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
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ABSTRACT |
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NaPi type IIb; vitamin D3
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-hydroxylase (1
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 Because low-Pi diet induces an upregulation of NaPi-IIb mRNA (23; Radanovic T, Wagner CA, Murer H, and Biber J, unpublished data), it could be envisaged that the action of 1,25(OH)2D is via a genomic mechanism that involves the vitamin D receptor (VDR) (2).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 1OHase-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.
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METHODS |
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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 107 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 -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
-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 -actin cDNA were described elsewhere (17). The relative expressions of NaPi-IIb mRNA to the
-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 -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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RESULTS |
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DISCUSSION |
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In the present study, we used VDR/ and 1OHase/ 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
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 1OHase/ 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
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
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 0.60.8% 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 1OHase and does not require the VDR. In accordance, it was reported that to blunt the stimulation of 1
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 1OHase-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
OHase.
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GRANTS |
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ACKNOWLEDGMENTS |
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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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