Internalization of renal type IIc Na-Pi cotransporter in response to a high-phosphate diet
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《美国生理学杂志》
Nutritional Science, Department of Nutrition, and General Laboratory for Medical Research, University of Tokushima School of Medicine, Tokushima
Department of Structural Pathology Institute of Nephrology Graduate School of Medical and Dental Sciences, Niigata University, Niigata, Japan
ABSTRACT
Dietary phosphate levels regulate the renal brush-border type IIa Na-Pi cotransporter. Another Na-Pi cotransporter, type IIc, colocalizes with type IIa Na-Pi cotransporter in the apical membrane of renal proximal tubular cells. The goal of the present study was to determine whether dietary phosphate levels also rapidly regulate the type IIc Na-Pi cotransporter. Type IIa and type IIc transporter protein levels were increased in rats chronically fed a low-Pi diet compared with those fed a normal-Pi diet. Two hours after beginning a high-Pi diet, type IIa transporter levels were decreased, whereas type IIc protein levels remained unchanged. Western blot analysis of brush-border membrane prepared 4 h after beginning a high-Pi diet showed a significant reduction in type IIc transporter protein levels, and immunohistochemistry showed translocation of the type IIc-immunoreactive signal from the entire brush border to subapical membrane. Membrane fractionation studies revealed a decrease in apical membrane type IIc protein without changes in total cortical type IIc protein, which is compatible with redistribution of type IIc protein from the apical membrane to the dense membrane fraction. The microtubule-disrupting reagent colchicine prevented this reduction in apical type IIc transporter at the apical membrane but had no effect on type IIa transporter levels. These data suggest that the type IIc Na-Pi cotransporter level is rapidly regulated by rapid adaptation to dietary Pi in a microtubule-dependent manner. Furthermore, the mechanisms of the internalization of the type IIc transporter are distinct from those of the type IIa transporter.
proximal tubule; dietary phosphate; regulation
INORGANIC PHOSPHATE (Pi) reabsorption in the renal proximal tubule is required for body Pi homeostasis (12–15). Evidence from physiological studies suggests that Na-dependent Pi transporters in the brush-border membrane (BBM) of proximal tubular cells mediate the rate-limiting step in the overall Pi-reabsorptive process (13, 14). Although the type IIa and type IIc Na-Pi cotransporters are expressed in the apical membrane of the proximal tubular cells and mediate Na-Pi cotransport (11–16, 21), the extent of Pi reabsorption in the proximal tubules is determined largely by the abundance of the type IIa Na-Pi cotransporter (12–15). In contrast, in weaning animals, the levels of type IIc Na-Pi cotransporter are increased, indicating that type IIc may play a role in the regulation of Pi reabsorption during growth (16, 21).
We previously demonstrated that expression of the type IIc Na-Pi cotransporter is increased in renal proximal tubular cells and that 30% of renal Na-Pi cotransport activity in Pi-deficient mice is mediated by the type IIc Na-Pi cotransporter (16). Furthermore, we reported that renal BBM type IIc protein levels were significantly increased in Npt2–/– mice compared with wild-type (Npt2+/+) littermates. Thus the type IIc Na-Pi cotransporter may account for the residual renal BBM Na-Pi cotransport in the type IIa knockout model (22).
Two major regulators of renal Pi reabsorption are dietary Pi and parathyroid hormone (PTH). Restriction of dietary Pi is associated with an adaptive increase in the overall capacity of the proximal tubule to reabsorb Pi. Reduced Pi reabsorption is achieved by a removal of the type IIa transporter from the apical membrane (5, 7–10, 12–15, 17). A high-Pi diet leads to transient accumulation of type IIa Na-Pi cotransporters in the subapical vacuolar apparatus (5, 10, 13, 14). Morphological and biochemical data suggest that internalized type IIa Na-Pi cotransporters are then directed to the lysosomes for degradation (8, 10, 24). Previous studies also suggest that microtubules are involved in this adaptive response (24). In addition, membrane insertion of the type IIa transporter, observed after acute administration of a low-Pi diet, requires an intact microtubular network (10). The goal of the present study was to determine whether the type IIc Na-Pi cotransporter undergoes rapid adaptation in response to dietary Pi manipulation.
MATERIALS AND METHODS
Animals and diets. Male Wistar rats (5 wk after birth) were purchased from SLC (Shizuoka, Japan), housed in plastics cages, and fed standard rat chow (Oriental, Osaka, Japan) ad libitum for 1 wk. After this period, a diet containing 0.6% calcium and 0.6% phosphorus (Pi) was administered for 5 days. On day 6, rats were either fed a diet containing 0.02% Pi. All animals were trained to consume their diets between 10 AM and 12 PM daily. On day 8, the following experimental groups were established: 1) chronic low-Pi diet group (CLP): rats were only fed a 0.02% Pi diet; 2) acute high-Pi diet groups (AHP): rats were chronically fed 0.02% Pi diet and, on the day of experiment, were acutely fed 1.2% Pi diet for 2, 4, 6, or 24 h. To study the role of microtubules in translocation of the type IIc by the acute high-Pi diet, rats were trained and adapted to the high-Pi diet as described above. On day 8, the rats were treated with colchicine (1.0 mg/kg body wt ip dissolved in saline) or vehicle (saline) 6 h before animal death and then fed a high-Pi diet 2, 4, or 6 h before animal death.
Experimental groups consisted of four or five individual rats for the BBM or cortical membrane isolation and of four individual rats for immunohistochemistry experiments.
Anti-peptide antibody. Oligopeptide CYENPQVIASQQL [corresponding to residues of rat type IIc Na-Pi cotransporter (589–601)] was synthesized. The NH2-terminal cysteine residues were introduced for conjugation with keyhole limpet hemocyanin. Guinea pigs were immunized with the peptide to prepare anti-type IIc Na-Pi cotransporter antibody, as previously described for preparation of rabbit anti-type IIa or type IIc Na-Pi cotransporter peptide antibodies (16, 21).
Preparation of membrane fraction. Methods for preparation of BBM vesicles (BBMVs) using Ca2+ precipitation and cortical membranes were described previously (7, 16, 21, 23).
The density separation of cellular membranes was accomplished by isopycnic centrifugation using OptiPrep (Nycomed Pharma, Oslo, Norway) density gradients (2). OptiPrep was diluted to appropriate concentrations from stocks using 20 mM Tricine (pH 8.7) and 8% sucrose according to the manufacturer’s protocols (2). Preformed OptiPrep gradients were made using a Gradient Maker (Pharmacia Biotech). One-five milligrams of cortical membranes in 5% OptiPrep were layered on the top of 15–25% OptiPrep gradients. Gradients were centrifuged to equilibrium (at least 2 h) at 100,000 g using an SW rotor in a HITACHI ultracentrifuge. One-milliliter fractions were manually collected from the top of the gradient preparation. Fractions 5-7 were used as the dense membranes (DM) for analysis by immunoblotting (2).
Immunoblotting. Protein samples were heated at 95°C for 5 min in sample buffer in the presence of 5% 2-mercaptoethanol and were subjected to SDS-polyacrylamide gel electrophoresis. The separated proteins were transferred electrophoretically to polyvinylidene difluoride transfer membranes (Hybond-P Amersham Pharmacia Biotech). The membranes were treated with diluted rabbit affinity-purified anti-type IIc (1:1,000), guinea pig affinity-purified anti-type IIc (1:500), or anti-type IIa (1:4,000) Na-Pi cotransporter antibodies, followed by treatment with horseradish peroxidase (HRP)-conjugated anti-rabbit or guinea pig IgG as the secondary antibody (Jackson ImmunoResearch Laboratories). Rabbit polyclonal antibody to megalin (1:10,000) was kindly provided by Dr. Saito (Niigata University, Niigata, Japan). Mouse monoclonal antibodies to clathrin heavy-chain (BD Transduction Laboratories; 1:10,000), villin (CHEMICON; 1:8,000), or NHE3 (CHEMICON; 1:3,000) were used, followed by treatment with HRP-conjugated anti-mouse IgG as secondary antibody (Jackson ImmunoResearch Laboratories). Signals were detected using the ECL Plus system (Amersham Pharmacia Biotech).
Immunohistochemistry. Rats were anesthetized with pentobarbital sodium (100 mg/kg body wt), and their kidneys were perfused via the left ventricle with PBS followed by 4% paraformaldehyde solution (pH 7.2) (16, 21). Kidneys were postfixed with the same solution overnight at 4°C, washed with PBS, cryoprotected with 10 and 20% sucrose at 4°C, and embedded in O.C.T. compound (Miles, Elkart), and frozen in hexane at –80°C. Frozen sections (5 μm) were collected onto silane-coated slides and air-dried. For immunofluorescence microscopy, serial sections were incubated with rabbit anti-type IIa (1:4,000), guinea pig anti-type IIc (1:200) Na-Pi cotransporter antibody overnight at 4°C. A mouse monoclonal antibody to clathrin heavy-chain (BD Transduction Laboratories; 1:1,000), EEA1 (BD Transduction Laboratories; 1:100), or GM130 (BD Transduction Laboratories; 1:100) and affinity-purified rabbit antibodies against anti-4F2 heavy chain (4F2hc)/CD98 antibody (1:1,000; 4F2hc antibody was kindly provided by Dr. Kanai, Kyorin University, Tokyo, Japan) were used for localization of the type IIc Na-Pi cotransporter. The Alexa Fluor568 anti-rabbit or mouse IgG (Molecular Probes) or the Alexa Fluor 488 anti-guinea pig IgG (Molecular Probes) was used as the secondary antibody, with incubation for 60 min at room temperature (16).
Data analysis. Data are expressed as means ± SE. Differences among multiple groups were analyzed by ANOVA. Differences between two experimental groups were determined by ANOVA followed by the Student’s t-test. P values < 0.05 were considered statistically significant.
RESULTS
Validation of guinea pig type IIc Na-Pi cotransporter antiserum. Western blot analyses were performed to determine whether type IIc proteins could be detected in BBMV prepared from rat kidney (Fig. 1). Rabbit type IIc antibody recognized two major polypeptide fragments with an apparent molecular mass of 150–160 and 75–80 kDa in the absence of 2-mercaptoethanol (2Me) (nonreducing condition). In the presence of 2Me (reducing condition), the 75- to 80-kDa band was prominent, suggesting that the 150- to 160-kDa band corresponds to a homodimeric complex. These bands (150–160 and 75–80 kDa) disappeared with absorption experiments, as described previously (Fig. 1A) (21). Similar results were observed with antibodies prepared from guinea pig COOH-terminal antibody.
Guinea pig type IIc COOH-terminal antibody recognized a major polypeptide fragment with an apparent molecular mass of 75–80 kDa in both the absence and presence of 2Me. The 150- to 160-kDa band was faint but present in the absence of the 2Me (Fig. 1B). Preincubation of guinea pig antibody with the appropriate immunizing synthetic peptide used for immunization prevented visualization of the respective bands on immunoblot (Fig. 1C). These data validate the reactivity and specificity of the antibodies employed in this study.
Expression of the type IIc Na-Pi cotransporter in proximal tubules of renal cortex. In mice, type IIc Na-Pi cotransporter-immunoreactive signals increase in the kidney after administration of a low-Pi diet (16, 21). In rats fed a normal-Pi diet, type IIc-immunoreactive signals were weaker in superficial nephrons compared with deep nephrons (Fig. 2A). In contrast, the intensity of type IIc-immunoreactive signals was clearly increased in the apical membrane of the proximal tubular cells in superficial and deep nephrons in rats chronically fed a low-Pi diet (Fig. 2B). Staining was absent in the outer and inner medulla and in the papilla.
To confirm localization of the type IIc Na-Pi cotransporter protein, the 4F2 heavy chain (4F2hc) was immunodetected. In the kidney, 4F2hc-immunoreactive signals were restricted to the proximal tubule at the lateral side and were most prominent in the initial portion of the proximal tubule (S1 segment) (20). Furthermore, type IIc Na-Pi cotransporter colocalized with the 4F2hc protein in the S1 segment (Fig. 3). No specific fluorescence of 4F2hc immunoreactivity was observed in the glomerulus or other tubular segments.
Adaptation of type IIc Na-Pi cotransporter in the kidney to dietary Pi. In rats chronically fed a low-Pi diet, the expression of type II Na-Pi cotransporters was observed in the proximal tubules of superficial and deep nephrons (Fig. 4). Type IIa transporter localized to the apical membrane of proximal tubules of all segment (S1, S2, S3) of rats fed a low-Pi diet (Fig. 4, A and D), whereas type IIc Na-Pi cotransporter localized to the apical membrane of proximal tubular cells in the S1 segment (Fig. 4, B and E).
Western blot analysis of the type IIc Na-Pi cotransporter in BBMV, cortical membrane, or DM fraction from the kidney of rats acutely fed a high-Pi diet. Acute feeding with a high-Pi diet induced translocation of the type IIa transporter from the apical membrane to the intracellular compartments as described previously (7, 8, 10). Next, adaptation of high-Pi diet on expression of type IIc Na-Pi cotransporter was examined via Western blot analysis of the type IIa and type IIc Na-Pi cotransporter proteins (Fig. 5). In BBMVs, the amount of the type IIa transporter protein at 2 h after switching to the high-Pi diet was 10% of that seen in rats chronically fed a low-Pi diet. Furthermore, the levels of the type IIa transporter were much lower at 4 and 6 h after the switch to a high-Pi diet than in rats fed a high-Pi diet (2 h) (Fig. 5A, top). In contrast, the amounts of the type IIc Na-Pi cotransporter were not significantly different at 2 h after a switch to a high Pi. Four and six hours after switching to the high-Pi diet, the amounts of the type IIc transporter were significantly decreased (55 and 30% of rats fed a low-Pi diet) (Fig. 5A, middle).
Next, the levels of the type IIa and type IIc transporter protein in the cortical membrane were determined (Fig. 5B). The levels of type IIa Na-Pi cotransporter protein were markedly decreased in rats acutely fed a high-Pi diet (Fig. 5B, top). However, the levels of the type IIc transporter were similar compared with rats acutely fed a high-Pi diet and in animals chronically fed a low-Pi diet (Fig. 5B, middle).
The distribution of type IIc Na-Pi cotransporter in renal cortical membrane was determined following their separation by density gradient centrifugation to further examine the subcellular location of type IIc Na-Pi cotransporters in the proximal tubule. In Fig. 5C, renal cortical microvilli (BBMV) and DM were isolated by centrifugation on an OptiPrep gradient (fractions 5-7) as described in MATERIALS AND METHODS (2). Equal quantities of protein from each preparation were separated by SDS-PAGE and subjected to immunoblotting. The microvillar protein, villin, and the type IIa transporter were enriched in the microvillar fraction and megalin was enriched in the DM as described previously (2). NHE3 and clathrin HC were present in approximately equal amounts in both membrane preparations (2). As shown in Fig. 5C, type IIa and type IIc Na-Pi cotransporters were equal in both membrane preparations in rats fed a chronic low-Pi diet. However, in rats acutely fed a high-Pi diet (6 h), the amount of type IIa Na-Pi cotransporter protein was markedly decreased in both membrane preparations. In contrast, the amount of type IIc Na-Pi cotransporter protein was decreased in the microvillar fraction and was only slightly increased in the dense membrane.
In addition, the levels of type II Na-Pi cotransporter proteins in BBMVs (Fig. 5, left) and cortical membranes (Fig. 5, right) were determined 24 h after administration of high-Pi diet. The levels of type IIc protein in the cortical membrane decreased to 20–30% those observed when rats were fed chronic low-Pi diet.
Immunofluorescence microscopy. Next, we performed immunocytochemical studies using the guinea pig anti-type IIc transporter polyclonal antibody. In rats fed a chronic low-Pi diet, type IIa and type IIc Na-Pi cotransporters colocalized to the apical membranes of the renal proximal cell (Fig. 6, A-C). The rabbit antibody was less useful for detecting the intracellular pool of the type IIc transporter (data not shown), but the guinea pig antibody stained two pools of type IIc transporter, which may be located either at the microvilli or in the intracellular compartment of proximal tubular cells (Fig. 6, B and C). Acute feeding of a high-Pi diet induced translocation of the type IIa transporter from the apical membrane to the intracellular compartments (2 h) (data not shown). In contrast, the localization of type IIc transporter-immunoreactive signals did not change at 2 h after administration of a high-Pi diet (data not shown). Four hours after administration of the high diet, type IIa transporter protein was detected as small dots in the intracellular compartments, probably in association with the Golgi and lysosomal membrane (Fig. 6D). In contrast, the intensity of type IIc transporter-immunoreactive signals was weaker in the apical membrane compared with those observed in rats fed a low-Pi diet (Fig. 6E). Six hours after administration of a high-Pi diet, the immunoreactive signals of the type IIa transporter were no longer present or barely detected in association with the intracellular organelles and were greatly decreased in the lysosomal fraction (Fig. 6G). The type IIc-immunoreactive signals were clearly detected in the intracellular compartment of the proximal tubular cells in rats acutely fed a high-Pi diet (6 h; Fig. 6H).
Localization of intracellular type IIc transporter. To confirm the specificity of the internalization of the type IIc Na-Pi cotransporter, double staining was performed with anti-clathrin heavy-chain (HC) antibody (Fig. 7). The type IIc Na-Pi cotransporter was detected in the apical membrane as described above (Fig. 6). Clathrin HC was detected at the base of the proximal tubule microvilli, as described previously (21). The type IIc Na-Pi cotransporter signals were gradually decreased in the apical membrane of the proximal tubular cells in rats acutely fed a high-Pi diet (4 and 6 h). Furthermore, colocalization of the clathrin HC and type IIc cotransporter was clearly detected in the subapical or the base of the proximal tubule microvilli regions in rats acutely fed a high-Pi diet (4 and 6 h; Fig. 7).
Further immunohistochemical staining for type IIc transporter, Golgi and endosome membranes were performed to determine the localization of the type IIc transporter protein in the rapid adaptation to dietary Pi. The staining of intracellular type IIc transporter protein was not correlated with the localization of the Golgi marker protein (GM130) or of the endosomal marker protein (EEA1). In addition, 24 h after administration of a high-Pi diet, the intensity of intracellular type IIc-immunoreactive signals was markedly decreased in the proximal tubular cells (data not shown).
Involvement of microtubules in downregulation of the type IIc transporter. Staining for -tubulin in control rats revealed a dense network of microtubules spanning the entire cytoplasm of proximal convoluted tubule cell, except for in the BBM (Fig. 8A). The administration of colchicine abolished the microtubules staining (Fig. 8B). Furthermore, colchicine did not prevent downregulation of the type IIa transporter (Fig. 8) (10) but did prevent downregulation of the apical type IIc Na-Pi cotransporter significantly (Fig. 8).
These results suggest that the internalization of the type IIc transporter from the apical membrane to the intracellular compartments is dependent on the microtubule network. Immunohistochemical analysis also showed similar observations (data not shown).
DISCUSSION
Previous studies characterized the phenomenon of transport membrane retrieval with high-Pi diet or medium-induced downregulation of BBMV Na-Pi cotransport activity and type IIa Na-Pi cotransporter levels (7–10, 12–14, 17). We previously demonstrated that type IIc Na-Pi cotransporter is also regulated by dietary Pi manipulation (16, 21) and that expression of the type IIc Na-Pi cotransporter protein was decreased in mice fed a chronic high-Pi diet. The present study showed that acute feeding of a high-Pi diet suppressed type IIc transporter expression in the apical membrane of the proximal tubular cell (S1 segment). Compared with the time course of the internalization of type IIa transporter, internalization of the type IIc transporter was slightly delayed. Previous immunoblotting and immunohistochemical analyses suggested that internalized type IIa Na-Pi cotransporters are directed to the lysosomes for degradation (7, 8, 10, 12–14). In contrast, the type IIc Na-Pi cotransporter was internalized to the intracellular pool but not degraded in the lysosomes. Immunoblot and immunohistochemical analyses have also demonstrated that the type IIc transporter is mainly localized to the subapical (or the base of the proximal tubule microvilli) regions after acute administration of a high-Pi diet. Indeed, type IIc transporter protein levels were not changed in cortical membrane fractions after acute feeding of the high-Pi diet in the present study, whereas type IIa Na-Pi cotransporter levels were significantly decreased. Furthermore, type IIc transporter levels were slightly increased in the subapical regions with a simultaneous decrease in immunoreactive intensity at the apical membrane. These observations suggest that translocation of the type IIc-immunoreactive signal from the entire brush border to the subapical (or base of the proximal tubule microvilli) regions. Furthermore, type IIc cotransporter, clathrin-HC, and megalin were localized to the DM after administration of a high-Pi diet. Further studies to clarify mechanisms of internalization of type IIc Na-Pi cotransporter and the involvement of the clathrin- or megalin-mediated endocytic pathways would be of benefit.
Hence, specific association of other protein and type IIc transporter in the proximal tubule is under study.
The present study also demonstrated that an intact microtubular system is required for the type IIc routing. Disruption of the microtubular network by colchicine prevented the adaptive downregulation of the type IIc transporter in the present study. These findings suggest that the microtubular network is necessary for the redistribution of type IIc Na-Pi cotransporter from the BBM to the intracellular compartment. Apical endocytosis in the renal epithelium is dependent on microtubule-based vesicle transport (4). A study of the role of microtubule in apical endocytosis was performed in renal proximal tubule cells from untreated vs. colchicine-treated rats (4). Comparison of untreated vs. colchicine-treated rats demonstrated that the apical surface of the proximal tubular epithelia contained numerous endocytic invaginations, small vacuoles, and dense apical tubules (which contain contents destined for insertion into apical plasma membrane) in untreated rats, whereas in colchicine-treated rats, the proximal tubular epithelia demonstrated absence of endocytic invaginations, an accumulation of large endocytic vacuoles, and a fivefold reduction in the dense apical tubules (4). Disruption in dense apical tubule function leads to reduction in the outgoing traffic, which leads to a reduction in insertion of apical proteins and promoting subsequent endocytosis (4). Further studies are needed to clarify whether internalization of the type IIc Na-Pi cotransporter is related to establishment of intact dense apical tubules.
The downregulation of type IIa transporter in response to a high-Pi diet occurs via endocytosis of the transporter and its subsequent degradation in lysosomes. Lotscher et al. (10) demonstrated that microtubule dynamics play a central role in the intracellular trafficking of the internalized type IIa transporter. In contrast, the rapid decrease in type IIa transporter levels from BBM was insensitive to microtubular network disruption by colchicines (10). Thus the mechanisms of the internalization of the type IIc transporter appear to be distinct from those of type IIa transporter regulation.
In summary, the present study demonstrated that the type IIc transporter was internalized and localized to intracellular compartments, including clathrin vesicles, following feeding of a high-Pi diet. Furthermore, this process was dependent on intact microtubule networks.
GRANTS
This work was supported by a grant (H. Segawa and K. Miyamoto) from the Ministry of Education, Science, Sports and Culture of Japan and Human Nutritional Science on Stress Control the 21st Century COE Program.
FOOTNOTES
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.
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Department of Structural Pathology Institute of Nephrology Graduate School of Medical and Dental Sciences, Niigata University, Niigata, Japan
ABSTRACT
Dietary phosphate levels regulate the renal brush-border type IIa Na-Pi cotransporter. Another Na-Pi cotransporter, type IIc, colocalizes with type IIa Na-Pi cotransporter in the apical membrane of renal proximal tubular cells. The goal of the present study was to determine whether dietary phosphate levels also rapidly regulate the type IIc Na-Pi cotransporter. Type IIa and type IIc transporter protein levels were increased in rats chronically fed a low-Pi diet compared with those fed a normal-Pi diet. Two hours after beginning a high-Pi diet, type IIa transporter levels were decreased, whereas type IIc protein levels remained unchanged. Western blot analysis of brush-border membrane prepared 4 h after beginning a high-Pi diet showed a significant reduction in type IIc transporter protein levels, and immunohistochemistry showed translocation of the type IIc-immunoreactive signal from the entire brush border to subapical membrane. Membrane fractionation studies revealed a decrease in apical membrane type IIc protein without changes in total cortical type IIc protein, which is compatible with redistribution of type IIc protein from the apical membrane to the dense membrane fraction. The microtubule-disrupting reagent colchicine prevented this reduction in apical type IIc transporter at the apical membrane but had no effect on type IIa transporter levels. These data suggest that the type IIc Na-Pi cotransporter level is rapidly regulated by rapid adaptation to dietary Pi in a microtubule-dependent manner. Furthermore, the mechanisms of the internalization of the type IIc transporter are distinct from those of the type IIa transporter.
proximal tubule; dietary phosphate; regulation
INORGANIC PHOSPHATE (Pi) reabsorption in the renal proximal tubule is required for body Pi homeostasis (12–15). Evidence from physiological studies suggests that Na-dependent Pi transporters in the brush-border membrane (BBM) of proximal tubular cells mediate the rate-limiting step in the overall Pi-reabsorptive process (13, 14). Although the type IIa and type IIc Na-Pi cotransporters are expressed in the apical membrane of the proximal tubular cells and mediate Na-Pi cotransport (11–16, 21), the extent of Pi reabsorption in the proximal tubules is determined largely by the abundance of the type IIa Na-Pi cotransporter (12–15). In contrast, in weaning animals, the levels of type IIc Na-Pi cotransporter are increased, indicating that type IIc may play a role in the regulation of Pi reabsorption during growth (16, 21).
We previously demonstrated that expression of the type IIc Na-Pi cotransporter is increased in renal proximal tubular cells and that 30% of renal Na-Pi cotransport activity in Pi-deficient mice is mediated by the type IIc Na-Pi cotransporter (16). Furthermore, we reported that renal BBM type IIc protein levels were significantly increased in Npt2–/– mice compared with wild-type (Npt2+/+) littermates. Thus the type IIc Na-Pi cotransporter may account for the residual renal BBM Na-Pi cotransport in the type IIa knockout model (22).
Two major regulators of renal Pi reabsorption are dietary Pi and parathyroid hormone (PTH). Restriction of dietary Pi is associated with an adaptive increase in the overall capacity of the proximal tubule to reabsorb Pi. Reduced Pi reabsorption is achieved by a removal of the type IIa transporter from the apical membrane (5, 7–10, 12–15, 17). A high-Pi diet leads to transient accumulation of type IIa Na-Pi cotransporters in the subapical vacuolar apparatus (5, 10, 13, 14). Morphological and biochemical data suggest that internalized type IIa Na-Pi cotransporters are then directed to the lysosomes for degradation (8, 10, 24). Previous studies also suggest that microtubules are involved in this adaptive response (24). In addition, membrane insertion of the type IIa transporter, observed after acute administration of a low-Pi diet, requires an intact microtubular network (10). The goal of the present study was to determine whether the type IIc Na-Pi cotransporter undergoes rapid adaptation in response to dietary Pi manipulation.
MATERIALS AND METHODS
Animals and diets. Male Wistar rats (5 wk after birth) were purchased from SLC (Shizuoka, Japan), housed in plastics cages, and fed standard rat chow (Oriental, Osaka, Japan) ad libitum for 1 wk. After this period, a diet containing 0.6% calcium and 0.6% phosphorus (Pi) was administered for 5 days. On day 6, rats were either fed a diet containing 0.02% Pi. All animals were trained to consume their diets between 10 AM and 12 PM daily. On day 8, the following experimental groups were established: 1) chronic low-Pi diet group (CLP): rats were only fed a 0.02% Pi diet; 2) acute high-Pi diet groups (AHP): rats were chronically fed 0.02% Pi diet and, on the day of experiment, were acutely fed 1.2% Pi diet for 2, 4, 6, or 24 h. To study the role of microtubules in translocation of the type IIc by the acute high-Pi diet, rats were trained and adapted to the high-Pi diet as described above. On day 8, the rats were treated with colchicine (1.0 mg/kg body wt ip dissolved in saline) or vehicle (saline) 6 h before animal death and then fed a high-Pi diet 2, 4, or 6 h before animal death.
Experimental groups consisted of four or five individual rats for the BBM or cortical membrane isolation and of four individual rats for immunohistochemistry experiments.
Anti-peptide antibody. Oligopeptide CYENPQVIASQQL [corresponding to residues of rat type IIc Na-Pi cotransporter (589–601)] was synthesized. The NH2-terminal cysteine residues were introduced for conjugation with keyhole limpet hemocyanin. Guinea pigs were immunized with the peptide to prepare anti-type IIc Na-Pi cotransporter antibody, as previously described for preparation of rabbit anti-type IIa or type IIc Na-Pi cotransporter peptide antibodies (16, 21).
Preparation of membrane fraction. Methods for preparation of BBM vesicles (BBMVs) using Ca2+ precipitation and cortical membranes were described previously (7, 16, 21, 23).
The density separation of cellular membranes was accomplished by isopycnic centrifugation using OptiPrep (Nycomed Pharma, Oslo, Norway) density gradients (2). OptiPrep was diluted to appropriate concentrations from stocks using 20 mM Tricine (pH 8.7) and 8% sucrose according to the manufacturer’s protocols (2). Preformed OptiPrep gradients were made using a Gradient Maker (Pharmacia Biotech). One-five milligrams of cortical membranes in 5% OptiPrep were layered on the top of 15–25% OptiPrep gradients. Gradients were centrifuged to equilibrium (at least 2 h) at 100,000 g using an SW rotor in a HITACHI ultracentrifuge. One-milliliter fractions were manually collected from the top of the gradient preparation. Fractions 5-7 were used as the dense membranes (DM) for analysis by immunoblotting (2).
Immunoblotting. Protein samples were heated at 95°C for 5 min in sample buffer in the presence of 5% 2-mercaptoethanol and were subjected to SDS-polyacrylamide gel electrophoresis. The separated proteins were transferred electrophoretically to polyvinylidene difluoride transfer membranes (Hybond-P Amersham Pharmacia Biotech). The membranes were treated with diluted rabbit affinity-purified anti-type IIc (1:1,000), guinea pig affinity-purified anti-type IIc (1:500), or anti-type IIa (1:4,000) Na-Pi cotransporter antibodies, followed by treatment with horseradish peroxidase (HRP)-conjugated anti-rabbit or guinea pig IgG as the secondary antibody (Jackson ImmunoResearch Laboratories). Rabbit polyclonal antibody to megalin (1:10,000) was kindly provided by Dr. Saito (Niigata University, Niigata, Japan). Mouse monoclonal antibodies to clathrin heavy-chain (BD Transduction Laboratories; 1:10,000), villin (CHEMICON; 1:8,000), or NHE3 (CHEMICON; 1:3,000) were used, followed by treatment with HRP-conjugated anti-mouse IgG as secondary antibody (Jackson ImmunoResearch Laboratories). Signals were detected using the ECL Plus system (Amersham Pharmacia Biotech).
Immunohistochemistry. Rats were anesthetized with pentobarbital sodium (100 mg/kg body wt), and their kidneys were perfused via the left ventricle with PBS followed by 4% paraformaldehyde solution (pH 7.2) (16, 21). Kidneys were postfixed with the same solution overnight at 4°C, washed with PBS, cryoprotected with 10 and 20% sucrose at 4°C, and embedded in O.C.T. compound (Miles, Elkart), and frozen in hexane at –80°C. Frozen sections (5 μm) were collected onto silane-coated slides and air-dried. For immunofluorescence microscopy, serial sections were incubated with rabbit anti-type IIa (1:4,000), guinea pig anti-type IIc (1:200) Na-Pi cotransporter antibody overnight at 4°C. A mouse monoclonal antibody to clathrin heavy-chain (BD Transduction Laboratories; 1:1,000), EEA1 (BD Transduction Laboratories; 1:100), or GM130 (BD Transduction Laboratories; 1:100) and affinity-purified rabbit antibodies against anti-4F2 heavy chain (4F2hc)/CD98 antibody (1:1,000; 4F2hc antibody was kindly provided by Dr. Kanai, Kyorin University, Tokyo, Japan) were used for localization of the type IIc Na-Pi cotransporter. The Alexa Fluor568 anti-rabbit or mouse IgG (Molecular Probes) or the Alexa Fluor 488 anti-guinea pig IgG (Molecular Probes) was used as the secondary antibody, with incubation for 60 min at room temperature (16).
Data analysis. Data are expressed as means ± SE. Differences among multiple groups were analyzed by ANOVA. Differences between two experimental groups were determined by ANOVA followed by the Student’s t-test. P values < 0.05 were considered statistically significant.
RESULTS
Validation of guinea pig type IIc Na-Pi cotransporter antiserum. Western blot analyses were performed to determine whether type IIc proteins could be detected in BBMV prepared from rat kidney (Fig. 1). Rabbit type IIc antibody recognized two major polypeptide fragments with an apparent molecular mass of 150–160 and 75–80 kDa in the absence of 2-mercaptoethanol (2Me) (nonreducing condition). In the presence of 2Me (reducing condition), the 75- to 80-kDa band was prominent, suggesting that the 150- to 160-kDa band corresponds to a homodimeric complex. These bands (150–160 and 75–80 kDa) disappeared with absorption experiments, as described previously (Fig. 1A) (21). Similar results were observed with antibodies prepared from guinea pig COOH-terminal antibody.
Guinea pig type IIc COOH-terminal antibody recognized a major polypeptide fragment with an apparent molecular mass of 75–80 kDa in both the absence and presence of 2Me. The 150- to 160-kDa band was faint but present in the absence of the 2Me (Fig. 1B). Preincubation of guinea pig antibody with the appropriate immunizing synthetic peptide used for immunization prevented visualization of the respective bands on immunoblot (Fig. 1C). These data validate the reactivity and specificity of the antibodies employed in this study.
Expression of the type IIc Na-Pi cotransporter in proximal tubules of renal cortex. In mice, type IIc Na-Pi cotransporter-immunoreactive signals increase in the kidney after administration of a low-Pi diet (16, 21). In rats fed a normal-Pi diet, type IIc-immunoreactive signals were weaker in superficial nephrons compared with deep nephrons (Fig. 2A). In contrast, the intensity of type IIc-immunoreactive signals was clearly increased in the apical membrane of the proximal tubular cells in superficial and deep nephrons in rats chronically fed a low-Pi diet (Fig. 2B). Staining was absent in the outer and inner medulla and in the papilla.
To confirm localization of the type IIc Na-Pi cotransporter protein, the 4F2 heavy chain (4F2hc) was immunodetected. In the kidney, 4F2hc-immunoreactive signals were restricted to the proximal tubule at the lateral side and were most prominent in the initial portion of the proximal tubule (S1 segment) (20). Furthermore, type IIc Na-Pi cotransporter colocalized with the 4F2hc protein in the S1 segment (Fig. 3). No specific fluorescence of 4F2hc immunoreactivity was observed in the glomerulus or other tubular segments.
Adaptation of type IIc Na-Pi cotransporter in the kidney to dietary Pi. In rats chronically fed a low-Pi diet, the expression of type II Na-Pi cotransporters was observed in the proximal tubules of superficial and deep nephrons (Fig. 4). Type IIa transporter localized to the apical membrane of proximal tubules of all segment (S1, S2, S3) of rats fed a low-Pi diet (Fig. 4, A and D), whereas type IIc Na-Pi cotransporter localized to the apical membrane of proximal tubular cells in the S1 segment (Fig. 4, B and E).
Western blot analysis of the type IIc Na-Pi cotransporter in BBMV, cortical membrane, or DM fraction from the kidney of rats acutely fed a high-Pi diet. Acute feeding with a high-Pi diet induced translocation of the type IIa transporter from the apical membrane to the intracellular compartments as described previously (7, 8, 10). Next, adaptation of high-Pi diet on expression of type IIc Na-Pi cotransporter was examined via Western blot analysis of the type IIa and type IIc Na-Pi cotransporter proteins (Fig. 5). In BBMVs, the amount of the type IIa transporter protein at 2 h after switching to the high-Pi diet was 10% of that seen in rats chronically fed a low-Pi diet. Furthermore, the levels of the type IIa transporter were much lower at 4 and 6 h after the switch to a high-Pi diet than in rats fed a high-Pi diet (2 h) (Fig. 5A, top). In contrast, the amounts of the type IIc Na-Pi cotransporter were not significantly different at 2 h after a switch to a high Pi. Four and six hours after switching to the high-Pi diet, the amounts of the type IIc transporter were significantly decreased (55 and 30% of rats fed a low-Pi diet) (Fig. 5A, middle).
Next, the levels of the type IIa and type IIc transporter protein in the cortical membrane were determined (Fig. 5B). The levels of type IIa Na-Pi cotransporter protein were markedly decreased in rats acutely fed a high-Pi diet (Fig. 5B, top). However, the levels of the type IIc transporter were similar compared with rats acutely fed a high-Pi diet and in animals chronically fed a low-Pi diet (Fig. 5B, middle).
The distribution of type IIc Na-Pi cotransporter in renal cortical membrane was determined following their separation by density gradient centrifugation to further examine the subcellular location of type IIc Na-Pi cotransporters in the proximal tubule. In Fig. 5C, renal cortical microvilli (BBMV) and DM were isolated by centrifugation on an OptiPrep gradient (fractions 5-7) as described in MATERIALS AND METHODS (2). Equal quantities of protein from each preparation were separated by SDS-PAGE and subjected to immunoblotting. The microvillar protein, villin, and the type IIa transporter were enriched in the microvillar fraction and megalin was enriched in the DM as described previously (2). NHE3 and clathrin HC were present in approximately equal amounts in both membrane preparations (2). As shown in Fig. 5C, type IIa and type IIc Na-Pi cotransporters were equal in both membrane preparations in rats fed a chronic low-Pi diet. However, in rats acutely fed a high-Pi diet (6 h), the amount of type IIa Na-Pi cotransporter protein was markedly decreased in both membrane preparations. In contrast, the amount of type IIc Na-Pi cotransporter protein was decreased in the microvillar fraction and was only slightly increased in the dense membrane.
In addition, the levels of type II Na-Pi cotransporter proteins in BBMVs (Fig. 5, left) and cortical membranes (Fig. 5, right) were determined 24 h after administration of high-Pi diet. The levels of type IIc protein in the cortical membrane decreased to 20–30% those observed when rats were fed chronic low-Pi diet.
Immunofluorescence microscopy. Next, we performed immunocytochemical studies using the guinea pig anti-type IIc transporter polyclonal antibody. In rats fed a chronic low-Pi diet, type IIa and type IIc Na-Pi cotransporters colocalized to the apical membranes of the renal proximal cell (Fig. 6, A-C). The rabbit antibody was less useful for detecting the intracellular pool of the type IIc transporter (data not shown), but the guinea pig antibody stained two pools of type IIc transporter, which may be located either at the microvilli or in the intracellular compartment of proximal tubular cells (Fig. 6, B and C). Acute feeding of a high-Pi diet induced translocation of the type IIa transporter from the apical membrane to the intracellular compartments (2 h) (data not shown). In contrast, the localization of type IIc transporter-immunoreactive signals did not change at 2 h after administration of a high-Pi diet (data not shown). Four hours after administration of the high diet, type IIa transporter protein was detected as small dots in the intracellular compartments, probably in association with the Golgi and lysosomal membrane (Fig. 6D). In contrast, the intensity of type IIc transporter-immunoreactive signals was weaker in the apical membrane compared with those observed in rats fed a low-Pi diet (Fig. 6E). Six hours after administration of a high-Pi diet, the immunoreactive signals of the type IIa transporter were no longer present or barely detected in association with the intracellular organelles and were greatly decreased in the lysosomal fraction (Fig. 6G). The type IIc-immunoreactive signals were clearly detected in the intracellular compartment of the proximal tubular cells in rats acutely fed a high-Pi diet (6 h; Fig. 6H).
Localization of intracellular type IIc transporter. To confirm the specificity of the internalization of the type IIc Na-Pi cotransporter, double staining was performed with anti-clathrin heavy-chain (HC) antibody (Fig. 7). The type IIc Na-Pi cotransporter was detected in the apical membrane as described above (Fig. 6). Clathrin HC was detected at the base of the proximal tubule microvilli, as described previously (21). The type IIc Na-Pi cotransporter signals were gradually decreased in the apical membrane of the proximal tubular cells in rats acutely fed a high-Pi diet (4 and 6 h). Furthermore, colocalization of the clathrin HC and type IIc cotransporter was clearly detected in the subapical or the base of the proximal tubule microvilli regions in rats acutely fed a high-Pi diet (4 and 6 h; Fig. 7).
Further immunohistochemical staining for type IIc transporter, Golgi and endosome membranes were performed to determine the localization of the type IIc transporter protein in the rapid adaptation to dietary Pi. The staining of intracellular type IIc transporter protein was not correlated with the localization of the Golgi marker protein (GM130) or of the endosomal marker protein (EEA1). In addition, 24 h after administration of a high-Pi diet, the intensity of intracellular type IIc-immunoreactive signals was markedly decreased in the proximal tubular cells (data not shown).
Involvement of microtubules in downregulation of the type IIc transporter. Staining for -tubulin in control rats revealed a dense network of microtubules spanning the entire cytoplasm of proximal convoluted tubule cell, except for in the BBM (Fig. 8A). The administration of colchicine abolished the microtubules staining (Fig. 8B). Furthermore, colchicine did not prevent downregulation of the type IIa transporter (Fig. 8) (10) but did prevent downregulation of the apical type IIc Na-Pi cotransporter significantly (Fig. 8).
These results suggest that the internalization of the type IIc transporter from the apical membrane to the intracellular compartments is dependent on the microtubule network. Immunohistochemical analysis also showed similar observations (data not shown).
DISCUSSION
Previous studies characterized the phenomenon of transport membrane retrieval with high-Pi diet or medium-induced downregulation of BBMV Na-Pi cotransport activity and type IIa Na-Pi cotransporter levels (7–10, 12–14, 17). We previously demonstrated that type IIc Na-Pi cotransporter is also regulated by dietary Pi manipulation (16, 21) and that expression of the type IIc Na-Pi cotransporter protein was decreased in mice fed a chronic high-Pi diet. The present study showed that acute feeding of a high-Pi diet suppressed type IIc transporter expression in the apical membrane of the proximal tubular cell (S1 segment). Compared with the time course of the internalization of type IIa transporter, internalization of the type IIc transporter was slightly delayed. Previous immunoblotting and immunohistochemical analyses suggested that internalized type IIa Na-Pi cotransporters are directed to the lysosomes for degradation (7, 8, 10, 12–14). In contrast, the type IIc Na-Pi cotransporter was internalized to the intracellular pool but not degraded in the lysosomes. Immunoblot and immunohistochemical analyses have also demonstrated that the type IIc transporter is mainly localized to the subapical (or the base of the proximal tubule microvilli) regions after acute administration of a high-Pi diet. Indeed, type IIc transporter protein levels were not changed in cortical membrane fractions after acute feeding of the high-Pi diet in the present study, whereas type IIa Na-Pi cotransporter levels were significantly decreased. Furthermore, type IIc transporter levels were slightly increased in the subapical regions with a simultaneous decrease in immunoreactive intensity at the apical membrane. These observations suggest that translocation of the type IIc-immunoreactive signal from the entire brush border to the subapical (or base of the proximal tubule microvilli) regions. Furthermore, type IIc cotransporter, clathrin-HC, and megalin were localized to the DM after administration of a high-Pi diet. Further studies to clarify mechanisms of internalization of type IIc Na-Pi cotransporter and the involvement of the clathrin- or megalin-mediated endocytic pathways would be of benefit.
Hence, specific association of other protein and type IIc transporter in the proximal tubule is under study.
The present study also demonstrated that an intact microtubular system is required for the type IIc routing. Disruption of the microtubular network by colchicine prevented the adaptive downregulation of the type IIc transporter in the present study. These findings suggest that the microtubular network is necessary for the redistribution of type IIc Na-Pi cotransporter from the BBM to the intracellular compartment. Apical endocytosis in the renal epithelium is dependent on microtubule-based vesicle transport (4). A study of the role of microtubule in apical endocytosis was performed in renal proximal tubule cells from untreated vs. colchicine-treated rats (4). Comparison of untreated vs. colchicine-treated rats demonstrated that the apical surface of the proximal tubular epithelia contained numerous endocytic invaginations, small vacuoles, and dense apical tubules (which contain contents destined for insertion into apical plasma membrane) in untreated rats, whereas in colchicine-treated rats, the proximal tubular epithelia demonstrated absence of endocytic invaginations, an accumulation of large endocytic vacuoles, and a fivefold reduction in the dense apical tubules (4). Disruption in dense apical tubule function leads to reduction in the outgoing traffic, which leads to a reduction in insertion of apical proteins and promoting subsequent endocytosis (4). Further studies are needed to clarify whether internalization of the type IIc Na-Pi cotransporter is related to establishment of intact dense apical tubules.
The downregulation of type IIa transporter in response to a high-Pi diet occurs via endocytosis of the transporter and its subsequent degradation in lysosomes. Lotscher et al. (10) demonstrated that microtubule dynamics play a central role in the intracellular trafficking of the internalized type IIa transporter. In contrast, the rapid decrease in type IIa transporter levels from BBM was insensitive to microtubular network disruption by colchicines (10). Thus the mechanisms of the internalization of the type IIc transporter appear to be distinct from those of type IIa transporter regulation.
In summary, the present study demonstrated that the type IIc transporter was internalized and localized to intracellular compartments, including clathrin vesicles, following feeding of a high-Pi diet. Furthermore, this process was dependent on intact microtubule networks.
GRANTS
This work was supported by a grant (H. Segawa and K. Miyamoto) from the Ministry of Education, Science, Sports and Culture of Japan and Human Nutritional Science on Stress Control the 21st Century COE Program.
FOOTNOTES
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.
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