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【摘要】 Sodium-dependent phosphate transport in NHERF-1 -/- proximal tubule cells does not increase when grown in a low phosphate media and is resistant to the normal inhibitory effects of parathyroid hormone (PTH). The current experiments employ adenovirus-mediated gene transfer in primary cultures of mouse proximal tubule cells from NHERF-1 null mice to explore the specific role of NHERF-1 on regulated Npt2a trafficking and sodium-dependent phosphate transport. NHERF-1 null cells have decreased sodium-dependent phosphate transport compared with wild-type cells. Infection of NHERF-1 null cells with adenovirus-GFP-NHERF-1 increased phosphate transport and plasma membrane abundance of Npt2a. Adenovirus-GFP-NHERF-1 infected NHERF-1 null proximal tubule cells but not cells infected with adenovirus-GFP demonstrated increased phosphate transport and Npt2a abundance in the plasma membrane when grown in low phosphate (0.1 mM) compared with high phosphate media (1.9 mM). PTH inhibited phosphate transport and decreased Npt2a abundance in the plasma membrane of adenovirus-GFP-NHERF-1-infected NHERF-1 null proximal tubule cells but not cells infected with adenovirus-GFP. Interestingly, phosphate transport is inhibited by activation of protein kinase A and protein kinase C in wild-type proximal tubule cells but not in NHERF-1 -/- cells. Together, these results highlight the requirement for NHERF-1 for physiological control of Npt2a trafficking and suggest that the Npt2a/NHERF-1 complex represents a unique PTH-responsive pool of Npt2a in renal microvilli.
【关键词】 renal phosphate transport regulation renal physiology
THE REABSORPTION OF FILTERED phosphate in the proximal convoluted tubule of the kidney is mediated, in large measure, by the apical membrane sodium-dependent phosphate transporter 2a (Npt2a, NaPi IIa) ( 2, 12 ). In response to a low-phosphate diet or incubation in low phosphate media, Npt2a is recruited to the brush-border membrane of renal proximal tubule cells where it mediates increased sodium-dependent phosphate uptake ( 4, 10, 16, 17, 25 ). Parathyroid hormone (PTH), on the other hand, facilitates the retrieval of Npt2a from the brush-border membrane and increases the urinary excretion of phosphate ( 1, 5, 16 ). Recent experiments have demonstrated that specific proteins that interact with the COOH terminus of Npt2a have significant effects on its trafficking ( 3, 7, 9, 18, 19 ). The best studied of these associated proteins is the sodium-hydrogen exchanger regulatory factor-1 (NHERF-1), a PDZ domain-containing adaptor that binds to the COOH terminus of Npt2a ( 7, 9, 19, 22, 23, 26 ). Inactivation of the NHERF-1 -/- gene in mice results in a decrease in the serum concentration of phosphate, an increase in the urinary excretion of phosphate, and a decrease in the renal proximal tubule brush-border membrane abundance of Npt2a ( 18 ). In response to culture in low phosphate media, wild-type renal proximal tubule cells but not NHERF-1 null cells recruit Npt2a to the plasma membrane resulting in increased sodium-dependent phosphate transport ( 4 ). PTH decreases brush-border membrane expression of Npt2a and phosphate transport in wild-type proximal tubule cells but does not affect either parameter in NHERF-1 -/- cells ( 5 ). Although these cellular studies suggest a potential role for NHERF-1 in regulating Npt2a function, the specific contribution of NHERF-1 in the physiological regulation of Npt2a in the apical membrane of renal proximal tubule cells has not been investigated.
To date, more than 40 proteins including many G protein-coupled receptors and other signaling proteins have been shown to bind NHERF-1 ( 19 ). The present studies were designed to differentiate between the direct effects of NHERF-1 compared with the indirect effects on other signaling pathways and/or other developmental alterations in renal tissues elicited by absence of this multifunctional protein on basal and regulated phosphate transport. Specifically, we used cultured renal proximal tubule cells from wild-type and NHERF-1 null cells and viral-mediated gene transfer to restore NHERF-1 expression in the mutant NHERF-1 null proximal tubule cells and demonstrate the recovery of Npt2a regulation by incubation in low phosphate media and by PTH. These studies highlight the key role played by NHERF-1 in transducing critical signals elicited by multiple physiological stimuli to regulate Npt2a function in the mammalian kidney.
METHODS
Animals and preparation of renal proximal tubule cells. Male NHERF-1 -/- mice (B6.129-Slc9a3r1 tmSsl /Ssl) bred into a C57BL/6 background for six generations and parental wild-type inbred control C57BL/6 mice age 12 to 16 wk were used in the current experiments ( 18 ). To prepare primary renal proximal tubule cell cultures, mice were euthanized by intraperitoneal injection of 100 mg/kg pentobarbital sodium followed by decapitation. The kidneys were removed, and the cortices were dissected, minced, and digested using 1% collagenase type II (Worthington) and 0.025% soy bean trypsin inhibitor, and sedimented on 45% Percoll ( 4, 5 ). The proximal tubule cells were grown in an incubator at 37°C in 5% CO 2 in DMEM-F12 media containing 50 U/ml penicillin, 50 µg/ml streptomycin, 10 ng/ml epidermal growth factor, 0.5 µM hydrocortisone, 0.87 µM bovine insulin, 50 µM prostaglandin E 1, 50 nM sodium selenite, 5 µg/ml human transferrin, and 5 pM 3,3±,5-triiodo- L -thyronine on Matrigel-coated coverslips or plastic cell culture dishes coated with Matrigel. The cultures were left undisturbed for 36 h after which the media were replaced every 2 days until the cells achieved confluence at 5 to 7 days after plating. No attempt was made to pass the cells.
Transport assay. Phosphate transport was measured by determination of the sodium-dependent uptake of 32 P-labeled phosphate ( 4, 5 ). The cells were washed three times and preincubated for 5 min in nonradioactive transport medium containing 137 mM NaCl, 5.0 mM KCl, 1.0 mM CaCl 2, 1.8 mM MgSO 4, and 0.1 mM KH 2 PO 4. Phosphate uptake was initiated by the addition of transport medium containing 32 P-radiolabeled orthophosphate. Uptake was continued for 10 min at room temperature, after which the cells were washed with ice-cold medium in which tetramethylammonium chloride was substituted for sodium chloride, [ 32 P]phosphate was omitted, and 0.5 mM sodium arsenate was added. 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. Where studied, proximal tubule cells incubated in normal-phosphate media (DMEM-F12, 0.9 mM) were treated with PTH 1-34 (10 -7 M) for 2 h, 8-bromo-cAMP (100 µM) for 45 min, or 1,2-dioctanoyl-sn-glycerol (DOG; 10 µM) for 45 min. To study adaptation to changes in the phosphate content of the incubation media, the cells were incubated in low (0.1 mM) or high (1.9 mM) phosphate media for 24 h before study. These media were prepared using phosphate-free DMEM media to which potassium phosphate was added to the indicated final concentrations. Potassium gluconate was added to the low phosphate media to equalize the potassium concentrations between the media.
Preparation and use of adenoviruses. Infective recombinant adenoviruses were produced using AdEasy (Stratagene) as described ( 4 ). Proximal tubule cells were incubated with 10 7 PFU adenovirus-green fluorescent protein (GFP) or adenovirus-GFP-NHERF-1 for 24 h. The virus was removed and functional studies were initiated 24 h later. This protocol results in the expression of GFP in 99% or more of exposed cells ( 4 ).
Other procedures. For confocal microscopy, cells were fixed in paraformaldehyde as previously described ( 21, 24 ). 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% beta-mercapto-ethanol, and Complete Protease Inhibitor Cocktail (Roche Applied Science). The cells were disrupted by three 20-s pulses with a probe sonicator followed by 100-min centrifugation at 1,000 g to remove large particulates. The supernatant was ultracentrifuged for 1 h at 100,000 g. The pellet was resuspended in 0.1% (wt/vol) SDS and prepared for electrophoresis by the addition of Lamelli's buffer. Western immunoblotting was performed using antibodies specific for Npt2a, GFP, or ezrin. Protein concentrations were determined and the gel lanes were loaded equally (generally, 5-10 µg/lane), concentrations that were found to be in the linear range of the relationship between protein concentration and densitometry readings. Individual bands were quantitated using laser densitometry and the measurements corrected for minor differences in protein loading by averaging six constant bands on Poseau S-stained gels. In some studies, staining for ezrin was used as an additional control for loading of the gels.
Total RNA from cultured proximal tubule cells infected with adenovirus-GFP or adenovirus-GFP-NHERF-1 was extracted using RNAqueous-Midi Kit (Ambion, Austin, TX). First-strand cDNA synthesis from total RNA was done using random hexamers with the RETROscript reverse transcription kit with SuperTaq polymerase (Ambion). Relative quantitation of Npt2a mRNA was done using the iCycler iQ tm (Bio-Rad) with 15S as an internal standard. For Npt2a, the sense primer was 5'-CTTCAACATCTCGGGCATCCTACTG and the anti-sense primer was 5'-TAGAGACGGGTGGCATTGTGGTGA-3'. For 15S RNA, the sense and anti-sense primers were 5'-GCAATTATTCCCCATGAACG-3' and 5'-GGCCTCACTAAACCATCCAA-3', respectively. PCR reactions in a volume of 25 µl, containing 400 nM gene-specific primers, were performed using iQ SYBR Green Supermix (Bio-Rad). Following activation of iTaq DNA polymerase (2 min at 94°C), the samples were amplified for 45 cycles at 94°C for 30 s, 62°C for 30 s, and 72°C for 30 s with a final extension of 72°C for 2 min. Relative expression was calculated according to the iCycler iQ Real-Time PCR Detection System Manual.
Protein concentrations were determined using the method of Lowery et al. ( 11 ). Statistical comparisons were performed using ANOVA ( 8 ).
RESULTS
In initial experiments, we examined the effect of infection of NHERF-1 null cells with either adenovirus-GFP or adenovirus-GFP-NHERF-1. Cells were exposed to virus for 24 h and studies were undertaken 24 h after removal of the viral particles. Sodium-dependent phosphate uptake averaged 4.9 ± 0.8 nmol·mg protein -1 ·10 min -1 in NHERF-1 -/- cells infected with control adenovirus-GFP and 6.9 ± 1.1 in cells infected with adenovirus-GFP-NHERF-1 ( P < 0.01, n = 9). As shown in Fig. 1 A, there was a significant increase in Npt2a abundance in the plasma membranes from adenovirus-GFP-NHERF-1-infected NHERF-1 -/- cells. In six separate experiments, there was an average increase of 40.0 ± 9.9% ( P < 0.01) in plasma membrane content of Npt2a in adenovirus-GFP-NHERF-1-infected cells compared with control adenovirus-GFP-infected cells. Western immunoblot analyses of whole cell lysates ( Fig. 1 B ) indicated no difference between NHERF-1 null cells infected with adenovirus-GFP and adenovirus-GFP-NHERF-1 cells (percent difference 0.3 ± 0.33%, P = not significant, n = 6). Using quantitative RT-PCR, Npt2a mRNA relative to 15S mRNA was not different in NHERF-1 -/- cells infected with adenovirus-GFP-NHERF-1 compared with adenovirus-GFP-infected cells (data not shown). With the use of an anti-GFP antibody, NHERF-1 expression in the plasma membrane fraction was readily detected in adenovirus-GFP-NHERF-1-infected cells but not in adenovirus-GFP-infected cells ( Fig. 1 C ). The identity of GFP band was confirmed using an antibody to NHERF-1 ( Fig. 1 D ). Figure 2 is representative confocal microscopic images of NHERF-1 -/- cells showing the presence of diffuse cellular Npt2a staining in cells infected with adenovirus-GFP but the presence of Npt2a on the surface of cells infected with adenovirus-GFP-NHERF-1.
Fig. 1. Representative Western immunoblots of NHERF-1 -/- cells infected with adenovirus-GFP ( lane 1 ) or adenovirus-GFP-NHERF-1 ( lane 2 ) using antibodies to Npt2a ( A and B ), GFP ( C ), or NHERF-1 ( D ). A, C, and D : plasma membrane fraction of the cells. B : whole cell lysates. Molecular weight markers (kDa) are shown.
Fig. 2. Confocal microscopy images of NHERF-1 -/- cells infected with adenovirus-GFP ( left ) or adenovirus-GFP-NHERF-1 ( right ). Cells were stained with antibody to Npt2a.
Our prior studies indicated that, by contrast to wild-type proximal tubule cells, NHERF-1 null cells grown in low phosphate media did not increase sodium-dependent phosphate transport or the plasma membrane content of Npt2a and were resistant to the inhibitory effects of PTH ( 4, 5 ). We next determined whether reexpression of NHERF-1 in NHERF-1 null cells could restore these adaptive responses. Where indicated, half of each individual preparation of NHERF-1 -/- proximal tubule cells was infected using the control adenovirus-GFP and the other half infected using adenovirus-GFP-NHERF-1. To minimize possible differences in the levels of expression between different cultures, cells from the same preparation infected with either adenovirus-GFP or adenovirus-GFP-NHERF-1 were studied following incubation in low (0.1 mM) or high (1.9 mM) phosphate media or in the presence or absence of PTH. Sodium-dependent phosphate uptake averaged 4.1 ± 0.2 nmol·mg protein -1 ·10 min -1 and 4.1 ± 0.3 ( P = not significant, n = 6) in null cells infected with adenovirus-GFP grown in low- and high-phosphate media, respectively ( Table 1 ). By contrast, sodium-dependent phosphate uptake averaged 7.4 ± 0.6 nmol·mg protein -1 ·10 min -1 in NHERF-1 null cells infected with adenovirus-GFP-NHERF-1 grown in low phosphate media ( P < 0.01 vs. adenovirus-GFP-infected NHERF-1 -/- cells grown in the same low phosphate media). In response to growth in high phosphate media, adenovirus-GFP-NHERF-1-infected NHERF-1 null cells demonstrated a significantly lower rate of sodium-dependent phosphate transport of 5.4 ± 0.3 nmol·mg protein -1 ·10 min -1 ( P < 0.01). There was no significant difference in plasma membrane expression of Npt2a in adenovirus-GFP-infected NHERF-1 null cells incubated in low- or high-phosphate media (% difference = 11.5 ± 8.4%, P = not significant, P = 4). Adenovirus-GFP-NHERF-1-infected cells grown in low-phosphate media had a 28.2 ± 2.5% greater abundance of Npt2a in the plasma membrane compared with cells grown in high-phosphate media ( P < 0.05; Fig. 3 ). The relative abundance of GFP-NHERF-1 in the plasma membrane was minimally but not statistically significantly higher in cells grown in low- vs. high-phosphate media (10.2 ± 3.5%, P = not significant). These experiments established the ability of NHERF-1 to restore the forward trafficking of Npt2a to the apical membrane to increase sodium-dependent phosphate transport in response to growth in low phosphate media.
Table 1. Effect of growth in low- or high-phosphate media on sodium-dependent phosphate transport in mouse NHERF-1 -/- proximal tubule cells infected with adenovirus-GFP or adenovirus-GFP-NHERF-1
Fig. 3. Representative Western immunoblots of plasma membranes from NHERF-1 -/- cells infected with adenovirus-GFP-NHERF-1. Cells were studied after growth in low-phosphate (0.1 mM) or high-phosphate (1.9 mM) media for 24 h. Top : Npt2a. Middle : GFP-NHERF-1. Bottom : ezrin as a loading control. In 4 separate experiments, the plasma membrane abundance of Npt2a but not GFP-NHERF-1 was significantly greater in cells grown in low compared with high-phosphate media (see RESULTS ). There was no statistical difference in Npt2a abundance in the plasma membrane of control adenovirus-GFP-infected NHERF-1 -/- cells grown in low- or high-phosphate media.
NHERF-1 -/- cells are resistant to the inhibitory effects of PTH ( 5 ). Accordingly, we examined whether expression of NHERF-1 in NHERF-1 null cells could restore the response to the hormone. Sodium-dependent phosphate uptake averaged 5.7 ± 0.5 nmol·mg protein -1 ·10 min -1 in the absence and 5.8 ± 0.4 in the presence of PTH ( P = not significant, n = 7; Table 2 ) in null cells infected with adenovirus-GFP. Sodium-dependent phosphate uptake averaged 9.5 ± 0.8 nmol·mg protein -1 ·10 min -1 in NHERF-1 null cells infected with adenovirus-GFP-NHERF-1 ( P < 0.01 vs. adenovirus-GFP-infected NHERF-1 -/- cells). In response to PTH, adenovirus-GFP-NHERF-1-infected NHERF-1 null cells demonstrated a significantly lower rate of sodium-dependent phosphate transport of 7.4 ± 0.5 nmol·mg protein -1 ·10 min -1 ( P < 0.01, n = 7). There was no significant difference in the abundance of Npt2a in the plasma membrane in adenovirus-GFP-infected NHERF-1 null cells treated with PTH compared with control cells (% difference = -1.0 ± 6.9, n = 4, P = not significant). By contrast, the decrease in sodium-dependent transport of phosphate in PTH-treated adenovirus-GFP-NHERF-1-infected NHERF-1 -/- cells was associated with a 29.6 ± 4.9% ( P < 0.05) lower plasma membrane expression of Npt2a ( Fig. 4 ). Plasma membrane abundance of GFP-NHERF-1 was 13.9 ± 4.7% lower in NHERF-1 null cells infected with adenovirus-GFP-NHERF-1 cells treated with PTH compared with non-PTH-treated cells ( P = not significant, n = 4).
Table 2. Effect of PTH on sodium-dependent phosphate transport in mouse NHERF-1 -/- proximal tubule cells infected with adenovirus-GFP or adenovirus-GFP-NHERF-1
Fig. 4. Representative Western immunoblots of plasma membranes from NHERF-1 -/- cells infected with adenovirus-GFP-NHERF-1. Cells were studied in the absence (-PTH) or presence of PTH (+PTH; 10 -7 M). Top : Npt2a. Middle : GFP-NHERF-1. Bottom : ezrin as a loading control. In 4 separate experiments using adenovirus-GFP-NHERF-1-infected cells, PTH significantly inhibited the plasma membrane abundance of Npt2a but not GFP-NHERF-1 (see RESULTS ). There was no statistical difference in Npt2a abundance in the plasma membrane of control adenovirus-GFP-infected NHERF-1 -/- cells in the absence or presence of PTH.
PTH is known to signal via the generation of cAMP leading to PKA activation and by the activation of protein kinase C ( 4, 5, 20 ). Prior cellular studies showed that NHERF-1 binds to the PTH1 receptor to modulate PTH signaling via the second messengers cAMP and diacylglycerol ( 13, 14 ). In our prior studies, we found no apparent differences in PTH signaling in proximal convoluted tubule cells from NHERF-1 null mice compared with wild-type animals, suggesting that the lack of response to PTH seen in the NHERF-1 null cells was the result of the inability of downstream second messengers to regulate Npt2a function ( 4, 5 ). To explore this question further using a different approach, wild-type and NHERF-1 -/- proximal tubule cells were treated with either 8-bromo-cAMP or DOG to activate protein kinase A or protein kinase C, respectively. Wild-type proximal tubule cells demonstrated decreased sodium-dependent phosphate transport from 8.1 ± 1.3 nmol·mg protein -1 ·10 min -1 in control conditions to 6.6 ± 1.1 (% decrease = 25.2 ± 1.4%, P < 0.05) in response to cAMP and to 5.7 ± 0.9 (% decrease = 31.0 ± 1.9%, P < 0.05) in response to DOG ( n = 6; Table 3 ). By contrast, neither cAMP nor DOG inhibited sodium-dependent phosphate transport in NHERF-1 null cells. As summarized in Table 3, sodium-dependent phosphate uptake averaged 5.4 ± 0.3 nmol·mg protein -1 ·10 min -1 in controls, 5.8 ± 0.6 in the presence of cAMP ( P = not significant), and 5.9 ± 0.5 in the presence of DOG ( P = not significant; n = 6) in NHERF-1 -/- cells. While infection of NHERF-1 null cells with adenovirus-GFP had no effect on the lack of response to cAMP or DOG, infection with adenovirus-GFP-NHERF-1 resulted in an inhibitory response to both cAMP (% decrease = 33.3 ± 5.2%, P < 0.05) and DOG (% decrease = 34.6 ± 6.0%, P < 0.05; Table 3 ). These data strongly suggest that NHERF-1 acts at postreceptor sites to transduce the second messenger signals by which PTH regulates Npt2a function in renal tissue.
Table 3. Effect of cAMP and DOG on sodium-dependent phosphate transport in mouse wild-type and NHERF-1 -/- proximal tubule cells (% decrease from control)
DISCUSSION
Prior studies from our laboratory indicated that sodium-dependent phosphate transport in proximal tubule cells in primary culture from NHERF-1 null mice has a lower basal rate of transport, fails to upregulate phosphate transport in response to incubation in a low phosphate media, and is resistant to the inhibitory effect of PTH ( 4, 5 ). These studies suggest that NHERF-1 may play a key role in processes that both recruit and retrieve Npt2a from the apical membrane of proximal tubule cells. On the other hand, dysfunction of the mouse NHERF-1 gene was reported to induce significant structural alterations in gut epithelia, inducing malformation of microvilli ( 16 ). While analyses of renal epithelia in our NHERF-1 null mice failed to show similar structural alterations, the potential indirect impact on Npt2a function and regulation of the loss of NHERF-1 that binds and regulates a multitude of receptors, transporters, scaffolds, and signaling proteins could not be discounted ( 18 ). Thus the current experiments that employ adenovirus constructs to affect highly efficient expression of NHERF-1 in NHERF-1 -/- renal proximal tubule cells are critical for assessing the direct functional impact of NHERF-1 expression on Npt2a localization and function. In initial experiments, we compared infection with control adenovirus-GFP with adenovirus-GFP-NHERF-1 and found that NHERF-1 -/- proximal tubule cells expressing GFP-NHERF-1 had significantly higher rates of phosphate transport associated with increased Npt2a abundance in the brush-border membrane. This indicates that, in large measure, the defect in phosphate transport and Npt2a trafficking in NHERF-1 -/- renal proximal tubule cells resides within these cells and is not the result of developmental defects or systemic factors associated with the absence of the NHERF-1 protein.
To fully understand the role of NHERF-1 in the physiological regulation of phosphate transport, we also examined the ability of NHERF-1 mutant cells to adapt phosphate transport in response to growth in low-phosphate media and to respond to PTH. As we previously reported and by contrast to wild-type proximal tubule cells, NHERF-1 null cells, herein infected with adenovirus-GFP, did not increase sodium-dependent phosphate transport or recruit Npt2a to the brush-border membrane in response to incubation in low-phosphate media ( 4 ). GFP-NHERF-1-expressing NHERF-1 null cells, on the other hand, demonstrated increased Npt2a abundance on the apical membrane and increased sodium-dependent phosphate transport in response to growth in low phosphate media. These findings provide direct experimental evidence that NHERF-1 plays a key role in regulating Npt2a trafficking in cultured proximal tubule cells in response to incubation in low-phosphate media and presumably following the feeding of a low-phosphate diet to intact animals ( 25 ). In cultured proximal tubule cells infected with GFP-NHERF-1 grown in low phosphate media, the increase in the plasma membrane abundance of Npt2a was not associated with a significant change in the abundance of GFP-NHERF-1. This finding is in accord with our prior observations in intact animals where feeding of a low-phosphate diet increased brush-border membrane abundance of Npt2a but not NHERF-1 ( 24 ). Taken together, these results indicate that within the sensitivity of these measurements, the recruitment of Npt2a to the plasma membrane is not obligatorily linked to the redistribution of NHERF-1. Accordingly, we favor the hypothesis that NHERF-1 functions as a membrane retention signal for Npt2a. Given the relative abundances of NHERF-1 and Npt2a, however, the current studies do not rule out the possibility that NHERF-1 could also function as a chaperone for the transporter.
Our current studies confirm that sodium-dependent phosphate transport in NHERF-1 null cells, by contrast to wild-type cells, is resistant to the inhibitory effect of PTH ( 5 ). Expression of NHERF-1 in NHERF-1 null cells results in the restoration of the inhibitory effect of PTH on sodium-dependent phosphate transport associated with a decrease in the plasma membrane abundance of Npt2a. Recent experiments using kidney slices from mice suggested that PTH decreases Npt2a abundance in brush-border membrane of the renal proximal tubule with lesser effects on the abundance of NHERF-1 ( 6 ). The present experiments would agree with these findings and indicate that treatment with PTH alters Npt2a abundance but does not significantly affect the abundance and/or distribution of GFP-NHERF-1 in NHERF-1 -/- proximal tubule cells.
Studies in cultured cells showed that the NHERF proteins, in addition to binding to Npt2a, also bind the PTH 1 receptor (PTH1R) to function as a molecular switch for downstream signaling ( 13, 14 ). When bound to NHERF-1, PTH1R preferentially signals through protein kinase C while its capacity to signal through cAMP is greatly decreased. The failure of PTH to inhibit phosphate transport in the NHERF-1 null cells, therefore, could arise from either the inability of PTH to regulate Npt2a or altered signaling of PTH1R. To address this question, we bypassed the PTH receptor and studied sodium-dependent phosphate transport in proximal cells treated with either cAMP to stimulate PKA or DOG to activate PKC. On average, PTH, cAMP, and DOG significantly inhibited sodium-dependent phosphate transport by 25 to 30% in wild-type cells but not in NHERF-1 null cells. Moreover, rescue of the NHERF-1 null cells with adenovirus-GFP-NHERF-1 restored the response to both cAMP and DOG, indicating that NHERF-1 was critical for the transduction of hormone signals mediated by both second messengers. Our prior biochemical studies had demonstrated no significant differences in the ability of PTH to increase cAMP levels or activate PKC in renal tissue from wild-type and NHERF-1 null mice ( 4, 5 ). When considered together, these data focus attention on the interaction between Npt2a and NHERF-1 rather than the interaction between NHERF-1 and PTH1R as the explanation for the resistance to PTH. This conclusion also fits the observed phenotype of the NHERF-1 -/- mouse that manifests hypophosphatemia and increased excretion of phosphate ( 18 ). If the interaction between NHERF-1 and PTH1R were the primary defect, we would have predicted that null animals would excrete less phosphate and tend toward hyperphosphatemia, reflecting inactivation of the receptor. It is worth noting that mouse proximal tubule cells express both NHERF-1 and NHERF-2 and that the relative abundance of NHERF-2 is not altered in the absence of a functional NHERF-1 gene ( 21 ). PTH1R appears to be able to use either NHERF-1 or NHERF-2 as the molecular switch while Npt2a shows greater selectively for NHERF-1. Npt2a trafficking is defective in the absence of NHERF-1 despite the continued expression of NHERF-2 in mouse tissues ( 18 ).
In summary, the present experiments indicate a requirement for NHERF-1 in the regulation of phosphate transport and Npt2a surface expression in response to growth in a low-phosphate media and to PTH in primary renal proximal tubule cells. NHERF-1 null cells are resistant not only to PTH but also to the major second-message signaling pathways normally used by the PTH1 receptor, namely, PKA and PKC. Expression of GFP-NHERF-1 in NHERF-1 null cells restores the adaptive response to growth in low-phosphate media and the inhibitory responses to PTH, cAMP, and DOG. From these findings, we hypothesize that NHERF-1 complexes with Npt2a in the apical membrane of renal proximal tubule cells, an interaction necessary to stabilize or anchor Npt2a recruited to the plasma membrane in response to incubation in low-phosphate media. Moreover, we would suggest that NHERF-1/Npt2a complexes represent the major PTH-responsive pool of membrane Npt2a and that PTH, acting via PKA and/or PKC, regulates the disassembly of Npt2a/NHERF-1 complexes ( 6 ). The disassembly of the Npt2a/NHERF-1 complexes may, in turn, increase the lateral mobility of Npt2a in the apical membrane necessary to engage clathrin and other elements that result in the internalization and subsequent degradation of the transporter.
GRANTS
These studies were supported by National Institutes of Health (NIH) Grants 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). Dr. Cunningham is a recipient of a Minority Career Development Award from the National Institutes of Health.
ACKNOWLEDGMENTS
The recombinant adenoviruses were kindly provided by Drs. J. Lederer and W. Randall, University of Maryland School of Medicine, Baltimore, MD. The Npt2a antibody was a gift from Drs. J. Biber and H. Murer, Institute of Physiology and Center for Integrative Human Physiology, University of Zürich, Zürich, Switzerland.
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作者单位：1 Department of Medicine, 2 Department of Physiology, University of Maryland School of Medicine, and 3 Medical Service, Department of Veterans Affairs Medical Center, Baltimore, Maryland; and 4 Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina