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X-linked Hyp rickets is the most common form of inherited vitamin D-resistant rickets in humans. This dominant disorder is characterized by impaired growth and skeletal abnormalities and is manifest as rickets and osteomalacia in children and adults, respectively (1). The hallmark of this disease is hypophosphatemia, secondary to a defect in the renal sodium phosphate (Na+-Pi) transporter that leads to phosphate wasting (2–4).

The Hyp mouse is an animal model of X-linked Hyp rickets, and it displays biochemical and phenotypical abnormalities similar to the human disease. The defective gene for Hyp was mapped to the short arm of the human X chromosome, Xp22.1-p22.2 (5) and the distal end of the mouse X chromosome (6,7). Study of this human disease has been greatly facilitated by discovery of this putative homolog in mouse. However, the mechanisms underlying the bone and renal defects are still unclear. Recent studies have shown that the bone abnormalities are due to low serum phosphate levels, which result from decreased renal phosphate reabsorption (8). However, the intestinal Na+-Pi transporter is shown to be normal in Hyp mice (9,10). The defect in Hyp mice has been attributed to a humoral factor that leads to decreased expression of the Na+-Pi transporter mRNA and protein in the kidney of adult mice (2,4). However, the ontogenic changes of the renal abnormalities and the developmental progression of this disease are still not known.

The PEX gene was identified as the gene defect in Hyp mice and it encodes a protein with homology to neutral endopeptidases (11). Multiple deletion and point mutations have been found in human patients (11,12). Recently, a 3′-end deletion of the PEX gene was found in Hyp mice (13). This observation provides a genetic basis for distinguishing Hyp and normal mice. The current investigation was designed to devise a rapid genetic screening test for Hyp mice and to characterize the ontogenic change in the renal Na+-Pi transporter. We developed a simple genomic DNA screening method by PCR to identify Hyp and normal mice. Using this PCR method, we distinguished Hyp from normal mice at 2 and 6 wk of age. We further studied phenotypical changes and expression of the Na+-Pi transporter in normal and Hyp mice during development.

The early detection of Hyp mice by a simple tail-tipped PCR method will significantly enhance our understanding of developmental aspects of vitamin D-resistant rickets. The change of serum Pi level and the defect in the renal Na+-Pi transporter in young Hyp mice without phenotypical changes may provide a unique opportunity for innovative therapies in the early stage of disease.

METHODS

PCR screening of genomic DNA isolated from Hyp and normal mice. Mutant Hyp heterozygote female (C57BI/6J Hyp/+, 6 wk) and normal male (C57BI/6J +/Y, 8 wk) mice were obtained from The Jackson Laboratories (Bar Harbor, ME). The mutant and normal mice were bred in the animal facility at the University of Arizona, College of Medicine. Female offspring mice were killed, and only male mice were used in the following experiments. The genotype of male Hyp and normal offspring of breeding pairs was determined by PCR screening. Body weight and tail length of each animal were determined before the experiments. The tail of the mouse was rinsed in water, then the tip of the tail (0.5 cm) was cut and digested in 700 µL of buffer (50 mM Tris, pH 8.0, 10 mM EDTA, 100 mM NaCl, and 1% SDS) with 17.5 µL of proteinase K (20 mg/mL) at 55°C for 4-5 h. Proteins were extracted with phenol/chloroform, and DNA was precipitated with ethanol. The DNA pellet was dissolved in the 100 µL of H2O, and 2 µL of genomic DNA were used for subsequent PCR analysis. PCR was carried out using two sets of oligonucleotide primers designed specifically for 5′ and 3′ regions of the mouse PEX gene. The primers for the 5′ region of the PEX gene (1-105 bp) were: upstream, 5′-TAGCAGACGAGCAAGAGAGT-3′; downstream, 5′-GGCTGGAGAGAAGACTTAGA-3′. The primers for the 3′ region of the PEX gene (2257-2341 bp) were: upstream, 5′-AGGGTCAATGGTGCCATTAG-3′; downstream: 5′-TGCACCTCTGTTCATAGTGG-3′. The PCR reaction was carried out for 30 cycles as follows: denaturing at 94°C for 1 min, annealing at 59°C for 2 min, and polymerization at 72°C for 2 min. The PCR products were then fractionated by gel electrophoresis on a 3% MetaPhor-agarose gel (FMC BioProducts, Rockland, ME). The normal mice showed both 105- and 84-bp products for the 5′ and 3′ regions of the PEX gene, respectively, whereas Hyp mice only had a PCR product of 105 bp. Both PCR products for the PEX gene were subloned into pGEM-T vectors (Promega, Madison, WI) and confirmed by sequence analysis (Applied Biosystem Division 373 stretch upgraded automated DNA sequencer) and showed 100% identity to the mouse PEX cDNA (13).

RT-PCR. Total RNA for RT-PCR was extracted from long bones (femur) of normal and Hyp mice (2 mo old) as described below. The mutant and normal mice for these experiments were purchased from The Jackson Laboratories. Tissues were excised from animals, snap frozen, homogenized, and placed in a guanidinium thiocyanate solution (4 M guanidinium thiocyanate, 42 mM sodium citrate, 0.83% SDS, and 0.2 mM β-mercaptoethanol in diethyl pyrocarbonate-treated water). Proteins were extracted by phenol/chloroform, and total RNA was precipitated by isopropanol at -20°C. To eliminate contaminating genomic DNA, the RNA sample was digested with RNase-free DNase (10-20 U/100 µg of DNA, Boehringer-Mannheim, Indianapolis, IN) at 37°C for 30 min. After treatment, proteins were extracted by phenol/chloroform, and RNA was precipitated again.

Total RNA (10 µg) from the bone was reversed transcribed using a mixture of random primers (500 ng), oligo(dT) primer (1 µg), RNAsin (40 U), and Moloney murine leukemia virus reverse transcriptase (200 U, Promega) in a total reaction volume of 25 µL. The reaction was performed at 37°C for 1 h; 2.5 µL of the RT mixture was used for subsequent PCR amplification using primers specific for 5′ and 3′ regions of the PEX gene and amplification parameters as described above.

Measurement of serum phosphate and alkaline phosphatase levels. Blood was drawn from the heart of normal and Hyp mice at 2 and 6 wk of age. Serum phosphate levels and alkaline phosphatase activity were measured using Sigma Chemical Co. diagnostic kits (Sigma Chemical Co., St. Louis, MO), and the procedures were done following the manufacturer's protocol. The reaction of phosphate with ammonium molybdate in the presence of H2SO4, produced a phosphomolybdate complex. The absorbance of this complex at 340 nm was measured by a spectrophotometer, and this value was used to calculate Pi levels. To assess serum alkaline phosphatase activity, serum was incubated with p-nitrophenyl phosphate, and the absorbance of converted complex was measured at 400-420 nm. This value was used to calculate alkaline phosphatase activity.

Northern blot analysis of renal Na+-Pi transporter mRNA levels. Total RNA was isolated from kidney cortex of individual normal and Hyp mice at 2 and 6 wk of age as described above. Total RNA (15 µg) from each animal was used for Northern blot analysis by a standard technique as described previously (14,15). RNA was fractionated by denaturing agarose gel electrophoresis, and transferred to a nitrocellulose membrane. The membrane was prehybridized at 65°C for 4 h with a buffer containing 6× SSC, 5× Denhardt's solution, 10% dextran sulfate, 1% SDS, and 100 µg/mL denatured salmon sperm DNA. Hybridization was carried out at 65°C overnight in the same solution with [α-32P]dCTP-labeled probes prepared from full-length mouse Na+-Pi transporter type II cDNA (13) by random prime labeling (Amersham, Arlington Heights, IL; specific activity, 1 × 107 dpm/mL). High stringency washes were carried out at 65°C with 0.1× SSC and 0.1% SDS. The blot was subsequently reprobed with 1B15 cDNA-specific probe, which is a constitutively expressed gene encoding rat cyclophilin (14). The blot was imaged and the signal intensities were measured by phosphorimage analysis (GS-525, Bio-Rad). Hybridization intensities for each sample were normalized for 1B15 levels on the same blot.

Western blot analysis of the Na+-Pi transporter protein levels. Kidney cortex was excised from 2- and 6-wk-old normal and Hyp mice. BBMs were purified by the MgCl2 precipitation technique as described previously (2). Protein was quantitated by Lowry protein assay (Bio-Rad kit). Purity and enrichment of one kidney cortex membrane preparation was assayed by measuring a BBM-specific marker, leucine amino-peptidase activity (Sigma Chemical Co. diagnostic kit). Protein samples (20 µg) were fractionated by 10% SDS-PAGE and transferred onto a nitrocellulose membrane. The membrane was blocked overnight in PBS/0.05% polyoxyethylene sorbitan monolaurate (Tween 20)/5% powdered milk. The strips were then incubated with primary antiserum at 1:4000 dilution in PBS/0.05% Tween 20/0.1% milk at room temperature for 1 h. This polyclonal antibody was raised in rabbit against a mouse Na+-Pi transporter-specific COOH-terminal peptide, and the specificity of this antiserum for Na+-Pi transporter has been extensively characterized previously (2,14,15). After several more washes, the strips were incubated with secondary antibodies (anti-rabbit immunoglobulin horseradish peroxidase-linked antibody) at 1:1500 dilution for 45 min at room temperature. After several washes, the strips were exposed to enhanced chemiluminescence reagents (ECL system; Amersham) for 1 min and placed to film for 30 s to 3 min. Subsequently, the blots were reacted with mouse monoclonal β-actin antiserum (Sigma Chemical Co.), which serves as an internal control for gel loading and transfer. Signal intensities for the Na+-Pi transporter were determined by densitometric analysis using the Bio-Rad GS-700 image densitometer and were normalized for β-actin signal intensities on the same blot.

Statistical analysis of results. All data were analyzed for statistical significance by factorial one-way ANOVA, using the StatView software (Abacus Concepts, Ins., version 4.53) and are presented as mean ±S.E.M.

RESULTS

Identification of normal and Hyp mice by PCR amplification of genomic DNA. To determine whether animals were normals or mutants, genomic DNA isolated from the tip of the mouse tail was used for PCR amplification. Figure 1A shows PCR amplification products from 2- and 6-wk-old normal and Hyp mouse genomic DNA using specific primers for 5′ and 3′ regions of the PEX gene. The expected band size for the 5′ region of the PEX gene is 105 bp and for the 3′ region is 84 bp. The results showed that both PCR products were obtained from normal mice and only the PCR product for the 5′ region of the PEX gene was obtained in Hyp mice. This lack of the 3′ region of the PEX gene in Hyp mice was further confirmed by RT-PCR amplification of cDNA generated from bone mRNA in which the PEX gene mRNA is expressed. Figure 1B shows that both bands were amplified from the bone RT reaction of normal mice, but only the 5′ region of the PEX gene was amplified from the bone RT reaction of Hyp mice. These results are consistent with the previous observation that 3′ region of the PEX gene was deleted in Hyp mice (13). Based on this deletion mutation in Hyp mice, the PCR detection of the PEX gene mutation from genomic DNA allowed us to identify the normal and Hyp mice at any developmental stage for subsequent experiments.

Figure 1
figure 1

Identification of normal and Hyp mice by PCR amplification. (A) Genomic DNA was isolated from the tip of the tail of individual normal (N) and Hyp (H) mice at 2 and 6 wk of age. PCR amplification was performed with primers specific for 5′ and 3′ regions of the PEX gene in separate reactions. PCR products from these two reactions (105 bp for the 5′ region and 84 bp for the 3′ region) were loaded in the same gel lane for each sample. N-C is negative PCR amplification without template DNA. (B) Total RNA was extracted from bone of normal (N) and Hyp (H) of adult mice (2 mo). PCR amplification was carried out using cDNA reverse transcribed from RNA. RT-PCR products for 5′ and 3′ regions of the PEX gene were loaded in the same gel lane. No-RT is PCR amplification using RNA as a template. N-C is a PCR amplification without DNA.

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Phenotypical changes in Hyp mice at 2 and 6 wk of age. Figure 2 shows the phenotypical changes in normal and Hyp mice at 2 and 6 wk of age. The body weight and tail length measured from each animal were compiled in Figure 2, A and B, respectively. In 2-wk-old animals, data points overlapped between normal and Hyp mice, and there are no significant differences in body weight or tail length between the two groups. The body weight was 8.23 ± 0.26 g for normals (n = 14) and 7.76 ± 0.41 g for Hyp mice (n = 11); tail length was 3.99 ± 0.08 cm for normals (n = 14) and 3.72 ± 0.10 for Hyp mice (n = 11). However, in 6 wk old Hyp mice, the body weight and tail length were significantly decreased as compared with normal mice. Body weight of 6-wk-old mice was 22.04 ± 0.38 g for normal mice (n = 12) and 18.53 ± 0.36 g for Hyp mice (n = 11; p < 0.0001). Tail length for 6-wk-old animals was 7.23 ± 0.06 cm for normal (n = 12) and 5.58 ± 0.08 cm for Hyp mice (n = 11; p < 0.001). These data indicated that Hyp mice did not develop any phenotypical changes at the early stage (2 wk), but manifest a shorter tail and lower body weight at the later stage (6 wk).

Figure 2
figure 2

Assessment of phenotypical changes in body weight and tail length in 2- and 6-wk-old Hyp mice. Body weight and tail length were measured from normal and Hyp mice at 2 and 6 wk of age. Data for body weight and tail are compiled in panels A and B, respectively. The graphs show the distribution of data points obtained from individual animals. 2w-Nor and 6w-Nor are 2- and 6-wk-old normal mice, respectively. 2w-Hyp and 6w-Hyp are 2- and 6-wk-old Hyp mice, respectively. The mean ± SEM is indicated only in the text.

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Changes in serum phosphate levels and alkaline phosphatase activity in 2- and 6-wk-old Hyp mice. Serum phosphate (Pi) levels and alkaline phosphatase activity from normal and Hyp mice at 2 and 6 wk of age are depicted graphically in Figure 3, A and B, respectively. In 2-wk-old animals, serum Pi levels were 14.62 ± 0.51 (mg/dL) for normals (n = 14) and 10.10 ± 0.61 mg/dL for Hyp mice (n = 7). In 6-wk-old mice, serum Pi levels were 11.78 ± 0.46 mg/dL for normals (n = 11) and 6.81 ± 0.25 mg/dL for Hyp mice (n = 11). Interestingly, although there are no phenotypical changes in 2-wk-old Hyp mice as described above, serum Pi levels were significantly decreased in both 2- and 6-wk-old Hyp mice (p < 0.0001 for both ages, normal versus Hyp). For serum alkaline phosphatase (Fig. 3B), activity levels were 18.73 ± 0.78 U/mL (Sigma Chemical Co.) for normals and 27.42 ± 1.13 U/mL for Hyp mice at 2 wk of age, and were 8.86 ± 0.56 U/mL for normals and 18.51 ± 0.80 U/mL for Hyp mice at 6 wk of age. These values showed significant differences between normal and Hyp mice at both age groups (p < 0.0001, normal versus Hyp for both ages). These data indicated that serum Pi levels and alkaline phosphatase activity were significantly altered in the young Hyp mice, although they have not yet developed any phenotypical changes. In addition, serum Pi levels were significantly decreased between 2- and 6-wk-old animals in both normal and Hyp groups (p < 0.0001 2 wk versus 6 wk for both), whereas alkaline phosphatase activity was significantly decreased between 2- and 6-wk-old mice in both normals and Hyp mice (p < 0.001, 2 wk versus 6 wk for both).

Figure 3
figure 3

Changes in serum Pi levels and alkaline phosphatase activity in 2- and 6-wk-old Hyp mice. Serum Pi levels (A) and alkaline phosphatase activity (B) were measured from normal and Hyp mice at 2 and 6 wk of age. Data are presented as mean ± SEM for each group. The asterisk (*) indicates a significant difference between normal and Hyp mice for both ages (p < 0.0001). In addition, for normal mice, there are significant differences between 2- and 6-wk groups for both serum Pi levels and alkaline phosphatase activity (p < 0.0001 for both, not indicated).

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Changes in Na+-Pi transporter mRNA levels in Hyp mice at 2 and 6 wk of age. The defect in the renal Na+-P1 transporter expression pathway in 2- and 6-wk-old Hyp mice was studied at the levels of mRNA and protein. Northern blot analysis was used to determine the level of Na+-Pi transporter mRNA in 2- and 6-wk-old normal and Hyp mice (Fig. 4). The 2.6-kb band is specific for the renal Na+-Pi transporter and the 1.0-kb band corresponds to rat 1B15 (Fig. 4A). In addition, a faint 4.6-bp band was also hybridized with the Na+-Pi probe as previously described (2) (data not shown). The signal intensity of the Na+-Pi transporter was normalized by the 1B15 signal for each sample. The data were averaged and presented graphically in Figure 4B. The results showed that Na+-Pi transporter mRNA levels in 2-wk-old mice were 1.47 ± 0.21 arbitrary densitometric units in normals (n = 6) and 0.68 ± 0.14 in Hyp mice (n = 7), whereas for 6-wk-old mice levels were 2.41 ± 0.42 in normals (n = 5) and 1.43 ± 0.33 (n = 5) in Hyp mice. Statistical analysis showed significant decreases in both 2- and 6-wk-old Hyp mice as compared with normal mice (p < 0.039, normal versus Hyp at 2 wk; p < 0.027, normal versus Hyp at 6 wk). In addition, in normal mice, the Na+-Pi transporter mRNA was significantly increased during ontogeny (p < 0.025, 2 versus 6 wk). Although statistical analysis showed no significance difference between ages in Hyp mice (p = 0.058), there was a strong trend that Na+-Pi transporter mRNA levels were developmentally increased in Hyp mice. These data demonstrated that a defect in the renal Na+-Pi transporter at the mRNA level in Hyp mice occurred at least as early as 2 wk.

Figure 4
figure 4

Northern blot analysis of renal Na+-Pi transporter mRNA levels in normal and Hyp mice at 2 and 6 wk of age. (A) Total RNA was prepared from kidney cortex of individual normal (N) and Hyp (H) mice at 2 and 6 wk of age. Hybridization to mouse renal Na+-Pi transporter-specific probes showed a predominant transcript at 2.6 kb and a minor transcript at 4.6 kb (the 4.6-kb band was revealed only after long exposure; data not shown). The hybridization signal at 1.0 kb corresponds to a constitutive 1B15 (cyclophilin) on the same blot. (B) Signal intensity for the Na+-Pi transporter was normalized by signal intensity for 1B15, and data are presented as mean ± SEM for each group. The asterisk (*) indicates a significant difference between normal and Hyp mice in both age groups (p < 0.03 for 2 wk; p < 0.02 for 6 wk). For normal mice, there is also a significant difference between 2 and 6 wk (p < 0.02; not indicated).

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Changes in Na+-Pi transporter protein levels in Hyp mice at 2 and 6 wk of age. Kidney BBM were obtained from individual mice. The membrane preparation was enriched 22-33-fold compared with crude membrane homogenate as determined by leucine aminopeptidase activity. Figure 5A shows Western blot analysis of renal BBM proteins from normal and Hyp mice at 2 and 6 wk of age. Antiserum specific for mouse renal Na+-Pi transporter showed a predominant 87-kD band, and antiserum against β-actin showed a 42-kD band. In addition, a minor 37-kD band was also detected with antiserum against the Na+-Pi transporter as previously described (2,14) (data not shown). The intensity of the signal for the 87-kD Na+-Pi transporter band was normalized by β-actin on the same blot, and results are depicted graphically in Figure 5B. For 2-wk-old animals, the signal intensity was significantly decreased in Hyp mice (0.22 ± 0.08 densitometric units, n = 5) as compared with normal mice (0.90 ± 0.10, n = 7; p < 0.0011). For 6-wk-old animals, the signal intensity was also significantly decreased in Hyp (0.30 ± 0.09, n = 5) compared with normal (1.47 ± 0.19, n = 6; p < 0.0001) mice. The normal mice showed ontogenic increase of Na+-Pi protein levels from 2 to 6 wk (p < 0.0001). However, Hyp mice showed no significant difference during development.

Figure 5
figure 5

Western blot analysis of renal Na+-Pi transporter protein levels in normal and Hyp mice at 2 and 6 wk of age. (A) BBM proteins were isolated from individual normal (N) and Hyp (H) mice at 2 and 6 wk. Protein (20 µg) from each sample was fractionated by 10% SDS-polyacrylamide gel and transferred onto nitrocellulose. Reaction with a mouse specific Na+-Pi transporter antiserum shows a predominant 87-kD band and a minor 33-kD band (33-kD band not shown). β-Actin serves as an internal control for loading. (B) Signal intensity for the Na+-Pi transporter was normalized by signal intensity for β-actin, and data are presented as mean ± SEM for each group. The asterisk (*) indicates a significant difference between normal and Hyp mice in both age groups (p < 0.001 for 2 wk; p < 0.0001 for 6 wk). For normal mice, there is also a significant difference between ages of 2 and 6 wk (p < 0.003; not indicated).

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DISCUSSION

X-linked hypophosphatemic rickets shows similar phenotypical changes in humans and mice, including growth retardation and skeletal abnormalities. In 6-wk-old mice, a 16% reduction in body weight and a 23% shortening in tail length were observed in Hyp mice compared with normal mice. However, these changes were not evident in the early age (suckling stage, 2 wk), suggesting that the skeletal abnormalities develop over a period of time and are manifest only in the older age group. Therefore, there were no phenotypical assessments that could be used in the early stage to distinguish Hyp from normal mice. Here we report a simple tail-tipped PCR method to screen for the genotype of male Hyp and normal mice. Using PCR to amplify genomic DNA isolated from mice allows us to accurately and rapidly segregate the normal and mutant (Hyp) mice in the male sex group. Both 5′ and 3′ regions of the PEX gene were amplified from normal mice, whereas only 5′ region of the PEX gene was amplified from Hyp mice, because a uniform deletion mutation has been identified in all Hyp mice (13). This method, however, cannot distinguish normal from heterozygote female animals, because most studies were carried out using male mice. This PCR method facilitated the studies of Hyp mice, particularly in the early stage when the phenotypical changes were not evident.

To further confirm this method, mice screened at 2 wk of age were followed into adulthood and physiologic parameters were assessed. We found that Hyp or normal mice that were genotyped by PCR at 2 wk of age indeed developed Hyp or normal phenotype in adulthood as expected.

Serum Pi levels and alkaline phosphatase activity were measured in 2- and 6-wk-old mice. Decreased serum Pi levels and increased alkaline phosphatase activities in adult Hyp mice have been shown previously (1,17). The current study additionally showed that, in 2-wk-old animals, Hyp mice have already developed these biochemical changes. The serum Pi level was decreased by 31% in 2-wk-old Hyp mice, whereas a reduction of 42% was observed in 6-wk-old mice. In contrast to serum Pi level, the activity of alkaline phosphatase was increased by 46% in 2-wk-old Hyp mice and 109% in 6-wk-old Hyp mice compared with the normals. Although the Hyp state occurred in the early stage (2 wk), the phenotypical features of Hyp mice were not evident until the later stage (6 wk), suggesting that the bone abnormalities resulted from low serum Pi levels and took place over a period of time.

The pathogenesis of the hypophosphatemic rickets has been linked to a defect in the renal Na+-Pi transporter (2–4,15). Previous studies have shown a decrease in renal BBM vesicle sodium-dependent phosphate uptake in Hyp mice (3,9). Molecular characterization of this defect showed a 3-fold decrease in mRNA level for the Na+-Pi transporter and 6-10-fold decrease in protein levels in adult Hyp mice (2,15). However, whether this Na+-Pi transporter defect occurs in the early stage of Hyp mice is not known. The present study showed that Na+-Pi transporter mRNA levels were decreased by 2.1-fold in 2-wk-old Hyp mice and 1.7-fold in 6-wk-old Hyp mice as determined by Northern blot analysis. Western blot analysis showed that Na+-Pi transporter protein levels were decreased by 4.5-fold in 2-wk-old Hyp mice and 4.9-fold in 6-wk-old mice. The discrepancy of decreased levels of the Na+-Pi transporter mRNA and proteins in older mice between the current and previous studies mentioned above may be due to the fact that individual animals were used here as opposed to groups of animals in the previous study, or that 6-wk-old animals were used in the current study, whereas 6-10-wk-old animals were used in the previous study. Overall, in the current study, the reduction of the Na+-Pi transporter protein is greater than mRNA, suggesting that a posttranscriptional mechanism in addition to a transcriptional mechanism may be involved in regulation of renal Na+-Pi transporter in Hyp mice. To date, there are no other studies that have been done to address this question.

Ontogenic changes in renal Na+-Pi transporter expression were studied only in rats previously. The abundance of renal Na+-Pi transporter mRNA analyzed by Northern blot was not significantly changed during development in the rat. However, the immunoreactive protein level analyzed by Western blot analysis showed a gradual increase from 2 wk of age to adulthood (18). However, the current study showed that expression of the Na+-Pi transporter mRNA was regulated during development in normal mice. In normal mice, the mRNA level for the Na+-Pi transporter was significantly increased by 1.7-fold, whereas the protein level was significantly increased by 1.6-fold between 2 to 6 wk of age. The correlated increases in levels of mRNA and protein during ontogeny of normal mice suggested a transcriptional regulation may be involved. However, it is still unclear why mice and rats showed different developmental expression patterns; that Na+-Pi mRNA levels were up-regulated in mice, but did not change in rats.

The PEX gene, a candidate gene for X-linked hypophosphatemic rickets, has been known to be expressed predominately in the mouse bone, lung, and embryo (13). The early expression of this gene may dictate an early onset of regulation of the renal Na+-Pi transporter in mice. In Hyp mice, a large 3′-end deletion (approximately 1 kb) of the PEX gene transcript has been identified (13). Therefore, most likely, this genetic defect affects the expression of the Na+-Pi transporter in the early stage of development. This hypothesis was indeed confirmed by an early occurrence of the renal Na+-Pi transporter defect in genetically identified Hyp mice observed in our study. The inhibition of expression of Na+-Pi transporter and changes in serum Pi and alkaline phosphatase were detected in 2-wk-old Hyp mice. These pathologic changes in Hyp mice possibly occur even earlier than the 2 wk of age studied here. However, phenotypical changes were developed only after 2 wk of age. Significant growth retardation as assessed by lower body weight and shorter tail length in Hyp mice can be identified in 6-wk-old Hyp mice. These results demonstrate the great potential and implications in seeking early intervention for human patients whose serum Pi levels are already reduced, but have not yet developed phenotypes. The early effective therapy in raising serum Pi levels may eventually prevent the skeletal abnormalities in the later stage of disease. However, conventional therapies with phosphate and vitamin D have not been effective, and future investigations should be directed at developing gene therapy strategies at the early stage. Osteoblast cell-specific delivery of the normal PEX gene or direct delivery of Na+-Pi gene to renal cortex of Hyp mice by intrarenal-pelvic infusion at the early stage will be suitable approaches to correct the defect in Hyp mice.