1 Institute of Physiology, University of Zurich-Irchel, CH-8057 Zurich, Switzerland; 2 Department of Medicine, Division of Nephrology, University of Louisville, Louisville, Kentucky 40292; and 3 Klinik B für Innere Medizin, Kantonsspital, CH-9007 St. Gallen, Switzerland
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ABSTRACT |
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The purpose of the present study was to determine whether isohydric changes in HCO3 concentration and PCO2 directly affect apical Na-dependent Pi (Na-Pi) cotransport in OK cells (opossum kidney cell line). Cells were kept at either 44 mM NaHCO3/10% CO2, pH 7.4 (high-HCO3/CO2 condition), or 22 mM NaHCO3/5% CO2, pH 7.4 (low-HCO3/CO2 condition) (for 14-24 h). Incubation in lower HCO3/CO2 concentrations increased Na-Pi cotransport 1.5-fold. The increased Na-Pi cotransport was paralleled by a two- to threefold increased expression of the NaPi-4 transporter protein and a two- to threefold increase in NaPi-4 mRNA abundance. The increase in NaPi-4 mRNA could be completely prevented by incubation in the presence of a transcriptional inhibitor, suggesting that the increase in NaPi-4 mRNA results from an increased NaPi-4 mRNA transcription. In agreement, the NaPi-4 promoter activity was stimulated by 50% at lower HCO3/CO2 concentrations. In conclusion, our data demonstrate that isohydric changes in HCO3 concentration and PCO2 exert a significant, direct cellular effect on Na-Pi cotransport and NaPi-4 protein expression in OK cells by affecting NaPi-4 mRNA transcription.
acidosis; alkalosis; parathyroid hormone; promoter; proximal tubule
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INTRODUCTION |
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THE INTERRELATIONSHIPS between acid-base and phosphate metabolism are manifold and complex. Acid-base disturbances have important effects on the renal handling of phosphate, whereas alterations in phosphate balance affect systemic acid-base homeostasis. Administration of phosphate has been demonstrated to induce metabolic alkalosis by increasing renal bicarbonate retention both in rats and humans (9, 15), whereas phosphate depletion induces metabolic acidosis by impairing renal proton secretory capacity (5).
Renal phosphate excretion is increased in chronic metabolic acidosis in humans resulting in renal hypophosphatemia (17). Metabolic alkalosis induced by bicarbonate infusion was also shown to increase renal phosphate excretion in parathyroidectomized rats (31). Respiratory acid-base disturbances also affect renal phosphate handling. Acute respiratory acidosis increased renal Pi excretion in rats (30), whereas chronic respiratory alkalosis was demonstrated to result in increased phosphate reabsorption and renal hyperphosphatemia in humans (16, 20).
Information on the mechanisms of regulation of renal phosphate transport by changes in acid-base balance is of great physiological and pathophysiological interest for at least two reasons: 1) alterations in renal phosphate transport by pH, HCO3, and/or CO2 concentrations are expected to (co)determine the renal systemic response to acid and alkali loads, that is, to affect the severity of acidemia or alkalemia; and 2) alterations in renal phosphate transport induced by changes in acid-base homeostasis change phosphate stores and, thereby, might affect multiple systemic processes and functions, including bone mineralization and functions of erythrocytes, leukocytes, and myocardium (12).
We have shown previously in opossum kidney (OK) cells that a proximal tubule-specific Na-dependent Pi transporter (Na-Pi cotransporter) is stimulated by low pH and low HCO3 concentrations by posttranscriptional mechanisms. This stimulation of the Na-Pi cotransport was prevented by dexamethasone (10), suggesting that the phosphaturia typical of metabolic acidosis might not be a direct consequence of acidemia but, at least in part, of associated systemic effects (enhanced glucocorticoid activity; Refs. 2 and 10). In addition, in vivo acid-base disturbances result in rather modest changes in hydrogen ion concentration due to compensatory mechanisms, which raises some doubts on the quantitative effects of increases in hydrogen ion concentration in vivo on Na-Pi cotransport activity.
The goals of the present studies were to evaluate whether Na-Pi cotransport is regulated by HCO3/CO2 independent of hydrogen ion concentration (isohydric environment). Previous studies on the regulation of intracellular pH in renal cells demonstrated that cell pH varies in direct linear proportion with extracellular hydrogen ion concentration (26). Only changes in extracellular pH (by changing HCO3 or CO2) affect intracellular pH significantly, and isohydric changes in HCO3/CO2 were shown to exert no significant effect on cell pH (14). Therefore, it appears that isohydric changes of HCO3/CO2 concentrations affect the cell and the regulation of its functions differently than do changes of extracellular pH (by changing HCO3 or CO2). For the present study, we used opossum kidney cells (OK cells, Ref. 13), which represent a well-characterized model with which to study the regulation of proximal tubular Na-Pi cotransport (1, 3, 4, 19, 23) and which contain an apically located type II Na-Pi cotransporter (NaPi-4, Ref. 5).
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MATERIALS AND METHODS |
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Cell cultures. All cell culture supplies were obtained from GIBCO-BRL (Basel, Switzerland). Opossum kidney cells (OK cells, clone 3B/2) were grown in DME + Hams's F-12 medium (DME-F12, 1:1) supplemented with 10% FCS, 22 mM NaHCO3, 20 mM HEPES, 2 mM L-glutamine, 50 IU/ml penicillin, and 50 µg/ml streptomycin in a humidified atmosphere of 5% CO2, 95% air at 37°C.
For experiments, cells were seeded on 35-mm petri dishes (Nunc) or 10-cm petri dishes (Corning) and grown to confluence in the media indicated above. Subsequently, cells were deprived of serum for 24 h in DME-F12 medium (1:1) supplemented with 44 mM NaHCO3, 50 IU/ml penicillin, and 50 µg/ml streptomycin in a humidified atmosphere of 10% CO2-90% air at 37°C. The pH was controlled with a microelectrode and adjusted to pH 7.4 with NaHCO3 or HCl. For experimental incubations (time interval as given in RESULTS), the cells were kept in DME-F12 medium (1:1) supplemented with 50 IU/ml penicillin and 50 µg/ml streptomycin and with 44 mM NaHCO3, pH 7.4, in a humidified atmosphere of 10% CO2-90% air at 37°C (control) or with 22 mM NaHCO3, pH 7.4, and additional 22 mM NaCl (for equalization of the osmolality difference) in a humidified atmosphere of 5% CO2-95% air at 37°C (experimental). Again, the pH was controlled with a microelectrode and adjusted to pH 7.4 for the control and experimental condition with NaHCO3 or HCl. Additionally, the pH was controlled a number of times at the end of the incubation periods. Even after prolonged incubation periods, the pH was never more than 0.04-0.06 pH units smaller than at the beginning of the experiments.
Membrane preparation. Cells grown in 10-cm petri dishes were washed with cold 0.9% NaCl and 10 mM Tris · HCl [pH 7.4, Tris-buffered saline (TBS)] and subsequently with 5 mM HEPES-KOH (pH 7.4). A volume of 15 ml of 5 mM HEPES-KOH supplemented with 4 mM EDTA and with the protease inhibitor phenylmethylsulfonyl fluoride (1 mM, Sigma) was added, and the cells were scraped off the dish. For homogenization, the cell suspension was passed five times through a 20-ml syringe connected to a 20-gauge needle. The homogenized suspension was centrifuged at 31,000 rpm for 40 min at 4°C (Sorvall ultracentrifuge OTD 50B, T 865 rotor). The pellet was resuspended in 400 µl of 50 mM mannitol with 10 mM HEPES-Tris (pH 7.2). The protein concentration was determined by the Bio-Rad protein assay.
SDS-PAGE and immunoblotting. Equal amounts of the membrane preparations were used for the SDS-PAGE (9% gels) (18). The separated proteins were transferred onto cellulose-nitrate (BA 83, Schleicher & Schuell) according to Towbin et al. (27). Nonspecific binding was blocked by incubating the nitrocellulose in TBS containing 5% nonfat dry milk and 1% Triton X-100 (Blotto-TX-100) at room temperature for 2 h. The NaPi-4 protein was detected using a polyclonal antiserum raised against the COOH-terminal 12 amino acids of the published NaPi-4 sequence (antibody dilution 1:2,000) (25). For antibody production (in New Zealand White rabbits), the NaPi-4 peptide was coupled to keyhole limpet hemocyanin. The specificity of this antiserum was previously established by a peptide protection assay (10). Incubation with the primary antibody took place overnight at 4°C. The nitrocellulose was washed four times with TBS + 10% Blotto-TX-100 and incubated for 1 h with Blotto-TX-100 at room temperature. Thereafter, the nitrocellulose was incubated with a 1:10,000 dilution of an anti-rabbit immunoglobulin labeled with horseradish peroxidase (Amersham) in Blotto-TX-100 for 2 h at room temperature. The nitrocellulose was washed four times with TBS. The signals were detected by the enhanced chemiluminescence system (Amersham) according to the manufacturer's protocol using Kodak X-OMAT AR films, and they were quantified by densitometry. Broad-range SDS-PAGE molecular protein markers (Bio-Rad) were run in parallel.
RNA isolation, Northern blot analysis, and
densitometry. Total RNA was isolated with TRIzol
Reagent (GIBCO) according to the manufacturer's protocol. The quantity
of the RNA was analyzed by absorption at 260 and 280 nm. After
extraction, RNA (8-10 µg) was size fractionated by
electrophoresis on 0.8% agarose gels containing formaldehyde.
Prehybridization (2-4 h at 68°C) and hybridization (overnight
at 68°C) of the RNA blots were performed in buffer consisting of
6× SSC (1× SSC is 0.15 M NaCl and 0.015 M sodium citrate,
pH 7.0), 5× Denhardt's solution, 0.1 mg/ml salmon sperm DNA,
0.5% SDS. Full-length cDNA probes of OK cell type II Na-Pi cotransporter (NaPi-4) (25)
and rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (28) were
labeled by random priming (Pharmacia) using
[-32P]dCTP
(DuPont-NEN). After hybridization, blots were washed twice for 5 min
each time in 2× SSC + 0.1% SDS at room temperature, twice for 30 min each time in 1× SSC + 0.1% SDS at 60°C, and once in
1× SSC at room temperature for 5 min. Northern blot signals were
quantitated using ImageQuant (Molecular Dynamics). Quantitated data are
presented as NaPi-4 mRNA-to-GAPDH mRNA ratios to correct unequal loading.
The OK-Npt2 promoter-luciferase constructs. Genomic DNA fragments of different sizes (327 bp and 4,700 bp including 40 bp of exon 1) of the 5'-flanking region of the OK-Npt2 (OK cell Npt2 gene = OK cell type II Na-Pi cotransporter gene) were subcloned into the pGL3 basic vector (luciferase reporter gene) from Promega as described by Hilfiker et al. (8).
Measurement of OK-Npt2 promoter
activity. The OK-Npt2
promoter activity was measured by reporter gene analysis using the
above mentioned constructs. For standardization of the promoter
activity the pGL3 (SV40) promoter (Promega) and the pGL3 basic vector
(Promega) were used. As internal standard for transfection efficiency a -galactosidase expressing vector pCMV-LacZ (kindly provided by Dr.
S. Rusconi, University of Zurich, Switzerland) was cotransfected. Transient cell transfections,
-galactosidase reaction and the reporter gene analysis were performed as described by Hilfiker et al.
(8).
Uptake measurements. Na-dependent uptake of Pi was measured on plastic dishes (35 mm; Nunc) as described previously (24).
Incubation in PTH. Parathyroid hormone
(PTH fragment 1-34; Sigma) was stored as a stock solution of
104 M in a solution of 10 mM acetic acid, 0.2 mM
1,4-dithio-DL-threitol, and 1%
bovine serum albumin. On the experimental day, this stock was diluted
100-fold in 10 mM acetic acid, and 20 µl of this PTH solution was
added to one dish (35 mm) containing 2 ml medium. The cells were
incubated in this 10
8 M PTH
solution for 3 h. In control cells, 20 µl of vehicle (10 mM acetic
acid) was used.
Incubation in dexamethasone.
Dexamethasone (Sigma) was dissolved in ethanol and stored as a stock
solution of 102 M. On the
experimental day, this stock solution was diluted in the cell culture
medium to the final solution of
10
6 M. Control cells
received the corresponding amount of vehicle.
Incubation in DRB.
5,6-Dichloro-1--D-ribofuranosylbenzimidazole
(DRB; Sigma) was stored as a stock solution of 13 mM in ethanol at
20°C. On the experimental day, this stock solution was
diluted 200-fold in the cell culture medium to a final concentration of 65 µM, which was shown to effectively block RNA polymerase II (7).
Control cells received the corresponding amount of vehicle.
Expression of data. All experiments were performed at least twice, and one representative experiment was chosen for publication. For the uptake experiments, Northern blots, and reporter gene analyses, statistic results are expressed as means ± SD. Significance of differences were calculated by the two-sided unpaired t-test (n = 4, unless indicated otherwise).
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RESULTS |
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Effect of HCO3/CO2 on Na-Pi cotransport. OK cells were grown to confluence and deprived of serum at 44 mM NaHCO3/10% CO2, pH 7.4 (for 24 h). Subsequently, cells were kept at either 44 mM NaHCO3/10% CO2, pH 7.4 (higher HCO3/CO2 condition; control), or 22 mM NaHCO3/5% CO2, pH 7.4 (lower HCO3/CO2 condition; experimental). Figure 1A illustrates that incubation in lower HCO3/CO2 concentrations for 24 h increased Na-dependent Pi uptake significantly by about a factor of 1.5. Na-dependent transport of L-glutamic acid, which was used as a control, was similar in the control and experimental condition (Fig. 1B). We did also inverse the experimental protocol in that the cells were deprived of serum at 22 mM NaHCO3/5% CO2, pH 7.4 (for 24 h), and subsequently the cells were left for additional 24 h at 22 mM NaHCO3/5% CO2, pH 7.4 or they were kept at 44 mM NaHCO3/10% CO2, pH 7.4. Using this experimental protocol, Na-Pi cotransport was also increased ~1.5-fold in cells kept at 22 mM NaHCO3/5% CO2, pH 7.4 compared with cells that were subsequently transferred to 44 mM NaHCO3/10% CO2, pH 7.4 (Fig. 1C). This response was also specific in that Na-dependent transport of L-glutamic acid was similar in both groups (data not shown).
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Action of PTH on
Na-Pi cotransport.
PTH inhibits type II Na-Pi
cotransport in OK cells (19, 22). We tested whether the increased
Na-Pi cotransport at the lower
HCO3/CO2
condition is sensitive to PTH (3 h exposure;
108 M). Figure
2 shows that the percent inhibition by PTH
was higher for the lower
HCO3/CO2
condition, but the "residual" transport activity was similar in
both groups. Thus a PTH-sensitive
Na-Pi cotransporter was induced by
the lower
HCO3/CO2
condition.
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Effect of dexamethasone on
Na-Pi cotransport.
Dexamethasone inhibits Na-Pi
cotransport in OK cells (290). Cells were incubated at either 44 mM
NaHCO3/10%
CO2 or 22 mM
NaHCO3/5% CO2 for 24 h in the absence or in
the presence of dexamethasone (106 M). Figure
3 shows that the inhibitory effect of
dexamethasone at the higher
HCO3/CO2
condition was relatively small, but significant. On the other hand
dexamethasone did not prevent the increase in Na-Pi cotransport induced by lower
HCO3/CO2
concentrations.
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Effect of HCO3/CO2 on NaPi-4 protein abundance. Western blot analyses of OK cells were performed using a polyclonal antibody. The antiserum recognized proteins with apparent molecular masses of 90-120 kDa, which are related to the NaPi-4 protein as previously demonstrated (10). Densitometric analysis of the staining intensities of NaPi-4 protein-related bands indicated a two- to threefold increase in NaPi-4 protein after incubation at lower HCO3/CO2 condition for 24 h compared with the control condition (Fig. 4).
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Effect of HCO3/CO2 on NaPi-4 mRNA abundance. In the following experiments, we tested whether the increased activity of Na-Pi cotransport and the increased amount of NaPi-4 protein at lower HCO3/CO2 concentrations are associated with alterations in NaPi-4 mRNA abundance. NaPi-4 mRNA was analyzed by Northern blots and quantitated by densitometry and normalized to the density of the corresponding GAPDH mRNA. Figure 5A shows a typical blot of six different mRNA preparations obtained after incubation for 24 h at either 44 mM NaHCO3/10% CO2, pH 7.4, or 22 mM NaHCO3/5% CO2, pH 7.4. Figure 5B depicts the quantitated results of the same blot showing an approximately threefold increase in NaPi-4 mRNA abundance after incubation in the lower HCO3/CO2 condition for 24 h compared with the control condition.
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Effect of HCO3/CO2 on the OK-Npt2 promoter activity. To further confirm that the increased NaPi-4 mRNA abundance at lower HCO3/CO2 concentrations is the result of an increased OK-Npt2 transcription, the promoter activity of OK-Npt2 was measured. Recently the promoter of the OK-Npt2 was cloned (8). Figure 7 shows the organization of the 5'-flanking region of the OK-Npt2 gene. For the reporter gene analysis, two promoter fragments of different sizes (327 and 4,700 bp) including 40 bp of exon 1 were cloned in front of a luciferase reporter gene of pGL3 to estimate transcriptional activity. Both promoter fragments comprised a typical TATA box and a conserved GCAAT element, which was shown to be very important for the promoter activity (8). As depicted in Fig. 8, both promoter constructs showed a higher activity if the cells were incubated at lower HCO3/CO2 concentrations compared with cells incubated at higher HCO3/CO2 concentrations. Thus the measurement of the OK-Npt2 promoter activity is in complete agreement with the experiments using the transcriptional inhibitor DRB and confirms that the increased mRNA abundance at the lower HCO3/CO2 condition is the result of an increased OK-Npt2 transcription.
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DISCUSSION |
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Systemic acid-base disturbances exert important influences on the regulation of phosphate balance in large part by affecting renal phosphate excretion, i.e., renal tubular phosphate reabsorption. To gain more information about the mechanisms by which HCO3/CO2 might affect the renal regulation of phosphate transport, we studied the effects of isohydric changes in HCO3/CO2 on Na-Pi cotransport in OK cells, a well-characterized model with which to study regulation of proximal tubule Na-Pi cotransport (1, 3, 4, 19, 23). The model allows regulatory studies without the confounding influences of other systemic factors. The apically located OK cell type II Na-Pi cotransporter studied in the present experiments (NaPi-4, Ref. 25) is homologous to the major Na-Pi cotransporter molecule present in the brush-border membrane of several different species (for review, see Ref. 21).
Using this model and the experimental conditions described, we obtained the following key findings in the present studies. 1) Incubation in the lower HCO3/CO2 milieu increased Na-Pi cotransport 1.5-fold. 2) Na-dependent transport of L-glutamic acid, which was measured as a control, was similar in both conditions. 3) The increased Na-Pi cotransport was sensitive to PTH inhibition. 4) Incubation in the presence of dexamethasone did not prevent the stimulation of Na-Pi cotransport. 5) The increased Na-Pi cotransport was paralleled by a two- to threefold increased expression of the NaPi-4 transporter protein and 6) a two- to threefold increase in NaPi-4 mRNA abundance. 7) The increase in NaPi-4 mRNA was prevented by incubation in the presence of DRB, which also reduced the increase in Na-Pi cotransport. 8) The NaPi-4 gene transcription was enhanced by 50% at lower HCO3/CO2 concentrations.
The data demonstrate that Na-Pi cotransport in OK cells is responsive to changes in HCO3/CO2 without concomitant changes in pH. Regulation of Na-Pi cotransport by HCO3/CO2 is specific since Na-dependent transport of L-glutamic acid was not changed significantly (Fig. 1, A-C).
PTH inhibition experiments show (Fig. 2) that the stimulation of Na-Pi cotransport in cells incubated at lower HCO3/CO2 concentrations was entirely attributable to a PTH-inhibitable Na-Pi cotransporter, i.e., the type II Na-Pi cotransporter (21). As PTH leads to a complete disappearance of NaPi-4 protein (22), NaPi-4 cannot account for the "residual" transport activity, which cannot be inhibited by PTH (Fig. 2) and which accounts for ~50% of the overall transport at the higher HCO3/CO2 condition (Fig. 2). If the data in Fig. 1 is corrected by the residual transport activity, then the increase in NaPi-4-related Na-Pi cotransport at lower HCO3/CO2 concentrations was approximately twofold.
The regulation of Na-Pi cotransport by HCO3/CO2 can be due to changes in the number of transporter sites and/or to altered activity of expressed transporters. Western blot analysis suggests that the activation of Na-Pi cotransport by lower HCO3/CO2 concentrations is the consequence of an increased number of transporter molecules (Fig. 4). The two- to threefold increase in NaPi-4 protein expression is in agreement with the approximately twofold increase in NaPi-4-related Na-Pi cotransport at the lower HCO3/CO2 condition. As incubation at lower HCO3/CO2 concentrations led to a threefold increase in NaPi-4 mRNA (Fig. 5), we suggest that the increased NaPi-4 protein abundance results from an increased de novo synthesis of NaPi-4 transporter molecules. Although preliminary experiments showed that cycloheximide prevented the increase in Na-Pi cotransport in response to the lower HCO3/CO2 condition (data not shown), the interpretation of these data is difficult, as in Western blots performed in parallel, NaPi-4 protein was hardly detectable for either condition after exposure to cycloheximide. These data can be explained by a high turnover of the type II Na-Pi cotransporter protein also under control conditions. For this reason, effects of isohydric changes in HCO3/CO2 on NaPi-4 protein synthesis and on NaPi-4 protein stability and membrane delivery should be addressed in further experiments.
The threefold increase in NaPi-4 mRNA after incubation for 24 h at lower HCO3/CO2 concentrations (Fig. 4) suggests a regulatory step at the level of transcription or mRNA stability. As a first step toward the clarification of the mechanisms leading to the increased NaPi-4 mRNA abundance at lower HCO3/CO2 concentrations, we tested the effect of the transcriptional inhibitor DRB on NaPi-4 mRNA abundance and Na-Pi cotransport. As DRB prevented the approximately twofold increase in NaPi-4 mRNA observed after 14 h (Fig. 6, A and B), we conclude that the more abundant NaPi-4 mRNA at lower HCO3/CO2 concentrations was the result of an increased OK-Npt2 (OK cell type II Na-Pi cotransporter gene) transcription and that the NaPi-4 mRNA stability was not altered in parallel. As depicted in Fig. 6C, the relative and absolute increase in Na-Pi cotransport was reduced but not completely prevented in the presence of DRB. This suggests the participation of an additional posttranscriptional regulatory mechanism in the regulation of Na-Pi cotransport by HCO3 and/or CO2 (e.g., regulation at the level of translation, protein stability or membrane delivery). DRB led to an increase in transport in both conditions; we have previously observed an increase in OK cell Na-Pi cotransport activity in response to actinomycin D (10). Although we have no satisfactory explanation, the reduction of a protein synthesis-dependent step within the "normal" turnover (degradation) of the NaPi-4 protein could account for this observation.
The OK-Npt2 promoter activity was also tested. As depicted in Fig. 8, a significant and reproducible upregulation for both promoter constructs was observed when cells were incubated at lower HCO3/CO2 concentrations. In agreement with the data on inhibitors, this indicates that the increased mRNA abundance at the lower HCO3/CO2 condition was determined to its major extent by increased activity of the OK-Npt2 promoter.
Recently, we demonstrated that variation of hydrogen ion concentration by changing HCO3 at constant PCO2 affects Na-Pi cotransport by regulating posttranscriptional, glucocorticoid-sensitive expression of the NaPi-4 cotransporter (10). In contrast, the increased Na-Pi cotransport by lower HCO3/CO2 concentrations is not prevented by incubation in the presence of dexamethasone (Fig. 3), and isohydric changes in PCO2 and HCO3 exert their major regulatory influence on NaPi-4 expression and activity by affecting gene transcription. The observations on transcriptional regulation could be explained either by counterbalancing effects of hydrogen ion and HCO3 concentrations on the transcriptional machinery of the NaPi-4 cotransporter or by direct and specific regulation of transcription by CO2 tension. A further explanation could be that some signal transduction pathway is activated and this modifies or sets the activity of transcription factors. Additional influences on posttranscriptional regulation of NaPi-4 cotransport are affected, however, by low hydrogen and/or low HCO3 concentration. Further studies on the regulation of promoter activity and the nature of response elements in the NaPi-4 promoter are needed to delineate which of the acid-base parameters and/or which intracellular messenger(s) is operative in affecting transcription and to determine the mechanisms by which it exerts transcriptional control.
Since cell pH could not be measured during the prolonged exposure times to different isohydric changes in HCO3 concentrations, some uncertainties exist on whether we induced significant changes in cell hydrogen ion concentration. However, we have demonstrated in studies on rabbit proximal tubule cells that only changes in extracellular pH (by changing HCO3 or CO2) affect intracellular pH significantly and that isohydric changes in HCO3/CO2 exert no significant effect on cell pH after prolonged exposure (14). Such behavior has also been assumed in studies on monkey kidney epithelial cells (BSC-1), characterizing the bicarbonate dependence of the sodium bicarbonate symport system (11). Furthermore, direct measurements of cell pH in isolated rat hepatocytes exposed to isohydric changes in HCO3/CO2 documented that cell pH reaches similar values within a few minutes after changing the superfusates (6). Thus it is fair to assume that the present observation is not related to changes in cell pH.
The present results provide novel insights into the mechanisms of renal regulation of phosphate transport in chronic respiratory alkalosis, probably the most frequent acid-base disturbance in humans. Increased tubular reabsorption of phosphate is characteristic of this disorder (16), which is defined by a primary decrease in PCO2 and a consecutive decrease in bicarbonate, resulting in only very small pH changes. Thus, in addition to the observed apparent renal PTH resistance (16), renal phosphate retention in chronic respiratory alkalosis might be due to CO2/HCO3-induced transcriptional stimulation of Na-Pi transport. Similarly, our results also provide a mechanistic explanation for the phosphaturia or decreased tubular phosphate reabsorption in respiratory acidosis (characterized by increases in PCO2 and bicarbonate, see Ref. 30).
In conclusion, the cellular response to lower HCO3/CO2 concentrations leads to a specific stimulation of the NaPi-4 cotransport that is paralleled by an increased expression of the NaPi-4 transporter protein. In contrast to the activation of Na-Pi cotransport by acidic pH (10), this stimulation is not prevented by incubation in the presence of dexamethasone and this stimulation is characterized by an increased expression of NaPi-4 mRNA, which was shown to be exclusively the result of an increased OK cell type II Na-Pi promoter activity without concomitant changes in NaPi-4 mRNA stability.
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ACKNOWLEDGEMENTS |
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We thank Norman P. Curthoys (Colorado State University) for helpful discussions and C. Gasser for professional assistance in preparing Figs. 1-8.
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FOOTNOTES |
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This work was supported by Swiss National Science Foundation Grant 31.46523.96 (to H. Murer). A. W. Jehle received a grant from the Olga Mayenfisch Stiftung (Zurich).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: J. Biber, Institute of Physiology, Univ. Zurich-Irchel, Winterthurerstr. 190, CH-8057 Zurich, Switzerland.
Received 14 May 1998; accepted in final form 17 September 1998.
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