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Endocrinology Vol. 148, No. 10 4974-4983
Copyright © 2007 by The Endocrine Society

Early Lethality in Hyp Mice with Targeted Deletion of Pth Gene

Xiuying Bai, Dengshun Miao, David Goltzman and Andrew C. Karaplis

Division of Endocrinology (X.B., A.C.K.), Department of Medicine and Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital, McGill University, Montreal, Canada H3T 1E2; Calcium Research Laboratory and Department of Medicine (D.M., D.G.), McGill University Health Centre and Royal Victoria Hospital, McGill University, Montreal, Canada H3A 1A1; and Department of Anatomy, Histology, and Embryology and Institute of Dental Research (D.M.), Nanjing Medical University, Nanjing, Jiangsu 210029, The People’s Republic of China

Address all correspondence and requests for reprints to: Andrew C. Karaplis, Department of Medicine and Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital, 3755 Côte Ste Catherine Road, Montréal, Québec, Canada H3T 1E2. E-mail: akarapli{at}ldi.jgh.mcgill.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Although increased circulating levels of PTH with mild hypocalcemia has been reported in Hyp mice, hyperparathyroidism in X-linked hypophosphatemic rickets is postulated to arise from the standard use of phosphate salts, which induce chronic stimulation of PTH secretion. In this study, we sought to examine the role of PTH in the metabolic derangements associated with Hyp by generating hemizygous hypophosphatemic (Hyp/Y) mice homozygous for the Pth-null allele (Pth–/–;Hyp/Y). Early postnatal lethality was observed in the Pth–/–;Hyp/Y mice. Within the first 6 h, postpartum serum phosphorus increased to levels comparable to those in the Pth–/– mice, whereas in Hyp mice, it decreased during the first 48 h after birth. Serum calcium concentration started low after birth and remained reduced in both Pth–/–;Hyp/Y and Pth–/– mice although more profoundly so in the former group, whereas in Hyp/Y mice, the levels were initially lower than but reached wild-type levels by 24 h. Circulating PTH levels in Hyp/Y mice were higher than wild-type levels throughout the first 48 h after birth and continued to be so well into adulthood. Twice-daily administration of PTH 1–34 to Pth–/–;Hyp/Y newborn mice increased serum calcium levels and prevented their early demise. The findings here indicate that the cause of death in the Pth–/–;Hyp/Y mice is severe hypocalcemia. A potential role for fibroblast growth factor 23 in promoting secondary hyperparathyroidism by suppressing renal 25-hydroxyvitamin D3-1{alpha}-hydroxylase (Cyp27b1) activity while increasing that of renal 25-hydroxyvitamin D3 24-hydroxylase (Cyp24) is proposed. Hyperparathyroidism, therefore, is an integral component in the pathophysiology of Hyp, and likely X-linked hypophosphatemic rickets and serves to prevent severe hypocalcemia in mice and perhaps in patients afflicted with the disorder.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
X-LINKED HYPOPHOSPHATEMIC RICKETS (XLH) is the most common inherited disorder of renal phosphate wasting (1). It is characterized by severe hypophosphatemia arising from a defect in the renal reabsorption of filtered inorganic phosphorus (Pi), elevated serum alkaline phosphatase activity, inappropriately low-normal serum concentration of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] levels for the degree of prevailing hypophosphatemia, and defective bone mineralization leading to clinical, radiographic, and histomorphometric changes of rickets and osteomalacia. Much of our understanding of XLH has been facilitated by the availability of two murine homologs, the Hyp and Gy mice, which exhibit many of the phenotypic and biochemical features of XLH (2, 3). Considerable effort has been placed in elucidating the molecular basis of the renal defect in Pi transport and altered vitamin D metabolism in these animal models.

Through positional cloning, a gene that spans the deleted region Xp22.1 in XLH patients or is mutated in nondeletion patients with the disorder has been identified (designated PEX and subsequently PHEX, for phosphate-regulating gene with homologies to endopeptidases on the X chromosome) (4, 5). Phex mRNA was also altered in both Hyp and Gy mice. Male Hyp mice lack the 3' end of the Phex message, whereas male Gy mice are missing the 5' end of the transcript (6). Taken together, the data provide unequivocal evidence that XLH and the homologous diseases in mice, Hyp and Gy, arise from loss of PHEX/Phex function. The predicted human PHEX gene product as well as its murine homolog (6) exhibit structural homology to a family of neutral endopeptidases involved in either activation or degradation of a number of peptide hormones. PHEX likely also functions as a protease processing factor involved in bone mineral metabolism (7, 8). Despite extensive work, however, little is presently known about the substrate specificity of PHEX. Interestingly, none of the newly identified phosphaturic substances or phosphatonins, such as fibroblast growth factor 23 (FGF-23), matrix extracellular phosphoglycoprotein (MEPE), secreted frizzled-related protein 4 (sFRP-4), and FGF-7 (reviewed in Ref. 9) have been demonstrated convincingly to act as substrates for PHEX. In addition to changes in phosphate homeostasis, FGF-23 and secreted frizzled-related protein 4 also inhibit the synthesis of 1,25(OH)2D3, leading to decreased intestinal phosphate absorption and further reduction in phosphate retention by the organism. FGF-23 has been reported to bind to FGFR1(IIIc), which is directly converted by Klotho, a senescence-related molecule, into the FGF-23 receptor (10). Thus, the concerted action of Klotho and FGFR1(IIIc) reconstitutes the FGF-23 receptor.

Recently, we described the generation of a murine model of FGF-23(R176Q) overexpression and the evolution of associated biochemical and bone histological changes at 1 and 2 months postnatally (11). We observed that, contrary to previous reports, increased circulating levels of FGF-23 alter parameters of phosphorus and vitamin D as well as calcium homeostasis and that hyperparathyroidism is an integral component of this renal phosphate wasting state. Interestingly, increased circulating levels of PTH with normocalcemia or mild hypocalcemia have also been reported in Hyp mice (12). Alternatively, hyperparathyroidism in XLH is postulated to arise from the standard use of phosphate salts, which induce chronic stimulation of PTH secretion (13). In this study, we have sought to use the mouse genetic approach to clarify the role of PTH in the metabolic derangements associated with Hyp. By generating hemizygous hypophosphatemic male mice (Hyp/Y) homozygous for the Pth-null allele (Pth–/–;Hyp/Y), we show that secondary hyperparathyroidism is an integral component of the pathophysiology of Hyp. In its absence, severe hypocalcemia ensues leading to early postnatal lethality.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Female Hyp mice were purchased from Jackson Animal Laboratories (Bar Harbor, ME), and Pth-null mice (Pth–/–) were generated in our laboratory (14). Mice were fed commercial rodent diet (5001 Rodent Laboratory Chow; Harlan, Indianapolis, IN) and drank tap water. All animal experiments were reviewed and approved by the institutional animal care committee.

Identification of test mice (Pth–/–;Hyp/Y)
Male Pth–/–;Hyp mice (Pth–/–;Hyp/Y) were identified by Southern blot analysis of tail-tip DNA. Genomic DNA (10 µg) was digested with the appropriate restriction endonuclease enzyme (BamHI digestion for Pth–/–;Hyp, EcoRI digestion for Sry), separated by 1% agarose gel electrophoresis, transferred to nitrocellulose membranes in 20x standard saline citrate (SSC) overnight, and hybridized to the radiolabeled DNA fragment (48% formamide, 10% dextran sulfate, 5x SSC, 1x Denhardt’s solution, and 100 µg/ml salmon sperm DNA) at 42 C overnight. The identification of Pth-null mice and Hyp mice was described previously (14, 15). To identify male newborn mice, a set of oligonucleotide primers was designed from the Sry gene (accession number X55491: forward primer, 5'-GAGAGCATGGAGGGCCAT-3', and reverse primer, 5'-CCACTCCTCTGTGACACT-3') to amplify a 265-bp fragment, corresponding to nucleotides 92–357 of Sry. The PCR product was sequenced using an automated ABI 310 sequencer for verification and then used as probe in Southern blot analysis of tail-tip genomic DNA restricted with EcoRI. The membranes were washed in 0.1% SDS plus 2x SSC for 15 min at room temperature with rotation and then in 0.1% SDS plus 0.1% x SSC for another 15 min at 60 C. The autoradiograms were prepared using Kodak BioMax film at –80 C with intensifying screens.

Histology
Long bones were removed, fixed in periodate-lysine-paraformaldehyde fixative overnight at 4 C, and decalcified in EDTA/glycerol solution for 5–7 d at 4 C. Decalcified tibiae were dehydrated and embedded in paraffin, after which 5-µm sections were cut on a rotary microtome. The sections were stained with hematoxylin and eosin or histochemically for tartrate-resistant acid phosphatase (TRAP) activity. Alternatively, undecalcified tibiae were embedded in LR White acrylic resin (London Resin Co. Ltd., London, UK), and 1-µm sections were cut on an ultramicrotome. These sections were stained for mineral with the von Kossa staining procedure and counterstained with toluidine blue.

Histomorphometric analysis
Histomorphometric measurements were made using a BIOQUANT Nova Prime image analysis system (BioQuant Image Analysis Corp., Nashville, TN). The following primary parameters were determined: bone area, bone perimeter, number of osteoblasts, and number of osteoclasts. From the primary data, the structural parameter of bone volume (bone volume/tissue volume) was calculated. All histomorphometric parameters were expressed according to the recommendations of the American Society for Bone and Mineral Research Nomenclature Committee.

Serum biochemistry
Serum concentration of calcium and phosphorus were determined using Sigma Diagnostics reagents (Sigma Chemical Co., St. Louis, MO). Serum-intact PTH was measured using an ELISA (Immutopics, Inc., San Clement), whereas 1,25(OH)2D3 determinations were performed using a commercially available RIA kit (Immunodiagnostic Systems Ltd., Boldon, UK). Mouse serum levels of FGF-23 were examined by an ELISA (Kainos Laboratories Inc., Tokyo, Japan).

Northern blot analysis
cDNA fragments corresponding to nucleotides 535-1586 of mouse 25-hydroxyvitamin D3 24-hydroxylase (Cyp24; accession number D49438), and to nucleotides 421-1471 of mouse 25-hydroxyvitamin-D3-1{alpha}-hydroxylase(1{alpha}-hydroxylase; Cyp27b1; accession number AB006034) were prepared by RT-PCR of mouse kidney total RNA, subcloned, and sequenced. DNA probes for Cyp24, Cyp27b1, and glyceraldehyde-3-phosphate dehydrogenase (Gapdh) were prepared by a random primed DNA labeling kit (Roche Molecular Biosciences, Indianapolis, IN) and [{alpha}-32P] dCTP (800 Ci/mmol; PerkinElmer Life Sciences, Norwalk, CT). Total RNA was isolated from kidney with Tripure Isolation Reagent (Roche), and 20-µg aliquots were fractionated by electrophoresis on a 1% formaldehyde agarose gel, transferred to nitrocellulose membranes, and hybridized to the radiolabeled cDNA fragment (48% formamide, 10% dextran sulfate, 5x SSC, 1x Denhardt’s solution, and 100 µg/ml salmon sperm DNA) at 42 C overnight. The membranes were washed in 0.1% SDS plus 2x SSC for 15 min at room temperature and then in 0.1% SDS plus 0.1% x SSC for another 15 min at 60 C. The autoradiograms were prepared using Kodak BioMax film at –80 C with intensifying screens. Quantification of signal intensity on autoradiograms was performed using NIH ImageJ 1.37 software.

Rescue with PTH (1–34)
Bovine PTH (1–34) was purchased from Calbiochem (La Jolla, CA). A stock solution of 100 µg/ml was prepared in 5% acetic acid and kept at –20 C. PTH (0.06 µg/g body weight in 0.89% NaCl, 0.1% BSA, 1 mM sodium acetate) injections were performed sc twice daily at 12-h intervals for 12 consecutive days. The first injection was administered within the first 12 h after birth because Pth–/–;Hyp/Y mice could survive 48–72 h without exogenous PTH. Because no genotype data were available at the beginning of each experiment, all mice in the litters were injected with PTH. The genotyping was performed at death or after killing the mice on d 13.

Immunohistochemistry
Immunohistochemical staining for 25-hydroxyvitamin D3-1{alpha}-hydroxylase (Cyp27b1) was performed using the avidin-biotin-peroxidase complex technique with a polyclonal sheep antibody against mouse protein (The Binding Site, Birmingham, UK), as described previously (11, 16). Briefly, dewaxed and rehydrated paraffin-embedded sections were incubated with methanol-hydrogen peroxide (1:100) to block endogenous peroxidase activity and then washed in Tris-buffered saline (pH 7.6). The slides were then incubated with Cyp27b1 antiserum (1:150) in 10% normal swine serum for 45 min at 25 C. After rinsing with Tris-buffered saline for 15 min, tissues were incubated with secondary antibody (biotinylated donkey antisheep IgG). Sections were then washed and incubated with the Vectastain Elite ABC reagent (Vector Laboratories, Burlington, Ontario, Canada) for 45 min. Staining was developed using 3,3'-diaminobenzidine (2.5 mg/ml) followed by counterstaining with Mayer’s hematoxylin.

Statistical analysis
The results were analyzed by GraphPad Prism 5.0 software (San Diego, CA). Student’s t test was used to compare data between two groups. Multiple group comparisons were performed using the one-way ANOVA, followed by the Bonferroni post test. All the data are presented as mean ± SEM. The value of P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Male Pth–/–/Hyp mice exhibit early neonatal lethality
Matings between female Hyp and male Pth-null mice (14) were conducted initially to obtain Pth+/–/Hyp progeny and subsequent intercrossings were used to derive Pth–/–;Hyp/Y animals (Fig. 1Go). Offspring from these matings were significantly decreased in numbers after weaning, indicating that a subset of these animals exhibit lethality early in the postnatal period. To determine the genotype and gender of newborn mice, tail-tip DNA was obtained, and Southern blot analysis for Pth, Phex, and Sry was conducted. The Pth–/–;Hyp/Y genotype was not observed in any of the 639 mice examined after weaning. However, immediately after birth, Pth–/–;Hyp/Y mice were present, but none of them were alive longer than 72 h after birth.


Figure 1
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  		FIG. 1. Genotyping of newborn mice. Southern blot analysis of tail-tip genomic DNA was used to identify male Pth–/–/Hyp mice (rectangle). The 6.0- and 4.0-kb bands indicate the presence of wild-type and null Pth alleles, respectively. Y indicates the presence of Y chromosome, as indicated by hybridization of the Sry probe. F, Female; M, male.

 
The skeletal phenotype of the Pth–/–;Hyp/Y mice
Long bones from neonatal Pth–/–;Hyp/Y compound mutants were longer in length than the corresponding Pth–/– or Hyp/Y long bones and exhibited longer proliferating and hypertrophic zones in the growth plate compared with wild-type controls (Fig. 2AGo). The mineralization of the hypertrophic zone was decreased in all three mutants but more so in the Pth–/–;Hyp/Y long bones (Fig. 2BGo). Both trabecular bone volume and osteoblast number were significantly decreased in Pth–/– and Pth–/–;Hyp/Y mice, but these changes were not evident in Hyp/Y mice (Fig. 2Go, C and D). Moreover, the number of TRAP-positive osteoclasts was reduced in all three mutant strains but more profoundly in the Pth–/– and Pth–/–;Hyp/Y long bones (Fig. 2EGo). These findings illustrate that the skeletal phenotype of the Pth–/–;Hyp/Y mice parallels that of the Pth–/– mice and suggests that the absence of PTH is the primary force behind the observed skeletal changes in the neonate double mutants.


Figure 2
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  		FIG. 2. Effect of PTH and Phex deficiency on endochondral skeletal growth. A, Representative micrographs of tibial longitudinal sections stained with von Kossa procedure from male newborn mice of each indicated genotype. Magnification, x25. B, The mineralized area of hypertrophic zone (HZ) was measured on sections stained with von Kossa procedure from three newborn mice of each genotype and presented as mean ± SEM. ***, P < 0.001, mutant relative to wild-type mice. C–E, Trabecular bone volume related to tissue volume (BV/TV, %) (C), osteoblast number related to tissue area (N.Ob/T.Ar, no./mm2) (D), and TRAP-positive osteoclasts related to tissue area (N.Oc/T.Ar, no./mm2) (E) were counted in the metaphyseal region from three newborn mice of each genotype and presented as mean ± SEM. **, P < 0.01; ***, P < 0.001, mutant mice relative to wild-type controls using the one-way ANOVA, followed by the Bonferroni posttest.

 
Male Pth–/–/Hyp mice die from severe hypocalcemia
To determine the cause of the early lethality of the Pth–/–;Hyp/Y mice that do not express functional PTH and PHEX proteins, analysis of serum biochemistry was undertaken. Interestingly, these mice exhibited serum phosphorus levels that paralleled those of the Pth–/– mice despite the concomitant greatly elevated circulating concentrations of FGF-23 (Fig. 3Go, A and E). On the other hand, whereas serum calcium levels were low and similar to those of the Pth–/– mice early on, they remained so until their demise, whereas Pth–/– mice were able to increase their serum calcium concentration starting at 1.5 d postpartum, which likely accounts for their survival (Fig. 3BGo). The ability of the Pth–/– mice to tolerate the hypocalcemia was reflected in circulating levels of 1,25(OH)2D3, which were significantly higher than those observed in the Pth–/–;Hyp/Y mice (125 vs. 35 pg/ml) (Fig. 3CGo). One major stimulus for synthesis of 1,25(OH)2D3 is PTH, which promotes the 1{alpha}-hydroxylation of 25(OH)D3, but the hormone was absent in both genotypes (Fig. 3DGo). Moreover, PTH levels remained elevated in Hyp/Y mice compared with wild-type littermates well into adulthood, thereby confirming previous observations of persistent hyperparathyroidism in this genetic background.


Figure 3
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  		FIG. 3. Serum biochemistry. A–D, Serum phosphorus (n = 4) (A), calcium (n = 4) (B), 1, 25(OH)2D3 (combination of 40 mouse sera) (C), PTH (combination of 20 mouse sera for newborn mice and n = 4 for adult group) (D), and FGF-23 (E and F) levels from 1-d-old (combination of 12 sera) (E) and 7-wk-old (n = 7) (F) mice. *, P < 0.05; **, P < 0.01; ***, P < 0.001, compared with wild-type mice. Student’s t test was used to compare data between two groups (D), whereas the one-way ANOVA followed by the Bonferroni posttest was used for multiple group comparisons (F).

 
FGF-23 is a potent inhibitor of Cyp27b1 expression and/or posttranslational modification of the gene product. Circulating FGF-23 levels were profoundly increased in the Pth–/–;Hyp/Y mice compared with the Pth–/– littermates (30,000 vs. 50 pg/ml) (Fig. 3EGo), and although these levels decreased significantly in adulthood, Hyp/Y mice continued to demonstrate higher and Pth–/– mice lower serum FGF-23 levels than wild-type controls (Fig. 3FGo). Consequently, 1,25(OH)2D3 levels were lower in the Pth–/–;Hyp/Y mice than in the Pth–/– animals (Fig. 3CGo), thereby explaining the inability of the former group of mice to counteract the profound hypocalcemia, itself a potent stimulus of the 1{alpha}-hydroxylation step.

Altered vitamin D metabolism in the Pth–/–;Hyp/Y mice
We next sought to verify that the high circulating levels of FGF-23 in the Pth–/–;Hyp/Y mice impair the ability of the renal hydroxylases to appropriately respond to the hypocalemic stimulus that prevails. Cyp27b1 mRNA expression in renal tissues at 1, 2, and 6 d of age was significantly higher in the Hyp/Y background but was almost completely undetectable when Pth was concurrently ablated (Fig. 4Go, A–C). We have previously reported that FGF-23 impairs the translation and/or posttranslational modification of Cyp27b1, and this was confirmed here with immunohistochemical staining in Hyp/Y renal tissue. In addition, staining for the protein was even more profoundly decreased in the Pth–/–;Hyp/Y background (Fig. 4DGo), confirming the supposition that rising circulating levels of PTH can rescue the Hyp mice by increasing the expression and activity of Cyp27b1 above a critical threshold, thereby increasing the circulating levels of 1,25(OH)2D3 sufficiently to maintain serum calcium levels compatible with survival.


Figure 4
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  		FIG. 4. Cyp27b1 expression in renal tissues. A–C, Representative Northern blot analysis of Cyp27b1 expression in mice aged 1 (A), 2 (B), and 6 (C) d old. Total RNA in each lane was derived from eight kidneys procured from four mice. Average blot intensity for each genotype was obtained from two samples, and the ratio Cyp27b1/Gapdh expression was calculated. D, Immunohistochemical staining for Cyp27b1 expression in renal tissues procured from 2-d-old mice of the indicated genotypes. Arrowheads indicate immunostained proximal convoluted tubules.

 
Using a similar approach, we assessed the renal expression of Cyp24, a cytochrome P450 enzyme that catalyzes the addition of a hydroxyl group on C24 of the vitamin D backbone. Studies in Cyp24–/– mice have confirmed its physiological importance in maintaining vitamin D homeostasis [catabolism of 1,25(OH)2D and inactivation of 25(OH)D thereby regulating production of 1,25(OH)2D] (17). Indeed, Cyp24 mRNA expression was greatly increased in Hyp/Y mice and was even more pronounced in Pth–/–;Hyp/Y animals (Fig. 5Go, A–C). This indicates that circulating PTH in the Hyp/Y background also serves to somewhat dampen the increased transcription of Cyp24 mRNA relative to the Pth–/–;Hyp/Y mice, as previously reported (18). The resulting increase in Cyp24 expression in the latter group of mice would in turn promote a further decrease in circulating levels of 1,25(OH)2D3.


Figure 5
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  		FIG. 5. Cyp24 expression in renal tissues. Representative Northern blot analysis of Cyp24 expression in mice aged 1 (A), 2 (B), and 6 (C) d old. The total RNA in each lane was from eight kidneys procured from four mice. Average blot intensity for each genotype was obtained from two samples, and the ratio Cyp24/Gapdh expression was calculated.

 
Rescue of the Pth–/–;Hyp/Y mice by PTH (1–34)
To further verify that PTH is indeed central in regulating the biochemical consequences of FGF-23, PTH (1–34) (0.06 µg/g body weight injections twice daily) was administered to Pth–/–;Hyp/Y newborn mice, and changes in serum calcium and phosphorus levels, renal hydroxylase expression, and overall survival were assessed. All together, nine Pth–/–;Hyp/Y mice were obtained for this experiment of 299 newborn mice derived from 37 mothers; six of them lived 12 d, two lived 10 d, and one lived 8 d. Longer periods were not investigated. After 2 d of PTH administration, serum phosphorus levels decreased (13 vs. 11 mg/dl), whereas serum calcium levels increased (4.5 vs. 6 mg/dl) (Fig. 6Go, A and B). These changes in serum calcium levels were sufficient in preventing the early demise of the Pth–/–;Hyp/Y mice well into the second week of life, when PTH (1–34) administration was continued (Fig. 6CGo).


Figure 6
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  		FIG. 6. Rescue of Pth–/–;Hyp/Y mice with administration of PTH (1–34). A and B, Serum phosphorus (A) and calcium (B) levels in 2-d-old mice (n = 4 for each group) without (vehicle) and 2 h after PTH (0.06 µg/g body weight) injections administered twice daily. *, P < 0.05; **, P < 0.01 using Student’s t test. C, The 12-d-old wild-type (WT) and surviving Pth–/–;Hyp/Y mice after PTH (1–34) administration twice daily. Both genotypes of mice were of the same age, 12 d old.

 
At 12 d of age, the average body weight of the surviving Pth–/–;Hyp/Y mice (n = 6) treated with PTH injections was 4.44 g, whereas average body and tail lengths were 3.54 and 2.57 cm, respectively. In contrast, wild-type mice (n = 6) of the same age had an average body weight of 6.94 g, body length of 5.1 cm, and tail length of 3.22 cm.

Next, we assessed whether the observed changes in serum calcium levels were in part related to altered expression of Cyp27b1 and Cyp24 in renal tissues after PTH (1–34) administration. Although Cyp27b1 mRNA levels were profoundly diminished in 2-d-old Pth–/–;Hyp/Y mice, PTH (1–34) administration decreased this difference significantly (34.1- vs. 7.0-fold less than wild-type mice), a change that was also reflected in the expression of the protein, as determined by immunostaining (Fig. 7Go, A and B). On the other hand, although Cyp24 mRNA expression in Pth–/–;Hyp/Y mice was increased, PTH (1–34) administration had a modest effect on decreasing these levels (6.1-vs. 4.6-fold higher than in wild-type mice) (Fig. 7CGo), suggesting that Cyp27b1 is the major target of PTH action by increasing enzyme expression, thereby raising serum calcium levels and preventing the early demise of the compound mutant mice.


Figure 7
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  		FIG. 7. Renal Cyp27b1 and Cyp24 expression after PTH (1–34) treatment. A–C, Cyp27b1 expression (A), Cyp27b1 immunohistochemistry (B), and Cyp24 expression (C) in 2-d-old WT and Pth–/–;Hyp/Y renal tissues with and without PTH (1–34) treatment. For Northern blot analysis (A and C), total RNA in each lane was from three mouse kidneys. Average blot intensity for each genotype was obtained from two samples, and the ratios Cyp27b1/Gapdh and Cyp24/Gapdh expression were calculated. Arrowheads indicate proximal convoluted tubules immunostained for Cyp27b1.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Parathyroid function has usually been described as normal in patients with XLH before initiation of therapy with phosphate salts. In fact, hyperparathyroidism is often regarded as a potential complication of treatment (13). Other studies, however, have reported a higher than expected frequency of hyperparathyroidism, even before treatment, in patients with phosphate-wasting rickets (19, 20, 21). This manifestation of hypophosphatemic rickets is therefore more widespread than previously recognized, and it has been speculated that it may precede the development of tertiary hyperparathyroidism.

The PTH status in Hyp mice also has been controversial. Under basal conditions, Hyp mice have been reported to manifest normocalcemia or mild hypocalcemia and have a plasma bioactive PTH concentration significantly greater than that of normal mice (12, 22). This hyperparathyroidism in the slightly hypocalcemic, osteomalacic Hyp mice is consistent with secondary hyperparathyroidism. In contrast, other laboratories have not observed this elevation with RIAs employing different antibodies.

In previous work, we have speculated on the probable cause of this secondary hyperparathyroidism that arises in the setting of high circulating FGF-23 levels (11). We proposed that under these conditions, the associated altered vitamin D metabolism leads to hypocalcemia and that the expected secondary rise in circulating PTH is an attempt to correct this deficit. The pathophysiology of the altered vitamin D metabolism resulting in inappropriately low-normal circulating 1,25(OH)2D3 levels despite significant hypophosphatemia in these states has also been investigated. Normally, the mitochondrial 25-hydroxyvitamin D3 24-hydroxylase (Cyp24) is responsible for the first step in the catabolism of 25(OH)D3 and 1,25(OH)2D3 via the C24 oxidation pathway. Studies have revealed that the enzyme, found in a variety of tissues including the kidney, intestine, and bone, is induced by 1,25(OH)2D3 itself, thus programming the hormone’s own breakdown (24). Increased renal Cyp24 mRNA and immunoreactive protein in phosphate-deprived Hyp mice has been proposed as one of the mechanisms for accelerated 1,25-(OH)2D3 catabolism in XLH (25). Moreover, Cyp24 activity and plasma concentrations of 24,25-dihydroxyvitamin D3 were reported to be significantly higher in Hyp mice than in normal mice when both groups were fed a normal diet (26). Hence, in Hyp mice, Cyp24 activity is reset such that it is inappropriately high for the prevailing serum phosphorus level over a wide range of concentrations, leading both to increased catabolism of 1,25-(OH)2D and decreased substrate availability for its formation.

Disordered regulation of renal Cyp27b1, the enzyme that converts 25(OH)D3 to 1,25(OH)2D3, has also been implicated in Hyp (27, 28). In previous work, we have shown that overexpression of FGF-23 in transgenic mice leads to decreased renal expression of Cyp27b1, whereas that of Cyp24 mRNA is greatly increased (11). The relative inability of PTH and hypophosphatemia to stimulate Cyp27b1 enzymatic activity has also been described recently in Hyp mice (29). The defect in Cyp27b1 activity does not result from aberrant transcriptional regulation but primarily from a defect in translational regulation (30). Nevertheless, rising circulating levels of PTH can rescue the Hyp mice by increasing modestly the expression and/or activity of Cyp27b1 above a critical threshold, although not to normal levels. As a consequence, the resulting increase in the circulating levels of 1,25(OH)2D3 is sufficient to maintain serum calcium levels compatible with survival, likely by, in part, increasing transient receptor potential channel vanilloid 5 (TRPV5) activity in epithelial cells of the renal distal convoluted tubule, thereby decreasing secretion of calcium by the kidney (31) while concurrently increasing absorption of calcium in the small intestine by potentiating intestinal TRPV6 activity (32). The absence of PTH in the Pth–/–;Hyp/Y mice makes this response impossible and leads to the demise of these animals, because in addition to the lack of the aforementioned effects on vitamin D metabolism, hypoparathyroidism also directly reduces renal TRPV5 expression (33).

Of noteworthy interest is our current observation that serum phosphorus is modulated extensively by PTH with very little contribution by circulating FGF-23. Hence, hyperphosphatemia ensues in the absence of PTH despite low serum 1,25(OH)2D3 and irrespective of FGF-23 levels, whether low as in Pth–/– mice or extremely high as in Pth–/–;Hyp/Y mice. It would appear therefore that FGF-23 influences Na gradient-dependent phosphate transporter 2a (Npt2a) activity indirectly via the secondary hyperparathyroidism that ensues from the dysregulated vitamin D metabolism. Interestingly, in a patient with XLH and autonomous hyperparathyroidism after chronic therapy with phosphorus and calcitriol, parathyroidectomy leads to an increase in both serum phosphorus concentration and renal phosphorus reabsorption (21).

Although the pathophysiology of hyperparathyroidism in XLH remains unclear at present, abnormal PTH regulation has been suggested (19). It was reported recently that elevated levels of circulating FGF-23 promote the development of secondary hyperparathyroidism in predialysis patients through suppression of CYP27B1 activity (34). In addition, given the high abundance of PHEX mRNA in parathyroid glands of patients with XLH (21), PHEX itself may also play a role in the regulation of synthesis and secretion of intact PTH. Functional FGF receptors and the coreceptor Klotho have been localized to parathyroid tissue (10). Consequently, it is conceivable that FGF-23 may have a direct influence on the parathyroid cell.

In the present study, we have used the mouse genetic approach to investigate the role of PTH in the biochemical derangements associated with Hyp. Ablation of the Pth locus leads to hyperphosphatemia, profound hypocalcemia, and early demise of the Pth–/–;Hyp/Y mice. This inescapable fate, however, can be prevented by daily administration of exogenous PTH (1–34), which ameliorates the underlying hyperphosphatemia and severe hypocalcemia in the newborn double-mutant mice. Our present findings therefore indicate that hyperparathyroidism is an integral and necessary part of the biochemical alterations in calcium homeostasis arising in Hyp/Y mice. In its absence, severe hypocalcemia and death ensue. Whether these findings are applicable to XLH, however, remains in question. A single case of XLH coexisting with idiopathic hypoparathyroidism has been described (35), and although the patient did exhibit severe hypocalcemia, serum calcium levels were sufficiently high to prevent tetany and death. Variations in the presentation of such a unique and rare condition could arise from a number of causes such as differences between humans and rodents in the renal and extrarenal synthesis and catabolism of 1,25(OH)2D3, disparity in the intestinal absorption of calcium (36), or more likely, differences in circulating FGF-23 levels. In XLH, many but not all affected individuals have elevated FGF-23 levels (23). In contrast, we show here that Hyp mice have high circulating FGF-23 levels compared with wild-type controls. This disparity notwithstanding, the elevated levels of circulating PTH in treatment-naive XLH patients (19) would indicate that hyperparathyroidism likely arises in response to the biochemical derangements in vitamin D metabolism associated with circulating FGF-23 excess or to direct effects of FGF-23 per se on the parathyroids. The ultimate goal is to prevent the occurrence of severe hypocalcemia resulting from the inappropriately low serum 1,25(OH)2D3 levels.


   Footnotes
 
This work was supported by grants to A.C.K. and D.G. from the Canadian Institutes of Health Research.

Disclosure Statement: X.B., D.M., D.G., and A.C.K. have nothing to declare.

First Published Online July 5, 2007

Abbreviations: Cyp24, 25-Hydroxyvitamin D3 24-hydroxylase; Cyp27b1, 25-hydroxyvitamin D3-1{alpha}-hydroxylase; FGF-23, Fibroblast growth factor 23; 1,25(OH)2D3, 1,25-dihydroxyvitamin D3; PHEX, phosphate-regulating gene with homologies to endopeptidases on the X chromosome; SSC, standard saline citrate; TRAP, tartrate-resistant acid phosphatase; TRPV5, transient receptor potential channel vanilloid 5; XLH, X-linked hypophosphatemic rickets.

Received February 21, 2007.

Accepted for publication June 28, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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