The effects of dietary rutin and/or ascorbic acid on high cholesterol diet (HCD) induced-nephrotoxicity in male and female Wistar albino rats were investigated. HCD administration for six consecutive weeks induced renal injury and nephrotoxicity, which was more in female than in male animals. Moreover, rutin and ascorbic acid were found to protect from HCD induced-nephrotoxicity. In addition, these nephro-protective effects were improved in our study by administration of both vitamins in combination to the HCD fed rats which was supported by results from histopathological investigation.
HCD is well known to cause nephrotoxicity and renal injury in different animal models. In the present study, signs of increased renal oxidative damage induced by HCD supplementation for six weeks were studied. Mean kidney weights were not significantly changed after HCD feeding and these results are in agreement with earlier reports [29, 30]. This may be because of HCD could not alter the body weights significantly. Plasma creatinine levels also remained same in HCD group as compared to control animals. Similar results are reported by Kasiske et al. (1990) that, HCD supplementations to rats did not change the creatinine levels although kidney injury was reported . Moreover, HCD feeding for eight weeks could not significantly altered the plasma creatinine levels in rats . In our study, HCD administration to male as well as female rats resulted in moderate degree of nephrotoxicity as confirmed by the histopathological investigation. Treatment with HCD for six weeks significantly altered kidney levels of total cholesterol and triglycerides. Moreover, kidney level of MDA, a specific lipid peroxidation marker, was elevated while GSH level was decreased after six weeks of HCD diet administration to rats. Previous data showed HCD administration to cause hyperlipidemia and to be associated with oxidative stress and nitric oxide inactivation by ROS, which diminishes nitric oxide (NO) bioavailability leading to renal dysfunction . Furthermore, HCD elevated brain, kidney and erythrocytes levels of lipid peroxidation products while decreased GSH content . It was reported that HCD induces modification in lipid composition of cell membranes and the extracellular matrix to be more prone to free radical generation . Therefore, we suggest that HCD induced-nephrotoxicity reported in our study were due to increased rate of oxidative stress and lipid peroxidation in the kidneys, which are kwon to potentiate generation of reactive oxygen species (ROS) and renal injury.
The present study also showed that HCD can induce more significant renal impairment and toxicity in female than male animals. This suggests that male antioxidant defense response to HCD-induced neohrotoxicity was higher than female response. It is well known that sexual steroid hormones can control many physiological and pathophysiological processes . Sexual hormones have a selective influence on gene transcription and RNA production, which explain their regulation of the biosynthesis of specific proteins . Therefore, these hormones can regulate endogenous pattern of antioxidant defense enzyme expression. Several studies have demonstrated the sexual dimorphism in the activity of antioxidant defense enzymes in different rat tissues [37–41]. One study conducted by Capel and Smallwood  did not find a noteworthy difference in glutathione peroxidase (GSH-Px), one of the antioxidant endogenous enzyme, activity in the brain between males and females, while in the liver the activity of this enzyme was significantly higher in females than in males. Similar results on the liver GSH-Px activity were noticed by in Prohaska and Sunde study . Interestingly, Finley and Kincaid  reported that plasma and kidney cytosol selenium content, an endogenous antioxidant, and GSH-Px activity were increased in male compared to the same tissues in female animals, although both were increased in female liver cytosol than male. Experimental data confirmed that males exhibited significantly higher levels of endogenose antioxidant defense parameters in kidney such as Mn, Cu, Zn, SOD, glutathione peroxidase, and catalase than females . Therefore, higher basal levels of ROS are measured by lucigenin chemiluminescence in the renal courtex of females . Furthermore, total renal superoxide dismutase and catalase activities were found to be higher in male spontaneously hypertensive rats than females . These findings are in agreement with our study results as male rats showed more renal antioxidant defense response to HCD-induced increase in MDA and decrease in RNA and total protein than did female rats.
One possible explanation of the degree of renal oxidative damage observed in female rats is that females are able to oxidize poly unsaturated fatty acids more than males. High fat diet was reported to reduce testosterone and to increase estrogen serum levels, which was associated with increased intra-abdominal fats in male than in female . That is why exogenous treatment of ovariectomized rats with estrogen was shown to limit the ovariectomy-induced increase of adiposity . In one study, male, but not female, rats showed a significant negative correlation with adipose poly unsaturated fatty acids content, which suggests that poly unsaturated fatty acids metabolism and oxidation is higher in female than in male . This high rate of lipid metabolism and oxidation could influence more lipid peroxidation and generation ROS. Another possible mechanism is that estrogen itself is able to induced oxidative stress . It was reported that, interaction between estrogen and its receptor (ER) involves an oxidative stress-mediated pathway . Some studies indicate that estrogen exposition damages DNA under acute conditions in cell culture, which was more significant in ER positive cells [47, 48]. Moreover, Wellejus and Loft 2002, suggested that the mechanism of estrogen induced oxidative stress may involve second messenger systems at which estrogen can bound to its cellular receptor and then transported to estrogen-sensitive genes in the nucleus, where redox cycling may take place .
In the present study, HCD induced renal impairment was significantly attenuated by administration of rutin and ascorbic acid. Our results revealed that the combination of both vitamins could have an additive, or sometimes synergistic, antioxidant effects. These speculations are supported by the histopathological evaluation that showed rutin and ascorbic acid combination to prevent HCD-induced nephrotoxicity especially in male animals. Flavonoids such as rutin are now widely accepted as physiologic antioxidants which are known to have effective free radical scavenging activity [50–52]. Previous data reported rutin, or vitamin P, to decrease the permeability of capillaries, scavenge free radicals, lower hepatic and blood cholesterol levels, and have antiplatelet activity [15, 53, 54]. Vitamin C (ascorbic acid) is a promising and effective agent in treating and preventing of allergic rhinitis , diabetes , heart disease  and cancer . Measurements of kidney level of nucleic acids revealed that rutin and ascorbic acid combination can prevent renal cytotoxic damage induced by HCD supplementation to the rats. However, kidney level of total cholesterol results showed that both vitamins had a reno-protective effect that was similar when they were supplemented alone or in combination. Furthermore, HCD induced elevation in kidneys triglyceride was significantly attenuated by vitamins combination with an extent that was much better than administration of each vitamin alone. ROS are well known to induce cyto-toxicity and their action on unsaturated fatty acids has been implicated in the pathogenesis of various diseases . Several studies reported both rutin and ascorbic acid to have a potent ability to damage free radicals produced through biological processes in many extracellular and intracellular reactions [15, 59]. Therefore, we suggest that the combined renal cyto-protective effects of both rutin and ascorbic acid are due to their free radical scavenging capability which seems to be increased by both vitamins combination.
In addition, both vitamins combination significantly decreased MDA and elevate GSH kidney levels as compared to their altered levels in HCD group suggesting that the antioxidant properties of rutin may be attributed to its protective effects on lipid peroxidation. Rutin can augment cellular antioxidant defense mechanisms leading to the protection against oxidative tissue injury . Rutin metabolism, inside the intestines by microflora, results in formation of its aglycone quercetin, which is responsible for rutin's in vivo antioxidant activity . Previous data showed that rutin possess membrane lipid peroxidation inhibitory effects . Furthermore, rutin was reported to increase the antioxidant capacity in the kidney of normal rats as well as in a liposomal model [61, 62]. Rutin can prevent lipid peroxidation by chelating metal ions such as ferrous cations . These ions are involved in the so called Fenton reaction, which generates ROS leading to lipid peroxidation. Interestingly, when red wine, which is known to contain rutin as one of its main components, was supplemented with HCD for 4 weeks, total cholesterol and lipid peroxidation products in the brain, kidney and erythrocytes were significantly decreased compared with the high-cholesterol diet alone, while GSH content and antioxidative enzymes activities were enhanced . In parallel, vitamin C functions as an electron donor to protect the body from radicals and pollutants [63, 64]. A study conducted by Grajeda-Cota et al.  reported that the extracellular ascorbic acid can scavenge ROS derived by the oxidation-reduction cycle which protected from tissue damages. Vitamin C is reported to improve diabetic nephro- and retino-pathy through its ability to induce remission of lipid peroxidation and lipid metabolism abnormalities in adult rats . Indeed, the protection against of oxidative modification of proteins and the alteration in endogenous antioxidants activities such as glutathione might be trough the chelating property of vitamin C to react with free radicals or with highly reactive by products of lipid peroxidation .