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Abstract

Currently, the number of imaging and interventional procedures that use contrast agents (CAs) is gradually increasing. Contrast-induced nephropathy (CIN) is the most important CA-related complication. Oxidative stress plays a significant role in its pathophysiology. Lycopene (LPN) is a natural substance with strong antioxidant capacity. The present study aimed to investigate the potential preventive effects of LPN against CIN. In total, 28 male Wistar albino rats were divided into 4 groups with 7 rats in each group; the groups include normal control group, LPN only group at a dose of 4 mg/kg/day for 10 days, CIN group by administering 10 mg/kg furosemide IM + 10 mg/kg indomethacin IP + 10 ml/kg iomeprol IV following 24-h dehydration, and CIN + LPN group. There were statistically significant increase in urea, creatinine, and malondialdehyde levels (p < 0.001, for all) but a significant decrease in glutathione, superoxide dismutase, catalase, and glutathione peroxidase levels (p < 0.001, for all) in the CIN group compared with the control group. On histological examination, a significant increase of infiltrated inflammatory cells and necrotic degenerative changes were observed in the CIN group and the immunohistochemical examination revealed a significant increase in inflammation (inducible nitric oxide synthase), autophagy (LC3/B), and apoptosis (cleaved caspase 3) in the CIN group compared with the control group (p < 0.05, for all). Significant improvements in these unfavorable parameters were observed with CIN + LPN group compared with the CIN only group. In conclusion, the favorable effects of LPN as an anti-inflammatory, antiautophagic, and antiapoptotic agent in an experimental model of CIN have been demonstrated.

Keywords

Contrast nephropathy oxidative stress inflammation autophagy apoptosis lycopene

Introduction

The use of contrast agents (CAs) have been gradually increasing simultaneously with an increase in imaging and interventional procedures. Despite advances in molecular structures, all CAs may have adverse effects, which range from mild to severe, with contrast-induced nephropathy (CIN) being the most important one. CIN is the third leading cause of hospital-acquired acute renal failure.¹ Although the incidence of CIN is 1–2% in the normal population, this incidence increases by up to 50% in patients with diabetic nephropathy. The development of CIN prolongs the duration of hospital stays and enhances the requirement for dialysis and increase mortality.²

Although the pathophysiology of CIN is unclear, at least four potential mechanisms have been suggested, including an alteration in renal perfusion, a direct tubular injury, oxidative stress, and an immunological mechanism.³ Oxidative stress due to inflammation and reactive oxygen species (ROS) production and direct tubular toxicity are thought to play basic roles in the development of CIN.⁴ Nephropathy occurs due to necrotic and apoptotic cell death because of these pathophysiological mechanisms at the cellular level.⁵ However, to date in the literature, there has been no study investigating autophagic cell death in CIN. Despite the use of various prophylactic therapies and low osmolar CAs with lesser adverse effects, intravenous isotonic fluid infusion is the only method that has been proven to be effective in the prevention of CIN in the clinic practice.⁶ Therefore, a novel kidney-protecting treatment model is required for CIN.

Lycopene (LPN) is a member of carotenoid, and it is an antioxidant substance found in tomato, and in other red fruits, and vegetables.⁷ LPN was shown to be protective against oxidation of lipids, proteins, and DNA in vivo. In previous studies, LPN has been studied together with many nephrotoxic agents, and its preventive effects have been demonstrated.⁸ However, it is not known whether LPN has a protective effect against CIN or not.

The present study aimed to investigate the extent of autophagy, apoptosis, and inflammation that result from oxidative stress in CIN, and the possible preventive effect of LPN against these unfavorable events.

Materials and methods

Animals

In total, 28 adult male Wistar albino rats, weighing 180–200 g were obtained from the Animal Laboratory at the Experimental Research Center at Atatürk University, Erzurum, Turkey. The animals were maintained at standard housing facilities (24 ± 1°C, 45 ± 5% humidity and 12-h light/12-h dark cycle). The animals were supplied with standard laboratory chow and water ad libitum and were left to acclimatize for 1 week before the experiments. The experimental protocol was approved by the Local Animal Care Committee at Atatürk University, Erzurum, Turkey, and experimental procedures were performed in accordance with the International Guidelines for Care and Use of Laboratory Animals.

Drugs and chemicals

Sevoflurane (Sevorane liquid 100%) was obtained from Abbott Laboratories (Istanbul, Turkey). Furosemide (Desal amp.) was obtained from Biofarma (Istanbul, Turkey). Indomethacin amp. was obtained from Sigma Chemical Co. (St Louis, Missouri, USA). Iomeprol (Iomeron flc. 400 mg/ml) was obtained from Bracco SpA (Milan, Italy). LPN %10 fluid suspension (FS) (Redivivo TM, Code 36275) was obtained from Sigma Chemical Company.

Experimental protocol

Rats were randomized into four groups with seven rats in each group. These groups include normal control rats (NC group), LPN-treated rats without CIN (LPN group), rats with CIN but not receiving LPN (CIN group) and LPN-treated rats with CIN (CIN + LPN group). The study period was 10 days. The application in each group was as follows:

NC group: 0.5 ml of corn oil was administered via gavage for 10 days. On day 5, 0.2 ml of saline intramuscular (IM) + 0.2 ml of saline intraperitoneal (IP) + 2 ml of saline intravenous (IV) was administered under mild sevoflurane anesthesia.

LPN group: Lycopene was given at a dose of 4 mg/kg/day as a suspension in corn oil via gavage for 10 days. The dose of LPN used in this study was selected on the basis of the previous studies.⁹

CIN group: 10 mg/kg furosemide IM + 10 mg/kg indomethacin IP + 10 ml/kg iomeprol IV were administered on day 5 following 24-h dehydration under mild sevoflurane anesthesia. The protocol of CIN in this study was selected on the basis of a previous study.¹⁰

CIN + LPN group: LPN was administered at a dose of 4 mg/kg/day as a suspension in corn oil via gavage for 10 days. 10 mg/kg Furosemide IM + 10 mg/kg indomethacin IP + 10 ml/kg iomeprol IV were administered on day 5 following 24 h dehydration under mild sevoflurane anesthesia.

Sample preparation and biochemical studies

All animals in the groups were decapitated under mild sevoflurane anesthesia 24 h after the last application. Blood samples were collected into plain tubes and centrifuged at 200g for 5 min, and serums were extracted. One of the kidneys was removed for biochemical analysis, washed with normal saline, and stored at −20°C until the day of analysis. The other kidney was removed for histopathological and immunohistochemical analyses and stored in 10% buffered formaldehyde.

Serum urea and creatinine concentrations were measured using photometric commercial kits (Diasis Diagnostic Systems, Istanbul, Turkey). The tissue homogenization was performed using a Teflon–glass homogenizer with a buffer that contained 1.15% potassium chloride to obtain 1:10 (w/v) whole homogenate.

The renal tissue catalase (CAT) enzyme activity was determined by measuring the decomposition of hydrogen peroxide (H₂O₂) at 240 nm according to the method described by Aebi¹¹ and was expressed as katal per gram of protein. In parallel, the protein concentration was also measured in the supernatants using the method described by Lowry et al.¹² The tissue-reduced glutathione (GSH) concentration was measured by a kinetic assay using dithionitrobenzoic acid recycling method that was previously described by Ellman¹³ and is expressed as micromole per gram of protein. GSH peroxidase (GSH-Px) enzyme activity was determined by the procedure that was previously described by Beutler.¹⁴ The analysis procedure was based on the oxidation of GSH by GSH-Px, which was coupled to the disappearance of nicotinamide adenine dinucleotide phosphate by GSH reductase, and it was measured at 37°C and 340 nm and expressed as units per gram of protein. Malondialdehyde (MDA) levels (as a marker of lipid peroxidation) in the renal homogenate were measured using thiobarbituric acid reaction according to the method described by Placer et al.¹⁵ The values of MDA were expressed as nanomoles per gram of tissue. The superoxide dismutase (SOD) enzyme activity determination was based on the production of H₂O₂ from xanthine by xanthine oxidase and on the reduction of nitroblue tetrazolium as previously described.¹⁶ The product was evaluated spectrophotometrically at 560 nm. The results are expressed as units per gram of protein.

Histopathological examination of renal tissue

At the end of the experiment, the necropsy of the rats was performed, and kidney tissue samples were fixed in 10% neutral-buffered formalin. Paraffin-embedded blocks were routinely processed; 5-µm thick sections were stained with hematoxylin–eosin and examined under a microscope, and 10 randomly selected microscopic fields were examined under 20× magnification. The histopathological findings in the sections were graded as 0 (none), 1 (mild), 2 (moderate), and 3 (severe).

Immunohistochemical examinations of renal tissue

To examine the protective effects of LPN on inflammation, autophagy, and apoptosis in the kidney, inducible nitric oxide synthase (iNOS), LC3/B, and cleaved caspase 3 expression in the kidney were assessed by immunohistochemical staining. Kidney sections on polylysine-coated slides were fixed in 10% neutral-buffered formalin, embedded in paraffin, and were treated with iNOS, LC3/B, and cleaved caspase 3 antibodies for immunohistochemical analysis. Sections were deparaffinized, rehydrated, and endogenous peroxidase activity was blocked with H₂O₂ (1%). Sections were pretreated in citrate buffer (pH 6.0) in a microwave. Sections were incubated at room temperature with polyclonal rabbit iNOS antibody (cat. no. ab48394, dilution 1:400; Abcam, UK), polyclonal rabbit LC3/B antibody (Abcam, cat. no. ab15323, dilution 1:400), and polyclonal rabbit active/cleaved saspase 3 antibody (Novus Biological, Littleton, Colorado, USA; cat no. NB600-1235, dilution 1:400). Expose mouse and rabbit specific HRP/DAB detection IHC kit was used as follows: sections were incubated with goat anti-mouse antibody, then with streptavidin peroxidase, and finally with 3,3′ diaminobenzidine + chromogen. Slides were counterstained with hematoxylin. The slides were visualized under light microscope, and the number of cell immunopositivity was assessed. Assessment was performed by counting the average percentage of immunopositive cells in 10 randomly selected microscopic fields (20×). Immunopositivity was grouped as 0 (no staining), group 1 (mild; below 25% of all cells), group 2 (moderate; 25–50% of all cells), group 3 (severe; 50–75% of all cells), group 4 (very severe; over 75% of all cells). Negative controls included staining tissue sections with the omission of the primary antibody.

Statistical analysis

All analyses were performed using the program Statistical Package for Social Sciences version 17 (SPSS Inc, Chicago, Illinois, USA). The data are presented as the mean ± the standard error of mean. A one-way analysis of variance and post hoc Tukey’s test were used to determine the differences between groups in terms of the studied parameters. A value of p < 0.05 was considered statistically significant.

Results

Serum biochemical analysis

There were no significant differences in serum urea and creatinine levels between NC and LPN groups (Table 1). CIN induction significantly increased both urea and creatinine levels compared with the NC and LPN groups (p < 0.001). LPN treatment resulted in a significant reduction in serum urea and creatinine levels (Table 1).

Table 1.

Effect of LPN on blood urea, creatinine levels, renal MDA and GSH levels, and catalase, SOD, and GSH-Px activities in rat exposed to CIN.^a

	NC	LPN	CIN	CIN + LPN
Serum urea (mg/dl)	65.00 + 1.56	68.37 + 1.32	92.09 + 1.98^b	75.80 + 1.34^c,d
Serum creatinine (mg/dl)	0.53 + 0.02	0.57 + 0.02	0.89 + 0.02^b	0.70 + 0.01^c.d
SOD (U/g protein)	1.62 + 0.01	1.74 + 0.01	1.05 + 0.03^b	1.55 + 0.02^c,d
Catalase (katal/g tissue)	60.17 + 0.82	60.02 + 0.45	27.48 + 0.79^b	56.76 + 0.98^c,d
GSH (µmol/g tissue)	25.37 + 0.45	27.53 + 0.26	16.26 + 0.24^b	21.57 + 0.66^c,d
GSH-Px (U/g protein)	14.11 + 0.30	15.11 + 0.12	7.06 + 0.22^b	11.19 + 0.32^c,d
MDA (nmol/g tissue)	61.73 + 1.06	60.97 + 0.36	130.26 + 2.17^b	71.71 + 1.00^c,d

NC: normal control; LPN: lycopene; MDA: malondialdehyde; GSH: glutathione; SOD: superoxide dismutase; GSH-Px: glutathione peroxidase; CIN: contrast-induced nephropathy.

^aAll the values are expressed as mean ± SEM, n = 7 in each group.

^b p < 0.05 versus control group.

^c p < 0.05 versus CIN group.

^d p < 0.05 versus control group.

Renal biochemical analysis

Kidney tissue SOD, CAT, and GSH-Px enzyme activities, GSH concentration levels were found to be significantly decreased in the CIN group compared with the NC and LPN groups (Table 1, p<0.001). Kidney tissue MDA levels were found to be significantly increased in the CIN group compared with the NC and LPN groups (Table 1, p<0.001). In CIN + LPN group, there was a significant increase in terms of tissue SOD, CAT, GSH, GSH-Px and a significant decrease in tissue MDA levels compared with the CIN group (Table 1, p < 0.001).

Renal histopathological examination

Histopathological appearances in the experimental groups and in the control group are shown in Figure 1. It was observed that the number of infiltrated inflammatory cells and necrotic degenerative changes were lower in the CIN + LPN group compared with the CIN group (1.42 ± 0.20 vs. 1.85 ± 0.17 and 0.85 ± 0.14 vs. 1.28 ± 0.18, p < 0.05, respectively; Table 2).

Figure 1.

Photomicrographs of rat kidney (×20) from (a) normal control group and (b) LPN-treated rats without CIN group showing normal renal architecture, (c) LPN-treated rats with CIN group showing inflamatory cell infiltration (*), (d) rats with CIN but not received LPN group showing necrotic and degenerative tubular cells (arrow), inflamatory cell infiltration (*). H&E: hematoxylin and eosin; LPN: lycopene; CIN: contrast-induced nephropathy.

Table 2.

Histopathological change rates in the groups.

	NC	LPN	CIN	CIN+LPN
Inflamatory cell infiltration	0.00 ± 0.00	0.00 ± 0.00	1.85 ± 0.17^a	1.42 ± 0.20^b
Necrosis and degenerative changes	0.00 ± 0.00	0.00 ± 0.00	1.28 ± 0.18^a	0.85 ± 0.14^b

NC: normal control; LPN: lycopene; CIN: contrast-induced nephropathy.

^a p < 0.05 versus control group.

^b p < 0.05 versus CIN group.

Renal immunohistochemical examination

iNOS expression in the control group and in the experimental groups are shown in Figure 2. Anti-iNOS-specific staining was observed predominantly in the tubular region and was rarely observed in podocytes in the glomerular region in the CIN group. There was a diffuse immunopositivity in some of the tubular epithelial cells, whereas immunopositivity was localized intensely on the luminal surface of tubular epithelial cells in other regions. It was observed that this specific staining was reduced in the CIN + LPN group which was mainly on the luminal surface of tubular epithelial cells compared with the CIN group (2.71 ± 0.18 vs. 3.28 ± 0.18, p < 0.05, respectively).

Figure 2.

Immunohistochemical staining of iNOS in rat kidney (×20) from (a) normal control group, (b) LPN-treated rats without CIN group showing no expression of iNOS, (c) LPN-treated rats with CIN group showing moderate immunopositivity in luminal surface of tubular cells (arrow) and inflamatory cell infiltration (*) (d) rats with CIN but not received LPN group showing severe immunopositivity tubular cells (arrow) and podocytes (arrowhead) and inflamatory cell infiltration (*). iNOS: inducible nitric oxide synthase; LPN: lycopene; CIN: contrast-induced nephropathy.

LC3/B expression in the control and in the experimental groups are illustrated in Figure 3. There was an anti-LC3/B-specific staining in tubular epithelial cells and in podocytes in the glomeruli as well as in cytoplasm and nucleus in the macula densa in the CIN group. This specific staining was observed to be reduced in the CIN + LPN group compared with the CIN group (2.42 ± 0.20 vs. 3.14 ± 0.14, p < 0.05, respectively).

Figure 3.

Immunohistochemical staining of LC3/B in rat kidney (×20) from (a) normal control group, (b) LPN-treated rats without CIN group showing no expression of LC3/B, (c) LPN-treated rats with CIN group showing moderate immunopositivity in tubular cells (arrow), podocytes (arrowhead), and (d) rats with CIN but not received LPN group showing severe immunopositivity in tubular cells (arrow), podocytes (arrowhead). LPN: lycopene; CIN: contrast-induced nephropathy.

Cleaved caspase 3 expression in the control and in the experimental groups are demonstrated in Figure 4. Anti-cleaved caspase 3-specific staining was predominant in the tubular region in the CIN group. Immunopositivity was observed to be intensive in the nuclei of the tubular epithelial cells and was rarely observed in infiltrated inflammatory cells. It was observed that this intense specific staining was reduced in the CIN + LPN group compared with the CIN group (3.00 ± 0.21 vs. 3.57 ± 0.20, p < 0.05, respectively). In CIN + LPN group, immunopositivity was detected in the tubular epithelial cells.

Figure 4.

Immunohistochemical staining of cleaved caspase 3 in rat kidney (×20) from (a) normal control group, (b) LPN-treated rats without CIN group showing no expression of cleaved caspase 3, (c) LPN-treated rats with CIN group showing moderate immunopositivity in tubular cells (arrow), and (d) rats with CIN but not received LPN group showing severe immunopositivity in tubular cells (arrow) and inflamatory cell infiltration (*). LPN: lycopene; CIN: contrast-induced nephropathy.

Apoptotic and autophagic cell deaths and the iNOS-induced inflammatory reactions were also reduced in the CIN + LPN group compared with the CIN group.

Discussion

The main findings in the present study were as follows: (i) oxidative stress, inflammation, lipid peroxidation, autophagy, and apoptotic cellular death are increased in rats with CIN and (ii) treatment with LPN significantly improved oxidative stress, inflammation, lipid peroxidation, autophagy, and apoptosis in rats with CIN. To our knowledge, this is the first study that shows the protective effects of LPN against oxidative stress, inflammation, lipid peroxidation, autophagy, and apoptosis in rats with CIN.

To date, the prevalence of CIN increases according to widely used imaging and interventional procedures that are performed with CA especially in patients with advanced age and comorbidities. Although the pathophysiology of CIN has not yet been clarified, studies have brought oxidative stress into the forefront.¹⁷ ROS production is increased because of the direct toxic effect of CA. Oxidative stress due to this increase in free radicals causes apoptosis in the renal tubular and glomerular cells.¹⁸ Necrotic and degenerative changes that are observed particularly in tubular cells in CIN¹⁹ are consistent with the present study (Figure 1).

Protective methods over these pathophysiological mechanisms have been explored in preclinical and clinical studies. For instance, an experimental study that was performed with melatonin demonstrated its protective and preventive effects against CIN.¹⁰ In the experimental studies that were performed with α-tocopherol and l-carnitine, the protective effects of these agents against CIN have also been demonstrated.^20,21 Although the benefits of N-acetylcysteine (NAC) have been shown in previous studies, one of the recent studies found that NAC has no benefit in patients with at least one risk factor.²² In the REMEDIAL trial, which was conducted in patients with renal failure, bicarbonate + NAC prevented CIN better than saline + NAC or saline + ascorbicacid.²³ Again, in a clinical study that was performed in patients with renal failure, ascorbic acid was shown to prevent CIN.²⁴ Despite all these studies, there is no method, except physiological saline, with a level proven efficacy in preventing CIN.⁶

LPN is a herbal agent with antioxidant and anticancer effects. The antioxidant activity of LPN is mainly dependent on its $O_{2}^{-}$ - and OH scavenging properties. LPN has been suggested to have strong antioxidant potency in vitro, almost being 100 times efficient in quenching singlet oxygen (¹O₂) than vitamin E. In an experimental study performed with ochratoxin A which is nephrotoxic, it was demonstrated that the natural antioxidant LPN might be partially protective against octratoxin A-induced nephrotoxicity and oxidative stress in rats.⁸ In a study performed with cyclosporine, LPN might have protective effects against cyclosporine-induced nephrotoxicity.²⁵

Serum creatinine is the most important parameter that is used in the diagnosis of CIN in clinical practice. Previous studies determined a significant increase in both urea and creatinine in the CIN groups compared with the NC group.^10,20,21 In the present study, the serum creatinine level significantly increased after CIN in the CIN group compared with the NC group . This result suggests that the protocol that we used has been successful in creating CIN. In the CIN + LPN group, the urea and creatinine levels were significantly lower, compared with the CIN group.

The increased levels of MDA in renal tissue are an indicator of an increase in lipid peroxidation due to nephrotoxicity. Previous studies of CIN and antioxidant agents demonstrated an increase in MDA levels in the renal tissue of the CIN groups.^20,21,26,27 In the present study, MDA levels were found to be significantly increased in the CIN group compared with the NC group. A significantly lower MDA levels were observed in CIN + LPN group compared with the CIN group . These results obtained from the present study are in accord with the previous studies.

There are two types of antioxidant systems in the cell: enzymatic and nonenzymatic. In the present study, SOD, CAT, GSH-Px enzyme activities were measured as enzymatic antioxidants, whereas the GSH levels were measured as a nonenzymatic antioxidant. Boyacioglu et al.²¹ found that SOD and CAT enzyme activities and the GSH levels significantly decreased in the CIN group, and these values improved significantly with l-carnitine therapy. Kongkham et al.²⁰ determined that SOD enzyme activity decreased in the CIN group but significantly improved with α-tocopherol therapy. In the present study, it was observed that tissue SOD, CAT, GSH-Px enzyme activities and GSH levels significantly decreased in the CIN group compared with the control group. A significant improvement was observed in these parameters in the CIN group that received LPN therapy. This improvement in biochemical parameters with LPN therapy can be explained by the antioxidant effects of LPN.

In a study that was conducted in rats, it was observed that nitric oxide (NO) production was increased with CA.²⁸ It has been reported that particularly the NOS, which has three different forms, such as endothelial NOS (eNOS), iNOS, and neuronal NOS (nNOS),²⁹ is produced in the presence of various cytokines and endotoxins as well as in impaired cellular media.³⁰ It has been stated that iNOS can be found in extremely low amounts or not at all in normal renal tissue but increases in nephropathy.³¹ In the present study, although iNOS expression was absent in the NC and LPN groups, iNOS expression was the highest in the CIN group and was moderate in the CIN + LPN groups. This result indicates that CIN-induced iNOS expression was decreased by LPN.

There are two different types of programmed cell death: autophagy and apoptosis.³² Autophagy is a complex and well-organized homostatic process that provides nutritional recycling by removing malfunctioned organelles and molecules to maintain life under stress. In contrast, changes in the rate of autophagy may lead to metabolic imbalance and cell death.³³ The appearance of the lipidated LC3 form among the membranes of autophagosomes is widely used as a marker of continuing autophagy.³⁴ In recent studies, it was stated that autophagy is present in nephrotoxin-associated acute renal injury and in nephropathic cystinosis.^35,36 In an experimental model of cisplatin-induced nephropathy, it was demonstrated that autophagy plays a role in cell death and that there is a precise balance between apoptosis and autophagy.³⁷ In the present study, LC3/B expression was the highest in the CIN group (Figure 3). Decreased expression of LC3/B in the CIN + LPN group indicates that LPN reduces autophagic cell death. Additionally, the fact that LC3/B expression is in line with cleaved caspase 3 indicates that both apoptosis and autophagy play a role in cell death.

Apoptosis is activated by apoptotic genes and by the executioner caspases. The activation of caspase 3 (cleaved caspase 3) plays an important role in the initiation and maintenance of apoptosis.³⁸ Some studies have demonstrated that CA has a cytotoxic or caspase-based apoptotic effect on renal tubular cells.³⁹ In the present study, the fact that cleaved caspase 3 is highly expressed in the CIN group but has decreased expression in the CIN + LPN group suggests that the apoptotic effect of CA on renal cells might be alleviated with LPN (Figure 4).

In conclusion, it was observed that LPN reduces inflammatory and programmed cell death in CA-induced renal injury; thus, it was concluded that LPN might have preventive effects in CIN. Further experimental and clinical studies investigating the effects of LPN in the pathways mentioned above are needed.

Footnotes

Conflict of interest

The authors declared no conflicts of interest.

Funding

This study was supported by the Scientific Research Fund of Erzincan University.

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