INTRODUCTION

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THE MAMMALIAN KIDNEY IS a primary organ involved in heavy metal excretion and accumulation. Chronic heavy metal intoxication of the kidney can lead to a number of reabsorptive and secretory defects. Inhibition of tubular reabsorption and secretion by heavy metals leads to proteinuria and polyuria, as well as conditions including glucosuria, aminoacidurias, calciurias, phosphaturia, and sulfaturias (16, 20, 22). Serum inorganic sulfate (S_i) concentrations are controlled to a large extent by the regulation of S_i reabsorption in the renal proximal tubule (3, 21). The cloning of the NaSi-1 cDNA (15), encoding the rat renal brush-border membrane Na-S_i cotransporter, has allowed us to study its role(s) during chronic renal changes as a consequence of heavy metal intoxication. Because the kidney is an important site for excretion of heavy metals, the function of several proximal tubular transporters has been recently shown to be affected by heavy metals. We have recently shown that mercury (Hg²⁺) and lead (Pb²⁺), but not cadmium (Cd²⁺), can inhibit amino acid transport by blocking the expression of the cloned amino acid transporter rBAT in Xenopus laevis oocytes (24). Similarly, the human renal Na-P_i cotransporter (NaPi-3) was recently shown to be inhibited by Hg²⁺, Pb²⁺, and Cd²⁺ in Xenopus oocytes (23). In a recent in vivo study, rats injected with CdCl₂ showed an inhibition in renal brush-border membrane vesicular Na/Pi and Na/glucose uptakes but not Na/sulfate uptake (9). These studies are of particular interest, since the heavy metals Hg²⁺, Cd²⁺, and Pb²⁺ have been shown to lead to nephrotoxicity and symptoms including phosphaturia, glucosuria, and aminoaciduria (16, 20, 22). Since to date no studies have examined the interaction of heavy metals with renal sulfate transporters, the aim of this study was to investigate the effects of Hg²⁺, Cd²⁺, Pb²⁺, and Cr⁶⁺ on the brush-border membrane Na-S_i cotransporter NaSi-1 protein expression in X. laevis oocytes.

	MATERIALS AND METHODS

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Oocytes and injections. Female X. laevis toads were obtained from African Xenopus Facility (Noordhoek, South Africa). Small clumps of oocytes (total ~500-1,500 oocytes) were treated for 60-90 min in collagenase type 4 (Worthington Biochemical, 2 mg/ml) in calcium-free OR II solution [in mM: 82.5 NaCl, 2 KCl, 1 MgCl₂, 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid-tris(hydroxymethyl)aminomethane (HEPES-Tris), pH 7.5]. Oocytes were then washed thoroughly five times with OR II solution and five times with modified Barth's solution [MBS, in mM: 88 NaCl, 1 KCl, 0.82 MgSO₄, 0.4 CaCl₂, 0.33 Ca(NO₃)₂, 2.4 NaHCO₃, 10 HEPES-Tris, pH 7.4, and 20 mg/l gentamicin sulfate]. The oocytes were sorted for morphologically intact, healthy-looking, stage V-VI oocytes, incubated in MBS at 17°C, and injected with either 50 nl water (control) or with 1 ng cRNA/oocyte derived from the NaSi-1 (15) and NaPi-3 (13) cDNAs, using a Nanoject automatic oocyte injector (Drummond Scientitfic, Broomall, PA). Oocytes were then kept at 17°C in MBS for 1-4 days, with daily changes of MBS solution.

In vitro transcription. NaSi-1 and NaPi-3 cRNA were synthesized in vitro, as described previously (13-15). Briefly, the transcription mixture [transcription buffer, 1× (40 mM Tris · HCl, pH 7.9, 2 mM spermidine and 6 mM MgCl₂), 0.5 mM ATP, 0.5 mM CTP, 0.5 mM UTP, 0.5 mM m7G(5')ppp(5')G, 0.1 mM GTP, 10 mM dithiothreitol, 50 units ribonuclease (RNase) inhibitor, and 50 units T7 RNA polymerase] was added to 1 µg of Not I linearized pSPORT-1 plasmid DNA. The reaction was incubated at 37°C for 1 h, followed by 50 units of RNase inhibitor, and 10 units of deoxyribonuclease I RNase free were added to the samples for a further 15 min at 37°C. cRNA was then extracted twice with phenol-chloroform-isoamylalcohol (25:24:1) and precipitated with 1 vol of ammonium acetate (7.5 M) and 2.5 vol of ethanol. cRNA was resuspended in 15 µl of water and used directly for injection.

Oocyte uptakes. Uptakes were performed as described previously (13-15, 19). In brief, oocytes (10 oocytes/individual data point) were first washed for 1-2 min in solution A (in mM: 100 choline chloride, 2 KCl, 1 CaCl₂, 1 MgCl₂, 10 HEPES-Tris, pH 7.5). This solution was then replaced by 100 µl of solution B (in mM: 100 NaCl, 2 KCl, 1 CaCl₂, 1 MgCl₂, 10 HEPES-Tris, pH 7.5) and supplemented with the desired concentration of cold substrate (K₂SO₄ or K₂HPO₄/KH₂PO₄; see Figs. 1-5) and labeled substrate Na₂³⁵SO₄ or H₃³²PO₄ (NEN) at the specific activity of 20 µCi/ml in the presence or absence of heavy metals HgCl₂, Pb(NO₃)₂, CdCl₂, or CrO₃ at the desired concentrations (see Figs. 1-5). Oocytes were incubated at room temperature 25°C for various times (1-60 min). Because of the nonlinearity of NaSi-1-induced transport activity up to 10 min, all transport assays (except in Figs. 2B-5B) were performed at uptake times 30 min. After incubation, the uptake solution was removed, and the oocytes were washed three times with 3 ml of ice-cold stop solution (solution A). Each single oocyte was then placed into a scintillation vial, dissolved in 250 µl of 1% sodium dodecyl sulfate, followed by the addition of 2 ml of scintillation fluid (Emulsifier Safe, Canberra Packard), and counted (2 min/oocyte), using liquid scintillation spectrometry. Reversibility of heavy metals was performed by preincubating oocytes with the heavy metals HgCl₂ (0.1 mM), Pb(NO₃)₂ (0.1 mM), CdCl₂ (0.5 mM), or CrO₃ (0.5 mM), independently, for 30 min at room temperature. Controls were subjected to solution A at room temperature for 30 min. Heavy metals were then removed, and oocytes were rinsed three times with (control) solution A at room temperature, then subjected to a standard ³⁵ uptake with 0.1 mM K₂SO₄ (substrate concentration) at room temperature for 30 min (as described above).

Data presentation and statistics. All experiments were repeated at least three times with different batches of oocytes. Each single point on the graphs is derived from a mean of 7-10 oocytes ± SE. Error bars not visible on graphs are smaller than the symbol used for that point. Statistical significance was tested using the paired student t-test, with P < 0.05 considered significant and any value greater considered nonsignificant (NS). The Michaelis-Menten equation was used to calculate K_m and V_max, using nonlinear regression.

	RESULTS

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Rat renal NaSi-1 (15) cRNA, when injected into X. laevis oocytes, leads to a strong (>50-fold) stimulation of Na-S_i cotransport (measured by ³⁵S uptake), compared with water injected oocytes (Fig. 1A). This stimulation was observed to be strongly inhibited by the heavy metals mercury (Hg²⁺) and lead (Pb²⁺) but not significantly by cadmium (Cd²⁺) and to a significant degree by the trace metal chromium (Cr⁶⁺), each at 0.1 mM final concentration, with the order of potency of NaSi-1 inhibition in descending order: Hg²⁺ > Pb²⁺ > Cr⁶⁺ > Cd²⁺ (Fig. 1). A similar order of potency of inhibition by these heavy metals was observed with the human renal NaPi-3 (13) cotransporter, when expressed in Xenopus oocytes, followed by ³²P uptake measurement in the presence of these same heavy metals (data not shown).

View larger version (12K): [in this window] [in a new window]   Fig. 1.   A: effects of various heavy metals on NaSi-1-induced transport in Xenopus oocytes. Oocytes were injected with 50 nl water or 50 nl water containing NaSi-1 cRNA (1 ng/oocyte). 35 uptake was performed on day 3 postinjection, with 0.1 mM K2SO4 (substrate concentration) at room temperature for 30 min with the heavy metals HgCl2, Pb(NO3)2, CdCl2, and CrO3 (each at 0.1 mM final concentration) added directly to the uptake medium. Uptake values (as %control) are HgCl2 (6.9 ± 0.2%), Pb(NO3)2 (40.9 ± 1.0%), CdCl2 (87.9 ± 11.2%), and CrO3 (60.6 ± 2.6%). B: reversibility of heavy metals on NaSi-1-induced transport in Xenopus oocytes. On day 3 postinjection, NaSi-1 cRNA (1 ng/oocyte)-injected oocytes were preincubated in the absence (control) or presence of heavy metals HgCl2 (0.1 mM), Pb(NO3)2 (0.1 mM), CdCl2 (0.5 mM), or CrO3 (0.5 mM), independently, for 30 min at room temperature, then followed by a 30 min 35 uptake with 0.1 mM K2SO4 (substrate concentration) at room temperature. Data are considered nonsignificant (NS) or significant at * P < 0.05.

To determine whether the heavy metals bound permanently to the NaSi-1 protein (and thus blocked transport activity), we tested the reversibility of these heavy metals to the NaSi-1 transporter by performing oocyte washout experiments (Fig. 1B). The effect of Hg²⁺ on NaSi-1 cotransport was not reversible, with the expressed NaSi-1 activity in oocytes being only 14.1 ± 1.7% on washout, whereas the effect of Pb²⁺, Cd²⁺, and Cr⁶⁺ on NaSi-1 cotransport was fully reversible (Fig. 1B).

Mercury interaction with the NaSi-1 transporter. To further characterize the inhibition of Hg²⁺ on NaSi-1 transport, we performed a series of transport kinetics in NaSi-1 cRNA-injected Xenopus oocytes. Hg²⁺ inhibition of NaSi-1 transport was both dose and time dependent (Fig. 2, A and B). Inhibition by Hg²⁺ was already observed at a concentration of 100 nM, and almost complete inhibition was observed at 100 µM (Fig. 2A). Half maximal inhibition (K_i) of Na-S_i cotransport by Hg²⁺ was determined as 7.1 ± 0.3 µM. Hg²⁺ inhibition of NaSi-1 transport was observed as early as 1 min (by 100 µM Hg²⁺) and continued in a time-dependent fashion up to 60 min (Fig. 2B); however, the slope did flatten after 10 min. NaSi-1-induced transport was not linear for the first 10 min (inset, Fig. 2B); thus all other assays were performed in the linear phase (30-min uptakes). The effect of Hg²⁺ not only decreased the maximal transport capacity (V_max) [49.74 ± 3.95 (control) vs. 38.00 ± 1.12 pmol S_i · oocyte¹ · min¹ (100 µM Hg²⁺); P < 0.01] but also the apparent affinity (K_m) of the NaSi-1 transporter for S_i [0.35 ± 0.12 (control) vs. 0.85 ± 0.08 mM (100 µM Hg²⁺); P < 0.05; Fig. 2C].

View larger version (19K): [in this window] [in a new window]   Fig. 2.   Transport characteristics of Hg2+ inhibition on NaSi-1-induced transport in Xenopus oocytes. A: dose-dependent inhibition by Hg2+. Oocytes were injected with 50 nl water () or 50 nl water containing NaSi-1 cRNA (1 ng/oocyte, ). 35 uptake was performed with 0.1 mM K2SO4 (substrate concentration) on day 3 postinjection at room temperature for 30 min in the presence of sodium (100 mM) and K2SO4 (0.1 mM), with various concentrations of HgCl2 (0-100 µM). Ki, half maximal inhibition. B: time-dependent inhibition by Hg2+. 35 uptake was performed with 0.1 mM K2SO4 (substrate concentration) on day 3 postinjection at room temperature for various lengths of incubation (1-60 min) in the presence or absence of 100 µM HgCl2. Data are presented as transport rate vs. time (inset) and as a percentage of control transport activity (NaSi-1 Hg/NaSi-1 control). C: sulfate interaction. 35 uptake was performed day 3 postinjection at room temperature for 35 min with various concentrations of K2SO4 (0.001-5 mM) with 100 µM HgCl2 () and no HgCl2 (control, ). Km, apparent affinity.

Lead interaction with the NaSi-1 transporter. Pb²⁺ inhibition of NaSi-1 transport was also dose and time dependent (Fig. 3, A and B). Significant inhibition was observed by 100 nM Pb²⁺ (and linear up to 100 µM; Fig. 3A), as early as 1 min (by 100 µM Pb²⁺), and increased with time up to 30 min, after which it remained constant up to 60 min (Fig. 3B). K_i of Na-S_i cotransport by Pb²⁺ was determined as 21.3 ± 1.8 µM. The effect of Pb²⁺ only decreased V_max [49.74 ± 3.95 (control) vs. 32.84 ± 3.09 pmol S_i · oocyte¹ · min¹ (100 µM Pb²⁺); P < 0.01], with no significant change in K_m of the NaSi-1 transporter for S_i [0.35 ± 0.12 mM (control) vs. 0.22 ± 0.09 mM (100 µM Pb²⁺); P > 0.05, Fig. 3C].

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Transport characteristics of Pb2+ inhibition on NaSi-1-induced transport in Xenopus oocytes. A: dose-dependent inhibition by Hg2+. Oocytes were injected with 50 nl water () or 50 nl water containing NaSi-1 cRNA (1 ng/oocyte, ). 35 uptake was performed with 0.1 mM K2SO4 (substrate concentration) on day 3 postinjection at room temperature for 30 min with various concentrations of Pb(NO3)2 (0-100 µM). B: time-dependent inhibition by Pb2+. 35 uptake was performed with 0.1 mM K2SO4 (substrate concentration) on day 3 postinjection at room temperature for various lengths of incubation (1-60 min) in the presence or absence of 100 µM Pb(NO3)2. Data are presented as transport rate vs. time (inset) and as a percentage of control transport activity (NaSi-1 Pb/NaSi-1 control). C: sulfate interaction. 35 uptake was performed day 3 postinjection at room temperature for 35 min with various concentrations of K2SO4 (0.001-5 mM), with 100 µM Pb(NO3)2 () and no Pb(NO3)2 (control, ).

Cadmium interaction with the NaSi-1 transporter. Cd²⁺ interaction with NaSi-1 transport was observed to be weakly dose dependent (from 1 to 1,000 µM; Fig. 4A); thus no K_i could be calculated for the interaction of Cd²⁺ with the NaSi-1 transporter. With 1 mM CdCl₂, inhibition of NaSi-1 transport could be observed after 1 min and increased up to 5 min, whereas afterward, there was very little additional inhibition (Fig. 4B). As with Pb²⁺, Cd²⁺ led to a decreased V_max [49.74 ± 3.95 (control) vs. 34.72 ± 2.37 pmol S_i · oocyte¹ · min¹ (500 µM Cd²⁺); P < 0.01] but no significant change in the K_m of the NaSi-1 transporter for S_i [0.35 ± 0.12 mM (control) vs. 0.48 ± 0.13 mM (500 µM Cd²⁺); P > 0.05, Fig. 4C].

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Transport characteristics of Cd2+ inhibition on NaSi-1-induced transport in Xenopus oocytes. A: dose-dependent inhibition by Cd2+. Oocytes were injected with 50 nl water () or 50 nl water containing NaSi-1 cRNA (1 ng/oocyte, ). 35 uptake was performed with 0.1 mM K2SO4 (substrate concentration) on day 3 postinjection at room temperature for 30 min with various concentrations of CdCl2 (0-1,000 µM). B: time-dependent inhibition by Cd2+. 35 uptake was performed with 0.1 mM K2SO4 (substrate concentration) on day 3 postinjection at room temperature for various lengths of incubation (1-60 min) in the presence or absence of 1 mM CdCl2. Data are presented as transport rate vs. time (inset) and as a percentage of control transport activity (NaSi-1 Cd/NaSi-1 control). C: sulfate interaction. 35 uptake was performed day 3 postinjection at room temperature for 35 min with various concentrations of K2SO4 (0.001-5 mM) with 500 µM CdCl2 () and no CdCl2 (control, ).

Chromium interaction with the NaSi-1 transporter. Cr⁶⁺ inhibition of NaSi-1 transport was both dose and time dependent (Fig. 5, A and B). Significant inhibition by Cr⁶⁺ was observed at a concentration of 50 µM (Fig. 5A), with K_i of Na-S_i cotransport by Cr⁶⁺ calculated as 0.52 ± 0.02 mM. Strong inhibition was observed after only 1 min (by 0.5mM Cr⁶⁺), which then flattened off and gradually continued until maximum inhibition was reached after 60 min (Fig. 5B). The effect of Cr⁶⁺ not only decreased the V_max [49.74 ± 3.95 (control) vs. 39.76 ± 3.60 pmol S_i · oocyte¹ · min¹ (500 µM Cr⁶⁺); P < 0.05] but also strongly reduced the K_m of the NaSi-1 transporter for S_i [0.35 ± 0.12 mM (control) vs. 2.42 ± 0.48 mM (100 µM Cr⁶⁺); P < 0.05, Fig. 5C].

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Transport characteristics of Cr6+ inhibition on NaSi-1-induced transport in Xenopus oocytes. A: dose-dependent inhibition by Cr6+. Oocytes were injected with 50 nl water () or 50 nl water containing NaSi-1 cRNA (1 ng/oocyte, ). 35 uptake was performed with 0.1 mM K2SO4 (substrate concentration) on day 3 postinjection at room temperature for 30 min with various concentrations of CrO3 (0-1,000 µM). B: time-dependent inhibition by Cr6+. 35 uptake was performed with 0.1 mM K2SO4 (substrate concentration) on day 3 postinjection at room temperature for various lengths of incubation (1-60 min) in the presence or absence of 0.5 mM CrO3. Data are presented as transport rate vs. time (inset) and as a percentage of control transport activity (NaSi-1 Cr/NaSi-1 control). C: sulfate interaction. 35 uptake was performed day 3 postinjection at room temperature for 35 min with various concentrations of K2SO4 (0.001-5 mM) with 500 µM CrO3 () and no CrO3 (control, ).

	DISCUSSION

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Since the kidney is one of the primary organs involved in excretion of metals, it is also the site for heavy metal intoxication. To date, very few studies have looked at the interaction of heavy metals on specific renal transporters. This is the first study that examines the interaction of heavy metals with the proximal tubular brush border Na-S_i cotransporter, NaSi-1. As we have previously demonstrated for the amino acid transporter, rBAT (24), and as was recently shown for the human Na-P_i cotransporter, NaPi-3 (23), heavy metals have the ability to inhibit the function of cloned renal transporters by mechanisms yet to be determined. In this study, we show that the Na-S_i cotransporter NaSi-1 is inhibited by heavy metals Hg²⁺, Pb²⁺, Cd²⁺, and the trace metal Cr⁶⁺.

Hg²⁺ showed a very strong inhibition of NaSi-1-induced Na-S_i cotransport in Xenopus oocytes (K_{i Hg}, 7.1 ± 0.3 µM) by reducing both the V_max and K_m for S_i: the reduction in NaSi-1 K_m for S_i was over twofold by Hg²⁺, suggesting that the metal may be interfering with Na-S_i cotransport by competitive inhibition. The Hg²⁺ inhibition of NaSi-1 cotransporter activity was not reversible, as previously described for the interaction of Hg²⁺, with both the NaPi-3 (23) and rBAT (24) transporters. NaPi-3 was also strongly inhibited by Hg²⁺ (data not shown; Ref. 23); however, only its V_max was altered, with no apparent change in K_m for P_i (23). This may suggest that indeed Hg²⁺ interaction with the NaSi-1 transporter is different than its interaction with the NaPi-3 transporter, in that Hg²⁺ could be competing for the S_i binding site on NaSi-1 and not for the P_i site on NaPi-3. At this stage, this is only speculation, since the S_i binding site on NaSi-1 protein has not yet been determined; however, we have recent evidence suggesting that it is not located within the first four transmembrane domains of the NaSi-1 protein (Pajor and Markovich; unpublished observations). A second mechanism by which Hg²⁺ may be inhibiting NaSi-1 transport is by oxidation: the NaSi-1 protein has several intracellularly located cysteine residues [at positions 318, 329, and 449; predicted by the hydropathy plot (Ref. 15)], which may have their thiol groups oxidized by the metal Hg²⁺, as postulated for the mechanism of Hg²⁺ inhibition of the NaPi-3 (23) and rBAT (24) transporters. In addition, since Hg²⁺ has been shown to interact with intracellular sulfhydryl groups (7, 8), this mechanism may also be responsible for inhibition of NaSi-1 transport, as postulated for rBAT (24). There may be a further possibility: since Hg²⁺ has been shown to block Na⁺-K⁺-adenosinetriphosphatase (Na⁺-K⁺-ATPase) activity by ligand binding (2), it could be that Hg²⁺ is indirectly inhibiting NaSi-1 transport by blocking the activity of the endogenous Na⁺-K⁺-ATPase pump and, as a consequence, inhibiting Na/S_i uptake into the oocyte. This type of inhibition was shown previously for the Na-P_i cotransporter, which was blocked indirectly via the inhibition of the Na⁺-K⁺-ATPase by hydrogen peroxide in LLC-PK₁ cells (1).

Lead interaction with the NaSi-1 transporter was different than Hg²⁺. Despite its strong inhibition (K_{i Pb}, 21.3 ± 1.8 µM), only its V_max was altered, with no apparent change in K_m for S_i, suggesting that the inhibition may be via a noncompetitive mechanism or allosteric fashion. This effect was analogous to the inhibitory effect of lead on rBAT transport (24) but was in contrast to its effect on NaPi-3 transport, in which Pb²⁺ decreased both V_max and K_m for P_i on the NaPi-3 transporter (23). The inhibition of NaSi-1 cotransporter activity by Pb²⁺ was fully reversible, as previously demonstrated with the NaPi-3 (23) and rBAT (24) transporters. This would suggest that Pb²⁺ may be interacting with NaSi-1 at an extracellular site. As with Hg²⁺, Pb²⁺ has been shown to interact with sulfhydryl groups of proteins (22), so this may be its mode of inhibition on NaSi-1 transport. Since Pb²⁺ does not affect NaSi-1 K_m for S_i, whereas Hg²⁺ does, this may suggest that mechanism of inhibition is different for the two metals and that their binding sites on NaSi-1 may be distinct. Another way that Pb²⁺ may be inhibiting NaSi-1 cotransport is by altering fatty acid composition of the plasma membrane by lipid peroxidation (6), leading to leakage of S_i out of the cell. Because lipid peroxidation releases free radicals, such as OH and H₂O₂, it may be that newly released hydrogen peroxide is blocking the Na⁺-K⁺-ATPase pump and thereby inhibiting NaSi-1 function, as demonstrated with the Na-P_i cotransporter (1).

Cadmium showed relatively little inhibiton of NaSi-1 transport, compared with the effects by Hg²⁺ and Pb²⁺: near millimolar quantities were needed to get inhibition of uptake. No K_i could be calculated for Cd²⁺ interaction with NaSi-1 transport, and the K_m for S_i was not significantly different than the control condition. Only a decrease in V_max was observed (using 0.5 mM CdCl₂), suggesting that its interaction with NaSi-1 may be via a noncompetitive mechanism, as postulated for the interaction with Pb²⁺. Cadmium inhibition of NaSi-1 cotransporter activity was fully reversible, as previously shown for Cd²⁺ with the NaPi-3 transporter (23). As with Hg²⁺ and Pb²⁺, Cd²⁺ has been shown to interact with sulfhydryl groups (20), and this may be the mechanism of inhibition of NaSi-1 transport. A recent study looking at brush-border membrane Na-S_i cotransport in rats treated chronically (14 days) with Cd²⁺ showed no inhibition of Na-S_i cotransport but strongly impaired Na-P_i cotransport compared with control rats (9). The lack of Cd²⁺ inhibition on Na-S_i cotransport in that study (in contrast to our present study) may be due to the different approach used to study the interaction between Cd²⁺ and Na-S_i cotransport. Our study looked at the interaction of Cd²⁺ on NaSi-1 protein expression in Xenopus oocytes, whereas the other study measured ³⁵S uptake in renal cortical brush-border membrane vesicles (BBMV) isolated from control rats and rats chronically treated with Cd²⁺ (9). The lack of an effect on ³⁵S uptake in BBMV from Cd²⁺-treated rats (compared with controls) may suggest that long-term exposure of Cd²⁺ may not lead to alterations in the overall number (or function) of sulfate transporters in the proximal tubules of rats. In our study, cadmium has a distinct effect on NaSi-1 protein expression when measured in a heterologous expression system (Xenopus oocytes), and this effect maybe more pronounced or more accurately quantitated when studying an individual protein (NaSi-1) than when studying a population of transporters present in renal cortical BBMV (9).

Cadmium showed no inhibition of rBAT amino acid transport in Xenopus oocytes (24), whereas it showed a dose-dependent inhibition of NaPi-3 transport [with maximal inhibition of P_i-induced current of 27.5 ± 2.6% with 1 mM Cd²⁺ (Ref. 23)]. With radiotracer uptake studies, our experiments show that 0.1 mM CdCl₂ produces ~10% inhibition of both NaSi-1 and NaPi-3 (data not shown)-induced transport activities, and 0.5 mM CdCl₂ shows a 30 ± 3% inhibition of NaSi-1 transport. This suggests that NaSi-1 transporter is inhibited by Cd²⁺ to a similar degree as the NaPi-3 transporter (data not shown; Ref. 23), most probably by a noncompetitive interaction. This is of particular importance, since Cd²⁺ is an occupational and environmental hazard having strong nephrogenic actions (20).

Chromium inhibition of the NaSi-1 transporter reduced both its V_max and K_m for S_i: the reduction in NaSi-1 K_m for S_i was nearly sevenfold by Cr⁶⁺, suggesting that it may be strongly competing for the S_i binding site on the NaSi-1 protein. This is in close agreement with the K_i of Cr⁶⁺ on Na-S_i cotransport being very close to the K_m value for NaSi-1 interaction with S_i. As with Pb²⁺ and Cd²⁺, the inhibition of NaSi-1 cotransporter by Cr⁶⁺ was fully reversible on washout, suggesting that the interaction is at an extracellular binding site on NaSi-1 protein. Chromium (VI) oxide forms an oxyanion (C) that has been reported to mimic the sulfate () anion, which is believed to cross plasma membranes using S_i transport systems (5, 12, 25). We have shown that chromium oxide can strongly inhibit NaSi-1 transport in Xenopus oocytes and believe that its mechanism of inhibition is by competitive binding for the S_i binding site on NaSi-1. This is of very significant importance, since chromium is an essential trace metal necessary for certain physiological functions, e.g., glucose metabolism (17). Overexposure to chromium has led to (among other things) renal tubular necrosis (11), and thus it is of special importance in determining the transport systems involved in its uptake in kidneys. NaSi-1 may play a key role in renal chromium intoxication, as well as in the degeneration of tubular function by other metals, e.g., mercury, lead, and cadmium, as analyzed in this study.

In summary, we have shown that mercury, lead, cadmium, and chromium can all inhibit NaSi-1-induced Na-S_i cotransport. This is of pathophysiological relevance, since the renal NaSi-1 transporter is essential for maintaining sulfate homeostasis and serum S_i levels. These metals are known to produce cell injury in the kidneys (and other organs) and may have a significant involvement during nephrotoxicity, which may be due to accumulation of the metals after systemic application (18, 26). Heavy metals have also been shown to impair the active transport of glucose and other substrates in the intestine (10). Since the NaSi-1 transporter is also localized in the gut and plays an important role in sulfate absorption in the small intestine (15, 19), its ability to transport sulfate across intestinal enterocytes would also be impaired by the heavy metals tested above. This is the first study showing heavy metal inhibition of the cloned NaSi-1 protein, involved in renal and small intestinal Na/S_i (re)absorption, which, as a consequence, may be responsible for the sulfaturia or Fanconi-type syndrome following heavy metal intoxication.