Biol Trace Elem Res DOI 10.1007/s12011-015-0314-7

Ameliorative Effects of Selenium on Cadmium-Induced Oxidative Stress and Endoplasmic Reticulum Stress in the Chicken Kidney Lili Liu 1,2 & Bingyou Yang 1 & Yupeng Cheng 1 & Hongjin Lin 3

Received: 5 February 2015 / Accepted: 13 March 2015 # Springer Science+Business Media New York 2015

Abstract The harmful influences of dietary cadmium (Cd) on the chicken kidney and the protective role of selenium (Se) against Cd-induced nephrotoxicity in the chicken are relatively unexplored subjects. The aim of this study was to investigate the ameliorative role of Se on the effects of Cd-induced oxidative stress, endoplasmic reticulum stress, and apoptosis in chicken kidneys. For this study, 100-day-old chickens received Se (as 10 mg Na2SeO3/kg dry weight of diet), Cd (as 150 mg CdCl2/kg dry weight of diet), or Cd+Se in their diets for 60 days. Then, the histopathological changes, Cd and Se contents, levels of oxidative stress, inducible nitric oxide synthase-nitric oxide (iNOS-NO) system activity, levels of endoplasmic reticulum (ER) stress, results of the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay of apoptosis, and expression levels of Bcl-2 and caspase 3 in the kidney were examined. The results Lili Liu holds a PhD degree, Heilongjiang University of Chinese Medicine, Northeast Agricultural University. Bingyou Yang holds a PhD degree, Heilongjiang University of Chinese Medicine. Yupeng Cheng holds a PhD degree, Heilongjiang University of Chinese Medicine. Hongjin Lin holds a Ph.D degree, Northeast Agricultural University. All other authors have read the manuscript and have agreed to submit it in its current form for consideration for publication in the journal. * Hongjin Lin [email protected] 1

College of Pharmacy, Heilongjiang University of Chinese Medicine, Harbin 150040, People’s Republic of China

2

College of Veterinary Medicine, Northeast Agricultural University, Harbin 150030, People’s Republic of China

3

College of Applied Technology, Northeast Agricultural University, Harbin 150030, People’s Republic of China

showed that Cd exposure caused histopathological and ultrastructural damage and apoptosis of the kidneys. Cd administration significantly increased the accumulation of Cd, the malondialdehyde (MDA) content, NO production, iNOS activity, iNOS expression levels, expression levels of ER stressrelated genes (GRP78, GRP94, ATF4, ATF6, and IRE) and the pro-apoptosis gene caspase 3, and the rate of apoptosis. Cd administration markedly decreased the Se content, superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities, and anti-apoptosis gene Bcl-2 expression levels. Co-treatment with Se and Cd obviously reduced the accumulation of Cd, Cd-induced histopathological and ultrastructural changes, oxidative stress, iNOS-NO system activity, ER stress, caspase 3 expression levels, and the rate of apoptosis in the kidneys. These results suggested that Cd exposure caused renal injury and that Se ameliorated Cd-induced nephrotoxicity in chickens.

Keywords Cadmium . Selenium . Oxidative stress . Endoplasmic reticulum stress . Apoptosis . Chicken kidney

Introduction Cadmium (Cd) is known to be one of the most toxic metals in the environment. The general population is exposed to Cd via contaminants found in water, food, and air. Cd has been widely reported to cause severe damage to multiple organs in mammals, such as the lung [1], the heart [2], the liver [3], the brain [4], the ovary [5], and the testes [6]. Moreover, recent studies have showed that dietary exposure to Cd gives rise to liver toxicity [7], testicular toxicity [8], and ovarian toxicity [9] in poultry. The kidneys is one of the most important target organs for Cd accumulation and intoxication in mammals [10], and the manifestations of Cd nephrotoxicity are usually reflected

Liu et al.

in proteinuria, calciuria, aminoaciduria, glycosuria, and tubular necrosis [11]. Increasing evidence has suggested that the Cd exposure-induced physiopathological mechanism in various organs is associated with oxidative stress, increased apoptosis, histopathology, and changes in the inducible nitric oxide synthase-nitric oxide (iNOS-NO) system [9, 12]. Overproduction of reactive oxygen species (ROS) after Cd exposure leads to oxidative stress, which is an important factor in Cdinduced apoptosis [13, 14]. A study found that the endoplasmic reticulum (ER) is a target organelle of Cd toxicity and that Cd could cause ER stress in vitro and in vivo, which also plays a critical role in the induction of apoptosis [15, 16]. Wang et al. [17] reported that ROS-mediated ER stress might participate in Cd-induced injury on placental and fetal development. An investigation by Jin [18] elucidated that a long period of exposure to Cd has the potential to elicit oxidative- and ER stress-mediated apoptosis in the livers of mice. In addition, Cd exposure could lead to apoptosis and a disorder of intracellular homeostasis induced by oxidative stress and mitochondrial dysfunction in rat proximal tubular cells [19]. Komoike et al. [20] indicated that Cd-induced apoptosis of human kidney 2 (HK-2) human renal proximal tubular cells is an ER stress-signaling event. However, the present studies about the nephrotoxicity of Cd mainly focused on mammals, and whether Cd exposure induced oxidative stress or ER stress or how these pathways regulate apoptosis have not been reported for the chicken kidney. Considering that Cd has been listed as a chemical substance that is potentially dangerous on a global level, prevention and/or therapeutic intervention after Cd intoxication are attracting tremendous attention. Selenium (Se), an essential micronutrient and a constituent of many antioxidase enzymes, has beneficial roles in a number of biological processes. Se significantly inhibited oxidative stress and oxidative stress-induced ER stress, resulting in decreased apoptosis [21]. In addition, some studies have reported that Se has ameliorative effects against Cd-induced tissue impairment by resisting oxidative stress [22, 23]. However, whether Se alleviates renal injury in chickens caused by Cd is unclear, and the protective mechanism of Se against Cdinduced nephrotoxicity in chickens also remains largely unexplored. In the present study, we designed an experiment to examine Cd and Se concentration, nephritic histopathology, oxidative stress, iNOS-NO system activity, ER stress, and apoptosis in the chicken kidney after subchronic Cd exposure and the synchronous administration of Cd and Se by diet. The aim of this study was to simultaneously illuminate the mechanism of nephrotoxicity caused by Cd and the possible protective roles of Se against Cd-induced renal toxicity in chickens.

Materials and Methods Poultry and Experimental Design All procedures used in this experiment were approved by the Institutional Animal Care and Use Committee of Northeast Agricultural University. The doses and durations of Cd and Se in this study were referred to in our previous study [7]. One hundred twenty 100-day-old Isa Brown chickens were divided randomly into four groups (n = 30 per group). Group I (control) was fed a basal diet; group II (Se-treated) was fed the basic diet supplemented with 10 mg/kg Na2SeO3; group III (Cd/Se co-treated) was fed the basic diet supplemented with 150 mg/kg CdCl2 +10 mg/kg Na2SeO3, and group IV (Cd-treated) was fed the basal diet supplemented with 150 mg/kg CdCl2. Chickens were maintained in the Laboratory Animal Center, College of Veterinary Medicine, Northeast Agricultural University, China, and kept under a 16/8 h light/dark cycle. The feed and water were supplied ad libitum. On the 60th day of the experiment, all of the chickens were fasted overnight. Following euthanasia with sodium pentobarbital, the kidneys were quickly collected and frozen immediately in liquid nitrogen and stored at −80 °C until required for further study.

Estimation of Cd and Se Concentrations The Cd content in the kidneys was detected by flame atomic absorption spectrometry (FAAS, Shanghai Huipu Analytical Instruments Company, Shanghai, China) according to the method used by Li et al. [7]. The wet tissue samples (1.0 g) were cut into small pieces and were transferred into beakers. For digestion, 25 ml of concentrated HNO3/HCl (4:1) was added to each beaker and warmed on a lowtemperature electric hot plate until solution transparence. The samples were metered to 10 ml by 0.5 % HNO3 and measured using FAAS. The Cd content was calculated from a standard curve. The Se content in the kidneys was estimated by the method described by Hasunuma et al. [24]. The assay is based on the principle that Se contained in the samples is converted to selenous acid in response to acid digestion. The Se content was calculated by reference to a standard curve.

Measurement of Oxidative Stress The content of malondialdehyde (MDA) and the activities of superoxide dismutase (SOD) and glutathione peroxidase (GPx) in the kidney homogenates were assayed using kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s protocol.

Effects of Selenium on Cadmium-Induced Oxidative Stress and Endoplasmic Reticulum Stress

NO Level and iNOS Activity Assay The NO level and iNOS activity in the kidney homogenates were measured using NO and iNOS activity assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions.

by 40 cycles of 95 °C for 15 s, 60 °C for 30 s, and 60 °C for 30 s. Dissociation curves were analyzed by Dissociation Curve 1.0 software (Applied Biosystems) for each PCR reaction to detect and eliminate possible primer-dimer and nonspecific amplification. The messenger RNA (mRNA) relative abundance was calculated according to the method of Pfaffl [25].

Quantitative Real-Time PCR Analysis Western Blot Analysis Total RNA was isolated from tissue samples using Trizol reagent according to the manufacturer ’s instructions (Invitrogen, Shanghai, China). The RNA concentrations were determined using the GeneQuant 1300 spectrophotometer (GE Healthcare Biosciences, Piscataway, NJ). The reverse transcription procedure was performed according to the manufacturer’s instructions (Invitrogen, Shanghai, China). Synthesized cDNA was stored at −20 °C for PCR. Oligo 7.0 Software was used to design specific primers for iNOS, GRP78, GRP94, ATF4, ATF6, IRE, B-cell lymphoma 2 (Bcl-2), caspase 3, and glyceraldehyde 3-phosphate dehydrogenase (GADPH; a housekeeping gene used as an internal reference) based on known chicken sequences (Table 1). Primers were synthesized by Invitrogen Biotechnology Co. Ltd. in Shanghai, China. Real-time quantitative reverse transcription PCR was used to detect the expression of the target genes in the kidney using SYBR Premix Ex TaqTM (Takara, Otsu, Japan), and real-time PCR work was performed using a ABI PRISM 7500 real-time PCR system (Applied Biosystems, Foster City, CA). The PCR procedure for iNOS, GRP78, GRP94, ATF4, ATF6, IRE, Bcl2, caspase 3, and GADPH consisted of 95 °C for 30 s followed Table 1 Gene-specific primers used in the real-time quantitative reverse transcription PCR

Protein extracts were subjected to 15 % SDS-polyacrylamide gel electrophoresis under reducing conditions. The separated proteins were then transferred to nitrocellulose membranes for 2 h at 100 mA in a transfer apparatus. Membranes were blocked with 5 % skim milk for 24 h and incubated overnight with diluted primary antibodies against iNOS (1:100, Santa Cruz Biotechnology, USA), GRP78 (1:1000, polyclonal antibody produced by our lab), Bcl-2 (1:500, Santa Cruz Biotechnology, USA), and caspase 3 (1:100, Santa Cruz Biotechnology, USA), and then, a horseradish peroxidase (HRP)-conjugated secondary antibody against goat (Bcl-2) or rabbit (iNOS, GRP78, and caspase 3) IgG was added (1:1000, Santa Cruz Biotechnology, USA). To verify equal loading of the samples, the membrane was incubated with a monoclonal βactin antibody (1:1000, Santa Cruz Biotechnology, USA), followed by an HRP-conjugated goat anti-mouse IgG (1:1000) secondary antibody. The signal was detected with X-ray films (TransGen Biotech Co., Beijing, China). The optical density (OD) of each band was determined using an Image VCD gel imaging system, and the relative abundance of the iNOS, GRP78, Bcl-2, and caspase 3 proteins was expressed as ratios of OD for each of these proteins to that of β-actin.

Gene

Primer sequence

Electron Microscopic Examination

iNOS

Forward 5′-CCTGGAGGTCCTGGAAGAGT-3′ Reverse 5′-CCTGGGTTTCAGAAGTGGC-3′ Forward 5′-GAATCGGCTAACACCAGAGGA-3′ Reverse 5′-CGCATAGCTCTCCAGCTCATT-3′ Forward 5′-CAAAGACATGCTGAGGCGAGT-3′ Reverse 5′-TCCACCTTTGCATCCAGGTCA-3′ Forward 5′-GAATCGGCTAACACCAGAGGA-3′ Reverse 5′-CGCATAGCTCTCCAGCTCATT-3′ Forward 5′-CGTCGTCTGAACCACTTACTGA-3′ Reverse 5′-CCTTCTTTCCTAACAGCCACAC-3′ Forward 5′-CTACAGGTCGCTCCTCACATC-3′ Reverse 5′-ATCAGTCCTTCTGCTCCCATCT-3′ Forward 5′-ATCGTCGCCTTCTTCGAGTT-3′ Reverse 5′-ATCCCATCCTCCGTTGTCCT-3′ Forward 5′-CATCT GCATCCGTGCCTGA-3′ Reverse 5′-CTCTCGGCTGTGGTGGTGAA-3′ Forward 5′-AGAACATCATCCCAGCGT-3′ Reverse 5′-AGCCTTCACTACCCTCTTG-3′

For electron microscopy, kidney tissue specimens were fixed with 2.5 % glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.2) for 3 h at 4 °C, washed in the same buffer for 1 h at 4 °C and postfixed with 1 % osmium tetroxide in sodium phosphate buffer for 1 h at 4 °C. The tissues were then dehydrated in graded series of ethanol, starting at 50 % each step for 10 min, after two changes in propylene oxide. The tissue specimens were embedded in Araldite. Ultrathin sections were stained with Mg-uranyl acetate and lead citrate for transmission electron microscope evaluation (GEM-1200ES, Japan).

GRP 78 GRP94 ATF4 ATF6 IRE Bcl-2 Caspase 3 GADPH

Light Microscopic Examination Parts of the kidney tissue obtained from each chicken were fixed in 10 % buffered neutral formalin, dehydrated in ascending grades of alcohol, and embedded in paraffin. Sections

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approximately 5 μm thick were taken, stained with hematoxylin and eosin (H&E), and examined under a light microscope (XDS-1B, Olympus, Japan). In Situ Apoptosis Detection We used an in situ cell death detection kit (Roche Diagnostics GmbH, Mannheim, Germany). The method distinguishes apoptotic cells from those cells undergoing necrosis because damaged DNA in the former cells leads to a different distribution of staining and nuclear morphology. Paraffin waxembedded tissue sections were treated with proteinase K, and the endogenous peroxidase activity was blocked with hydrogen peroxide. The sections were incubated at 37 °C with a terminal TdT/nucleotide mixture for 1 h. Then, the reaction was stopped, and the slides were rinsed with phosphatebuffered saline. Nuclear labeling was performed with horseradish peroxidase and diaminobenzidine. Hematoxylin was used for counterstaining. Quantitative evaluation of the apoptotic index was performed by manual counting of positively stained nuclei at 400× magnification. Apoptosis was determined in five kidneys from each group by counting at least 1000 cells from five to six sections of each kidney. The results are expressed as the percentage of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells among the total number of cells counted. Statistical Analysis Statistical analyses of all of the data were performed using SPSS for Windows (version 13; SPSS Inc., Chicago, IL, USA). When a significant value (p