Cadmium Inhibits Albumin Endocytosis in Opossum Kidney Epithelial Cells

Toxicology and Applied Pharmacology 161, 146 –152 (1999) Article ID taap.1999.8797, available online at http://www.idealibrary.com on

Cadmium Inhibits Albumin Endocytosis in Opossum Kidney Epithelial Cells Jong Soo Choi, Kyoung Ryong Kim, Do Whan Ahn, and Yang Saeng Park Department of Physiology, Kosin Medical College, Pusan, Korea 602-030 Received March 26, 1999; accepted September 14, 1999

Cadmium Inhibits Albumin Endocytosis in Opossum Kidney Epithelial Cells. Choi, J. S., Kim, K. R., Ahn, D. W., and Park, Y. S. (1999). Toxicol. Appl. Pharmacol. 161, 146 –152. Chronic exposure to cadmium results in proteinuria. To gain insights into the mechanism by which cadmium inhibits the protein transport in the renal proximal tubule, we investigated the effects of cadmium on the receptor-mediated endocytosis of albumin, using fluorescein isothiocyanate-labeled bovine serum albumin (FITC-albumin) as a model substrate and opossum kidney cell line (OK cell) as a proximal tubular cell model. Cell monolayers grown to confluence were treated with 100 mM CdCl 2 for 60 min at 37°C, washed, and tested for FITC-albumin uptake (37°C) and surface binding (4°C). The amounts of FITC-albumin uptake and binding were quantified by fluorimetrically determining the cell-adherent fluorescence. Both the binding and uptake of FITCalbumin by OK cells appeared to be saturable and inhibitable by unlabeled albumin in the medium, indicating that specific receptor sites were involved. The uptake of FITC-albumin was inhibited by agents that interfere with the formation of endocytotic vesicle (hypertonic mannitol), endosomal acidification (NH 4Cl), and vesicular trafficking (cytochalasin D and nocodazole), confirming that the uptake occurred via the process of receptor-mediated endocytosis. In cells treated with cadmium, the specific FITCalbumin uptake was significantly attenuated, and this was due to a reduction in V max and a rise in K m. These changes in kinetic parameters were similar to those induced by NH 4Cl. The binding of FITC-albumin to the apical surface of OK cells was inhibited by cadmium treatment, and this was attributed to a reduction in B max. The values of K d and its pH dependency were not altered by cadmium treatment. The formation of endocytotic vesicles, as judged by fluid phase endocytosis of FITC-inulin, was not changed by cadmium treatment. These results indicate that the receptor-mediated endocytosis of albumin is impaired in cadmium-treated OK cells most likely due to a defect in endosomal acidification and the attendant fall in ligand-receptor dissociation, which impairs receptor recycling and the overall efficiency of endocytosis. © 1999 Academic Press Key Words: albumin; endocytosis; kidney; cell cultures; cadmium.

Cadmium is an important occupational and environmental pollutant generated in zinc and lead mines (Tsuchiya, 1978), metallurgical and plating industries, and manufacturing pro0041-008X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.

cesses of various pigments and compounds (Schroeder, 1965; Flick et al., 1971). Humans are exposed to cadmium via food, water, air, and dust. Chronic exposure to cadmium via inhalation or ingestion may result in renal functional changes. Proteinuria, glycosuria, aminoaciduria, and phosphaturia are among the most prominent changes (Friberg, 1950; Kazantzis et al., 1963; Axelsson and Piscator, 1966; Piscator, 1966; Adams et al., 1969; Goyer et al., 1972; Nordberg and Piscator, 1972; Nomiyama et al., 1973, 1975, 1982; Gieske and Foulkes, 1974; Bernard et al., 1979, 1981; Iwao et al., 1980; Kim et al., 1988; Mason et al., 1988). To elucidate the mechanisms underlying these changes, we have carried out a series of experiments using animal models and found that the cadmiuminduced glycosuria, aminoaciduria, and phosphaturia are attributed to an alteration of sodium-dependent cotransport systems for glucose, amino acids, and inorganic phsophate in the proximal tubular brush-border membranes (Kim et al., 1990; Lee et al., 1990, 1991; Ahn and Park, 1995; Kim and Park, 1995; Park et al., 1997). With respect to the mechanism of cadmium-induced proteinuria, no systematic studies have been conducted yet. Therefore, in the present study, we evaluated the effect of cadmium on the renal protein transport system. Since the basic mechanism for protein transport in the renal tubular cells is the receptor-mediated endocytosis (Maack et al., 1992), we investigated the effects of cadmium on the process of receptor-mediated endocytosis of protein, using albumin as a model substrate, in opossum kidney (OK) cell cultures. The OK cell is a continuous cell line derived from the proximal tubule of American opossum kidney (Koyama et al., 1978). When grown to confluence, these cells express many characteristics of proximal tubular epithelia, including albumin transport, and thus have been widely used as a model system to study renal tubular protein transport (Schwegler et al., 1991; Gekle et al., 1995a,b, 1996, 1997; Brunskill et al., 1996). MATERIALS AND METHODS Cell culture. OK cell line was obtained from the American Type Culture Collection (ATCC) and maintained by serial passages in 75-cm 2 plastic culture flasks (Corning, NY). The cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum and 1% antibiotic/antimycotic solution (Gibco) in an atmosphere of 5% CO 2–95% air at

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37°C (Montrose and Murer, 1990). The culture was fed with fresh medium every 3 days. When cell growth reached saturation density, subcultures were prepared by 0.5% trypsin– 0.2% ethylenediaminetetraacetic acid (EDTA) treatment in Ca 21- and Mg 21-free Hank’s balanced salt solution (HBSS, Gibco) for 5–10 min at 37°C. The cells were suspended by gently shaking and seeded in 12-well plates at 1:10 dilution with culture medium. Confluent cell monolayers obtained after 7 days in culture were used for the experiments. All experiments were performed on passages 85–105. Determination of cell viability. Monolayers of OK cells were detached by incubating them in Ca 21- and Mg 21-free HBSS containing 0.5% trypsin and 0.2% EDTA for 5–10 min at 37°C. The viability was assessed by counting viable cells in the presence of trypan blue using hemocytometer and quantified by measuring the amount of protein in the viable cells after treatment with detergents (0.1% Triton X-100) (Baldew et al., 1992). The protein concentration was determined by the method of Bradford (1976) using the Bio-Rad Protein Assay Kit, with bovine b-globulin as a standard. Uptake and binding experiments. Cell monolayers were washed twice with HBSS and then exposed to DMEM medium containing CdCl 2 (100 mM) for 60 min in 5% CO 2-95% air (pH 7.4) at 37°C. Upon completion of the exposure, the monolayers were washed three times with Ringer’s solution (in mM: 122.5 NaCl, 5.4 KCl, 1.2 CaCl 2, 0.8 MgCl 2, 0.8 Na 2HPO 4, 0.2 NaH 2PO 4, 5.5 glucose, and 10 HEPES, pH 7.4) to remove the residual drugs. In control experiments, monolayers were exposed to the vehicle (DMEM medium). For uptake study, cell monolayers were incubated with serum-free Ringer solution containing FITC-albumin or FITC-inulin at 37°C in 5% CO 2 atmosphere for an appropriate time period. Incubation was stopped by rinsing the monolayer eight times with 2 ml ice-cold Ringer solution. The cells were disintegrated by detergents (Triton X-100, 0.1% v/v in 3-(N-morpholino) propanesulfonic acid [Mops] solution, 20 mM, pH 7.4). The cell-adherent fluorescence was measured using a spectrofluorometer (Hitachi 650 –10S, xenon lamp) at an excitation wavelength of 480 nm and an emission wavelength of 520 nm, according to Schwegler et al. (1991). Binding of FITC-albumin to the plasma membrane was determined by incubating cell monolayers at ;4°C, according to Gekle et al. (1994, 1995a, 1996). Cell monolayers were washed three times with pH 6 Ringer solution and then incubated in Ringer solution containing FITC-albumin on ice for 45 min. Gekle et al. (1994) have shown that at this low temperature the substrates bind to plasma membrane but are not internalized. Unbound FITC-albumin was removed by rinsing eight times with ice-cold Ringer solution. Nonspecific binding was determined by including 200-fold excess of unlabeled bovine serum albumin (BSA). To ensure that incubation at 4°C led to binding of FITC-albumin and incubation at 37°C to endocytotic uptake, binding and uptake experiments were also performed on cells grown on glass coverslips. Cells were fixed with 3:1 methanol– glacial acetic acid and examined for fluorescence under the fluorescence microscope (Nikon Labophot, oil immersion). Incubation at 4°C led to staining of the plasma membrane only, whereas after incubation at 37°C most of fluorescence was found inside the cell, as observed by others (Gekle et al., 1994, 1995). Chemicals. DMEM, fetal bovine serum, trypsin–EDTA, and antibacterial/ antimycotic solution were purchased from Gibco (Grand Island, NY). We obtained FITC-albumin, FITC-inulin, cytochalasin D, nocodazole, CdCl 2, and Mops from Sigma Chemicals (St. Louis, MO). All other chemicals were of analytical grade. Statistical analysis. Statistical evaluation of data was done by the Student’s t test and covariance analysis. The differences with p , 0.05 were considered statistically significant.

RESULTS

Characterization of Albumin Uptake by OK Cells Figure 1 shows the time courses of FITC-albumin uptake by OK cells at 37°C. FITC-albumin uptake rose linearly for the

FIG. 1. Time courses of FITC-albumin uptake by OK cells. The uptake was determined at 37°C. The medium concentration of FITC-albumin was 30 mg/l. FITC-albumin uptake was determined both in the absence and presence of 200-fold excess of unlabeled bovine serum albumin. Data represent the means 6 SE of four determinations. The SE bars smaller than the symbols are not illustrated.

initial 30 min and then gradually leveled off after 2 h. Nonspecific uptake of FITC-albumin determined in the presence of 200-fold excess of unlabeled albumin also changed with time, but the amount of steady state uptake was less than 15% of that without unlabeled albumin. These results indicate that the major portion of albumin uptake in OK cells is mediated by specific receptors and a small amount of nonspecific uptake is due to a trapping of extracellular fluid by fluid phase endocytosis. The difference between FITC-albumin uptakes in the absence and presence of unlabelled albumin is, therefore, a measure of the specific receptor-mediated albumin uptake. In order to confirm that the FITC-albumin uptake is achieved via receptor-mediated endocytosis, we examined the effects of various agents that are known to alter the process of receptormediated endocytosis on FITC-albumin uptake. The results summarized in Fig. 2 indicate that the initial (20 min) rate of FITC-albumin uptake was significantly attenuated by hypertonic (750 mOsm/l) mannitol, which interferes with clathrincoated pit formation (Heuser and Anderson, 1989); by NH 4Cl, a weak base that accumulates in acidic endocytic vesicles and prevents vesicular acidification; by cytochalasin D, an actincytoskeleton depolymerizer; and by nocodazole, a microtubule disrupter that impair vesicle trafficking, as observed by others in OK cells (Schwegler et al., 1991; Gekle et al., 1997). Effect of Cadmium Treatment on Albumin Uptake by OK Cells In order to investigate the effect of cadmium on albumin endocytosis, OK cell monolayers were first preincubated in media containing various concentrations (10, 25, 50, 100, 200, and 400 mM) of CdCl 2 for 60 min at 37°C, washed, and then tested for albumin uptake. Figure 3 shows that FITC-albumin uptake was progressively inhibited as the CdCl 2 concentration in the preincubation medium increased from 25 to 100 mM,

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FIG. 2. Effects of hypertonic medium, NH 4Cl, cytochalasin D, and nocodazole on FITC-albumin uptake by OK cells. The FITC-albumin uptake was significantly inhibited by incubation in a hypertonic (750 mOsm/l) medium, a maneuver that interferes with clathrin-coated vesicle formation and in media containing NH 4Cl (20 mM), a weak base that prevents vesicular acidification; cytochalasin D (5 mg/l), an actin-cytoskeleton depolymerizer; and nocodazole (6 mg/l), a microtubule disrupter. The medium concentration of FITC-albumin was 30 mg/l. Data represent the means 6 SE of six determinations. *Significantly (p , 0.05, unpaired t test) different from the control.

above which no further inhibition was observed. The viability of the cell judged by trypan blue staining was not apparently changed by cadmium up to 200 mM. On the basis of these results, we fixed the cadmium concentration in the preincubation medium at 100 mM in the following experiments. This concentration of cadmium is similar to that of unbound cadmium (13 mg/g, ;115 mM) measured in the renal cortical tissues of cadmium-exposed rabbits at the time of onset of proteinuria (Nomiyama and Nomiyama, 1986). Figure 4 depicts initial (20 min) rates of FITC-albumin and FITC-inulin uptake by control and cadmium-treated cells as a function of substrate concentration. In both control and cadmium group, the uptake of FITC-albumin displayed saturation

FIG. 4. Initial rates of FITC-albumin and FITC-inulin uptake by control and cadmium-treated OK cells at various substrate concentrations. In the cadmium group, cell monolayers were preincubated in a medium containing 100 mM CdCl 2 for 60 min at 37°C, washed, and incubated in substrate containing medium for 20 min. Note that the uptake of FITC-albumin tends to saturate at high substrate concentrations, whereas that of FITC-inulin, a marker for fluid-phase endocytosis, changes linearly with the substrate concentration. Data represent the means 6 SE of six determinations. The SE bars smaller than the symbols are not illustrated. *Significantly (p , 0.05, unpaired t test) different from the corresponding control value.

kinetics, whereas that of FITC-inulin, a marker for fluid phase endocytosis, showed a linear dose–response relationship. The cadmium (100 mM) treatment significantly attenuated FITCalbumin uptake, but it had no effect on FITC-inulin uptake, indicating that the receptor-mediated endocytosis was specifically impaired. The amount of receptor-mediated FITC-albumin uptake was estimated by subtracting the uptake by fluidphase endocytosis measured in the presence of 200-fold excess of unlabeled albumin from the total uptake at each substrate concentration. Hofstee plots of the data (Fig. 5) indicated that V max was reduced, but K m was increased in cadmium group. These changes in kinetic parameters were similar in direction to those observed in NH 4Cl-treated cells (Fig. 6). To evaluate if the effect of cadmium on the albumin uptake was specific, we also studied effects of various other heavy metals. OK cell monolayers were preincubated in a medium containing 100 mM CdCl 2, ZnCl 2, Pb(CH 3COO) 2, or CuSO 4 for 60 min and then tested for FITC-albumin uptake. The results summarized in Table 1 indicated that the FITC-albumin uptake was markedly reduced in Cd-treated cells but not in the cells exposed to other heavy metals. Effect of Cadmium Treatment on Albumin Binding to the Apical Membrane of OK Cells

FIG. 3. FITC-albumin uptake by OK cells as a function of CdCl 2 concentration in the preincubation medium. OK cell monolayers were first preincubated in media containing various concentrations (10, 25, 50, 100, 200, and 400 mM) of CdCl 2 for 60 min at 37°C, washed, and then incubated in an uptake medium containing FITC-albumin (30 mg/l) for 20 min at 37°C. Data represent the means 6 SE of six determinations. All values for CdCl 2 concentrations above 25 mM are significantly (p , 0.05, unpaired t test) different from the control.

Figure 7 depicts the effect of cadmium treatment on FITCalbumin binding to OK cells. The data represent steady-state binding determined for 60 min at 4°C. In both the control and cadmium-treated cells, the FITC-albumin binding increased curvilinearly with the FITC-albumin concentration in the medium, providing evidence for saturability. The value of binding

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TABLE 1 Effects of Heavy Metals on FITC-Albumin Uptake by OK Cells Heavy metal Control 100 mM 100 mM 100 mM 100 mM

FIG. 5. Changes in kinetics of FITC-albumin uptake in cadmium-treated OK cells. Receptor-mediated component of FITC-albumin uptake was obtained by subtracting the fluid-phase endocytotic component measured in the presence of 200-fold excess of unlabeled albumin from the total uptake at each substrate concentration, and the values are presented as Hofstee plots. The units for v and S are mg/mg protein/20 min and mg/ml, respectively. Data are based on Fig. 4. The SE bars smaller than the symbols are not illustrated. The regression lines for the control and cadmium group are significantly different (p , 0.05, analysis of covariance) both in the slope (K m, 52 6 11 for control; 84 6 5 for Cd group) and the y-intercept (V max, 2.21 6 0.27 for control; 1.59 6 0.06 for Cd group).

CdCl 2 ZnCl 2 Pb(CH 3COO) 2 CuSO 4

Uptake (mg/mg/20 min) 0.693 6 0.029 0.405 6 0.032* 0.609 6 0.088 0.645 6 0.022 0.665 6 0.044

Note. OK cell monolayers were preincubated in a medium containing 100 mM CdCl 2, ZnCl 2, Pb(CH 3COO) 2, or CuSO 4 for 60 min at 37°C, washed, and then incubated in an uptake medium containing 30 mg FITC-albumin for 20 min at 37°C. Data represent the means of six determinations 6 SE. *Significantly ( p , 0.05, unpaired t test) different from the control.

at a given concentration was, however, significantly lower in cadmium-treated cells than in controls. Nonspecific binding of FITC-albumin determined by including 200-fold excess of unlabeled albumin changed linearly with the medium concentration of FITC-albumin in both the control and cadmium group with an identical slope. Thus, only the specific binding appeared to be inhibited by cadmium treatment. Scatchard plot of the specific bindings estimated by subtracting the nonspecific binding from the total binding at each substrate concen-

tration yielded a single regression line in both the control and cadmium group (Fig. 8), which indicates that a single type of binding site was involved. Figure 8 also shows that cadmium treatment attenuated B max (maximum binding capacity), but had no effect on K d (apparent dissociation constant). Figure 9 compares pH effects on albumin binding kinetics in the control and cadmium-treated OK cells. Lowering the extracellular pH from 7.4 to 5.0 caused an increase in K d with no change in B max of albumin binding, which is in accordance with a previous report (Gekle et al., 1996). This pattern of pH dependency was not altered by cadmium treatment. At any pH, the K d value was not significantly different between the control and cadmium-treated cells, and the B max was consistently lower in the latter than in the former. These data suggest that, although the number of binding sites were reduced, their substrate affinity and its pH dependency were not altered by cadmium treatment.

FIG. 6. Effect of NH 4Cl on kinetics of FITC-albumin uptake by OK cells. Receptor-mediated components of FITC-albumin uptake determined in the absence and presence of 20 mM NH 4Cl were presented as Hofstee plots. The units for v and S are mg/mg protein/20 min and mg/ml, respectively. Data represent the means 6 SE of six determinations. The SE bars smaller than the symbols are not illustrated. The regression lines for the control and NH 4Cl group are significantly different (p , 0.05, analysis of covariance) both in the slope (K m, 52 6 11 for control; 124 6 11 for NH 4Cl group) and the y-intercept (V max, 2.21 6 0.27 for control; 1.31 6 0.08 for NH 4Cl group).

FIG. 7. Changes in albumin binding to the apical surface of control and cadmium-treated OK cells as a function of substrate concentration. Steadystate (60 min) bindings of FITC-albumin determined at various FITC-albumin concentrations (10 –200 mg/l) in the absence (total binding) and presence (nonspecific binding) of 200-fold excess of unlabeled albumin are plotted against the substrate concentration. The incubation temperature was 4°C. Data represent the means 6 SE of four determinations. The SE bars smaller than the symbols are not illustrated. *Significantly (p , 0.05, unpaired t test) different from the corresponding control value.

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FIG. 8. Changes in albumin binding kinetics in cadmium-treated OK cells. The specific component of FITC-albumin binding was computed by subtracting the nonspecific component from the total binding at each concentration and presented as Scatchard plots. Data are based on Fig. 7. The SE bars smaller than the symbols are not illustrated. The regression lines for control and cadmium group are significantly different (p , 0.05, analysis of covariance) in the x-intercept (B max, the maximal binding capacity, 0.706 6 0.015 for control; 0.577 6 0.027 for Cd group) but not in the slope (K d, the apparent dissociation constant, 45.4 6 1.7 for control; 48.1 6 4.1 for Cd group).

DISCUSSION

During the process of ultrafiltration in the kidney, a substantial amount of plasma albumin reaches the proximal tubular lumen. It has been estimated that 500-5000 mg of albumin is filtered per day in humans (Gekle et al., 1996). The urinary excretion of albumin amounts to only 30 –100 mg/day (Gekle, 1996), thus the major fraction of filtered albumin is reabsorbed in the renal tubule. The mechanism of albumin reabsorption is an endocytosis in the proximal tubule (Maunsbach, 1966; Park and Maack, 1984; Clapp et al., 1988; Ishidate et al., 1992). Following uptake by the tubular cell, albumin undergoes intracellular trafficking through a number of vesicular structures culminating in its entry into lysosome, where it is broken down to amino acids and small peptides (Park and Maack, 1984; Maack et al., 1992). Recent studies by Schwegler et al. (1991) and Gekle et al. (1995a,b, 1996, 1997) have shown that the opossum kidney proximal tubule-derived cell line (OK cell) is a suitable model for investigating the albumin endocytosis. These studies have shown that the endocytotic uptake of albumin in OK cells occurs via a receptor-mediated mechanism from the apical (not the basolateral) surface. A receptor-mediated endocytosis is known to proceed with the following sequence (Mellman, 1987; Lodish et al., 1995): the ligand binds to a specific receptor on the plasma membrane. The receptor–ligand complex is internalized in a clathrincoated pit that pinches off to become a coated vesicle. The clathrin coat is then depolymerized to triskelions, resulting in an early endosome. This endosome fuses with a sorting vesicle, known as CURL or late endosome, which contains V-class H 1–ATPase (Melman et al., 1986), where the low pH causes the ligand particle to dissociate from the receptor. A receptorrich region buds off to form a separate vesicle that recycles the

receptors back to the plasma membrane. The remaining portion of the vesicle containing ligand particles (transport vesicle) ultimately fuses with a lysosome to form a large lysosome where the endocytosed molecule is hydrolyzed. The FITC-albumin uptake by OK cells in the present study was consistent with a receptor-mediated endocytosis. As depicted in Fig. 2, the uptake was inhibited by extracellular hypertonicity, a condition that interferes with the formation of clathrin lattice (Heuser and Anderson, 1989; Wang et al., 1993); by NH 4Cl, a compound that interferes with vesicular acidification (Gekle et al., 1995b; Batuman and Guan, 1997); and by cytochalasin D and nocodazole, agents that impair vesicle trafficking by disrupting actin-cytoskeltons and microtubules, respectively (Gottlieb et al., 1993; Brown and Stow, 1996; Gekle et al., 1997). The present study clearly demonstrated that the albumin endocytosis is inhibited in cells exposed to inorganic cadmium (Figs. 3 and 4). Such an inhibition may be resulted from 1) an impaired ligand-receptor interaction at the cell surface, 2) a decrease in clathrin-coated vesicle formation, 3) defects in intracellular vesicle trafficking, 4) a reduction in ligand-receptor dissociation in the endosome, or 5) alterations in endosome–lysosome fusion process. Which of these possibilities was responsible for the cadmium inhibition of albumin endocytosis is not entirely certain. Cell surface albumin binding experiments indicated that the number of binding sites (B max) was reduced in cadmium-exposed cells (Fig. 8). Their ligand binding affinity (1/K d) and its pH dependency were not changed by cadmium exposure (Fig. 9), thus the biochemical nature of the receptor was probably not altered. Thus, it seems

FIG. 9. Effects of pH on kinetic constants (B max and K d) of FITC-albumin binding to control and cadmium-treated OK cells. Data represent the means 6 SE of four determinations. *Significantly (p , 0.05, unpaired t test) different from the respective control value. †Significantly (p , 0.05, unpaired t test) different from the respective value at pH 7.4.

CADMIUM ON RENAL ALBUMIN ENDOCYTOSIS

that the cadmium-induced inhibition of albumin endocytosis was attributed, in part, to a loss of functional receptor sites. Endocytotic vesicle formation at the cell surface was not significantly altered by cadmium, as demonstrated by the lack of change in fluid phase endocytosis (FITC-inulin uptake) (Fig. 4). In the kinetic analysis of albumin endocytosis, the V max was reduced, but the K m was increased in cadmium-treated cells (Fig. 5). Such changes in kinetic parameters were similar to those observed in cells treated with NH 4Cl, a weak base that impairs endosomal acidification (Fig. 6). The changes in albumin uptake kinetics by NH 4Cl in the present study are comparable to those observed by others (Gekle et al., 1995b). Since the pH dependence of albumin-receptor dissociation was not changed by cadmium exposure (Fig. 9), we suspect that the endosomal acidification was impaired by cadmium, as induced by NH 4Cl. The acidic pH is important in the ligand-receptor dissociation (Mellman et al., 1986) and thus, the prevention of endosomal acidification would cause reexocytosis of albumin, lowering the efficiency of endocytosis. Enhanced reexocytosis would also reduce the number of free receptor sites returning to the cell surface, thus the fall in B max of albumin binding in cadmium-exposed cells (Fig. 8) may also be attributed, in part, to an impaired endosomal acidification. Whether the endosomal proton pump activity was indeed impaired by cadmium in the present study is not certain. However, such effect of cadmium has recently been documented in rat renal cortical endocytic vesicles (Herak-Kramberger et al., 1998). The changes in albumin endocytosis induced by cadmium may be distinct in nature from those induced by nocodazole. Unlike the cadmium-treated group in this study, Gekle et al. (1997) have reported that nocodazole does not induce a change in albumin binding to OK cell. It is, therefore, unlikely that the processes of vesicle trafficking associated with microtubules were affected by cadmium treatment. The effect of cadmium on the endosome–lysosome interaction is not certain at present. In summary, the present study demonstrated that the receptor-mediated endocytosis of albumin is significantly inhibited in OK cells exposed to cadmium. The inhibition seems to be associated with a prevention of endosomal acidification and the attendant fall in ligand-receptor dissociation, which impairs receptor recycling and the overall efficiency of endocytosis. Such changes in receptor-mediated endocytosis, if occurred in the intact kidney, would lead to proteinuria, as documented in cadmium-intoxicated patients and animals. ACKNOWLEDGMENT The authors acknowledge the financial support of the Korea Research Foundation made in the program year of 1998 (1998 – 021-D00101).

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