Omnia E Hussein1, Mousa O Germoush2 and Ayman M Mahmoud1*
1Physiology Division, Zoology Department, Faculty of Science, Beni-Suef University, Beni-Suef, Egypt
2Department of Biology, Faculty of Science, Aljouf University, Sakaka, Saudi Arabia
Received: 22 December, 2015; Accepted: 20 February, 2016; Published: 22 February, 2016
Ayman M. Mahmoud, PhD Physiology Division, Zoology Department, Faculty of Science, Beni-Suef University, Salah Salim St, 62514, Beni-Suef, Egypt. Tel.: +201144168280; E-mail:
Hussein OE, Germoush MO, Mahmoud AM (2016) Ruta graveolensProtects Against Isoniazid/Rifampicin-Induced Nephrotoxicity through Modulation of Oxidative Stress and Inflammation. Glob J Biotechnol Biomater Sci 1(1): 017-022.
© 2016 Hussein OE, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Background and Aim: Drug-induced nephrotoxicity is a renal dyfunction that arises as a result of exposure to nephrotoxic drugs. Anti-tuberculosis therapy can cause nephrotoxicity and permanent kidney damage. The current study was designed to evaluate the possible protective effects of Ruta graveolens L. leaves extract against isoniazid/rifampicin-induced nephrotoxicity in rats..
Methods: The experimental rats received isoniazid and rifampicin at dose level of 50 mg/kg, and 50 or 100 mg/kg/day Ruta graveolens leaves extract orally for 45 days.
Results: Isoniazid/rifampicin administration induced kidney injury evidenced by the histopathological alterations as well as significant (P<0.001) increase in serum creatinine, urea and uric acid. Isoniazid/rifampicin-intoxicated rats exhibited a significant increase in serum tumor necrosis factor alpha (P<0.001), and renal lipid peroxidation (P<0.01) and nitric oxide (P<0.001) levels. On the other hand, reduced glutathione level, and activity of superoxide dismutase and glutathione peroxidase were markedly (P<0.001) declined in kidney of isoniazid/rifampicin-induced rats. Concomitant supplementation with either 50 or 100 mg/kg dose of Ruta graveolens leaves extract prevented the isoniazid/rifampicin-induced biochemical and histopathological alterations.
Conclusion: The present investigation confers new information on the protective mechanism of Ruta graveolens leaves extract on anti-tuberculosis therapy-induced nephrotoxicity. Ruta graveolens protects against isoniazid/rifampicin-induced renal injury through attenuation of inflammation and oxidative stress, and potentiation of the antioxidant defense system.
The kidney is a vital organ required to essential functions including regulation of the extracellular environment, maintenance of homeostasis, and detoxification and excretion of drugs and toxic metabolites . The kidneys are therefore vulnerable to drug-induced toxicity. Drug-induced nephrotoxicity is a renal dyfunction that arises as a direct or indirect result of exposure to drugs . Nephrotoxic drugs are therapeutic agents that have the potential to induce adverse effects on renal function due to direct toxicity or compromised renal perfusion . Previous studies showed that nephrotoxic drugs were responsible for 19%–25% of acute kidney injury in critically ill patients [4-6]. Nephrotoxic drug-induced kidney dysfunction include acute tubular necrosis, haemodynamically mediated damage, glomerular and tubulointerstitial injury and obstructive nephropathy .
The use of anti-tuberculosis drugs, isoniazid and rifampicin, has been reported to be associated with hepatotoxicity that could result in acute liver injury and a high mortality rate [8,9]. The rate of their hepatotoxic effects is much higher in developing countries than that in developed countries . We have demonstrated the contribution of oxidative stress and inflammation in isoniazid-induced hepatotoxicity . In addition, studies have reported that reactive oxygen species and oxidative stress play a key role in the pathogenesis of drug-induced renal damage [11,12]. Although hepatotoxicity of the anti-tuberculosis drugs has been extensively studied, their induced nephrotoxicity has been poorly documented. Recently, we reported that isoniazid and rifampicin combination induced nephrotoxicity associated with oxidative stress and inflammation in rats .
Medicinal plants are broadly used in the treatment of several diseases owing their cheapness, safety and nontoxicity when compared with the synthetic drugs . Ruta graveolens L. (Family: Rutaceae) is an ancient medicinal plant. It is commonly known as rue or sadab and currently used for treatment of eye problems, dermatitis, aching pain, rheumatism, psoriasis, multiple sclerosis, leucoderma and cutaneous lymphomas . Multiple studies have reported that rue possesses hepatoprotective, anticancer, anti-inflammatory, antioxidant, anti-hyperammonemic and antidiabetic activities [16-19]. Recently, we demonstrated the protective effect of R. graveolens leaves extract against diethylnitrosamine-induced kidney damage in rats . Although the beneficial effects of R. graveolens in multiple disease cases have been reported, its protective effect against nephrotoxicity associated with the use of anti-tuberculosis drugs has not been studied. Therefore, the present study was designed to scrutinize the protective effects of R. graveolens leaves extract against isoniazid/rifampicin-induced renal injury in rats, focusing on oxidative stress and inflammation.
Materials and Methods
Isoniazid (INH) was supplied by El Nasr Company for Chemicals and Drugs (Cairo, Egypt), and rifampicin (RIF) was purchased from Novartis Pharma Company (Cairo, Egypt). Reduced glutathione (GSH), pyrogallol, trichloroacetic acid (TCA), thiobarbituric acid (TBA) and 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) were purchased from Sigma (USA). All other chemicals were of analytical grade and obtained from standard commercial supplies.
Collection of plant and extract preparation
R. graveolens (sadab) was collected and the extract was prepared as we reported previously . Briefly, sadab was obtained from the Experimental Station of Medical Plants (ESMP), Faculty of Pharmacy, Cairo University (Egypt). The plant leaves were collected, cleaned, air dried and ground with an electric grinder. The powdered leaves were macerated in 80% aqueous ethanol for 24 h at room temperature. Following filtration, the filtrate was concentrated under vacuum in a rotary evaporator. The residue obtained was stored frozen till use.
Animals and treatments
Male Wistar rats weighing 140-160 g, obtained from animal house of the National Research Centre (El-Giza, Egypt) were included in the present investigation. The animals were housed in plastic well-aerated cages (6 rats/cage) at normal atmospheric temperature (25 ± 2°C) and normal 12 h light/dark cycle. Rats had free access to water and were supplied daily with laboratory standard diet of known composition ad libitum. All animal procedures were undertaken with the approval of Institutional Animal Ethics Committee of Beni-Suef University (Egypt).
Twenty-four rats were divided to four groups (N = 6) and were subjected to the following treatments:
Group I (Control): Rats received the vehicle 0.5% carboxymethylcellulose (CMC) via oral gavage for 45 days and served as control.
Group II (INH/RIF): Rats received isoniazid (50 mg/kg body weight), rifampicin (50 mg/kg body weight) and 0.5% CMC orally for 45 days .
Group III (INH/RIF + 50 mg R. graveolens): Rats received isoniazid (50 mg/kg), rifampicin (50 mg/kg) and 50 mg/kg body weight R. graveolens leaves extract dissolved in 0.5% CMC orally for 45 days .
Group IV (INH/RIF + 100 mg R. graveolens): Rats received isoniazid (50 mg/kg) and rifampicin (50 mg/kg) dissolved in water and 100 mg/kg body weight R. graveolens leaves extract dissolved in 0.5% CMC orally for 45 days .
The doses of INH, RIF and R. graveolens were balanced consistently over the entire period of study, as indicated by any change in body weight.
By the end of the experiment, rats were sacrificed under ether anesthesia and blood samples were collected, left to coagulate and centrifuged at 3000 rpm for 15 min to separate serum. Kidneys were immediately excised, perfused with ice-cold saline, and samples were kept frozen or fixed in 10% buffered formalin for histological processing. Frozen samples (10% w/v) were homogenized in chilled phosphate buffered saline and the homogenates were centrifuged at 3000 rpm for 10 min. The clear homogenates were collected and used for assaying oxidative stress and antioxidant defenses.
Determination of serum creatinine, urea and uric acid: Serum creatinine, urea and uric acid levels were assayed using reagent kits purchased from Biosystems (Spain), following the methods of Young , Kaplan  and Fossati et al. , respectively.
Determination of tumor necrosis factor-alpha (TNF-α): Serum levels of TNF-α were determined by specific ELISA kits purchased from R&D Systems (USA), according to the manufacturer's instructions. The concentration of TNF-α was determined spectrophotometrically at 450 nm. Standard plot was constructed by using standard cytokine and the concentration for unknown samples was calculated from the standard plot.
Determination of oxidative stress and antioxidant defenses: Lipid peroxidation, assayed as malondialdehyde (MDA), was determined in kidney homogenate according to the method of Preuss et al. . Nitric oxide level was estimated in the kidney homogenate as nitrite according to the method of Montgomery and Dymock , using reagent kit purchased from Biodiagnostics (Egypt). The assay is based on the Griess method which is a simple colorimetric reaction between nitrite, N-(1-naphthyl) ethylenediamine and sulfonamide to produce a pink product with maximum absorbance at 543 nm. Reduced glutathione (GSH) content was assayed according to the method of Beutler et al. . Glutathione peroxidase (GPx) and superoxide dismutase (SOD) activities were measured according to the methods of Matkovics et al.  and Marklund and Marklund , respectively.
Kidney samples were flushed with cold saline and then fixed in 10% buffered formalin for at least 24 h. The specimens were then dehydrated in ascending series of ethanol, cleared in xylene and embedded in paraffin wax. Blocks were prepared and 4 μm thick sections were cut by a sledge microtome. The paraffin embedded sections were deparaffinized, washed and stained with hematoxylin and eosin (H&E). The stained slides were examined under light microscope.
Statistical analysis was performed using GraphPad Prism 5 software (GraphPad Software, San Diego, CA, USA). Results were expressed as mean ± standard deviation (SD) and all statistical comparisons were made by means of the one-way ANOVA test followed by Tukey's test post hoc analysis. A P value <0.05 was considered significant.
Data summarized in Table 1 show the effect of isoniazid/rifampicin administration and treatment with R. graveolens leaves extract on renal function markers. The administration of isoniazid/rifampicin produced marked impairment of kidney function as showed by the significant (P<0.001) increase in serum creatinine, urea and uric acid levels. Concurrent oral administration of 50 mg/kg R. graveolens leaves extract significantly decreased the elevated levels of serum creatinine (P<0.001), urea (P<0.001) and uric acid (P<0.01) when compared with the isoniazid/rifampicin control group. Similarly, administration of 100 mg/kg R. graveolens significantly (P<0.001) alleviated serum levels of creatinine, urea and uric acid in isoniazid/rifampicin-induced rats.
Serum levels of TNF-α exhibited a significant (P<0.001) increase in isoniazid/rifampicin-intoxicated rats when compared with the control group, as depicted in Figure 1. Although non-significant (P>0.05), concomitant supplementation of the 50 mg/kg R. graveolens leaves extract decreased serum TNF-α. On the other hand, administration of the higher R. graveolens dose (100 mg/kg) produced a significant (P<0.001) decrease in circulating levels of TNF-α when compared with the isoniazid/rifampicin-administered rats.