Acute study of interaction among cadmium, calcium, and zinc transport along the rat nephron in vivo
O. Barbier,
G. Jacquillet,
M. Tauc,
P. Poujeol, and
M. Cougnon
Unité Mixte de Recherche-Centre National de la Recherche Scientifique 6548, Université de Nice-Sophia Antipolis, 06108 Nice Cedex 2, France
Submitted 5 April 2004
; accepted in final form 22 July 2004
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ABSTRACT
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This study investigates the effect in rats of acute CdCl2 (5 µM) intoxication on renal function and characterizes the transport of Ca2+, Cd2+, and Zn2+ in the proximal tubule (PT), loop of Henle (LH), and terminal segments of the nephron (DT) using whole kidney clearance and nephron microinjection techniques. Acute Cd2+ injection resulted in renal losses of Na+, K+, Ca2+, Mg2+, PO42, and water, but the glomerular filtration rate remained stable. 45Ca microinjections showed that Ca2+ permeability in the DT was strongly inhibited by Cd2+ (20 µM), Gd3+ (100 µM), and La3+ (1 mM), whereas nifedipine (20 µM) had no effect. 109Cd and 65Zn2+ microinjections showed that each segment of nephron was permeable to these metals. In the PT, 95% of injected amounts of 109Cd were taken up. 109Cd fluxes were inhibited by Gd3+ (90 µM), Co2+ (100 µM), and Fe2+ (100 µM) in all nephron segments. Bumetanide (50 µM) only inhibited 109Cd fluxes in LH; Zn2+ (50 and 500 µM) inhibited transport of 109Cd in DT. In conclusion, these results indicate that 1) the renal effects of acute Cd2+ intoxication are suggestive of proximal tubulopathy; 2) Cd2+ inhibits Ca2+ reabsorption possibly through the epithelial Ca2+ channel in the DT, and this blockade could account for the hypercalciuria associated with Cd2+ intoxication; 3) the PT is the major site of Cd2+ reabsorption; 4) the paracellular pathway and DMT1 could be involved in Cd2+ reabsorption along the LH; 5) DMT1 may be one of the major transporters of Cd2+ in the DT; and 6) Zn2+ is taken up along each part of the nephron and its transport in the terminal segments could occur via DMT1.
heavy metals; epithelial calcium channel; divalent metal transporter 1; kidney
CADMIUM (CD2+) IS ONE OF THE most commonly found toxic metals present in our environment. The major sources of exposure to Cd2+ are contaminated food and water, tobacco, and industrial fumes and dusts (16). Cd2+ accumulates in the body, and chronic exposure causes severe nephrotoxicity in humans (16) and animals (2, 4). The renal dysfunction may be due to proximal tubular damage affecting the passive paracellular pathway (14, 27) and decreasing active transcellular ion transport (30). With the use of in vitro models, deleterious effects of Cd2+ have been described on several solute transporters, such as stretch-activated ion channels (24), the epithelial Ca2+ channel (ECaC) transporter (32), the NaPi-II transporter (19), the Na/glucose transporter (1), and the NaSi-1 transporter (25). These acute effects of Cd2+ suggest the involvement of ion transporters in Cd2+-induced nephropathy. Therefore, the question arises as to whether these transporters are affected in vivo after Cd2+ exposure. To answer this question, studies have been performed in animal models chronically intoxicated with Cd2+ (22, 31). Unfortunately, severe intoxication induces renal solute wasting, as well as nephrotoxic damage to glomeruli and proximal tubular structures, which could mask more subtle alterations in transporter function.
It has been clearly demonstrated that free cytosolic Cd2+ is responsible for the toxicological damage to cells and that the bound form of Cd, complexed with proteins like albumin and metallothionein, plays a protective role (8). Cd2+ increases urine Ca2+ excretion (28), which can affect bone metabolism and cause severe bone pathology (29). A blockade of the distal Ca2+ transporter (ECaC) was proposed to explain Cd2+-induced hypercalciuria and associated renal stone formation (26). In light of these findings, it is evident that Cd2+ may interact with several different transporters. Such interactions suggest the participation of pathways for free Cd2+ reabsorption. Therefore, the free ionized Cd2+ might be, with CdMT, Cd-GSH, and Cys-Cd, one of the reabsorbed forms of Cd. Although the uptake of free Cd2+ accounts for a minor portion of cellular Cd2+ uptake in vivo, it could be of interest to identify the transporters involved because the Cd2+ complexed to small peptides may be released at the brush border and then taken up as a free ion by renal cells (27). For this purpose, we have chosen to use acute perfusion of Cd2+ and microinjections of 109Cd to study the renal handling of Cd. These protocols minimized the participation of bound Cd forms in Cd reabsorption because the de novo metallothionein or glutathione synthesis induced by Cd2+ takes several hours and very little Cd2+-albumin complexes are present in the tubular fluid.
Initial data were reported by Felley-Bosco and Diezi (9). Using in vivo micropuncture and microinjection techniques in the rat, these authors showed that Cd2+ was mainly reabsorbed in the proximal tubule and that a sodium-cysteine cotransporter was involved. Since their study, a divalent metal transporter (DMT1) has been cloned (18) and immunolocalized to the apical membrane of the thick ascending limb (TAL), distal tubule (DT), and the cortical collecting tubule (CCT) of the rat nephron (11), suggesting a role for this transporter in the renal handling of divalent metal cations. In a recent study, Wareing et al. (34) have clearly demonstrated in the rat that DMT1 can transport Fe2+ along the loop of Henle (LH) and distal tubule. Besides Fe2+, DMT1 also transports Cd2+ and Zn2+ (18). In view of these findings, we decided to re-investigate Cd2+ transport along the rat nephron and its relationship to other cations.
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MATERIALS AND METHODS
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Three types of experimental techniques were used: clearance, tracer microinjection, and micropuncture. The experiments were carried out in female Wistar rats weighing 180220 g. The animals were fed a standard laboratory diet. They had free access to water until the start of the experiment and were starved for 18 h before the surgical procedure. Anesthesia was induced by injection of pentobarbital sodium (Nembutal, 5 mg/100 g body wt ip) and maintained by additional 1-mg doses administered when necessary. The animals were then placed on a heated table to maintain their body temperature between 37 and 39°C. A tracheotomy was performed leaving the thyroid gland untouched. One catheter (PE-20) was inserted into the right jugular vein for perfusion of experimental solutions and another (PE-10) into the left ureter for urine collection. For clearance experiments, a third catheter (PE-50) was inserted into the right femoral artery for blood sampling and arterial blood pressure recording (Research BP Transducer, Harvard Apparatus). For tracer microinjection experiments, the kidney was ventrally exposed as described by Gottschalk and Mylle (14). The use of animals was in accordance with the ILAR Guide for Care and Use of Laboratory Animals.
Clearance Experiments
These experiments were performed to analyze the effect of an acute load of Cd2+ on whole kidney function. Because it is necessary to induce diuresis when microinjection techniques are used, it was important to determine the effect of Cd2+ under the chosen diuretic conditions. Therefore, clearance experiments were carried out in rats intravenously (iv) infused with a 2% NaCl solution at a rate of 100 µl/min. [3H]metoxy-inulin (0.53 Ci/mmol) was used to measure the glomerular filtration rate (GFR). Urine samples were collected serially every 10 min, and blood samples were taken halfway through each urine collection. In all experiments a loading dose of [3H]inulin (4 µCi) was given iv, followed by a continuous infusion of 0.4 µCi/min for the duration of each experiment. Urine collection began 1 h after the administration of the [3H]inulin priming dose. After three control clearance periods, Cd2+ was added and infused continuously at a rate of 880 µM/min to maintain a plasma concentration of
5 µM. After a 40-min equilibration period, urine was collected during three additional 10-min clearance periods.
Tracer Microinjections
Droplets of isotonic buffered solutions containing traces of [3H]inulin and the radioactive isotope of the divalent metal ion under investigation were prepared for injection into early proximal, late proximal, early distal, or late DT sites. The volume injected was 3 nl, and the duration of the injection ranged between 20 and 90 s. The method used to identify the structure was identical to that reported previously (21). Briefly, the tubule segment was selected from its shape, location, and refringence and identified by injecting a small volume (
2 nl of isotonic NaCl solution colored with 0.05% of erioglaucine dye) just before the microinjection. At the start of each microinjection, four 60-s, three 1-min, and two 2-min urine samples were collected serially directly into counting vials containing 2 ml of scintillant. This was followed by a 2-min urine collection to determine the urine flow rate. The amount of injected radioactivity was measured by counting the radioactivity of the injected droplet. Background in the urine was calculated from the activity of the urine samples collected just before each microinjection. Urine recovery for each tracer was expressed as a percentage of the amount injected. This amount in the urine reflects reabsorption of the tracer in all downstream segments.
Control of Injection Site
To ensure that the chosen microinjection site was reproducible from one tubule to another, we have performed parallel micropuncture experiments to measure the F/P inulin at the injection site. Diuretic rats were continuously infused with [3H]inulin (3 µCi/min), and, after a 1-h equilibration period, tubular fluid was collected for 12 min in proximal and distal tubules identified as described above.
Late Proximal Micropunctures
The protocol of [3H]inulin infusion and the method used to identified the structure were the same as that for the microinjection technique. Late proximal tubular fluid samples were collected. under free-flow conditions. Each collection lasted 60120 s. Three successive 30-min clearance periods were performed to determine [3H]inulin and Cd2+ concentrations in urine and plasma. Ultrafiltrable plasma Cd2+ concentration was estimated by measuring Cd2+ after the plasma was spun through a 30-kDa filter (Centrifree micropartition system; Amicon).
Analytic Procedure
3H, 109Cd, 45Ca, and 65Zn radioactivities were measured by liquid scintillation counting (Packard). In plasma and urine samples, Na+, K+, Mg2+, Ca2+, Cl, and Pi concentrations were determined by ion exchange chromatography (AS50/BioLC, Dionex), and Cd2+ was measured by atomic absorption spectrometry using a Zeeman furnace system (Solaar 969, Thermo Optek).
Student's t-test was used for statistical analysis. The data are expressed as means ± SE. P < 0.05 was considered significant.
Radioactive Material
All radioactive elements were produced by Amersham Pharmacia Biotech UK: [3H]inulin (TRA324), specific radioactivity 120 µCi/mg inulin; calcium-45 (CES.3), specific radioactivity 550 mCi/mg 45Ca; cadmium-109 (CUS.1), specific radioactivity 501,000 µCi/µg 109Cd; and zinc-65 (ZAS.2), specific radioactivity 83 µCi/mg 65Zn.
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RESULTS
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Clearance Experiments
The effect of an acute load of Cd2+ on whole kidney function was assessed using the clearance technique. Arterial blood pressure was similar during the control and Cd2+ infusions (126.2 ± 0.5 and 128.5 ± 4.4 mmHg, respectively; P > 0.05, n = 8). Sham-treated animals did not show any significant changes over time in plasma Na+, K+, Ca2+, Cl, or PO42 concentrations, whereas plasma Mg2+ concentration decreased slightly during the saline infusion (Fig. 1). Concomitantly, urine flow rate, GFR, and electrolyte fractional excretion (FE) remained stable (Fig. 2). In experimental animals, the infusion of Cd2+ increased plasma Cd2+ concentration to 3.03 ± 0.32 µM and was associated with a significant fall in plasma Ca2+, Mg2+, K+, Na+, and PO42 concentrations (Fig. 1); although urine flow rate increased and GFR was unchanged (Fig. 2). Furthermore, Cd2+ infusion induced significant increases in the FE of Ca2+, Mg2+, K+, Na+, Cl, and PO42 (Fig. 2); most of the filtered Cd2+ was reabsorbed by the kidney (FE = 0.0018 ± 0.0005%, n = 15).

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Fig. 2. Effects of 5 µM Cd2+ infusion on glomerular filtration (expressed in ml/min), U/P inulin, urine flow rate (expressed in µl/min), and fractionnal excretions (FE) of Ca2+, Mg2+, Na+, K+, Cl, and PO42 (expressed %) in Wistar rats during mild diuretic conditions. , Control rats (n = 5); , experimental rats (n = 5) exposed to Cd2+ throughout the 30-min period. Time 0 corresponds to the start of the first urine collection period after 1 h of equilibration. Values are means ± SE calculated for each clearance period, refer to the difference in the change in glomerular filtration rate (GFR), U/P inulin, urine flow rate, and fractional excretions between periods without and with Cd2+ by Student's t-test (*P < 0.05, **P < 0.001).
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Tracer Microinjection Experiments
First, as shown in Fig. 3A, there was a correlation between the F/P inulin values and the injection site, indicating that the morphological criteria used to estimate the injection site did identify the tubular segment appropriately. Furthermore, in all experiments no site of injection modified the [3H]inulin urine recovery, which was similar to the amount injected (Table 1; see Tables 3 and 4). In a separate series (n = 3, Fig. 3B), we evaluated nonspecific binding of the tracer to plasma membranes. No change in urine recovery of radioactivity from background occurred after the perfusion of a high concentration of the nonradioactive ion after 109Cd or 45Ca microinjection.

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Fig. 3. Correlation between the identification of injected tubules at the kidney surface and the value of F/P inulin (A). Measurement of nonspecific binding of cadmium (Cd). A microinjection of 109Cd was performed in early proximal tubule followed by perfusion (arrow) at the same site of 500 µM CdCl2 (B). Values are means ± SE. The nos. of microinjections are in parentheses.
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45Ca.
Because it has been postulated that Cd2+ may affect Ca2+ reabsorption, it was first necessary to investigate the tubular transport of Ca2+ in the different nephron segments. Results of 45Ca microinjections are shown in Table 1. Ca2+ concentration of the injected solution was 4.5 µM. In this experimental series, the Ca2+ injection rate ranged between 1 and 3 pmol/min, indicating that Ca2+ was introduced into the tubular lumen at tracer doses, because from free-flow micropuncture studies it can be calculated that the mean flow rate of Ca2+ varies from 80 pmol/min at the beginning of the proximal tubule to 15 pmol/min at the end of the DT in rats (30). In our experiments, the total amount of Ca2+ injected did not significantly increase the normal free-flow delivery rate of Ca2+ to the segment located downstream of the injection site, because no significant correlation between the injection rate and the 45Ca recoveries has been found and the cumulative curves of [3H]inulin and 45Ca excretions show that both isotopes appeared in urine and reached their maximum excretion in parallel (data not shown). Table 1 shows that recovery of 45Ca, expressed as a percentage of 45Ca microinjected, depended on the injection site: the more proximal the injection, the lower the 45Ca urine recovery. The differences between the 45Ca recovery at each microinjection site allow an estimate of the unidirectional 45Ca fluxes (expressed as percent delivered load) and provide a measure of Ca2+ reabsorption occurring between the early and late proximal tubules, the late proximal and early DTs (including the LH), the early and late DT [corresponding to the distal convoluted tubule (DCT)], and the late DT and final urine [including the connecting tubule (CNT) and the convoluted tubule (CT)]. This mode of calculation is valid for calcium because injected 45Ca was in a tracer-dose condition and because a sufficient load of filtered Ca2+ was delivered to each segment downstream of the proximal tubule. Under control conditions, >65% of the injected Ca2+ was reabsorbed between the late proximal and the early DT, whereas >20% was reabsorbed between the early and late DTs (Tables 1 and 2). The addition of Cd2+ (20 µM), Gd3+ (100 µM), or La3+ (1 mM) significantly increased 45Ca urine recovery when microinjections were performed at the early proximal tubule site (Table1). This increase was mainly due to a decrease in unidirectional 45Ca flux between the early and late distal segments (Table 2). In contrast, nifedipine did not modify 45Ca recovery at each injection site (Tables 1 and 2). The percent inhibition of distal 45Ca reabsorption produced by each agent used is shown in Fig. 4.

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Fig. 4. Inhibitory effect of Cd (20 µM), Gd3+ (100 µM), La3+ (1 mM), and nifedipine (20 µM) on Ca2+ reabsorption in terminal part of the nephron. Percentages of inhibition were calculated from 45Ca microinjection in early distal tubule. The nos. of microinjections are above each bar.
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109Cd.
The same concentration of Cd2+ (5 µM) was used as for a clearance study. This concentration corresponded to that determined during mild chronic exposure (36, 38). The absence of a significant correlation between the Cd2+ injection rates and urine recovery (data not shown) has been verified, indicating that under these conditions, the percentage of urine 109Cd recovery reflected Cd2+ unidirectional reabsorption. The urine excretion profiles of [3H]inulin and 109Cd were roughly parallel (data not shown) at the three injection sites. Table 3 shows that the urine 109Cd recovery after early proximal microinjection was close to 5% of the amount injected, indicating an important transport of Cd2+ in segments beyond this site. The 109Cd excretion was greater when the injections were performed in late proximal and early DTs. Using Cd2+ concentrations ranging between 0.005 and 50 mM, microinjections indicate saturation at high Cd2+ concentrations in each segment (Fig. 5). These results showed that the segment between early and late proximal sites exhibited an important reabsorptive capacity for Cd2+ because in the presence of 50 mM Cd2+, 45% of Cd is reabsorbed. In contrast, the DT exhibited a lower reabsorptive capacity because 5 mM Cd2+ is sufficient to saturate the uptake in this segment. To characterize further the transport mechanisms involved in 109Cd fluxes, microinjections were performed in the presence of different cations (Table 3). Unfortunately, in contrast to calcium, the difference between the 109Cd recovery at each microinjection site could not be used to estimate the unidirectional 109Cd in each segment of the nephron flux. This is due to the fact that 109Cd microinjections were not performed in the tracer-dose condition (an endogenous pool of Cd2+ does not exist) and because most of Cd2+ was taken up by the proximal tubule (see micropuncture results below) so that the amount of 109Cd delivered to the downstream segments after early proximal microinjection was too low to allow mathematical calculation performed for calcium. Therefore, the results were interpreted on the basis of 109Cd urinary recovery after microinjection in early proximal, late proximal, and early DT, respectively. In early, late proximal, and early distal microinjections, the addition of Gd3+ (90 µM), Co2+ (100 µM), or Fe2+ (100 µM) induced a strong increase in 109Cd urine excretion. These data are consistent with a decrease in unidirectional 109Cd fluxes in proximal tubule, LH, and terminal nephron segments (Table 3). The effect of Zn2+ is more complicated, because it clearly depends on the concentration used: at a low concentration (50 µM), the addition of Zn2+ increased 109Cd excretion only in early distal injections; at a high concentration (500 µM), Zn2+ enhanced 109Cd excretion at every injected site. The diuretic bumetanide had an effect only at late proximal sites, suggesting that blockade of the Na+-K+-2Cl transporter in the LH induces a decrease in 109Cd transport in this segment (Table 3).

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Fig. 5. Influence of Cd2+ concentration on urine excretion of 109Cd after microinjection in early (PT) and late proximal (PCT) tubule and late distal tubule (DCT). Cd2+ concentrations were increased from 5 µM to 50 mM. 109Cd excreted amounts are expressed in % of injected amount. The number of microinjections is in parentheses.
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65Zn.
As for 45Ca and 109Cd, 65Zn microinjections were performed in early, late proximal, and early DTs. With the specific radioactivity of 65Zn available commercially, the minimal concentration in the microinjected solution ranged between 17 and 34 µM, which is higher than the plasma free Zn2+ concentration (15 µM). The absence of a positive correlation between the injection rate and 65Zn urine recovery satisfies tracer-dose requirements (data not shown). As shown in Table 4, 65Zn urine excretion increased from early proximal to early distal microinjection sites, indicating significant Zn2+ reabsorption along the proximal tubule, LH, and terminal nephron segments. 65Zn microinjections performed in the presence of 50 µM Cd2+ showed a slight inhibitory effect of Cd2+ on zinc transport along the early proximal tubule and the LH (Table 4). These results also indicate an important inhition of 65Zn transport along the terminal segments of the nephron.
Cd Micropuncture Experiments
Micropuncture experiments were performed to investigate the amount of Cd delivered to the late proximal tubule during an acute Cd loading. To be consistent with clearance and microinjection experiments, total plasma concentration of Cd2+ was maintained at 5 µM throughout the experiments. Under these conditions, the estimated ultrafiltrable plasma Cd concentration was 2.8 ± 0.2 µM (n = 7). Flow rate and single-nephron glomerular filtration were 34 ± 9 and 38 ± 4 nl/min (n = 7), respectively. The fractional delivery of Cd at the end of the accessible proximal tubule was 14.6 ± 1.4% (n = 7). Thus most of the 85% of the filtered Cd2+ was reabsorbed between the glomerulus and the late proximal tubule.
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DISCUSSION
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The aim of this study was to investigate specifically the transport of free Cd2+ in each segment of the nephron and to identify the putative transporters involved in the renal transport of Cd2+. First, it was important to check the effect of acute Cd2+ intoxication on renal function. To do this, clearance experiments were carried out and infusion of CdCl2 increased plasma free Cd2+ concentration to 3.03 ± 0.32 µM. Such a Cd2+ concentration corresponds to a 90 µg/kg body wt, equivalent to a low dose of acute Cd2+ intoxication (21). During Cd2+ infusion, GFR remained stable, indicating that glomerular function was unaffected. This is in agreement with light microscopy studies by Uriu et al. (31) showing that a single intraperitoneal dose of 0.4 mg/kg Cd2+ did not cause any identifiable glomerular pathology. Acute infusion of Cd2+ was associated with an increase in urine flow rate, a significant increase in FE of all the ions measured, and a parallel decrease in their plasma concentrations. The rapid and excessive renal losses of PO42, Na+, K+, Mg2+, Ca2+, and water were probably due to an effect of Cd2+ mainly on the proximal tubule, and the fall in plasma ion concentrations probably relates to this. Thus, although Cd2+ is already known to cause a Fanconi-like wasting of many solutes normally reabsorbed by the proximal tubule (30), this is the first study to show that acute injection of Cd2+ can produce a Fanconi-like pattern of ion excretion. In the TAL segment of the LH, Cd2+ could reduce reabsorption by blocking ROMK channels (22). An effect on K+ recycling in the apical membrane might account for reduced Na+ and K+ reabsorption, as well a decrease in paracellular transport of divalent cations. A dysfunction of the terminal segments of the nephron could also be involved in Cd2+-induced salt wasting. In the DT, Ca2+ transport occurs via a transcellular pathway (3). Recently, many experiments have confirmed the involvement of the ECaC ion channel in Ca2+ reabsorption in the DT. In heterologous expression systems, ECaC generates Ca2+ currents inhibited by several divalent cations with a blocking order of Gd3+ >> Cd2+ > La3+ and also slightly by L-type voltage-dependent Ca2+ channel antagonists/agonists (25, 32). In the present study, the unidirectional flux of 45Ca estimated by microinjection in the DT shared the known pharmacological properties of ECaC. This suggests that ECaC might be the main transporter involved in distal reabsorption of Ca2+ and highlights a functional role for ECaC in the kidney. Another important finding was the nature of the distal Ca2+ transport inhibition induced by Cd2+. Under diuretic conditions, the Ca2+ concentration at the beginning of the DT ranged between 0.5 and 1 mM. The fact that only 20 µM Cd2+ was sufficient to inhibit >60% of Ca2+ reabsorption indicates that Cd2+ acted as a channel blocker and did not compete directly with Ca2+ on the transporter. This observation corroborates the hypothesis of Peng et al. (25), who proposed an inhibition of ECaC via a binding of Cd2+ to a high-affinity inhibitory site. Thus inhibition of distal Ca2+ reabsorption by Cd2+ could explain the hypercalciuria elicited by acute intoxication.
In contrast to Ca2+, renal handling of Cd2+ is not well understood. Cd is an environmental pollutant, and its presence in tissues and biological fluids usually results from industrial contamination. The 109Cd microinjections performed in the presence of various Cd2+ concentrations (ranging from 5 µM to 50 mM) indicate that the proximal tubule exhibits the highest reabsorptive capacity for Cd2+. These results were corroborated by micropuncture data demonstrating that filtered Cd2+ is strongly reabsorbed by the early proximal tubule and that very little Cd2+ is delivered to the downstream segments (S3, LH, DT). Therefore, the difference between 109Cd urinary recovery after late and early proximal microinjection did not reflect the 109Cd transport between these two sites. The urinary recovery after early proximal microinjection probably corresponds to a best estimate of proximal transport. Finally, the present results indicated that 95% of Cd2+ was taken up by the proximal tubule. Using a similar microinjection technique, Felley-Bosco and Diezi (8) calculated that 70% of injected inorganic Cd2+ was reabsorbed in the proximal tubule. Taken together, these data underline the important role of the proximal tubule in free Cd2+ reabsorption.
If we accept that Cd2+ is almost completely reabsorbed along the proximal tubule, the inhibition of Cd2+ transport in downstream segments should not affect the percentage of excreted 109Cd after proximal injection. This is the case, because the presence of bumetanide (50 µM) or zinc (50 µM) did not change the percentage of Cd2+ recovered in the urine. Finally, although they exhibited permeability to Cd2+, the segments beyond the proximal tubule did not significantly participate in Cd2+ transport at least at low Cd2+ concentrations. It is different at high Cd2+ concentrations and in the presence of factors that decrease proximal reabsorption of Cd2+. The studies performed with Fe2+ (100 µM), Co2+ (100 µM), and Zn2+ (500 µM) revealed an inhibitory effect of these metals in the proximal tubule. Consequently, a significant amount of Cd2+ is delivered to the S3 segment, the LH, and terminal parts of the nephron, and the contribution of these segments to Cd2+ reabsorption then becomes significant.
The observation that Fe2+ and Co2+ clearly decrease Cd2+ transport in all segments suggests that the divalent metal cation transporter DMT1 is involved in the transcellular pathway. DMT1 can transport Fe2+, Zn2+, Mn2+, Co2+, Cd2+, Cu2+, Ni2+, and Pb2+ (17) and has been localized to the proximal S3 segment, the LH, the DCT, and collecting ducts (10). In the proximal tubule, DMT1 is probably present in the endosomal compartment (10), whereas in downstream nephron segments it is located in the apical plasma membrane. Interestingly, Fe2+ and Co2+, and a high concentration of Zn2+, decreased proximal Cd2+ reabsorption, indicating that DMT1 might be involved in its transport. This result is different from the finding of Ferguson et al. (10) that DMT1 was not involved in the translocation of Fe2+ across the brush-border membrane of the proximal tubule. Actually, the transport systems involved in proximal reabsorption of Cd2+ remain unclear. However, the transporters belonging to the zinc-related transport-like protein (ZIP) family are possible candidates. Mouse ZIP1 is expressed in kidney tissue and located in the plasma membrane (6). Several other metals, such as Cd2+, Fe2+ Co2+, or Cu2+ inhibit Zn2+ uptake by this protein (6). It is tempting to speculate that a ZIP transporter might be involved in the transport of Zn2+ and Cd2+ along the proximal tubule. The blocking effect of Gd3+ indicates that stretch-activated cation channels could be also involved in Cd2+ uptake in the proximal tubule. However, the absence of the effect of Gd3+ on proximal 45Ca transport suggests that stretch-activated cation channels play only a minor role in both Ca2+ and Cd2+ transport.
The present study demonstrated that Fe2+ and Co2+ decreased Cd2+ transport in segments downstream to the proximal tubule. Using microperfusion of 55Fe, Wareing et al. (34) demonstrated significant reabsorption of Fe2+ in the LH and that this reabsorption could be mediated by DMT1 (10, 34). Thus it is reasonable to conclude that, like with Fe2+, Cd2+ is transported, at least in part, by DMT1 in the apical membrane of the LH, in its TAL segment where DMT1 has been shown to be present (10).
In the LH and terminal nephron segments, 109Cd microinjections in the presence of bumetanide highlight the role of a different pathway from the transcellular route for Cd2+ transport. In the TAL, the paracellular pathway plays an important role in divalent cation reabsorption. Bumetanide, by inhibiting Na+-K+-2Cl cotransport, decreases the transepithelial voltage and diminishes cation paracellular reabsorption (5, 16). The fact that this loop diuretic decreased Cd2+ transport along LH and DT indicates that in these segments Cd2+ transport may also occur via the paracellular pathway.
The inhibitory effect of Gd3+ on 109Cd and 45Ca uptake suggests that Cd2+ might be reabsorbed through Ca2+ channels. ECaC could be one of these channels, because it participates in distal Ca2+ transport and it is blocked by low Cd2+ concentrations.
Using cells derived from distal portions of the nephron, Friedman and Gesek (11) have proposed that Cd2+ transport involves Ca2+ channels and a membrane transport protein that can be inhibited by Fe2+. In light of more recent experiments, it is likely that this membrane transporter is DMT1 (11). Our data also support this conclusion, because distal Cd2+ permeability was strongly blocked by Fe2+, Co2+, and Zn2+.
As far as zinc transport is concerned, the present study shows that Zn2+ is transported along the proximal tubule, the LH, and the terminal segments of the nephron. The finding that a low Zn2+ concentration (50 µM) did not modify Cd2+ transport in the proximal tubule and LH strongly suggests that Zn2+ and Cd2+ do not share the same uptake pathways in these segments. In the proximal tubule, Zn2+ could be transported in the apical membrane as a free ion via a saturable carrier-mediated process and an unsaturable pathway, or complexed with histidine or cysteine via a sodium-amino acid cotransport mechanism (12); however, the interaction of Cd2+ with such processes is unclear. In the LH, the lack of competition between Zn and Cd is perhaps surprising, because DMT1 has a strong affinity for Zn2+ (17) and may be one of the Cd2+ transporters in this segment. However, studies of Fe2+ transport by DMT1 in this segment by Wareing et al. (34) have shown a lack of competition between Zn2+ and Fe2+. According to these authors, it is possible that in the LH, DMT1 does not transport Zn2+, or transports it with only low efficiency. This possibility is consistent with our data, because a high Zn2+ concentration (500 µM) only produced a modest decrease in Cd2+ absorption along the LH.
Along the terminal nephron segments, Zn2+ inhibited Cd2+ transport and vice versa, suggesting that DMT1 may play an important role in Zn2+ reabsorption in this part of the nephron. Interestingly, four DMT1 isoforms have been identified in renal tissue (19). Thus the difference between the effects of Zn2+ action on Cd2+ uptake in the LH and more distal segments could be due to the pattern of expression of these different isoforms (33).
The results obtained in the present study suggest several ways in which Cd2+ might be eliminated: 1) in the proximal tubule by blocking paracellular and transcellular pathways (unknown for the moment); 2) in the LH by use of loop diuretics (such as bumetanide and furosemide) and DMT1 inhibition; and 3) in the distal tubule by DMT1 inhibition. Our findings on Cd2+ transport highlight the need to characterize the properties in vivo of these different transport pathways and to develop pharmacological tools with which to manipulate DMT1 function.
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ACKNOWLEDGMENTS
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We are grateful to Dr. Robert Unwin for constructive criticism of this manuscript, corrections and for helpful discussions.
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FOOTNOTES
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Address for reprint requests and other correspondence: P. Poujeol, UMR-CNRS 6548, BÂtiment Sciences Naturelles, Université de Nice-Sophia Antipolis, Parc Valrose, 06108 Nice Cedex 2, France (E-mail: poujeol{at}unice.fr)
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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