Glucocorticoid regulation and glycosylation of mouse intestinal type IIb Na-Pi cotransporter during ontogeny

Kayo Arima, Eric R. Hines, Pawel R. Kiela, Jason B. Drees, James F. Collins, and Fayez K. Ghishan

Departments of Pediatrics, Physiology, and Nutritional Sciences, Steele Memorial Children's Research Center, University of Arizona Health Sciences Center, Tucson, Arizona 85724


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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We sought to characterize expression of an apically expressed intestinal Na-Pi cotransporter (Na-Pi-IIb) during mouse ontogeny and to assess the effects of methylprednisolone (MP) treatment. In control mice, Na-Pi uptake by intestinal brush-border membrane vesicles was highest at 14 days of age, lower at 21 days, and further reduced at 8 wk and 8-9 mo of age. Na-Pi-IIb mRNA and immunoreactive protein levels in 14-day-old animals were markedly higher than in older groups. MP treatment significantly decreased Na-Pi uptake and Na-Pi-IIb mRNA and protein expression in 14-day-old mice. Additionally, the size of the protein was smaller in 14-day-old mice. Deglycosylation of protein from 14-day-old and 8-wk-old animals with peptide N-glycosidase reduced the molecular weight to the predicted size. We conclude that intestinal Na-Pi uptake and Na-Pi-IIb expression are highest at 14 days and decrease with age. Furthermore, MP treatment reduced intestinal Na-Pi uptake approximately threefold in 14-day-old mice and this reduction correlates with reduced Na-Pi-IIb mRNA and protein expression. We also demonstrate that Na-Pi-IIb is an N-linked glycoprotein and that glycosylation is age dependent.

methylprednisolone; development; Na-Pi-IIb


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Pi HOMEOSTASIS IS PARTIALLY maintained by type II Na-Pi cotransporters, which include types IIa and IIb. Na-Pi-IIa is expressed in apical membranes of epithelial cells in the renal proximal tubules, and it represents the major Na-Pi cotransporter in the kidney. The Na-Pi-IIa cotransporter is regulated by several physiological effectors, including glucocorticoids (GCs) (18), epidermal growth factor (EGF) (2, 3), parathyroid hormone (26, 32, 37), thyroid hormone (1, 41), vitamin D3 (42), and dietary phosphate (11, 15, 25, 28, 32, 44, 46). Meanwhile, the Na-Pi-IIb cotransporter is expressed in several tissues, including the brush-border membranes (BBMs) of the small intestinal epithelium, where it is thought to be the major Na-Pi cotransporter (19, 24). Similar to Na-Pi-IIa, intestinal Na-Pi-IIb is also regulated by EGF (47) and by vitamin D3 and dietary phosphate (19, 27). Both isoforms have similar functional properties, and they show striking homology in their amino acid sequences with eight predicted transmembrane domains and a large hydrophilic loop between the third and forth transmembrane domains. The hydrophilic loop in the renal type IIa protein contains two N-linked glycosylation sites located at Asn-298 and Asn-328, and glycosylation of these sites is likely important for plasma membrane expression (20).

Genes encoding these proteins have very distinct 5'-flanking regions that include functional promoters (4). Thus their gene expression is likely controlled by different transcriptional mechanisms. For example, renal Na-Pi-IIa gene expression is enhanced by vitamin D3 administration and a low-phosphate diet (28, 42). In contrast, these treatments posttranscriptionally increase intestinal Na-Pi-IIb protein abundance and Na-Pi transport activity but do not alter Na-Pi-IIb mRNA expression (19, 27).

Age-related plasma Pi levels may result from decreased renal and intestinal Pi absorption (5). In intestine, Na-Pi absorption across the apical and endoplasmic reticulum membranes of enterocytes is higher in suckling animals than in young adult animals (7, 9, 17). Conversely, renal Na-Pi cotransport is lowest in suckling rats, highest in weanling rats, and declines with age (43). Furthermore, the age-related decline in renal Pi absorption is likely due to lower Na-Pi-IIa expression (1, 18, 43). However, a correlation between ontogeny of intestinal Na-Pi cotransport and expression of the Na-Pi-IIb cotransporter has not yet been documented.

During the suckling/weaning transition in rats and mice, the intestinal mucosa rapidly matures in conjunction with the dietary change from milk to carbohydrate-based solid food. Skeletal growth is also most rapid in this period. Thus it is not surprising that the suckling rat intestine shows elevated absorptive capacities for a number of nutrients, including phosphate (21).

Among physiological factors involved in intestinal maturation, GCs are the most potent regulators (21). Indeed, direct measurement of rat plasma concentration of endogenous GCs demonstrated that total plasma corticosterone, the principal GC in rats and mice, was <0.5 µg/ml on days 6-12 (corresponding to the suckling period), rose to 5 µg/ml on days 17-24 (corresponding to the weaning period), and then gradually decreased into adulthood (22).

Exogenous GC administration induces precocious intestinal maturation in the first and second postnatal weeks by modulating gene expression, membrane fluidity, and patterns of protein glycosylation (6, 10, 16, 23, 35, 45). Meanwhile, adrenalectomy and GC antagonists delay intestinal maturation (21). Clinical observations showed that chronic GC administration enhances urinary Pi excretion and reduces plasma Pi levels in patients (29, 30). Pharmacological doses of GCs have also been reported to reduce renal and intestinal Pi absorption and renal Na-Pi-IIa expression (9, 18). Furthermore, long-term GC treatment increases the risk of bone demineralizaion in children and is known to increase the risk of developing osteoporosis in adults (16, 36). Although GCs appear to play a crucial role in regulating intestinal Pi absorption, the effects of GCs on Na-Pi-IIb expression have not been addressed.

The purpose of the current studies was to investigate molecular mechanisms involved in intestinal Na-Pi-IIb expression during postnatal development and to look at the effect of GC administration. Our results showed that Na-Pi-IIb is differentially expressed during mouse ontogeny. We further demonstrate that Na-Pi-IIb expression is decreased by GC treatment, with the effect being most pronounced in the suckling period. Additionally, we provide evidence that Na-Pi-IIb is an N-linked glycoprotein and that glycosylation occurs around the time of the suckling/weaning transition.


    MATERIALS AND METHODS
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Animals. Male C57BL/6 mice (Jackson Laboratory, Bar Harbor, ME) were used in groups of four to six animals for all experiments. Age groups were as follows: suckling mice at 14 days of age, weanling mice at 21 days of age, young adult mice at 8 wk of age, and old adult mice at 8-9 mo of age. Animals were subcutaneously injected with methylprednisolone (MP; Solu-Medrol; Upjohn, Kalamazoo, MI) at a dose of 30 µg/g body wt or equal volumes of saline once every 12 h for a total of four injections. The animals were killed by cervical dislocation 3 h after the last injection after CO2 narcosis. Animals were supplied with food and water ad libitum. The proximal two-thirds of the small intestine was used for all experiments. All animal procedures were approved by the University of Arizona Institutional Animal Care and Use Committee.

Chemicals and reagents. Total RNA was isolated from tissues using TRIzol reagent (GIBCO-BRL, Bethesda, MD). KH232PO4 (1 Ci/nmol) for uptake studies and [alpha -32P]dATP (3,000 Ci/mmol) for Northern blot analyses were purchased from New England Nuclear (Boston, MA). Strip-EZ PCR kit, used to generate antisense, radioactive probes for Northern blot analyses and Ultrahyb buffer were from Ambion (Austin, TX). Nitrocellulose membranes (Nitroplus) were from Osmonics (Westboro, MA). SuperSignal chemiluminescent regent, Western blot stripping buffer and X-ray film were from Pierce (Rockford, IL). SDS-PAGE precast gels and protein molecular weight standards were from Bio-Rad (Hercules, CA). Horseradish peroxidase-linked secondary antibodies (anti-rabbit IgG) were from Amersham (Piscataway, NJ). Mouse anti-beta -actin monoclonal antibody was purchased from Sigma (St. Louis, MO). Plasmid DNA was isolated from bacterial cultures with the QIAfilter Plasmid Maxi Prep kit, and DNA fragments were gel purified by the use of Qiaquick gel extraction kit from Qiagen (Valencia, CA). Protein was quantitated by using Bio-Rad protein assay reagent. Peptide N-glycosidase F (PNGase F) and endoglycosidase H (Endo H) were from New England BioLabs (Beverly, MA). Herculase proofreading, high-temperature DNA polymerase was from Stratagene (San Diego, CA). Taq polymerase and restriction enzymes were from Promega (Madison, WI). All other chemicals and reagents were purchased from Fisher Biotechnology (Pittsburgh, PA) or Sigma.

BBM and basolateral membrane vesicle preparations. BBM vesicles were prepared from the intestinal mucosa from groups of five to six animals for 14- and 21-day-old mice and from groups of four to five animals for 8-wk-old and 8- to 9-mo-old mice, by the MgCl2 precipitation technique, as previously described (33, 34). Additionally, basolateral membrane (BLM) vesicles were isolated from 8-wk-old mice by a well-established method in our lab (14). The final BBM and BLM vesicle pellets were resuspended in resuspension buffer (280 mM mannitol, 20 mM HEPES-Tris, pH 7.4) and homogenized with a 26-gauge needle, and protein was quantitated. For uptake analyses, BBM preps were used on the day of purification and were never frozen.

Uptake analysis of intestinal BBM vesicles. 32PO<UP><SUB>4</SUB><SUP>2−</SUP></UP> transport was measured by a rapid filtration technique as described previously (11-14, 18). The Na-dependent component of Pi uptake was obtained by subtracting uptake values in the presence of KCl from uptake values in the presence of NaCl. Values are means ± SE for each age group and represent the results of three separate experiments with samples isolated from different groups of animals.

Northern blot analysis. Total RNA was isolated from at least four mice per group. To generate Na-Pi-IIb-specific probes, the Na-Pi-IIb open reading frame was amplified from mouse intestinal RNA by RT-PCR with sense primer at 15 bp (15L; 5'-TGTCCCTTACCTGCGA-3') and antisense primer at 2,264 bp (2264R; 5'-TAGGAGAGCTAGAGTTGGTG-3') in mouse Na-Pi-IIb cDNA (GenBank accession no. AF081499) using a high-fidelity DNA polymerase. PCR products were A-tailed, subcloned into the pCR 3.1-Uni vector (Invitrogen, Carlsbad, CA), and transformed into Escherichia coli DH-5alpha . The entire amplicon was confirmed by sequence analysis. Then, plasmid DNA was digested with NheI and EcoRI, and the fragment containing the entire Na-Pi-IIb open reading frame was gel purified and used as the template to make mouse Na-Pi-IIb-specific probes. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) template was also amplified from mouse intestinal RNA by RT-PCR with sense primer at 563 bp (563L; 5'-ATGACCACAGTCCATGCCAT-3') and antisense primer at 832 bp (832R; 5'-CTGCTTCACCACCTTCTTGA-3') in mouse GAPDH cDNA (GenBank accession no. NM 008084). The PCR fragment was gel purified and confirmed by sequence analysis.

Antisense probes were synthesized by the use of [alpha -32P]dATP and Na-Pi-IIb template and 2264R primer, or mouse GAPDH template with 763R primer (5'-CAGTGAGCTTCCCGTTCA-3'). Both radioactive probes were purified using G-50 columns (Bio-Rad). Northern blots were carried out according to the manufacturer's protocol (Ambion). In preliminary experiments, it was noted that expression of Na-Pi-IIb mRNA in 14-day-old mice was much higher than in older mice and was severely reduced with MP treatment. Furthermore, Na-Pi-IIb hybridization bands in older mice (i.e., 8 wk and 9 mo) were not visible when 10 µg of total RNA was loaded, whereas those in suckling mice exceeded the linear range of phosphorimage analysis. Therefore, to exemplify the band in each age group, it was necessary to load variable amounts of total RNA (10-40 µg) per lane, resulting in different apparent intensities of the reference mRNA band (GAPDH). This unequal loading was corrected by taking ratios between Na-Pi-IIb and GAPDH hybridization band intensities. Quantitation of hybridization signals was done by phosphorimage analysis by the use of volume integration (FX molecular image; Bio-Rad) or by scanning densitometry (GS-700 imaging densitometer; Bio-Rad). The Northern blot experiment was performed three times with total RNA samples isolated from different groups of animals. Na-Pi-IIb hybridization intensities were normalized for GAPDH levels on the same blot and the ratios were averaged.

Production of Na-Pi-IIb-specific antiserum. A multiple-antigen peptide corresponding to the COOH-terminal region of the mouse Na-Pi-IIb protein was synthesized by Research Genetics (Huntsville, AL). The antigenic peptide is 95% homologous to the rat Na-Pi-IIb protein (GenBank accession no. AAF76291). This region of the molecule showed no amino acid sequence homology to any other proteins in GenBank. The peptide was injected into rabbits for polyclonal antibody production (Research Genetics). Antibody specificity was assessed by Western blots using antiserum, preimmune serum, or immunogenic peptide-pretreated serum, and also by immunohistochemical analyses (as described below).

Western blot analysis of mouse intestinal BBM proteins with Na-Pi-IIb-specific antiserum. Intestinal BBM and BLM proteins were purified from mice and rats as described. Protein (20-60 µg) was placed in a twofold excess of Laemmli solubilization buffer (2% SDS, 10% glycerol, 1 mM EDTA, and 2 mM beta -mercaptoethanol, pH 6.8), boiled for 4 min, and placed on ice. Protein samples were fractionated by 7.5% SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were blocked for 1 h in PBS with 0.05% Tween 20 (PBST) and 10% nonfat dry milk, rinsed with PBST-0.1% milk, and incubated overnight at 4°C with a 1:500 dilution of Na-Pi-IIb-specific antiserum in PBST-0.1% milk. Additionally, some blots were reacted with preimmune serum, and serum that was pretreated with antigenic peptide (1,000 µg/ml at 4°C overnight) at 1:500 dilution. The membranes were washed with PBST-0.1% milk two times for 5 min each and then incubated with the secondary antibody (anti-rabbit IgG) at 1:20,000 dilution for 40 min. Finally, the membranes were washed with PBST-0.1% milk four times for 15 min each, reacted with chemiluminescence reagent for 5 min, and then exposed to film. Membranes were stripped and subsequently reacted with beta -actin antiserum at a 1:5,000 dilution. Na-Pi-IIb-specific band intensities were determined by densitometric analysis and were normalized for beta -actin band intensities on the same blot. This experiment was repeated three times with protein samples isolated from different groups of animals.

Immunohistochemical analysis of rat intestine with Na-Pi-IIb-specific antiserum. Proximal intestinal tissue was harvested from a 3-wk-old rat, fixed in paraformaldehyde, and embedded in paraffin, and sections were cut and affixed to slides. After deparrafinization, slides were blocked by overnight incubation with 5% normal goat serum (Vector Labs, Burlingame, CA) at room temperature in a humidified chamber. Na-Pi-IIb antiserum was reacted with sections for 30 min at a 1:250 dilution in PBS. Some sections were reacted with secondary antibody only at a 1:400 dilution. Slides were subsequently reacted with secondary antiserum (Alexa Fluor 568 goat anti-rabbit IgG; Molecular Probes, Eugene, OR) at a 1:400 dilution of the company stock solution (2 mg/ml) and visualized by confocal microscopy (MRC-1024ES laser scanning confocal; Bio-Rad) equipped with a Nikon TE-300 research grade microscope using the HQ-598-40 emission filter and an excitation wavelength of 568 nm. All slides were visualized with the exact same confocal settings.

Prediction of glycosylation sites in Na-Pi-IIb. A database search for N-linked (N-X-S/T) and O-linked glycosylation sites (GGS/T) was performed with the mouse Na-Pi-IIb protein sequence. Also, the largest extracellular loop of mouse Na-Pi-IIb was aligned with mouse Na-Pi-IIa (GenBank accession no. AAC42026) and Na-Pi-IIb cloned from other species including rat (AAF76291), human (AAF31328), bovine (S49228), chicken (AAG35801), carp (AAG35803), dogfish (AAG35795), flounder (AAB16821), rainbow trout (AAG35798), zebrafish (AAG35356), and little skate (AAG35797). Single and multiple amino acid alignments were performed with the GenBank BLASTP (http://www.ncbi.nlm.nih.gov/BLAST), DNAMAN (Lynnon BioSoft, Vaudreuil, Quebec, Canada), and LALIGN (http://fasta.bioch.virginia.edu/fasta/lalign.htm) computer programs.

Treatment of mouse intestinal BBMs with glycosidases. Mouse intestinal BBM protein (20-60 µg) isolated from 2- and 8-wk-old mice was mixed with 1 µl of denaturing buffer (5% SDS and 10% beta -mercaptoethanol) and boiled for 10 min. Samples were subsequently incubated with 5.00 U of PNGase F or Endo H for 2 h at 37°C. Digested proteins were fractionated by 7.5% SDS-PAGE and analyzed by immunoblotting with Na-Pi-IIb-specific antiserum.

Statistical analysis of results. Data were analyzed for statistical significance by Student's t-tests or ANOVA followed by Fisher's protected least significant difference post hoc test by using the StatView software package (version 4.53; SAS Institute, Cary, NC). Data are expressed as means ± SE.


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Uptake analysis of intestinal BBM vesicles. Intestinal BBM vesicles were prepared from animals treated with MP or saline at various ages. Uptake of radioactive phosphate was measured by a rapid filtration technique with or without sodium at pH 7.4. In control animals (saline treated), Na-Pi uptake (in nmol · mg protein-1 · 10 s-1) was highest in 14-day-old mice (1.63 ± 0.43; n = 3 for all groups) compared with the other three age groups (P < 0.0001 for 14 days vs. other ages). Uptake levels diminished with age until adulthood as indicated by the higher level in 21-day-old animals (0.24 ± 0.02) than in 8-wk-old (0.09 ± 0.05; P < 0.001) and 8- to 9-mo-old (0.04 ± 0.03; P < 0.001) mice. No significant difference in uptake was present between the 8-wk-old and 8- to 9-mo-old mice. To determine the age-specific response to GCs on intestinal Na-Pi uptake, MP-injected mice were utilized for uptake analysis and compared with saline injected (control) animals. MP administration significantly decreased intestinal Na-Pi uptake in 14-day-old (1.63 ± 0.43 control and 0.48 ± 0.1 MP; P < 0.05) and 21-day-old (0.24 ± 0.02 control and 0.04 ± 0.02 MP; P < 0.05) mice but had no effect in older animals (0.09 ± 0.05 control and 0.05 ± 0.04 MP in 8-wk-old mice; 0.04 ± 0.03 control and 0.10 ± 0.03 MP in 8- to 9-mo-old mice). Data are expressed graphically in Fig. 1.


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Fig. 1.   Effect of methylprednisolone (MP) on Na-Pi uptake. Brush border membrane vesicles (BBMVs) were isolated from intestinal mucosa of suckling (14-day-old), weanling (21-day-old), young (8-wk-old), and old (adult; 8- to 9-mo-old) mice and assayed for Na-Pi uptake at 10 s (initial rate conditions). The Na-dependent component of Pi uptake was obtained by subtracting uptake in the presence of KCl from uptake in the presence of NaCl. Values are means ± SE of 3 experiments. C, control (saline injected). P < 0.0001 for a vs. b and a vs. c; P < 0.0001 for b vs. c. * P < 0.05 between control and MP-treated mice at that age.

Northern blot analysis. Total RNA was isolated from intestine of MP- or saline-injected mice from each age group, fractionated on denaturing agarose gels, and transferred to nylon membranes. Blots were hybridized with radiolabeled antisense Na-Pi-IIb- and GAPDH-specific probes. Na-Pi-IIb hybridization levels were quantitated by densitometry and normalized for GAPDH levels (Fig. 2). Intestinal Na-Pi-IIb hybridization levels in 14-day-old suckling mice (7.35 ± 1.66 random densitometric units) were approximately threefold higher than in 21-day-old weanlings (2.53 ± 1.15) and older mice (8-wk-old = 2.22 ± 1.28, 8- to 9-mo-old = 1.71 ± 0.24; P < 0.0001 for 14-day-old vs. other groups). MP administration significantly decreased Na-Pi-IIb hybridization levels in 14-day-old (7.35 ± 1.66 control vs. 2.02 ± 1.55 MP; P < 0.05), 21-day-old (2.53 ± 1.15 control vs. 0.44 ± 0.09 MP; P < 0.05), and 8- to 9-mo-old (1.71 ± 0.24 control vs. 0.71 ± 0.26 MP; P < 0.05) animals. The difference in 8-wk-old mice, however, was not significant (2.22 ± 1.28 control vs. 1.00 ± 0.42 MP).


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Fig. 2.   Effect of MP on Na-Pi-IIb mRNA expression in suckling, weaning, young, and old adult mice. Total RNA was isolated from the intestine of groups of mice, fractionated by denaturing agarose gel electrophoresis and transferred onto nitrocellulose membranes. Different amounts of total RNA (10-40 µg) were loaded per lane because band intensities were highly variable between groups (see MATERIALS AND METHODS). A: Na-Pi-IIb hybridization band is shown at ~4 kb and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) hybridization band is shown at ~1.2 kb. B: graphical representation of data obtained from 3 independent Northern blot experiments. Values are means ± SE. P < 0.0001 between a and b; * P < 0.05 between control and MP-treated mice at that age.

Characterization of Na-Pi-IIb-specific antiserum. Polyclonal antibodies were developed against a mouse Na-Pi-IIb-specific peptide. Intestinal BBM and BLM proteins were purified from 8-wk- and 14-day-old mice. Additionally, BBM protein was isolated from 14-day-old rats. Western blot analyses with mouse BBM protein and the immune serum resulted in a single band at ~110 kDa, which is consistent with a previous report (24) (Fig. 3A). No band was present on blots with preimmune serum or with the serum pretreated with the antigenic peptide. Furthermore, no band was present on blots of mouse intestinal BLM proteins using the Na-Pi-IIb-specific antiserum. In addition, the antiserum recognized an ~88 kDa band in 14-day-old mice and two major bands (~110 and ~78 kDa) in intestinal BBM protein from 14-day-old rats (Fig. 3B).


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Fig. 3.   Na-Pi-IIb antiserum characterization. Brush-border membrane (BBM) or basolateral membrane (BLM) proteins were isolated from intestinal mucosa in mice and rats. For immunohistochemistry, proximal intestinal tissue was harvested from a 3-wk-old rat. A: Western blot analysis of BBM proteins from 8-wk-old mice with Na-Pi-IIb-specific antiserum, preimmune serum, and antigenic protein-pretreated serum (blocked). B: Western blot analysis of BBM or BML proteins from mice and rats with Na-Pi-IIb-specific antiserum. C and D: immunohistochemical analysis of proximal intestine from a 3-wk-old rat. Magnification in both C and D is ×40. C: Na-Pi-IIb antiserum reaction. D: secondary antibody only.

To further exemplify the specificity of the immune serum, proximal intestinal tissue was reacted with Na-Pi-IIb-specific antiserum. Results showed specific staining of the apical membranes of enterocytes (Fig. 3C), whereas secondary antibody alone showed no staining.

Western blot analyses of intestinal BBM proteins with Na-Pi-IIb-specific antiserum. To determine the pattern of Na-Pi-IIb protein expression during ontogeny, 20-60 µg of BBM protein from MP- or saline- injected mice was used for Western blot analyses with Na-Pi-IIb-specific antiserum (Fig. 4). Na-Pi-IIb protein levels (presented as a Na-Pi-IIb/beta -actin ratio) were markedly higher in suckling animals (1.44 ± 0.29 random densitometric units) compared with the other age groups (21-day-old = 0.26 ± 0.16, 8-wk-old = 0.29 ± 0.09, 8- to 9-mo-old = 0.15 ± 0.11; P < 0.0001 for 2-wk-old vs. other groups). MP administration decreased Na-Pi-IIb protein levels more than threefold in 14-day-old mice (1.44 ± 0.29 control vs. 0.38 ± 0.17 MP; P < 0.05) but had no effect in 8-wk-old (0.29 ± 0.09 control vs. 0.29 ± 0.06 MP), and 8- to 9-mo-old animals (0.15 ± 0.11 control vs. 0.05 ± 0.03 MP). Although the data were not statistically significant, Na-Pi-IIb protein levels in 21-day-old mice appeared also to be downregulated by MP (0.26 ± 0.16 control vs. 0.11 ± 0.10 MP). Additionally, the molecular mass of the immunogenic band in 14-day-old mice was smaller than in the other age groups (~88 vs. ~110 kDa), and more than one band was present in MP-treated 14-day-old suckling mice.


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Fig. 4.   Effect of MP on Na-Pi-IIb protein abundance in suckling, weanling, young, and old adult mice. BBM proteins were purified from groups of mice, fractionated by SDS-PAGE, and transferred to nitrocellulose membranes. Blots were reacted with Na-Pi-IIb-specific antiserum at 1:500 dilution. A: Na-Pi-IIb bands are shown at ~110 or ~88 kDa, and the beta -actin band is shown at ~42 kDa. B: graphical representation of data obtained from 3 independent Western blot experiments. Values are means ± SE. P < 0.0001 between a and b; * P <0.05 between control and MP treated mice at that age.

Treatment of mouse intestinal BBMs with glycosidases. Different molecular weights of the immunoreactive bands of 14-day-old mice versus older mice suggested that the Na-Pi-IIb protein is differentially modified with age. Analysis of the mouse Na-Pi-IIb amino acid sequence identified six potential consensus sequences for N-linked glycosylation (N-X-S/T located at Asn-295, -308, -313, -321, -340, and -356) within a large putative extracellular loop. There were no putative O-linked glycosylation sites (G-G-S/T) identified. A GenBank BLASTP search found that the Na-Pi-IIb cDNA had been cloned from 11 species. Amino acid alignments indicated that three N-linked glycosylation sites at Asn-295, -313, and -340 are conserved in all known Na-Pi-IIb sequences and the site at Asn-321 is present in 9 of 11 species (Fig. 5). Furthermore, the putative sites at Asn-295 and -340 are conserved with the identified N-linked glycosylation sites in mouse Na-Pi-IIa (20).


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Fig. 5.   Multiple sequence alignment of mouse Na-Pi-IIa (GenBank accession no. AAC42026) and Na-Pi-IIb isoforms from several species. Amino acids associated with putative N-linked glycosylation sites are boxed, and the conserved amino acids are shaded. ** Conserved potential N-linked glycosylation sites in the Na-Pi-IIb family and in mouse Na-Pi-IIa. * Conserved potential N-linked glycosylation sites in the Na-Pi-IIb family.

To investigate the variation in molecular mass of the Na-Pi-IIb protein, we performed enzymatic deglycosylation studies. Treatment of BBM proteins with PNGase F decreased the molecular mass of immunoreactive Na-Pi-IIb from both 14-day-old (~88 kDa) and 8-wk-old mice (~110 kDa) to ~78 kDa, which is the calculated molecular mass based on the predicted amino acid sequence (Fig. 6). Digestion with Endo H had no apparent effect on the molecular mass of the protein.


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Fig. 6.   Analyses of N-glycosylation of the Na-Pi-IIb protein with peptide N-glycosidase F (PNGase F) and endoglycoside H (Endo H) using BBMs isolated from suckling and young adult mice. BBMs (40-60 µg) were incubated without enzyme or with PNGase F or Endo H for 2 h at 37°C. Proteins were then analyzed by Western blot analysis with Na-Pi-IIb-specific antiserum.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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Intestinal Na-Pi-IIb cotransporter likely plays a major role in the regulation of Pi homeostasis. Although Pi homeostasis is a tightly controlled process, lower plasma Pi levels have been observed in old age and in the case of chronic GC treatment (5, 30, 38). In both cases, decreased renal Pi absorption was reported and was associated with reduced Na-Pi-IIa expression, (1, 18, 43). To date, the downregulation of intestinal Pi absorption has not been investigated with respect to possible alterations in Na-Pi-IIb cotransporter expression, although reduced intestinal Pi absorption has been reported in old animals. We hypothesized that the age-related and GC-induced reductions of total intestinal Na-Pi transport would correlate with decreases in intestinal Na-Pi-IIb expression. Therefore, the present work focused on the ontogeny of intestinal Na-Pi uptake and Na-Pi-IIb expression at distinct periods throughout the lifespan and how pharmacological doses of GCs affect the same processes.

In laboratory animals, Na-Pi absorption across jejunal, apical, and endoplasmic reticular membranes of enterocytes was significantly greater in suckling (2-wk-old) rats compared with adolescent (6-wk-old) rats (7, 17). A study with rabbits at various ages (2, 4, and 6 wk and 3 mo) demonstrated similar results in which duodenal and jejunal Na-Pi uptake was greatest in 2-wk-old animals and severely decreased in older (3-mo-old) animals (9). Conversely, renal Na-Pi cotransport increases during the suckling/weaning transition and decreases with age. It is unclear why changes in intestinal and renal Pi (re)absorption during ontogeny are opposite during the suckling/weaning transition. Data from our laboratory previously demonstrated that renal Na-Pi cotransport was lowest in 2-wk-old rats, highest in 3-wk-old rats, and declined in 6-wk- and 4-mo-old rats (43). We further demonstrated that age-related decreases in Na-Pi uptake were paralleled by changes in Na-Pi-IIa protein levels, but no changes were seen in Na-Pi-IIa mRNA levels. Another study compared young (3 mo) and old (24 mo) adult rats and found that serum Pi concentrations, renal Na-Pi cotransport, and Na-Pi-IIa protein and mRNA abundance were markedly higher in younger than in older rats (1). Furthermore, age-related decreases in plasma Pi levels may result from decreased renal and intestinal Pi absorption (5). In the kidney, the age-related decline in renal Pi absorption is likely due to lower Na-Pi-IIa expression (1, 43); however, a correlation between ontogeny of intestinal Na-Pi cotransport and expression of the Na-Pi-IIb cotransporter has not yet been documented.

Total intestinal Na-Pi uptake in the current studies was highest in 14-day-old suckling mice, lower in 21-day-old weanlings, and lowest in older age groups. This observation agreed with previous reports that intestinal Na-Pi transport was higher in suckling than in adult rats and rabbits (7, 9, 17). Moreover, in the current studies, the decline in uptake activity was not constant with age, but instead was rapid (sevenfold) from 14- to 21-day-old mice and then gradually decreased (threefold) from weanling to young adult mice (8-wk-old). No difference was seen between the younger (8-wk-old) and older adult animals (8- to 9-mo-old). Therefore, the suckling/weaning transition seems to be a critical period for changes in intestinal Na-Pi transport activity.

Similar patterns of decline were observed in Na-Pi-IIb protein levels during the suckling/weaning transition. Na-Pi-IIb protein levels in the suckling mouse intestine were sixfold higher than in the weanling and adult groups. Thus the decrease in total intestinal Na-Pi uptake seems to parallel the decrease in Na-Pi-IIb protein expression. These results support the previous suggestion that Na-Pi-IIb protein is the major intestinal Na-Pi cotransporter. In addition to decreases in protein abundance, Na-Pi-IIb mRNA levels were also altered with age. Na-Pi-IIb mRNA in suckling mouse intestine was markedly higher than in the older groups, but the difference between suckling and older animals was only threefold. Because mRNA levels did not decrease as severely as uptake and protein levels, it is conceivable that part of the observed decreases in functional protein expression may be due to posttranscriptional mechanisms. Alternatively, the discrepancies may be due to the different methodologies used and their differential sensitivities.

To characterize Na-Pi-IIb-specific antiserum, we performed Western blots and immunohistochemical analyses. The antiserum detected Na-Pi-IIb protein in mouse BBM as a single band of ~110 kDa, which was consistent with a previous report (24). Before immunohistochemistry studies, rat Na-Pi-IIb protein was confirmed to be detectable with the antiserum on Western blots. Proximal intestine from a 3-wk-old rat was alternatively used for immunohistochemistry because of technical difficulties in obtaining intact tissue samples from young mouse intestine. Immunohistochemical analysis exemplified the specificity of the antiserum because it only reacted with the intestinal apical membranes.

In Western blot analyses from mice of different ages, the molecular mass of Na-Pi-IIb in suckling mice was lower (~88 kDa) than the protein in older animals (~110 kDa). We found three conserved N-linked glycosylation sites among the known Na-Pi-IIb cotransporter family members, and two of the three sites were conserved with sites confirmed to be glycosylated in the mouse renal Na-Pi-IIa cotransporter (20). Because these results suggested that mouse Na-Pi-IIb cotransporter may also be an N-glycoprotein, we performed enzymatic deglycosylation studies with PNGase F and Endo H. PNGase F cleaves between the innermost N-acetylglucosamine and asparagine residues of high mannose, hybrid, and complex oligosaccharides of N-linked glycoproteins (31), whereas Endo H cleaves the chitobiose core of high mannose and some hybrid oligossacharides of N-linked glycoproteins (39). Treatment with PNGase F decreased the molecular mass of both Na-Pi-IIb-specific bands (~88 and ~110 kDa) to its predicted size (~78 kDa), whereas treatment with Endo H had no apparent effect. Therefore, our findings strongly suggest that the type IIb Na-Pi cotransporter is an N-linked glycoprotein containing complex oligosaccharides and that partial glycosylation occurs during the suckling/weaning transition.

To date, effects of GCs on Na-Pi-IIb expression have not been addressed, although GC-induced downregulation of intestinal Na-Pi transport has been reported (8). Therefore, in the present study we demonstrate that MP injection reduced Na-Pi uptake (3.4-fold), Na-Pi-IIb protein levels (3.8-fold), and mRNA (3.7-fold) in suckling animals. So, our results indicate that the GC-induced decrease in intestinal Na-Pi transport correlates well with Na-Pi-IIb protein and mRNA level reductions in suckling animals. The parallel decline in mRNA abundance implicates a possible genomic effect of GCs. Indeed, several studies have suggested maturation of intestinal proteins by genomic action of GCs (23, 40, 45). However, one previous study (6) suggested that GCs modulate patterns of glycosylation in suckling rat intestine through enhanced fucosyl-transferase activity, and thus we cannot eliminate the possibility that Na-Pi-IIb glycosylation is enhanced by nongenomic actions of GCs during the suckling period. Furthermore, the 21-day-old animals showed MP responsiveness in intestinal Na-Pi uptake and Na-Pi-IIb mRNA expression, although no significant alteration was seen in Na-Pi-IIb protein levels.

There are clear differences between Na-Pi-IIb and its renal homologue Na-Pi-IIa in expression during ontogeny and hormonal response. Our findings indicate that: 1) intestinal Na-Pi uptake activity and Na-Pi-IIb protein and mRNA levels are all highest during the suckling period, 2) Na-Pi-IIb GC responsiveness is also highest during the suckling period, and 3) the Na-Pi-IIb protein is not fully processed (glycosylated) until weaning. Accumulated evidence regarding renal Na-Pi-IIa have indicated that: 1) renal Na-Pi uptake activity and Na-Pi-IIa protein are lower during the suckling period than during weaning, 2) Na-Pi-IIa GC responsiveness is not seen during the suckling period but is observed in adults, and 3) there is no evidence of not fully glycosylated Na-Pi-IIa at any age in rodents.

Because the intestine matures very rapidly during the suckling/weaning transition (21), it is not surprising that suckling animals have a higher capacity to absorb nutrients than older animals. The rapid decline in Na-Pi uptake activity and Na-Pi-IIb expression between the suckling and weaning periods suggests that the Na-Pi transport system matures early in postnatal life. In conclusion, we found that Na-Pi-IIb mRNA expression rapidly decreases during the suckling/weaning transition and that mRNA expression is regulated by GCs in the suckling period. This decrease in mRNA expression is likely responsible for the observed decrease in Na-Pi-IIb protein expression and the concomitant decrease in intestinal Na-Pi transport in early life. Further investigation is necessary to decipher maturational mechanisms of Na-Pi-IIb cotransporter expression and function during the suckling/weaning transition.


    ACKNOWLEDGEMENTS

This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant 2R01-R37-DK-33209 and by the W. M. Keck Foundation.


    FOOTNOTES

Address for reprint requests and other correspondence: Fayez K. Ghishan, Dept. of Pediatrics, Steele Memorial Children's Research Center, University of Arizona Health Sciences Center, 1501 N. Campbell Ave., Tucson, AZ 85724 (E-mail: fghishan{at}peds.arizona.edu).

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.

October 3, 2001;10.1152/ajpgi.00319.2001

Received 24 July 2001; accepted in final form 21 September 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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