Introduction

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The haloalkane cysteine conjugate S-(1-chloro-1,2,2-trifluoroethyl)-L-cysteine (CTFC) produces cell injury and death to suspensions of rabbit renal proximal tubules (RPTs) (Groves, 1991; Groves et al., 1991a). Differences were found to exist in the mechanism by which the haloalkane cysteine conjugates CTFC and S-(1,1,2,2-tetrafluoroethyl)-L-cysteine (TFEC) produce cell injury in suspensions of rabbit RPT compared with the haloalkene cysteine conjugates S-(1,2-dichlorovinyl)-L-cysteine (DCVC) and S-(1,2,3,4,4-pentachlorobutadienyl)-L-cysteine (PCBC) (Groves, 1991; Groves et al., 1991a,b, 1993). In spite of their structural similarities, aminooxyacetic acid, the inhibitor of cysteine conjugate -lyase, completely prevented TFEC- and CTFC-induced cytotoxicity to tubule suspensions. However, aminooxyacetic acid had no effect on DCVC-induced toxicity and delayed but did not prevent PCBC-induced toxicity to tubule suspensions (Groves et al., 1991a,b, 1993). The differences in the response of the renal cell to these toxicants may exist at the level of transport as well. The ability of these toxicants to cross the renal membrane and enter the renal cell appears to be paramount to the production of cytotoxicity. Thus, the presence of various transport pathways within the RPT for toxicant uptake and accumulation may play an important role in the target-organ selectivity of cysteine conjugates.

Cysteine conjugates enter the renal cell by various transport pathways in the RPT membrane (Schaeffer and Stevens, 1987a,b; Lash and Anders, 1989; Mertens et al., 1990; Chakrabarti et al., 1991). One such pathway, the organic anion transport system, serves as a mechanism to transport and accumulate various exogenous and potentially toxic substances by the RPT cell. The peritubular membrane organic anion transporter mediates the transport of the cysteine conjugate DCVC in rabbit S2 segments (Dantzler et al., 1998). In contrast, apical DCVC transport in rabbit and rat renal cortical brush-border membrane vesicles (BBMVs), as well as in LLC-PK1 cells, involves the neutral amino acid transporter (Schaeffer and Stevens, 1987a,b; Wright and Wunz, 1998). The ability of CTFC to block the uptake of DCVC in LLC-PK1 cells or rat RPT cells suggests that these cysteine conjugates may interact with a common transport pathway (Schaeffer and Stevens, 1987a; Lash and Anders, 1989). However, studies have not demonstrated whether the pathways that transport DCVC also transport CTFC.

Although the presence of probenecid blocked the toxicity of DCVC in rat RPT cells (Lash and Anders, 1986), no protective effect of probenecid on CTFC- and TFEC-induced cytotoxicity in this model was detected (Boogaard et al., 1989). In contrast, probenecid was reported to reduce the cytotoxicity associated with exposure of rat RPT cells to the mercapturic acid conjugates of CTFC and TFEC (Boogaard et al., 1989). Thus, the differences in the response of the renal cell to these toxicants may also exist at the level of transport. The objective of this study was to examine the interaction of the cysteine conjugate CTFC with the organic anion and neutral amino acid transport pathways in the basolateral and apical membranes of the RPT cell. The model systems used in the study were primary cultures of rabbit RPT cells grown on permeable and impermeable membrane surfaces and suspensions of rabbit RPT.

	Experimental Procedures

Top Abstract Introduction Experimental Procedures Results Discussion References

Animals and Materials. New Zealand White rabbits of either sex were purchased from Big D Rabbitry (Dade City, FL). Unlabeled phenylalanine, [³H]phenylalanine (50 Ci/mmol), p-aminohippurate (PAH), and fluorescein were purchased from Sigma Chemical Co. (St. Louis, MO). S-(1-Chloro-1,2,2-trifluoroethyl)-L-cysteine was a generous gift from Dr. Stephen Hasal (Tucson, AZ). All other chemicals were purchased from standard sources as reported previously (Groves et al., 1994, 1999) and were of the highest quality available.

Isolation of Proximal Tubule Suspensions and Culture Conditions. An in vitro perfusion with iron oxide as described previously (Groves and Schnellmann, 1996) was used for the isolation and purification of rabbit RPT. For transport studies with suspensions of rabbit RPT, the final tubule pellet was resuspended at a protein concentration of 1 mg/ml. Tubules were resuspended in an incubation medium containing: 110 mM NaCl, 25 mM NaHCO₃, 5 mM KCl, 2 mM NaH₂PO₄, 1 mM MgSO₄, 1.8 mM CaCl₂, 10 mM sodium acetate, 8.3 mM D-glucose, 5 mM alanine, 0.9 mM glycine, 1.5 mM lactate, 1 mM malate, and 1 mM sodium citrate (pH 7.4, 295 mOsm/kg). Tubular protein was measured with a Bio-Rad Laboratories, Inc. (Richmond, CA) protein assay with a BSA standard.

Measurement of Paracellular Inulin Flux in Primary Cultures. The movement of fluorescein isothiocyanate-inulin (FL-I) diffusion across the monolayer was measured to evaluate monolayer permeability. Culture medium was removed, incubation medium containing 100 µM (final concentration) FL-I was added to the basal or apical compartments, and incubation medium alone was added to the opposite compartment. Cultures were incubated at 37°C as described above. At timed intervals up to 2 h, aliquots (100 µl) were removed from both compartments, and FL-I fluorescence (filter pair: 485 nm excitation, 538 nm emission) determined with a Molecular Devices (Sunnyvale, CA) F_max fluorescence microplate reader.

Measurement of Transport in Suspensions of RPT. Tubule suspensions (1 mg/ml) were preincubated in Erlenmeyer flasks for 15 min at 37°C and were gassed with 95% O₂/5% CO₂. To measure tubular efflux, fluorescein (4 µM final concentration) was added to the suspension and incubated for 5 min. After the desired incubation, 0.25-ml aliquots of the suspension were removed and added to a 15-ml polypropylene tube containing 5 ml of ice-cold incubation buffer to stop uptake. Samples were immediately centrifuged for ~30 s at 1480g to pellet the tubules. The supernatant fraction was aspirated, and the pellet was rinsed a second time. The samples were frozen for ~12 h at 20°C to lyse the cells and subsequently thawed by adding 1.5 ml of ddH₂O. The pellet was vortexed for ~60 s and centrifuged at 1480g for 5 min. Aliquots (100 µl) were transferred to a 96-well plate, and fluorescein fluorescence was then measured with an F_max fluorescence microplate reader (filter pair: 485 nm excitation, 538 nm emission). The amount of accumulated fluorescence represented time 0 fluorescein content at the start of the efflux period. A 0.25-ml aliquot of the tubule suspension was transferred to a tube containing 2.5 ml of either incubation medium alone or 1 mM PAH or CTFC. The incubation was continued for 1 min, during which fluorescein was typically lost from the tubules to the bath. At the end of the efflux period, 5 ml of ice-cold incubation buffer was added to terminate the reaction, and the tubule pellets were prepared for measuring intracellular fluorescence as described.

Measurement of Transport in Primary Cultures. The interaction of CTFC with the transepithelial transport of fluorescein or [³H]phenylalanine was studied in confluent cultures (day 4-5) grown on permeable membranes. Confluence was assessed by phase-contrast microscopy and measurement of the diffusion of fluorescein-inulin as described by Groves et al. (1999). For basal-to-apical transepithelial organic anion flux studies, 2.5 ml of an incubation medium containing 4 µM 4 µM fluorescein in the presence or absence of 1 mM CTFC or 2.5 mM PAH was added to the basal compartment, and incubation buffer alone was added to the trans-compartment. Cultures were incubated in a humidified incubator under a 95% air/5% CO₂ atmosphere at 37°C and were constantly swirled on an orbital shaker (80-85 rpm). At timed intervals up to 2 h, 100-µl aliquots of the incubation medium were removed from the apical and basal compartments and transferred to an F_max fluorescence microplate reader for measurement of fluorescein fluorescence. To measure intracellular accumulation of fluorescein at the end of the incubation for transepithelial flux measurements, cultures were washed twice with ice-cold incubation buffer, and the membranes were removed and transferred to scintillation vials. Cells were frozen at 20°C for 12 h and lysed by thawing in the presence of 1 ml of ddH₂O. A 100-µl aliquot was removed, and the intracellular accumulation of fluorescein by the cultures was determined with an F_max microplate reader as described. The autofluorescence of control cultures not incubated with fluorescein or CTFC was subtracted from the total fluorescence of treated cultures.

Calculations of Fluorescein and Phenylalanine Clearance. The transepithelial flux of fluorescein and [³H]phenylalanine in the presence and absence of CTFC, PAH, or phenylalanine was expressed as clearance units. The clearance is defined as the volume of medium that is totally cleared of the test substrate at a time t, normalized to the surface area (4.7 cm²) of the permeable membrane. The transepithelial movement of substrate from the basal-to-apical or apical-to-basal compartments was calculated and expressed as clearance by the following equations:

(1)

where C_b-a is the clearance from the basal to apical side, F_a is the arbitrary fluorescent units per milliliter or radioactivity (dpm per milliliter) measured in the apical compartment, V_a is the volume of incubation medium in the apical compartment, and F_b is arbitrary fluorescent units per milliliter or radioactivity (dpm per milliliter) measured in the basal compartment.

(2)

where C_a-b is the clearance from the apical to basal side, F_b is arbitrary fluorescence units per milliliter or radioactivity (dpm per milliliter) measured in the basal compartment, V_b is the volume of incubation medium in the basal compartment, and F_a is arbitrary fluorescent units per milliliter or radioactivity (dpm per milliliter) measured in the apical compartment. The clearance data were normalized to the surface area of the Transwell culture membrane (4.7 cm²), and the results were expressed as microliters per centimeter squared. All data were corrected for paracellular leakage by subtracting inulin diffusion from total transport measured in separate cultures at each examined time point.

Statistics. Data are presented as means ± S.E. Each preparation of tubules from a single rabbit represented a separate experiment. Data from three or four separate experiments were compared for statistical significance with a paired t test, ANOVA, and a posttest with Fisher's protected least-significant-difference method and a value of p < .05.

	Results

Top Abstract Introduction Experimental Procedures Results Discussion References

Inhibition of Basolateral PAH Uptake by CTFC. As shown in Fig. 1, increasing concentrations of CTFC progressively reduced the uptake of [³H]PAH with maximal inhibition produced by CTFC concentrations >0.5 mM. However, the highest concentrations of CTFC did not completely block the uptake of [³H]PAH, which is consistent with the presence of diffusion, and/or nonspecific binding. The inhibition of PAH uptake with only 1 min of exposure to various CTFC concentrations supports the conclusion that CTFC inhibited the carrier-mediated transport of PAH. The inhibition of [³H]PAH uptake by CTFC was described by the kinetics of competitive inhibition with the isotope-dilution procedure as described previously by Groves et al. (1994, 1995) and Dantzler et al. (1998). With the data in Fig. 1, the calculated inhibition constant (K_i) value for CTFC inhibition of [³H]PAH uptake was 105 ± 3 µM, which is similar to the K_m of 165 µM for basolateral PAH transport in S2 segments of rabbit RPT (Dantzler et al., 1995). Also with concentrations of DCVC similar to those used for CTFC in this study, Dantzler et al. (1995) measured a K_i of 86 µM for the inhibition of PAH uptake by DCVC in rabbit S2 segments. Because both PAH and DCVC are substrates for the organic anion transporter (Dantzler et al., 1995, 1998), these observations collectively suggest that CTFC also interacts with the organic anion transport pathway in the basolateral membrane.

View larger version (11K): [in this window] [in a new window]   Fig. 1.   Inhibition of [3H]PAH uptake by increasing concentrations of CTFC in rabbit RPT suspensions. The concentration of [3H]PAH was 25 nM. Uptakes were determined from 1-min incubations. Each point is the mean ± S.E. of triplicate measurements from a representative experiment. The line fit to the data was calculated and kinetic parameters derived with a nonlinear regression algorithm (Enzfitter; Biosoft, Cambridge, U.K.).

CTFC Interaction with Basal-to-Apical Flux and Basolateral Uptake of Fluorescein. Based on a K_i of 105 µM as measured for CTFC inhibition of PAH uptake, a concentration of 1 mM CTFC would be expected to reduce the basal-to-apical flux and intracellular accumulation of organic anion substrates by ~70% or more through a specific interaction with the basolateral organic anion transporter. As shown in Fig. 2, the flux of 4 µM fluorescein through ~2 h was reduced ~75 to 80% at all time points examined in the presence of 2.5 mM PAH or 1 mM CTFC. Because transepithelial flux involves substrate transport across two separate membranes, blocking its entry into the cell or its exit from the cell can reduce the transepithelial flux of fluorescein. The intracellular uptake and accumulation of fluorescein from the basal compartment, measured after 2 h of incubation, also was reduced ~70% by the addition of 1 mM CTFC or 2.5 mM PAH to the basal compartment (Fig. 3). The inhibition of the basal-to-apical flux and accumulation of fluorescein produced by 1 mM CTFC was not statistically different from the inhibition produced by 2.5 mM PAH, which suggests that CTFC, like PAH, reduced the basal-to-apical flux and uptake of fluorescein by blocking the basolateral membrane organic anion pathway. The basal-to-apical transepithelial flux of fluorescein was more than 10-fold greater than the reabsorptive flux (data not shown). Because the apical uptake of fluorescein was minimal, this event did not contribute significantly to organic anion transport in primary cultures and would likely play only a minor role in CTFC transport.

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Time course of the basal-to-apical transepithelial flux of fluorescein in the presence and absence of 2.5 mM PAH and 1 mM CTFC in 4- to 5-day-old monolayers of primary cultures of rabbit RPT cells grown on CoStar Transwell permeable membranes. The concentration of fluorescein added to the basal compartment was 4 µM. Each clearance value was corrected for the paracellular diffusion of fluorescein-inulin. Data are expressed as means ± S.E. (n = 3).

View larger version (24K): [in this window] [in a new window]   Fig. 3.   cis-Inhibition of the intracellular accumulation of fluorescein by 2.5 mM PAH and 1 mM CTFC in 4- to 5-day-old monolayers of primary cultures of rabbit RPT cells grown on CoStar Transwell permeable membranes. The intracellular accumulation of fluorescein was measured after a 2-h incubation in the presence and absence of PAH or CTFC. The concentration of fluorescein added to the basal compartment was 4 µM. Data are expressed as means ± S.E. (n = 3). *p < .05 versus control.

CTFC Interaction with Apical-to-Basal Flux and Apical Uptake of Phenylalanine. The flux of [³H]phenylalanine (0.1 µCi/ml) from the apical to the basal compartments increased with time and was linear for ~2 h. In the presence of 10 mM phenylalanine or 1 mM CTFC, the apical-to-basal flux of [³H]phenylalanine was reduced ~75 to 80% during the 2-h incubation (Fig. 4). To determine whether the inhibition of [³H]phenylalanine flux by CTFC was caused by inhibition of apical uptake or basal efflux, the effect of CTFC on the intracellular accumulation of [³H]phenylalanine was measured. The intracellular accumulation of [³H]phenylalanine from the apical compartment, measured after 2 h of incubation, also was reduced ~70 and 95% by the addition of 1 mM CTFC or 10 mM phenylalanine, respectively, to the apical compartment (Fig. 5). In contrast, the addition of 1 mM CTFC to the basal compartment had no effect on the intracellular uptake and accumulation of [³H]phenylalanine from the basal compartment by primary cultures, whereas accumulation was reduced ~95% by unlabeled phenylalanine (Fig. 5).

View larger version (14K): [in this window] [in a new window]   Fig. 4.   Time course of the apical-to-basal transepithelial flux of [3H]phenylalanine in the presence and absence of 10 mM phenylalanine and 1 mM CTFC in 4- to 5-day-old monolayers of primary cultures of rabbit RPT cells grown on CoStar Transwell permeable membranes. [3H]Phenylalanine was added to the apical compartment at 2 nM (0.1 µCi/ml) for transepithelial flux measurements. Each clearance value was corrected for the paracellular diffusion of fluorescein-inulin. Data are expressed as means ± S.E. (n = 3).

View larger version (29K): [in this window] [in a new window]   Fig. 5.   cis-Inhibition of the apical and basal intracellular accumulation of [3H]phenylalanine by 10 mM phenylalanine and 1 mM CTFC in 4- to 5-day-old monolayers of primary cultures of rabbit RPT cells grown on CoStar Transwell permeable membranes. The intracellular accumulation of [3H]phenylalanine was measured after a 2-h incubation in the presence and absence of phenylalanine or CTFC. [3H]Phenylalanine was added to the apical or basal compartments at 2 nM (0.1 µCi/ml) for uptake measurements. Data are expressed as means ± S.E. (n = 3). *p < .05 versus control.

Trans-Stimulation of Substrate Efflux by CTFC. The ability of a substrate to inhibit the uptake or transepithelial flux of a second substrate is merely indicative of some type of interaction of the inhibitor with a transporter. An indicator used frequently to determine whether two substrates share a common transport pathway is to demonstrate the ability of a substrate present on one face of the membrane to stimulate the flux of a second substrate from the opposite face (i.e., trans-membrane surface) of the membrane. Because rabbit RPT suspensions provide an excellent model for study of basolateral transport due to the presence of collapsed lumens (Groves et al., 1994; Groves and Wright, 1995; Dantzler et al., 1998), tubule suspensions were used for fluorescein efflux studies (Fig. 6). The ability of CTFC present in the external bath to trans-stimulate the basolateral membrane efflux of fluorescein was examined with fluorescein-preloaded suspensions of rabbit RPT (Fig. 6). When either 1 mM CTFC or 1 mM PAH was present on the trans-membrane surface, ~70% of the accumulated fluorescein was lost during the 1-min efflux period, compared to ~50% fluorescein loss from tubules incubated with buffer containing no substrate (0 trans). The efflux of fluorescein in the presence of 1 mM CTFC or 1 mM PAH was significantly greater than the 0-trans condition, which reflects a trans-stimulation of efflux by these substrates, presumably because of increased turnover of the carrier caused by the mediated uptake of the external substrate.

View larger version (39K): [in this window] [in a new window]   Fig. 6.   Effect of 1 mM PAH and 1 mM CTFC on the efflux of fluorescein from freshly isolated suspensions of rabbit renal proximal tubules. Tubules were loaded with 4 µM fluorescein for 5 min before measurement of 1-min efflux. Data are expressed as means ± S.E. (n = 3). *p < .05 versus 0-trans condition.

View larger version (39K): [in this window] [in a new window]   Fig. 7.   Effect of phenylalanine and CTFC on the efflux of [3H]phenylalanine from 5-day-old primary cultures of rabbit RPT cells grown in Petri dishes (apical membrane exposed). Primary cultures grown in Petri dishes (impermeable surface) were incubated with 2 nM [3H]phenylalanine (0.1 µCi/ml) for 30 min before measurement of 30-min efflux. Data are expressed as means ± S.E. (n = 3). *p < .05 versus 0-trans condition.

	Discussion

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In primary cultures of rabbit RPT cells, the cysteine conjugate CTFC interacts with two functionally and anatomically distinct transport processes located in the apical and basolateral membranes. The inhibition of the basal-to-apical and apical-to-basal transepithelial fluxes of fluorescein and phenylalanine, respectively, by CTFC were similar to those produced by saturating concentrations of PAH and phenylalanine, respectively. Thus, organic anion or amino acid transport across both poles of the renal cell is significantly reduced by the addition of CTFC to the basal or apical compartment and suggests that CTFC interacts with these transport systems.

Although the observed inhibition of transport could reflect an indirect effect on transport such as disruption of the Na gradient, the ability of increasing CTFC concentrations to progressively inhibit the 1-min uptake of PAH at the basolateral membrane in RPT suspensions also demonstrates a specific interaction of CTFC with the organic anion transporter. During such a short incubation with CTFC, toxicity is unlikely. However, this conjugate was a potent inhibitor of [³H]PAH uptake in RPT suspensions with a K_i of 105 µM, a value similar to the K_i of 86 µM for inhibition of PAH uptake by the cysteine conjugate DCVC, which is also a substrate organic anion transporter (Dantzler et al., 1995, 1998). The 1-min uptake of PAH in tubule suspensions was reduced ~75% or more in the presence of CTFC concentrations >0.5 mM. An interaction of 1 mM CTFC with the organic anion pathway would be expected to produce a similar inhibition of transepithelial fluorescein flux. As expected, 1 mM CTFC reduced the flux of the organic anion substrate fluorescein by ~75% at all time points examined. Thus, the inhibition of the basal-to-apical transepithelial flux of fluorescein by CTFC, respectively, is probably caused by the inhibition of carrier-mediated organic anion transport by CTFC during the experimental observations.

Transepithelial flux involves the movement of substrate across both poles of the renal cell. Thus, an inhibitor can block flux by preventing substrate movement across one or both poles. The intracellular accumulation of fluorescein was significantly reduced by the addition of PAH or CTFC to the basal compartment, which also suggests that CTFC interacts with basolateral organic anion transport to block fluorescein transepithelial flux. The inhibition produced by PAH was similar to the inhibition of fluorescein flux seen with CTFC and demonstrates that CTFC is a potent inhibitor of basolateral organic anion transport. Because fluorescein transport by the rabbit proximal tubule appears to be mediated solely by the PAH pathway (Sullivan et al., 1990; Sullivan and Grantham, 1992), the trans-stimulation of fluorescein efflux across the peritubular membrane of suspensions of rabbit RPT by CTFC suggests that CTFC is a substrate for the organic anion transporter. In contrast, the minimal reabsorptive flux and hence the apical uptake of fluorescein suggests that apical membrane accumulation of CTFC by the organic anion pathway appears to play little functional role in CTFC accumulation (data not shown). These results are in contrast to the findings from an earlier study in which organic anion transport was concluded to play no role in CTFC uptake, because probenecid failed to block CTFC toxicity to rat RPT cells (Boogaard et al., 1989). Because multiple pathways are involved in CTFC transport by the RPT cell, the lack of probenecid inhibition may simply reflect CTFC transport by a second pathway.

The uptake of the related cysteine conjugate DCVC by freshly isolated rat RPT cells or LLC-PK1 cells was reduced ~60 and 96% by 5 and 1 mM CTFC, respectively, which suggests these conjugates may share a common transporter in these model systems (Schaeffer and Stevens, 1987a; Lash and Anders, 1989). In rabbit S2 segments, PAH (5 mM) and probenecid (1 mM) reduced the basolateral membrane transport of DCVC ~60%. Also DCVC stimulated PAH efflux in this model, which indicates that this conjugate shares the organic anion pathway (Dantzler et al., 1998). Rat basolateral membrane vesicle transport of S-(1,2-phenylhydroxyethyl)-L-cysteine (PEC) also is mediated by the organic anion transporter (Chakrabarti et al., 1991). In contrast, 1 mM probenecid had no effect on basolateral DCVC uptake by suspensions of rat RPT (Zhang and Stevens, 1989). Thus, the organic anion transport system appears to mediate the basolateral uptake of various cysteine conjugates, but the presence of a second transport system that may be involved in cysteine conjugate uptake in the basolateral membrane cannot be excluded. Just as CTFC had no effect on the basolateral uptake of phenylalanine by rabbit RPT cells in this study, phenylalanine also had no effect on the basolateral DCVC uptake by rabbit S2 segments (Dantzler et al., 1998). These data suggest that the second basolateral membrane transport site may not be the phenylalanine neutral amino acid pathway.

Previous studies have shown that the apical transport of the cysteine conjugates DCVC and PCBC by proximal tubule cells and BBMV involves amino acid transport pathways (Schaeffer and Stevens, 1987a,b; Mertens et al., 1990; Wright and Wunz, 1998), whereas PEC accumulation is mediated by apical organic anion transport (Chakrabarti et al., 1991). The apical-to-basal flux of phenylalanine was significantly reduced by the addition of either unlabeled phenylalanine or CTFC to the apical compartment. A concentration of 10 mM phenylalanine produced similar inhibition to that produced by 1 mM CTFC, which suggests that CTFC is a potent inhibitor of phenylalanine flux. The decreased intracellular accumulation of phenylalanine indicates that CTFC primarily blocks the influx of phenylalanine into the cell to reduce transepithelial flux. Interestingly, as mentioned earlier, no effect of CTFC on the basolateral uptake of phenylalanine was observed. These data suggest that the type of interaction between CTFC and phenylalanine transport differs in the apical and basolateral membranes. Phenylalanine efflux across the apical membrane of cultures grown on impermeable membranes (plastic) was trans-stimulated by CTFC, which implies that these substrates share a common transport system in the apical membrane. The apical membrane transport of phenylalanine under physiological conditions appears to be mediated primarily by the Na-dependent neutral amino acid pathway (Silbernagl, 1988). The interactions of CTFC with phenylalanine transport are therefore probably due to competition at a common binding site on the apical membrane neutral amino acid carrier.

Amino acid transport also has been reported to play a role in the uptake of other cysteine conjugates. The uptake of PCBC reportedly involves Na⁺-independent system T amino acid transport in LLC-PK1 monolayers (Mertens et al., 1990). On the other hand, the transport of DCVC in LLC-PK1 cells is inhibited by substrates for the Na⁺-independent system L amino acid transporter and neutral amino acid substrates such as phenylalanine. Similar to LLC-PK1 cells, the Na⁺-dependent neutral amino acid pathway transports DCVC in both rat and rabbit BBMV (Schaeffer and Stevens, 1987a,b; Wright and Wunz, 1998). In contrast, the apical uptake of PEC in isolated rat BBMV was mediated by organic anion transport (Chakrabarti et al., 1991). Apical organic anion uptake was minimal in the current culture system, indicating that toxicant accumulation by this pathway also would be minimal. These observations show that variability, perhaps associated with structural differences, does seem to exist in the pathways by which various cysteine conjugates cross the renal membranes to enter the renal cell. However, some differences may also be associated with species and or model system differences. In spite of differences in mechanisms of toxicity in the rabbit RPT, similarities do exist between the transport pathways by which DCVC and CTFC access the RPT from the apical and basolateral compartments.

The haloalkane cysteine conjugate CTFC is biotransformed by the enzyme cysteine conjugate -lyase present within the cytoplasm and mitochondria of renal cells (Hayden and Stevens, 1990; Groves, 1991; Groves et al., 1991a, 1993). The entry of this toxicant into the RPT cell is therefore paramount to the production of injury. Our study demonstrates that CTFC uptake is mediated by both the neutral amino acid and organic anion transport pathways in the apical and basolateral membranes within the intact RPT cell. Any CTFC present in the systemic circulation may access the RPT by glomerular filtration and/or the peritubular capillaries in vivo to result in luminal or peritubular exposure. These pathways may also serve as avenues for the efflux of CTFC. However, further studies are required to understand the role of these pathways in the overall influx, efflux, and intracellular accumulation of CTFC within the RPT cell, as well as the role other pathways may play in transport.

These data illustrate the usage of primary cultures grown on permeable supports to examine within one model system the interaction and transport of nephrotoxic cysteine conjugates with transport pathways in the apical and basolateral membrane of the RPT cell. The neutral amino acid transport pathway in the luminal membrane and the organic anion transporter in the basolateral membrane both appear to play a role in CTFC transport. These pathways may be critical to the entrance of cysteine conjugates to the renal cell and should be considered a prime site at which to concentrate efforts directed at the establishment of therapeutic protocols to prevent toxicity.