Protective effect of rutin supplementation against cisplatin-induced Nephrotoxicity in rats

Cisplatin (CP) is a chemotherapy commonly used in cancer treatment including head, neck, ovarian, and testicular cancers [1, 2] but is associated with nephrotoxicity in 28–36% of patients receiving an initial dose (50–100 mg/m²) of cisplatin [3]. The accumulation of high concentrations of cisplatin in the kidneys caused nephrotoxicity [4]. This serious complication is contributed to limiting its clinical use. The intermission of cisplatin remains the only choice in the case of progressive renal failure [5]. Cisplatin-induced nephrotoxicity through apoptosis and necrosis [6], vascular factors [7], and inflammation of the tubules [8]. The development of renal tubule injury is caused by the oxidative stress induced by cisplatin [9‐12]. The reactive oxygen species (ROS) and reactive nitrogen species (RNS) production [13] alter the structure and function of cellular membranes [14]. In addition to their accumulation in kidney and lysosomes [15] explained the mechanisms for CP-induced acute nephropathy [13]. Although numerous mechanisms for CP-induced nephrotoxicity such as mitochondrial dysfunction, inflammation, DNA damage, oxidative stress and apoptosis had been studied, the precise mechanism is not well understood [16, 17]. Therefore, the free radical scavengers and the antioxidants agent can prevent cisplatin-induced nephrotoxicity.

Cisplatin damages the DNA resulting in apoptosis induction [18]. In response to cisplatin, several signaling pathways, which can be activated by lipid peroxidation and oxidative stress, modulate the cell survival or apoptosis [18, 19]. The mitogen-activated protein kinase (MAPK) pathways regulate differentiation, proliferation, apoptosis and are activated by chemical and physical stresses [20]. The three major MAPK pathways terminate in ERK, p38, and JNK/SAPK enzymes. Cisplatin is known to activate these three pathways in various cell lines including renal epithelial cells [21, 22]. p38 MAPK was involved in inflammation, cell cycle regulation, and differentiation [23] but its role in cancer therapy is not clear. Recently, some investigator suggests that p38 MAPK is able to control the p53-mediated response to cisplatin [24].

The interleukin-1 (IL-1) made up of 11 proteins encoded by 11 different genes [25] and its main function, in response to tissue injury or damage, is to control the pro-inflammatory reactions [26]. Activation of IL-1 lead to activation of some genes such as Mitogen-activated protein kinase kinase 4 (MKK4) and (MKK7) which activate JNK [27, 28], and MKK4, MKK3, and MKK6 activate p38 MAPK [29].

Flavonoids are a group of natural poly-phenolic compounds found in plants and have a variety of biological effects and play important role in detoxification of free radicals [30]. Rutin is flavonoid glycosides that are present in herbs and plant foods and possessed different protective effects in vitro as well as in vivo [31, 32] against lipid peroxidation and oxidative stress-mediated diseases [33]. Rutin is an immuno-modulator and has anti-oxidant, anti-diarrheal, anti-tumor, and anti-inflammatory effect, myocardial protection, and has renal protective effects against the ischemia-reperfusion-induced renal injury [34]. Therefore, this study investigated the possible protective effects of rutin against cisplatin-induced nephrotoxicity in rats.

Cisplatin is an anticancer drug used in the treatment of many types of cancer such as head and neck, lung, testis, ovary, and breast cancers [1, 2]. Nephrotoxicity is the dose-limiting side effect of cisplatin [43] such as acute kidney injury was found in about 20–30% of patients receiving CP [44], Hypo-magnesemia in about 40–100% of patients [45], Fanconi-like syndrome, distal renal tubular acidosis, hypo-calcemia, renal salt wasting and hyper-uricemia [46].

Nephrotoxicity induced by CP is characterized by a reduction in renal function that leads to increasing in serum creatinine and blood urea levels [47]. In the current study, creatinine and BUN serum levels were significantly high in CP-treated rats compared to untreated rats suggesting that CP produced nephrotoxicity as evidenced by the glomerular filtration rate reduction. The elevated serum creatinine and BUN levels induced by CP were significantly restored to their normal levels as in control group by rutin. The rutin protective effect against nephrotoxicity can be attributed to its antioxidant and anti-inflammatory effect on ROS and some cytokines may be involved in the glomerular filtration rate damage [48]. Although the accurate mechanism of CP-induced nephrotoxicity is not well understood, previous study suggested that cisplatin interacts with DNA, through the formation of covalent adducts between certain DNA bases and the platinum compound leading to cell cytotoxicity [49]. Other studies suggest that CP-induced ROS and immune response which are mediators of nephrotoxicity [50‐52]. In the present study, the MDA and GSH were measured as biomarkers for the oxidative stress. In the kidney tissue, the MDA level was significantly increased and GSH level was decreased by the effect of cisplatin. However, rutin administration caused significant decreases in lipid peroxidation and promoted increases in GSH content in the kidney. Therefore, rutin can protect the kidney from CP-induced injury via improvement in oxidant status. A similar study found that rutin pre-treatment attenuates renal inflammation and apoptosis induced by cisplatin through reducing TNF-α, NF_kB and caspase-3 levels [18, 25].

The p38-MAPK stress pathway, stimulated with inflammatory cytokines such as TNF-α or IL-1, act as a key regulator of apoptosis in cells [53]. The expression of the number of inflammatory cytokines and chemokines is increased in the kidney after cisplatin injury [54]. In the present study, CP increased the expression levels of both TNF-α or IL1-α. Similarly, other study found that the single injection of cisplatin in mice induced nephrotoxicity. In the kidneys of cisplatin-treated mice, the nephrotoxicity caused up-regulation in TNF-α, IL-1β, macrophage inflammatory protein-2 (MIP-2), monocyte chemoattractant protein-1 (MCP-1), ICAM-1, and TGF-β [55].

The present study showed that the rutin supplementation improved the CP-induced increased in the expression levels of IL-1 and TNF-α that were in agreement with previous reports. Rutin acts as antioxidant and anti-inflammatory and improves renal abnormality induced by several factors or chemotherapeutic agents like doxorubicin or cisplatin [56‐58]. TNF-α induced by cisplatin is highly dependent upon the production of ROS, activation of NFκB, and p38 MAPK. However, the activation of TNF-α and IL-1 are involved in several signal transduction mechanisms, including the NF _K B and AP-1 pathways. In fact, the stress-activated group of MAPKs (JNK and p38) is strongly activated by TNF-α and IL-1 [54]. This was in agreement with the present study in which single dose of CP increase the expression levels of JNK and P38. The activation of JNK by TNF-α mediated by the TNF receptor-associated factor (TRAF) group of adapter proteins [59].

In the current study, the overexpression of TRAF2 as a result of cisplatin may be the cause of nephrotoxicity and apoptosis. The decrease in the expression level of TRAF2 in kidney tissues after rutin supplementation in CP-treated rats suggests that rutin may protect against CP-induced nephrotoxicity by regulating apoptotic pathways. Activation of TNF receptors leads to recruitment of the TRAF2 adapter protein [60, 61]. The activation of the TRAF2 expression is required for JNK activation by TNF [62]. A study showed that in nephrotoxicity induced by chemotherapy, genes for JNK play an essential role in modulating the pro- and anti-apoptotic proteins located in the mitochondria [63]. JNK with ROS can promote apoptosis by inhibiting anti-apoptotic proteins [64]. Also, JNK can be activated through its phosphorylation by MKK4 and MKK7 at threonine, tyrosine. MAPKKK activate both MKK4 and MKK7 protein kinases by dual phosphorylation at two sites in the T-loop [65]. The MKK7 protein kinase is primarily activated by cytokines (e.g TNF-α and IL-1) and MKK4 is primarily activated by environmental stress [66]. In the current study CP- induced the expression levels of MKK4 and MKK7 and these alterations attenuated by rutin supplementation in CP-treated rats. P38 MAPK is activated by MKK3, MKK4 and MKK6 [67]. In the present study, P38 expression levels were increased after a single dose of cisplatin. Similarly, several studies suggested that the inhibition of p38 MAPK, ERK or JNK with specific pharmacologic or genetic inhibitors reduced inflammation and renal injury [17, 68, 69].

Rutin administration in CP-treated rat restored the expression levels of P38 and reduced the apoptosis. Therefore, cisplatin-induced nephrotoxicity can be ameliorated by free radical scavengers [70], iron chelators [71], superoxide dismutase [48] and Vitamin E [72].

In conclusion, single dose of cisplatin-induced nephrotoxicity through the activation of P38 MAPK pathway. Our data may help in understanding the molecular mechanisms of rutin in CP nephrotoxicity. Rutin attenuates CP nephrotoxicity might be through its antioxidant as well as p38-MAPK inhibitor properties.