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【关键词】 phosphate
Department of Medicine, Department of Physiology, University of Maryland School of Medicine, and Medical Service, Department of Veterans Affairs Medical Center, Baltimore, Maryland
Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina
ABSTRACT
The present experiments using primary cultures from renal proximal tubule cells examine two aspects of the regulation of sodium-dependent phosphate transport and membrane sodium-dependent phosphate transporter (Npt2a) expression by parathyroid hormone (PTH). Sodium-dependent phosphate transport in proximal tubule cells from wild-type mice grown in normal-phosphate media averaged 4.4 ± 0.5 nmol?mg protein1?10 min1 and was inhibited by 30.5 ± 8.6% by PTH (107 M). This was associated with a 32.7 ± 5.2% decrease in Npt2a expression in the plasma membrane. Proximal tubule cells from Na+/H+ exchanger regulatory factor-1 (NHERF-1)/ mice had a lower rate of phosphate transport compared with wild-type cells and a significantly reduced inhibitory response to PTH. Wild-type cells incubated in low-phosphate media for 24 h had a higher rate of phosphate transport compared with wild-type cells grown in normal-phosphate media but a significantly blunted inhibitory response to PTH. These data indicate a role for NHERF-1 in mediating the membrane retrieval of Npt2a and the subsequent inhibition of phosphate transport in renal proximal tubules. These studies also suggest that there is a blunted phosphaturic effect of PTH in cells adapted to low-phosphate media.
Na+/H+ exchanger regulatory factor-1; renal phosphate transport; sodium-dependent phosphate transporter IIa; mouse kidney
THE SODIUM-DEPENDENT PHOSPHATE transporter IIa (Npt2a, NaPi IIa) located in the apical membrane of the renal proximal convoluted tubule is the major determinant of urinary phosphate excretion (11, 22, 30). This transporter is regulated by physiological stimuli such that the abundance of Npt2a in the apical membrane is increased in response to dietary restriction of phosphate and decreased in response to parathyroid hormone (PTH) (1, 3, 9, 17, 18, 21, 25). The processes that determine the trafficking of Npt2a to and from the apical brush-border membrane (BBM), however, have not been completely elucidated. While the transporter has a number of modifiable sites on its COOH terminus, phosphorylation of these residues has little influence on its cellular distribution or activity. These findings resulted in a continued search for regulatory factors and the identification of two novel pathways. One regulatory pathway involves a series of proteins collectively called phosphatonins (30). The other pathway ensued the recognition that the COOH terminus of Npt2a constituted a class 1 PDZ binding motif (8, 12, 27). The renal proximal tubule expresses at least three PDZ proteins that bind Npt2a, namely, Na+/H+ exchanger regulatory factor-1 (NHERF-1), NHERF-2, and PDZK1. Prior studies have indicated that disruption of the NHERF-1 interaction with ezrin resulted in loss of Npt2a from the apical membrane surface of opossum kidney (OK) cells (12). Moreover, compared with wild-type mice, NHERF-1 null mice demonstrate hypophosphatemia and an increase in the urinary excretion of phosphate associated with decreased abundance of Npt2a in the apical membrane of the cells of the proximal tubule (27).
The present experiments were designed to address two questions related to Npt2a trafficking and phosphate transport in response to PTH. In OK cells using an ezrin binding domain-deficient NHERF-1 as a dominant-negative reagent, we found that basal phosphate transport was decreased but that the response to PTH remained intact (17). On the other hand, Mahon and co-workers (21), using a subclone of OK cells that had reduced NHERF-1, found a striking decrease in the inhibitory effect of PTH on phosphate transport. OK cells, in contrast to proximal renal tubules of mice, express only NHERF-1. Accordingly, the first objective of the present experiments was to study the role of NHERF-1 in modulating the effect of PTH on phosphate transport and membrane expression of Npt2a using primary proximal tubule cell cultures from wild-type and NHERF-1/ mice. The second goal of the present experiments was to determine the effect of PTH on phosphate transport in proximal tubule cells from wild-type mice adapted to growth in low-phosphate media. A number of prior studies in intact animals have suggested that adaptation to a low-phosphate diet decreases the phosphaturic effect of PTH (23, 29, 36). The mechanism of this response is unknown but may reflect increased trafficking of Npt2a to the apical membrane in response to phosphate restriction (3, 18). While it is not certain that the biochemical pathways are identical, incubation of proximal tubule-like cells in culture in low-phosphate media also increases Npt2a membrane expression and phosphate uptake. In intact animals, the changes in transport and BBM expression of Npt2a could be the result of specific signals read by proximal tubule cells or the result of systemic and/or hormonal changes associated with reduced dietary intake of phosphate. To address this question, we have examined the effect of PTH in cultured wild-type renal proximal tubule cells from mice that have been adapted to low-phosphate media. The results of these experiments demonstrate that the inhibitory effect of PTH is blunted in NHERF-1 null cells, indicating a role for this adaptor protein in the PTH signaling pathways that regulate Npt2a expression. In addition, we demonstrate that the inhibitory effect of PTH on phosphate transport and Npt2a expression is decreased in wild-type proximal tubule cells adapted to low-phosphate media. This suggests that the mechanism of the blunted phosphaturic effect of PTH in animals on a low-phosphate diet can be explained, at least in part, by biochemical changes in the cells of the proximal tubule.
MATERIALS AND METHODS
Animals and preparation of renal proximal tubule cells. Male NHERF-1/ mice (B6.129-Slc9a3r1tmSsl/Ss1) bred into a C57BL/6 background for six generations and parental wild-type inbred control C57BL/6 mice age 1216 wk were used in the current experiments (27). All animal protocols and procedures were approved by Institutional Animal Care and Use Committee of the University of Maryland School of Medicine. Primary renal proximal tubule cell cultures were prepared as recently described from this laboratory (5). Kidney cortices were dissected, minced, and digested using 1% Worthington collagenase type II and 0.25% soybean trypsin inhibitor. The samples were then resuspended in 35 ml of 45% Percoll and centrifuged at 26,891 g for 15 min at 4°C. The 5- to 10-ml layer containing the proximal tubule cells was aspirated, centrifuged, washed to remove the remaining Percoll, and resuspended in 610 ml of DMEM/F-12 containing 50 U/ml of penicillin, 50 μg/ml streptomycin, 10 ng/ml epidermal growth factor, 0.5 μM hydrocortisone, 0.87 μM bovine insulin, 50 μM prostaglandin E1, 50 nM sodium selenite, 50 μg/ml human transferrin, and 5 pM 3,3',5-triiodo-L-thyronine. The proximal tubule cells were plated on Matrigel-coated coverslips or plastic cell culture dishes coated with Matrigel and maintained in an incubator at 37°C in 5% CO2. The cultures were left undisturbed for 36 h, after which the media was replaced every 2 days until the cells achieved confluence.
Transport assays. Phosphate transport was measured by determination of the sodium-dependent uptake of radiolabeled phosphate (5, 17). Except in the phosphate adaptation studies, the cells were grown in DMEM/F-12 containing 0.9 mM phosphate. In the adaptation experiments, cells were grown in DMEM/F-12 containing 0.3 mM phosphate for 24 h. One-half of the wells of a given culture were incubated with DMSO for 2 h, whereas the other half were treated with PTH (107 M) for the same duration. The cells were then washed in serum-free medium, followed by incubation in a transport medium containing (in mM) 137 NaCl or 137 tetramethylammonium chloride (TMA-Cl), 5.4 KCl, 2.8 CaCl2, 1.2 MgSO4, and 0.1 KH2PO4. Phosphate uptake was determined by the addition of transport medium containing 32P-radiolabeled orthophosphate. Uptake was continued for 10 min at room temperature, after which the cells were washed with ice-cold medium in which TMA-Cl was substituted for sodium chloride, 32P was omitted, and 0.5 mM sodium arsenate was added. The uptake of 32P from the TMA-Cl solution was used to determine sodium-independent uptake and was subtracted to yield the sodium-dependent uptake of 32P. The cells were solubilized in 1% Triton X-100 for 90 min at 4°C, and an aliquot was analyzed by liquid scintillation spectroscopy. Each assay was performed in triplicate and averaged to provide a single data point.
Other methods. To obtain membrane preparations from the cultured cells, the cells were washed with sterile ice-cold phosphate-buffered saline, detached by scraping, and centrifuged for 5 min at 800 g. The supernatant was discarded, and the pellet was resuspended in 1.5 ml of buffer containing 50 mM Tris (pH 7.4), 0.1 mM EDTA, 0.1% -mercaptoethanol, and Complete Protease Inhibitor Cocktail (Roche Applied Science). The cells were then disrupted by three 20-s bursts from a probe sonicator followed by 10-min centrifugation at 1,000 g to remove large particulates. This supernatant was ultracentrifuged for 1 h at 100,000 g. The pellet was then resuspended in 0.1% SDS and prepared for electrophoresis by the addition of Laemmli buffer. Western immunoblotting was performed using antibodies specific for Npt2a and ezrin (5).
The production of intracellular cAMP in cultured cells in response to 107 M PTH was measured by nonacetylation enzyme immunoassay (cAMP Biotrak Assay Kit, Amersham) in the presence of 0.4 mM 3-isobutyl-1-methylxanthine. PKC activity was assayed using Promega's SignaTECT PKC Assay System containing a specific PKC substrate and capture membrane. ERK activation was determined using a rabbit polyclonal antibody specific for ERK1/2 phosphorylated at tyrosine 204. Total ERK1/2 loading was determined using an anti-ERK1/2 antibody.
Protein concentrations were determined by the method of Lowry et al. (19). Densitometry scanning of protein-stained gels and of ezrin was used to confirm equal loading. Statistical comparisons were performed using Peritz' ANOVA (10).
RESULTS
The primary proximal tubule cells in culture had a morphological appearance similar to that recently reported from this laboratory (5). In control cells grown in DMEM/F-12 containing 0.9 mM phosphate, sodium-dependent phosphate uptake averaged 4.4 ± 0.5 nmol?mg protein1?10 min1 and was decreased by 30.5 ± 8.6 to 3.1 ± 0.6% (P < 0.05) after treatment with PTH (107 M) for 2 h (Table 1). Sodium-independent phosphate transport averaged 0.47 ± 0.10 and 0.44 ± 0.07 nmol?mg protein1?10 min1 in the absence or presence of PTH, respectively, indicating that PTH inhibited the sodium-dependent component of phosphate uptake. The decrease in PTH-associated sodium-dependent phosphate transport was associated with a 32.7 ± 5.2% decrease in membrane expression of Npt2a (Fig. 1, Table 2). Sodium-dependent phosphate transport in proximal tubule cells from NHERF-1/ mice grown in DMEM/F-12 was significantly lower than in wild-type cells grown in the same media and averaged 2.9 ± 0.4 nmol?mg protein1?10 min1 (P < 0.05) (Table 1). Treatment of the NHERF-1/ cells with PTH for 2 h resulted in a small but significant decrease in sodium-dependent phosphate uptake to 2.7 ± 0.4 nmol?mg protein1?10 min1 (P < 0.05; Table 1). There were no significant differences in sodium-independent phosphate transport, which averaged 0.47 ± 0.10 and 0.44 ± 0.07 nmol?mg protein1?10 min1 in the absence or presence of PTH, respectively. The decrease in sodium-dependent phosphate transport was associated with a nonsignificant decrease in membrane expression of Npt2a of 12.8 ± 6.3% (Fig. 1, Table 2). The percent decrease in Npt2a expression in response to PTH was significantly less in NHERF-1/ proximal tubule cells compared with wild-type cells (P < 0.05).
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Animals fed a low-phosphate diet have been reported to have a blunted phosphaturic response to PTH (23, 29, 36). To determine whether this response was intrinsic to the cells, wild-type cells were incubated in low-phosphate media (0.3 mM phosphate) for 24 h. Our prior studies indicated that the adaptation to low-phosphate media is complete by 24 h. Sodium-independent phosphate transport did not differ between wild-type proximal tubule cells grown in DMEM/F-12 (0.39 ± 0.09 and 0.34 ± 0.08 nmol?mg protein1?10 min1 in the absence or presence of PTH, respectively) and wild-type cells grown in low-phosphate media (0.32 ± 0.06 and 0.39 ± 0.10 nmol?mg protein1?10 min1 in the absence or presence of PTH, respectively). Compared with wild-type cells incubated in DMEM/F-12, cells grown in low-phosphate media had a higher rate of sodium-dependent phosphate transport of 5.7 ± 0.3 nmol?mg protein1?10 min1 (P < 0.05; Table 1). Treatment of phosphate-adapted wild-type cells with PTH resulted in a small but significantly lower rate of sodium-dependent phosphate transport of 5.0 ± 0.4 nmol?mg protein1?10 min1 (P < 0.05; Table 1) associated with a significant decrease in Npt2a expression in the membrane of 12.5 ± 5.0% (P < 0.05; Fig. 1, Table 2). The percent decrease in Npt2a expression in response to PTH was significantly less in the low-phosphate media-adapted proximal tubule cells compared with wild-type cells grown in DMEM/F-12 (P < 0.05). We also examined NHERF-1/ cells grown in low-phosphate media. Sodium-dependent phosphate transport averaged 2.8 ± 0.2 nmol?mg protein1?10 min1 in the absence of PTH and 2.50 ± 0.2 nmol?mg protein1?10 min1 in the presence of the hormone (P = not significant).
In parallel studies, the signaling pathways of the parathyroid 1R receptor (PTH1R) were examined. PTH (107 M) stimulated cAMP generation 43.3 ± 6.8-fold and PKC activity by 36.6 ± 0.8% in wild-type proximal tubule cells grown in DMEM/F-12 (Table 3). The response to PTH was not significantly different in NHERF-1 null proximal tubule cells grown in the same media in which PTH stimulated cAMP generation 42.7 ± 6.2-fold and PKC activity by 32.3 ± 3.3%. In wild-type cells grown in the low-phosphate media, PTH stimulated cAMP generation by 45.8 ± 6.5-fold and PKC activity by 33.2 ± 2.2%. These values were not significantly different from the results obtained in wild-type cells grown in DMEM/F-12 (Table 3). Prior studies in OK cells have indicated that PTH significantly increases activation of ERK1/2 (2, 4, 14, 16). In the current studies, PTH stimulated ERK1/2 phosphorylation in OK cells by 1.6 ± 0.3 fold. By contrast, PTH did not stimulate ERK1/2 phosphorylation at 1530 min in wild-type mouse proximal tubule cells grown in normal phosphate media but actually inhibited ERK1/2 by 27.5 ± 4.2%. Similar results were observed in NHERF-1/ cells grown in normal phosphate media where ERK1/2 phosphorylation was decreased by 26.9 ± 7.7%. In wild-type cells grown in low-phosphate media PTH inhibited ERK1/2 activation by 20.5 ± 3.7% (Table 3).
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DISCUSSION
The factors that regulate BBM Npt2a expression and, as a consequence, renal phosphate excretion are under intense study. Recently, a role for the PDZ domain adaptor protein NHERF-1 in the regulation of Npt2a expression has been advanced (8, 12, 27). Initially, NHERF-1 was identified as an interacting protein in a yeast two-hybrid screen using the Npt2a tail as bait (8). Consistent with these findings, NHERF-1/ mice demonstrated hypophosphatemia, an increase in the urinary excretion of phosphate, and decreased Npt2a expression in BBM of the renal proximal tubule (27). In intact animals, we have recently demonstrated that the adaptive response to a low-phosphate diet is impaired in NHERF-1/ mice (35). Moreover, we have reported that NHERF-1 was required for the adaptive increase in sodium-dependent phosphate transport in primary cell cultures of renal proximal tubule cells of mice grown in low-phosphate media (5). In the present experiments, we address two aspects of the regulation of phosphate transport by PTH in the renal proximal tubule, namely, the role of NHERF-1 and the effect of adaptation to low-phosphate media.
Two prior studies have addressed the potential role of NHERF-1 on the inhibitory effect of PTH on Npt2a expression and sodium-dependent phosphate transport (17, 21). In OK cells, expression of an ezrin binding domain-deficient NHERF-1 as a dominant-negative reagent resulted in decreased basal phosphate transport but no change in the relative magnitude of the inhibitory effect of PTH (17). By contrast, more recent studies using a subclone of OK cells that expresses low levels of NHERF-1 demonstrated a blunted effect of PTH (21). OK cells, while a favored proximal tubule cell model for study of PTH signaling and phosphate transport, may not mirror events in all proximal tubule cells. Like the proximal tubule of the rat, OK cells express NHERF-1 but not NHERF-2 (32). Human and mice proximal tubules cells, on the other hand, express both NHERF-1 and NHERF-2, and the potential interaction between these proteins may affect the response to physiological stimuli (33, 34). In wild-type proximal tubule cells, PTH inhibited membrane Npt2a expression and sodium-dependent phosphate transport by 30%. By contrast, the inhibitory effect of PTH on Npt2a expression and phosphate transport was significantly less in NHERF-1/ cells. When considered with our prior studies, the present results indicate involvement of NHERF-1 in the two major regulatory controllers of phosphate excretion: limitation of the dietary intake of phosphate or incubation of cultured cells in low-phosphate media, which increases phosphate transport, and the response to PTH, which inhibits phosphate transport (5). The precise interaction between NHERF-1 and Npt2a is not known, but we have previously speculated that NHERF-1 serves as a retention signal for Npt2a in BBM. We favor this speculation rather than postulating that NHERF-1 is required for the likely independent processes that recruit Npt2a to the apical membrane in response to restriction of dietary phosphate intake as well as the processes that retrieve Npt2a from the BBM in response to PTH. We would also propose that the NHERF-1/Npt2a pool of Npt2a is the PTH-responsive pool. We recognize that these hypotheses await experimental verification.
The adaptation to dietary restriction of phosphate intake is rapid and associated with recruitment of Npt2a to the BBM and, as a consequence, decreased urinary excretion of phosphate (3, 18, 25, 35). Several prior studies in intact animals have indicated that the phosphaturic effect of PTH is blunted in animals adapted to a low-phosphate diet (23, 29, 36). The mechanism of this relative resistance to the phosphaturic effect of PTH is unknown but may be the result of specific biochemical changes in renal proximal tubule cells or changes in systemic factors that alter PTH signaling. Proximal tubule cells from wild-type mice demonstrate increased phosphate transport when incubated in low- compared with high-phosphate media (5). This model system, then, permits examination of the effect of PTH on phosphate transport and Npt2a expression in the absence of changes in systemic factors. Our findings indicate that while basal phosphate transport is higher in cells grown in the low- compared with normal-phosphate media, there is a decrease in the inhibitory effect of PTH on Npt2a expression and phosphate transport in cells adapted to low-phosphate media. It is acknowledged that the adaptive response to incubation of cells in low-phosphate media may not be identical to restriction of the dietary intake of phosphate. To the degree that both types of responses utilize similar mechanisms, however, the present studies indicate that the biochemical changes reside, at least in part, in the cells of the proximal tubule and are not the consequence of systemic factors. Our prior studies have indicated that the abundance of NHERF-1 and NHERF-2 was not different between mice proximal tubule cells grown in low- vs. high-phosphate media, making it unlikely that the NHERF proteins are involved in the blunted response to PTH in adapted cells (5). We did observe a modest increase in PDZK1 expression in cells grown in low-phosphate media (5). The role of PDZK1 in phosphate transport is not fully elucidated. PDZK1 mRNA is increased in rats fed a low-phosphate diet, and initially it was believed that this protein might be involved in the recruitment of Npt2a to the BBM (6). On the other hand, changes in PDZK1 protein have not been consistently observed, and PDZK1/ mice on a normal-phosphate diet have no apparent abnormalities in Npt2a targeting in the kidney, or in blood or urine phosphate concentrations (15). Nonetheless, a role for PDZK1 cannot be excluded. In our view, it remains possible that PDZK1 recruited to the BBM in mice proximal cells grown in low-phosphate media may interfere with the mechanisms subserving PTH-mediated reduction of Npt2a abundance in the BBM.
The relationship among NHERF-1, PTH signaling, and control of Npt2a activity is complex. PTH (PTH1R) receptors are located on both the apical as well as the basolateral side of renal proximal tubule cells (7, 26, 31). The basolateral receptor signals through adenylate cyclase and PKC, whereas the apical receptor is believed to signal exclusively through PKC (31). Recent studies have indicated that NHERF-1 acts as a molecular switch by binding to and facilitating PTH receptor signaling through the PKC rather than the PKA pathway (20). In addition, NHERF-1 also regulates PTH receptor kinetics in response to PTH fragments (28). Because NHERF-1 is localized predominantly at the apical membrane of the renal proximal tubule, it is believed that it interacts with apical PTH1R (32, 33). In our prior studies in mice proximal tubule cells in culture, however, we were unable to demonstrate differences in cAMP generation or PKC activation in response to PTH in wild-type cells compared with NHERF-1/ cells, results which are confirmed herein (5). At the present time, it is not known whether the normal cAMP and PKC responses represent PTH interaction exclusively with the basolateral receptor or the equivalent NHERF-2 interaction with the apical PTH1R receptor (20). The signaling pathways utilized by PTH to inhibit phosphate transport and apical membrane Npt2a expression include activation of PKA, PKC, and, as recently reported, ERK1/2 (2, 4, 14, 16). In OK cells, PTH causes a vigorous two- to fivefold increase in ERK1/2 activation when assayed as activity or by phosphorylation of serine 204 (4, 14, 16). Lederer and co-workers (16) studied the effect of a specific inhibitor of ERK1/2 in OK cells. Curiously, these studies showed significant attenuation of PTH inhibition of phosphate uptake but not PTH-associated downregulation of the abundance of NaPi-4, the OK cell sodium-dependent phosphate transporter (16). Other experiments in kidney slices of mice demonstrated that PTH treatment decreased apical membrane expression of Npt2a and that this effect was blocked by an inhibitor of ERK1/2 (2). Assays of stimulation of ERK1/2 in response to PTH, however, indicated only modest (3050%) stimulation at early time points rather than the several-fold stimulation reported in OK cells. In the present experiments, we confirm the activation of ERK1/2 in response to PTH in OK cells. In contrast, in cultured proximal tubules from mice, PTH did not stimulate ERK1/2 and, in fact, decreased ERK1/2 phosphorylation below baseline. Interestingly, there is precedence for an inhibitory effect of PTH on ERK1/2 activation in osteoblastic and bone marrow-derived cells (13, 24). Although the mechanism and biological significance remain to be elucidated, it would appear that the effect of PTH on ERK1/2 is clearly different in mouse proximal tubule cells compared with OK cells. The fact that PTH inhibited phosphate transport and decreased plasma membrane expression of Npt2a in cultured wild-type mouse proximal tubule cells grown in normal-phosphate media suggests that activation of ERK1/2 is not an absolute requirement for mediating the inhibitory effect of PTH. Moreover, the inhibitory effect of PTH on ERK1/2 was the same in wild-type cells grown in normal- or low-phosphate media, and in NHERF-1 null cells, indicating that the differential responses to PTH are not the result of differences in ERK1/2 kinetics.
GRANTS
These studies were supported by grants from the National Institutes of Health (NIH) DK-55881 (E. J. Weinman and S. Shenolikar), Research Service, Department of Veterans Affairs (E. J. Weinman), the University of Maryland (R. Cunningham), and the Kidney Foundation of Maryland (R. Cunningham). R. Cunningham is a recipient of a Minority Career Development Award from NIH.
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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