Increased renal catabolism of 1,25-dihydroxyvitamin D3 in murine X-linked hypophosphatemic rickets.

The hypophosphatemic (Hyp) mouse, a murine homologue of human X-linked hypophosphatemic rickets, is characterized by renal defects in brush border membrane phosphate transport and vitamin D3 metabolism. The present study was undertaken to examine whether elevated renal 25-hydroxyvitamin D3-24-hydroxylase activity in Hyp mice is associated with increased degradation of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] by side chain oxidation. Metabolites of 1,25(OH)2D3 were separated by HPLC on Zorbax SIL and identified by comparison with standards authenticated by mass spectrometry. Production of 1,24,25-trihydroxyvitamin D3, 24-oxo-1,25-dihydroxyvitamin D3, and 24-oxo-1,23,25-trihydroxyvitamin D3 was twofold greater in mitochondria from mutant Hyp/Y mice than from normal +/Y littermates. Enzyme activities, estimated by the sum of the three products synthesized per milligram mitochondrial protein under initial rate conditions, were used to estimate kinetic parameters. The apparent Vmax was significantly greater for mitochondria from Hyp/Y mice than from +/Y mice (0.607 +/- 0.064 vs. 0.290 +/- 0.011 pmol/mg per protein per min, mean +/- SEM, P less than 0.001), whereas the apparent Michaelis-Menten constant (Km) was similar in both genotypes (23 +/- 2 vs. 17 +/- 5 nM). The Km for 1,25(OH)2D3 was approximately 10-fold lower than that for 25-hydroxyvitamin D3 [25(OH)D3], indicating that 1,25(OH)2D3 is perhaps the preferred substrate under physiological conditions. In both genotypes, apparent Vmax for 25(OH)D3 was fourfold greater than that for 1,25(OH)2D3, suggesting that side chain oxidation of 25(OH)D3 may operate at pharmacological concentrations of substrate. The present results demonstrate that Hyp mice exhibit increased renal catabolism of 1,25(OH)2D3 and suggest that elevated degradation of vitamin D3 hormone may contribute significantly to the clinical phenotype in this disorder.

Abnormal regulation of renal vitamin D catabolism by dietary phosphate in murine X-linked hypophosphatemic rickets.

Hyp mice exhibit increased renal catabolism of vitamin D metabolites by the C-24 oxidation pathway (1988. J. Clin. Invest. 81:461-465). To examine the regulatory influence of dietary phosphate on the renal vitamin D catabolic pathway in Hyp mice, we measured C-24 oxidation of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) in renal mitochondria isolated from Hyp mice and normal littermates fed diets containing 0.03% (low-Pi), 1% (control-Pi), and 1.6% (high-Pi) phosphate. In normal mice the low-Pi diet led to a rise in serum 1,25(OH)2D (22.2 +/- 1.8 to 48.1 +/- 6.8 pg/ml, P less than 0.05) and no change in C-24 oxidation products (0.053 +/- 0.006 to 0.066 +/- 0.008 pmol/mg protein per min) when compared with the control diet. In Hyp mice the low-Pi diet elicited a fall in serum 1,25(OH)2D (21.9 +/- 1.2 to 8.0 +/- 0.2 pg/ml, P less than 0.05) and a dramatic increase in C-24 oxidation products (0.120 +/- 0.017 to 0.526 +/- 0.053 pmol/mg protein per min, P less than 0.05) when compared with the control diet. The high-Pi diet did not significantly alter serum levels of 1,25(OH)2D or C-24 oxidation products in normal mice. Hyp mice on the high-Pi diet experienced a rise in serum 1,25(OH)2D (21.9 +/- 1.2 to 40.4 +/- 7.3, P less than 0.05) and a fall in C-24 oxidation products (0.120 +/- 0.017 to 0.043 +/- 0.007 pmol/mg protein per min, P less than 0.05). The present results demonstrate that the defect in C-24 oxidation of 1,25(OH)2D3 in Hyp mice is exacerbated by phosphate depletion and corrected by phosphate supplementation. The data suggest that the disorder in vitamin D metabolism in the mutant strain is secondary to the perturbation in phosphate homeostasis.

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.

Effects of Npt2 gene ablation and low-phosphate diet on renal Na(+)/phosphate cotransport and cotransporter gene expression.

The renal Na(+)/phosphate (Pi) cotransporter Npt2 is expressed in the brush border membrane (BBM) of proximal tubular cells. We examined the effect of Npt2 gene knockout on age-dependent BBM Na(+)/Pi cotransport, expression of Na(+)/Pi cotransporter genes Npt1, Glvr-1, and Ram-1, and the adaptive response to chronic Pi deprivation. Na(+)/Pi cotransport declines with age in wild-type mice (Npt2(+/+)), but not in mice homozygous for the disrupted Npt2 allele (Npt2(-/-)). At all ages, Na(+)/Pi cotransport in Npt2(-/-) mice is approximately 15% of that in Npt2(+/+) littermates. Only Npt1 mRNA abundance increases with age in Npt2(+/+) mice, whereas Npt1, Glvr-1, and Ram-1 mRNAs show an age-dependent increase in Npt2(-/-) mice. Pi deprivation significantly increases Na(+)/Pi cotransport, Npt2 protein, and mRNA in Npt2(+/+) mice. In contrast, Pi-deprived Npt2(-/-) mice fail to show the adaptive increase in transport despite exhibiting a fall in serum Pi. We conclude that (a) Npt2 is a major determinant of BBM Na(+)/Pi cotransport; (b) the age-dependent increase in Npt1, Glvr-1, and Ram-1 mRNAs in Npt2(-/-) mice is insufficient to compensate for loss of Npt2; and (c) Npt2 is essential for the adaptive BBM Na(+)/Pi cotransport response to Pi deprivation.

Pex/PEX tissue distribution and evidence for a deletion in the 3' region of the Pex gene in X-linked hypophosphatemic mice.

PEX, a phosphate-regulating gene with homology to endopeptidases on the X chromosome, was recently identified as the candidate gene for X-linked hypophosphatemia. In the present study, we cloned mouse and human Pex/PEX cDNAs encoding part of the 5' untranslated region, the protein coding region, and the entire 3' untranslated region, determined the tissue distribution of Pex/PEX mRNA, and characterized the Pex mutation in the murine Hyp homologue of the human disease. Using the reverse transcriptase/polymerase chain reaction (RT/PCR) and ribonuclease protection assays, we found that Pex/PEX mRNA is expressed predominantly in human fetal and adult mouse calvaria and long bone. With RNA from Hyp mouse bone, an RT/PCR product was generated with 5' but not 3' Pex primer pairs and a protected Pex mRNA fragment was detected with 5' but not 3' Pex riboprobes by ribonuclease protection assay. Analysis of the RT/PCR product derived from Hyp bone RNA revealed an aberrant Pex transcript with retention of intron sequence downstream from nucleotide 1302 of the Pex cDNA. Pex mRNA was not detected on Northern blots of poly (A)+ RNA from Hyp bone, while a low-abundance Pex transcript of approximately 7 kb was apparent in normal bone. Southern analysis of genomic DNA from Hyp mice revealed the absence of hybridizing bands with cDNA probes from the 3' region of the Pex cDNA. We conclude that Pex/PEX is a low-abundance transcript that is expressed predominantly in bone of mice and humans and that a large deletion in the 3' region of the Pex gene is present in the murine Hyp homologue of X-linked hypophosphatemia.

Renal Na(+)-phosphate cotransport in murine X-linked hypophosphatemic rickets. Molecular characterization.

The X-linked Hyp mouse is characterized by a specific defect in proximal tubular phosphate (Pi) reabsorption that is associated with a decrease in Vmax of the high affinity Na(+)-Pi cotransport system in the renal brush border membrane. To understand the mechanism for Vmax reduction, we examined the effect of the Hyp mutation on renal expression of Na(+)-Pi cotransporter mRNA and protein. Northern hybridization of renal RNA with a rat, renal-specific Na(+)-Pi cotransporter cDNA probe (NaPi-2) (Magagnin et al. 1993. Proc. Natl. Acad. Sci. USA. 90:5979-5983.) demonstrated a reduction in a 2.6-kb transcript in kidneys of Hyp mice relative to normal littermates (NaPi-2/beta-actin mRNA = 57 +/- 6% of normal in Hyp mice, n = 6, P < 0.01). Na(+)-Pi cotransport, but not Na(+)-sulfate cotransport, was approximately 50% lower in Xenopus oocytes injected with renal mRNA extracted from Hyp mice when compared with that from normal mice. Hybrid depletion experiments documented that the mRNA-dependent expression of Na(+)-Pi cotransport in oocytes was related to NaPi-2. Western analysis demonstrated that NaPi-2 protein is also significantly reduced in brush border membranes of Hyp mice when compared to normals. The present data demonstrate that the specific reduction in renal Na(+)-Pi cotransport in brush border membranes of Hyp mice can be ascribed to a proportionate decrease in the abundance of Na(+)-Pi cotransporter mRNA and protein.

Phorbol myristate acetate activates protein kinase C, stimulates the phosphorylation of endogenous proteins and inhibits phosphate transport in mouse renal tubules.

Calcium-activated, phospholipid-dependent protein kinase (protein kinase C) has been implicated in the regulation of transport processes in a variety of tissues and cell lines. To establish whether protein kinase C participates in the regulation of renal phosphate transport, we examined the effect of phorbol myristate acetate (PMA), a potent activator of protein kinase C, on phosphate uptake in fresh preparations of mouse renal tubules, and we correlated the changes in transport activity with protein kinase C activation and phosphorylation of endogenous proteins. PMA inhibited Na+-dependent phosphate transport, elicited a rapid translocation of protein kinase C from the cytosolic to the particulate fraction and stimulated the phosphorylation of endogenous substrates in the cytosolic and brush border membrane fractions. Effects of PMA were maximal after a 10 min incubation of the tubules with the activator. 4 alpha-Phorbol, an inert analogue of PMA, did not elicit any of these effects. The present results demonstrate a temporal correlation between inhibition of Na+-dependent phosphate transport, translocation and activation of protein kinase C, and phosphorylation of endogenous proteins in mouse renal tubules. These data suggest that protein kinase C may play a regulatory role in phosphate transport in mammalian kidney.

Effect of phosphonoformic acid, dietary phosphate and the Hyp mutation on kinetically distinct phosphate transport processes in mouse kidney.

We examined the kinetics of phosphate transport in mouse renal brush-border membrane vesicles under initial rate (6 s), trans zero, voltage clamp conditions. Two kinetically distinct Na+-dependent phosphate transport processes were identified: a high-affinity, low-capacity system (Km, 0.09 +/- 0.02 mM; Vmax, 539 +/- 50 pmol/mg protein per 6 s) and a low-affinity, high-capacity system (Km, 1.28 +/- 0.35 mM; Vmax, 1677 +/- 198 pmol/mg protein per 6 s). The high-affinity system was inhibited competitively by 1 mM phosphonoformic acid (PFA) (apparent Ki, 0.31 +/- 0.03 mM) and completely abolished by 20 mM PFA; the low-affinity system was insensitive to 1 mM PFA and was inhibited competitively by 20 mM PFA (apparent Ki, 9.03 +/- 1.21 mM). Dietary phosphate deprivation elicited a significant increase in Vmax of both high- and low-affinity phosphate transport systems whereas the X-linked Hyp mutation caused a 50% decrease in Vmax of the high-affinity system with no change in the low-affinity system. Phosphate deprivation of Hyp mice elicited a 3.5-fold increase in Vmax of the high-affinity system. Neither diet nor mutation significantly altered the apparent Km values of either phosphate transport process. We conclude that (1) mouse kidney brush-border membranes have two distinct Na+-dependent phosphate transport systems which differ in affinity and capacity; (2) both processes participate in the adaptive response to dietary phosphate restriction; (3) only the high-affinity system is impaired by the X-linked Hyp mutation.

Different molecular sizes for Na(+)-dependent phosphonoformic acid binding and phosphate transport in renal brush border membrane vesicles.

We compared several features of Na(+)-dependent phosphono[14C]formic acid (PFA) binding and Na(+)-dependent phosphate transport in rat renal brush border membrane vesicles. From kinetic analyses, we estimated an apparent Km for PFA binding of 0.86 mM, an order of magnitude greater than that for phosphate and the high-affinity phosphate transport system. A hyperbolic Na(+)-saturation curve for PFA binding and a sigmoidal Na(+)-saturation curve for phosphate transport were demonstrated; based on these data, we estimated stoichiometries of 1:1 for Na+/PFA and 2:1 for Na+/phosphate. By radiation inactivation analysis, target sizes for brush border membrane protein(s) mediating Na(+)-dependent PFA binding and Na(+)-dependent phosphate transport corresponded to molecular masses of 555 +/- 32 kDa and 205 +/- 36 kDa, respectively. Similar analysis of the phosphate-inhibitable component of Na(+)-dependent PFA binding gave a target size of 130 +/- 28 kDa. We also demonstrated that phosphate deprivation, which elicits a 2.6-fold increase in brush border membrane Na(+)-dependent phosphate transport, had no effect on either Na(+)-dependent PFA binding or on the target size for PFA binding. However, phosphate deprivation appeared to increase the target size for phosphate transport (from 255 +/- 32 to 335 +/- 75 kDa (P less than 0.01]. In summary, we present evidence for several differences between Na(+)-dependent PFA binding and Na(+)-dependent phosphate transport in rat renal brush border membrane vesicles and suggest that PFA may not interact exclusively with the proteins mediating Na(+)-phosphate co-transport.

Renal Na(+)-phosphate cotransport in X-linked Hyp mice responds appropriately to Na+ gradient, membrane potential, and pH.

To investigate the mechanism for the 50% decrease in Vmax of the high-affinity phosphate transport system in the renal brush-border membrane of X-linked Hyp mice, we compared the effects of external Na+ concentration, membrane potential, pH, phosphonoformic acid (PFA), and arsenate on Na(+)-Pi cotransport in brush-border membrane vesicles prepared from normal mice and Hyp littermates. The affinity of the Na(+)-Pi cotransport system for Na+ (apparent Km = 60 +/- 7 and 64 +/- 2 mM for normal and Hyp mice, respectively) and the Na(+)-Pi stoichiometry estimated from Hill plots (2.5 +/- 0.2 and 2.9 +/- 0.6 for normal and Hyp mice, respectively) were similar in brush-border membranes of both strains. Inside-negative membrane potential, generated by anions of different permeabilities, stimulated Na(+)-Pi cotransport and inside-positive membrane potential generated by valinomycin, and a K+ gradient (outside greater than inside) inhibited Na(+)-Pi cotransport to the same extent in brush-border membranes derived from normal mice and Hyp littermates. The pH dependence of Na(+)-Pi cotransport was similar in brush-border membrane vesicles of normal and Hyp mice. The ratio of Na(+)-Pi cotransport measured at pH 7.5 relative to that at pH 6.5 was 2.9 +/- 0.6 in normal mice and 2.9 +/- 0.7 in Hyp mice. PFA was a competitive inhibitor of Na(+)-Pi cotransport in brush-border membranes of both normal and Hyp mice. However, the apparent Ki for PFA was significantly lower in Hyp mice (0.31 +/- 0.01 and 0.19 +/- 0.02 mM in normal and Hyp mice, respectively, P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Growth hormone normalizes renal 1,25-dihydroxyvitamin D3-24-hydroxylase gene expression but not Na+-phosphate cotransporter (Npt2) mRNA in phosphate-deprived Hyp mice.

The murine X-linked Hyp mutation is characterized by decreased renal expression of type II Na+-phosphate (Pi) cotransporter (Npt2) mRNA and an abnormal vitamin D response to Pi deprivation. The latter is manifest by an aberrant fall in serum 1,25-dihydroxyvitamin D3 (1,25(OH)2D) levels that is associated with an increase in renal 1,25(OH)2D-24-hydroxylase (24-hydroxylase), the first enzyme in the C-24 oxidation pathway. Because growth hormone (GH) enhances renal Na+-Pi cotransport and permits the adaptive 1,25(OH)2D response in Pi-deprived hypophysectomized rats, we examined the effects of GH on vitamin D metabolism and renal Npt2 mRNA abundance in Hyp mice fed control and low Pi diets. GH significantly decreased renal 24-hydroxylase activity (0.202+/-0.020 to 0.098+/-0.008 pmol/mg of protein/minute, p < 0.05) and mRNA abundance, relative to beta-actin mRNA (299+/-13 to 78+/-14, p < 0.05), in Hyp mice fed the low Pi diet but had no effect on either parameter in mutants fed the control diet. Moreover, after GH treatment, renal 24-hydroxylase gene expression was no longer elevated in Pi-deprived Hyp mice relative to mutants fed control diet. In contrast, GH did not correct the serum concentration of 1,25(OH)2D in Pi-deprived Hyp mice. We also demonstrate that GH did not normalize renal Npt2 mRNA expression, relative to beta-actin mRNA, in Hyp mice fed either control or low Pi diets. The present data demonstrate that normalization of renal 24-hydroxylase gene expression in Pi-deprived Hyp mice by GH is not sufficient to correct the serum concentration of 1,25(OH)2D and is not associated with an alteration in renal Npt2 mRNA expression.

Comparative effects of 1,25-dihydroxyvitamin D3 and EB 1089 on mouse renal and intestinal 25-hydroxyvitamin D3-24-hydroxylase.

EB 1089 is a vitamin D analog that is less potent than 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) in its calcemic action but more potent in its antiproliferative action. We characterized the interaction of 1,25(OH)2D3 and EB 1089 with renal 25-hydroxyvitamin D3-24-hydroxylase (24-hydroxylase), the first enzyme in the C-24 oxidation pathway, and compared the effects of 1,25(OH)2D3 and EB 1089 on induction of 24-hydroxylase mRNA in mouse kidney and intestine. 1,25(OH)2D3 and EB 1089 were competitive inhibitors of 24-hydroxylase activity. However, the Ki for 1,25(OH)2D3 (5.2 +/- 2.5 nM) was significantly lower than that for EB 1089 (286 +/- 59 nM). In the kidney, the time course and extent of 24-hydroxylase mRNA induction, relative to 18S rRNA, was similar for 1,25(OH)2D3 and EB 1089 with a peak response at approximately equal to 6 h that was sustained for at least 16 h. In the intestine, however, induction of 24-hydroxylase mRNA, relative to 18S rRNA, was approximately 50% lower for EB 1089 than for 1,25(OH)2D3 at 3 h (p < 0.05) and 6 h (p < 0.05) while at 16 h 24-hydroxylase mRNA was no longer detectable. Moreover, while both 1,25(OH)2D3 and EB 10898 elicited a similar dose-dependent induction of 24-hydroxylase mRNA in the kidney (EC50 = 0.4 +/- 0.13 and 0.3 +/- 0.08 ng/g for EB 1089 and 1,25(OH)2D3, respectively), the EC50 for EB 1089 (6.6 +/- 1.7 ng/g) was significantly higher than that for 1,25(OH)2D3 (0.9 +/- 0.32 ng/g) in the intestine (p < 0.01). EB 1089 was also less effective than 1,25(OH)2D3 in the induction of intestinal but not renal calbindin-D9k mRNA. To determine the mechanism for tissue-specific differences in potency, we determined the binding affinity of 1,25(OH)2D3 and EB 1089 for the vitamin D receptor. In the kidney, Kd values for 1,25(OH)2D3 (0.40 +/- 0.95 nM) and EB 1089 (0.48 +/- 0.04 nM) were not different. However, in the intestine, the Kd for EB 1089 (1.43 +/- 0.19 nM) was significantly higher than that for 1,25(OH)2D3 (0.85 +/- 0.06 nM; p < 0.05). Our results demonstrate that: (i) EB 1089 has a 50-fold lower affinity than 1,25(OH)2D3 for renal 24-hydroxylase, suggesting that it is more resistant to catabolism by the C-24 oxidation pathway; and (ii) EB 1089 and 1,25(OH)2D3 exhibit tissue-specific differences in vitamin D receptor-mediated responses in vivo that may be ascribed, at least in part, to differences in binding affinities for the vitamin D receptor.

Phosphate transport in immortalized cell cultures from the renal proximal tubule of normal and Hyp mice: evidence that the HYP gene locus product is an extrarenal factor.

Whether renal phosphate wasting in X-linked hypophosphatemia (XLH) results from an intrinsic renal or humoral defect remains controversial. In studies of the murine homolog of XLH, harboring the Simian Virus 40 (SV40) large T antigen, we obviated the influence of renal cell heterogeneity and impressed memory by comparing Na(+)-phosphate cotransport in immortalized cells from the S1 segment of the proximal tubule. Cells from SV40 transgenic normal and Hyp mice exhibit characteristics of differentiated proximal tubule cells including gluconeogenesis and alkaline phosphatase activity. Surprisingly, however, we found two distinct populations of cells from the S1 proximal tubule of both normal and Hyp mice. In one, PTH treatment increases cAMP accumulation, while in the other both PTH and thyrocalcitonin enhance cAMP production. Kinetic parameters for Na(+)-phosphate cotransport were similar in both subpopulations of cells from normal (Km, 0.29 +/- 0.03 vs. 0.39 +/- 0.04 mM; Vmax, 4.6 +/- 0.6 vs. 5.2 +/- 0.4 nmol/mg/5 minutes) and Hyp mice (0.33 +/- 0.02 vs. 0.26 +/- 0.04; 6.0 +/- 0.7, 4.8 +/- 0.6). More importantly, phosphate transport in S1 cells of either subpopulation from Hyp mice is no different than that of normals. These data indicate that renal proximal tubule cells from Hyp mice have intrinsically normal phosphate transport and support the hypothesis that a humoral abnormality underlies renal phosphate wasting in XLH.

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)

Association of a polymorphism in the TNFR2 gene with low bone mineral density.

Previous genetic linkage data suggested that a gene on chromosome 1p36.2-36.3 might be linked to low bone mineral density (BMD). Here, we examine the gene for tumor necrosis factor receptor 2 (TNFR2), a candidate gene within that interval, for association with low BMD in a group of 159 unrelated individuals. We assess two polymorphic sites within the gene, a microsatellite repeat within intron 4, and a three-nucleotide variation in the 3' untranslated region (UTR) of the gene. The latter has five alleles of which the rarest allele is associated with low spinal BMD Z score (p = 0.008). Lowest mean spinal BMD Z scores were observed for individuals having genotypes that were heterozygous for the rarest allele. No homozygotes for the rarest allele were observed. Preliminary analysis suggests that there is a difference in the genotype frequency distribution between the group with low BMD and a control group.

Na+ -phosphate cotransport in mouse distal convoluted tubule cells: evidence for Glvr-1 and Ram-1 gene expression.

While there is considerable evidence for phosphate (Pi) reabsorption in the distal tubule, Pi transport and its regulation have not been well characterized in this segment of the nephron. In the present study, we examined Na+-dependent Pi transport in immortalized mouse distal convoluted tubule (MDCT) cells. Pi uptake by MDCT cells is Na+-dependent and, under initial rate conditions, is inhibited by phosphonoformic acid (41 +/- 3% of control), a competitive inhibitor of Na+-Pi cotransport. The transport system has a high affinity for Pi (Km = 0.46 mM) and is stimulated by lowering the extracellular pH from 7.4 to 6.4 and inhibited by raising the pH from 7.4 to 8.4. Exposure to Pi-free medium for 21 h increased Na+-Pi cotransport from 2.1 to 5.5 nmol/mg of protein/5 minutes (p < 0.05) while parathyroid hormone, forskolin, and phorbol 12-myristate 13-acetate failed to alter Pi uptake in MDCT cells. Reverse transcriptase polymerase chain reaction of MDCT cell RNA provided evidence for the expression of the Npt1 but not the Npt2 Na+-Pi cotransporter gene. However, preincubation of MDCT cells with Npt1 antisense oligonucleotide led to only 20% inhibition of Na+-Pi cotransport, suggesting that other Na+-Pi cotransporters are operative in MDCT cells. Indeed, we showed, by ribonuclease protection assay, that MDCT cells express the ubiquitous cell surface receptors for gibbon ape leukemia virus (Glvr-1) and amphoteric murine retrovirus (Ram-1) that also function as Na+-Pi cotransporters. In summary, we demonstrate that the pH dependence and regulation of Na+-Pi cotransport in MDCT cells is distinct from that in the proximal tubule and suggest that different gene products mediate Na+-Pi cotransport in the proximal and distal segments of the nephron.

The renal phosphate transport defect in normal mice parabiosed to X-linked hypophosphatemic mice persists after parathyroidectomy.

The X-linked hypophosphatemic (Hyp) mouse is a model for human X-linked hypophosphatemia. Surgical joining of normal to Hyp mice by parabiosis results in the normal mice developing low renal retention of phosphate and hypophosphatemia. These results suggest a humoral component to the renal defect. To test whether this component could be parathyroid hormone, surgical parathyroidectomy (PTX) or sham surgery was performed in mice 3 weeks after parabiotic union (n greater than 20 per group). After an overnight fast, PTX mice were hypocalcemic and hyperphosphatemic relative to sham-operated control mice. PTX normal mice joined to PTX Hyp mice were significantly lower in plasma phosphate and higher in fractional excretion of phosphate [U/P phosphate/(U/P creatinine)] when compared with PTX normal mice joined to other PTX normals. To test for more specific evidence of altered renal transport function, renal brush-border membrane vesicles (BBMV) were prepared from these mice, and phosphate and glucose uptakes were measured. The phosphate/glucose transport ratio was lower in BBMV from Hyp mice, joined to either normal mice or to Hyp mice, when compared with that from normal-normal pairs. Moreover, BBMV from normal mice joined to Hyp mice had a significantly lower phosphate/glucose uptake ratio than BBMV from normal mice joined to other normal mice, and their activity approached that of BBMV derived from Hyp mice. Glucose uptake in BBMV was unaffected by parabiosis or genotype. In summary, parabiosis of normal mice to Hyp mice resulted in the development of phosphaturia and decreased BBMV phosphate transport in the normal mice. The persistence of the phosphate transport defect in parathyroidectomized mice suggests that parathyroid hormone is not the humoral factor contributing to these results.

Developmental expression and tissue distribution of Phex protein: effect of the Hyp mutation and relationship to bone markers.

Mutations in PHEX, a phosphate-regulating gene with homology to endopeptidases on the X chromosome, are responsible for X-linked hypophosphatemia (XLH). The murine Hyp homologue has the phenotypic features of XLH and harbors a large deletion in the 3' region of the Phex gene. We characterized the developmental expression and tissue distribution of Phex protein, using a monoclonal antibody against human PHEX, examined the effect of the Hyp mutation on Phex expression, and compared neprilysin (NEP), osteocalcin, and parathyroid hormone/parathyroid hormone-related protein (PTH/PTHrP) receptor gene expression in bone of normal and Hyp mice. Phex encodes a 100- to 105-kDa glycoprotein, which is present in bones and teeth of normal mice but not Hyp animals. These results were confirmed by in situ hybridization (ISH) and ribonuclease protection assay. Phex protein expression in femur and calvaria decreases with age, suggesting a correlation between Phex expression and bone formation. Immunohistochemical studies detected Phex protein in osteoblasts, osteocytes, and odontoblasts, but not in osteoblast precursors. In contrast to Phex, the abundance of NEP messenger RNA (mRNA) and protein is not significantly altered in Hyp bone. Similarly, osteocalcin and PTH/PTHrP receptor gene expression are not compromised in bone of Hyp mice. Our results are consistent with the hypothesis that loss of Phex function affects the mineralizing activity of osteoblasts rather than their differentiation.

Ontogeny of Phex/PHEX protein expression in mouse embryo and subcellular localization in osteoblasts.

PHEX, a phosphate-regulating gene with homologies to endopeptidases on the X chromosome, is mutated in X-linked hypophosphatemia (XLH) in humans and mice (Hyp). Although recent observations indicate that Phex protein is expressed primarily in bone and may play an important role in osteoblast function and bone mineralization, the pattern of the Phex protein expression in the developing skeleton and its subcellular localization in osteoblasts remain unknown. We examined the ontogeny of the Phex protein in the developing mouse embryo and its subcellular localization in osteoblasts using a specific antibody to the protein. Immunohistochemical staining of mouse embryos revealed expression of Phex in osteogenic precursors in developing vertebral bodies and developing long bones on day 16 postcoitum (pc) and thereafter. Calvaria from day 18 pc mice showed Phex epitopes in osteoblasts. No Phex immunoreactivity was detected in lung, heart, hepatocytes, kidney, intestine, skeletal muscle, or adipose tissue of mouse embryos. Interestingly, embryonic mouse skin showed moderate amounts of Phex immunostaining. In postnatal mice, Phex expression was observed in osteoblasts and osteocytes. Moderate expression of Phex was seen in odontoblasts and slight immunoreactivity was observed in ameloblasts. Confocal microscopy revealed the presence of immunoreactive PHEX protein in the Golgi apparatus and endoplasmic reticulum of osteoblasts from normal mice and in osteoblasts from Hyp mice transduced with a human PHEX viral expression vector. PHEX protein was not detected in untransduced Hyp osteoblasts. These data indicate that Phex protein is expressed in osteoblasts and osteocytes during the embryonic and postnatal periods and that within bone, Phex may be a unique marker for cells of the osteoblast/osteocyte lineage.

Metabolism of 25-hydroxyvitamin D3 in renal slices from the X-linked hypophosphatemic (Hyp) mouse: abnormal response to fall in serum calcium.

The effect of the X-linked Hyp mutation on 25-hydroxyvitamin D3 (25-OH-D3) metabolism in mouse renal cortical slices was investigated. Vitamin D replete normal mice and Hyp littermates fed the control diet synthesized primarily 24,25-dihydroxyvitamin D3 (24,25-(OH)2D3); only minimal synthesis of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) was detected in both genotypes and 1,25-(OH)2D3 formation was not significantly greater in Hyp mice relative to normal littermates, despite hypophosphatemia and hypocalcemia in the mutants. Calcium-deficient diet fed to normal mice reduced serum calcium (p less than 0.01), increased renal 25-hydroxyvitamin D3-1-hydroxylase (1-OHase) activity (p less than 0.05), and decreased 25-hydroxyvitamin D3-24-hydroxylase (24-OHase) activity (p less than 0.05). In contrast, Hyp littermates on the calcium-deficient diet had decreased serum calcium (p less than 0.01), without significant changes in the renal metabolism of 25-OH-D3. Both normal and Hyp mice responded to the vitamin D-deficient diet with a fall in serum calcium (p less than 0.01), significantly increased renal 1-OHase, and significantly decreased renal 24-OHase activities. In Hyp mice, the fall in serum calcium on the vitamin D-deficient diet was significantly greater than that observed on the calcium-deficient diet. Therefore the ability of Hyp mice to increase renal 1-OHase activity when fed the vitamin D-deficient diet and their failure to do so on the calcium-deficient diet may be related to the resulting degree of hypocalcemia. The results suggest that although Hyp mice can respond to a disturbance of calcium homeostasis, the in vivo signal for the stimulation of renal 1-OHase activity may be set at a different threshold in the Hyp mouse; i.e. a lower serum calcium concentration is necessary for Hyp mice to initiate increased synthesis of 1,25(-OH)2D3.