Human X-linked Hypophosphatemia Research Articles

FGF receptor inhibitor BGJ398 partially rescues osteoarthritis-like phenotype in older high molecular weight FGF2 transgenic mice via multiple mechanisms

We have used Basic Fibroblast Growth Factor (FGF2) transgenic mice as experimental models for human X-linked hypophosphatemia (XLH)-related degenerative osteoarthritis (OA) to investigate the pathogenesis of the disease and to test potential pharmacotherapies for treatment. This study tested the efficacy of BJG398, a small molecule fibroblast growth factor receptor tyrosine kinase (FGFRTK) inhibitor, to rescue the knee joint osteoarthritis phenotype in High Molecular Weight fibroblast growth factor 2 transgenic (HMWTgFGF2) mice. BJG398 was administered in vivo to 8-month-old female HMWTgFGF2 mice for six weeks. Histomorphometry, immunohistochemistry and micro-CT were used to examine the knee joints in BGJ398-treated and control mice. We assessed: Fibroblast Growth Factor 23 (FGF23) expression and FGFR1 activity; Matrix metalloproteinase 13 (MMP13) and Aggrecanase2 (ADAMTS5) expression; then signaling by SMAD1/5/8-pSMAD6, pERK1/2 and Runt-related transcription factor 2 (RUNX2). Using PrimePCR arrays, we identified a contributing role for major target genes in the TGFB/BMP2 signaling pathway that were regulated by BGJ398. BGJ398 inhibited HMWFGF2/FGF23-induced increase in bone morphogenic protein receptor-1, bone morphogenic protein-2 and 4 and Serine peptidase inhibitor, clade E, member 1. The results from Micro-CT and histology show BGJ398 treatment rescued the OA changes in subchondral bone and knee articular cartilage of HMWTgFGF2 mice. The gene expression and signal transduction results provide convincing evidence that HMWFGF2 generates OA through FGFRTK with characteristic downstream signaling that defines OA, namely: increased FGF23-FGFR1 activity with BMP-BMPR, activation of pSMAD1/5/8-RUNX2 and pERK signaling pathways, then upregulation of MMP13 and ADAMTS5 to degrade matrix. BGJ398 treatment effectively reversed these OA molecular phenotypes, providing further evidence that the OA generated by HMWFGF2 in the transgenic mice is FGFR-mediated and phenocopies the OA found in the Hyp mouse homolog of XLH with a spontaneous mutation in the Phex (phosphate regulating endopeptidase on the X chromosome) gene and human XLH-OA. Overall, the results obtained here explain how the pleotropic effects of FGF2 emanate from the different functions of HMW protein isoforms for cartilage and bone homeostasis, and the pathogenesis of XLH-degenerative osteoarthropathy. BGJ398 inhibits HMWFGF2-induced osteoarthritis via multiple mechanisms. These results provided important scientific evidence for the potential application of BGJ398 as a therapeutic agent for osteoarthritis in XLH.

1,25-Dihydroxyvitamin D Maintains Brush Border Membrane NaPi2a and Attenuates Phosphaturia in Hyp Mice.

Phosphate homeostasis is critical for many cellular processes and is tightly regulated. The sodium-dependent phosphate cotransporter, NaPi2a, is the major regulator of urinary phosphate reabsorption in the renal proximal tubule. Its activity is dependent upon its brush border localization that is regulated by fibroblast growth factor 23 (FGF23) and PTH. High levels of FGF23, as are seen in the Hyp mouse model of human X-linked hypophosphatemia, lead to renal phosphate wasting. Long-term treatment of Hyp mice with 1,25-dihydroxyvitamin D (1,25D) or 1,25D analogues has been shown to improve renal phosphate wasting in the setting of increased FGF23 mRNA expression. Studies were undertaken to define the cellular and molecular basis for this apparent FGF23 resistance. 1,25D increased FGF23 protein levels in the cortical bone and circulation of Hyp mice but did not impair FGF23 cleavage. 1,25D attenuated urinary phosphate wasting as early as one hour postadministration, without suppressing FGF23 receptor/coreceptor expression. Although 1,25D treatment induced expression of early growth response 1, an early FGF23 responsive gene required for its phosphaturic effects, it paradoxically enhanced renal phosphate reabsorption and NaPi2a protein expression in renal brush border membranes (BBMs) within one hour. The Na-H+ exchange regulatory factor 1 (NHERF1) is a scaffolding protein thought to anchor NaPi2a to the BBM. Although 1,25D did not alter NHERF1 protein levels acutely, it enhanced NHERF1-NaPi2a interactions in Hyp mice. 1,25D also prevented the decrease in NHERF1/NaPi2a interactions in PTH-treated wild-type mice. Thus, these investigations identify a novel role for 1,25D in the hormonal regulation of renal phosphate handling.

CRISPR/Cas9-mediated mutation of PHEX in rabbit recapitulates human X-linked hypophosphatemia (XLH).

X-linked hypophosphatemia (XLH) is the most common cause of inheritable rickets, with an incidence of 1/20 000 in humans. Inactivation or mutation of the gene PHEX, a phosphate-regulating endopeptidase, leads to hypophosphatemia and defective bone mineralization in XLH patients. Presently, there is no adequate animal model for safety assessments of physiotherapies and drug screening for XLH rickets. In this study, an XLH model was generated via PHEX gene knockout (KO) through coinjection of clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9)/sgRNA mRNA into rabbit zygotes. The typical phenotypes of growth retardation, hypophosphatemia, elevated serum FGF23 and bone mineralization were observed in the PHEX KO rabbits but not in normal controls. In summary, for the first time, we have successfully obtained PHEX KO rabbits and recapitulated human XLH using the CRISPR/Cas9 system. This novel XLH rabbit model could be utilized as a drug screening model for XLH prevention and preclinical therapy.

Cellular ATP Synthesis Mediated by Type III Sodium-dependent Phosphate Transporter Pit-1 Is Critical to Chondrogenesis

Disturbed endochondral ossification in X-linked hypophosphatemia indicates an involvement of P(i) in chondrogenesis. We studied the role of the sodium-dependent P(i) cotransporters (NPT), which are a widely recognized regulator of cellular P(i) homeostasis, and the downstream events in chondrogenesis using Hyp mice, the murine homolog of human X-linked hypophosphatemia. Hyp mice showed reduced apoptosis and mineralization in hypertrophic cartilage. Hyp chondrocytes in culture displayed decreased apoptosis and mineralization compared with WT chondrocytes, whereas glycosaminoglycan synthesis, an early event in chondrogenesis, was not altered. Expression of the type III NPT Pit-1 and P(i) uptake were diminished, and intracellular ATP levels were also reduced in parallel with decreased caspase-9 and caspase-3 activity in Hyp chondrocytes. The competitive NPT inhibitor phosphonoformic acid and ATP synthesis inhibitor 3-bromopyruvate disturbed endochondral ossification with reduced apoptosis in vivo and suppressed apoptosis and mineralization in conjunction with reduced P(i) uptake and ATP synthesis in WT chondrocytes. Overexpression of Pit-1 in Hyp chondrocytes reversed P(i) uptake and ATP synthesis and restored apoptosis and mineralization. Our results suggest that cellular ATP synthesis consequent to P(i) uptake via Pit-1 plays an important role in chondrocyte apoptosis and mineralization, and that chondrogenesis is ATP-dependent.

Expression and distribution of SIBLING proteins in the predentin/dentin and mandible of hyp mice

Human X-linked hypophosphatemia (XLH) and its murine homologue, Hyp are caused by inactivating mutations in PHEX gene. The protein encoded by PHEX gene is an endopeptidase whose physiological substrate(s) has not been identified. Dentin matrix protein 1 (DMP1) and dentin sialophosphoprotein (DSPP), two members of the Small Integrin-Binding LIgand, N-linked Glycoprotein (SIBLING) family are proteolytically processed. It has been speculated that PHEX endopeptidase may be responsible for the proteolytic cleavage of DMP1 and DSPP. To test this hypothesis and to analyse the distribution of SIBLING proteins in the predentin/dentin complex and mandible of Hyp mice, we compared the expression of four SIBLING proteins, DMP1, DSPP, bone sialoprotein (BSP) and osteopontin (OPN) between Hyp and wild-type mice. These SIBLING proteins were analysed by protein chemistry and immunohistochemistry. (1) Dentin matrix protein 1 and DSPP fragments are present in the extracts of Hyp predentin/dentin and bone; (2) the level of DMP1 proteoglycan form, BSP and OPN is elevated in the Hyp bone. The PHEX protein is not the enzyme responsible for the proteolytic processing of DMP1 and DSPP. The altered distribution of SIBLING proteins may be involved in the pathogenesis of bone and dentin defects in Hyp and XLH.

Phex Mutation Causes the Reduction of Npt2b mRNA in Teeth

Hyp mice (murine homologue of human X-linked hypophosphatemia) have a disorder in phosphate homeostasis, and display hypomineralization in bones and teeth. We investigated whether a mutation of Phex (phosphate regulating gene homologies to endopeptidase on the X chromosome) has an effect on the expression level of type II sodium-dependent phosphate co-transporter (Npt2) in the developing teeth of the Hyp mouse. Quantitative RT-PCR analyses revealed that the amount of Npt2b mRNA, an isoform of Npt2, in Hyp mouse tooth germs was significantly lower than that in wild-type mice, in both in vivo and in vitro experiments. In addition, tooth germs from wild-type mice cultured in medium supplemented with antisense oligo-deoxynucleotide for Phex also showed a reduction of Npt2b mRNA expression. These findings suggest that the loss of Phex function is related to the defect of Npt2b expression in teeth, and Npt2b reduction is an intrinsic defect of Hyp murine teeth.

Phosphorylated acidic serine–aspartate-rich MEPE-associated motif peptide from matrix extracellular phosphoglycoprotein inhibits phosphate regulating gene with homologies to endopeptidases on the X-chromosome enzyme activity

Inactivating PHEX (phosphate regulating gene with homologies to endopeptidases on the X chromosome) mutations cause X-linked hypophosphatemia in humans and mice (Hyp) through overproduction of fibroblast growth factor 23 (FGF23) a phosphaturic factor, by osteocytes. Matrix extracellular phosphoglycoprotein (MEPE) is also elevated in Hyp and other hypophosphatemic disorders. In addition, the administration of an ASARM (acidic serine-aspartate rich MEPE-associated motif) peptide derived from MEPE causes phosphaturia and inhibits bone mineralization in mice, suggesting that MEPE also plays a role in phosphate homeostasis. Since recent studies found that MEPE binds specifically to PHEX in vitro, we tested the effect of recombinant-MEPE and its ASARM peptide on PHEX enzyme activity in vitro and FGF23 expression in bone marrow stromal cell cultures ex vivo. We found that both recombinant MEPE and synthetic phosphorylated ASARM peptide (ASARM-PO(4)) inhibit PHEX enzyme activities in an in vitro fluorescent-quenched PHEX enzyme activity assay. The ASARM-PO(4) peptide inhibits PHEX enzyme activity in a dose-dependent manner with a K(i) of 128 nM and V(max-i) of 100%. Recombinant MEPE also inhibits PHEX activity (K(i) = 2 nM and V(max-i) = 26%). Long-term bone marrow stromal cell cultures supplemented with 10 microM ASARM-PO(4) peptide resulted in significant elevation of FGF23 transcripts and inhibition of mineralization. These findings suggest that MEPE inhibits mineralization and PHEX activity and leads to increased FGF23 production. The resulting coordination of mineralization and release of a phosphaturic factor by MEPE may serve a physiological role in regulating systemic phosphate homeostasis to meet the needs for bone mineralization.

Overexpression of Human PHEX Under the Human β-Actin Promoter Does Not Fully Rescue the Hyp Mouse Phenotype

XLH in humans and the Hyp phenotype in mice are caused by inactivating Phex mutations. Overexpression of human PHEX under the human beta-actin promoter in Hyp mice rescued the bone phenotype almost completely, but did not affect phosphate homeostasis, suggesting that different, possibly independent, pathophysiological mechanisms contribute to hyperphosphaturia and bone abnormalities in XLH. Mutations in PHEX, a phosphate-regulating gene with homologies to endopeptidases on the X chromosome, are responsible for X-linked hypophosphatemia (XLH) in humans, and its mouse homologs, Hyp, Phex(Hyp-2J), Phex(Hyp-Duk), Gy, and Ska1. PHEX is thought to inactivate a phosphaturic factor, which may be fibroblast growth factor 23 (FGF)-23. Consistent with this hypothesis, FGF-23 levels were shown to be elevated in most patients with XLH and in Hyp mice. The aim of this study was, therefore, to examine whether transgenic overexpression of PHEX under the human beta-actin promoter would rescue the Hyp phenotype. We tested this hypothesis by generating two mouse lines expressing human PHEX under the control of a human beta-actin promoter (PHEX-tg). With the exception of brain, RT-PCR analyses showed transgene expression in all tissues examined. PHEX protein, however, was only detected in bone, muscle, lung, skin, and heart. To assess the role of the mutant PHEX, we crossed female heterozygous Hyp mice with male heterozygous PHEX-tg mice to obtain wildtype (WT), PHEX-tg, Hyp, and Hyp/PHEX-tg offspring, which were examined at 3 months of age. PHEX-tg mice exhibited normal bone and mineral ion homeostasis. Hyp mice showed the known phenotype with reduced body weight, hypophosphatemia, hyperphosphaturia, and rickets. Hyp/PHEX-tg mice had almost normal body weight relative to WT controls, showed a dramatic improvement in femoral BMD, almost normal growth plate width, and, despite remaining disturbances in bone mineralization, almost normal bone architecture and pronounced improvements of osteoidosis and of halo formation compared with Hyp mice. However, Hyp and Hyp/PHEX-tg mice had comparable reductions in tubular reabsorption of phosphate and were hypophosphatemic relative to WT controls. Our data suggest that different, possibly independent, pathophysiological mechanisms contribute to renal phosphate wasting and bone abnormalities in Hyp and XLH.

Increased cathepsin D release byHypmouse osteoblast cells

The X-linked hypophosphatemia (XLH), the most common form of hereditary rickets, is caused by loss-of-function mutations of PHEX (phosphate-regulating gene with homology to endopeptidases on the X chromosome) leading to rachitic bone disease and hypophosphatemia. Available evidence today indicates that the bone defect in XLH is caused not only by hypophosphatemia and altered vitamin D metabolism but also by factor(s) locally released by osteoblast cells (ObCs). The identity of these ObC-derived pathogenic factors remains unclear. In our present study, we report our finding of a prominent protein in the culture media derived from ObC of the hypophosphatemic (Hyp) mice, a murine homolog of human XLH, which was identified as the murine procathepsin D (Cat D). By metabolic labeling studies, we further confirmed that Hyp mouse ObCs released greater amount of Cat D into culture media. This increased Cat D release by Hyp mouse ObCs was unlikely to be due to nonspecific cell damage or heterogeneous cell population and was found to be associated with an increased Cat D expression at the protein level, possibly due to a reduced Cat D degradation. However, we were not able to detect a direct effect of PHEX protein on Cat D cleavage. In support of the involvement of Cat D in mediating the inhibitory effect of Hyp mouse ObC-conditioned media on ObC calcification, we found that exposure to Cat D inhibited ObC (45)Ca incorporation and that inhibition of Cat D abolished the inhibitory effect of Hyp mouse-conditioned media on ObC calcification. In conclusion, results from our present study showed that Hyp mouse ObCs release a greater amount of Cat D, which may contribute to the inhibitory effect of Hyp mouse ObC-conditioned media on ObC mineralization.

Hyper-expression of Osteocalcin mRNA in Odontoblasts of Hyp Mice

The Hyp mouse is a murine homologue of human X-linked hypophosphatemia that displays hypo-mineralization in bone and dentin. In this study, we tested the hypothesis that the defect in Hyp mice leads to alterations in the expression of dentin matrix proteins that may be associated with the hypo-mineralization changes in the tissues. Quantitative RT-PCR analyses showed that expression of the osteocalcin gene in Hyp mice tooth germ samples was significantly higher than in wild-type mice, whereas the gene expressions of osteonectin, osteopontin, dentin matrix protein 1, and type I collagen in both types of mice were similar. Further, cultured Hyp mice tooth germ samples exhibited a higher expression of the osteocalcin gene than did those from wild-type mice, which was in accord with the results of our in vivo analysis. These findings suggest that osteocalcin mRNA is highly expressed in Hyp mice odontoblasts and may be associated with dentin hypo-mineralization.

Up-regulation of liver glucose-6-phosphatase in x-linked hypophosphatemic mice.

We previously showed that a phosphate-deficient diet resulting in hypophosphatemia upregulated the catalytic subunit p36 of rat liver glucose-6-phosphatase, which is responsible for hepatic glucose production. A possible association between phosphate and glucose homeostasis was now further evaluated in the Hyp mouse, a murine homologue of human X-linked hypophosphatemia. We found that in the Hyp mouse as in the dietary Pi deficiency model, serum insulin was reduced while glycemia was increased, and that liver glucose-6-phosphatase activity was enhanced as a consequence of increased mRNA and protein levels of p36. In contrast, the Hyp model had decreased mRNA and protein levels of the putative glucose-6-phosphate translocase p46 and liver cyclic AMP was not increased as in the phosphate-deficient diet rats. It is concluded that in genetic as in dietary hypophosphatemia, elevated glucose-6-phosphatase activity could be partially responsible for the impaired glucose metabolism albeit through distinct mechanisms.

The X chromosome deletion in Hyp mice extends into the intergenic region but does not include the Sat gene downstream from Phex

The murine Hyp mutation is a model for X-linked hypophosphatemia (XLH), the most prevalent form of inherited rickets in humans. Although mutations in the murine Phex gene and the human PHEX gene have been identified in both murine and human disorders, the extent of the Hyp deletion on the mouse X chromosome has not been delineated. In the present study we demonstrate that the Hyp deletion starts in the middle of Phex intron 15 and includes ∼48 kb of the 3′ region of the Phex gene and ∼10 kb of intergenic sequence on the mouse X chromosome. In addition, we show that the Hyp deletion does not involve the downstream spermidine/spermine N1-acetyl transferase (Sat; formerly Ssat) gene and thus is not a contiguous gene deletion syndrome. Our data indicate that the Hyp mouse is a true homolog of XLH in humans and underscore the validity of this murine model in studies of XLH pathophysiology and for testing novel treatment modalities.

MEPE, the Gene Encoding a Tumor-Secreted Protein in Oncogenic Hypophosphatemic Osteomalacia, Is Expressed in Bone

The MEPE (matrix extracellular phosphoglycoprotein) gene is a strong candidate for the tumor-derived phosphaturic factor in oncogenic hypophosphatemic osteomalacia (OHO). X-linked hypophosphatemia (XLH) is phenotypically similar to OHO and results from mutations in PHEX, a putative metallopeptidase believed to process a factor(s) regulating bone mineralization and renal phosphate reabsorption. Here we report the isolation of the murine homologue of MEPE, from a bone cDNA library, that encodes a protein of 433 amino acids, 92 amino acids shorter than human MEPE. Mepe, like Phex, is expressed by fully differentiated osteoblasts and down-regulated by 1,25-(OH)2D3. In contrast to Phex, Mepe expression is markedly increased during osteoblast-mediated matrix mineralization. Greater than normal Mepe mRNA levels were observed in bone and osteoblasts derived from Hyp mice, the murine homologue of human XLH. Our data provide the first evidence that MEPE/Mepe is expressedby osteoblasts in association with mineralization.

MRNA expression of Phex in mice and rats: the effect of low phosphate diet.

Phex is the gene whose mutation is the cause of X-linked hypophosphatemia in humans and mice. The organs expressing Phex in normal animals, and their possible sensitivity to stimulation by low phosphate diets, are unknown. In this study, Phex expression was measured in 6-wk-old normal B6C3H male and female mice and in 135 g Sprague-Dawley rats fed a normal phosphate diet or a low phosphate diet with deionized water ad libitum for 7 d. The animals were then anesthetized, and a variety of organs were collected and frozen in liquid nitrogen. Phex mRNA expression was measured in each organ by reverse transcription-polymerase chain reaction (RT-PCR) with primers specific for both Phex and glyceraldehyde-3-phosphate dehydrogenase (G3PDH). Southern blots were prepared, hybridized with 32P-labeled internal oligonucleotides, and quantified with a phosphor imager. The Phex/G3PDH ratio was computed, and the data were compiled as the mean +/- SEM. In these growing animals, the highest Phexexpression levels were found in the gonads, brain, and lung. In contrast, Phex expression in calvaria and femur was markedly less. Two significant changes were found in animals that were fed a low phosphate diet. Spleen showed a significant decrease in Phex mRNA levels on low phosphate diet (60+/-10% of normal P diet, n = 12/group, p = 0.002). The pituitary gland showed a significant increase in Phex expression with low phosphate diet (851+/-127% of G3PDH) over normal P diet (569+/-78%, n = 24 - 25/group, p = 0.03). No significant change was found in femur, calvaria, or a variety of soft tissues. In summary, Phex mRNA was found in most tissues examined. Expression levels varied by two orders of magnitude from highest to lowest with more in gonads, brain, and lung and with less in bone. Increased Phex mRNA was found in the pituitary gland of animals that were fed a low phosphate diet.

Osteocalcin Production in Primary Osteoblast Cultures Derived from Normal and Hyp Mice

Rickets and osteomalacia are characteristic features of the Hyp mouse model of human X-linked hypophosphatemia. Hyp mice demonstrate elevated circulating osteocalcin levels, as well as altered regulation of osteocalcin by 1,25(OH)2D3. Whether this osteocalcin abnormality is intrinsic to the osteoblast, or mediated by the in vivo milieu, has not been established. We therefore characterized osteocalcin production and its regulation by 1,25(OH)2D3 in primary cultures of murine osteoblasts and examined osteocalcin and its messenger RNA in response to 1,25(OH)2D3 in cultures of Hyp mouse-derived osteoblasts. Cell viability and osteocalcin production are optimal when murine cells are harvested within 36 h of age. Murine primary osteoblast cultures mineralize and produce osteocalcin in a maturation-dependent fashion (as demonstrated in other species), and continuous exposure to 1,25(OH)2D3, beginning at day 9 of culture, inhibits osteoblast differentiation and osteocalcin production and prevents mineralization of the culture. However, in contrast to other species, exposure to 1,25(OH)2D3, added later (days 17-25) in culture, does not stimulate osteocalcin but arrests osteocalcin production at current levels. Ambient media levels of osteocalcin were no different in cultures from Hyp mice and their normal litter mates, and the down-regulatory response to 1,25(OH)2D3 was comparable in cultures from normal and Hyp mice. Furthermore, expression of osteocalcin messenger RNA in murine cultures is reduced with exposure to 1,25(OH)2D3, and there is no difference between normal and Hyp cultures in this response. Thus, primary murine osteoblasts manifest a species-specific effect of 1,25(OH)2D3 on osteocalcin production. Furthermore, the increased serum osteocalcin production seen in intact Hyp mice, and the altered response to 1,25(OH)2D3 in Hyp mice, are not observed in osteoblast cultures derived from the mutant strain. These data indicate that abnormalities of osteocalcin described in intact Hyp mice require factors other than those present in cultured cells.

Skeletal casein kinase activity defect in the HYP mouse.

The Hyp mouse, a model for human X-linked hypophosphatemia (XLH), is characterized by phosphate wasting and defective mineralization. Since osteopontin (OPN) is considered pivotal for biological mineralization, we examined the biosynthesis of OPN in osteoblasts of +/Y and Hyp/Y mice. Immunoprecipitation analyses using a specific antibody to OPN revealed that Hyp/Y and +/Y osteoblasts secrete similar levels of OPN as determined by [35S]-methionine biosynthetic labeling, but a reduced phosphorylation was noted after 32P-PO4 biosynthetic labeling. Northern blot hybridization analysis of +/Y and Hyp/Y mice osteoblast mRNAs, using a cDNA probe for mouse OPN, revealed no difference in the steady state levels of osteopontin mRNA. Analysis of casein kinase II activity in +/Y and Hyp/Y mice osteoblast, kidney, heart and liver membrane fractions revealed that casein kinase II activity in the Hyp/Y mice osteoblasts and kidney is only 35%-50%, respectively, of that of the +/Y mice tissues. The accumulated data are consistent with a post-translation defect in the Hyp/Y mouse osteoblast which results in the under-phosphorylation of osteopontin and subsequent under-mineralization of bone matrix.

Expression and Cloning of the Human X-Linked Hypophosphatemia Gene cDNA

X-linked hypophosphatemia (XLH), which is a heritable metabolic bone disease characterized biochemically by selective renal phosphate (Pi) wasting, is associated with mutations in the PEX (Phosphate-regulating gene with homologies toEndopeptidases on theX-chromosome) gene. To further explore the physiologic role of PEX and define its effect in XLH we have determined the expression and tissue distribution. Northern analysis found abundant PEX mRNA in a restricted pattern, predominantly in adult ovary and fetal lung. In addition, PEX expression was also found in adult lung and fetal liver. A PEX cDNA of 2550 basepairs, which contains the full PEX coding region, was isolated from a human ovary cDNA library. The PEX cDNA shows high homology to other membrane-bound zinc metallopeptidases. The presence of PEX in non-osseous tissues strongly suggests features of a systemic role, rather than a unique function in bone development.

Direct demonstration of a humorally-mediated inhibition of renal phosphate transport in the Hyp mouse

The murine Hyp model reproduces the characteristics of human X-linked hypophosphatemia (XLH), an inherited disease causing renal loss of phosphate (Pi), severe rickets and osteomalacia. A current hypothesis considers that a humoral factor may be responsible for the renal Pi loss, although in vitro experiments with renal cell models have failed to demonstrate the presence of such a factor in XLH or in the Hyp mouse model. To test this hypothesis directly, we prepared primary mouse proximal tubule cell cultures (MPTC), expressing normal features of proximal tubule cells. These cells possess high alkaline phosphatase activity, and respond to human parathyroid hormone fragment 1-34 (PTH) with a four- to sixfold increase in cAMP production but do not respond to either arginine vasopressin (AVP) or to salmon calcitonin (sCT). They also show sodium-dependent phosphate, glucose and amino acid uptake. The presence of 10% Hyp mouse serum in HAMF12/DMEM media (1 mM Pi) for the last 48 hours of culture of MPTC reduced Pi uptake (0.1 mM 32P-Pi in the presence of 140 mM NaCl) by 45.7 +/- 3.9% (P < 0.01) as compared to normal mouse serum. This effect of Hyp mouse serum was dose-dependent between 5 to 20% (final concentration) in culture media for the last 48 hours of culture (P < 0.01 by analysis of variance). This effect of Hyp mouse serum was also time-dependent, with a lag time of at least 12 hours. Indeed, no significant inhibition of Pi uptake could be detected with incubations less than 12 hours in the presence of 10% Hyp mouse serum, whereas a maximal effect was obtained after 24 hours of incubation and remained unchanged after 36 and 48 hours. The inhibition of phosphate uptake by Hyp mouse serum was specific, since neither sodium-dependent glucose nor alpha-aminobutyric acid uptake was modified under these conditions. MPTC cells showed a very nice adaptation to Pi concentration in the media; low Pi (0.4 mM final concentration in the presence of 10% serum) stimulated Pi uptake, whereas high Pi concentration (3 mM) reduced Pi uptake by these cells as compared to regular HAMF12/DMEM media containing 1 mM Pi. Normal and Hyp mouse serum both inhibited Pi uptake by MPTC following adaptation in low or normal Pi media, however, Hyp mouse serum always showed a stronger inhibition than normal serum. In contrast, adaptation of MPTC in high Pi media resulted in no inhibition of phosphate uptake either in the presence of normal or Hyp mouse serum. We next questioned whether conditioned media from confluent Hyp mouse primary osteoblast-like cell cultures could affect Pi uptake by MPTC. These osteoblast-like cells expressed high alkaline phosphatase and produced the bone specific protein, osteocalcin. When MPTC were treated for 48 hours with Hyp mouse bone cell media conditioned for the last 48 hours of cultures, Pi uptake was specifically inhibited by 30.5 +/- 4.1% (P < 0.025) as compared to normal mouse bone cell-conditioned media. This effect of primary Hyp mouse bone cell-conditioned media is specific for these cells since it was not observed with CHO cell-conditioned media, nor with either mouse fibroblast (NCTC), normal mouse Kupffer cell- or Hyp mouse Kupffer cell-conditioned media. This effect also persisted through a number of passages of Hyp mouse bone cells, since conditioned-media from cells at their third passage still resulted in a 32 +/- 9.4% inhibition (P < 0.02). These results are the first to show an effect of Hyp mouse serum on Pi uptake by primary renal cell cultures in vitro. This effect is dose- and time-dependent, requiring 24 hours for maximum response, and is blocked in Pi rich media. These results also suggest that a specific intrinsic cellular defect, present in Hyp mouse osteoblasts, is responsible for the release of and/or the modification of a factor that can reach the circulation and which inhibits renal phosphate reabsorption. The molecular nature of this factor and its mode of action remains to be determined.

Effect of phosphate supplementation on the expression of the mutant phenotype in murine X-linked hypophosphatemic rickets

The X-linked Hyp mouse, a murine homologue of X-linked hypophosphatemia in humans, is characterized by rachitic bone disease, hypophosphatemia, impaired renal brushborder membrane Na +-phosphate cotransport and abnormal regulation of renal vitamin D metabolism. We demonstrated that short-term phosphate supplementation decreases renal 1,25-dihydroxyvitamin D 3 (1,25-(OH) 2D) catabolism and increases serum 1,25-(OH) 2D levels in Hyp mice (Tenenhouse & Jones 1990). In the present study, we compared several other parameters in normal and Hyp mice fed control (1%) and high (1.6%) phosphate diets for 4 days. Phosphate supplementation significantly raised serum phosphate levels and decreased renal brush-border membrane Na +-phosphate, but not Na +-glucose, cotransport in both genotypes (67% of control diet, p < 0.05). However, under both dietary conditions, the phosphate/glucose transport ratio was significantly reduced in Hyp mice (58% of normal littermates, p < 0.05). Renal PTH-stimulated cAMP accumulation, which was significantly blunted in Hyp mice compared to normal mice under control dietary conditions ( p < 0.05), was not altered by phosphate supplementation in either genotype. Serum alkaline phosphatase activity was significantly higher than normal in Hyp mice on the control diet and was further increased in mutants but not in normals fed the high phosphate diet ( p < 0.05). Measurements of serum bilirubin and electrophoresis of serum alkaline phosphatase suggested that the elevation in serum alkaline phosphatase activity in phosphate-supplemented Hyp mice represents the bone-derived isozyme. The present study demonstrates that, in contrast to the abnormality in vitamin D metabolism, the abnormalities in renal brush-border membrane Na +-phosphate cotransport, PTH-stimulated cAMP accumulation and serum alkaline phosphatase activity are not corrected by phosphate supplementation of Hyp mice. We suggest that the increase in serum alkaline phosphatase activity in phosphatesupplemented Hyp mice represents a stimulation of osteoblastic activity in response to the increase in available phosphate and elevated serum 1,25-(OH) 2D levels.

Renal adaptation to phosphate deprivation: lessons from the X-linkedHyp mouse

The X-linked Hyp mutation, a murine homologue of X-linked hypophosphatemia in humans, is characterized by renal defects in phosphate reabsorption and vitamin D metabolism. In addition, the renal adaptive response to phosphate deprivation in mutant Hyp mice differs from that of normal littermates. While Hyp mice fed a low phosphate diet retain the capacity to exhibit a significant increase in renal brush-border membrane sodium-phosphate cotransport in vitro, the mutants fail to show an adaptive increase in maximal tubular reabsorption of phosphate per volume of glomerular filtrate (TmP/GFR) in vivo. Moreover, unlike their normal counterparts, Hyp mice respond to phosphate restriction with a fall in the serum concentration of 1,25-dihydroxyvitamin D [1,25(OH)2D] that can be ascribed to increased renal 1,25(OH)2D catabolism. The dissociation between the adaptive brush-border membrane phosphate transport response and the TmP/GFR and vitamin D responses observed in Hyp mice is also apparent in X-linked Gy mice and hypophysectomized rats. Based on these findings and the notion that transport across the brush-border membrane reflects proximal tubular function, we suggest that the adaptive TmP/GFR response requires the participation of 1,25(OH)2D or a related metabolite and that a more distal segment of the nephron is the likely target for the 1,25(OH)2D-dependent increase in overall tubular phosphate conservation.