Departments of 1Human Genetics, 2Pediatrics, 4Biology, and 5Surgery, McGill University, 3The McGill University-Montreal Children's Hospital Research Institute, Montreal H3Z 2Z3, and 6Shriners Hospital for Children, Montreal, Quebec, Canada H3G 1A6
Submitted 15 October 2003 ; accepted in final form 1 December 2003
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ABSTRACT |
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phosphate wasting; hypophosphatemia; hypercalciuria; nephrocalcinosis; 1,25-dihydroxyvitamin D
Hypophosphatemia, induced by feeding a low-Pi diet, is an important stimulus for increased renal synthesis of 1,25-dihydroxyvitamin D3 [1,25(OH)2D] by the cytochrome P-450 25-hydroxyvitamin D3-1-hydroxylase (CYP27B1, hereafter referred to as 1
OHase) (9). We showed that hypophosphatemia, secondary to renal Pi wasting in Npt2-/- mice, also elicits a significant increase in the serum concentration of 1,25(OH)2D (2, 22) and that the latter is attributable to an increase in renal 1
OHase activity and mRNA abundance (22). Furthermore, we demonstrated that the adaptive increase in serum 1,25(OH) D in Npt2-/- mice is associated with significantly increased intestinal Ca absorption (21), hypercalcemia, decreased circulating levels of PTH, and hypercalciuria (2).
Consistent with the presence of chronic renal Pi wasting and hypercalciuria in Npt2 knockout mice (2), mineral deposits, composed of Ca and Pi with an apatitic mineral phase, were detected in renal sections from Npt2-/- mice but not in those from wild-type littermates (5). The extent of renal calcification, determined by microcomputed tomography (µCT) of intact kidneys, was significantly greater in newborn and weanling Npt2-/- mice than in adult mutants (5). Although the precise mechanism for the decrease in renal calcification with aging is not clear, it correlates with the age-dependent decrease in urinary Ca excretion that is evident in Npt2-/- mice (5). In addition, the degree of hypophosphatemia in Npt2-/- mice, i.e., the difference in serum Pi concentration between wild-type and Npt2-/- mice, decreases with age (2), a finding consistent with the decrease in renal Npt2a gene expression with increasing age (20, 25). Thus the trigger for the adaptive increase in renal 1OHase activity, which leads to increased renal production and serum concentration of 1,25(OH)2D (22), hypercalcemia, hypercalciuria, and renal calcification (5), is likely to be less robust in adult Npt2-/- mice than in weanlings and newborns.
The present study was undertaken to assess the contribution of the increased serum 1,25(OH)2D concentration to the development of hypercalcemia, hypercalciuria, and nephrocalcinosis in Npt2-/- mice. To achieve this goal, we examined the effects of 1OHase gene ablation on urinary Ca excretion and renal calcification in Npt2-/- mice. In addition, we investigated the impact of long-term Pi supplementation on urinary Ca excretion and renal calcification in Npt2-/- mice. The rationale for the latter study was based on the well-known finding that high-Pi diets elicit a decrease in the renal production and serum concentration of 1,25(OH)2D (17). Moreover, we demonstrated that P supplementation of Npt2-/- mice over a 4-day period corrects renal 1
OHase activity and the serum concentration of 1,25(OH)2D, as well as the associated hypercalciuria (22), which is a known risk factor for renal calcification (18, 19). In the present study, we report that both 1
OHase gene ablation and Pi supplementation lead to a significant reduction in urinary Ca excretion and a dramatic decrease in renal calcification in Npt2-/- mice. These findings underscore the importance of 1,25(OH)2D in the development of hypercalciuria and renal calcification in Npt2-/- mice.
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MATERIALS AND METHODS |
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Mouse genotyping. Mouse genotyping was accomplished by PCR amplification of genomic DNA extracted from tail tissue, using Taq polymerase (Qiagen, Mississauga, Ontario, Canada). To test for disruption of the Npt2a gene, we used three primers [sense primer 3F (5'-TGCCCAGGTTGGCACGAAGC-3'), antisense primer 4R (5'-AGTCCTGTCCCCTGCCTGCA-3') both in the Npt2a gene, and antisense primer PGKR (5'-TGCTACTTCCATTTGTCACGTCC-3') in the neor gene cassette], as described previously (2), and fragments of 1,800 and 1,400 bp, respectively, were amplified from the wild-type and mutant alleles. To test for disruption of the 1OHase gene, we used two primers in the 1
OHase gene (sense primer, 5'-CCCCTGTTAAAGGCTGTGAT-3' and antisense primer 5'-GGTCATGGGCTTGATAGGAA-3'), and amplified fragments of 1,277 and 650 bp, respectively, were generated from the wild-type and mutant alleles. The amplified fragments from the Npt2a and 1
OHase PCR reactions were then mixed, fractionated on 1% agarose gels, and visualized with ethidium bromide (Fig. 1).
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Tissue processing and von Kossa staining. Kidneys from wild-type, Npt2-/-, 1OHase-/-, and Npt2-/-/1
OHase-/- mice were split in half transversely with a razor blade and immediately immersed in 10% neutral-buffered formalin and dehydrated to 70% ethanol. Sections from paraffin-embedded kidneys were cut on a rotary microtome at a thickness of 7 µm, and von Kossa staining for mineral was performed by the application of 5% silver nitrate to the sections and exposure to UV light for 30 min. Sections were counterstained for tissue and cell morphology using safranin. Light micrographs were obtained using a Sony DXC-950 3-CCD camera mounted on a Leica DMRBE light microscope (5).
µCT. Kidney scans were performed on a standard desktop Skyscan µCT instrument (model 1072, Skyscan, Aartselaar, Belgium, from Soquelec, Montreal, Quebec, Canada), as described previously (5). This instrument has an 100-KeV sealed, air-cooled, microfocus X-ray source with a polychromatic beam derived from a tungsten target and having a spot size of less than 5 µm. For these analyses, the X-ray source was operated at maximum power (100 KeV) and at 100 µA. Images were captured using a 12-bit, cooled CCD camera (1,024 x 1,024 pixels) coupled by a fiber optics taper to the scintillator. Kidneys from wild-type, Npt2-/-, 1OHase-/-, and Npt2-/-/1
OHase-/- mice were fixed overnight in 10% neutral-buffered formalin, dehydrated to 70% ethanol, wrapped in parafilm (to prevent drying), and scanned by µCT at a magnification resulting in a pixel size of 14.4 µm. With the use of a rotation step of 0.9°, the total scanning time for each rotated kidney was
35 min. This was followed by reconstruction of
300 cross-sections (slice-to-slice distance of 16.5 µm) to give a three-dimensional (3-D) distribution of the calcified tissue and soft tissue with a square voxel size of 28.8 µm. Quantitative data for calcified and total kidney volumes were derived from the 3-D images. Scans were performed on kidneys from 3 to 6 newborn and 3 to 6 weanling Npt2-/- and Npt2-/-/1
OHase-/- mice. Kidneys from age-matched Npt2+/+ (5) and 1
OHase-/- mice were devoid of mineral deposits and were used to set the threshold for analysis. The representative cross-sections shown are in a 1,024 x 1,024-pixel image format, and 3-D reconstruction was performed using the Skyscan tomography software based on triangular surface rendering.
BBM preparation and Western blot analysis. Renal BBMs were prepared from kidney cortex by the MgCl2 preparation method as reported previously (24). The BBMs (50 µg protein) were suspended in gel buffer (14), heated at 55°C for 3 min, electrophoresed on 10% PAGE-SDS gels, transferred to nitrocellulose membranes, and probed with rabbit polyclonal antibodies generated against Npt2a (24) and actin (Sigma, Oakville, Ontario, Canada). Immune complexes on Western blots were visualized by chemiluminescence using an ECL kit (Amersham Biosciences, Montreal, Quebec).
Serum and urine parameters. Commercial kits (Stanbio Laboratory, Boerne, TX) were used to determine serum Ca and Pi and urine Ca, Pi, and creatinine. The fractional excretion index (FEI) for Pi was determined as follows: urine Pi/(urine creatinine x serum Pi). Millimolar concentrations of Pi, Ca, and creatinine were used to calculate the ratios indicated. The serum concentration of PTH was determined using the Mouse Intact PTH ELISA Kit (Immunotopics International, San Clemente, CA), as recommended by the supplier. The serum concentration of 1,25(OH)2D was measured using a Gamma-B Radioimmunoassay kit from Immunodiagnostic Systems (Medicorp, Royalmount, Quebec, Canada).
Statistical analysis. The number of samples examined per group is indicated for each experiment, and the means ± SE are depicted. Differences between two groups were tested for significance using Student's t-test. Differences between more than two groups were evaluated by multivariate analysis of variance, and post hoc analyses of differences between individual groups were performed by Tukey's test using SPSS software. A probability of P < 0.05 was considered to be significant.
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RESULTS |
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The serum Ca concentration is significantly increased in Npt2-/- mice and decreased in 1OHase-/- mice, compared with wild-type littermates (Table 1), as reported previously (2, 7). In Npt2-/-/1
OHase-/- mice, the serum Ca concentration is similar to that in wild-type mice but significantly lower than that in Npt2-/- mice (Table 1). The urine Ca/creatinine ratio is increased in Npt2-/- mice and unchanged in 1
OHase-/- mice compared with wild-type littermates (Table 1). Of relevance to the present study is the demonstration that urine Ca/creatinine is significantly decreased in Npt2-/-/1
OHase-/- mice compared with Npt2-/- mice (Table 1).
The serum concentration of 1,25(OH)2D is significantly increased in Npt2-/- mice compared with wild-type mice and undetectable in 1OHase-/- mice (Table 1), as reported previously (2, 7). Moreover, 1,25(OH)2D is not detectable in the serum of Npt2-/-/1
OHase-/- mice (Table 1). The serum PTH concentration is significantly decreased in Npt2-/- mice and increased in 1
OHase-/- mice compared with wild-type mice (Table 1), as reported previously (2, 7). In Npt2-/-/1
OHase-/- mice, serum PTH is significantly higher than that in Npt2-/- mice (Table 1).
Consistent with the significant increase in serum PTH in 1OHase-/- mice, the renal BBM abundance of immunoreactive Npt2a protein is decreased compared with wild-type littermates, whereas differences in BBM actin abundance are not apparent (Fig. 2). Densitometric analysis revealed that the abundance of Npt2a protein, relative to actin, in 1
OHase-/- mice is 52% of that in wild-type mice. These findings can be attributed to the well-known action of PTH on the endocytosis of Npt2a from the BBM and its subsequent lysosomal degradation (16). Npt2a protein was not detected in BBMs derived from Npt2-/- mice (Fig. 2), as described (2).
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Serum and urine parameters in newborn Npt2-/- and Npt2-/-/1OHase-/- mice are compared in Table 2. Relevant to the present study is the significant decrease in urinary Ca/creatinine in Npt2-/-/1
OHase-/- mice compared with Npt2-/- mice.
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Effect of 1OHase gene ablation on renal calcification in Npt2-/- mice. von Kossa-stained renal sections demonstrated the presence of renal calcification in kidneys of weanling Npt2-/- mice, as reported previously (5), as well as a significant reduction in the number of mineral deposits in kidneys of Npt2-/-/1
OHase-/- mice compared with Npt2-/- mice (data not shown). Given that the mineral deposits are frequently displaced during renal sectioning, the extent of renal calcification in Npt2-/- and Npt2-/-/1
OHase-/- mice was determined by µCT of intact kidneys. Representative images of kidneys from weanling and newborn Npt2-/- and Npt2-/-/1
OHase-/- mice are depicted in Fig. 3 and clearly confirm the impression from von Kossa-stained sections, namely, that the number of mineral deposits is reduced in kidneys of Npt2-/-/1
OHase-/- mice compared with Npt2-/- mice. The data in Table 3 demonstrate that whereas the total tissue volume (TV) is not significantly different in the mutant strains, both the calcified volume (CV) and the CV/TV ratio are significantly decreased in weanling and newborn Npt2-/-/1
OHase-/- mice compared with age-matched Npt2-/- mice.
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Effect of Pi supplementation on serum and urine parameters and renal calcification in Npt2-/- mice. We reported that the serum concentration of 1,25(OH)2D and the urinary Ca excretion are completely normalized in Npt2-/- mice by feeding a high-Pi (1.65% Pi) diet for 4 days (22). In the present study, we examined the long-term effects of Pi supplementation on renal calcification in Npt2-/- weanling and newborn mice. Weanling Npt2-/- mice fed a 1.65% Pi diet exhibited an increase in serum Pi, a decrease in serum Ca, a 21-fold increase in urine Pi/creatinine, and an eightfold decrease in urine Ca/creatinine compared with counterparts fed the 0.6% Pi diet (Table 4). Moreover, with the exception of serum Ca, similar changes were observed in newborn Npt2-/- mice fed a 1.65% Pi diet (Table 4). However, despite the decrease in urinary Ca/creatinine, von Kossa-stained renal sections revealed no apparent changes in renal calcification in weanling and newborn Npt2-/- mice supplemented with the 1.65% Pi compared with age-matched controls fed the 0.6% Pi diet (data not shown).
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We then examined the effects of Pi supplementation, achieved by feeding a 1% Pi diet, on serum and urine parameters in Npt2-/- mice. In both weanling and newborn Npt2-/- mice, serum Pi and urine Pi/creatinine were significantly increased, and urine Ca/creatinine significantly decreased, in mice fed the 1% Pi diet, compared with mutants on the 0.6% diet (Table 4). In addition, the serum 1,25(OH)2D concentration was significantly decreased in Npt2-/- mice maintained on the 1% Pi diet compared with counterparts fed the 0.6% Pi diet [114 ± 17 (n = 4) vs. 239 ± 26 (n = 3) pg/ml, P < 0.05]. Moreover, serum 1,25(OH)2D values in Pi-supplemented Npt2-/- mice [114 ± 17 pg/ml (n = 4)] were not significantly different from those in Npt2+/+ mice fed the 0.6% diet [118 ± 28 pg/ml (n = 6)].
The decrease in urinary Ca/creatinine in weanling and new-born Npt2-/- mice fed the 1% Pi diet (Table 4) was accompanied by a decrease in renal calcification, which was evident by both von Kossa staining of renal sections (data not shown) and µCT of intact kidneys (Fig. 4). Although total kidney volume was not affected by feeding the 1% Pi diet, both CV and CV/TV were significantly reduced in weanling and new-born Npt2-/- mice on the 1% Pi diet compared with counterparts on the 0.6% Pi diet (Table 5). Of interest was the finding that CV/TV is significantly greater in Npt2-/- mice fed the 0.6% Pi test diet (Table 5) than in aged-matched Npt2-/- mice raised on Lab Chow (Table 3). Although the 0.6% Pi test diet and Lab Chow contain the same amounts of Pi and Ca, their content of vitamin D, and of protein and fat, and the source of the latter are markedly different (see MATERIALS AND METHODS). Consistent with these differences are the differences in the urinary Pi/creatinine values in Npt2-/- mice fed the 0.6% test diet (Table 4) and the Lab Chow (Table 1).
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DISCUSSION |
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To abrogate renal 1,25(OH)2D production in Npt2-/- mice, the mutants were crossed with mice in which the 1OHase gene was disrupted by targeted mutagenesis. Previous studies demonstrated that 1
OHase-/- mice exhibit the features of pseudovitamin D deficiency rickets in humans, including hypocalcemia and secondary hyperparathyroidism, which is responsible for increased urinary Pi loss and the development of hypophosphatemia in this model (7, 15). In the present study, we report that the fractional excretion of Pi is significantly elevated in 1
OHase-/- mice and that the latter can be ascribed to a decrease in the abundance of immunoreactive Npt2a protein in the renal BBM. Our findings are consistent with earlier studies demonstrating that the retrieval of Npt2a from the BBM and its subsequent lysosomal degradation are stimulated by PTH (16). Finally, we demonstrate that the circulating concentrations of 1,25(OH)2D are undetectable in mice deficient in both Npt2a and 1
OHase genes and are decreased to normal values in P-supplemented Npt2-/- mice. These findings serve to validate the utility of the Npt2-/-/1
OHase-/- and P-supplemented Npt2-/- mouse models to assess the contribution of 1,25(OH)2D to the development of mineral deposits in kidneys of Npt2-/- mice.
We demonstrate that renal calcification is decreased in both newborn and weanling Npt2-/-/1OHase-/- mice compared with age-matched Npt2-/- mice and suggest that 1,25(OH)2D can play an important physiological role before weaning. In this regard, we reported that both renal 1
OHase (5) and the 1,25(OH)2D receptor (H. Chau, M.Sc. Thesis, McGill University, 2003) are expressed as early as embryonic day 16.5 and that expression is sustained in the postnatal period (5), in agreement with an earlier report (26). These studies predict that 1,25(OH)2D deficiency should have a negative impact on Ca homeostasis in newborn 1
OHase-/- mice. However, the phenotypic consequences of 1
OHase gene ablation have not been examined before weaning (7, 15).
The importance of hypercalciuria as a predisposing factor for the development of renal calcification is underscored in the present study. We show that both strategies tested, namely 1OHase gene ablation and Pi supplementation with a 1% Pi diet, significantly decrease urinary Ca excretion in Npt2-/- mice and that this decrease is associated with a concomitant decrease in renal calcification, estimated by µCT. On the other hand, we also demonstrate that the reduction in urinary Ca/creatinine in Npt2-/- mice supplemented with the 1.65% Pi diet is not associated with a decrease renal calcification. The latter findings may be explained by the marked increase in urinary Pi excretion under these conditions, leading to urinary Ca/Pi supersaturation. Thus the urinary excretion of both Ca and Pi plays an important role in the development of renal calculi.
The present findings are relevant to the pathophysiology and treatment of patients with Ca nephrolithiasis. Prié et al. (18) reported that 30% of stone formers with normal PTH status exhibit a low tubular maximum for Pi reabsorption and, on the basis of these findings, proposed that the renal Pi leak in these patients predisposed them to calcium stone formation by increasing serum 1,25(OH)2D levels, Ca excretion, and urinary saturation. In this regard, the present data suggest that a modest increase in dietary Pi intake may be sufficient to prevent the increase in serum 1,25(OH)2D levels and the recurrence of stones in this subset of patients. Although dietary manipulation is one of the most important strategies for the prevention of recurrent nephrolithiasis (8), the impact of Pi supplementation has not yet been assessed in stone formers with renal Pi wasting.
The most prevalent type of renal calculus in the human population is composed of Ca oxalate (8). However, a high proportion of renal stones in humans consists of both Ca oxalate and Ca/Pi. Moreover, it has been proposed that the formation of Ca oxalate stones is accelerated on a nidus of Ca/Pi. Thus the Npt2-/- mouse, which forms renal mineral deposits consisting of Ca and Pi, may serve as a useful model to study the pathophysiology and treatment of nephrolithiasis in humans.
An unexpected finding in the present study was that renal CV/TV is significantly lower in weanling and newborn Npt2-/- mice fed standard lab chow (Table 3) than in age-matched Npt2-/- mice raised on the 0.6% test diet (Table 5), despite a similar content of Ca and Pi in both diets. Although the underlying basis for the difference in renal calcification is not clear, it may be related to differences in the composition of the two diets. In this regard, there is considerable evidence to suggest that differences in urinary lipids (13), dietary intake of animal protein (3), and dietary acid load (8) can have a profound influence on renal stone formation in patients. Further work is necessary to identify which dietary factors are responsible for the observed differences in renal calcification in Npt2-/- mice reported in the present study.
It has been suggested that hypercalciuria, elicited by Pi depletion in rats, could be explained by an increase in Ca efflux from bone, associated with an increase in osteoclast number and efficiency (1, 4). The contribution of bone to the development of hypercalciuria in Npt2-/- mice has not been assessed. However, the bone phenotype in Npt2-/- mice is markedly different from that in Pi-deprived rats. The mild skeletal abnormalities evident in weanling Npt2-/- mice, when the demand for Pi is high, are completely corrected postweaning (2). Furthermore, histomorphometric analysis revealed that indexes of bone formation are increased and of bone resorption decreased in Npt2-/- mice compared with wild-type littermates (10). This is clearly not the case in Pi-depleted rats where several characteristics of bone, as measured by histomorphometry, are significantly compromised compared with Pi-replete controls (12). Taken together, the data suggest that increased bone resorption is not a major contributor to the hypercalcemia and hypercalciuria in Npt2-/- mice. The reduced serum PTH levels in Npt2-/- mice (Ref. 2 and Table 1) and the well-documented action of PTH on bone resorption are consistent with this hypothesis. However, given that PTH also stimulates renal calcium reabsorption, we cannot exclude the possibility that the reduction in circulating PTH levels, secondary to increased serum 1,25(OH)2D concentrations, contributes to the hypercalciuria in Npt2a knockout mice.
In summary, we demonstrate that strategies aimed at decreasing the serum concentration of 1,25(OH)2D and urinary Ca excretion inhibit renal calcification in Npt2-/- mice. Our findings underscore the importance of 1,25(OH)2D in the development of hypercalciuria and renal calcification in this mouse model and are relevant to a subset of stone-forming patients with renal Pi wasting.
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ACKNOWLEDGMENTS |
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GRANTS
This work was supported by a grant from the Canadian Institutes of Health Research (GR-13297 to H. S. Tenenhouse).
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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