JOURNAL OF MEDICINAL FOOD J Med Food 18 (5) 2015, 1–11 # Mary Ann Liebert, Inc., and Korean Society of Food Science and Nutrition DOI: 10.1089/jmf.2014.3242

FULL COMMUNICATION

Protective Effects of Quercetin Against HgCl2-Induced Nephrotoxicity in Sprague-Dawley Rats Yu Jin Shin,1 Jeong Jun Kim,2 Ye Ji Kim,3 Won Hee Kim,4 Eun Young Park,1 In Young Kim,5 Han-Seung Shin,6 Kyeong Seok Kim,7 Eui-Kyung Lee,7 Kyu Hyuck Chung,7 Byung Mu Lee,7 and Hyung Sik Kim 7 1

College of Pharmacy, Pusan National University, Busan, South Korea. 2 Groton School, Groton, Massachusetts, USA. 3 The Hockaday School, Dallas, Texas, USA. 4 Sehwa High School, Seoul, South Korea. 5 Korea Food and Drug Administration, Busan, South Korea. 6 Department of Food Science and Biotechnology, Dongguk University, Seoul, South Korea. 7 School of Pharmacy, Sungkyunkwan University, Suwon, South Korea. ABSTRACT Mercury is a well-known environmental pollutant that can cause nephropathic diseases, including acute kidney injury (AKI). Although quercetin (QC), a natural flavonoid, has been reported to have medicinal properties, its potential protective effects against mercury-induced AKI have not been evaluated. In this study, the protective effect of QC against mercury-induced AKI was investigated using biochemical parameters, new protein-based urinary biomarkers, and a histopathological approach. A 250 mg/kg dose of QC was administered orally to Sprague-Dawley male rats for 3 days before administration of mercury chloride (HgCl2). All animals were sacrificed at 24 h after HgCl2 treatment, and biomarkers associated with nephrotoxicity were measured. Our data showed that QC absolutely prevented HgCl2-induced AKI, as indicated by biochemical parameters such as blood urea nitrogen (BUN) and serum creatinine (sCr). In particular, QC markedly decreased the accumulation of Hg in the kidney. Urinary excretion of protein-based biomarkers, including clusterin, kidney injury molecule-1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL), monocyte chemoattractant protein-1 (MCP-1), tissue inhibitor of metalloproteinases 1 (TIMP-1), and vascular endothelial growth factor (VEGF) in response to HgCl2 administration were significantly decreased by QC pretreatment relative to that in the HgCl2-treated group. Furthermore, urinary excretion of metallothionein and Hg were significantly elevated by QC pretreatment. Histopathological examination indicated that QC protected against HgCl2-induced proximal tubular damage in the kidney. A TUNEL assay indicated that QC pretreatment significantly reduced apoptotic cell death in the kidney. The administration of QC provided significant protective effects against mercury-induced AKI.

KEY WORDS:  acute kidney injury  mercury  metallothionein  quercetin  protective effect

human exposure to inorganic mercury, which may indirectly affect human health.8 To understand the AKI induced by mercury and to find a means of protection against this nephropathy, it is essential to understand the mechanisms associated with intracellular uptake, accumulation, and elimination of mercury in the kidney, specifically in the proximal tubular epithelial cells. Various mechanisms have been suggested to be protective against mercury-induced nephrotoxicity.9,10 Previous studies have indicated that mercury chloride (HgCl2) exposure markedly reduces glutathione (GSH) levels, and that intracellular mercury reduces the number of sulfhydryl groups on proteins, interfering with protein function and disturbing both protein synthesis and energy metabolism.11,12 In addition, an imbalance in antioxidant status induces oxidative stress in the proximal tubules after HgCl2 exposure.13 A dramatic decrease in

INTRODUCTION

M

ercury is among the major environmental pollutants responsible for nephrotoxicity in both humans and animals.1–3 Mercury is a strong nephrotoxicant that has been widely used in animal models for investigating acute kidney injury (AKI), because the kidney is the location where mercury accumulates after acute exposure.4,5 Mercury is rapidly transferred to the kidney via binding to metallothionein (MT), and is subsequently released as a free form that accumulates in the proximal tubules of the kidneys.6,7 Environmentally contaminated foods are an important source of Manuscript received 18 May 2014. Revision accepted 26 November 2014. Address correspondence to: Hyung Sik Kim, PhD, School of Pharmacy, Sungkyunkwan University, Suwon 440-746, South Korea, E-mail: [email protected]

1

2

SHIN ET AL.

antioxidant enzymes such as catalase, superoxide dismutase (SOD), and glutathione peroxidase (GPx), was observed after HgCl2 exposure in animals.10,14 However, the mechanism has not yet been clarified, because complex factors may be involved in mercury-induced AKI.15,16 Numerous studies have demonstrated the protective effects of natural compounds extracted from a variety of plants against acute mercury toxicity.17–19 Quercetin (QC), which is one of the most abundant flavonoids, is widely present in vegetables, fruits, nuts, and beverages derived from them,20–22 has potent antioxidant activity, and exerts protective effects against cadmium-induced kidney toxicity by inhibiting apoptosis and oxidative damage as shown in both in vivo and in vitro studies.23–25 QC also exhibits metal chelation and free radical scavenging activity and protects against cisplatin-induced oxidative damage in kidneys of rats.26 However, the protective effect of QC against mercury-induced AKI has not been fully investigated. It would be of great interest to find an effective and natural compound to prevent mercury-caused nephrotoxicity. The aim of this study was to evaluate the possible use of QC as a herbal medication for protection against renal damages induced by HgCl2 in rats. Biomarkers of renal function, such as blood urea nitrogen (BUN) and serum creatinine (sCr), were measured. Furthermore, pathological changes were also observed and mercury concentrations in the kidneys and urine were determined. We also compared the levels of protein-based biomarkers associated with AKI in rats exposed to HgCl2 with or without QC pretreatment. To determine whether the protective effect of QC against HgCl2-induced AKI is associated with MT-mediated Hg excretion in the kidney urinary MT levels were measured after pretreatment with QC. MATERIALS AND METHODS Chemicals HgCl2, QC, and 3-indoxyl sulfate (3-IS) were purchased from Sigma-Aldrich Biotechnology (St. Louis, MO, USA). Primary antibodies specific for clusterin, b2-macroglobulin, NGAL, monocyte chemoattractant protein-1 (MCP-1), and tissue inhibitor of metalloproteinases 1 (TIMP-1), in addition to MT and a horseradish peroxidase (HRP)-conjugated secondary antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies specific for vascular endothelial growth factor (VEGF), calbindin, osteopontin (OPN), and b-actin were purchased from Abcam (Cambridge, MA, USA). A KIM-1 antibody was purchased from R&D Systems Incorporated (Minneapolis, MN, USA). Experimental design Male Sprague-Dawley rats were obtained from Charles River Laboratory Animal Resources (Seoul, Korea) and maintained in a specific pathogen free-conditioned room with a 12 h light/dark cycle. The ambient air temperature and relative humidity were set at 23C – 2C and 55%, respectively. Before experimentation, all animals were checked for

any overt signs of illness and only healthy animals were selected. Tap water and rodent chow were provided ad libitum. The experimental protocol was approved by Pusan National University Laboratory Animal Care Service (PNU2011-000221), in accordance with the Ministry of Food and Drug Safety (MFDS) Animal Protection of Korea (Oh-Song, Korea). The animals were randomly divided into the following four groups, with six animals per group: control, QC (250 mg/kg), HgCl2 (20 mg/kg), and QC pretreatment (250 mg/kg) plus HgCl2 (20 mg/kg). A single dose of HgCl2 was administered orally to rats, and QC (suspended in 0.5% carboxylmethyl cellulose) was administered orally to the appropriate groups for 3 days before HgCl2 administration. Throughout the experimental period, all animals were observed at least once daily for clinical signs of toxicity related to the treatment. All cages were checked in the morning and in the afternoon for dead or moribund animals. The body weights of each animal were recorded daily. Urinalysis and serum biochemical examinations For 24 h before sacrifice on day 3, each animal was kept in a metabolic cage overnight. On day 3, 24 h urine samples were collected and the total urine volume was recorded. The samples were immediately centrifuged at 4C for 10 min at 900 g. A 5 mL sample of urine from each animal was immediately frozen and stored at - 80C. At the time of the assay, the samples were thawed and centrifuged for 10 min at 3000 g. Urinalysis was performed, and various parameters, including lactate dehydrogenase, urinary total protein, BUN, sCr, and glucose levels, were measured using a TBA200FR NEO urine chemistry analyzer (Toshiba, TochigiKen, Japan). The rats were fasted overnight and anesthetized with CO2. Blood was collected from the abdominal aorta and immediately centrifuged at 1500 g for 10 min. The serum samples were immediately frozen and stored at - 80C until the time of analysis. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities, and sCr, BUN, glucose, and uric acid levels were analyzed using a Hitachi 912 Automatic Analyzer (Roche Diagnostics, Sandhofer, Mannheim, Germany). The kidneys were immediately removed and weighed. Histological evaluation The right kidneys were fixed in 10% neutral phosphatebuffered formalin, embedded in paraffin wax, cut into sections of a 5-lm thickness, and stained with hematoxylin and eosin (H&E). Several consecutive 5-lm paraffin sections were deparaffinized three times with xylene for 7 min each. After gradual rehydration in a series of graded alcohol and washing with deionized water, the sections were stained with H&E for 1 min, rinsed with deionized water, and developed in tap water for 5 min. The tissue sections were destained by dipping the slides in acidified ethanol and rinsing in tap water. After washing with deionized water, the sections were stained with eosin for 30 sec, dehydrated, and mounted. Histopathological findings were obtained using a

QUERCETIN AMELIORATES HGCL2-INDUCED KIDNEY INJURY

Zeiss Axiphot light microscope (Zeiss, Carl Zeiss, Oberkochen, Germany). Western blot analysis Preserved urine samples were mixed with an activation/ wash buffer and the resulting slurry with bound proteins was loaded onto a Mini Filter Spin Columns (NORGEN, Thorold, Ontario, Canada). The bound urine proteins were washed to remove any remaining impurities. Finally, the purified total urine proteins were eluted into 150–300 lL of the provided elution buffer. The method employed removes highly concentrated salts and metabolic wastes to allow for the isolation of high quality proteins. The protein concentration in the tissue extract was determined using Bio-Rad Protein Assay Reagent (Bio-Rad, Hercules, CA, USA). Extracted proteins were denatured by boiling in sample buffer (0.5 M Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 0.1% bromophenol blue, and 10% b-mercaptoethanol) at 96C for 5 min. A total of 50 lg protein was separated via 8–15% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) for 90 min at 100 V using running buffer, and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA) for 90 min at 100 V in transfer buffer. The membranes were blocked for 1 h in TNT buffer (10 mM Tris-Cl, pH 7.6, 100 mM NaCl, and 0.5% Tween 20) containing 5% skim milk and incubated overnight at 4C with specific primary antibodies. After washing for 1 h with TNT buffer, the membranes were incubated with HRP-conjugated anti-rabbit, anti-mouse, or anti-goat antibodies (1:10,000) for 1 h at room temperature and subsequently washed for 1 h with TNT buffer. Proteins were detected using ECL Plus western blotting reagents (Amersham Biosciences, Buckinghamshire, United Kingdom). An image analyzer was used to determine relative band intensities. Immunohistochemistry To verify the expression of clusterin and MCP-1 in the kidney, 5 lm thick paraffin sections were exposed to anticlusterin (MBL, Nagoya, Japan) and anti-MCP-1 antibodies. Briefly, after incubating the sections in 3% H2O2 for 10 min, the sections were blocked with 3% goat serum containing 0.3% Triton X-100 and incubated overnight with monoclonal anti-mouse clusterin (1:150) and MCP-1 (1:200) antibodies. After incubation with a biotinylated goat anti-mouse secondary antibody, the sections were treated with HRP-streptavidin (Golden Bridge International, Mukilteo, WA, USA). The sections were analyzed using an Ultravision detection system and the 3,30 -diaminobenzidine (DAB) plus substrate system (Thermo Fisher Scientific, Inc., Pittsburgh, PA, USA) and counterstained with hematoxylin. All immunohistochemical analyses were performed using the Zeiss Axiphot Light Microscope System (Zeiss, Oberkochen, Germany). Analysis of 3-IS Plasma and kidney samples were extracted by protein precipitation, and 3-IS concentrations were determined

3

using high-performance liquid chromatography (HPLC) analysis. A solution for protein precipitation with an added internal standard (internal standard/protein precipitation solution) was prepared by adding 1 mL of a 1.0 mg/mL isatin stock solution to 1 L of acetonitrile/methanol (90:10) to yield a final concentration of 1 lg/mL. HPLC analysis was performed on an Alltech Econosphere C18 column (250 · 4.6 mm, 5 lm) using isocratic elution with an 88:12 solution of water:methanol containing 50 mM KH2PO4, 1.0 mM 1-octylamine, and 50 mM NH4Cl. The pH of the solution was adjusted to 5.5 before the addition of methanol. The analysis of all reaction mixtures was conducted at a flow rate of 1.0 mL/min, and absorbance at 280 nm was measured using a Gilson UV detector. Analysis of Hg concentrations Hg concentrations in urine and in the renal cortex were determined using a DMA 80 Direct Mercury Analyzer (Milestone, Inc., Shelton, CT, USA). To determine Hg concentrations, 1.0 mL of urine was combined with 1.0 mL of concentrated sulfuric acid and 2.0 mL of a saturated potassium permanganate mixture. The samples were heated in a water bath and maintained at 45–50C for 2 h. When that procedure was complete, 20% hydroxylamine hydrochloride was added to reach achromatism. Finally, the volume was brought up to a total of 10 mL by the addition of distilled water. To determine mercury concentrations in the renal cortex, the samples were first digested with 2.0 mL of nitric acid for 12 h, and then 1:1 sulfuric acid and 3.0 mL of saturated potassium permanganate were added. The mixture was heated in boiling water for 1 h and cooled. Finally, hydroxylamine hydrochloride was added dropwise to achieve achromatism and the volume was brought up to a total of 10 mL by the addition of distilled water. One milliliter of the sample was removed from each tube, and 1.0 mL of dehydrated alcohol and 2.0 mL of 20% stannous chloride were added. The samples were analyzed against standards representing a linear range of concentrations using a Direct Mercury Analyzer. Analysis of urinary excreted MT levels Flat-bottom, 96-well polystyrene ELISA plates were coated with rabbit MT (250 ng/mL) in 100 mM sodium bicarbonate buffer (pH 9.6), stored overnight at 4C, decanted, and washed thrice with PBST buffer (phosphate buffered saline [PBS] containing 0.5% Tween-20). The free surface of the well was blocked with PBS containing 1% bovine serum albumin for 1 h. Sheep anti-rat MT antibody was diluted (1:1000) with PBST buffer. Diluted primary antibody was added to 125 lL of sample or MT standard in microtubes and incubated overnight at 4C. An aliquot (200 lL) of the incubated mixture was transferred to the MTcoated plates. After 15 min at room temperature, the plates were decanted, and washed thrice with PBST buffer, and 100 lL of the secondary antibody (1:3000 diluted rabbit anti-sheep IgG) conjugated to alkaline phosphatase (Sigma, St. Louis, MO, USA) was added to each well. The plates

4

SHIN ET AL.

were incubated at room temperature for 1 h, and then 100 lL of 40 mM p-nitrophenol phosphate was added to each well followed by incubation at 37C for 30 min. The absorbance was measured at 405 nm using a Synergy HT ELISA reader (BioTek Instruments, Winooski, VT, USA). TUNEL assay To investigate the induction of apoptosis, TUNEL assay was performed using a TUNEL assay kit (Merck KGaA, Frankfurt, Germany). Kidney tissue sections were deparaffinized in xylene, rehydrated using a graded alcohol series and water, digested with 20 lg/mL of proteinase K for 15 min, washed twice in PBS, and incubated with equilibration buffer for at least 10 sec. dUTP-digoxigenin was then incorporated in the presence of the working-strength TdT provided in the TUNEL assay kit for 1 h in a humidified chamber at 37C. The reaction was terminated, the antidigoxigen conjugate was applied, and the sections were incubated for 30 min in the dark. The sections were washed four times with PBS, and 0.5 lg/mL of propidium iodide (PI) was applied for counterstaining. The sections were mounted under a glass cover slip, and fluorescein was detected by confocal laser-scanning microscopy (Olympus FV10iLIV, Tokyo, Japan). Analysis of reactive oxygen species production ROS production in kidney was measured using a 2,7dichlorofluorescin diacetate (DCFH-DA) assay. Briefly, freshly detached kidney samples were weighed and homogenized with 5 mL of 40 mM Tris/HCl buffer, pH 7.4. The protein concentration was measured using the BioRad protein assay kit. The samples were diluted 1:5000 with 40 mM Tris/HCl buffer and incubated with 10 lmol (final concentration) DCFH-DA for 15 min at 37C. After loading, DCF fluorescence was measured with excitation at 485 nm and emission at 535 nm using a VICTORX2 fluorescence plate reader (PerkinElmer, Santa Clara, CA, USA). ROS levels were expressed as fluorescence intensity normalized to grams of protein. All measurements were performed in triplicate. Statistical analysis Data are expressed as the mean – SD. Statistical analyses were performed using one-way analysis of variance to compare experimental groups followed by Duncan’s multiple range tests when appropriate. For Hg concentrations in the urine and kidney, a nonparametric Kruskal–Wallis test was applied, depending on the properties of data sets, followed by Dunn’s multiple comparison tests. Differences between groups were considered to be statistically significant when P values were < .05. RESULTS Changes in body weight and organ weights The effects of QC on the body and organ weight changes in HgCl2-treated rats are presented in Table 1. Oral ad-

Table 1. Effect of Various Treatments on Body Weight Changes Body wt. (g) Groups Control QC HgCl2 QC + HgCl2

Initial 227.8 – 0.44 221.8 – 0.57 234.7 – 0.45 244.3 – 0.61

Final

Change (%)

235.4 – 0.46 3.7 – 0.33 232.9 – 0.95 5.0 – 0.41 205.4 – 2.25 - 11.98 – 0.85* 233.3 – 1.72 - 4.63 – 0.48

Kidney wt (g) 2.12 – 0.19 2.05 – 0.15 2.08 – 0.09 2.05 – 0.12

Data are expressed as mean – SD of n = 6 rats/group. *Significant difference from the control group at P < .05. HgCl2, mercury chloride; QC, quercetin.

ministration of HgCl2 (20 mg/kg) caused a significant decrease in body weight gain and kidney weight. However, pretreatment with QC significantly attenuated the adverse effects of HgCl2 by restoring body weight and kidney weight to levels near those observed in control animals. Changes in serum biochemistry The serum biochemistry results obtained in this study are presented in Table 2. The mean BUN, sCr, and AST levels were significantly increased in the HgCl2-treated group compared with the control group. These results suggest that severe renal damage occurred in rats treated with 20 mg/kg of HgCl2. In addition, uric acid and glucose levels were significantly lower in the HgCl2-treated group than in the control group. However, ALT levels exhibited no significant differences among the treatment groups. Pretreatment with QC significantly reduced HgCl2-induced BUN, sCR, and AST levels. No changes in BUN, sCr, AST, ALT, uric acid, or glucose levels occurred in rats that received QC alone (Table 2). Changes in urinalysis Urine volume was significantly increased, but urinary pH was significantly lowered in the HgCl2-treated group compared to the control group. However, pretreatment with QC restored urine volume and urinary pH to approximately control levels (Table 3). This result was similar to a previously reported data that intracellular or proximal tubular acidosis was attributed to the appearance of acidic amino acids by HgCl2 exposure.27 Urinary excretion of BUN, and sCr levels, was significantly decreased in the HgCl2-treated group compared with the control group. Creatinine clearance was significantly reduced in the HgCl2 group (62.81 – 9.36 mL/h) compared with the control group (84.37 – 12.54 mL/h). In rats pretreated with QC, the increases in urinary BUN and sCr levels were much higher than those observed in the HgCl2-treated group that did not receive QC, and these values were significantly different from basal values. Urinary glucose levels were significantly increased in rats treated with HgCl2, with a more than 20-fold increase over control values. In contrast, pretreatment with QC significantly decreased urinary glucose concentration compared with treatment with HgCl2 alone (Table 3).

5

QUERCETIN AMELIORATES HGCL2-INDUCED KIDNEY INJURY Table 2. Effects of Quercetin on Serum Biochemical Parameters Against HgCl2-Induced Nephrotoxicity in Sprague-Dawley Rats Groups BUN (mg/dL) Creatinine (mg/dL) AST (IU/L) ALT (IU/L) Uric acid (mg/dL) Glucose (mg/dL)

Control

QC

HgCl2

QC + HgCl2

16.62 – 0.27 0.21 – 0.02 71.53 – 0.95 24.51 – 0.48 4.42 – 0.16 220.25 – 4.23

17.52 – 3.81 0.25 – 0.08 69.83 – 6.24 23.26 – 0.57 4.20 – 0.09 225.93 – 13.57

68.9 – 4.41** 1.15 – 0.13** 119.84 – 5.83** 26.00 – 0.64 2.42 – 0.07** 181.61 – 3.01

32.2 – 1.51# 0.34 – 0.02# 86.51 – 1.81# 22.60 – 0.82 3.20 – 0.13# 197.83 – 3.75

Data are expressed as mean – SD (six animals per group). *Significant difference from the control group at P < .05. **Significant difference from the control group at P < .01. # Significant difference from the HgCl2-treated group at P < .05. ALT, alanine aminotransferase; AST, aspartate aminotransferase; BUN, blood urea nitrogen.

Histological examination The apparent protective effect of QC on HgCl2-induced AKI was observed by histopathological examination. Light microscopy revealed that control kidneys contained normal glomeruli, proximal tubular cells, and vessels. Marked tubular necrosis and medullary vascular congestion were observed in HgCl2-treated rats compared with the control group. However, pretreatment with QC protected against proximal tubular damage and inhibited the observed HgCl2mediated disruption of renal architecture (Fig. 1). In addition, tubular epithelial cell damage was apparently prevented by QC pretreatment. No alterations in tubular cell structure were observed in samples from rats treated with QC alone.

cifically, the protein-based these biomarkers induced by HgCl2 were significantly reduced in rats pretreated with QC. Furthermore, MCP-1 was predominantly expressed in the cytoplasm of renal tubular epithelial cells, but a low level of expression was observed in renal tissue from the control group. MCP-1 expression in renal tissue was significantly increased in the HgCl2 group compared with that of the control group, particularly in the tubular epithelial cells. However, MCP-1 expression in the QC pretreatment group was significantly reduced compared with that of the HgCl2 group (Fig. 2B). Clusterin-positive cells indicate renal tubular cell damage, and a number of clusterin-positive cells were observed in renal sections after HgCl2 administration. In contrast, a significantly lower number of clusterin-positive cells were observed in rats that were pretreated with QC before HgCl2-treatment (Fig. 2B).

Changes in urinary biomarkers associated with AKI Urinary excretion of protein-based biomarkers was measured by western blot analysis. As shown in Figure 2A, HgCl2 (20 mg/kg) exposure significantly increased urinary excretion of protein-based biomarkers, namely clusterin, b2-macroglobin (b2-Mg), KIM-1, MCP-1, NGAL, TIMP-1, and VEGF, compared with control. However, pretreatment with QC markedly reduced urinary excretion of proteinbased biomarkers associated with AKI in HgCl2-exposed rats compared with the HgCl2 alone group (Fig. 2A). Spe-

Changes in 3-IS levels The endogenous 3-IS concentrations in the urine, serum, and kidneys of rats treated with HgCl2 are presented in Figure 3. Urinary excretion of 3-IS was significantly decreased in rats treated with HgCl2 compared with the control group. In contrast, a significant elevation in 3-IS level was observed in serum (3.8-fold) and in the kidney (2.4-fold). However, pretreatment with QC maintained urinary excretion and serum and kidney accumulation of 3-IS at control

Table 3. Effects of Quercetin on Urinary Parameters Against HgCl2-Induced Nephrotoxicity in Sprague-Dawley Rats Groups Urine volume (mL) Urinary pH BUN (g/dL) Creatinine (mg/dL) Creatinine clearance (mL/h) Total protein (mg/dL) Glucose (mg/dL)

Control

QC

HgCl2

QC + HgCl2

5.75 – 0.21 6.93 – 0.20 3499.1 – 67.7 86.75 – 2.36 84.37 – 12.54 154.41 – 6.52 63.48 – 2.53

6.78 – 1.54 7.01 – 0.49 3153.8 – 57.6 83.28 – 3.52 86.59 – 15.21 162.37 – 4.31 68.31 – 11.54

15.24 – 1.31** 5.89 – 0.26** 970.4 – 77.4** 28.91 – 1.76** 62.81 – 9.36* 139.8 – 4.31 1266.6 – 111.5**

8.7 – 0.24## 6.83 – 0.26## 1850 – 61.4## 50.24 – 1.33## 74.59 – 9.85# 202.3 – 11.0# 448.7 – 11.8##

Data are expressed as mean – SD (six animals per group). *Significant difference from the control group at P < .05. **Significant difference from the control group at P < .01. # Significant difference from the HgCl2-treated group at P < .05. ## Significant difference from the HgCl2-treated group at P < .01.

6

SHIN ET AL.

FIG. 1. Histological findings obtained from light microscopy of H&E-stained renal tissue from HgCl2-treated rats. (A) In rats treated with corn oil, the renal tubules exhibited a normal appearance. (B) In rats treated with 250 mg/kg of quercetin (QC), the renal tubules exhibited normal architecture, indicating that QC caused no nephrotoxicity. (C) In rats treated with 20 mg/kg of HgCl2, the tubules exhibited severe and extensive necrosis (arrows), particularly in the tubular region. (D) In rats pretreated with 250 mg/kg of QC for 3 days, followed by treatment with 20 mg/kg of HgCl2; QC effectively protected against kidney damage associated with HgCl2-induced acute kidney injury. H&E, hematoxylin and eosin; HgCl2, mercury chloride.

values. No significant changes in 3-IS levels were observed in the urine, kidney, or serum of rats treated with QC alone.

was observed in HgCl2-treated rats after pretreatment with QC (Fig. 4C). Thus, urinary MT levels were markedly elevated in response to QC in rats treated with HgCl2.

Changes in Hg and MT levels in the kidney or urine Urinary Hg content was measured using urine collected during the 24 h following HgCl2 treatment. A single 20 mg/kg dose of HgCl2 caused a significant accumulation of Hg in the renal cortex (Fig. 4A). The kidney and urinary Hg concentrations in control rats were below the limit of detection of our system. Pretreatment with QC significantly reduced Hg accumulation in the kidney and increased urinary excretion of Hg compared to HgCl2-treated rats without QC pretreatment (Fig. 4B). The urinary MT level in control rats was 0.43 – 0.12 mg/g creatinine, and this level significantly increased in HgCl2-treated rats. No significant change in urinary MT level was observed in rats treated with 250 mg/kg of QC alone. A two-fold increase in urinary MT

TUNEL assay and ROS production in the kidney Apoptosis in the kidney is associated with DNA fragmentation that is measurable by the TUNEL. As shown in Figure 5, TUNEL analysis revealed a significant increase in the number of TUNEL-positive cells in the renal cortex in the HgCl2-treated group compared with the control group. The mean number of TUNEL-positive apoptotic cells in the renal cortex is presented in Figure 5B. QC administration alone did not cause a significant difference in the mean number of TUNEL-positive cells (data not shown). The mean number of apoptotic cells was significantly decreased after pretreatment with QC compared with the HgCl2 group. The data pertaining to ROS production in the kidney are

FIG. 2. Comparison in levels of protein-based biomarkers indicative of HgCl2-induced renal injury. (A) Male Sprague-Dawley rats were treated with 20 mg/kg of HgCl2, 250 mg/kg of QC, or both, and urine, serum, and kidney tissues were collected 24 h after HgCl2 administration. Urinary excretion levels of clusterin, b2-macroglobulin (b2-Mg), KIM-1, MCP-1, NGAL, TIMP-1, and VEGF were measured by western blot analysis. Representative blots from three separate experiments are shown. The presented data are representative of three independent experiments. (B) Immunohistochemical analysis showing expression and immunolocalization of MCP-1 (arrowheads) and clusterin (arrows) in kidneys of rats treated with HgCl2, QC, or QC + HgCl2. Images were acquired at · 200 magnification using an Olympus CH-2 microscope fitted with an Olympus E330 digital camera. KIM-1, kidney injury molecule-1; MCP-1, monocyte chemoattractant protein-1; NGAL, neutrophil gelatinase-associated lipocalin; TIMP-1, tissue inhibitor of metalloproteinases 1; VEGF, vascular endothelial growth factor.

QUERCETIN AMELIORATES HGCL2-INDUCED KIDNEY INJURY

7

FIG. 3. Changes in 3-indoxyl sulfate (3-IS) levels in urine (A), serum (B), and kidney tissue (C). Male Sprague-Dawley rats were treated with 20 mg/kg of HgCl2, 250 mg/kg of QC, or both, and urine, serum, and kidney tissues were collected 24 h after HgCl2 treatment. In this experiment, a single dose of HgCl2 was orally administered, and QC was orally administered to rats for 3 days before HgCl2 treatment. 3-IS levels were measured by HPLC analysis. Statistically significant changes compared with the HgCl2 group are indicated (*P < .05). HPLC, high-performance liquid chromatography.

presented in Figure 5C. Compared with the control group, the generation of ROS was significantly increased in HgCl2-treated rats. In contrast, HgCl2-induced intracellular ROS generation was significantly attenuated (by 27.6%) in rats pretreated with QC compared with rats treated with HgCl2 alone. A previous study suggested that HgCl2 induces ROS generation in the rat kidney.17 These findings indicate that HgCl2 can induce oxidative stress by increasing ROS production in the kidneys of rats, and that pretreatment with QC decreases HgCl2-mediated ROS generation in rat kidneys. DISCUSSION Mercury is a well-known environmental contaminant associated with an increased risk of acute renal failure.6 Mercury compounds have been shown to cause severe renal damage via their ability to induce formation of ROS.13 Many different substances, including chelating agents, have been investigated for their potential protective effects against heavy metal-induced toxicity.28,29 However, most of them have not yet been proven safe for clinical application.

The use of natural compounds for prevention of nephrotoxicity induced by heavy metal exposure has been garnering interest.30 In this study, we evaluated the potential protective effects of QC against mercury-induced nephrotoxicity. Our data demonstrated that QC has a marked protective effect in rats against AKI induced by HgCl2, as indicated by changes in serum or urinary biomarkers. Pretreatment with QC significantly reduced BUN, sCr, and protein-based biomarker levels in rats with mercury-induced renal tubular damage. This protective effect of QC was confirmed by histological examination, which demonstrated that Hg-induced tubular damage was markedly attenuated by pretreatment with QC. Furthermore, urinary levels of protein-based biomarkers were measured to investigate the mechanisms by which QC exerts its protective effects against mercury-induced AKI. Early diagnosis of AKI is made difficult by the delay in appearance of clinical symptoms and elevation of serum levels of BUN or sCr in response to tissue damage. Recently, the U.S. Food and Drug Administration (FDA) approved the use of urinary excreted low molecular weight proteins as biomarkers for diagnosis of acute renal

FIG. 4. Effects of QC on observed changes in mercury and metallothionein levels. (A) Total mercury concentration in the kidney, and urinary excretion (B) of mercury and (C) metallothionein after administration of 20 mg/kg of HgCl2, 250 mg/kg of QC, or both to male SpragueDawley rats. In this experiment, a single dose of HgCl2 was administered orally and QC was administered orally for 3 days before HgCl2 treatment. Each point represents the mean – SD for six rats. Statistically significant changes compared with the HgCl2 group are indicated (*P < .05).

8

SHIN ET AL.

FIG. 5. Effect of QC on apoptotic cell death and reactive oxygen species generation in the kidney. (A) Photomicrographs of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) staining. Control group (Con); HgCl2 group (Hg); quercetin + HgCl2 group (QC + Hg). Green TUNEL-positive apoptotic cells were counted in 10 random fields for each renal region, including the cortex and the medulla, at a magnification of 400 · . (B) Bars represent the mean – SD of six animals. (C) Effect of quercetin on HgCl2-induced ROS production in rat kidneys. Statistically significant changes compared with the HgCl2 group are indicated (*P < .05).

damage.31,32 These biomarkers include b2-Mg, clusterin, cystatin-C, KIM-1, NGAL, osteopontin (OPN), TIMP-1, and VEGF. A previous study indicated that increased levels of most of these protein-based biomarkers were observed in urine following injury to the proximal tubule, where loss of reabsorption generally indicates injury to the tubular epithelium.33 Clusterin, b2-Mg, KIM-1, NGAL, TIMP-1, and VEGF were previously characterized as early and sensitive urinary protein biomarkers that significantly increase after AKI.34–36 In this study, clusterin, KIM-1, MCP-1, NGAL, TIMP-1, and VEGF levels were significantly elevated in urine of rats treated with HgCl2, whereas pretreatment with QC markedly reduced the urinary excretion of these biomarkers in HgCl2-treated rats. Previous study also indicated that these protein-based biomarkers were significantly increased in urine of rats with proximal tubule injury after treatment with nephrotoxicants.32,36 Similarly, elevated levels of TIMP-1 have been observed in urine of kidney injury models and of patients with renal disease as compared to healthy controls.37–39 Furthermore, increased TIMP-1 and TIMP-2 expressions were found in patients with glomerulosclerosis and urinary levels of TIMP-1 were increased in patients with chronic kidney disease.38 VEGF expression and activity could be linked with indicators of renal injury such as glomerular proliferation and creatinine, respectively. In the

previous study, urinary excretion of b2-microglobulin, GST-a, KIM-1, TIMP-1, VEGF, and calbindin, clusterin, cystatin C, NGAL, osteopontin levels were significantly increased in the urine of animals with AKI.40 In particular, urinary concentrations of KIM-1, NAG, NGAL, and VEGF were significantly higher in patients with AKI than in those without AKI.35 The high increases in these protein-based biomarkers following HgCl2 treatment reflect a severe insult to the renal proximal tubular cells. Data from this study indicate the protective role of QC against HgCl2-induced kidney injury. The magnitude of increase in these protein-based urinary biomarkers was larger than that observed for BUN or sCr, and the correlation of these biomarkers with kidney injury was confirmed by immunohistochemical staining of MCP-1 and clusterin. As shown in Figure 4B, the expressions of MCP-1 and clusterin in kidney tissue significantly increased after HgCl2 exposure, particularly in the tubular epithelial cells. However, pretreatment with QC markedly decreased MCP-1 and clusterin expression, and thus we suggest that MCP-1 and clusterin might be highly excreted in the urine in an early stage of kidney damage. We measured 3-IS levels in the urine, serum, and kidney tissue of rats after HgCl2 exposure as metabolite biomarkers for AKI. 3-IS is a dietary protein metabolite and a metabolite of tryptophan. 3-IS accumulates in the blood of animals

QUERCETIN AMELIORATES HGCL2-INDUCED KIDNEY INJURY

with AKI or patients with nephrotoxicity as a consequence of decreased or absent urinary excretion, and is believed to be indicative of renal tubular injury.36,41 After HgCl2 exposure, a marked increase in 3-IS was observed in serum and kidney, whereas urinary excretion of 3-IS were significantly decreased. This finding is particularly relevant to its identification as a biomarker, because the observed decrease in 3-IS preceded any histopathological evidence of kidney damage. On the other hand, pretreatment with QC significantly reduced 3-IS levels in the kidney and serum of rats treated with HgCl2. Thus, the lower levels of 3-IS noted in the urine may be indicative of renal tubular damage. In addition, the higher urinary excretion of Hg and MT that was observed in QC-pretreated animals indicates that QC increases the urinary excretion of Hg bound to MT and inhibits the kidney damage induced by HgCl2 exposure in rats. Previous studies have demonstrated that low doses of inorganic mercury accumulate in the kidney, with approximately half of the dose accumulating in the kidney during the first 24 h following HgCl2 exposure in rats.42,43 Approximately 75% of the Hg accumulates in the tubular epithelial cells after exposure.42,44 Similarly, we found that Hg significantly accumulated in the kidneys of rats, but pretreatment with QC markedly reduced the concentration of Hg in the kidney, and significantly increased urinary excretion of Hg. These results indicate that pretreatment with QC markedly reduced the levels of the free form of Hg in the kidney via metal chelation or upregulation of MT. This finding is consistent with the results of a previous study, which reported that QC is a potent chelator.26 Therefore, we suggest that the protective effect of QC might be mediated by increased urinary excretion of Hg. These results also suggest that MT is an important protective factor against Hg-induced AKI, and that MT may play a pivotal role in the retention of Hg in the kidney. A progressive increase in MT-1 and MT-2 gene expression was previously reported in rats treated with Cd + QC.23 Exogenous MT protects against Cd-induced acute renal toxicity in rats, and this effect is likely mediated by the antioxidant properties of MT.45,46 Therefore, overexpression of MT after Cd + QC treatment can also explain the protective effect of QC against Cd-induced renal injury. Similarly, the kidney damage caused by inorganic mercury is prevented by preinduction of renal MT, because MT traps intracellular mercury in the kidney.43,47 Thus, our data suggest that QC exerts a synergistic effect against Hg by increasing the expression of MT and potentiating the urinary excretion of Hg. The results of our study strongly indicate that accumulation of Hg in the kidney can generate ROS, resulting in oxidative damage. Pretreatment with QC markedly reduced Hg-induced ROS generation and oxidative damage in the kidney. Previous studies have indicated that potent antioxidants have protective effects against heavy metal-induced target organ toxicity.12,48,49 These changes in biochemical parameters correlate with renal histological findings. Both necrotic and apoptotic mechanisms have been implicated in the death of proximal tubule epithelial cells during exposure to Hg. A previous study demonstrated that chronic Cd exposure induces apoptosis and subsequent regeneration of the

9

renal tubular epithelium in rats and dogs.50 Our data suggest that the protective effect of QC is mediated at least in part by its antioxidant properties. Consistent with this finding, Sanhueza et al.51 reported a protective effect of QC against oxygen free radical production after renal ischemia/reperfusion in the rat, and Shoskes52 demonstrated a protective effect of QC and curcumin against postischemic renal injury. A recent study indicated that the number of TUNEL-labeled cells was increased in the renal cortex of rats treated with Cd for long periods.53 In this study, the number of TUNEL-positive cells was higher in the Hg-treated group than in the control group. Pretreatment with QC markedly reduced the number of TUNEL-positive cells in the kidney. Thus, these findings suggest that QC attenuates Hg-induced renal toxicity. Our findings demonstrate that QC prevents mercury toxicity, as indicated by decreased levels of protein-based biomarkers, of 3-IS in the serum and kidney, and of Hg in the kidney. In conclusion, the results of these investigations indicate that QC has a protective effect against HgCl2-induced AKI in rats. The mechanism by which QC exerts this effect is associated with high urinary excretion of a Hg-MT complex, which reduces proximal tubule accumulation of Hg. This was the first study to explore AKI by mercury exposure and its amelioration by QC in the rat kidney. Much attention has been recently focused on protective biochemical functions of naturally occurring substances against renal toxicity of mercury. However, further investigation will be necessary to determine the exact mechanism of QC protection against mercury-induced AKI.

ACKNOWLEDGMENTS We are grateful for financial support from the National Institute of Food and Drug Safety Evaluation (12162KFDA736) of Korea. We thank Profs. Tae Chun Chung and Whan Soo Choi for their helpful suggestions and comments, which enabled us to further improve this article. AUTHOR DISCLOSURE STATEMENT The authors declare that there are no conflicts of interest. REFERENCES 1. Miller S, Pallan S, Gangji AS, Lukic D, Clase CM: Mercuryassociated nephrotic syndrome: A case report and systematic review of the literature. Am J Kidney Dis 2013;62:135–138. 2. Rana SV: Metals and apoptosis: Recent developments. J Trace Elem Med Biol 2008;22:262–284. 3. Ye X, Qian H, Xu P, Zhu L, Longnecker MP, Fu H: Nephrotoxicity, neurotoxicity, and mercury exposure among children with and without dental amalgam fillings. Int J Hyg Environ Health 2009;212:378–386. 4. Agarwal R, Behari JR: Effect of selenium pretreatment in chronic mercury intoxication in rats. Bull Environ Contam Toxicol 2007;79:306–310. 5. Zalups RK: Molecular interactions with mercury in the kidney. Pharmacol Rev 2000;52:113–143. 6. Bridges CC, Zalups RK: Transport of inorganic mercury and methylmercury in target tissues and organs. J Toxicol Environ Health B Crit Rev 2010;13:385–410.

10

SHIN ET AL.

7. Chan HM, Satoh M, Zalups RK, Cherian MG: Exogenous metallothionein and renal toxicity of cadmium and mercury in rats. Toxicology 1992;76:15–26. 8. Bose-O’Reilly S, McCarty KM, Steckling N, Lettmeier B: Mercury exposure and children’s health. Curr Probl Pediatr Adolesc Health Care 2010;40:186–215. 9. Huang YL, Cheng SL, Lin TH: Lipid peroxidation in rats administrated with mercuric chloride. Biol Trace Elem Res 1996; 52:193–206. 10. Mahboob M, Shireen KF, Atkinson A, Khan AT: Lipid peroxidation and antioxidant enzyme activity in different organs of mice exposed to low level of mercury. J Environ Sci Health B 2001;36:687–697. 11. Jan AT, Ali A, Haq Q: Glutathione as an antioxidant in inorganic mercury induced nephrotoxicity. J Postgrad Med 2011;57:72–77. 12. Patrick L: Mercury toxicity and antioxidants: Part 1: Role of glutathione and alpha-lipoic acid in the treatment of mercury toxicity. Altern Med Rev 2002;7:456–471. 13. Valko M, Morris H, Cronin MTD: Metals, toxicity and oxidative stress. Cur Med Chem 2005;12:1161–1208. 14. Hussain S, Atkinson A, Thompson SJ, Khan AT: Accumulation of mercury and its effect on antioxidant enzymes in brain, liver, and kidneys of mice. J Environ Sci Health B 1999;34:645–660. 15. Aslamkhan AG, Han YH, Yang XP, Zalups RK, Pritchard JB: Human renal organic anion transporter 1-dependent uptake and toxicity of mercuric-thiol conjugates in Madin-Darby canine kidney cells. Mol Pharmacol 2003;63:590–596. 16. Berndt WO, Baggett JM, Blacker A, Houser M: Renal glutathione and mercury uptake by kidney. Fundam Appl Toxicol 1985;5:832–839. 17. Agarwal R, Goel SK, Behari JR: Detoxification and antioxidant effects of curcumin in rats experimentally exposed to mercury. J Appl Toxicol 2010;30:457–468. 18. Augusti PR, Conterato GM, Somacal S, Einsfeld L, Ramos AT, Hosomi FY, Grac¸a DL, Emanuelli T: Effect of lycopene on nephrotoxicity induced by mercuric chloride in rats. Basic Clin Pharmacol Toxicol 2007;100:398–402. 19. Cavusoglu K, Oruc E, Yapar K, Yalcin E: Protective effect of lycopene against mercury-induced cytotoxicity in albino mice: Pathological evaluation. J Environ Biol 2009;30:807–814. 20. Noh HJ, Kim CS, Kang JH, Park JY, Choe SY, Hong SM, Yoo H, Park T, Yu R: Quercetin suppresses MIP-1a-induced adipose inflammation by downregulating its receptors CCR1/CCR5 and inhibiting inflammatory signaling. J Med Food 2014;17:550–557. 21. Lin Q, Zhang L, Yang D, Chunjie Z: Contribution of phenolics and essential oils to the antioxidant and antimicrobial properties of Disporopsis pernyi (Hua) Diels. J Med Food 2014;17:714–722. 22. Choi EJ, Bae SC, Yu R, Youn J, Sung MK: Dietary vitamin E and quercetin modulate inflammatory responses of collageninduced arthritis in mice. J Med Food 2009;12:770–775. 23. Morales AI, Vicente-Sa´nchez C, Sandoval JM, Egido J, Mayoral P, Are´valo MA, Ferna´ndez-Tagarro M, Lo´pez-Novoa JM, Pe´rezBarriocanal F: Protective effect of quercetin on experimental chronic cadmium nephrotoxicity in rats is based on its antioxidant properties. Food Chem Toxicol 2006;44:2092–2100. 24. Renugadevi J, Prabu SM: Quercetin protects against oxidative stress-related renal dysfunction by cadmium in rats. Exp Toxicol Pathol 2010;62:471–481. 25. Wang L, Lin SQ, He YL, Liu G, Wang ZY: Protective effects of quercetin on cadmium-induced cytotoxicity in primary cultures of rat proximal tubular cells. Biomed Environ Sci 2013;26:258–267.

26. Vlachodimitropoulou E, Sharp PA, Naftalin RJ: Quercetin-iron chelates are transported via glucose transporters. Free Radic Biol Med 2011;50:934–944. 27. Abdennour C, Khelili K, Boulakoud MS, Nezzal A, Boubsil S, Slimani S: Urinary markers of workers chronically exposed to mercury vapor. Environ Res 2002;89:245–249. 28. Joshi D, Mittal DK, Shukla S, Srivastav AK, Srivastav SK: Nacetyl cysteine and selenium protects mercuric chloride-induced oxidative stress and antioxidant defense system in liver and kidney of rats: A histopathological approach. J Trace Elem Med Biol 2014;28:218–226. 29. Bhadauria M: Combined treatment of HEDTA and propolis prevents aluminum induced toxicity in rats. Food Chem Toxicol 2012;50:2487–2495. 30. Rizwan S, Naqshbandi A, Farooqui Z, Khan AA, Khan F: Protective effect of dietary flaxseed oil on arsenic-induced nephrotoxicity and oxidative damage in rat kidney. Food Chem Toxicol 2014;68:99–107. 31. Hoffmann D, Adler M, Vaidya VS, Rached E, Mulrane L, Gallagher WM, Callanan JJ, Gautier JC, Matheis K, Staedtler F, Dieterle F, Brandenburg A, Sposny A, Hewitt P, Ellinger-Ziegelbauer H, Bonventre JV, Dekant W, Mally A: Performance of novel kidney biomarkers in preclinical toxicity studies. Toxicol Sci 2010;116:8–22. 32. Hoffmann D, Fuchs TC, Henzler T, Matheis KA, Herget T, Dekant W, Hewitt P, Mally A: Evaluation of a urinary kidney biomarker panel in rat models of acute and subchronic nephrotoxicity. Toxicology 2010;277:49–58. 33. Sieber M, Hoffmann D, Adler M, Vaidya VS, Clement M, Bonventre JV, Zidek N, Rached E, Amberg A, Callanan JJ, Dekant W, Mally A: Comparative analysis of novel noninvasive renal biomarkers and metabonomic changes in a rat model of gentamicin nephrotoxicity. Toxicol Sci 2009;109: 336–349. 34. Alge JL, Arthur JM: Biomarkers of AKI: A review of mechanistic relevance and potential therapeutic implications. Clin J Am Soc Nephrol 2015;10:147–155. 35. Brott DA, Adler SH, Arani R, Lovick SC, Pinches M, Furlong ST: Characterization of renal biomarkers for use in clinical trials: Biomarker evaluation in healthy volunteers. Drug Des Devel Ther 2014;8:227–237. 36. Fuchs TC, Hewitt P: Biomarkers for drug-induced renal damage and nephrotoxicity-an overview for applied toxicology. AAPS J 2011;13:615–631. 37. Chromek M, Tullus K, Hertting O, Jaremko G, Khalil A, Li YH, Brauner A: Matrix metalloproteinase-9 and tissue inhibitor of metalloproteinases-1 in acute pyelonephritis and renal scarring. Pediatr Res 2003;53:698–705. 38. Ho¨rstrup JH, Gehrmann M, Schneider B, Plo¨ger A, Froese P, Schirop T, Kampf D, Frei U, Neumann R, Eckardt KU: Elevation of serum and urine levels of TIMP-1 and tenascin in patients with renal disease. Nephrol Dial Transplant 2002;17: 1005–1013. 39. Wasilewska AM, Zoch-Zwierz WM: Urinary levels of matrix metalloproteinases and their tissue inhibitors in nephrotic children. Pediatr Nephrol 2008;23:1795–1802. 40. Fuchs TC, Frick K, Emde B, Czasch S, von Landenberg F, Hewitt P: Evaluation of novel acute urinary rat kidney toxicity biomarker for subacute toxicity studies in preclinical trials. Toxicol Pathol 2012;40:1031–1048.

QUERCETIN AMELIORATES HGCL2-INDUCED KIDNEY INJURY 41. Kirkland JL, Vargas E, Lye M: Indican excretion in the elderly. Postgrad Med J 1983;59:717–719. 42. Zgoda-Pols JR, Chowdhury S, Wirth M, Milburn MV, Alexander DC, Alton KB: Metabolomics analysis reveals elevation of 3indoxyl sulfate in plasma and brain during chemically-induced acute kidney injury in mice: Investigation of nicotinic acid receptor agonists. Toxicol Appl Pharmacol 2011;255:48–56. 43. Zalups RK, Koropatnick J: Temporal changes in metallothionein gene transcription in rat kidney and liver: Relationship to content of mercury and metallothionein protein. J Pharmacol Exp Ther 2000;295:74–82. 44. Zalups RK, Cherian MG: Renal metallothionein metabolism after a reduction of renal mass. II. Effect of zinc pretreatment on the renal toxicity and intrarenal accumulation of inorganic mercury. Toxicology 1992;71:103–117. 45. Zalups RK: Progressive losses of renal mass and the renal and hepatic disposition of administered inorganic mercury. Toxicol Appl Pharmacol 1995;130:121–131. 46. Kara H, Karatas F, Canatan H, Servi K: Effects of exogenous metallothionein on acute cadmium toxicity in rats. Biol Trace Elem Res 2005;104:223–232. 47. Lazo JS, Kondo Y, Dellapiazza D, Michalska AE, Choo KHA, Pitt BR: Enhanced sensitivity to oxidative stress in cultured

48.

49.

50.

51.

52.

53.

11

embryonic cells from transgenic mice deficient in metallothionein I and II genes. J Biol Chem 1995;270:5506–5510. Rao MV, Sharma PS: Protective effect of vitamin E against mercuric chloride reproductive toxicity in male mice. Reprod Toxicol 2001;15:705–712. Sener G, Sehirli AO, Ayanoglu-Du¨lger G: Melatonin protects against mercury(II)-induced oxidative tissue damage in rats. Pharmacol Toxicol 2003;93:290–296. Takaki A, Jimi S, Segawa M, Iwasaki H: Cadmium-induced nephropathy in rats is mediated by expression of senescenceassociated beta-galactosidase and accumulation of mitochondrial DNA deletion. Ann NY Acad Sci 2004;1011:332–338. Sanhueza J, Valdes J, Campos R, Garrido A, Valenzuela A: Changes in the xanthine dehydrogenase/xanthine oxidase ratio in the rat kidney subjected to ischemia-reperfusion stress: Preventive effect of some flavonoids. Res Commun Chem Pathol Pharmacol 1992;78:211–218. Shoskes DA: Effect of bioflavonoids quercetin and curcumin on ischemic renal injury: A new class of renoprotective agents. Transplantation 1998;66:147–152. Aoyagi T, Hayakawa K, Miyaji K, Ishikawa H, Hata M: Cadmium nephrotoxicity and evacuation from the body in a rat modeled subchronic intoxication. Int J Urol 2003;10:332–338.