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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)2D] production, and rachitic bone disease. Increased serum fibroblast growth factor-23 concentration is responsible for the disordered metabolism of Pi and 1,25(OH)2D. 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)2D 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)2D metabolism and bone mineralization.

Synthesis of the hormone 1,25-dihydroxyvitamin D, the biologically active form of vitamin D, occurs in the kidney and is catalyzed by the mitochondrial cytochrome P450 enzyme, 25-hydroxyvitamin D-1α-hydroxylase (1α-hydroxylase). We sought to characterize the effects of changes in dietary phosphorus on the kinetics of renal mitochondrial 1α-hydroxylase activity and the renal expression of P450c1α and P450c24 mRNA, to localize the nephron segments involved in such regulation, and to determine whether transcriptional mechanisms are involved. In intact mice, restriction of dietary phosphorus induced rapid, sustained, approximately 6- to 8-fold increases in renal mitochondrial 1α-hydroxylase activity and renal P450c1α mRNA abundance. Immunohistochemical analysis of renal sections from mice fed the control diet revealed the expression of 1α-hydroxylase protein in the proximal convoluted and straight tubules, epithelial cells of Bowman’s capsule, thick ascending limb of Henle’s loop, distal tubule, and collecting duct. In mice fed a phosphorusrestricted diet, immunoreactivity was significantly increased in the proximal convoluted and proximal straight tubules and epithelial cells of Bowman’s capsule, but not in the distal nephron. Dietary phosphorus restriction induced a 2-fold increase in P450c1α gene transcription, as shown by nuclear run-on assays. Thus, the increase in renal synthesis of 1,25-dihydroxyvitamin D induced in normal mice by restricting dietary phosphorus can be attributed to an increase in the renal abundance of P450c1α mRNA and protein. The increase in P450c1α gene expression, which occurs exclusively in the proximal renal tubule, is due at least in part to increased transcription of the P450c1α gene.

Several reports have suggested that the regulation of renal 1,25-dihydroxyvitamin D [1,25-(OH)(2)D] synthesis by extracellular phosphate (Pi) is dependent on normal transepithelial Pi transport by the renal tubule. Mice homozygous for the disrupted Na/Pi cotransporter gene Npt2 (Npt2(-/-)) exhibit renal Pi wasting, an approximately 85% decrease in renal brush border membrane Na/Pi cotransport, hypophosphatemia, and an increase in serum 1,25-(OH)(2)D concentration. We undertook 1) to determine the mechanism for the increased circulating levels of 1,25-(OH)(2)D in Npt2(-/-) mice and 2) to establish whether renal 1alpha-hydroxylase was appropriately regulated by dietary Pi in the absence of Npt2 gene expression. On a control diet, the 2.5-fold increase in the serum 1,25-(OH)(2)D concentration in Npt2(-/-) mice, relative to that in Npt2(+/+) littermates, is associated with a corresponding increase in renal mitochondrial 25-hydroxyvitamin D-1 alpha-hydroxylase (1 alpha-hydroxylase) activity and messenger RNA (mRNA) abundance. A low Pi diet elicits an increase in serum 1,25-(OH)(2)D concentration, renal 1alpha-hydroxylase activity, and mRNA abundance in Npt2(+/+) and Npt2(-/-) mice to similar levels in both mouse strains. A high Pi diet has no effect on serum 1,25-(OH)(2)D concentration, renal 1 alpha-hydroxylase activity, or mRNA abundance in Npt2(+/+) mice, but normalizes these parameters in Npt2(-/-) mice. In addition, renal 24-hydroxylase mRNA abundance is significantly reduced in Npt2(-/-) mice compared with that in Npt2(+/+) mice under all dietary conditions. In summary, we demonstrate that 1) increased renal synthesis of 1,25-(OH)(2)D is responsible for the increased serum 1,25-(OH)(2)D concentration in Npt2(-/-) mice; and 2) renal 1alpha-hydroxylase gene expression is appropriately regulated by dietary manipulation of serum Pi in both Npt2(+/+) and Npt2(-/-) mice. Thus, intact renal Na/Pi cotransport is not required for the regulation of renal 1alpha-hydroxylase by Pi.

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 (r2 = 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 1α-hydroxylase activity and renal 1α-hydroxylase (P450c1α) mRNA abundance were significantly higher than in mice fed the higher Pi diets and varied inversely with serum FGF-23 concentrations (r2 = 0.86 and r2 = 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.

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.