Zinc protects renal function during cadmium intoxication in the rat
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《美国生理学杂志》
1Unite Mixte de Recherche-Centre National de la Recherche Scientifique 6548, Universite de Nice-Sophia Antipolis, Nice, France
2Departamento de Fisiologia y Biofisica, Centro de Investigacion y de Estudios Avanzados del Instituto Politecnico Nacional, Mexico City, Mexico
ABSTRACT
This study investigates the effect in the rat of chronic CdCl2 intoxication (500 μg Cd2+/kg, daily ip injection for 5 days) on renal function and the changes in tight junction proteins claudin-2, claudin-3, and claudin-5 present in rat kidney. We also studied the effect of coadministration of ZnCl2 (500 μg Zn2+/kg) during chronic CdCl2 intoxication. Our results indicate that 1) most of the filtered Cd2+ is reabsorbed within the kidney; 2) chronic Cd2+ intoxication can induce a change in renal handling of ions without altering glomerular filtration rate; 3) a delayed nephropathy, showing Fanconi-like features, appears more than 5 days after the end of CdCl2 exposure; 4) epithelial integrity is altered by chronic Cd2+ intoxication affecting the expression and localization of claudin tight junction proteins; and 5) cotreatment with Zn2+ protects against the renal toxic effects of Cd2+, preventing altered claudin expression and inhibiting apoptosis. In conclusion, these results show that Cd2+ toxicity and cellular toxic mechanisms are complex, probably affecting both membrane transporters and tight junction proteins. Finally, Zn2+ supplementation may provide a basis for future treatments.
heavy metals; kidney; protective treatment; tight junction
CADMIUM (CD2+) IS ONE OF THE most common toxic metals in our environment. The major sources of exposure to Cd2+ in the general population are contaminated food and water, tobacco and industrial smoke, and dust (18). Cd2+ accumulates in the body and has a very long biological half-life (10–30 yr) in humans (15, 18).
It is known that chronic exposure to Cd2+ can induce severe nephropathy in humans (18) and animals (4, 9). This nephrotoxicity causes reabsorptive and secretory dysfunction of the renal tubule. The main signs include proteinuria, ion losses, glucosuria, aminoaciduria, and polyuria (1, 2, 21, 24, 29, 31). Experimental chronic intoxication with Cd2+ has been performed at various doses over several weeks (9, 30, 48) and causes a Fanconi-like syndrome with predominant tubular dysfunction that develops into renal failure (47, 48). However, to our knowledge no detailed study of renal function during chronic Cd2+ intoxication has been carried out using clearance methods. Thus a documented time course for the appearance of the deleterious effects of Cd2+ exposure has not been described and would be useful in understanding the toxic mechanisms involved.
Several mechanisms have been proposed to explain the toxic effect of Cd2+ on renal cells. Cd2+ may cause nephrotoxicity by generating free radicals (20, 42) and by inducing necrosis and apoptosis (13). Interestingly, a protective effect of zinc (Zn2+) has been reported in vitro against the cellular toxicity due to Cd2+. Zn2+ protection is probably due to an action on oxidative stress and apoptosis (8, 17, 23, 43). In addition, exposure of renal cultured cells (LLC-PK1 line) to Cd2+ caused a decrease in transepithelial electrical resistance and in the number of domes, suggesting alterations of the tight junctions (41).
For the reasons outlined above, we decided to study the renal effects of Cd2+ during chronic exposure to Cd2+ and the putative protective effects of zinc.
MATERIALS AND METHODS
Clearance Experiments
These experiments were performed to analyze the effect of chronic intoxication with Cd2+ on whole kidney function and were carried out in female Wistar rats weighing 180–220 g. The animals were fed a standard laboratory diet. They had free access to water until the beginning of the experiments and were starved for 18 h before surgery. Anesthesia was induced by intraperitoneal (ip) injection of pentobarbital sodium (Nembutal, 5 mg/100 g body wt) and maintained by additional 1-mg doses when necessary. The animals were placed on a heated table to maintain their body temperature between 37 and 38°C. A tracheotomy was performed leaving the thyroid gland untouched. One catheter (Clay Adams, PE-20) was inserted into the right jugular vein for infusion and another (Clay Adams, PE-10) in the left ureter for urine collection. A third catheter (Clay Adams, PE-50) was inserted into the right femoral artery for blood sampling and arterial blood pressure recording (Research BP Transducer, Harvard Apparatus). Clearance experiments were carried out in rats infused with 0.9% NaCl solution at a rate of 20 μl/min. [3H]methoxy-inulin (TRA.324 Specific radioactivity, 120 μCi/mg inulin, 0.53 Ci/mmol; Amersham Pharmacia Biotech) was used to estimate the glomerular filtration rate (GFR). Urine samples were collected serially during 20-min periods, and blood samples were taken halfway through each urine collection. In all experiments, a loading dose of [3H]inulin (4 μCi) was given, 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.
At the end of the experiment, the rats were killed by an overdose of Nembutal, and the liver and kidneys were removed for measurement of cadmium content. The use of animals was in accordance with the Institute for Laboratory Animal Research Guide for Care and Use of Laboratory Animals.
Chronic Intoxication with Cd2+ and Protection by Zn2+
In the Cd2+-treated groups, rats were intoxicated by daily ip injection for 5 days with 500 μg Cd2+·kg–1·day–1. There was a recovery period of 15 days, without further Cd2+ exposure. Clearance experiments were performed on days 0 (control group), 1, 3, 5, 7, 10, and 20. To study the protective effect of Zn2+, Wistar rats were injected daily ip with a solution containing both 500 μg Cd2+·kg–1·day–1 and 500 μg Zn2+·kg–1·day–1 for 5 days. Clearance experiments were performed on day 10 and day 20. Experiments were also performed in a control group and a treated group with 500 μg Zn2+·kg–1·day–1 for 5 days. Each group comprised five rats.
Rat body weights were recorded daily to determine the effect of Cd2+ on the general health of the animals.
Analytic Procedures
3H radioactivity was 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).
Cd2+ Measurement in Tissue
The technique used to measure Cd2+ was described earlier by Playle et al. (40). Liver and kidney samples were taken, weighed, and digested in a solution of 1 N nitric acid (TraceMetal Grade HNO3; Fisher Scientific) at a temperature of 80°C for 4 h. At the end of this time, the supernatant was taken, and its volume was measured to calculate Cd2+ concentration. The supernatant was then diluted 1:1,000 with deionized water, and Cd2+ content was determined by atomic absorption spectrometry using a Zeeman furnace system (Solaar 969, Thermo Optek).
Apoptosis Measurement in Tissue
Rats were intoxicated with one ip injection of either NaCl as a control, 500 Cd2+/kg, both 500 μg Cd2+/kg and 500 μg Zn2+/kg, or only 500 μg Zn2+/kg. Twenty-four hours after the injection, the kidneys were perfused with 1x PBS and then dissected from euthanized animals. The kidney cortexes were then removed and homogenized immediately in lysis buffer (Euromedex). Protein was quantified using the Bradford assay (Bio-Rad) using BSA as a standard. Apoptosis measurement in cortex samples was monitored by measuring the color generated due to hydrolysis of a chromogenic caspase-3 substrate (Apopain, Euromedex) added to the assay medium. Color was measured using a plate reader.
Rat Frozen Kidney Tissue Sections
Kidneys of experimental rats were removed at the end of each experiment and washed with PBS. Cubes (0.5 cm/side) were cut and immediately immersed for 2 min in 2-methylbutane (263–1, Aldrich), which was previously cooled in liquid nitrogen. The cubes were then transferred for 10 min to liquid nitrogen. Eight-micrometer sections were cut in an IEC minotome cryostat (International Equipment). For immunofluorescence, the sections were fixed for 20 min in 70% ethanol at –20°C, washed with PBS, and quenched for unspecific staining with 0.2% IgG-free albumin (1331-A, Research Organics) in PBS for 20 min at 4°C. The immunofluorescence protocol used is described below.
Immunofluorescence. The frozen sections were incubated overnight with one of the following rabbit polyclonal antibodies: claudin-2 (51–6,100, dilution 1 μg/ml, Zymed); claudin 3 (34–1,700, dilution 5 μg/ml, Zymed); and claudin-5 (34–1,600, dilution 20 μg/ml, Zymed). The sections were washed three times with PBS and incubated for 1 h with a FITC-conjugated goat anti-rabbit IgG (65–6,111, 6 μg/ml, Zymed) or with anti-mouse IgG TRITC conjugate developed in goat (T-5393, 7.5 μg/ml, Sigma). After three more washes with PBS, the sections were transferred to glass coverslips and mounted with the antifade reagent Fluogard (170–3140, Bio-Rad). The fluorescence of the sections was examined using a confocal microscope (Leica DMRI2). The images collected had an optical thickness of 1 μm. The image shown represents a projection of the sections made for each slide.
Statistical Analyses
Student's t-test and ANOVA were used to analyze differences in quantitative variables between control and experimental groups. Results were expressed as means ± SE. A P value <0.05 was considered significant. Statistical analysis was performed using a statistical software package (SigmaStat software, SPSS).
RESULTS
Chronic Cd2+ Intoxication
Renal function. Data of the effect of a daily dose of 500 μg Cd2+/kg for 5 days are shown in Figs. 1 and 2.
Urinary flow decreased on day 5 and then had an overshoot on days 10 and 20 (Fig. 1A). The urinary-to-plasma inulin ratio (U/P) remained unchanged until day 7 and then decreased significantly (Fig. 1B). GFR was not significantly modified from day 1 to day 7. It decreased from 1.01 ± 0.04 (n = 3) in controls to 0.72 ± 0.04 (n = 3) on day 10 and to 0.77 ± 0.07 ml·min–1·kidney–1 (n = 3) on day 20 in Cd2+-treated animals.
Fractional excretion and plasma concentration of Na+, K+, Ca2+, Mg2+, Cl– and PO4 for each experimental group are shown in Fig. 2.
Regarding phosphate handling, its fractional excretion did not change from day 1 to day 7, but significantly increased on day 10 and remained higher than control until day 20. Plasma phosphate concentration was increased from day 5 to day 10 and then decreased below control at day 20.
Cl–, Na+, and Ca2+ handling followed a similar pattern: at day 10, the fractional excretions of Cl–, Na+, and Ca2+ increased, and by day 20 they were higher than control. Plasma concentrations of Cl–, Na+, and Ca2+ showed different changes over time. Cl– concentration in plasma was constant until days 10 and 20, when it became higher than control. Plasma Na+ concentration was not changed. Plasma Ca2+ concentration started to decrease at day 5 and thereafter remained low until day 20.
Interestingly, the excretory pattern for renal K+ and Mg2+ was similar. From day 1 to day 7, fractional excretions of K+ and Mg2+ decreased until days 10 and 20, when they became higher than control. Plasma concentration of K+ increased significantly since the first day of intoxication and decreased with time but stayed still higher than control. Mg2+ concentration in plasma was constant until day 5, when it became to decrease significantly.
It is important to emphasize that the fractional excretions of each ion increased significantly on days 10 and 20 and that these changes occurred 5 days after the end of Cd2+ intoxication, indicating a delayed deleterious effect of Cd2+ on renal function. All changes suggested the induction of a Fanconi-like syndrome by Cd2+.
Zn2+ protection. Experiments were also performed to investigate the effect of Zn2+ on Cd2+ nephrotoxicity. Urine flow rate, inulin U/P, GFR, plasma ion concentrations, and fractional excretion of ions were measured at days 10 and 20 after exposure to Cd2+ alone, to Zn2+ alone, and to exposure to both Cd2+ and Zn2+. The results are shown in Figs. 3 and 4. Chronic treatment by zinc (500 μg·kg–1·day–1) alone did not modify renal function or plasma ion concentrations, indicating that at this concentration Zn2+ did not cause any detectable nephrotoxic effect (Figs. 3 and 4). When the intoxication by Cd2+ was performed in the presence of an equivalent amount of Zn2+, the effects observed on urine flow rate, inulin U/P, and GFR were significantly less that the effects observed with Cd2+ alone. Thus at day 10, the decrease in inulin U/P, the increase in urinary flow rate, and the decrease in GFR induced by Cd2+ alone were partially attenuated by Zn2+. At day 20, the presence of Zn2+ completely prevented the effect of Cd2+, because no modification of inulin U/P, urine flow rate, or GFR was observed compared with the values obtained in the absence of Cd2+ or in the presence of Zn2+ alone.
Figure 4 shows the plasma concentrations and fractional excretions of Na+, K+, Ca2+, Mg2+, Cl–, and PO of rats given Cd2+ and/or Zn2+. In Cd2+-intoxicated animals, the presence of Zn2+ also prevented the changes in plasma concentrations and fractional excretions of Na+, K+, Ca2+, Mg2+, Cl–, and PO induced by Cd2+. Interestingly, the effect of Zn2+ was observed from day 10 and persisted until day 20.
Our data indicate that in the presence of Zn2+, the renal function in Cd2+-intoxicated rats remains normal and therefore Zn2+ showed a protective effect against the nephrotoxicity due to Cd2+.
Cd2+ accumulation. Figure 5A illustrates the time course of Cd2+ accumulation in the kidney and liver during chronic intoxication. In these organs, Cd2+ content gradually increased after each ip injection of Cd2+ from day 1 to day 5 and reached a maximal value at the end of the intoxication period (day 5). Cd2+ content remained at this level during the recovery period, from day 7 to day 20. This combined accumulation was also observed in the experimental groups intoxicated with 500 μg Cd2+·kg–1·day–1 and 500 μg Zn2+·kg–1·day–1, demonstrating that Zn2+ did not prevent Cd2+ accumulation in these organs.
Body weight gain. The gain in body weight of rats during chronic exposure to Cd2+, Cd2++Zn2+, and in control conditions is shown in Fig. 5B. Cd2+ ip injection reduced body weight gain. At the end of the experiment (day 20), the increase in body weight in the Cd2+-intoxicated group was reduced by 23% compared with control. In the Cd2++Zn2+-intoxicated group, there was no significant difference between animals exposed to Cd2++Zn2+ and control animals.
Apoptosis. Figure 6 shows results of caspase 3 activity, as a marker of apoptosis, measured by the degradation of a caspase 3 substrate. Data show an increase in the caspase 3 enzymatic reaction compatible with an increase in apoptosis in Cd2+-treated compared with control or Zn2+-treated animal. In Cd2+-intoxicated animals, the simultaneous injection of Zn2+ has prevented the increase in cell apoptosis.
Intercellular junctions. Immunofluorescence of renal cryosections detected claudins 2 and 5 of the tight junction complex. We analyzed the tubular distribution of claudin-2 in kidney slices from control rats, Cd2+-treated rats, and Cd2++Zn2+-treated rats (Fig. 7). As expected, in control, claudin-2 was only detectable in proximal tubules (Fig. 7, A and B), showing a clear "chicken fence" pattern at the cell borders. In Fig. 7B, a merged image shows that two tubules do not depict fluorescence as would be expected for these segments. In the presence of Cd2+, after 5 days of ip injection (Fig. 7C), claudin-2 was mainly detectable in the cytoplasm and barely at the cell border, indicating an alteration in its cellular expression and trafficking to the cell plasma membrane. When a linear fluorescence pattern was present at the cell borders, it was disrupted. These abnormalities were observed in most of the tubules that showed fluorescence for claudin-2. In rats treated with Cd2+ and Zn2+, renal sections obtained after 5 days of treatment and 5 days of recovery (Fig. 7D) showed partial preservation of the pattern of claudin-2, albeit with evident damage. After 10 days, 5 days of intoxication, and 5 days of recovery (Fig. 7E), the expression of claudin-2 was sparse and only a few proximal tubules expressed claudin-2 with a cytoplasmic pattern, indicating progression of the toxic effects of Cd2+ on proximal cells. Figure 7, D and F, shows the claudin-2 pattern in rats intoxicated with Cd2++Zn2+ at day 5 and day 10, respectively. In these two cases, the cell border pattern of claudin-2 was preserved, indicating a protective effect of Zn2+. Sections obtained from a rat after 5 days of Cd2+ treatment and 15 days of recovery show that the alterations observed in animals at days 5 and 10 were less severe but still evident (Fig. 7G). In contrast to the observations in Fig. 7G, sections from animals treated with Cd2++Zn2+ obtained after 15 days of recovery showed an almost complete recovery of the pattern for claudin-2 at the cell borders (Fig. 7H). These findings indicate that treatment with Zn2+ partially protected tubules from the damage caused by Cd2+ and accelerated recovery.
Figure 8 shows immunofluorescence for claudin-5. Claudin-5 had been demonstrated to be specific for endothelial cells. Normally, in control rats (Fig. 8A), claudin-5 stains the endothelia of glomeruli with a dotted pattern, whereas in renal vessels a linear cell border pattern is observed. In the presence of Cd2+ on day 5, vascular expression of claudin-5 was altered, and it was possible to see the disrupted elastin band, which appeared as strong autofluorescence in vessel walls (Fig. 8B). This change was not seen in the presence of Zn2+ (Fig. 8C). After the presence of Cd2+ for 5 days and after 5 days of recovery, glomeruli and a small blood vessels (Fig. 8D) showed thicker fluorescence than control, indicating vascular damage and disruption of the claudin-5 pattern. Surprisingly, with Zn2+ (Fig. 8E), glomerular capillaries were still thicker than in control, suggesting that the protective effect of Zn2+ observed in the tubules was not present in endothelial cells.
In control animals, claudin-3 fluorescence was located at the distal segments of the nephron (Fig. 9). In contrast to claudin-5, in glomerular capillaries only very scant dotted fluorescence was detectable (Fig. 9B). As for claudin-2, the epithelial pattern of claudin-3 was disrupted in Cd2+-treated animals, both after 5 days of treatment with Cd2+ alone (Fig. 9C) and after 5 days of Cd2+ treatment and 5 days of recovery (Fig. 9E). There was partial rearrangement of claudin-3 at the cell borders in the animals treated 5 days with Cd2+ and then killed 15 days later (Fig. 9F). Treatment with Zn2+ partially protected against those alterations (Fig. 9, D, F, and H).
DISCUSSION
In recent years, a large number of studies have examined the renal effects of Cd2+ intoxication. These investigations have clearly demonstrated that Cd2+ toxicity depends on the dose, the route of administration, and duration of exposure (31). However, several crucial points remain unclear. The aim of our study was to determinate the time course of renal damage during chronic intoxication and to determine whether the kidney is able to recover normal function after intoxication. Moreover, we assessed the possible therapeutic benefit of Zn2+ on the nephrotoxic effect of Cd2+. This approach has not been previously assayed.
First, to better understand the chronic effect of Cd2+ on renal function, we chose to intoxicate Wistar rats with 0.5 mg Cd2+/kg by daily ip injection for 5 days. According to Liu et al. (31), Dudley et al. (14), and Goyer et al. (19), such a dose corresponds to low-dose chronic intoxication. The present study showed that during intoxication and at the beginning of the recovery period, GFR was not affected, although significant changes in renal handling of the measured ions occurred between days 1 and 7: fractional excretions of K+, Ca2+, and Mg2+ were decreased, suggesting that, in the early stage of chronic intoxication, Cd2+ acts on ion transports without affecting renal tissue. Evidence of renal damage was only apparent at day 10, during the recovery period, and 5 days after the end of the exposure to Cd2+; GFR was decreased, and fractional excretions of all ions were increased, indicating both glomerular and proximal tubular damages. This delayed toxic effect might be explained by the long biological half-life of Cd2+. Indeed, each new daily dose was cumulative, because this heavy metal is poorly eliminated by the kidney. These results are in accordance with the data of Aoyagi et al. (3), who reported that the concentration in the kidney reached 120 μg Cd2+/g after 8 wk of daily subcutaneous injections with 0.6 mg Cd2+/kg body wt and that this treatment was nephrotoxic. These authors also described severe damage to the tubular structure due to necrotic and apoptotic cell death, which could also explain the deleterious renal effects we observed.
Another phenomenon could be responsible for tubular dysfunction and renal tissue damage observed after day 10: tubular transport occurs through a transcellular route and paracellular pathways. Thus the proximal tubulopathy might be due to a defect in tight junction organization. The tight junction constitutes the main barrier in epithelia to the passive movement of electrolytes and macromolecules through the paracellular pathway. It also functions as a barrier that maintains a polarized distribution of lipids and proteins between the apical and basolateral plasma membrane (10, 28). Claudins are constitutive junction proteins of epithelia. Reyes et al. (42) showed that in the kidney claudin-2 is predominantly located at the leaky portions of the nephron (proximal segments), whereas claudin-5 is present in the renal vasculature. Immunofluorescence experiments demonstrated that the expression of claudin-2 and claudin-5 was strongly modified after exposure to Cd2+: proximal epithelium and renal endothelium showed disorganization of the pattern of expression of proteins of the tight junction in rat kidney. To our knowledge, these alterations of claudin-2 and claudin-5 induced by Cd2+ have not been previously reported. In agreement with our findings, Prozialeck et al. (41) reported that, in rats intoxicated with 0.6 mg Cd2+/kg, cadherin-dependent cell-cell junctions in the proximal tubule were disrupted. Cadherin and catenin constitute the zonula adherens, which is present in all epithelia and located just below the tight junction complex in the proximal segment. Our results confirmed that proteins of the functional complexes like claudins, cadherins, and catenins are early targets of Cd2+ toxicity. Moreover, it must be pointed out that we observed alterations in glomerular claudin-5, whereas Prozialeck et al. did not show such modifications. These early changes in endothelial tight junctions could announce more damage to the glomerulus, leading later to severe renal failure.
The cryosections also showed dilatation of the luminal compartment of many tubules and severe damage to glomeruli and vascular endothelia. Concomitantly, this change in renal structures induced severe losses of all ions. These observations confirm the data of Brzoska et al. (9), who described that chronic exposure to Cd2+ induced injuries affecting the main reabsorptive parts (proximal convoluted tubules and straight tubules) and the filtering part (glomeruli) of the nephron. These authors also demonstrated an increase in urinary excretion of enzymes that are markers of cytotoxicity, such as N-acetyl--F-glucoseaminidase, isoenzyme B, and alkaline phosphatase. Finally, as previously reported, exposure to Cd2+ causes a Fanconi-like wasting of many filtered solutes (49).
Another goal of our study was to explore a way of protecting renal function against Cd2+ intoxication. Because Zn2+ had been demonstrated in vitro to decrease cellular damage induced by Cd2+ (12, 31, 47), it seemed reasonable to test this issue in vivo. The most noteworthy findings of the present study are that the effect of cotreatment with Zn2+ during Cd2+ administration completely prevented the changes in renal function produced by the toxic metal. Among the possible mechanisms, it might occur that Zn2+ reduced the renal uptake of Cd2+ by competition for a common transporter. Although it as been demonstrated that both metals can share the DMT1 transporter in epithelial cells, this explanation seems unlikely because we showed that Cd2+ accumulation in the kidney was not decreased by Zn2+. Perhaps a better explanation for this protection is that Zn2+ plays a role in preventing apoptosis and necrosis. Kondoh et al. (25) have demonstrated that Cd2+ induces cytochrome c release from mitochondria, leading to apoptosis via the activation of the caspase 3 and 9 cascade (25, 27, 32). The work of Perry et al. (39), who demonstrated that Zn2+ inhibited caspase-3, suggests that Zn2+ protection against Cd2+ could be due to an inhibition of caspase-3 and apoptosis. This is in agreement with our results showing an increase in apoptosis in a kidney cortex sample from a Cd2+-treated animal, measured by the activity of caspase 3, and prevented by Zn2+ treatment. Moreover, it should also be noticed that we have recently shown an increase in apoptosis in a proximal cell line incubated in the presence of Cd2+ and the prevention of necrosis phenomena when cells were incubated in the presence of Zn2+ (5).
In rabbit and mouse kidney, claudin-2 is expressed mainly in the proximal tubule, whereas claudin-3 is in the distal segments of the nephron and claudin-5 in the endothelium (26, 42). In the rat, we found that claudin-2, claudin-3, and claudin-5 are also expressed in proximal segments, distal segments, and renal vessels, respectively. As for claudin-2, expression of claudin-3 was altered in Cd2+-treated animals and Zn2+ afforded partial protection against those alterations.
These data suggest that in chronic Cd2+ contamination, Cd2+ has an effect in the proximal as well as in distal tubule, leading to a pronounced renal defect. The protective effect of Zn2+ on renal function could be used as the basis of preventive treatment for potential Cd2+ intoxication.
GRANTS
This work was supported by ECOS-Nord, a cooperative program between France and Mexico (M02-S02).
ACKNOWLEDGMENTS
We are grateful to Dr. Robert Unwin for constructive criticism of this manuscript, corrections, and for helpful discussions.
FOOTNOTES
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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2Departamento de Fisiologia y Biofisica, Centro de Investigacion y de Estudios Avanzados del Instituto Politecnico Nacional, Mexico City, Mexico
ABSTRACT
This study investigates the effect in the rat of chronic CdCl2 intoxication (500 μg Cd2+/kg, daily ip injection for 5 days) on renal function and the changes in tight junction proteins claudin-2, claudin-3, and claudin-5 present in rat kidney. We also studied the effect of coadministration of ZnCl2 (500 μg Zn2+/kg) during chronic CdCl2 intoxication. Our results indicate that 1) most of the filtered Cd2+ is reabsorbed within the kidney; 2) chronic Cd2+ intoxication can induce a change in renal handling of ions without altering glomerular filtration rate; 3) a delayed nephropathy, showing Fanconi-like features, appears more than 5 days after the end of CdCl2 exposure; 4) epithelial integrity is altered by chronic Cd2+ intoxication affecting the expression and localization of claudin tight junction proteins; and 5) cotreatment with Zn2+ protects against the renal toxic effects of Cd2+, preventing altered claudin expression and inhibiting apoptosis. In conclusion, these results show that Cd2+ toxicity and cellular toxic mechanisms are complex, probably affecting both membrane transporters and tight junction proteins. Finally, Zn2+ supplementation may provide a basis for future treatments.
heavy metals; kidney; protective treatment; tight junction
CADMIUM (CD2+) IS ONE OF THE most common toxic metals in our environment. The major sources of exposure to Cd2+ in the general population are contaminated food and water, tobacco and industrial smoke, and dust (18). Cd2+ accumulates in the body and has a very long biological half-life (10–30 yr) in humans (15, 18).
It is known that chronic exposure to Cd2+ can induce severe nephropathy in humans (18) and animals (4, 9). This nephrotoxicity causes reabsorptive and secretory dysfunction of the renal tubule. The main signs include proteinuria, ion losses, glucosuria, aminoaciduria, and polyuria (1, 2, 21, 24, 29, 31). Experimental chronic intoxication with Cd2+ has been performed at various doses over several weeks (9, 30, 48) and causes a Fanconi-like syndrome with predominant tubular dysfunction that develops into renal failure (47, 48). However, to our knowledge no detailed study of renal function during chronic Cd2+ intoxication has been carried out using clearance methods. Thus a documented time course for the appearance of the deleterious effects of Cd2+ exposure has not been described and would be useful in understanding the toxic mechanisms involved.
Several mechanisms have been proposed to explain the toxic effect of Cd2+ on renal cells. Cd2+ may cause nephrotoxicity by generating free radicals (20, 42) and by inducing necrosis and apoptosis (13). Interestingly, a protective effect of zinc (Zn2+) has been reported in vitro against the cellular toxicity due to Cd2+. Zn2+ protection is probably due to an action on oxidative stress and apoptosis (8, 17, 23, 43). In addition, exposure of renal cultured cells (LLC-PK1 line) to Cd2+ caused a decrease in transepithelial electrical resistance and in the number of domes, suggesting alterations of the tight junctions (41).
For the reasons outlined above, we decided to study the renal effects of Cd2+ during chronic exposure to Cd2+ and the putative protective effects of zinc.
MATERIALS AND METHODS
Clearance Experiments
These experiments were performed to analyze the effect of chronic intoxication with Cd2+ on whole kidney function and were carried out in female Wistar rats weighing 180–220 g. The animals were fed a standard laboratory diet. They had free access to water until the beginning of the experiments and were starved for 18 h before surgery. Anesthesia was induced by intraperitoneal (ip) injection of pentobarbital sodium (Nembutal, 5 mg/100 g body wt) and maintained by additional 1-mg doses when necessary. The animals were placed on a heated table to maintain their body temperature between 37 and 38°C. A tracheotomy was performed leaving the thyroid gland untouched. One catheter (Clay Adams, PE-20) was inserted into the right jugular vein for infusion and another (Clay Adams, PE-10) in the left ureter for urine collection. A third catheter (Clay Adams, PE-50) was inserted into the right femoral artery for blood sampling and arterial blood pressure recording (Research BP Transducer, Harvard Apparatus). Clearance experiments were carried out in rats infused with 0.9% NaCl solution at a rate of 20 μl/min. [3H]methoxy-inulin (TRA.324 Specific radioactivity, 120 μCi/mg inulin, 0.53 Ci/mmol; Amersham Pharmacia Biotech) was used to estimate the glomerular filtration rate (GFR). Urine samples were collected serially during 20-min periods, and blood samples were taken halfway through each urine collection. In all experiments, a loading dose of [3H]inulin (4 μCi) was given, 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.
At the end of the experiment, the rats were killed by an overdose of Nembutal, and the liver and kidneys were removed for measurement of cadmium content. The use of animals was in accordance with the Institute for Laboratory Animal Research Guide for Care and Use of Laboratory Animals.
Chronic Intoxication with Cd2+ and Protection by Zn2+
In the Cd2+-treated groups, rats were intoxicated by daily ip injection for 5 days with 500 μg Cd2+·kg–1·day–1. There was a recovery period of 15 days, without further Cd2+ exposure. Clearance experiments were performed on days 0 (control group), 1, 3, 5, 7, 10, and 20. To study the protective effect of Zn2+, Wistar rats were injected daily ip with a solution containing both 500 μg Cd2+·kg–1·day–1 and 500 μg Zn2+·kg–1·day–1 for 5 days. Clearance experiments were performed on day 10 and day 20. Experiments were also performed in a control group and a treated group with 500 μg Zn2+·kg–1·day–1 for 5 days. Each group comprised five rats.
Rat body weights were recorded daily to determine the effect of Cd2+ on the general health of the animals.
Analytic Procedures
3H radioactivity was 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).
Cd2+ Measurement in Tissue
The technique used to measure Cd2+ was described earlier by Playle et al. (40). Liver and kidney samples were taken, weighed, and digested in a solution of 1 N nitric acid (TraceMetal Grade HNO3; Fisher Scientific) at a temperature of 80°C for 4 h. At the end of this time, the supernatant was taken, and its volume was measured to calculate Cd2+ concentration. The supernatant was then diluted 1:1,000 with deionized water, and Cd2+ content was determined by atomic absorption spectrometry using a Zeeman furnace system (Solaar 969, Thermo Optek).
Apoptosis Measurement in Tissue
Rats were intoxicated with one ip injection of either NaCl as a control, 500 Cd2+/kg, both 500 μg Cd2+/kg and 500 μg Zn2+/kg, or only 500 μg Zn2+/kg. Twenty-four hours after the injection, the kidneys were perfused with 1x PBS and then dissected from euthanized animals. The kidney cortexes were then removed and homogenized immediately in lysis buffer (Euromedex). Protein was quantified using the Bradford assay (Bio-Rad) using BSA as a standard. Apoptosis measurement in cortex samples was monitored by measuring the color generated due to hydrolysis of a chromogenic caspase-3 substrate (Apopain, Euromedex) added to the assay medium. Color was measured using a plate reader.
Rat Frozen Kidney Tissue Sections
Kidneys of experimental rats were removed at the end of each experiment and washed with PBS. Cubes (0.5 cm/side) were cut and immediately immersed for 2 min in 2-methylbutane (263–1, Aldrich), which was previously cooled in liquid nitrogen. The cubes were then transferred for 10 min to liquid nitrogen. Eight-micrometer sections were cut in an IEC minotome cryostat (International Equipment). For immunofluorescence, the sections were fixed for 20 min in 70% ethanol at –20°C, washed with PBS, and quenched for unspecific staining with 0.2% IgG-free albumin (1331-A, Research Organics) in PBS for 20 min at 4°C. The immunofluorescence protocol used is described below.
Immunofluorescence. The frozen sections were incubated overnight with one of the following rabbit polyclonal antibodies: claudin-2 (51–6,100, dilution 1 μg/ml, Zymed); claudin 3 (34–1,700, dilution 5 μg/ml, Zymed); and claudin-5 (34–1,600, dilution 20 μg/ml, Zymed). The sections were washed three times with PBS and incubated for 1 h with a FITC-conjugated goat anti-rabbit IgG (65–6,111, 6 μg/ml, Zymed) or with anti-mouse IgG TRITC conjugate developed in goat (T-5393, 7.5 μg/ml, Sigma). After three more washes with PBS, the sections were transferred to glass coverslips and mounted with the antifade reagent Fluogard (170–3140, Bio-Rad). The fluorescence of the sections was examined using a confocal microscope (Leica DMRI2). The images collected had an optical thickness of 1 μm. The image shown represents a projection of the sections made for each slide.
Statistical Analyses
Student's t-test and ANOVA were used to analyze differences in quantitative variables between control and experimental groups. Results were expressed as means ± SE. A P value <0.05 was considered significant. Statistical analysis was performed using a statistical software package (SigmaStat software, SPSS).
RESULTS
Chronic Cd2+ Intoxication
Renal function. Data of the effect of a daily dose of 500 μg Cd2+/kg for 5 days are shown in Figs. 1 and 2.
Urinary flow decreased on day 5 and then had an overshoot on days 10 and 20 (Fig. 1A). The urinary-to-plasma inulin ratio (U/P) remained unchanged until day 7 and then decreased significantly (Fig. 1B). GFR was not significantly modified from day 1 to day 7. It decreased from 1.01 ± 0.04 (n = 3) in controls to 0.72 ± 0.04 (n = 3) on day 10 and to 0.77 ± 0.07 ml·min–1·kidney–1 (n = 3) on day 20 in Cd2+-treated animals.
Fractional excretion and plasma concentration of Na+, K+, Ca2+, Mg2+, Cl– and PO4 for each experimental group are shown in Fig. 2.
Regarding phosphate handling, its fractional excretion did not change from day 1 to day 7, but significantly increased on day 10 and remained higher than control until day 20. Plasma phosphate concentration was increased from day 5 to day 10 and then decreased below control at day 20.
Cl–, Na+, and Ca2+ handling followed a similar pattern: at day 10, the fractional excretions of Cl–, Na+, and Ca2+ increased, and by day 20 they were higher than control. Plasma concentrations of Cl–, Na+, and Ca2+ showed different changes over time. Cl– concentration in plasma was constant until days 10 and 20, when it became higher than control. Plasma Na+ concentration was not changed. Plasma Ca2+ concentration started to decrease at day 5 and thereafter remained low until day 20.
Interestingly, the excretory pattern for renal K+ and Mg2+ was similar. From day 1 to day 7, fractional excretions of K+ and Mg2+ decreased until days 10 and 20, when they became higher than control. Plasma concentration of K+ increased significantly since the first day of intoxication and decreased with time but stayed still higher than control. Mg2+ concentration in plasma was constant until day 5, when it became to decrease significantly.
It is important to emphasize that the fractional excretions of each ion increased significantly on days 10 and 20 and that these changes occurred 5 days after the end of Cd2+ intoxication, indicating a delayed deleterious effect of Cd2+ on renal function. All changes suggested the induction of a Fanconi-like syndrome by Cd2+.
Zn2+ protection. Experiments were also performed to investigate the effect of Zn2+ on Cd2+ nephrotoxicity. Urine flow rate, inulin U/P, GFR, plasma ion concentrations, and fractional excretion of ions were measured at days 10 and 20 after exposure to Cd2+ alone, to Zn2+ alone, and to exposure to both Cd2+ and Zn2+. The results are shown in Figs. 3 and 4. Chronic treatment by zinc (500 μg·kg–1·day–1) alone did not modify renal function or plasma ion concentrations, indicating that at this concentration Zn2+ did not cause any detectable nephrotoxic effect (Figs. 3 and 4). When the intoxication by Cd2+ was performed in the presence of an equivalent amount of Zn2+, the effects observed on urine flow rate, inulin U/P, and GFR were significantly less that the effects observed with Cd2+ alone. Thus at day 10, the decrease in inulin U/P, the increase in urinary flow rate, and the decrease in GFR induced by Cd2+ alone were partially attenuated by Zn2+. At day 20, the presence of Zn2+ completely prevented the effect of Cd2+, because no modification of inulin U/P, urine flow rate, or GFR was observed compared with the values obtained in the absence of Cd2+ or in the presence of Zn2+ alone.
Figure 4 shows the plasma concentrations and fractional excretions of Na+, K+, Ca2+, Mg2+, Cl–, and PO of rats given Cd2+ and/or Zn2+. In Cd2+-intoxicated animals, the presence of Zn2+ also prevented the changes in plasma concentrations and fractional excretions of Na+, K+, Ca2+, Mg2+, Cl–, and PO induced by Cd2+. Interestingly, the effect of Zn2+ was observed from day 10 and persisted until day 20.
Our data indicate that in the presence of Zn2+, the renal function in Cd2+-intoxicated rats remains normal and therefore Zn2+ showed a protective effect against the nephrotoxicity due to Cd2+.
Cd2+ accumulation. Figure 5A illustrates the time course of Cd2+ accumulation in the kidney and liver during chronic intoxication. In these organs, Cd2+ content gradually increased after each ip injection of Cd2+ from day 1 to day 5 and reached a maximal value at the end of the intoxication period (day 5). Cd2+ content remained at this level during the recovery period, from day 7 to day 20. This combined accumulation was also observed in the experimental groups intoxicated with 500 μg Cd2+·kg–1·day–1 and 500 μg Zn2+·kg–1·day–1, demonstrating that Zn2+ did not prevent Cd2+ accumulation in these organs.
Body weight gain. The gain in body weight of rats during chronic exposure to Cd2+, Cd2++Zn2+, and in control conditions is shown in Fig. 5B. Cd2+ ip injection reduced body weight gain. At the end of the experiment (day 20), the increase in body weight in the Cd2+-intoxicated group was reduced by 23% compared with control. In the Cd2++Zn2+-intoxicated group, there was no significant difference between animals exposed to Cd2++Zn2+ and control animals.
Apoptosis. Figure 6 shows results of caspase 3 activity, as a marker of apoptosis, measured by the degradation of a caspase 3 substrate. Data show an increase in the caspase 3 enzymatic reaction compatible with an increase in apoptosis in Cd2+-treated compared with control or Zn2+-treated animal. In Cd2+-intoxicated animals, the simultaneous injection of Zn2+ has prevented the increase in cell apoptosis.
Intercellular junctions. Immunofluorescence of renal cryosections detected claudins 2 and 5 of the tight junction complex. We analyzed the tubular distribution of claudin-2 in kidney slices from control rats, Cd2+-treated rats, and Cd2++Zn2+-treated rats (Fig. 7). As expected, in control, claudin-2 was only detectable in proximal tubules (Fig. 7, A and B), showing a clear "chicken fence" pattern at the cell borders. In Fig. 7B, a merged image shows that two tubules do not depict fluorescence as would be expected for these segments. In the presence of Cd2+, after 5 days of ip injection (Fig. 7C), claudin-2 was mainly detectable in the cytoplasm and barely at the cell border, indicating an alteration in its cellular expression and trafficking to the cell plasma membrane. When a linear fluorescence pattern was present at the cell borders, it was disrupted. These abnormalities were observed in most of the tubules that showed fluorescence for claudin-2. In rats treated with Cd2+ and Zn2+, renal sections obtained after 5 days of treatment and 5 days of recovery (Fig. 7D) showed partial preservation of the pattern of claudin-2, albeit with evident damage. After 10 days, 5 days of intoxication, and 5 days of recovery (Fig. 7E), the expression of claudin-2 was sparse and only a few proximal tubules expressed claudin-2 with a cytoplasmic pattern, indicating progression of the toxic effects of Cd2+ on proximal cells. Figure 7, D and F, shows the claudin-2 pattern in rats intoxicated with Cd2++Zn2+ at day 5 and day 10, respectively. In these two cases, the cell border pattern of claudin-2 was preserved, indicating a protective effect of Zn2+. Sections obtained from a rat after 5 days of Cd2+ treatment and 15 days of recovery show that the alterations observed in animals at days 5 and 10 were less severe but still evident (Fig. 7G). In contrast to the observations in Fig. 7G, sections from animals treated with Cd2++Zn2+ obtained after 15 days of recovery showed an almost complete recovery of the pattern for claudin-2 at the cell borders (Fig. 7H). These findings indicate that treatment with Zn2+ partially protected tubules from the damage caused by Cd2+ and accelerated recovery.
Figure 8 shows immunofluorescence for claudin-5. Claudin-5 had been demonstrated to be specific for endothelial cells. Normally, in control rats (Fig. 8A), claudin-5 stains the endothelia of glomeruli with a dotted pattern, whereas in renal vessels a linear cell border pattern is observed. In the presence of Cd2+ on day 5, vascular expression of claudin-5 was altered, and it was possible to see the disrupted elastin band, which appeared as strong autofluorescence in vessel walls (Fig. 8B). This change was not seen in the presence of Zn2+ (Fig. 8C). After the presence of Cd2+ for 5 days and after 5 days of recovery, glomeruli and a small blood vessels (Fig. 8D) showed thicker fluorescence than control, indicating vascular damage and disruption of the claudin-5 pattern. Surprisingly, with Zn2+ (Fig. 8E), glomerular capillaries were still thicker than in control, suggesting that the protective effect of Zn2+ observed in the tubules was not present in endothelial cells.
In control animals, claudin-3 fluorescence was located at the distal segments of the nephron (Fig. 9). In contrast to claudin-5, in glomerular capillaries only very scant dotted fluorescence was detectable (Fig. 9B). As for claudin-2, the epithelial pattern of claudin-3 was disrupted in Cd2+-treated animals, both after 5 days of treatment with Cd2+ alone (Fig. 9C) and after 5 days of Cd2+ treatment and 5 days of recovery (Fig. 9E). There was partial rearrangement of claudin-3 at the cell borders in the animals treated 5 days with Cd2+ and then killed 15 days later (Fig. 9F). Treatment with Zn2+ partially protected against those alterations (Fig. 9, D, F, and H).
DISCUSSION
In recent years, a large number of studies have examined the renal effects of Cd2+ intoxication. These investigations have clearly demonstrated that Cd2+ toxicity depends on the dose, the route of administration, and duration of exposure (31). However, several crucial points remain unclear. The aim of our study was to determinate the time course of renal damage during chronic intoxication and to determine whether the kidney is able to recover normal function after intoxication. Moreover, we assessed the possible therapeutic benefit of Zn2+ on the nephrotoxic effect of Cd2+. This approach has not been previously assayed.
First, to better understand the chronic effect of Cd2+ on renal function, we chose to intoxicate Wistar rats with 0.5 mg Cd2+/kg by daily ip injection for 5 days. According to Liu et al. (31), Dudley et al. (14), and Goyer et al. (19), such a dose corresponds to low-dose chronic intoxication. The present study showed that during intoxication and at the beginning of the recovery period, GFR was not affected, although significant changes in renal handling of the measured ions occurred between days 1 and 7: fractional excretions of K+, Ca2+, and Mg2+ were decreased, suggesting that, in the early stage of chronic intoxication, Cd2+ acts on ion transports without affecting renal tissue. Evidence of renal damage was only apparent at day 10, during the recovery period, and 5 days after the end of the exposure to Cd2+; GFR was decreased, and fractional excretions of all ions were increased, indicating both glomerular and proximal tubular damages. This delayed toxic effect might be explained by the long biological half-life of Cd2+. Indeed, each new daily dose was cumulative, because this heavy metal is poorly eliminated by the kidney. These results are in accordance with the data of Aoyagi et al. (3), who reported that the concentration in the kidney reached 120 μg Cd2+/g after 8 wk of daily subcutaneous injections with 0.6 mg Cd2+/kg body wt and that this treatment was nephrotoxic. These authors also described severe damage to the tubular structure due to necrotic and apoptotic cell death, which could also explain the deleterious renal effects we observed.
Another phenomenon could be responsible for tubular dysfunction and renal tissue damage observed after day 10: tubular transport occurs through a transcellular route and paracellular pathways. Thus the proximal tubulopathy might be due to a defect in tight junction organization. The tight junction constitutes the main barrier in epithelia to the passive movement of electrolytes and macromolecules through the paracellular pathway. It also functions as a barrier that maintains a polarized distribution of lipids and proteins between the apical and basolateral plasma membrane (10, 28). Claudins are constitutive junction proteins of epithelia. Reyes et al. (42) showed that in the kidney claudin-2 is predominantly located at the leaky portions of the nephron (proximal segments), whereas claudin-5 is present in the renal vasculature. Immunofluorescence experiments demonstrated that the expression of claudin-2 and claudin-5 was strongly modified after exposure to Cd2+: proximal epithelium and renal endothelium showed disorganization of the pattern of expression of proteins of the tight junction in rat kidney. To our knowledge, these alterations of claudin-2 and claudin-5 induced by Cd2+ have not been previously reported. In agreement with our findings, Prozialeck et al. (41) reported that, in rats intoxicated with 0.6 mg Cd2+/kg, cadherin-dependent cell-cell junctions in the proximal tubule were disrupted. Cadherin and catenin constitute the zonula adherens, which is present in all epithelia and located just below the tight junction complex in the proximal segment. Our results confirmed that proteins of the functional complexes like claudins, cadherins, and catenins are early targets of Cd2+ toxicity. Moreover, it must be pointed out that we observed alterations in glomerular claudin-5, whereas Prozialeck et al. did not show such modifications. These early changes in endothelial tight junctions could announce more damage to the glomerulus, leading later to severe renal failure.
The cryosections also showed dilatation of the luminal compartment of many tubules and severe damage to glomeruli and vascular endothelia. Concomitantly, this change in renal structures induced severe losses of all ions. These observations confirm the data of Brzoska et al. (9), who described that chronic exposure to Cd2+ induced injuries affecting the main reabsorptive parts (proximal convoluted tubules and straight tubules) and the filtering part (glomeruli) of the nephron. These authors also demonstrated an increase in urinary excretion of enzymes that are markers of cytotoxicity, such as N-acetyl--F-glucoseaminidase, isoenzyme B, and alkaline phosphatase. Finally, as previously reported, exposure to Cd2+ causes a Fanconi-like wasting of many filtered solutes (49).
Another goal of our study was to explore a way of protecting renal function against Cd2+ intoxication. Because Zn2+ had been demonstrated in vitro to decrease cellular damage induced by Cd2+ (12, 31, 47), it seemed reasonable to test this issue in vivo. The most noteworthy findings of the present study are that the effect of cotreatment with Zn2+ during Cd2+ administration completely prevented the changes in renal function produced by the toxic metal. Among the possible mechanisms, it might occur that Zn2+ reduced the renal uptake of Cd2+ by competition for a common transporter. Although it as been demonstrated that both metals can share the DMT1 transporter in epithelial cells, this explanation seems unlikely because we showed that Cd2+ accumulation in the kidney was not decreased by Zn2+. Perhaps a better explanation for this protection is that Zn2+ plays a role in preventing apoptosis and necrosis. Kondoh et al. (25) have demonstrated that Cd2+ induces cytochrome c release from mitochondria, leading to apoptosis via the activation of the caspase 3 and 9 cascade (25, 27, 32). The work of Perry et al. (39), who demonstrated that Zn2+ inhibited caspase-3, suggests that Zn2+ protection against Cd2+ could be due to an inhibition of caspase-3 and apoptosis. This is in agreement with our results showing an increase in apoptosis in a kidney cortex sample from a Cd2+-treated animal, measured by the activity of caspase 3, and prevented by Zn2+ treatment. Moreover, it should also be noticed that we have recently shown an increase in apoptosis in a proximal cell line incubated in the presence of Cd2+ and the prevention of necrosis phenomena when cells were incubated in the presence of Zn2+ (5).
In rabbit and mouse kidney, claudin-2 is expressed mainly in the proximal tubule, whereas claudin-3 is in the distal segments of the nephron and claudin-5 in the endothelium (26, 42). In the rat, we found that claudin-2, claudin-3, and claudin-5 are also expressed in proximal segments, distal segments, and renal vessels, respectively. As for claudin-2, expression of claudin-3 was altered in Cd2+-treated animals and Zn2+ afforded partial protection against those alterations.
These data suggest that in chronic Cd2+ contamination, Cd2+ has an effect in the proximal as well as in distal tubule, leading to a pronounced renal defect. The protective effect of Zn2+ on renal function could be used as the basis of preventive treatment for potential Cd2+ intoxication.
GRANTS
This work was supported by ECOS-Nord, a cooperative program between France and Mexico (M02-S02).
ACKNOWLEDGMENTS
We are grateful to Dr. Robert Unwin for constructive criticism of this manuscript, corrections, and for helpful discussions.
FOOTNOTES
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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