Regulation of rat intestinal Na-dependent phosphate transporters by dietary phosphate

Abstract

Hyperphosphatemia associated with chronic kidney disease is one of the factors that can promote vascular calcification, and intestinal P(i) absorption is one of the pharmacological targets that prevents it. The type II Na-P(i) cotransporter NaPi-2b is the major transporter that mediates P(i) reabsorption in the intestine. The potential role and regulation of other Na-P(i) transporters remain unknown. We have identified expression of the type III Na-P(i) cotransporter PiT-1 in the apical membrane of enterocytes. Na-P(i) transport activity and NaPi-2b and PiT-1 proteins are mostly expressed in the duodenum and jejunum of rat small intestine; their expression is negligible in the ileum. In response to a chronic low-P(i) diet, there is an adaptive response restricted to the jejunum, with increased brush border membrane (BBM) Na-P(i) transport activity and NaPi-2b, but not PiT-1, protein and mRNA abundance. However, in rats acutely switched from a low- to a high-P(i) diet, there is an increase in BBM Na-P(i) transport activity in the duodenum that is associated with an increase in BBM NaPi-2b protein abundance. Acute adaptive upregulation is restricted to the duodenum and induces an increase in serum P(i) that produces a transient postprandial hyperphosphatemia. Our study, therefore, indicates that Na-P(i) transport activity and NaPi-2b protein expression are differentially regulated in the duodenum vs. the jejunum and that postprandial upregulation of NaPi-2b could be a potential target for treatment of hyperphosphatemia.

Fig. 1.

Expression profile of Na-P_i transporter activity, protein, and mRNA along the small intestine of rats (n = 6) fed the 0.6% P_i diet ad libitum. A: brush border membrane (BBM) vesicle (BBMV) Na-dependent P_i uptake activity in small intestinal segments. B: BBMV expression of NaPi-2b and PiT-1 protein showing specific signal in duodenum and jejunum, but not ileum, of rats; 20 μg of protein were loaded per well. C: type II (NaPi-2b) and type III (PiT-1, PiT-2) Na-P_i transporter mRNA abundance determined by quantitative PCR in rats. AU, arbitrary units. *P < 0.05. **P < 0.01. ***P < 0.001.

Fig. 2.

Immunofluorescence microscopy of rat small intestine sections showing NaPi-2b expression on apical membrane of enterocytes. NaPi-2b signal is weak in duodenum (0–10 cm), intense in jejunum (20–40 cm), and negligible in ileum (50–80 cm). Sections (0.5 cm) were obtained from rats that were chronically fed the low-P_i (0.1%) diet, fixed with methanol, stained with anti-NaPi-2b antibody, and examined at 10-cm intervals along the small intestine.

Fig. 3.

Effects of dietary P_i on intestinal and renal BBMV P_i transport activity. Na-P_i transporter uptake was measured in BBMV of duodenum (A) and jejunum (B) of rats chronically fed high-P_i (1.2%), normal-P_i (0.6%), or low-P_i (0.1%) diet. P_i uptake in BBMV from jejunum was increased 67% in rats fed the low-P_i diet compared with rats fed the high-P_i diet (59.6 ± 14.2, 46.3 ± 11.4, and 123.9 ± 14.3 pmol P_i·mg protein⁻¹·30 s⁻¹ for rats fed 1.2%, 0.6%, and 0.1% P_i, respectively). No significant regulation is observed in BBMV from duodenum. C: progressive upregulation of phosphate uptake in BBMV isolated from kidney superficial cortex (SC-BBMV) in rats fed normal-P_i and low-P_i diet compared with rats fed high-P_i diet. *P < 0.05. **P < 0.01. ***P < 0.001.

Fig. 4.

BBMV Na-P_i protein regulation in rats chronically fed high-, normal-, or low-P_i diet. BBMV NaPi-2b and PiT-1 protein abundance was determined by Western blot analysis in duodenum (A) and jejunum (B). NaPi-2b protein abundance is markedly and significantly increased in response to chronic low-P_i diet in jejunal BBMV. There is a gradual increase in duodenal BBMV NaPi-2b abundance that did not reach statistical significance. C: upregulation of NaPi-2a, NaPi-2c, and PiT-2 protein abundance in kidney SC-BBMV in response to chronic low-P_i diet. Densitometry analysis was performed for each section; results were normalized to actin expression. ***P < 0.001.

Fig. 5.

Intestinal Na-P_i mRNA regulation in rats chronically fed high- or low-P_i diet. In jejunum, NaPi-2b mRNA abundance is increased in parallel with increases in BBM NaPi-2b protein abundance and BBM P_i uptake in response to chronic low-P_i diet. There are no changes in jejunal PiT-1 mRNA or duodenal NaPi-2b or PiT-1 mRNA abundance in response to low-P_i diet. *P < 0.05.

Fig. 6.

Immunofluorescence microscopy of NaPi-2b protein in intestinal sections corresponding to rat jejunum (30–50 cm). Abundance of NaPi-2b protein is increased in apical membrane of enterocytes in rats chronically fed low-P_i diet. Sections were embedded in optimal cutting temperature compound, fixed with methanol, and stained with anti-NaPi-2b antibody.

Fig. 7.

Effects of dietary P_i on serum P_i levels. A: serum P_i levels of rats chronically fed high-, normal-, or low-P_i diet. Serum P_i concentration is significantly lower in rats chronically fed low-P_i diet than in rats fed high- or normal-P_i diet. B: serum P_i concentration is markedly increased (to levels as high as 16 mg/dl) in rats chronically fed low-P_i diet when they are acutely fed high-P_i diet as early as 2 h after the high-P_i diet. There are no significant changes in serum P_i concentrations between rats chronically low-P_i diet and those trained to eat a low-P_i diet for 4 h/day for 7 days and then acutely fed the same low-P_i diet (chronic low-P_i diet to acute low-P_i diet). **P < 0.01. ***P < 0.001.

Fig. 8.

Na-dependent P_i uptake in BBMV of rats chronically adapted to a low-P_i diet and then fed low- or high-P_i diet for 4 h. Duodenal and jejunal BBMV show an unexpected reverse adaptation to the change from low- to high-P_i diet compared with renal BBMV. Duodenal and jejunal BBMV demonstrate an increased Na-P_i transport activity, whereas kidney superficial cortex BBMV (SC BBMV) show a decreased Na-P_i transport activity, in response to an acute challenge with high-P_i diet. **P < 0.01. ***P < 0.001.

Fig. 9.

Duodenal (A), jejunal (B), and renal (C) BBMV Na-Pi protein regulation in rats chronically adapted to low-P_i diet and then fed low- or high-P_i diet for 4 h. There is a significant increase in duodenal BBMV NaPi-2b protein abundance. No significant changes in NaPi-2b protein expression in jejunal BBMV or duodenal and jejunal PiT-1 protein expression were observed. In kidney superficial cortex BBMV, there is a marked and significant decrease in NaPi-2a protein abundance, whereas there are no changes in NaPi-2c or PiT-2 protein abundance in response to acute high-P_i diet for 4 h. *P < 0.05. ***P < 0.001.

Fig. 10.

NaPi-2b and PiT-1 mRNA regulation in rats chronically adapted to low-P_i diet and then fed low- or high-P_i diet for 4 h. There are no significant changes in NaPi-2b or PiT-1 mRNA abundance in response to acute (4 h) changes in dietary P_i.