Intestinal and renal adaptation to a low-Pi diet of type II NaPi cotransporters in vitamin D receptor- and 1{alpha}OHase-deficient mice

Paola Capuano,1 Tamara Radanovic,1 Carsten A. Wagner,1 Desa Bacic,1 Shigeaki Kato,2 Yasushi Uchiyama,3 René St.-Arnoud,4 Heini Murer,1 and Jürg Biber1

1Institute of Physiology; University of Zurich, Zurich, Switzerland; 2Institute of Molecular and Cellular Biosciences, University of Tokyo, Tokyo, Japan; 3Fuji Gotemba Research Laboratories, Shizuoka, Japan; and 4Genetic Unit, Shriners Hospital, Montreal, Quebec, Canada

Submitted 8 July 2004 ; accepted in final form 20 September 2004


    ABSTRACT
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 ABSTRACT
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Intake of a low-phosphate diet stimulates transepithelial transport of Pi in small intestine as well as in renal proximal tubules. In both organs, this is paralleled by a change in the abundance of the apically localized NaPi cotransporters NaPi type IIa (NaPi-IIa) and NaPi type IIb (NaPi-IIb), respectively. Low-Pi diet, via stimulation of the activity of the renal 25-hydroxyvitamin-D3-1{alpha}-hydroxylase (1{alpha}OHase), leads to an increase in the level of 1,25-dihydroxy-vitamin D3 [1,25(OH)2D]. Regulation of the intestinal absorption of Pi and the abundance of NaPi-IIb by 1,25(OH)2D has been supposed to involve the vitamin D receptor (VDR). In this study, we investigated the adaptation to a low-Pi diet of NaPi-IIb in small intestine as well as NaPi-IIa in kidneys of either VDR- or 1{alpha}OHase-deficient mice. In both mouse models, upregulation by a low-Pi diet of the NaPi cotransporters NaPi-IIa and NaPi-IIb was normal, i.e., similar to that observed in the wild types. Also, in small intestines of VDR- and 1{alpha}OHase-deficient mice, the same changes in NaPi-IIb mRNA found in wild-type mice were observed. On the basis of the results, we conclude that the regulation of NaPi cotransport in small intestine (via NaPi-IIb) and kidney (via NaPi-IIa) by low dietary intake of Pi cannot be explained by the 1,25(OH)2D-VDR axis.

NaPi type IIb; vitamin D3


IN SMALL INTESTINE AND RENAL proximal tubules, transepithelial transport of Pi is initiated by members of the type II Na+-dependent phosphate cotransporter family SLC34 (18), which are localized at the apical membrane of the respective epithelial cells. In adult mice, renal reabsorption of Pi is determined largely by the abundance of NaPi type IIa (NaPi-IIa) in the brush-border membrane vesicles (BBMV) of proximal tubular cells and in small intestine by NaPi type IIb (NaPi-IIb), the only apical NaPi cotransporter known to be involved in the absorption of Pi (14, 19). Thus regulation of intestinal absorption and renal reabsorption of Pi is mainly explained by alterations in the apical abundances of NaPi-IIb and NaPi-IIa, respectively (12, 15, 19, 28). Low dietary intake of Pi is a well-known stimulator of both renal and small intestinal Pi handling (3, 6, 16). In both organs, low-Pi diet provokes an increase of the abundance of both type II NaPi cotransporters. In mouse intestine, the increase in NaPi-IIb protein is also paralleled by an increase in NaPi-IIb mRNA (23; Radanovic T, Wagner CA, Murer H, and Biber J, unpublished data), whereas in proximal tubules of adult mice, an increase in NaPi-IIa mRNA was not observed (17).

Low dietary intake of Pi stimulates the synthesis of 1,25-dihydroxy-vitamin D3 [1,25(OH)2D] via stimulation of the renal 25-hydroxyvitamin-D3-1{alpha}-hydroxylase (1{alpha}OHase; for review, see Ref. 2). In turn, the stimulation of intestinal Pi absorption by a low-Pi diet has been attributed to an increased level of 1,25(OH)2D (3, 6). In agreement with these findings, it was shown that the status of 1,25(OH)2D influences the abundance of the apical NaPi-IIb protein (12, 28). Because low-Pi diet induces an upregulation of NaPi-IIb mRNA (23; Radanovic T, Wagner CA, Murer H, and Biber J, unpublished data), it could be envisaged that the action of 1,25(OH)2D is via a genomic mechanism that involves the vitamin D receptor (VDR) (2). The role of 1,25(OH)2D in the renal handling of Pi is less clear, notably also because of the complexity rooted in an interrelationship with the status of parathyroid hormone (PTH) (8).

In the present study, we investigated the regulation of NaPi-IIb in enterocytes and NaPi-IIa in proximal tubules by a low-Pi diet in VDR- and renal 1{alpha}OHase-deficient mice. Our results demonstrate that in both mouse models, NaPi-IIb (protein and mRNA) and NaPi-IIa (protein) are upregulated by a low-Pi diet to a degree similar to that in wild-type animals. On the basis of the presented results, we conclude that in neither intestine nor kidney does the VDR or 1,25(OH)2D play a central role in the adaptive phenomenon induced by a low-Pi diet.


    METHODS
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Animals. All experiments were performed with male mice aged 10–12 wk. Two different batches of VDR–/– mice (originally described in Ref. 29) were used. As controls, wild-type C57BL/6 mice were purchased from Charles River Laboratories (Sulzfeld, Germany). After weaning, VDR–/– mice were fed a high-Ca2+ diet (2%) to rescue the abnormalities observed in these animals (16a). (For a description of 1{alpha}OHase-deficient mice, see Ref. 7.) As controls, the corresponding heterozygotes were used because it has been demonstrated that 1{alpha}OHase heterozygotes do not show any differences from the wild types (7). Before experiments, all animals were maintained for 2 wk on a standard diet (0.8% Pi, 1.2% Ca2+). Afterward, the animals were fed diets containing either high Pi (1.1%) or low Pi (0.1%) (Kliba; NAFAG, Kaiseraugst, Switzerland) for 5 days. Both diets contained 1.0% Ca2+. During all feeding periods, the animals had free access to normal tap water. Animal care and experimentation were performed according to the regulations of the Veterinary Authority of Zurich (Zurich, Switzerland).

Determinations. Plasma and urinary concentrations of phosphate and creatinine were determined on the basis of spontaneous urine samples and heparinized serum using commercial kits (Sigma Diagnostics, Münich, Germany, and Wako Chemicals, Reinach, Switzerland) according to the manufacturers' protocols. Total protein was determined using Coomassie blue reagent (Bio-Rad).

Incubation of kidney slices and immunofluorescence. Incubation and processing of kidney slices was performed according to a previously described method (1). After a 45-min incubation in either the absence or the presence of 10–7 M bovine 1-34 PTH (Sigma), slices were fixed for 4 h, rinsed with PBS, and immunostained for NaPi-IIa as described elsewhere (1, 5).

Isolation of membranes, Western blot analysis, and NaPi transport measurements. Kidney cortex tissue was homogenized, and crude membranes were prepared by performing differential centrifugation. From whole small intestines, BBMV were isolated using a Mg2+ precipitation technique described previously (24). Before this step, intestines were rinsed with cold 0.9% NaCl and inverted, and the mucosa was scraped off. For each preparation, the intestinal mucosa of two or three animals was pooled.

For Western blot analysis, 50 µg of total protein were separated by SDS-PAGE and analyzed for the abundance of NaPi-IIa, NaPi-IIb, calbindin D9k, and {beta}-actin after transfer onto nitrocellulose membranes according to standard procedures using 5% fat-free milk powder as a blocking reagent (5, 14). The following primary antibodies were used: anti NaPi-IIa and anti NaPi-IIb, as described previously (5, 14), anti-calbindin D9k (SWANT, Bellinzona, Switzerland), and anti {beta}-actin (no. 5316; Sigma). Immunoreactions were detected using the corresponding secondary HRP-conjugated IgG (Amersham) and enhanced chemiluminescence (SuperSignal; Pierce Biotechnology). Quantitative analysis was performed with digital imaging (Diana III; Raytest, Straubenhardt, Germany).

Na+-dependent transport of Pi into isolated small intestine BBMV was determined at 25°C and a final concentration of 0.3 mM Pi in the presence of inwardly directed gradients of either 100 mM NaCl or 100 mM KCl (13).

Real-time PCR. Small intestines were rinsed with cold 0.9% NaCl and inverted, and mucosa from late jejunum up to the early ileum was frozen immediately. Total RNA was extracted using the RNAeasy kit (Qiagen). Reverse transcription was performed using random hexamers (TaqMan reverse transcription reagents; Applied Biosystems). The sequences of TaqMan probes (Applied Biosystems) used for the amplification of NaPi-IIb were as follows: 5'-(6-FAM) TGGTCAGAGAGAGACAC (BHQ-1)-3' (probe), 5'-CCTGGGACCTGCCTGAACT-3' (forward primer), and 5'-AATGCAGAGCGTCTTCCCTTT-3' (reverse primer).

The probes used to amplify {beta}-actin cDNA were described elsewhere (17). The relative expressions of NaPi-IIb mRNA to the {beta}-actin mRNA were calculated according to the method described in User Bulletin 2 for the ABI Prism 7700 Sequence Detection System (http://docs.appliedbiosystems.com/pebiodocs/04303859.pdf).

For each condition, mucosa of three animals was collected. Three independent rounds comprising reverse transcription and real-time (RT)-PCR were performed. In each round, samples from one animal for each condition were analyzed. Because the values of the relative NaPi-IIb mRNA contents (expressed as the ratio of NaPi-IIb mRNA to {beta}-actin mRNA) varied between the different rounds, the results (see Fig. 6) are expressed as relative upregulation (low- vs. high-Pi diet) of the relative NaPi-IIb mRNA contents.



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Fig. 6. Real-time PCR of NaPi-IIb mRNA. Total RNA was isolated from late jejunum and early ileum of mice fed a high or a low-Pi diet for 5 days. Bars represent relative (-fold) upregulation by a low-Pi diet of the relative contents of NaPi-IIb mRNA (ratios of NaPi-IIb mRNA to {beta}-actin mRNA). Each bar represents the result obtained from 1 animal.

 
Statistical analysis. Data are presented as means ± SD. Student's t-test was used to test for significance of differences between mice of the same strain fed a high- or a low-Pi diet. P < 0.05 was considered significant.


    RESULTS
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Urinary excretion of phosphate of wild-type animals, VDR-deficient mice, and 1{alpha}OHase-deficient mice was determined over a period of several days during which the dietary content of Pi was varied as indicated in Fig. 1. As illustrated, after switching from a normal to a low-Pi diet, urinary excretion was reduced to almost zero and there was no difference between the wild types and both strains of knockout mice. In contrast, while both knockout groups were fed a high-Pi diet, their normalized urinary excretion of Pi was significantly reduced compared with that of the corresponding wild types. Similarly, reduced Pi excretion was observed while mice were fed a normal laboratory chow diet containing 0.8% Pi. No difference was observed with regard to urinary Pi excretion of VDR–/– and 1{alpha}OHase–/– mice fed a high-Pi diet.



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Fig. 1. Urinary excretion of Pi (in mg/dl) and creatinine (in mg/dl) of wild-type and VDR–/– mice (A) and 25-hydroxyvitamin-D3-1{alpha}-hydroxylase (1{alpha}OHase)+/– and 1{alpha}OHase–/– mice (B). The animals were fed diets with different Pi contents as indicated. Each point represents the mean ± SD urinary excretion of 4 mice. After being fed low-Pi diet, all mice strains examined showed no significant differences in Pi excretion. After being fed a high-Pi diet or a normal diet to both knockout groups, Pi excretion levels were reduced compared with the respective controls. *P < 0.05, significant differences.

 
The concentrations of Pi in the serum of animals fed a high- or a low-Pi diet for 5 days are listed in Table 1. After 5 days on a high-Pi diet, serum Pi in the VDR–/– animals did not differ from that of the wild types, whereas the 1{alpha}OHase–/– animals showed moderate hypophosphatemia, which is in agreement with earlier reports (7, 26). In contrast to data published by others (23), the serum Pi concentration in VDR-deficient mice fed a high-Pi diet was not different from that of the wild-type mice. In all animals, low-Pi diet resulted in a hypophosphatemia that was more pronounced in both knockouts compared with the wild types. After low-Pi diet, serum Pi levels of the VDR–/– and the 1{alpha}OHase–/– mice did not differ significantly. The values for the fractional excretion of Pi (, %) in animals fed a high-Pi diet were not significantly different between the wild-type and VDR–/– mice and OHase+/– and OHase–/– mice, respectively (Table 1). values of all animals fed a low-Pi diet were close to zero.


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Table 1. Urine and serum parameters and fractional excretion of Pi

 
Because renal excretion of Pi is determined largely by the abundance of the type IIa NaPi cotransporter in the apical membranes of proximal tubular cells (19), the total abundance of NaPi-IIa contained in a crude membrane fraction of renal cortex tissue was analyzed using immunoblotting (Fig. 2). In VDR–/–, 1{alpha}OHase–/– mice fed a high-Pi diet, the abundance of NaPi-IIa did not differ significantly. After feeding both knockout strains a low-Pi diet, upregulation of NaPi-IIa was observed, and the relative increase in NaPi-IIa abundance was similar to that observed in kidney cortex tissue of wild-type and 1{alpha}OHase+/– animals. In agreement with these findings, upregulation of NaPi-IIa by a low-Pi diet also was observed on the basis of immunofluorescence performed with kidney slices that were prepared from wild-type and VDR-deficient animals (Fig. 3). Furthermore, it was recognized that the segmental distribution and cellular localization of NaPi-IIa in the VDR–/– samples were normal; i.e., NaPi-IIa was almost exclusively localized to the apical membrane, and its abundance was highest in the S1 segments. In parallel, kidney slices obtained from animals fed a low-Pi diet were incubated with 1-34 PTH for 45 min. As illustrated, PTH led to downregulation of NaPi-IIa in slices derived from the wild types as well as in slices derived from the VDR–/– mice. Similar observations were made with kidney slices obtained from 1{alpha}OHase-deficient mice (data not shown). In addition, the cellular localization of the NaPi-IIa-interacting proteins NHERF-1 and PDZK1 (11) was analyzed using immunofluorescence. The apical localization of those proteins was not disturbed by the deficiency in VDR or 1{alpha}OHase (data not shown).



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Fig. 2. Immunoblot analysis of NaPi-IIa of kidney membrane preparations prepared from single animals: Left, wild-type vs. vitamin D receptor (VDR)–/– mice; right, heterozygotes vs. 1{alpha}OHase–/– mice. In addition, each blot was analyzed for the abundance of {beta}-actin, which served as loading control. Ratios of the densities [NaPi type IIa (NaPi-IIa) signal over the {beta}-actin signal] are presented in the bar graphs as means ± SD. Significant (P < 0.05) upregulation of the NaPi-IIa abundance was observed in all mice strains after they were fed a low-Pi diet. No significant difference in NaPi-IIa abundance was observed in the knockout mice compared with the corresponding wild-type mice.

 


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Fig. 3. Immunostaining for NaPi-IIa of kidney slices after in vitro incubation for 45 min. In slices derived from wild-type and VDR-deficient mice, the increases of the NaPi-IIa contents mediated by a low-Pi diet were clearly visible and were not distinguishable. Treatment of slices obtained from the adapted animals with 10–7 M 1-34 PTH for 45 min led to downregulation of NaPi-IIa in kidney slices derived from both wild-type and VDR-deficient mice.

 
In the small intestine, the type II NaPi cotransporter NaPi-IIb is involved in the absorption of Pi (12, 14). To investigate whether VDR or 1{alpha}OHase is required for the upregulation of NaPi-IIb by a low-Pi diet, BBMV were isolated from small intestines of animals fed a high- or a low-Pi diet and analyzed for the abundance of NaPi-IIb by performing Western blot analysis and for NaPi cotransport. As illustrated in Fig. 4, the low-Pi diet provoked an increase in NaPi-IIb abundance in both knockout models that was comparable to the upregulation of NaPi-IIb in BBMV obtained from the corresponding control animals. In VDR–/– as well as 1{alpha}OHase–/– animals fed a high-Pi diet, the abundance of NaPi-IIb tended to be less than that of the wild types. The same BBMV preparations were analyzed for calbindin D9k, which was previously described to be regulated by 1,25(OH)2D (2). As expected, upregulation of calbindin D9k by a low-Pi diet was detected in BBMV of wild types but not in BBMV derived from VDR-deficient mice. In agreement with previously published data (7), calbindin D9K was absent in the 1{alpha}OHase–/– mice (data not shown). Na+-dependent uptake of Pi was determined with BBMV isolated from wild-type and VDR–/– animals.



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Fig. 4. Western blot analysis of NaPi-IIb in brush-border membrane vesicles (BBMV) isolated from small intestines of mice fed with either a high- or a low-Pi diet for 5 days. For each isolation, the small intestines of 3 animals were pooled. Two isolations of membranes were performed for each condition. The results shown were obtained with BBMV of 1 isolation and are representative of both isolations. In all experiments, NaPi-IIb was upregulated between 3- and 5-fold by feeding the mice a low-Pi diet. Equal loadings are indicated by the abundance of {beta}-actin. In addition, BBMV from wild-type and VDR-deficient mice were analyzed for the presence of calbindin D9k.

 
In agreement with the data obtained by Western blot analysis, NaPi cotransport into BBMV derived from VDR–/– animals was upregulated by a low-Pi diet (Fig. 5). Similar results were obtained with BBMV isolated from and OHase–/– mice (data not shown).



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Fig. 5. Net Na+-dependent transport of Pi in BBMV prepared from small intestines of wild-type and VDR–/– mice. In both BBMV populations, Na+-independent Pi uptake (in the presence of 100 mM KCl) and equilibrium values were similar (45 pmol/mg of protein/90 s and 110 pmol/mg of protein/90 min, respectively). Data represent the means ± SD (n = 4) of transport rates obtained in 1 of 3 experiments. *P < 0.05, significant upregulation of Na+-dependent transport of Pi.

 
Because a low-Pi diet leads to an increase in the abundance of NaPi-IIa mRNA in the small intestines of wild-type mice (23; Radanovic T, Wagner CA, Murer H, and Biber J, unpublished results), the contents of NaPi-IIb mRNA were determined by performing RT-PCR and compared with the contents of the {beta}-actin mRNA. As displayed in Fig. 6, in both VDR–/– and 1{alpha}OHase–/– mice, NaPi-IIb mRNA was similarly upregulated by a low-Pi diet as observed in wild-type and OHase+/– mice.


    DISCUSSION
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Low-Pi diet represents a classic stimulator of transepithelial transport of Pi in renal proximal tubules and the small intestine. In both organs, a low-Pi diet provokes an increase in the abundance of apically located type II NaPi cotransporters: NaPi-IIa and NaPi-IIc in proximal tubules and NaPi-IIb in small intestine (12, 16, 21). The precise mechanisms of regulation are still not entirely clear. One prominent event elicited by a low-Pi diet is an increase in the activity of the renal mitochondrial 1{alpha}OHase, which results in an increased level of 1,25(OH)2D (reviewed in Ref. 2). Thus part of the restoration of phosphate homeostasis, when impaired by a low-Pi diet, has been explained by a stimulation of intestinal absorption of Pi and bone resorption by 1,25(OH)2D (3, 6, 22). In fact, it has been demonstrated that the abundance of the NaPi cotransporter NaPi-IIb is altered by changes in vitamin D status (12, 15, 28). Only recently it was reported that a low-Pi diet leads to an increase in the abundance of NaPi-IIb mRNA as well (23; Radanovic T, Wagner CA, Murer H, and Biber J, unpublished results). Therefore, the latter finding suggests that an increased level of 1,25(OH)2D would result in increased transcription of NaPi-IIb mRNA, possibly involving the VDR (2). In the kidney, the role of 1,25(OH)2D in Pi reabsorption, and more specifically its effect on the abundance of NaPi-IIa cotransporter in proximal tubules, is less clear. Conflicting results have been reported indicating that a possible action of 1,25(OH)2D on the NaPi-IIa cotransporter is dose dependent and depends on alterations in the level of PTH (8, 19). As demonstrated recently, a genomic effect of low-Pi diet on the upregulation of NaPi-IIa seems unlikely, because in cortical as well as juxtamedullary proximal tubular S1 and S3 segments of adult mice, the abundance of NaPi-IIa mRNA was not altered by a low-Pi diet (17).

In the present study, we used VDR–/– and 1{alpha}OHase–/– mice to investigate the roles of the VDR and elevated levels of 1,25(OH)2D in the adaptation of NaPi-IIa in kidney and NaPi-IIb in small intestine to a low-Pi diet. Our results demonstrate that in VDR–/– and 1{alpha}OHase–/– mice, the adaptation to a low-Pi diet of the NaPi-IIa and NaPi-IIb proteins and NaPi-IIb mRNA was normal, i.e., comparable to that of the respective wild-type animals. Similar results were reported recently for VDR-deficient mice (23).

As indicated by the determinations of urinary excretion of Pi and by Western blot analysis of NaPi-IIa, VDR–/– and 1{alpha}OHase–/– mice adapted to a low-Pi diet similarly to the wild types. On the other hand, under high-Pi diet conditions, urinary excretion of Pi was less in both knockout models compared with the wild types, and similar values for the fractional excretion of Pi were observed in the knockout mice and the corresponding wild-type mice. Decreased urinary excretion of Pi was not paralleled by detectable changes in the abundance of the NaPi-IIa protein as one would assume to occur because of increased levels of PTH reported for VDR- and 1{alpha}OHase-deficient mice (7, 23). In contrast to our findings, slight decreases in NaPi-IIa abundance have been reported in VDR-deficient mice (23), 1,25(OH)2D-deficient rats (25), and 1{alpha}OHase-deficient weanling mice (26). Because the regulation of NaPi-IIa by PTH may be impaired by the lack of 1,25(OH)2D (10), kidney slices of adapted mice were incubated with PTH. These experiments demonstrated that the signaling machinery involved in the downregulation of NaPi-IIa by PTH was intact and did not depend on the presence of the VDR or on an increased level of 1,25(OH)2D. Therefore, the lack of a decreased abundance of NaPi-IIa in the knockout mice may be explained by the fact that, in our study, mice were fed a high-Pi diet, a condition that leads to complete downregulation of NaPi-IIa (see Fig. 3). Under such conditions, additional downregulation of NaPi-IIa by PTH may be too small to be detected. Of note, reported reductions in NaPi-IIa content (23, 25, 26) were observed in animals fed a normal Pi diet containing 0.6–0.8% Pi.

Another type II NaPi cotransporter, NaPi-IIc, was recently described and localized to the renal proximal tubules (21). The observed reduced urinary Pi excretion of mice fed a high-Pi diet may therefore be explained by a change in the content of the NaPi-IIc cotransporter. However, NaPi-IIc was related to growth and is of very low abundance in adult mice. Furthermore, the abundance of NaPi-IIc was reported not to be altered in VDR-deficient mice (23).

In the aggregate, our results suggest that the expression of the NaPi-IIa protein in the renal proximal tubules of mice fed a high-Pi diet is not affected by the status of 1,25(OH)2D or by the VDR. However, in both knockout mice, less urinary excretion of Pi was observed, despite similar values for the fractional excretion of Pi in the knockout mice compared with the corresponding controls. As mentioned above, this finding can be explained by small, undetectable changes in NaPi-IIa abundance in mice fed a high-Pi diet. Furthermore, our results demonstrate that the upregulation of NaPi-IIa due to a low-Pi diet does not depend on an increase in the level of 1,25(OH)2D via stimulation of renal 1{alpha}OHase and does not require the VDR. In accordance, it was reported that to blunt the stimulation of 1{alpha}OHase after hypophysectomy, the adaptation of NaPi cotransport by feeding a low-Pi diet to rats was normal (27).

Numerous studies have shown that intestinal absorption of Pi is regulated by 1,25(OH)2D (3, 6). Furthermore, it has been demonstrated that upregulation of intestinal Pi absorption can be explained by an alteration of the apical abundance of the NaPi-IIb protein (12, 15, 28). In adult mice, an increase in the NaPi-IIb protein is paralleled by an increase in NaPi-IIb mRNA (23; Radanovic T, Wagner CA, Murer H, and Biber J, unpublished results). The latter finding suggests that upregulation of NaPi-IIb occurs via a genomic mechanism possibly involving the VDR. On the other hand, it has been postulated that 1,25(OH)2D may act on the basis of rapid, nongenomic mechanisms (9). In this context, a protein of chicken enterocytes that belongs to the superfamily of glucose-regulated and redox-sensitive proteins was recently described as a 1,25(OH)2D-binding protein (20). However, in mouse small intestine, a change in the abundance of the NaPi-IIb protein was not observed 3 h after the onset of a low-Pi diet (12), indicating that a rapid nongenomic mechanism may not be involved in the regulation of NaPi-IIb in mouse small intestine.

As shown in the present study, upregulation of the NaPi-IIb protein by a low-Pi diet in VDR–/– mice (see also Ref. 23) and 1{alpha}OHase-deficient mice is comparable to that in wild-type mice. Also, upregulation of NaPi-IIb mRNA by feeding a low-Pi diet to the knockout mice strains was comparable to that observed in the wild-type mice. Therefore, we conclude that the upregulation of the NaPi-IIb protein by feeding mice a low-Pi diet involves a genomic mechanism that does not include the 1,25(OH)2D-VDR axis; i.e., it does not require the VDR and is not dependent on an increase in the level of 1,25(OH)2D via stimulation of the renal 1{alpha}OHase.


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We thank the Swiss National Science Foundation for financial support under Grant 31-61438 (to J. Biber).


    ACKNOWLEDGMENTS
 
We thank C. Madjdpour for performing the initial RT-PCR experiments.


    FOOTNOTES
 

Address for reprint requests and other correspondence: J. Biber, Institute of Physiology, Univ. of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland (E-mail: JuergBiber{at}access.unizh.ch)

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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