Introduction

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The multidrug transporter P-glycoprotein has been identified in many resistant tumor cells. This 170- to 180-kdalton membrane glycoprotein is part of the ABC family of transporters and mediates the extrusion of chemotherapeutic agents, decreasing their accumulation within cells and consequently conferring MDR to cancer cells (Kane, 1996). The protein also transports peptides (Saeki et al., 1993a), steroids (Ueda et al., 1992) and a variety of structurally unrelated, hydrophobic substances including calcium channel blockers and immunosuppressive agents (Saeki et al., 1993a; Tsuruo, 1991)

P-glycoprotein also has been identified in several normal human tissues and is most abundant on the luminal surface of polarized transporting epithelia of kidney proximal tubule, small intestine and colon, liver biliary hepatocytes and the secretory gland of the pregnant mouse endometrium, as well as in the adrenals and capillary endothelial cells such as those of the brain and testis (Thiebaut et al., 1987, 1989; Sugawara et al., 1988; Cordon-Cardo et al., 1990). The location of P-glycoprotein expression suggests that one of the physiological roles of this protein in normal tissues may be the excretion of xenobiotics (Gottesman and Pastan, 1993); in particular, in the kidney it is unclear whether P-glycoprotein is involved in the transepithelial drug transport pathway or whether it simply has a protective role for those cells lining the nephron.

The anthracycline antibiotic doxorubicin is a well-known substrate for P-glycoprotein in MDR cells. Although many reports have studied the transport of anthracyclines in various tumor cells extensively, only few studies have examined the transport mechanism of these anticancer agents in normal cells and in particular in the renal proximal tubular cells, which are particularly rich in P-glycoprotein. Charuk et al. (1994) studied the interactions of anthracyclines with renal P-glycoprotein and observed that both doxorubicin and daunomycin interact with this protein, even if their relative affinities (half-maximal inhibition constant, 10 µM) were lower than those of several other drugs tested.

Renal elimination is not a hallmark of anthracycline pharmacology; indeed, dose modification is not required in patients with compromised renal function (Pratt and Ruddon, 1979) and renal clearance approximates that of creatinine (Krarup-Hansen et al., 1988). However, urinary excretion of daunomycin has been observed in mice and rats (Finkel et al., 1969); the drug is accumulated in the kidney after intravenous administration (Bachur et al., 1970) and is metabolized by several tissues primarily to daunorubicinol (Bachur and Gee, 1971), which also is excreted in the urine of humans (Bachur, 1971). In the rabbit, in which the pharmacokinetics of doxorubicin have been found to be closest to those in humans (Koren et al., 1992), the highest tissue levels of doxorubicin and doxorubicinol were found in the kidneys (Bachur, 1975).

The studies presented here were therefore designed to clarify the transport mechanism of doxorubicin in the proximal tubular cells and in particular to examine the effects of a series of compounds that are known to interact with P-glycoprotein or to interfere with the transporters of organic anions and cations in renal tubules. We have used the LLC-PK₁ cell line, derived from pig kidney (Hull et al., 1976), which has structure and function similar to those of renal proximal tubular cells (Handler et al., 1980). The LLC-PK₁ cells form an oriented monolayer with microvilli and tight junctions and have been shown to express P-glycoprotein (Horio et al., 1990).

	Materials and Methods

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Cell cultures. LLC-PK₁ cells obtained from the American Type Culture Collection (Rockville, MD) (ATCC-CRL-1392) were grown in medium 199 containing 3% fetal bovine serum without antibiotics under an atmosphere of 95% air and 5% CO₂ at 37°C, and subcultured twice weekly with 0.02% EDTA and 0.05% trypsin. The cells were used in the passages 199 to 230. For experiments, 35-mm dishes were inoculated with 2 × 10⁴ cells/ml in 2 ml of complete culture medium. Unless otherwise stated, the uptake of doxorubicin was measured on confluent cells on the 5th day after inoculation.

Microscopic studies. LLC-PK₁ cells were seeded in Leighton tubes at a cell density of 2 × 10⁴ cells/ml in 1 ml of complete culture medium, and the uptake of doxorubicin was observed on confluent cells, on the 5th day after inoculation. Cells on glass slides were examined under a Zeiss Axioskop microscope; the instrument contained two illumination sources, a tungsten bulb for bright-field observation and a mercury lamp (Osram HBO 100W, Germany) for epifluorescence examination. The fluorescence setting was equipped with fluorescein/rhodamin optics. Cells were studied using a ×100 Plan-Neofluar N.A. 1.30 objective at oil immersion, by alternative bright field and fluorescence observations.

	Results

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First, doxorubicin accumulation in LLC-PK₁ cells was examined as a function of growth in culture. As shown in figure 1, on day 3, when the cells were subconfluent, doxorubicin uptake was relatively low, and a marked increase in the uptake was observed on day 4 when the cells reached a confluent monolayer. After the development of confluence, the uptake was steady until day 6 and then started to decrease. Based on the above findings, all subsequent uptake studies were carried out with the LLC-PK₁ cells on day 5. 

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Effect of culture time on doxorubicin uptake. Cells were plated in 35-mm wells at a density of 2 · 104 cells/ml in 2 ml of complete culture medium and were cultured for up to 11 days. On the day indicated in the graph, cells were incubated with 50 µM doxorubicin in complete culture medium for 1 hr. Each point represents mean ± S.E. of data from three to six wells.

Figure 2 shows the time course of doxorubicin uptake at 37 and 4°C for up to 4 hr by renal tubular cells. At 37°C, doxorubicin uptake reached the equilibrium within about 4 hr. At 4°C, the uptake increased with time but was drastically lower than that at 37°C. Thus, temperature is a very important determinant for doxorubicin uptake.

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Effect of temperature on doxorubicin uptake. Cells were incubated with 50 µM doxorubicin in complete culture medium for up to 4 hr at 37°C () or 4°C (). Each point represents mean ± S.E. of data from three to six wells.

Figure 3 shows the relationship between drug concentration and the initial doxorubicin uptake (15 min). At 4°C the relation was linear; when the experiments were carried out at 37°C, the uptake of doxorubicin was greater and showed a trend for saturation. According to other reports (Gachot et al., 1991; Takano et al., 1992), linear accumulation at 4°C and saturable accumulation at 37°C appear to correspond with uptakes by simple diffusion and by the sum of simple diffusion and carrier-mediated transport, respectively; thus the subtraction of doxorubicin uptake at 4°C from that at 37°C (dotted line in fig. 3) suggested a saturable process in doxorubicin uptake. The curve obtained from subtraction of doxorubicin uptake at 4°C from that at 37°C was analyzed further by the Lineweaver-Burk plot (fig. 4), and a linear relationship was clearly observed.

View larger version (23K): [in this window] [in a new window]   Fig. 3.   Concentration dependence of doxorubicin uptake. Cells were incubated with different concentrations of doxorubicin in complete culture medium at 37°C () or 4°C () for 15 min. The dotted line indicates the difference of doxorubicin uptake at 37°C and 4°C. Each point represents mean ± S.E. of data from three to six wells.

View larger version (13K): [in this window] [in a new window]   Fig. 4.   The saturable uptake shown in figure 3 was plotted by the Lineweaver-Burk plot. The parameters (Km, Vmax) were calculated. The correlation coefficient was 0.999.

We next studied the kinetics of doxorubicin efflux from LLC-PK₁ cells cultured for 2 hr with 50 µM doxorubicin and, after washing to remove free doxorubicin, incubated for various times in drug-free medium. As shown in figure 5, doxorubicin was released slowly, and 75.7 ± 1.7% remained associated with the cells after 180 min, which suggests a strong cellular binding or trapping. The fluorescence microscopy observations revealed that doxorubicin was trapped and concentrated in intracellular organelles; in particular the fluorescent drug was confined to cytoplasmic perinuclear localization, with a faint fluorescence in the nucleus (fig. 6).

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Kinetics of doxorubicin release from LLC-PK1 cells incubated in complete medium containing 50 µM doxorubicin. After 2 hr of incubation the cells were washed with ice-cold PBS to remove free doxorubicin (time 0, total uptake) and incubated at 37°C () or 4°C () in fresh PBS buffer. Doxorubicin remaining in the cells was measured at determined times. Each point represents mean ± S.E. of data from three to six determinations from a typical experiment.

View larger version (124K): [in this window] [in a new window]   Fig. 6.   Fluorescence photomicrograph showing doxorubicin distribution in LLC-PK1 cells exposed to 50 µM drug for 2 hr. Calibration bar, 5 µm.

Doxorubicin is a well-known substrate for P-glycoprotein; hence, the effect of several MDR reversing agents on the concentration of the drug was studied. When cells were preincubated with 500 µM verapamil, doxorubicin concentration increased significantly to approximately 3-fold; other P-glycoprotein substrates, vinblastine (100 µM), vincristine (100 µM) and quinidine (200 µM), also significantly increased doxorubicin concentration (fig. 7).

View larger version (38K): [in this window] [in a new window]   Fig. 7.   Effect of 500 µM verapamil (), 100 µM vinblastine (), 100 µM vincristine () and 200 µM quinidine () on doxorubicin uptake in LLC-PK1 cells. The cells were preincubated in medium containing the inhibitors at 37°C for 30 min, then in medium containing 50 µM doxorubicin and the inhibitors for 30 additional min. Each point represents mean ± S.E. of data from three to six determinations from a typical experiment. *P < .05; **P < .01; Student's t test for independent data.

Transport mediated by the MDR transporter depends on cellular metabolism and thus should be sensitive to metabolic inhibitors. Figure 8 shows the effect of 2,4-dinitrophenol (4 mM) and sodium metavanadate (10 µM) on doxorubicin uptake by LLC-PK₁ cells. The substances significantly increased the concentrations of doxorubicin in the cells.

View larger version (32K): [in this window] [in a new window]   Fig. 8.   Effect of the metabolic inhibitors 10 µM sodium metavanadate () and 4 mM 2,4-dinitrophenol () on doxorubicin uptake in LLC-PK1 cells. The cells were preincubated in PBS containing the inhibitors at 37°C for 30 min, then in PBS containing 50 µM doxorubicin and the inhibitors for 30 additional min. Each point represents mean ± S.E. of data from three to six determinations from a typical experiment. **P < .01; Student's t test for independent data.

The effect of classic organic cation transport drugs, such as cimetidine (200 µM) and the cyanine derivatives decynium 22 (5 µM) and decynium 24 (5 µM), and of the organic anion drug probenecid (50 µM) also was tested, but none of the substances modified doxorubicin concentrations in LLC-PK₁ cells (fig. 9).

View larger version (49K): [in this window] [in a new window]   Fig. 9.   Effect of the organic anion 50 µM drug probenecid () and of the organic cation drugs 200 µM cimetidine (), 5 µM decynium 22 (), and 5 µM decynium 24 () on doxorubicin uptake in LLC-PK1 cells. The cells were preincubated in medium containing the inhibitors at 37°C for 30 min, then in medium containing 50 µM doxorubicin and the inhibitors for 30 additional min. Each point represents mean ± S.E. of data from three to six determinations from a typical experiment.

	Discussion

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P-glycoprotein is expressed in renal tissue and in particular in the proximal tubular epithelium. Charuk et al. (1994) showed that the anthracyclines doxorubicin and daunorubicin block ³H-azidopine photolabeling of renal P-glycoprotein. The relative affinities (half-maximal inhibition constant, 10 µM) of the anthracyclines were quite low, but high tissue, plasma and urinary levels attained during chemotherapy probably allow some interactions of these drugs with renal P-glycoprotein. Therefore we studied the uptake characteristics of doxorubicin in the LLC-PK₁ cell line, a cell line derived from the pig kidney (Hull et al., 1976) that possesses structure and function similar to those of renal proximal tubules (Handler et al., 1980) and expresses the P-glycoprotein (Horio et al., 1990). A transepithelial drug transport pathway involving P-glycoprotein was demonstrated in several kidney-derived cell lines (Horio et al., 1989, 1990, 1991). In particular, several authors showed that, in a canine kidney cell line (MDCK) carrying the human MDR-1 gene, P-glycoprotein is expressed in a polarized manner similar to the kidney (Pastan et al., 1988) and plays a role in the transepithelial transport of anticancer agents such as vinblastine, vincristine, daunorubicin, actinomycin D and verapamil (Horio et al., 1989). Several other studies were conducted in renal cell cultures transfected with the MDR-1 cDNA (Ito et al., 1993; Pan et al., 1994; Saeki et al., 1993a, b; Tanaka et al., 1997). These transfectant cells have overexpressed human P-glycoprotein on the apical membrane. However, in these cells the levels of the protein are extremely high, whereas to evaluate the possible clinical effects of modulators, cells with a physiological level of expression of P-glycoprotein should be used. In our study we used the wild-type of LLC-PK₁ cells, that, similarly to normal mammalian proximal renal cells, express endogenous P-glycoprotein in low amounts.

When doxorubicin uptake was studied as a function of growth in culture, it was shown that the uptake strictly depends on cell growth and was maximal when cells reached a confluent monolayer, which suggests that the increased doxorubicin uptake corresponds to attainment of a confluent cell density and to the development of functional properties for apical membranes of the LLC-PK₁ cells.

The transport of doxorubicin in various neoplastic cell types has been studied; however, how the drug enters epithelial cells such as the renal proximal tubule cells is not yet completely clear. In cancer cells, the main route of entry is passive diffusion across the plasma membrane of the uncharged form of the drug, which is a weak base with a pKa of 8.3 (Frezard and Garnier-Suillerot, 1991; Harrigan et al., 1993; Madden and Redelmeier, 1994); however, differences in the specific parameters of membrane transport systems between tumor and normal cells are likely and are suspected to be associated closely with the determinants of cytotoxicity. In our study with the LLC-PK₁ cells, the subtraction of doxorubicin uptake at 4°C from that at 37°C suggested a saturable process and so was analyzed by the Lineweaver-Burk plot. A linear relationship was observed, which suggests a carrier-mediated transport. The values of Michaelis constant (K_m) and maximum uptake rate (V_max) were estimated with the aid of a computer to be 0.779 nmol doxorubicin/mg protein/min and 319.96 µM respectively. In addition, doxorubicin was accumulated against the concentration gradient, and, assuming the intracellular volume of LLC-PK₁ cells as 7 µl/mg of protein (Saito et al., 1986), the cellular concentration of doxorubicin at the equilibrium was approximately 50-fold compared with external concentrations.

The concentration-dependent uptake, along with the temperature dependent transport is evocative of the presence of a specific mechanism, such as facilitated diffusion; however, nonspecific factors also can be invoked as alternative explanations. Indeed, the relative increase in the rate of drug uptake as the temperature is raised by 10°C (the Q₁₀), in LLC-PK₁ cells is <2; this value is in the range expected for passive diffusion, whereas it is >6 for facilitated diffusion or active transport (Dalmark and Storm, 1981). On the other hand, doxorubicin could enter cells by passive diffusion and the concentration dependence can, for example, originate from the saturation of nonspecific binding sites on the cell membrane, or from ion trapping in acidic compartments of the cell. The fluorescence microscopy observations agree with this latter hypothesis. The fluorescence is indeed primarily punctate, an observation that is indicative of doxorubicin localization to intracellular organelles, primarily Golgi and lysosomes. The fluorescent drug is confined to cytoplasmic perinuclear localization, whereas the nucleus presents a fainter fluorescence. This picture is very similar to that observed by various authors in doxorubicin-resistant cancer cell lines (Coley et al., 1993; de Lange et al., 1992; Rutherford and Willingham, 1993; Simon and Schindler, 1994), and in these cells it has been suggested that trapping of doxorubicin in intracellular organelles decreases drug concentration in the cytoplasm and in the nucleus, and facilitates cell survival. Nephrotoxicity is not a major side effect of anthracyclines; doxorubicin usually induces a glomerular damage characterized by proteinuria, ipoproteinemia and peripheral edemas in animals (Burke, 1977; Hayashi et al., 1984). It has been suggested that the drug also can induce a tubular toxicity (Landwehr et al., 1977); however, the effects on tubular cells are usually not very important. In addition, in vitro studies have shown that fragments of proximal tubule are less sensitive to the cytotoxic effects of doxorubicin than glomerular tissue (Kastner et al., 1990, 1991).

When the efflux of doxorubicin after incubation for 2 hr with 50 µg/ml was studied, more than 75% remained associated with the cells after 180 min, which confirms the very strong cellular binding or trapping in intracellular cell compartments.

To elucidate the role of P-glycoprotein, which is expressed in LLC-PK₁ cells, in the handling of doxorubicin, cells were treated with high concentrations of agents that interfere with the transport by this protein. The tested substances verapamil, vinblastine, vincristine and quinidine significantly increased doxorubicin concentrations, which suggests therefore that in this cell line, P-glycoprotein could play a role in the extrusion of the antineoplastic drug.

Because P-glycoprotein actively pumps out drugs in an ATP-dependent manner, higher uptake could be expected in LLC-PK₁ cells in the presence of metabolic inhibitors, and this was indeed the case. The effect was particularly evident with sodium metavanadate; as a matter of fact, P-glycoprotein belongs to a family of ATPases that are particularly sensitive to this substance.

The localization of P-glycoprotein in the brush-border membranes of proximal tubular cells and the observation that many P-glycoprotein substrates are organic cations, led to the hypothesis of a physiological role for the kidney P-glycoprotein in the energy-dependent renal secretion of organic cations (Nelson, 1988). Indeed, the mammalian kidney, like P-glycoprotein, has pleiotropic drug transport capacity, best characterized by the organic cation and anion secretory systems. These systems are considered distinct because probenecid inhibits the anion carrier, and cimetidine inhibits the cation carrier. Therefore it was decided to test the effect of probenecid, an organic anion transport inhibitor, and the effect of cimetidine and of two cyanine derivatives, decynium 22 and decynium 24, which recently have been shown to exert a potent inhibitory effect on the renal transport of organic cations (Schömig et al., 1993) and were extremely effective in reducing the tubular uptake of L-DOPA (Pinto-do-Ó and Soares-da-Silva, 1996), but none of the tested substances interfered with the uptake of doxorubicin.

Our studies were conducted on cells reaching a confluent monolayer and hence it is very probable that the entrance via simple diffusion occurs across the luminal membrane; similarly, the efflux also probably is limited to the luminal site where the P-glycoprotein is located. Luminal entrance of doxorubicin in the readsorptive direction after glomerular filtration was demonstrated in dogs (Daoud and Huang, 1988), and the authors suggested that both readsorption and secretion of the drug occurred in the proximal tubular section. Hence, it seems that P-glycoprotein could have an important protecting role for cells lining the nephron.

In conclusion, doxorubicin enters LLC-PK₁ probably by passive diffusion and is then trapped and accumulated into intracellular organelles. P-glycoprotein seems to be very important in extruding doxorubicin from these cells, even if the levels of the protein expressed in wild-type LLC-PK₁ as well as in mammalian proximal tubules are quite low. On the contrary, the renal organic anion and cation secretory system is not involved in doxorubicin renal handling. It remains to be elucidated if interactions between doxorubicin and P-glycoprotein substrates also could occur in clinical practice, hence increasing the risk of drug-induced nephrotoxicity.