Impact of various doses of curcumin on contrast media induced nephrotoxicity: a histopathological and biochemical study

1Water and Electrolytes Research Center & Department of Physiology, Isfahan University of Medical Sciences, Isfahan, Iran 2Department of Internal Medicine, Isfahan University of Medical Sciences, Isfahan, Iran 3Department of Community Nutrition, School of Nutrition and Food Sciences, Isfahan University of Medical Sciences, Isfahan, Iran 4Cellular and Molecular Research Center, Department of Anatomical Sciences, Faculty of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran 5Medical Plants Research Center, Shahrekord University of Medical Sciences, Shahrekord, Iran


Introduction
Radiocontrast agents are common substances used to enhance the visibility of internal organs structures in X-raybased imaging techniques such as computed tomography (CT), radiography, and fluoroscopy. Radiocontrast agents are typically iodine or barium compounds. Recently, the number of imaging methods using contrast agents (radiocontrast media) has been increased. Applying these methods has led to increased prevalence of renal failure (RF), especially in high-risk populations including patients with history of RF, congestive heart failure, and diabetes mellitus as well as the elderly. The incidence rate of RF caused by contrast agents (contrast-induced nephropathy [CIN]) following these imaging procedures is about 3%-5% in populations without risk factors while this rate may reach approximately 100% in people at risk (1,2). Several studies have revealed the effectiveness of medicinal plant extracts as a phytotherapeutic agent or as a protective agent in various diseases. Curcumin (CUR) (diferuloylmethane) is one of these compounds and is obtained from the milled Rhizomes of Curcuma longa Linn. It is consumed as a spice to give desirable flavor and color to food (3,4). CUR has numerous pharmaceutical properties such as antioxidant, anticancer, and antimicrobial (5)(6)(7). CUR also helps to reduce free radicals, inhibit lipids peroxidation, and increase the activity of superoxide dismutase (8,9). In addition, it has anti-inflammatory properties (10) and can reduce lipidemia and glycemia and improve Alzheimer disease (11).

Objectives
The purpose of this study was to investigate the protective effects of CUR in improving biochemical and histopathological parameters in CIN.

Experimental design
This study was performed on 36 Wistar rats weighing 200-250 g. Rats were purchased from the animal house of the Isfahan University of Medical Sciences. Animals were kept in standard conditions and maintained at room temperature (25 ± 2 º C) and had free access to water and food. All the group's animals were left for 5 days without any intervention for environmental compatibility and climatic conditions and then underwent their respective interventions. They were divided randomly into six groups of 6 each; negative control (NC) did not receive any drugs. Positive control (PC) was intraperitoneally injected with 10 ml/kg/day contrast media. CUR 50 and 250 mg/kg/d (CUR50; T1, CUR250; T2) groups were received intraperitoneally CUR suspended in corn oil for 10 days. CUR50 or CUR250 plus contrast media (CUR50 + CM; T3, CUR250 + CM; T4 ) groups were received intraperitoneally CUR + CM with 10 mL/kg/d of contrast media and CUR suspension for 10 days.
Drug doses were selected according to a previous study (6). At the end of study, the rats were anesthetized as their blood samples were taken by heart puncture, and the animals were sacrificed. The kidney was removed for histopathological investigation.

Assessment of renal function
Blood serum was collected after centrifuging at 4000 rpm for 10 minutes. Serum urea and creatinine (Cr) were measured by a biochemical analyzer. Then, serum Cr and blood urea nitrogen (BUN) levels were measured using a commercially available kit (Pars Azmon, Iran) by an autoanalyzer (Hitachi 902, Japan) in accordance with the manufacturers' guidelines.

Histopathology analysis
Kidneys were removed placed in 10% formalin buffer for 24 hours. The specimens were subjected to sectioning and staining with hematoxylin & eosin method. Then, they were examined using an optical microscope (Olympus CH-2, Japan) by a nephropathologist who was blind to the grouping of the rats, and the percentage of flattening, degeneration, renal tubular dilatation, debris, and vacuolization was determined.

Ethical issues
The research was approved by Ethics Committee of Isfahan University of Medical. Prior to the experiment, the protocols were confirmed to be in accor dance with the guidelines of Animal Ethics Committee of Isfahan University of Medical Sciences (code# IR.MUI. REC.1395.3.823). This study was extracted from internal medicine residency of Hossein Safi (Thesis# 395823).

Statistical analysis
The data were expressed as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) was used to compare differences between groups and followed by Tukey's multiple comparison test using SPSS 20 software. P ≤ 0.05 was considered as the level of statistical significance.

Biochemical parameters
It is apparent from Table 1 that serum Cr did not significantly increase in rats after contrast media injection compared to negative control group, but BUN concentration increased significantly after contrast media injection (P = 0.023). BUN concentration was decreased significantly in rat treated with 250 mg of CUR before and simultaneously with contrast media as compared to positive control group (P < 0.05). Also treatment with 250 mg/kg CUR caused a significant decrease in Cr in rats with nephrotoxicity (P = 0.001).

Histopathology
According to Figure 1, there was significant increase in the percentage of debris, degeneration, vacuolization of renal tubular cells and dilation of tubular lumen, in kidneys of group receiving contrast media compared to control group (P = 0.002, 0.002, 0.004, and 0.024, respectively), while there was no significant difference in flattening of renal tubular cells between the NC group and the PC group (P > 0.05). The percentage of tubular cell debris, degeneration, vacuolization and tubular lumen dilatation in nephrotoxic rats receiving 50 mg/kg and 250 mg/kg CUR decreased insignificantly compared to group receiving contrast media, while flattening did not change. Also, there was no significant difference in all lesions between healthy rats receiving 50 mg/kg or 250 mg/kg of CUR and other healthy rats (P > 0.05).

Discussion
In the recent years, the frequency of experiments using contrast media has increased with the growth of the elderly population. The current study found that serum Cr was increased significantly after contrast media injection, however, BUN concentration did not significantly increase. The findings of the current study are consistent with previous study by Nasri et al, that found the contrast media contributed to increase in BUN, Cr, and renal lesions (12). The mechanisms of renal damage following exposure to contrast media have not yet been known. However, previous studies reported that renal hemodynamics disturbances and direct damage to the renal tubular cells are involved in contrast media-induced renal damage. Contrast media reduces renal circulation especially in the medulla by increasing osmotic load and blood viscosity, and the resulting hypoxia leads to the production of oxygen free radicals. Free radicals cause damage to different cells including tubular cells and their components such as mitochondria that could ultimately induce cell necrosis (13). The oxygen free radicals play a role in tissue damage for various causes including tissue ischemia, toxins' effects, and immune-mediated damage all of which contribute to increasing the production of these harmful radicals (14).
Another important finding of our study was that CUR reduced serum Cr, BUN, and renal tissue lesions in rats with contrast media-induced nephropathy. Our finding confirmed the result of a previous study that investigated the effect of CUR on CIN in rats, and showed CUR increased the levels of glutathione, superoxide dismutase, catalase, and glutathione peroxidase that are important factors for oxidative stress at glomerular level, and finally cause cell apoptosis and RF (15). Venkatesan study showed that CUR intake in rats with adriamycin-induced nephrosis increased the urinary excretion of N-acetyl-β-D-glucosaminidase, fibrinogen, and glycosaminoglycan as well as the levels of glutathione and glutathione peroxidase in the kidney, and therefore protected the kidney (16). Some previous studies showed CUR exerted protective effects on the kidney and used to treat of diabetic nephropathy, chronic RF, and nephrotoxicity induced by gentamicin, adriamycin, chloroquine, and cisplatin (17)(18)(19). In a study, the administration of CUR in rats with nephropathy reduced the expression of chemokine genes regulated upon activation of normal T cell, MCP-1 (monocyte chemotactic protein 1), and AIF (allograft inflammatory factor-1) in the renal tubules, Figure 1. Data are expressed as mean ± SD. The percentage of tubular cell debris, degeneration, vacuolization and tubular lumen dilatation of kidney, in different groups; NC; negative control (healthy rats); PC; positive control (nephrotoxic rats); CUR 50 (T1) and CUR250 (T2) groups; nephrotoxic rats that were intraperitoneally injected with 50 mg/kg and 250 mg/kg CUR, respectively; CUR50 + CM (T3) and CUR250 + CM (T4); normal rats that were intraperitoneally injected with 50 mg/kg and 250 mg/kg CUR, respectively. a, b, and c: Dissimilar letters in each column represent significant difference (P < 0.05).  (20). Various studies support the role of antioxidant agents in preventing contrast induced nephropathy and other causes of kidney damage (1). Today, because of the protective effects of phenolic compounds against reactive oxygen species and improving antioxidant system of the body, herbal treatments, such as applying supplements and plant extracts rich in polyphenols especially flavonoids and phenolic acids, are common throughout the world (21,22). CUR (C21H20O6) is an active compound of turmeric (23,24), and its effects are due to anti-inflammatory activity by proinflammatory cytokines and inhibiting NF-Kβ and its antioxidant properties. In addition, studies revealed that CUR had the antiapoptotic properties and prevented the development of renal tubular cell apoptosis by reducing the activation of proteins (25). Therefore CUR may consider an important factor in preventing CIN in rats.

Conclusion
Based on the results of this study, CUR was effective to improve contrast media induced nephropathy by decreasing of BUN, Cr, the percentage of tubular cell debris, degeneration, vacuolization and tubular lumen dilatation in kidney.

Authors' contribution
HS, HN and MN conducted the experimental procedures and helped to prepare the first draft of the article. MRF conducted the statistical analysis and prepared the primary draft. MK and EM revised the manuscript. MN designed and supervised the research and completed the final manuscript. All authors read and signed the paper.

Conflicts of interest
The authors declare no conflict of interest.

Ethical considerations
Ethical issues (including plagiarism, data fabrication, double publication) have been completely observed by the authors.

Funding/Support
This study was supported by Isfahan University of Medical Sciences. This study was extracted from internal medicine residency of Hossein Safi (Thesis# 395823).