1 -Hydroxylase gene ablation and P i supplementation inhibit renal calcification in mice homozygous for the disrupted Npt2a gene

【摘要】  Disruption of the major renal Na-phosphate (P i ) cotransporter gene Npt2a in mice leads to a substantial decrease in renal brush-border membrane Na-P i cotransport, hypophosphatemia, and appropriate adaptive increases in renal 25-hydroxyvitamin D 3 -1 -hydroxylase (1 OHase) activity and the serum concentration of 1,25-dihydroxyvitamin D 3 [1,25(OH) 2 D]. The latter is associated with increased intestinal Ca absorption, hypercalcemia, hypercalciuria, and renal calcification in Npt2 - / - mice. To determine the contribution of elevated serum 1,25(OH) 2 D levels to the development of hypercalciuria and nephrocalcinosis in Npt2 - / - mice, we examined the effects of 1 OHase gene ablation and long-term P i supplementation on urinary Ca excretion and renal calcification by microcomputed tomography. We show that the urinary Ca/creatinine ratio is significantly decreased in Npt2 - / - /1 OHase - / - mice compared with Npt2 - / - mice. In addition, renal calcification, determined by estimating the calcified volume to total renal volume (CV/TV), is reduced by 80% in Npt2 - / - /1 OHase - / - mice compared with that in Npt2 - / - mice. In Npt2 - / - mice derived from dams fed a 1% P i diet and maintained on the same diet, we observed a significant decrease in urinary Ca/creatinine that was also associated with 80% reduction in CV/TV when compared with counterparts fed a 0.6% diet. Taken together, the present data demonstrate that both 1 OHase gene ablation and P i supplementation inhibit renal calcification in Npt2 - / - mice and that 1,25(OH) 2 D is essential for the development of hypercalciuria and nephrocalcinosis in the mutant strain.

phosphate wasting; hypophosphatemia; hypercalciuria; nephrocalcinosis; 1,25-dihydroxyvitamin D

【关键词】  Hydroxylase ablation supplementation calcification homozygous disrupted

THE TYPE IIA Na-phosphate (P i ) cotransporter Npt2a is the most abundant Na-P i cotransporter in mouse kidney ( 23 ) and is expressed in the brush-border membrane (BBM) of proximal tubular cells ( 6 ) where the bulk of filtered P i is reabsorbed. Studies in our laboratory demonstrated that mice homozygous for the disrupted Npt2a gene ( Npt2 - / - ) exhibit an increase in urinary P i excretion, an 80% loss in BBM Na-P i cotransport, and significant hypophosphatemia ( 2 ). In addition, Npt2 - / - mice fail to respond to P i deprivation with an adaptive increase in BBM Na-P i cotransport ( 11 ) and to parathyroid hormone (PTH) with a decrease in transport ( 27 ). These findings underscore the significant role of Npt2a in renal P i reabsorption and its regulation by dietary P i and PTH.

Hypophosphatemia, induced by feeding a low-P i diet, is an important stimulus for increased renal synthesis of 1,25-dihydroxyvitamin D 3 [1,25(OH) 2 D] by the cytochrome P -450 25-hydroxyvitamin D 3 -1 -hydroxylase (CYP27B1, hereafter referred to as 1 OHase) ( 9 ). We showed that hypophosphatemia, secondary to renal P i wasting in Npt2 - / - mice, also elicits a significant increase in the serum concentration of 1,25(OH) 2 D ( 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 P i wasting and hypercalciuria in Npt2 knockout mice ( 2 ), mineral deposits, composed of Ca and P i 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 P i 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 1 OHase activity, which leads to increased renal production and serum concentration of 1,25(OH) 2 D ( 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) 2 D concentration to the development of hypercalcemia, hypercalciuria, and nephrocalcinosis in Npt2 - / - mice. To achieve this goal, we examined the effects of 1 OHase gene ablation on urinary Ca excretion and renal calcification in Npt2 - / - mice. In addition, we investigated the impact of long-term P i 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-P i diets elicit a decrease in the renal production and serum concentration of 1,25(OH) 2 D ( 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) 2 D, 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 P i 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) 2 D in the development of hypercalciuria and renal calcification in Npt2 - / - mice.

MATERIALS AND METHODS

Mice. Mice homozygous for the disrupted Npt2a ( 2 ) and 1 OHase ( 7 ) genes were established by targeted mutagenesis and backcrossed for several generations onto a C57BL/6J background. Npt2 - / - /1 OHase - / - mice were generated by three consecutive breeding strategies: 1 ) Npt2 - / - females x 1 OHase + / - male mice, 2 ) Npt2 + / - /1 OHase + / - females x Npt2 + / - /1 OHase + / - male mice, and 3 ) Npt2 - / - /1 OHase + / - females x Npt2 - / - /1 OHase + / - male mice. Unless otherwise specified, all mice were maintained on Rodent Lab Chow (5001, Ralston Purina, St. Louis, MO) containing 0.6% P i, 1.0% Ca, 4.5 IU vitamin D/g, 23.4% protein, and 4.5% fat. For the P i supplementation study, Npt2 - / - female and male breeders were maintained on test diets (TD) containing either 0.6, 1, or 1.65% P i (TD-98243, TD-86129, and TD-88345, respectively, Harlan Teklad, Madison, WI) and offspring derived therefrom were kept on the respective test diets until death. Other than the Pi content, the test diets were identical in every respect and contained 1.0% Ca, 2.2 IU vitamin D/g, 16% protein, and 10% fat. Newborn (1-5 days of age) and weanling (19-30 days of age) mice were used for the present studies. All animal studies were conducted in accordance with the guidelines of the Canadian Council of Animal Care and with the approval of the local Institutional Animal Care and Use Committee.

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 neo r 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 1 OHase 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 ).

Fig. 1. PCR genotyping of mice homozygous for the disrupted Npt2a and 1 OHase alleles. PCR amplification of genomic DNA extracted from tail tissue was accomplished as described in MATERIALS AND METHODS. Fragments of 1,800 and 1,400 bp, respectively, were amplified from the wild-type and disrupted Npt2a alleles, and fragments of 1,277 and 650 bp, respectively, were generated from the wild-type and disrupted 1 OHase alleles.

Tissue processing and von Kossa staining. Kidneys from wild-type, Npt2 - / -, 1 OHase - / -, 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 ).

µ CT. 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 - / -, 1 OHase - / -, 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 MgCl 2 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 P i and urine Ca, P i, and creatinine. The fractional excretion index (FEI) for P i was determined as follows: urine P i /(urine creatinine x serum P i ). Millimolar concentrations of P i, 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) 2 D 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.

RESULTS

Effect of 1 OHase gene ablation on serum and urine parameters in Npt2 -/- mice. In weanling animals, the serum P i concentration is significantly decreased in both Npt2 - / - and 1 OHase - / - mice compared with wild-type littermates ( Table 1 ), as reported previously ( 2, 7 ). Serum P i is also significantly decreased in weanling mice in which both the Npt2a and 1 OHase genes have been disrupted ( Table 1 ). The FEI Pi is similar in all three mutant genotypes ( Npt2 - / -, 1 OHase - / -, and Npt2 - / - /1 OHase - / - mice) and significantly increased compared with wild-type mice ( Table 1 ). These data provide evidence for renal P i wasting in Npt2 - / -, 1 OHase - / -, and Npt2 - / - /1 OHase - / - mice.

Table 1. Effect of 1 OHase gene ablation on serum and urine parameters in weanling Npt2 -/- mice

The serum Ca concentration is significantly increased in Npt2 - / - mice and decreased in 1 OHase - / - 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) 2 D is significantly increased in Npt2 - / - mice compared with wild-type mice and undetectable in 1 OHase - / - mice ( Table 1 ), as reported previously ( 2, 7 ). Moreover, 1,25(OH) 2 D 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 1 OHase - / - 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 ).

Fig. 2. Npt2a protein abundance in renal brush-border membranes (BBMs) prepared from wild-type, Npt2 + / + /1 OHase - / -, and Npt2 - / - /1 OHase + / + mice. Renal BBM vesicles were prepared from kidneys derived from Npt2 + / + /1 OHase + / + ( n = 3), Npt2 + / + /1 OHase - / - ( n = 2), and Npt2 - / - /1 Ohase + / + ( n = 1) mice, subjected to 10% SDS-PAGE and analyzed by immunoblotting with antibodies against Npt2a and actin, as described in MATERIALS AND METHODS.

Serum and urine parameters in newborn Npt2 - / - and Npt2 - / - /1 OHase - / - 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.

Table 2. Effect of 1 OHase gene ablation on serum and urine parameters in newborn Npt2 -/- mice

Effect of 1 OHase 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.

Fig. 3. Effect of 1 OHase gene ablation on renal calcification in Npt2 - / - mice. Images were derived by microcomputed tomography of intact kidneys from newborn and weanling Npt2 - / - /1 OHase + / + and Npt2 - / - /1 OHase - / - mice using a Skyscan instrument as described in MATERIALS AND METHODS. Dense black spots are indicative of renal calcification. Representative images of kidneys from 2 mice per group are depicted.

Table 3. Effect of 1 OHase gene ablation on renal calcification in Npt2 -/- mice

Effect of P i supplementation on serum and urine parameters and renal calcification in Npt2 -/- mice. We reported that the serum concentration of 1,25(OH) 2 D and the urinary Ca excretion are completely normalized in Npt2 - / - mice by feeding a high-P i (1.65% P i ) diet for 4 days ( 22 ). In the present study, we examined the long-term effects of P i supplementation on renal calcification in Npt2 - / - weanling and newborn mice. Weanling Npt2 - / - mice fed a 1.65% P i diet exhibited an increase in serum P i, a decrease in serum Ca, a 21-fold increase in urine P i /creatinine, and an eightfold decrease in urine Ca/creatinine compared with counterparts fed the 0.6% P i diet ( Table 4 ). Moreover, with the exception of serum Ca, similar changes were observed in newborn Npt2 - / - mice fed a 1.65% P i 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% P i compared with age-matched controls fed the 0.6% P i diet (data not shown).

Table 4. Effect of P i supplementation on serum and urine parameters in Npt2 -/- mice

We then examined the effects of P i supplementation, achieved by feeding a 1% P i diet, on serum and urine parameters in Npt2 - / - mice. In both weanling and newborn Npt2 - / - mice, serum P i and urine P i /creatinine were significantly increased, and urine Ca/creatinine significantly decreased, in mice fed the 1% P i diet, compared with mutants on the 0.6% diet ( Table 4 ). In addition, the serum 1,25(OH) 2 D concentration was significantly decreased in Npt2 - / - mice maintained on the 1% P i 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) 2 D values in P i -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% P i 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% P i diet, both CV and CV/TV were significantly reduced in weanling and new-born Npt2 - / - mice on the 1% P i diet compared with counterparts on the 0.6% P i diet ( Table 5 ). Of interest was the finding that CV/TV is significantly greater in Npt2 - / - mice fed the 0.6% P i test diet ( Table 5 ) than in aged-matched Npt2 - / - mice raised on Lab Chow ( Table 3 ). Although the 0.6% P i test diet and Lab Chow contain the same amounts of P i 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 P i /creatinine values in Npt2 - / - mice fed the 0.6% test diet ( Table 4 ) and the Lab Chow ( Table 1 ).

Fig. 4. Effect of P i supplementation on renal calcification in Npt2 - / - mice. Images were derived by microcomputed tomography of intact kidneys from newborn and weanling Npt2 - / - mice derived from dams fed either 0.6 or 1% P i diets and raised on the same diets. Images were obtained using a Skyscan instrument as described in MATERIALS AND METHODS. Dense black spots are indicative of renal calcification. Representative images are depicted.

Table 5. Effect of P i supplementation on renal calcification in Npt2 -/- mice

DISCUSSION

Mice homozygous for the disrupted Npt2a gene exhibit renal calcification that is associated with significant increases in the renal synthesis and serum concentration of 1,25(OH) 2 D, which are accompanied by intestinal Ca hyperabsorption, hypercalcemia, a decrease in serum PTH levels, and hypercalciuria ( 2, 5, 21, 22 ). To establish the role of 1,25(OH) 2 D in the development of renal calcification, we examined the effects of 1 OHase gene ablation and P i supplementation on urinary Ca excretion and renal stone formation in Npt2 - / - mice. We report that disruption of renal 1,25(OH) 2 D synthesis significantly decreases urinary Ca excretion and inhibits renal calcification in Npt2 - / - mice. Furthermore, we demonstrate that P i supplementation also reduces serum 1,25(OH) 2 D, urinary Ca loss, and nephrocalcinosis in Npt2 - / - mice. Our data support the hypothesis that the adaptive increase in serum 1,25(OH) 2 D, in response to hypophosphatemia, serves as a trigger for the cascade of events leading to the development of renal mineral deposits in Npt2 - / - mice. Moreover, our data are relevant to the finding that a significant proportion of stone-forming patients with hypercalciuria exhibit hypophosphatemia, secondary to a decrease in renal tubular P i reabsorption ( 18 ).

To abrogate renal 1,25(OH) 2 D production in Npt2 - / - mice, the mutants were crossed with mice in which the 1 OHase 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 P i loss and the development of hypophosphatemia in this model ( 7, 15 ). In the present study, we report that the fractional excretion of P i 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) 2 D 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) 2 D to the development of mineral deposits in kidneys of Npt2 - / - mice.

We demonstrate that renal calcification is decreased in both newborn and weanling Npt2 - / - /1 OHase - / - mice compared with age-matched Npt2 - / - mice and suggest that 1,25(OH) 2 D can play an important physiological role before weaning. In this regard, we reported that both renal 1 OHase ( 5 ) and the 1,25(OH) 2 D 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) 2 D 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 1 OHase gene ablation and P i supplementation with a 1% P i 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% P i diet is not associated with a decrease renal calcification. The latter findings may be explained by the marked increase in urinary P i excretion under these conditions, leading to urinary Ca/P i supersaturation. Thus the urinary excretion of both Ca and P i 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 P i reabsorption and, on the basis of these findings, proposed that the renal P i leak in these patients predisposed them to calcium stone formation by increasing serum 1,25(OH) 2 D levels, Ca excretion, and urinary saturation. In this regard, the present data suggest that a modest increase in dietary P i intake may be sufficient to prevent the increase in serum 1,25(OH) 2 D 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 P i supplementation has not yet been assessed in stone formers with renal P i 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/P i. Moreover, it has been proposed that the formation of Ca oxalate stones is accelerated on a nidus of Ca/P i. Thus the Npt2 - / - mouse, which forms renal mineral deposits consisting of Ca and P i, 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 P i 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 P i 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 P i -deprived rats. The mild skeletal abnormalities evident in weanling Npt2 - / - mice, when the demand for P i 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 P i -depleted rats where several characteristics of bone, as measured by histomorphometry, are significantly compromised compared with P i -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) 2 D 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) 2 D and urinary Ca excretion inhibit renal calcification in Npt2 - / - mice. Our findings underscore the importance of 1,25(OH) 2 D in the development of hypercalciuria and renal calcification in this mouse model and are relevant to a subset of stone-forming patients with renal P i wasting.

ACKNOWLEDGMENTS

We thank S. Aubin, J. Martel, and Dr. X. Ying He for technical assistance and M. Charlebois and M. Gratton at the Montreal Centre for Bone and Peridontal Research, McGill University, for help with µCT imaging and 1,25(OH) 2 D analysis, respectively.

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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作者单位：Departments of 1 Human Genetics, 2 Pediatrics, 4 Biology, and 5 Surgery, McGill University, 3 The McGill University-Montreal Children‘s Hospital Research Institute, Montreal H3Z 2Z and 6 Shriners Hospital for Children, Montreal, Quebec, Canada H3G 1A6