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J Am Soc Nephrol 15: 2972-2980, 2004
© 2004 American Society of Nephrology
doi: 10.1097/01.ASN.0000144207.44469.BE
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BASIC SCIENCE

Calcineurin A{beta} Is Central to the Expression of the Renal Type II Na/Pi Co-transporter Gene and to the Regulation of Renal Phosphate Transport

Yulia Moz*, Ronen Levi*, Vardit Lavi-Moshayoff*, Keith B. Cox{dagger}, Jeffery D. Molkentin{dagger}, Justin Silver* and Tally Naveh-Many*

*Minerva Center for Calcium and Bone Metabolism, Nephrology and Hypertension Services, The Hadassah Hebrew University Medical Center, Jerusalem, Israel; and {dagger}Department of Pediatrics, University of Cincinnati, Children’s Hospital Medical Center, Cincinnati, Ohio

Correspondence to Dr. Tally Naveh-Many, Nephrology Services, Hadassah Hospital, P.O. Box 12000, Jerusalem Israel 91120. Phone: +972-2-6776789; Fax: +972-2-6421234; E-mail: tally{at}cc.huji.ac.il


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The sensing and response to extracellular phosphate (Pi) concentration is preserved from prokaryotes to mammals and ensures an adequate supply of Pi in the face of large differences in its availability. In mammals, the kidneys are central to Pi homeostasis. Renal Pi reabsorption is mediated by a Na/Pi co-transporter that is regulated by a renal Pi sensing system and humoral factors. The signal transduction by which Pi regulates type II Na/Pi activity is largely unknown. It is shown that calcineurin inhibitors specifically and dramatically decrease type II Na/Pi gene expression in a proximal tubule cell line and in vivo. Mice with genetic deletion of the calcineurin A{beta} gene had a marked decrease in type II Na/Pi mRNA levels and remarkably did not show the expected increase in type II Na/Pi mRNA levels after the challenge of a low-Pi diet. In contrast, the regulation of renal 25(OH)-vitamin D 1{alpha}-hydroxylase gene expression by Pi was intact. This is the first demonstration that calcineurin has a crucial role in the signal transduction pathway regulating renal Pi homeostasis both in vitro and in vivo. These results suggest that the use of calcineurin inhibitors contributes to the renal Pi wasting seen in renal transplant patients.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Phosphate (Pi) homeostasis is essential to life and is dependent on active renal reabsorption. In diseases of phosphorus (P) homeostasis, such as X-linked hypophosphatemia or oncogenic osteomalacia, there is a tremendous renal P loss with severe bone disease (1). In contrast, in chronic renal failure, the P retention leads to secondary hyperparathyroidism with disabling bone disease and vascular calcification associated with a high mortality (2). The kidney has an intrinsic P sensing system that regulates the activity of apical brush border membrane type II Na/Pi co-transporters (3). However, it is still not known how the organism senses changes in serum P.

There are three mammalian Na/Pi co-transporter families: types I through III. Type II Na/Pi activity is responsible for >80% of renal P reabsorption and contains three isoforms—IIa through c—of which IIa is the major factor in renal P reabsorption and regulation in adult mice (4). Type II Na/Pi activity is increased by a low serum P and decreased by parathyroid hormone (PTH) and FGF23 (4–8). The regulation of type II Na/Pi is at the level of recruitment of new transporters to the apical proximal tubule membrane, the level of synthesis of new transporters and their breakdown (5,9). In addition, there is regulation at the level of type II Na/Pi gene expression, which in vivo is mainly posttranscriptional (10,11). In the rat, a cis acting element in the type II Na/Pi co-transporter (Na/Pi-2) mRNA at the junction of the coding region and the 3'-untranslated region (UTR) interacts with trans acting renal cytosolic proteins and determines Na/Pi-2 mRNA stability in response to dietary P restriction (12). An additional level of regulation of Na/Pi-2 is its translational control by a low-P diet (10).

The renal type II Na/Pi co-transporter interacts with a number of other intracellular proteins that determine its localization to the apical cell membrane. Type II Na/Pi functions as part of a heteromeric protein complex, organized by the PDZ proteins Na/Pi-Cap 1 and Na+/H+ exchange-regulatory factor 1 (NHERF-1) as well as regulatory proteins such as AKAP2 (4,13). PEX19 also interacts with Na/Pi-II and is involved in the internalization and trafficking of the co-transporter (14). These proteins are important to the proper positioning of the co-transporter as well as its recycling. Many of these proteins are protein kinases, and we therefore hypothesized that kinases or phosphatases may have a role in the regulation of Na/Pi II gene expression and transport activity. In addition, we have preliminary studies on the involvement of calcineurin in the regulation of PTH gene expression by a low-P diet in the rat.

We now show in vitro using OK cells that express the type II Na/Pi co-transporter, Na/Pi-4, that the protein phosphatase type 2B, calcineurin (15), has an important regulatory role in the activity of Na/Pi-4 as well as in its regulation by a low extracellular Pi. The calcineurin inhibitor cyclosporin A (CsA) decreased Na/Pi type II mRNA levels both in vitro in OK cells that were grown in a low-P medium and in vivo in kidneys of rats that were fed a low-P diet. We also show in mice with genetic deletion of calcineurin A{beta} that type II Na/Pi mRNA levels are decreased and do not increase after the stimulation of a low-P diet. This effect was specific to Na/Pi type II gene expression because these mice exhibited the expected increase in renal 25(OH)-vitamin D 1{alpha}-hydroxylase gene expression after a low-P diet. These results demonstrate the important role of the protein phosphatase type 2B calcineurin in determining type II Na/Pi activity and underscore the role of protein phosphorylation in renal P handling.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Tissue Culture and Cells
OK cells were grown in DMEM supplemented with 1% glutamine, 1% penicillin-streptomycin, and 10% FCS. For transport experiments, cells were plated in six-well plates in triplicate. When the cells reached confluence of 95 to 100%, the medium was changed to DMEM that contained 3 mM (high P), 1 mM (normal P), or no Pi (low Pi) and supplemented with 1% glutamine, 1% penstrep, and 0.1% BSA. The medium also contained the different compound or vehicle, as indicated. All compounds were purchased from Sigma (St. Louis, MO) and used at a 1:1000 dilution. CsA and okadaic acid were dissolved in ethanol and added to final concentrations of 10–5 to 10–6 and 10–7 M, respectively. PD 98059 was dissolved in methanol and studied at 10–5 M. SB 203580 and Forskolin were dissolved in DMSO and added at 10–5 M. For transcription inhibition, 5,6-dichlorobenzimidazole riboside (DRB) dissolved in DMSO was added (25 µg/ml) for 6, 16, and 24 h, together with CsA.

Pi and Alanine Transport
Sodium-dependent Pi transport was measured by a modification of the method described by Lederer et al. (16). Cells were grown in six-well plates, and after incubation of the cells in different media for the indicated times, the medium was aspirated or collected for measurements of Pi levels in the medium. The cells were washed with unlabeled transport medium that contained 10 mM HEPES, 10 mM Tris (pH 7.4), 137 mM NaCl, 5.4 mM KCl, 2.8 mM CaCl2, 1.2 mM MgSO4, and 0.1 mM KH2PO4. For measuring P uptake, the cells were exposed to labeled transport buffer that contained 0.5 µCi/ml [32P] KH2PO4 (200 mCi/mmol; Amersham, Little Chalfont, UK) for 15 min at room temperature with gentle shaking. Uptake was terminated by aspiration of the transport medium and washing the cells three times with ice-cold buffer that contained 137 mM NaCl and 14 mM Tris (pH 7.4). The cells were then collected in 1 N NaOH, and the radioactivity representing Pi incorporation was measured in a scintillation counter using Opti-flour cocktail scintillation fluid for aqueous samples (Packard Bioscience, Meriden, CT) after a 60-min incubation of the vials in the dark at room temperature. Incorporation of alanine into OK cells was performed similarly with the unlabeled transport buffer that contained 0.1 mM alanine and the labeled buffer that contained 2.5 mCi/ml [3H] alanine (54 Ci/mmol; Amersham). Each experiment was performed in triplicate, and the mean values of counts per minute were calculated. The experiments were repeated at least three times. Protein determination was performed by Bradford reagent (Biorad, Hercules, CA) on randomly selected wells from each experiment and was shown to be constant in all measurements.

Mice and Diets
Weanling Sprague-Dawley rats (Harlan, Jerusalem, Israel) or weanling calcineurin A{beta}–/– mice and wild-type mice (17–19) were fed a normal-calcium (0.6%), normal-P (0.6%) diet (normal; TD 92158); a low-P (0.02%), normal-calcium (0.6%) diet (low Pi; TD 92157); or a low-calcium (0.02%), normal-P diet (0.6%; TD 03547; Teklad, Madison, WI) for 2 wk. At 2 wk, the kidneys were removed under pentobarbital anesthesia for RNA extraction. CsA (Novartis, Basel, Switzerland) was prepared in olive oil and given by oral gavage at a concentration of 10 mg/kg. All animal experiments were approved by the Institutional Animal Care and Use Committees.

Northern Blot Analysis
For Northern blots, cells were plated in 24-well plates in triplicate in parallel with plating of the cells in six-well plates for transport analysis. In experiments in which cells were treated with DRB, cells were grown in six-well plates to increase the amount of total RNA loaded on the gel and by that the Na/Pi mRNA signal in Northern blots. Mouse kidneys were snap-frozen in liquid nitrogen and stored at –80°C until RNA extraction. Total RNA was extracted with Tri reagent, and Northern blots were performed as described previously (8). For opossum Na/Pi-4 mRNA, a NotI-SalI Na/Pi-4 cDNA fragment in pSPORT (20) was used. For mouse Na/Pi-2 mRNA, a fragment for the rat Na/Pi cDNA was used (8). For the 25(OH) vitamin D 1-{alpha} hydroxylase (1{alpha}-OHase) mRNA, a PCR fragment was provided by T. Yamashita (Pharmaceutical Research Laboratories, KIRIN Brewery Co., Gunma, Japan) (6).

Western Blots
A total of 50 µg of Cytoplasmic S-100 extracts were run on 10% SDS-PAGE and transferred to a nitrocellulose membrane (Schleicher and Schuel, Dassel, Germany). The membranes were washed in TBS 0.05% Tween, blocked in 5% skim milk powder in TBS 0.05% Tween, and analyzed with a goat anti-rat protein phosphatase type 2B (PP2B) A{beta} antibody (Santa Cruz Biotechnologies, Santa Cruz, CA) diluted 1:100 in TBS and 5% milk powder by incubation overnight at 4°C, according to the manufacturer’s instructions. The membrane was then washed and incubated for 1 h with a secondary antibody, and bound antibody was detected by chemiluminescence. For identifying the PP2B A{beta} band, a parallel gel was run and Western blot was performed with a PP2B A{beta} antibody that was first preincubated with a PP2B A{beta} peptide for 2 h at room temperature, before it was added to the membrane. This incubation completely eliminated the PP2B A{beta} band, demonstrating the specificity of the relevant band.

Statistical Analyses
Results are presented as mean ± SE. Statistical significance of differences was analyzed using t test for unpaired values. Values were considered different from each other at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
OK cells respond to a low extracellular P by a two- to threefold increase in P transport compared with a high extracellular Pi. The signal transduction pathway that transduces the message of extracellular P to the regulation of Na/Pi gene expression and P transport is not clear. For studying this, OK cells were cultured in a low-P medium and treated with different agonists and inhibitors of classical signal transduction pathways. The effects of these compounds on Na/Pi-4 mRNA levels and P transport were measured. CsA inhibits the PP2B calcineurin. Addition of CsA to cells that were grown in a low-P medium led to a marked decrease in Na/Pi mRNA levels (Figure 1, A and C) with no effect on total RNA levels as measured by ribosomal 28S and 18S levels (Figure 1A) or on a specific L32 ribosomal protein control mRNA (Figure 1C). CsA also markedly decreased P transport in these cells (Figure 1, B and D). Okadaic acid is a specific inhibitor of phosphatases PP2A and PP1 but at the concentrations used here (1 x 10–7 M) also inhibits PP2B. The addition of okadaic acid led to a decrease in Na/Pi mRNA levels and P transport (Figure 1, C and D). As a control, the transport of alanine was measured under the same conditions. Alanine transport was not decreased by CsA or okadaic acid (Figure 1E). In fact, in some experiments, alanine transport was increased by these compounds. Although we do not know the reason for this effect, it shows that the decrease in Na/Pi transport by CsA and okadaic acid is not a result of a general decrease in transport in these cells. Forskolin, the PKA stimulator, and PD98059, the ERK 1/2 inhibitor, had no effect on Na/Pi mRNA levels or on Na/Pi transport at 24 h (Figure 1, A and B). In some but not all OK cell lines, forskolin decreased P transport at 4 h, when its effect has been shown to be dependent on the presence of NHERF-1 (21). Forskolin (22) and the inhibitors of the ERK 1/2 and mitogen-activated protein kinase (MAPK) pathways (23) are stable at 24 h, at which time the present studies were performed when an effect on Na/Pi mRNA would have been evident. There was also no effect of SB 203580, the p38 MAPK inhibitor, when given separately or together with PD98059 (data not shown). CsA had no effect on Na/Pi-4 mRNA levels in OK cells that were grown in high-P (3 mM) medium (Figure 1F). P transport was decreased approximately twofold in the high-P medium and increased by approximately threefold in the low-P medium compared with (1 mM) normal-P medium (Figure 1G). There was no effect of CsA on P transport in both normal- and high-P media (Figure 1G). This may be because calcineurin mediates the stimulatory effect of low P and is not involved in basal P transport or in the inhibition by a high P.



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  		Figure 1. Cyclosporine A (CsA) but not PD98059 and forskolin regulate Na/Pi-4 mRNA levels and phosphorus (P) transport in OK cells that were grown in a low-P medium. OK cells were plated in DMEM in triplicate, and at confluence, the medium was changed to a low-P (A through G) or a normal- (G) or high-P medium (F and G) that contained different compounds for an additional 24 h. (A) Northern blot for Na/Pi-4 mRNA of cells that were grown in a low-P medium and treated with vehicle (ethanol or DMSO), PD98059 (1 x 10–5 M), CsA (1 x 10–5 M), or forskolin (1 x 10–5 M). Each lane contains RNA from one plate. Ethidium bromide staining of the membrane showing 28S and 18S ribosomal RNA demonstrates equal loading of the gels. (B) P transport shown as percentage of transport in untreated low-P controls in the same cells as in A. (C) Northern blot for Na/Pi-4 mRNA of cells that were grown in a low-P medium and treated with vehicle (ethanol), okadaic acid (1 x 10–7 M), or CsA (1 x 10–5 M) for an additional 24 h. Rehybridization to L 32 ribosomal protein mRNA demonstrates equal loading of the gels. (D and E) P transport and alanine transport shown as percentage of transport in untreated low-P controls in the same cells as in C. (F) Northern blot for Na/Pi-4 mRNA of cells that were grown in low- or high-P media and treated with vehicle (ethanol) or CsA (1 x 10–5 M) and ethidium bromide staining of the membrane showing 28S and 18S ribosomal RNA. (G) P transport shown as percentage of transport in untreated normal P controls in the same cells as in F and in cells that were treated with a normal-P medium (control). CsA only decreased P transport in a low-P medium. *P < 0.05 versus low-P controls. Similar results were obtained in three to five repeat experiments.

 
The effect of CsA on Na/Pi gene expression and transport in a low-P medium was dose and time dependent. P transport was decreased already at 4 h of CsA with a larger decrease at 24 h (10–6 and 10–5 M) and with a maximal effect at 48 h (10–5 M; Figure 2). CsA dramatically de-creased Na/Pi mRNA levels at 16 h (data not shown) and 24 h (Figure 1, A and C). Therefore, CsA decreases Na/Pi-4 mRNA Na/Pi-4 gene expression and P transport at 16 and 24 h in a dose-dependent manner.



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  		Figure 2. CsA decreases P transport in OK cells that were grown in a low-P medium in a dose- and time-dependent manner. OK cells were grown in triplicate in a low-P medium with 1 x 10–6 and 1 x 10–5 M CsA or vehicle for 4, 24, and 48 h, when P transport was measured and expressed as percentage of control at each time point. *P < 0.05 versus controls. Similar results were obtained in three repeat experiments.

 
For determining whether the regulation of Na/Pi-4 mRNA by CsA is transcriptional or posttranscriptional, DRB was added to OK cells that were grown in a low-P medium and treated with CsA or vehicle. Addition of the transcriptional inhibitor DRB to OK cells demonstrated that the half-life of Na/Pi-4 mRNA was ~8 h (data not shown). CsA decreased Na/Pi mRNA to nearly undetectable levels (Figure 1), so we could not measure the decay rate of Na/Pi-4 mRNA in CsA-treated cells after DRB (data not shown). We therefore studied the effect of transcription inhibition on P transport, with and without CsA given simultaneously with the DRB, in OK cells that were grown in a low-P medium. If the effect of CsA on Na/Pi-4 mRNA levels and P transport were transcriptional, then DRB would be expected to eliminate it. OK cells were grown in a low-P medium, and DRB was added together with CsA for 6, 16, and 24 h, at which times P transport was measured. CsA, in the absence of DRB, led as before to a threefold decrease in P transport at 24 h (Figure 3). DRB alone led to an ~40% decrease in P transport, representing the effect of transcriptional inhibition on Na/Pi transport. The addition of CsA still led to a further threefold decrease in P transport (Figure 3). This result indicates that the effect of CsA on P transport is not dependent on transcription of new Na/Pi-4 mRNA, suggesting a posttranscriptional regulation by CsA. It is interesting that the regulation of Na/Pi-2 gene expression by low P in vivo in rats is also posttranscriptional (10).



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  		Figure 3. The transcription inhibitor DRB does not affect the decrease in P transport by CsA. OK cells were grown in triplicate in a low-P medium and treated without and with CsA 1 x 10–5 M and DRB (25 mg/ml) for 24 h. P transport is shown as percentage of transport in cells that were grown in low-P medium without addition of DRB or CsA. *P < 0.05 versus controls. Similar results were obtained in three repeat experiments.

 
Because calcineurin inhibitors specifically and dramatically decrease Na/Pi-4 mRNA levels and Pi transport in OK cells, we studied the effect of CsA on Na/Pi-2 gene expression in vivo. Weanling rats (four rats in each group) were fed a low-P diet for 2 wk to increase Na/Pi-2 mRNA levels (24) and then administered either CsA (10 mg/kg) or vehicle by oral gavage. Kidney total RNA was extracted at 2, 4, and 20 h and analyzed by Northern blot for Na/Pi-2 mRNA levels and a control gene, the Na+/Ca+ exchanger. There was a marked decrease in Na/Pi-2 mRNA levels after CsA at 2 (Figure 4) and 5 h but not at 20 h (data not shown). As expected. the low-P diet led to a decrease in serum P levels compared with rats that were fed a normal diet (1.3 ± 0.3:3.2 ± 0.4 mmol/L; P < 0.01) and an increase in serum calcium levels compared with rats that were fed a control diet (3.3 ± 0.2:2.6 ± 0.1 mmol/L; P < 0.05). There was no significant difference in serum P in rats that were given CsA or vehicle (1.5 ± 0.1:1.3 ± 0.3 mmol/L; NS) despite the large differences in Na/Pi-2 mRNA levels. This may be because of the transient effect of CsA on Na/Pi-2 mRNA levels. These results show that CsA decreases Na/Pi mRNA levels both in vivo and in vitro.



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  		Figure 4. CsA decreases Na/Pi-2 mRNA levels in vivo in rats that were fed a low-P diet. Weanling rats were fed a low-P diet for 2 wk and then given CsA by oral gavage. After 2 h, kidneys were analyzed for Na/Pi-2 mRNA and the Na/Ca exchanger as a control gene by Northern blot. Each lane represents RNA from a single rat. Similar results were obtained in a repeat experiment.

 
CsA inhibits the PP2B calcineurin. To study the role of calcineurin on Na/Pi-2 gene expression in vivo more directly, we used mice with genetic deletion of the calcineurin A{beta} gene (calcineurin A{beta}–/–) (25). We first showed the expression of calcineurin A{beta} in rat renal cortex. It has been shown by immunohistochemistry that calcineurin A{beta} is expressed in the rat renal tubules and calcineurin A{alpha} in the glomeruli (26). A Western blot confirmed the expression of calcineurin A{beta} in rat renal cortex as in brain and also in the parathyroid (Figure 5A). The calcineurin A{beta} antibody also identified a band in OK cells that was slightly larger than the 60-kD calcineurin A{beta} band (Figure 5A). For showing specificity of the calcineurin A{beta} band in the Western blot, proteins were analyzed on a parallel gel using the calcineurin A{beta} antibody preincubated with a calcineurin A{beta} peptide. The calcineurin A{beta} peptide eliminated the smaller specific calcineurin A{beta} band in all extracts tested (data not shown), thus identifying it as the relevant band. These results show that calcineurin A{beta} is expressed in the kidney cortex as well as in OK cells. We therefore studied the effect of ablation of the calcineurin A{beta} gene on NaPi-2 gene expression.



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  		Figure 5. Calcineurin A{beta} is expressed in rat renal cortex, and calcineurin A{beta}–/– mice have decreased Na/Pi-2 mRNA levels that are not increased after a low-P diet. (A) Protein extracts from rat brain (B), renal cortex (K), parathyroid (PT), and OK cells (OK) were run on SDS-PAGE and analyzed by Western blot for calcineurin A{beta}. The 60-kD calcineurin A{beta} is shown by the arrow on the left, and the position of molecular weight markers are shown on the right. (B) Weanling calcineurin (Cn) A{beta}+/+ and calcineurin A{beta}–/– mice were fed a control or a low-P diet for 2 wk when kidneys were removed, and total RNA was extracted and analyzed for Na/Pi-2 mRNA and 18S ribosomal RNA by Northern blots. Representative Northern blot for Na/Pi-2 mRNA in kidneys from three mice in the wild-type control group and four mice in all other groups, with each lane containing RNA from the kidney of a single mouse. (C) Quantification of three Northern blots for Na/Pi mRNA normalized to 18S ribosomal RNA, derived from a total of seven mice in each group fed control, low-P, or low-calcium diets for 2 wk. Results are expressed as percentage of control of calcineurin A{beta}+/+ mice on a control diet. (D) Northern blot for 1{alpha}-hydroxylase (1{alpha}-OHase) in kidneys of Cn A{beta}+/+ or Cn A{beta}+/+ weanling mice that were fed control, low-calcium, or low-P diets for 2 wk. Ethidium bromide staining of the membrane is shown below and demonstrates equal loading. *P < 0.05 versus calcineurin A{beta}+/+ mice fed a control diet.

 
Calcineurin A{beta}–/– mice and wild-type mice (calcineurin A{beta}+/+) were fed normal, low-P or low-calcium diets, and after 2 wk, renal Na/Pi 2 mRNA was analyzed by Northern blots. In the wild-type mice, a low-P diet led to the expected increase in Na/Pi-2 mRNA levels (Figure 5, B and C), similar to our previous results in mice (27) and rats (8). Calcineurin A{beta}–/– mice had decreased Na/Pi-2 mRNA levels and remarkably showed no increase in Na/Pi-2 mRNA levels after the low-P diet (Figure 5, B and C). Dietary calcium does not regulate Na/Pi-2 mRNA levels in rats (8). A low-calcium diet had no effect on Na/Pi-2 mRNA levels in kidneys of calcineurin Ab+/+ mice and calcineurin A{beta}–/– mice (Figure 5C).

Another renal gene that is increased by a low-P diet is 25(OH)-vitamin D 1-{alpha} hydroxylase (1{alpha}-OHase) (28–30). It therefore was of interest to study whether the 1{alpha}-OHase mRNA levels were affected by genetic deletion of the calcineurin A{beta} gene. mRNA levels for 1{alpha}-OHase as measured by Northern blots were undetectable in both calcineurin A{beta}+/+ and calcineurin A{beta}–/– mice kidneys (Figure 5D). However, a low-P diet led to an increase in 1{alpha}-OHase mRNA levels in both the wild-type and knockout mice (Figure 5D), indicating that this regulation is not affected by calcineurin gene deletion. The expression of the 1{alpha}-OHase gene is also regulated by low calcium (31,32) in contrast to NaPi-2 gene expression that is not regulated by low calcium. A low-calcium diet led to an increase in 1{alpha}-OHase mRNA levels in the wild-type and knockout mice (Figure 5D). Therefore, the regulation of the 1{alpha}-OHase gene expression by low P and Ca2+ does not involve calcineurin A{beta} in contrast to the critical role of calcineurin A{beta} in the regulation of type II Na/Pi gene expression and transport by low P. Together, our in vitro and in vivo data underline the importance and specificity of calcineurin in the signal transduction pathway regulating renal type II Na/Pi co-transporter gene expression and thereby renal P transport and renal P homeostasis.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Using OK cells, we studied several inhibitors of signal transduction and showed that calcineurin inhibitors were effective in decreasing Na/Pi-4 mRNA levels and P transport in cells that were grown in a low-P medium. Therefore, the PP2B calcineurin is necessary for the low-P-induced increase in P transport. There was no effect of CsA on Na/Pi-4 mRNA levels and P transport in cells that were grown in a medium with normal or high P concentrations. This suggests that calcineurin is necessary only for the stimulatory effect of low P in these cells. Changes in extracellular fluid (ECF) P regulate P transport by OK cells but not Na/Pi-4 mRNA levels (33). This is in contrast to in vivo, where rats that are fed a low-P diet have an increase in both P transport and Na/Pi-2 mRNA levels (8,10). It is of interest that in OK cells, Na/Pi-4 mRNA levels responded to CsA but not to the stimulus of low P in the medium. The mechanism for this difference is not clear.

In the kidney, Na/Pi type II is localized to the proximal tubule, but the administration of CsA could be acting on calcineurin isoforms in different segments of the renal tubule (34,35). We have shown that the calcineurin A{beta} isoform is expressed in the proximal tubule OK cell line and renal cortex, suggesting that it could be functional in the proximal tubule. Prie et al. (36) showed in rats that were fed a normal-P diet that CsA increased the expression of the Na/Pi-2 protein and P transport. However, we now show in weanling rats that were fed a low-P diet that CsA decreased renal NaPi-2 mRNA levels at short time intervals (2 and 5 h), suggesting a physiologic relevance for the effect of CsA. This suggests that calcineurin may be involved only in the renal response to the stress of low P. Mice with genetic deletion of calcineurin A{beta} provided an exciting tool to study the physiologic relevance of our in vitro findings using OK cells and our in vivo studies on Na/Pi mRNA levels. The calcineurin A{beta}–/– mice had been used to determine the role of calcineurin A{beta} in a mouse model of cardiac hypertrophy (18,19,37). Calcineurin A{beta}–/– mice showed that calcineurin is involved both in basal type II Na/Pi co-transporter mRNA levels and in its increase after a low-P diet. The results on Na/Pi-2 gene expression in vivo support the physiologic relevance of our in vitro results in OK cells, where the calcineurin inhibitor CsA decreased Na/Pi-4 mRNA levels and P transport. The specificity of the effect of calcineurin gene deletion on Na/Pi-2 gene expression was shown by the intact gene expression of 1{alpha}-OHase in calcineurin A{beta}–/– mice as well as its increase after a low-P diet in these mice. It has been shown that low P increases 1{alpha}-OHase gene transcription (30) in contrast to the posttranscriptional effect of low P on Na/Pi-2 gene expression (12). Therefore, low P regulates these two genes by different mechanisms. These two genes also respond differently to a low-calcium diet. A low-calcium diet has no effect on Na/Pi mRNA levels in rats (24) and in the calcineurin A{beta}+/+ mice as well as the calcineurin A{beta}–/– mice. However, a low-calcium diet increases 1{alpha}-OHase mRNA levels in rats (31,32) and in both the calcineurin wild-type and knockout mice (Figure 5D). Therefore, calcineurin is essential to the expression and regulation of the Na/Pi gene, and this effect is specific because it is not necessary for the regulation of the1{alpha}-OHase gene by both P and calcium. The role of calcineurin A{beta} is not at the level of P sensing because the response to low P of the 1{alpha}-OHase gene is intact. The results indicate, therefore, that calcineurin A{beta} has a crucial role in the signal transduction that responds to the changes in ECF P concentration and determines type II Na/Pi co-transporter mRNA levels.

The enigma of how the organism recognizes and responds to changes in ECF P is not restricted to eukaryotes. The cellular response to changes in ECF P concentration is preserved from bacteria to humans. In the budding yeast Saccharomyces cerevisiae, P starvation induces the transcription of several genes involved in P metabolism (38,39). It first increases the production of both a high-affinity P transporter and secreted phosphatases, which scavenge P from the environment. If starvation persists, then S. cerevisiae arrest both their growth and cell division. In mammals, P depletion not only has a local effect on the kidney but also has a separate effect to markedly decrease PTH gene expression and PTH secretion, which in turn results in an increased renal P reabsorption (40,41). In the parathyroid, P deprivation results in a marked inhibition of parathyroid cell proliferation (42). This is similar to the effect of P depletion in yeast to decrease cell proliferation. In the kidney, the Na/Pi co-transporter itself is not involved in P sensing, because mice with knockouts of Na/Pi-2 gene retain an intact P responsive system (28). These Na/Pi-2 knockout mice still respond to low P in the regulation of intracellular processes, such as increased renal 1,25(OH)2 vitamin D3 synthesis and the regulation of the 1{alpha}-OHase gene expression (28).

The type II Na/Pi co-transporter is regulated by changes in dietary P but also by factors such as PTH and FGF23 (7). The phosphaturic response to PTH has been well studied. It involves lysosomal breakdown of Na/Pi-II proteins as a result of activation of several protein kinases (PK) (5). These include PKA, PKC, and PKG and the ERK/MAPK pathway (4,21,43,44). The NHERF-1 interacts directly with the Na/Pi-4 transporter and is involved in the signaling pathway of PTH inhibition of P uptake by OK cells (21).

The well-documented changes in intracellular Ca2+ induced by a low ECF P are particularly relevant to our findings showing the centrality of calcineurin A{beta} to the response to a low-P diet. P deficiency results in a secondary increase in intracellular Ca2+ derived from a decreased efflux of Ca2+ from the cell (45,46). The intracellular Ca2+ might then increase the activity of calcium-responsive pathways, such as cPLA2 or Ca2+-calmodulin-dependent protein phosphatases. Our results demonstrate that the PP2B calcineurin is necessary for the regulation of type II Na/Pi gene expression and P transport by low P. The decrease in basal levels of Na/Pi-2 mRNA in the calcineurin A{beta}–/– mice suggests that calcineurin may also be involved in basal Na/Pi gene expression. They show that calcineurin is critical both to type II Na/Pi gene expression and to the signal transduction from P sensing to type II Na/Pi mRNA levels. There may also be a clinical relevance to our findings. Patients who are given CsA after organ transplantation often have a marked phosphaturia and resultant hypophosphatemia that may require supplementation with oral P (47–49). The studies presented here provide insight into the mechanisms involved.


   Acknowledgments
 
This work was supported in part by grants from the Israel Academy of Sciences, the Hadassah Women’s Health program, Chugai Pharmaceuticals, and the Minerva Foundation. Minerva is funded through the BMBF.

We thank Miriam Offner for skilled technical assistance and T. Yamashita for the 1{alpha}-OHase PCR fragment.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received for publication June 30, 2004. Accepted for publication August 20, 2004.




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