Introduction

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It has been well established that chronic exposure to cadmium (Cd) causes irreversible damage to the liver and the kidneys (Cronin and Henrich, 1996). Chronic Cd intoxication has been a serious problem for workers in Cd industries as well as for residents of areas of environmental Cd pollution (Kazantis et al., 1963; Tsuchiya, 1976; Nogawa et al., 1979; Wedeen, 1984). In Japan, the chronic, endemic Cd intoxication is represented by "Itai-Itai" disease, which is literally "ouch-ouch" disease in English, standing for the cardinal symptom of pain from osteomalacia due to chronic renal functional deterioration (Tsuchiya, 1976; Nogawa et al., 1979).

Although the mechanisms by which Cd causes renal dysfunction have been extensively studied by many investigators, detailed cellular mechanisms remain to be established. Relatively early manifestations of renal dysfunction after chronic exposure to Cd are renal glucosuria, aminoaciduria, enzymuria, and microglobulinuria (Adams et al., 1969; Bernard et al., 1979; Gonick et al., 1980; Raghavan and Gonick, 1980; Cronin and Henrich, 1996), which are assumed to represent the proximal tubular dysfunction.

Cd in the tissue is mainly bound to metallothionein (MT). Although the intracellular MT protects tissues from acute toxicity of heavy metals, it has been reported that the injection of cadmium-metallothionein (Cd-MT) exhibited acute renal toxicity (Klaassen and Liu, 1997). Wang et al. (1993) demonstrated in rats that nephrotoxicity of Cd is related to urinary excretion of Cd-MT and that renal cell injury may be independent of Cd in the renal cortex. They demonstrated that the first injection of Cd-MT caused urinary excretion of low molecular weight protein, suggesting an early onset of proximal tubular dysfunction. These findings lead us to speculate that filtered Cd-MT may play a primary role in initiating nephrotoxicity by acting from the tubular lumen in the proximal tubule (Nomiyama and Nomiyama, 1998).

To provide direct evidence in support of this hypothesis, we used an in vitro microperfusion technique to examine effects of Cd-MT added to the lumen of the rabbit proximal straight tubule (S2 segment) on glucose and amino acid transport across the apical brush-border membrane. The results indicate that the addition of Cd-MT, rather than CdCl₂, to the lumen inhibited glucose and amino acid transport across the proximal tubule instantaneously, followed by gradual inhibition of active Na⁺ transport across the basolateral membrane.

	Materials and Methods

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In Vitro Microperfusion. In vitro microperfusion of isolated renal tubules developed by Burg et al. (1966) was used as modified previously (Tsuruoka et al., 1993, 1994). Female Japanese White rabbits weighing 1.5 to 2.2 kg were maintained on regular laboratory chow and allowed free access to tap water. On the day of experiments, animals were anesthetized with sodium pentobarbital (35 mg/kg, i.v.). Left kidneys were removed, and coronal slices were made. Slices were transferred to a chilled dish containing modified Collins' solution of the following composition: 14 mM KH₂PO₄, 44 mM K₂HPO₄, 15 mM KCl, 9 mM NaHCO₃, and 160 mM sucrose, with pH maintained at 7.4. Segments of proximal tubule (S2), with lengths ranging from 0.7 to 1.2 mm, were isolated from the medullary ray of renal cortex by fine forceps under a stereomicroscope. Isolated tubules were transferred to the perfusion chamber mounted on an inverted microscope (IMT-2; Olympus, Tokyo, Japan) and perfused in vitro at 37°C. Tubules were hooked up to the holding pipette and a single-barreled perfusion pipette was inserted into the tubular lumen. Triple-barreled polyethylene tubing was inserted into the perfusion pipette to allow rapid exchange of the perfusion fluid. The perfusion rate was controlled at 10 to 20 nl/min by adjusting the height of the fluid reservoir, which was connected to the back end of the perfusion pipette. A system of a flow-through bath was used to permit rapid exchange of the bathing fluid. The bathing fluid was maintained at 37°C by using a warm water jacket. The flow rate of the bathing fluid ranged from 3 to 5 ml/min, allowing the bath fluid to be exchanged within 2 s. The composition of the basal solution used in this study was as follows: 110 mM NaCl, 5 mM KCl, 25 mM NaHCO₃, 0.8 mM Na₂HPO₄, 0.2 mM NaH₂PO₄, 10 mM sodium acetate, 1.8 mM CaCl₂, 1.0 mM MgCl₂, 8.3 mM glucose, and 5 mM alanine. In some experiments, solutions without either alanine or glucose were also used. The pH of the solutions was maintained at 7.4 by bubbling with 95% O₂, 5% CO₂ gas.

Electrophysiological Studies. Transmural voltage (V_t) was measured by connecting a 1 M KCl agar bridge to a saturated KCl reservoir where a calomel half-cell electrode was placed. The electrode was connected to a dual channel electrometer (Duo 773; WP Instruments, New Haven, CT), and measured voltage was recorded on a two-pen recorder (R301; Rikadenki, Tokyo, Japan). A flowing-boundary 1 M KCl agar bridge connected to a calomel half-cell was placed at the outflow of the bath and served as a ground.

Glucose and Fluid Transport. To measure net volume flux (J_v) and lumen-to-bath glucose flux (J_glucose), 10 µCi/ml of predialyzed [methoxy-³H]inulin and [¹⁴C]glucose (NEN, Boston, MA) were added to the perfusate (Tsuruoka et al., 1994). After V_t was stabilized, each of three samples of tubular effluent were collected into a constant volume pipette (40-50 nl) under water-equilibrated mineral oil. They were transferred into vials containing 1 ml of water, to which was added 6 ml of scintillation solution containing OMNIFLUOR (New England Nuclear, Boston, MA) per milliliter toluene:Triton X-100 (2:1, v/v) solution to permit measurement of -emission of ¹⁴C and ³H on a -counter (LSC-3500; Aloka, Tokyo, Japan) simultaneously. Net J_v (nl/min/mm) was calculated as:
where V_o is collection rate (nl/min), L is tubular length (mm), and C*_o and C*_i are concentrations of [methoxy-³H]inulin outlet and inlet of the renal tubule of collected and perfused fluid, respectively. J_glucose (pmol/min/mm) was calculated as:

where V_i is perfusion rate (nl/min), G*_i and G*_o are concentrations of [¹⁴C]glucose inlet and outlet of perfusate of the renal tubule, respectively, and G_i is concentration of glucose in the perfusate. A positive J_glucose value meant absorption.

Chemicals. All solutions containing bicarbonate were bubbled with 95% O₂, 5% CO₂ to adjust pH to 7.4. All reagent grade chemicals were purchased from Sigma. Concentration of Cd-MT was expressed as grams of Cd per milliliter to represent Cd concentration contained in the molecule.

Statistics. All the data are expressed as means ± S.E. Statistical analysis was performed by using the Student's t test and ANOVA where appropriate. P < .05 was regarded as significant.

	Results

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Electrophysiological Evaluation of Apical Na⁺-Glucose and Na⁺-Amino Acid Cotransporters. First, we examined the effects on V_t and V_a of glucose or alanine removal from the lumen in the isolated perfused S2 segment. Mean basal V_t was 2.9 ± 0.4 mV (n = 80) and mean basal V_a was 67.9 ± 1.4 mV (n = 80). As shown in Figs. 1a and 2a, abrupt removal of glucose from the perfusate hyperpolarized V_a from 66.7 ± 1.1 to 75.3 ± 0.9 mV (P < .001; n = 58). This observation is in good agreement with the findings in previous reports that the magnitude of hyperpolarization reflects the activity of Na⁺-dependent glucose transporter in the apical membrane (Maruyama and Hoshi, 1972; Fromter, 1982; Bello and Weber, 1986). Mean V_a change after glucose removal (V_aglu) was 8.5 ± 1.1 mV (n = 58). Glucose removal also caused positive deflection of V_t from 3.1 ± 0.3 to 0.5 ± 0.3 mV (P < .001; n = 58). Removal of alanine from the lumen also hyperpolarized V_a from 67.4 ± 1.3 to 74.6 ± 1.1 mV (P < .001; n = 46; Figs. 1b and 2b). Again, this observation is compatible with the findings of previous reports that the magnitude of the voltage deflection represents the activity of Na⁺-dependent neutral amino acid transporter (Hoshi et al., 1976; Fromter, 1982; Bello and Weber, 1986). V_a by alanine removal (V_aala) was slightly lower than that by removal of glucose (mean V_aala = 7.3 ± 0.9; n = 46). This effect was also reversible. Removal of alanine from the lumen also deflected V_t in a positive direction from 2.9 ± 0.3 to 0.6 ± 0.3 mV (P < .001; n = 58). This finding is similar to that observed on glucose removal.

View larger version (45K): [in this window] [in a new window]   Fig. 1.   Representative tracing of the effects of glucose (-G) or alanine (-A) removal from lumen and the effect of Cd-MT (106 g Cd/ml) to lumen on Vt and Vb. Voltage deflection was observed on abrupt removal of glucose (a) or alanine (b) from the lumen before and 10 min after Cd-MT was added to the lumen. Although the membrane potential is represented by Vb, changes of Vb induced by elimination of glucose or alanine from the lumen reflect those of Va generated by circular current through paracellular shunt pathway.

View larger version (10K): [in this window] [in a new window]   Fig. 2.   Summary of Va deflection by glucose (a) or alanine (b) removal from the lumen. Va was calculated as Vb  Vt; *P < .05 as compared with the control period.

Effects of Cd-MT and CdCl₂ on V_aglu and V_aala. After we confirmed effects of removal of glucose or alanine from the lumen on V_t and V_a, we examined effects of Cd-MT or CdCl₂ added to the lumen on these parameters. Following addition of Cd-MT to the lumen, both V_a and V_t were gradually depolarized after 5 to 7 min and reached plateaus within 10 min (Fig. 1, a and b). After the voltage was stabilized at depolarizing levels, the effect of glucose removal was examined again. As shown in Fig. 1a, V_aglu was markedly decreased after luminal addition of Cd-MT. This effect was exerted in a dose-dependent manner in the range from 10⁹ to 10⁵ g Cd/ml. When 10⁸ g Cd/ml Cd-MT was added to the lumen, V_aglu was decreased from 8.4 ± 0.4 to 5.0 ± 0.3 mV (P < .001) (Fig. 3a). On the other hand, addition of Cd-MT to the bath did not affect the basal V_a (data not shown) or V_aglu with exception at 10⁵ g Cd/ml (Fig. 3b). Figure 4a summarizes the effect of Cd-MT added to the lumen or to the bath on the percentage change of V_aglu. V_t depolarization by glucose removal (V_tglu) was also reduced after luminal exposure to Cd-MT (data not shown). For V_aala similar changes of voltage were observed. Figure 4b shows the luminal and basolateral effects of Cd-MT on V_aala. Luminal Cd-MT diminished V_aala in a dose-dependent manner, whereas basolateral administration showed only a minor effect on voltage deflection. These results indicate that the sensitivity to Cd-MT in inhibiting glucose and amino acid transport in the proximal tubule was more than 100-fold greater in the lumen as compared with that of the basolateral exposure. Therefore, it is highly possible that the action of filtered Cd-MT on the luminal membrane is more critical in causing proximal tubular dysfunction than the action on the basolateral site. To determine whether Cd²⁺ itself has a similar effect on the proximal tubule, we examined the effects of CdCl₂, compared with the effects of Cd-MT, on V_aglu and V_aala. Figure 5a and b show that CdCl₂ has less of an effect on V_aglu and V_aala than does Cd-MT even when it was added to the lumen. The same holds true for the effects on baseline V_a (data not shown). These observations suggest that Cd-MT is more toxic than Cd²⁺ itself to the renal proximal tubule cells.

View larger version (13K): [in this window] [in a new window]   Fig. 3.   Effects of Cd-MT added to the lumen (a, left) and to the bath (b, right) on Va deflection on glucose removal from the lumen (Vaglu). Cd-MT, at concentrations indicated, was added for about 10 min in all experiments. *P < .05 as compared with the control.

View larger version (27K): [in this window] [in a new window]   Fig. 4.   Dose-dependent effects of Cd-MT on relative change of glucose-dependent Vaglu (a) and alanine-dependent Vaala (b). Closed circles represent Cd-MT added to the lumen, whereas open circles represent Cd-MT added to the bath. *P < .01 as compared with preceding dose.

View larger version (26K): [in this window] [in a new window]   Fig. 5.   Summary of relative change of Vaglu (a) and Vaala (b) by CdCl2. Closed circles represent Cd-MT added to the lumen, whereas open circles represent Cd-MT added to the bath. *P < .01 as compared with preceding dose.

Acute Effect of Cd-MT on V_aglu and V_aala. Because the data shown above indicate that both glucose and amino acid transport were impaired when V_t had already been suppressed by Cd-MT, it could be interpreted that Cd-MT from the lumen, after entering the cell, somehow inhibits primarily the Na⁺-K⁺ pump in the basolateral membrane, followed by secondary inhibition of glucose and amino acid transport in the brush-border membrane. Alternatively, it is also possible that Cd-MT primarily acts on the cotransporters in the brush-border. Thus, we were curious to know whether these cotransporters were affected by Cd-MT before V_t was inhibited. To examine whether Cd-MT exerts a more rapid effect on these cotransporters, we performed intermittent pulse of luminal glucose removal soon after 10⁷ g Cd/ml Cd-MT was added to the lumen. As shown in Fig. 6a, V_aglu diminished within 1 min, whereas the depolarization of basal V_a as well as V_t occurred at 5 to 7 min after the exposure to Cd-MT. Figure 6b summarizes the time courses of V_a and V_aglu for 9 min after the exposure. The result indicates that the reduction of apical Na⁺-dependent glucose transport activity was preceded the inhibition of Na⁺-K⁺-ATPase activity. Similar results were also obtained on rapid intermittent removal of alanine (data not shown).

View larger version (39K): [in this window] [in a new window]   Fig. 6.   Time course of the effects of luminal Cd-MT (106 g Cd/ml) on glucose-dependent voltage deflections. a, representative tracings. b, summary of the time course. *P < .01 as compared with the control.

Effects of Ouabain and Phloretin on V_tglu To define the effects of Cd-MT on glucose or amino acid transport and on Na⁺-K⁺ pump more precisely, we examined effects of inhibitors of Na⁺-K⁺-ATPase (ouabain) and glucose transporter (phloretin) on these elecrophysiological parameters and compared them with those of Cd-MT.

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View larger version (35K): [in this window] [in a new window]   Fig. 7.   Effect of ouabain (104 M) added to the bath on glucose-dependent voltage deflections. a, representative tracings. b, summary of the effects of ouabain alone and addition of luminal Cd-MT (106 g Cd/ml) in combination with ouabain. *P < .01 as compared with the control.

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View larger version (30K): [in this window] [in a new window]   Fig. 8.   Effect of phloretin (104 M) added to the lumen on glucose-dependent voltage deflections. a, representative tracings. b, summary of the effects of phloretin alone and time-dependent effect of luminal Cd-MT (106 g Cd/ml) in the presence of phloretin. *P < .01 as compared with the control.

Prevention of the Initial Effect of Cd-MT on V_aglu by Dithiothreitol (DTT). Because the initial effect of Cd-MT on glucose transport is so rapid, we hypothesized that the effect might be mediated by a direct interaction of Cd-MT with Na⁺-glucose cotransporter, rather than by some toxic effects of Cd that is taken up in the cytoplasm. To test this hypothesis, we challenged whether the SH group in the glucose-transporter protein moiety could prevent the initial effect of Cd-MT. For this purpose, we examined the effects of DTT, which protects SH residues of the protein. The results are summarized in Fig. 9, a and b. The pretreatment with DTT did not alter both V_aglu and V_a (P > .1). It prevented the initial reduction of V_aglu (0.1 ± 0.05-0.1 ± 0.05 mV; P > .1), but not the later effect of Cd-MT on V_a and V_aglu (V_a: 9.0 ± 0.9-0.7 ± 0.4 mV, P < .01; V_aglu: 0.1 ± 0.05-8.6 ± 1.1 mV, P < .01).

View larger version (34K): [in this window] [in a new window]   Fig. 9.   Prevention by DTT (103 M) added to the lumen of early inhibitory effect of luminal Cd-MT (106 g Cd/ml) on glucose-dependent voltage deflections. a, representative tracings. b, summary of time-dependent effect of DTT to Cd-MT action. *P < .01 as compared with the preceding experiments.

Inhibition of Glucose Transport by Luminal Addition of Cd-MT. Finally, we measured J_glucose to confirm that electrophysiological changes by luminal Cd-MT represent inhibition of glucose transport. The mean basal glucose flux was 32.4 ± 1.1 pmol/min/mm (n = 32) and addition of Cd-MT (10⁷ g Cd/ml) to lumen significantly decreased glucose flux to 8.7 ± 0.8 pmol/min/mm (P < .001; Fig. 10). The same amount of Cd-MT added to the bath and an equivalent dose of CdCl₂ in the lumen or bath did not affect the glucose transport. Addition of vehicle did not change J_glucose. These results are compatible with our findings obtained by the electrophysiological technique as reported above.

View larger version (25K): [in this window] [in a new window]   Fig. 10.   Effect of Cd-MT or CdCl2 on Jglucose. a, effect of Cd-MT (109 g Cd/ml) added to the lumen. b, effect of Cd-MT (107 g Cd/ml) added to the lumen. c, effect of Cd-MT (107 gCd/ml) added to the bath. (d) Effect of CdCl2 (107 g Cd/ml) added to the lumen. e, effect of CdCl2 (107 g Cd/ml) added to the bath. f, effect of vehicle.

	Discussion

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Chronic toxicity of Cd, causing hepatic and renal involvement, is a major concern of industrial hygiene as well as environmental pollution (Kazantis et al., 1963; Tsuchiya, 1976; Wedeen, 1984; Cronin and Henrich, 1996). In spite of extensive studies on chronic renal toxicity of Cd, no detailed cellular mechanism in the initial stage of Cd intoxication has been clearly defined yet.

The MT production is induced in the liver on chronic Cd exposure. Cd-MT is liberated from the liver to the circulation, and then delivered to the kidney (Klaassen and Liu, 1997) and filtered at the glomerulus (Abel et al., 1987). It has been postulated that the hepatic involvement precedes the renal injury and is responsible for the Cd-induced nephrotoxicity (Klaassen and Liu, 1997; Nomiyama and Nomiyama, 1998). It has been reported that administration of Cd-MT caused renal damage (Wang et al., 1993; Klaassen and Liu, 1997; Nomiyama and Nomiyama, 1998). Wang et al. (1993) showed that repeated injection of Cd-MT to rats caused renal tubular damage, which was related to urinary excretion of Cd rather than Cd concentration in the renal cortex. Chan et al. (1993) reported that liver transplantation from Cd-exposed rats to normal recipients caused nephrotoxicity associated with accumulation of Cd and MT in the kidney. These observations are in accord with the view that Cd initially causes hepatic injury, inducing an increase in Cd-MT in the liver. Cd-MT liberated from the liver is transferred to the kidney, where filtered Cd-MT, probably acting from the luminal side, causes renal proximal tubular damage.

This study provides, for the first time, direct evidence in support of the view that Cd-MT, rather than CdCl₂, acts directly on the brush-border membrane of the proximal tubule to inhibit both glucose and amino acid transport. In this study, we used voltage deflections of the apical membrane that were generated on abrupt elimination of glucose or alanine from the perfusate as indices for Na⁺-glucose and Na⁺-amino acid cotransporter activities, respectively. The rationale for the use of these parameters to represent glucose or amino acid transport across the brush-border membrane has already been established by detailed electrophysiological micropuncture studies in the newt (Maruyama and Hoshi, 1972; Hoshi et al., 1976) and rat kidney (Frömter and Gessner, 1975; Bello and Weber, 1986). Elimination of glucose or alanine from the perfusate caused hyperpolarization of the apical membrane as well as the basolateral membrane. The latter phenomenon is explained by circular current through paracellular shunt pathway (Maruyama and Hoshi, 1972; Frömter, 1982). Because the reference electrode was placed in the bath, we usually recorded V_b rather than V_a. Therefore, all representative tracings reported under Results (Figs. 1, 6a, 7a, 8a, and 9a) show V_b. Because V_a is calculated as V_b  V_t, and V_t is small compared with V_b, one can imagine that the tracings of V_a should be qualitatively similar to those of V_b. The observations that either V_aglu or V_aala was diminished immediately after luminal application of Cd-MT indicate that the inhibitory effects of Cd-MT on these cotransporters are very rapid. By measuring J_glucose, we confirmed that the electrical response to glucose elimination reflects glucose transport across the apical membrane of the proximal tubule.

It should be noted that the effect of Cd-MT was more potent than that of CdCl₂ in inhibiting glucose transport of the renal proximal tubule. This is compatible with in vivo observation that nephrotoxicity of Cd-MT is more potent than that of CaCl₂ (Nordberg et al., 1975), supporting the view that the generation of Cd-MT is critical for the Cd-induced nephrotoxicity.

ED₅₀ of Cd-MT to the luminal exposure was roughly estimated to be 6 × 10⁸ g Cd/ml, whereas that of basolateral exposure was higher than 10⁵ g Cd/ml. This clearly indicates that the toxic effect of Cd-MT is exerted from the tubular lumen, but not from the basolateral side. Thus, Cd-MT that is filtered through the glomerulus is critical for the initiation of the Cd-induced nephrotoxicity. This finding is in good agreement with previous results by Nomiyama and Nomiyama (1984) that nephrotoxicity by chronic Cd administration developed when serum Cd-MT concentration was higher than 5 × 10⁸ g Cd/ml. Thus, when Cd-MT concentration in the luminal fluid exceeds a critical level, Cd-MT affects the proximal tubule cells from luminal membrane, leading to cellular damage.

In this study, we observed the time course of the effect of Cd-MT on membrane voltage. It is important to note that the effect of Cd-MT on Na⁺-glucose cotransport was manifested immediately after addition of Cd-MT to the perfusate. This finding was unexpected because Cd nephrotoxicity is known to be a chronic process. On the other hand, V_t as well as baseline V_a gradually decreased 10 to15 min after administration of Cd-MT. Although the exact cellular mechanisms of the sequential events of voltage changes by Cd-MT remain to be determined, it is clear that direct inhibition of the transporters in the apical membrane by Cd-MT is an initial step in the process that leads to the subsequent cellular functional deterioration.

One of the possible mechanisms of the depolarization of V_a (and V_t) observed in the later period is a reduction of Na⁺-K⁺-ATPase activity. It has been reported that addition of CdCl₂ in vivo decreases renal Na⁺-K⁺-ATPase activity (Nechay and Sauners, 1977; Gonick et al., 1980). To simulate the situation of inhibition of Na⁺-K⁺-ATPase, we observed the effect of ouabain on the electrophysiological parameters. Addition of ouabain to the bath depolarized V_t and V_a within 1 to 2 min and decreased V_tglu in parallel with change in V_a. These observations are compatible with those previous reported by Maruyama and Hoshi (1972) and Nomiyama and Nomiyama (1984). Addition of Cd-MT in the lumen after the addition of ouabain did not cause further change in V_a. Thus, ouabain mimicked at least the later effects of Cd-MT added to the lumen, supporting the view that the later effect of Cd-MT is associated with an inhibition of Na⁺-K⁺-ATPase.

It should be noted that in the initial period after administration of Cd-MT, the decrease in V_tglu was observed even under the condition where V_t and V_a were not yet decreased. This phenomenon lead us to speculate that in the initial period Cd-MT inhibits only cotransporters in the brush-border membrane without affecting Na⁺-K⁺-ATPase. To explore whether an inhibition of cotransporter could cause an inhibition of Na⁺-K⁺-ATPase, we observed the effect of phloretin, an inhibitor of Na⁺-glucose cotransporter, on electrophysiological parameters. We found that, in spite of marked inhibition of Na⁺-glucose cotransporter, phloretin added to the bath did not necessarily inhibit V_t and V_a. Thus, a simple inhibition of Na⁺-glucose cotransporter does not necessarily cause a secondary inhibition of Na⁺-K⁺-ATPase. Therefore, it is possible that the inhibition of cotransporters is independent from that of Na⁺-K⁺-ATPase.

Although our results clearly show that Cd-MT acts on renal proximal cells from the luminal side, it was unclear whether Cd-MT directly inhibits Na⁺-glucose cotransporter in the apical membrane or affects the transporter after entrance into the cell. The observation that the reduction of V_aglu occurred within a relatively short period (2-3 min) after addition of Cd-MT to the lumen suggests that Cd-MT may act directly on the transporter at the luminal site of the membrane. To confirm this possibility, we examined whether DTT, which protects sulfhydryl groups of proteins, can prevent the Cd-MT-induced potential change. We found that DTT protected the initial, but not later, effect of Cd-MT. This suggests that Cd-MT initially affects Na⁺-glucose transporter at the luminal side but that the later effect that leads to an inhibition of active transport may be caused by Cd-MT that has entered the cell. At this time, the mechanism by which Cd-MT enters cells across the brush-border membrane is unknown.

Our observation of the protective effect of DTT against inhibition of glucose transport by Cd-MT is, in part, compatible with the observation by Ahammadsahib, Jinna, and Desaiah (Ahammadsahib et al., 1989), who reported that DTT prevents reduction of renal Na⁺-K⁺-ATPase activity and improves mortality of Cd-intoxication in rats. These investigators, however, speculated that chelation of Cd ion by DTT may be responsible for prevention of the toxicity because DTT chelates heavy metals. This speculation is unlikely because other chelators, such as EDTA, were not effective for prevention of Cd-induced nephrotoxicity in their report. Our observations are distinct from those of Ahammadsahib et al. in that DTT could not prevent the later inhibitory effect on active transport. The reason for this discrepancy is unknown at present time. Further study may be necessary to determine whether the protective effect of DTT is dependent on the dose of Cd-MT. Our observation suggests that prevention of the entry of Cd-MT from the apical membrane into the proximal tubular cell, rather than protection of cotransporters, is more important for the protection of the kidney from Cd intoxication. Clarification of the detailed mechanisms of Cd-MT action from the apical membrane of the proximal tubule must await further investigation.

In summary (Fig. 11), we provided direct evidence that Cd-MT reduces glucose and amino acid reabsorption from the luminal side of the proximal tubule by 1) initial direct inhibition of apical Na⁺-glucose and Na⁺-amino acid transporter activities, and 2) subsequent secondary effect associated with decrease in basolateral Na⁺-K⁺-ATPase activity. Furthermore, Cd-MT, rather than CdCl₂, is more toxic to renal proximal tubular cells. DTT prevents the initial reduction of Na⁺-glucose transport by Cd-MT, suggesting that Cd-MT may have an effect not only after it entered the cells but that it also directly affects the transporters at the apical membrane.

View larger version (23K): [in this window] [in a new window]   Fig. 11.   Schematic illustration showing that filtered Cd-MT acts initially on the Na+-glucose and Na+-alanine cotransporters existing in the brush-border membrane of the proximal tubule cells. These effects are prevented by DTT. Free Cd2+ is without effect. In the latter stage, Cd-MT may be incorporated into cell. Cd2+ splitted from Cd-MT in the cytoplasm may affect Na+-K+-ATPase in the basolateral membrane. This latter effect is reversible. Cd-MT does not act from the basolateral side.