Endocrinology. Journal of Diabetes Care &

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1 OPEN ACCESS Journal of Diabetes Care & Endocrinology Research Article Menthol Attenuates Hyperglycemia Induced Renal Oxidative Stress Damage via Amending Renal Biomarkers in Streptozotocin-Nicotinamide Induced Experimental Rats Udaiyar Muruganathan 1, Subramani Srinivasan 1, 2, *, Veerasamy Vinothkumar 1 1 Department of Biochemistry and Biotechnology, Faculty of Science, Annamalai University, Annamalainagar , Tamilnadu, India 2 Postgraduate and Research Department of Biochemistry, Government Arts College For Women, Krishnagiri , Tamil Nadu, India *Corresponding author: S. Srinivasan, Department of Biochemistry and Biotechnology, Faculty of Science, Annamalai University, Annamalainagar , Tamilnadu, India, Tel: ; Fax: ; sivaseenu77@gmail.com Published: 30 November 2017 Reviewed by: Dr. Palsamy P and Dr. Poyomtip T Abstract Diabetes mellitus is characterised by hyperglycemia associated with the enhance of oxidative stress. Nephropathy is one of the most frequent complication in diabetes. With an objective to develop complementary and alternative medicine for the treatment of diabetic nephropathy, the present study explored the protective effect of menthol in streptozotocin-nicotinamide (STZ-NA) induced diabetic nephropathy. Diabetes was induced by single intraperitoneal (i.p.) injection of STZ (50 mg/kg/b.w) and NA (110 mg/kg/b.w). Diabetic rats were treated with menthol (50 mg/kg/b.w) and glibenclamide (600 µg/kg/b.w) daily for 45 days. Our results showed an improved levels of insulin, with significant decline in glucose, serum urea, uric acid and creatinine. The altered activities of superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST) and glutathione peroxidase (GPx) were restored to near normal. Histological evaluation revealed that abnormalities in renal tissues were significantly ameliorated by menthol intervention. In conclusion, our results demonstrated that menthol may rescue STZ-NA-induced diabetic nephropathy, through suppressing the oxidative stress and improved renal biomarkers. Keywords: Menthol, Enzymatic antioxidants, Streptozotocin-nicotinamide, Diabetes mellitus

2 Abbreviation CAT: Catalase, GFR: Glomerular filtration rate, Gpx: Glutathione peroxidase, GST: Glutathione S-Transferase, ROS: Reactive oxygen species, SOD: Superoxide Dismutase, STZ-NA: Streptozotocin-Nicotinamide Introduction Diabetes mellitus is a metabolic disorder of multiple etiologies, is characterized by hyperglycemia, in which diabetes without treatment commonly results in severe disability or diffuse glomerulosclerosis. Diabetic nephropathy is one of the major complications of diabetes mellitus and a leading cause of end-stage renal failure in the world, which is characterized by a series of renal structure abnormalities, such as mesangial expansion, glomerulosclerosis and basement membrane thickening [1]. The etiology of diabetes is multiple; however, studies have related that oxidative stress plays a central role in the pathogenesis and complications of the disease. Preveous study reported that free radicals induced by hyperglycemia cause oxidative stress in the body that contributes to the development and progression of diabetes and other complications predominantly diabetic nephropathy [2]. Thereby, the amelioration of the imbalanced condition by enhancing the cellular antioxidant capacity may make some difference for a variety of pathology in diabetic nephropathy [3]. About % of type 1 diabetes patients and 30-40% of patients with type 2 diabetes suffer from diabetic nephropathy. Approximately one-third to one-half of patients with diabetes develops diabetic nephropathy, which remains a major cause of morbidity and mortality [4]. Hence, the targets of managing diabetic nephropathy are to optimize the control of blood glucose, reduce the effects of oxidative stress and normalize disturbances in renal markers [5]. The complications in the existing diabetic treatment have led to the employment of natural resources either as a food supplement or as a medicinal formulation. Plant-based nutraceuticals and supplements have been used in traditional medicine since antiquity. Indian traditional medicine formulates several herbs and the active principles are used in the treatment of diabetes since time immemorial [6]. Chemical and pharmacological research studies have identified numerous bioactive and nontoxic compounds in naturally occurring nutraceuticals and botanical drugs in efforts to find novel therapeutic agents for treatment of diabetic nephropathy [7]. Mono terpenoids receiving considerable attention across the world for the potential health benefits in relation to various diseases including diabetes and its complications. Menthol, a naturally occurring monocyclic terpene alcohol, is a key component of pepper mint oil, popular flavoring and cooling additive in several common household remedies (topical pain gels, throat lozenges, toothpaste), foods (gum, candies, teas). It has been widely used in cosmetics, pharmaceutical products and as flavoring in food [8]. Recently, we have reported the antihyperglycemic nature of menthol by determining its ameliorating role on the activities of hepatic key glucose metabolic enzymes and attenuates pancreatic β-cell apoptosis in streptozotocinnicotinamide (STZ-NA)- induced experimental rats [9]. Given the above findings, it is plausible, we can speculate beneficial effect of menthol against hyperglycemiainduced renal oxidative stress. Therefore, the aim of this study was to evaluate the effect of menthol on biochemical and histological parameters in the kidney of STZ-NA-induced experimental rats. Materials and methods Chemicals Menthol, STZ and NA were purchased from Sigma Chemical Co (St. Louis, Mo. USA). All other chemicals and solvents were of analytical grade and purchased from Himedia Laboratories Pvt.Ltd, Mumbai, India. Animals Male albino Wistar rats ( g) were bred in the Central Animal House, Department of Pharmacology, Rajah Muthiah Medical College and Hospital, Annamalai University, Tamil Nadu, India. The rats had free access to water and a commercial standard pelleted diet (Lipton India Ltd, Mumbai, India). The animals were housed in standard polypropylene cages and maintained under controlled room temperature (22 ± 2 C) and humidity (55 ± 5%) with 12:12 h light and dark cycle. The rats used in the present study were maintained in accordance with the guidelines of the National Institute of Nutrition, Indian Council of Medical Research, Hyderabad, India. The study protocol was approved (Reg No.: 160/1999/CPCSEA, Proposal No.: 1122/2015) by the Committee for the Purpose of Control and Supervision on Experimental Animals at Annamalai University, Annamalainagar, Tamil Nadu, India. Induction of diabetes in experimental animals After 12 h of fasting, rats were given single injection of NA (dissolved in normal saline), intraperitoneally (i.p), at dose of 110 mg/kg. 15 min later, STZ at 50 mg/kg (in 0.1 M sodium citrate bu ffer,

3 ph 4.5) was also injected i.p. STZ-NA injected rats were given 5% glucose for 24 h to prevent drug-induced hypoglycemia. After 72 h, glucose levels were measured with a glucose test meter (accucheck glucometer). Only rats with fasting glycemia over 250 mg/dl were considered diabetic. Treatment was commenced on the fourth day after STZ-NA injection which was considered as day one. Experimental design The rats were randomly segregated into five Assay of kidney enzymatic antioxidant status Renal tissues were homogenized in 0.1 moi/l Tris HCI buffer, ph 7.4, and centrifuged at g for 30 min at 4 C. The activity of antioxidant enzymes were evaluated with standard protocols with minor modification. Kidney supernatant was collected and used for assays of enzymatic antioxidants. The activity of superoxide dismutase (SOD) determined by the activity was measured by the method of Aebi [13]. The activity glutathione-s-transferase (GST) was deliberated using Group I: Group II: Group III: Group IV: Group V: Normal control (vehicle treated) Normal rats received intragastrically menthol (50 mg/kg b.w.) dissolved in 1 ml of corn oil Diabetic control (vehicle treated) Diabetic rats received intragastrically menthol (50 mg/kg b.w.) dissolved in 1 ml of corn oil Diabetic rats received intragastrically glibenclamide (600 µg/kg b.w.) dissolved in 1 ml of water Table 1. Experimental protocol. groups of six rats in each group. Menthol were dissolved in a vehicle solution of 1 ml of corn oil and glibenclamide was diluted in water and administered orally to experimental groups using intragastric tube daily morning for a period of 45 days. The experimental protocol (Table 1) was shown below. On the last day of treatment, after an overnight fast, blood samples were collected from the orbital sinus and immediately centrifuged at 3500 g for 15 min to obtain the serum. All the rats were then killed by cervical dislocation. The kidneys were excised, blotted dry, and quickly weighed and used for pathological histology by hematoxylin and eosin (H&E) staining. The kidneys and serum were stored at 70 C for further analyses. Biochemical estimations Plasma glucose levels were estimated using a commercial kit (Sigma Diagnostics Pvt. Ltd., Baroda, India) by the method of Trinder [10] and plasma insulin was determine using a rat ELISA kit (Lincoplex Ltd, St. Charles, MO) respectively. Plasma protein was determined by the method of Lowry et al. [11], using bovine serum albumin as the standard. Serum urea, uric acid, and creatinine were evaluated using commercially available diagnostic kits (Agappe Diagnostic Pvt. Ltd., India). method of Kakkar et al. [12]. Catalase (CAT) Rotruck et al. [14] and glutathione peroxidase (GPx) estimated according to method described previously [15]. Histopathological observation H&E staining was done to analyse the protective role of menthol in the renal cells of all groups of rats as portrayed by Fischer et al. [16]. The kidney tissues were fixed in normal 10% neutral-buffered formalin for 48 h. Thereafter, the samples were dehydrated in a graded alcohol, embedded in paraffin wax and stained with H&E according to the standard protocol. After that, the pathological alterations in the kidney tissues were observed with a light microscope in a blinded manner. Statistical analysis The statistical significance of the data has been determined using one-way analysis of variance (ANOVA) and significant difference among treatment groups were evaluated by Duncan s multiple range test (DMRT) [17]. The results were considered statistically significant at p<0.05. All statistical analyses were made using SPSS 16.0, SPSS Inc, and Cary, NC. Results Efficacy of menthol on plasma glucose and insulin levels To examine the efficacy of menthol on hyperglycemia, the levels of plasma glucose and insulin

4 were detected. As depicted in Figure 1, the levels of plasma glucose and insulin in the diabetic rats were significantly higher than those of control animals. Meanwhile, treatments with menthol (50 mg/kg) and glibenclamide (600 µg/kg b.w.) effectively suppressed the levels of plasma glucose and enhanced the levels of insulin in STZ-NA-induced diabetic rats. Effect of menthol on STZ-NA-induced oxidative stress in kidney This paragraph talks about the effect of menthol on renal oxidative stress of the experimental groups was shown in Table 2. The activities of SOD, CAT, GST and GPx in the kidneys was significantly lowered in STZ-NA-induced diabetic rats compared to normal rats. Treatment with menthol and glibenclamide significantly up regulated the activities of enzymatic antioxidants in diabetic rats. Efficiency of menthol on STZ-NA-induced renal dysfunction in diabetic rats Serum levels of kidney function markers such Figure 1. 1A Effect of menthol on plasma glucose. 1B Effect of menthol on insulin levels. Each value is mean ± S.D. for 6 rats in each group. In each graph line, means with different superscript letter (a-c) differ significantly at p<0.05 (DMRT). NC: Normal control, DC: Diabetic control, ML: Menthol, GE: Glibenclamide.

5 Parameters SOD NC N + ML D + ML D + GE DC (50 mg/kg b.w) (50 mg/kg b.w) (600 µg/kg b.w) kidney (U a /mg protein) 7.10 ± 0.54 a 7.50 ± 0.57 a 3.10 ± 0.24 b 5.23 ± 0.40 c 5.37± 0.41 c CAT kidney (U b /mg protein) ± 3.35 a ± 3.45 a ± 1.77 b ± 2.74 c ± 2.85 c GST kidney (U/mg protein) GPx kidney (U c /mg protein) 6.39 ± 0.49 a 6.91 ± 0.53 a 2.91 ± 0.22 b 5.21 ± 0.40 c 5.24 ± 0.40 c ± 3.01 a ± 3.07 a ± 1.50 b ± 2.31 c ± 2.46 c Values are given as mean ± S.D from six rats in each group. Values not sharing a common superscript letter (a-c) differ significantly at p<0.05 (DMRT) NC: Normal control, DC: Diabetic control, ML: Menthol, GE: Glibenclamide. U a - The amount of enzyme required to inhibit 50 % NBT reduction/ min for SOD. U b - Micromoles of H 2 O 2 utilized/ per mg of protein for catalase. U c - Micromoles of glutathione oxidized /mg of protein for GPx Table 2. Effect of menthol on the activities of renal enzymatic antioxidant in STZ-NA experimental rats. as urea, uric acid and creatinine serve as the diagnostic tools for the assessment of kidney function. Increased levels of these markers in serum are the indicators of renal dysfunction. Significant increase in the levels of urea, uric acid and creatinine were detected in diabetes group (Table 3). Administration of menthol significantly restored the serum levels of urea, uric acid and creatinine near to normal. However, treatment with menthol to normal rats did not cause significant alterations in the renal function markers. Efficacy of menthol on STZ-NA-induced kidney histology changes To evaluate the protective role of menthol on physiological impairment, H&E staining was performed (Figure 2). Histological examination of renal tissue from control rats showed normal cell architecture (Figure 2A). Normal rats treated with menthol illustrated normal architecture and tubules with regular morphology (Figure 2B). By contrast, STZ-NA-induced damaged rats revealed severe tubular degeneration or necrosis, glomerular hypertrophy, narrowing of the lumen and interstitial inflammation (Figure 2C). Nevertheless, the severity of renal injury was attenuated by menthol and glibenclamide (Figure 2D and E). Discussion The current exploration reveals the renoprotective potential of menthol against hyperglycemia-mediated oxidative stress in STZ-NAinduced diabetic nephropathy. In our study, we found the pathophysiological alterations and deteriorated renal functions due to chronic hyperglycemia-mediated

6 Parameters Total protein (mg/dl) NC N + ML DC D + ML D + GE (50 mg/kg b.w) (50 mg/kg b.w) (600 µg/kg b.w) 8.5 ± 0.65 a 8.6 ± 0.66 a 3.91 ± 0.30 b 7.24 ± 0.55 c 7.58 ± 0.58 c Urea (mg/dl) ± 2.0 a ± 1.98 a ± 4.33 b ± 2.44 c ± 2.34 c Uric acid (mg/ dl) 2.64 ± 0.20 a 2.59 ± 0.20 a 7.10 ± 0.54 b 3.42 ± 0.26 c 3.35 ± 0.26 c Creatinine (mg/ 0.48 ± 0.04 a 0.46 ± 0.04 a 1.87 ± 0.14 b 0.74 ± 0.06 c 0.72 ± 0.06 c Values are given as mean ± S.D from six rats in each group. Values not sharing a common superscript letter (a-c) differ significantly at p<0.05 (DMRT) NC: Normal control, DC: Diabetic control, ML: Menthol, GE: Glibenclamide Table 3. Effect of menthol on the levels of total protein, urea, uric acid, and creatinine in STZ-NA-induced experimental rats. oxidative stress in the experimental rats. STZ-induced diabetic rats pretreated with NA have been reported as models for the evaluation of insulin secretagogues. STZ- NA experimental rats exhibited moderate hyperglycemia associated with the loss of postprandial earlyphase insulin secretion and the condition displays constant hyperglycemia, glucose intolerance and a significantly altered glucose-stimulated insulin secretion and contributes a number of features similar with human diabetes [18]. In the current hypothesis, we observed an increase in the level of plasma glucose and decrease in the level of insulin. Interestingly, treatment with menthol (50 mg/kg b.w.) significantly reduce plasma glucose levels in diabetic rats is due to its potential to secrete insulin from existing islet β-cells. The dose was determined based on our previous work [9] where the 50 mg/kg dose has been found to be therapeutically effective in fabricating antihyperglycemic efficacy in STZ-NA experimental rats. The observed increase in the level of plasma insulin indicates that menthol stimulates insulin secretion by the closure of K + -ATP channels, membrane depolarization and stimulation of Ca 2+ influx, an initial key step in insulin secretion from the remnet β-cells. We substantiated aforementioned declaration that the effect by menthol might be stimulation of survival β-cells from islets Langerhans by closure of K + -ATP channels leading to more insulin release was proved by earlier study [19]. SOD catalyzes the dismutation of superoxide anion (O2 ) into hydrogen peroxide (H 2 O 2 ), which is then degraded to H 2 O by CAT. A decrease in the activity of these antioxidants may lead to an excess of availability of O2 and H 2 O 2, which in turn generates hydroxyl radicals, resulting in SOD and CAT are reduced in renal tissues of STZ-NA rats found by current study. This may result in a number of injurious effects due to the accretion of reactive oxygen species (ROS) [20]. The probable mechanism for this diminution in SOD and CAT activities may be due to the inactivation caused by the excess of free radicals and/or by non-enzymatic glycation due to the persistent hyperglycemia. We originated that supplementation with menthol augmented the activities of SOD and CAT in STZ-NA rats. The results of our hypothesis demonstrated the increased activities these enzymes clearly illustrate that menthol has free radical scavenging property, which exerts a beneficial action against pathological alterations caused by ROS. In addition, we found that the activity of GST was also declined in the kidney of diabetic rats. GST is an enzyme of crucial importance, mainly in the kidney, which catalyses the conjugation of reduced glutathione with toxic substances [21]. Inhibition of GST activity in STZ-NA rats can cause alterations in the major cellular defence strategies against xenobiotics. GPx protects the cells from lipid peroxidation and reduces the loss of membrane integrity by metabolizing H 2 O 2, to H 2 O by using reduced glutathione as a hydrogen donor. The activity of GST and GPx were diminished due to

7 the inactivation and glycation of the enzyme in STZ- NA-induced diabetic rats. Intervention with menthol restores the activities of these enzymatic antioxidants to near normal levels. Taken together, these findings suggest that menthol can strengthen the antioxidant enzymatic protection system, reduce free radicals and alleviate kidney damage caused by oxidative stress in STZ-NA experimental rats. Meanwhile, next we determined the serum urea level, accumulation of urea nitrogen in experimental diabetes may due to the enhanced breakdown of both liver and plasma proteins. Alterations in nitrogen homeostasis may leads to increased hepatic elimination of urea nitrogen and increased peripheral release of nitrogenous substances. In the current study, diabetic rats exhibited renal damages that were evidenced by the elevation in serum urea levels, which are considered as significant markers of renal dysfunction [22]. Thus measurement of urea in serum is extremely useful for diagnostic purpose. Treatment with menthol decreased the levels of urea in STZ-NA-induced experimental rats and the findings suggest that menthol possesses the potential to attenuate renal injury caused by hyperglycemic state. Uric acid, one of the major endogenous water-soluble antioxidants of the body, has been thought to be a metabolically inert end product of purine metabolism [23]. Our data showed that uric acid levels were increased in STZ-NA hyperglycemic rats. This may be due to metabolic disturbance in hyperglycemia reflected in high activities of xanthine oxidase [24]. Moreover, protein glycation in diabetes may lead to muscle wasting and increased release of purine, the main source of uric acid as well as in activity of xanthine oxidase [25]. Thus, the elevated levels of circulating uric acid may be an indicator that the body is trying to protect itself from the deleterious effect of free radicals by increasing the products of endogenous antioxidants, such as uric acid. In the present study, the improved levels of uric acid observed in STZ-NA experimental rats were restored to near normalcy by the administration of menthol indicating the free radical scavenging and hypoglycemic activity of menthol. Hence, we subsequently attempted effect menthol on serum creatinine levels in STZ-NA-induced diabetic rats. Creatinine is excreted by kidneys into the urine and in normal conditions, its excretion is relatively constant, the amount of creatinine produced being proportional to the muscle mass of the individual. It is reported that hyperglycemia leads to an increased production of glomerular matrix proteins [26], the accumulation of which decreases the surface area for filtration leading to decreased glomerular filtration rate (GFR). Increased serum creatinine in diabetic rats is taken as an index of altered GFR in diabetic nephropathy [27]. Intervention with menthol reduced elevated levels of creatinine in diabetic rats, it possesses the potential to attenuate renal injury caused by hyperglycemic state and this can be associated directly with the antioxidant capacity of this monoterpene, which protects the kidneys against oxidative damage, as evidenced in this study. The histopathology evaluation of kidney in normal control rats illustrated normal architectures of renal tissue. However, deterioration of kidney functions as observed in STZ-NA experimental rats was consistent with the histopathological changes in the kidneys as featured by altered renal architectures, glomerular atrophy and reduced surface area of the Bowman capsule. The STZ-NA induced histological alteration are in concord with previously reported study [28]. Our findings on menthol also strengthen the cutting edge research on monoterpene and notion that monoterpene have great potential in the management of diabetic nephropathy [29]. Menthol administration extensively improved the STZ-NA-induced histopathological changes and showed minimum tubular damage and less necrotic damage. The histopathological findings support other biochemical findings in experimental groups and indicate nephroprotective potential of menthol. Conclusion In conclusion, the present study exhibited that menthol may provide effective protection against oxidative stress damage in the kidney of STZ-NA experimental diabetes. Menthol may reduce free radical levels and enhanced enzymatic antioxidant defense in renal tissues and also improved renal damage markers in diabetic rats. These findings provide evidence that menthol may be useful for the treatment of renal complications associated with diabetes and can serve as an attractive therapeutical substitution for managing diabetic nephropathy. However, more molecular

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