e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 9 ( 2 0 1 5 ) 125–136
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.elsevier.com/locate/etap
Nephroprotection of plantamajoside in rats treated with cadmium Ha-Young Jung a,1 , Dong-Won Seo a,b,1 , Chung-Oui Hong a , Ji-Yeon Kim a , Sung-Yong Yang a , Kwang-Won Lee a,∗ a
Department of Food Bioscience and Technology, College of Life Science & Biotechnology, Korea University, Seoul 136-713, Republic of Korea b Food Analysis Center, Korea Food Research Institute, 516, Baekhyeon, Bundang, Seongnam, Gyeonggi 463-746, Republic of Korea
a r t i c l e
i n f o
a b s t r a c t
Article history:
Cadmium (Cd), an environmental and industrial pollutant, generates free radicals responsi-
Received 1 July 2014
ble for oxidative stress. Cd can also lead to various renal toxic damage such as the proximal
Received in revised form
tubules and glomerulus dysfunction. Plantamajoside (PMS), a major compound of Plantago
16 November 2014
asiatica (PA), was reported to have the antioxidant effects. In this study, we investigated
Accepted 21 November 2014
the protective effects of PMS on Cd-induced renal damage in the NRK-52E cell and rat
Available online 30 November 2014
kidney tissue. Cd exposure increased the ROS generation, lipid peroxidation, serum bio-
Keywords:
in vivo. The significant reduction in glutathione (GSH)/glutathione disulfide (GSSG) ratio and
chemical values of renal damage, and mRNA and protein expressions of KIM-1 in vitro and Cadmium
activities of antioxidant enzymes were also observed in the rats treated with Cd. PMS signifi-
Kidney injury molecule-1
cantly decreased the ROS generation and lipid peroxidation, thus enhancing GSH/GSSG ratio,
Lipid peroxidation
antioxidant enzyme activities in the cells and rats, and improved histochemical appear-
Nephrotoxicity
ances, indicating that PMS has protective activities against Cd-induced renal injury.
Oxidative stress
© 2014 Elsevier B.V. All rights reserved.
Plantamajoside
1.
Introduction
Recently, with economic growth and industrial development, heavy metal contaminations in foods are of major concerns.
The human body may be exposed to heavy metals through food, water, air or the skin contact (Singh, 2005). Heavy metals become toxic when they are not metabolized, and accumulate in the body. As a heavy metal, cadmium (Cd) is exposed via inhalation or ingestion from the environment in the industry,
Abbreviations: BUN, blood urea nitrogen; BW, body weight; CAT, catalase; Cd, cadmium; DCFDA, 2,7-dichlorofluorescein diacetate; DMEM, Dulbecco’s modified eagle medium; DMSO, dimethyl sulfoxide; PADTNB, 5,5 -dithiobis (2-nitrobenzoic); GAPDH, glyceraldehyde3-phosphate dehydrogenase; GPx, GSH peroxidase; GSH, glutathione; GSSG, glutathione disulfide; KIM-1, kidney injury molecule-1; LDH, lactate dehydrogenase; MTT, 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide; NADPH, -nicotinamide adenine dinucleotide phosphate reduced form; NEM, N-ethylmaleimide; PA, Plantago asiatica; qRT-PCR, quantitative RT-PCR; ROS, reactive oxygen species; TBARs, thiobarbituric and reactive substances; SOD, superoxide dismutase. ∗ Corresponding author at: Department of Food Bioscience & Technology, College of Life Science and Biotechnology, Korea University, 145 Anam-Ro, Sungbuk-Gu, Seoul 136-713, Republic of Korea. Tel.: +82 2 3290 3027; fax: +82 2 953 0737. E-mail address: [email protected] (K.-W. Lee). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.etap.2014.11.012 1382-6689/© 2014 Elsevier B.V. All rights reserved.
126
e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 9 ( 2 0 1 5 ) 125–136
and causes acute and chronic poisonings (Johri et al., 2010). Approximately 7% of the human population develops kidney dysfunction from Cd exposure (Friberg et al., 1986). Cd is easily accumulated, and has a relatively long half-life in the kidney of human body (Bernard, 2008). The kidney is one of the major target organs for Cd, and exposure to Cd causes various renal toxic effects such as the proximal tubules and glomerulus dysfunction (Akesson et al., 2005). It has been suggested that one of mechanisms involved in metal-induced toxicity can be due to the ability of metals to generate reactive oxygen species (ROS) (Ercal et al., 2001). In contrast to redox-active metals, such as iron and copper catalyzing the Fenton reactions producing hydoxy radicals, redox-inactive metal, Cd induces oxidative stress indirectly by diminishing the endogenous antioxidants including glutathione, other sulfhydryl-containing antioxidants and protein-bound thiols (Stohs and Bagchi, 1995) and activities of antioxidant enzymes including superoxide dismutase resulting in ROS accumulation in cells (Hussain et al., 1987). In addition, Cd is a source shown to indirectly produce ROS. Heme oxygenase induction by Cd in cell is shown to contribute to generation of ROS (Müller et al., 1994; Ossola and Tomaro, 1995) also detected that Cd-metallothionein can induce ROS in vitro extracellular condition. Plantago asiatica (PA) has been traditionally used as a folk medicine (Chiang et al., 2003), and has anti-inflammatory and antioxidant activities to protect kidney against ferric nitrilotriacetic-induced renal injury. Bioactive compounds such as PMS, phenylethanoids, plantaginin, and acetoside have been isolated from PA (Hong et al., 2011). To the best of our knowledge, this is the first report on the renal protective effect of PMS on the Cd-induced renal injury. In this study, kidney proximal tubule epithelial (NRK-52E) cells and rats were treated with Cd in the presence of PMS to investigate the potential of the compound to improve Cd-induced nephrotoxicity.
2.
Materials and methods
2.1.
Materials
Plantamajoside was isolated and obtained in our laboratory (Choi et al., 2008). Cadmium chloride (CdCl2 , purity ≥99.9%), 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), 2,7-dichlorofluorescein diacetate (DCFDA), GSH, Nethylmaleimide (NEM), 5,5 -dithiobis (2-nitrobenzoic) (DTNB), 2-vinylpyridine, triethanolamine, -nicotinamide adenine dinucleotide phosphate reduced form (NADPH), and glutathione reductase were purchased from Sigma–Aldrich (St. Louis, MO, USA). Dulbecco’s modified eagle medium (DMEM) cell culture media, bovine serum albumin, and BCA protein assay kit were purchased from Thermo scientific (IL, USA), and trizol, penicillin streptomycin and trypsin-EDTA were purchased from Life technologies (Grand Island, NY, USA). Penicillin streptomycin and trypsin-EDTA were purchased from GIBCO (Grand Island, NY, USA). Antibody against kidney injury molecule-1 (KIM-1) was purchased from Abcam (Cambridge, MA, USA), and horseradish
Fig. 1 – Chemical structure of plantamajoside (PMS) (C29 H38 O16 ).
peroxidase-conjugated secondary antibody was purchased from Millipore (Billerica, MA, USA). Chemical structure of PMS is shown in Fig. 1.
2.2.
Cell culture and viability assay
Rat kidney proximal tubule epithelial (NRK-52E) cells were obtained from ATCC (Manassas, VA, USA) and cultured in DMEM medium containing 10% (v/v) FBS, 4 mM l-glutamine, 1.5 g/L sodium bicarbonate, and 4.5 g/L of glucose. The cells were maintained at 37 ◦ C in a 5% CO2 humidified atmosphere. Cell viability was measured by the MTT assay (Mosmann, 1983). NRK-52E cells were seeded at a density of 5 × 104 cells/well in 24 well plates and cultured in DMEM medium for 24 h. After confirming cell attachment, because the treatment of 5 M CdCl2 gave 50% of cell viability on MTT assay, cells were treated with 5 M CdCl2 and 3.125, 6.25, 12.5, 25, 50, and 100 M PMS in serum free medium for 24 h. Then, the medium was changed to 5 mg/mL MTT reagent in DMEM. After incubation for 3 h at 37 ◦ C, the medium was removed, and 200 L of DMSO was added to each well to dissolve formazan crystals. The absorbance was measured at 540 nm wavelength using a multiplate spectrometer. The results were determined as percentages of the control.
2.3. assay
Reactive oxygen species (ROS) scavenging activity
NRK-52E cells were seeded at a density of 5 × 104 cell/well in 96 well plates for 24 h. After cell detachment, the cells were incubated with 100 M of DCF-DA in DMEM medium for 30 min. After washing with phosphate buffer saline (PBS) at pH 7.4, the cells were treated with Cd (5 M) and PMS (25, 50 and 100 M). After incubation for 1 h, the fluorescence intensity was measured using a spectrofluorometer.
2.4.
Animals and administration
Male Sprague–Dawley rats (7-week-old, about 250 g of body weight (BW)) were purchased from Samtako Bio Korea Co. (Gyeonggi, Korea) and were housed in cages with free access to standard diet (Samyang Feed Co. Ltd., Incheon, Korea) and tap water. The experimental animals were treated in compliance with the instructions of the Committee for Ethical Usage of Experimental Animals of Korea University. This experimental protocol was reviewed, and approved by the Korea University Animal Care Committee (No. KUIACUC-20130704-1).
e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 9 ( 2 0 1 5 ) 125–136
Thirty-two rats were divided randomly into four groups consisting of eight rats in each group. The group I was the control without treatment. In our preliminary experiment, some rats administered with 10 mg/kg BW of cadmium chloride showed bloody nose secretions after 3-day treatment. Based on reports on Cd-induced nephrotoxicity and oxidative stress using rat animals (Renugadevi and Milton Prabu, 2010; Renugadevi and Prabu, 2009), they administered rats with 5 mg/kg BW of cadmium chloride for 4 weeks. Thus, the animals in the group II were orally intubated with 5 mg/kg BW of Cd for 4 weeks. Animals in the group III and IV were orally intubated with 5 mg/kg BW of Cd and two different levels of PMS (10 mg/kg or 40 mg/kg BW) for 4 weeks. PMS was given 3 h prior to the administration of Cd. After 18 h from the last treatment, the rats were sacrificed, and blood samples were collected from the vena cava for measuring serum creatinine blood urea nitrogen (BUN) and lactate dehydrogenase (LDH) activity. After collecting the bloods, the kidneys were removed and rinsed with PBS and stored at −80 ◦ C prior to the biochemical analysis.
2.5.
was determined by the BCA protein assay using bovine serum albumin (BSA) as the standard.
2.7. Measurement of catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx) activity Frozen kidney tissues (0.5 g) were homogenized in 1 mL of ice-cold 0.1 M potassium phosphate buffer. The homogenate was centrifuged at 12,000 × g for 30 min. Then, the supernatants were collected in a fresh 1.5 mL tube for determining antioxidant enzyme activities. The CAT activity was measured colorimetrically by the modification of a previously reported (Sinha, 1972) using dichromate-acetic acid reagent. The superoxide dismutase (SOD) activity was determined by the modified xanthine/xanthine oxidase method (McCord and Fridovich, 1969). GSH peroxidase (GPx) activity was assayed using the Paglia and Valentine method (Paglia and Valentine, 1967) with a slight modification. The rate of oxidation of NADPH caused by the addition of GSH reductase was monitored.
Lipid peroxidation assay
Lipid peroxidation was estimated by measuring thiobarbituric and reactive substances (TBARs). Intracellular lipid peroxidation was determined by the modified thiobarbituric acid (TBA) fluorometric method (Lee et al., 2000). For the TBARs assay, the solutions were homogenized on ice using homogenizing buffer of 10% volume of kidney weight. TBA reagent (1 mL) was added to 100 L of homogenized solutions and heated at 90 ◦ C in water bath for 20 min. Fluorescence intensity was measured at the excitation wavelength of 530 nm and emission of 590 nm using a spectrofluorometer (VICTOR2TM , Perkin-Elmer, USA). Protein concentration was determined by the BCA protein assay using bovine serum albumin (BSA) as the standard.
2.6.
127
GSH contents analysis
The GSH contents of NRK-52E cells and rat kidney tissues were measured by the modification of previously reported method (Moron et al., 1979; Smith et al., 1988; Zhu et al., 1991). GSH was determined by the reaction with DTNB to afford a compound absorbing at 412 nm. The reaction mixture containing 0.1 M potassium phosphate buffer and 0.1 M DTNB in DMSO was added to the sample. The absorbance at 412 nm was measured using a spectrophotometer. Glutathione disulfide (GSSG) was measured using a recycling reaction of GSH with DTNB in the presence of excess GSH reductase. 100 L of samples was derivatized with 2 L of 2-vinylpyridine and 6 L of triethanolamine in the pH range 6–7. After reaction for 60 min, 190 L of the reaction buffer (0.3 mM NADPH in potassium phosphate buffer (pH 7.4) and 6 mM DTNB in DMSO) and 10 L of derivatized sample were mixed and incubated at 30 ◦ C for 15 min. Then, 20 L of GSH reductase was added to the mixture. GSH reductase was kept on ice during the preparation of the reaction buffer. Change in the absorption was measured using a multiplate reader at 412 nm. Protein concentration
2.8. tissue
Quantifications of Cd and total thiols in rat kidney
All the solvents which are used for washing of all the glasses and preparation of all the test solutions were tertiary distilled water which passed Milli-Q system (Millipore; MA, USA). Standard stock solution containing 10 mg/kg of cadmium was purchased from Perkin-Elmer (New York, USA). These were used to prepare calibration standards. Ultrapure grade carrier (argon [Ar], 99.9995% pure) was supplied by Shin-Yang (Korea). Samples were digested by Ethos1 microwave digestion system (Milestone, Millan, Italy). About 0.2 g of kidney tissue sample, 7 mL of nitric acid and 2 mL of hydrogen peroxide were added to vessel and performed microwave digestion. One randomly selected vessel was filled with reagents only and taken through the entire procedure as a blank. After cooling at room temperature, sample solutions were quantitatively transferred into 25 mL flasks. Then, the digested samples were filled with ultrapure water to the final volume before analysis by ICP-MS. Cadmium analysis was performed using NEXION 300D ICP-MS system (Perkin-Elmer, USA). The isotopes 112 Cd was selected as analytical mass in ICP-MS standard mode. Specific isotope was monitored regarding to the sensibility of the element and/or possible isobaric and polyatomic interferences. Torch position, ion lenses and gases yield were optimized daily with the tuning solution (1 g/kg) to implement a short-term stability test of the instrument, to maximize ion signals and to minimize interference effects from polyatomic ions and doubly charged ions. Total thiol groups of the rat kidney tissue were measured using modified DTNB reagent method (Ellman, 1959). Briefly, a working solution was prepared by mixing 0.5 mL of DTNB solution (50 mM NaAc and 2 mM DTNB), 1 mL of 1 M Tris–HCl, pH 8.0 and 8.4 mL of distilled water. This 190 L of working solution and 10 L of sample were mixed, incubated 5 min at 25 ◦ C, and its absorbance was read at 412 nm using spectrophotometer.
128
2.9.
e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 9 ( 2 0 1 5 ) 125–136
Pathological histology in kidney tissue
After removal of the rat kidney, the tissues were fixed in 10% buffered formaldehyde. The formaldehyde-fixed tissue samples were embedded in parafilm, and 3–4-m sections were stained with periodic acid-Schiff (PAS). Tubular injury was observed at 200× magnification for each sample from all the group. Tubular necrosis was evaluated on an arbitrary semiquantitative assessment as follows: no cell necrosis; mild usually single-cell necrosis in sparse tubules; moderate more than one cell involved in sparse tubules; and massive necrosis in every power field.
2.10.
RNA isolation, RT-PCR and qRT-PCR analysis
Total RNA was isolated using trizol reagent according to the manufacture’s instruction. Total RNA (3 g) and the First Strand cDNA synthesis kit (Legene Biosciences Inc., USA) were used to synthesize cDNA. PCR primers of kidney injury molecule-1 (KIM-1) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used. Sequences of RT-PCR primers were as follows: KIM-1 sense 5 -CGGTGCCTGTGAGTAAATAGATand antisense 5 -CTGGCCATGACACAAATAAGAC-3 ; 3 GAPDH sense 5 -GCCATCAACGACCCCTTCATT-3 and antisense 5 -CGCCTGCTTCACCACCTTCTT-3 . Sequences of quantitative RT-PCR (qRT-PCR) primers used are as follows: KIM-1 sense 5 -TGGCACTGTGACATCCTCAGA-3 and antisense 5 -GCAACGGACATGCCAACATA-3 ; GAPDH sense 5 -ACTGCCAGCCTCGTCTCATAG-3 and antisense 5 -CCTTGACTGTGCCGTTGAACT-3 . RT-PCR was conducted according to the manufacturer’s protocol (DreamTaq DNA polymerase, Thermo scientific, Pittsburgh, PA, USA), and the optimal PCR results were secured at an annealing temperature of 60.5 ◦ C with 30 cycles. PCR products were separated electrophoretically on 1.5% agarose gel stained with red-safe nucleic acid staining solution (INTRON Biotechnology, Korea). Quantitative data normalized to GAPDH were obtained from a densitometer and analyzed using the Quantity One software (Bio-Rad, USA). Validation of the RT-PCR results was determined by quantitative real time-PCR (qRT-PCR). Amplification and detection of cDNA were performed with SYBR® Green supermix. Quantitative real-time PCR data were expressed as a threshold cycle (Ct) value by iCycler iQ® real-time PCR detection system (BioRad, Korea), and GAPDH was used to normalize the mRNA expression level.
2.11.
Western-blot analysis
Samples containing 20 g of protein from each lysate were dissolved in the loading buffer, and then denatured by boiling for 5 min. Protein lysates were separated on 10% SDSpolyacrylamide gel and transferred to polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA). After blocking with 5% skim milk in TBST (0.25 M Trisbase pH 7.4, 1.5 M NaCl, 1% Tween-20) at room temperature for 1 h, membranes were incubated with primary anti-KIM-1 antibody (1:1000) in skim milk overnight at 4 ◦ C. After washing in TBST for 30 min, the membranes were incubated with suitable secondary antibody for 45 min at room temperature. Then, protein bands
Fig. 2 – Effects of cadmium (Cd) and PMS on cell viability in NRK-52E cells. The results are expressed as mean ± SD. Different letters indicate significant differences at the p < 0.05 by the Duncan’s multiple range tests.
were detected using ECL solution (Abclon, Seoul, Korea). Protein expression levels were determined using a developer, and the intensity was calculated using the image analysis software.
2.12.
Statistical analysis
All the data were expressed as mean ± standard deviation, and the analysis was performed using the SAS software version 9.3 (SAS Institute Inc., NC, USA). Statistical analysis was conducted by the Duncan’s multiple range tests.
3.
Results
3.1.
Effect of PMS on Cd-induced cytotoxicity
Cell viability of NRK-52E cells was measured to evaluate the protective effect of PMS against Cd cytotoxicity using the MTT assay. Cells were treated with 5 M of CdCl2 and 3.1, 6.3, 12.5, 25, 50 and 100 M PMS. The cell viability of PMS-treated group was more than 90%, indicating that PMS has no toxic effect in the range 3.1–100 M (data not shown). The effects of various concentrations of PMS on Cd cytotoxicity are shown in Fig. 2. The treatment of cells with 5 M Cd only reduced cell viability to ∼57% after 24 h incubation. The presence of PMS at the concentrations of 3.1–25 M did not show any protective effects on Cd cytotoxicity; however, the treatment with ≥50 M PMS significantly decreased the Cd induced cytotoxicity (p < 0.05).
3.2. Effect of PMS on ROS generation induced by Cd in NRK-52E cells DCF-DA, a sensitive fluorescent probe, is useful for the detection of the ROS generation in cells (Bartosz, 2006). The intracellular generation of the ROS was measured to investigate the antioxidant effect of PMS on Cd-induced oxidative damage in cells. As seen in Fig. 3, DCF fluorescence intensity
e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 9 ( 2 0 1 5 ) 125–136
129
Fig. 3 – Effects of PMS on ROS generation induced by Cd in NRK-52E cells. The results are expressed as mean ± SD. Different letters indicate significant differences at the p < 0.05 by the Duncan’s multiple range tests.
increased for the cells treated with Cd compared to the untreated cells. However, ROS levels significantly decreased in all plantamajoside co-treatment groups (p < 0.05).
3.3.
Effect of PMS on lipid peroxidation induced by Cd
The evaluation of the formation of TBARs provides an estimation of the extent of lipid oxidation originated by the treatment with Cd. The TBAR values (expressed as M MDA mg−1 of protein) were measured to evaluate the effect of PMS on the lipid peroxidation induced by Cd in the NRK-52E cells and rat kidney tissue. The treatment of NRK-52E cells with Cd significantly increased the MDA level compared to the control group (Fig. 4A); however, Cd-treated cells in the presence of PMS dose dependently showed decreased level of MDA (p < 0.05). The treatment with 50 and 100 M of PMS reduced MDA levels compared to that of untreated control cells. Moreover, the lowering effect of PMS on the lipid peroxidation was evaluated in vivo. As seen in Fig. 4B, compared to the control group, the administration of Cd in rats showed a significantly increased concentration of MDA in the kidney tissues. However, the MDA level decreased significantly by the treatment with PMS. Therefore, we demonstrated that Cd-induced oxidative stress in kidney was effectively prevented by the treatment with PMS.
3.4.
Effects of Cd and PMS on glutathione (GSH) levels
The kidney NRK-52 cells treated with 5 M Cd showed a decrease in GSH level, an increase in GSSG level, and decreasing GSH/GSSG ratio (Fig. 5A–C). Significant (p < 0.05) recovery was noted in the groups treated with PMS supplementation. These antioxidant effects of PMS on kidney oxidative stress were also confirmed in post Cd exposed rats (Fig. 5D–F).
Fig. 4 – Effect of PMS on the lipid peroxidation induced by Cd in NRK-52E cells (A) and rat kidney tissue (B). The results are expressed as mean ± SD. Different letters indicate significant differences at the p < 0.05 by the Duncan’s multiple range tests.
3.5. Measurement of catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx) activity As seen in Fig. 6, all the CAT, SOD and GPx activities showed significant (p < 0.05) decrease in kidney tissues upon Cd exposure. High level of PMS (40 mg/kg BW) supplementation provided recovery, whereas low level of PMS (10 mg/kg BW) was marginally effective (non-significant) (p < 0.05).
3.6. Serum biochemical values in rats treated with Cd and PMS The blood samples from the rats treated with Cd and PMS supplementation were collected to determine serum creatinine, serum blood urea nitrogen (BUN) and lactate dehydrogenase (LDH). Cd-treated group showed significantly
130
e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 9 ( 2 0 1 5 ) 125–136
Fig. 5 – Effects of Cd and PMS on the glutathione levels in NRK-52E cells (A-C) and rat kidney tissue (D-F). (A, D) GSH, (B, E) GSSG, and (C, F) GSH/GSSG ratio. The results are expressed as mean ± SD. Different letters indicate significant differences at the p < 0.05 by the Duncan’s multiple range tests.
(p < 0.05) increased serum creatinine, BUN, and LDH in comparison to the control group (Table 1). In contrast, PMS supplementation significantly (p < 0.05) recovered serum biochemical values to the levels of control values.
3.7.
Levels of Cd and total thiols in rat kidney tissue
Cd concentrations in rat kidney tissues were measured by ICP-MS system. The standard curve linearity for ICP-MS
131
e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 9 ( 2 0 1 5 ) 125–136
Table 1 – Effects of plantamajoside (PMS) on serum creatinine, BUN and LDH in the rats treated with cadmium (Cd). Treatment Control Cd (5 mg/kg BW) PMS (10 mg/kg BW) PMS (40 mg/kg BW)
Creatinine (mg/dL) 0.42 0.48 0.44 0.42
± ± ± ±
BUN (mg/dL)
0.03a 0.04b 0.02a 0.02a
13.5 16.3 14.9 13.8
± ± ± ±
0.54a 0.76b 0.63c 1.38ac
LDH (U/L) 1783.0 2018.4 1751.0 1061.2
± ± ± ±
117.3a 112.4b 177.6a 60.8c
BUN: blood urea nitrogen; LDH: lactate dehydrogenase; BW: body weight. Values are expressed as mean ± SD of five independent experiments. Means with different superscript letters within a column were determined to be significantly different at p < 0.05 in the same column when applied to the Duncan’s multiple range tests.
method was determined at seven points ranging from 0.001 to 0.20 mg/L of CdCl2 solution (R2 > 0.9999) (data not shown). As seen in Fig. 7A, Cd was barely detectable in control group. And Cd level (11.7 ± 0.7 g of Cd/g tissue) showed significant (p < 0.05) increase in kidney tissue upon Cd exposure. High level (40 mg/kg BW) and low one (10 mg/kg BW) of PMS supplementations showed significantly reduced Cd levels to 8.3 ± 0.5 and 6.8 ± 1.1, respectively (p < 0.05). In addition, we measured the total thiols in kidney tissue. The control group showed 1.46 ± 0.12 mM thiols/g protein in kidney tissue (Fig. 7B). However, Cd-treated group significantly reduced the total thiols to 0.38 ± 0.13 mM thiols/g protein (p < 0.05). And low level of PMS supplementation recovered the thiol level to normal (1.44 ± 0.12), and even high level of PMS showed higher total thiols (1.79 ± 0.19) than control (p < 0.05).
3.8.
Histology of the renal tubule
Fig. 8A shows the histological results. The kidney from the untreated control group showed normal appearance. The Cdtreated rats had a massive degree of renal tubular necrosis (Fig. 8B). The treatment with a low level of PMS (10 mg/kg BW) mitigated the severity of the Cd-induced renal necrosis (Fig. 8C), and the rats treated with high level of PMS (40 mg/kg BW) had only slight degree of tubular necrosis (Fig. 8D).
3.9. Effects of PMS on the expression of kidney injury molecule-1 (KIM-1) mRNA and protein in Cd-treated kidney in vitro and in vivo To determinate the effect of PMS pretreatment on KIM-1 mRNA expression in the NRK-52E cells and rat kidney tissue treated with Cd, RT-PCR and quantitative real-time PCR were used to analyze mRNA levels. As seen in Fig. 9A and B, KIM-1 mRNA level was significantly (p < 0.05) upregulated in the cells treated 5 M Cd alone. However, KIM-1 mRNA level was significantly (p < 0.05) reduced in the groups supplemented with PMS. Moreover, the rats administered with 5 mg/kg BW of Cd also showed significantly (p < 0.05) enhanced KIM-1 mRNA level compared to the control rats (Fig. 9C and D). However, the mRNA expression of KIM-1 significantly (p < 0.05) decreased in the rats pretreated with various concentrations of PMS. In concordance with the kidney KIM-1 mRNA levels, which increased after Cd treatment, KIM-1 protein levels were significantly (p < 0.05) increased in the NRK-52E cells and kidney tissues (Fig. 10A and B). The exposure to Cd
upregulated KIM-1 protein in the NRK-52E cells is shown in Fig. 10A. In 25 and 50 M of PMS pretreatment groups, no inhibitory activities of PMS on KIM-1 expression by Cd was observed. However, the pretreatment with 100 M of PMS reduced KIM-1protein expression. As seen in Fig. 10B, Cd exposure upregulated the KIM-1 protein expression in the rat kidney tissue; however, the pretreatment with PMS significantly reduced KIM-1 protein expression level in the control group.
4.
Discussion
Cd, a toxic metal, occurring in the environment causes negative health effects (Choong et al., 2014), and is classified as a class I carcinogen by The World Health Organization’s International Agency for Research on Cancer (Cancer et al., 1993). Long half-life of Cd exerts a continuously increasing environmental burden resulting in entry into the food chain (Choong et al., 2014). Because of a relatively long half-life of Cd (10–30 years in human), it affects the major organs such as the kidney and liver accounting for onethird of the total Cd in the body (Satarug and Moore, 2004). Cd-induced renal damage causes the proximal tubular reabsorptive dysfunction (Jarup and Akesson, 2009). Therefore, nephrotoxic injury is indicated to be an underlying factor for establishing important biomarkers for Cd toxicity. The oral exposure of Cd leads to its absorption from gastrointestinal tract up to 5–10%, and its systemic accumulation into the bloodstream makes Cd to be delivered to liver where it induces the synthesis of metallothionein, binding Cd and buffering its toxic effects in the liver cells (Prozialeck and Edwards, 2012). Due to normal turnover or a result of Cd injury the Cd-metallothionein complex from the liver cells is released into blood stream (Klaassen et al., 2009), and is filtered at the glomerulus and taken up by the epithelial cells of the proximal tubule. Additionally, proximal tubule cells can uptake lower molecular-weight cadmium-thiol conjugates such as GSH (Bridges and Zalups, 2005). However, the interaction between Cd and proteins/low-molecular thiols depending upon various conditions could not be enough, and free Cd dissociating from the complex, could enter the cell. It is generally accepted that Cd form not Cd-metallothionein injures proximal tubule epithelial cells (Klaassen et al., 2009). Another concern regarding Cd toxicity is the concentration of Cd that is typically achieved in vivo. The kidney
132
e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 9 ( 2 0 1 5 ) 125–136
Fig. 7 – Levels of Cd and total thiols in the rat kidney tissue. (A) Cd, and (B) total thiols. The results are expressed as mean ± SD. Different letters indicate significant differences at the p < 0.05 by the Duncan’s multiple range tests.
Fig. 6 – Effects of Cd and PMS on the activities of CAT, superoxide dismutase (SOD), and glutathione peroxidase (GPx) in the rat kidney tissue. (A) CAT, (B) SOD, and (C) GPx. The results are expressed as mean ± SD. Different letters indicate significant differences at the p < 0.05 by the Duncan’s multiple range tests.
tissue level of Cd is associated with the onset of renal injury is reported to be 150–200 g/g tissue (Prozialeck and Edwards, 2012). In our study, oral administration of Cd (5 mg/kg BW) for 4 weeks increased Cd concentration (11.7 ± 0.7 g of Cd/g tissue) in kidney tissue. Also in vitro study, the kidney cells were exposed to 5 M of Cd for 24 h. This concentration is much higher than the Cd level in blood (5–10 nM), but it is well below the millimolar concentrations of Cd that are attained in renal cortical tissue in vivo. The localized Cd level in the immediate vicinity could surpass 1 mM (Prozialeck and Edwards, 2012). Cd is not a redox-active and essential element, and is unable to directly produce ROS (Cuypers et al., 2010). However, Cd is clearly in capable of inducing oxidative stress, this mechanism plays a role in Cd-induced kidney injury (Gobe and Crane, 2010). The Cd-specific binding to sulfhydryl group of GSH depletes the major antioxidant thiol, thus disturbing the redox equilibrium toward an oxidative condition (Lopez et al., 2006). GSH contributes to cellular antioxidant defense system against oxidative stress (Cnubben et al., 2001),
e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 9 ( 2 0 1 5 ) 125–136
133
Fig. 8 – Representative photographs of sections of renal tubule under light microscope of a rat treated with saline (A), Cd (5 mg/kg BW) (B), Cd + PMS (10 mg/kg BW) and (C), Cd + PMS (40 mg/kg BW) (D). Note the normal structure in (A) and the massive necrosis in (B), the relatively moderate necrosis in necrosis in (C) and mild necrosis in (D). PAS 200X.
scavenges ROS directly and is oxidized to form GSSG under the catalysis of GSH peroxidation. GSH/GSSG, a major redox couple, plays an important role in antioxidant defense (Wu et al., 2004). Moreover, iron in various proteins is replaced by Cd resulting in an increase in iron free cells, enhancing Fenton reaction, thus increasing ROS generation in mitochondria (Wang et al., 2004) and NADPH oxidase dependent ROS (Rockwell et al., 2004). Also, Cd interferes with antioxidant defense systems, for instance, an alteration in the activity of antioxidant enzymes (Chandran et al., 2005; Waisberg et al., 2003). Cd exposure inactivates most of the antioxidant enzymes, relating to the binding of Cd directly to the active sites of enzymes containing sulfhydryl groups or displacement of metal cofactors from the active sites (Casalino et al., 2000) resulting in the diminished activities of antioxidant enzymes. This indicates destruction of antioxidant defense system, and increase in ROS because of the exposure to Cd. This Cd-induced oxidative stress causes the kidney proximal tubular injury (Cuypers et al., 2010). Through these indirect mechanisms, Cd can significantly intensify the actions of normal oxidative processes within the cell, which results in oxidative stress. Kidney is highly susceptible to toxic injury because of high blood perfusion and filtration of the blood plasma at a high rate (Mohamed et al., 2003). The levels of serum creatinine and serum BUN are generally used as the biomarkers of renal function (Urbschat et al., 2011). Therefore, use of antioxidants is considered as a meaningful approach for the suppression of Cd-induced kidney toxicity. P. asiatica extract provides renal protective effects against oxidative stress (Hong et al., 2011).
In our experiment, kidney cells and animals were pretreated with PMS prior to Cd intoxication because PMS, a caffeic acid derivatives has been shown to have an antioxidant providing scavenging activity of ROS and protective action on low-density lipoprotein against oxidative damage (Cardinali et al., 2012). We thought that post-treatment schedule for PMS in Cd-induced oxidative stress was not effective. The significant enhancement in the GSH/GSSG ratio by the treatment with PMS in the presence of Cd indicates its potential role as an antioxidant. The decreased levels of lipid peroxidation in PMS-supplemented group indicate the inhibitory effect of PMS against Cd-induced oxidative stress. Meanwhile, histochemical examination revealed that Cd caused the degeneration of tubules in renal ultrastucture indicating marked tubular damage. It has been reported that proximal tubular epithelial cells are susceptible to oxidative stress damage (Wilmes et al., 2011). In our study, rat kidney proximal tubule epithelial (NRK-52E) cells were used for assessing Cd intoxication. The exposure to Cd results in the dysfunction of the kidney proximal tubule. KIM-1 is a sensitive and early biomarker of Cd-induced nephrotoxicity, and is a type I transmembrane protein that is less detectable in normal kidney tissue (Prozialeck et al., 2007). This protein it is detected at high levels in the proximal tubule epithelial cells after toxic injury. In the present in vitro and in vivo study also demonstrated that the treatment with Cd activates KIM-1 mRNA and protein expression. However, we observed that PMS can downregulate KIM-1 expression, indicating that PMS had a protective effect on Cd-induced nephrotoxicity.
134
e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 9 ( 2 0 1 5 ) 125–136
Fig. 9 – Effects of Cd and PMS on the mRNA expression of KIM-1 in NRK-52E cells (A, B) and rat kidney tissue (C, D). (A, C) KIM-1 mRNA expression level by RT-PCR. (B, D) Real time PCR was performed, and the relative KIM-1 mRNA expression is normalized by GAPDH. The fold induction is expressed as the relative mRNA expression of the treatment groups divided by the control group. The results are expressed as mean ± SD. Different letters indicate significant differences at the p < 0.05 by the Duncan’s multiple range tests.
Fig. 10 – Effects of Cd and PMS on the KIM-1 protein expression in NRK-52E cells (A) and rat kidney tissue (B). Intensity of each band was quantified by densitometry. KIM-1 protein expression is normalized by GAPDH. The fold induction is expressed as the relative protein expression of the treatment groups divided by the control group. The results are expressed as mean ± SD. Different letters indicate significant differences at the p < 0.05 by the Duncan’s multiple range tests.
e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 9 ( 2 0 1 5 ) 125–136
5.
Conclusion
In this study, we confirmed that PMS exhibited protective effects against Cd-induced kidney injury as confirmed by the suppression of ROS generation, enhancement of antioxidant status, improved histochemical appearances, and inhibition of KIM-1 activation in the cells and rat kidney tissues. These results suggest that PMS is effective in inhibiting Cd-induced kidney injury.
Conflict of interest The authors declare that there are no conflicts of interest.
Transparency document The Transparency document associated with this article can be found in the online version.
Acknowledgement This research was a part of the project (Grant No. R1111343) titled ‘Material development and commercialization of Korean seaweed alga (Capsosiphon fulvescens) conferring whitening function, anti-diabetes, and inhibitory effects on diabetic complications’, funded by the Ministry of Oceans and Fisheries, Korea.
references
Akesson, A., Lundh, T., Vahter, M., Bjellerup, P., Lidfeldt, J., Nerbrand, C., Samsioe, G., Stromberg, U., Skerfving, S., 2005. Tubular and glomerular kidney effects in Swedish women with low environmental cadmium exposure. Environ. Health Perspect. 113, 1627–1631. Bartosz, G., 2006. Use of spectroscopic probes for detection of reactive oxygen species. Clin. Chim. Acta 368, 53–76. Bernard, A., 2008. Cadmium & its adverse effects on human health. Indian J. Med. Res. 128, 557–564. Bridges, C.C., Zalups, R.K., 2005. Molecular and ionic mimicry and the transport of toxic metals. Toxicol. Appl. Pharmacol. 204, 274–308. Cancer, I.A.f.R.o., Organization, W.H., Humans, I.W.G.o.t.E.o.C.R.t., 1993. Beryllium, Cadmium, Mercury, and Exposures in the Glass Manufacturing Industry. IARC. Cardinali, A., Pati, S., Minervini, F., D’Antuono, I., Linsalata, V., Lattanzio, V., 2012. Verbascoside, isoverbascoside, and their derivatives recovered from olive mill wastewater as possible food antioxidants. J. Agric. Food Chem. 60, 1822–1829. Casalino, E., Calzaretti, G., Sblano, L., Landriscina, C., 2000. Cadmium dependent enzyme activity alteration is not imputable to lipid peroxidation. Arch. Biochem. Biophys. 383, 288–295. Chandran, R., Sivakumar, A.A., Mohandass, S., Aruchami, M., 2005. Effect of cadmium and zinc on antioxidant enzyme activity in the gastropod, Achatina fulica. Comp. Biochem. Phys. Toxicol. Pharmacology: CBP 140, 422–426. Chiang, L.C., Chiang, W., Chang, M.Y., Lin, C.C., 2003. In vitro cytotoxic, antiviral and immunomodulatory effects of Plantago major and Plantago asiatica. Am. J. Chin. Med. 31, 225–234.
135
Choi, S.Y., Jung, S.H., Lee, H.S., Park, K.W., Yun, B.S., Lee, K.W., 2008. Glycation inhibitory activity and the identification of an active compound in Plantago asiatica extract. Phytother. Res. PTR 22, 323–329. Choong, G., Liu, Y., Templeton, D.M., 2014. Interplay of calcium and cadmium in mediating cadmium toxicity. Chem. Biol. Interact. 211, 54–65. Cnubben, N.H., Rietjens, I.M., Wortelboer, H., van Zanden, J., van Bladeren, P.J., 2001. The interplay of glutathione-related processes in antioxidant defense. Environ. Toxicol. Pharmacol. 10, 141–152. Cuypers, A., Plusquin, M., Remans, T., Jozefczak, M., Keunen, E., Gielen, H., Opdenakker, K., Nair, A.R., Munters, E., Artois, T.J., Nawrot, T., Vangronsveld, J., Smeets, K., 2010. Cadmium stress: an oxidative challenge. Biometals 23, 927–940. Ellman, G.L., 1959. Tissue sulfhydryl groups. Arch. Biochem. Biophys. 82, 70–77. Ercal, N., Gurer-Orhan, H., Aykin-Burns, N., 2001. Toxic metals and oxidative stress part I: mechanisms involved in metal-induced oxidative damage. Curr. Top. Med. Chem. 1, 529–539. Friberg, L., Elinder, C., Kjellstrom, T., Nordberg, G., 1986. Cadmium and Health: A Toxicological and Epidemiological Approach, vols. 1 and 2. CRC Press, Boca Raton. Gobe, G., Crane, D., 2010. Mitochondria, reactive oxygen species and cadmium toxicity in the kidney. Toxicol. Lett. 198, 49–55. Hong, C.O., Hong, S.T., Koo, Y.C., Yang, S.Y., Lee, J.Y., Lee, Y., Ha, Y.M., Lee, K.W., 2011. Protective effect of Plantago asiatica L. extract against ferric nitrilotriacetate (Fe-NTA) induced renal oxidative stress in wistar rats. J. Food Hyg. Saf. 26, 107–113. Hussain, T., Shukla, G.S., Chandra, S., 1987. Effects of cadmium on superoxide dismutase and lipid peroxidation in liver and kidney of growing rats: in vivo and in vitro studies. Pharmacol. Toxicol. 60, 355–358. Jarup, L., Akesson, A., 2009. Current status of cadmium as an environmental health problem. Toxicol. Appl. Pharmacol. 238, 201–208. Johri, N., Jacquillet, G., Unwin, R., 2010. Heavy metal poisoning: the effects of cadmium on the kidney. Biometals 23, 783–792. Klaassen, C.D., Liu, J., Diwan, B.A., 2009. Metallothionein protection of cadmium toxicity. Toxicol. Appl. Pharmacol. 238, 215–220. Lee, Y.T., Chiang, L.Y., Chen, W.J., Hsu, H.C., 2000. Water-soluble hexasulfobutyl[60]fullerene inhibit low-density lipoprotein oxidation in aqueous and lipophilic phases. Proc. Soc. Exp. Biol. Med. 224, 69–75. Lopez, E., Arce, C., Oset-Gasque, M.J., Canadas, S., Gonzalez, M.P., 2006. Cadmium induces reactive oxygen species generation and lipid peroxidation in cortical neurons in culture. Free Radic. Biol. Med. 40, 940–951. Müller, T., Schuckelt, R., Jaenicke, L., 1994. Evidence for radical species as intermediates in cadmium/zinc-metallothionein-dependent DNA damage in vitro. Environ. Health Perspect. 102, 27. McCord, J.M., Fridovich, I., 1969. Superoxide dismutase. An enzymic function for erythrocuprein (hemocuprein). J. Biol. Chem. 244, 6049–6055. Mohamed, M., Abdellatif, M.D., Sabar, A., Elglammal, M.D., 2003. Sodium fluoride ion and renal function after prolonged sevoflurane or isoflurane anaesthesia. Br. J. Anaesth. 19, 79–83. Moron, M.S., Depierre, J.W., Mannervik, B., 1979. Levels of glutathione, glutathione reductase and glutathione S-transferase activities in rat lung and liver. Biochim. Biophys. Acta 582, 67–78. Mosmann, T., 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 65, 55–63.
136
e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 9 ( 2 0 1 5 ) 125–136
Ossola, J.O., Tomaro, M.L., 1995. Heme oxygenase induction by cadmium chloride: evidence for oxidative stress involvement. Toxicology 104, 141–147. Paglia, D.E., Valentine, W.N., 1967. Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J. Lab. Clin. Med. 70, 158–169. Prozialeck, W.C., Edwards, J.R., 2012. Mechanisms of cadmium-induced proximal tubule injury: new insights with implications for biomonitoring and therapeutic interventions. J. Pharmacol. Exp. Ther. 343, 2–12. Prozialeck, W.C., Vaidya, V.S., Liu, J., Waalkes, M.P., Edwards, J.R., Lamar, P.C., Bernard, A.M., Dumont, X., Bonventre, J.V., 2007. Kidney injury molecule-1 is an early biomarker of cadmium nephrotoxicity. Kidney Int. 72, 985–993. Renugadevi, J., Milton Prabu, S., 2010. Quercetin protects against oxidative stress-related renal dysfunction by cadmium in rats. Exp. Toxicol. Pathol. 62, 471–481. Renugadevi, J., Prabu, S.M., 2009. Naringenin protects against cadmium-induced oxidative renal dysfunction in rats. Toxicology 256, 128–134. Rockwell, P., Martinez, J., Papa, L., Gomes, E., 2004. Redox regulates COX-2 upregulation and cell death in the neuronal response to cadmium. Cell. Signal. 16, 343–353. Satarug, S., Moore, M.R., 2004. Adverse health effects of chronic exposure to low-level cadmium in foodstuffs and cigarette smoke. Environ. Health Perspect. 112, 1099–1103. Singh, V.P., 2005. Metal Toxicity and Tolerance in Plants and Animals. Sarup & sons.
Sinha, A.K., 1972. Colorimetric assay of catalase. Anal. Biochem. 47, 389–394. Smith, I.K., Vierheller, T.L., Thorne, C.A., 1988. Assay of glutathione reductase in crude tissue homogenates using 5,5 -dithiobis(2-nitrobenzoic acid). Anal Biochem. 175, 408–413. Stohs, S., Bagchi, D., 1995. Oxidative mechanisms in the toxicity of metal ions. Free Radic. Biol. Med. 18, 321–336. Urbschat, A., Obermuller, N., Haferkamp, A., 2011. Biomarkers of kidney injury. Biomarkers 16 (1), S22–S30. Waisberg, M., Joseph, P., Hale, B., Beyersmann, D., 2003. Molecular and cellular mechanisms of cadmium carcinogenesis. Toxicology 192, 95–117. Wang, Y., Fang, J., Leonard, S.S., Rao, K.M., 2004. Cadmium inhibits the electron transfer chain and induces reactive oxygen species. Free Radic. Biol. Med. 36, 1434–1443. Wilmes, A., Crean, D., Aydin, S., Pfaller, W., Jennings, P., Leonard, M.O., 2011. Identification and dissection of the Nrf2 mediated oxidative stress pathway in human renal proximal tubule toxicity. Toxicol. In Vitro 25, 613–622. Wu, G., Fang, Y.Z., Yang, S., Lupton, J.R., Turner, N.D., 2004. Glutathione metabolism and its implications for health. J. Nutr. 134, 489–492. Zhu, P., Frei, E., Bunk, B., Berger, M.R., Schmahl, D., 1991. Effect of dietary calorie and fat restriction on mammary tumor growth and hepatic as well as tumor glutathione in rats. Cancer Lett. 57, 145–152.
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.elsevier.com/locate/etap
Nephroprotection of plantamajoside in rats treated with cadmium Ha-Young Jung a,1 , Dong-Won Seo a,b,1 , Chung-Oui Hong a , Ji-Yeon Kim a , Sung-Yong Yang a , Kwang-Won Lee a,∗ a
Department of Food Bioscience and Technology, College of Life Science & Biotechnology, Korea University, Seoul 136-713, Republic of Korea b Food Analysis Center, Korea Food Research Institute, 516, Baekhyeon, Bundang, Seongnam, Gyeonggi 463-746, Republic of Korea
a r t i c l e
i n f o
a b s t r a c t
Article history:
Cadmium (Cd), an environmental and industrial pollutant, generates free radicals responsi-
Received 1 July 2014
ble for oxidative stress. Cd can also lead to various renal toxic damage such as the proximal
Received in revised form
tubules and glomerulus dysfunction. Plantamajoside (PMS), a major compound of Plantago
16 November 2014
asiatica (PA), was reported to have the antioxidant effects. In this study, we investigated
Accepted 21 November 2014
the protective effects of PMS on Cd-induced renal damage in the NRK-52E cell and rat
Available online 30 November 2014
kidney tissue. Cd exposure increased the ROS generation, lipid peroxidation, serum bio-
Keywords:
in vivo. The significant reduction in glutathione (GSH)/glutathione disulfide (GSSG) ratio and
chemical values of renal damage, and mRNA and protein expressions of KIM-1 in vitro and Cadmium
activities of antioxidant enzymes were also observed in the rats treated with Cd. PMS signifi-
Kidney injury molecule-1
cantly decreased the ROS generation and lipid peroxidation, thus enhancing GSH/GSSG ratio,
Lipid peroxidation
antioxidant enzyme activities in the cells and rats, and improved histochemical appear-
Nephrotoxicity
ances, indicating that PMS has protective activities against Cd-induced renal injury.
Oxidative stress
© 2014 Elsevier B.V. All rights reserved.
Plantamajoside
1.
Introduction
Recently, with economic growth and industrial development, heavy metal contaminations in foods are of major concerns.
The human body may be exposed to heavy metals through food, water, air or the skin contact (Singh, 2005). Heavy metals become toxic when they are not metabolized, and accumulate in the body. As a heavy metal, cadmium (Cd) is exposed via inhalation or ingestion from the environment in the industry,
Abbreviations: BUN, blood urea nitrogen; BW, body weight; CAT, catalase; Cd, cadmium; DCFDA, 2,7-dichlorofluorescein diacetate; DMEM, Dulbecco’s modified eagle medium; DMSO, dimethyl sulfoxide; PADTNB, 5,5 -dithiobis (2-nitrobenzoic); GAPDH, glyceraldehyde3-phosphate dehydrogenase; GPx, GSH peroxidase; GSH, glutathione; GSSG, glutathione disulfide; KIM-1, kidney injury molecule-1; LDH, lactate dehydrogenase; MTT, 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide; NADPH, -nicotinamide adenine dinucleotide phosphate reduced form; NEM, N-ethylmaleimide; PA, Plantago asiatica; qRT-PCR, quantitative RT-PCR; ROS, reactive oxygen species; TBARs, thiobarbituric and reactive substances; SOD, superoxide dismutase. ∗ Corresponding author at: Department of Food Bioscience & Technology, College of Life Science and Biotechnology, Korea University, 145 Anam-Ro, Sungbuk-Gu, Seoul 136-713, Republic of Korea. Tel.: +82 2 3290 3027; fax: +82 2 953 0737. E-mail address: [email protected] (K.-W. Lee). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.etap.2014.11.012 1382-6689/© 2014 Elsevier B.V. All rights reserved.
126
e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 9 ( 2 0 1 5 ) 125–136
and causes acute and chronic poisonings (Johri et al., 2010). Approximately 7% of the human population develops kidney dysfunction from Cd exposure (Friberg et al., 1986). Cd is easily accumulated, and has a relatively long half-life in the kidney of human body (Bernard, 2008). The kidney is one of the major target organs for Cd, and exposure to Cd causes various renal toxic effects such as the proximal tubules and glomerulus dysfunction (Akesson et al., 2005). It has been suggested that one of mechanisms involved in metal-induced toxicity can be due to the ability of metals to generate reactive oxygen species (ROS) (Ercal et al., 2001). In contrast to redox-active metals, such as iron and copper catalyzing the Fenton reactions producing hydoxy radicals, redox-inactive metal, Cd induces oxidative stress indirectly by diminishing the endogenous antioxidants including glutathione, other sulfhydryl-containing antioxidants and protein-bound thiols (Stohs and Bagchi, 1995) and activities of antioxidant enzymes including superoxide dismutase resulting in ROS accumulation in cells (Hussain et al., 1987). In addition, Cd is a source shown to indirectly produce ROS. Heme oxygenase induction by Cd in cell is shown to contribute to generation of ROS (Müller et al., 1994; Ossola and Tomaro, 1995) also detected that Cd-metallothionein can induce ROS in vitro extracellular condition. Plantago asiatica (PA) has been traditionally used as a folk medicine (Chiang et al., 2003), and has anti-inflammatory and antioxidant activities to protect kidney against ferric nitrilotriacetic-induced renal injury. Bioactive compounds such as PMS, phenylethanoids, plantaginin, and acetoside have been isolated from PA (Hong et al., 2011). To the best of our knowledge, this is the first report on the renal protective effect of PMS on the Cd-induced renal injury. In this study, kidney proximal tubule epithelial (NRK-52E) cells and rats were treated with Cd in the presence of PMS to investigate the potential of the compound to improve Cd-induced nephrotoxicity.
2.
Materials and methods
2.1.
Materials
Plantamajoside was isolated and obtained in our laboratory (Choi et al., 2008). Cadmium chloride (CdCl2 , purity ≥99.9%), 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), 2,7-dichlorofluorescein diacetate (DCFDA), GSH, Nethylmaleimide (NEM), 5,5 -dithiobis (2-nitrobenzoic) (DTNB), 2-vinylpyridine, triethanolamine, -nicotinamide adenine dinucleotide phosphate reduced form (NADPH), and glutathione reductase were purchased from Sigma–Aldrich (St. Louis, MO, USA). Dulbecco’s modified eagle medium (DMEM) cell culture media, bovine serum albumin, and BCA protein assay kit were purchased from Thermo scientific (IL, USA), and trizol, penicillin streptomycin and trypsin-EDTA were purchased from Life technologies (Grand Island, NY, USA). Penicillin streptomycin and trypsin-EDTA were purchased from GIBCO (Grand Island, NY, USA). Antibody against kidney injury molecule-1 (KIM-1) was purchased from Abcam (Cambridge, MA, USA), and horseradish
Fig. 1 – Chemical structure of plantamajoside (PMS) (C29 H38 O16 ).
peroxidase-conjugated secondary antibody was purchased from Millipore (Billerica, MA, USA). Chemical structure of PMS is shown in Fig. 1.
2.2.
Cell culture and viability assay
Rat kidney proximal tubule epithelial (NRK-52E) cells were obtained from ATCC (Manassas, VA, USA) and cultured in DMEM medium containing 10% (v/v) FBS, 4 mM l-glutamine, 1.5 g/L sodium bicarbonate, and 4.5 g/L of glucose. The cells were maintained at 37 ◦ C in a 5% CO2 humidified atmosphere. Cell viability was measured by the MTT assay (Mosmann, 1983). NRK-52E cells were seeded at a density of 5 × 104 cells/well in 24 well plates and cultured in DMEM medium for 24 h. After confirming cell attachment, because the treatment of 5 M CdCl2 gave 50% of cell viability on MTT assay, cells were treated with 5 M CdCl2 and 3.125, 6.25, 12.5, 25, 50, and 100 M PMS in serum free medium for 24 h. Then, the medium was changed to 5 mg/mL MTT reagent in DMEM. After incubation for 3 h at 37 ◦ C, the medium was removed, and 200 L of DMSO was added to each well to dissolve formazan crystals. The absorbance was measured at 540 nm wavelength using a multiplate spectrometer. The results were determined as percentages of the control.
2.3. assay
Reactive oxygen species (ROS) scavenging activity
NRK-52E cells were seeded at a density of 5 × 104 cell/well in 96 well plates for 24 h. After cell detachment, the cells were incubated with 100 M of DCF-DA in DMEM medium for 30 min. After washing with phosphate buffer saline (PBS) at pH 7.4, the cells were treated with Cd (5 M) and PMS (25, 50 and 100 M). After incubation for 1 h, the fluorescence intensity was measured using a spectrofluorometer.
2.4.
Animals and administration
Male Sprague–Dawley rats (7-week-old, about 250 g of body weight (BW)) were purchased from Samtako Bio Korea Co. (Gyeonggi, Korea) and were housed in cages with free access to standard diet (Samyang Feed Co. Ltd., Incheon, Korea) and tap water. The experimental animals were treated in compliance with the instructions of the Committee for Ethical Usage of Experimental Animals of Korea University. This experimental protocol was reviewed, and approved by the Korea University Animal Care Committee (No. KUIACUC-20130704-1).
e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 9 ( 2 0 1 5 ) 125–136
Thirty-two rats were divided randomly into four groups consisting of eight rats in each group. The group I was the control without treatment. In our preliminary experiment, some rats administered with 10 mg/kg BW of cadmium chloride showed bloody nose secretions after 3-day treatment. Based on reports on Cd-induced nephrotoxicity and oxidative stress using rat animals (Renugadevi and Milton Prabu, 2010; Renugadevi and Prabu, 2009), they administered rats with 5 mg/kg BW of cadmium chloride for 4 weeks. Thus, the animals in the group II were orally intubated with 5 mg/kg BW of Cd for 4 weeks. Animals in the group III and IV were orally intubated with 5 mg/kg BW of Cd and two different levels of PMS (10 mg/kg or 40 mg/kg BW) for 4 weeks. PMS was given 3 h prior to the administration of Cd. After 18 h from the last treatment, the rats were sacrificed, and blood samples were collected from the vena cava for measuring serum creatinine blood urea nitrogen (BUN) and lactate dehydrogenase (LDH) activity. After collecting the bloods, the kidneys were removed and rinsed with PBS and stored at −80 ◦ C prior to the biochemical analysis.
2.5.
was determined by the BCA protein assay using bovine serum albumin (BSA) as the standard.
2.7. Measurement of catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx) activity Frozen kidney tissues (0.5 g) were homogenized in 1 mL of ice-cold 0.1 M potassium phosphate buffer. The homogenate was centrifuged at 12,000 × g for 30 min. Then, the supernatants were collected in a fresh 1.5 mL tube for determining antioxidant enzyme activities. The CAT activity was measured colorimetrically by the modification of a previously reported (Sinha, 1972) using dichromate-acetic acid reagent. The superoxide dismutase (SOD) activity was determined by the modified xanthine/xanthine oxidase method (McCord and Fridovich, 1969). GSH peroxidase (GPx) activity was assayed using the Paglia and Valentine method (Paglia and Valentine, 1967) with a slight modification. The rate of oxidation of NADPH caused by the addition of GSH reductase was monitored.
Lipid peroxidation assay
Lipid peroxidation was estimated by measuring thiobarbituric and reactive substances (TBARs). Intracellular lipid peroxidation was determined by the modified thiobarbituric acid (TBA) fluorometric method (Lee et al., 2000). For the TBARs assay, the solutions were homogenized on ice using homogenizing buffer of 10% volume of kidney weight. TBA reagent (1 mL) was added to 100 L of homogenized solutions and heated at 90 ◦ C in water bath for 20 min. Fluorescence intensity was measured at the excitation wavelength of 530 nm and emission of 590 nm using a spectrofluorometer (VICTOR2TM , Perkin-Elmer, USA). Protein concentration was determined by the BCA protein assay using bovine serum albumin (BSA) as the standard.
2.6.
127
GSH contents analysis
The GSH contents of NRK-52E cells and rat kidney tissues were measured by the modification of previously reported method (Moron et al., 1979; Smith et al., 1988; Zhu et al., 1991). GSH was determined by the reaction with DTNB to afford a compound absorbing at 412 nm. The reaction mixture containing 0.1 M potassium phosphate buffer and 0.1 M DTNB in DMSO was added to the sample. The absorbance at 412 nm was measured using a spectrophotometer. Glutathione disulfide (GSSG) was measured using a recycling reaction of GSH with DTNB in the presence of excess GSH reductase. 100 L of samples was derivatized with 2 L of 2-vinylpyridine and 6 L of triethanolamine in the pH range 6–7. After reaction for 60 min, 190 L of the reaction buffer (0.3 mM NADPH in potassium phosphate buffer (pH 7.4) and 6 mM DTNB in DMSO) and 10 L of derivatized sample were mixed and incubated at 30 ◦ C for 15 min. Then, 20 L of GSH reductase was added to the mixture. GSH reductase was kept on ice during the preparation of the reaction buffer. Change in the absorption was measured using a multiplate reader at 412 nm. Protein concentration
2.8. tissue
Quantifications of Cd and total thiols in rat kidney
All the solvents which are used for washing of all the glasses and preparation of all the test solutions were tertiary distilled water which passed Milli-Q system (Millipore; MA, USA). Standard stock solution containing 10 mg/kg of cadmium was purchased from Perkin-Elmer (New York, USA). These were used to prepare calibration standards. Ultrapure grade carrier (argon [Ar], 99.9995% pure) was supplied by Shin-Yang (Korea). Samples were digested by Ethos1 microwave digestion system (Milestone, Millan, Italy). About 0.2 g of kidney tissue sample, 7 mL of nitric acid and 2 mL of hydrogen peroxide were added to vessel and performed microwave digestion. One randomly selected vessel was filled with reagents only and taken through the entire procedure as a blank. After cooling at room temperature, sample solutions were quantitatively transferred into 25 mL flasks. Then, the digested samples were filled with ultrapure water to the final volume before analysis by ICP-MS. Cadmium analysis was performed using NEXION 300D ICP-MS system (Perkin-Elmer, USA). The isotopes 112 Cd was selected as analytical mass in ICP-MS standard mode. Specific isotope was monitored regarding to the sensibility of the element and/or possible isobaric and polyatomic interferences. Torch position, ion lenses and gases yield were optimized daily with the tuning solution (1 g/kg) to implement a short-term stability test of the instrument, to maximize ion signals and to minimize interference effects from polyatomic ions and doubly charged ions. Total thiol groups of the rat kidney tissue were measured using modified DTNB reagent method (Ellman, 1959). Briefly, a working solution was prepared by mixing 0.5 mL of DTNB solution (50 mM NaAc and 2 mM DTNB), 1 mL of 1 M Tris–HCl, pH 8.0 and 8.4 mL of distilled water. This 190 L of working solution and 10 L of sample were mixed, incubated 5 min at 25 ◦ C, and its absorbance was read at 412 nm using spectrophotometer.
128
2.9.
e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 9 ( 2 0 1 5 ) 125–136
Pathological histology in kidney tissue
After removal of the rat kidney, the tissues were fixed in 10% buffered formaldehyde. The formaldehyde-fixed tissue samples were embedded in parafilm, and 3–4-m sections were stained with periodic acid-Schiff (PAS). Tubular injury was observed at 200× magnification for each sample from all the group. Tubular necrosis was evaluated on an arbitrary semiquantitative assessment as follows: no cell necrosis; mild usually single-cell necrosis in sparse tubules; moderate more than one cell involved in sparse tubules; and massive necrosis in every power field.
2.10.
RNA isolation, RT-PCR and qRT-PCR analysis
Total RNA was isolated using trizol reagent according to the manufacture’s instruction. Total RNA (3 g) and the First Strand cDNA synthesis kit (Legene Biosciences Inc., USA) were used to synthesize cDNA. PCR primers of kidney injury molecule-1 (KIM-1) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used. Sequences of RT-PCR primers were as follows: KIM-1 sense 5 -CGGTGCCTGTGAGTAAATAGATand antisense 5 -CTGGCCATGACACAAATAAGAC-3 ; 3 GAPDH sense 5 -GCCATCAACGACCCCTTCATT-3 and antisense 5 -CGCCTGCTTCACCACCTTCTT-3 . Sequences of quantitative RT-PCR (qRT-PCR) primers used are as follows: KIM-1 sense 5 -TGGCACTGTGACATCCTCAGA-3 and antisense 5 -GCAACGGACATGCCAACATA-3 ; GAPDH sense 5 -ACTGCCAGCCTCGTCTCATAG-3 and antisense 5 -CCTTGACTGTGCCGTTGAACT-3 . RT-PCR was conducted according to the manufacturer’s protocol (DreamTaq DNA polymerase, Thermo scientific, Pittsburgh, PA, USA), and the optimal PCR results were secured at an annealing temperature of 60.5 ◦ C with 30 cycles. PCR products were separated electrophoretically on 1.5% agarose gel stained with red-safe nucleic acid staining solution (INTRON Biotechnology, Korea). Quantitative data normalized to GAPDH were obtained from a densitometer and analyzed using the Quantity One software (Bio-Rad, USA). Validation of the RT-PCR results was determined by quantitative real time-PCR (qRT-PCR). Amplification and detection of cDNA were performed with SYBR® Green supermix. Quantitative real-time PCR data were expressed as a threshold cycle (Ct) value by iCycler iQ® real-time PCR detection system (BioRad, Korea), and GAPDH was used to normalize the mRNA expression level.
2.11.
Western-blot analysis
Samples containing 20 g of protein from each lysate were dissolved in the loading buffer, and then denatured by boiling for 5 min. Protein lysates were separated on 10% SDSpolyacrylamide gel and transferred to polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA). After blocking with 5% skim milk in TBST (0.25 M Trisbase pH 7.4, 1.5 M NaCl, 1% Tween-20) at room temperature for 1 h, membranes were incubated with primary anti-KIM-1 antibody (1:1000) in skim milk overnight at 4 ◦ C. After washing in TBST for 30 min, the membranes were incubated with suitable secondary antibody for 45 min at room temperature. Then, protein bands
Fig. 2 – Effects of cadmium (Cd) and PMS on cell viability in NRK-52E cells. The results are expressed as mean ± SD. Different letters indicate significant differences at the p < 0.05 by the Duncan’s multiple range tests.
were detected using ECL solution (Abclon, Seoul, Korea). Protein expression levels were determined using a developer, and the intensity was calculated using the image analysis software.
2.12.
Statistical analysis
All the data were expressed as mean ± standard deviation, and the analysis was performed using the SAS software version 9.3 (SAS Institute Inc., NC, USA). Statistical analysis was conducted by the Duncan’s multiple range tests.
3.
Results
3.1.
Effect of PMS on Cd-induced cytotoxicity
Cell viability of NRK-52E cells was measured to evaluate the protective effect of PMS against Cd cytotoxicity using the MTT assay. Cells were treated with 5 M of CdCl2 and 3.1, 6.3, 12.5, 25, 50 and 100 M PMS. The cell viability of PMS-treated group was more than 90%, indicating that PMS has no toxic effect in the range 3.1–100 M (data not shown). The effects of various concentrations of PMS on Cd cytotoxicity are shown in Fig. 2. The treatment of cells with 5 M Cd only reduced cell viability to ∼57% after 24 h incubation. The presence of PMS at the concentrations of 3.1–25 M did not show any protective effects on Cd cytotoxicity; however, the treatment with ≥50 M PMS significantly decreased the Cd induced cytotoxicity (p < 0.05).
3.2. Effect of PMS on ROS generation induced by Cd in NRK-52E cells DCF-DA, a sensitive fluorescent probe, is useful for the detection of the ROS generation in cells (Bartosz, 2006). The intracellular generation of the ROS was measured to investigate the antioxidant effect of PMS on Cd-induced oxidative damage in cells. As seen in Fig. 3, DCF fluorescence intensity
e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 9 ( 2 0 1 5 ) 125–136
129
Fig. 3 – Effects of PMS on ROS generation induced by Cd in NRK-52E cells. The results are expressed as mean ± SD. Different letters indicate significant differences at the p < 0.05 by the Duncan’s multiple range tests.
increased for the cells treated with Cd compared to the untreated cells. However, ROS levels significantly decreased in all plantamajoside co-treatment groups (p < 0.05).
3.3.
Effect of PMS on lipid peroxidation induced by Cd
The evaluation of the formation of TBARs provides an estimation of the extent of lipid oxidation originated by the treatment with Cd. The TBAR values (expressed as M MDA mg−1 of protein) were measured to evaluate the effect of PMS on the lipid peroxidation induced by Cd in the NRK-52E cells and rat kidney tissue. The treatment of NRK-52E cells with Cd significantly increased the MDA level compared to the control group (Fig. 4A); however, Cd-treated cells in the presence of PMS dose dependently showed decreased level of MDA (p < 0.05). The treatment with 50 and 100 M of PMS reduced MDA levels compared to that of untreated control cells. Moreover, the lowering effect of PMS on the lipid peroxidation was evaluated in vivo. As seen in Fig. 4B, compared to the control group, the administration of Cd in rats showed a significantly increased concentration of MDA in the kidney tissues. However, the MDA level decreased significantly by the treatment with PMS. Therefore, we demonstrated that Cd-induced oxidative stress in kidney was effectively prevented by the treatment with PMS.
3.4.
Effects of Cd and PMS on glutathione (GSH) levels
The kidney NRK-52 cells treated with 5 M Cd showed a decrease in GSH level, an increase in GSSG level, and decreasing GSH/GSSG ratio (Fig. 5A–C). Significant (p < 0.05) recovery was noted in the groups treated with PMS supplementation. These antioxidant effects of PMS on kidney oxidative stress were also confirmed in post Cd exposed rats (Fig. 5D–F).
Fig. 4 – Effect of PMS on the lipid peroxidation induced by Cd in NRK-52E cells (A) and rat kidney tissue (B). The results are expressed as mean ± SD. Different letters indicate significant differences at the p < 0.05 by the Duncan’s multiple range tests.
3.5. Measurement of catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx) activity As seen in Fig. 6, all the CAT, SOD and GPx activities showed significant (p < 0.05) decrease in kidney tissues upon Cd exposure. High level of PMS (40 mg/kg BW) supplementation provided recovery, whereas low level of PMS (10 mg/kg BW) was marginally effective (non-significant) (p < 0.05).
3.6. Serum biochemical values in rats treated with Cd and PMS The blood samples from the rats treated with Cd and PMS supplementation were collected to determine serum creatinine, serum blood urea nitrogen (BUN) and lactate dehydrogenase (LDH). Cd-treated group showed significantly
130
e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 9 ( 2 0 1 5 ) 125–136
Fig. 5 – Effects of Cd and PMS on the glutathione levels in NRK-52E cells (A-C) and rat kidney tissue (D-F). (A, D) GSH, (B, E) GSSG, and (C, F) GSH/GSSG ratio. The results are expressed as mean ± SD. Different letters indicate significant differences at the p < 0.05 by the Duncan’s multiple range tests.
(p < 0.05) increased serum creatinine, BUN, and LDH in comparison to the control group (Table 1). In contrast, PMS supplementation significantly (p < 0.05) recovered serum biochemical values to the levels of control values.
3.7.
Levels of Cd and total thiols in rat kidney tissue
Cd concentrations in rat kidney tissues were measured by ICP-MS system. The standard curve linearity for ICP-MS
131
e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 9 ( 2 0 1 5 ) 125–136
Table 1 – Effects of plantamajoside (PMS) on serum creatinine, BUN and LDH in the rats treated with cadmium (Cd). Treatment Control Cd (5 mg/kg BW) PMS (10 mg/kg BW) PMS (40 mg/kg BW)
Creatinine (mg/dL) 0.42 0.48 0.44 0.42
± ± ± ±
BUN (mg/dL)
0.03a 0.04b 0.02a 0.02a
13.5 16.3 14.9 13.8
± ± ± ±
0.54a 0.76b 0.63c 1.38ac
LDH (U/L) 1783.0 2018.4 1751.0 1061.2
± ± ± ±
117.3a 112.4b 177.6a 60.8c
BUN: blood urea nitrogen; LDH: lactate dehydrogenase; BW: body weight. Values are expressed as mean ± SD of five independent experiments. Means with different superscript letters within a column were determined to be significantly different at p < 0.05 in the same column when applied to the Duncan’s multiple range tests.
method was determined at seven points ranging from 0.001 to 0.20 mg/L of CdCl2 solution (R2 > 0.9999) (data not shown). As seen in Fig. 7A, Cd was barely detectable in control group. And Cd level (11.7 ± 0.7 g of Cd/g tissue) showed significant (p < 0.05) increase in kidney tissue upon Cd exposure. High level (40 mg/kg BW) and low one (10 mg/kg BW) of PMS supplementations showed significantly reduced Cd levels to 8.3 ± 0.5 and 6.8 ± 1.1, respectively (p < 0.05). In addition, we measured the total thiols in kidney tissue. The control group showed 1.46 ± 0.12 mM thiols/g protein in kidney tissue (Fig. 7B). However, Cd-treated group significantly reduced the total thiols to 0.38 ± 0.13 mM thiols/g protein (p < 0.05). And low level of PMS supplementation recovered the thiol level to normal (1.44 ± 0.12), and even high level of PMS showed higher total thiols (1.79 ± 0.19) than control (p < 0.05).
3.8.
Histology of the renal tubule
Fig. 8A shows the histological results. The kidney from the untreated control group showed normal appearance. The Cdtreated rats had a massive degree of renal tubular necrosis (Fig. 8B). The treatment with a low level of PMS (10 mg/kg BW) mitigated the severity of the Cd-induced renal necrosis (Fig. 8C), and the rats treated with high level of PMS (40 mg/kg BW) had only slight degree of tubular necrosis (Fig. 8D).
3.9. Effects of PMS on the expression of kidney injury molecule-1 (KIM-1) mRNA and protein in Cd-treated kidney in vitro and in vivo To determinate the effect of PMS pretreatment on KIM-1 mRNA expression in the NRK-52E cells and rat kidney tissue treated with Cd, RT-PCR and quantitative real-time PCR were used to analyze mRNA levels. As seen in Fig. 9A and B, KIM-1 mRNA level was significantly (p < 0.05) upregulated in the cells treated 5 M Cd alone. However, KIM-1 mRNA level was significantly (p < 0.05) reduced in the groups supplemented with PMS. Moreover, the rats administered with 5 mg/kg BW of Cd also showed significantly (p < 0.05) enhanced KIM-1 mRNA level compared to the control rats (Fig. 9C and D). However, the mRNA expression of KIM-1 significantly (p < 0.05) decreased in the rats pretreated with various concentrations of PMS. In concordance with the kidney KIM-1 mRNA levels, which increased after Cd treatment, KIM-1 protein levels were significantly (p < 0.05) increased in the NRK-52E cells and kidney tissues (Fig. 10A and B). The exposure to Cd
upregulated KIM-1 protein in the NRK-52E cells is shown in Fig. 10A. In 25 and 50 M of PMS pretreatment groups, no inhibitory activities of PMS on KIM-1 expression by Cd was observed. However, the pretreatment with 100 M of PMS reduced KIM-1protein expression. As seen in Fig. 10B, Cd exposure upregulated the KIM-1 protein expression in the rat kidney tissue; however, the pretreatment with PMS significantly reduced KIM-1 protein expression level in the control group.
4.
Discussion
Cd, a toxic metal, occurring in the environment causes negative health effects (Choong et al., 2014), and is classified as a class I carcinogen by The World Health Organization’s International Agency for Research on Cancer (Cancer et al., 1993). Long half-life of Cd exerts a continuously increasing environmental burden resulting in entry into the food chain (Choong et al., 2014). Because of a relatively long half-life of Cd (10–30 years in human), it affects the major organs such as the kidney and liver accounting for onethird of the total Cd in the body (Satarug and Moore, 2004). Cd-induced renal damage causes the proximal tubular reabsorptive dysfunction (Jarup and Akesson, 2009). Therefore, nephrotoxic injury is indicated to be an underlying factor for establishing important biomarkers for Cd toxicity. The oral exposure of Cd leads to its absorption from gastrointestinal tract up to 5–10%, and its systemic accumulation into the bloodstream makes Cd to be delivered to liver where it induces the synthesis of metallothionein, binding Cd and buffering its toxic effects in the liver cells (Prozialeck and Edwards, 2012). Due to normal turnover or a result of Cd injury the Cd-metallothionein complex from the liver cells is released into blood stream (Klaassen et al., 2009), and is filtered at the glomerulus and taken up by the epithelial cells of the proximal tubule. Additionally, proximal tubule cells can uptake lower molecular-weight cadmium-thiol conjugates such as GSH (Bridges and Zalups, 2005). However, the interaction between Cd and proteins/low-molecular thiols depending upon various conditions could not be enough, and free Cd dissociating from the complex, could enter the cell. It is generally accepted that Cd form not Cd-metallothionein injures proximal tubule epithelial cells (Klaassen et al., 2009). Another concern regarding Cd toxicity is the concentration of Cd that is typically achieved in vivo. The kidney
132
e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 9 ( 2 0 1 5 ) 125–136
Fig. 7 – Levels of Cd and total thiols in the rat kidney tissue. (A) Cd, and (B) total thiols. The results are expressed as mean ± SD. Different letters indicate significant differences at the p < 0.05 by the Duncan’s multiple range tests.
Fig. 6 – Effects of Cd and PMS on the activities of CAT, superoxide dismutase (SOD), and glutathione peroxidase (GPx) in the rat kidney tissue. (A) CAT, (B) SOD, and (C) GPx. The results are expressed as mean ± SD. Different letters indicate significant differences at the p < 0.05 by the Duncan’s multiple range tests.
tissue level of Cd is associated with the onset of renal injury is reported to be 150–200 g/g tissue (Prozialeck and Edwards, 2012). In our study, oral administration of Cd (5 mg/kg BW) for 4 weeks increased Cd concentration (11.7 ± 0.7 g of Cd/g tissue) in kidney tissue. Also in vitro study, the kidney cells were exposed to 5 M of Cd for 24 h. This concentration is much higher than the Cd level in blood (5–10 nM), but it is well below the millimolar concentrations of Cd that are attained in renal cortical tissue in vivo. The localized Cd level in the immediate vicinity could surpass 1 mM (Prozialeck and Edwards, 2012). Cd is not a redox-active and essential element, and is unable to directly produce ROS (Cuypers et al., 2010). However, Cd is clearly in capable of inducing oxidative stress, this mechanism plays a role in Cd-induced kidney injury (Gobe and Crane, 2010). The Cd-specific binding to sulfhydryl group of GSH depletes the major antioxidant thiol, thus disturbing the redox equilibrium toward an oxidative condition (Lopez et al., 2006). GSH contributes to cellular antioxidant defense system against oxidative stress (Cnubben et al., 2001),
e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 9 ( 2 0 1 5 ) 125–136
133
Fig. 8 – Representative photographs of sections of renal tubule under light microscope of a rat treated with saline (A), Cd (5 mg/kg BW) (B), Cd + PMS (10 mg/kg BW) and (C), Cd + PMS (40 mg/kg BW) (D). Note the normal structure in (A) and the massive necrosis in (B), the relatively moderate necrosis in necrosis in (C) and mild necrosis in (D). PAS 200X.
scavenges ROS directly and is oxidized to form GSSG under the catalysis of GSH peroxidation. GSH/GSSG, a major redox couple, plays an important role in antioxidant defense (Wu et al., 2004). Moreover, iron in various proteins is replaced by Cd resulting in an increase in iron free cells, enhancing Fenton reaction, thus increasing ROS generation in mitochondria (Wang et al., 2004) and NADPH oxidase dependent ROS (Rockwell et al., 2004). Also, Cd interferes with antioxidant defense systems, for instance, an alteration in the activity of antioxidant enzymes (Chandran et al., 2005; Waisberg et al., 2003). Cd exposure inactivates most of the antioxidant enzymes, relating to the binding of Cd directly to the active sites of enzymes containing sulfhydryl groups or displacement of metal cofactors from the active sites (Casalino et al., 2000) resulting in the diminished activities of antioxidant enzymes. This indicates destruction of antioxidant defense system, and increase in ROS because of the exposure to Cd. This Cd-induced oxidative stress causes the kidney proximal tubular injury (Cuypers et al., 2010). Through these indirect mechanisms, Cd can significantly intensify the actions of normal oxidative processes within the cell, which results in oxidative stress. Kidney is highly susceptible to toxic injury because of high blood perfusion and filtration of the blood plasma at a high rate (Mohamed et al., 2003). The levels of serum creatinine and serum BUN are generally used as the biomarkers of renal function (Urbschat et al., 2011). Therefore, use of antioxidants is considered as a meaningful approach for the suppression of Cd-induced kidney toxicity. P. asiatica extract provides renal protective effects against oxidative stress (Hong et al., 2011).
In our experiment, kidney cells and animals were pretreated with PMS prior to Cd intoxication because PMS, a caffeic acid derivatives has been shown to have an antioxidant providing scavenging activity of ROS and protective action on low-density lipoprotein against oxidative damage (Cardinali et al., 2012). We thought that post-treatment schedule for PMS in Cd-induced oxidative stress was not effective. The significant enhancement in the GSH/GSSG ratio by the treatment with PMS in the presence of Cd indicates its potential role as an antioxidant. The decreased levels of lipid peroxidation in PMS-supplemented group indicate the inhibitory effect of PMS against Cd-induced oxidative stress. Meanwhile, histochemical examination revealed that Cd caused the degeneration of tubules in renal ultrastucture indicating marked tubular damage. It has been reported that proximal tubular epithelial cells are susceptible to oxidative stress damage (Wilmes et al., 2011). In our study, rat kidney proximal tubule epithelial (NRK-52E) cells were used for assessing Cd intoxication. The exposure to Cd results in the dysfunction of the kidney proximal tubule. KIM-1 is a sensitive and early biomarker of Cd-induced nephrotoxicity, and is a type I transmembrane protein that is less detectable in normal kidney tissue (Prozialeck et al., 2007). This protein it is detected at high levels in the proximal tubule epithelial cells after toxic injury. In the present in vitro and in vivo study also demonstrated that the treatment with Cd activates KIM-1 mRNA and protein expression. However, we observed that PMS can downregulate KIM-1 expression, indicating that PMS had a protective effect on Cd-induced nephrotoxicity.
134
e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 9 ( 2 0 1 5 ) 125–136
Fig. 9 – Effects of Cd and PMS on the mRNA expression of KIM-1 in NRK-52E cells (A, B) and rat kidney tissue (C, D). (A, C) KIM-1 mRNA expression level by RT-PCR. (B, D) Real time PCR was performed, and the relative KIM-1 mRNA expression is normalized by GAPDH. The fold induction is expressed as the relative mRNA expression of the treatment groups divided by the control group. The results are expressed as mean ± SD. Different letters indicate significant differences at the p < 0.05 by the Duncan’s multiple range tests.
Fig. 10 – Effects of Cd and PMS on the KIM-1 protein expression in NRK-52E cells (A) and rat kidney tissue (B). Intensity of each band was quantified by densitometry. KIM-1 protein expression is normalized by GAPDH. The fold induction is expressed as the relative protein expression of the treatment groups divided by the control group. The results are expressed as mean ± SD. Different letters indicate significant differences at the p < 0.05 by the Duncan’s multiple range tests.
e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 9 ( 2 0 1 5 ) 125–136
5.
Conclusion
In this study, we confirmed that PMS exhibited protective effects against Cd-induced kidney injury as confirmed by the suppression of ROS generation, enhancement of antioxidant status, improved histochemical appearances, and inhibition of KIM-1 activation in the cells and rat kidney tissues. These results suggest that PMS is effective in inhibiting Cd-induced kidney injury.
Conflict of interest The authors declare that there are no conflicts of interest.
Transparency document The Transparency document associated with this article can be found in the online version.
Acknowledgement This research was a part of the project (Grant No. R1111343) titled ‘Material development and commercialization of Korean seaweed alga (Capsosiphon fulvescens) conferring whitening function, anti-diabetes, and inhibitory effects on diabetic complications’, funded by the Ministry of Oceans and Fisheries, Korea.
references
Akesson, A., Lundh, T., Vahter, M., Bjellerup, P., Lidfeldt, J., Nerbrand, C., Samsioe, G., Stromberg, U., Skerfving, S., 2005. Tubular and glomerular kidney effects in Swedish women with low environmental cadmium exposure. Environ. Health Perspect. 113, 1627–1631. Bartosz, G., 2006. Use of spectroscopic probes for detection of reactive oxygen species. Clin. Chim. Acta 368, 53–76. Bernard, A., 2008. Cadmium & its adverse effects on human health. Indian J. Med. Res. 128, 557–564. Bridges, C.C., Zalups, R.K., 2005. Molecular and ionic mimicry and the transport of toxic metals. Toxicol. Appl. Pharmacol. 204, 274–308. Cancer, I.A.f.R.o., Organization, W.H., Humans, I.W.G.o.t.E.o.C.R.t., 1993. Beryllium, Cadmium, Mercury, and Exposures in the Glass Manufacturing Industry. IARC. Cardinali, A., Pati, S., Minervini, F., D’Antuono, I., Linsalata, V., Lattanzio, V., 2012. Verbascoside, isoverbascoside, and their derivatives recovered from olive mill wastewater as possible food antioxidants. J. Agric. Food Chem. 60, 1822–1829. Casalino, E., Calzaretti, G., Sblano, L., Landriscina, C., 2000. Cadmium dependent enzyme activity alteration is not imputable to lipid peroxidation. Arch. Biochem. Biophys. 383, 288–295. Chandran, R., Sivakumar, A.A., Mohandass, S., Aruchami, M., 2005. Effect of cadmium and zinc on antioxidant enzyme activity in the gastropod, Achatina fulica. Comp. Biochem. Phys. Toxicol. Pharmacology: CBP 140, 422–426. Chiang, L.C., Chiang, W., Chang, M.Y., Lin, C.C., 2003. In vitro cytotoxic, antiviral and immunomodulatory effects of Plantago major and Plantago asiatica. Am. J. Chin. Med. 31, 225–234.
135
Choi, S.Y., Jung, S.H., Lee, H.S., Park, K.W., Yun, B.S., Lee, K.W., 2008. Glycation inhibitory activity and the identification of an active compound in Plantago asiatica extract. Phytother. Res. PTR 22, 323–329. Choong, G., Liu, Y., Templeton, D.M., 2014. Interplay of calcium and cadmium in mediating cadmium toxicity. Chem. Biol. Interact. 211, 54–65. Cnubben, N.H., Rietjens, I.M., Wortelboer, H., van Zanden, J., van Bladeren, P.J., 2001. The interplay of glutathione-related processes in antioxidant defense. Environ. Toxicol. Pharmacol. 10, 141–152. Cuypers, A., Plusquin, M., Remans, T., Jozefczak, M., Keunen, E., Gielen, H., Opdenakker, K., Nair, A.R., Munters, E., Artois, T.J., Nawrot, T., Vangronsveld, J., Smeets, K., 2010. Cadmium stress: an oxidative challenge. Biometals 23, 927–940. Ellman, G.L., 1959. Tissue sulfhydryl groups. Arch. Biochem. Biophys. 82, 70–77. Ercal, N., Gurer-Orhan, H., Aykin-Burns, N., 2001. Toxic metals and oxidative stress part I: mechanisms involved in metal-induced oxidative damage. Curr. Top. Med. Chem. 1, 529–539. Friberg, L., Elinder, C., Kjellstrom, T., Nordberg, G., 1986. Cadmium and Health: A Toxicological and Epidemiological Approach, vols. 1 and 2. CRC Press, Boca Raton. Gobe, G., Crane, D., 2010. Mitochondria, reactive oxygen species and cadmium toxicity in the kidney. Toxicol. Lett. 198, 49–55. Hong, C.O., Hong, S.T., Koo, Y.C., Yang, S.Y., Lee, J.Y., Lee, Y., Ha, Y.M., Lee, K.W., 2011. Protective effect of Plantago asiatica L. extract against ferric nitrilotriacetate (Fe-NTA) induced renal oxidative stress in wistar rats. J. Food Hyg. Saf. 26, 107–113. Hussain, T., Shukla, G.S., Chandra, S., 1987. Effects of cadmium on superoxide dismutase and lipid peroxidation in liver and kidney of growing rats: in vivo and in vitro studies. Pharmacol. Toxicol. 60, 355–358. Jarup, L., Akesson, A., 2009. Current status of cadmium as an environmental health problem. Toxicol. Appl. Pharmacol. 238, 201–208. Johri, N., Jacquillet, G., Unwin, R., 2010. Heavy metal poisoning: the effects of cadmium on the kidney. Biometals 23, 783–792. Klaassen, C.D., Liu, J., Diwan, B.A., 2009. Metallothionein protection of cadmium toxicity. Toxicol. Appl. Pharmacol. 238, 215–220. Lee, Y.T., Chiang, L.Y., Chen, W.J., Hsu, H.C., 2000. Water-soluble hexasulfobutyl[60]fullerene inhibit low-density lipoprotein oxidation in aqueous and lipophilic phases. Proc. Soc. Exp. Biol. Med. 224, 69–75. Lopez, E., Arce, C., Oset-Gasque, M.J., Canadas, S., Gonzalez, M.P., 2006. Cadmium induces reactive oxygen species generation and lipid peroxidation in cortical neurons in culture. Free Radic. Biol. Med. 40, 940–951. Müller, T., Schuckelt, R., Jaenicke, L., 1994. Evidence for radical species as intermediates in cadmium/zinc-metallothionein-dependent DNA damage in vitro. Environ. Health Perspect. 102, 27. McCord, J.M., Fridovich, I., 1969. Superoxide dismutase. An enzymic function for erythrocuprein (hemocuprein). J. Biol. Chem. 244, 6049–6055. Mohamed, M., Abdellatif, M.D., Sabar, A., Elglammal, M.D., 2003. Sodium fluoride ion and renal function after prolonged sevoflurane or isoflurane anaesthesia. Br. J. Anaesth. 19, 79–83. Moron, M.S., Depierre, J.W., Mannervik, B., 1979. Levels of glutathione, glutathione reductase and glutathione S-transferase activities in rat lung and liver. Biochim. Biophys. Acta 582, 67–78. Mosmann, T., 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 65, 55–63.
136
e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 9 ( 2 0 1 5 ) 125–136
Ossola, J.O., Tomaro, M.L., 1995. Heme oxygenase induction by cadmium chloride: evidence for oxidative stress involvement. Toxicology 104, 141–147. Paglia, D.E., Valentine, W.N., 1967. Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J. Lab. Clin. Med. 70, 158–169. Prozialeck, W.C., Edwards, J.R., 2012. Mechanisms of cadmium-induced proximal tubule injury: new insights with implications for biomonitoring and therapeutic interventions. J. Pharmacol. Exp. Ther. 343, 2–12. Prozialeck, W.C., Vaidya, V.S., Liu, J., Waalkes, M.P., Edwards, J.R., Lamar, P.C., Bernard, A.M., Dumont, X., Bonventre, J.V., 2007. Kidney injury molecule-1 is an early biomarker of cadmium nephrotoxicity. Kidney Int. 72, 985–993. Renugadevi, J., Milton Prabu, S., 2010. Quercetin protects against oxidative stress-related renal dysfunction by cadmium in rats. Exp. Toxicol. Pathol. 62, 471–481. Renugadevi, J., Prabu, S.M., 2009. Naringenin protects against cadmium-induced oxidative renal dysfunction in rats. Toxicology 256, 128–134. Rockwell, P., Martinez, J., Papa, L., Gomes, E., 2004. Redox regulates COX-2 upregulation and cell death in the neuronal response to cadmium. Cell. Signal. 16, 343–353. Satarug, S., Moore, M.R., 2004. Adverse health effects of chronic exposure to low-level cadmium in foodstuffs and cigarette smoke. Environ. Health Perspect. 112, 1099–1103. Singh, V.P., 2005. Metal Toxicity and Tolerance in Plants and Animals. Sarup & sons.
Sinha, A.K., 1972. Colorimetric assay of catalase. Anal. Biochem. 47, 389–394. Smith, I.K., Vierheller, T.L., Thorne, C.A., 1988. Assay of glutathione reductase in crude tissue homogenates using 5,5 -dithiobis(2-nitrobenzoic acid). Anal Biochem. 175, 408–413. Stohs, S., Bagchi, D., 1995. Oxidative mechanisms in the toxicity of metal ions. Free Radic. Biol. Med. 18, 321–336. Urbschat, A., Obermuller, N., Haferkamp, A., 2011. Biomarkers of kidney injury. Biomarkers 16 (1), S22–S30. Waisberg, M., Joseph, P., Hale, B., Beyersmann, D., 2003. Molecular and cellular mechanisms of cadmium carcinogenesis. Toxicology 192, 95–117. Wang, Y., Fang, J., Leonard, S.S., Rao, K.M., 2004. Cadmium inhibits the electron transfer chain and induces reactive oxygen species. Free Radic. Biol. Med. 36, 1434–1443. Wilmes, A., Crean, D., Aydin, S., Pfaller, W., Jennings, P., Leonard, M.O., 2011. Identification and dissection of the Nrf2 mediated oxidative stress pathway in human renal proximal tubule toxicity. Toxicol. In Vitro 25, 613–622. Wu, G., Fang, Y.Z., Yang, S., Lupton, J.R., Turner, N.D., 2004. Glutathione metabolism and its implications for health. J. Nutr. 134, 489–492. Zhu, P., Frei, E., Bunk, B., Berger, M.R., Schmahl, D., 1991. Effect of dietary calorie and fat restriction on mammary tumor growth and hepatic as well as tumor glutathione in rats. Cancer Lett. 57, 145–152.
