Introduction

Cisplatin (cis-diamminedichloroplatinum II, CDDP) is a potent antineoplastic agent. However, its main dose-limiting side effects include nephrotoxicity, neurotoxicity, ototoxicity, allergic reactions, and hemorrhage (Dasari and Tchounwou 2014; Wang and Lippard 2005). Among these side effects, nephrotoxicity remains the most common, manifesting as acute kidney injury (AKI) in 20 to 30% of patients (Goldstein and Mayor 1983) and limiting the use of CDDP in cancer therapy (Thadhani et al. 1996). The mechanisms by which CDDP exerts its anti-carcinogenic or cytotoxic effects in normal tissues involve inflammation, oxidative stress, and apoptosis. Cisplatin can directly bind to DNA, forming inter- and intra-strand cross-links, which arrests DNA synthesis and replication, especially in rapidly proliferative cells (Jamieson and Lippard 1999; Zamble and Lippard 1995). The main nephrotoxic effect of CDDP occurs by generation of reactive oxygen species (ROS) (Arany and Safirstein 2003). Moreover, CDDP is bio-transformed by the microsomal cytochrome-P450 system into a highly reactive thiol form, depleting glutathione during this reaction. Further, it causes mitochondrial dysfunction, leading to exhaustion of the mitochondrial antioxidants and oxidative stress-mediated cytotoxicity (Siddik 2003).

Ceftriaxone (CTX) is a third-generation cephalosporin with a potent activity against several Gram-positive and Gram-negative bacteria (Bertram 2009). It is clinically used to treat acute bacterial otitis media, bone and joint infections, gonorrhea, intra-abdominal and urinary tract infections, and bacterial septicemia (Perry and Schentag 2001). The antibacterial activity of CTX is due to inhibition of the mucopeptide synthesis in the bacterial cell wall (Rothstein et al. 2005). Former studies reported that CTX protects against neurodegenerative disorders, including cerebral ischemia, amyotrophic lateral sclerosis, and epilepsy (Rothstein et al. 2005), and alleviates neuropathic pain in diabetic rats (Gunduz et al. 2011). Other authors showed that CTX can protect against the nephrotoxicity of tobramycin (Beauchamp et al. 1994), isepamicin (Yoshiyama et al. 1998), cyclosporine A (Yılmaz et al. 2011), and cadmium (Dwivedi et al. 2012) in rats.

Vitamin E (Vit.E) is a naturally occurring, fat-soluble molecule that has a potent, chain-breaking antioxidant activity. It can scavenge ROS and lipid peroxyl radicals (Arreola-Mendoza et al. 2006; Burton et al. 1982; Traber and Stevens 2011). Moreover, it protects the cell membrane integrity by inhibiting the peroxidation of polyunsaturated fatty acids (Ellie and Rolfes 2011). In a randomized controlled trial on cancer patients treated with CDDP, Vit.E supplementation significantly increased the plasma antioxidant enzymatic levels (Weijl et al. 2004). Further, high doses of Vit.E were shown able to protect against CDDP-induced oxidative damage to renal, hepatic (Nazıroǧlu et al. 2004), and neural tissues in rats (Younan and Rashed 2013).

To our knowledge, no previous studies investigated the chemopreventive effects of CTX against acute CDDP nephrotoxicity. Thus, this study was performed to evaluate the antioxidant and nephroprotective efficacy of CTX and/or Vit.E against CDDP-induced AKI.

Materials and methods

Animals

Fifty-six Wistar albino rats (males, weighing 170–200 g) were obtained from The Egyptian Organization for Biological Products and Vaccines (Giza, Egypt). The animals were acclimatized for 10 days in propylene cages with wire mesh cover and sawdust bedding and housed in a well-ventilated room with normal temperature (22 ± 2 °C) and light/dark cycle. They received balanced diet and tape water ad libitum. The Research Ethical Committee at Suez Canal University approved our experimental protocol (approval no. 20147). All animal stress conditions were taken into consideration and hardly avoided.

Chemicals

Cisplatin (Cisplatin® vial, CAS 15663-27-1) was purchased as a clinical formulation from Merck Co. (Lyon, France). Ceftriaxone (250 mg vial as crystalline powder) was obtained from Sandoz-Novartis, Egypt, while vitamin E was provided by Adwia Pharmaceuticals (Cairo, Egypt). The analytical kits were obtained from Biodiagnostics Co. (Dokki, Giza, Egypt) except for TNF-α assay kits (Assay Designs Inc., Ann Arbor, MI, USA). The other used chemicals in this study were of the highest analytical grade, available commercially.

Animal grouping and work design

After acclimatization, rats were randomly allocated into seven groups (n = 8 per group). Eight rats served as controls (group I) and were injected with normal saline. Group II rats received CTX (100 mg/kg bw intraperitoneal [i.p] injection) (Gunduz et al. 2011), while group III rats received Vit.E (100 mg/kg bw orally) (Stojiljkovic et al. 2018). Group IV rats received normal saline and cisplatin (5 mg/kg bw i.p injection) (de Oliveira et al. 2003), while rats in groups V and VI received CDDP plus CTX and CDDP plus Vit.E, respectively. Lastly, group VII rats received cisplatin plus CTX with Vit.E in combination. All treatments were administered daily for 10 days except CDDP [a single i.p. injection at the sixth day of the treatment schedule]. Each treatment was given at the same dose and via the same route in different groups.

Sample collection and preparation of tissue homogenate

After completing the dosing schedules, all rats were fasted overnight. Then, blood samples were obtained from the retro-orbital plexus under isoflurane anesthesia in centrifugation tubes without anticoagulants. The samples were allowed to clot at 25 °C and then centrifuged for 15 min (at 3000 rpm). The obtained sera were then stored at − 20 °C as aliquots for biochemical assays.

The animals were sacrificed by cervical dislocation. The kidneys were then removed, cleaned of connected tissue and blood clots, and washed with normal saline. Next, kidneys were perfused with sodium phosphate (Na2HPO4/NaH2PO4) buffer (50 mM, pH 7.4) mixed with heparin (0.16 mg/ml) to remove any blood clots. Following that, 1 g tissue was homogenized in 5–10 ml cold PBS (PH 7.4) in Teflon Dounce Tissue Grinder (Omni International, Kennesaw Georgia), surrounded by ice media then centrifuged at 4 °C, 10,000 rpm for 15 min in a cooling centrifuge. The resulting supernatant was collected in Eppendorf tubes and stored at − 80 °C.

Serum and tissue biochemical analyses

Serum creatinine, urea, and uric acid levels were measured according to Larsen (1972), Coulombe and Favreau (1963), and Whitehead et al. (1991), respectively. The renal tissue concentrations of malondialdehyde (MDA: lipid peroxidation biomarker) and nitric oxide (NO: nitrosative stress biomarker) were measured according to Mihara and Uchiyama (1978) and Green et al. (1982), respectively. The non-enzymatic antioxidant biomarker (reduced glutathione (GSH)) was assessed according to Beutler et al. (1963). While the enzymatic antioxidants superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) were evaluated according to Nishikimi et al. (1972), Paglia and Valentine (1967), and Aebi (1984), respectively. Further, the total antioxidant capacity (TAC) was evaluated according to Koracevic et al. (2001). The serum and renal tissue concentrations of TNF-α were measured using commercial kits from R&D Systems (Minneapolis, MN, USA) according to the manufacturer’s protocol.

Statistical analysis

The obtained data were expressed as means ± standard error of means (SEM). The statistical significance of between-group differences was analyzed using the Statistical Package for Social Sciences (SPSS, version 22, IBM, Chicago, IL). For comparison, the ANOVA was used, followed by post hoc Tukey’s test. The statistical significance was set at p ≤ 0.05.

Results

Serum markers of renal injury

Biochemical analysis of renal injury biomarkers showed significant elevations in urea, uric acid, and creatinine levels by 186.4, 189.5, and 927.5%, respectively, in CDDP-intoxicated rats, compared to the untreated control group. Treatment with CTX (100 mg/kg bw i.p) and Vit.E (100 mg/kg bw oral) daily for 5 days before and after CDDP injection significantly reduced (p ≤ 0.05) serum uric acid, urea, and creatinine by about 26.7, 28.5, and 51%, respectively, in the CTX group and 44.1, 43.9, and 61.3%, respectively, in the Vit.E group, compared to the CDDP-intoxicated rats. The most significant reduction (p ≤ 0.05) in the serum levels of these parameters was obtained with CTX + Vit.E combination (57.4, 62.3, and 87.3%, respectively, in comparison to the CDDP group) (Figs. 1, 2, and 3).

Fig. 1
figure 1

The effects of ceftriaxone (CTX) and/or vitamin E on serum uric acid in CDDP-induced nephrotoxicity in rats (mean ± SEM). Different superscript letters indicate statistical significance at p ≤ 0.05

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Fig. 2
figure 2

Effects of ceftriaxone (CTX) and/or vitamin E on serum urea in CDDP-induced nephrotoxicity in rats (mean ± SEM). Different superscript letters indicate statistical significance at p ≤ 0.05

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Fig. 3
figure 3

Effects of ceftriaxone (CTX) and/or vitamin E on serum creatinine in CDDP-induced nephrotoxicity in rats (mean ± SEM). Different superscript letters indicate statistical significance at p ≤ 0.05

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Renal lipid peroxidation and antioxidant biomarkers

Compared to normal control rats, CDDP-intoxicated rats exhibited significant increases (p ≤ 0.05) in renal MDA and NO levels (273 and 104.9%, respectively), as well as significant decreases in renal GSH, GPx, SOD, CAT, and TAC (28.8, 38.7, 52.2, 52.2, and 31.4%, respectively). In contrast, treatment of CDDP-intoxicated rats with CTX or Vit.E significantly reduced renal MDA (24.6 and 50.4%, respectively) and NO (26.3 and 32.5%, respectively), as well as increased renal GSH (23.6 and 32.7%, respectively), GPx (34 and 52%, respectively), SOD (56.2 and 77.8%, respectively), CAT (31.9 and 61.3%, respectively), and TAC levels (25.6 and 38.5, respectively), compared to CDDP-intoxicated rats. The most significant improvements were recorded when CTX and Vit.E were given in combination with CDDP: reduction of renal MDA and NO levels (by 68.6 and 47.2%, respectively) and increase in GSH, GPx, SOD, CAT, and TAC levels (by 40, 59.8, 113.5, 97.4, and 44.9%, respectively) (Table 1).

Table 1 Effects of ceftriaxone (100 mg/kg bw i.p) and/or vitamin E (100 mg/kg bw orally) treatment on oxidant/antioxidant parameters in cisplatin (5 mg/kg bw i.p)-intoxicated rats
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Serum and renal TNF-α assays

Cisplatin intoxication was associated with significant increases (p ≤ 0.05) in serum and renal tissue TNF-α concentrations (339.6 and 371.4%, respectively), compared to control rats. Treatment with CTX or Vit.E was associated with significant reductions (p ≤ 0.05) in serum (45.7% and 53.9%, respectively) and renal TNF-α (66.3% and 70.7%, respectively) levels. Further, when CTX and Vit.E were administered together, more significant (p ≤ 0.05) reductions in serum and TNF-α levels were recorded (73.2 and 76.5%, respectively), compared to CDDP-intoxicated rats (Figs. 4 and 5).

Fig. 4
figure 4

Effects of ceftriaxone (CTX) and/or vitamin E on serum tumor necrosis factor-α in CDDP-induced nephrotoxicity in rats (mean ± SEM). Different superscript letters indicate statistical significance at p ≤ 0.05

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Fig. 5
figure 5

Effects of ceftriaxone (CTX) and/or vitamin E on renal tumor necrosis factor-α in CDDP-induced nephrotoxicity in rats (mean ± SEM). Different superscript letters indicate statistical significance at p ≤ 0.05

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In all investigated parameters, no significant changes were detected in rats that received CTX (second group) or Vit.E alone (third group) in comparison to normal control rats (first group), highlighting the safety of both agents at the tested doses, routes, and durations of administration.

Discussion

Cisplatin may be used alone or with radiotherapy to treat several solid tumors, especially treatment-resistant ones. The therapeutic potency of CDDP is dose-dependent; however, its clinical use is jeopardized by cumulative neurotoxicity and nephrotoxicity (O’Dwyer et al. 1999). Although other platinum-based drugs as carboplatin are non-nephrotoxic derivatives, CDDP remains the first choice in several platinum-based chemotherapeutic regimens. Various experiments were performed to identify novel agents that would mitigate CDDP nephrotoxicity without diminishing its anticancer efficacy. In this direction, this study was performed to investigate the chemoprotective effects of Vit.E and/or CTX against CDDP-induced acute renal injury.

Treatment of rats with a single i.p. dose of cisplatin induced oxidative stress-mediated renal injury as marked by (a) the significant elevation in serum urea, uric acid, and creatinine; (b) augmented renal tissue levels of lipid peroxidation (MDA) and nitrosative stress (NO) markers; (c) drop in tissue GSH concentration, TAC, and activities of antioxidant enzymes, indicating disturbed oxidant/antioxidant balance; and (d) elevated serum and renal tissue TNF-α concentrations. These results confirm those of previous studies on acute and sub-acute CDDP nephrotoxicity in mice and rats (Hassan et al. 2014; Ibrahim et al. 2016; Kawai et al. 2009).

Multiple mechanisms have been postulated for CDDP-induced renal cell injury; among them, oxidative stress and inflammation are the most important (Hanigan and Devarajan 2003; Miller et al. 2010). Proximal tubular cells express the transporters and enzymes, needed in each step of CDDP bio-activation into a more potent nephrotoxin (Park et al. 2002; Townsend et al. 2003). This metabolic activation process starts in the kidneys with the formation of glutathione conjugates, causing GSH depletion and formation of the reactive thiol nephrotoxin (Townsend et al. 2009). The latter induces oxidative stress and cellular apoptosis (Baliga et al. 1997). Moreover, CDDP significantly increases NADPH oxidase gene expression, a membrane-bound enzyme that generates high levels of O2·− (Babior 2004; Palipoch et al. 2014). The generated free radicals cause membrane lipid peroxidation and denaturation of DNA and proteins, which lead to enzymatic inactivation and mitochondrial dysfunction, enhancing ROS production via the disruption of the respiratory chain (Kruidering et al. 1997; Yilmaz et al. 2004). In addition, CDDP induces microsomal free radicals (superoxide anion (O2·), hydrogen peroxide (H2O2), and hydroxyl radical (·OH)) formation via the cytochrome-P450 (CYP) system, overwhelming the endogenous antioxidant mechanisms (Liu and Baliga 2003). Further, the renal injury, caused by CDDP, is partly mediated by a series of inflammatory changes and enhancement in renal expression of TNF-α (Deng et al. 2001). The latter can induce apoptosis and ROS generation and coordinate the activation of a large network of cytokines in the kidney (Ramesh and Reeves 2002, 2004).

Daily treatment with CTX antibiotic and Vit.E solely or in combination daily for 5 days before and after CDDP treatment significantly ameliorated all CDDP-induced alterations and relatively restored the normal cellular redox status. These findings confirm the results of previous studies, which examined the renoprotective effects of CTX against xenobiotics-induced nephrotoxicity (Beauchamp et al. 1994; Dwivedi et al. 2012; Yılmaz et al. 2011; Yoshiyama et al. 1998). Abdel-Daim and El-Ghoneimy (2015) referred the renoprotective effect of CTX against diazinon and deltamethrin-induced oxidative stress to its ability to scavenge ROS and enhance antioxidant enzymes’ activities. Moreover, cephalosporins can act as multidentate chelating agents and contain thio-ether groups, which prevent free radical-mediated oxidation (Anacona and Osorio 2008).

Vitamin E is an exogenous antioxidant that exerts its function through the GPx pathway (Wefers and Sies 1988). As a lipid-soluble antioxidant, it prevents the propagation of lipid peroxidation in the cell membrane by free radicals scavenging (Ellie and Rolfes 2011) and interference with oxidase enzymes, which initiate the production of free radicals (Palipoch et al. 2014). Previous studies proved that Vit.E can mitigate CDDP-induced ototoxicity and nephrotoxicity without interfering with its anti-tumor efficacy (Kalkanis et al. 2004; Leonetti et al. 2003). Moreover, Azzi et al. (2002) concluded that Vit.E reduced the production of NO and superoxide radicals (by endothelial cells and neutrophils, respectively) via inhibiting protein kinase C. In parallel, Vit.E was found to have anti-inflammatory effect beside its antioxidant effect. Several studies have shown that pre-treatment with Vit.E reduces TNF-α production in dichromate-induced nephrotoxicity (Mehany et al. 2013) and acetic acid colitis (Tahan et al. 2011). Future investigators are recommended to perform additional studies on urine osmolarity and chemical properties, histopathological studies of the kidneys, and exploration of the involved inflammatory pathways with the tested pharmacological compounds.

In conclusion, pre-treatment of rats with CTX and Vit.E, alone or in combination, can protect against CDDP nephrotoxicity and should be considered in future trials on nephroprotection against chemotherapeutic drugs.