2016 Volume 39 Issue 6 Pages 1042-1046
Carbon tetrachloride (CCl4) is commonly used as a chemical inducer of experimental liver injury. In addition, many studies showed that CCl4 can induce kidney damage. In the current study, we evaluated the protective effect of zinc (Zn) against CCl4-induced nephrotoxicity. We hypothesized that this protective effect would result from the ability of Zn to serve as an inducer of metallothionein (MT), a known endogenous scavenger of free radicals. We administered Zn (as ZnSO4) 50 mg/kg subcutaneously once daily for 3 successive days prior to a single intraperitoneal administration of CCl4 4 g/kg in male ddY mice. Our results showed that Zn pretreatment significantly decreased creatinine and blood urea nitrogen levels and reduced renal histopathological damage at 6 h post-CCl4 injection, observations consistent with enhanced antioxidative activity in the kidney. Moreover, kidney MT levels in the Zn+CCl4-treated group decreased by greater than 70% compared with levels in the Zn-alone group, implying that MT was consumed by CCl4-induced radicals. These findings suggest that prophylaxis with Zn protects mice from CCl4-induced acute nephrotoxicity, presumably by induction of MT, which in turn scavenges radicals induced by CCl4 exposure.
The kidney is the waste filtering and disposal system of the animal body. This organ is also the target of numerous xenobiotic toxicants. Carbon tetrachloride (CCl4) is a haloalkane, a class of chemcials that were widely used as grain fumigants, anthelmintics and intermediates in the synthesis of chlorofluorocarbons.1) CCl4 is one of the most extensively studied hepatotoxicants, and the mechanism by which CCl4 causes hepatotoxicity is well documented. Nonetheless, toxic effects of CCl4 also are observed in other tissues, including kidney,2–11) testis,3,5) and brain.4) Especially, kidney is likely to be targeted to CCl4 as well as liver because much amount of CCl4 is distributed to kidney compared to liver when CCl4 is administered systemically.12)
The molecular mechanism of CCl4-induced renal toxicity has been examined in detail. Specifically, CCl4 is metabolized through the cytochrome P450 CYP2E1 to produce the highly toxic trichloromethyl free radical and/or trichloromethyl peroxyl free radical. These radicals induce renal injury because free radicals bind to intracellular proteins, lipids of cell membrane, or DNA, leading in turn to protein denaturation, lipid peroxidation in the cell membrane, and oxidative DNA damage (respectively), resulting in the death of kidney cells.7,13)
Many studies have reported that antioxidants can prevent hepatic damage and nephropathy by counteracting free radicals and preventing lipid peroxidation.2,4,6,11) Zinc (Zn) is well known as an essential trace element for a variety of biological activities. Zn is a component of more than 300 metalloenzymes and contributes to the maintenance of structure and catalytic activity in these enzymes. In addition, Zn plays regulatory roles in diverse cellular processes such as signaling transduction and gene expression.14,15) Aside from these physiological roles, Zn also may reduce oxidative stress by inducing metallothionein (MT), a small, cysteine-rich protein lacking aromatic amino acids and histidine16) that acts as a chelator or scavenger of heavy metals, reactive oxygen species (ROS), and free radicals.17) We previously reported that pretreatment with Zn prevented CCl4-induced lethal toxicity in mice and that Zn-induced MT may be involved in the detoxification mechanism18); however, we did not evaluate the potential renal protective effects of Zn prophylaxis. To our knowledge, previous studies have not examined the potential protective effect of Zn or MT against CCl4-induced nephrotoxicity.
Therefore, in the current study, we investigated whether pretreatment with Zn is sufficient to attenuate CCl4-induced acute nephrotoxicity.
Male ddY mice were purchased from Japan SLC (Shizuoka, Japan). Following arrival at our facility, mice were maintained under standard conditions of controlled temperature (24±1°C), humidity (55±5%), and light (12 : 12 h light/dark cycles) with free access to water and food. Experimental treatments were performed using eight-week-old animals. Following the experiment, any surviving mice were sacrificed using pentobarbital. All experiments were approved by the Institutional Animal Care and Experiment Committee of Kinjo Gakuin University (No. 110).
Experimental ProtocolMice were divided into four groups. At −72, −48, and −24 h, animals of the Group 2 (Zn group) and Group 4 (Zn+CCl4 group) were administered (by subcutaneous (s.c.) injection at 24-h intervals) with 50 mg/kg ZnSO4 (Nacalai Tesque, Kyoto, Japan). Animals of Group 1 (control group) and Group 3 (CCl4 group) were injected s.c. with equivalent volumes of saline vehicle. Twenty-four hours after the final ZnSO4 or saline injection, both the CCl4 group and Zn+CCl4 group were administered intraperitoneally (i.p.) with 4 g/kg (at 5 mL/kg) CCl4. The control and Zn groups were injected i.p. with equivalent volumes of olive oil. At 6 h after the CCl4 or olive oil injection, mice from each group were euthanized and bled for plasma. The resulting plasma samples were stored at −80°C pending creatinine and blood urea nitrogen (BUN) assays. Kidneys were harvested from each of these animals, and separate samples from each kidney were stored at −80°C (pending anti-oxidant power assay and MT determination) or fixed in 15% neutral buffered formalin (pH 7.2) (pending processing for histological evaluation).
Measurement of Creatinine and BUNConcentrations of plasma creatinine and BUN were measured using Creatinine Liquid Reagents Assay (DIAZYME, Poway, CA, U.S.A.) and BUN Wako Test (Wako Pure Chemical Industries, Ltd., Osaka, Japan) (respectively) according to the manufacturer’s instructions. The absorbance of the assay reaction products was measured at 550 or 570 nm, respectively. For relative quantification, calibration curves were prepared using standard solutions.
Determination of Total Antioxidant Power in the KidneyTotal antioxidant power levels in the kidney were examined by colorimetric total antioxidant power assay kit (TA02, Oxford Biochemical Research, Oxford, MI, U.S.A.) according to the manufacturer’s protocol. Aliquots (0.1 g each, including mixed cell types) of right renal tissue were homogenized with 900 µL ice-cold phosphate-buffered saline (PBS) containing protease inhibitor (Nacalai Tesque) and centrifuged at 18000×g for 20 min at 4°C. The resulting supernatant (diluted to yield consistent total protein concentrations) for each sample was used for further steps. The antioxidant level was determined by the reduction of Cu2+ to Cu+ to permit assessment of the combined action of all antioxidants present in the sample. Generated Cu+ was detected by determining the formation of complexes between Cu+ and bathocuproine; stable complex was detected at 450 nm. The obtained absorbance values were compared to a standard curve obtained using uric acid as the reductant.
Measurement of Malondialdehyde (MDA) Levels in KidneysThe total MDA levels in the kidney were examined by colorimetric thiobarbituric acid-reactive substance microplate assay kit (FR40, Oxford Biochemical Research, Oxford, MI, U.S.A.) according to the manufacturer’s protocol. Aliquots (0.1 g each, including mixed cell types) of right renal tissue were homogenized with 900 µL ice-cold PBS containing protease inhibitor and centrifuged at 18000×g for 20 min at 4°C. An aliquot (200 µL) of the resulting supernatant was combined with saturated ammonium sulfate (40 µL; Wako Pure Chemical Industries, Ltd.) and trichloroacetic acid (35 mg; Nacalai Tesque) and vortexed. After centrifugation (3000×g, 10 min, 4°C), supernatants were incubated at 65°C for 45 min. The absorbance of this reaction product was measured at 532 nm. For relative quantification, calibration curves were prepared using a standard solution of MDA.
Measurement of MT Levels in the KidneyThe renal MT protein levels were determined by the cadmium saturation-hemolysate method (Cd-hem method). Aliquots (0.2–0.3 g each, including mixed cell types) of right kidney tissue were homogenized with 5 volumes of 0.25 M sucrose (Nacalai Tesque). The homogenates were centrifuged at 18000×g for 20 min at 4°C to isolate the postmitochondrial supernatant; suitable aliquots then were used for MT assay by the Cd-hem method, as described previously.19,20)
Histopathological FindingsFor histological analysis, each left kidney was perfused with 15% phosphate-buffered neutral formalin (pH 7.2), dehydrated, and embedded in paraffin. Embedded tissues were sectioned at 4-µm thicknesses and stained with hematoxylin and eosin (H&E). Histopathological features in the slices were observed via light microscope.
Statistical AnalysisAll data from the control and treatment groups were obtained from the same numbers of replicated experiments. All experiments were performed independently at least two times. The results were analyzed using one-way ANOVA with post-hoc Tukey–Kramer’s test. All statistical analyses were performed using SPSS 19.0 J software (Chicago, IL, U.S.A.). Values of p<0.05 were considered statistically significant.
Plasma concentrations of creatinine and BUN are known as markers for injury and dysfunction of the kidney. In the present study, the effect of CCl4 administration on the blood concentration of these markers was tested. Systemic (i.p.) exposure of mice to CCl4 significantly increased the plasma concentrations of creatinine (Fig. 1A) and BUN (Fig. 1B). In contrast, three-time pretreatment with Zn significantly attenuated the CCl4-induced acute increases in creatinine and BUN levels. There was no significant difference in these parameters between the control group and the Zn group (i.e., in the absence of CCl4 exposure).
Mice were injected s.c. with 50 mg/kg ZnSO4 (three times, q24h). Twenty-four hours after final pretreatment, mice were injected i.p. with 4 g/kg CCl4. Creatinine (A) and BUN (B) levels in plasma were determined at 6 h after i.p. injection. Data are presented as the mean±S.D. of five or six mice. ** Significant difference between compared values (** p<0.01).
In parallel with the measurement of creatinine and BUN (Fig. 1), we evaluated histopathological changes in renal tissue. The morphology of tubules, glomeruli, and tubulointerstitial cells in the Zn-treated group (Fig. 2B) were similar to that seen in the control group (Fig. 2A). Exposure to CCl4 induced swelling, degeneration, and the appearance of protein columna (penetration of protein) in the renal proximal tubules (Fig. 2C). In contrast to the CCl4-treated group, Zn+CCl4-treated group showed almost normal morphology of the kidney (Fig. 2D).
Mice were injected s.c. with 50 mg/kg ZnSO4 (three times, q24h). Twenty-four hours after final pretreatment, mice were injected i.p. with 4 g/kg CCl4. Kidneys were harvested 6 h after i.p. injection, fixed, processed, and stained with H&E. (A), (B), (C), and (D) provide 20× magnification images of representative H&E-stained sections from kidneys obtained from control, Zn, CCl4, and Zn+CCl4 animals. Section for (C) reveals swelling, degeneration, and the appearance of protein columna (penetration of protein) in renal proximal tubules in a CCl4-exposed animal, in contrast to the mostly normal renal structure seen in (A), (B), and (D). Green arrows, black arrows and orange arrows indicate swelling, degeneration, and protein columna, respectively.
To investigate Zn-induced nephroprotective activity against CCl4 in greater detail, we tested oxidative stress in the kidney tissue. CCl4 treatment significantly increased renal MDA levels compared with those detected in the control group (Fig. 3A). In contrast, pretreatment with Zn of CCl4-treated mice significantly attenuated the CCl4-induced increase in MDA levels. No significant difference in the levels of MDA was detected between the control and Zn-treated groups.
Mice were injected s.c. with 50 mg/kg ZnSO4 (three times, q24h). Twenty-four hours after final pretreatment, mice were injected i.p. with 4 g/kg CCl4. MDA levels (A) and the antioxidant power (B) in kidney were determined 6 h after i.p. injection. Data are presented as the mean±S.D. of five or six mice. ** Significant difference between compared values (** p<0.01).
Total antioxidant level is commonly used as an indicator of oxidative stress because this parameter inversely correlates with oxidant level. As shown in Fig. 3B, the total antioxidant level in CCl4-treated mice was markedly decreased compared with that in the control group. In addition, Zn pretreatment indicated upregulation of antioxidant power. In contrast, pretreatment of CCl4-treated mice with Zn significantly increased the renal total antioxidant level compared with that seen in mice exposed to CCl4 alone.
Zn Attenuation of CCl4 Acute Toxicity as Measured by MT LevelsTo examine the involvement of MT in protection from CCl4-induced nephrotoxicity, we determined MT protein levels by the Cd-hem assay (Fig. 4). Pretreatment with Zn increased the amount of MT protein up to 370 µg/g kidney (about 24 times higher than that seen in control animals). Notably, this elevation of MT level by Zn prophylaxis was attenuated more than 70% by subsequent CCl4 exposure.
Mice were injected s.c. with 50 mg/kg ZnSO4 (three times, q24h). Twenty-four hours after final pretreatment, mice were injected i.p. with 4 g/kg CCl4. MT levels in kidney were determined 6 h after i.p. injection. Data are presented as the mean±S.D. of five or six mice. ** Significant difference between compared values (** p<0.01).
Many studies have demonstrated that CCl4 can induce dysfunction and histopathological changes in the kidney.2–11) The present study also showed that plasma levels of creatinine and BUN were markedly increased in the CCl4-treated mice. As in previous studies, we detected acute histological changes in kidney morphology in CCl4-treated mice, although the renal damage observed in the current study was milder than that reported in previous studies by others. Hermenean et al. reported that CCl4 (at 1.6 g/kg) induced severe renal damage at 24 h after injection, in spite of using a lower dose than that of the present study (4 g/kg).8) This histological difference implies that the renal injury observed in our study (at 6 h post-CCl4) was an initial phase, with more serious damage accumulating by 24 h post-dose. In addition, we previously showed that severe hepatic disorder was observed at 6 h after CCl4 administration,18) suggesting that initial toxicity to kidney was lower than that in liver, even though CCl4 distribution in kidney is higher than that in liver.12) The initial phase of CCl4-induced toxicity is mediated by CYP2E1. Wong et al. reported that the CYP2E1 KO mouse is resistant to CCl4 toxicity.21) Although CYP2E1 is expressed in the kidney, the level of expression in kidney is much lower than in liver.22,23) This difference in CYP2E1 protein level may be one cause of the difference in organ sensitivity.
In the present study, pretreatment with Zn effectively protected mice from CCl4-induced acute kidney damage by attenuating the CCl4-induced elevation in function markers (creatinine and BUN) and alleviating renal histological changes. These results showed that Zn has protective effect against radical-induced renal dysfunction and histopathologic damage. In addition, Zn attenuated increases in MDA levels and restored total antioxidant power in CCl4-treated mouse kidney. These protective effects may be due to the powerful antioxidative activity of Zn itself.14,15) These results also suggest that Zn may attenuate oxidative stress by decreasing levels of lipid peroxide in CCl4-exposed mouse kidney, an effect that may (at least partially) prevent renal damage.
The antioxidative activities of Zn may reflect the increased MT level that results from pretreatment with Zn, given that MT is a potent radical scavenger. It is well known that MT exhibits antioxidative activity against ROS via scavenging of free radicals,24–27) with MT estimated to exhibit activity some 300-fold higher than that of glutathione (GSH). In addition, we showed in the present study that the Zn-induced increase of kidney MT levels was attenuated by more than 70% following CCl4 injection, suggesting that MT is consumed by CCl4-derived radicals. The Cd-hem assay, used to determine MT in our present study, is based on quantification of metal ions (Cd) bound to MT molecules. Since oxidized MT does not bind to Cd and is not detected in this assay, we speculate that CCl4-derived free radicals preferentially attacked MT, resulting in a loss of Cd-binding activity.
Indeed, many researchers have reported that CCl4 treatment significantly reduces the renal total content of reduced GSH, and that CCl4 metabolites can react with sulfhydryl groups of GSH and protein thiols to alter the redox status of cells.28–30) Under oxidative stress (such as that induced under our experimental conditions), reduced GSH is likely to be oxidized directly and is converted to the oxidized forms of GSH.31) Therefore, antioxidant level is reduced in our current study (change 0.44±0.01 to 0.35±0.03 mM by CCl4-injection). In contrast, although Zn-induced kidney MT levels was reduced by more than 70% by CCl4-injection, no significant change in total antioxidant power between the Zn and Zn+CCl4 groups was observed (0.50±0.01, 0.49±0.04 mM). In this context, total antioxidant level was poorly elevated despite marked MT increase in Zn-pretreatment group. Although we cannot exhibit rational assumption about the causative factor of low detecting sensitivity, a possible cause is that the detecting sensitivity to antioxidant potential of MT is considerably low. Hence, we speculate that total antioxidant power was comparable between Zn-treated group and control group because vast of antioxidant power of MT is not reflected in total antioxidant power, that also supports this hypothesis.
In conclusion, we have demonstrated that pretreatment with Zn suppresses CCl4-induced acute nephrotoxicity; we hypothesize that the nephroprotective effect of Zn is due to the antioxidative role and free radical scavenging activity of MT, the expression of which is induced by Zn pretreatment. To our knowledge, this is the first evidence that Zn prophylaxis protects against CCl4-induced acute nephrotoxicity. Although further investigation is needed to clarify the involvement of MT, these findings are expected to contribute to improved understanding of the protective effect of Zn against radical-induced organ injury and disease.
The authors thank Dr. Kenichi Saeki (Kinjo Gakuin University, Japan) for his kind suggestions.
The authors declare no conflict of interest.
