Introduction

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Organisms use two general strategies to modify the biological activity of potentially toxic xenobiotics: altering chemical reactivity through metabolism and removing biologically active molecules and their metabolites from sensitive sites by excretory transport. A major function of renal proximal tubule is the transport from blood to urine of a wide variety of potentially toxic metabolic wastes, drugs, pollutants, and drug and pollutant metabolites. Anionic xenobiotics are handled by a long-studied, classical, sodium-dependent system (Pritchard and Miller, 1993, 1996) and by a newly discovered, sodium-independent system (Masereeuw et al., 1996b). These systems are similar in that both involve two transport steps arranged in series: the first at the basolateral membrane of renal epithelial cells and a second at the luminal membrane. Substrates for both systems also accumulate in intracellular compartments (Miller et al., 1993; Masereeuw et al., 1994, 1996b). The systems differ in the specificities and energetics of the carriers involved (Fig. 1). Renal secretion on the classical system is driven by indirect coupling of organic anion influx to sodium at the basolateral membrane followed by carrier-mediated transport at the luminal membrane. Secretion on the sodium-independent system is driven by an as yet uncharacterized carrier at the basolateral membrane followed by cell-to-lumen transport on a carrier with specificity characteristics similar to that of a xenobiotic-transporting ATPase (a multidrug resistance-associated protein isoform, Mrp2). This transport protein has been localized to the canalicular membrane of hepatocytes (Paulusma et al., 1996) and the luminal membrane of rat renal proximal tubule cells (Schaub et al., 1997).

View larger version (64K): [in this window] [in a new window]   Fig. 1.   Model of organic anion transport in renal proximal tubule. Basolateral uptake may be mediated by 1) the classical sodium-dependent transport system, which includes sodium--ketoglutarate (-KG) cotransport and -ketoglutarate organic anion (OA) exchange via the organic anion transport protein Oat (Sweet et al., 1997), or 2) the sodium-independent system. Luminal secretion may involve facilitated diffusion at the brush-border membrane or be mediated by a transporting ATPase, Mrp2. Accumulation occurs for substrates for both transport pathways.

Masereeuw et al. (unpublished data) not only demonstrated the presence of Mrp2 in killifish renal proximal tubules, but also provided experimental criteria by which the route of secretion from bath to tubular lumen could be determined for fluorescent substrates (Masereeuw et al., 1996b). The defining criteria were sodium dependence and ouabain sensitivity at the basolateral membrane and leukotriene C₄ (LTC₄) and cyclosporin A sensitivity at the luminal membrane. By these criteria, the small organic anion, fluorescein (FL), crossed the epithelium using carriers associated with the sodium-dependent system, because uptake by cells and secretion into the lumen were abolished by ouabain but LTC₄ was without effect. In contrast, the large organic anion, fluorescein methotrexate (FL-MTX), entered the cells on the basolateral carrier for the sodium-independent system (lack of inhibition by ouabain) and was transported into the lumen primarily on Mrp2 (inhibition by LTC₄). Sulforhodamine 101, intermediate in size, exhibited intermediate sensitivities to ouabain and LTC₄, indicating partitioning of transport between the two systems. These results imply that one could simply consider selectivity as a function of transport system rather than as a function of the individual carriers involved, i.e., the transport systems could be treated as if they were competing metabolic pathways.

In the present study, we tested this inference by examining the transport of the fluorescent organic anion, lucifer yellow (LY). This dye has been used extensively in cell biology as a tool to trace cell lineage and to investigate gap junction function. Little is known about the mechanisms of LY transport. It is clear that macrophages possess a potent mechanism for LY efflux, and, based on the probenecid sensitivity of that process, it was proposed that a transport system comparable to the organic anion secretory system in renal proximal tubule mediates LY efflux (Steinberg et al., 1987). However, no evidence for specific transport of LY was found in monolayers of canine proximal tubular cells in primary culture, although these monolayers did exhibit a net reabsorptive paracellular flux of LY (Goligorsky and Hruska, 1986). Here, we used fluorometry and confocal microscopy to examine the renal handling of LY. Our results indicate that: 1) LY undergoes net secretion by the isolated perfused rat kidney (IPK), 2) LY is secreted in killifish renal proximal tubules by a process that is inhibited by small organic anions and nearly abolished by ouabain, but 3) a substantial fraction of the transport of LY from cell to tubular lumen is blocked by LTC₄. A similar inhibition pattern also was found for a fluorescent mercapturic acid derivative. Together, the data indicate that these organic anions enter renal proximal tubule cells on the sodium-dependent organic anion system, but that a substantial component of transport from cell to lumen is on the carrier for the sodium-independent system Mrp2.

	Experimental Procedures

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Materials. Sodium pentobarbital was obtained from Apharmo (Arnhem, the Netherlands), pluronic F-108 was obtained from BASF (Arnhem, the Netherlands), and heparin, aldosterone, and inulin were obtained from Organon (Oss, the Netherlands). LY-CH dilithium salt, leukotriene C₄, and chlorodinitrobenzene were purchased from Sigma (St. Louis, MO). Lysine-vasopressin was obtained from Sandoz Pharma Ltd. (Basel, Switzerland), angiotensin II was obtained from Beckman (Palo Alto, CA), and synthamin 14 was obtained from Travenol (Thetford, Norfolk, UK). The mercapturic acid derivative of monochlorobimane (MCB) was synthesized by reacting MCB (Molecular Probes) with an excess of N-acetylcysteine; the derivative was purified by HPLC as described previously (Miller et al., 1996). All other chemicals were purchased from either Sigma or Merck (Darmstadt, Germany) and were of analytical grade.

Kidney Isolation and Perfusion. Rat kidneys were isolated and perfused as described in detail previously (Cox et al., 1990). Briefly, male Wistar-Hannover rats (225-275 g) were anesthetized i.p. with pentobarbital (6 mg/100 g) and furosemide was injected i.p. (1 mg/100 g) to prevent deterioration of the distal nephron. Heparin (125 I.U./100 g) was injected in the femoral vein. The ureter of the right kidney was cannulated as well as the renal artery via the mesenteric artery without interruption of the blood flow. The kidney then was excised and placed in a fluid bath with a constant temperature of 37.5°C. The perfusate reservoir also was placed in a water bath of 37.5°C, and fluid was gassed with 95% O₂/5% CO₂. The perfusion fluid had the following composition: 114.0 mM NaCl, 5.2 mM KCl, 1.8 mM CaCl₂, 1.0 mM MgCl₂, 22.5 mM NaHCO₃, 0.84 mM Na₂HPO₄, 0.28 mM KH₂PO₄, 5.0 mM glucose, 4.0 mM urea, 25.0 g/liter pluronic F108, 0.33 mM glutathione, 0.083 mM inositol, 0.50 mM cysteine, 2.3 mM glycine, 2.0 mM sodium-pyruvate, 1.22 mM sodium-acetate, 0.21 mM sodium-propionate, 1.0 mM inosine, 5.0 mM alanine, 0.11 mM glutamine, 2.0 mM L-glutamine acid, 0.01 mM ascorbic acid, 1.0 mM sodium-lactate, 1.0 mg/liter choline chloride, 4 I.U./liter insulin, 2.0 µg/liter aldosterone, 0.01 I.U./liter lysine-vasopressin, and 15.0 ng/liter angiotensin II. To this solution 1.0% synthamin 14, a mixture of 15 amino acids, was added. Pluronic F-108 was used as oncotic agent in the albumin-free perfusion fluid. For the determination of glomerular filtration rate (GFR), inulin was added to the perfusion fluid (100 µg/ml).

Analytical Methods. Inulin was determined according to a previously published method (Heyrovski, 1956). The concentration of LY in perfusate, urine, and kidney samples was determined by fluorescence spectrophotometry. To this end, an aliquot of 50 µl of the perfusate sample was taken and adjusted to 600 µl with analysis buffer (Sörensen buffer, pH 7.36). Urine samples were diluted 10 times with buffer, from which an aliquot of 25 µl was taken and adjusted to 600 µl with 50 µl of blank perfusion fluid and 525 µl buffer. Fluorescence in these prepared samples was measured using a Perkin-Elmer LS50 luminescence spectrophotometer (Perkin-Elmer Ltd., Beaconsfield, Buckinghamshire, UK). The excitation wavelength was set to 425 nm, the emission wavelength was set to 525 nm, and, for both wavelengths, a band width of 5 nm was used. Concentrations were calculated by comparing fluorescence intensity (in photomultiplier units) with a calibration curve of spiked samples of blank perfusion fluid with different concentrations of LY. The concentration of LY in kidney tissue extracts was determined similarly. The kidneys were homogenized in 2.5 ml of distilled water with a Polytron homogenizer (Braun Melsungen, Germany) on setting 10 for 2 × 60 s. Subsequently, 100 µl of 6 N HCl was added to 500 µl of the kidney homogenate, vortexed, and centrifuged for 10 min at 2000g. Of the supernatant, an aliquot of 100 µl was taken and adjusted to 600 µl with buffer, and fluorescence intensities of quadruplicate samples were measured and averaged. Concentrations were calculated by comparing fluorescence intensity with a calibration curve of spiked samples of blank kidney homogenates with various concentrations of LY.

Killifish Proximal Tubule Isolation. Killifish (Fundulus heteroclitus) were collected near Duke University Marine Laboratory (Beaufort, NC) and maintained in tanks with recirculating, artificial sea water (18°C) at the National Institute of Environmental Health Sciences. Killifish proximal tubules were isolated by dissection of renal tubular masses with forceps, as described previously (Miller and Pritchard, 1991, 1994; Masereeuw et al., 1996b). Proximal tubules were maintained in a marine teleost saline based on that of Forster and Taggart (1950), containing 140 mM NaCl, 2.5 mM KCl, 1.5 mM CaCl₂, 1.0 mM MgCl₂, and 20 mM Tris, pH 8.0.

Confocal Microscopy. For microscopy, killifish proximal tubules were transferred to a Teflon chamber (Bionique) with a 4 × 4-cm glass coverslip floor containing 1.5 ml of marine teleost saline. Killifish proximal tubules were preincubated for 15 min (18°C) in the absence (controls) and presence of various inhibitors under an atmosphere of 95% oxygen and 5% carbon dioxide. Then, LY (final concentration of 2 µM) was added to the tubules and incubation took place for 30 min at room temperature.

Data Analysis. Data of isolated perfused kidney are expressed as mean ± S.D. All other data are expressed as mean ± S.E., unless indicated otherwise. Student's t test was used to evaluate statistical significance in the renal clearance studies. Statistical differences between multiple means were determined with one-way ANOVA followed by the least significant difference post test. Means were considered significantly different when P < .05.

	Results

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Transport of LY in the Isolated Perfused Rat Kidney. The viability of perfused rat kidneys was assessed by following fractional excretion of sodium and glucose, fractional reabsorption of water, urine flow and pH, GFR, and renal perfusate pressure. Based on these criteria, kidneys showed excellent transport function over the 2-h time course of the LY clearance experiments (Table 1). Pretreating kidneys with probenecid or -ketoglutarate did not alter kidney function.

View this table: [in this window] [in a new window]   TABLE 1 Renal functional parameters of the isolated perfused rat kidney during perfusion with 10 µM LY without or with pretreatment with probenecid or -ketoglutarate

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Renal clearance over GFR as a function of time. A concentration of 10 µM LY was used for control () and, after pretreatment, with 1.15 mM -ketoglutarate () or 0.5 mM probenecid (). Data are means of four experiments ± S.D. *Significantly different from controls (P < .05). **Significantly different from controls (P < .01).

LY Uptake and Secretion by Killifish Renal Proximal Tubules. Isolated renal proximal tubules from certain marine teleost fish offer several advantages for the study of mechanisms of xenobiotic secretion (Miller and Pritchard, 1991). The nephron of these animals is composed primarily of proximal tubules, which are isolated easily and retain viability for long periods of time when maintained in a simple physiological saline. During tubule isolation, broken ends reseal and form a closed, fluid-filled luminal compartment that is separated from the medium by the epithelium. Finally, using fluorescent substrates, confocal microscopy and image analysis xenobiotic uptake by cells and secretion into the lumen can be visualized and measured (Miller and Pritchard, 1994; Masereeuw et al., 1996b; Miller et al., 1996).

View larger version (128K): [in this window] [in a new window]   Fig. 3.   Confocal micrograph of killifish proximal tubules incubated to steady state with 2 µM LY. Tubules were viewed through a 40× Plan-Neofluar objective with a numerical aperture of 1.3 (0.625 µm/pixel) without using a zoom factor. All other settings were as described in Experimental Procedures. A, control uptake of LY. B, high-magnification image of control uptake. C, preincubation for 15 min with 1 mM PAH, after which incubation with LY took place. D, preincubation for 15 min with 0.5 mM probenecid. Scale bar, 10 µm.

View larger version (15K): [in this window] [in a new window]   Fig. 4.   Time course of LY transport in killifish proximal tubules in the absence or presence of 100 µM PAH. Luminal values for control (ctrl, ) or PAH () and cellular values (control, ; PAH, ) were measured as described in Experimental Procedures. PAH treatment reduced both cellular and luminal fluorescence intensity significantly (P < .01). Data are presented as means ± S.E. for four to eight tubules from two to three fish.

View larger version (13K): [in this window] [in a new window]   Fig. 5.   Inhibition of LY (A) or FL (B) transport by ouabain (100 µM). Tubules were incubated at room temperature for 30 min in medium containing 2 µM LY or 1 µM FL without or with treatment with ouabain, as described in Experimental Procedures. Data are presented as means ± S.E. for 7 to 11 tubules from two to three fish. *Significantly different from controls (P < .01).

View larger version (19K): [in this window] [in a new window]   Fig. 6.   Inhibition of LY (A) or FL (B) transport by different doses of LTC4 (LTC). Tubules were incubated at room temperature for 30 min in medium containing 2 µM LY or 1 µM FL without or with treatment with LTC4, as described in Experimental Procedures. Luminal secretion of LY was reduced significantly by LTC4 treatment at a concentration of 200 or 300 nM, whereas FL secretion was not affected by LTC4. Data are presented as means ± S.E. for 10 to 13 tubules from two fish. *Significantly different from controls (P < .01).

View larger version (20K): [in this window] [in a new window]   Fig. 7.   Effects of different doses of CDNB on LY (A) and FL (B) transport in killifish renal proximal tubules. Tubules were incubated at room temperature for 30 min in medium containing 2 µM LY or 1 µM FL without or with treatment with CDNB, as described in Experimental Procedures. Luminal secretion of LY was reduced significantly by CDNB treatment at a concentration of 10 or 25 µM, whereas FL secretion was not affected by the same concentrations of CDNB. Data are presented as means ± S.E. for 9 to 13 tubules from two fish. *Significantly different from controls (P < .01).

View larger version (14K): [in this window] [in a new window]   Fig. 8.   Effects of LTC4, CDNB, and both in combination on LY transport in killifish renal proximal tubules. Tubules were incubated at room temperature for 30 min in medium containing 2 µM LY without or with treatment with LTC4 (LTC) and CDNB, as described in Experimental Procedures. Luminal secretion of LY was reduced significantly by LTC4 and CDNB, but both inhibitors were not additive in their effects. Data are presented as means ± S.E. for 9 to 12 tubules from two fish. *Significantly different from control (P < .01).

Transport of a Bimane Mercapturic Acid Derivative. MCB is a nonfluorescent, uncharged compound that enters cells by simple diffusion and is conjugated to GSH by GSH-transferase. The GSH derivative of MCB and all bimane-S metabolites are negatively charged and fluorescent. MCB has been used as a tool to study organic anion transport mechanisms in hepatocytes and renal proximal tubules. The mercapturic acid derivative of MCB (MCB-cys-Nac) is the final product of the renal metabolism of the GSH-conjugate of MCB. Like other sulfur-linked conjugates of MCB, MCB-cys-Nac is anionic and fluorescent. Previous experiments have shown that the mercapturic acid derivative of MCB is formed when isolated killifish renal tubules are exposed to MCB and that this derivative inhibits the uptake and secretion of FL (Miller et al., 1996).

View larger version (13K): [in this window] [in a new window]   Fig. 9.   Time course of luminal NAC-cys-MCB secretion in killifish renal proximal tubules with or without treatment with 100 µM probenecid (PROB). Luminal values for control (ctrl, ) or PROB () were measured as described in Experimental Procedures. PROB treatment reduced luminal fluorescence intensity significantly (P < .01). Data are presented as means ± S.E. for six tubules from one fish.

View larger version (13K): [in this window] [in a new window]   Fig. 10.   Effects of transport inhibitors on NAC-cys-MCB secretion in killifish renal proximal tubules. Tubules were incubated at room temperature for 30 min in medium containing 5 µM NAC-cys-MCB without or with treatment with PAH (10 or 100 µM), ouabain (100 µM), or LTC4 (LTC, 300 nM) as described in Experimental Procedures. Luminal secretion of LY was reduced significantly by 100 µM PAH, ouabain, and LTC4. Data are presented as means ± S.E. for 34 to 79 tubules from nine fish. *Significantly different from control (P < .05). **Significantly different from control (P < .01).

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

By mediating active drug excretion, xenobiotic transporters play a major role in determining drug concentrations reaching sensitive sites within an organism. Along with drug metabolizing enzymes, these transporters are important determinants of drug effectiveness on the one hand and of xenobiotic toxicity on the other. Moreover, because of their wide specificity limits, these transporters also provide a mechanism, competition for transport, by which chemicals with very different structures may interact to alter xenobiotic-excretion rates, plasma-concentration profiles, and tissue-distribution patterns. Thus, it is important to characterize the individual transporters that drive excretion, understand how xenobiotics interact with these membrane proteins, and identify the routes that chemicals follow during transport from blood to urine.

The results of the present study provide the first evidence for active secretion of LY in renal proximal tubule. This conclusion is supported by two types of experiments. First, in isolated perfused rat kidney, LY clearance exceeded the GFR by a factor of nearly 2. LY clearance, but not GFR, was reduced significantly by the organic anions, probenecid and -ketoglutarate. LY accumulation in kidney tissue was reduced similarly by probenecid and -ketoglutarate. Second, in isolated killifish renal proximal tubules, confocal microscopy showed that LY was transported from bath to tubular lumen by a process that was concentrative and sensitive to inhibition by probenecid and PAH and by the Na,K-ATPase inhibitor, ouabain. At present, it is not clear why specific LY transport was not seen in a previous study with monolayers of canine proximal tubular cells (Goligorsky and Hruska, 1986), but species differences or differences in the transport characteristics of cells in culture versus cells in an intact tubule are possible explanations.

Based on the ability of organic anions, such as PAH and probenecid, to inhibit LY cellular accumulation in rat renal tissue and killifish proximal tubule cells and to inhibit LY secretion in perfused rat kidneys and killifish proximal tubules, LY transport appeared to be mediated by a renal organic anion system. Two such transport systems have been identified. The classical organic anion transport system, which appears to be present in the renal systems of all animals studied (Pritchard and Miller, 1993), is sodium-dependent and ouabain-sensitive. The system for large organic anions is sodium-independent and ouabain-insensitive. It was first demonstrated in killifish proximal tubules (Masereeuw et al., 1996b; unpublished data), but at least one component of that system, the luminal carrier (Mrp2), also has been shown to be present in rat proximal tubule (Schaub et al., 1997).

Each system appears to use different transporters at the basolateral and luminal membranes (Fig. 1). Thus, at least four transporters can be involved in organic anion secretion. For fluorescent substrates, these can be sorted out in killifish tubules by simple inhibition experiments using ouabain and LTC₄ (Fig. 1). The present data show that LY entered proximal tubule cells on the sodium-dependent basolateral carrier for small organic anions, because LY uptake and secretion, like FL, were nearly abolished by ouabain. LY was transported into the tubular lumen by the luminal transporters for both systems, because LTC₄ reduced secretion by about 50% and because, with 200 to 300 nM LTC₄, luminal fluorescence still exceeded cellular fluorescence by a factor of 2 to 3. We assume that the LTC₄-insensitive component of secretion was on the luminal carrier for small organic anions. Supporting evidence for Mrp2 involvement in renal LY secretion is given by Klein et al. (1997), showing Mrp2-like transport of LY into plant vacuoles, and by R. Van Aubel, who used LY to inhibit ATP-dependent transport of 17-estradiol-17--D-glucuronide in Mrp2-expressed insect cells (personal communication). The mixed mechanism of transport was not unique for LY. A similar inhibition pattern was found for a fluorescent mercapturic acid derivative, MCB-cys-Nac, although some aspects of the data may be open to interpretation because of our inability to measure cellular accumulation of MCB-cys-Nac. Nevertheless, our result suggests that a similar mixed mechanism of transport would be seen with other GSH conjugates. The lack of increase in cellular fluorescence seen for both LY and MCB-cys-Nac is consistent with earlier findings using similar experimental conditions (Schramm et al., 1995; Masereeuw et al., 1996) and indicates, most likely, that steady-state cellular levels are set independently of events at the luminal membrane.

The teleost proximal tubule is one of the few preparations for which we possess enough information about xenobiotic transporters and for which we have the experimental tools needed to dissect carrier-mediated pathways in the intact tubule. Taken together, the present data are the first to demonstrate that xenobiotics can enter renal proximal tubule cells on the carrier associated with one organic anion transport system and exit into the tubular lumen on multiple carriers, one of which is associated with a second system. This pattern of substrate crossover is not unique to organic anions. Daunomycin, a weak organic base, enters killifish proximal tubules both by simple diffusion and transport mediated by the basolateral carrier for the organic cation system; it is transported from cell to lumen by the luminal carrier for the organic cation system and by p-glycoprotein (Miller, 1995). Thus, a significant fraction of secreted daunomycin crosses over from the organic cation system to p-glycoprotein.

Much of current discussion of renal xenobiotic excretion is still couched in terms of transport systems for organic anions and organic cations. This is true even though it is clear that the basolateral and luminal carriers associated with a given system are not mechanistically or spatially linked but, rather, are separated by a cytoplasmic compartment. These carriers are only related through common specificity characteristics. These considerations and the mounting physiological and molecular evidence for the presence of multiple carriers with overlapping specificities in each of the membranes suggest that when we are able to follow the transport of individual substrates through the tubular epithelium, the concept of transport systems no longer will be tenable. Note that in transiting the tubular epithelium, organic anions and organic cations must cross two membrane barriers arranged in series. Because of this series arrangement, inhibition of the first step in transport not only blocks entry into the cell but may also reduce transport into the lumen (secretion). This presents a problem in interpreting previous studies of renal secretion, because transport pathways were defined primarily based on sensitivity to compounds that, at least to some extent, affected the uptake step, e.g., probenecid, PAH, and ouabain for organic anions. To better understand how chemicals are excreted in the proximal tubule, we need to identify additional agents that affect luminal transport but have only minimal effects on basolateral transport.