ToxSci Advance Access originally published online on March 28, 2008
Toxicological Sciences 2008 103(2):397-408; doi:10.1093/toxsci/kfn052
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Renal Anemia Induced by Chronic Ingestion of Depleted Uranium in Rats
* Institut de Radioprotection et de Sûreté Nucléaire, Direction de la RadioProtection de l'Homme, Service de Radiobiologie et d'Epidémiologie, F-92262 Fontenay-aux-Roses Cedex, France
Unité des Maladies Métaboliques et Micronutriments, Institut National de la Recherche Agronomique, Centre de Clermont-Ferrand/Theix, F63122 Saint-Genes Champanelle, France
1 To whom correspondence should be addressed at Institut de Radioprotection et de Sûreté Nucléaire, Direction de la RadioProtection de l'Homme, Service de Radiobiologie et d'Epidémiologie, Laboratoire de Radiotoxicologie Expérimentale, IRSN, B. P. n°17, F 92262 Fontenay-aux-Roses Cedex, France. Fax: +33-1-58-35-84-67. E-mail: isabelle.dublineau{at}irsn.fr.
Received December 5, 2007; accepted March 2, 2008
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSResultsDiscussionFUNDINGREFERENCES |
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Kidney disease is a frequent consequence of heavy metal exposure and renal anemia occurs secondarily to the progression of kidney deterioration into chronic disease. In contrast, little is known about effects on kidney of chronic exposure to low levels of depleted uranium (DU). Study was performed with rats exposed to DU at 40 mg/l by chronic ingestion during 9 months. In the present work, a
20% reduction in red blood cell (RBC) count was observed after DU exposure. Hence, three hypotheses were tested to determinate origin of RBC loss: (1) reduced erythropoiesis, (2) increased RBC degradation, and/or (3) kidney dysfunction. Erythropoiesis was not reduced after exposure to DU as revealed by erythroid progenitors, blood Flt3 ligand and erythropoietin (EPO) blood and kidney levels. Concerning messenger RNA (mRNA) and protein levels of spleen iron recycling markers from RBC degradation (DMT1 [divalent metal transporter 1], iron regulated protein 1, HO1, HO2 [heme oxygenase 1 and 2], cluster of differentiation 36), increase in HO2 and DMT1 mRNA level was induced after chronic exposure to DU. Kidneys of DU-contaminated rats had more frequently high grade tubulo-interstitial and glomerular lesions, accumulated iron more frequently and presented more apoptotic cells. In addition, chronic exposure to DU induced increased gene expression of ceruloplasmin (x12), of DMT1 (x2.5), and decreased mRNA levels of erythropoietin receptor (x0.2). Increased mRNA level of DMT1 was associated to decreased protein level (x0.25). To conclude, a chronic ingestion of DU leads mainly to kidney deterioration that is probably responsible for RBC count decrease in rats. Spleen erythropoiesis and molecules involved in erythrocyte degradation were also modified by chronic DU exposure. Key Words: metal; iron homeostasis; chronic; ingestion; kidney; depleted uranium.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSResultsDiscussionFUNDINGREFERENCES |
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The properties of kidney to reabsorb and accumulate divalent metals make these tissues the first target of heavy metal intoxication. For instance, acute exposure to chromium leads to tubular necrosis, and tubular proteinuria observed in workers suggests that chronic chromium exposure might also induce tubular lesions (Wedeen and Qian, 1991
). In a clinical study, chronic exposure to dietary cadmium (Cd) was associated with chronic end-stage renal failure (Satarug and Moore, 2004). Experimentally, it has been shown that chronic contamination to low levels of Cd induces tubular damages in rat (Brzoska et al., 2003). Lead is well known to induce, among others, renal insufficiency (Patrick, 2006).
Kidney is also particularly sensitive to uranium, a radioactive heavy metal. In 1909, it was shown histologically for the first time that acute uranium exposure induced nephrotoxicity (Dickson, 1909). Because then, several authors evidenced the consequences of acute exposure to high uranium concentrations on kidney at histological, cellular, and molecular levels (Bencosme et al., 1960; Goldman et al., 2006). However, though the effects of acute uranium exposure on kidney are well documented, the renal response to chronic contamination with small depleted uranium (DU) doses remains unknown. Nowadays the biological effects of chronic exposure to DU are becoming an increasing concern. Extensive civil and military applications using DU lead to increased environmental contamination which means it is important to address the consequences of its ingestion via the food chain and/or drinking water on human health (Abu-Qare and Abou-Donia, 2002). There is accumulating evidence that shows effects of chronic exposure to small doses of DU on the central nervous system (Lestaevel et al., 2005), liver (Gilman et al., 1998; Pellmar et al., 1999a, b; Souidi et al., 2005; Tissandie et al., 2007), intestine (Dublineau et al., 2007), lung (Souidi et al., 2005), and kidney (Donnadieu-Claraz et al., 2007; Taulan et al., 2004). In light of the previous data concerning kidney and acute uranium exposure, investigations about the effects of daily DU exposure on kidney are of public health interest. As it is broadly accepted that renal deterioration may lead to the progression of chronic kidney disease responsible for anemia, it could be hypothesized that chronic long-term ingestion of DU may result in progressive kidney deterioration, which would induce hematological changes. To test this hypothesis, rats were subjected to 9-month contamination with 40 mg DU/l in their drinking water. Their blood cell count was then examined. A decrease was observed in their red blood cell (RBC) content. To explain this reduction, three hypotheses were tested: reduced erythropoiesis, elevated RBC degradation and renal deterioration. The present study demonstrates that the diminution of RBC number induced by chronic exposure to DU was mainly due to renal deterioration. In spleen, erythropoiesis was slightly increased, as well as iron recycling (via increased messenger RNA [mRNA] levels of DMT1 [divalent metal transporter 1]).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSResultsDiscussionFUNDINGREFERENCES |
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Animals.
Sprague–Dawley male rats (Charles River, France) weighed 250 g at the beginning of the experiment. The rats were housed in pairs, with a 12-h light/12-h dark cycle (light on: 08:00 h/20:00 h) and a temperature of 22 ± 1°C. Animals were given ad libitum standard diet (Safe, R04 chow, France). Drinking mineral water was also delivered ad libitum. All experimental procedures were approved by the Animal Care Committee of the Institute of Radiation protection and Nuclear Safety and complied with French regulations for animal experimentation (Ministry of Agriculture Act No. 87-848, October 19, 1987, modified 29 May 2001).
DU exposure.
Rats (3 months old) were divided into two groups: an experimental group exposed to DU (DU-contaminated rats) in their drinking mineral water for 9 months and a second group of control rats that received the same drinking mineral water without DU. The mineral water used for DU exposure has the following composition (in mg/l): Ca2+, 78; Mg2+, 24; Na+, 5; K+, 1; SO42–, 10; HCO3–, 357; Cl–, 4,5. The DU concentration in water was 40 mg/l (specific activity, 25.103 Bq/g; 238U, 99.28%; 235U, 0.72%; 234U, 0.0056%; Merck, Strasbourg, France). The DU dose chosen in the present study was twice the highest environmental concentrations found in Finland (20 mg/l; Juntunen, 1991). In rat, this concentration corresponded to a daily ingestion of 1 mg per animal. DU-contaminated and control rats were raised in the same conditions with weekly measurement of their body weight, food, and water intake during the whole experiment.
Plasma and organ sampling.
Rats were anesthetized by inhalation (TEM anesthesia, Angers, France) of 95% air/5% isoflurane (Forène, Abbott, Rungis, France) and killed by intracardiac puncture with a 10-ml syringe to collect blood. Spleen and kidney were then excised, weighed, quickly frozen in liquid nitrogen and stored at –80°C. One third of the spleen and one femur were placed in washing medium composed of RPMI (Roswell Park Memorial Institute) 1640 medium supplemented with penicillin, streptocin, and 1% fetal calf serum (all from Invitrogen, le Pont de Claix, France) until they were processed for cell culture.
Blood analysis.
A complete blood cell count was carried out immediately after blood collection on an automated cell counter, MS9 (Melet Schloesing Laboratoires, Osny, France). Blood was centrifuged at 4000 x g at 4°C for 10 min to collect serum or plasma. Serum and plasma were aliquoted and stored at –80°C.
The serum unsaturated iron binding capacity (UIBC) of control and DU-contaminated rats was estimated colorimetrically at 600 nm using a colorimetric kit (UIBC-Test or Fer-CTF, Biolabo, Maizy, France) according to the manufacturer's instructions. The total iron binding capacity (TIBC) was then calculated as the sum of the UIBC and serum iron concentration.
Plasma concentrations of Flt3 ligand (Flt3l) and erythropoietin (EPO) were measured by a sandwich enzyme-linked immunosorbent assays according to the manufacturer's recommendation (R&D Systems, Abington, UK). The sensitivity of the assays was 5 pg/ml for Flt3 ligand and 12.5 pg/ml for EPO.
Plasma urea and creatinine of DU-contaminated and control rats were measured using kit reagents and automated Konelab 20 apparatus (Thermo Electron Corporation, Courtaboeuf, France).
Colony-forming cell assays.
Spleen was crushed into a tissue grinder and femurs were flushed using a 10-ml syringe mounted with a 19-G needle with washing medium. Cell suspensions were centrifuged 10 min at 400 x g. Cells were counted in the presence of 1:10 dilution of trypan blue dye. This allowed the determination of cell viability by trypan blue exclusion. Spleen and bone marrow cells were then plated at 5 x 105 and 5 x 104 cells, respectively, in 1.1 ml of complete methyl cellulose medium with recombinant cytokines (Stem Cell Technologies, Vancouver, Canada). Cultures were incubated at 37°C in 95% air/5% CO2 in a humidified atmosphere. Colony-forming units–granulocyte macrophage (CFU-GM), burst-forming units–erythroid (BFU-E), and CFU-granulocyte erythrocyte monocyte megakaryocytes were scored when composed of more than 50 white cells and/or red cells onto an inverted microscope on day 12 of culture.
Gene expression analysis.
Total RNA from spleen and kidney of both control and DU-contaminated rats were extracted with the RNeasy total RNA isolation Kit (Qiagen, Courtaboeuf, France) according to the manufacturer's instructions. Firstly, a lysis buffer containing 1% beta-mercaptoethanol was added to tissues that were then crushed using ribolyser (Hybaid, Thermoscientific, Courtaboeuf, France). After a 3-min centrifugation at 11,000 x g, the tissue lysates were homogenized with 70% ethanol and distributed on RNeasy column with silica resin. Different steps of elution and centrifugation were then applied. ARN was finally eluted with 40 µl of RNAse free water. The RNA quality was assessed by electrophoresis on ethidium bromide-stained agarose gel and by A260/A280 nm absorption ratio. One microgram of total RNA was reverse transcribed in complimentary DNA (cDNA) using BD Sprint PowerScript PrePrimed 96 Plate (BD Biosciences Clontech, Erembodegem, Belgium).
The following genes were studied: DMT1, Ireg1 (iron regulated protein 1), HO1, HO2 (heme oxygenase 1 and 2), CD36 (cluster of differentiation 36), EPO, erythropoietin receptor (EPOR), CP (ceruloplasmin), and the housekeeping gene hypoxanthine–guanine phosphoribosyltransferase (HPRT). The sequences for the forward and reverse primers used in the present study are listed in Table 1. Except for Ireg1 primers which were chosen from literature (Collins et al., 2005), primers were designed using PrimerTool (http://biotools.umassmed.edu/bioapps/primer3_www.cgi, funded by Howard Hughes Medical Institute and by the National Institutes of Health, National Human Genome Research Institute). Experiments were performed with the ABI prism 7000 apparatus (Applied Biosystems, Courtaboeuf, France). Relative mRNA levels were quantified using the comparative 
CT method. The relative quantification of the target, normalized to an endogenous reference (HPRT) and a relevant uncontaminated control, equals 
, with CT defined as the difference between the mean CT (contaminated sample) and mean CT (control sample) and CT as the difference between mean CT (interest gene) and CT (HPRT) as the endogenous control. Each sample was monitored for fluorescent dyes, and signals were regarded as significant if the fluorescence intensity significantly exceeded (10-fold) the standard deviation of the baseline fluorescence, defined as threshold cycles (CT). The data were thus expressed as the ratio of each specific gene expression to HPRT used as housekeeping gene.
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Protein analysis.
The following proteins were studied: DMT1, HO1, HO2, CD36, and the housekeeping protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Tissues extracted (
30 mg) from spleen of control and DU-contaminated rats were homogenized in a cold cell lysis buffer (radio immunoprecipitation assay buffer) containing protease-inhibitor cocktail (Sigma Aldrich, St-Quentin-Fallavier, France). After 20 min of incubation on ice, samples were centrifuged at 12,500x g at 4°C. Supernatants were aliquoted and stored at –80°C. Protein concentrations were determined using the Bio-Rad protein assay kit (Bio-Rad, Marnes-la-Coquette, France). Tissue lysates (50 µg) were subjected to electrophoresis and Western blotted using anti-rat DMT1 polyclonal rabbit antibody (Interchim, Montluçon, France), polyclonal goat anti-rat HO1, polyclonal goat anti-human HO2, polyclonal goat anti-human CD36 (Tebu-bio, Le Perray-en-Yvelines, France) and anti-human GAPDH rabbit polyclonal antibodies (Tebu-bio) used as the internal reference antibody. Chemiluminescence was detected according to manufacturer's protocol (ECL, Millipore, Saint-Quentin-en-Yvelynes, France). Band densities were quantified using the LAS3000 apparatus (Fujifilm, Raytest, Courbevoie, France) and normalized to the total amount of control protein (GAPDH).
Determination of iron level in rat kidney.
Quantification of iron concentration was performed in kidneys of both control and DU-contaminated rats. This assay was based on a methodology kindly communicated by Dr Schumacher (Mok et al., 2004). Tissue samples were digested in 3N HCl with 10% trichloroacetic acid at 65°C overnight. Colorimetric iron determination of supernatants was performed using the Direct Method reagents (Biolabo) according to the manufacturer's instructions.
Histological analyses.
Renal segments were fixed in a 4% formaldehyde solution (Carlo Erba, Rueil Malmaison, France) at room temperature. Kidney samples were then dehydrated, embedded in paraffin, and cut in 5-µm-thick sections. A hematoxylin–eosin–saffron (HES) staining of paraffined slides was then performed.
The histological analysis was performed in a single-blind manner by a histopathologist (Dr Dudoignon). Glomerular lesions were semiquantitatively scored as none (0+), mild (1+), moderate (2+), or severe (3+). Tubulo-interstitial lesions were scored on the basis of tubular atrophy, dilation, hyaline casts and interstitial fibrosis as follows: 0 = no lesion; 1 = very minor dilation; 2 = larger presence of dilated tubules; 3 = marked tubular dilation and interstitial fibrosis.
The proliferative cells in the renal section were estimated following the immunohistochemical staining of Ki67, antigen present on a 36-kDa nuclear protein (dilution = 1/100, Dako, Trappes, France).
Cell apoptosis in the kidney was analyzed using immunohistochemical staining (in situ cell death detection kit with the TUNEL [terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end labeling] technique, Roche Diagnostics, Meylan, France) following the manufacturer's instructions. The number of apoptotic cells was estimated in the whole section.
Iron deposition was detected using Prussian blue staining according to the manufacturer's recommendations (Accustain, Sigma). Iron deposition was classified as follows: small deposits (point staining), intermediate iron deposits (splash staining), and clustered iron deposits (aggregate staining) (see Fig. 4). The extension of each class of iron deposition was estimated semiquantitatively using the following scales: 0 = none, 1 = mild; 2 = moderate; 3 = marked.
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Statistical analysis.
The results are expressed as mean ± SEM for seven animals unless otherwise indicated. Comparisons between groups were performed using Student's t-test for nonpaired data or the nonparametric Mann–Whitney test (SigmaStat3.0, Systat software). The text of this report comments on significant differences (p
0.05) or strong trends (p 0.10) when appropriated within control and DU-contaminated groups, with relevant p values quoted. |
Results |
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Changes in General Hematological Parameters
The blood cell count and global iron status (serum iron content and iron binding capacity) were measured in both control and DU-contaminated rats (Table 2). The RBC amount was decreased by about 20% in DU-contaminated animals as compared with control rats (p < 0.05). This reduction was associated with a similar 20% diminution of hematocrit (p = 0.051) and hemoglobin levels (p = 0.056). Other hematological parameters (leucocytes, mean corpuscular volume and platelets) were similar between the two groups (data not shown). Iron concentration and iron total and unsaturated binding capacities were not modified statistically by DU contamination.
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DU Effects on Hematopoiesis
The origin of RBC changes was first investigated within the hematopoietic system: a decrease in erythropoiesis may lead to a reduced RBC count. Hematopoietic activity was compared between control and DU groups using CFC assays in both spleen and bone marrow as well as blood cytokine measurements. The chronic ingestion of DU increased the frequency of CFU-GM and BFU-E from spleen whereas it did not affect bone marrow progenitors (Fig. 1a). BFU-GM progenitors were increased by 2.7-fold and BFU-E by 1.5-fold in spleen after DU exposure. Hematopoietic activity was also assessed by measuring two markers, Flt3 ligand and EPO (Fig. 1b). Chronic exposure to DU did not induce changes in blood concentrations of the studied cytokines. Reduced erythropoiesis may also be reflected by changes in EPO which mRNA levels are informative of EPO synthesis. After 9-month DU exposure, EPO mRNA relative levels remained unchanged (Fig. 1c). These results indicated that DU contamination did not induce significant changes in general hematopoiesis and erythropoiesis.
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Alterations in Spleen Iron Recycling
RBC reduction could also be a consequence of an increased rate of erythrocyte degradation that increases iron recycling from heme degradation. The modifications in iron recycling from RBC degradation were thus estimated in the spleen by measuring the expression levels of proteins involved in iron flux (DMT1, Ireg1), heme degradation (HO1 and HO2) and RBC adhesion (CD36) (Fig. 2). Chronic exposure to DU enhanced the expression of iron transport. DMT1 mRNA levels were increased by threefold (p = 0.05), meanwhile the twofold augmentation in the Ireg1 gene expression was not significant (Fig. 2a). DU exposure affected also heme degradation through an increase in mRNA levels of HO2 (threefold, p = 0.034) whereas HO1 gene expression was not affected. Expression of the RBC adhesion receptor CD36 remained unchanged with chronic DU ingestion. The relative protein levels of DMT1, HO1, HO2, and CD36 are indicated in the Figure 2b. These protein levels were not modified statistically by DU exposure.
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These data suggested that an increase in RBC degradation for iron recycling may probably not be at the origin of RBC diminution observed after 9 months of chronic exposure to DU. It is broadly accepted that renal deterioration may lead to the progression of chronic kidney disease responsible for anemia. Renal deterioration was thus the last tested hypothesis to explain reduction in RBC.
Modifications in the Expression of Renoprotective Genes
Renal dysfunction would be reflected—among others—by changes in renoprotective genes mRNA levels. The renoprotective genes measured here were EPOR because of its antiapoptotic role in kidney (Westenfelder, 2002), HO1 and 2 which have been shown to play a key role in cellular defenses (Maines and Panahian, 2001), and CP the so-called "Superoxide scavenger" that oxidizes toxic ferrous iron to non toxic ferric iron (Goldstein et al., 1982). The mRNA levels of EPO and renoprotective genes were quantified relatively to the HPRT reference gene (Fig. 3). The EPOR expression was reduced by about 90% after chronic ingestion of DU. The relative mRNA levels of heme degradation enzymes HO1 and HO2 were not modified by exposure to DU. CP mRNA relative levels of DU-contaminated rat kidneys were 12-fold higher than those of control animals.
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DU and Iron Accumulation in Kidney
The tissue iron content in kidney was evaluated in control and DU-contaminated rats. There was no significant difference in the renal iron concentration between control (8.86 ± 5.93 µg/g tissue; n = 7) and DU-contaminated animals (7.96 ± 1.74; n = 8).
Iron deposits in tissue were observed using Prussian blue staining (Fig. 4). As shown in Figure 4a, iron deposits were classified in small, intermediate and clustered iron deposits. The observed kidney sections were divided in three distinct parts for analysis: cortex, outer medulla and inner medulla for which the different levels of iron deposition were estimated semiquantitatively (Fig. 4b). Concerning small iron deposits, statistical analysis showed that there was no significant difference between control and DU rats regardless of kidney section part. Intermediate size iron deposits were increased after chronic exposure to DU in the whole kidney section: they were augmented by 1.5-fold in the cortex, (non significant); approximately fourfold in the outer medulla (p = 0.02) and approximately ninefold in the inner medulla (p = 0.02). Iron aggregates—referred as clustered iron deposits—were observed more frequently in DU rats than in control rats: approximately twofold in the renal cortex (p = 0.02); approximately threefold in the outer medulla (p = 0.02) and approximately twofold in the inner medulla (nonsignificant).
These data indicate that DU exposure modified the distribution of iron in kidney, but not the total content in this tissue.
Kidney Iron Transport is Altered by DU Contamination
The expression of DMT1, apical iron transporter, was quantified at both mRNA and protein levels. Relative DMT1 gene expression was increased by approximately threefold in kidney (Fig. 5a). On the contrary, renal DMT1 protein levels were decreased by 80% in DU-contaminated rats as compared with control rats (Fig. 5b).
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Histological Changes of Kidney Tissue with following Contamination
Creatinine and urea blood concentrations were evaluated in control and DU-contaminated rats. The values of blood creatinine were 55.56 ± 2.56 µmol/l and 52.91 ± 2.33 for control (n = 6) and DU-exposed (n = 6) rats respectively, indicating no functional alterations of renal function after DU exposure. This was confirmed by the lack of significant difference of blood urea levels between control rats (6.45 ± 0.50; n = 6) and DU-contaminated animals (6.02 ± 0.36; n = 6).
Representative histological slides of kidney sections are presented in Figure 6. This figure indicated clearly tubular dilation and presence of hyaline casts in kidney of DU-contaminated animals. The degree of lesion observed in renal glomerular and tubulo-interstitial tissues was scored in control and DU rats. In the control group, glomerular lesion scores were equally dispersed between grades 0 and 1, whereas in the DU group, glomerular lesions were mostly found to be grade 1. However, no clear distinction was observed between the two experimental groups. Tubulo-interstitial lesion scores of control rats were distributed homogenously between 0 and 3, whereas a major part of the DU-contaminated group had higher grades of tubulo-interstitial lesions: 70% of animals had a lesion degree 
2. These results suggest that chronic DU exposure induced especially tubulo-interstitial lesions.
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DU Affects Kidney Apoptosis
The influence of DU exposure on kidney apoptosis and proliferation was evaluated by immunohistochemistry. On light microscopy, apoptotic (TUNEL) and proliferative (Ki67) cells were observed in control and DU-contaminated rat kidneys. Representative sections are shown in Figure 7a. Quantification of TUNEL-positive cell count indicated that the number of apoptotic cells was enhanced by a factor of 2 in the corticomedullary junction after DU chronic ingestion (p = 0.026) whereas in the inner medulla, number of apoptotic cells was not influenced by DU contamination (Fig. 7b). Concerning proliferation, there was no significant difference in the number of Ki67 positive proliferative cells in the corticomedullary junction (-36%) and in the inner medulla (-51%) of DU-contaminated rats when compared with control rats.
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Discussion |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSResultsDiscussionFUNDINGREFERENCES |
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The present report shows for the first time that chronic exposure to DU at dose similar to this found in the environment induces renal deterioration responsible for a decrease in RBC numbers. Renal anemia is known to be an early symptom in the progression of chronic kidney disease. The present results thus suggest that chronic DU ingestion may lead to renal anemia consecutive to the progression of a DU-induced chronic kidney disease. Previous records of hematological parameters after chronic exposure to DU concerned the epidemiological survey of human populations and proved to be contradictory (Pinney et al., 2003
; Shawky et al., 2002; Squibb and McDiarmid, 2006). For instance, hematocrit, hemoglobin and RBC content from uranium processing site workers remained within the normal ranges (Shawky et al., 2002) whereas surveyed residents living around nuclear plant area revealed increases in these hematological parameters (Pinney et al., 2003). Similarly to the present work, a clinical study of Gulf War veterans showed that soldiers exposed to uranium had a reduction in hemoglobin and hematocrit levels (Squibb and McDiarmid, 2006). This discrepancy between these studies may be due to a difference in the exposure pathway (inhalation for workers, ingestion for populations living on contaminated territories and injury for soldiers), in the physical–chemical form of uranium (particulate for workers and soluble for civilian populations), in the received doses, in the duration of exposure and the time postexposure, as well as in the occurrence of exposure in these different cases of uranium exposure. It can be noticed that Gulf War veterans presented a similar decrease in hemoglobin and hematocrit levels than rats chronically exposed to DU by ingestion, probably because of the continuous liberation of uranium from embedded DU fragments. The mechanisms leading to the reduction in RBC amounts obtained following chronic exposure to DU were then explored. Three hypotheses could explain RBC decrease: reduced erythropoiesis, increased erythrocyte degradation for iron recycling or renal dysfunction.
Concerning erythropoiesis, spleen erythroid progenitors were increased with the chronic ingestion of DU. This is contradictory with the transient depression in erythropoiesis observed after the intravenous injection of 1 mg uranium/kg (Giglio et al., 1989). This previous report showed a reduction in plasmatic EPO concentrations, whereas in the current work no change was observed either in EPO plasmatic concentrations or EPO kidney mRNA levels. Furthermore, the results obtained in the present study indicate that the RBC productions in spleen and bone marrow were not reduced by chronic exposure to uranium. Consequently, a diminution in the erythropoiesis could not explain the RBC decrease observed after chronic exposure to DU.
The second possible explanation of RBC diminution in DU-contaminated rats was the increase in RBC degradation, which implies an increase in spleen iron recycling. Chosen indicators to study iron recycling were those implied in RBC attachment (CD36), heme catabolism (HO1 and HO2) and iron flux (DMT1, Ireg1) (Beaumont and Canonne-Hergaux, 2005). The increase in DMT1 gene expression induced by DU exposure could indicate an increase in iron transport. The increase in mRNA level of molecules of DMT1 could indicate an increase in iron flux, but this was not associated to increased protein level. HO2 mRNA relative levels were also enhanced. However these mRNA variations were not reflected at protein level. These data indicate thus that iron recycling rate changes could not explain the reduction in RBC after 9-month exposure to DU.
Renal dysfunction was thus the third hypothesis tested to explain the reduction in RBC content after 9-month chronic exposure to DU. Uranium-induced renal injury was first described in the early 20th century following acute exposure to high doses of uranium (Dickson, 1909). In 1915, Oliver, observed a tubular dilation and hyaline casts in histological kidney sections of guinea pigs and rats acutely contaminated with 5 mg uranium (Oliver, 1915). More recently, extensive tubular damage was induced following the intravenous injection of 5 mg uranium/kg in rats (Fujigaki et al., 2003). In this previous report, the tubules recovered 7 days after the uranium injection, probably due to the increase in cell proliferation. Chronic exposure to uranium was also demonstrated to induce renal injury. A 4-week experimental chronic exposure to uranium with doses ranging from 0.96 to 600 mg/l in drinking water evidenced that uranium-treated rat kidneys were more likely to suffer from tubular dilation than those of control rats (Gilman et al., 1998). These different data are consistent with the tubular lesions seen in the current report after chronic exposure to 40 mg uranium/l. It is well known that functional renal deterioration correlates more closely with the extent of tubulo-interstitial injury than glomerular pathology both in humans and in animal models of kidney disorders (Alfrey and Hammond, 1990; Risdon et al., 1968). This tubular localization of renal deterioration is thus in accordance with the tubulo-interstitial lesions observed after chronic exposure to DU. A decade ago, Savill suggested that defects in apoptosis might be involved in the pathogenesis of several renal diseases (Savill, 1994). It appeared that kidneys of DU-treated rats had more apoptotic cells than those of controls. This was not really surprising because several in vitro and in vivo experiments showed an association between kidney damages and apoptosis following acute uranium exposure (Prat et al., 2005; Thiebault et al., 2007). In addition, it has been shown recently that uranium exposure induces the activity of the apoptotic agent caspase-9 in renal cells (Thiebault et al., 2007). As tubulo-interstitial lesions are more frequent in DU-contaminated rats and proliferation is not augmented to regenerate damaged tubules, one can thus suppose that DU-induced tubular injury may lead to early progressive renal defect. However, the effects of 9-month DU exposure on kidney are very subtle as no change was observed in blood renal marker creatinin and urea. This is consistent with a recent clinical finding that showed no significant change in these blood markers in adults drinking water from uranium contaminated well (Magdo et al., 2007). Nevertheless, an elevated creatinin level was noted for a child thus highlighting the potential hazard of uranium contamination on kidney.
Concerning the molecular effects of chronic exposure to DU, the present study demonstrated that iron transporters, notably DMT1, were protein targets for uranium in kidney. However, a discrepancy was noted between mRNA level (x3) and protein level (reduced by 80%) of this transporter after DU ingestion. These results were not in accordance with a previous study that demonstrated increase in both mRNA and protein levels of DMT1 after acute short-term contamination with manganese (Wang et al., 2006). The discrepancy between mRNA levels and protein levels suggests that chronic DU contamination had an impact on DMT1 regulation, not only at transcriptional level but also at translational level. However, the protein processing stage—translational or post-translational level—at which this inhibitory effect of chronic exposure to DU occurred, remains to be determined.
Recently characterized in the kidney, EPOR was proved to be antiapoptotic. Indeed EPOR expressing cells had reduced apoptosis under EPO treatment (Westenfelder, 2002). The main role of the EPO signaling pathway in kidney is to protect this tissue from ischemic injury by preventing excess apoptosis (Sharples and Yaqoob, 2006). Further developments made it possible to understand EPO antiapoptotic signaling pathway: EPO binding to EPOR leads to a cascade of phosphorylations that inactivates proapoptotic factors such as caspase-9 (Rossert and Eckardt, 2005). In the present investigation, the dramatic reduction in EPOR mRNA of 90% observed in DU-contaminated rats may thus explain the increased apoptosis in these animals probably due to impairment of the inactivation of apoptotic factors.
To date, no report describes EPOR downregulation in kidney. The rare studies dealing with EPOR mRNA reduction were made in cultured cells (Pontikoglou et al., 2006; Yoon et al., 2006). These previous reports indicated that EPOR mRNA reduction may be due to hypoxia or inflammation, suggesting a certain complexity in the mechanisms regulating EPOR expression. Within the context of the current investigation, this raises the question of EPOR regulation in kidney after chronic exposure to DU.
The relative expression of mRNA or protein levels of heme oxygenases was not markedly modified by chronic DU exposure neither in spleen nor in kidney. Such lack of changes after chronic exposure to DU was not quite surprising, because previous study demonstrated a transient induction of HO1 between 24 and 48 h after ischemia (Villanueva et al., 2007). Paradoxally, the mRNA level of the constitutively expressed isoform HO2 was increased in spleen after DU contamination. This augmentation was not associated with similar increase in protein level of HO2, suggesting an additional post-transcriptional change. Contrary to EPOR, CP mRNA levels were dramatically augmented after 9-month DU contamination. The CP antioxidant role is known to mimic super oxide dismutase activity (Goldstein et al., 1982). Owing to CP higher expression in developing kidney than in mature kidney, it was proposed that CP protected kidney from growth-induced oxidative damages (Gupta et al., 1999). Hence, it can be assumed that CP is enhanced in the present study to protect kidney from DU-induced permanent oxidative stress. The primordial role of CP in case of uranium contamination was confirmed by recent in vitro blood experiments that demonstrated the capacity of CP to bind 2 mol of uranium per mole of protein (Vidaud et al., 2005). It can be thus hypothesized that CP plays a protector role with regard to DU nephrotoxicity via uranium sequestration. The presence of DU-triggered oxidative stress in kidney is demonstrated in previous reports showing an induction of oxidative stress markers in rat kidney after chronic uranium treatment (Linares et al., 2006; Taulan et al., 2004). For instance, 3-month chronic exposure to 10, 20, or 40 mg uranium/kg/day increased levels of oxidative stress markers such as thiobarbituric acid–reactive substances content, super oxide dismutase and oxidized glutathione activities in rat kidney (Linares et al., 2006). These various lines of evidence show thus the induction of several oxidative stress markers following uranium exposure.
It is well known that iron catalyzes the Fenton reaction, which generates highly reactive cytotoxic hydroxyl radicals. In rat kidney, it has been proved that iron accumulation obtained by experimental chronic hemosiderosis leads to oxidative stress generation whereas iron-depleted rats showed normal levels of oxidative stress markers (Zhou et al., 2000). Iron deposition in DU-contaminated rats presented more "hemosiderosis-like" figures of iron deposition that is, they had extended and numerous iron deposits. Iron deposition was already recorded in a similar experiment with DU (Donnadieu-Claraz et al., 2007). Iron induced oxidative stress is a pathway incriminated in chronic renal disease (Nath et al., 1994). Furthermore, recent advances showed that excess iron inhibited cell proliferation and increased apoptosis (Carlini et al., 2006). In addition, the tissue iron accumulation has been shown in vivo and in vitro to lead to tubular damages (Agarwal et al., 2004; Sponsel et al., 1996). These data suggests that iron accumulation plays a direct key role in renal apoptosis and injury. Furthermore, these data indicate that disturbances of iron metabolism may be reflected by changes in iron accumulation rather than by tissue iron content. These underlying iron mechanisms may explain the effects of chronic exposure to DU on rat kidney.
As reviewed by Nurko (2006), anemia in kidney disease include: RBC loss, decreased RBC life span, EPO deficiency, altered iron transport, iron deficiency and inflammation. In the present study, a reduction in RBC content, a deficiency in EPO pathway and kidney iron disorders were observed after chronic exposure to DU. Thus we propose the following scheme to interpret the effects of 9-month DU ingestion (Fig. 7): in rat kidney, DU leads to perturbation of iron transport, inducing iron accumulation, which itself generates an oxidative stress. This may trigger excess apoptosis responsible for renal injury. This renal dysfunction may be the reason for the RBC loss observed in DU-treated rats. The repeated DU insult to kidney may at least lead to chronic kidney disease which would lead to early renal anemia. Iron transport and accumulation seem thus to be the first link that results in renal injury. This would mean that iron metabolism is a critical target of DU exposure. However, it is difficult to estimate if the perturbations of iron metabolism were responsible for kidney deterioration or, in the contrary, if the uranium nephrotoxicity led to the alteration of tissue iron homeostasis. To answer this question, it would be necessary to develop further experiments with time-course of DU exposure in rats. In conclusion, the findings of the present work indicate that chronic exposure to DU may induce subtle effects on kidney with hematological consequences. The effects observed in spleen (increased erythroid progenitors and increased iron transport) suggest the setting up of a compensatory process in this tissue. Thus, the results of this investigation contribute to understanding the consequences and the mechanisms underlying the biological effects of DU on the kidney function.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSResultsDiscussionFUNDINGREFERENCES |
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ENVIRHOM research program supported by the Institut de Radioprotection et de Sûreté Nucléaire (IRSN, Institute for Radioprotection and Nuclear Safety, FRA).
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We are thankful to T. Loiseau and F. Voyer for providing the animal care.
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