Impact of d -pinitol on the attenuation of proinflammatory cytokines, hyperglycemia-mediated oxidative stress and protection of kidney tissue ultrastructure in streptozotocin-induced diabetic rats

Chemico-Biological Interactions 188 (2010) 237–245

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Impact of d-pinitol on the attenuation of proinflammatory cytokines, hyperglycemia-mediated oxidative stress and protection of kidney tissue ultrastructure in streptozotocin-induced diabetic rats Selvaraj Sivakumar, Periyasamy Palsamy, Sorimuthu Pillai Subramanian ∗ Department of Biochemistry, University of Madras, Guindy Campus, Chennai 600025, Tamil Nadu, India

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Article history: Received 15 May 2010 Received in revised form 2 July 2010 Accepted 12 July 2010 Available online 17 July 2010 Keywords: d-Pinitol Diabetic kidney Lipid peroxidation Oxidative stress Streptozotocin

a b s t r a c t Oxidative stress plays a crucial role in the progression and development of diabetes and its complications due to chronic hyperglycemia. The present study was aimed to investigate the kidney tissue protective nature of d-pinitol, a cyclitol present in soybean, by assessing the key markers of hyperglycemia-mediated oxidative stress, proinflammatory cytokines and ultrastructural alterations in streptozotocin-induced diabetic rats. Oral administration of d-pinitol (50 mg/kg body weight/day) for 30 days to diabetic group of rats showed a significant elevation in the level of total protein and significant decline in the levels of blood urea, serum uric acid, creatinine and advanced glycation endproducts (AGEs) and kidney proinflammatory cytokines such as TNF-␣, IL-1␤, IL-6, NF-␬B p65 subunit and nitrite. Further, d-pinitol administration elicited a significant attenuation in the activities of kidney enzymatic antioxidants such as superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx), glutathione-S-transferase (GST) and glutathione reductase (GR) and the levels of kidney non-enzymatic antioxidants such as vitamin E, vitamin C and reduced glutathione (GSH) in the diabetic group of rats, with a concomitant decline in the levels of kidney lipid peroxides, hydroperoxides and protein carbonyls. The histological and ultrastructural observations on the kidney tissues also confirmed the renoprotective nature of d-pinitol. Thus the present study demonstrated the renoprotective nature of d-pinitol by attenuating the hyperglycemia-mediated proinflammatory cytokines and antioxidant competence in kidney tissues of streptozotocin-induced diabetic rats. © 2010 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Diabetes mellitus is one of the most common chronic metabolic disorder and a major contributor to the development of cardiovascular disease. The total number of people with diabetes is projected to rise from 171 million in 2000 to 366 million in 2030 [1]. It is characterized by supraphysiological glucose due to the deficiency in insulin secretion or insulin receptor or postreceptor events leading to the disturbance in the metabolism of carbohydrates, proteins and fats [2]. Moreover, the impaired metabolism leads to the progression and aggravation of oxidative stress through several mechanisms, such as glucose autoxidation, protein glycation and

Abbreviations: AGEs, advanced glycation end-products; ANOVA, analysis of variance; ELISA, enzyme linked immunosorbent assay; GPx, glutathione peroxidase; GR, glutathione reductase; GST, glutathione-S-transferase; GSH, reduced glutathione; LPO, lipid peroxidation; LSD, least significant difference; SEM, standard error of mean; SOD, superoxide dismutase. ∗ Corresponding author. Tel.: +91 44 22300488; fax: +91 44 22300488. E-mail addresses: [email protected], [email protected] (S.P. Subramanian). 0009-2797/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.cbi.2010.07.014

AGEs formation leading to the development of secondary diabetic complications such as nephropathy, retinopathy, neuropathy and macro and microvascular damages [3]. Diabetic nephropathy is a single leading cause of end stage renal disease and is a medical catastrophe worldwide in which oxidative stress plays a decisive role in the kidney impairments such as acute and chronic renal failure, glomerular injury and obstructive nephropathy [4,5]. Various studies have demonstrated that the supplementation of antioxidants such as vitamin E, ␣-lipoic acid and plant polyphenols are known to attenuate free radical mediated diabetic nephropathy [6–9]. Thus, the agent that restrains the detonated production of reactive oxygen species might alleviate the oxidative stress thereby protecting the kidney tissues. d-Pinitol, 3-methoxy analogue of d-chiroinositol, is one of the most abundant cyclitol present in soybean seeds, legumes and soy food [10,11]. The role of d-pinitol in plants is often associated with salt and drought stress [12], osmoprotectant [13], embryo development [14] and nodulation [15]. In addition, d-pinitol has been suggested to possess multifunctional properties including anti-inflammatory [16], antihyperlipidemic [17], cardioprotective [18] and inhibition of ovalbumin-induced airway inflammation

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[19]. It also modulates the immune response by interacting with maturation of dendritic cells [20]. Recently, we have reported the antihyperglycemic potential of d-pinitol by assessing the activities of hepatic key carbohydrate metabolizing enzymes [21] and the pancreatic and hepatic tissue protective nature by assessing the markers of oxidative stress and proinflammatory cytokines in streptozotocin-induced diabetic rats. However, none of the study reported the beneficial effect of d-pinitol on the protection of diabetic kidney in experimental animals. In this context, the present study was designed to elucidate the renoprotective nature of d-pinitol by assessing the markers of oxidative stress as well as proinflammatory cytokines in streptozotocin-induced diabetic rats.

by cervical decapitation. The blood was collected with or without anticoagulant for plasma or serum separation, respectively. Fasting blood glucose, glycosylated hemoglobin and plasma insulin levels were determined to confirm the antihyperglycemic property of d-pinitol [21–23]. 2.5. Biochemical estimations Blood urea was determined by the method of Natelson et al. [26]. Serum uric acid was estimated according to the method of Caraway [27] and creatinine was estimated according to the method of Brod and Sirota [28]. The concentration of serum AGEs was measured by ELISA (Abcam, Cambridge, UK) as per the manufacturer’s instructions.

2. Materials and methods 2.6. Preparation of kidney tissue homogenate 2.1. Chemicals d-Pinitol and streptozotocin were purchased from Sigma Chemicals Co., St Louis, MO, USA. All other chemicals used in the present study were of analytical grade available commercially. 2.2. Animals and diet All the animal experiments compiled with the ethical norms approved by Ministry of Social Justices and Empowerment, Government of India and Institutional Animal Ethics Committee Guidelines (approval no. 01/017/08). Male, Wistar rats weighing 160–180 g, procured from Tamil Nadu Veterinary and Animal Sciences University, Chennai, India were used in this study. The rats were housed in standard laboratory condition under a 12 h light/dark cycle at an ambient temperature of 20–25 ◦ C with relative humidity of 50 ± 15%. The animals were acclimatized to the laboratory conditions 2 week prior to the commencement of experiments. Throughout the experimental period, the rats were fed with balanced commercial pellet diet (Hindustan Lever Ltd., Bangalore, India) with a composition of 5% fat, 21% protein, 55% nitrogen-free extract, and 4% fiber (w/w) with adequate mineral and vitamins to the animals. Diet and water were provided ad libitum.

A portion of the kidney tissue was dissected out, rinsed with icecold saline and homogenized in 0.1 M Tris–HCl buffer (pH 7.4) with a Teflon homogenizer at 4 ◦ C. The homogenate was centrifuged at 13,000 × g to remove the debris and the supernatant was used for the estimations. The protein content in the tissue homogenate was estimated by the method of Lowry et al. [29]. 2.7. Determination of proinflammatory cytokines The levels of proinflammatory cytokines such as TNF-␣, IL-1␤ and IL-6 in kidney tissue homogenate were determined by using specific ELISA kits (Biosource, California, US). The analyses were performed according to instructions of the manufacturer’s. Standard plots were constructed by using standard cytokines and the concentrations for unknown samples were calculated from the standard plot. The level of NF-␬B p65 unit was determined in the nuclear fraction of kidney tissues by using ActivELISA (Imgenex, San Diego, USA) kit. The kidney nitrite level was indirectly estimated by determining the nitrite levels in the kidney tissue homogenate using a colorimetric method based on the Griess reaction [30]. 2.8. Assessment of antioxidant status

2.3. Induction of experimental diabetes The overnight fasted rats received a single intraperitoneal injection of streptozotocin (50 mg/kg body weight) dissolved in freshly prepared 0.1 M cold citrate buffer, pH 4.5 [24]. As streptozotocin is capable of inducing fatal hypoglycemia as a result of massive pancreatic insulin release, the streptozotocin-treated rats were provided with 10% glucose solution after 6 h for the next 24 h to prevent hypoglycemia [25]. After a week in time for the development and aggravation of diabetes, rats with moderate diabetes (i.e. blood glucose concentration, >14 mM) were selected for the experiment.

The levels of kidney non-enzymatic antioxidants such as vitamin C [31], vitamin E [32] and reduced glutathione (GSH) [33] were estimated in the control and experimental groups of rats. Further, the levels of lipid peroxides [34], hydroperoxides [35] and protein carbonyls [36] were determined in the kidney homogenate. The activities of kidney enzymatic antioxidants such as superoxide dismutase (SOD) [37], catalase [38], glutathione peroxidase (GPx) [39], glutathione-S-transferase (GST) [40] and glutathione reductase (GR) [41] were determined in the control and experimental groups of rats. 2.9. Histological study

2.4. Experimental design The rats were divided into four groups, each group comprised of six rats, as follows; Group 1 served as control rats; Group 2 served as streptozotocin-induced diabetic rats; Group 3 served as diabetic rats orally treated with d-pinitol (50 mg/kg body weight/day) for 30 days; and Group 4 served as diabetic rats orally treated with glyclazide (5 mg/kg body weight/day) for 30 days. During the experimental period, body weight, respiratory changes, distress, abnormal locomotion and catalepsy were monitored at regular intervals in all the rats and the blood glucose level was estimated twice a week. At the end of the experimental period, the rats were fasted overnight, anaesthetized and sacrificed

A portion of the kidney tissue was fixed in 10% formalin for a week at room temperature. Then the specimens were dehydrated in a graded series of ethanol, cleared in xylene and embedded in paraffin wax. The blocks were then sectioned into 5 ␮m thick using a rotary microtome. Sections were stained with hematoxylin and eosin and photomicrographs were obtained under light microscope. 2.10. Transmission electron microscopic study A portion of the kidney (about 1 mm3 ) were excised from the control and experimental groups of rats and fixed in 3% glutaralde-

S. Sivakumar et al. / Chemico-Biological Interactions 188 (2010) 237–245 Table 1 Levels of urea, uric acid and creatinine in control and experimental groups of rats. Groups

Urea (mg/dl)

Control Diabetic control Diabetic + d-pinitol Diabetic + glyclazide

22.77 45.18 26.25 25.64

± ± ± ±

0.78 1.34a 0.41b 0.22b

Creatinine (mg/dl) 0.41 1.23 0.57 0.63

± ± ± ±

0.31 0.61a 0.25b 0.19b

Uric acid (mg/dl) 2.62 7.06 2.98 3.31

± ± ± ±

0.19 0.28a 0.11b 0.14b

Results are mean ± SEM (n = 6). One way ANOVA followed by post hoc test LSD. Values are statistically significant at p < 0.05, when compared with (a) control rats, (b) diabetic control rats.

hyde in sodium phosphate buffer (0.2 M, pH 7.4) for 3 h at 4 ◦ C. Tissue samples were washed with the same buffer, post-fixed in 1% osmium tetroxide and sodium phosphate buffer (0.2 M, pH 7.4) for 1 h at 4 ◦ C. The samples were again washed with the same buffer for 3 h at 4 ◦ C, dehydrated with graded series of ethanol and embedded in Araldite. Thin sections were cut with LKBUM4 ultramicrotome using a diamond knife, mounted on a copper grid and stained with 2% uranyl acetate and Reynolds lead citrate. The grids were examined under a Philips EM201C transmission electron microscope. 2.11. Statistical analysis The results were expressed as mean ± SEM of six rats per group and the statistical significance was evaluated by one-way analysis of variance (ANOVA) using the SPSS/16.0 software followed by the post hoc test LSD. Values were considered statistically significant at p < 0.05. 3. Results 3.1. Effect of d-pinitol on the levels of blood urea, serum uric acid and creatinine levels Table 1 shows the effect of d-pinitol on the blood urea, serum uric acid and creatinine in control and experimental groups of rats. The blood urea, serum uric acid and creatinine levels in streptozotocin-induced diabetic group of rats were significantly (p < 0.05) increased, when compared with control group of rats. The oral administration of d-pinitol as well as glyclazide significantly (p < 0.05) decreased blood urea, serum uric acid and creatinine when compared with diabetic group of rats.

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3.2. Effect of d-pinitol on the level of serum AGEs levels The effect of d-pinitol on the level of AGEs in control and experimental groups of rats is depicted in Fig. 1. The serum AGEs levels in streptozotocin-induced diabetic group of rats were significantly (p < 0.05) increased, when compared with control group of rats. The oral administration of d-pinitol as well as glyclazide significantly (p < 0.05) decreased serum AGEs when compared with diabetic group of rats. 3.3. Effect of d-pinitol on the attenuation of kidney proinflammatory cytokines Fig. 2A–C summarizes the levels of kidney proinflammatory cytokines such as TNF-␣, IL-1␤ and IL-6 of control and experimental groups of rats. The levels of TNF-␣, IL-1␤ and IL-6 were significantly (p < 0.05) elevated in streptozotocin-induced diabetic group of rats when compared to control group of rats. A significant (p < 0.05) decline in the levels of TNF-␣, IL-1␤ and IL-6 was observed on oral administration of d-pinitol as well as glyclazide. The level of kidney NF-␬B p65 subunit and renal nitrite in control and experimental groups of rats is depicted in Fig. 3A and B. The levels of kidney NF-␬B p65 subunit and renal nitrite were significantly (p < 0.05) escalated in diabetic group of rats when compared to control group of rats. However the diabetic rats administered with d-pinitol as well as glyclazide for 30 days showed a marked decline in the levels of kidney NF-␬B p65 subunit and renal nitrite. 3.4. Effect of d-pinitol on the augmentation of antioxidant competence Fig. 4A–C depicts the levels of LPO, hydroperoxides and protein carbonyls in the kidney tissues of the control and experimental groups of rats. The diabetic group of rats showed a significant (p < 0.05) increase in the levels of LPO, hydroperoxides and protein carbonyls when compared to the control group of rats. These elevated levels were significantly (p < 0.05) declined in the diabetic rats administered with d-pinitol as well as glyclazide. Table 2 shows the levels of kidney non-enzymatic antioxidants such as vitamin C, vitamin E and reduced glutathione in control and experimental groups of rats. The levels of non-enzymatic antioxidants were significantly (p < 0.05) declined in diabetic rats as compared with control rats and oral administration of d-pinitol as well as glyclazide elevated these levels to near normalcy.

Fig. 1. Levels of AGEs in control and experimental groups of rats. Results are mean ± SEM (n = 6). One way ANOVA followed by post hoc test LSD. Values are statistically significant at p < 0.05, when compared with (a) control rats, (b) diabetic control rats.

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Fig. 2. Levels of (A) TNF-␣, (B) IL-1␤ and (C) IL-6 in the renal tissues of control and experimental groups of rats. Results are mean ± SEM (n = 6). One way ANOVA followed by post hoc test LSD. Values are statistically significant at p < 0.05, when compared with (a) control rats, (b) diabetic control rats, (c) diabetic + glyclazide. Table 2 Levels of non-enzymatic antioxidants such as vitamin C, vitamin E and reduced glutathione in kidney tissue of control and experimental groups of rats. Groups

Vitamin C (␮g/mg of protein)

Control Diabetic control Diabetic + d-pinitol Diabetic + glyclazide

1.46 0.49 0.95 1.01

± ± ± ±

0.51 0.33a 0.36b 0.35b

Vitamin E (␮g/mg of protein) 1.11 0.44 0.88 0.94

± ± ± ±

0.29 0.21a 0.37b 0.45b

Reduced glutathione (mg/100 g of wet tissue) 37.26 20.75 30.83 31.09

± ± ± ±

1.14 0.88a 0.69b 0.59b

Results are mean ± SEM (n = 6). One way ANOVA followed by post hoc test LSD. Values are statistically significant at p < 0.05, when compared with (a) control rats, (b) diabetic control rats.

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Fig. 3. Levels of (A) renal p65 unit of NF-␬B and (B) renal NO in control and experimental groups of rats. Results are mean ± SEM (n = 6). One way ANOVA followed by post hoc test LSD. Values are statistically significant at p < 0.05, when compared with (a) control rats, (b) diabetic control rats, (c) diabetic + glyclazide.

The activities of kidney enzymatic antioxidants such as SOD, catalase, GPx, GST and GR in control and experimental groups of rats are depicted in Table 3. There was a significant (p < 0.05) decline in the activities of enzymatic antioxidants in kidney tissues of streptozotocin-induced diabetic rats when compared to the control group of rats. Conversely, these activities were significantly (p < 0.05) elevated in diabetic rats treated with d-pinitol as well as glyclazide. 3.5. Effect of d-pinitol on the normalization of renal histology and ultrastructure The photomicrograph of hematoxylin-eosin stained kidney tissues of control and experimental groups of rats are represented in Fig. 5A–D. Fig. 5A illustrates the section of kidney tissue of control

rats showing normal convoluted tubules, corticomedullary junction, renal hilum and glomeruli within the cortex. The diabetic group of rats (Fig. 5B) shows the enlargement of lining cells of tubules, fatty infiltration, capillary tufts and large area of hemorrhage and lymphocyte infiltration. These changes were normalized notably in the diabetic group of rats treated with d-pinitol as well as glyclazide (Fig. 5C and D). The ultrastructural changes occurred in the kidney tissues of control and experimental groups of rats are exemplified in Fig. 6A–D. The electron micrograph of control rat’s kidney shows a normal ultrastructure (Fig. 6A). In the diabetic rats, the glomeruli appears larger and dilated, Bowman’s capsule is partially thickened, swollen mitochondria, broadened podocyte, and vacuolization (Fig. 6B). Oral administration of d-pinitol as well as glyclazide normalized the above said alterations significantly (Fig. 6C and D).

Table 3 Activities of enzymatic antioxidants such as SOD, catalase, GPx, GST and GR in kidney tissue of control and experimental groups of rats. Groups

SOD

Control Diabetic control Diabetic + d-pinitol Diabetic + glyclazide

17.75 9.44 14.34 14.88

Catalase ± ± ± ±

0.71 0.73a 0.43b 0.44b

44.40 20.93 34.49 30.70

± ± ± ±

GPx 1.65 1.03a 1.10b 0.83bc

8.57 4.04 6.93 7.22

GST ± ± ± ±

0.34 0.21a 0.09b 0.15b

7.20 2.44 6.20 6.31

GR ± ± ± ±

0.28 0.17a 0.13b 0.27b

33.76 11.21 26.72 26.87

± ± ± ±

1.01 1.07a 1.63b 1.49b

Results are mean ± SEM (n = 6). One way ANOVA followed by post hoc test LSD. Values are statistically significant at p < 0.05, when compared with (a) control rats, (b) diabetic control rats, (c) diabetic + glyclazide. Activity is expressed as: 50% of inhibition of epinephrine autoxidation/min for SOD; ␮mol of hydrogen peroxide decomposed/min/mg of protein for catalase; ␮mol of glutathione oxidized/min/mg of protein for GPx; units/min/mg of protein for GST; ␮mol of DTNB-GSH conjugate formed/min/mg of protein for GR.

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Fig. 4. Levels of (A) lipid peroxide, (B) hydroperoxide and (C) protein carbonyls in kidney tissues of control and experimental groups of rats. Results are mean ± SEM (n = 6). One way ANOVA followed by post hoc test LSD. Values are statistically significant at p < 0.05, when compared with (a) control rats, (b) diabetic control rats.

4. Discussion Streptozotocin is a glucopyranose derivative of l-methyl-lnitrosourea and the existence of 2-deoxy-d-glucose in its structure expedites the preferential uptake of streptozotocin through GLUT2 into the pancreatic ␤-cells [42]. The nitrosourea moiety of streptozotocin is known to be involved in the alkylation of DNA, especially at the O6 position of guanine [43]. This induces a chain of cellular processes including poly-ADP-ribose synthetase activation

followed by nicotinamide adenine dinucleotide depletion resulting in the necrosis of pancreatic ␤-cells [44]. Further, streptozotocin is also known to provoke reactive oxygen species especially nitric oxide along with superoxide anion, hydroxyl radicals and hydrogen peroxide [45] thereby exerts its toxic effects on the pancreas, liver and kidneys [46]. Pancreatic ␤-cells are one among the least endowed cells because of their limited antioxidant competence than that of other tissues and hence they are selectively destroyed by streptozotocin.

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Fig. 5. Light micrographs of hematoxylin and eosin staining of kidney tissues of control and experimental groups of rats. Photomicrographs of (A) control, (B) diabetic control, (C) diabetic + d-pinitol and (D) diabetic + glyclazide stained by hematoxylin and eosin at 100× magnification, scale bar: 0.1 mm.

As a result, the expression and secretion of insulin is declined notably thereby leading to hyperglycemia, a clinical hallmark of diabetes. The declined level of plasma insulin also reflects its negative impact on hepatic tissue leading to the overproduction of hepatic glucose through accelerated glycogenolysis and gluconeogenesis. This supraphysiologic glucose non-enzymatically reacts with the free amino groups of globulin component of hemoglobin leading to the increased formation of glycosylated hemoglobin [47]. However, oral administration of d-pinitol to diabetic rats is reported to increase the plasma insulin level and normalizes the chronic hyperglycemia thereby reduces the formation of glycosylated hemoglobin in streptozotocin-induced experimental diabetic rats [21–23]. The ambient blood glucose level in diabetics induces notable decline in the total proteins and significant elevation in the levels

of blood urea, serum creatinine and uric acid which are standard markers of renal dysfunction [48]. These alterations might be due to the metabolic disturbances during diabetes by elevation in the activities of xanthine oxidase, increased levels of lipid peroxides, triglycerides and cholesterol. One of the major metabolic products of protein metabolism is urea. The protein glycation during diabetes is associated with muscle wasting and thereby, an increased release of purines. The elevated levels of purine nucleotides are the main source of uric acid, a water soluble antioxidant, by the increased activity of xanthine oxidase [49]. The creatinine is a byproduct of creatine and phosphocreatine catabolism, which is an energy storage compound in muscles. Creatinine is endogenously produced and released into body fluids and its clearance measured as an indicator of glomerular filtration rate. The oral administration of d-pinitol decreased the levels of blood urea, serum creatinine and

Fig. 6. Transmission electron micrographs of kidney tissues of control and experimental groups of rats. Transmission electron micrographs of (A) Control (×10,000), (B) diabetic control (×20,000), (C) diabetic + d-pinitol (×20,000) and (D) diabetic + glyclazide (×10,000). Brush border (BB), basement membrane (BM), capillary loop (CL), endothelial fenestrations (EF), mesangial cell (MC), mitochondria (M), podocytes (P), urinary space (US), scale bar: 1 ␮m.

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uric acid in streptozotocin-induced diabetic group of rats, thereby elicits the renoprotective nature of d-pinitol. Chronic hyperglycemia is one of the chief causative factors involved in the irreversible formation of AGEs through a reaction between sugars and the free amino groups on proteins, lipids, and nucleic acids [50]. Various studies reported that the AGEs are known to possess a variety of chemical, cellular and tissue damaging potential implicated in the development and progression of diabetic nephropathy [51]. However, oral administration of d-pinitol to diabetic group of rats showed a significant reduction in the formation of AGEs. The observed decrease in the levels of AGEs in diabetic rats might be one of the ameliorative ways by which d-pinitol recovers diabetic kidney from oxidative glycation. Moreover, AGEs are also involved in the activation of various transcription factors including NF-␬B which are implicated in the development of diabetic nephropathy [50]. The activation of NF-␬B in diabetic state mediates a cascade of signaling pathway leading to the renal dysfunction which is positively correlated with elevated oxidative-nitrosative stress and inflammation. Moreover, NF-␬B enhances the production of nitric oxide, which is reported to be involved in the pathogenesis of diabetic nephropathy, especially, cell destruction and progression of kidney tubular damage [52]. During hyperglycemia-mediated oxidative stress, NF-␬B activation obviously provokes the elevation of various proinflammatory cytokines such as TNF-␣, IL-1␤ and IL-6, which signify its role in diabetic nephropathy [53]. TNF-␣ is a pleiotropic proinflammatory cytokine produced by mesangial, glomerular, endothelial, dendritic and renal tubular cells [54]. TNF-␣ induced cytotoxicity occurs through varying mechanisms including the overproduction of reactive oxygen species which in turn damages the cellular components such as protein, lipids and DNA. Elevated production of TNF-␣ along with several inflammatory cytokines including IL-1␤ and IL-6 has been revealed to play a central role in the development and progression of diabetic nephropathy. IL-1␤ is able to stimulate the production of prostaglandins and nitric oxide in mesangial cells [55]. IL-6 disturbs extracellular matrix dynamics at mesangial and podocyte levels, stimulates mesangial cell proliferation, increases fibronectin expression and enhances endothelial permeability [56]. The overproduction of IL-6 and TNF-␣ are associated with deterioration of glycemic control, increased insulin resistance and dyslipidemia, which contributes to the dysfunction of the metabolic status of diabetics [57]. However, the oral administration of d-pinitol to diabetic group of rats reduced the TNF-␣, IL-1␤ and IL-6 levels suggesting its anti-inflammatory role in the attenuation of proinflammatory cytokines mediated toxicity. The elevated level of nitric oxide reacts with superoxide anion to produce peroxynitrite, a strong oxidant that in turn hastens the elevation of lipid peroxidation, which is one of the crucial cellular complications of chronic diabetes [58]. Hydroperoxides also causes cellular damage by itself or by disintegrating to highly toxic hydroxyl radicals. Thus prevention of the formation of hydroxyl radicals reduces the hydroxyl radical induced damage. Protein carbonyls are the secondary products of oxidative stress which are presumably formed in response to metal catalyzed Fenton reaction on proline, arginine, lysine or threonine residues [59]. The elevated level of protein carbonyls in addition with lipid peroxides and hydroperoxides resulted in the functional loss of specific membrane proteins and membrane permeability or destruction of cell or whole cell system [60]. The elevated levels of lipid peroxides, hydroperoxides and protein carbonyls in the diabetic group of rats are significantly reduced by the oral administration of d-pinitol by protecting the cells from peroxidative stress and lipid peroxidation. These results signify the free radical scavenging potential and antioxidant nature of d-pinitol.

The extent of oxidative stress is measured by elevation in the levels of lipid peroxidation, diminution of non-enzymatic antioxidant and enzymatic antioxidant activities in tissues and these modification leads to the increased susceptibility of tissues to oxidative damage. The GSH acts as a first line defense against the proxidant status [61]. It is a major intracellular redox system, acts as a direct scavenger as well as a co-substrate for GPx [62]. Vitamin E, a fat soluble vitamin, is regenerated from its oxidized form and its level is preserved in the presence of vitamin C. Vitamin E plays a pivotal role in the suppression of the propagation of lipid peroxidation. It inhibits the hydroperoxide formation along with vitamin C [63]. The levels of non-enzymatic antioxidants such as vitamin E, vitamin C and GSH are significantly decreased in streptozotocin-induced diabetic rats. However, oral administration of d-pinitol elevated the levels of these non-enzymatic antioxidants to near normalcy in streptozotocin-induced diabetic rats, thus signifying its role in the free radical scavenging property, which in turn readily accounts for its antidiabetic nature. The elevated level of oxidative stress leads to damage of membrane lipids, cellular proteins and nucleic acids which eventually leads to cell death. One of the sources of free radicals is oxidation of glucose in a transition-metal dependant reaction to enediol radicals; this is further converted into reactive ketoaldehydes and superoxide anion. The superoxide dismutase converts superoxide radical into hydrogen peroxide, subsequently catalase and GPx convert hydrogen peroxide into water [64]. Catalase catalyzes the reduction of hydroperoxide, that in turn protects the tissue from hydroxyl radical mediated damage [65]. Oxidized glutathione is recycled back to glutathione by GR, through an NADPH consuming process [66]. The observed decline in these enzymatic antioxidants in streptozotocin-induced diabetic rat is normalized by the administration of d-pinitol signifying its role in free radical quenching and antioxidant defense. Further, the renoprotective nature of d-pinitol was demonstrated by the hampering of oxidative stress, brought about by the amelioration of antioxidant defense and restraining peroxides in the streptozotocin-induced diabetic group of rats. The histological observation made on the kidney tissues further substantiates the claim that d-pinitol has renoprotective nature. Thus it may be concluded that d-pinitol possesses antioxidant activity against hyperglycemia-mediated oxidative stress in kidney. Conflict of interest The authors declare that there are no conflicts of interest. Acknowledgements The Research Fellowship of the University Grant Commission (UGC), New Delhi, India, to the first author, Mr. S. Sivakumar is gratefully acknowledged. The authors wish to record sincere thanks to Ms. B. Rita and Mr. P. Srinivasan, The Wellcome Trust Research Laboratory, Department of Gastrointestinal Sciences, Christian Medical College and Hospital, Vellore-632004, India, for their help in transmission electron microscopic and histopathological studies. References [1] S. Wild, G. Roglic, A. Green, R. Sicree, H. King, Global prevalence of diabetes: estimates for the year 2000 and projections for 2030, Diabetes Care 27 (2004) 1047–1053. [2] J.W. Baynes, Role of oxidative stress in development of complications in diabetes, Diabetes 40 (1991) 405–412. [3] J.W. Baynes, S.R. Thorpe, Role of oxidative stress in diabetic complications: a new perspective on an old paradigm, Diabetes 48 (1999) 1–9. [4] R. Baliga, N. Ueda, P.D. Walker, S.V. Shah, Oxidant mechanisms in toxic acute renal failure, Drug Metab. Rev. 31 (1999) 971–997.

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