YTAAP-13409; No of Pages 10 Toxicology and Applied Pharmacology xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

Toxicology and Applied Pharmacology journal homepage: www.elsevier.com/locate/ytaap

Metallothionein deficiency aggravates depleted uranium-induced nephrotoxicity Yuhui Hao a, Jiawei Huang a, Ying Gu a, Cong Liu a, Hong Li a, Jing Liu a, Jiong Ren a, Zhangyou Yang a, Shuangqing Peng b, Weidong Wang c,⁎, Rong Li a,⁎ a State Key Laboratory of Trauma, Burns and Combined Injury, Institute of Combined Injury, Chongqing Engineering Research Center for Nanomedicine, College of Preventive Medicine, Third Military Medical University, No. 30 Gaotanyan Street, Shapingba District, Chongqing 400038, China b Evaluation and Research Center for Toxicology, Institute of Disease Control and Prevention, Academy of Military Medical Science, 20 Dongdajie Street, Fengtai District, Beijing 100071, China c Department of Radiation Oncology, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai 200233, China

a r t i c l e

i n f o

Article history: Received 2 January 2015 Revised 29 April 2015 Accepted 27 June 2015 Available online xxxx Keywords: Depleted uranium Metallothionein Oxidative stress Apoptosis Sodium-glucose cotransporter Nephrotoxicity

a b s t r a c t Depleted uranium (DU) has been widely used in both civilian and military activities, and the kidney is the main target organ of DU during acute high-dose exposures. In this study, the nephrotoxicity caused by DU in metallothionein-1/2-null mice (MT −/−) and corresponding wild-type (MT +/+) mice was investigated to determine any associations with MT. Each MT −/− or MT +/+ mouse was pretreated with a single dose of DU (10 mg/kg, intraperitoneal injection) or an equivalent volume of saline. After 4 days of DU administration, kidney changes were assessed. After DU exposure, serum creatinine and serum urea nitrogen in MT−/− mice significantly increased than in MT+/+ mice, with more severe kidney pathological damage. Moreover, catalase and superoxide dismutase (SOD) decreased, and generation of reactive oxygen species and malondialdehyde increased in MT−/− mice. The apoptosis rate in MT−/− mice significantly increased, with a significant increase in both Bax and caspase 3 and a decrease in Bcl-2. Furthermore, sodium-glucose cotransporter (SGLT) and sodium-phosphate cotransporter (NaPi-II) were significantly reduced after DU exposure, and the change of SGLT was more evident in MT−/− mice. Finally, exogenous MT was used to evaluate the correlation between kidney changes induced by DU and MT doses in MT−/− mice. The results showed that, the pathological damage and cell apoptosis decreased, and SOD and SGLT levels increased with increasing dose of MT. In conclusion, MT deficiency aggravated DU-induced nephrotoxicity, and the molecular mechanisms appeared to be related to the increased oxidative stress and apoptosis, and decreased SGLT expression. © 2015 Elsevier Inc. All rights reserved.

Introduction Depleted uranium (DU), which has a relatively low radiation capacity and chemical properties similar to natural uranium, is a by-product formed by refining natural uranium to enriched uranium (235U) (Bleise et al., 2003). Exposure to DU is primarily from nuclear waste and the manufacture and use of DU weapons. In the process of DU weapon production and use, uranium may enter the body through the respiratory tract, gastrointestinal tract or skin (ATSDR, 2013), after which DU is mainly distributed into the kidneys, bone and liver, posing possible

Abbreviations: Bax, Bcl-2 associated X protein; Bcl-2, B-cell lymphoma/leukemia-2; BUN, blood urea nitrogen; CAT, catalase; CBMIDA, catechol-3,6-bis (methyleiminodiacetic acid); Cr, creatinine; DU, depleted uranium; ELISA, enzyme-linked immunosorbent assay; FITC, fluorescein isothiocyanate; GSH-Px, glutathione peroxidase; H & E, hematoxylin and eosin; NaPi-II, sodium-phosphate cotransporter; PBS, phosphate buffer solution; PI, propidium iodide; MDA, malondialdehyde; MT, metallothionein; ROS, reactive oxygen species; SD, standard deviation; SOD, superoxide dismutase; SGLT, sodium-glucose cotransporter. ⁎ Corresponding authors. E-mail addresses: [email protected] (W. Wang), [email protected] (R. Li).

serious threats to human health (Brugge and Buchner, 2011). The kidney is the main target organ of the acute chemical toxicity caused by DU in high-dose exposures, and DU exposure (single intramuscular doses of 0.1, 0.3 or 1.0 mg/kg uranium) has been shown to elicit dosedependent increases in kidney concentrations of the metal (Jortner, 2008). DU toxicity can result in severe renal tubular necrosis, which can lead to renal failure and death (Vicente-Vicente et al., 2010; Cheng et al., 2010). A common, initial event in the action of all toxic metals seems to be the generation of oxidative stress that is characterized by the following effects: (a) depletion of intracellular antioxidants and free-radical scavengers, (b) inhibition of the activities of various enzymes that contribute to the metabolism and detoxification of reactive oxygen species (ROS), such as glutathione peroxidase (GSH-Px), catalase (CAT) and superoxide dismutase (SOD), and (c) increased production of ROS (Thiebault et al., 2007). To date, chelation therapy that uses ligands with strong metal-binding capabilities remains the only way to accelerate the excretion of a poisonous metal. Many chelating agents have been examined in animal studies over the years to determine their efficacies for removing uranium (Fukuda et al., 2008; Zhang et al., 2011; Bao et al., 2013). Catechol-3,6-bis

http://dx.doi.org/10.1016/j.taap.2015.06.019 0041-008X/© 2015 Elsevier Inc. All rights reserved.

Please cite this article as: Hao, Y., et al., Metallothionein deficiency aggravates depleted uranium-induced nephrotoxicity, Toxicol. Appl. Pharmacol. (2015), http://dx.doi.org/10.1016/j.taap.2015.06.019

2

Y. Hao et al. / Toxicology and Applied Pharmacology xxx (2015) xxx–xxx

(methyleiminodiacetic acid) (CBMIDA), which may be administered orally, has been shown to reduce the uranium burden without causing renal damage (Fukuda et al., 2009). However, CBMIDA has low gastrointestinal absorption and showed no uranium removal effect when the uranium was dissolved in a solution of pH 7 (Fukuda et al., 2007). In our previous study (Hao et al., 2012), pretreatment with zinc significantly increased the survival rates at 30 days post-DU administration and alleviated acute toxicity of DU in rats, and the induction of metallothionein (MT) by zinc might play an important role in DU detoxification. MT is a family of low molecular weight, sulphur-containing proteins that is broadly distributed throughout the body. The family consists of four isoforms, MT-1, MT-2, MT-3 and MT-4, and MT-1 and MT-2 are the major contributors in the prevention of heavy metal toxicity (Kang, 2006; Maria et al., 2014; Gu et al., 2014; He et al., 2014). Jiang et al. (2009) has shown that MT exerts significant protective effects in nematodes exposed to DU, demonstrating a new avenue for the prevention and treatment of DU-induced injuries. We also found exogenous MT effectively inhibited DU-induced human kidney cells (HK-2) apoptosis (Hao et al., 2014). Nevertheless, the role and detoxification mechanisms of MT-1 and MT-2 in mice are not well understood. In the present study, MT-1 and MT-2 double-knockout mice (MT −/−) and wild-type mice (MT +/+) were used to evaluate the relationship between MT and DU toxicity. Exogenous MT was also used to evaluate the correlation between kidney injuries and MT doses. The pathological changes and antioxidant activity were assessed. We also determine whether DU-induced cytotoxicity is mediated partly by the activation of apoptotic pathways, as our previous in vitro studies have suggested (Hao et al., 2014). In addition, studies (Muller et al., 2008; Vicente-Vicente et al., 2010) have reported that alterations in solute transport related to the mechanism involved in uranium nephrotoxicity. Therefore, we hypothesized that DU lead to dysfunction of transporters, and that the presence of MT protects the transporters and thus prevents DU nephrotoxicity. The relationship between MT and transporters was evaluated to explore the protective mechanisms of MT in countering DU toxicity, which would lay the foundation for future studies about the treatment of DU exposure. Materials and methods Animals. MT-null mice, which are deficient in MT-1 and MT-2 genes, and homozygous wild-type mice were originally obtained from the Murdoch Institute of the Royal Children's Hospital (Parkville, Australia) and bred as previously reported (Shuai et al., 2007). Male mice aged 6–8 weeks were acclimated to the laboratory for 5 days prior to the experiment, then mice with an approximately equal mean body weight were selected for experiment. Food intake, water intake, body weight, and health status were recorded daily. The study was conducted in accordance with Chinese legislation and “Principles of laboratory animal care” (NIH publication No. 85-23, revised 1985) regarding the care of animals used for experimental purposes. Treatments. Both MT +/+ and MT −/− mice were randomly divided into two groups, each consisting of 8 animals, as detailed below. In the DU group, the mice were exposed to uranyl nitrate [the source and constituent of DU was the same as in a previous study (Hao et al., 2009), composed of 99.25% DU and 0.75% titanium by weight, with 238 U = 99.75%, 235U = 0.20%, trace 234U] at a single dose (10 mg/kg body weight, intraperitoneal injection). Tris-maleate buffer pH 7.0 (Sigma-Aldrich, Santa Clara, CA, USA) was used to dissolve uranyl nitrate (weight/volume = 0.1%). The final concentration of uranyl nitrate solution was 1.0 mg/ml, and the volume of injection was 0.25 ml for 25 g mice. The second group, which was the saline group, received an equal volume of saline as the DU group. The animals were killed 4 days after DU or saline injection. Blood and kidney samples were collected and analyzed as described below. In order to further prove the correlation of DU-induced nephrotoxicity and MT, another experiment

was conducted. MT −/− mice were administrated with or without exogenous MT (Sigma-Aldrich, Santa Clara, CA, USA) once at three different doses (10, 20, 30 μmol/kg body weight, intraperitoneal injection) 1 h before DU exposure, respectively. The working concentration of MT solution was 1.0 μmol/ml. The high dose of MT (30 μmol/kg) was based on relevant references (Kukner et al., 2007; Helal and Helal, 2009). The control group received an equal volume of saline as the DU group (10 mg/kg) as described above. Therefore, the mice were randomly divided into five groups [control, DU, MT(10) + DU, MT(20) + DU, MT(30) + DU], each consisting of 8 animals. After 4 days of DU exposure, the kidney pathological changes, serum concentrations of creatinine (Cr) and urea nitrogen (BUN), cell apoptosis, the levels of SOD and SGLT were analyzed as described below. Uranium analyses in kidney tissue. The uranium content in the kidney tissue was determined using an inductively coupled plasma mass spectrometer (ICP-MS, Thermo Finnigan MAT, Bremen, Germany), as described previously (Hao et al., 2013). Values are expressed as ng/g tissue. MT measurements in kidney tissue. Total kidney tissue MT concentrations were determined by cadmium–hemoglobin affinity assay (Eaton and Cherian, 1991), as described previously (Shuai et al., 2007). In brief, kidney tissues were homogenized in Tris–HCl buffer (50 mM, pH = 8.0), and the supernatant was collected by centrifugation at 18,000 ×g for 15 min at 4 °C. The supernatant was determined for the total protein and MT concentrations. The total protein concentrations were measured with a Bradford Protein Assay kit (Beyotime, Haimen, Jiangsu, China). The MT concentrations in the kidney were expressed as mg/g protein. Biochemical assays. Femoral artery blood samples were obtained while the mice were sedated with xylazine hydrochloride. Serum concentrations of Cr and BUN were measured via an automated Konelab 20 (Thermo Electron Corporation, Cergy-Pontoise, France), as previously reported (Hao et al., 2013). Histopathology and light microscopy. At the 4th day after the administration of DU, kidneys were dissected and fixed with 4% formaldehyde for 48 h, dehydrated and embedded in paraffin, then sliced into 5 μm sections. Hematoxylin and eosin (H & E, Beyotime) staining were used to observe pathomorphological changes. During the evaluation of tissue damage, vacuolization, necrosis, cast formation in renal proximal and distal tubules and mononuclear cell infiltration in interstitial spaces were scored by an assessment in randomly selected areas. The damage was scored as follows: 0 = none, 1 = 0–10%, 2 = 11–25%, 3 = 26–50%, and 4 = 51–100% (Inal et al., 2014). The scoring of the histological data was blinded and measured by an independent laboratory. Analysis of malondialdehyde (MDA) and antioxidant enzymes. Kidney tissues were placed in ice-cold PBS and homogenized at 16,000 rpm for 3 min. The homogenates were centrifuged at 1000 g for 10 min at 4 °C. The supernatants were stored at − 80 °C until analysis. Tissue MDA content was measured using the thibabituric acid method (Yoshioka et al., 1979). The activities of SOD and CAT were measured using the methods of Wang et al. (2008). All of these measurements were performed using commercial kits (Jian Cheng Institute of Bioengineering, Nanjing, China), and absorbance readings were obtained using a microplate Reader (Bio-rad 550, BioRad Laboratories, California, USA). ROS determination. Kidneys were removed aseptically from euthanized mice of each group (n = 8) and single cell suspensions prepared as our previously described (Hao et al., 2012). Intracellular ROS generation was measured by the reactive oxygen species assay kit (Beyotime) according to the manufacturer's instructions. The ROS

Please cite this article as: Hao, Y., et al., Metallothionein deficiency aggravates depleted uranium-induced nephrotoxicity, Toxicol. Appl. Pharmacol. (2015), http://dx.doi.org/10.1016/j.taap.2015.06.019

Y. Hao et al. / Toxicology and Applied Pharmacology xxx (2015) xxx–xxx

generation was monitored by intracellular conversion of 2,7dichlorodihydrofluorescein diacetate (DFCH-DA) into the fluorescent product dichlorofluorescein (DCF). DCF fluorescence was detected by excitation at 488 nm and emission at 525 nm under fluorescence microscope (IX51, Olympus, Tokyo, Japan). The relative fluorescence intensity was analyzed and quantified using Image Pro-Plus 6.0 (Media Cybernetics, Silver Spring, MD, USA). Apoptosis detection. Kidney cell suspensions prepared as described above. Kidney cells (5 × 105) were washed in cold phosphate buffer solution (PBS) and stained with annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI, BD Biosciences Pharmingen, San Jose, CA, USA) according to the manufacturer's protocol, as our previous report (Hao et al., 2014). Analysis of caspase-3 activity. Caspase-3 activity was measured using a caspase activity kit (Beyotime) according to the manufacturer's instructions as our previous report (Hao et al., 2014). Briefly, kidney tissues were lysed, and the total protein content was measured with the Bradford method as described above. The activity of caspase-3 was measured by determining the absorbance at 405 nm with a microplate reader. All the experiments were conducted in triplicate.

3

Results DU exposure increased the accumulation of uranium and decreased the MT content of kidney tissues After 4 days of DU exposure, the concentrations of uranium were both significantly increased in MT−/− mice and MT+/+ mice (p b 0.05), and there were no significant differences between MT−/− and MT+/+ mice (p N 0.05). The nephrotoxic limit of DU is 3 μg/g tissue in humans (Lu and Zhao, 1990), and uranium accumulations in MT−/− mice and MT+/+ mice were more than 60 μg/g tissue (Fig. 1A). To elucidate the dose-dependent correlation between kidney injuries and MT content, we established a kidney injury model using MT−/− and MT+/+ mice. The renal MT concentrations in MT+/+ mice in the saline group were approximately 6.0 mg/g protein (Fig. 1B). Four days after DU injection (10 mg/kg), MT levels were significantly reduced to about half of the levels observed in the saline group (p b 0.05). As expected, renal MT levels in MT−/− mice in each group were relatively low (approximately 2.0 mg/g protein) and showed no significant correlation with DU treatment. In our previous study (Shuai et al., 2007), the basal cardiac MT contents of MT+/+ and MT−/−mice were about 5 μg/g tissue and 1.5 μg/g tissue, which also showed significant differences. Moreover, the MT levels in the MT−/− mice of the DU group were significantly lower than those obtained for the corresponding MT+/+ mice (p b 0.05).

Enzyme-linked immunosorbent assay (ELISA). Similarly, kidney tissues were lysed, and the total protein content was measured. The levels of B-cell lymphoma/leukemia-2 (Bcl-2) and Bcl-2 associated X protein (Bax) were estimated according to the ELISA kit procedures as our previously described (Hao et al., 2014). The Bcl-2 ELISA kit was purchased from R&D Systems, and the Bax ELISA kit was purchased from Cusabio Biotech (Barksdale, DE, USA). The limit of detection was 62.5 pg/ml for Bcl-2 and 1.25 ng/ml for Bax. Western blotting. Western blot analysis was used to determine various protein levels, as described previously (Xu et al., 2014). Briefly, tissue lysates (50 μg) were subjected to electrophoresis and western blotted using anti-mouse SGLT polyclonal rabbit antibody and polyclonal goat anti-mouse NaPi-II (Abcam, Cambridge, MA, USA) and anti-mouse β-actin rabbit polyclonal antibodies (Beyotime) 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 (β-actin). Analysis for subcellular localization of SGLT. Kidney were removed and homogenized by pressing the organs gently through a metal net, and cell suspension was collected. Kidney cells were fixed and labeled as previously described (Gaudreault et al., 2008). Briefly, cells were fixed and probed with antibodies against SGLT and a secondary antibody conjugated with the fluorescent dye (red). The cells' nuclei were labeled with Hoechst 33342 (Life Technologies Corporation; CA, USA). Fluorescence was captured using laser confocal microscopy (Leica TCS-SP5, Bensheim, Germany). Negative control experiment was conducted (blank). The cells were labeled with a secondary antibody conjugated with the fluorescent dye without the primary antibody. Data analysis. All data were analyzed using SPSS 11.5 (SPSS Inc., Chicago, IL, USA). The indicators were evaluated by a two-way ANOVA for two independent variables and by a one-way ANOVA for one variable. Least significant difference (LSD) test was used to compare data obtained from the different groups. All data are expressed as means ± standard deviations (SD). Results were considered statistically significant at p b 0.05 (two-sided).

Fig. 1. After 4 days of depleted uranium (DU) acute exposure, the content of uranium and metallothionein (MT) in kidney tissues changed in different degrees between MT+/+ and MT−/− mice. This figure illustrates the content of uranium (A) and MT (B) in each group of MT−/− and MT+/+ mice, as measured by inductively coupled plasma mass spectrometry (ICP-MS) and cadmium–hemoglobin affinity assay. Data are expressed as means ± SD (n = 8), and the error bars represent the SD. ⁎p b 0.05, compared with the saline group of MT +/+ mice; #p b 0.05, compared with the saline group of MT −/− mice; @p b 0.05, with a two-way ANOVA.

Please cite this article as: Hao, Y., et al., Metallothionein deficiency aggravates depleted uranium-induced nephrotoxicity, Toxicol. Appl. Pharmacol. (2015), http://dx.doi.org/10.1016/j.taap.2015.06.019

4

Y. Hao et al. / Toxicology and Applied Pharmacology xxx (2015) xxx–xxx

BUN levels were similar in both saline-treated MT −/− and MT +/+ mice. The concentrations of Cr and BUN were both significantly increased in MT−/− mice and MT+/+ mice 4 days after DU exposure (p b 0.05). Interestingly, there were significant differences between MT −/− and MT +/+ mice following DU (p b 0.05). In fact, after 4 days of DU exposure, the Cr levels in MT +/+ and MT −/− mice were approximately 1.5 and 2 times higher, respectively, than corresponding saline group, while the BUN levels in MT +/+ and MT−/− mice were approximately 3.5 and 4 times higher, respectively, than corresponding saline group. DU induced greater renal pathological damage in MT−/− mice compared with MT+/+ mice We analyzed renal tissue samples under a light microscope and observed that the extent of injuries varied among the experimental groups (Fig. 3). Similar to the saline group in MT+/+ mice, the saline group in MT−/− mice did not show renal damage (Fig. 3A and B). In the renal tissue of MT −/− mice of the DU group (Fig. 3D), however, we observed a large number of tube-shaped structures with hyaline degeneration in the renal tubular epithelial cells, cell vacuolization, abscission and necrosis and mononuclear cell infiltration in some interstitial tissues. Interestingly, these pathological injuries seemed milder in MT+/+ mice (Fig. 3C). The histopathological scores were shown in Table 1. There were significant differences in the scores of tubular damage and mononuclear cell infiltration between MT+/+ and MT−/− mice after exposure to DU (p b 0.05).

Fig. 2. Metallothionein (MT) deficiency increased serum biochemical parameters at 4 days post-depleted uranium (DU) exposure. This figure illustrates the contents of creatinine (Cr, A) and urea nitrogen (BUN, B) in each group of MT−/− and MT+/+ mice, as measured by automated Konelab 20. Data are expressed as means ± SD (n = 8), and the error bars represent the SD. ⁎p b 0.05, compared with the saline group of MT+/+ mice; #p b 0.05, compared with the saline group of MT−/− mice; @p b 0.05, with a two-way ANOVA.

MT deficiency increased serum Cr and BUN after DU exposure After 4 days of DU exposure, renal-function test results were assessed by measuring changes in serum Cr and BUN (Fig. 2). Cr and

MT deficiency aggravated the oxidative stress of kidney tissue after DU exposure Hexavalent uranium was the major contributor to DU toxicity, and the cytotoxicity of DU with intracellular damage to mitochondria and lysosomes was caused primarily by the generation of ROS, lipid peroxidation and the reduction of biological metabolism (Pourahmad et al., 2006). In the present study, the activities of SOD, CAT and MDA in kidney tissue were measured to evaluate changes in these kidney antioxidant enzyme systems and the level of lipid peroxidation (Fig. 4A–C). Interestingly, changes in SOD and CAT showed the

Fig. 3. Metallothionein (MT) deficiency significantly aggravated pathological damage at 4 days post-depleted uranium (DU) exposure. This figure depicts kidney pathological sections for each group (n = 8) of MT−/− and MT+/+ mice, stained by H & E. A and B: Saline group; C and D: DU group; A and C were from MT+/+ mice; B and D were from MT−/− mice.

Please cite this article as: Hao, Y., et al., Metallothionein deficiency aggravates depleted uranium-induced nephrotoxicity, Toxicol. Appl. Pharmacol. (2015), http://dx.doi.org/10.1016/j.taap.2015.06.019

Y. Hao et al. / Toxicology and Applied Pharmacology xxx (2015) xxx–xxx Table 1 Histopathological scores in kidney after exposure to depleted uranium (DU) in metallothionein-1/2-null mice (MT−/−) and corresponding wild-type (MT+/+) mice.a Parameters

Saline group MT+/+

Tubular damage Mononuclear cell infiltration

0.63 ± 0.52 0.38 ± 0.52

5

intensity, was significant higher in MT−/− mice than in MT+/+ mice after 4 days of DU exposure (p b 0.05). There was low fluorescence signal with low ROS level in saline group of MT+/+ or MT−/− mice.

DU group MT−/−

MT+/+

MT−/−

0.75 ± 0.46 0.50 ± 0.53

2.75 ± 0.46⁎ 1.75 ± 0.71⁎

3.38 ± 0.74⁎⁎,⁎⁎⁎ 2.63 ± 0.52⁎⁎,⁎⁎⁎⁎

a Values are expressed as the mean ± SD (n = 8). Statistical analysis was performed by a two-way ANOVA. ⁎ p b 0.01, compared with the saline group of MT+/+ mice. ⁎⁎ p b 0.01, compared with the saline group of MT−/− mice. ⁎⁎⁎ p b 0.05, compared with the DU group of MT+/+ mice. ⁎⁎⁎⁎ p b 0.01, compared with the DU group of MT+/+ mice.

same trend in each group. After 4 days of DU exposure, the SOD levels in MT +/+ and MT −/− mice were decreased to approximately 63.6% and 28.3%, respectively, of the levels in corresponding saline group, while there were significant differences in DU group between MT +/+ and MT −/− mice (p b 0.05). Similarly, after 4 days of DU exposure, the CAT levels in MT +/+ and MT −/− mice were decreased to approximately 35.4% and 21.0%, respectively, of the levels in corresponding saline group, while there were significant differences in DU group between MT+/+ and MT−/− mice (p b 0.05). With regard to MDA, the MDA content was significantly increased (approximately 4-fold) in the DU group of MT+/+ mice compared with the saline group (p b 0.05), while increases in MDA levels were nearly 7-fold greater in the DU group of MT −/− mice compared with the corresponding saline group (p b 0.05). There was no difference about the activity of SOD, CAT or MDA in saline group between MT+/+ and MT−/− mice (p N 0.05). As expected, the intracellular ROS level (Fig. 4D and E), as detected by DCF fluorescence

MT deficiency increased DU-induced apoptosis in kidney cells Given that DU is known to induce apoptosis in a variety of cell types and tissues, we studied the effects of DU on kidney cell apoptosis using annexin V-FITC/PI double staining (Fig. 5). The percentages of normal, early-apoptotic, late-apoptotic, and necrotic cells in each group were calculated by flow cytometry. The total percentage of apoptotic cells (early and late) in MT +/+ mice and MT −/− mice in the saline group were approximately 3.2% and 3.5%, respectively. Four days after DU exposure, higher percentages of apoptotic cells were observed compared to the saline group, especially in MT −/− mice. Moreover, the total percentage of apoptotic cells in MT −/− mice was higher than that in MT+/+ mice (p b 0.05). We assessed expression levels of the key markers of apoptosis, Bax, Bcl-2 and caspase-3 to explore molecular mechanisms underlying DU-induced cell apoptosis (Fig. 6). After exposure to DU, levels of both Bax and caspase-3 proteins were higher (p b 0.05), and level of Bcl-2 were lower (p b 0.05). These changes were more evident in MT−/− mice. DU induced lower SGLT expression in MT −/− mice compared with MT +/+ mice To determine whether DU administration affects kidney cells transport function, we studied the effects of DU on the expression of two important transport proteins, namely SGLT and NaPi-II. SGLT is the main glucose transporter in the rodent placenta, and its reduced expression is often associated with uranyl acetate exposure (Goldman et al., 2006). NaPi-II participated in the reabsorption of organic

Fig. 4. Metallothionein (MT) deficiency decreased the activities of antioxidation enzymes, and increased the lipid peroxidation levels and the generation of reactive oxygen species (ROS) at 4 days post-depleted uranium (DU) exposure. This figure illustrates the contents of catalase (CAT, A), superoxide dismutase (SOD, B), malondialdehyde (MDA, C) and ROS (D) in each group of MT−/− and MT+/+ mice, as measured by colorimetry or fluorescence microscopy as described in the Materials and methods section. The fluorescence intensity of ROS (E) was quantified using Image Pro-Plus 6.0. Data are expressed as means ± SD (n = 8), and the error bars represent the SD. ⁎p b 0.05, compared with the saline group of MT+/+ mice; #p b 0.05, compared with the saline group of MT−/− mice; @p b 0.05, with a two-way ANOVA.

Please cite this article as: Hao, Y., et al., Metallothionein deficiency aggravates depleted uranium-induced nephrotoxicity, Toxicol. Appl. Pharmacol. (2015), http://dx.doi.org/10.1016/j.taap.2015.06.019

6

Y. Hao et al. / Toxicology and Applied Pharmacology xxx (2015) xxx–xxx

phosphate and its expression is also regulated by uranium (Muller et al., 2006). We found that DU exposure decreased levels of SGLT and NaPi-II protein in kidney tissues of either MT−/− or MT+/+ mice (Fig. 7A–C). Moreover, the level of SGLT protein in MT+/+ and MT−/− mice were decreased to approximately 20.0% and 50.0%, respectively, of the levels in corresponding saline group, and there were significant differences in DU group between MT+/+ and MT−/− mice (p b 0.05), while there was no difference between MT −/− mice and MT +/+ mice in the level of NaPi-II protein (p N 0.05). SGLT labeling was observed in the cytosol in saline group of MT+/+ or MT−/− mice (Fig. 7D), consistent with relevant study (Merigo et al., 2012). No fluorescence about SGLT was observed in the blank, demonstrating the specificity of the secondary antibodies. What's more, the immunoreactivity for SGLT in MT −/− mice was lower than that in MT +/+ mice after 4 days of DU exposure.

Fig. 6. Metallothionein (MT) deficiency influenced depleted uranium (DU)-induced caspase-3 activation, and expression of the key apoptotic proteins at 4 days post-DU exposure. Kidney tissues were lysed, and the total protein concentrations were measured using the Bradford method. (A) The activities of caspase-3 in cell lysates were measured using a caspase activity kit according to the manufacturer's instructions. (B) Bcl-2 and Bax in cell lysates were analyzed by ELISA as described in the Materials and methods section. The data are expressed as the mean ± SD (n = 8), and the error bars represent the SD. ⁎p b 0.05, compared with the saline group of MT+/+ mice; #p b 0.05, compared with the saline group of MT−/− mice; @p b 0.05, with a two-way ANOVA.

Exogenous MT protected against depleted uranium-induced nephrotoxicity in MT−/− mice To verify the protective role of MT against DU, exogenous MT was used to estimate the effect on DU-induced nephrotoxicity. The results showed that exogenous MT alleviated DU-induced kidney pathological damage in MT −/− mice with the increasing dose of MT (Fig. 8A and Table 2). Especially in the MT(30) + DU group, there were almost no obvious pathological changes. Meanwhile, the total percentage of apoptotic cells and serum Cr and BUN levels caused by DU reduced as the increasing dose of MT (Fig. 8B–E). Furthermore, treatment of DU with exogenous MT increased the activity of SOD and level of SGLT protein in kidney tissue in a dose-dependent manner (Fig. 8F and G).

Discussion

Fig. 5. Metallothionein (MT) deficiency increased depleted uranium (DU)-induced apoptosis in kidney cells. At 4 days post-DU exposure, the kidney cells were collected as described in the Materials and methods section. (A) Apoptosis was measured by flow cytometry, followed by Annexin V-FITC (FL 1 channels) and PI (FL 2 channels) double staining. (B) The percentage of apoptosis was counted including early apoptosis (in the lower-right quadrants) and late apoptosis (in the upper-right quadrant). The data are expressed as the mean ± SD (n = 8), and the error bars represent the SD. ⁎p b 0.05, compared with the saline group of MT+/+ mice; #p b 0.05, compared with the saline group of MT−/− mice; @p b 0.05, with a two-way ANOVA.

DU has been used with increasing frequency in both civilian and military activities, and might lead to a wide range of health problems (Squibb et al., 2005; Milacic and Simic, 2009). Our previous results (Hao et al., 2012, 2014) showed that zinc could effectively alleviate kidney damage in vitro and vivo, and zinc-induced MT expression might play an important role in detoxification processes. In the present study, we compared DU-induced nephrotoxicity between MT −/− mice and MT +/+ mice. The results showed that after 4 days of DU acute exposure, serum Cr and BUN levels were up-regulated, and

Please cite this article as: Hao, Y., et al., Metallothionein deficiency aggravates depleted uranium-induced nephrotoxicity, Toxicol. Appl. Pharmacol. (2015), http://dx.doi.org/10.1016/j.taap.2015.06.019

Y. Hao et al. / Toxicology and Applied Pharmacology xxx (2015) xxx–xxx

pathological damage was more severe in MT−/− mice compared with MT+/+ mice. Additionally, MT deficiency induced down-regulation of the activities of antioxidation enzymes, increased ROS and lipid peroxidation levels in kidney tissue and increased DU-induced apoptosis in kidney cells. Moreover, SGLT and NaPi-II were significantly reduced after exposure to DU, and the change of SGLT was more evident in MT −/− mice. Furthermore, exogenous MT could also reduce the pathological damage, serum Cr and BUN levels and cell apoptosis, and increase SOD and SGLT levels after DU exposure. In conclusion, MT deficiency aggravated DU-induced nephrotoxicity, and the molecular mechanisms appeared to be related to the increased oxidative stress and apoptosis, and decreased SGLT expression. Characterized by a large number of thiol groups, MT plays an important role in essential trace element homeostasis and in metal detoxification (He et al., 2014). In this study, we found that the MT levels in MT+/+ mice declined about 50% in the DU group, compared with the saline group. It demonstrated that a short exposure to a high dose of DU can consume a lot of MT. The results further confirmed our early reports (Hao et al., 2012, 2014). In fact, low MT levels theoretically would predispose people to cadmium toxicity (Nordberg, 2004). By contrast, Andrews (2000) reported that cadmium exposure induced MT gene expression. We think different materials, different exposure time and different doses would lead to different MT levels. This hypothesis needs further study. In addition, after exposure to arsenic, the susceptible population of Guizhou, China showed reduced MT expression (Liu et al., 2007). We also found that in MT−/− mice, the mean MT content of each group was relatively low. The low level of MT content might be attributed to a small amount of MT-3, MT-4 and some thiol-containing compounds which did not significantly decrease after exposure to DU.

7

When a body's uranium intake is higher than 2 mg/kg, kidney damage is caused (Vicente-Vicente et al., 2010). Rats were pretreated with DU (8 mg/kg), and serum Cr and BUN levels increased after 6 days of DU administration (Fukuda et al., 2009). In this study, the dose of DU (10 mg/kg) and uranium accumulation (N60 μg/g tissue) both suggest the kidney damage. Using MT+/+ and MT−/− mice, the present study further demonstrated after 4 days of DU acute exposure, MT deficiency aggravated the kidney functional and pathological damage. The histopathological scores of tubular damage and mononuclear cell infiltration in MT+/+ mice were lower than that in MT−/− mice. What's more, exogenous MT also reduced DU-induced kidney functional and pathological damage in MT−/− mice. With the increasing dose of MT, the histopathological scores of tubular damage and mononuclear cell infiltration were declining. Jiang et al. (2009) reported that Caenorhabditis elegans MT protected against toxicity induced by DU. MT−/− cell lines and MT−/− mice are sensitive to heavy metal toxicity, while cells and mice that express an excess amount of MT are resistant to cadmium (Palmiter, 1994; Liu et al., 1995). Our results also agree with the conclusions from other relevant studies about cadmium (Loebus et al., 2013; Xiang et al., 2013). In addition, MT deficiency decreased the CAT and SOD activities, and increased the MDA and ROS levels at 4 days post-DU exposure, which indicates that MT plays a protective role in preserving the activities of CAT and SOD and suppressing the generation of MDA and ROS. What's more, exogenous MT also increased the activity of SOD in a dosedependent manner in MT −/− mice. Studies (Thiebault et al., 2007; Hao et al., 2014) showed that uranium stimulated the production of ROS and lipid peroxidation. Oxidative stress was induced on isolated liver mitochondria of rats After 1 h of DU (2 mg/kg) administration

Fig. 7. Metallothionein (MT) deficiency down-regulate expression of sodium-glucose cotransporter (SGLT) at 4 days post-DU exposure. Kidney tissues were lysed, and the total protein concentrations were measured using the Bradford method. Levels of sodium-phosphate cotransporter (NaPi-II, B) and SGLT (C) proteins were determined by western blotting. One representative western blot result is shown in A. The data are expressed as the mean ± SD (n = 8), and the error bars represent the SD. ⁎p b 0.05, compared with the saline group of MT+/+ mice; #p b 0.05, compared with the saline group of MT−/− mice; @p b 0.05, with a two-way ANOVA. In addition, at 4 days post-DU exposure, the kidney cells were collected. Then the cells were fixed and probed with SGLT antibody to detect subcellular localization of SGLT, and there was no SGLT primary antibody in the blank (negative control). Nuclei were visualized by staining with Hoechst. Intracellular localization of SGLT was visualized by confocal microscopy (D). The results are representative of three independent experiments.

Please cite this article as: Hao, Y., et al., Metallothionein deficiency aggravates depleted uranium-induced nephrotoxicity, Toxicol. Appl. Pharmacol. (2015), http://dx.doi.org/10.1016/j.taap.2015.06.019

8

Y. Hao et al. / Toxicology and Applied Pharmacology xxx (2015) xxx–xxx

Fig. 8. The effect of exogenous metallothionein (MT) on depleted uranium (DU)-induced nephrotoxicity in MT−/− mice at 4 days post-DU exposure. The mice were administrated with or without MT once at three different doses (10, 20, 30 μmol/kg) 1 h before DU exposure, respectively. (A) The kidney pathological changes in each group (n = 8) were evaluated by H & E staining. (B) At 4 days post-DU exposure, the kidney cells were collected as described in the Materials and methods section. Apoptosis was measured by flow cytometry, followed by Annexin V-FITC and PI double staining. (C) The percentage of apoptosis was counted including early apoptosis (in the lower-right quadrants) and late apoptosis (in the upper-right quadrant). (D) The level of superoxide dismutase (SOD) was measured by colorimetry (n = 8). (E) The level of sodium-glucose cotransporter (SGLT) proteins was determined by western blotting. The results are representative of three independent experiments (n = 8 mice/group/experiment). The data are expressed as the mean ± SD, and the error bars represent the SD. ⁎p b 0.05, compared with the DU group; @p b 0.05, with a one-way ANOVA and LSD test for multiple comparisons.

Table 2 Histopathological scores in kidney after exposure to depleted uranium (DU) in metallothionein-1/2-null mice (MT−/−).a Parameters

Control

DU

MT(10) + DU

MT(20) + DU

MT(30) + DU

Tubular damage Mononuclear cell infiltration

0.71 ± 0.49⁎ 0.50 ± 0.53⁎

3.29 ± 0.76 2.63 ± 0.52

2.57 ± 0.53⁎⁎ 2.00 ± 0.53⁎⁎

2.14 ± 0.69⁎ 1.88 ± 0.64⁎

1.86 ± 0.38⁎,⁎⁎⁎ 1.25 ± 0.46⁎,⁎⁎⁎⁎

a

⁎ ⁎⁎ ⁎⁎⁎ ⁎⁎⁎⁎

Values are expressed as the mean ± SD (n = 8). Statistical analysis was performed by a one-way ANOVA and LSD test for multiple comparisons. p b 0.01, compared with the DU group. p b 0.05, compared with the DU group. p b 0.05, compared with the MT(10) + DU group. p b 0.01, compared with the MT(10) + DU group.

Please cite this article as: Hao, Y., et al., Metallothionein deficiency aggravates depleted uranium-induced nephrotoxicity, Toxicol. Appl. Pharmacol. (2015), http://dx.doi.org/10.1016/j.taap.2015.06.019

Y. Hao et al. / Toxicology and Applied Pharmacology xxx (2015) xxx–xxx

(Shaki et al., 2013). MT could protect against various injuries resulting from ROS (Chiaverini and De Ley, 2010). Intracellular MT-1 and MT-2 were distributed in cytoplasm and subcellular organelles like lysosomes and mitochondria where they might function as expendable targets for oxidants due to their highly enriched cysteine residue structures (Formigari et al., 2007). Thus, the detoxification mechanisms of DU may be related to antagonizing the cellular production of ROS and inhibiting the generation of MDA by MT. Moreover, MT deficiency increased DU-induced apoptosis in kidney cells and influenced apoptotic protein expression. In normal rat kidney proximal cells, uranium induces caspase dependent apoptosis cell death from 200 μM mainly through activation of caspase-9 and caspase-3 (Thiebault et al., 2007). Periyakaruppan et al. (2009) reported that, the activity of caspase-8 and caspase-3 in rat lung epithelial cells were increased in dose and time-dependent manner after exposure to uranium (0.25, 0.5, 1 mmol/l). In our previous study (Hao et al., 2014), we found DU (500 μM) induce HK-2 cell apoptosis and MT might play an important role in protecting against DU induced apoptosis. To explore the molecular mechanisms underlying DU-induced apoptosis in mice, we examined the expression of the key markers of apoptosis, namely Bcl-2, Bax and caspase-3. Consistent with increased apoptosis, the amounts of both Bax and caspase-3 were elevated and the level of Bcl-2 decreased after exposure to DU in either MT−/− or MT+/+ mice, while these changes were more evident in MT−/− mice. Consistent with our conclusions, Formigari et al. (2007) have reported that MT+/+ cells exhibit a lower level of apoptosis and necrosis factor production after treatment with copper when compared with MT−/− cells, which indicates that MT plays an important role in apoptosis. In the present study, we also found with the increasing dose of exogenous MT, the total percentage of apoptotic cells caused by DU reduced. Furthermore, MT deficiency down-regulated expression of SGLT at 4 days post-DU exposure. In addition to causing increased cell death that compromises kidney function, DU has also been implicated in dysfunction of kidney cells transporters (Vicente-Vicente et al., 2010). Selvaratnam et al. (2013) found that after exposure to cadmium, levels of glucose transporter (GLUT1) protein were decreased in MT−/− but not MT+/+ placentas, and reduced GLUT1 in the placenta might be an important factor contributing to the molecular mechanisms underlying cadmium-induced fetal growth restriction in MT−/− mice. Therefore, we chose to examine two transporters of specific interest in this study, SGLT and NaPi-II. SGLT and NaPi-II expressions were deemed to be strongly correlated with DU exposure (Goldman et al., 2006; Muller et al., 2006). Our results showed that SGLT and NaPi-II proteins were sensitive proteins in kidney tissues of either MT−/− or MT+/+ mice after exposure to DU. Previous studies (Muller et al., 2006, 2008) reported that, uranium cytotoxicity was directly dependent on the formation of the phosphate complexes of uranyl, which suppressed the NaPi-II participating in the reabsorption of organic phosphate. In the present study, the low-expression of NaPi-II caused by DU would also influence reabsorption of organic phosphate. Taulan et al. (2006) found that the gene expression of NaPi-II decreased through comprehensive analysis of the renal transcriptional response to acute uranyl nitrate exposure. Moreover, there was no difference between MT−/− mice and MT+/+ mice in the level of NaPi-II protein, suggesting that altered NaPi-II expression is unlikely to be involved in MT protecting against DU. However, the level of SGLT protein in MT−/− mice was lower than that in MT+/+ mice, and exogenous MT also increased SGLT levels in a dose-dependent manner after DU exposure, suggesting that SGLT may be an important causal factor contributing to MT antagonizing the nephrotoxicity caused by DU. SGLT has a key role in the reabsorption of glucose in the kidney (Chao and Henry, 2010), and in the apical membrane utilizes a Na+ gradient to catalyze active glucose transport into the cell. Therefore, DU exposure will decrease glucose uptake, and MT will prevent or slow down the process. In fact, a study (Hori et al., 1985) reported that uranyl nitrate (10 mg/kg, subcutaneous injection) induced a decrease in the sodium-dependent

9

glucose gradient and changed the transport properties of renal brush border membranes in rats. Uranyl acetate (140 μg/mg protein of renal brush border membrane vesicles) reduced the maximal capacity of the system to transport glucose because of a reduction in the number of SGLT (Goldman et al., 2006). In summary, our results clearly showed that after 4 days of DU acute exposure, MT deficiency significantly aggravated kidney functional and morphological damage caused DU. Moreover, MT deficiency induced more ROS and kidney cells apoptosis after DU exposure. Furthermore, our results verified that SGLT and NaPi-II proteins were sensitive proteins in kidney tissues after exposure to DU. In addition, MT is associated with the level of SGLT protein. It may provide a novel direction for MT protecting against DU. What's more, exogenous MT could also reduce the pathological damage, serum Cr and BUN levels and cell apoptosis, and increase SOD and SGLT levels in a dose-dependent manner after DU exposure. These suggest that MT might be a novel therapeutic target for the treatment of DU-induced nephrotoxicity. Further research is required to elucidate the specific interactions between MT and SGLT, and the roles of different MT isoforms in DU detoxification. Transparency document The Transparency document associated with this article can be found, in the online version. Conflict of interest statement None. Funding This work was supported by the National Science & Technology Pillar Program during the 12th Five-year Plan Period (no. 2013BAK03B05-02), the National Natural Science Foundation of China (no. 81472913) and the State Key Laboratory of Trauma, Burns and Combined Injury, Institute of Combined Injury, China (no. SKLZZ200809). Acknowledgment We thank Huiqin Sun as well as members of the Institute of Combined Injury, Third Military Medical University for the technical assistance in some aspects of this study. References Agency for Toxic Substances and Disease Research (ATSDR), 2013. Toxicological Profile for Uranium. Department of Health and Human Services. Public Health Service, Atlanta, GA. Andrews, G.K., 2000. Regulation of metallothionein gene expression by oxidative stress and metal ions. Biochem. Pharmacol. 59, 95–104. Bao, Y., Wang, D., Li, Z., Hu, Y., Xu, A., Wang, Q., Shao, C., Chen, H., 2013. Efficacy of a novel chelator BPCBG for removing uranium and protecting against uranium-induced renal cell damage in rats and HK-2 cells. Toxicol. Appl. Pharmacol. 269, 17–24. Bleise, A., Danesi, P.R., Burkart, W., 2003. Properties, use and health effects of depleted uranium (DU): a general overview. J. Environ. Radioact. 64, 93–112. Brugge, D., Buchner, V., 2011. Health effects of uranium: new research findings. Rev. Environ. Health 26, 231–249. Chao, E.C., Henry, R.R., 2010. SGLT2 inhibition—a novel strategy for diabetes treatment. Nat. Rev. Drug Discov. 9, 551–559. Cheng, K.L., Hogan, A.C., Parry, D.L., Markich, S.J., Harford, A.J., van Dam, R.A., 2010. Uranium toxicity and speciation during chronic exposure to the tropical freshwater fish, Mogurnda mogurnda. Chemosphere 79, 547–554. Chiaverini, N., De Ley, M., 2010. Protective effect of metallothionein on oxidative stress-induced DNA damage. Free Radic. Res. 44, 605–613. Eaton, D.L., Cherian, M.G., 1991. Determination of metallothionein in tissues by cadmium–hemoglobin affinity assay. Methods Enzymol. 205, 83–88. Formigari, A., Irato, P., Santon, A., 2007. Zinc, antioxidant systems and metallothionein in metal mediated-apoptosis: biochemical and cytochemical aspects. Comp. Biochem. Physiol. C 146, 443–459. Fukuda, S., Ikeda, M., Nakamura, M., Yan, X., Xie, Y., 2007. Effects of pH on du intake and removal by CBMIDA and EHBP. Health Phys. 92, 10–14.

Please cite this article as: Hao, Y., et al., Metallothionein deficiency aggravates depleted uranium-induced nephrotoxicity, Toxicol. Appl. Pharmacol. (2015), http://dx.doi.org/10.1016/j.taap.2015.06.019

10

Y. Hao et al. / Toxicology and Applied Pharmacology xxx (2015) xxx–xxx

Fukuda, S., Ikeda, M., Nakamura, M., Katoh, A., Yan, X., Xie, Y., Kontoghiorghes, G.J., 2008. The effects of bicarbonate and its combination with chelating agents used for the removal of depleted uranium in rats. Hemoglobin 32, 191–198. Fukuda, S., Ikeda, M., Nakamura, M., Yan, X., Xie, Y., 2009. Efficacy of oral and intraperitoneal administration of CBMIDA for removing uranium in rats after parenteral injections of depleted uranium. Radiat. Prot. Dosim. 133, 12–19. Gaudreault, N., Scriven, D.R.L., Laher, I., Moore, E.D.W., 2008. Subcellular characterization of glucose uptake in coronary endothelial cells. Microvasc. Res. 75, 73–82. Goldman, M., Yaari, A., Doshnitzki, Z., Cohen-Luria, R., Moran, A., 2006. Nephrotoxicity of uranyl acetate: effect on rat kidney brush border membrane vesicles. Arch. Toxicol. 80, 387–393. Gu, C.S., Liu, L.Q., Zhao, Y.H., Deng, Y.M., Zhu, X.D., Huang, S.Z., 2014. Overexpression of Iris. lactea var. chinensis metallothionein llMT2a enhances cadmium tolerance in Arabidopsis thaliana. Ecotoxicol. Environ. Saf. 105, 22–28. Hao, Y., Li, R., Leng, Y., Ren, J., Liu, J., Ai, G., Xu, H., Su, Y., Cheng, T., 2009. A study assessing the genotoxicity in rats after chronic oral exposure to a low dose of depleted uranium. J. Radiat. Res. 50, 521–528. Hao, Y., Ren, J., Liu, J., Luo, S., Ma, T., Li, R., Su, Y., 2012. The protective role of zinc against acute toxicity of depleted uranium in rats. Basic Clin. Pharmacol. 111, 402–410. Hao, Y., Ren, J., Liu, J., Yang, Z., Liu, C., Li, R., Su, Y., 2013. Immunological changes of chronic oral exposure to depleted uranium in mice. Toxicology 309, 81–90. Hao, Y., Ren, J., Liu, C., Li, H., Liu, J., Yang, Z., Li, R., Su, Y., 2014. Zinc protects human kidney cells from depleted uranium-induced apoptosis. Basic Clin. Pharmacol. 114, 271–280. He, Y., Ma, W., Li, Y., Liu, J., Jing, W., Wang, L., 2014. Expression of metallothionein of freshwater crab (Sinopotamon henanense) in Escherichia coli enhances tolerance and accumulation of zinc, copper and cadmium. Ecotoxicology 23, 56–64. Helal, G.K., Helal, O.K., 2009. Metallothionein attenuates carmustine-induced oxidative stress and protects against pulmonary fibrosis in rats. Arch. Toxicol. 83, 87–94. Hori, R., Takano, M., Okano, T., Inui, K., 1985. Transport of p-aminohippurate, tetraethylammonium and D -glucose in renal brush border membranes from rats with acute renal failure. J. Pharmacol. Exp. Ther. 233, 776–781. Inal, S., Koc, E., Ulusal-Okyay, G., Pasaoglu, O.T., Isik-Gonul, I., Oz-Oyar, E., Pasaoglu, H., Guz, G., 2014. Protective effect of adrenomedullin on contrast induced nephropathy in rats. Nefrologia 34, 724–731. Jiang, G., Hughes, S., Stürzenbaum, S., Evje, L., Syversen, T., Aschner, M., 2009. Caenorhabditis elegans metallothioneins protect against toxicity induced by depleted uranium. Toxicol. Sci. 111, 345–354. Jortner, B.S., 2008. Effect of stress at dosing on organophosphate and heavy metal toxicity. Toxicol. Appl. Pharmacol. 233, 162–167. Kang, Y.J., 2006. Metallothionein redox cycle and function. Exp. Biol. Med. 231, 1459–1467. Kukner, A., Colakoglu, N., Kara, H., Oner, H., Özogul, C., Ozan, E., 2007. Ultrastructural changes in the kidney of rats with acute exposure to cadmium and effects of exogenous metallothionein. Biol. Trace Elem. Res. 119, 137–146. Liu, Y., Liu, J., Iszard, M.B., Andrews, G.K., Palmiter, R.D., Klaassen, C.D., 1995. Transgenic mice that overexpress metallothionein-I are protected from cadmium lethality and hepatotoxicity. Toxicol. Appl. Pharmacol. 135, 222–228. Liu, J., Cheng, M.L., Yang, Q., Shan, K.R., Shen, J., Zhou, Y., Zhang, X., Dill, A.L., Waalkes, M.P., 2007. Blood metallothionein transcript as a biomarker for metal sensitivity: low blood metallothionein transcripts in arsenicosis patients from Guizhou, China. Environ. Health Perspect. 115, 1101–1106. Loebus, J., Leitenmaier, B., Meissner, D., Braha, B., Krauss, G.J., Dobritzsch, D., Freisinger, E., 2013. The major function of a metallothionein from the aquatic fungus Heliscus lugdunensis is cadmium detoxification. J. Inorg. Biochem. 127, 253–260. Lu, S., Zhao, F.Y., 1990. Nephrotoxic limit and annual limit on intake for natural U. Health Phys. 58, 619–623. Maria, V.L., Ribeiro, M.J., Amorim, M.J., 2014. Oxidative stress biomarkers and metallothionein in Folsomia candida—responses to Cu and Cd. Environ. Res. 133, 164–169.

Merigo, F., Benati, D., Cristofoletti, M., Amarù, F., Osculati, F., Sbarbati, A., 2012. Glucose transporter ⁄T1R3-expressing cells in rat tracheal epithelium. J. Anat. 221, 138–150. Milacic, S., Simic, J., 2009. Identification of health risks in workers staying and working on the terrains contaminated with depleted uranium. J. Radiat. Res. 50, 213–222. Muller, D., Houpert, P., Cambar, J., Hengé-Napoli, M.H., 2006. Role of the sodium-dependent phosphate co-transporters and of the phosphate complexes of uranyl in the cytotoxicity of uranium in LLC-PK1 cells. Toxicol. Appl. Pharmacol. 214, 166–177. Muller, D.S., Houpert, P., Cambar, J., Hengé-Napoli, M.H., 2008. Role of the sodium-dependent phosphate cotransporters and absorptive endocytosis in the uptake of low concentrations of uranium and its toxicity at higher concentrations in LLC-PK1 cells. Toxicol. Sci. 101, 254–262. Nordberg, G.F., 2004. Cadmium and health in the 21st century—historical remarks and trends for the future. Biometals 17, 485–489. Palmiter, R.D., 1994. Regulation of metallothionein genes by heavy metals appears to be mediated by a zinc-sensitive inhibitor that interacts with a constitutively active transcription factor, MTF-1. Proc. Natl. Acad. Sci. U. S. A. 91, 1219–1223. Periyakaruppan, A., Sarkar, S., Ravichandran, P., Sadanandan, B., Sharma, C.S., Ramesh, V., Hall, J.C., Thomas, R., Wilson, B.L., Ramesh, G.T., 2009. Uranium induces apoptosis in lung epithelial cells. Arch. Toxicol. 83, 595–600. Pourahmad, J., Ghashang, M., Ettehadi, H.A., Ghalandari, R., 2006. A search for cellular and molecular mechanisms involved in depleted uranium (DU) toxicity. Environ. Toxicol. 21, 349–354. Selvaratnam, J., Guan, H., Koropatnick, J., Yang, K., 2013. Metallothionein-I- and -II-deficient mice display increased susceptibility to cadmium-induced fetal growth restriction. Am. J. Physiol. Endocrinol. Metab. 305, E727–E735. Shaki, F., Hosseini, M.J., Shahraki, J., Ghazi-Khansari, M., Pourahmad, J., 2013. Toxicity of depleted uranium on isolated liver mitochondria: a revised mechanistic vision for justification of clinical complication of depleted uranium (DU) on liver. Toxicol. Environ. Chem. 95, 1221–1234. Shuai, Y., Guo, J.B., Peng, S.Q., Zhang, L.S., Guo, J., Han, G., Dong, Y.S., 2007. Metallothionein protects against doxorubicin-induced cardiomyopathy through inhibition of superoxide generation and related nitrosative impairment. Toxicol. Lett. 170, 66–74. Squibb, K.S., Leggett, R.W., McDiarmid, M.A., 2005. Prediction of renal concentrations of depleted uranium and radiation dose in Gulf War veterans with embedded shrapnel. Health Phys. 89, 267–273. Taulan, M., Paquet, F., Argiles, A., Demaille, J., Romey, M.C., 2006. Comprehensive analysis of the renal transcriptional response to acute uranyl nitrate exposure. BMC Genomics 7, 2. Thiebault, C., Carriere, M., Milgram, S., Simon, A., Avoscan, L., Gouget, B., 2007. Uranium induces apoptosis and is genotoxic to normal rat kidney (NRK-52E) proximal cells. Toxicol. Sci. 98, 479–487. Vicente-Vicente, L., Quiros, Y., Pérez-Barriocanal, F., López-Novoa, J.M., López-Hernández, F.J., Morales, A.I., 2010. Nephrotoxicity of uranium: pathophysiological, diagnostic and therapeutic perspectives. Toxicol. Sci. 118, 324–347. Wang, X.B., Liu, Q.H., Wang, P., Tang, W., Hao, Q., 2008. Study of cell killing effect on S180 by ultrasound activating protoporphyrin IX. Ultrasonics 48, 135–140. Xiang, D.F., Zhu, J.Q., Jin, S., Hu, Y.J., Tan, F.Q., Yang, W.X., 2013. Expression and function analysis of metallothionein in the testis of Portunus trituberculatus exposed to cadmium. Aquat. Toxicol. 140–141, 1–10. Xu, Y., Wang, S., Shen, M., Zhang, Z., Chen, S., Chen, F., Chen, M., Zeng, D., Wang, A., Zhao, J., Cheng, T., Su, Y., Wang, J., 2014. hGH promotes megakaryocyte differentiation and exerts a complementary effect with c-Mpl ligands on thrombopoiesis. Blood 123, 2250–2260. Yoshioka, T., Kawada, K., Shimada, T., Mori, M., 1979. Lipid peroxidation in maternal and cord blood and protective mechanism against activated-oxygen toxicity in the blood. Am. J. Obstet. Gynecol. 135, 372–376. Zhang, X.F., Ding, C.L., Liu, H., Liu, L.H., Zhao, C.Q., 2011. Protective effects of ion-imprinted chitooligosaccharides as uranium-specific chelating agents against the cytotoxicity of depleted uranium in human kidney cells. Toxicology 286, 75–84.

Please cite this article as: Hao, Y., et al., Metallothionein deficiency aggravates depleted uranium-induced nephrotoxicity, Toxicol. Appl. Pharmacol. (2015), http://dx.doi.org/10.1016/j.taap.2015.06.019

Metallothionein deficiency aggravates depleted uranium-induced nephrotoxicity.

Depleted uranium (DU) has been widely used in both civilian and military activities, and the kidney is the main target organ of DU during acute high-d...
4MB Sizes 2 Downloads 9 Views