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Experimental Biology and Medicine 227:626-631 (2002)
© 2002 Society for Experimental Biology and Medicine

ORIGINAL ARTICLE

Basolateral Phosphate Transport in Renal Proximal-Tubule-Like OK Cells1

M. Barac-Nieto*,{dagger},2, M. Alfred* and A. Spitzer*

* Department of Pediatrics, Albert Einstein College of Medicine, New York, New York; and
{dagger} Department of Physiology, Kuwait University Faculty of Medicine, Kuwait


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
It is generally assumed that phosphate (Pi) effluxes from proximal tubule cells by passive diffusion across the basolateral (BL) membrane. We explored the mechanism of BL Pi efflux in proximal tubule-like OK cells grown on permeable filters and then loaded with 32P. BL efflux of 32P was significantly stimulated (P < 0.05) by exposing the BL side of the monolayer to 12.5 mM Pi, to 10 mM citrate, or by acid-loading the cells, and was inhibited by exposure to 0.05 mM Pi or 25 mM HCO3; by contrast, BL exposure to high (8.4) pH, 40 mM K+, 140 mM Na gluconate (replacing NaCl), 10 mM lactate, 10 mM succinate, or 10 mM glutamate did not affect BL 32P efflux. These data are consistent with BL Pi efflux from proximal tubule-like cells occurring, in part, via an electro-neutral sodium-sensitive anion transporter capable of exchanging two moles of intracellular acidic H2PO4- for each mole of extracellular basic HPO4= or for citrate.

Key Words: phosphate • transport • mineral metabolism • kidney • renal • reabsorption • anion • citrate exchange • acid extrusion • opossum • OK cells


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Data from experiments in renal cell monolayers in culture and from renal cortical membrane vesicles indicate the existence of both Na+-independent and Na+-dependent phosphate (Pi) transport systems at the basolateral (BL) membrane of proximal tubule cells (1–5). We have recently shown that in monolayers of OK cells, the apical Na-dependent Pi transporters are upregulated in response to Pi depletion, but neither the effective basal-lateral Pi permeability nor transepithelial Pi transport are altered (6). A basal-lateral Na+-independent Pi transport system was reported to be inhibited by 4,4`-diisothiocyanostilbene-2,2`-disulphonic acid (DIDS), suggesting the presence of Pi-anion exchange (3, 4, 7). The counter anion for this exchanger has not been yet identified. The Na+-dependent system most probably mediates entry of Pi from the extracellular fluid into the cells, rather than Pi efflux from the cells (5, 8).

Although it is widely accepted that the apical NaPi co-transport system is the rate-limiting step for epithelial Pi transport (9), the aim of this study was to explore the mechanism by which Pi exits across the BL membrane of proximal tubule-like OK cells.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Cell Cultures.
OK cells were grown in monolayers on 75-cm3 plastic Falcon flasks in 6 ml of Dulbecco’s modified Eagle’s medium (DMEM):Ham F-12 (1:1) containing 1 mM Pi, 22 mM NaHCO3, 20 mM Hepes, 2 mM L-glutamine, 50 IU/ml penicillin, 50 µg/ml streptomycin, and 10% fetal bovine serum (FBS). To prepare subsequent cultures, the confluent monolayers grown on 75-cm3 flasks were trypsinized (0.25% trypsin and 1 mM EDTA.4Na) for 3–5 min, washed three times by repeated centrifugations in the above described growing medium, and seeded on CycloporeR Falcon inserts at a density of 5 x 105 cells/well.

The top (apical) compartment of permeant polyester membrane filter inserts (25 mm diameter, 0.45 µm pore diameter) was filled with 2 ml of DMEM:F-12. Each insert was placed in a plastic plate well containing 2 ml of growing medium. This system provides access to both sides of the cell monolayer growing on the top surface of the insert membrane. The cells were kept in an incubator gassed with 5% CO2 in air at 37°C . The medium was changed 72 hr after seeding and every 24 hr thereafter. Cell monolayers reached confluence over a period of 7–10 days as verified under the microscope. As shown previously, in these monolayers, apical uptake of Pi is upregulated in response to Pi depletion, there is net apical-to-BL Pi transport, but net BL-to-apical mannitol flux, and asymmetry in BL (33% ± 5% in 60 min) and apical (10% ± 5% in 60 min) efflux of 32Pi from the cells (6).

Assay of 32P Efflux.
The confluent monolayers were incubated overnight (14–16 hr) in FBS-free Ham F12 medium. Monolayers on inserts were then exposed at the apical side to 2 ml of Hepes-buffered saline (HBS) containing (in millimoles/liter): 140 NaCl, 5.4 KCl, 2.8 CaCl2, 1.2 MgCl2, 15 Hepes, and 0.1 32Pi (2–4 µci/ml), pH 7.4, for 1 hr to load the cells with 32P. The side facing the well (BL side) remained in Ham F12 medium with 1 mM Pi. After loading with 32P, the media were aspirated from both sides of the inserts, and the inserts were rinsed three times with 100 mM ice-cold Mg gluconate. The inserts were then placed at 37°C in the wells of a clean 6-well culture plate containing 2 ml/well of either control or experimental HBS-like solution; 2 ml of the 1 mM Pi, Ham F12 medium were placed on the apical side. A 1-ml sample of the solution in the BL compartment was collected 5, 10, 30, and 60 min after the last Mg gluconate wash. The remaining solution was aspirated and replaced by 2 ml of fresh medium. The collected sample was mixed with 10 ml of scintillation fluid (Aquasol) for counting of radioactivity. At 60 min, and in some experiments at other sampling times, the apical solution was collected, the cell monolayer was lysed in 2 ml of 0.2 N KOH containing 0.25% Triton X-100, and was then mixed by shaking for at least 30 min. The radioactivity in the monolayers was measured by counting 1.5 ml of the KOH-Triton extract in 10 ml of scintillation fluid. The remaining extract was used to measure the cell protein concentration using the Coomassie blue procedure (Bio-Rad, Hercules, CA) and the Pi specific activity. The fractional BL efflux of cellular 32P was calculated as the ratio of the 32P collected at each time point in the BL solution to the total 32P present measured as the sum of that collected in BL and apical solutions and that recovered in the cell monolayer. Transepithelial fluxes of 32P were minimized by the frequent changes in BL solution and by the relatively large pools (usually 2 µMoles) of unlabeled Pi present in apical and BL compartments. In some experiments, to measure the cell Pi specific activity and calculate absolute rates of Pi efflux, the cell proteins were precipitated with 6% perchloric acid (PCA), and the organic 32P present in the PCA soluble fraction was adsorbed on charcoal (Norit A in 1 N HCl, 1:1, v:v). The concentration of Pi in this adsorbed extract was measured using a phosphomolybdate assay (10); the 32P concentration in another aliquot of the same extract was determined by liquid scintillation counting.

BL Pi efflux was measured under the following experimental conditions: when the concentration of Pi in the HBS solution bathing the BL side of the monolayer was altered from 1 to 0.05 or to 12.5 mM Na2HPO4 (reducing NaCl to 127.5 mM); to investigate the electrical potential dependence of Pi efflux, cells were depolarized by exposing their BL side to a HBS-like solution (containing 1 mM Pi) in which the KCl was increased to 40 mM while the NaCl was reduced to 100 mM; when 10 of the 140 mM NaCl in a HBS solution bathing the BL side was replaced by 10 mM of either sodium lactate, glutamate, succinate, or citrate (Pi remaining at 1 mM); when all the Cl- in a HBS solution containing 1 mM Pi was replaced by gluconate, an impermeant anion; the sensitivity of Pi efflux to changes in pH of the solution bathing the BL side of the cells was tested by comparing efflux into HBS solutions containing 1 mM Pi at pH 8.4 and 7.4, respectively; when the concentration of NaHCO3 at the BL side was increased from 0 to 25 mM (while the NaCl was decreased to 115 mM in the nominal absence of CO2); and finally, the effect of intracellular acidification on BL Pi efflux was studied in cell monolayers exposed for 1 hr to an apical HBS solution with 0.1 mM 32Pi and 10 mM NH4Cl and was exposed thereafter, on both apical and BL sides, to a 15-mM Hepes-buffered (pH 7.4) Na+-free solution containing 140 mM choline Cl and 1 mM potassium Pi.

Materials.
The OK cell line was kindly provided by Drs. J. Biber and H. Murer (Zurich, Switzerland). Cell culture supplies were purchased from Falcon (BD Bioscience, Bedford, MA) or Gibco (Grand Island, NY). Radio-labeled materials were obtained from New England Nuclear (Boston, MA). Organic and inorganic chemicals used for transport assays were purchased from either Sigma (St. Louis, MO) or Baker (Sanford, ME).

Statistics.
Data were analyzed using paired or unpaired t test. A value of P < 0.05 was considered statistically significant. All values are presented as means ± SE.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Exposure of the BL side of the monolayers to a solution containing a high concentration of Pi accelerated absolute as well as fractional efflux of 32P (Fig. 1, A and BGo). Efflux was a multiexponential function of washout time. A rapid phase (0–5 min) could be distinguished from a slower phase (10–60 min). At 5 min, 19.2% ± 2.3% of the cell 32P (14.6 x 104 dpm/mg protein) had exited through the BL membranes of cells exposed to 12.5 mM Pi in the BL solution compared with 15% ± 2.6% in cells exposed to 1 mM Pi solution (P < 0.005). At 60 min of incubation, 48.6% ± 4.4% of the cellular 32P had exited through the BL side of cells exposed to 12.5 mM Pi compared with 33.5% ± 3.0% (10 x 104 dpm/mg) in cells exposed to a solution containing 1 mM Pi (P < 0.005; Fig. 1AGo). Exposure of the BL side of the monolayers to a solution containing 0.05 mM instead of 1 mM Pi reduced 32P efflux to 2.7% ± 0.05% at 5 min and to 7% ± 1% at 60 min (P < 0.01; Fig 1AGo). Absolute rates of Pi efflux (Fig. 1BGo) were calculated from the sums of the 32Pi recovered from the BL solution divided by the cell Pi-specific activity measured in the PCA-soluble charcoal-adsorbed extracts of the cell monolayers. Kinetic analysis of the trans-stimulatory effect of BL Pi on the Pi efflux rates (Fig. 1CGo) were based on the data obtained after 5 min of washout and were: Vmax = 6.64 ± 0.03 nmol/mg at 5 min; Km 0.323 ± 0.003 mM BL Pi.



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  		Figure 1. Trans-stimulation of BL Pi efflux by phosphate. Monolayers were incubated for 1 hr in uptake medium containing 0.1 mM 32Pi to load the cells with 32Pi. The time course of 32P efflux into a BL solution containing either 0.05, 1, or 12.5 mM Pi was measured. (A) Each point is the mean ± SE of the cumulated fraction of intracellular 32P (at time 0) effluxing into the solution bathing the BL side of the cells, for three separate experiments, performed in duplicate. (B) Absolute rates of Pi efflux were calculated using the data in A, the cell Pi uptake, and the specific activity of cell Pi, measured as described in `METHODS." (C) Kinetics of trans-stimulation of Pi efflux across the BL membrane by the Pi concentration in the solution to which it was exposed. Data were derived from those presented in B. *P < 0.025; **P < 0.05.

 
Exposure of the BL side of the cells to solutions containing 40 mM KCl had no effect on 32P efflux (Table IGo). Relative rates of BL 32P efflux were 15.0% ± 2.6% vs 15.3% ± 2.5% (P > 0.8) at 5 min and 33.5% ± 3% vs 35.5% ± 2.7% (P > 0.4) at 60 min, for control and experimental conditions, respectively.


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  		Table I. Percentage of the Intracellular 32P That Effluxed Across the Basolateral Cell Membrane
 
BL efflux of 32P into an HBS solution containing physiological concentration of bicarbonate (25 mM replacing NaCl in the nominal absence of CO2) was 8.5% ± 1.1% at 5 min and 17.1% ± 1.2% (5 x 104 dpm/mg) at 60 min compared with 14.2% ± 1.7% efflux at 5 min and 21.7% ± 3.0% at 60 min (6.5 x 104 dpm/mg, P < 0.01) into a bicarbonate-free solution (Fig. 2Go) of the same pH (140 mM NaCl and 15 mM Hepes-Tris, pH 7.4; P < 0.05).



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  		Figure 2. Inhibition of BL Pi efflux by HCO3-. 32Pi efflux into a solution containing 1 mM Pi, 25 mM HCO3-, 15 mM Hepes, and 115 mM NaCl, pH 7.4, and into a HCO3-free solution containing 1 mM Pi, 15 mM Hepes, and 140 mM NaCl, pH 7.4, *P < 0.025. Data are for three experiments performed in duplicate. Note that a 15 mM Hepes, 140 mM NaCl, HCO3--free, alkaline solution (pH 8.4) had no significant effect on BL 32Pi efflux (Table IGo).

 
When exposed to a BL solution with an alkaline pH (8.4), 12.3% ± 1.6% (P > 0.05) of the 32P in the cells (32 x 104 dpm/mg) effluxed in 5 min; at a neutral pH (7.4), 32P efflux was 11.4% ± 1.04%. At 60 min, the corresponding values were 40.0% ± 4.4% and 38.1% ± 4.2% (P > 0.1; Table IGo).

To evaluate the influence of intracellular pH changes, we measured BL efflux of 32P from acid-loaded (10 mM NH4Cl) and control OK cells into a solution containing 140 mM choline chloride, 1 mM Pi, and 15 mM Hepes, pH 7.4 (Fig. 3Go). An identical solution bathed the apical cell surface. Cell 32P uptake (30 ± 2 x 104 dpm/mg) was not significantly altered by ammonium loading (29 ± 2 x 104 dpm/mg, P > 0.8). In the presence of 140 mM choline chloride, BL efflux of 32Pi was 5% ± 1.1% at 5 min and 12% ± 1.2% at 60 min, lower (P < 0.05) than observed into 140 mM NaCl-containing HBS solutions (Table IGo, Controls) where it was 10.5%–15% at 5 min and 28.5%–38.1% at 60 min. In addition, BL efflux of 32P was significantly higher when occurring from acid-loaded than from control cells (8.1% ± 0.4% vs 5.2% ± 1.4% at 5 min, 16.4% ± 1.2% vs 12.9% ± 1.5% at 60 min, P < 0.05).Replacement of Cl-, the major extracellular anion, by gluconate, an impermeant anion, or by permeant anions such as glutamate, succinate, and lactate in the solution bathing the BL side did not affect 32P efflux (Table IGo). Only citrate accelerated the absolute as well as the fractional efflux of 32P (Fig. 4Go). Because citrate is a calcium chelator, we tested the effect of citrate on 32P efflux in the absence of extracellular Ca2+(Ca-free HBS containing 1 mM EGTA). The cell 32P (35 x 104 dpm/mg) effluxed into a citrate-containing Ca2+-free HBS solution at a significantly higher rate than into a Ca2+-free, 1 mM EGTA HBS solution without citrate (52.1% ± 1.6% vs 41.2% ± 3.4% at 60 min, P < 0.05; Fig. 4Go). The absence of extracellular Ca2+ (and presence of 1 mM EGTA) had no significant effect on 32P efflux (P > 0.5; Fig. 4Go). These results indicate that 32P efflux across the BL membrane of OK cells is stimulated specifically by citrate but not by succinate nor by a low [Ca2+]o.



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  		Figure 3. Stimulation of BL Pi efflux by intracellular acidification. To load the cells with 32P and NH4Cl, the apical side was exposed for 60 min to the HBS solution described in "METHODS" in which 10 mM NaCl were replaced by 10 mM NH4Cl. The values represent BL 32Pi efflux from NH4Cl-loaded and control cell monolayers exposed on both sides to a solution containing 140 mM choline chloride, 1 mM Pi, and 15 mM Hepes, pH 7.4, *P < 0.025. Data are from three separate experiments performed in duplicate.

 


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  		Figure 4. Effects of Ca2+ and citrate on BL Pi efflux. The time course of 32P efflux was measured in a control solution containing 140 mM NaCl, 2.8 mM Ca2+, and in two experimental solutions: one containing 120 mM NaCl, 10 mM citrate, and 0 Ca2+, the other containing 140 mM NaCl, 0 citrate, 0 Ca2+, and 1 mM EGTA. Measurements were performed over 60 min. Values are the means ± SE of fractional 32Pi efflux from three separate experiments performed in duplicate. *P < 0.05.

 
The efflux of Pi from the cells to the apical compartment after 60 min was 10% ± 5% of initial cell Pi and did not significantly vary in the various experimental conditions.


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
Our data show (Fig. 1Go) that phosphate in the solution bathing the BL membrane trans-stimulates Pi efflux across this cell membrane, consistent with the existence of a carrier-like Pi transport system capable of Pi-Pi exchange at this membrane. Similar changes in BL Pi efflux were observed at 5 and 60 min of incubation, indicating that the influence of transepithelial 32P fluxes on these data was minimal.

The trans-stimulation had a Km of 0.32 mM. Thus, 1 mM BL Pi exerts a near maximal trans-stimulatory effect (Fig. 1, A and BGo). The Vmax was 6.6 nMoles/mg at 5 min, severalfold higher than the Vmax reported for apical sodium-Pi co-transport in OK cells (3). Schwab et al. (4) demonstrated trans-stimulation of 32P uptake in rat BL membrane vesicles preloaded with 2 mM Pi. A carrier, exchanging sulfate for HPO4=, was found in BL membrane vesicles of rat kidney cortex (7). Experiments performed on isolated proximal tubule cells obtained from chicks revealed that the phosphate analog vanadate inhibits Pi efflux (11), further supporting the presence of a specific carrier protein for Pi exit from the cells.

The fact that changes in the BL K+ concentration did not alter Pi efflux in OK cells suggests that the exchanger is not affected by changes in cell membrane potential and that it is electrically neutral. This observation is concordant with that of Butterworth et al. (11) who reported that addition of the K+ ionophore valinomycin to isolated proximal tubule chick cells suspended in K+-free medium (to increase electronegativity within the cell) had no effect on Pi efflux. However, Na+-independent, electrogenic Pi transport has been reported to occur in canine renal cortical BL membrane vesicles (4).

The addition of HCO3- to the media bathing the BL membrane (in the nominal absence of CO2) decreased Pi efflux in the absence of extracellular alkalinization. An inhibitory effect of HCO3- on Pi transport has been shown to occur in intact proximal tubules (12) and in renal cortical BL membrane vesicles (13). It may be due to a reduction in cellular acid phosphate [H2PO4-]i when the cell alkalinizes due to HCO3- entry, as has previously been observed in proximal tubule and other epithelial cells where basal-lateral HCO3 transport has a dominant influence on cell pH (14, 15). By contrast, acidification of the cell interior induced by NH3 exit from NH4+-loaded cells, the major process determining OK cell pH changes when in the absence of extracellular sodium (15), increased Pi efflux, probably by raising the intracellular acid phosphate [H2PO4-]I concentration. However, changing the pH of the BL solution from 7.4 to 8.4, which produces only a small (22%) increase in the extracellular basic phosphate [HPO4=] concentration (to 0.8 mM), did not significantly affect 32P efflux, as expected from the low Km of the exchanger for extracellular Pi (0.32 mM). These observations are consistent with an exchange of extracellular basic phosphate for intracellular acid phosphate. It should be noted that an electroneutral exchange of 2 moles of H2PO4- for 1 mole of HPO4= results in a net extrusion of phosphate and of acid from the cell, and therefore may contribute not only to net BL efflux of Pi, but also to the regulation of cell (pH), similar to the cell alkalinizing effect of predominant apical influx of basic HPO4= via NaPi co-transport (16).

The presence of a Pi exchanger in the BL membrane of the proximal tubule (17) capable of exchanging 2 moles of intracellular H2PO4- for 1 mole of extracellular HPO4= may explain why Pi does not leak rapidly out of the cells, and the [Pi]i remains stable for several hours, when the kidney is perfused with a Pi-free solution (18, 19).

The finding that BL 32P efflux is slower into choline than into sodium-containing solutions suggests that this BL Pi exchanger may be modulated by extracellular sodium, similar to other anion exchangers.

Our study confirms the presence of a Pi-anion exchange mechanism across the BL membrane of proximal tubule-like cells (4, 7, 20). The fact that Pi efflux is accelerated by extracellular Pi and citrate, but not by Cl-, lactate, succinate, or glutamate suggests that this anion transport system is specific. Oxidation or metabolism of citrate as well as that of succinate (21), added as Na+ salts, results in net cell alkalinization, which was found to reduce rather than enhance 32P efflux. Only citrate, but not succinate, another metabolized anion, promoted Pi efflux. Thus, it is likely that the effect of citrate is due to a specific direct trans-stimulation of Pi efflux rather than to the metabolism of citrate. Citrate is produced mostly by bone (22) and is utilized preferentially by kidneys (23, 24). Its plasma concentration is strongly influenced by the bone turnover rate, and thus, it may serve to link renal Pi reabsorption to bone accretion.

Based on the information described herein, it is reasonable to surmise that efflux of Pi across the BL cell membrane of OK cells occurs, in part, via an electroneutral Pi transporter capable of exchanging 2 moles of intracellular acidic Pi for each mole of extracellular basic Pi or citrate. Such system is stimulated by intracellular acidity and is inhibited by intracellular alkalinity. It is stimulated by extracellular sodium and may contribute to net basal-lateral Pi and acid extrusion from these cells.


   Acknowledgments
 
We gratefully acknowledge the kindness of Drs. Jurgen Biber and Heini Murer who provided us with the OK cells. We also thank Mr. Ashley Singleton for his technical assistance.


   Footnotes
 
1 This work was supported in part by the National Institutes of Health (grant RO1 DK 28477), by United States Public Health Service (training grant DK07110 to M.A.), and by Kuwait University Research Administration MD030. Back

2 To whom requests for reprints should be addressed at Department of Physiology, Health Sciences Center, Kuwait University, P.O. Box 24923, Safat 13110, Kuwait. E-mail: mario{at}hsc.kuniv.edu.kw Back


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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Received for publication January 4, 2002. Accepted for publication April 18, 2002.




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