice. Histology of tibiae displayed a disorganized and widened growth plate and peripheral quantitative computerized tomography analysis revealed reduced bone mineral density in transgenic mice. The data indicate that FGF-23 induces phenotypic changes in mice resembling those of patients with ADHR, OOM, and XLH and that FGF-23 is an important determinant of Pi homeostasis and bone mineralization.     

Hypophosphatemia: mouse model for human familial hypophosphatemic (vitamin D-resistant) rickets.

A new dominant mutation in the laboratory mouse, hypophosphatemia (gene symbol Hyp), has been identified. The Hyp gene is located on the X-chromosome and maps at the distal end. Mutant mice are characterized by hypophosphatemia, bone changes resembling rickets, diminished bone ash, dwarfism, and high fractional excretion of phosphate anion (low net tubular reabsorption). Phosphate supplementation of the diet from wearning prevents the appearance of severe skeletal abnormalities. The hypophosphatemic male mouse resembles human males with X-linked hypophosphatemia and the Hyp gene is presemably homologous with the X-linked human gene. The mouse model should facilitate study of the defect in transport of plasma inorganic phosphate anion.     

Elevated levels of vitamin D-dependent calcium-binding protein (calbindin-D9k) in the osteosclerotic (oc) mouse

The osteosclerotic (oc) mouse is an osteopetrotic mutation that has recently been identified as having rickets associated with its osteopetrosis. The presence of this rachitic lesion, unexplainable from a nutritional standpoint, prompted an investigation into the vitamin D endocrine system in these animals. The developmental appearance of vitamin D-dependent calcium-binding protein (calbindin-D9k) and alkaline phosphatase was studied in oc mutant and normal mice from birth to weaning, as were serum concentrations of 25-hydroxyvitamin D3 (25OHD3), 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], calcium, and phosphorus. Intestinal and renal calbindin-D9k levels were markedly and precociously elevated (4- to 9-fold) in young suckling, but not newborn, mutant mice compared to values in normal controls. Serum 25OHD3 levels were very low to undetectable in 2-week-old mutant mice compared to normal values, while 1,25-(OH)2D3 levels were 6 times higher in mutants. The exact cause of this premature induction in mutants is unknown, but may be due to elevated circulating levels of 1,25-(OH)2D. Alkaline phosphatase activity was similar between phenotypes at all ages. These studies indicate that the rachitic lesion present in oc mutants may be the result of some inherited disorder in vitamin D metabolism in these animals. Alternatively, these data are also consistent with a normal appropriate response to hypocalcemia and hypophosphatemia resulting from decreased osteoclastic bone resorption.     

Hemangiopericytoma-induced osteomalacia: tumor transplantation in nude mice causes hypophosphatemia and tumor extracts inhibit renal 25-hydroxyvitamin D 1-hydroxylase activity

Although more than 50 patients with the tumor-induced osteomalacia syndrome, characterized by remission of unexplained osteomalacia after resection of a coexisting tumor, have been reported, the pathogenesis of this syndrome is still not clear. We investigated the cause of biopsy-confirmed osteomalacia which was resistant to treatment with 1 alpha-hydroxyvitamin D3 in a 54-yr-old man. He had severe hypophosphatemia, a high serum alkaline phosphatase level, a low plasma 1,25-dihydroxyvitamin D level, and remarkably increased urinary phosphorus excretion. A tumor, with histological characteristics of a hemangiopericytoma, was found on his left thigh. After surgical removal of this tumor, his plasma 1,25-dihydroxyvitamin D and serum phosphorus levels increased to normal levels, and his bone pain subsided. The tumor was transplanted to athymic nude mice. A nodule formed in each mouse, with histological features identical to those of the original tumor, and the tumor-bearing mice had hypophosphatemia, high serum alkaline phosphatase levels, and increased urinary phosphorus excretion. When extracts of the original tumor were added to primary cultures of renal tubular cells, renal cAMP levels did not change, but 25-hydroxyvitamin D-1 alpha-hydroxylase activity was significantly inhibited. These data indicate tumoral production of some humoral factor(s) inhibiting 25-hydroxyvitamin D-1 alpha-hydroxylase activity and phosphorus reabsorption unrelated to adenylate cyclase-cAMP production in proximal renal tubules.     

Tumor-induced osteomalacia. Kinetics of calcium, phosphorus, and vitamin D metabolism and characteristics of bone histomorphometry

A patient with a mesenchymal tumor and hypophosphatemic osteomalacia was studied before and after tumor excision. Initial laboratory values included normal serum calcium, decreased serum phosphorus and tubular reabsorption of phosphate, undetectable 1,25-dihydroxyvitamin D, and normal parathyroid hormone. Histomorphometry of a bone biopsy specimen showed evidence of increased osteoclastic bone resorption. By 16 hours after tumor removal, 1,25-dihydroxyvitamin D level had normalized, but serum phosphorus level was unchanged; at 28 hours, both serum phosphorus value and tubular reabsorption of phosphate were within normal limits. It is concluded that tumor removal is associated with rapid correction both of 1,25-dihydroxyvitamin D production and of renal phosphate wasting. Increased bone resorption suggests the production of an osteoclast activator by the tumor and may explain the typically normal serum calcium value in this disorder.     

Vitamin D-dependent calcium binding proteins in the kidney and intestine of the X-linked hypophosphatemic mouse: changes with age and responses to 1,25-dihydroxycholecalciferol

We have previously observed decreased intestinal 9 kilodalton (kd) vitamin D-dependent calcium binding protein (CaBP) and decreased calcium absorption in juvenile X-linked hypophosphatemic (Hyp) mice. The present studies were undertaken to examine whether the kidney CaBPs (9 kd and 28 kd) are also affected in young Hyp mice and to investigate the ability of 1,25-dihydroxycholecalciferol [1,25-(OH)2D3] to increase CaBP in the intestine and kidney. The 28 kd CaBP and the 9 kd CaBP were measured in the kidneys and the 9 kd CaBP in the intestines of normal and Hyp mice from 1 week to 40 weeks of age. At all times between 3 and 6 weeks, intestinal CaBP in Hyp mice was decreased by more than 50% (P less than 0.005-0.001) and no significant decrease was present in the adult Hyp mice (12 and 40 weeks of age). By contrast, both kidney CaBPs were decreased only slightly in young Hyp mice. Between 1 and 6 weeks of age, the 9 kd CaBP in Hyp mice was 82% +/- 4% of control (P less than 0.001) and the 28 kd protein was 89% +/- 3% of control (P less than 0.001). Minipumps containing 1,25-(OH)2D3 or vehicle were implanted in 4-week and 13-week-old Hyp mice for 3 days to provide a dose of 0.12 micrograms/kg mouse X day. The 9 kd CaBP was increased approximately 3-fold (P less than 0.001) by 1,25-(OH)2D3 in the intestines of Hyp mice at both ages. The 9 kd kidney CaBP in Hyp mice also was increased by 1,25-(OH)2D3 treatment at both ages, but only by 33-52%. The 28 kd CaBP in the kidney was not affected by 1,25-(OH)2D3 treatment of Hyp mice at either age. We conclude that (9 kd and 28 kd) CaBPs levels in both intestine and kidney are decreased in juvenile Hyp mice although to much different degrees. The administration of 1,25-(OH)2D3 to Hyp mice increases the 9 kd CaBP in both intestine and kidneys, whereas the renal 28 kd CaBP is unaffected.     

Abnormal regulation of 25-hydroxyvitamin D3-1 alpha-hydroxylase activity by calcium and calcitonin in renal cortex from hypophosphatemic (Hyp) mice

25-Hydroxyvitamin D3-1 alpha-hydroxylase activity was assayed in primary serum-free monolayer tissue culture of renal cortical cells from hypophosphatemic (Hyp) mice and normal litter mates. Morphological and growth characteristics of cells from the two genotypes were indistinguishable. Basal enzyme activity was not significantly different in either type of cell over a wide range of substrate concentration. The enzyme from both genotypes was stimulated by PTH and suppressed by increased phosphate concentration in the culture medium. Whereas 1 alpha-hydroxylase activity in cells from normal mice was increased in low calcium medium and suppressed in high calcium medium, the enzyme in cells from Hyp mice was not altered by similar changes in the medium calcium concentration. Salmon calcitonin caused a significant increase in 1 alpha-hydroxylase in cells from normal mice, but did not stimulate enzyme activity in cells from Hyp mice. These studies indicate that control of 1 alpha-hydroxylase activity is abnormal in renal cortical cells from Hyp mice. Impaired control of this enzyme could result in the inappropriately low circulating concentrations of 1,25-dihydroxyvitamin D3 that have been observed in humans with hypophosphatemic rickets and in the relatively low activity of 1 alpha-hydroxylase in renal cortical homogenates of Hyp mice compared to that in normal mice on a low phosphate diet.     

The Gy mutation: another cause of X-linked hypophosphatemia in mouse.

An X-linked dominant mutation (gyro, gene symbol Gy) in the laboratory mouse causes hypophosphatemia, rickets/osteomalacia, circling behavior, inner ear abnormalities, and sterility in males and a milder phenotype in females. Gy maps closely (crossover value 0.4-0.8%) to another X-linked gene (Hyp) that also causes hypophosphatemia in the mouse. Gy and Hyp genes have similar quantitative expression in serum phosphorus values, renal excretion of phosphate, and impairment of Na+/phosphate cotransport by renal brush-border membrane vesicles. These findings indicate that independent translation products of two X-linked genes serve phosphate transport in mouse kidney and thereby control phosphate content of extracellular fluid. The Gy translation product, unlike the Hyp product, is also expressed in the inner ear. These findings have implications for our understanding of the human counterpart known as "X-linked hypophosphatemia."     

Effects of the administration of phosphate on nuclear 1,25-dihydroxyvitamin D3 uptake by duodenal mucosal cells of Hyp mice

Effects of the administration of phosphate on nuclear 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] uptake by duodenal mucosal cells of Hyp mice were investigated. In Hyp mice fed a high phosphate diet (1.1% Ca and 2.0% phosphate) for 2 weeks, maximal nuclear 1,25-(OH)2D3 binding by duodenal mucosal cells is significantly increased from 5.01 +/- 0.49 x 10(3) to 8.23 +/- 1.10 x 10(3) sites/cell (P less than 0.05). No significant change was observed in normal mice fed the same diet. The serum phosphate concentration of Hyp mice increased significantly (P less than 0.01), whereas no significant change was found in normal mice. On this regimen, serum calcium, urinary cAMP to creatinine ratio, and cytosolic 1,25-(OH)2D3 receptor number in Hyp mice were not changed significantly. On the basis of these data, we speculate that the recovery of serum phosphate in Hyp mice fed a high phosphate diet affects the recovery of nuclear 1,25-(OH)2D3 uptake by duodenal mucosal cells. The mechanism for this recovery is not related to either the secondary hyperparathyroidism or the change in cytosolic 1,25-(OH)2D3 receptor content but, rather, to increased binding of 1,25-(OH)2D3-receptor complex to nuclei. Hypophosphatemia, therefore, appears to play a role in the vitamin D resistance in Hyp mice.     

Parathyroid hormone sensitivity in familial X-linked hypophosphatemic rickets

X-Linked hypophosphatemic rickets is associated with low or normal serum 1,25-dihydroxyvitamin D [1,25-(OH)2D] concentrations despite low circulating levels of inorganic phosphate. It is generally believed that the enzyme 25-hydroxyvitamin D 1 alpha-hydroxylase does not respond appropriately to either hypophosphatemia or PTH in this condition. We performed 6-h human PTH-(1-34) infusions in five patients with X-linked hypophosphatemic rickets who were receiving vitamin D and phosphate therapy. We measured changes in serum 1,25-(OH)2D, serum calcium, urinary nephrogenous cAMP, and phosphate clearance. Human PTH-(1-34) caused a rise in serum calcium, a rise in nephrogenous cAMP, a fall in renal phosphate reabsorption, and, in particular, a rise in serum 1,25-(OH)2D. All of these responses were indistinguishable from those in normal subjects or patients with surgical or idiopathic hypoparathyroidism. The population studied was not homogeneous, and in one elderly man with mild renal impairment serum 1,25-(OH)2D concentrations did not increase. Nevertheless, these results suggest that absolute PTH resistance is not a feature of X-linked hypophosphatemic ricket, although subtle forms of resistance at the level of the 25-hydroxyvitamin D 1 alpha-hydroxylase enzyme are not excluded by these data.