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

Annina Stauber,* Tamara Radanovic,* Gerti Stange, Heini Murer, Carsten A. Wagner,* and Jürg Biber*

Institute of Physiology, University of Zurich, Zurich, Switzerland

Submitted 16 April 2004 ; accepted in final form 21 October 2004


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During metabolic acidosis, Pi serves as an important buffer to remove protons from the body. Pi is released from bone together with carbonate buffering protons in blood. In addition, in the kidney, the fractional excretion of phosphate is increased allowing for the excretion of more acid equivalents in urine. The role of intestinal Pi absorption in providing Pi to buffer protons and compensating for loss from bone during metabolic acidosis has not been clarified yet. Inducing metabolic acidosis (NH4Cl in drinking water) for 2 or 7 days in mice increased urinary fractional Pi excretion twofold, whereas serum Pi levels were not altered. Na+-dependent Pi transport in the small intestine, however, was stimulated from 1.89 ± 3.22 to 40.72 ± 11.98 pmol/mg protein (2 days of NH4Cl) in brush-border membrane vesicles prepared from total small intestine. Similarly, the protein abundance of the Na+-dependent phosphate cotransporter NaPi-IIb in the brush-border membrane was increased 5.3-fold, whereas mRNA levels remained stable. According to immunohistochemistry and real-time PCR NaPi-IIb expression was found to be mainly confined to the ileum in the small intestine, and this distribution was not altered during metabolic acidosis. These results suggest that the stimulation of intestinal Pi absorption during metabolic acidosis may contribute to the buffering of acid equivalents by providing phosphate and may also help to prevent excessive liberation of phosphate from bone.

phosphate


SEVERAL MECHANISMS CONTRIBUTE to the buffering and elimination of excessive protons and acid equivalents during metabolic acidosis. Besides respiration, increased release of buffer substances from bone and stimulated reabsorption of bicarbonate and increased excretion of protons by the kidneys are the major mechanisms to restore acid-base balance (11, 16). Excretion of protons by the kidney requires so called titratable acids, i.e., ammonia, citrate, and phosphate, buffering protons in urine in the collecting duct and thus increasing the maximal acid excretion rate (16). The renal excretion of ammonia and phosphate is highly increased during metabolic acidosis, whereas excretion of citrate is decreased (3). Ammonia (NH3) is synthetized from glutamine metabolism in the kidney proximal tubule in response to metabolic acidosis (27). In addition, it is thought that inhibition of renal reabsorption of phosphate contributes mainly to increased phosphate excretion (1). In a rat model for metabolic acidosis, a decrease in mRNA and protein of the major Na+-Pi cotransporter NaPi-IIa as well as in Na+-dependent Pi uptake into brush border membrane (BBM) vesicles (BBMV) has been found (1). Systemic Pi levels are only slightly decreased during metabolic acidosis, an effect that has been attributed to the acid-stimulated release of Pi from bone together with Ca2+ and carbonate (23). Indeed, the release of phosphate from bone during short- and long-term metabolic acidosis in in vivo and in in vitro models has been extensively documented and investigated (7–9, 23).

The role of intestinal phosphate absorption during metabolic acidosis has received only little attention, and available data are conflicting and suggest either a stimulation or a decrease in intestinal Pi absorption without information at a molecular level (5, 13, 14). To date, only one apical Na+-dependent Pi cotransporter, NaPi-IIb, has been identified in the small intestine, and shown to be expressed on the apical side of enterocytes (18). Intestinal Na+-dependent Pi uptake and NaPi-IIb abundance are regulated by a number of factors including dietary Pi content, age, or several hormones such as estrogen, glucocorticoids, and 1,25 (OH)2-dihydroxyvitamin D3 (2, 6, 17, 21, 25, 30, 31).

In the present study, we investigated whether metabolic acidosis affects intestinal Pi absorption focusing on Na+-dependent Pi transport across the apical membrane using BBMV and examining mRNA and protein abundance and localization of NaPi-IIb in a mouse model of metabolic acidosis. Our results demonstrate a stimulation of Na+-dependent Pi uptake and a concomitant increase of NaPi-IIb protein without affecting its mRNA levels or segmental distribution along the small intestine.


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Animal studies. NMRI mice (Charles River Laboratories, Germany; male, 12 wk, 35–45 g) were maintained on standard chow and had access to drinking water ad libitum. For some control experiments, male Sprague-Dawley rats, 250 g, were used. To induce metabolic acidosis, mice were given 2% sucrose/0.28 M NH4Cl in the drinking water for 48 h or 7 days as described previously (3, 28). Each group consisted of four animals for each time point and treatment, and experiments were repeated three times, respectively. For blood analysis, mice were anesthetized with ketamine/xylazine, and heparinized mixed arterial-venous blood samples were collected and analyzed immediately for blood gases and electrolytes on a Radiometer ABL 505, (Radiometer, Copenhagen, Denmark) blood gas analyzer. Urine was collected as spot urine, and pH was measured immediately using a pH microelectrode (Lazar Research Laboratories, CA) connected to Thermo Orion 290 pH meter. Phosphate in serum and urine was measured using a commercial kit (Sigma, St. Louis, MO), creatinine in serum was measured using an enzymatic kit (Wako Chemicals, Neuss, Germany) and in urine using the Jaffe reaction (Sigma) according to the manufacturers’ instructions. All animal studies were approved by the Swiss Kantonales Veterinäramt, Zurich.

Isolation of BBMs, transport studies, and Western blot analysis. For preparation, transport studies and Western blot analysis of small intestinal BBMV, see Radanovic et al. (27a).

BBMs from total rat and mouse kidneys were prepared as described previously (4). Each preparation contained membranes from both kidneys of each individual animal (4 animals in each group).

The transport rate of phosphate into BBMV was determined at 90 s and 90 min (equilibrium value) as described (17, 29) at 25°C in the presence of inward gradients of 100 mM NaCl or 100 mM KCl and 0.1 mM K2HPO4.

Western blot analysis of small intestinal BBMV and analysis of NaPi-llb and {beta}-actin content were performed as described by Radanovic et al. (27a). Before gel electrophoresis, renal BBMV (10 µg) were denaturated by heating for 2 min in the presence of 100 mM DTT. For detection of NaPi-lla, a rabbit polyclonal antiserum raised against a synthetic NH2-terminal peptide rat NaPi-lla was used (18).

Kidney BBMs were analyzed by Western blot analysis. Purified BBMs (10 µg) were separated by 10% SDS-PAGE. Before being loaded on gel, denaturation of kidney BBMs was performed at 96°C for 2 min in 2% SDS, 1 mM EDTA, 10% glycerol, 100 mM DTT, 85 mM Tris·HCl (pH 6.8). After transfer, nitrocellulose membranes containing transferred proteins were horizontally cut at the level of ~66 kDa, and each half was treated separately (the upper halves for NaPi-IIa detection and lower halves for {beta}-actin detection). The membranes were blocked with 5% nonfat milk powder and 1% Triton X-100 in TBS (150 mM NaCl, 25 mM Tris, pH 7.3) for 2 h. Immunodetection of electrotransferred protein was performed according to standard procedures. For detection of NaPi-IIa, rabbit polyclonal antisera raised against NH2-terminus of NaPi-IIa at a dilution of 1:6,000 (10) was used. {beta}-actin was detected with a mouse monoclonal anti-{beta}-actin antibody (Sigma; dilution 1:6,000). For detection of antibody binding and densitometric analysis, see Radanovic et al. (27a).

Immunofluorescence and real-time PCR. Both techniques were performed as described in the preceeding paper.

Statistics. Data are presented as means ± SD. All data were tested for significance using the unpaired Student’s t-test and ANOVA. Only P values <0.05 were considered significant.


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Induction of metabolic acidosis. Animals receiving NH4Cl in their drinking water for 2 or 7 days developed metabolic acidosis as evident from blood and urine analysis (Table 1). Urinary pH acidified from 7.61 ± 0.16 to 5.54 ± 0.04 and 5.61 ± 0.04 after 2 or 7 days, respectively. Blood gas analysis demonstrated lower pH (7.35 ± 0.02 under control and after NH4Cl loading 7.15 ± 0.06 and 7.27 ± 0.03 for 2 and 7 days, respectively) and bicarbonate as well as elevated Cl concentrations after acid loading with NH4Cl as described previously (Table 1) (15, 22, 28). Thus animals receiving NH4Cl in their drinking water had profound metabolic acidosis. Metabolic acidosis was less severe in animals receiving NH4Cl for 7 days as evident from blood pH and serum bicarbonate levels consistent with a partially compensated metabolic acidosis. PCO2 was lower in 7-day acidosis pointing to a respiratory compensation.


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Table 1. Blood and urine analysis after 2 and 7 days

 
Renal phosphate wasting and regulation of the renal type IIa Na+-phosphate cotransporter. Serum phosphate levels during metabolic acidosis were only slightly reduced. In contrast, urine analysis demonstrated that the fractional excretion of phosphate (FEPi, i.e., the fraction of filtered phosphate that is not reabsorbed) increased approximately twofold in animals receiving NH4Cl as described previously in metabolic acidosis (Table 1) (1). Na+-dependent phosphate uptake into BBMVs prepared from kidneys from the same animals was not significantly reduced in animals with 2 or 7 days of metabolic acidosis (Fig. 1). There was a considerable scattering of phosphate uptake rates between different animals in the same group. Western blot analysis showed a small, not significant increase in abundance of the major renal Na+-Pi cotransporter NaPi-IIa in the BBM (Fig. 1, B and C). These findings are somewhat in contrast to what has been shown in rat where a reduction in Na+-dependent phosphate uptake and NaPi-IIa protein abundance in the BBM was observed (1). To test whether this discrepancy could be due to a species difference, Na+-dependent Pi uptake was measured using BBMVs obtained from control rats and rats made acidotic with 0.28 M NH4Cl in their drinking water for 2 days. Na+-dependent Pi uptake was reduced by 42.7 ± 4.2% in kidneys from acidotic rats (n = 3 for each group).



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Fig. 1. Effect of metabolic acidosis on renal Pi handling and type IIa Na+-Pi-cotransporter abundance. A: Na+-dependent Pi-uptake rates into renal brush- border membrane vesicles (BBMV) from control and acidotic animals showed a small decrease. The results from 2 independent groups of animals for each condition are shown where each data point represents a single animal. No significant difference between all groups. B: protein abundance of the type IIa Na+-Pi cotransporter in BBMVs was slightly increased in acidotic animals. {beta}-Actin was tested on the same membranes to control for loading. The ratio of NaPi-IIa to {beta}-actin was not significantly different between control and treated groups. C: corresponding ratios of NaPi-IIa to {beta}-actin showed a small, not significant increase in acidotic animals.

 
Stimulation of Na+-dependent Pi transport and the type IIb Na+-Pi cotransporter in small intestine. BBMVs were prepared from total small intestine, and Na+-dependent Pi uptake was measured. Na+-dependent Pi uptake was low in BBMV from control animals (3.25 ± 5.31 pmol/mg protein, n = 4 experiments with each 4 animals). In contrast, after 2 days of metabolic acidosis, Na+-dependent Pi uptake was stimulated up to 20-fold (55.48 ± 28.23 pmol/mg protein, n = 3 groups of animals, each with 4 animals; Fig. 2). After 7 days of metabolic acidosis, stimulation of Na+-dependent Pi uptake was found in one group of animals (4 animals) but not in two other groups (each with 4 animals; average: 11.29 ± 8.69 pmol/mg protein; Fig. 2). The occurrence of diarrhea was noted in these animals, which may have been due to the treatment with NH4Cl, because no diarrhea was seen in control animals from the same batches. Western blot analyses for the intestinal Na+-dependent Pi cotransporter NaPi-IIb showed similar results. After 2 days of metabolic acidosis, a strong increase (~5-fold) in NaPi-IIb protein abundance was found, whereas after 7 days, only one group of animals showed an increase (corresponding to BBMV uptakes; Fig. 3, A and B).



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Fig. 2. Effect of metabolic acidosis on intestinal Na+-dependent Pi-uptake rates into BBMVs. Na+-dependent Pi-uptake rates in isolated BBMVs prepared from the whole small intestine of control and acidotic animals from 3 independent groups of animals. Each data point represents the Pi-uptake rate into the pooled BBMVs prepared from 4 animals. The increase in Na+-dependent Pi uptake was significant only for 2-day acidosis.

 


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Fig. 3. Metabolic acidosis increased intestinal NaPi-IIb-cotransporter protein abundance. A: NaPi-IIb protein abundance was assessed in brush border membrane preparations from total small intestine of the same groups as analyzed for Pi uptake and compared with actin abundance. NaPi-IIb and {beta}-actin were tested on the same Western blot membranes. B: ratio of NaPi-IIb to {beta}-actin increased strongly in animals with metabolic acidosis for 2 days and showed a stronger variance in animals with 7 days metabolic acidosis corresponding to varying Pi-transport rates. The increase in NaPi-IIb protein abundance was significant for both periods of treatment and did not differ between days 2 and 7.

 
In mouse intestine, NaPi-IIb is mainly found in the ileum, and adaptation to low-Pi diet occurs predominantly in this segment (see Ref. 27a). To test whether metabolic acidosis affects this segmental localization and regulation of NaPi-IIb abundance, real-time PCR and Western blot analysis were performed on mucosa harvested from jejunum and ileum. NaPi-IIb mRNA (Fig. 4A) and protein abundance (Fig. 4B) were several fold higher in ileum than in jejunum under all conditions. Induction of metabolic acidosis did not significantly alter NaPi-IIb mRNA levels both in jejunum and ileum but increased NaPi-IIb protein abundance strongly in ileum (Fig. 4). In addition, immunohistochemistry was performed on samples that had been collected every 5 cm from the stomach pylorus to the cecum. NaPi-IIb-related immunostaining was observed as described previously on the apical side of enterocytes in the ileum. Only a very weak signal was seen in the jejunum and a stronger signal in the duodenum, with some but not all enterocytes being positive for NaPi-IIb staining. No change in this segmental distribution was observed after 2 or 7 days of metabolic acidosis; the intensity of the NaPi-IIb signal, however, appeared to be stronger after 2 and 7 days of metabolic acidosis (Fig. 5).



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Fig. 4. Relative NaPi-IIb mRNA and protein abundance in jejunum and ileum. A: real-time PCR was used to assess NaPi-IIb mRNA levels in the mucosa of jejunum and ileum from control and acidotic animals (n = 3) and related to mRNA levels of {beta}-actin. No change in the relative abundance of NaPi-IIb could be detected, but NaPi-IIb mRNA levels were much higher in ileum. B: Western blot analysis of brush border membrane. Seventy micrograms of brush border membrane from jejunum or 35 µg of BBM from ileum were loaded. In jejunum, a very faint signal for NaPi-IIa could be detected after 2 days of metabolic acidosis. In ileum, NaPi-IIb protein abundance was increased 2.3-fold after 2 days of metabolic acidosis. All membranes were also stained for {beta}-actin to control for loading.

 


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Fig. 5. Segmental distribution of NaPi-IIb localization in small intestine of control and acidotic animals. Samples were taken every 5 cm starting from stomach pylorus to cecum, and immunohistochemistry for NaPi-IIb was performed. Shown here are representative examples from duodenum (0 cm from pylorus), jejunum (10 cm from pylorus), and ileum (35 cm from pylorus). In control animals, only a faint NaPi-IIb-related signal was detected in duodenum but not in jejunum. In the ileum, NaPi-IIb was localized on the apical side of enterocytes (35 cm). NaPi-IIb segmental distribution and localization in small intestines from acidotic animals was not altered, but the staining appeared to be enhanced in the ileum. All pictures were taken with identical camera settings.

 

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Metabolic acidosis is associated with several changes in metabolism aiming to restore acid-base homeostasis. These compensatory changes occur on different levels such as increasing respiration (exhalation of CO2), increasing renal elimination of acid equivalents (i.e., excretion of H+ and titratable acids, stimulation of HCO3 reabsorption), liberating carbonate and phosphate from bone, and stimulating bicarbonate reabsorption from the small and large intestine (11, 15, 16, 23, 24). Pi plays a key role in the compensation of metabolic acidosis, both as a buffer in blood and as a titratable acid in urine. In the kidney, the fractional excretion of phosphate is increased, an effect that has been attributed to downregulation of the major renal Na+-Pi cotransporter NaPi-IIa (1). Our data confirm the strong increase in the fractional urinary excretion of phosphate during metabolic acidosis. However, no significant reduction in Na+-dependent phosphate transport in isolated BBMVs and in BBM abundance of NaPi-IIa could be detected in three independent groups of mice. Because this result was in clear contrast to previously published data, we have tested the same treatment also with rats where a significant reduction in Na+-dependent Pi uptake into kidney BBMVs was found after 2 days of acidosis. Thus the observed discrepancy could be due to a species difference (mouse vs. rat). However, this was not further investigated because it was beyond the scope of this project.

Intestinal phosphate absorption is closely linked to the expression of the Na+-Pi cotransporter NaPi-IIb (see Refs. 12, 18, 21, 26, 27a, 31). Our data demonstrate that metabolic acidosis leads to an increase in intestinal Na+-dependent Pi uptake due to an increased abundance of NaPi-IIb protein in the BBM of the small intestine. This adaptation occurs in the ileum, as shown by immunohistochemistry, and does not alter the segmental distribution of NaPi-IIb protein localization. Furthermore, the increase in NaPi-IIb protein abundance is not associated with an increase in NaPi-IIb mRNA abundance in the small intestine, as evident from real-time PCR. In contrast, adaptation to a low-Pi-containing diet leads to stimulation of Na+-dependent Pi uptake and is associated with an increase in both NaPi-IIb mRNA and protein abundance (see Ref. 27a). This suggests that the mechanisms by which NaPi-IIb protein abundance is increased during adaptation to either low-Pi intake or metabolic acidosis may differ. The signal(s) mediating intestinal adaptation of Pi uptake may involve hormones such as vitamin D3, which has been shown to stimulate intestinal Pi uptake (6, 13, 17, 21).

Adaptation of intestinal NaPi-IIb expression during acidosis was strongest after 2 days of acidosis. At this time point, serum Pi levels were lower than in the control group and the 7-day acidosis group. However, it is unclear whether this small fall in serum Pi levels is enough to serve as a stimulus for intestinal adaptation, because much lower serum Pi levels during dietary Pi reduction have been found, which in turn stimulated NaPi-IIa or NaPi-IIb expression (19). At the moment, it remains to be examined whether such a small fall in serum Pi levels stimulates NaPi-IIb protein expression without elevating mRNA levels. The fact that after 7 days of NH4Cl loading, only little adaptation of NaPi-IIb was found may also be due to the less severe acidosis and/or development of diarrhea in this group.

Interestingly, metabolic acidosis resulted in a strong increase in fractional Pi excretion with urine, which was paralleled by no change in Na+-dependent Pi uptake and NaPi-IIa expression in BBMVs, suggesting alternative mechanisms of renal Pi excretion or inactivation of NaPi-IIa in the BBM in vivo. In fact, a recent report (20) has shown that NaPi-IIa may be inactivated due to a change in lateral mobility in the plasma membrane during systemic K+ deprivation.

Serum Pi levels during metabolic acidosis fell only minimally despite massive renal loss of Pi pointing to compensatory mechanisms. Because serum Pi is in balance with Pi in other body compartments such as bone or muscle and determined by intestinal uptake as well as by renal loss, it is difficult to identify the source of Pi contributing to the maintenance of serum Pi levels. Release of Pi from bone has been demonstrated during metabolic acidosis and contributes to systemic serum Pi levels under this condition (23). The functional consequence of stimulated intestinal Na+-dependent Pi uptake for systemic Pi levels may be severalfold. Increased intestinal Pi availability may provide Pi for buffering of acid equivalents during renal excretion or may prevent excessive liberation of Pi from bone. The latter would help to reduce the massive bone loss that is associated with chronic metabolic acidosis and represents one of the major clinical problems (23).

In summary, our results demonstrate a strong stimulation of Na+-dependent Pi uptake in the BBM of small intestine of acidotic animals and an increase of NaPi-IIb protein abundance. Increased Pi uptake and delivery may support the compensation of metabolic acidosis and be part of a concerted regulation of mechanisms aiming to restore normal acid-base balance.


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This study was supported by Swiss National Foundation Grants 31–65397.01 (to H. Murer), 31–61438.00 (to J. Biber), and 31–68318.02 (to C. A. Wagner).


    FOOTNOTES
 

Address for reprint requests and other correspondence: Carsten A. Wagner, Institute of Physiology, Univ. of Zurich, Winterthurerstr. 190, CH-8057 Zurich, Switzerland (E-mail: Wagnerca{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.

* A. Stauber and T. Radanovic contributed equally to this study. C. A. Wagner and J. Biber equally share last authorship. Back


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