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Experimental Physiology 91.3 pp 531-537
DOI: 10.1113/expphysiol.2005.032516
© The Physiological Society 2006
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Intestinal phosphate absorption and the effect of vitamin D: a comparison of rats with mice

Joanne Marks1,2, Surjit K. Srai3, Jürg Biber4, Heini Murer4, Robert J. Unwin1,2 and Edward S. Debnam1

1 Department of Physiology2 Centre for Nephrology3 Department of Biochemistry & Molecular Biology, Royal Free and University College Medical School, London, UK4 Institute of Physiology, University of Zurich, Zurich, Switzerland


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Previously, it was thought that intestinal phosphate transport occurred exclusively in the proximal small intestine of rodents and humans. However, a recent study has demonstrated that the ileum of mice contributes significantly to the absorption of dietary phosphate, but it is not known whether this region is also an important site of phosphate absorption in the rat. In the present study, we have investigated the mRNA and protein levels of the sodium–phosphate cotransporter, NaPi-IIb, in three regions of rat and mouse small intestine, and related its expression levels to the rate of net phosphate absorption, as measured using the in situ intestinal loop technique. 1,25-Dihydroxyvitamin D3 is an important physiological regulator of intestinal phosphate absorption that increases phosphate transport in both the duodenum and jejunum of the rat. Based on the recently proposed regional profile of phosphate absorption along the mouse small intestine, we have re-evaluated the effects of 1,25-dihydroxyvitamin D3 using three distinct regions of the mouse and rat small intestine. Our studies have revealed important differences in the intestinal handling of phosphate between mice and rats. In mice, maximal phosphate absorption occurs in the ileum, which is paralleled by the highest expression levels of NaPi-IIb mRNA and protein. In contrast, in rats maximal absorption occurs in the duodenum with very little absorption occurring in the ileum, which is similar to the pattern reported in humans. However, in both rodent species only the jejunum shows an increase in phosphate absorption in response to treatment with 1,25-dihydroxyvitamin D3.

(Received 7 October 2005; accepted after revision 23 January 2006; first published online 23 January 2006)
Corresponding author J. Marks: Department of Physiology, Royal Free and University College Medical School, Royal Free Campus, Rowland Hill Street, London NW3 2PF, UK. Email: j.marks{at}medsch.ucl.ac.uk


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Earlier studies, carried out mainly in vitro, using rat small intestine have shown that the duodenum and jejunum are responsible for the bulk of intestinal phosphate (Pi) absorption (Walling, 1977; Danisi & Murer, 1991). More recently, the protein responsible for regulated phosphate transfer across the enterocyte brush border membrane has been identified as the sodium–phosphate transporter, NaPi-IIb (Hilfiker et al. 1998; Murer et al. 2001), a member of the solute carrier family SLC34 (http://www.gene.ucl.ac.uk/nomenclature). This transporter is thought to be the rate-limiting step for transepithelial phosphate absorption (Murer et al. 2001, 2004). Recent studies have localized this transport protein to the ileum of mice (Radanovic et al. 2005; Stauber et al. 2005), which until now was not considered to be a major site of phosphate absorption.

In addition to sodium-dependent phosphate transport, experiments using brush border membrane (BBM) vesicles have revealed a sodium-independent mechanism of phosphate transport. This route of absorption has been proposed to contribute significantly to phosphate flux across the enterocyte BBM (Lee et al. 1986; Borowitz & Ghishan, 1989; Borowitz & Granrud, 1992b), although it appears to be unregulated (Danisi et al. 1980; Katai et al. 1999a,b).

At present, little is known about the transporters involved in the transfer of phosphate across the basolateral membrane (BLM) of absorptive epithelial cells. Early studies using BLM vesicles demonstrated the presence of both sodium-dependent and -independent efflux pathways. The latter route may use an as yet unidentified anion exchanger, but this has not been confirmed. Owing to the relatively small contribution of the sodium-dependent mechanism, and the fact that the electrochemical gradient for sodium across the BLM does not favour efflux via a sodium–phosphate cotransporter, this mechanism may be involved in the housekeeping of cellular phosphate (reviewed by Hammerman, 1986). More recent studies have demonstrated the presence of a sodium-dependent transporter, Pit-2, in the small intestine (Katai et al. 1999a; Bai et al. 2000), that is likely to be targeted to the BLM of jejunal enterocytes, where it may be involved in phosphate influx.

Early studies investigating the mechanisms of phosphate homeostasis concluded that phosphate reabsorption across the proximal tubule is acutely regulated and plays a critical role in controlling the circulating levels of phosphate. Conversely, control of intestinal phosphate absorption is believed to be chronically regulated (reviewed by Murer et al. 1994, 2001). 1,25-Dihydroxyvitamin D3 and dietary phosphate depletion are thought to be the most important physiological stimuli of intestinal phosphate absorption (Hattenhauer et al. 1999; Murer et al. 2004) and act by increasing the abundance of the transporter protein. Epidermal growth factor (EGF) (Xu et al. 2001, 2003a) and glucocorticoids (Borowitz & Granrud, 1992a; Arima et al. 2002) reduce intestinal phosphate absorption, and other factors like oestrogens (Xu et al. 2003b) and systemic metabolic acidosis (Gafter et al. 1986; Stauber et al. 2005) can also affect intestinal phosphate absorption.

Since it has been shown recently that the ileum is responsible for the bulk of intestinal phosphate absorption in the mouse, the aim of the present study was to re-evaluate the regional profile of phosphate absorption in vivo in rats and mice. Although the effect of 1,25-dihydroxyvitamin D3 on phosphate absorption is already well established in the proximal small intestine, the responsiveness of the ileum has not been determined. Therefore, we also determined the effects of 1,25-dihydroxyvitamin D3 on phosphate absorption in three distinct regions of the small intestine in both rodent species.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Animals

The study used 230 g male Sprague–Dawley rats and 25 g male C57BL/6 mice bred at the Comparative Biology Unit at the Royal Free and University College Medical School. To determine the effect of vitamin D on intestinal phosphate handling, animals received two I.P. injections of 1 µg kg –1 1,25-dihydroxyvitamin D3 dissolved in ethanol, 24 and 12 h prior to experimentation. Control animals received two injections of ethanol alone. Animals were allowed ad libitum access to water and a standard rodent chow containing 0.73% calcium, 0.52% phosphorous and 0.62 IU g–1 vitamin D3 (Diet RM1, SDS Ltd, Witham, UK) until the time of experimentation. All procedures were carried out in accordance with the Animals (Scientific Procedures) Act 1986 and animals were humanely killed by cervical dislocation at the end of the experiment.

In vivo uptake studies

Male Sprague–Dawley rats (230 g) and male C57BL/6 mice (25 g) were anaesthetized with an intraperitoneal injection of 60 mg kg–1 pentobarbitone sodium (Pentoject, Animalcare Ltd, York, UK) and maintained at 37°C on a thermostatically controlled heating blanket (Harvard Apparatus Ltd, Edenbridge, Kent, UK). Five-centimetre-long segments of duodenum (first 5 cm from pylorus), jejunum (5 cm from the ligament of Treitz) or ileum (last 10 cm) were cannulated and flushed with warm 0.9% saline, followed by air. Uptake buffer (500 µl for rats and 200 µl for mice) containing (in mmol l–1): 16 Na-Hepes, 140 NaCl, 3.5 KCl, 0.1 KH2PO4 and approximately 1.5 MBq 32Pi (PerkinElmer, Beaconsfield, Bucks, UK) was instilled into the lumen and the segment tied off. The volumes instilled into the lumen were chosen in order to fill the intestinal segment whilst avoiding distension. Blood (0.5 ml) was collected after 10 min by cardiac puncture and centrifuged at 1500g for 15 min to obtain plasma. The segment of small intestine was removed and washed with 0.9% saline, blotted and the segment length recorded. From scintillation counting of the plasma and the initial uptake solution, phosphate transfer from the lumen into 1 ml of blood by 5 cm of small intestine was calculated.

Brush border membrane vesicle preparation

The methods used to prepare intestinal BBM vesicles have been described previously (Kessler et al. 1978). Male Sprague–Dawley rats (230 g) and male C57BL/6 mice (25 g) were anaesthetized with an intraperitoneal injection of 60 mg kg–1 pentobarbitone sodium (Pentoject) before removal of the distinct regions of the small intestine. Intestinal segments were opened longitudinally, and the mucosa scraped off using glass slides. The resulting mucosa was suspended in buffer containing (in mmol l–1): 50 mannitol, 2 Hepes (pH 7.1) and 0.25 phenylmethylsulphonyl fluoride (PMSF), and homogenized three times at half-speed for 20 s using a Ultra Turrax homogenizer (VWR, lutterworth, Leicestershire, UK), followed by the addition of MgCl2 to a concentration of 10 mmol l–1, and then stirred on ice for 20 min. The suspension was then centrifuged at 3000g for 20 min, and the supernatant then re-centrifuged at 27000g for 30 min. The pellet was suspended in buffer containing (in mmol l–1): 300 mannitol, 20 Hepes, 0.1 MgSO4 (pH 7.2) and 0.25 PMSF, by passing six times through a 21 gauge needle. The suspension was then centrifuged for 15 min at 6000g, and the resulting supernatant centrifuged at 27000g for a further 30 min. The purified BBM pellet was finally resuspended in the same buffer to a protein concentration of 3–6 mg ml–1 using five or six passes through a 21 gauge needle. All steps were carried out at 4°C. The concentration of protein in the BBM vesicles was determined using the Bradford method (Bradford, 1976).

Western blotting

Polyclonal antibodies for NaPi-IIb were raised in female New Zealand white rabbits against a synthetic peptide corresponding to the first 21 residues of the N-terminus of the mouse NaPi-IIb protein (GenBank accession number AF081499 [GenBank] ). Mouse monoclonal antibodies raised against the first 14 residues of Xenopus laevisß-actin were used as a loading control (Abcam, Cambridge, UK). For Western blotting, BBM samples (30 µg protein) were solubilized in Laemmli sample buffer containing 5% SDS and 5% mercaptoethanol, and electrophoresed on a 10% SDS polyacrylamide gel. The proteins were transferred to nitrocellulose membranes by semidry electrophoretic blotting for 1 h at a constant current of 1 mA cm–2. Non-specific protein-binding sites were blocked with PBS-T (PBS containing 0.1% Tween 20) and 5% fat-free milk for 1 h at room temperature. The membranes were incubated with NaPi-IIb (1:2500) and ß-actin antibodies (1:5000) for 16 h at 4°C. The filters were then washed (2 x 15 min) with PBS-T containing 1% fat-free milk and incubated with either anti-rabbit (Amersham Pharmacia Biotech UK Ltd, Amersham, UK) or anti-mouse IgG antibody (Sigma Ltd, Poole, UK) conjugated to horseradish peroxidase for 1 h at room temperature and finally washed again with PBS-T. Bound antibodies were detected by an enhanced chemiluminescence system (Amersham Pharmacia Biotech UK Ltd) and visualized and quantified using a Fluor-S MultiImager System (Biorad, Hemmel Hempstead, UK). The ratio of NaPi-IIb to ß-actin was established for each sample and expressed in arbitrary units (a.u.).

Real-time PCR

Total RNA was extracted from mucosal scrapes of the distinct regions of the rat and mouse small intestine using a QIAamp RNA blood mini kit according to the manufacturer's instructions (Qiagen, Crawley, UK). RNA was reverse transcribed with 0.5 µg of oligo-dT 12–18 primer and a First Strand cDNA synthesis kit (Superscript II RNase H-reverse transcriptase; Life Technologies, Paisley, UK). NaPi-IIb transporter transcripts were analysed by Real-time PCR using QuantiTech SYBR® Green PCR kit (Qiagen) on a LightCycler Real-Time PCR instrument (version 3.5, Roche Diagnostics, Lewes, UK) using specific primers designed from the published sequence of the mouse NaPi-IIb transporter (GenBank accession number AF081499 [GenBank] ; forward position 1402–1418, reverse position 1631–1616). The levels of ß-actin transcripts were assessed in parallel experiments using primers designed from the sequence of rat ß-actin (GenBank accession number NM031144; forward position 937–955, reverse position 1223–1208). For both primers, cycling conditions were as follows: 95°C for 10 min followed by 45 cycles of 95°C for 15 s, 60°C for 20 s and 72°C for 40 s with transition rates of 20°C s–1 and a single fluorescence acquisition at 81°C. A standard curve for each gene was established by performing the above procedure with serially diluted DNA samples of known concentrations. The relative amounts of the target and reference genes in each sample were then calculated based on the crossing-point analysis (Relquant, version 1.01, Roche Diagnostics); the second derivative maximum method was used to automatically determine the crossing point for individual samples. The sequences of the PCR products were confirmed by DNA sequencing.

Statistics

Data are presented as means ±S.E.M. Normal distribution was determined by Kolmogrov–Smirnov test of normality and statistical comparisons made using either Student's unpaired t test or a one-way analysis of variance (ANOVA) with post hoc comparisons performed using either the Bonferroni multiple comparisons test or the Kruskal–Wallis test. All analyses were performed using Graphpad Instat software with statistical significance taken as P < 0.05. Each group consists of n= 6.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
The profile of phosphate absorption and the expression of NaPi-IIb mRNA and protein along the rat and mouse small intestine were examined using three distinct intestinal regions. The first 5 cm of small intestine was taken to represent the duodenum, the jejunum was taken 5 cm distal from the ligament of Treitz and the ileum represented the final 10 cm of the small intestine. In vivo uptake studies demonstrated that phosphate transfer from lumen to blood by the rat small intestine was highest in the duodenum followed by the jejunum, with relatively little absorption occurring in the ileum (Fig. 1). By contrast, in the mouse small intestine, absorption was significantly higher in the ileum than in the duodenum and jejunum (Fig. 1), with the latter two regions having the ability to absorb equivalent amounts of phosphate. Interestingly, uptake by the mouse small intestine is considerably higher than in the rat.


Figure 1
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  		Figure 1.  In vivo phosphate absorption by distinct regions of the rat ({blacksquare}) and mouse small intestine ({square})
Results are expressed as the means ±S.E.M. of 6 individual experiments per region as a percentage of the initial counts transferred from 5 cm of small intestine into 1 ml of blood over 10 min. **P < 0.01, ***P < 0.001 compared to the duodenum and jejunum for each species using a one-way ANOVA.

 
Quantification of NaPi-IIb mRNA expression in the mouse small intestine using real-time PCR demonstrated that the transcript for this protein is expressed at highest levels in the ileum followed by the jejunum, but is undetectable in the duodenum (Fig. 2). By comparison, highest levels of NaPi-IIb mRNA were detected in the rat jejunum followed by the duodenum, with the transcript being undetectable in the rat ileum (Fig. 2). In addition, comparisons of NaPi-IIb mRNA expression levels in the rat with mice showed that the transporter mRNA is present at much higher levels in the mouse small intestine.


Figure 2
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  		Figure 2.  Real-time PCR quantification of NaPi-IIb mRNA in distinct regions of the rat ({blacksquare}) and mouse small intestine ({square})
Results are expressed as the means ±S.E.M. of duplicate PCR reactions performed on 6 individual samples. The abundance of NaPi-IIb is given as a ratio of NaPi-IIb mRNA to ß-actin mRNA, in arbitrary units (a.u.). {dagger}P < 0.05 and {dagger}{dagger}P < 0.01 compared with rat ileum using a one-way ANOVA with Bonferroni multiple comparisons post hoc test. ***P < 0.001 compared with mouse ileum using a one-way ANOVA with Kruskal–Wallis post hoc test.

 
The protein levels of NaPi-IIb were examined using Western blotting of intestinal BBM vesicles isolated from the distinct regions of the mouse small intestine (Fig. 3A). The ratio of this protein to ß-actin enabled us to access the regional distribution of the sodium–phosphate cotransporter. In accordance with the in vivo uptake data and the mRNA expression profile, quantification of NaPi-IIb protein in relation to ß-actin protein levels revealed that the transporter was most abundant in the ileum and that lower levels were evident in the jejunum; however, the protein was undetectable in the duodenum (Fig. 3B).


Figure 3
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  		Figure 3.  Western blot analysis of NaPi-IIb protein in distinct regions of the mouse small intestine
A, detection of NaPi-IIb protein in BBM vesicles prepared from intestinal mucosa of mouse duodenum, jejunum and ileum. B, quantification of NaPi-IIb protein relative to ß-actin; results are expressed as the means ±S.E.M. of Western blots performed in duplicate on 6 individual BBM vesicle samples. The abundance of NaPi-IIb is given as a ratio of NaPi-IIb protein to ß-actin protein, in a.u. **P < 0.01 compared with the jejunum and ***P < 0.001 compared with the duodenum using a one-way ANOVA.

 
The polyclonal antibodies used in this study were raised in rabbits against a synthetic peptide corresponding to the first 21 residues of the N-terminus of the mouse NaPi-IIb protein. The transcript for NaPi-IIb in rats has two residues different within this region and consequently the antibody was unable to detect NaPi-IIb protein in BBM vesicles isolated from the rat small intestine. An antibody raised against a synthetic peptide corresponding to a region close to the C-terminus of mouse NaPi-IIb was also used in this study. There is only one residue different in this region between the rat and mouse transcript and, although the antibody successfully detected the protein in mouse small intestine (results not shown), we were unable to identify the protein in the rat small intestine. Various reducing and denaturing conditions were tried in an attempt to get these antibodies to recognize rat NaPi-IIb protein, but without success. Unfortunately, at present there is no commercial antibody available against the rat NaPi-IIb.

1,25-Dihydroxyvitamin D3 did not affect phosphate uptake by the rat and mouse duodenum in vivo (percentage of initial luminal 32P counts detected in 1 ml of plasma, for rats: control, 0.089 ± 0.025 versus vit D, 0.079 ± 0.167; and for mice: control, 0.143 ± 0.042 versus vit D, 0.132 ± 0.047) nor by the rat and mouse ileum (percentage of initial luminal 32P counts detected in 1 ml of plasma, for rats: control, 0.0067 ± 0.001 versus vit D, 0.0072 ± 0.003; and for mice: control, 0.29 ± 0.036 versus vit D, 0.34 ± 0.046). However, it did significantly increase phosphate absorption by the jejunum in both rodent species (Fig. 4) which, in the mouse at least, was accompanied by a significant increase in NaPi-IIb protein levels (Fig. 5B) but not in mRNA expression (Fig. 5A). As stated above, we were unable to examine protein levels of NaPi-IIb in rat small intestine owing to difficulties with the available antibody; however, examination of mRNA levels in rat jejunum following 1,25-dihydroxyvitamin D3 treatment showed no change in transcript expression (levels of NaPi-IIb relative to ß-actin (a.u.): control, 0.0266 ± 0.003 versus vit D, 0.028 ± 0.005).


Figure 4
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  		Figure 4.  Effect of 1,25-dihydroxyvitamin D3 onin vivophosphate absorption by rat and mouse jejunum
Results are expressed as a percentage of the initial counts transferred from 5 cm of small intestine into 1 ml of blood over 10 min. *P < 0.05 using Student's unpaired t test, n= 6.

 

Figure 5
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  		Figure 5.  Effect of 1,25-dihydroxyvitamin D3 on NaPi-IIb in distinct regions of the mouse small intestine
A, real-time PCR quantification of NaPi-IIb mRNA in distinct regions of the mouse small intestine following 24 h treatment with 1,25-dihydroxyvitamin D3. B, quantification of NaPi-IIb protein in distinct regions of the mouse small intestine following 24 h treatment with 1,25-dihydroxyvitamin D3. **P < 0.01 using Student's unpaired t test, n= 6.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
In agreement with studies previously published (Walling, 1977; Yeh & Aloia, 1987; Danisi & Murer, 1991), we have shown that the bulk of intestinal phosphate absorption in the rat occurs in the duodenum and jejunum. When results are expressed in terms of centimetre length of small intestine, the duodenum has the greater capacity for phosphate absorption but, owing to the relative shortness and the sojourn time of the contents in this segment (Bronner, 2003), the jejunum would in fact be responsible for the bulk of the dietary phosphate absorption. Our studies have revealed that the ileum of the rat has a very limited capacity to absorb phosphate. Indeed, it has been proposed that the rat ileum is a secretory site for calcium and phosphate, and that this occurs paracellularly (Peters & Binswanger, 1988). The factors controlling paracellular transport of phosphate are unknown, although ileal calcium secretion may be influenced by 1,25-dihydroxyvitamin D3 (Peters & Binswanger, 1988).

In contrast, the mouse small intestine has the ability to absorb phosphate in all segments examined, with the highest rate occurring in the ileum. This finding is in agreement with a recent study showing that NaPi-IIb mRNA and protein levels are also highest in this region (Radanovic et al. 2005).

Studies of the processes involved in intestinal phosphate absorption have concentrated on the sodium-dependent transfer of phosphate across the BBM. This process is mediated by the protein NaPi-IIb, which has been extensively characterized and is reviewed elsewhere (Murer et al. 2001, 2004). At present, little is known about the mechanism involved in the transfer of phosphate across the basolateral membrane of absorptive epithelial cells. Our studies, using the in situ intestinal loop technique, provide information on the transepithelial absorption process, rather than exclusively on transport across the BBM. However, owing to the lack of information on the transporters involved in the efflux of phosphate at the enterocyte BLM, and the fact that uptake of phosphate across the BBM is deemed to be the rate-limiting step for intestinal phosphate absorption (Murer et al. 2001, 2004), we have studied the expression levels of NaPi-IIb mRNA and protein and correlated these with our in vivo data. We have demonstrated that in the mouse, the transcript for NaPi-IIb is expressed at highest levels in the ileum, which correlates with high levels of the protein in this region, as well as maximal phosphate absorption. Interestingly, the rates of in vivo phosphate transfer by the mouse duodenum and jejunum are comparable, although both NaPi-IIb mRNA transcript and protein are undetectable in the mouse duodenum. Therefore, uptake in this region may occur via a sodium-independent mechanism (Lee et al. 1986; Borowitz & Ghishan, 1989; Borowitz & Granrud, 1992b) or by a novel phosphate transporter. Moreover, a recent study has demonstrated that a low-phosphate diet induces an increase in mRNA expression of NaPi-IIb along the entire length of the small intestine, including the duodenum, but without corresponding changes in protein levels or phosphate uptake by BBM vesicles prepared from the duodenum (Radanovic et al. 2005).

In contrast to mice, the highest levels of NaPi-IIb mRNA were detected in the rat jejunum, but this finding differed from the in vivo uptake data, which showed that absorption was highest in the duodenum. This inconsistency might be a consequence of regional differences in the stability of the mRNA, or the rate of translation and/or degradation of the protein, or might relate to differences in post-translational modification or the intrinsic activity of the protein in the different segments of the small intestine. However, owing to the lack of a suitable antibody capable of detecting NaPi-IIb protein in rat small intestine, we cannot address this problem at present.

1,25-Dihydroxyvitamin D3 is an important physiological regulator of intestinal phosphate absorption (Hattenhauer et al. 1999; Murer et al. 2004). Several studies have confirmed that this hormone increases phosphate transport in the proximal small intestine via an increase in sodium-dependent phosphate transport (Danisi et al. 1980; Kabakoff et al. 1982; Lee et al. 1986; Hattenhauer et al. 1999; Xu et al. 2002). However, a specific regional effect of this hormone has not been defined. Our in vivo data have demonstrated that 1,25-dihydroxyvitamin D3 treatment promotes transepithelial phosphate flux only in the jejunum, which correlates with an increase in protein content, but not mRNA expression, at least in the mouse. This finding is in accordance with previous studies that provided evidence in adult rats and mice that increased phosphate absorption following treatment with 1,25-dihydroxyvitamin D3 occurs in a non-transcriptional manner (Hattenhauer et al. 1999; Xu et al. 2002). This effect of 1,25-dihydroxyvitamin D3 in adult rats is in contrast to the effect in suckling rats, which is mediated partly by increased gene transcription (Xu et al. 2002). Of importance is the finding that in humans the jejunum is also responsive to this steroid hormone, although a specific regional effect has not been established (Davis et al. 1983).

Finally, studies of the regional profile of phosphate absorption in humans indicate that the proximal small intestine has a greater capacity for absorption of this anion than the distal small intestine (Walton & Gray, 1979; Borowitz & Ghishan, 1989). Thus, according to our present findings, the profile of phosphate absorption (and its likely regulation) along the rat small intestine is much closer to that in humans than is the mouse. The rat is therefore a more appropriate animal model in which to study the processes and regulatory mechanisms influencing phosphate handling along the human small intestine.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Arima K, Hines ER, Kiela PR, Drees JB, Collins JF & Ghishan FK (2002). Glucocorticoid regulation and glycosylation of mouse intestinal type IIb Na-Pi cotransporter during ontogeny. Am J Physiol Gastrointest Liver Physiol 283, G426–G434.[Abstract/Free Full Text]

Bai L, Collins JF & Ghishan FK (2000). Cloning and characterization of a type III Na-dependent phosphate cotransporter from mouse intestine. Am J Physiol Cell Physiol 279, C1135–C1143.[Abstract/Free Full Text]

Borowitz SM & Ghishan FK (1989). Phosphate transport in human jejunal brush-border membrane vesicles. Gastroenterology 96, 4–10.[Medline]

Borowitz SM & Granrud GS (1992a). Glucocorticoids inhibit intestinal phosphate absorption in developing rabbits. J Nutr 122, 1273–1279.[Abstract/Free Full Text]

Borowitz SM & Granrud GS (1992b). Ontogeny of intestinal phosphate absorption in rabbits. Am J Physiol 262, G847–G853.

Bradford MM (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72, 248–254.[CrossRef][Medline]

Bronner F (2003). Mechanisms of intestinal calcium absorption. J Cell Biochem 88, 387–393.[CrossRef][Medline]

Danisi G, Bonjour JP & Straub RW (1980). Regulation of Na-dependent phosphate influx across the mucosal border of duodenum by 1,25-dihydroxycholecalciferol. Pflugers Arch 388, 227–232.[CrossRef][Medline]

Danisi G & Murer H (1991). Inorganic phosphate absorption in small intestine. In Handbook of Physiology, section 6, vol IV, chapter 12, The Gastrointestinal System, Intestinal absorbtion & secretion, pp. 323–336, (eds. Schutt SG, Field M and Frizzell RA). American Physiological Society, Bethesda, MD, USA.

Davis GR, Zerwekh JE, Parker TF, Krejs GJ, Pak CY & Fordtran JS (1983). Absorption of phosphate in the jejunum of patients with chronic renal failure before and after correction of vitamin D deficiency. Gastroenterology 85, 908–916.[Medline]

Gafter U, Edelstein S, Hirsh J & Levi J (1986). Metabolic acidosis enhances 1,25(OH)2D3-induced intestinal absorption of calcium and phosphorus in rats. Miner Electrolyte Metab 12, 213–217.[Medline]

Hammerman MR (1986). Phosphate transport across renal proximal tubular cell membranes. Am J Physiol 251, F385–F398.

Hattenhauer O, Traebert M, Murer H & Biber J (1999). Regulation of small intestinal Na-Pi type IIb cotransporter by dietary phosphate intake. Am J Physiol 277, G756–G762.

Hilfiker H, Hattenhauer O, Traebert M, Forster I, Murer H & Biber J (1998). Characterization of a murine type II sodium-phosphate cotransporter expressed in mammalian small intestine. Proc Natl Acad Sci U S A 95, 14564–14569.[Abstract/Free Full Text]

Kabakoff B, Kendrick NC & DeLuca HF (1982). 1,25-Dihydroxyvitamin C3-stimulated active uptake of phosphate by rat jejunum. Am J Physiol 243, E470–E475.[Medline]

Katai K, Miyamoto K, Kishida S, Segawa H, Nii T, Tanaka H, Tani Y, Arai H, Tatsumi S, Morita K, Taketani Y & Takeda E (1999a). Regulation of intestinal Na+-dependent phosphate co-transporters by a low-phosphate diet and 1,25-dihydroxyvitamin D3. Biochem J 343, 705–712.

Katai K, Tanaka H, Tatsumi S, Fukunaga Y, Genjida K, Morita K, Kuboyama N, Suzuki T, Akiba T, Miyamoto K & Takeda E (1999b). Nicotinamide inhibits sodium-dependent phosphate cotransport activity in rat small intestine. Nephrol Dial Transplant 14, 1195–1201.[Abstract/Free Full Text]

Kessler M, Acuto O, Storelli C, Murer H, Muller M & Semenza G (1978). A modified procedure for the rapid preparation of efficiently transporting vesicles from small intestinal brush border membranes. Their use in investigating some properties of D-glucose and choline transport systems. Biochim Biophys Acta 506, 136–154.[Medline]

Lee DB, Walling MW & Corry DB (1986). Phosphate transport across rat jejunum: influence of sodium, pH, and 1,25-dihydroxyvitamin D3. Am J Physiol 251, G90–G95.

Murer H, Forster I & Biber J (2004). The sodium phosphate cotransporter family SLC34. Pflugers Arch 447, 763–767.[CrossRef][Medline]

Murer H, Hernando N, Forster L & Biber J (2001). Molecular mechanisms in proximal tubular and small intestinal phosphate reabsorption (plenary lecture). Mol Membr Biol 18, 3–11.[Medline]

Murer H, Markovich D & Biber J (1994). Renal and small intestinal sodium-dependent symporters of phosphate and sulphate. J Exp Biol 196, 167–181.[Abstract/Free Full Text]

Peters J & Binswanger U (1988). Calcium and inorganic phosphate secretion of rat ileum in vitro. Influence of uremia and 1,25(OH)2D3 inhibition. Res Exp Med (Berl) 188, 139–149.

Radanovic T, Wagner CA, Murer H & Biber J (2005). Regulation of intestinal phosphate transport. I. Segmental expression and adaptation to low-Pi diet of the type IIb Na+-Pi cotransporter in mouse small intestine. Am J Physiol Gastrointest Liver Physiol 288, G496–G500.[Abstract/Free Full Text]

Stauber A, Radanovic T, Stange G, Murer H, Wagner CA & Biber J (2005). Regulation of intestinal phosphate transport. II. Metabolic acidosis stimulates Na+-dependent phosphate absorption and expression of the Na+-Pi cotransporter NaPi-IIb in small intestine. Am J Physiol Gastrointest Liver Physiol 288, G501–G506.[Abstract/Free Full Text]

Walling MW (1977). Intestinal Ca and phosphate transport: differential responses to vitamin D3 metabolites. Am J Physiol 233, E488–E494.

Walton J & Gray TK (1979). Absorption of inorganic phosphate in the human small intestine. Clin Sci (Lond) 56, 407–412.[Medline]

Xu H, Bai L, Collins JF & Ghishan FK (2002). Age-dependent regulation of rat intestinal type IIb sodium-phosphate cotransporter by 1,25-(OH)2 vitamin D3. Am J Physiol Cell Physiol 282, C487–C493.[Abstract/Free Full Text]

Xu H, Collins JF, Bai L, Kiela PR & Ghishan FK (2001). Regulation of the human sodium-phosphate cotransporter NaPi-IIb gene promoter by epidermal growth factor. Am J Physiol Cell Physiol 280, C628–C636.[Abstract/Free Full Text]

Xu H, Inouye M, Hines ER, Collins JF & Ghishan FK (2003a). Transcriptional regulation of the human NaPi-IIb cotransporter by EGF in Caco-2 cells involves c-myb. Am J Physiol Cell Physiol 284, C1262–C1271.[Abstract/Free Full Text]

Xu H, Uno JK, Inouye M, Xu L, Drees JB, Collins JF & Ghishan FK (2003b). Regulation of intestinal NaPi-IIb cotransporter gene expression by estrogen. Am J Physiol Gastrointest Liver Physiol 285, G1317–G1324.[Abstract/Free Full Text]

Yeh JK & Aloia JF (1987). Effect of glucocorticoids on the passive transport of phosphate in different segments of the intestine in the rat. Bone Miner 2, 11–19.[Medline]


   Acknowledgements
 
We are grateful to Kidney Research UK (grant RP15/1/2003) for financial support.




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