Introduction

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DCVC is a member of the cysteine conjugate family of nephrotoxicants which are derived from the halogenated alkanes and alkenes (HAs), a broad class of environmental toxicants that have become increasingly prevalent in the workplace (U.S. Environmental Protection Agency, 1975; Birner et al., 1993). Treatment of animals with DCVC and other HAs typically results in the relatively rapid development of renal insufficiency, associated with elevated blood urea nitrogen and decreased creatinine clearance (Weinberg, 1993). Exposure to DCVC, in particular, results in lesions in the proximal tubule, initially in the S3 segment (Lock, 1988).

The cytotoxicity caused by cysteine conjugates is dependent on metabolic events that occur within target cells (Lash and Anders, 1989), so the mechanism(s) by which these toxicants enter cells is a critical element in the development of toxicity. Because DCVC is a zwitterionic organic electrolyte and, therefore, electrically charged at physiological pH, it is unlikely that diffusion across plasma membranes plays a significant role in the entry of this toxicant, at least from the very low (<micromolar) concentrations that are likely to arise from environmental exposure to the parent compound (Birner et al., 1993). A number of studies have characterized aspects of the renal transport of DCVC (Lash and Anders, 1989; Schaeffer and Stevens, 1987b; Wolfgang et al., 1989a; Zhang and Stevens, 1989; Schaeffer and Stevens, 1987a) and other cysteine conjugates (Heuner et al., 1991; Lock and Ishmael, 1985; Lock et al., 1986; Inoue et al., 1984; Boogaard et al., 1989) using different experimental preparations. Most of these studies examined the influence on DCVC uptake of transport processes in the basolateral (peritubular) membrane of proximal cells. Although some of these studies implicated the activity of one or more amino acid transport pathways in the accumulation of DCVC into renal cells (Heuner et al., 1991; Schaeffer and Stevens, 1987a; and b; Lash and Anders, 1989), other studies have shown that DCVC can access the organic anion transporter in these cells (Lash and Anders, 1989; Dantzler et al., 1995). By comparison, relatively few studies have attempted to characterize directly the pathway or pathways by which DCVC crosses the luminal membrane of proximal cells, despite evidence showing that cysteine conjugates are actively reabsorbed from the renal filtrate within the proximal tubule (Heuner et al., 1991). Observations of active reabsorption of cysteine conjugates support the contention that DCVC transport in the luminal membrane may involve cotransport with Na. In fact, studies of DCVC transport in isolated cells (Lash and Anders, 1989) and membranes (Schaeffer and Stevens, 1987b) from rat kidney have implicated Na-cotransport processes in the uptake of DCVC into renal cells, although conclusions concerning the identity and cellular location of these processes differed in these studies.

We undertook the present study to test the hypothesis that luminal transport of DCVC involved interaction with one or more secondary active Na-cotransport processes in the luminal membrane of renal proximal cells. Using a preparation of isolated luminal BBMV, we found that DCVC transport is effectively limited to a single Na-cotransport process that also transports phenylalanine and other non-polar amino acids. The kinetics of this process suggest that uptake of DCVC from the tubular filtrate could represent an important site of toxicant entry into proximal cells.

	Materials and Methods

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Preparation of brush-border membrane vesicles. BBMV were prepared from cortices isolated from kidneys of New Zealand White rabbits using a modified Ca⁺⁺-Mg⁺⁺ precipitation procedure (Wright and Wunz, 1987). Compared to the initial homogenate, these vesicles are routinely enriched approximately 10-fold in trehalase and alkaline phosphatase activity, and 1-fold or less in Na,KATP-ase and K-dependent p-nitrophenylphosphatase activity (Wright and Wunz, 1987). BBMV were used within 24 hr of preparation if held on ice, or within 2 wk if stored in liquid nitrogen. There was no significant decrease in transport activity compared to fresh vesicles when stored in these ways.

Measurement of transport. Uptake of labeled substrates was measured using a rapid filtration procedure (Wright et al., 1983). Briefly, the transport reaction was started by rapidly mixing 10 µl of the BBMV suspension with 90 µl of transport buffer containing salts and radiolabeled substrate (see figure legends for a description of solutions used in individual experiments). The transport reaction was terminated by adding 1 ml of an ice-cold "stop" solution, which typically was identical in composition to the BBMV suspension solution. One ml of the stopped reaction mixture was filtered under vacuum through a .45 µm filter (type HAWP; Millipore, Bedford, MA). The trapped BBMV were rinsed with 4 ml of stop solution and the radioactivity left on the filter was measured using a liquid scintillation counter (Beckman model LS3801, Beckman, Irvine, CA). Samples were corrected for variable quench. Each sample was also corrected for nonspecific binding of the labeled substrate to the filters or vesicles by subtracting the number of counts remaining on the filters following filtration and washing of vesicles that were simultaneously exposed to a mixture of transport buffer and ice-cold stop solution. Uptakes were expressed as moles of labeled substrate accumulated per milligram of membrane protein (Bio-Rad, Richmond, CA; -globulin standard). Experimental observations were performed at room temperature (21-23^o C).

Chemicals. [³⁵S]DCVC (50-53 mCi mmol¹) was synthesized using the procedure described by (Hayden et al., 1987). [¹⁴C]phenylalanine (456 mCi mmol¹) was purchased from ICN (Costa Mesa, CA). [¹⁴C]succinate (58.2 mCi mmol¹) was purchased from Du Pont NEN (Boston, MA). Unlabeled DCVC and NAC-DCVC were a gift from A. J. Gandolfi (University of Arizona). All other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO) or other routine suppliers and were the highest grade available.

	Results

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Effect of Na⁺ on the time course of DCVC uptake. Transport of DCVC into rat renal BBMV has been shown to involve Na-DCVC cotransport (Schaeffer and Stevens, 1987b). Consequently, we first determined the effect of Na⁺ on DCVC accumulation into rabbit renal BBMV. In the absence of Na⁺ in the extravesicular solution, the initial rate of DCVC accumulation into renal BBMV, i.e., the accumulation noted after a 1- to 3-sec exposure to 17 µM [³⁵S]DCVC, was comparatively slow and uninfluenced by the presence of a 5 mM concentration of unlabeled DCVC in the medium (fig. 1). In the presence of an inwardly-directed Na⁺ gradient (150 mM_out:0 mM_in) the initial rate of [³⁵S]DCVC uptake was increased 17-fold (fig. 1). After 15 sec of incubation, the Na-sensitive uptake of labeled DCVC reached a maximum, approximately 10-times that supported by K⁺. The presence of 5 mM unlabeled DCVC reduced both the initial rate of uptake in the presence of Na⁺ and the substantial accumulation of labeled substrate noted at 15 sec (fig. 1). These data suggest that DCVC transport in renal BBMV involves a saturable process sensitive to the presence of Na⁺ in the medium.

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Effect of Na+ gradient on the time course of [35S]DCVC uptake in BBMV. Vesicles were preloaded in 150 mM KCl, 339 mM mannitol and 10 mM HEPES taken to pH 7.5 with KOH (HEPES-KOH). Uptakes were measured in transport buffers with final concentrations of either 150 mM KCl or 150 mM KCl plus 150 mM NaCl, 10 mM HEPES-KOH (pH 7.5), 20 µM valinomycin, 17 µM [35S]DCVC, with or without 5 mM unlabeled DCVC and osmotically balanced with mannitol. Points are means ± S.E. of triplicate uptakes measured in three separate membrane preparations.

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Effect of membrane potential on the rate of [³⁵S]-DCVC transport. DCVC is a zwitterion and at pH 7.5 is effectively electroneutral. Cotransport of DCVC with Na⁺, regardless of the coupling stoichiometry, should result in a net movement of positive charge and, therefore be sensitive to the transmembrane electrical potential difference. The observations presented in figure 2 support this prediction. When the membrane PD was clamped to 0 mV (by means of equal intra- and extravesicular K⁺ concentrations in the presence of the K⁺ ionophore, valinomycin), the 5-sec rate of [³⁵S]DCVC transport was increased 4.6-fold by the presence of an inwardly directed Na gradient, compared to the uptake measured in the absence of Na⁺ (fig. 2). Imposition of a 60 mV inside-negative PD while in the presence of the inwardly directed Na⁺ gradient, increased the 5-sec rate of labeled DCVC transport by an additional 2.4-fold (fig. 2), consistent with the electrogenicity of Na-DCVC cotransport.

View larger version (22K): [in this window] [in a new window]   Fig. 2.   Effect of membrane potential on the time course of [35S]DCVC uptake in BBMV. Vesicles were preloaded in 150 mM KCl, 10 mM HEPES-KOH (pH 7.5) and 350 mM mannitol. Uptakes were measured in transport buffers with final concentrations of 10 mM HEPES-KOH (pH 7.5), 150 mM NaCl, 20 µM valinomycin, [35S]DCVC (30 µM in two experiments and 98 µM in the other), either with 150 mM KCl (0 mV) or without KCl (60 mV inside-negative) and osmotically balanced with mannitol. Points are the means ± S.E. of duplicate or triplicate uptakes measured in three separate membrane preparations.

Kinetics of Na-DCVC cotransport. To determine the capacity of the Na-dependent transport pathway(s) in renal brush border membranes, we tested the effect of increasing DCVC concentrations on the rate of DCVC uptake. Based on the observations presented in figures 1 and 2, we used three second uptakes as estimates of the initial rate of DCVC transport in the presence of an inwardly directed Na⁺ gradient and with electrical PD held at 0 mV. Figure 3A shows the relationship between increasing concentration of DCVC and rate of total DCVC uptake into BBMV. This relationship was adequately described by the Michaelis-Menten equation by assuming the presence of a single, saturable process and a distinct, non-saturable process that presumably represented some combination of diffusion or non-specific binding.

(1)

where J is the rate of substrate (in this case, DCVC) transport from a substrate concentration of [S]; J_max is the maximal rate of mediated transport when the carrier is saturated with substrate; K_t (the apparent Michaelis constant) is the substrate concentration resulting in half-maximal transport by the carrier; and D is a first-order constant consisting of those components of total substrate transport that do not approach saturation over the concentration range studied (including diffusion, binding, or a combination of these processes). In experiments with three separate membrane preparations the average values for J_max was 3.1 ± .51 nmol mg¹ 3 sec¹ and the average K_t was 0.51 ± 0.15 mM. The nonsaturable component of [³⁵S]DCVC transport was relatively small, as emphasized by figure 3B which shows the relationship between increasing concentrations of unlabeled DCVC on the transport of 50 µM [³⁵S]DCVC, a relationship described by the following equation (Malo and Berteloot, 1991):

(2)

where [T] is the concentration of the radiolabeled substrate (in this case, [³⁵S]DCVC); [S_unlab] is the concentration of unlabeled substrate that competes with the labeled substrate for binding at the transport site(s); and C is a constant reflecting the transport of unlabeled substrate that cannot be inhibited by the presence of unlabeled substrate (a consequence of a combination of diffusion and binding). The values for J_max and K_t calculated using this method, 3.0 ± 0.15 nmol mg¹ 3 sec¹, with a K_t of 0.45 ± 0.01 mM, respectively, were quite similar to those obtained using equation 1, consistent with the conclusion that DCVC transport was well described by a single site model adhering to Michaelis-Menten kinetics.

View larger version (13K): [in this window] [in a new window]   Fig. 3.   Kinetics of [35S]DCVC uptake in BBMV measured in the presence of a Na+ gradient. Vesicles were preloaded in 150 mM KCl, 10 mM HEPES-KOH (pH 7.5), and 350 mM mannitol. Three-second uptakes were measured in transport buffer with final concentration of 150 mM KCl, 150 mM NaCl, 10 mM HEPES-KOH (pH 7.5), 0-9950 µM unlabeled DCVC, 50 µM [35S]DCVC, 20 µM valinomycin and osmotically balanced with mannitol. Data were analyzed with a nonlinear regression algorithm (SigmaPlot; Jandel Scientific, San Rafael, CA). Points are the means ± S.E. of triplicate uptakes measured in three separate membrane preparations. A, Data expressed as total DCVC uptake vs. total substrate concentrations (i.e., labeled plus unlabeled DCVC) in standard Michaelis-Menton plot. B, Berteloot representation of data expressed as [35S]DCVC uptake vs. unlabeled DCVC concentrations.

Specificity of Na-DCVC cotransport. To determine if DCVC transport across the renal brush border membrane involves interaction with one or more pathways used by other organic electrolytes, we tested the effect of a 10 mM concentration of each of several potential competitive inhibitors on DCVC transport. As shown in figure 4, unlabeled DCVC was the most effective inhibitor of uptake of 49 µM [³⁵S]DCVC, reducing transport by approximately 90%, a degree of inhibition that was used as a measure of the fraction of total DCVC accumulation that was carrier-mediated. The first category of compounds tested was amino acids. DCVC is an amino acid (has both -amino and -carboxyl residues) and transport of DCVC in rat renal BBMV is inhibited, at least partially, by the presence of several amino acids (Schaeffer and Stevens, 1987b). We determined the inhibitory effectiveness of 12 amino acids known to interact with one or more different transport pathways in renal brush border membranes (Mircheff et al., 1982; Silbernagl, 1992; Kilberg et al., 1993). Of these nine, L-phenylalanine, L-leucine and L-cysteine substantially exhibited the greatest inhibition of DCVC transport, with each reducing transport by about 80% (fig. 4). L-Alanine and L-serine displayed moderate effects on DCVC transport, reducing transport by 50 to 60%. The other amino acids tested (i.e., glycine, proline, MeAIB, taurine, BCH, aspartate and lysine had minimal effects (<20% inhibition) on transport of DCVC.

View larger version (43K): [in this window] [in a new window]   Fig. 4.   Inhibitory effects of 10 mM concentrations of several organic electrolytes on uptake of [35S]DCVC in BBMV. Vesicles were preloaded in 150 mM KCl, 10 mM HEPES-KOH (pH 7.5) and 350 mM mannitol. Uptakes were measured in transport buffers with final concentration of 150 mM KCl, 10 mM HEPES-KOH (pH 7.5), 150 mM NaCl, 10 mM test compound, 20 µM valinomycin, 49 µM [35S]DCVC and osmotically balanced with mannitol. Bars are means ± S.E. of triplicate 5-sec uptakes measured in three separate membrane preparations except for the studies with taurine, cysteine and leucine (n = 2), and MeAIB, alanine and serine (n = 1).

Effect of DCVC on transport of phenylalanine in renal BBMV. Except for unlabeled DCVC, of the substrates tested, phenylalanine, leucine and cysteine were the most effective inhibitors of DCVC transport. To understand more fully the basis of the interaction of these substrates with transport of DCVC, we examined the effect of DCVC on transport of phenylalanine. We first confirmed that transport of phenylalanine into rabbit renal BBMV involves Na-cotransport. As shown in figure 5, uptake of [¹⁴C]phenylalanine into BBMV was stimulated by the presence of an inwardly-directed Na⁺ gradient: compared to uptake measured in the absence of Na⁺, the five sec uptake of phenylalanine was increased 8.8-fold in the presence of the Na gradient. Moreover, at 60 sec the Na gradient supported an intravesicular concentration of [¹⁴C]phenylalanine 5.3-fold more than that observed at 1 hr. These observations support the conclusion that, as in the case of DCVC uptake, phenylalanine transport in renal BBMV involved cotransport with Na⁺.

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Effect of Na+ gradient on the time course of [14C]phenylalanine uptake in BBMV. Vesicles were preloaded in 100 mM KCl, 300 mM mannitol and 10 mM HEPES taken to pH 7.4 with Tris (HEPES-Tris). Uptakes were measured in transport buffers with final concentrations of either 100 mM KCl or 100 mM KCl plus 100 mM NaCl, 10 mM HEPES-Tris (pH 7.4), 50 µM unlabeled phenylalanine, 2.2 µM [14C]phenylalanine and osmotically balanced with mannitol. Points are means ± S.E. of duplicate or triplicate uptakes measured in three separate membrane preparations.

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View larger version (18K): [in this window] [in a new window]   Fig. 6.   Kinetics of [14C]phenylalanine uptake in BBMV measured in the presence of a Na+ gradient. Vesicles were preloaded in 100 mM KCl, 10 mM HEPES-Tris (pH 7.4) and 300 mM mannitol. Five-second uptakes were measured in transport buffer with final concentration of 100 mM KCl, 100 mM NaCl, 10 mM HEPES-Tris (pH 7.4), 0.05-50 mM unlabeled phenylalanine, either with (open circles) or without (solid circles) 1 mM unlabeled DCVC, 2.5 to 7.5 µM [14C]phenylalanine and osmotically balanced with mannitol. Lines were fit to the data using a nonlinear regression algorithm (SigmaPlot; Jandel Scientific) according to the model presented in equation 1. Points are the means ± S.E. of triplicate measurements of uptake from three separate membrane preparations.

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	Discussion

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Three conclusions concerning the transport of DCVC across the luminal membrane of proximal cells of rabbit kidney are supported by the evidence presented here. First, DCVC transport is effectively limited to a mediated process involving cotransport with Na⁺. Second, DCVC transport appears to share only one of the several pathways in the luminal membrane that accept neutral amino acids. Third, the kinetics of DCVC transport support the contention that luminal uptake of this cysteine conjugate could represent an important avenue for entry of this nephrotoxicant into renal cells.

Evidence in the literature concerning the first two of these issues, i.e., the Na-dependency and structural specificity of luminal DCVC transport, includes a number of apparently contradictory observations, the interpretation of which is frequently complicated by the use of different experimental systems. Following is a brief outline of the evidence pertaining to each of these two issues within the context of our results.

We found that a Na-gradient stimulated DCVC transport into luminal membrane vesicles from rabbit kidney and provided the energy capable of supporting a transient accumulation of DCVC above that in the surrounding solution (fig. 1). Although DCVC is a zwitterion at the pH used in these studies, imposition of an inside-negative membrane potential stimulated the initial rate of DCVC uptake in the presence of Na⁺, consistent with an electrogenic mode of transport (fig. 2). These observations support the conclusion that DCVC transport involved secondary active Na-DCVC cotransport. We found no evidence for a Na-independent component of DCVC transport in luminal membrane vesicles from rabbit kidney (figs. 1 and 2).

The Na-dependency of DCVC transport has also been examined in cultured renal cells and in several different experimental preparations using rat kidney. Mediated transport of DCVC transport into confluent monolayers of the cultured renal cell line, LLC-PK1, was not influenced by the removal of Na⁺ from the medium (Schaeffer and Stevens, 1987a). That observation, in conjunction with evidence that transport was inhibited by the presence of large, nonpolar amino acids, including BCH, the specific inhibitor of the Na-independent "system L" transporter, (Christensen et al., 1969), led to the conclusion that DCVC transport into LLC-PK1 cells is largely restricted to a pathway or pathways with characteristics attributed to system L. Although system L appears to be restricted to the basolateral membrane of native proximal tubules, it is found in both apical and basolateral membranes of LLC-PK1 cells (Hensley and Mircheff, 1994). A study using isolated proximal tubule cells from rat kidney (Lash and Anders, 1989) found that DCVC transport has both Na-dependent and Na-independent components. The authors suggested that the Na-dependent component of DCVC transport reflected interaction with the organic anion transporter of the basolateral membrane, rather than the influence of Na-dependent transport across the luminal membrane. This conclusion runs counter, however, to the failure of probenecid to influence transport of DCVC into isolated, intact, rat renal proximal tubules (Zhang and Stevens, 1989). Moreover, DCVC transport into isolated luminal membranes from rat kidney was shown to be dominated by a Na-dependent process(es) (Schaeffer and Stevens, 1987b), leading to the conclusion that luminal DCVC transport in the rat kidney involves the Na-dependent neutral amino acid transporter described for this membrane (Evers et al., 1976). As noted earlier, our results support the conclusion that luminal DCVC transport involves cotransport with Na⁺ and we suggest that, at least in rabbit proximal tubule, transport of DCVC across the luminal membrane is effectively limited to a Na-dependent pathway.

Conclusions concerning the structural specificity of DCVC transport are even more likely to be influenced by the use of different test species and experimental preparations. Nevertheless, several previous studies showed that amino acids, particularly phenylalanine, can inhibit luminal transport of DCVC (Schaeffer and Stevens, 1987a, 1987b; Heuner et al., 1991). In our study we found that DCVC transport across the luminal membrane of rabbit renal proximal cells showed a rather narrowly defined substrate specificity. Indeed, the profile of substrate inhibition supported the conclusion that DCVC transport was limited to a single pathway: System 1 as defined by Mircheff et al. (1982). This conclusion was based on the following observations. The influence of System ASC ["alanine/serine/cysteine-preferring" (Christensen, 1975)] was eliminated because glycine, an effective substrate for this system (Kilberg et al., 1993), had little effect on DCVC transport. Similarly, System A ["alanine-preferring" (Christensen, 1975)] is unlikely to play a role in DCVC transport because MeAIB, a frequently used test substrate for System A (Christensen, 1975; Kilberg et al., 1993), had no effect on DCVC transport. The lack of effect by proline and lysine indicated that DCVC transport involves neither the IMINO System nor System y+, respectively (Kilberg et al., 1993; Mircheff et al., 1982). The failure of BCH and taurine to inhibit DCVC uptake eliminated System L and the  System as pathways for DCVC transport (Mircheff et al., 1982). Finally, none of the organic anions tested had a direct inhibitory effect on DCVC transport, eliminating the Na-dependent monocarboxylate (Nord et al., 1983) and dicarboxylate pathways (Wright et al., 1980) and the luminal organic anion transporter as avenues for DCVC transport (fig. 4). Phenylalanine (as well as leucine and cysteine) was an effective inhibitor of DCVC transport (figs. 4, 6 and 7). Mircheff et al. (1982) identified two major pathways for phenylalanine transport in the luminal membrane of rabbit proximal cells: System 1 and System 3. System 3 was eliminated as a pathway for DCVC transport because of its interaction with lysine and beta-amino acids (Mircheff et al., 1982). System 1, however, is a Na-dependent pathway that transports phenylalanine and is inhibited by nonpolar neutral amino acids (e.g., leucine and cysteine), but is not inhibited by MeAIB, proline, lysine or the -amino acid -alanine. The profile of inhibition of DCVC uptake into rabbit BBMV (figs. 4, 6 and 7) supports the conclusion that DCVC transport across the luminal membrane of rabbit proximal cells is limited to System 1. 

View larger version (19K): [in this window] [in a new window]   Fig. 7.   Alternative analytical method, based on the model represented by equation 2, for the assessment of the effect of DCVC on the kinetics of phenylalanine transport in BBMV. Data presented are those presented in figure 6, except the rate of [14C]phenylalanine transport is reported as a function of increasing concentration of unlabeled phenylalanine (0.05 to 50 mM). In the case marked "control" (solid circles), transport was measured in the absence of DCVC; in the case marked "+1 mM DCVC" (open circles), transport was measured in the presence of 1 mM unlabeled DCVC. The curves describing each set of points were calculated using estimates of Jmax and Kt for each experimental condition determined from a fit of equation 2 to these data. Each point is the mean ± S.E. of triplicate measurements of uptake determined in three separate membrane preparation.

View larger version (21K): [in this window] [in a new window]   Fig. 8.   Woolf-Augustinsson-Hofstee plot (J vs. J/[S]) of the "control" data from figure 6 describing the kinetics of phenylalanine transport in BBMV. The dotted line was drawn using the values for Jmax and Kt derived from the model represented by equation 1; the solid line was drawn using the values for Jmax and Kt derived from the model represented by equation 2.

The last issue raised by the present set of observations involves the kinetics of DCVC transport across the luminal membrane. As emphasized below, we believe that the rate of DCVC transport across the luminal membrane makes this site of entry potentially significant in the development of toxicity after exposure to this agent. The nephrotoxic effects of DCVC and other cysteine conjugates are the result of metabolic activation by enzymes, particularly -lyase, located within the cytoplasm of proximal cells (Stevens et al., 1985). Consequently, the entry of these toxicants into proximal cells is believed to be a necessary precursor to their toxicity (Lash and Anders, 1989). Most in vitro studies on the toxic effects of cysteine conjugates have involved use of either isolated proximal tubules or renal cortical slices, preparations that effectively limit access of the toxicant to the basolateral membrane. Glomerular filtration will, however, result in in vivo exposure of luminal membranes to cysteine conjugates. Although most of the absorptive flux of amino acids (and, presumably, DCVC) occurs in the early proximal tubule, luminal exposure to and transport of amino acid occurs along the entire length of the proximal tubule (Silbernagl, 1992). Nevertheless, the role of the luminal membrane as an avenue for entrance of toxicants has received little attention (Heuner et al., 1991). Thus, it was instructive to compare the rate of DCVC transport measured in rabbit renal BBMV to rates of luminal transport for other substrates. Because the absolute rate of transport in a preparation of isolated membrane vesicles is dependent on issues such as preparative method, protocol for measuring substrate uptake, and procedure for normalizing measured rates of transport, comparisons of results obtained in different studies are often suspect. However, measurement of transport kinetics for several other organic electrolytes in previous studies from our laboratory offers the opportunity to make comparisons of results obtained using a common set of techniques.

Table 2 presents a set of values for the J_max and K_t for transport of several compounds, including the kinetics for DCVC and phenylalanine transport measured in our study. The most instructive comparison, we believe, is the ratio of J_max to K_t, which represents the "carrier-mediated permeability" of the membrane to substrate when the carrier is exposed to comparatively low concentrations of substrate (K_t), i.e., the concentrations to which transporters are typically exposed. Using this means of comparison, the luminal membrane was observed to have a carrier-mediated permeability to DCVC as high or higher than that for any other substrate. It is worth noting that, in both rabbit (Wolfgang et al., 1989b) and rat (Terrancini and Parker, 1965), cysteine conjugate toxicity tends to be noted first in cells of the late proximal tubule (S3 segment). Because of the "upstream" transport activity of the early proximal tubule it is not clear to what extent the luminal membrane of cells in the S3 segment is exposed to DCVC. Indeed, it may be that peritubular exposure to and accumulation of cysteine conjugates may be critical in the development of toxicity in late proximal cells, although the relative rate of luminal and peritubular transport of these compounds is not clear (for any segment of proximal tubule). Nevertheless, our results indicate that the luminal membrane can play a role in facilitating entry of cysteine conjugates into renal cells.

In conclusion, DCVC transport into luminal membrane vesicles isolated from rabbit renal cortex was shown to involve cotransport with Na⁺. Transport appeared to be limited to a single pathway that also carries phenylalanine and other nonpolar neutral amino acids. The kinetics of DCVC transport revealed that the carrier-mediated permeability of the luminal membrane to DCVC is comparatively large, suggesting that active luminal uptake can represent a significant avenue of entry of this toxicant into proximal cells.