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J. Biol. Chem., Vol. 277, Issue 22, 19665-19672, May 31, 2002

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Growth-related Renal Type II Na/P_i Cotransporter^*

Hiroko Segawa, Ichiro Kaneko, Akira Takahashi, Masashi Kuwahata, Mikiko Ito, Ichiro Ohkido, Sawako Tatsumi, and Ken-ichi Miyamoto

From the Department of Nutrition, School of Medicine, Tokushima University, Kuramoto-Cho 3, Tokushima City 770-8503, Japan

Received for publication, January 29, 2002, and in revised form, March 1, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Growth is critically dependent on the retention of a variety of nutrients. The kidney contributes to this positive external balance. In the present study, we isolated a cDNA from the human and rat kidney that encodes a growth-related Na⁺-dependent inorganic phosphate (P_i) cotransporter (type IIc). Microinjection of type IIc cRNA into Xenopus oocytes demonstrated sodium-dependent P_i cotransport activity. Affinity for P_i was 0.07 mM in 100 mM Na⁺. The transport activity was dependent on extracellular pH. In electrophysiological studies, type IIc Na/P_i cotransport was electroneutral, whereas type IIa was highly electrogenic. In Northern blotting analysis, the type IIc transcript was only expressed in the kidney and highly in weaning animals. In immunohistochemical analysis, the type IIc protein was shown to be localized at the apical membrane of the proximal tubular cells in superficial and midcortical nephrons of weaning rat kidney. Hybrid depletion experiments suggested that type IIc could function as a Na/P_i cotransporter in weaning animals, but its role is reduced in adults. The finding of the present study suggest that the type IIc is a growth-related renal Na/P_i cotransporter, which has a high affinity for P_i and is electroneutral.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Inorganic phosphate (P_i) is of critical importance to body functions, particularly during periods of growth. The kidneys contribute to the maintenance of the positive P_i balance required for growth by reabsorbing a high fraction of the filtered P_i (1). The capacity for Na⁺-dependent phosphate cotransport across the luminal brush border membrane of renal proximal tubular cells is higher in juveniles than in adults (2, 3).

Several mammalian renal Na⁺-dependent P_i cotransporters have recently been isolated and characterized (4). The cDNAs of these transporters can be divided into three types (types I-III) in the kidney cortex (4). Type II Na/P_i cotransporters belong to a unique class of Na⁺-coupled cotransport proteins. They can be further subdivided into two subgroups, type IIa and type IIb (4). Type IIa cotransporters are expressed in the proximal tubule of the kidney, whereas type IIb are expressed in several tissues such as the lung and small intestine (4). Functional characteristics, proximal tubular localization of the mRNAs, and apical expression of type IIa Na/P_i cotransporters suggest that this protein represents the most likely pathway of proximal tubular apical Na⁺-dependent entry of P_i (4).

Age dependence was observed at the level of the type IIa Na/P_i cotransporter protein expression (5, 6). In addition, a specific type IIa-related Na/P_i cotransporter protein was postulated to account for high P_i transport rates in weaning animals (7). Evidence for this was obtained by antisense experiments and transport expression in Xenopus oocytes (1, 7). When mRNA isolated from the kidney cortex of rapidly growing rats was treated with type IIa transporter antisense oligonucleotides or was depleted of type IIa-specific mRNA by a subtractive hybridization procedure, Na⁺-dependent P_i uptake was still detected in injected oocytes (1, 7). The type IIa transporter-depleted mRNA contained a mRNA species that showed some sequence homology to the type IIa transporter encoding message. This conclusion is compatible with the observation that young type IIa (Npt2) knock-out mice lacking type IIa mRNA and protein retain the capacity to reabsorb P_i at a rate that cannot be explained by the presence of type I and III Na/P_i transporter (8, 9). In the present study, we isolated a growth-related type II Na/P_i cotransporter in human and rat kidneys.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Animals and Diets-- Male Wister rats (3 weeks after birth) were purchased from Shizuoka Laboratory Animal Center (Shizuoka, Japan). They were housed in plastic cages and fed standard rat chow diet (Oriental, Osaka, Japan) ad libitum for the first week. After that period, they received a diet containing 1.2% calcium and 0.6% phosphorus for 5 days. On the 6th day, the following three groups of six rats each were established: the control P_i group, rats that were chronically fed a diet containing 0.6% P_i; the low P_i group, rats that received a diet containing a low percentage (0.02%) of P_i; and the high P_i group, in which the rats received a high percentage (1.2%) P_i diet. After 7 days of the given diet, all of the rats were anesthetized with intraperitoneal pentobarbital, and their kidneys were removed rapidly.

cDNA Cloning-- cDNAs for human expressed sequence tags (EST) (GenBank^TM/EBI/DDBJ accession no. AI792826), which we found in the course of EST database searches to show nucleotide sequence similarity to human type IIa Na/P_i cotransporter, were obtained using IMAGE (integrated and molecular analysis of genomes and their expression). The ~0.8-kb SacI fragment was excised from human cDNA (IMAGE cDNA clone 1535299) and labeled with ³²P using the Megaprime DNA labeling system, dCTP (Amersham Biosciences) for use as a probe to screen a human kidney 5'-Stretch Plus cDNA library (CLONTECH). Screening of the cDNA library and isolation of positive plaques were performed as described previously (10, 11).

The human type IIc Na/P_i cotransporter fragment (corresponding to nucleotides 89-600 of the nucleotide sequence) was used to isolate a rat cDNA for type IIc Na/P_i cotransporter. The oligo(dT)-primed cDNA library was prepared from rat kidney poly(A)⁺ RNA using the Superscript Choice system (Invitrogen) (12). The synthesized cDNA was ligated to ZIPLOX EcoRI arms (Invitrogen). Screening of the cDNA library and isolation of the positive plaques were performed as described previously (12).

Xenopus Oocyte Expression-- cRNAs obtained by in vitro transcription using T7 RNA polymerase for the human type IIc cDNA (hNPIIc) and rat type IIa (NaPi-2) in plasmid pBluescript SK (Stratagene) were linearized with XbaI as described previously (12). Xenopus oocyte expression studies and uptake measurements were performed as described previously (11, 12). The uptake rates of [³²P]phosphorus were measured 2-3 days after injection of cRNA. For expression experiments, 25 ng of cRNA was injected into each oocyte. Xenopus oocyte expression was performed as described previously (11, 12).

P_i Uptake Measurements-- Groups of six to eight oocytes were incubated in 500 µl of standard uptake solution (100 mM NaCl, 2 mM KCl, 1 mM CaCl₂, 1 mM MgCl₂ 10 mM HEPES, and 5 mM Tris, pH 7.4) or Na⁺-free uptake solution in which NaCl in standard uptake solution was replaced by choline chloride containing 0.1 µCi of radiolabeled compounds (10, 11).

Electrophysiology and Data Acquisition-- Electrophysiological measurements were performed at room temperature using oocytes 3 days after cRNA injection. The oocytes were impaled with two 3 M KCl-filled electrodes with resistances of 0.5-2 M. The electrodes were connected to a commercial two-electrode voltage clamp amplifier (CEZ 1250, Nihon Koden, Tokyo Japan) via Ag-AgCl pellet electrodes and referenced to an Ag-AgCl pellet that was connected to the bath via a 3 M KCl-agar bridge. The voltage clamp was controlled by an analog-to-digital-to-analog interface board (Digidata 1200, Axon Instruments, Foster City, CA) using pCLAMP 6 software (Axon Instruments). The voltage clamp protocol was for 2 s at -80 to +80 mV membrane potential. The external control solution (superfusate) contained (mM): 96 NaCl, 2 KCl, 1.8 CaCl₂, 1 MgCl₂, and 5 HEPES, pH 7.4. Phosphate was added to this solution at the indicated concentrations. The final experimental solutions were adjusted to pH 7.4. The flow rate of the superfusate was 20 ml/min, and complete exchange of the bath solution was reached within about 10 s.

Antisense Hybrid Depletion-- For hybrid depletion experiments, rat kidney poly(A)⁺ RNA (5 µg/µl) was denatured at 65 °C for 5 min in solution A (50 mM NaCl and a 20 µM concentration of a 16-mer oligonucleotide complementary to rat type II phosphate transporters) and further incubated at 42 °C for 30 min (13). The positions of sense oligonucleotide type IIa (5'-GTCCAGGGTAGAGGCC-3', nucleotides +1004-1019), antisense type IIa (5'-GGCCTCTACCCTGGAC-3', nucleotides +1004-1019), sense type IIc (5'-ATTGGCCTGGTGGACT-3', nucleotides +134-149), and antisense type IIc (5'-AGTCCACCAGGCCAA-3', nucleotides +134-149) are complementary to the rat type IIa and type IIc mRNA sequence (7). The sample of poly(A)⁺ RNA was injected into the oocytes, and uptake measurements were performed as described previously (10, 11).

Immunoblotting Analysis-- Brush-border membrane vesicles (BBMVs)¹ were prepared from rat kidney by the Ca²⁺ precipitation method as described previously (14). The levels of leucine aminopeptidase, Na⁺K⁺-ATPase, and cytochrome c oxidase were measured to assess the purity of the membranes. Protein samples were heated at 95 °C for 5 min in sample buffer in either the presence or absence of 5% 2-mercaptoethanol and subjected to SDS-polyacrylamide gel electrophoresis. The separated proteins were transferred electrophoretically on Hybond-P polyvinylidene difluoride transfer membranes (Amersham Biosciences). The membranes were treated with diluted affinity-purified anti-type IIa (1:4000) (14) or type IIc (1:1000) Na/P_i cotransporter antibody and then with horseradish peroxidase-conjugated anti-rabbit IgG as the secondary antibody (Jackson ImmunoResearch Laboratories, Inc.). The signals were detected using the ECL Plus system (Amersham Biosciences) (15).

Immunohistochemistry-- Immunohistochemical analysis of the rat kidney was performed as described previously with minor modification (15). For immunostaining, serial sections (5 µm) were incubated with affinity-purified anti-type IIa (1:4000) or type IIc (1:1000) Na/P_i cotransporter antibodies overnight at 4 °C. Thereafter, they were treated with Envision(+) rabbit peroxidase (Dako) for 30 min. To detect immunoreactivity, the sections were treated with diaminobenzidine (0.8 mM).

Anti-peptide Antibody-- An oligopeptide (CYENPQVIASQQL) corresponding to amino acid residues 590-601 of rat type IIc Na/P_i cotransporter was synthesized. The C-terminal cysteine residues were introduced for conjugation with keyhole limpet hemocyanin. Rabbit anti-peptide antibodies were produced as described previously (14).

Statistical Analysis-- Data are expressed as the mean ± S.E. Differences between experimental groups were determined by analysis of variance, and p values of <0.05 were accepted as indicating a significant difference.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cloning of Type IIc Na/P_i Cotransporter-- The human type IIc cDNA was 2020 bp long with an open reading frame of 1797 bp encoding 599 amino acids. Hydropathy analysis of the predicted amino acid sequence revealed the presence of eight putative transmembrane domains. The extracellular segments of human type IIc cotransporter contained four putative N-linked glycosylation sites. Potential intracellular phosphorylation sites for protein kinase C was detected at residues 24, 152, 481, and 581 (Fig. 1a).

View larger version (48K): [in this window] [in a new window]   Fig. 1.   Cloning of Na/Pi cotransporter (type IIc). a, sequence alignment of type II Na/Pi cotransporters. The deduced amino acid sequence of type IIc Na/Pi cotransporter (human) is shown aligned with those of types IIa, IIb, and IIc cotransporters. Residues identical in at least two sequences are shaded. Lines under the sequences show predicted transmembrane regions of type IIc Na/Pi cotransporter, numbered 1-8. In type IIc Na/Pi cotransporter, putative N-linked glycosylation sites are marked by the # sign. Putative protein kinase C-dependent phosphorylation sites are located at residues 24, 152, 481, and 581 (labeled with *). The residue numbers are indicated beside the aligned sequences. b, Northern blotting analysis in human tissues. High stringency Northern hybridization analysis using a human type IIc probe was performed against poly(A)+ RNA from human tissues. c, Northern blotting analysis in rat tissues. High stringency Northern hybridization analysis using a rat type IIc probe was performed against poly(A)+ RNA from rat tissues. d, developmental changes in rat renal type IIc mRNA levels. Lane 1, 5 days old; lane 2, 15 days; lane 3, 22 days; lane 4, 60 days.

Amino acids in the membrane-spanning regions were especially well conserved among the three isoforms. Amino acid comparisons revealed that the newly identified protein was 36-38% homologous to Na/P_i cotransporters identified in human type IIa and type IIb amino acid sequences, respectively (16, 17). Overall homology to types I and III Na/P_i cotransporters was ~10% (10, 18). The highest degrees of homology were detected in regions that have been suggested to be the membrane-spanning domains. The most striking difference in the newly identified protein compared with the type II Na/P_i cotransporters was found in the C-terminal region containing clusters of cysteine residues. A similar clustering of cysteine residues was also present in the type IIb Na/P_i cotransporters of human mouse, and flounder kidney.

Tissue Distribution of type IIc Na/P_i Cotransporter-- The expression of type IIc mRNA was analyzed by Northern blotting using human multiple tissue Northern blot and poly(A)⁺ RNA from rat tissues (Fig. 1, b and c). Using the type IIc cDNA as a probe, a strong 2.4-kb signal was observed only in the kidney. No signals were detected in the brain, heart, skeletal muscle, thymus, spleen, lung, or peripheral blood leukocytes. In addition, the expression of the type IIc mRNA was significantly higher in weaning animals (22 days old) compared with those in adults (60 days old) (Fig. 1d). The levels of type IIc mRNA were lowest in suckling animals.

Functional Analysis of Type IIc Na/P_i Cotransporter-- The functional properties of human type IIc Na/P_i cotransporter were examined in Xenopus oocytes. As shown in Fig. 2, the microinjection of Xenopus oocytes with human type IIc Na/P_i cotransporter resulted in a marked increase relative to the level apparent in water-injected oocytes (Fig. 2a). [³²P]Phosphate uptake mediated by human type IIc was dependent on Na⁺ but not Cl (Fig. 2b), and it increased in a concentration-dependent manner in the presence of Na⁺ (Fig. 2b). The uptake was saturable, and the Michaelis-Menten constant (K_m) for P_i was 70 µM (Fig. 2c). Type IIc-mediated Na/P_i uptake was stimulated by a more alkaline pH, a hallmark of proximal tubular Na/P_i cotransport (Fig. 2d). The apparent K_d and Hill coefficient for Na interaction was K_d = 48 ± 9 mM and n = 1.73, respectively (Fig. 2e).

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Characterization of the type IIc Na/Pi cotransporter in Xenopus oocytes. a, oocytes injected with either water (open bar), cRNA of human type IIc Na/Pi cotransporter (closed bar), or cRNA of rat type IIa Na/Pi cotransporter (open bar) (10) were assayed after 2 days for uptake of Pi (100 µM) in 96 mM NaCl medium (n = 8 experiments). Values are means ± S.E. b, ion dependence of Pi transport in oocytes expressing human type IIc Na/Pi cotransporter. The uptake of 100 µM Pi measured in the standard uptake solution (Na) was not increased in the Na+-free uptake solution in which Na+ was replaced with choline. In contrast, it was not altered in the Cl-free uptake solution in which Cl was replaced with gluconate. Values are means ± S.E. (n = 3). c, Pi concentration dependence of human type IIc Na/Pi cotransporter-mediated Pi uptake. The type IIc Na/Pi cotransporter-mediated Pi uptake was measured at 3, 10, 30, 100, 300, and 1000 µM Pi in standard uptake solution and plotted against the Pi concentration. The Pi uptake was saturable and fit the Michaelis-Menten curve. Values are means ± S.E. (n = 6 experiments). d, sodium concentration dependence of type IIc Na/Pi cotransporter-mediated Pi uptake. The type IIc Na/Pi cotransporter-mediated Pi uptake was measured at 10, 25, 50, 75, and 100 mM sodium. Choline was used for isoosmotic ionic replacement. Values are means ± S.E. (n = 5 experiments). e, pH dependence of type IIc Na/Pi cotransporter-mediated Pi uptake. The type IIc cotransporter-mediated uptake of Pi (100 µM) was measured in the standard uptake solution at various pH values. The uptake value was greatest at pH 7.5. Values are means ± S.E. (n = 5 experiments).

Electrophysiology of Type IIc Na/P_i Cotransporter-- Fig. 3 shows typical time courses of currents at a membrane potential of -60 mV during the addition of Pi. Superfusion of oocytes expressing the type IIa Na/P_i cotransporter with P_i exhibited currents that depended on the presence of external Na⁺. Such currents were not observed when the same protocol was applied to water or noninjected oocytes (data not shown). Washout of P_i was also accompanied by a similar biphasic return to the base-line values. Reversal potential shifted from -22 mV to +16 mV during stimulation with 1 mM P_i in type IIa Na/P_i cotransporter-expressing oocytes. These observations suggest that the currents stimulated by 1 mM P_i were Na⁺ currents. These findings confirmed that the previous observation that the Na/P_i cotransport by the type IIa cotransporter was electrogenic (19). In contrast, a superinfusion of oocytes expressing the type IIc cotransporter with P_i (0.1-3 mM) did not exhibit the currents. These observations suggested that, unlike type IIa, Na/P_i cotransport by the type IIc Na/P_i cotransporter is electroneutral.

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Voltage-dependent Pi-induced currents in an oocytes expressing type IIa and type IIc Na/Pi cotransporters. A, time course changes in membrane current during stimulation by Pi. Pi perfusion was performed with the indicated concentration and for the indicated times. The holding membrane potential was -60 mV. B, current-voltage curves. The current-voltage relationship was recorded by the voltage clamp protocol before stimulation, during perfusion with Pi, and after washout of Pi. C, Pi dose-response relation for membrane currents. The membrane current was measured at Vm = 60 mV. Values were recorded at the steady-state response of membrane current and are means ± S.E. (n = 6). I, oocytes were injected with cRNA of type IIa Na/Pi cotransporter. II, oocytes were injected with cRNA of type IIc Na/Pi cotransporter.

Western Blotting Analysis-- The molecular weight of type IIc Na/P_i cotransporter protein was determined by Western blotting analysis (Fig. 4a). In BBMVs isolated from the rat kidney (22 days old), the specific antibody reacted with a band of 80-85 kDa under reducing conditions (Fig. 4a). As measured by the presence of antigen peptides in the absorption experiments, the 80-85 kDa band disappeared (Fig. 4a). In addition, FLAG-fused type IIc Na/P_i cotransporter in COS-7 cells was observed as 85- and 160-kDa bands using FLAG-specific monoclonal antibody (Fig. 4b). The type IIc antibodies reacted with the 80-85 kDa protein band (data not shown). In addition, we examined whether the type IIc antibodies react with type IIa Na/P_i cotransporter protein. The type IIc antibodies did not react with any bands in the COS 7 cells expressing the type IIa or type IIb Na/P_i cotransporters (Fig. 4b).

View larger version (29K): [in this window] [in a new window]   Fig. 4.   Western blotting analysis under reducing conditions. a, Western blotting analyses were performed on the BBMVs prepared from rat kidney in the presence of 2-mercaptoethanol. Lane 1, type IIc antibodies; lane 2, results from peptide absorption experiments. b, The type IIc antibodies did not react with the type IIa Na/Pi cotransporter. To generate FLAG-tagged type IIc transporter cDNA, PCR amplification was performed with the rat type IIc clone as a template. Fragments were subcloned into the pFLAG-CMV-2 expression vector (Sigma) for transient transfection. Total protein homogenates from COS-7 cells are shown. Lane 1, control cells (transfected with empty vector); lane 2, transient in transfected with FLAG-type IIc transporter vector. The type IIa (lane 3) or IIb (lane 4) Na/Pi cotransporter cDNA was subcloned for the pCDNA3.1(+) for transient transfection. The membranes were treated with diluted affinity-purified anti-type IIc (1:1,000) Na/Pi cotransporter antibody. Findings indicate that the type IIc antibodies are not reacted with type IIa and IIb Na/Pi cotransporter protein. c, developmental changes in rat renal type IIc protein levels. Renal BBMVs from each aged rat were prepared, and 20 µg of the protein was analyzed by Western blot analysis. Lane 1, 5 days old; lane 2, 15 days; lane 3, 22 days; lane 4, 60 days. d, relative intensity of the type IIc transporter protein in developmental rats. **, p < 0.01. e, effects of dietary Pi on the amounts of type IIc protein. Brush-border membrane vesicles were isolated from 40-day-old rats fed the test diet for 6 days. Lane 1, low Pi (0.02%) diet; lane 2, control Pi (0.6%) diet; lane 3, high Pi (1.2%) diet. **, p < 0.01. f, relative intensity of the type IIc transporter protein in rats fed a low, normal, or high Pi diet.

Next, we investigated developmental changes in rat renal type IIc protein levels (Fig. 4c). Western blotting demonstrated that the amount of type IIc protein in the BBMVs was highest in weaning rat, lower in adult rats, and lowest in suckling rats. In Fig. 4e, BBMVs isolated from the kidney of a rat (40 days old) fed a diet low in P_i for 7 days were prepared and used for Western blotting. The amounts of type IIc transporter protein (80-85-kDa band) were significantly increased (by about 5.0-fold for the 80-85-kDa band) compared with those in rats fed the control diet. In contrast, the high P_i diet markedly suppressed the level of type IIc transporter protein.

Immunohistochemistry-- Immunolocalization of type IIc Na/P_i cotransporter protein was performed with the kidneys of weaning rats (22 days old). In Fig. 5, a and b, expression of type IIc cotransporter immunoreactive protein was detected exclusively in the superficial and juxtamedullary nephron. The control antibodies did not stain it (data not shown). The highest expression was observed in convoluted proximal tubules. At higher magnification, it was evident that type IIc antibody-mediated immunoreactivity was localized in the brush border of proximal tubular cells and was completely absent in the basolateral membrane domain (Fig. 5g). Brush-border staining was slightly weaker in superficial nephrons than in juxtamedullary nephrons. In contrast, in weaning rats, type IIa-related immunoreactivity was detected only in juxtamedullary nephrons (Fig. 5, c and d) but not in the superficial and midcortical regions. Type IIa-related immunostaining was observed in a subapical vesicular structure, which likely belongs to the vacuolar endocytic apparatus, in weaning rat kidney (Fig. 5h). In the adult kidney (Fig. 5, e and f), type IIc-related immunoreactivity was detected only in juxtamedullary nephrons and not in the superficial and midcortical regions. Type IIa-related immunostaining was observed in midcortical and juxtamedullary nephrons in adult rats.

View larger version (111K): [in this window] [in a new window]   Fig. 5.   Localization of type IIc-immunoreactive protein in weaning and adult kidneys. Type II Na/Pi cotransporter proteins detected by diaminobenzidine staining using rabbit anti-type IIc antibodies (a and b) or rabbit anti-type IIa antibodies (c and d) in cryostat sections of weaning rat kidneys. The type IIc transporter protein in adult kidneys is shown in panels e and f. At higher magnification, type IIc (g) or type IIa (h) antibody-mediated immunoreactivity is shown.

Hybrid Depletion-- Evidence for the type IIc Na/P_i cotransporter was obtained by antisense experiment (Fig. 6). As described under "Experimental Procedures," when poly (A)⁺ RNA isolated from the kidney of adult rats was treated with type IIa transporter antisense oligonucleotides of type IIa-specific mRNA, Na⁺-dependent P_i uptake was completely suppressed in injected oocytes (Fig. 6a). In contrast, when poly(A)⁺ RNA isolated from the kidney of weaning rats was treated with type IIa antisense oligonucleotides, P_i uptake was still detected in injected oocytes (Fig. 6b). In contrast, type IIc antisense oligonucleotides significantly suppress P_i uptake in oocytes expressing poly(A)⁺ RNA from weaning rat kidney (Fig. 6d). However, similar treatment did not affect P_i uptake in oocytes expressing poly(A)⁺ RNA from adult rat kidney (Fig. 6c).

View larger version (21K): [in this window] [in a new window]   Fig. 6.   Hybrid depletion of type II Na/Pi cotransporter in Xenopus oocytes. Pi uptake in oocytes injected with renal poly(A)+ RNA from weaning and adult rat kidneys. Hybrid depletion analyses by antisense oligonucleotide were performed as described under "Experimental Procedures." a and c, poly(A)+ RNA from adult rat kidney (60 days old). b and d, poly(A)+ RNA from weaning rat kidney (22 days old). Values are mean ± S.D. (n = 8-10 oocytes). **, p < 0.01; *, p < 0.05.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Tubular P_i reabsorption decreases during aging as has been indicated by metabolic balance studies, clearance studies, and studies with isolated vesicles (1-3). This decrease is due to a reduction in the V_max without a change in the apparent K_m for P_i of the brush border membrane Na/P_i cotransport. Kinetic properties and pH dependence of type IIc-mediated Na/P_i cotransport favor this protein as a candidate for a Na/P_i cotransporter involved in a high P_i transport activity in weaning animals (4).

In addition, our characterization of the kinetics of the type IIc transporter gave findings consistent with those reported in the BBMV studies (20, 21). As reported for the renal type IIa Na/P_i cotransporter, superfusion of oocytes expressing the type IIa Na/P_i cotransporter with P_i exhibited an inwardly directed current that was dependent on the presence of Na⁺ and the steady-state holding potential (19). However, type IIc mediated Na/P_i cotransport was electroneutral. The apparent K_d and Hill coefficient for Na⁺ interaction were obtained using the Hill equation (for human type IIc-mediated uptake, K_d = 48 + 9 mM, and n = 1.73). Busch et al. (22) characterized the electrogenicity by expressing the type IIa Na/P_i cotransporter (NaPi-2) cloned from rat kidney in Xenopus oocytes. They showed that in the mandatory presence of extracellular Na⁺, P_i induced an inward current (I_p) for membrane potentials (V) in the range of -80 < V < +10 mV. Consistent with the findings from BBMVs, the magnitude of I_p depended on the substrate concentrations, extracellular pH, and membrane potential. However, in contrast to the 2:1 stoichiometry to Na/P_i at pH 7.4 proposed from BBMV studies (23, 24), findings of a Hill slope close to 3 for the Na⁺ dose response at saturating P_i suggested a 3:1 stoichiometry for type IIa Na/P_i cotransport at -50 mV. In contrast, the present findings suggest that type IIc has the 2:1 stoichiometry to Na/P_i at pH 7.4 as proposed from BBMV studies (23, 24).

The physiological significance of an electroneutral Na/P_i cotransporter during growth is unknown. An electroneutral transporter would transport less P_i across the apical membrane of the proximal tubule, as the driving force for Na⁺ would be less. Two factors oppose the entry of P_i from the tubular lumen into the cell, the inside negative cell potential and the high intracellular P_i concentration. The intracellular P_i concentration measured in isolated perfused kidneys using nuclear magnetic resonance (NMR) was significantly lower in growing animals than in adults (25). This provides a greater driving force for an electroneutral Na/P_i cotransporter in growing animals. However, further studies are needed to clarify the role of the electroneutral Na/P_i cotransporter in P_i transport.

The type IIc transporter protein is detected in the apical membrane of renal proximal tubular cell in adult rats. Western blot analysis also shows that type IIc Na/P_i cotransporter is present in the BBMVs from adult rat kidneys. However, in the hybrid depletion experiment, type IIc antisense oligonucleotide did not affect the P_i uptake in oocytes induced by microinjection of renal poly(A)⁺ RNA from adult rats. These observations suggest that post-transcriptional regulation (e.g. regulatory protein) of type IIc is responsible for the discrepancy observed in the adult between the levels of type IIc mRNA and those of type IIc-like protein.

Age dependence was also observed at the level of type IIa Na/P_i cotransporter protein expression (1, 26-28). In the kidneys of newborn rats, expression of the type IIa Na/P_i cotransporter was observed in differentiated juxtamedullary and intermediate nephrons only and was absent in the outer cortex (28). After completion of nephron formation, during suckling, expression of the transporter was similarly high in the brush-border membrane of all nephron generations. In weaning, the expression pattern resembled that in adults, i.e. type IIa abundance decreased in the brush-border membrane of superficial and midcortical nephrons (28). Traebert et al. (28) reported that the abundance of type IIa protein in the BBMVs of 22-day-old rats was decreased to ~70% of that in 13-day-old rats. In contrast, renal type IIc protein was not detected in newborn and suckling rats, and was markedly increased in the superficial and midcortical regions of the kidney in weaning animals. The type IIc immunoreactive protein in those regions was gradually decreased and was detected in the deep nephrons in the adult kidney. The present findings suggest that the high expression of type IIc Na/P_i cotransporter in the kidney of weaning rats may support high P_i transport activity in weaning animals during down-regulation of the type IIa Na/P_i cotransporter.

It is possible that the induction of type IIc protein in superficial nephrons in the weaning rats not only may be related to the developmental stage but also may be affected by the different P_i contents of the available food (28). The suckling rats were fed exclusively with rat milk (0.2% P_i), and from day 20, the rats were fed the standard laboratory diet with a P_i content of 0.6% (29). Supplementing the P_i content may induce the type IIc protein in the weaning kidney. We therefore investigated the effect of dietary P_i on the amount of type IIc protein. The findings of the present study suggest that a high P_i diet suppresses the expression of type IIc protein, whereas a low P_i diet increases the amount of type IIc protein in 40-day-old rats. It is suggested that the different levels of P_i food intake are not involved in the induction of type IIc protein in weaning rats. Hormonal changes occurring around the time of weaning might contribute to the observed changes in type IIa and type IIc abundance in the BBMVs.

Recently, Hoag et al. (9) examined the effect Npt2 gene knock-out on age-dependent BBMVs Na/P_i cotransport and expression of Na/P_i cotransporter genes Npt1, Glvr-1, and Ram-1 (9). At all ages, Na/P_i cotransport in Npt2/ mice is ~15% of that in Npt2+/+ littermates. They concluded that Npt2/ mice cannot be compensated for by the age-dependent increase in renal expression of type I and type III transporters (8, 9). They also provided evidence that differences in the Npt2 protein abundance alone could account for the age-dependent decrease in Na/P_i cotransport in renal proximal tubules (8, 9). However, the present findings suggest that type IIc is highly expressed in the renal cortex in weaning animals and may support a demand of high P_i intake. A low P_i transport activity in the BBMV from Npt2/ mice kidney may be due to the low expression of the type IIc Na/P_i cotransporter. Further studies are needed to clarify the regulation of type IIc Na/P_i cotransporter in Npt2/ mice.

Finally, the findings presented herein illustrate the mechanism by which the weaning kidney achieves the high rates of P_i reabsorption required for the maintenance of a positive external balance. In this study, the type IIc was a growth-related renal Na/P_i cotransporter, which is highly expressed in the weaning kidney.

	ACKNOWLEDGEMENTS

We thank Dr. Hidekazu Arai, Misako Kawahara, R. Yanagida, K. Yano, R. Saito, K. Shinohara, and N. Yata.

	FOOTNOTES

^* This work was supported by Grant 11557202 (to K. M.) from the Ministry of Education, Science, Sports and Culture of Japan.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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AB055000 and AB077042.

To whom correspondence should be addressed: Dept. of Nutrition, School of Medicine, Tokushima University, Kuramoto-Cho 3, Tokushima City 770-8503, Japan. Tel.: 81-886-7081; Fax: 81-886-33-7082; E-mail: miyamoto@nutr.med.tokushima-u.ac.jp.

Published, JBC Papers in Press, March 5, 2002, DOI 10.1074/jbc.M200943200

	ABBREVIATIONS

The abbreviation used is: BBMVs, brush border membrane vesicles..

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