PKB/SGK-Resistant GSK3 Enhances Phosphaturia and Calciuria

Abstract

Insulin and IGF1-dependent signaling activates protein kinase B and serum and glucocorticoid inducible kinase (PKB/SGK), which together phosphorylate and inactivate glycogen synthase kinase GSK3. Because insulin and IGF1 increase renal tubular calcium and phosphorus reabsorption, we examined GSK3 regulation of phosphate transporter activity and determined whether PKB/SGK inactivates GSK3 to enhance renal phosphate and calcium transport. Overexpression of GSK3 and the phosphate transporter NaPi-IIa in Xenopus oocytes decreased electrogenic phosphate transport compared with NaPi-IIa–expressing oocytes. PKB/SGK serine phosphorylation sites in GSK3 were mutated to alanine to create gsk3^KI mice resistant to PKB/SGK inactivation. Compared with wildtype animals, gsk3^KI animals exhibited greater urinary phosphate and calcium clearances with higher excretion rates and lower plasma concentrations. Isolated brush border membranes from gsk3^KI mice showed less sodium-dependent phosphate transport and Na-phosphate co-transporter expression. Parathyroid hormone, 1,25-OH vitamin D levels, and bone mineral density were decreased in gsk3^KI mice, suggesting a global dysregulation of bone mineral metabolism. Taken together, PKB/SGK phosphorylation of GSK3 increases phosphate transporter activity and reduces renal calcium and phosphate loss.

The kidneys play a central role in the regulation of mineral homeostasis by mediating excretion or reabsorption, respectively, of phosphate, calcium, and magnesium. Phosphate reabsorption occurs in the proximal tubule and is mediated by at least three distinct sodium-dependent phosphate cotransporters, namely NaPi-IIa (SLC34A1), NaPi-IIc (SLC34A3), and Pit-2 (SLC20A2), located in the apical brush border membrane.^1–3 Renal phosphate reabsorption is regulated by various factors including dietary phosphate intake, acid-base status, and various hormones, such as parathyroid hormone (PTH), 1,25-(OH)₂ vitamin D₃, fibroblast growth factor 23, insulin, and insulin-like growth factor 1 (IGF1).^4–10 Active calcium reabsorption is mediated by the transient receptor potential channel V5 (TRPV5) calcium channel expressed in the luminal membrane of the distal convoluted tubule and connecting tubule.^11–13 The expression and activity of TRPV5 is regulated by several factors similarly regulating renal phosphate transport, such as dietary calcium intake, PTH, klotho, acid-base status, and 1,25-(OH)₂ vitamin D₃.^11–13 The role of insulin and IGF1 has remained controversial.^14,15

The intracellular signaling cascades mediating the effects of these hormones on renal phosphate transporters are still incompletely understood. Signaling of the phosphaturic hormone PTH involves the protein kinases A and C and extracellular signal-regulated kinase,¹⁶ leading to the internalization and degradation of the NaPi-IIa cotransporter in the mouse and rat kidney.¹⁷ Signaling mediating the stimulating effect of insulin and IGF1 on renal phosphate reabsorption^4–6 has remained ill-defined. Signaling of insulin includes stimulation of the PI3 kinase pathway with subsequent activation of protein kinase B (PKB/Akt) and the serum- and glucocorticoid-inducible kinase (SGK) isoforms.^18,19 Both, PKB^20,21 and SGK^22,23 isoforms are known to phosphorylate and thus to inhibit the glycogen synthase kinase GSK3. However, nothing is known about the regulation of epithelial phosphate and Ca²⁺ transport by GSK3.

This study aimed to define the role of PKB/SGK-dependent regulation of GSK3 in the control of renal tubular calcium and phosphate transport. To this end, renal mineral excretion was analyzed in gene-targeted mice in which the serine residues within the respective PKB/SGK phosphorylation sites of GSKα and GSK3β had been replaced by alanin residues (GSK3α^21A/21A, GSK3ß^9A/9A). In those mice (gsk3^KI), GSKα and GSK3β are resistant against inactivation by PKB/SGK.²⁴ As shown before, gsk3^KI mice are resistant to the effect of insulin on muscle glycogen synthase.²⁴

RESULTS

A first series of experiments analyzed the in vitro influence of GSK3β on NaPi-IIa, a major renal tubular phosphate transporter. Exposure of water-injected Xenopus oocytes to phosphate (2 mM) in the bath solution did not induce any significant current, indicating that these oocytes do not express significant endogenous electrogenic phosphate transport (Figure 1A). In oocytes injected with cRNA encoding NaPi-IIa, however, the addition of phosphate induced an inward current (intraperitoneally_i) of 58 ± 10 nA (n = 21 oocytes). Coexpression of GSK3β significantly decreased intraperitoneally_i in NaPi-IIa–expressing oocytes (27 ± 5 nA; n = 17 oocytes). A chemiluminescence-based assay was used to study whether coexpression of GSK3β altered the membrane abundance of NaPi-IIa. As shown in Figure 1B, the surface expression of NaPi-IIa was indeed significantly reduced by coexpression of GSK3β. The decrease of NaPi-IIa activity is not simply caused by the expression of an additional protein. The phosphate-induced current in NaPi-IIa–expressing oocytes is, for instance, similar with and without coexpression of mTOR, if the kinase is inhibited by rapamycin.²⁵

Figure 1.

Coexpression of GSK3 inhibits electrogenic phosphate transport in NaPi-IIa–expressing Xenopus oocytes. (A) Arithmetic means ± SEM (n = 13 to 21) of phosphate (2 mM)-induced inward currents (I_Pi) in Xenopus oocytes injected with water (left bar), NaPi-IIa cRNA (middle bar), or NaPi-IIa and GSK3β cRNA (right bar). ***Statistically significant difference from absence of NaPi-IIa cRNA (P < 0.001). #Difference from absence of GSK3β cRNA (P < 0.05). (B) Arithmetic means ± SEM (n = 42 to 60) of the normalized chemiluminescence intensity of NaPi-IIa expression in Xenopus oocytes injected with water (left bar), NaPi-IIa cRNA (middle bar), or NaPi-IIa and GSK3β cRNA (right bar). ***Statistically significant difference from absence of NaPi-IIa cRNA (P < 0.001). ###Difference from absence of GSK3β cRNA (P < 0.001).

As a next step, activity and expression of renal sodium-dependent phosphate transporters was assessed in brush border membrane vesicles (BBMVs) from GSK3αβ knockin (KI) mice (gsk3^KI) and corresponding wild-type (WT) mice (gsk3^WT). Total sodium-dependent uptake of phosphate into isolated BBMVs was significantly reduced in gsk3^KI mice (Figure 2). After inhibition of SLC34 type transporters (i.e., NaPi-IIa and NaPi-IIc) with phosphonoformic acid (PFA, 6 mM), phosphate transport was significantly reduced in both genotypes (Figure 2). In the presence of PFA, the residual sodium-dependent phosphate transport activity was still lower in gsk3^KI mice than in gsk3^WT mice (Figure 2). The protein expression of NaPi-IIa, NaPi-IIc, and Pit-2 in the brush border membrane fraction of WT and KI murine kidneys is shown in Figure 3. The abundance of NaPi-IIa and of NaPi-IIc protein was significantly reduced in gsk3^KI mice compared with gsk3^WT mice (Figure 3). Semiquantitative real-time PCR analysis showed significantly increased mRNA abundance of NaPi-IIa and Pit-2 in gsk3^KI mice, suggesting that GSK3 acts partially upstream of the transcription of these transporters (all data expressed as mRNA expression normalized to HPRT mRNA abundance; see Concise Methods; NaPi-IIa: gsk3^WT: 29.0 ± 3.2, n = 6; gsk3^KI: 59.97 ± 11.12, n = 5; P < 0.05; NaPi-IIc: gsk3^WT: 0.22 ± 0.01, n = 6; gsk3^KI: 0.22 ± 0.03, n = 5; not significant; Pit-2: gsk3^WT: 0.16 ± 0.01, n = 6; gsk3^KI: 0.34 ± 0.04, n = 5; P < 0.01).

Figure 2.

Sodium-dependent phosphate transport activity in BBMVs from gsk3^KI and gsk3^WT mice. Arithmetic means ± SEM (n = 5 to 6 each group) of the sodium-dependent transport rates into isolated BBMVs after 1 minute in the absence (left bars) and presence (right bars) of 6 mM PFA to block phosphate transport mediated by SLC34 family members. *P < 0.05.

Figure 3.

Protein abundance of renal sodium-dependent phosphate cotransporters in the brush border membrane in kidneys from gsk3^KI and gsk3^WT mice. Western blots for NaPi-IIa, NaPi-IIc, and Pit-2. All membranes were stripped and reprobed for β-actin to control for loading. Densitometry was performed and the ratio of the protein of interest over β-actin calculated. Bar graphs present data as arithmetic means ± SEM (n = 5 to 6 each group).

Next, in vivo experiments were performed in gsk3^KI and gsk3^WT mice. As reported earlier,²⁶ the body weight was similar in gsk3^KI mice (29.1 ± 0.3 g, n = 10) and gsk3^WT mice (28.9 ± 1.4 g, n = 10). The urinary flow rate was significantly higher in gsk3^KI mice (95.2 ± 10.1 μl/24 h per gram body weight, n = 6) than in gsk3^WT mice (24.1 ± 4.8 μl/24 h per gram body weight, n = 6). Similarly, the creatinine clearance was significantly higher in gsk3^KI mice (0.39 ± 0.06 ml/24 h, n = 6) than in gsk3^WT mice (0.19 ± 0.02 ml/24 h, n = 6).

As shown in Figure 4 (top right panel), the urinary phosphate excretion was significantly larger in gsk3^KI than in gsk3^WT mice. In a further series of experiments, the mice were fed a control diet followed by a phosphorus-deficient diet for 3 days. Before the phosphorus-deficient diet, the fractional phosphate excretion was 31.1 ± 6.9% (n = 5) in gsk3^WT and 63.9 ± 13.6% (n = 6) in gsk3^KI mice (Figure 4, bottom panel). On the third day of the phosphorus-deficient diet, the fractional phosphate excretion amounted to 1.4 ± 0.1% (n = 5) in gsk3^WT mice and to 31.8 ± 11.1% (n = 6) in gsk3^KI mice, values significantly (P < 0.05) different (Figure 4, bottom panel).

Figure 4.

Plasma phosphate concentration, urinary phosphate excretion, and fractional phosphate excretion in gsk3^KI and gsk3^WT mice. Arithmetic means ± SEM (n = 6 to 10) of plasma phosphate concentration (top left panel) and urinary phosphate excretion (top right panel) in GSK3 knockin mice (gsk3^KI, closed bars) and corresponding wild-type mice (gsk3^WT, open bars). The bottom panel depicts the arithmetic means ± SEM (n = 5 to 6) of the fractional phosphate excretion of GSK3 knockin mice and wild type mice maintained under a normal diet (left bars) or maintained for 3 days under a phosphorus-deficient diet (right bars). *P < 0.05 versus respective value of gsk3^WT mice.

In theory, the enhanced urinary phosphate excretion could have been secondary to enhanced plasma phosphate concentration. However, the plasma phosphate concentration was significantly lower in gsk3^KI than in gsk3^WT mice (Figure 4, top left panel).

The phosphaturia could further have been caused by inhibition of renal tubular phosphate reabsorption by PTH. However, as shown in Figure 5A (left panel), the plasma concentration of PTH was significantly lower in gsk3^KI than in gsk3^WT mice. A decreased plasma PTH concentration is expected to reduce the formation of 1,25-(OH)₂ vitamin D₃. As shown in Figure 5A (right panel), the plasma concentration of 1,25-(OH)₂ D₃ was indeed significantly lower in gsk3^KI than in gsk3^WT mice. The transcript levels of CYP27B1 (25-hydroxyvitamin D₃ 1α-hydroxylase), which catalyzes the last step in the synthesis of the active 1,25-(OH)₂D₃, tended to be higher in gsk3^KI than in gsk3^WT mice; this difference, however, did not reach statistical significance (Figure 5B).

Figure 5.

Plasma PTH and 1,25-(OH)₂D₃ concentration in gsk3^KI and gsk3^WT mice. (A) Arithmetic means ± SEM of plasma PTH (left panel, n = 6) and 1,25-(OH)₂D₃ (right panel, n = 17 to 19) concentration in GSK3 knockin mice (gsk3^KI, closed bars) and corresponding wild-type mice (gsk3^WT, open bars). *P < 0.05 versus respective value of gsk3^WT mice. (B) mRNA expression of CYP27B1 (25-hydroxyvitamin D₃ 1a-hydroxylase). Arithmetic means ± SEM (n = 5 to 6).

A decrease of the plasma PTH and 1,25-(OH)₂ vitamin D₃ concentration is further expected to blunt the renal tubular Ca²⁺ reabsorption. As shown in Figure 6 (right panel), the renal Ca²⁺ excretion was indeed larger in gsk3^KI than in gsk3^WT mice. The plasma Ca²⁺ concentration tended to be lower in gsk3^KI than in gsk3^WT mice; this difference, however, did not reach statistical significance (Figure 6, left panel). Further experiments addressed the fractional calcium excretion before and after a phosphorus-deficient diet for 3 days. Before the low phosphate diet, the fractional calcium excretion was 1.6 ± 0.2% (n = 4) in gsk3^WT mice and 2.4 ± 0.2% (n = 5) in gsk3^KI mice, values significantly (P < 0.05) different. On the third day of the phosphorus-deficient diet, the fractional calcium excretion amounted to 0.8 ± 0.1% (n = 4) in gsk3^WT mice and to 11.7 ± 5.4% (n = 5) in gsk3^KI mice.

Figure 6.

Plasma calcium concentration and urinary calcium excretion in gsk3^KI and gsk3^WT mice. Arithmetic means ± SEM (n = 6 to 19) of plasma calcium concentration (left panel) and urinary calcium excretion (right panel) in GSK3 knockin mice (gsk3^KI, closed bars) and corresponding wild-type mice (gsk3^WT, open bars). *P < 0.05 versus respective value of gsk3^WT mice.

Renal loss of both Ca²⁺ and phosphate together with low PTH and 1,25-(OH)₂ vitamin D₃ levels is expected to compromise the mineralization of bone. Thus, bone density was determined. As shown in Figure 7, femur bone density was indeed significantly lower in gsk3^KI than in gsk3^WT mice.

Figure 7.

Bone density of gsk3^KI and gsk3^WT mice. (A) Arithmetic means ± SEM (n = 4 each group) of bone density in GSK3 knockin mice (gsk3^KI, closed bar) and corresponding wild-type mice (gsk3^WT, open bar). *P < 0.05 versus respective value of gsk3^WT mice. (B) Original microCT image of the leg of a GSK3 knockin mice (gsk3^KI, right image) and of a corresponding wild-type mouse (gsk3^WT, left image). (C) Cuts from microCT images in tranaxial, coronal, and sagittal directions through the femur and knee. These high-resolution microCT images display the degradation of trabecular bone and compacta in GSK3 knockin mice (gsk3^KI, right image) and in wild-type mice (gsk3^WT, left image).

DISCUSSION

This study confirmed the previous observation²⁶ that creatinine clearance and urinary flow rate are higher in mice expressing protein kinase B(PKB)/serum and glucocorticoid-inducible kinase (SGK)-resistant glycogen synthase kinase GSK3 (gsk3^KI). PKB/SGK resistance was accomplished by introducing a mutation of GSK3ß in which the serine of the PKB phosphorylation site was replaced by an alanine (GSK3ß^9A/9A) and at the same time a mutation in GSK3α in which the serine of the PKB phosphorylation site was replaced by an alanine (GSK3ß^12A/12A). Because inhibition of GSK3 mediates the effect of insulin on glycogen synthase,^24,27 the effect of insulin on muscle glycogen synthase is abrogated in gsk3^KI mice.²⁴

More importantly, these observations showed a completely novel function of GSK3, i.e., a powerful role in the regulation of renal mineral excretion. Compared with wild-type mice (gsk3^WT), gsk3^KI mice are both hypercalciuric and hyperphosphaturic. Renal phosphate excretion is mostly determined by the expression and activity of Na⁺-dependent phosphate transporters in the brush border membrane of the proximal tubule. Their expression and activity is regulated by various hormones and factors, but only very little is known about intracellular signaling events mediating the control of these transporters.^1,2,8,10 Our observations showed that GSK3 plays an important role as evident from the stimulation of NaPi-IIa activity in Xenopus oocytes by GSK3 and the decreased phosphate fluxes in isolated BBMVs from gsk3^KI mice. Reduced activity of SLC34 transporters (NaPi-IIa and NaPi-IIc) and other PFA-resistant phosphate transporters was detected. The expression of Pit2, resistant to PFA, was, however, not altered, suggesting that either only its activity is affected by GSK3 or that other unknown PFA-resistant phosphate transporters contribute to the residual activity in the presence of PFA.

The phosphaturia of gsk3^KI mice cannot be explained by an increased plasma phosphate concentration, which was actually significantly decreased. Moreover, the phosphaturia of gsk3^KI mice contrasts the decreased plasma level of PTH, a major regulator of renal phosphate transport.⁸ PTH stimulates the internalization and subsequent degradation of NaPi-IIa.^8,17 The hormone thus enhances renal phosphate excretion and decreases plasma phosphate concentration. A decreased plasma PTH concentration is thus expected to enhance renal phosphate reabsorption and to increase plasma phosphate concentration, the opposite of what was observed. A decreased plasma phosphate concentration inhibits PTH release.²⁸ However, the decrease of the plasma phosphate level is very discrete and hardly accounts for the low plasma PTH levels. The plasma 1,25-(OH)₂ D₃ concentration were decreased in gsk3^KI mice. Low plasma PTH levels are expected to decrease the plasma 1,25-(OH)₂ D₃ concentration, because PTH stimulates the renal 1α-hydroxylase,²⁹ the rate-limiting enzyme of 1,25-(OH)₂ D₃ formation.^29,30 On the other hand, formation of 1,25-(OH)₂ D₃ is stimulated in a PTH-independent manner by cellular phosphate depletion.³¹ The transcript levels of the 1α-hydroxylase tended to be enhanced in gsk3^KI mice. The possibility must be considered that GSK3 influences 1α-hydroxylase and 1,25-(OH)₂ D₃ formation by further mechanisms in addition to its influence on phosphate balance and PTH plasma concentration.

Because 1,25-(OH)₂ D₃ stimulates intestinal phosphate absorption,³² decreased 1,25-(OH)₂ D₃ formation is expected to decrease intestinal phosphate absorption. Impaired intestinal phosphate uptake could contribute to the development of hypophosphatemia.

PTH and 1,25-(OH)₂D₃ stimulate renal tubular Ca²⁺ reabsorption via upregulation of the TRPV5 channel.^12,13,33 The decreased plasma PTH and 1,25-(OH)₂D₃ concentration in gsk3^KI mice could thus contribute to the calciuria of gsk3^KI mice by affecting translation, trafficking, and/or activity of TRPV5 channels.

Despite the decreased PTH plasma concentration, the bone mass was significantly decreased in gsk3^KI mice. Thus, the renal Ca²⁺ and phosphate loss of gsk3^KI mice may impair mineralization of bone. Phosphate is known to inhibit the generation of new osteoclasts and to stimulate apoptosis of mature osteoclasts.³⁴ Moreover, Akt/PKB-dependent regulation of GSK3 may more directly participate in the regulation of bone cells. Akt/PKB regulates the survival of both osteoblasts³⁵ and osteoclasts.^36,37

In conclusion, Akt/PKB-dependent regulation of GSK3 participates in the control of renal tubular phosphate transport. Loss of Akt/PKB-dependent inhibition of GSK3αβ leads to renal phosphate wasting, which presumably contributes to or even accounts for the decrease of PTH release with resulting calciuria, decreased formation of 1,25-(OH)₂ D₃, and decreased mineralization of bone. These observations thus show a novel, powerful element in the regulation of mineral metabolism.

CONCISE METHODS

In Vitro Expression

For generation of cRNA, constructs were used encoding wild-type human NaPi-IIa³⁸ and human GSK3β. The cRNA was generated as described previously.³⁹ GSK3β cDNA was from the “Deutsches Ressourcenzentrum für Genomforschung,” Berlin, Germany. For electrophysiology, Xenopus oocytes were prepared as described previously.^40,41 Ten nanograms of NaPi-IIa cRNA was injected on the first day and 7.5 ng of GSK3β cRNA was injected on the second day after preparation of Xenopus oocytes. All experiments were performed at room temperature 3 days after the second injection. Two electrode voltage-clamp recordings were performed at a holding potential of −50 mV. The data were filtered at 10 Hz and recorded with a Digidata A/D-D/A converter and Chart V.4.2 software for data acquisition and analysis (Axon Instruments). The control solution (superfusate/ND96) contained 96 mM NaCl, 2 mM KCl, 1.8 mM CaCl₂, 1 mM MgCl₂, and 5 mM HEPES, pH 7.4. Phosphate (2 mM) was added to induce NaPi-IIa–dependent currents. The flow rate of the superfusion was 20 ml/min, and a complete exchange of the bath solution was reached within about 10 seconds. For the determination of NaPi-IIa surface expression, defolliculated oocytes were incubated with primary rabbit anti-NaPi-IIa antibody (diluted 1:500; Lifespan Biosciences, Seattle, WA) and secondary, peroxidase-conjugated goat anti-rabbit antibody (diluted 1:1000; Cell Signaling, Danvers, MA). Individual oocytes were placed in 96-well plates with 10 μl of Super Signal ELISA Femto Maximum Sensitivity Substrate (Pierce, Rockford, IL). The chemiluminescence of the oocytes was quantified in a luminometer (WalterWallac2 plate reader; Perkin Elmer, Jügesheim, Germany) by integrating the signal over a period of 1 second. Results display normalized arbitrary light units that are proportional to the detector voltage.

In Vivo Experiments

All animal experiments were conducted according to the guidelines of the American Physiologic Society and approved by the respective authorities.

Mice were generated in which the codon encoding Ser9 of the GSK3ß gene was changed to encode nonphosphorylatable alanine (GSK3ß^9A/9A), and simultaneously, the codon encoding Ser21 of GSK3α was changed to encode the nonphosphorylatable GSK3α^21A/21A, thus yielding the GSK3α/β^{21A/21A/9A/9A} double knockin mouse (gsk3^KI) as described previously.²⁴ The mice were compared with corresponding wild-type mice (gsk3^WT).

The mice were fed a control diet (Altromin, Lage, Germany) containing 7523 mg/kg phosphorus or a phosphorus-deficient diet containing 131 mg/kg phosphorus, as indicated. They had free access to tap drinking water.

To determine creatinine clearance, urinary flow rate, and urinary excretion of Ca²⁺ and phosphate, the mice were placed individually in metabolic cages (Techniplast, Hohenpeissenberg, Germany) for 24-hour urine collection as described previously.⁴² They were allowed a 2-day habituation period, during which food and water intake, urinary flow rate, and excretion of electrolytes were recorded every day to ascertain that the mice were adapted to the new environment. Subsequently, 24-hour collection of urine was performed for 3 consecutive days to obtain the urinary parameters. To assure quantitative urine collection, metabolic cages were siliconized, and urine was collected under water-saturated oil.

The phosphate concentration was determined colorimetrically using commercial diagnostic kits (Roche Diagnostics, Mannheim, Germany). The urinary concentration of Ca²⁺ was measured by flame photometry (ELEX 6361; Eppendorf) or by a photometric method according to the manufacturer's instructions (dri-chem clinical chemistry analyzer FUJI FDC 3500i; Sysmex, Norsted, Germany). The creatinine concentration in urine was determined using the Jaffe reaction (Sigma, St. Louis, MO); creatinine and calcium concentration in plasma were measured using a photometric method. The plasma intact parathormone concentration was measured using an ELISA kit (Immunotopics, San Clemante, CA). A radioimmunassay kit was used to determine the concentration of 1,25-(OH)₂D₃ (IDS, Boldon, UK) in plasma.

For the analysis of bone density, animals were killed, and legs were amputated and fixated in formalin. The samples were scanned with a high-resolution microCAT-II (Siemens Preclinical Solutions) small animal computed tomography (CT) scanner using a field of view of 3.1 × 3.1 × 4.8 cm³. The x-ray tube parameters were set at 80 kVp and 400 μA. The images were acquired with 720 angular projections (exposure time, 1200 ms per projection) over 360° and binned with a factor of two, yielding a spatial resolution of approximately 38 μm. The total scan time was 24 minutes. Reconstructed CT images were analyzed with the Inveon Research Workplace software (Siemens Preclinical Solutions) by drawing a standard-sized container around the femur and applying a region growth routine to segment the trabecular bone structure. For all samples, the same upper and lower density threshold was applied, and the relative numbers of trabecular bone density were compared.

RNA Extraction and Real-Time RT-PCR

Snap-frozen kidneys (five kidneys for each condition) were homogenized in RLT-Buffer (Qiagen, Basel, Switzerland) and supplemented with ß-mercaptoethanol, resulting in a final concentration of 1%. Total RNA was extracted and reverse transcribed as described previously.⁹ Quantitative real-time qRT-PCR was performed on the ABI PRISM 7700 Sequence Detection System (Applied Biosystems). Primers for all genes of interest were as described.⁹ For analysis of the data, the threshold was set to 0.06 because this value had been determined to be in the linear range of the amplification curves for all mRNAs in all experimental runs. The expression of the gene of interest was calculated in relation to hypoxanthine guanine phosphoribosyl transferase. Relative expression ratios were calculated as R = 2^{[Ct(HPRT) − Ct(test gene)]}, where Ct represents the cycle number at the threshold 0.06.

BBMV Preparation and Phosphate Transport Assays

BBMVs were prepared from rat kidney cortex and outer medulla using the Mg²⁺ precipitation technique as described previously.^43,44 The phosphate transport rate into BBMVs was measured in freshly prepared BBMVs at 25°C in the presence of inward gradients of 100 mM NaCl or 100 mM KCl and 0.1 mM K-phosphate. The substrate P_i was prepared with 0.125 mM K₂HPO₄ and ³²P (1 μCi/ml) to yield a final concentration of 0.1 mM, close to the expected apparent K_mPi for Na⁺-dependent transport in renal BBMVs. The stop solution contained 100 mM mannitol, 5 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 5 mM P_i. The Na⁺ dependence was established by incubating BBMVs in solutions in which KCl replaced NaCl equimolarly. Phosphate uptake was determined after 60 seconds, representing initial linear conditions, and after 120 minutes, to determine the equilibrium values. To distinguish between Na⁺-dependent P_i uptake mediated by SLC34 family members (e.g., NaPi-IIa and NaPi-IIc) and other Na⁺-dependent phosphate transporters such as SLC20 family members (e.g., Pit-1 and Pit-2), we used trisodium PFA (final concentration, 6 mM) added to the same solution with 107 mM NaCl. PFA has previously been shown to have a higher selectivity for SLC34 than for SLC20 phosphate transporters at this concentration.³ The total protein concentration was measured using the Bio-Rad Protein Assay kit (Bio-Rad, Hercules, CA). BBMVs were stored at −80°C until further use.

Western Blotting

After measurement of the protein concentration (Bio-Rad), 10 μg of renal brush border membrane proteins was solubilized in loading buffer containing dithiothreitol and separated on 8% polyacrylamide gels. For immunoblotting, the proteins were transferred electrophoretically to polyvinylidene fluoride membranes (Immobilon-P; Millipore, Bedford, MA). After blocking with 5% milk powder in Tris-buffered saline/0.1% Tween-20 for 60 minutes, the blots were incubated with the primary antibodies: rabbit polyclonal anti-NaPi-IIa (1:6000),⁴⁵ rabbit polyclonal anti-NaPi-IIc (1:10,000),⁹ rabbit polyclonal anti-Pit-2 (1:3000; kindly provided by Dr. V. Sorribas, University of Zaragoza, Spain),³ and mouse monoclonal anti-β-actin antibody (42 kD; Sigma; 1:5000) either for 2 hours at room temperature or overnight at 4°C. Membranes were incubated for 1 hour at room temperature with secondary goat anti-rabbit or donkey anti-mouse antibodies 1:5000 linked to alkaline phosphatase (Promega) or to horseradish peroxidase (Amersham). The protein signal was detected with the appropriate substrates (Millipore) using the DIANA III-chemiluminescence detection system (Raytest, Straubenhardt, Germany). All images were analyzed using the software Advanced Image Data Analyzer AIDA, Raytest to calculate the protein of interest/β-actin ratio.

Statistical Analysis

Data are provided as means ± SEM; n represents the number of independent experiments. All data were tested for significance using paired or unpaired t tests or ANOVA. GraphPad InStat version 3.00 for Windows 95 (GraphPad Software, San Diego, CA) was used. Only results with P < 0.05 were considered statistically significant.

DISCLOSURES

None.