Hyp Mice Research Articles

Parathyroid hormone gene expression in Hyp mice fed a low-phosphate diet.

The murine analogue of X-linked hypophosphataemia is the Hyp mouse; it has chronic phosphate depletion from an inherited defect of renal tubular reabsorption. Phosphate directly regulates the parathyroid (PT) in normal rats and it is of interest whether this regulation is intact in Hyp mice. Hyp mice were fed either a low-phosphate diet or control diet and PTH mRNA levels were measured. In addition changes in NMR-visible kidney and muscle intracellular phosphate potentials in normal and Hyp mice were determined. Mice were maintained on a low-phosphate (0.02%) or normal-phosphate (0.6%) diet for 24 and 72 h. On the normal diet, Hyp mice had hypophosphataemia, normocalcaemia, and normal PTH mRNA levels. Phosphate deprivation for 72 h led to a profound fall in plasma phosphate, a slight but significant rise in plasma calcium, and a dramatic decrease in PTH mRNA, similar to that of normal mice fed this diet. Changes in kidney and muscle intracellular phosphate measured by NMR spectroscopy were not affected by diet or genotype. Dietary phosphate deprivation decreased Hyp mice PTH mRNA levels and caused no change in intracellular phosphate potentials. Therefore Hyp mice parathyroids' adapt appropriately to phosphate deprivation albeit at a lower threshold compared to normal mice.

Medical management and complications of X-linked hypophosphatemic vitamin D resistant rickets.

To improve the growth failure, bowed legs, and biochemical and radiological abnormalities in patients with X-linked hypophosphatemic vitamin D resistant rickets (XLH), combined therapy of phosphate and calcitriol is the best therapeutic approach at present. However, the complications involving combined therapy, such as hypercalcemia, nephrocalcinosis and hyperparathyroidism, are not fully solved. To achieve better control, new therapeutic approaches have been reported recently, for example, growth hormone (GH) or new vitamin D analogs. GH improved linear growth, decreased phosphate reabsorption and increased 1-alpha-hydroxylase activity. Furthermore, 24R,25-dihydroxyvitamin D3 (24,25) improved the bone lesions in hypophosphatemic (Hyp) mice, and also in XLH, without the adverse effects such as hypercalcemia or hypercalciuria compared with 1,25-dihydroxyvitamin D3. These new approaches should be considered for the treatment of patients with XLH.

Phosphate transport in renal cell cultures of gy mice: evidence of a single defect in X-linked hypophosphatemia.

Although current theory holds that the murine homologs of X-linked hypophosphatemia represent mutations of two closely linked genes with distinct pathophysiological consequences, insufficient data are available to support this hypothesis. We investigated whether an intrinsic defect in renal sodium (Na+)-dependent Pi cotransport truly distinguishes gy from hyp mice. We compared Pi transport in immortalized cells from S1 and S2 segments of the renal proximal convoluted tubule (PCT) of normal and gy mice. Cells from both murine models exhibit characteristics of differentiated PCT cells including gluconeogenesis, alkaline phosphatase activity, and parathyroid hormone (PTH)- and thyrocalcitonin (TCT)-dependent adenosine 3',5'-cyclic monophosphate production. More importantly, kinetic studies reveal that cells from the PCT of gy mice have intrinsically normal Pi transport and support the hypothesis that, as in hyp mice, a humoral abnormality is likely responsible for the renal Pi wasting in this mouse model. These observations are consistent with the conclusion that gy and hyp mice do not represent mutations of two closely linked genes but rather two separate mutations of the same gene.

Microassay for the assessment of low levels of hydroxyproline.

Microassay for the assessment of low levels of hydroxyproline.

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

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

Pex gene deletions in Gy and Hyp mice provide mouse models for X-linked hypophosphatemia.

X-linked hypophosphatemic rickets in humans is caused by mutations in the PEX gene which codes for a protein homologous to neutral endopeptidases. Hyp and Gy mice both have X-linked hypophosphatemic rickets, although genetic data and the different phenotypic spectra observed have previously suggested that two different genes are mutated. In addition to the metabolic disorder observed in Hyp mice, male Gy mice are sterile and show circling behavior and reduced viability. We now report the cloning of the mouse homolog of PEX which is highly conserved between man and mouse. The 3' end of this gene is deleted in Hyp mice. In Gy mice, the first three exons and the promotor region are deleted. Thus, Hyp and Gy are allelic mutations and both provide mouse models for X-linked hypophosphatemia.

Osteocalcin abnormalities in Hyp mice reflect altered genetic expression and are not due to altered clearance, affinity for mineral, or ambient phosphorus levels.

The Hyp mouse manifests rickets and renal wasting of phosphorus. We previously reported elevated circulating osteocalcin in Hyp mice, and a paradoxical decrease in response to 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. To investigate these abnormalities further, we characterized in detail the response of circulating osteocalcin to 1,25-(OH)2D3 and compared skeletal osteocalcin in normal and Hyp mice. The affinity of osteocalcin for hydroxyapatite and the protein's clearance were compared in Hyp and normal animals. Finally, the response of osteocalcin messenger RNA (mRNA) to 1,25-(OH)2D3 was examined in normal mice, Hyp mice, and normal mice subjected to dietary phosphorus deprivation. Multiple (n = 3-6) daily doses of 1,25-(OH)2D3 are required to increase serum osteocalcin levels in normal C57 BL/6 mice; the effect is apparent by 6 h and persists for at least 24 h after injection. Single doses of up to 50 ng have no significant effect. In contrast, an approximately 50% decrement in circulating osteocalcin occurs after a single dose of 1,25-(OH)2D3 in Hyp mice. Osteocalcin clearance in Hyp mice appears to be normal. Bone osteocalcin per U calcium or phosphorus is normal in Hyp mice, suggesting that its affinity for hydroxyapatite is normal. Osteocalcin mRNA from Hyp mice is expressed in greater abundance than that from normal animals, reflecting the differences in circulating levels of the protein. Similarly, osteocalcin mRNA from Hyp mice decreases in response to 1,25-(OH)2D3, whereas an increase in osteocalcin message is seen in normal animals. These studies indicate that normal mice are relatively resistant to 1,25-(OH)2D3 stimulation of osteocalcin production. Furthermore, the differences between Hyp and normal mice in circulating osteocalcin reflect differences in the regulation of gene expression.

Osteocalcin abnormalities in Hyp mice reflect altered genetic expression and are not due to altered clearance, affinity for mineral, or ambient phosphorus levels

Osteocalcin abnormalities in Hyp mice reflect altered genetic expression and are not due to altered clearance, affinity for mineral, or ambient phosphorus levels

P35. Changes in tenascin distribution are associated with rachitic lesions: localisation studies in vitamin D-deficient chick and hyp mouse bone

P35. Changes in tenascin distribution are associated with rachitic lesions: localisation studies in vitamin D-deficient chick and hyp mouse bone

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.

Transcriptional regulation and renal localization of 1,25-dihydroxyvitamin D3-24-hydroxylase gene expression: effects of the Hyp mutation and 1,25-dihydroxyvitamin D3.

X-Linked hypophosphatemic (HYP) mice respond to low phosphate (Pi) intake with a fall in the serum concentration of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] and an increase in the renal activity of 1,25-(OH)2D3-24-hydroxylase (24-hydroxylase), the first enzyme in the C-24 oxidation pathway that degrades 1,25-(OH)2D3 to its final inactivation product. In contrast, normal mice respond to a low Pi diet with an adaptive increase in serum 1,25-(OH)2D3 levels and no change in renal 24-hydroxylase. The low Pi response in Hyp mice involves a 3-fold increase in renal 24-hydroxylase maximum velocity and a corresponding increase in 24-hydroxylase immunoreactive protein and messenger RNA (mRNA). To determine the mechanism for the increase in 24-hydroxylase mRNA in the mutant strain, we examined the effect of actinomycin D on renal 24-hydroxylase mRNA abundance and measured renal 24-hydroxylase gene transcription by nuclear run-off assay in Hyp mice fed control and low Pi diets for 4 h. Vehicle and 1,25-(OH)2D3-treated normal mice were also studied 2-4 h post-treatment. Actinomycin D abrogated the increase in renal 24-hydroxylase mRNA elicited by a low Pi diet in Hyp mice and by 1,25-(OH)2D3 in both normal and Hyp mice. 24-Hydroxylase gene transcription, relative to that of glyceraldehyde-3-phosphate dehydrogenase, was increased 2-fold by feeding the low Pi diet to Hyp mice (n = 4; P < 0.05) and 5.4-fold by 1,25-(OH)2D3 administration to normal mice (n = 3; P < 0.01). In situ hybridization localized 24-hydroxylase transcripts to the proximal tubule of normal and mutant mice fed control and low Pi diets and showed that the 1,25(OH)2D3-induced increase in 24-hydroxylase mRNA occurred in the same nephron segment. The present study demonstrates that 1) transcriptional activation can account for the increase in renal 24-hydroxylase mRNA in Pi-deprived Hyp mice and for 24-hydroxylase mRNA induction by 1,25-(OH)2D3; and 2) the renal proximal tubule is the primary site of increased expression of 24-hydroxylase mRNA.

24R,25-dihydroxyvitamin D3 promotes bone formation without causing excessive resorption in hypophosphatemic mice.

To clarify the differences in the action of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] and 24,25-(OH)2D3 in hypophosphatemic (Hyp) mice, a model for familial X-linked hypophosphatemic rickets in humans, we carried out histomorphometric examinations of the effects of these agents in the lumbar vertebra of these mice. The Hyp mice received 1-1000 micrograms/kg.day 24,25-(OH)2D3, 0.01-0.1 micrograms/kg.day 1,25.(OH)2D3, or vehicle alone given daily for 28 days by ip injection. Histomorphometrically, 1,25-(OH)2D3 and 24,25-(OH)2D3 showed similar effects on bone formation. The parameters of bone formation, mineralized bone volume/bone volume, mineral apposition rate, and bone formation rate/bone surface, were improved to a similar extent in a dose-dependent manner by 1,25-(OH)2D3 and 24,25-(OH)2D3, but there were remarkable differences in the indexes of the bone resorption between these two metabolites. In 24,25-(OH)2D3-treated Hyp mice, osteoclast number/bone perimeter and osteoclast surface/bone surface, the parameters of bone resorption, increased to control levels and did not change according to the dose of 24,25-(OH)2D3. However, in 1,25-(OH)2D3-treated Hyp mice, these values increased remarkably, exceeding the control level. That is, 24,25-(OH)2D3 normalized bone resorption in the rachitic mice, whereas 1,25-(OH)2D3 caused excessive stimulation of bone resorption. This qualitative difference between the two compounds contributes to the superior effects exerted by 24,25-(OH)2D3 in improving the bone lesion in Hyp mice. At doses from 1-1000 micrograms/kg.day, 24,25-(OH)2D3 had dose-dependent effects in increasing bone formation without promoting excessive bone resorption, as shown by histomorphometric analysis.

The molecular defect in the renal sodium-phosphate transporter expression pathway of Gyro (Gy) mice is distinct from that of hypophosphatemic (Hyp) mice.

Two animal models of the human disorder hypophosphatemic vitamin D-resistant rickets exist, the Hyp and Gy mice. Affected mice and humans both manifest an X-linked phenotype, and show decreased Na+/Pi transport activity in the renal proximal tubules, which is characterized by a decreased maximal velocity (Vmax). The defect in Hyp mice is most likely due to a decreased transcription rate of the renal Na+/Pi transporter gene. The current studies were designed to define the molecular defect in the Gy mice. Sodium-dependent uptake of phosphate (Pi) in renal BBMV showed uptake levels of 170.58 +/- 25 and 66.00 +/- 11 pmol x mg protein-1 x 6 s-1 in normal and Gy mice, respectively (n=3, P=0.0102). Glucose uptake levels in the BBMV were 1.94 +/- 0.87 and 1.91 +/- 0.35 pmol x mg protein-1 x 6 s-1 in normal and Gy mice, respectively (n=3). Northern blot analysis of kidney cortex in both mice revealed nearly equivalent message levels (normal/Gy=1.01 +/- 0.12, n=3). In situ hybridization localized the mRNA to the renal cortex in both mice and confirmed equal message levels. Western blot analysis of renal BBM proteins, using a polyclonal antiserum, showed one predominant band at 87 kDa in both mouse samples, with intensities being decreased in the Gy mice (normal/Gy=4.129 +/- 0.70, n=4, P< 0.04). Immunohistochemical analysis localized the protein to the apical membrane of proximal tubules in both mice. These results suggest that the molecular defect in the Gy mice is distinct from that in the Hyp mice, and furthermore, that the manifestation of the diseased phenotype in Gy mice is related to a different defect in the renal Na+/Pi transporter expression pathway. The molecular mechanism of the defect likely relates to protein processing, metabolic turnover rate, or translocation to the brush-border membrane. These results further suggest that two distinct X-linked factors modulate different steps in the expression pathway of the Na+/Pi transporter gene.

Renal Na+-phosphate cotransporter gene expression in X-linked Hyp and Gy mice

The X-linked Hyp and Gy mutations are murine homologues of X-linked hypophosphatemia (XLH), a dominant disorder of phosphate (Pi) homeostasis characterized by growth retardation, rickets, hypophosphatemia and decreased renal tubular maximum for Pi reabsorption relative to glomerular filtration rate (Tmp/GFR). In Hyp and Gy mice, the decrease in Tmp/GFR is associated with a reduction in renal brush-border membrane (BBM) Na(+)-Pi cotransport that can be ascribed to a decrease in renal-specific, Na(+)-Pi cotransporter (NPT2) mRNA and protein abundance. Although renal NPT2 gene expression is reduced in Hyp and Gy mice, the NPT2 gene does not map to the X chromosome. These findings exclude NPT2 as a candidate gene for murine and human X-linked hypophosphatemias and suggest that genes at the Hyp, Gy and XLH (HYP) loci are involved in regulation of NPT2 gene expression. Both Hyp and Gy mice respond to low Pi diet with an increase in BBM Na(+)-Pi cotransport, NPT2 mRNA and protein. The increase in NPT2 protein in Pi-depleted mice far exceeds the increase in NPT2 mRNA, suggesting that translational or post-translational mechanisms are involved in the adaptive process. NPT2 protein is localized to the apical surface of the proximal tubule, where immunostaining in both normal and Hyp mice is increased in response to low Pi diet. Pi-deprived Hyp and Gy mice fail to show an increase in Tmp/GFR, indicating that adaptation at the BBM is not sufficient for the overall increase in Tmp/GFR in response to low Pi diet.

Effects of serum phosphate level on formation of incisor dentine in hypophosphatemic mice.

The effects of serum phosphate level on the formation of incisor dentine were investigated in hypophosphatemic (Hyp) mice fed a diet high in calcium and phosphorus (high Ca/P diet). Feeding a high Ca/P diet for more than 10 days resulted in an increase in the serum phosphate level in Hyp mice to one similar to that of normal mice. Lower incisors were cut transversely at the centre of the length of the incisor, a point that had taken approximately 40 days to be reached from the start of dentine formation in Hyp mice. Transverse views of the incisors showed a triangle-like outline in Hyp mice fed a control diet, while the outline became rounded in Hyp mice fed the high Ca/P diet for more than 40 days. In Hyp mice fed the high Ca/P diet for 40 days interglobular dentine was still observed and fluorescent lines produced by tetracycline showed a diffuse and wavy pattern in incisor dentine; however, interglobular dentine became indistinct and fluorescent lines showed a relatively smooth pattern in the incisor dentine of Hyp mice fed the diet for more than 60 days.

Positional cloning of the HYP gene: A review

X-linked hypophosphatemic rickets (HYP) is the most common form of familial hypophosphatemic rickets. Patients variably prcsent with lower extremity deformities, rickets, short stature, bone pain, dental abscesses, enthesopathy, and osteomalacia [1]. It is an X-linked dominant disorder [21 characterized by decreased renal tubular phosphate reabsorption and consequent hypophosphatemia. Observations in the Hyp mouse, one of the murine homologues of the human diseasc, indicate that the disorder is not due to an intrinsic renal defect [3—6]. However, the HYP gene may directly or indirectly regulate the expression of the sodium dependent phosphate cotransporter (NPT2) in the renal proximal tubule [7]. Despite extensive study, the pathophysiology of the disorder has not been elucidated. To gain better understanding of this disorder we have used the positional cloning approach to locate and clone the HYP gene.

Normal phosphate transport in cells from the S2 and S3 segments of Hyp-mouse proximal renal tubules.

An intrinsic phosphate (Pi) transport defect in the proximal tubule (PT) presumably underlies X-linked hypophosphatemic rickets. We recently reported normal Pi transport in the S1 segment of the Hyp mouse PT. Whether Pi wasting results from an abnormality in the S2 or S3 segment remains unknown. Thus, we compared Pi transport in S2 and S3 immortalized cells from transgenic (simian virus 40) normal and Hyp mice. These cells display biochemical features of PT cells, including alkaline phosphatase- and hormone- stimulated cAMP activity as well as gluconeogenesis. Moreover, kinetic studies in S2 cells reveal a similar Km[0.26 +/- 0.03 (+/-SEM) vs. 0.22 +/- 0.03 mM] and maximum velocity (Vmax; 5.5 +/- 0.66 vs. 5.9 +/- 0.72 nmol/mg x 5 min) in normal and Hyp mice, respectively. Km and Vmax were also similar in cells from the S3 segment; however, the Vmax values in S3 cells in normal and Hyp mice (2.8 +/- 0.45 and 3.0 +/- 0.56 nmol/mg x 5 min) were reduced in both animal models compared to those in S2 cells (P < 0.001), whereas the Km values in S3 cells from normal and Hyp mice (0.10 +/- 0.02 and 0.11 +/- 0.04 mM) were increased relative to those in S2 cells (P < 0.001). These data indicate that Pi transport throughout the PT of Hyp mice is intrinsically normal. Such observations exclude the presence of a nascent defect in renal Pi transport in the kidneys of Hyp mice and support the hypothesis that a humoral abnormality underlies X-linked hypophosphatemic rickets.

Fine Genetic Mapping of theHypMutation on Mouse Chromosome X

The hypophosphatemic (Hyp) mouse is the murine homolog of hypophosphatemic vitamin-D-resistant rickets (HYP) in human. Despite extensive investigations in theHypmouse, the pathophysiology of this X-linked dominant disorder remains unclear. As a first step toward cloning theHypgene, we have generated a high-resolution linkage map in the vicinity of theHyplocus using two independent backcross panels segregating theHypmutation, one generated from an interspecific mating between C57BL/6J-Hyp/HypandMus spretusand the other from an intrasubspecific mating between C57BL/6J-Hyp/HypandMus musculus castaneus.Linkage analyses in 1101 backcross progeny using a total of 23 DNA markers favor the following gene order from the centromere:DXMit13–(DXMit11,DXMit34)–(DXMit36,Alas2)–(Hyp,DXMit80)–DXMit98–(DXMit28,DXMit33,DXMit70)–Pdha1–DXMit20. This study has localizedHypto a region of approximately 1 cM flanked by the proximal markersDXMit36andAlas2and the distal markerDXMit98.One microsatellite marker,DXMit80,was found to be very tightly linked toHyp,as it was nonrecombinant withHypamong all the progeny of both backcrosses corresponding to 1101 meioses.

Expression of Na&lt;SUP&gt;+&lt;/SUP&gt;/Phosphate Cotransporter in Hypophosphatemic Mouse Kidney

The hypophosphatemic (Hyp) mouse is a useful model for studying certain genetic defects in renal phosphate (Pi) handling in humans. Sodium-dependent phosphate transport activity was measured in brush border membrane vesicle (BBMV) prepared from renal cortex and in Xenopus oocytes that had been injected with renal poly (A) +RNA. Pi transport activity in Hyp mice was significantly reduced compared with the control. Northern blot analysis revealed that the level of NaPi-7 mRNA was reduced in Hyp mice. Hybrid depletion study showed that the decrease in Pitransport activity in Hyp mice was primarily due to reduced expression of NaPi-7 mRNA. Immunohistochemical staining for control mice confirmed the localization of NaPi-7 protein in the brush border membrane (BBM) of proximal convoluted tubules. In contrast, the staining was decreased in the membrane, but increased in intracellular structures of Hyp mice, suggesting that an abnormality of endocytosis/exocytosis in BBM is also involved in Hyp mutation. In conclusion, the defect in renal phosphate reabsorption in Hyp mice resulted from a reduction in mRNA and protein for NaPi-7 in the proximal convoluted tubules of midcortical and superficial nephrons.

Decreased transcription of the sodium-phosphate transporter gene in the hypophosphatemic mouse.

Recently, it has been hypothesized that the proximal tubular Na(+)-Pi transporter may play a role in murine X-linked hypophosphatemic vitamin D-resistant rickets. In the present investigation, Western blot analysis of renal brush-border membrane proteins, utilizing polyclonal antisera raised against the mouse Na(+)-Pi transporter, revealed a predominant band at 87 kDa in normal and hypophosphatemic (Hyp) mice. The intensity of this band was reduced in the Hyp mouse by 4.5-fold (Hyp/normal = 0.22 +/- 0.04, n = 3, P < 0.05). Additionally, immunohistochemical analysis of kidney cortex in both mice localized the protein to the apical membrane of the proximal tubules. Relative transcription rates of the Na(+)-Pi transporter gene in the normal and Hyp mouse were then investigated. Nuclear run-on assays showed a 51 +/- 0.02% decreased rate of transcription of the Na(+)-Pi transporter gene in the Hyp mice (n = 3). Thus abnormal transcriptional control of this gene in the Hyp mouse likely plays a role in X-linked hypophosphatemia.