parathyroid hormone (PTH) has a multitude of effects on the renal proximal tubule. Specifically, PTH causes inhibition of sodium-dependent phosphate (NaPi) uptake (3, 4, 39, 48, 53) and sodium-hydrogen exchange (NHE) at the apical membrane (2, 7, 30, 53), inhibition of Na⁺-K⁺-ATPase ion transport (Na-K) at the basolateral membrane (17, 34), stimulation of 1-α hydroxylase to produce 1,25 dihydroxy vitamin D₃ (1, 31, 36, 45, 46), stimulation of ammoniagenesis (15, 47), and stimulation of gluconeogenesis (42, 47). The study of the effects of PTH on proximal tubule function is complicated by the fact that PTH receptors are expressed on both apical [brush-border membranes (BBM)] and basolateral membranes (BLM) (32). We have shown in a cell culture model of the proximal renal tubule that the PTH receptor is associated with the type IIa sodium- phosphate cotransporter (Npt2a) at the BBM in a signaling complex that also includes Na-H exchanger regulatory factor 1 (NHERF-1), an A kinase anchoring protein (AKAP79/150), protein kinase A (PKA), a receptor for activated C kinase (RACK1), and protein phosphatase 2a (35). The fact that the PTH receptor is localized in a protein complex with one of its target proteins suggests one potential mechanism for the specificity of PTH effects on proximal tubule functions; however, whether PTH could exert discrete and differential effects on each of its known targets of action has not been examined.

Studies of PTH-dependent mechanisms have largely been confined to studies of acute PTH stimulation. However, individuals with primary hyperparathyroidism, a common clinical condition characterized by chronic elevation in PTH secretion, do not exhibit laboratory values that would correspond to these acute effects. Patients with primary hyperparathyroidism or subjects given PTH on a chronic basis commonly present with hypophosphatemia, while volume depletion and/or metabolic acidosis is infrequently observed (24, 26, 27, 29, 47, 49). The absence of metabolic acidosis in the presence of chronic hyperparathyroidism has been attributed to PTH-stimulated release of alkali from bone and to stimulation of hydrogen ion secretion by hypercalcemia. Furthermore, micropuncture studies in rats have shown that although PTH decreases proximal tubule bicarbonate reabsorption, this effect is countered by increases in ammonia and net acid excretion, resulting in minimal change in systemic pH balance (5). Whether sodium balance changes with acute or chronic PTH stimulation has not been examined. Importantly, the studies of chronic effects by PTH on renal function have been descriptive studies based on patient data and have not investigated mechanisms at a molecular level.

The discrepancy between the clinical observations and the predictions based on studies of acute PTH effects on the proximal tubule suggested to us the hypothesis that chronic stimulation with PTH may result in differential desensitization of PTH-stimulated responses in the proximal renal tubule. To test this hypothesis, we compared the effects of acute and chronic stimulation with PTH on Npt2a and Na⁺-K⁺-ATPase in a cell culture model of proximal tubule [opossum kidney (OK) cells], transgenic mice displaying hyperparathyroidism (PTH-D1), and in Sprague-Dawley rats. We show that the regulation of expression and function of NpT2a by PTH differs from the regulation of other proximal tubule proteins and that the mechanism for the chronic regulation of NpT2a by PTH occurs at the posttranscriptional level.

Experimental Procedures

Materials.

PTH (1–34) was purchased from Bachem Biosciences (King of Prussia, PA). Antibodies against NpT2a were described previously (38). NHERF-1 antibody was a gift from Dr. E. J. Weinman (University of Maryland) (16). Antibodies against actin (horseradish peroxidase-conjugated) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). PTH receptor antibody was purchased from Covance. Monoclonal antibodies against the Na⁺-K⁺-ATPase α₁-subunit were purchased from Sigma-RBI (Natick, MA). Monoclonal antibodies against γ-glutamyl transferase were purchased from Neomarkers (Rockford, IL). Ambion MirVana miRNA isolation kits and Applied Biosystems PCR TaqMan Pre-Developed Assay Reagents were purchased from Life Technologies (Grand Island, NY). Immunohistochemistry reagents were purchased from Vector Laboratories (Burlingame, CA). All other chemicals were purchased from Sigma, unless otherwise specified.

Animal models.

All animal experiments were performed in accordance with the guidelines established by Institutional Animal Care and Use Committee (IACUC) at the University of Louisville. Sprague-Dawley rats, weighing 200–250 g, were stabilized on standard rat chow and water ad libitum for 1 wk before the experiments. Rats were subcutaneously injected with 5 μg/kg body wt PTH 1–34 for 6 h or 4 days. Rats were euthanized under anesthesia, and kidneys were removed and collected in ice-cold PBS. Kidneys were decapsulated, the cortex was carefully separated from the medulla, and membranes (BBM or BLM) were prepared from whole cortex by standard protocols.

Kidneys from 8-mo-old transgenic mice featuring parathyroid-specific cyclin D1 overexpression (PTH-D1) were obtained from Charles River Laboratories (Wilmington, MA). These mice gradually develop biochemical characteristics of primary hyperparathyroidism, as first described by Imanishi et al. (28) and later by Mallya et al. (40). Kidneys either were frozen in liquid nitrogen for later analysis or were fixed overnight in paraformaldehyde and embedded in paraffin for analysis by immunohistochemistry. Kidneys from control mice of background strain and similar age were used for control immunohistochemical and immunoblot studies. mRNA from 8-mo-old control mice was a generous gift from Paul Epstein, University of Louisville.

Cell culture.

OK cells, a continuous proximal tubule cell line derived from Virginia opossum, were maintained in minimal essential medium with Earle's salts (EMEM) supplemented with 10% FCS, and 1% penicillin/streptomycin and cultured to 90–95% confluence. Cells were washed with serum-free medium 24 h before use.

BBM isolation.

BBM vesicles (BBMV) from Sprague-Dawley rat kidney cortex were prepared at 4°C using the MgCl₂ precipitation method, exactly as previously described (35). Briefly, freshly minced cortical slices were homogenized in 50 mM mannitol and 5 mM Tris-HEPES buffer, pH 7.0 (20 ml/g), in a glass teflon homogenizer with four complete strokes. The homogenate was then subjected to high-speed [20,500 revolutions/min (rpm)] homogenization using a polytron homogenizer for three strokes of 15 s each with an interval of 15 s between each stroke. MgCl₂ was added to the homogenate to a final concentration of 10 mM and slowly stirred for 20 min. The homogenate was spun at 2,000 g in a Sorvall high-speed centrifuge using an SS-34 rotor. The supernatant was centrifuged at 35,000 g for 30 min using the SS-34 rotor. The pellet was resuspended in 300 mM mannitol and 5 mM Tris-HEPES, pH 7.4, with four passes by a loose-fitting Dounce homogenizer (Wheaton, IL) and centrifuged at 35,000 g for 20 min in 15-ml corex tubes using the SS-34 rotor. The outer fluffy pellet was resuspended carefully in a small volume of buffered 300 mM mannitol. Aliquots of homogenates were also saved to check the membrane purity by enzyme analysis. For OK cell BBM preparation, cells were grown on 0.1-μm Transwell inserts. At 95% confluence, cells were serum-starved overnight and treated with PTH for 2 h or 3 days before processing. The protocol was the same as for rat cortex BBM isolation.

BLM isolation.

BLM were prepared by the method of Sacktor et al. (50) with slight modifications. All steps were performed at 4°C unless otherwise stated. Briefly, 3-mm slices of kidney cortex was carefully separated and minced in 250 mM sucrose, 1 mM PMSF, and 10 mM Tris·HCl, pH 7.4, by 20 strokes in a glass teflon homogenizer. The homogenate was subjected to high-speed homogenization in a polytron type homogenizer at maximum speed for three strokes of 30 s each with a 30-s interval. The homogenates were incubated with 15 mM MgCl₂ on ice with constant shaking for 20 min to precipitate other membrane organelles. The homogenate was centrifuged at 2,500 g for 10 min in a Sorvall centrifuge using a SS-34 rotor. The supernatant was centrifuged at 24,000 g in a Sorvall centrifuge using the SS-34 rotor. The pellet was resuspended in 32.2 ml of homogenization buffer and mixed vigorously with 2.8 ml Percoll. The samples were centrifuged at 30,000 g for 35 min, and the middle layer (8 ml) containing BLM was diluted with 100 mM mannitol, 100 mM KCl, 10 mM Tris-HEPES buffer, pH 7.1, and centrifuged at 34,000 g for 30 min as above. The white fluffy BLM were resuspended in the KCl-mannitol buffer and centrifuged again at 38,000 g for 30 min, and the final pellet was resuspended in 300 mM mannitol, 5 mM Tris-HEPES, pH 7.4, at 1 ml/g starting tissue.

Protein determination.

Protein concentration was determined using a bicinchoninic acid protein kit (Sigma) using a BSA standard.

Western blot analysis.

The membrane proteins were separated by 10% SDS-PAGE and then transferred to nitrocellulose membranes. The nitrocellulose membranes were incubated in Tris-buffered saline containing 0.5% Tween 20 (TTBS) containing 5% milk at room temperature (RT) for 1 h to block nonspecific binding, followed by overnight incubation at 4°C with antibodies diluted in TTBS containing 5% milk. After being washed, the membranes were incubated at RT for 1 h in horseradish peroxidase-conjugated secondary antibodies diluted in TTBS containing 5% milk. Bands were detected by chemiluminescence (Pierce), and visualized on X-ray film.

ATP hydrolysis assay.

Na⁺-K⁺-ATPase activity in BLM was assayed as ouabain (4 mM)-sensitive ATP hydrolysis as first described by Szczepanska-Konkel et al. (56). BLM were frozen and thawed on ice before assay for Na⁺-K⁺-ATPase. Briefly, 50 μg of BLM protein was incubated for 15 min at 37°C in 1.5 ml of media containing 4.8 mM ATP, 120 mM NaCl, 5 mM KCl, 7.2 mM MgSO₄, and 48 mM Tris·HCl, pH 7.6, with or without 4 mM ouabain. To stop the reaction, 0.3 ml 30% TCA was used. Na⁺-K⁺-ATPase measurement was determined by the difference in the ATPase activity assayed in the absence and presence of ouabain. Na⁺-K⁺-ATPase activity is expressed as nanomoles P_i released per milligram protein per hour.

Immunohistochemistry.

Immunohistochemistry staining for NpT2a, Na⁺-K⁺-ATPase, NHERF-1, and the PTH receptor was performed, as previously described (14). Briefly, kidneys from PTH-D1 and control mice were fixed in paraffin blocks. Four-micrometer-thick sections were shaved from blocks using a Leica RM2125RT microtome and were mounted on glass slides (Fisher, Pittsburgh, PA). Paraffin was cleared from slides with xylene twice (all washing steps were performed for 5 min unless otherwise noted). Samples were rehydrated in graded ethanol to TTBS (100% EtOH, 95%, 70%, water twice, TTBS). Antigen retrieval was performed by incubating slides in a citrate buffer solution (Vector) for 20 min in a 95°C water bath. Slides were cooled at RT for 20 min and washed three times with water. Endogenous peroxidase was quenched with 3% H₂O₂, followed by three more washes with water and one wash with TTBS. Nonspecific binding was blocked with 2.5% horse serum for 30 min at RT. Sections were incubated with NpT2a (1:5,000), Na⁺-K⁺-ATPase (1:2,000), NHERF-1 (1:1,000), or PTH receptor (1:200) antibodies overnight at 4°C (diluted with 1% BSA, 0.5% Triton X-100, 0.5% Tween 20 in water). Slides were washed three times with TTBS and incubated with biotin-conjugated secondary antibodies for 30 min at RT. Slides were again washed three times with TTBS. 3,3'-Diaminobenzidine (DAB) reagent was prepared according to the Vector protocol. Sections were incubated with DAB and monitored under a microscope. Following visualization of chromatographic tubule staining, slides were quenched in water to stop the reaction and then counterstained with methyl green. Digital Images were obtained with a Q Color 5 camera attached to an Olympus BX51 microscope using ImagePro software. Immunostaining was quantified with ImagePro 6.2 software (Media Cybernetics, Silver Spring, MD). Five visual fields of cortex/kidney section were randomly selected and captured with a ×40 objective. Positive DAB staining was defined in a color profile by selecting a range of DAB staining intensities, and the profile was applied to all images collected. Values for total staining area/field were obtained and presented as the sum of staining area for each group.

Isolation of RNA.

OK cells were seeded on six-well plates and grown to 80% confluence. After treatment with either 100 nM PTH, actinomycin D (1 μg/ml), or cycloheximide (1 μg/ml) for the indicated time (0–24 h), cells were washed twice with PBS and then processed to isolate total RNA according to the manufacturer's instructions (MirVana miRNA isolation kit, Life Technologies). For transgenic mice, the kidney cortex was dissected from decapsulated kidneys and homogenized with a pestle. Once homogenized, total RNA was isolated as described above. RNA concentrations were determined using a Beckman Coulter DU730 UV/Vis Spectrophotometer (Brea, CA).

Quantitative real-time PCR.

One microgram of RNA was reverse-transcribed to cDNA using a Bio-Rad MyCycler thermal cycler with the High Capacity RNA-to-cDNA Master Mix system (Applied Biosystems, Foster City, CA). Reverse transcription was carried out at 25°C for 5 min, 42°C for 45 min, and heat inactivated at 85°C for 5 min. Two hundred nanograms of cDNA was PCR-amplified using FAM detection and the TaqMan Gene expression system with 50 nM NpT2a primers. 18S rRNA was used as a control housekeeping gene reference. For OK cells, the NpT2a forward primer sequence was 5′-TCTGAGAGTGCTGATGTACCTAAGT-3′, and the reverse primer sequence was 5′-AGGTACTCATCCAACACCAGGTAT-3′. Real-time PCR was then conducted in the Applied Biosystems 7500 Real Time PCR System with thermocycling setting of 50°C for 2 min, 95°C for 10 min, and 40 reps of 95°C for 15 s followed by 60°C for 1 min. Relative quantitation was achieved by normalizing to 18S rRNA reference values, as detected through FAM-MGB and by calibrating to control cells using the 2^−ΔΔCt method as described in Applied Biosystems User Bulletin 2: Rev B. For mouse RNA samples, the Δ^CT value was determined using 18S and ΔΔ^CT values determined using the Δ^CT value from one FVB control as the calibrator for all samples (FVB and PTH-D1). For OK cell time course, the Npt2a mRNA levels were expressed relative to control (t = 0) that was set to 100 for each experiment. The average values from four to six independent experiments ± SE were plotted on a semilogarithmic scale, and the half-life was calculated by linear regression.

Phosphate uptake assays.

Phosphate transport was measured by determination of radiolabeled phosphate uptake into OK cell monolayers. Cells were seeded onto 12-well plates and grown to 100% confluence, washed free of serum the night before the experiment, and treated with PTH for the designated period at 37°C. They were washed three times with transport medium consisting of (in mM) 137 NaCl, 5.4 KCl, 2.8 CaCl₂, 1.2 MgSO₄, and 0.1 KH₂PO₄. Phosphate uptake was initiated by the addition of transport medium containing ³²P-radiolabeled phosphoric acid. Uptake was continued for 10 min at room temperature. The cell monolayers were then washed three times with ice-cold medium in which N-methylglucamine was substituted for sodium chloride and ³²P_i was omitted. The cells were solubilized in 300 μl 0.5% Triton X-100 for 1.5 h at room temperature. A 100-μl aliquot from each well was aspirated into a scintillation vial to which scintillant was added and analyzed by liquid scintillation spectroscopy. From each separate assay, nonspecific binding was determined and subtracted. Each assay of OK cells was performed in triplicate and averaged, with the mean considered as a single data point. The data are expressed as percent inhibition compared with control uptake.

Ouabain-sensitive ⁸⁶Rb uptake.

Ouabain-sensitive ⁸⁶Rb uptake in OK cells was measured exactly as previously described (33). Cells treated with 100 nM PTH 1–34 for different times (see results) were pretreated with 5 μM monensin for 30 min. Half the cells received 4 mM ouabain. A trace amount of ⁸⁶Rb (∼1 μCi/ml ⁸⁶Rb) in serum-free MEM was added, and uptake was carried out for 10 min. The cells were washed five to six times with PBS and lysed overnight in 0.5 N NaOH containing 0.1% Triton X-100 at 37°C. An aliquot (100 μl) of the lysate was used to measure radioactivity. The difference between ⁸⁶Rb uptake measured in the presence of 4 mM ouabain was used as a measure of Na⁺-K⁺-ATPase-mediated transport activity. Uptake data are expressed as nanomoles rubidium accumulated per milligram of protein per 10 min.

Luciferase reporter gene assays.

NpT2a promoter-luciferase reporter gene constructs (NpT2a-luc) containing −208 and −4,400 bp were a generous gift of Heine Murer (25). NpT2a-luc or pGL3-basic and Renilla luciferase reporter (pRL-TK, Promega) plasmids were cotransfected into OK-WT cells using Lipofectamine 2000 (Invitrogen). At 24 h posttransfection, firefly luciferase and Renilla luciferase activities were determined using Dual-Glo Luciferase Assay System (Promega) according to the manufacturer's protocol. The luciferase:Renilla ratios were determined per well. For each independent experiment, the cells were transfected in triplicate and the values were averaged.

Statistics.

Data are shown as means ± SE. The n values represent the number of independent experiments. Each experiment was performed in triplicate unless otherwise indicated. P values were calculated by Student's t-test or by one-way ANOVA, followed by Bonferroni analysis using GraphPad Prism software. A P value <0.05 was a priori considered statistically significant.

RESULTS

Effect of acute vs. chronic PTH treatment on transport in Sprague-Dawley rats.

To determine whether the acute and chronic effects of PTH on apical membrane transporters (NpT2a and γ-glutamyl transferase) or BLM transporters (Na⁺-K⁺-ATPase) in kidney proximal tubules differed, Sprague-Dawley rats were treated subcutaneously with vehicle or with PTH [5 μg·kg body wt⁻¹·day⁻¹] for 6 h (acute) or for 4 days (chronic), with PTH injected every 24 h. BBM or BLM were prepared, and transporter expression was measured by Western blot analysis. As shown in Fig. 1, both acute and chronic treatment with PTH decreased expression of two apical membrane proteins, NpT2a (Fig. 1A) and γ-glutamyl transferase (Fig. 1B). Expression of the BLM protein, the Na⁺-K⁺-ATPase α-subunit, decreased in BLM from rats acutely treated with PTH but recovered to greater than control levels in BLM from rats chronically treated with PTH (Fig. 2A). Correspondingly, Na⁺-K⁺-ATPase activity significantly decreased in BLM prepared from rats acutely treated with PTH and recovered to control levels in BLM prepared from rats chronically treated with PTH (Fig. 2B).

Effect of PTH on NHERF-1 expression in Sprague-Dawley rats.

Recent reports suggest that NHERF-1 abrogates PTH receptor desensitization by displacing β-arrestin2 binding to the PTH receptor (13, 19, 54, 58, 60). Therefore, we measured the expression of NHERF-1 in BBM and BLM isolated from kidney cortex of rats treated with PTH for 6 h or 4 days. As shown in Fig. 3A, neither acute nor chronic PTH treatment affected NHERF-1 expression in BBM. However, NHERF-1 expression increased in BLM of rats treated with PTH for either 6 h or 4 days (Fig. 3B).

Transporter expression in renal tubules of PTH-D1 mice.

To confirm our findings in rats treated with exogenous PTH, we used a model of excessive endogenous PTH production. In this mouse model, parathyroid-specific cyclin D1 overexpression causes a gradual development of primary hyperparathyroidism, with parathyroid hyperplasia at 6 mo of age followed by development of biochemical features of hyperparathyroidism (28). With this model, we first measured transporter expression by immunohistochemistry. The kidneys of PTH-D1 animals displayed decreased levels of NpT2a expression in the proximal tubules compared with kidneys from FVB control mice (Fig. 4A). The expression of the α-subunit of Na⁺-K⁺-ATPase did not differ between the control and PTH-D1 mouse kidneys (Fig. 4B). No apparent differences in the expression of NHERF-1 in the BBM or BLM of the proximal tubules were observed (Fig. 4C), and similarly PTH-D1 mice displayed no discernible differences in the expression of the PTH receptor in either the BBM or BLM vs. control (Fig. 4D). To quantify these changes observed in immunohistochemistry, we measured the area occupied by the staining for NpT2a (Fig. 5A) and performed immunoblot analysis of crude membrane isolated from kidney cortex of PTH-D1 and background FVB animals for the Na-K α-subunit and NHERF-1 (Fig. 5B). Quantitative data confirmed our immunohistochemical data. We next isolated RNA from the cortex of control and PTH-D1 mice, which showed that, compared with control animals, PTH-D1 mice express ∼50% less NpT2a mRNA (Fig. 6).

Effect of acute and chronic PTH treatment on OK cell proximal tubule proteins.

To examine the mechanism for the sustained reduction of NpT2a during chronic PTH treatment, we turned to OK cells, a cell culture model of the renal proximal tubule. To confirm that the responses to acute and chronic PTH treatment in OK cells replicated our findings in rodent kidneys, we examined transporter activity and expression in OK cells. To examine activity, we treated OK cells with PTH for 2 h (acute effect) or for 1–7 days (chronic effect) and measured ³²P_i uptake as a measure of NpT2a function. As shown in Fig. 7A, PTH decreased ³²P_i uptake at all time points. This was coupled with a decreased NpT2a BBM expression with 2-h and 3-day PTH treatment (Fig. 7B). NHERF-1 BBM expression did not alter with either acute or chronic PTH (data not shown). Ouabain-sensitive ⁸⁶Rb uptake in OK cells decreased with acute PTH and returned to control levels with chronic PTH treatment (data not shown). These findings mimic observations from rats acutely and chronically treated with PTH (Fig. 1).

Effect of chronic PTH treatment on NpT2a mRNA expression.

To test for possible mechanisms for PTH-dependent downregulation of NpT2a mRNA expression, we determined the NpT2a mRNA half-life in OK cells. The basal half-life of NpT2a mRNA was determined by inhibiting transcription with actinomycin D. As shown in Fig. 8A, the half-life was 8.65 h. Treatment with PTH resulted in a significantly decreased NpT2a mRNA half-life of 2.22 h (Fig. 8A). We chose the 2- and 6-h time points to test whether the mechanism for PTH-mediated degradation of NpT2a mRNA is dependent on either transcription or translation. As shown in Fig. 8, B and C, inhibiting either transcription or translation in the presence of PTH blocked the PTH-mediated degradation of NpT2a mRNA at both time points.

To determine whether PTH affects NpT2a promoter function in addition to NpT2a mRNA stability, we measured NpT2a promoter activity in OK cells using luciferase reporter gene assays. We found no effect of PTH on NpT2a promoter function in OK cells (Fig. 9).

DISCUSSION

Our studies demonstrate that the actions of PTH on the proximal tubule are differentially regulated and suggest mechanisms to explain the discrepancies reported in the experimental models of the acute effects of PTH vs. the clinically observed effects of primary hyperparathyroidism, a naturally occurring human model of chronic PTH stimulation (26, 27, 29, 30, 49). Animals and proximal tubule cells treated with PTH for extended periods of time show loss of the PTH effect on the sodium pump but persistent inhibition of phosphate transport. These effects are seen at both the expression level for those transporters and at the functional level. While the BLM expression and the activity of the α-subunit of the sodium pump were transiently reduced after PTH stimulation, the apical membrane expression and function of the sodium-phosphate cotransporter were persistently suppressed. This persistent suppressive effect was specific for Npt2a as the expression of Na⁺-K⁺-ATPase, another membrane transporter whose function is inhibited by PTH, actually showed an increase in expression, followed by a return to control levels. Wang et al. (60) showed persistently decreased Na⁺-K⁺-ATPase α-subunit expression after 48-h treatment with high-dose but not low-dose PTH. The current study employed a protocol resembling the lower dose PTH arm of their study and examined Na transporter expression after a longer treatment. Collectively, our findings suggest that desensitization of transporter function and expression to PTH is determined specifically for each transporter. The Na⁺-K⁺-ATPase escaped regulation by PTH with chronic stimulation and is involved in global transepithelial solute transport, especially sodium transport. In contrast, Npt2a, which has a minor contribution to proximal renal tubule Na transport but a predominant role in determination of renal phosphate handling, showed persistent regulation by PTH.

The renal proximal tubule expresses two additional sodium-dependent phosphate transporters, Npt2c and PiT2 (9, 21). Npt2c is the third member of the type II sodium phosphate cotransporter family and is expressed exclusively on the apical membrane of the renal proximal tubule. PiT2 is a member of the type III sodium-phosphate cotransporter family which was initially described as a viral receptor and is expressed ubiquitously. The respective roles of Npt2a, Npt2c, and PiT2 in phosphate reabsorption have not been entirely clarified, especially with regard to human physiology, but in rodent models Npt2a plays the predominant role, being responsible for 70% of phosphate uptake (6, 57). Animals not expressing Npt2a show a distinctive phenotype characterized by hypophosphatemia, phosphate wasting, hypercalciuria, and bone abnormalities. Npt2c is expressed most highly in immature animals, waning as the animals age. The absence of Npt2c in rodents results in a minimal discernible phenotype (51). In humans, however, Npt2c mutations produce the specific syndrome of hereditary hypophosphatemic rickets with hypercalciuria, while Npt2a mutations have been associated with less remarkable phenotypes, suggesting species differences in respective roles of these proteins (37). Like Npt2a, Npt2c is regulated by PTH (52). Whether Npt2c inhibition is persistent or mediated through a similar mechanism as for Npt2a was not examined in this study but clearly warrants further investigation. Very little is known about the role of PiT2 or its regulation in the renal proximal tubule (59).

The mechanisms for desensitization of PTH responses are not well understood (8, 12, 13, 18–20, 23, 24, 55, 58, 60–62). In both PTH-D1 mice and Sprague-Dawley rats, PTH receptor expression in BBM and BLM did not change over the time of PTH stimulation, indicating that loss of PTH receptor expression did not account for the loss of the sodium pump responsiveness to PTH (Sprague-Dawley data not shown). We also showed in PTH-treated Sprague-Dawley rats that BBM NHERF-1 expression remained stable with both acute and chronic PTH stimulation while BLM NHERF-1 expression increased. These results suggest that NHERF-1 is potentially involved in the desensitization of BLM transporters to the effects of PTH, whereas PTH regulation of the apical NpT2a persists.

Given the results of the membrane protein expression experiments, we turned our attention to possible pathways for the chronic downregulation of NpT2a protein expression. In PTH-D1 animals, a 50% reduction in NpT2a mRNA was observed. This decrease in NpT2a mRNA in vivo is consistent with another study by Moe et al. (44), showing that mice with secondary hyperparathyroidism due to chronic kidney disease express levels of Npt2a mRNA that are ∼50% that of control Npt2a mRNA levels. Friedlaender et al. (22) were also able to demonstrate a decrease in Npt2a mRNA in PTH-infused parathyroidectomized rats vs. control rats. These studies, in addition to ours, suggest that PTH is capable of regulating NpT2a not only at the protein level but also at the mRNA level. The decrease in NpT2a mRNA could result from either a PTH-stimulated decrease in transcription, an increase in mRNA degradation, or both. Our data demonstrate for the first time that PTH treatment decreases NpT2a mRNA half-life. The PTH effect was abrogated by both actinomycin D and cycloheximide, indicating that transcription and translation are required for PTH-mediated degradation of NpT2a mRNA.

Future directions will include identifying the pathway responsible for chronic regulation of NpT2a mRNA by PTH. Multiple studies have identified PKA and protein kinase C (PKC) as major signal transduction pathways activated by PTH and integral to the regulation of both the transport activities and the metabolic activities stimulated by PTH (2, 10, 11, 31, 34, 38, 41, 43, 48). PTH-activated signaling pathways appear to exert redundant effects. For example, stimulation of OK cells with either a direct PKA or PKC activator results in inhibition of NaPi transport, sodium hydrogen exchange, and sodium pump activity. The significance of this redundancy and the mechanisms for coordinating the activation and subsequent effects of these signaling pathways remain largely unknown. Identifying the pathway involved in NpT2a mRNA regulation will elucidate some of the mechanisms of PTH signaling.

In summary, we have demonstrated differential regulation and desensitization of proximal renal tubule functions by PTH. The effects of PTH on Na⁺-K⁺-ATPase desensitize rapidly, while the effects on phosphate transport do not, conforming to the clinical picture seen in patients with chronic hyperparathyroidism. Persistent inhibition of phosphate transport appears to occur at least in part at the posttranscriptional level.

GRANTS

The work was supported by a Veterans Affairs Merit Review (E. D. Lederer), a Grant-in-Aid, Great River Affiliate, and a Scientist Development Grant from The American Heart Association (S. J. Khundmiri).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

Author contributions: R.M., K.H., S.A.S., and S.J.K. performed experiments; R.M., K.H., B.J.C., M.T.B., S.J.K., and E.D.L. analyzed data; R.M., B.J.C., S.J.K., and E.D.L. interpreted results of experiments; R.M. prepared figures; R.M. drafted manuscript; B.J.C., S.J.K., and E.D.L. provided conception and design of research; B.J.C., S.J.K., and E.D.L. edited and revised manuscript; B.J.C., S.J.K., and E.D.L. approved final version of manuscript.