Effects of Quercetin on Tubular Cell Apoptosis and Kidney Damage in Rats Induced by Titanium Dioxide Nanoparticles

Background Recent studies have demonstrated that many nanoparticles have an adverse or toxic effect on the kidney. Objective To investigate the nephroprotective effect of quercetin (QT) against renal injury induced by titanium dioxide nanoparticles (NTiO2) in rats. Methods NTiO2-intoxicated rats received 50 mg/kg of NTiO2 for seven days. The QT + NTiO2 group was pretreated with QT for seven days before being administered NTiO2. Uric acid, creatinine, and blood urea nitrogen were considered to be biomarkers of nephrotoxicity. Catalase (CAT) and superoxide dismutase (SOD) activities and renal levels of malondialdehyde (MDA) were measured to assess the oxidative stress caused by NTiO2. Results NTiO2 significantly increased the plasma level of the biomarkers. It also significantly decreased the activities of CAT (P = 0.008) and SOD (P = 0.004), and significantly increased the MDA levels (P = 0.007). NTiO2 caused proximal tubule damage, the accumulation of red blood cells, the infiltration of inflammatory cells, and reduced the glomerular diameters, as well as induced apoptosis in the proximal tubules. Pre-treatment with QT attenuated the histological changes, normalised the plasma biomarkers, suppressed oxidative stress, ameliorated the activities of CAT (P = 0.007) and SOD (P = 0.006), and reduced apoptosis (P < 0.001). Conclusion QT was found to have a potent protective effect against nephrotoxicity induced by NTiO2 in rats. It also reduced apoptosis caused by NTiO2.


Introduction
The kidney is one of the most sensitive organs to toxic substances in the body because of its high blood flow and its ability to concentrate waste (1). Previous studies demonstrated that the administration of nanoparticles (NPs) to rodents resulted in the particles accumulating in various tissues, including the liver, brain, kidney, and spleen (2). An NP is a microscopic particle that has at least one dimension that is less than 100 nm. Toxicological studies have confirmed that NPs are potentially harmful because of their high surface area to volume ratio and unique physicochemical properties (3). Among the various metal nanomaterials, titanium dioxide nanoparticles (NTiO 2 ) are used in a variety of consumer products, including sunscreens, cosmetics, clothing, electronics, paints, and surface coatings (4). NTiO 2 is also widely used in toothpaste, food colourants, and nutritional supplements. However, NTiO 2 can accumulate in the renal tissue and induce renal injuries (5).
In recent years, herbal medicines, such as flavonoids, have been shown to have protective effects against chemically induced toxicities (6). For example, quercetin (QT) is a powerful flavonoid that has a wide range of health benefits because of its anti-hypertensive, anti-diabetic, anti-inflammatory, anti-oxidant, anti-cancer, neuroprotective, and anti-viral activities (7). QT is found in lettuce, onion, tomato, red grapes, olive oil, apple skin, tea, coffee, bracken fern, and citrus fruits (8). QT also has a protective effect against some toxic materials, such as arsenic, bisphenol A, and lead, in kidney tissue (9)(10)(11). QT has anti-apoptotic effects against some drugs and chemicals, such as doxorubicin, cyclophosphamide, and lead (11,12). In the present work, the effect of QT on NTiO 2 -induced nephrotoxicity and apoptosis in rats was investigated.

Animals
Thirty-two healthy adult female Wistar rats, 8-10 weeks old and 180 g-200 g, were used in this work. The estrous cycle was not synchronised in the rats. The rats were purchased from the Experimental Research Center of Ahvaz Jundishapur University of Medical Sciences. The rats were maintained in individual cages and kept under a light-dark cycle of 12:12 at a temperature of 22 °C ± 3 °C and a humidity of 50% ± 5%. They were also given free access to commercial food (pellets) and water.
This work was performed according to the guidelines of the Animal Ethics Committee of Ahvaz Jundishapur University of Medical Sciences (approval number: IR.AJUMS. REC.1395.650).

Experimental Design
The study was based on the block randomisation method. Before the experiment, the 32 rats were divided into four groups. Based on the power calculation and estimation of attrition from our pilot study and previous studies (6,13), the sample size was determined to be eight rats per group. The control group was given saline for three weeks. The QT group received 75 mg/kg QT for three weeks (14). The NTiO 2 -intoxicated group received 50 mg/kg NTiO 2 on day 7 after a daily administration of saline and was followed for two weeks. The QT + NTiO 2 group received 75 mg/kg QT for one week followed by the concomitant administration of 50 mg/kg NTiO 2 for two weeks. All treatments were given by gavage. The doses of NTiO 2 and QT were determined based on our pilot study and previous studies (13,14). The stock solution of NTiO 2 (Sigma-Aldrich) was prepared in Milli-Q water as previously described (6). The particle size and morphology of the prepared NTiO 2 was determined using an atomic force microscope (AFM). The NPs had a spherical morphology, homogeneous particle size distribution, and a mean size less than 100 nm.
Twenty-four hours after the last administration, blood samples were collected, the rats were euthanised, and the renal tissues were removed and weighed. The left kidneys were maintained in a freezer at -80 °C to assess the superoxide dismutase (SOD) and catalase (CAT) activities and malondialdehyde (MDA levels. The right kidneys were fixed in 10% formalin for the histological assessments.

Biochemical Tests
Blood samples from the tail vein of the rats were collected and centrifuged. The plasma concentrations of uric acid, creatinine (Cr), and blood urea nitrogen (BUN) were determined spectrophotometrically using suitable kits.

Lipid Peroxidation
Lipid peroxidation was measured as previously described (15). Briefly, kidney tissue homogenates were prepared and 500 μL of supernatant from each sample was added to 1.5 mL 10% trichloroacetic acid and centrifuged for 15 min at 5000× g. Then, 1.5 mL of the supernatant was mixed with 2 mL 0.67% Thiobarbituric acid (TBA) and boiled for 0.5 h. After cooling, 2 mL n-butanol was added to each sample and centrifuged at 5000× g for 20 min. The absorbance was read at 535 nm using a spectrophotometer.

Superoxide Dismutase (SOD) Activity
The SOD activity was determined using a Ransod kit (Randox Laboratories Ltd., UK) as previously described (15). This method produces a water-soluble formazan dye upon reduction with the superoxide anion. The rate of the reduction is linearly related to the xanthine oxidase (XO) activity, which is inhibited by SOD.
The inhibition activity of SOD can be measured using a spectrophotometer at 505 nm.

CAT Activity
CAT activity was measured as described (16) in a previous study. Briefly, the CAT present in the sample reacts with hydrogen peroxide (H 2 O 2 ) to generate H 2 O and O 2 . The unconverted H 2 O 2 can be determined colorimetrically at the optical density (OD) of 570 nm.

Histological Changes
In this study, six sections per rat were stained with hematoxylin and eosin (H & E) and assessed for the following histological criteria: nuclear pyknosis, infiltration of inflammatory cells, brush border loss, and accumulation of red blood cells (RBC). The average percentage of each feature was determined. The infiltration of inflammatory cells and the accumulation of RBCs were divided into four categories and the averages were considered. The categories were normal (0), weak (1), moderate (2), and intense (3). Two researchers, who were blinded to the control and experimental groups, analysed the slides independently (17).

TUNEL Staining
The in situ Cell Death Detection kit (POD, Roche) was used for TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) staining. The paraffin sections were dewaxed and incubated with proteinase K for 0.5 h at 24 °C. The sections were exposed to the TUNEL reaction mixture in a humidity chamber at 37 °C for 1 h. The sections were first incubated with Anti-Fluorescein-AP for 0.5 h at 37 °C, rinsed in deionised water, and incubated with DAB substrate (Sigma-Aldrich) for 5 min. Cells with a homogeneous dark brown nucleus were considered to be TUNEL-positive cells (18).

Statistical Analysis
To make comparisons between the four groups, a statistical analysis was performed using one-way ANOVA followed by a post hoc least significant difference (LSD) or Tukey's test for multiple pairwise comparisons. Data were expressed as the mean and standard deviation (SD). A P-value less than 0.05 was considered statistically significant.

Body and Renal Weights
The body weights in the control and experimental groups were similar and there were no significant differences between them. The body and renal weights in the NTiO 2 -intoxicated group significantly decreased (P = 0.009 and P = 0.006, respectively). The QT-pretreated group had significantly higher body and renal weights compared with the NTiO 2 -intoxicated group (P = 0.042 and P = 0.036, respectively). The results are shown in Table 1.

Biochemical Tests
In the QT group, the blood levels of uric acid, Cr, and BUN were slightly decreased. However, the blood concentrations of uric acid, Cr, and BUN were significantly elevated in the NTiO 2 group (P < 0.001). In the QT + NTiO 2 group, the levels of uric acid, Cr, and BUN were significantly reduced compared with the NTiO 2intoxicated group (P = 0.006, P = 0.003, and P = 0.007, respectively). The results are shown in Figure 1.   Values expressed as mean ± SD for eight rats. * P < 0.05, ** P < 0.001, † P < 0.01; * and † symbols respectively indicate comparison to control and NTiO2-intoxicated groups

MDA Level, and SOD and CAT Activities
NTiO 2 significantly increased the renal level of MDA (P = 0.007). In the QT + NTiO 2 group, the renal level of MDA was significantly decreased compared with the NTiO 2 -intoxicated group (P = 0.08). The CAT and SOD activities were significantly reduced in the NTiO 2 group (P = 0.008 and P = 0.004, respectively). The CAT and SOD activities were significantly elevated in the QT-pretreated group compared with the NTiO 2 -intoxicated group (P = 0.006 and P = 0.007, respectively). The results are shown in Figure 2.

Histological Changes
The kidney sections in both the control and QT groups all had a normal appearance. The administration of NTiO 2 significantly increased the infiltration of inflammatory cells, accumulation of RBCs, brush border loss, and fat deposits in the proximal cells, while the diameter of the glomerulus was significantly decreased. The administration of QT + NTiO 2 ameliorated the proximal tubule damage, infiltration of inflammatory cells, accumulation of RBCs, and the diameter of the glomerulus compared with the NTiO 2 -intoxicated group (Figure 3 and Table 2).

TUNEL Staining
Apoptosis was observed only in the proximal cells. As shown in Figure 4, a few proximal cells in some of the tubules in the control and QT groups showed TUNEL-positive staining. In the NTiO 2 -intoxicated group, TUNEL-positive cells were observed in most of the proximal tubules, while the percentage of apoptotic cells was significantly increased (P < 0.001). In the QT + NTiO 2 group, the percentage of TUNEL-positive cells was significantly less than in the NTiO 2 group (P < 0.001).  Values expressed as mean ± SD for eight rats. * P < 0.05, ** P < 0.01, *** P < 0.001, † P < 0.01; * and † symbols respectively indicate comparison to control and NTiO 2 -intoxicated groups.

Discussion
The results of our study demonstrated that QT protected against kidney damage induced by NTiO 2 . The results showed that the weight of the kidneys decreased in the NTiO 2 group. This change may have been the result of damage to the proximal tubule and the glomerulus. However, the body weight of the NTiO 2intoxicated group also decreased. This suggested that NTiO 2 has a toxic effect on the kidney and may induce apoptosis in other tissues. Hong and Zhang showed that NTiO 2 induced liver damage and enhanced the apoptotic index of hepatocytes in rats (19). Another study found that NTiO 2 can damage mouse testicular tissue and induce germ cell apoptosis (14). Zhao et al. showed that NTiO 2 induced follicular atresia in female mice (20).
In this study, QT reversed the renal weight loss induced by NTiO 2 . Sangai et al. showed that QT mitigated the effects of bisphenol A on the body and organ weights of mice (10).
One study showed that zinc oxide NP (ZNP) significantly elevated the levels of BUN, Cr, and uric acid biomarkers. When the kidney is damaged, these biomarkers, which are inside the proximal cells of the nephrons, are released into the bloodstream. Hence, elevated concentrations of the biomarkers indicate damage to the proximal cells (21). QT caused the levels of the biomarkers to decrease, which indicates that this flavonoid has beneficial effects on the proximal cells.
This study showed that NTiO 2 has necrotic effects on the kidney as evidenced by the destruction of tubular structure, vacuolation (fat deposit), nuclear pyknosis of the proximal cells, and the accumulation of RBCs and inflammatory cells. In a study by Fartkhooni et al., the administration of NTiO 2 induced damage to the proximal tubules and glomerulus (22). In this study, histological changes that were caused by NTiO 2 were significantly attenuated by QT. The improvement of renal tissue was accompanied by a significant reduction in the plasma levels of the biomarkers and a significant increase in renal weight.
Thus, due to the anti-inflammatory and anti-oxidant properties of QT, it may inhibit NTiO 2 -induced renal damage in rats. Liu et al. showed that QT also suppressed the inflammatory response induced by lead in the kidneys of rats (23).
In this study, the MDA concentration in kidney tissue was significantly increased by NTiO 2 . MDA is an indicator of the peroxidation process. Fat deposits in the proximal cells indicate abnormal fat metabolism. Previous studies demonstrated that NTiO 2 induced lipid peroxidation (fat deposit) and oxidative stress in the kidney of rodents (24). Another study found that NTiO 2 generates apoptosis and oxidative stress in human nephrons (25). Zhao et al. suggested that NTiO 2 causes the accumulation of reactive oxygen species, suppresses the antioxidative systems, and triggers nephritis in the kidney (26).
As discussed in the results, QT attenuated the fat deposits in proximal cells induced by NTiO 2 . Another study found that QT improved the fat deposits in the proximal tubules of doxorubicin-intoxicated rats (27).
In this study, the significantly reduced activities of SOD and CAT indicated that NTiO 2 induced oxidative stress in renal tissue. However, pre-treatment with QT significantly reversed the activities of CAT and SOD, which indicated the anti-oxidant property of QT.
In this study, both apoptosis and necrosis were observed at the same time in the NTiO 2 group. Wilhelmi et al. demonstrated that ZNP induced both apoptosis and necrosis in macrophages (28). Liu et al. showed that QT inhibited apoptosis and DNA damage induced by lead in the kidney of rats (11). We also observed that NTiO 2 increased apoptosis in the proximal cells. Pre-treatment with QT effectively decreased the apoptotic index in kidney tissue. Özyurt et al. showed that QT prevented apoptosis in the kidney tissue induced by radiation (29). It has also been reported that QT protects the kidney of mice against oxidative stress induced by arsenic (9).
The beneficial effects of QT on nephrotoxicity have also been reported by other researchers. For example, Faddah et al. showed that QT reduced nephrotoxicity induced by ZNP in rats (30). QT also reduced cisplatin-induced nephrotoxicity in rats (31), and was found to attenuate the damage to kidneys caused by pesticides (32).

Conclusion
This study showed that QT prevents NTiO 2 -induced apoptosis and nephrotoxicity in female rats. QT protected kidney cells due to its antioxidant and anti-apoptotic effects. Future research should be done to confirm the antiapoptotic effects of QT against NPs in animals.