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Endocrinology Vol. 142, No. 3 1124-1129
Copyright © 2001 by The Endocrine Society

ARTICLES

Renal Expression of the Sodium/Phosphate Cotransporter Gene, Npt2, Is Not Required for Regulation of Renal 1{alpha}-Hydroxylase by Phosphate1

H. S. Tenenhouse, J. Martel, C. Gauthier, M. Y. H. Zhang and A. A. Portale

Departments of Pediatrics and Human Genetics, McGill University-Montreal Children’s Hospital Research Institute (H.S.T., J.M., C.G.), Montréal, Québec, Canada H3H 1P3; and Department of Pediatrics, University of California (M.Y.H.Z., A.A.P.), San Francisco, California 94143

Address all correspondence and requests for reprints to: Harriet S. Tenenhouse, Ph.D., Montreal Children’s Hospital, 2300 Tupper Street, Montréal, Québec, Canada H3H 1P3. E-mail: mdht{at}www.debelle.mcgill.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Several reports have suggested that the regulation of renal 1,25-dihydroxyvitamin D [1,25-(OH)2D] synthesis by extracellular phosphate (Pi) is dependent on normal transepithelial Pi transport by the renal tubule. Mice homozygous for the disrupted Na/Pi cotransporter gene Npt2 (Npt2-/-) exhibit renal Pi wasting, an approximately 85% decrease in renal brush border membrane Na/Pi cotransport, hypophosphatemia, and an increase in serum 1,25-(OH)2D concentration. We undertook 1) to determine the mechanism for the increased circulating levels of 1,25-(OH)2D in Npt2-/- mice and 2) to establish whether renal 1{alpha}-hydroxylase was appropriately regulated by dietary Pi in the absence of Npt2 gene expression. On a control diet, the 2.5-fold increase in the serum 1,25-(OH)2D concentration in Npt2-/- mice, relative to that in Npt2+/+ littermates, is associated with a corresponding increase in renal mitochondrial 25-hydroxyvitamin D-1{alpha}-hydroxylase (1{alpha}-hydroxylase) activity and messenger RNA (mRNA) abundance. A low Pi diet elicits an increase in serum 1,25-(OH)2D concentration, renal 1{alpha}-hydroxylase activity, and mRNA abundance in Npt2+/+ and Npt2-/- mice to similar levels in both mouse strains. A high Pi diet has no effect on serum 1,25-(OH)2D concentration, renal 1{alpha}-hydroxylase activity, or mRNA abundance in Npt2+/+ mice, but normalizes these parameters in Npt2-/- mice. In addition, renal 24-hydroxylase mRNA abundance is significantly reduced in Npt2-/- mice compared with that in Npt2+/+ mice under all dietary conditions. In summary, we demonstrate that 1) increased renal synthesis of 1,25-(OH)2D is responsible for the increased serum 1,25-(OH)2D concentration in Npt2-/- mice; and 2) renal 1{alpha}-hydroxylase gene expression is appropriately regulated by dietary manipulation of serum Pi in both Npt2+/+ and Npt2-/- mice. Thus, intact renal Na/Pi cotransport is not required for the regulation of renal 1{alpha}-hydroxylase by Pi.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
VITAMIN D3 MUST undergo two hydroxylation reactions, the first in the liver to 25-hydroxyvitamin D3 (25OHD) and the second in the kidney to 1,25-dihydroxyvitamin D3 [1,25-(OH)2D], before it can assume its role in the regulation of calcium (Ca) and phosphate (Pi) homeostasis (1, 2, 3). The renal synthesis of 1,25-(OH)2D is catalyzed by 25OHD-1{alpha}-hydroxylase (1{alpha}-hydroxylase), a mitochondrial cytochrome P450 mixed function oxidase whose catalytic activity requires molecular O2, ferredoxin, and ferredoxin reductase. 1,25-(OH)2D production in the kidney is subject to tight homeostatic regulation by PTH, calcitonin, calcium, Pi, and 1,25-(OH)2D itself (1, 2, 3). 1,25-(OH)2D can be further hydroxylated in kidney and vitamin D target tissues by vitamin D 24-hydroxylase (P450c24), a mitochondrial cytochrome P450 that is involved in catabolism of the hormone to its final inactivation product, calcitroic acid (4).

We and others recently cloned the 1{alpha}-hydroxylase complementary DNA (cDNA) and gene (P450c1{alpha}) (5, 6, 7, 8, 9, 10) and identified mutations in the human P450c1{alpha} gene in patients with vitamin D-dependent rickets type I (5, 11, 12, 13, 14). It has been demonstrated that the renal abundance of P450c1{alpha} messenger RNA (mRNA) is significantly increased by the administration of PTH and calcitonin (15, 16) and by feeding diets deficient in vitamin D (8), Ca, or Pi (10). Further studies suggest that both PTH (16, 17) and calcitonin (16) increase P450c1{alpha} gene transcription and that the protein kinase A signaling pathway is necessary for the response to PTH, but not that to calcitonin (16). Administration of 1,25-(OH)2D can suppress renal P450c1{alpha} mRNA abundance and prevent the increase induced by PTH and calcitonin by a mechanism that requires participation of the vitamin D receptor (16, 18).

The importance of Pi as a regulator of renal 1,25-(OH)2D synthesis is well established. Dietary Pi restriction stimulates the renal production of 1,25-(OH)2D (19, 20, 21, 22). Although the mechanism for the adaptive response to low Pi intake is not clear, it appears to be independent of PTH (19, 20). Several groups have postulated that normal transepithelial Pi transport is essential for the regulation of 1,25-(OH)2D synthesis by Pi in the proximal renal tubule, the nephron segment where the bulk of filtered Pi is reabsorbed (23, 24). This hypothesis is based on the finding that the serum concentration of 1,25-(OH)2D is not appropriately increased despite significant hypophosphatemia in patients with renal Pi wasting disorders, either Mendelian (X-linked hypophosphatemia and autosomal dominant hypophosphatemic rickets) or acquired (oncogenic hypophosphatemic osteomalacia) (25, 26, 27), or in murine Hyp and Gy homologs of X-linked hypophosphatemia (28, 29, 30). Moreover, both X-linked hypophosphatemia patients (31, 32) and Hyp mice (28, 29, 33, 34) fail to increase renal 1,25-(OH)2D production in response to dietary Pi restriction or infusion of PTH.

To test the hypothesis that normal Pi transport in the proximal renal tubule is necessary for the regulation of renal 1,25-(OH)2D synthesis by dietary Pi, we examined the effect of Npt2 gene ablation (35) on the regulation of renal 1{alpha}-hydroxylase. Npt2 is expressed exclusively in the proximal tubule (36), is the most abundant of the Na/Pi cotransporters in mouse kidney (37) and is a target for regulation by dietary Pi and PTH (38). Mice homozygous for Npt2 gene disruption (Npt2-/-) exhibit renal Pi wasting, an approximately 85% loss in renal brush border membrane Na/Pi cotransport, hypophosphatemia, increased serum 1,25-(OH)2D levels, and associated hypercalcemia and hypercalciuria (35). Moreover, brush border membrane Na/Pi cotransport in Npt2-/- mice is not responsive to Pi deprivation (39) or PTH (40). We demonstrate here that the regulation of renal P450c1{alpha} gene expression by Pi is normal in Npt2-/- mice, indicating that Npt2-dependent renal Pi reabsorption is not required for the regulation of renal 1,25-(OH)2D synthesis by Pi.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice
Npt2 knockout mice were established in our laboratory by targeted mutagenesis (35). Wild-type (Npt2+/+) and homozygous mutant (Npt2-/-) mice, generated by crossing heterozygous (Npt2+/-) male and female mice, were genotyped by PCR amplification of genomic DNA obtained from tail tissue, using Taq polymerase (QIAGEN, Mississauga, Canada) and three primers [sense primer 3F (5'-TGC CCA GGT TGG CAC GAA GC-3') in exon 4 of Npt2, antisense primer 4R (5'-AGT CCT GTC CCC TGC CTG CA-3') in exon 6 of Npt2, and antisense primer PGKR (5'-TGC TAC TTC CAT TTG TCA CGT CC-3') in the neor gene cassette] as described previously (35). The expected sizes of amplified fragments are 1.8 kb for the wild-type allele (primers 3F and 4R) and 1.4 kb for the disrupted allele (primers 3F and PGKR). The mice (60 ± 10 days of age) were maintained on a 0.6% Pi diet (5001, Purina Lab Chow, Ralston Purina Co., St. Louis, MO) unless otherwise indicated. To examine the effect of dietary Pi on serum and renal parameters, Npt2+/+ and Npt2-/- mice were fed low Pi (0.02% Pi), control Pi (0.6% Pi), or high Pi (1.65% Pi) diets for 4 days (test diets TD 86128, TD 98243, and TD 88345, respectively, Harlan Teklad, Madison, WI). The test diets were otherwise identical. All animal studies were conducted in accordance with the guidelines of the Canadian Council on Animal Care.

Renal 1{alpha}-hydroxylase activity
Mitochondria were prepared from renal cortex of individual mice (1.5 kidneys/mouse) and incubated (2–3 mg mitochondrial protein/ml) with 0.5 µM HPLC-purified 25OHD in 125 mM KCl, 20 mM HEPES, 10 mM malic acid, 2 mM MgSO4, 1 mM dithiothreitol, and 0.25 mM EDTA, adjusted to pH 7.4, for 15 min at 25 C, as described previously (29). The mitochondria and medium were extracted with acetonitrile, and the 1{alpha}-hydroxylated product was determined in duplicate by RRA after C18 and silica Sep-Pak chromatography (5).

Ribonuclease protection analysis
cDNA fragments corresponding to nucleotides 176–626 of mouse P450c1{alpha} cDNA (7) and nucleotides 1262–1637 of mouse P450c24 cDNA (41) were prepared by RT-PCR of mouse kidney RNA, subcloned, and sequenced. Riboprobes for P450c1{alpha}, P450c24, and {beta}-actin (42) were prepared by transcription of subcloned cDNA fragments using either T7 or T3 RNA polymerases and [{alpha}-33P]UTP (3000 Ci/mmol; ICN Biomedicals, Inc., Mississauga, Canada), and the ribonuclease protection assay was performed as described previously (37, 42, 43). Briefly, total RNA (5–20 µg), isolated from kidney with TRIzol reagent (BRL-Life Technologies, Inc., Burlington, Canada), was hybridized with the appropriate labeled riboprobes (5 x 105 cpm) at 50 C for 18 h and treated with 2 µg/ml ribonuclease T1 for 1 h at 30 C. The protected fragments were precipitated, heat denatured, and electrophoresed on 6% denaturing polyacrylamide gels. The gels were dried and exposed to a PhosphorImager screen for quantification of radioactive signals under conditions where linearity is achieved. Results are expressed as the ratio of 1{alpha}- or 24-hydroxylase mRNA to {beta}-actin mRNA.

Serum and urine parameters
Serum Pi and Ca and urinary Ca and creatinine concentrations were assayed using phosphorus, calcium, and creatinine kits (Stanbio Laboratories, San Antonio, TX) as described previously (29). The serum concentration of 1,25-(OH)2D was measured by Roche Bioscience (Palo Alto, CA) using a calf thymus RRA (DiaSorin, Inc., Stillwater, MN), as described previously (29).

Statistical analysis
The number of mice studied per group is indicated for each experiment, and the mean ± SEM are depicted. Statistical analysis was performed using two-way ANOVA and t test where appropriate, and P < 0.05 was taken to be statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
We previously reported that the serum concentration of 1,25-(OH)2D is significantly increased in mice homozygous for the disrupted Npt2 gene compared with that in wild-type littermates (35). To elucidate the mechanism for this increase, we examined the effect of Npt2 gene disruption on renal mitochondrial 1{alpha}-hydroxylase activity and mRNA abundance. The 2.5-fold increase in the serum 1,25-(OH)2D concentration in Npt2-/- mice, compared with Npt2+/+ mice (Fig. 1AGo), was associated with a 2-fold increase in renal 1{alpha}-hydroxylase activity (Fig. 1BGo) and a corresponding 3-fold increase in P450c1{alpha} mRNA abundance (Fig. 1CGo). These data indicate that increased renal production of 1,25-(OH)2D is responsible for its increased serum levels in Npt2-/- mice. Thus, renal 1{alpha}-hydroxylase activity in Npt2-/- mice increases appropriately in response to the hypophosphatemia that results from renal Pi wasting (35).



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  		Figure 1. Effect of Npt2 gene disruption on serum concentration of 1,25-(OH)2D (A), and renal 1{alpha}-hydroxylase activity (B) and mRNA abundance (C). Npt2+/+ and Npt2-/- mice fed the control diet (0.6% Pi) were anesthetized, blood samples were collected by cardiac puncture, and the kidneys were removed. The serum concentration of 1,25-(OH)2D was determined, as described in Materials and Methods, in pooled samples if sufficient serum (0.5 ml) was not available from each mouse. Renal mitochondria were prepared and incubated with 25OHD3 to measure 1{alpha}-hydroxylase activity, and renal total RNA was prepared to estimate the abundance of P450c1{alpha} mRNA, relative to {beta}-actin mRNA, as described in Materials and Methods. Data displayed are the mean ± SEM from 11–29 mice/group. The effect of Npt2 gene ablation (#) was determined by ANOVA (P < 0.05).

 
We also examined the effect of Npt2 gene disruption on the regulation of renal 1,25-(OH)2D production by dietary Pi. In both wild-type and Npt2-/- mice, the low Pi (0.02%) diet induced a significant decrease in the serum Pi concentration and a significant increase in serum calcium and urinary calcium/creatinine (Table 1Go). Under these conditions, serum Pi levels were similar in Npt2+/+ and Npt2-/- mice, whereas serum calcium and urinary calcium/creatinine values continued to be significantly increased in the mutants relative to those in wild-type mice (Table 1Go). The low Pi diet induced an increase in serum 1,25-(OH)2D levels (Fig. 2AGo) and a corresponding increase in renal 1{alpha}-hydroxylase activity (Fig. 2BGo) and mRNA abundance (Fig. 2CGo) in both Npt2+/+ and Npt2-/- mice. Of interest was the observation that although serum 1,25-(OH)2D levels, renal mitochondrial 1{alpha}-hydroxylase activity, and mRNA abundance were significantly higher in Npt2-/- mice than in Npt2+/+ mice on the control diet (Fig. 1Go), these parameters were similar in Pi-deprived Npt2+/+ and Pi-deprived Npt2-/- mice (Fig. 2Go), consistent with the idea that maximal stimulation had been achieved in both groups of mice.


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  		Table 1. Effect of dietary Pi on serum Pi and calcium concentrations and urinary calcium/creatinine ratio in Npt2+/+ and Npt2-/- mice

 


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  		Figure 2. Effects of low and high Pi diets on the serum concentration of 1,25-(OH)2D (A), and renal 1{alpha}-hydroxylase activity (B) and mRNA abundance (C) in Npt2+/+ and Npt2-/- mice. Npt2+/+ and Npt2-/- mice were fed diets containing 0.6% (control), 0.02% (low), or 1.65% (high) Pi for 4 days. The mice were anesthetized, blood samples were collected by cardiac puncture, and the kidneys were removed. The serum concentration of 1,25-(OH)2D was determined, as described in Materials and Methods, on pooled samples if sufficient serum was not available from each mouse. Renal mitochondria were prepared and incubated with 25OHD3 to determine 1{alpha}-hydroxylase activity, and renal total RNA was prepared to estimate the abundance of P450c1{alpha} mRNA, relative to {beta}-actin mRNA, as described in Materials and Methods. Data displayed are the mean ± SEM from 6–29 mice/group. The effects of diet (*) and Npt2 gene ablation (#) were determined by ANOVA (P < 0.05).

 
The high Pi (1.65%) diet elicited a significant increase in the serum Pi concentration in both Npt2+/+ and Npt2-/- mice, and under these conditions, genotype differences were no longer apparent (Table 1Go). In addition, on the high Pi diet the serum calcium concentration and urinary calcium/creatinine ratio decreased in Npt2-/- mice to values not different from those in wild-type littermates (Table 1Go). In wild-type mice, the high Pi had no effect on the serum 1,25-(OH)2D concentration (Fig. 2AGo), renal 1{alpha}-hydroxylase activity (Fig. 2BGo), or P450c1{alpha} mRNA abundance (Fig. 2CGo), whereas in Npt2-/- mice, the high Pi diet decreased all three parameters to values not different from those in Npt2+/+ mice (Fig. 2Go, A–C).

Figure 3Go demonstrates that in both Npt2+/+ and Npt2-/- mice there was a significant inverse relationship between renal P450c1{alpha} mRNA abundance and the serum Pi concentration. Both the serum 1,25-(OH)2D concentration and the renal 1{alpha}-hydroxylase activity also were inversely related to serum Pi levels in Npt2+/+ and Npt2-/- mice (data not shown). The data clearly demonstrate that the regulation of renal P450c1{alpha} gene expression by Pi is not abrogated by disruption of Npt2 gene expression.



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  		Figure 3. Relationship between serum Pi concentration and renal P450c1{alpha} mRNA abundance in Npt2+/+ and Npt2-/- mice. Data were obtained from wild-type and mutant mice fed diets containing 0.6% (control), 0.02% (low), or 1.65% (high) Pi for 4 days. The serum Pi concentration and renal abundance of P450c1{alpha} mRNA, relative to {beta}-actin mRNA, were determined as described in Materials and Methods. Each point depicts data from individual Npt2+/+ (•) and Npt2-/- ({circ}) mice. Slopes of regression lines for Npt2+/+ and Npt2-/- mice are significantly different from zero (P < 0.0001).

 
To determine whether expression of the enzyme responsible for renal catabolism of 1,25-(OH)2D is influenced by Npt2 gene disruption and dietary Pi, we estimated the renal abundance of P450c24 mRNA in Npt2+/+ and Npt2-/- mice fed control, low, and high Pi diets. On the control diet, P450c24 mRNA abundance was significantly decreased in Npt2-/- mice relative to that in wild-type littermates (Fig. 4Go). The low Pi diet elicited a decrease in renal P450c24 mRNA abundance in Npt2+/+ mice, but not in Npt2-/- mice, where it appeared to be maximally reduced (Fig. 4Go). The high Pi diet had no effect on 24-hydroxylase mRNA abundance in either mouse strain (Fig. 4Go). On both low and high diets, however, renal P450c24 mRNA abundance was also significantly lower in Npt2-/- mice than in Npt2+/+ littermates (Fig. 4Go).



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  		Figure 4. Effects of low and high Pi diets on renal P450c24 mRNA abundance in Npt2+/+ and Npt2-/- mice. Npt2+/+ and Npt2-/- mice were fed diets containing 0.6% (control), 0.02% (low), or 1.65% (high) Pi for 4 days. The kidneys were removed for the preparation of total RNA, and the abundance of P450c24 mRNA, relative to {beta}-actin mRNA, was estimated by ribonuclease protection assay as described in Materials and Methods. Data displayed are the mean ± SEM from 21–36 mice/group. The effects of diet (*) and Npt2 gene ablation (#) were determined by ANOVA (P < 0.01).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study we demonstrate that in mice homozygous for the disrupted Na/Pi cotransporter gene, Npt2, the increase in the serum concentration of 1,25-(OH)2D can be ascribed to an increase in renal P450c1{alpha} gene expression. These changes in vitamin D metabolism are consistent with an appropriate adaptive response to hypophosphatemia that results from an 85% loss in renal brush border membrane Na/Pi cotransport secondary to Npt2 gene ablation (35, 39, 40). In addition, the homozygous mutant mice are able to respond to a low Pi diet with a further increase in serum 1,25-(OH)2D levels and renal 1{alpha}-hydroxylase activity and mRNA abundance and to a high Pi diet with a decrease in serum 1,25-(OH)2D, 1{alpha}-hydroxylase activity, and mRNA abundance. Furthermore, we demonstrate that the relationship between serum Pi concentration and parameters of renal 1,25-(OH)2D synthesis is similar in both Npt2-/- mice and wild-type littermates. Our results demonstrate that the regulation of renal mitochondrial P450c1{alpha} by Pi is not dependent on normal renal Pi reabsorption, thereby dispelling the idea that renal Pi wasting in itself contributes to dysregulated 1,25-(OH)2D production.

The present data in Npt2-/- mice are in sharp contrast to those reported in mutant X-linked hypophosphatemic (Hyp and Gy) mice, which harbor large deletions in the 3'- and 5'-regions, respectively, of the Phex gene (42, 44). Both Hyp and Gy mice are characterized by a 50% reduction in Na/Pi cotransport across the renal brush border membrane (45, 46, 47), hypophosphatemia (47, 48), and a normal plasma concentration of 1,25-(OH)2D, which is inappropriate for the degree of hypophosphatemia (28, 29, 30). Moreover, Hyp and Gy mice respond to dietary Pi restriction with a paradoxical decrease in serum 1,25-(OH)2D levels and renal 1{alpha}-hydroxylase activity and to Pi supplementation with a paradoxical increase in serum 1,25-(OH)2D levels and renal 1{alpha}-hydroxylase activity (29, 30, 33). In normal littermates, however, both serum 1,25-(OH)2D and 1{alpha}-hydroxylase activity were increased by Pi deprivation and unchanged by Pi supplementation (29, 30, 33), consistent with the present data in wild-type mice.

Renal 1{alpha}-hydroxylase activity in X-linked Hyp mice also is abnormally regulated by vitamin D deficiency (49, 50), calcium restriction (51), and infusion of PTH (34), cAMP (52), or PTH-related peptide (53). The precise mechanism for abnormal 1,25-(OH)2D production in Hyp and Gy mice is not understood. As the bulk of Pi reabsorption (23, 24) and 1,25-(OH)2D production (54, 55) are both localized to the proximal renal tubule, it was postulated that in Hyp and Gy mice, an impairment in Na/Pi cotransport across the brush border membrane results in an altered intracellular milieu, and this is responsible for the disordered regulation of 1,25-(OH)2D synthesis in the mutant strains (52, 56). However, in view of the present results in Npt2-/- mice, this hypothesis is no longer tenable. A more likely explanation is that loss of Phex function arising from deletions in the Phex gene either directly or indirectly is responsible for both impaired renal Na/Pi cotransport and disordered regulation of renal 1,25-(OH)2D synthesis in Hyp and Gy mice.

In the present study we demonstrate that Pi supplementation in Npt2-/- mice reversed the hypophosphatemia and normalized renal 1{alpha}-hydroxylase activity and mRNA abundance and thereby the serum 1,25-(OH)2D concentration. It is likely that normalization of 1,25-(OH)2D production was responsible for correcting the hypercalcemia and increased urinary excretion of calcium in Npt2-/- mice. These findings provide further evidence that regulation of renal vitamin D metabolism by dietary and serum Pi is independent of Npt2 gene expression. Similar findings were reported in patients with hereditary hypophosphatemic rickets with hypercalciuria (HHRH) (25, 26, 57), in whom the increased serum 1,25-(OH)2D concentration and urinary calcium excretion were both corrected by Pi supplementation (57). Notwithstanding that both HHRH patients and Npt2-/- mice have similar biochemical features and an identical vitamin D response to Pi supplementation, we recently demonstrated, by DNA sequencing and linkage analysis, that NPT2 in not a candidate gene for HHRH (58).

To determine whether Npt2 gene disruption interferes with expression of the enzyme responsible for renal catabolism of 1,25-(OH)2D, we compared the effect of dietary Pi on renal P450c24 mRNA abundance in Npt2+/+ and Npt2-/- mice. In Npt2+/+ mice, P450c24 mRNA abundance is significantly decreased on the low Pi diet and unchanged on the high Pi diet. By contrast, renal P450c24 mRNA abundance is lower in Npt2-/- mice than in wild-type counterparts under all dietary conditions and is unaffected by changes in dietary Pi. Although our data suggest that renal catabolism of 1,25-(OH)2D is decreased by Npt2 gene ablation, and that such a decrease may contribute to the increase in serum 1,25-(OH)2D levels in Npt2-/- mice, further studies are necessary to determine whether a stoichiometric increase in renal 24-hydroxylase activity is indeed apparent. It is of interest that Npt2-/- mice also differ from Hyp and Gy mice, in which the catabolism of 1,25-(OH)2D is increased relative to that in normal littermates on control and low Pi diets and is corrected by Pi supplementation (29, 30).

We recently reported that Npt2-/- mice fail to exhibit an adaptive increase in renal brush border membrane Na/Pi cotransport in response to Pi deprivation, indicating that Npt2 protein is essential for brush border membrane adaptation (39). These results are in contrast to the present findings that Npt2 gene ablation does not interfere with the adaptive increase in renal 1,25-(OH)2D synthesis in response to a low Pi diet. Taken together, our data support the idea that the diet-induced adaptation in brush border membrane Na/Pi cotransport and regulation of renal P450c1{alpha} gene expression are not interdependent and occur by separate distinct mechanisms. Further studies are necessary to define the molecular mechanisms involved. Of interest in this regard are the observations that the increase in Npt2 gene expression induced by a low Pi diet appears to occur by both posttranscriptional (59) and transcriptional (60) mechanisms. Furthermore, the decrease in PTH mRNA abundance in parathyroid glands of Pi-depleted rats is attributed to a decrease in PTH mRNA stability that results from the binding of cytoplasmic proteins to the 3'-untranslated region of the PTH transcript (61).

In summary, using a mouse model in which the Npt2 gene was disrupted by targeted mutagenesis, we provide evidence that normal renal Na/Pi cotransport is not necessary for the regulation of renal 1,25-(OH)2D production by Pi. Our data suggest that changes in serum Pi concentration per se are sufficient to initiate the signaling pathways involved in the up-regulation and down-regulation of the P4501{alpha} gene by restriction and supplementation of dietary Pi.


   Acknowledgments
 
We thank Danielle Boulais and Ghislaine Sabbagh for technical support, Diana Yang at Roche Bioscience (Palo Alto, CA) for the assay of serum 1,25-(OH) 2D, and Hoffman-LaRoche Inc. (Mississauga, Canada) for 25OHD3.


   Footnotes
 
1 This work was supported by grants from the Medical Research Council of Canada (GR-13297 to H.S.T.), Roche Bioscience (to H.S.T.), and the NIH (DK-54433 to A.A.P.) and gifts from the David Carmel Trust (to A.A.P.). Back

Received October 6, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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