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J Am Soc Nephrol 13:2628-2638, 2002
© 2002 American Society of Nephrology

The Renal Na+-Dependent Dicarboxylate Transporter, NaDC-3, Translocates Dimethyl- and Disulfhydryl-Compounds and Contributes to Renal Heavy Metal Detoxification

Birgitta C. Burckhardt*, Britta Drinkuth*, Christine Menzel*, Angela König*, Jürgen Steffgen*, Stephen H. Wright{dagger} and Gerhard Burckhardt*

*Zentrum Physiologie und Pathophysiologie, Abteilung Vegetative Physiologie und Pathophysiologie, Georg-August Universität, Göttingen, Germany; and {dagger}Department of Physiology, College of Medicine, University of Arizona, Tucson, Arizona.

Correspondence to Dr. Birgitta C. Burckhardt, Zentrum Physiologie u. Pathophysiologie, Abteilung Vegetative Physiologie und Pathophysiologie, Georg-August Universität Göttingen, Humboldtallee 23, D-37073 Göttingen, Germany, Phone: 49-551-395880; Fax: 49-551-395883; E-mail: bcburckhardt{at}veg-physiol.med.uni-goettingen.de


   Abstract
Top
 Abstract
Introduction
 Material and Methods
 Results
 Discussion
 References
 
ABSTRACT. The active transport of Krebs cycle intermediates, such as succinate, {alpha}-ketoglutarate, and citrate, is mediated by sodium-coupled transporters found in the luminal (NaDC-1) and basolateral plasma membranes (NaDC-3) of proximal tubule cells. This study used the two-electrode voltage clamp technique to examine steady-state currents associated with the influx of three sodium ions and one divalent dicarboxylate into oocytes expressing the sodium-dicarboxylate transporter from winter flounder kidney, fNaDC-3. The substrate concentration, where half-maximal current was observed (K0.5), was 30 µM for succinate. Besides 2,2-dimethylsuccinate, fNaDC-3 also accepted 2,3-dimethylsuccinate and the oral lead-chelating agent, meso-2,3-dimercaptosuccinate (DMSA or Succimer). Whereas the K0.5 for succinate and 2,2-dimethylsuccinate was independent of membrane voltage within -90 and -10 mV, K0.5 for 2,3-dimethylsuccinate and 2,3-dimercaptosuccinate increased with decreasing voltage, indicating a critical role of the position of the methyl- or sulfhydryl-group in voltage-sensitive affinity. In addition to meso-2,3-dimercaptosuccinate, fNaDC-3 translocated dimercaptopropane-1-sulfonate (DMPS or Dimaval), an oral chelator for the treatment of mercury intoxication. The chelates formed by HgCl2 and DMSA or DMPS and by Pb(NO3)2 and DMSA, however, were not translocated by fNaDC-3. The data suggest that NaDC-3 is an essential component in the delivery of uncomplexed antidotes for renal heavy metal detoxification.


   Introduction
Top
 Abstract
 Introduction
Material and Methods
 Results
 Discussion
 References
 
Di- and tricarboxylates, such as succinate, {alpha}-ketoglutarate, and citrate, are actively taken up by renal proximal tubule cells both from the peritubular capillaries and the glomerular filtrate. These compounds serve as metabolic fuels and as substrates for gluconeogenesis (1,2,3). In addition, intracellular {alpha}-ketoglutarate drives the uptake of organic anions by an organic anion/dicarboxylate exchanger, OAT1, located in the basolateral membrane (4,5). This exchange process constitutes the first step in the proximal tubular excretion of a large number of organic anions, including widely used drugs such as {beta}-lactam antibiotics, antiviral drugs, diuretics, and nonsteroidal antiinflammatory drugs (6–9).

The transport of di- and tricarboxylates across the luminal and the basolateral cell membrane of proximal tubule cells is mediated by two distinct Na+-dependent dicarboxylate transporters, NaDC-1 and NaDC-3 [reviewed in references 10 and 11]. Both translocate three sodium ions and a di- or tricarboxylate in its divalent form. NaDC-1 has been cloned from rabbit, rat, mouse, and human. Except for rat NaDC-1 (or SDCT1), these transporters exhibit a comparatively low affinity for succinate (Km above 0.2 mM). By immunohistochemical studies, rabbit and rat NaDC-1 were located to the luminal membrane of proximal tubule cells. NaDC-3 (or SDCT2) cloned from human (12), rat (13,14), mouse (15), and flounder (16) showed a comparatively high affinity for succinate in radiotracer uptake experiments (Km: 20 ± 1 µM for human, 15 ± 1.3 µM for rat, and 30.4 ± 13.5 µM for flounder NaDC-3). In the flounder kidney, NaDC-3 is located at the basolateral membrane of proximal tubule cells (Steffgen et al., unpublished results).

Na+-diacarboxylate cotransporters in the luminal and basolateral membrane of proximal tubule cells do not only differ in their affinity for succinate, but also in their pH dependence and substrate specificity. Methylsuccinate transport by the cotransporter present in the luminal membrane was largely pH-independent between pH 6 and 8, whereas a decrease in pH from 7.5 to 6.0 attenuated transport in basolateral membrane vesicles (17). The dependence of succinate transport on pH was reciprocated in studies on the expressed NaDC-3 (13–16). Earlier microperfusion experiments on the intact kidney (18,19) and subsequent studies on the expressed NaDC-3 (12–16) revealed that Na+-dicarboxylate transporters in the basolateral membrane, but not those in the luminal membrane, accept a succinate homolog with methyl substitutions at carbon 2 and 3, i.e., 2,3-dimethylsuccinate, with high affinity. Similar results were obtained with respect to 2,3-dimercaptosuccinate (DMSA), which did not inhibit luminal dicarboxylate transport but markedly suppressed the uptake of dicarboxylates across the basolateral membrane (13,18,19).

Meso-2,3-dimercaptosuccinic acid (DMSA or Succimer) is an orally effective chelating agent for the treatment of heavy metal poisoning (20,21). The two vicinal SH-groups in DMSA complex with Hg2+, whereas one oxygen of the carboxyl group and one sulfur of the neighboring SH-group form complexes with Cd2+ and Pb2+ (22). DMSA has been successfully used in children (23,24,25) and adults (26) to increase urinary lead excretion and to deplete body lead stores. Lead intoxication is still a concern, particularly in children living in old houses contaminated with lead-based paints (27,28). Blood lead levels exceeding 10 µg/dl are considered to cause irreversible health defects including mental disorders (28). As opposed to EDTA, another lead-chelating agent, the urinary loss of the essential metals, zinc and copper, was small, and severe adverse reactions were not observed under the therapy, rendering DMSA a safe drug (25).

2,3-dimercapto-1-propane sulfonate (DMPS) is another orally effective heavy metal chelator, which binds Hg2+ with an extremely high affinity by formation of complexes with the two vicinal SH-groups (21,29). DMPS is formally derived from the relatively toxic dimercaprol (2,3-dimercatopropane; British anti-lewisite or BAL) by adding a hydrophilic sulfonate group and is now considered to replace BAL whenever the duration of therapy is not critical (30). In humans, urinary mercury excretion was enhanced by a factor of more than 15 from 106 µg/24 h to 1754 µg/24 h when DMPS was applied (31). In rats, DMPS was more effective than DMSA in decreasing the renal Hg2+ burden by strongly increasing urinary Hg2+ excretion (29). Similar results with respect to DMPS and HgCl2 were obtained in isolated S2-segments of rabbits (32). In the isolated, perfused rat kidney (33), net tubular secretion of DMPS was saturable and blocked by p-aminohippurate and probenecid, substrate and inhibitor of the organic anion/dicarboxylate exchanger, OAT1. In rat kidney in situ, DMPS inhibited radiolabeled PAH uptake, but not dimethylsuccinate uptake (34). Recently, an inhibition of the heterologously expressed human OAT1 by DMPS was shown (35). Translocation of DMPS by the organic anion transporter was directly demonstrated in isolated rabbit kidney tubules; DMPS in the medium trans-stimulated the efflux of the model organic anion OAT1, fluorescein (36).

In this study, we used the two-electrode voltage clamp technique to demonstrate translocation of 2,2- and 2,3-dimethylsuccinate (DMS) as well as of DMSA by fNaDC-3, the Na+-dependent dicarboxylate transporter from flounder kidney. Our studies show that fNaDC-3 not only transports DMSA, but surprisingly, also the monovalent DMPS. Hence this transporter should play an important role in renal heavy metal detoxification.


   Material and Methods
Top
 Abstract
 Introduction
 Material and Methods
Results
 Discussion
 References
 
Oocyte Preparation and Storage
Stage V and VI oocytes from Xenopus laevis (Nasco, Fort Atkinson, WI) were treated with collagenase (Type CLS II, Biochrom, Berlin, Germany), subsequent washing in Ca2+-free control solution and maintained at 18°C in control solution containing (in mM): 110 NaCl, 3 KCl, 2 CaCl2, 5 HEPES/Tris, pH 7.5. One day after removal from the frog, individual oocytes were injected with 30 nl of cRNA (1 µg/µl) encoding for fNaDC-3 and maintained at 16 to 18°C in control solution supplemented with 100 kU/L penicillin, 0.1 mg/L streptomycin, and 2.5 mM sodium pyruvate. After 3 to 4 d of incubation with daily medium changes and discarding damaged oocytes, the remaining healthy oocytes were used for electrophysiologic studies. Oocytes injected with water served as control.

Electrophysiologic Studies
Electrophysiologic studies were performed by the conventional two-microelectrode voltage clamp method using a commercial amplifier (OC 725, Warner, CT). The microelectrodes were filled with 3 M KCl and had resistances of less than 1 M{Omega}. The membrane potential of the oocytes was clamped at -60 mV, and the current induced by 1 mM succinate was measured to assure functional expression of the fNaDC-3 protein. Voltage pulses between -90 and +10 mV, in 10 mV increments, were applied for 5 s each, and steady-state currents were recorded to obtain current-voltage (I-V) relations. In general, the I-V protocol was applied first under control conditions and then 30 s after changing the perfusion to the test solution. The difference between the steady-state currents measured in the presence and absence of substrates was considered as substrate-induced current ({Delta}I). The substrate was washed away with control solution, and experiments were continued only when the current had returned to baseline. Data were expressed as mean ± SEM. Statistical analyses were performed with t test with statistical significance set at P <= 0.01. Michaelis-Menten parameters, the substrate concentration at half-maximal current (K0.5) and the maximum current observed at saturating substrate concentrations ({Delta}Imax), were obtained by Eadie-Hofstee analysis.

Chemicals
All chemicals, including succinate, 2,2- and 2,3-dimethylsuccinate, 2,3-dimercaptosuccinate, 2,3-dimercaptopropane-1-sulfonate, and dithiotreitol, as well as HgCl2 and Pb(NO3)2 were of analytical grade and purchased from Merck (Darmstadt, FRG) or Sigma (Deisenhofen, FRG). The oxidized form of DMPS, DMPSS (10 mM), was prepared by drop-by-drop addition of saturated iodine solution to a slightly alkaline solution of DMPS (20 mM, pH 8). Addition of the iodine reagent was stopped when the brownish-yellow color of the reaction persisted. Reduction of oxidized DMPSS to DMPS was performed by mixing dithiothreitol (DTT, 10 mM) and 5 mM DMPSS in a 1:1 ratio and stirring the solution under nitrogen for at least 30 min. This procedure was sufficient to reduce all DMPSS to DMPS as confirmed by others (37). Mercury-DMSA and mercury-DMPS as well as lead-DMSA complexes were formed by equimolar addition of HgCl2 and the chelating agent, i.e. 1 mM DMPS plus 1 mM HgCl2, 5 mM DMPS, and 5 mM HgCl2. The Pb complexes of DMSA were prepared accordingly.


   Results
Top
 Abstract
 Introduction
 Material and Methods
 Results
Discussion
 References
 
Translocation of 2,2- and 2,3-dimethylsuccinate and 2,3-dimercaptosuccinate by fNaDC-3
fNaDC-3 is a high-affinity Na+-dependent dicarboxylate transporter cloned from winter flounder kidney (16,38). Due to a 3:1 stoichiometry of Na+:succinate2-, the Na+-coupled transport of succinate via fNaDC-3 is electrogenic. If a similar mechanism operates for the dimethyl-substituted succinates, 2,2-dimethylsuccinate (2,2-DMS) and 2,3-dimethylsuccinate (2,3-DMS), and also for the sulfhydryl-substituted succinate, 2,3-dimercaptosuccinate (DMSA), translocation should be detectable with electrophysiological methods in fNaDC-3-expressing oocytes.

The currents evoked by equimolar concentrations of succinate (Figure 1, A through C, closed circles) and 2,2-DMS (Figure 1A, open circles), as well as of 2,3-DMS (Figure 1B, open circles) and DMSA (Figure 1C, open circles) were comparable, indicating that all three substituted succinates are indeed translocated by the Na+-dependent dicarboxylate transporter from winter flounder kidney. A direct comparison of the succinate and 2,2- and 2,3-DMS-elicited currents on single oocytes in random order revealed that the currents induced by 1 mM 2,2-DMS were not significantly different from those evoked by 1 mM succinate, whereas the currents induced by 1 mM 2,3-DMS and 1 mM DMSA were smaller than those evoked by 1 mM succinate on the same oocyte (Figure 1D). No inward currents were detected in control oocytes when succinate, 2,2-DMS, 2,3-DMS, or DMSA was added to the medium (Figure 1, A through C, closed and open squares), indicating that native oocytes do not possess a Na+-dependent dicarboxylate transporter.



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  		Figure 1. Translocation of 2,2-dimethylsuccinate (DMS), 2,3-DMS, and 2,3-dimercaptosuccinate (DMSA) by fNaDC-3. Current-voltage (I-V) relations were obtained in oocytes injected with 30 ng of cRNA coding for fNaDC-3 without or with substrates (each 1 mM). Substrate-dependent currents were determined as the difference between these two conditions. (A) 2,2-DMS-evoked (open circles) and (B) 2,3-DMS-evoked currents (open circles) were measured in random order on six oocytes from three different donors and compared with the succinate-evoked current (closed circles) determined on the same oocyte. (C) DMSA-evoked (open circles) and succinate-evoked currents (closed circles) as measured in 11 oocytes from six different donors. Water-injected oocytes when superfused with succinate (closed squares), 2,2-DMS, 2,3-DMS, or DMSA (open squares) showed no currents. (D) Comparison of currents relative to those induced by succinate (100%). Clamp potential was -60 mV, substrate concentrations were 1 mM each.

 
In the next set of experiments, we tested whether the currents evoked by 2,2- and 2,3-DMS and DMSA were sensitive to Na+ and Li+. As shown here for DMSA, in the presence of Na+, DMSA-induced currents were linear within the potential range tested (Figure 2, , A and B, closed circles) and were abolished when all NaCl was replaced by N-methyl-D-glucamine chloride (Figure 2A, open circles). In addition, the DMSA-induced currents were inhibited by Li+ (2 mM) (Figure 2B, open triangles), indicating Na+-dependent, Li+-inhibitable translocation of DMSA via fNaDC-3. Similar results with respect to Na+-dependency and inhibition by Li+ were obtained for 2,2- and 2,3-DMS (data not shown).



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  		Figure 2. Sodium and lithium dependency of the DMSA-induced currents. I-V relations of DMSA-induced currents were determined (A) in the presence of sodium (closed circles) and when all sodium was substituted by N-methyl-D-glucamine (open circles) in eight oocytes from four different frogs and (B) in the absence (closed circles) and presence of 2 mM lithium (open triangles) in six oocytes from four different frogs.

 
Comparable to succinate (reference 38 and this study), steady-state inward currents produced by DMSA were concentration- and voltage-dependent. Eadie-Hofstee plots obtained from a representative experiment for succinate and DMSA at -90 and -30 mV are shown in Figures 3A 3B, respectively. As compared with succinate (Figure 3A), where Eadie-Hofstee plots showed similar slopes at -90 (closed circles) and -30 mV (closed squares), the Eadie-Hofstee plots for DMSA (Figure 3B) differed with respect to the slopes at -90 (open circles) and -30 mV (open squares). The slope at -30 mV was steeper than the slope at -90 mV, indicating that the K0.5 increased with decreasing potential.



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  		Figure 3. Eadie-Hofstee plots for succinate (A) and 2,3-dimercaptosuccinate (B). The analysis was performed using identical substrate concentrations for succinate as well as for DMSA: 0.02, 0.05, 0.1, 0.2, 0.5, and 1 mM. Experiments were performed at -30 and at -90 mV to show increase in slope at depolarizing potentials when substituting a sulfhydryl-group at C3.

 
Using a similar analysis, the K0.5 values were determined for succinate, 2,2- and 2,3-DMS, and DMSA at various clamp potentials. At -60 mV, the rank order of K0.5 was 2,2-DMS < succinate <= 2,3-DMS < 2,3-DMSA (Table 1), demonstrating an approximately 13 times higher affinity for 2,2-DMS than for DMSA. 2,2-DMS stimulated fNaDC-3-mediated currents not only with high affinity, but it was also translocated with high efficiency, shown by the rank order of {Delta}Imax/K0.5: 2,2-DMS > 2,3-DMS >= succinate > DMSA.


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  		Table 1. Substrate specificity of fNaDC-3a
 
Whereas K0.5 for 2,2-DMS and succinate (Figure 4, A and B) was independent of membrane voltage within the range of -90 to -10 mV, the K0.5 for 2,3-DMS and 2,3-DMSA (Figure 4, C and D) increased with decreasing voltage, demonstrating that the position of the methyl- or sulfhydyl-groups is critical for the voltage-dependence of fNaDC-3.



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  		Figure 4. The influence of membrane voltage on the apparent affinity constant K0.5 for succinate (A), 2,2-DMS (B), 2,3-DMS (C), and DMSA (D). The K0.5 was determined from Eadie-Hofstee analysis from the potential- and concentration-dependent substrate-evoked currents based on three to six determinations for each experimental condition. Concentrations used for determination of K0.5 were (A) for succinate: 0.05, 0.1, 0.5, 1, 2.5, and 5 mM; (B) for 2,2-DMS: 0.005, 0.01, 0.05, 0.1, and 0.5 mM; (C) for 2,3-DMS: 0.01, 0.025, 0.05, 0.1, and 0.5 mM; and (D) for DMSA: 0.02, 0.05, 0.1, 0.2, 0.5, and 1 mM.

 
Translocation of 2,3-dimercaptopropane-1-sulfonate and its disulfide, DMPSS, by fNaDC-3
Besides DMSA, DMPS is used to reduce the renal burden of mercury within the body. Reduced DMPS is oxidized to DMPSS at high rates (39). Both, DMPS (Figure 5A) and DMPSS (Figure 5B) showed concentration-dependent inward currents at -60 mV. The currents evoked by DMPSS tended to saturate at concentrations >2 mM, whereas no saturation occurred for DMPS up to >5 mM. By Eadie-Hofstee analysis, the K0.5 for DMPSS obtained at -60 mV was in the range of 0.7 mM with an {Delta}Imax of approximately -60 to -70 nA (4 oocytes from 3 donors). Albeit translocated by fNaDC-3, DMPSS and DMPS turned out to be substrates with only medium (DMPSS) and low affinity (DMPS) to fNaDC-3.



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  		Figure 5. Translocation of 2,3-dimercaptopropane-1-sulfonate (DMPS) and of oxidized DMPSS by fNaDC-3. DMPS-induced currents, {Delta}I, were measured at a clamp potential of -60 mV (A) and of increasing DMPS concentrations (B). Experiments were performed on four oocytes from three donors. DMPSS was oxidized before the experiment started, and it was assumed that most of the DMPSS was present as cyclic dimer (54). Experiments were performed on four oocytes from three donors.

 
To verify that DMPS was present in our experiments as monovalent, reduced sulfhydryl group-containing DMPSH and not as divalent, oxidized disulfide, DMPSS, dithiothreitol (DTT) was added to the solution in excess to reduce SH groups (37,39). In paired experiments on six oocytes from three frogs, the currents induced by DMPS (5 mM) in the absence (open circles) and presence of DTT (10 mM, open triangles) were not significantly different (Figure 6). DTT (10 mM, closed triangles) was not able to induce a significant current throughout the tested potential range. These results indicate that DMPS was present in the reduced state and that the currents shown in Figure 6 as well as in Figure 5A were evoked by monovalent DMPS.



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  		Figure 6. Current-voltage (I-V) relation of succinate, dimercaptopropane-1-sulfonate (DMPS), and dithiotreitol (DTT) in fNaDC-3-expressing oocytes. Currents induced by succinate (1 mM, closed circles) were compared with those evoked by DMPS (5 mM, open circles), DMPS plus DTT (5 mM, 10 mM, open triangles), and DTT (10 mM, closed triangles). Substrates were applied to the oocytes in random order. Results were obtained from seven oocytes of four donors.

 
So far, an interaction of monovalent anions with the high affinity Na+-dependent dicarboxylate transporters was not reported; therefore, a more detailed investigation of the currents evoked by DMPS in fNaDC-3-expressing oocytes was performed. The currents induced by 1 mM DMPS were sensitive to lithium and sodium. As tested on five oocytes from 3 donors, lithium (2 mM) inhibited the DMPS-induced current by 46 ± 8%. When all sodium was replaced by N-methyl-D-glucamine (NMDG), the DMPS-induced currents were nearly abolished. As measured in three oocytes from three different donors, the DMPS-sensitive current was inhibited by 86, 90, and 93%, respectively. These results are summarized in Figure 7, where the DMPS-induced currents in the presence of Li+ (2 mM) or total replacement of Na+ by NMDG are plotted as percentage of the currents under control (110 mM Na+).



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  		Figure 7. Effect of cations on DMPS-induced currents. Currents induced by DMPS (2 mM) were measured in the presence of Na+ (white column), when all Na+ was substituted by N-methyl-D-glucamine+ (black column) and in the presence of Li+ (2 mM, hatched column). Currents are indicated as a percentage of the DMPS-evoked current under control in the same oocyte. Experiments demonstrating inhibition of the DMPS-induced current by Li+ were obtained on six oocytes of three donors, those demonstrating Na+-dependency on three oocytes of three donors.

 
The pKa values of DMPS are as follows: pK1 1.2 (carboxyl group), pK2 8.7 to 9.4, and pK3 11.2 to 12.6 (sulfhydryl groups) (40). At physiologic pH, approximately 10% of DMPS will be present as a divalent anion, and lowering pH to 6.0 will decrease the concentration of divalent DMPS. As measured in paired experiments at -60 mV, a decrease in pH from 7.5 to 6.0 decreased both succinate- and DMPS-induced current (black and white column in Figure 8A). The relative decrease in current was significantly larger for DMPS, which may be due to the decrease in divalent DMPS concentration. Moreover, propane sulfonate, which does not contain dissociable SH-groups, produced at pH 7.5 very small, if any, currents. As compared to DMPS (5 mM, white column) which exhibited a current of -11 ± 3 nA, propane-sulfonate (5 mM, hatched column), showed only a current of -1 ± 1 nA at -60 mV (5 oocytes from 3 frogs), which is 12 ± 10% of the DMPS-evoked current (Figure 8B). These data suggest that DMPS is translocated as a divalent anion, the sulfonate- and one sulfhydryl-group being negatively charged.



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  		Figure 8. Effect of pH on succinate-induced and DMPS-induced currents. (A) Currents induced upon application of succinate (1 mM) at pH 7.5 and pH 6.0 and DMPS (5 mM) at pH 7.5 and pH 6.0 obtained in paired experiments on five oocytes from four frogs. Currents are indicated as a percentage of the succinate-evoked (black column) and DMPS-evoked (white column) current under control (pH 7.5) in the same oocyte. The relative decrease in current is significantly larger for DMPS. (B) Comparison of the currents evoked by DMPS (5 mM, white column) and propane-sulfonate (5 mM, hatched column). Results were obtained in paired experiments on five oocytes from three donors and plotted as percentage of the DMPS-evoked current. Experiments shown in panels A and B were performed at -60 mV.

 
Finally, we tested whether mercury-DMSA, lead-DMSA, and mercury-DMPS complexes were translocated by fNaDC-3. Mercury-DMSA (Figure 9A, open circles) as well as the lead-DMSA complexes (Figure 9B, open triangles) were not translocated by fNaDC-3-expressing oocytes. At -60 mV, as observed in paired experiments on six oocytes from four donors, DMSA (1 mM, Figure 9A, closed circles) evoked a current of -83 ± 16 nA. Application of mercury-DMSA (each 1 mM, open circles) yielded a current of -5 ± 1 nA. Similarily, the lead-DMSA complex did not evoke an inward current, independent of clamp potential (Figure 9B, open triangles). A comparable effect was observed for DMPS on seven oocytes of three donors. At -60 mV, DMPS (5 mM) induced a current of -33 ± 5 nA (Figure 9C, closed squares). The mercury-complex itself evoked a current of -8 ± 3 nA (Figure 9C, open squares), demonstrating no or only weak translocation.



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  		Figure 9. Currents induced by DMSA and DMPS and their heavy metal complexes. The currents evoked by DMSA (1 mM, closed circles in panel A, closed triangles in panel B) and their respective Hg2+ (panel A, open circles) or Pb2+ complexes (panel B, open triangles) are plotted as a function of membrane potential. In panel C, the currents induced by DMPS (5 mM, closed squares) and its 1:1 DMPS-mercury complex (open squares) are shown. The number of oocytes where paired experiments were performed were (A) six oocytes from four frogs, (B) four oocytes from three donors, and (C) seven oocytes from three donors.

 

   Discussion
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Discussion
References
 
Na+-dependent dicarboxylate transporters, which transport succinate and other Krebs cycle intermediates, fall into two categories on the basis of their molecular structure and their substrate affinities (10,11). The Na+-dependent dicarboxylate transporter located at the brush-border membrane of intestinal and renal epithelial cells exhibits a K0.5 of 0.2 to 1 mM for succinate. This transporter, designated NaDC-1, has been cloned from various species and functionally characterized (41–45). The high-affinity Na+-dependent dicarboxylate transporter with a K0.5 < 0.2 mM for succinate is located at the basolateral membrane of renal proximal tubular cells, sinusoidal membrane of hepatocytes, brain synaptosomes, and the maternal-facing brush border membrane of the placental syncytiotrophoblast. This transporter, designated NaDC-3, has also been cloned from different species and functionally characterized (12–16). We have recently reported the cloning of fNaDC-3 from winter flounder kidney (16). fNaDC-3 showed an affinity for succinate ranging between 30 (16) and 60 µM (38) as determined by radiotracer or electrophysiologic measurements, respectively. Up to now, all cloned NaDC-3 showed nearly identical patterns with respect to substrate affinity, dependence on sodium, inhibition by lithium, and electrogenicity. Thus, it is likely that experiments on the flounder NaDC-3 can be extrapolated to NaDC-3 s from other species.

In this study, we investigated to the translocation of methyl- or sulfhydryl-substituted succinate derivatives in fNaDC-3-expressing oocytes. 2,2- and 2,3-dimethylsuccinate (DMS) as well as 2,3-dimercaptosuccinate (DMSA) induced concentration- and voltage-dependent inward currents in fNaDC-3-expressing oocytes. These inward currents were suppressed by removal of Na+ from the bath, indicating the Na+ dependence of transport and the electrogenic translocation of three Na+ and one divalent succinate analog. The inhibition of currents induced by substituted succinates by Li+ is a further hallmark of the NaDC transporters.

The currents evoked by all four substrates tended to saturate at concentrations higher than 0.5 mM. No large differences in currents between succinate and its three analogs were detected, suggesting that the 2,3-substitutions do not have an impact on the maximal rate of dicarboxylate transport. As determined at -60 mV, half maximal currents were achieved at 7 ± 2 µM for 2,2-DMS, at 40 ± 27 µM for 2,3-DMS, and at 31 ± 9 µM for succinate. These results reveal 2,2-DMS as a high-affinity substrate for fNaDC-3. The comparable affinities for 2,3-DMS and succinate suggest that a 2,3-disubstitution of succinate does not measurably change the interaction with the transporter. In rat kidney in situ, 2,3-DMS inhibited methylsuccinate uptake across the basolateral membrane with a Ki of 0.19 mM. In contrast, 2,3-DMS was a very weak inhibitor of dicarboxylate transport at the luminal membrane (Ki, 3.76 ± 0.73 mM) (19). Within the cloned NaDC-1 orthologs, DMS analogs left either radiolabeled succinate uptake unaffected (41,45,46) or induced currents comparable in magnitude to succinate under two-electrode voltage clamp conditions (44,47,48). In contrast, NaDC-3 orthologs were generally sensitive to DMS analogs (12–16). Thus, 2,3-DMS can be used as a discriminator between luminal and basolateral Na+-dependent dicarboxylate transporters. In addition, 2,3-DMS could be a useful tool for future delineation of the substrate binding site, e.g., by constructing chimera between luminal and basolateral NaDCs.

Whereas K0.5 for succinate was determined only at -50 mV in one study (14), studies on mNaDC-3 (15) and fNaDC-3 (reference 38 and this study) were performed at various clamp potentials. When calculated at -50 mV, K0.5 for succinate was comparable in rat kidney (27.2 ± 2.2 µM [14]), mouse brain (15.5 ± 2.7 µM [15]), and flounder kidney (59.3 ± 15 µM [38]), and 30 ± 7 µM [this study]). In our previous study (38) and again in this study, K0.5 for succinate was independent of voltage within the range between -90 and 0 mV. Over a larger range of potentials (-50 to +50 mV), the K0.5 for succinate of mouse brain NaDC-3 increased by a factor of 3 from 16 at -50 mV to 51 µM at +50 mV (15). Similar to succinate, the K0.5 for 2,2-DMS was independent of voltage. In contrast, K0.5 for 2,3-DMS increased with decreasing voltage, indicating that the position of the methyl-group at the succinate molecule determined the dependency on voltage. Changing the substitution of the methyl-group from the 2,2- to the 2,3-position may require an additional voltage-sensitive conformational change of the transporter to accommodate the substrate. The other 2,3-substituted succinate, 2,3-dimercaptosuccinate (DMSA), showed a similar voltage-dependency as 2,3-DMS, indicating that the substitution at the 3-position per se is critical for the voltage dependency of the apparent affinity. NaDC-1 does not appreciably interact with 2,3-disubstituted succinates; therefore, this conformational change may not be possible in the low-affinity Na+-dependent dicarboxylate transporters. Accordingly, 2,3-dimercaptosuccinate (DMSA) showed different sensitivities versus luminal and basolateral Na+-dependent dicarboxylate transporters in rat kidney in situ. DMSA inhibited the uptake of [3H]methylsuccinate from the peritubular capillaries with a Ki of 2.2 ± 1.6 µM (19), whereas DMSA was ineffective in inhibiting {alpha}-ketoglutarate uptake across the luminal membrane (18). In HRPE cells transfected with NaDC-3 from rat placenta, DMSA inhibited [3H]succinate uptake by 69% (13), indicating that DMSA interacted preferentially with the high-affinity Na+-dependent dicarboxylate transporter, NaDC-3.

Meso-2,3-dimercaptosuccinate (DMSA, Succimer) is an important orally active heavy metal chelator that is used to increase renal excretion of lead and mercury in humans (20,21,29). DMSA contains a backbone of four carbon atoms, two carboxyl-groups, and two vicinal sulfhydryl-groups, which are highly active in chelating free heavy metals within cells or blood (20,21,29). Heavy metals are predominantly deposited within renal proximal tubule cells (49). Therefore, renal detoxification is only possible if uncomplexed DMSA enters proximal tubule cells via a suited transport system. Glomerular filtration is very low due to a high degree of protein binding; therefore, DMSA uptake can occur only across the basolateral membrane. In the present study, we provide conclusive evidence for a translocation of this heavy metal antidote by fNaDC-3. Once within the cells, DMSA can chelate free and protein-bound heavy metals and thus contributes to decrease the renal burden of e.g. mercury. The DMSA-heavy metal complex must leave the proximal tubule cell via the luminal membrane, probably by MRP2 (50,51), peptide- and amino acid-transporting proteins (33), or by other as yet unknown transporters.

2,3-dimercapto-propane-1-sulfonate (DMPS, Dimaval) is also clinically used to increase urinary mercury excretion and to reduce the renal burden of mercury (21,31,52). In the isolated, perfused rat kidney (53), net tubular secretion of DMPS was saturable and blocked by p-aminohippurate and probenecid, indicating the involvement of the organic anion secretion system. Indeed, DMPS was recently shown to interact with the rabbit (36) and human (35) organic anion transporter 1, OAT1. In vivo, DMPS is oxidized to the disulfide DMPSS possessing two negatively charged sulfonate-groups. Several metabolites of DMPSS with different chemical structures, including cyclic and acyclic DMPSS as well as mixed conjugates with cysteine, were proposed (54). Despite the fact that DMPSS is a divalent anion, the Na+-dependent currents induced by DMPSS in fNaDC-3-expressing oocytes were smaller in magnitude than those evoked by DMSA or succinate, indicating that only a small amount of the DMPSS metabolites were in a chemical structure favoring translocation by fNaDC-3. It is surprising that the monovalent DMPS induced a current in fNaDC-3-expressing oocytes. By using dithiothreitol to avoid oxidation of DMPS to DMPSS, we secured that the antidote was present as monovalent DMPS. In fNaDC-3-expressing oocytes, DMPS-induced currents vanished when all sodium was replaced by N-methyl-D-glucamine. In addition, Li+ inhibited DMPS-evoked currents. Both features clearly identify these currents as fNaDC-3-mediated currents. The ability of DMPS to be transported by fNaDC-3 may be due to the dissociation of the sulfhydryl-group at C2 (55); hence, the occurrence of a second negative charge in the molecule. At physiologic pH approximately 10% of the total DMPS is present as an divalent anion and therefore possibly suited for the interaction with and transport by fNaDC-3. At lower pH, less DMPS must be present in the divalent form; hence the current should decrease. Such a decrease was indeed observed, although the current evoked by succinate also decreased at lower pH, which is due to the known pH-dependence of NaDC-3. The percent of decrease of DMPS-induced currents, however, was significantly larger than that of succinate. In addition, propane sulfonate does not possess sulfhydryl groups and is therefore a monovalent anion at pH 7.5. We therefore propose that DMPS can be translocated by fNaDC-3 as a divalent anion. The monovalent DMPS is a substrate of OAT1. The relative importance of these transporters remains to be established in intact tubules or in the intact kidney.

In blood plasma, mercury is bound extensively to albumin, the most abundant sulfhydryl. Due to the very high affinity (20,40) of DMPS and DMSA to mercury, formation of mercury-DMPS or mercury-DMSA complexes is possible also in the plasma. These complexes, however, were not translocated by fNaDC-3. Binding of Hg2+ to the two vicinal SH-groups of either DMPS or DMSA or of Pb2+ to the carboxyl- and SH-groups obviously renders the complexes unacceptable for NaDC-3.

Our data provide evidence that the Na+-dependent dicarboxylate transporter in the basolateral membrane of proximal tubule cells, fNaDC-3, is involved in the uptake of uncomplexed antidotes used for heavy metal detoxification. Because the antidotes are tightly bound to protein (29,54) and therefore not accessible to the Na+-dependent dicarboxylate transporter located in the luminal membrane of the proximal tubule, the basolateral Na+-dependent dicarboxylate transporter is an important route for uptake of antidotes for renal heavy metal detoxification. Accumulation of mercury within the kidney occurs predominantely in S2 and S3 segment of the proximal tubule (49,55), and these nephron segments are the locus of the Na+-dependent dicarboxylate transporters (11). This implies a new, clinical role for the high-affinity Na+-dependent dicarboxylate transporter in the kidney.


   Acknowledgments
 
The authors want to thank I. Markmann for excellent technical assistance and E. Thelen for the artwork. This study was supported by the Deutsche Forschungsgemeinschaft, grant Bu 998/2–2 to BC Burckhardt. The authors also acknowledge support from PHS award DK56224 to SH Wright.


   References
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Discussion
 References
 

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Received for publication February 19, 2002. Accepted for publication July 20, 2002.




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