Introduction

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The nephrotoxic cysteine S-conjugates (e.g., DCVC) and their N-acetyl derivatives must enter the proximal tubule cells to produce nephrotoxicity (Dantzler and Wright, 1997). Although entry of filtered toxicant could occur across the luminal membrane, entry from the blood across the basolateral membrane could also be of considerable importance. However, the pathway or pathways for this basolateral entry are not yet clearly established. Early studies on rat kidneys in vivo (Lock and Ishmael, 1985) and rat proximal tubule cells in vitro (Lash and Anders, 1986) showing reduction in toxicity of cysteine conjugates with the administration of probenecid, a well-known inhibitor of the basolateral organic anion transport pathway, suggested that the nephrotoxic cysteine conjugates might enter via this pathway. However, the in vivo data are also compatible with entry of only the negatively charged N-acetyl derivative of the cysteine conjugate, not the zwitterionic cysteine conjugate itself, via the organic anion transport pathway and this was the interpretation of the authors (Lock and Ismael, 1985). Moreover, later work by Zhang and Stevens (1989) with rat proximal tubule suspensions in vitro and by Wolfgang et al. (1989a) with rabbit renal cortical slices in vitro indicated that probenecid only inhibited the basolateral uptake of N-acetyl-DCVC, not DCVC itself.

In contrast, our previous study (Dantzler, et al., 1995) on the kinetics of the interaction of DCVC and N-acetyl-DCVC with the transport of PAH, the prototype for substances transported by the basolateral organic anion transporter, in individual isolated S2 segments of rabbit proximal tubules provided different information. The data showed that DCVC cis-inhibited ³ and trans-stimulated basolateral PAH transport at least as well as N-acetyl-DCVC and suggested that both compounds were transported by the basolateral organic anion transport system.

In the present study, we examined directly the basolateral transport of radiolabeled DCVC, the inhibition of such transport by PAH, probenecid, L-phenylalanine and a nontoxic cysteine S-conjugate, and the effects of inhibition of transport on the toxicity produced by DCVC in isolated individual S2 segments of rabbit proximal tubules. The results indicate that in isolated rabbit tubules basolateral entry of DCVC can occur to a major extent via the organic anion transporter and that inhibition of such entry reduces toxicity to approximately the extent that transport is reduced.

	Methods

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Preparation of individual isolated tubules. New Zealand White rabbits were killed by i.v. injection of pentobarbital sodium. The kidneys were flushed via the renal artery with a solution containing 250 mM sucrose and 10 mM HEPES at pH 7.4. They were then removed gently and placed in chilled (4°C) medium for dissection. The standard Ringer solution used for dissecting and bathing the tubules contained (in mM): 110 NaCl, 25 NaHCO₃, 5.0 KCl, 2.0 NaH₂PO₄, 1.0 MgSO₄, 1.8 CaCl₂, 10 Na-acetate, 8.3 D-glucose, 5.0 L-alanine, 0.9 glycine, 1.5 lactate, 1 malate and 1 sodium citrate. This solution was gassed continuously with 95% O₂/5% CO₂ to maintain the pH at 7.4. For a few experiments involving the effects of preloading with KG, the bicarbonate was replaced with 25 mM HEPES and the solution was gassed with 100% O₂. The bathing medium also contained 3 g/100 ml neutral dextran (40,000 ± 3,000 mol wt) to approximate the plasma protein concentration. The osmolality of the solutions averaged 290 mOsmol/kg H₂O.

Determination of rate of DCVC uptake across basolateral membrane of individual nonperfused tubules. These experiments were performed in a manner similar to that used previously (Chatsudthipong and Dantzler, 1992; Groves et al., 1994). Briefly, an appropriate number of tubule segments (three for each condition to be studied) were teased from fresh renal tissue and maintained in oxygenated (95% O₂/5% CO₂), oil-covered Ringer at 4°C until the start of each experiment. For most experiments, the Ringer was warmed to 37°C for 5 min before the start of the uptake periods. This was done so that the tubules would be functioning at 37°C at the start of rapid uptake measurements. In some series of experiments, isolated tubules were preincubated for 20 to 30 min at 37°C in the absence (controls) or presence (experimentals) of KG (100 µM or 1 mM) to load the tubules with this substrate before the start of the uptake experiments. At the end of either the 5-min warming period or the 20- to 30-min preincubation period, each tubule was transferred to oil-covered incubation medium at 37°C containing [³⁵S]- DCVC (13-14 µM) without (control) or with (experimental) a putative inhibitor to measure uptake. This concentration of [³⁵S]DCVC was the minimum concentration consistent with obtaining adequate counts over short time periods in individual tubules. The incubations were stopped by transferring each tubule into 10 µl of 1 N NaOH. The concentration of intracellular [³⁵S]DCVC was determined as described below.

Determination of effects of DCVC on ⁸⁶Rb content of individual nonperfused tubules. Tubule segments (3-6 for each condition studied, depending on the nature of the experiment) were teased out and maintained as described above. Initially, they were incubated with ⁸⁶Rb (~20 cpm/nl) until a steady-state was reached. An extensive series of preliminary experiments showed that for all three tubule segments (S1, S2 and S3) a steady-state cell/bath ratio was always reached within 30 min and, therefore, this time period was used for all initial incubations. After this initial incubation period, each tubule was transferred as described above to oil-covered incubation medium containing ⁸⁶Rb alone, ⁸⁶Rb plus DCVC alone, or ⁸⁶RB plus DCVC and an inhibitor of DCVC uptake for an additional 30 min. The incubations were stopped by transferring each tubule into 10 µl of 3% TCA. The concentration of ⁸⁶Rb cpm in cell water relative to the concentration in the incubation medium was determined as described below.

Determination of cellular concentration of [³⁵S]DCVC and ⁸⁶Rb. The concentration of [³⁵S]DCVC or ⁸⁶Rb in the cells was determined at the end of the incubations by methods described in detail previously (Dantzler, 1973, 1974, Dantzler and Brokl, 1987, Dantzler et al., 1989). Briefly, each tubule was pulled through the oil layer covering the bathing medium to minimize transfer of extracellular fluid and was then immersed in 10 µl of an appropriate solution for extraction of the radioactivity. In the case of radiolabeled DCVC, this solution was 1 N NaOH. Although this extraction procedure completely dissolved the tubule, it was necessary to obtain all the radioactivity associated with DCVC bound inside the cells. Because the tubules dissolved in the extraction solution, they were photographed prior to incubation to determine their length. In the case of ⁸⁶Rb, the extraction solution used was 3% TCA, as in our previous studies (Dantzler, 1973, 1974; Dantzler, et al., 1989). These tubules, which were not dissolved by TCA, were weighed on a quartz fiber balance and the concentration of ⁸⁶Rb in the cell water was determined as described in detail previously (Dantzler, 1973, 1974, Dantzler et al., 1989).

Isolation of tubule suspensions. Suspensions of renal proximal tubules were isolated and purified from New Zealand White rabbits by an enzymatic (collagenase) procedure based on the method of Vinay et al. (1981) as modified by Groves et al. (1991). Briefly, this method involves collagenase digestion of the renal cortex followed by differential centrifugation in 50% Percoll. The tubule pellet was resuspended at a protein concentration of 1 mg/ml in the same Ringer solution as that used for the individual tubules. Tubule protein was measured using a Bio-Rad (Richmond, CA) protein assay with a -globulin standard.

Measurement of rate of DCVC uptake in tubule suspensions. Tubule suspensions (1 mg/ml) were preincubated in Erlenmeyer flasks for 15 min at 37°C or in an ice bath and were gassed with 95% O₂/5% CO₂. To measure the uptake of DCVC, [³⁵S]DCVC (4-5 µM) alone or with 2 mM unlabeled DCVC was added to the suspension. This concentration of [³⁵S]DCVC was the minimum concentration consistent with obtaining counts over short time periods in tubule suspensions. At timed intervals from 10 sec to 60 min, 0.5 ml aliquots of the suspension were removed and were added to a 15-ml polypropylene tube containing 5 ml of ice-cold incubation buffer to stop uptake. Samples were centrifuged for ~25 sec at 1480 × g to pellet the tubules. The supernatant fraction was aspirated, and the pellet was rinsed a second time. The pellet was dissolved in 1 N NaOH and aliquots were taken for liquid scintillation counting. Uptake measurements were based on triplicate determinations for each time point or experimental condition.

Determination of radioactivity. The activity of ³⁵S and ⁸⁶Rb was determined by counting in a liquid scintillation system. The scintillation fluid was EcoLite (ICN Biomedicals, Inc., Irvine, CA) and water in a ratio of 15:1 (v/v).

Chemicals. [³⁵S]DCVC (50-53 mCi mmol¹) was synthesized using the procedure described by Hayden et al. (1987). ⁸⁶Rb was purchased from NEN. Unlabeled DCVC and BTC were a gift from Dr. A. Jay Gandolfi, University of Arizona. All other chemicals were purchased from standard sources and were of the highest purity available.

Statistical analysis. Results are summarized as means ± S.E. The n value is the number of experiments. Differences between means were determined by Student's t test for paired or unpaired values, as appropriate. Differences were assumed to be significant when P < .05.

	Results

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Time course of DCVC uptake in tubule suspensions. The uptake of [³⁵S]DCVC (4-5 µM) increased with time, reaching a steady-state after about 30 min (fig. 1A). In tubule suspensions, measurement of DCVC uptake can be made readily over periods of seconds, permitting a reasonable estimate of initial rates of transport. Uptake of DCVC (4-5 µM) was linear over 5 min (fig. 1B). Therefore, the 1-min uptakes used in the subsequent kinetic analyses appear to be a reasonable approximation of the initial rate of transport. The presence of 2 mM unlabeled DCVC in the incubation medium inhibited the uptake of [³⁵S]DCVC (4-5 µM) by ~70% at 1 min, by ~85% at 5 min and ~90% at 60 min. Although 2 mM unlabeled DCVC is probably toxic after 15 min of exposure, inhibition of uptake with 5 min or less of exposure certainly supports the conclusion that accumulation of DCVC involves a carrier-mediated process.

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Time course of uptake of [35S]DCVC (4-5 µM) by suspensions of rabbit proximal renal tubules in the absence (solid circles) and presence (open circles) of 2 mM unlabeled DCVC. A, Full 60-min time course of uptake. B, First 5 min of the time course. Each point with vertical lines represents mean ± S.E. of triplicate measurements of three separate tubule preparations.

Kinetics of DCVC uptake in tubule suspensions. The kinetics of peritubular DCVC uptake were determined to help evaluate the transport process (fig. 2). Increasing concentrations of unlabeled DCVC in the incubation medium reduced the tubular 1-min accumulation of [³⁵S]DCVC. The highest concentration of unlabeled DCVC used failed to completely block the uptake of radiolabeled DCVC, a finding consistent with the presence of some passive diffusion and/or nonspecific binding of DCVC. Inhibition of uptake of radiolabeled DCVC by unlabeled DCVC was described by the kinetics of competitive inhibition using the isotope dilution procedure of Malo and Berteloot (1991) in which the data are plotted as the rate of uptake of the radiolabeled substrate, J, vs. the concentration of the unlabeled substrate, [S] (fig. 2). The kinetics of peritubular uptake can then be expressed by the following nonlinear regression equation:

(1)

where J is the rate of DCVC uptake into tubule suspensions from an extracellular concentration of radiolabeled DCVC, [T*], in the presence of unlabeled DCVC, [S]. The kinetic parameters, J_max, K_t and C are defined, respectively, as the maximal capacity of the carrier for DCVC, the concentration of DCVC at one-half J_max and a coefficient describing the nonsaturable accumulation of DCVC (passive diffusion and/or nonspecific binding). The J_max and K_t generated from the analysis of the kinetics of DCVC transport in three experiments were 2.3 ± 0.21 nmol mg of protein¹ min¹ and 283 ± 23.3 µM, respectively.

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Inhibition of [35S]DCVC (4-5 µM) uptake by increasing concentrations of unlabeled DCVC in rabbit proximal renal tubule suspensions. Uptakes were determined from 1-min incubations. Each point with vertical lines represents mean ± S.E. of triplicate measurements from three separate tubule preparations. The line fitted to the data was calculated from equation 1, and the kinetic parameters (Kt and Jmax) were derived using a nonlinear regression algorithm (Enzfitter, Biosoft).

Time course of DCVC uptake by individual isolated tubules. The uptake of [³⁵S]DCVC (13-14 µM) by isolated individual S2 segments of proximal tubules increased with time and was essentially linear up to 30 min (fig. 3). Because it was difficult in individual tubules to get enough radioactivity for accurate counting with low concentrations of radiolabeled DCVC at very short time periods and because the uptake was linear with time, we performed detailed studies of inhibition for a 5-min uptake period as an approximation of the initial rate.

View larger version (11K): [in this window] [in a new window]   Fig. 3.   Time course of uptake of [35S]DCVC (12-13 µM) by individual S2 segments of rabbit proximal renal tubules. Each point with vertical lines represents mean ± S.E. of three experiments (three tubules per experiment).

Effects of unlabeled DCVC, BTC, PAH, probenecid and L-phenylalanine on uptake of radiolabeled DCVC by individual isolated tubules. To examine the degree to which the basolateral transport of DCVC involved specific transport pathways, we examined the cis-inhibition of [³⁵S]DCVC uptake by unlabeled substances that might be substrates for the same transporter. We first began by determining the inhibitory effect of unlabeled DCVC itself. As shown in figure 4, 1 mM unlabeled DCVC (about 3.5 times the K_t for basolateral DCVC transport determined in tubule suspensions above) inhibited the uptake of [³⁵S]DCVC (13-14 µM) by ~80%. Similarly, as shown in figure 4, 1 mM BTC, a nontoxic cysteine S-conjugate, inhibited [³⁵S]DCVC (13-14 µM) by ~80%.

View larger version (39K): [in this window] [in a new window]   Fig. 4.   Effects of possible transport inhibitors on the uptake of [35S]DCVC (13-14 µM) by individual S2 segments of rabbit proximal renal tubules. Each bar and straight line represents mean ± S.E. of [35S]DCVC uptake at 5 min in the presence of the substance indicated below it. Number above each bar indicates number of experiments and asterisk indicates significant difference from control. Values are given as combined data in percents of control (as indicated on the ordinate) for convenience of comparing them in a single figure. However, each experiment was performed with its own controls and statistics were performed by paired analysis using the actual uptake values.

View larger version (29K): [in this window] [in a new window]   Fig. 5.   Effect of increasing concentrations of DCVC on 86Rb content in S1, S2 and S3 segments of rabbit renal proximal tubules. Key on figure indicates the tubule segment studied. Each bar and straight line represents mean ± S.E. for 86Rb content, as percent of control, in the presence of the concentration of DCVC given below it. Number above each bar indicates number of experiments and asterisk indicates significant difference from control. Values are given as percents of control for convenience of presentation in a single figure. However, each experiment with each segment at each DCVC concentration was performed with its own controls and statistical difference from controls was determined by paired analysis using the actual data.

Effect of preloading individual isolated tubules with KG on DCVC uptake. The transport of organic anions into renal tubule cells across the basolateral membrane apparently involves, as a final step, the countertransport of the organic anion (e.g., PAH) for KG (Pritchard, 1987, 1988; Shimada et al., 1987). We had previously shown that preloading single isolated S2 segments of rabbit proximal tubules with KG stimulated PAH uptake into the cells and net transepithelial transport (Chatsudthipong and Dantzler, 1992; Dantzler et al., 1995). Therefore, because our data suggested that DCVC was transported into the cells via the basolateral organic anion transporter, we examined the effect of preloading isolated tubules with KG on radiolabeled DCVC uptake in a few experiments. In one experiment in bicarbonate-buffered medium, preloading the tubules with 100 µM KG for 30 min (the protocol used in the previous studies on PAH transport) had no effect on the uptake of [³⁵S]DCVC (18 µM) at 1, 2 or 5 min (data not shown). Because we had observed in previous studies that the stimulatory effect of preloading with KG was twice as great in HEPES-buffered as in bicarbonate-buffered medium (Chatsudthipong and Dantzler, 1992; Dantzler et al., 1995), we switched to HEPES-buffered medium and examined the effects of preloading the tubules with either 100 µM or 1 mM KG on the uptake of [³⁵S]DCVC (18-19 µM) at 2, 5 and 15 min in three experiments. Even under these circumstances, there was no significant effect of KG preloading on DCVC uptake (average percent of control: 102 ± 6.5).

Effects of DCVC alone and in the presence of possible inhibitors of DCVC transport on cellular content of ⁸⁶Rb. As a measure of DCVC toxicity in S2 segments of renal proximal tubules, we determined its effect on ⁸⁶Rb content in lieu of K⁺ content. As indicated in "Methods," tubules were incubated for 30 min with ⁸⁶Rb and then incubated for an additional 30 min in the presence of both ⁸⁶Rb and DCVC. We first examined the effects of a number of concentrations of DCVC on ⁸⁶Rb content in proximal S1, S2 and S3 segments. As shown in figure 5, the ⁸⁶Rb content was significantly reduced with DCVC concentrations of 10 µM and higher in all three segments and there was no statistically significant difference in this effect between segments. The 10 µM concentration was approximately that used to study DCVC uptake and inhibition of uptake described above, and the effect of this concentration on ⁸⁶Rb content was great enough so that changes in the effect could be readily assessed. Therefore, we chose 10 µM DCVC as our toxic dose of the agent and determined its effect on ⁸⁶Rb content in the presence of various apparent inhibitors of its basolateral transport. Because there was no difference between the three proximal segments in the effect of DCVC on ⁸⁶Rb content and because our studies of DCVC transport and inhibition had been performed on S2 segments, we also performed all these studies on inhibition of toxicity with S2 segments.

View larger version (28K): [in this window] [in a new window]   Fig. 6.   Effect of 10 µM DCVC in absence and presence of 1 mM BTC on 86Rb content of S2 segments of rabbit renal proximal tubules. Each bar and line above it represents mean ± S.E. of 86Rb cell-to-bath (C/B) ratio (on ordinate) under the circumstances shown below the bar. Results are from five experiments. 86Rb C/B ratio is significantly (P < .05) higher in presence of BTC plus DCVC than in presence of DCVC alone.

View larger version (28K): [in this window] [in a new window]   Fig. 7.   Effect of 10 µM DCVC in absence and presence of 5 mM PAH on 86Rb content of S2 segments of rabbit renal proximal tubules. Symbols are the same as in figure 6. Results are from five experiments. 86Rb C/B ratio is significantly (P < .05) higher in presence of PAH plus DCVC than in presence of DCVC alone.

View larger version (26K): [in this window] [in a new window]   Fig. 8.   Effect of 10 µM DCVC in absence and presence of 1 mM probenecid on 86Rb content of S2 segments of rabbit renal proximal tubules. Symbols are the same as in figure 6. Results are from four experiments. 86Rb C/B ratio in presence of probenecid plus DCVC is significantly (P < .05) higher than in presence of DCVC alone but is not significantly different from control.

	Discussion

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Our study supports directly the suggestion from previous studies (Dantzler et al., 1995; Ullrich et al., 1990) that cysteine S-conjugates, not simply their negatively charged N-acetyl derivatives, are transported into rabbit renal proximal S2 tubules at the basolateral membrane to a significant extent by the organic anion transporter. Our previous work with these tubule segments indicated that DCVC effectively cis-inhibited PAH uptake and trans-stimulated PAH efflux across the basolateral membrane (Dantzler et al., 1995). In the present study, both PAH and probenecid, at concentrations about 50 times their K_t values for the organic anion transport system, cis-inhibited the basolateral transport of radiolabeled DCVC into the tubule cells to an equal extent. Although probenecid might have had effects other than inhibition of the organic anion transport system (e.g., possible interference with intracellular covalent binding of DCVC), PAH should only have interacted with the organic anion transport system. The failure of preloading with KG to stimulate DCVC uptake by individual tubules is the only observation in our study that does not further support transport of DCVC via the basolateral organic anion transporter. However, the degree of such stimulation in isolated tubules, even for PAH, can vary substantially among preparations, apparently depending on the level of metabolic production of KG.

These general results contrast with the earlier findings of Zhang and Stevens (1989) and Wolfgang et al. (1989a) in which probenecid failed to inhibit the uptake of radiolabeled DCVC by rat proximal tubule suspensions or rabbit renal cortical slices. It appeared possible that, in these earlier studies, the effect of probenecid was being masked by continued uptake of DCVC by an additional transport pathway. Indeed, in the study with rabbit renal cortical slices, L-phenylalanine significantly inhibited DCVC uptake at 5 min, suggesting that an amino acid transport pathway might be involved (Wolfgang et al., 1989a). In our study, the maximum cis-inhibition of uptake of radiolabeled DCVC by PAH or probenecid was ~70%. Some of the apparent remaining 30% certainly involves passive diffusion and/or nonspecific binding, but about ~10 to 15% (judging by the ~80-85% inhibition produced by DCVC itself and BTC) could involve a different transport pathway. However, in our present study with isolated individual rabbit proximal tubules, in contrast to the earlier study with rabbit kidney slices (Wolfgang et al., 1989a), there is no convincing evidence that a basolateral amino acid transporter is involved because there was no cis-inhibition of radiolabeled DCVC transport at 5 min even by an L-phenylalanine-to-DCVC concentration ratio more than seven times that used in the earlier study (Wolfgang et al., 1989a). Moreover, the kinetic data provide no evidence for more than one transporter in these isolated rabbit tubules.

The difference between our work with individual rabbit renal tubules and the previous work with rabbit kidney slices may result from the fact that the slices are more complex structures with numerous tubule types. It is also possible that in the studies with slices some of the DCVC accessed luminal amino acid transporters and that transport via these transporters was inhibited by L-phenylalanine but not by probenecid. How DCVC might have accessed the luminal membrane is not clear. However, studies with brush-border membrane vesicles from both rats and rabbits clearly indicate that DCVC is transported across this membrane by an apparent amino acid transporter that is significantly inhibited by L-phenylalanine (Schaeffer and Stevens, 1987; Wright et al., 1998, in press). In addition, the difference between the individual rabbit tubules and the rat tubule suspensions may reflect species differences in basolateral transporters between rabbit and rat tubules.

The kinetics of basolateral DCVC uptake were determined in proximal tubule suspensions because such data can be obtained much more easily with this preparation than with individual tubules. The tubule suspensions probably consisted largely of S1 segments whereas the individual tubules were S2 segments. However, previous studies on organic cation (TEA) and organic anion (PAH) transport gave comparable K_t values for these transporters in tubule suspensions and isolated individual S2 segments as well as in individual isolated segments from different regions of rabbit proximal tubules (Groves et al., 1994, 1998; Shpun et al., 1995).

In our present study, inhibiting basolateral uptake of DCVC in individual isolated rabbit tubules inhibited toxicity, as measured by ⁸⁶Rb loss. In the case of either nontoxic BTC or PAH the percent of inhibition of ⁸⁶Rb loss was very close to the percent of inhibition of DCVC uptake. These data indicate that preventing entry of DCVC into tubule cells via the basolateral organic anion transporter can reduce toxicity to the extent that DCVC entry is reduced. However, a concentration of probenecid that blocked DCVC entry to the same extent as PAH completely prevented toxicity (at least as measured by ⁸⁶Rb loss). An apparent inhibitory effect of probenecid on toxicity produced by DCVC has been described previously in studies on rat proximal tubule cells in vitro (Lash and Anders, 1986). These data were interpreted as indicating interference of probenecid with transport of DCVC via the basolateral organic anion transporter. In contrast, studies on DCVC transport and toxicity with rabbit renal cortical slices showed no effect of probenecid on DCVC transport or toxicity (Wolfgang et al., 1989a). Nevertheless, the present study using individual S2 segments of rabbit renal tubules has clearly shown that DCVC can enter the tubule cells to a significant extent via the basolateral organic anion transporter and that inhibition of this entry can reduce toxicity. These observations agree with the observations on rat renal tubule cells (Lock and Ismael, 1985; Lash and Anders, 1986) but not with those on rabbit kidney slices (Wolfgang et al., 1989a). Again, the differences from the study with rabbit kidney slices may reflect the greater structural specificity of individual tubules compared to kidney slices or probenecid-insensitive luminal DCVC uptake in kidney slices. The differences also may partially reflect the much higher concentrations of DCVC required to produce measurable toxicity in kidney slices (Wolfgang et al., 1989a).

In our study, probenecid appears to have an ability to reduce nephrotoxicity beyond its ability to reduce DCVC transport into the cells. The nature of this additional protective effect of probenecid is unknown, but it may reflect some antioxidant effects or interference with covalent binding of DCVC.

Our present study clearly demonstrates in individual S2 segments of rabbit proximal tubules that toxic cysteine S-conjugates (e.g., DCVC) can enter the cells via the general basolateral organic anion (PAH) pathway and that inhibition of such entry reduces toxicity to the extent that entry is inhibited. However, previous histological studies indicate that the S3 segment of rabbit and rat proximal tubules is more susceptible initially to DCVC-induced toxicity than the S2 segment (Hassall et al., 1983; Lock, 1988; Terracini and Parker, 1965; Wolfgang et al., 1989b, 1990). In our study, this was not the case. A given dose of DCVC had the same toxic effect in all three segments, at least as measured by ⁸⁶Rb loss during a 30-min exposure in vitro. This observation might not actually contradict the findings of the earlier studies with kidney slices, which also examined the effect of DCVC on intracellular K⁺ content (Wolfgang et al., 1990), if it had been possible in those studies to determine K⁺ loss immediately after a 30-min exposure and to localize the site of loss at that time.

In addition, with regard to segmental effects of DCVC, our previous kinetic data on PAH transport by isolated individual S2 and S3 segments of rabbit proximal tubules indicate that the basolateral organic anion transporter is the same in both segments but that there are fewer of them in the S3 segment than in the S2 segment (Dantzler et al., 1995; Shpun et al., 1995). Therefore, we suggest that DCVC can enter the S3 segments via the same transport system as in the S2 segments. Any differences between segments in the degree of toxicity occurring with time, as described by others (Wolfgang et al., 1990), would thus apparently relate to differences in the metabolic effects of DCVC once it has entered the cells.