Water transport by the renal Na+-dicarboxylate cotransporter

Anne-Kristine Meinild1, Donald D. F. Loo1, Ana M. Pajor2, Thomas Zeuthen3, and Ernest M. Wright1

1 Department of Physiology, University of California, Los Angeles, School of Medicine, Los Angeles, California 90095-1751; 2 Department of Physiology and Biophysics, University of Texas Medical Branch, Galveston, Texas 77555; and 3 Department of Medical Physiology, The Panum Institute, University of Copenhagen, Blegdamsvej 3, DK-2200N Copenhagen, Denmark


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

This study investigated the ability of the renal Na+-dicarboxylate cotransporter, NaDC-1, to transport water. Rabbit NaDC-1 was expressed in Xenopus laevis oocytes, cotransporter activity was measured as the inward current generated by substrate (citrate or succinate), and water transport was monitored by the changes in oocyte volume. In the absence of substrates, oocytes expressing NaDC-1 showed an increase in osmotic water permeability, which was directly correlated with the expression level of NaDC-1. When NaDC-1 was transporting substrates, there was a concomitant increase in oocyte volume. This solute-coupled influx of water took place in the absence of, and even against, osmotic gradients. There was a strict stoichiometric relationship between Na+, substrate, and water transport of 3 Na+, 1 dicarboxylate, and 176 water molecules/transport cycle. These results indicate that the renal Na+-dicarboxylate cotransporter mediates water transport and, under physiological conditions, may contribute to fluid reabsorption across the proximal tubule.

passive water transport; solute-coupled water flow; Xenopus laevis oocyte


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

A MAJOR FUNCTION OF the kidney is the reabsorption of salt and water from the glomerular filtrate. The mechanism of water transport is not well understood, although it is generally accepted that water transport is secondary to active solute transport (23). Of the 180 liters of plasma filtered by the glomerular apparatus every day, >80% is reabsorbed in the proximal tubule. Because aquaporins have been shown to be present both in the apical and basolateral membranes of proximal tubular cells, transcellular water transport is thought to be mediated through these water channels. However, in a recent study on aquaporin-1 knockout mice (21), the transepithelial water permeability of the proximal tubule was reduced 78%, whereas the fluid reabsorption was reduced only 50%. One interpretation of this result is that other membrane proteins are involved in fluid reabsorption. It has been proposed that ion-coupled solute transporters can mediate the transport of water, and there is evidence for the involvement of the intestinal Na+-glucose cotransporter (SGLT1) in transport of water (10, 11, 14, 31). We speculate that renal cotransport proteins may contribute to water reabsorption in the proximal tubule.

The goal of this study was to investigate whether, in general, cotransport proteins could play a role in renal water reabsorption. Specifically, we tested the renal Na+-dicarboxylate cotransporter (NaDC-1) for water transport. NaDC-1 belongs to a family of Na+-dependent anion transporters, which includes the Na+-dependent dicarboxylate transporters (1, 3, 16, 17) and the renal Na+-sulfate cotransporter (13). NaDC-1 is primarily found in the apical membrane of the kidney proximal tubules where it reabsorbs tricarboxylic acid cycle intermediates (24). Defects in this transporter may play a role in the development of idiopathic hypocitraturia (see Ref. 18). NaDC-1 is electrogenic, with a stoichiometry of 3 Na+ transported/divalent carboxylate ion (6, 19). We measured substrate transport using the two-electrode voltage clamp and water transport as the changes in cell volume by an optical method on the cloned NaDC-1 expressed in Xenopus laevis oocytes. Our results indicate that NaDC-1 mediates both passive and solute-coupled water transport, and under physiological conditions, the cotransporter could contribute to fluid reabsorption across the proximal tubule.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

X. laevis oocytes were defolliculated, injected with 50 ng rabbit NaDC-1 cRNA (19), and maintained in Barth's medium supplemented with 50 mg/ml gentamycin at 18°C for 3-8 days before use (11, 20). During the experiments, the oocyte was normally perfused with a NaCl control solution containing (in mM) 90 NaCl, 20 mannitol, 2 KCl, 1 MgCl2, 1 CaCl2, and 10 HEPES (pH 7.5, 212 mosM). In some experiments, 90 mM NaCl was exchanged with 90 mM LiCl or 90 mM choline chloride. Solutions containing substrates were made by isosmotic replacement of mannitol in the control solution with either 10 mM sodium citrate or 10 mM sodium succinate. In experiments studying lithium inhibition of water transport, the test solution contained 80 mM NaCl, 10 mM LiCl, and 10 mM citrate; the osmolarity was the same as the control solution (212 mosM). Hyperosmotic solutions were prepared by addition of 20 mM mannitol to the appropriate solutions. The osmolarity of the solutions was measured using a vapor pressure osmometer (Wescor, Logan, UT).

The experimental protocol allowed simultaneous measurements of substrate transport and the oocyte volume. The experiments were performed under continuous superfusion of the chamber (functional volume ~15 µl), and the half time for solution exchange was ~1 s. Electrogenic substrate transport by NaDC-1 was studied using the two-electrode voltage-clamp method (8, 19). Because 3 Na+ are transported with each divalent carboxylate ion, the activity of NaDC-1 can be monitored as the inward current evoked by the substrate. The number of positive charges (Q) transported into the oocyte was obtained from the integral of the substrate-induced inward current.

Two general protocols were used to measure water fluxes. The osmotic water flux was estimated from the rate of change of cell volume when an osmotic gradient (-20 mosM) was imposed by removal of mannitol from the superfusing solution. These experiments were performed in the absence of substrates (citrate and succinate) in the external solution. To measure water flow occurring during Na+-succinate (or citrate) cotransport, the oocyte membrane potential was held at -110 mV and the external solution was abruptly exchanged with a test solution in which mannitol in the control solution was isosmotically replaced by a transported substrate (succinate or citrate). Water fluxes across the oocyte membrane were calculated from the changes in cell volume. The oocyte cross-sectional area was measured every 0.5 s from bright-field images by a charged coupled device camera, and the images were digitized (11, 14, 31). The relative volume of the oocytes (V/Vo) is related to the relative cross-sectional area (A/Ao) by the following equation: V/Vo=(A/Ao)3/2, where V is the volume, Vo is the initial volume, A is the cross-sectional area, and Ao is the initial cross-sectional area. Volume flow (Jv) was obtained from the following relation: Jv = Vo(d/dt)(V/Vo), where (d/dt)(V/Vo) is the relative volume change with time. Osmotic water permeability (Lp) of the oocyte is related to the volume flow by the following equation: Lp = Jv/SovwDelta pi , where So is the oocyte surface area (0.4 cm2 , Ref. 11), vw is the partial molar volume of water (18 cm3/mol), and Delta pi is the osmotic difference. All experiments were repeated on at least three oocytes from different donor frogs. Statistics are given as means ± SE, and n is the number of experiments. When values were obtained from linear regression of the data, their error was obtained as the SE of the fits.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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REFERENCES

Osmotic water transport. Oocytes injected with NaDC-1 cRNA showed an increase in osmotic water permeability compared with control noninjected oocytes. This is illustrated in Fig. 1A where the volume changes in response to an osmotic challenge of -20 mosM (created by removal of mannitol from the superfusing control solution) to an oocyte injected with NaDC-1 cRNA and a control oocyte from the same batch are shown. Upon imposing the osmotic gradient in the absence of substrate, the NaDC-1 cRNA injected oocyte swelled at the rate of 53 pl/s. This rate was three times greater than that of the control oocyte (18 pl/s). When the osmotic gradient was removed, the swelling in both oocytes stopped. The osmotic water permeability Lp of the oocyte injected with NaDC-1 cRNA was 3.6 × 10-4 cm/s, compared with 1.3 × 10-4 cm/s for the control oocyte. Figure 1B shows that, on average, oocytes injected with NaDC-1 cRNA had an Lp of 2.8 ± 0.1 × 10-4 cm/s (n = 20 oocytes), which was twofold higher than the Lp of control oocytes (1.2 ± 0.1 × 10-4 cm/s, n = 6 oocytes).


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Fig. 1.   Osmotic water permeability (Lp) is increased in oocytes injected with NaDC-1 cRNA. A: an osmotic gradient (-20 mosM) was imposed on an oocyte injected with NaDC-1 cRNA (NaDC-1) and a noninjected control oocyte (control) from the same batch, by removal of 20 mM mannitol from the superfusing control solution. The experiment was performed in the absence of substrates. In the oocyte expressing NaDC-1 (in a separate experiment, the current induced by 10 mM citrate was 830 nA at -110 mV), the rate of volume change was 53 pl/s. In the control oocyte, the rate of volume change was 18 pl/s. Delta V, volume change. B: comparison of the osmotic water permeability in oocytes expressing NaDC-1 and in control oocytes. Lp was calculated from volume flow (Jv) by relationship Lp= Jv/SovwDelta pi . Lp was 2.8 ± 0.1 × 10-4 cm/s (n = 20) in oocytes expressing NaDC-1 and 1.2 ± 0.1 × 10-4 cm/s (n = 6) in control oocytes. A t test showed that the difference is statistically significant (P << 0.01).

The increase in Lp of oocytes injected with NaDC-1 cRNA was proportional to the number of transporters in the oocyte plasma membrane. In a series of experiments, the expression levels of NaDC-1 and osmotic water transport (expressed as Lp) were measured in the same oocyte (Fig. 2). Expression level of NaDC-1 was determined as the total number transporters in the oocyte plasma membrane. This was estimated by measuring the maximal inward current (Imax) induced by a saturating concentration of citrate (10 mM) in NaCl solution at the membrane potential of -110 mV. Imax is the product of the number of transporters in the plasma membrane and the turnover rate of each transporter (50/s). Osmotic water transport was then measured in the absence of substrates. In Fig. 2, the contribution to the Lp from the background (control oocytes) has been subtracted. There was a direct relationship between the number of transporters in the plasma membrane and the Lp when the oocyte was bathed in the NaCl solution (Fig. 2, ). Because the function of NaDC-1 depends on the cations (19), osmotic water transport was also studied when the bath Na+ was replaced with an equimolar concentration of Li+ (Fig. 2, open circle ) and choline (Fig. 2, black-down-triangle ). The linear relationship between Lp and the transporter density was similar in Na+, Li+, and choline. Pooled data gave a slope of 1.5 ± 0.1 × 10-14 cm · s-1 · transporter-1.


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Fig. 2.   Relationship between passive (osmotic) water permeability Lp and the expression level of NaDC-1. The expression level was determined from the maximal transport rate (Imax) generated by a saturating concentration (10 mM) of citrate in the NaCl solution at -110 mV membrane potential. The number of transporters in the oocyte plasma membrane was obtained by dividing maximal transport rate Imax by the turnover rate of NaDC-1, which was assumed to be 50/s. The volume flow experiments were performed as described in Fig. 1A, with the external solution containing Na+ (), Li+ (open circle ), or choline (black-down-triangle ). The background Lp, which was determined in control oocytes (1.2 ± 0.1 × 10-4 cm/s), has been subtracted. All data points were used to determine the slope of the regression line, 1.5 ± 0.1 × 10-14 cm · s-1 · protein-1. The Lp obtained in choline (2.2 ± 0.1 × 10-14 cm · s-1 · protein-1) was higher than those obtained in Na+ (1.4 ± 0.1 × 10-14 cm · s-1 · protein-1) or Li+ (1.4 ± 0.2 × 10-14 cm · s-1 · protein-1).

Solute-coupled water transport. We tested whether NaDC-1 exhibited solute-coupled water transport. The experiments were performed in the absence of an osmotic gradient, so the only driving force for water transport would be derived from the NaDC-1-dependent solute flux. In these experiments, transporter activity, which was measured as the inward current induced by citrate, and the oocyte volume were monitored simultaneously. Figure 3 shows the results of such an experiment. Initially, the oocyte was perfused with the control solution, and the membrane potential was held at -110 mV. The solution was abruptly exchanged with a test solution, in which mannitol in the control solution was isosmotically replaced by 10 mM citrate. The isosmotic addition of citrate evoked an inward current, reaching a maximum of 1,280 nA (Fig. 3B). Concurrent with the increase in citrate-induced current, there was a linear increase in the volume of the oocyte (Fig. 3A, jagged trace) at a rate of 40 pl/s. The initial volume change was directly correlated with the inward charge (Q) obtained as the integral of the citrate-induced inward current. The smooth curve of Fig. 3A was drawn with each inward-positive charge accompanied by the transport of 165 water molecules (see below and Fig. 4). The curve, which represents the water flow coupled to Na+-citrate cotransport, superimposed on the curve describing the volume change of the oocyte. The ratio between water and substrates (3 Na, 1 dicarboxylate, 165 H2O) resulted in the transport of fluid which was hyperosmotic, and one would anticipate a small transient osmotic influx of water after removal of the substrate. This was observed when oocytes were exposed to citrate for periods longer than 30 s (see Fig. 3).


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Fig. 3.   NaDC-1-coupled water transport measured under isosmotic conditions. In an NaDC-1-expressing oocyte, water transport (volume change; Delta V) and substrate transport (inward current) were measured concurrently during isosmotic solution changes. The membrane potential was held at -110 mV during the experiment. At the arrow (+citrate), the superfusing control solution was rapidly changed to an isosmotic citrate-containing solution, by replacement of 20 mM mannitol with 10 mM citrate. Addition of citrate immediately evoked a linear increase in oocyte volume (jagged line in A) at a rate 40 pl/s and an inward citrate-dependent current (B), reaching a maximum of 1,280 nA. Removal of citrate (-citrate) resulted in a decrease in current to the initial value and a stop in swelling of the oocyte. The number of charges (Q) transported into the oocyte (obtained by integration of the current) is plotted as a function of time (smooth line in A). The relationship observed between water and charge transport implied that transport of 1 inward positive charge is accompanied by transport of 165 H2O molecules.



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Fig. 4.   Relationship between volume flow and the rate of substrate transport under isosmotic conditions. Data were obtained by exchanging the superfusing control solution with different isosmotic solutions. The experimental solutions contained the following (in mM): , 90 Na+ and 10 citrate; open circle , 80 Na+, 10 Li+, and 10 citrate; black-down-triangle , 90 Na+ and 10 succinate. The volume change and substrate transport rate Imax were measured simultaneously. Membrane potential was maintained at -110 mV. The data were pooled from all experiments and showed a linear relationship between volume flow and Na+-dicarboxylate transport rate. The line was drawn by linear regression and gave a slope of 33 ± 2 × 10-3 pl · s-1 · nA-1, corresponding to 176 ± 12 H2O/charge (41 trials on 14 oocytes). Experiments performed with citrate and succinate were performed on different oocytes.

The volume flow under isosmotic conditions was directly correlated with the rate of Na+-dicarboxylate cotransport. This is illustrated in Fig. 4. The data were obtained from a series of experiments where the volume flow and substrate transport rate were measured simultaneously. The solid circles in Fig. 4 represent the volume flow that resulted from the transport of citrate in the presence of Na+ solution. The slope as determined from a linear regression through the data was 32 ± 1 × 10-3 pl · s-1 · nA-1 (24 trials on 10 oocytes). Citrate and succinate are both substrates of NaDC-1; however, a higher maximal transport rate is observed with citrate (19), indicating that the turnover rate of NaDC-1 is higher when citrate is transported. We also examined volume flow when NaDC-1 was transporting succinate (Fig. 4, black-down-triangle ). The transport rate of NaDC-1 in succinate was ~60% of that in citrate (present study; Ref. 19), but the coupling ratio between water and succinate cotransport was slightly higher, 42 ± 5 × 10-3 pl · s-1 · nA-1 (9 trials on 4 oocytes).

In the presence of Na+, Li+ inhibits substrate transport by NaDC-1 (6). Presumably, Na+ and Li+ are competing for the same binding site, and Li+ is a poor activator of substrate transport (19, 27). The presence of 10 mM Li+ (together with 80 mM Na+ and 10 mM citrate) reduced both the substrate-evoked inward current and the water flux to 33 ± 2 and 31 ± 3%, respectively (8 trials on 4 oocytes) (data not shown). The relationship between water flow and NaDC-1 transport rate is shown in Fig. 4 (open circle ). The coupling ratio between water and current in the presence of 10 mM Li+ was 34 ± 3 × 10-3 pl · s-1 · nA-1 (8 trials on 4 oocytes).

The data from the experiments with citrate, succinate, and Li+ could be described by the same linear function (Fig. 4). The slope of the regression line, 33 ± 2 × 10-3 pl · s-1 · nA-1, corresponds to the transport of 176 ± 12 H20 per inward positive charge, or 176 H2O:3 Na+:1 dicarboxylate (since the stoichiometry of NaDC-1 is 3 Na+ per divalent dicarboxylate, Refs. 6 and 19).

Uphill water transport. NaDC-1 was capable of transporting water uphill, against the osmotic driving force (Fig. 5). In Fig. 5A, bottom trace, an oocyte expressing NaDC-1 was subjected to an osmotic challenge of +20 mosM, obtained by addition of 20 mM mannitol to the control solution. The oocyte volume immediately started to decrease at a rate (Jvpassive) predicted by the osmotic water permeability (Lp). When the same osmotic gradient was applied but under the condition that NaDC-1 was transporting citrate (addition of 20 mM of mannitol to the isosmotic citrate solution), then the rate of shrinkage of the oocyte and the volume flow Jvcitrate was decreased (Fig. 5A, top trace). The difference between the two volume flows (NaDC-1-coupled volume flow) is represented by the jagged line in Fig. 5B and corresponds to the water transport that was coupled to the influx of Na+ and citrate by NaDC-1. The smooth curve of Fig. 5B was obtained from the integral of the citrate-induced inward current with a coupling ratio of 245 water molecules/inward charge. The smooth curve superimposed on the volume curve, indicating that NaDC-1 transported water together with Na+ and citrate against the osmotic gradient. On average, the coupling ratio was 182 ± 12 water molecules/inward charge (7 trials on 4 oocytes). Thus the stoichiometry for uphill water transport is 182 H2O:3 Na+:1 dicarboxylate.


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Fig. 5.   Uphill water transport by NaDC-1. Volume changes and inward current were measured during hyperosmotic solution changes in an NaDC-1-expressing oocyte. Introduction of an osmotic gradient of +20 mosM (first arrow) induced a volume change of -56 pl/s (A, mannitol). There were no changes in inward current (data not shown). Introduction of an osmotic gradient of +20 mosM in the presence of substrate (10 mM citrate) induced both a volume change of -18 pl/s (A, citrate) and an inward current, with Imax of 820 nA (data not shown). NaDC-1-coupled water flow was obtained from the difference between the two volume changes (Delta Vcitrate - Delta Vmannitol). The volume change was 46 pl/s, shown as the jagged trace in B. The number of charges transported into the oocyte (the integral of the inward current) was plotted in B as a smooth line. The relationship between the inward charge and the volume flow suggested cotransport of 246 H2O molecules with 1 charge.


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INTRODUCTION
MATERIALS AND METHODS
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The increase in Lp of oocytes expressing NaDC-1 and the linear relationship between expression level and the Lp is due to a NaDC-1-specific water permeability. This increase is not simply due to an increase in protein density in the plasma membrane because specific inhibitors block passive transport through other cotransporters, for instance, phlorizin blocks the SGLT1 Lp and SKF blocks the GAT1 (Na+-Cl--GABA transporter) Lp (10). The rate of water transport through the passive pathway is determined by the direction of the imposed osmotic gradient (Figs. 1A and 5A). The passive water permeability per protein can be obtained from the turnover rate of the cotransporter. In general, Na+-coupled cotransporters have turnover rates in the range of 10-100 s-1 (12). Assuming a turnover rate for NaDC-1 of 50 per s would give an Lp of 1.5 ×10-14. cm · s-1 · protein-1. This is comparable to the Lp per protein found for the Na+-glucose cotransporter SGLT1 (1.4 × 10-14 cm · s-1 · protein-1) (10, 28). The Lp per protein found for cotransporters is ~100 times lower than that of the aquaporins (15). The activation energy for passive water transport by SGLT1 is 5 kcal/mol and is comparable to those of the aquaporins (10). Therefore, Na+-dependent cotransporters may be described as being low-conductance water channels (10, 11, 28). In addition to a similar Lp, the passive water permeability was the same in Na+, Li+, and choline for both NaDC-1 and SGLT1, independent of the cation. These similarities suggest that SGLT1 and NaDC-1 share a common mechanism for passive water transport.

Solute-coupled water flow by NaDC-1 is summarized by the following observations: 1) secondary active transport of Na+ and citrate was accompanied by an increase in oocyte volume (Fig. 3); 2) the time courses of volume change and citrate-induced current (inward charge) were superimposable (Fig. 3); 3) there was a strict stoichiometric relationship between Na+, citrate (or succinate), and water, and the coupling ratio was 3 Na+, 1 dicarboxylate, and 176 water molecules per transport cycle (Figs. 3 and 4); 4) Li+ inhibited both water flow and NaDC-1 transport to the same extent, with no change in the coupling ratios (Fig. 4); and 5) the solute-coupled water flow occurred in the absence of, and even against, an osmotic gradient (Figs. 3 and 5). These findings suggest that there is a direct coupling between water flow and secondary active transport of the substrates via NaDC-1. Coupling may be related to conformational changes of the protein during the transport cycle (11, 14).

Solute-coupled water transport mediated by NaDC-1 shares a number of properties with that observed in SGLT1 (11, 14, 31): 1) independence from osmotic gradients, 2) strict stoichiometric relationship between water and substrates, and 3) both components of water transport (osmotic and solute coupled) are present under substrate transporting conditions. Water transport has also been observed in cotransport proteins from native tissue, such as the choroid plexus K+-Cl- and the retinal pigment epithelium H+-lactate cotransporters (29, 30), and in the cloned human brain Na+-Cl--GABA, rat thyriod Na+-iodide, and the plant H+-amino acid cotransporters expressed in oocytes (11). This is consistent, in view of the hypothesis that cotransporters from different families share common kinetic mechanisms (26), and suggests that there may be an essential role for water in the cotransport process.

The following observations support the hypothesis that solute-coupled water flow is not due to a local osmotic mechanism: 1) transport through ion channels, such as connexin 50, nystatin, and gramicidin, does not result in initial solute-coupled water flow (14, 25, 31); 2) the osmotic effects have also been ruled out by theoretical considerations (11); and 3) if solute-coupled water transport were driven by local osmotic gradients, then the ratio of water molecules to solutes would be the same for all transporters. In fact, this ratio varies from transporter to transporter. In human SGLT1, 210 water molecules are coupled to 3 solutes (2 Na+ and 1 glucose) (14), whereas in rabbit SGLT1, the ratio was 360 H2O:2 Na+:1 glucose (31). In both the K+-Cl- (29) and in the H+-lactate cotransporter (30), the water-to-solute ratio was 500:2. The ratio for NaDC-1 was 175 water coupled to 4 solutes: 3 Na+ and 1 dicarboxylate. In preliminary experiments, the plant H+-amino acid cotransporter had a ratio of 50:2 (1 H+ and 1 amino acid) (unpublished data).

Does NaDC-1 play a role in renal proximal tubular water transport? We note that citrate stimulates fluid absorption in rabbit isolated proximal tubule (2). In the case of human, the plasma concentration of all tricarboxylic acid cycle intermediates is ~0.25 mM (7), and on the basis of a glomerular filtration rate of 180 liters/day and a reabsorption efficiency of 90% (5), 40 mmol are reabsorbed in the proximal tubule per day. One millimole of tricarboxylic acid cycle intermediate is coupled to 175 mmol water, equivalent to 3.15 ml. Thus NaDC-1 could mediate the reabsorption of ~100 ml of solute-coupled water per day. The transport of 176 water molecules with 3 Na+ and 1 dicarboxylate is equivalent to a coupling ratio of 46 water molecules/solute molecule. Because isotonicity requires 275 water molecules/solute molecule (4), this results in a fluid that is hypertonic. The hypertonic fluid transported across the apical membrane by NaDC-1 per se would slowly create an osmotic gradient, which could then provide a driving force for water movement across the apical membrane. Most of the water is probably transported by aquaporin-1. With the fact that the overall water reabsorption is isotonic taken into account, a total of 800 ml of water could be reabsorbed as a result of the reabsorption of dicarboxylates by NaDC-1. Furthermore, because water transport appears to be a common property of cotransporters, the coupling of water to the absorption of all organic acids (which includes tricarboxylic acid cycle intermediates, short-chain fatty acids, and ketone bodies; in total, 2-3 mM) could account for ~6% of the total daily fluid reabsorption in the kidney proximal tubule.


    ACKNOWLEDGEMENTS

We thank Daisy Leung and Elsa Gallardo for preparations of oocytes and cRNA and Bruce Hirayama and Sepehr Eskandari for helpful comments on the manuscript.


    FOOTNOTES

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-44602 and DK-46469 and by the Danish Research Council.

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 and other correspondence: A.-K. Meinild, Dept. of Physiology, UCLA School of Medicine, Center for the Health Sciences, 10833 LeConte Ave., Los Angeles, CA 90095-1751 (E-mail: smeinild{at}mednet.ucla.edu).

Received 11 August 1999; accepted in final form 30 November 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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