d -Saccharic acid 1,4-lactone protects diabetic rat kidney by ameliorating hyperglycemia-mediated oxidative stress and renal inflammatory cytokines via NF-κB and PKC signaling

Toxicology and Applied Pharmacology 267 (2013) 16–29

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D-Saccharic

acid 1,4-lactone protects diabetic rat kidney by ameliorating hyperglycemia-mediated oxidative stress and renal inflammatory cytokines via NF-κB and PKC signaling Semantee Bhattacharya a, Prasenjit Manna b, Ratan Gachhui a, Parames C. Sil b,⁎ a b

Department of Life Sciences & Biotechnology, Jadavpur University, 188, Raja S C Mullick Road, Kolkata 700 032, India Division of Molecular Medicine, Bose Institute, P-1/12, CIT Scheme VII M, Kolkata-700054, India

a r t i c l e

i n f o

Article history: Received 12 September 2012 Revised 4 December 2012 Accepted 6 December 2012 Available online 19 December 2012 Keywords: Diabetic renal injury Aldose reductase PKC NF-κB and inflammation D-Saccharic acid 1,4-lactone Antioxidant

a b s t r a c t Increasing evidence suggests that oxidative stress is involved in the pathogenesis of diabetic nephropathy (DN) and this can be attenuated by antioxidants. D-Saccharic acid 1,4-lactone (DSL) is known for its detoxifying and antioxidant properties. Our early investigation showed that DSL can ameliorate alloxan (ALX) induced diabetes mellitus and oxidative stress in rats by inhibiting pancreatic β-cell apoptosis. In the present study we, therefore, investigated the protective role of DSL against renal injury in ALX induced diabetic rats. ALX exposure (at a dose of 120 mg/kg body weight, i. p., once) elevated the blood glucose level, serum markers related to renal injury, the production of reactive oxygen species (ROS), and disturbed the intra-cellular antioxidant machineries. Oral administration of DSL (80 mg/kg body weight) restored all these alterations close to normal. In addition, DSL could also normalize the aldose reductase activity which was found to increase in the diabetic rats. Investigating the mechanism of its protective activity, we observed the activation of different isoforms of PKC along with the accumulation of matrix proteins like collagen and fibronectin. The diabetic rats also showed nuclear translocation of NF-κB and increase in the concentration of inflammatory cytokines in the renal tissue. The activation of mitochondria dependent apoptotic pathway was observed in the diabetic rat kidneys. However, treatment of diabetic rats with DSL counteracted all these changes. These findings, for the first time, demonstrated that DSL could ameliorate renal dysfunction in diabetic rats by suppressing the oxidative stress related signalling pathways. © 2012 Elsevier Inc. All rights reserved.

Introduction Oxidative stress is increased in diabetes mellitus and plays an important role in the onset and progression of diabetic vascular complications including nephropathy (Oberley, 1988). Diabetic nephropathy (DN) is characterized by functional as well as structural abnormalities. Within the glomeruli, there is thickening of basement

Abbreviations: ALX, Alloxan monohydrate; AR, Aldose reductase; BSA, Bovine serum albumin; BUN, Blood urea nitrogen; CAT, catalase; CDNB, 1-chloro 2,4-dinitrobenzene; DAB, 3,3′-diaminobenzidine tetrahydrochloride; DNPH, 2,4-dinitrophenyl hydrazine; DSL, D-saccharic acid 1,4-lactone; DTNB, 5,5′-dithiobis(2-nitrobenzoic acid); FBG, Fasting blood glucose; GPx, glutathione peroxidase; GR, glutathione reductase; GSH, Reduced glutathione; GST, glutathione-S-transferase; IL, Interleukin; MDA, malonaldehyde; NBT, nitroblue tetrazolium; PARP, Poly (ADP-ribose) polymerase; ROS, reactive oxygen species; SOD, superoxide dismutase; TNF, tumor necrosis factor; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labelling. ⁎ Corresponding author at: Division of molecular medicine, Bose Institute, P-1/12, CIT Scheme VII M, Calcutta-700054, West Bengal, India. Fax: + 91 33 2355 3886. E-mail addresses: [email protected], [email protected] (P.C. Sil). 0041-008X/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.taap.2012.12.005

membranes and progressive accumulation of extracellular matrix components in the mesangium (Kang et al., 2008; Makino et al., 2006). The hyperglycemic environment in diabetes mellitus is the backbone of the pathophysiology of diabetic nephropathy (Vasavada and Agarwal, 2005). Hyperglycemia usually contributes to the oxidative stress either by the direct generation of reactive oxygen species (ROS) or by altering the redox balance (Shena and Gesualdo, 2005). This is believed to occur through several mechanisms, including increased polyol pathway flux (Hodgkinson et al., 2001), increased intracellular advanced glycation end products (AGEs) formation, protein kinase C activation (Inoguchi et al., 2000), poly ADP-ribose polymerase (PARP) over-activation (Drel et al., 2009) or mitochondrial dysfunction (Nishikawa et al., 2000). Besides, these highly active species can also induce cellular pathophysiology by up-regulating various transcriptional factors like NF-κB (Nuclear factor kappa-light-chain-enhancer of B cells), AP-1 (Activator protein-1), HIF-1 (Hypoxia-inducible factor-1), etc. involved in inflammatory pathways (Kanwar et al., 2008). It has now been established that inflammatory cytokines, mainly, IL-1, IL-6, IL-18 and TNF-α play important roles in the development of diabetic nephropathy leading to various associated complications (Navarro-Gonzalez and Mora-Fernandez, 2008). In the present study, alloxan (ALX), a

S. Bhattacharya et al. / Toxicology and Applied Pharmacology 267 (2013) 16–29

derivative of uric acid, has been used as a diabetogenic agent that induces renal injury by the formation of excessive free radicals (Sotoa et al., 2010) and thus provides a useful model to study the effectiveness of antioxidant compounds. D-Saccharic acid 1,4-lactone (DSL) is a derivative of D-glucaric acid and is present in dietary plants like cruciferous vegetables, citrus fruits, apples, etc. (Walaszek et al., 1996). It is a beta-glucuronidase inhibitor (Horton and Walaszek, 1982) and is known to possess antioxidative (Olas et al., 2007; Saluk-Juszcak et al., 2008), anticarcinogenic (Hanausek et al., 2003) and cholesterol lowering properties (Walaszek et al., 1996). DSL has also been found to reduce the activation of blood platelets (Saluk-Juszcak et al., 2008). Recently, we have also shown that DSL could ameliorate TBHP (tertiary butyl hydroperoxide) -induced oxidative stress in murine hepatocytes through mitochondria dependent pathways (Bhattacharya et al., 2011a). In our earlier investigations, we reported that DSL plays a beneficial role in reducing ALX-induced type 1 diabetes by inhibiting the apoptotic death of pancreatic β-cells (Bhattacharya et al., 2011b) and is also effective in attenuating the oxidative stress in the spleen tissue of diabetic rats (Rashid et al., 2012). In the present study, we investigated whether DSL could attenuate the renal injury and oxidative stress in the kidney tissues of diabetic rats by evaluating serum creatinine, blood urea nitrogen, antioxidant enzymes and glutathione levels. In addition, we explored the underlying molecular mechanisms involved in its protective action by investigating the role of aldose reductase, NF-kB and the inflammatory cytokines, protein kinase C and poly ADP-ribose polymerase, and the role of mitochondrial pathway. Thus the present study assesses the role of DSL as a potential agent for the treatment of renal tissue damage in hyperglycemic condition.

17

normal control (received only water as vehicle) and toxin control (received ALX intraperitoneally at a dose of 120 mg/kg body weight), respectively. The remaining four groups of ALX-induced diabetic rats were separately treated (orally) with four different doses of DSL — 20, 40, 80, 120 mg/kg body weight daily for 6 weeks. The effective dose of DSL for ameliorating the renal injury was selected by studying their effects on fasting serum glucose and serum creatinine levels.

Determination of time dependent effect of DSL for post treatment study. To determine the time dependent effects of DSL, experiments were carried out with eight groups of animals each consisting of six animals. DSL was administered to ALX-induced diabetic rats at a dose of 80 mg/kg body weight for 1, 2, 3, 4, 5 and 6 weeks respectively. In addition, two other groups of rats were kept as normal control (received only water as vehicle) and toxin control (received ALX intraperitoneally at a dose of 120 mg/kg body weight), respectively. The time of DSL administration was selected by studying its effect on fasting serum glucose and serum creatinine levels. Determination of time dependent preventive effect of DSL. To determine whether DSL could prevent diabetes by exposing rats for several weeks to DSL prior to administration of alloxan, a time dependent study was carried out with five groups of rats each consisting of six animals. First two groups were served as normal control (received only water as vehicle) and toxin control (received ALX intraperitoneally at a dose of 120 mg/kg body weight), respectively. DSL was administered to the remaining three groups of rats at a dose of 80 mg/kg body weight for 1, 2 and 3 weeks respectively. Then the rats were injected with ALX and after 1 week of ALX injection the fasting serum glucose level was measured to study the effect of DSL for preventing diabetes.

Materials and methods Chemicals. Alloxan monohydrate (ALX) (2,4,5,6-tetraoxypyrimidine), D-saccharic acid 1,4-lactone (DSL), bovine serum albumin (BSA) and Bradford reagent were purchased from Sigma-Aldrich Chemical Company, (St. Louis, MO) USA. All other reagents were bought from Sisco research laboratory, India. The antibodies were purchased from Abcam-Antibodies and Reagents Suppliers (PKC-α, PKC-β, PKC-δ and PKC-ε) and Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Animal. Swiss albino male rats (4 weeks old), weighing approximately 120–130 g, were purchased from M/S Ghosh Enterprises, Kolkata, India. They were acclimatized under laboratory conditions for two weeks prior to the experiments. They were fed standard pellet diet (Agro Corporation Private Ltd., Bangalore, India) and water ad libitum. All the experiments with animals were carried out according to the guidelines of the institutional animal ethical committee and full details of the study was approved by the CPCSEA, Ministry of Environment & Forests, New Delhi, India (the permit number is: 95/99/CPCSEA). Experimental induction of diabetes. After overnight fasting diabetes was induced in the experimental animals with an intraperitoneal injection of ALX dissolved in sterile normal saline at a dose of 120 mg/kg body weight (Kim et al., 2008). Diabetes was confirmed in the rats by measuring the fasting blood glucose concentration after 1 week of ALX injection. Blood for blood glucose was obtained by tail incision and measured using an Advanced Accu-check glucometer (Boehringer Mannheim, Indianapolis, IN, USA). The rats with blood glucose above 240 mg/dL were considered to be diabetic and then they were used for the experiments as necessary. Determination of dose dependent effect of DSL for post treatment study. For this study, rats were randomly divided into six groups each consisting of six animals. First two groups were served as

Experimental design. Experimental design needed for the present in vivo study has been summarized as follows: The animals were randomly assigned to five groups each consisting of six rats. Group 1 (Cont) consisted of normal animals that received vehicle only. Group 2 (DSL) consisted of normal rats that were administered DSL orally at a dose of 80 mg/mL to know whether any toxic effect was produced by DSL. Group 3 (ALX) consisted of ALX treated diabetic animals. Group 4 (ALX + DSL) consisted of animals that were given DSL orally at a dose of 80 mg/kg body weight for 6 weeks after diabetic induction. Group 5 (ALX+Insulin) consisted of animals that were administered insulin at a dose of 2 IU/kg body weight after diabetic induction (Kumar et al., 2012). The experimental protocol has been represented schematically in Fig. 1. After 6 weeks of treatment the animals were anesthetized first and then sacrificed. Blood was collected from the experimental animals by cardiac puncture and the serum was separated by centrifugation at 3000 ×g for 10 min. Kidney tissue collection and preparation of homogenates. The kidneys were aseptically taken out from the experimental rats, decapsulated, blotted dry, weighed and kept at −80 °C. Before each assay, the tissue homogenates (10%) were prepared in 100 mM potassium phosphate buffer (pH 7.4) containing 1 mM EDTA, 0.25 mM sucrose, 1 mM PMSF (protease inhibitor phenylmethane sulfonyl fluoride) and phosphatase inhibitor cocktail and centrifuged at 12,000 ×g for 30 min at 4 °C. The resultant supernatant was collected; the protein contents were measured

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Fig. 1. Schematic diagram of in vivo experimental protocol.

by the method of Bradford using crystalline BSA as standard (Bradford, 1976) and used for the experiments. Assessment of serum glucose and insulin levels. The serum glucose level in the experimental animals was estimated by using a commercially available glucose kit (Autospan, Span Diagnostics, India) based on the glucose oxidase method. Serum insulin was determined by ELISA using standard kits (Span diagnostic Ltd., India). Estimation of serum creatinine and blood urea nitrogen (BUN). Serum creatinine and BUN levels were estimated by using standard kits (Span diagnostic Ltd., India). Histological studies. Kidneys from the normal and experimental animals were fixed in 10% buffered formalin and were processed for paraffin sectioning. Sections of about 5 μm thickness were stained with hematoxylin and eosin to evaluate the pathophysiological changes under light microscope. Measurement of renal ROS level. Renal ROS level was estimated by using 2,7-dichlorofluorescein diacetate (DCFDA) as a probe following the method of Onozato et al. (2002) with slight modifications. DCFH-DA diffuses through the cell membrane where it is enzymatically deacetylated by intracellular esterases to the more hydrophilic nonfluorescent reduced dye dichlorofluorescin. In the presence of reactive oxygen metabolites, nonfluorescent DCFH rapidly oxidized to highly fluorescent product DCF. Briefly, 500 μl kidney homogenate (freshly prepared) was mixed with 4.4 mL 100 mM potassium phosphate buffer (pH7.4) and incubated with DCFH-DA at final concentration of 16 μg/ml for 20 min at 37 °C. After centrifuging at 10,000 ×g for 10 min at 4 °C, the pellet was suspended in 5 mL potassium phosphate buffer (pH7.4) on ice and incubated for 60 min at 37 °C. Fluorescence was measured fluorescence spectrometer (HITACHI, Model No F7000) at wavelengths of 485 nm for excitation and 535 nm for emission. Estimation of lipid and protein damage. Lipid peroxidation in the renal tissue homogenate of the experimental animals was assessed by a colorimetric method with thiobarbituric acid (TBA) as described by Esterbauer and Cheeseman (1990). The absorbance of thiobarbituric acid reactive substance (TBARS) formed was measured at 532 nm and

its concentration was calculated using the extinction coefficient of MDA (1.56× 105 M−1 cm−1) since 99% of TBARS exists as MDA. Protein carbonyl contents in the tissue homogenates were determined according to the method of Uchida and Stadtman (1993). The assessment was done based on the formation of protein hydrazone by reaction with 2,4-DNPH. The absorbance was recorded at 365 nm. The results were expressed as nmol of DNPH incorporated/mg protein based on the molar extinction coefficient of 22000 M −1 cm−1 for aliphatic hydrazones. Assay of antioxidant enzymes. The activities of antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT), glutathione-S-transferase (GST), glutathione reductase (GR) and glutathione peroxidase (GPx) were measured in the renal tissue of all the experimental rats following the method of Sinha et al. (2007). SOD activity (mainly Cu-Zn-SOD) was measured by the following ways. The sample containing 5 μg proteins was mixed with sodium pyrophosphate buffer, PMT and NBT. The reaction was started by the addition of NADH. Reaction mixture was then incubated at 30 °C for 90 seconds and stopped by the addition of 1 ml of glacial acetic acid. The absorbance was measured at 560 nm. One unit of SOD activity is defined as the enzyme concentration required to inhibit chromogen production by 50% in 1 min under the assay conditions. CAT activity was measured by mixing 5 μg proteins from the tissue homogenate with 2.1 ml of 7.5 mM H2O2 and the decrease in absorbance at 240 nm was monitored spectrophotometrically for about 10 min at 25 °C. One unit of CAT activity is defined as the amount of enzyme, which reduces 1 μmol of H2O2 per minute. GST activity was measured by mixing suitable amount of enzyme (25 μg of protein in renal tissue homogenate), KH2PO4 buffer, EDTA, CDNB and GSH. The reaction was carried out at 37 °C and monitored spectrophotometrically at 340 nm for 5 min. One unit of GST activity is defined as 1 μmol product formation per minute. GR activity was measured by mixing 50 μl of sample with 1 ml of 0.2 M KH2PO4 buffer containing 1 mM EDTA (pH 7.5), 500 μl of 0.3 mM DTNB, 250 μl water, 100 μl of 2 mM NADPH in water and 100 μl of 20 mM GSSG solution. The increase in absorbance at 412 nm was monitored spectrophotometrically for 3 min at 24 °C. The enzyme activity was calculated using molar extinction coefficient of 13,600 M −1 cm −1. One unit of enzyme activity is defined as the amount of enzyme which catalyzes the oxidation of 1 μmol NADPH per minute.

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Dose dependent

400

Time dependent

a

a

a

b

b b

200

b

b b

b

Blood glucose level (mg/dL)

300

b b 200

b 100 i

Fig. 2. Dose and time dependent effects of DSL on blood glucose level of ALX-induced diabetic rats. Cont: Blood glucose level in normal rats, ALX: Blood glucose level in ALX treated rats, ALX + DSL-20, ALX + DSL-40, ALX + DSL-80, ALX + DSL-120: Blood glucose level in DSL treated diabetic rats for 6 weeks at a dose of 20, 40, 80 and 120 mg/kg body weight, ALX + DSL-1, ALX + DSL-2, ALX + DSL-3, ALX + DSL-4, ALX + DSL-5, ALX + DSL-6: Blood glucose level in DSL treated diabetic rats for 1, 2, 3, 4, 5, 6 weeks at a dose 80 mg/kg body weight. Each column represents mean ± SD, n = 6. “a” indicates the significant difference between the normal control and diabetic control groups and “b” indicates the significant difference between the diabetic control and DSL treated diabetic groups (Pa b 0.05, Pb b 0.05).

GPx activity was assayed using H2O2 and NADPH as substrates. The conversion of NADPH to NADP+ was observed by recording the changes in absorption intensity at 340 nm. One unit of enzyme activity is defined as the amount of enzyme that catalyzes the oxidation of 1 μmol NADPH per minute.

Assay of aldose reductase activity. Aldose reductase (AR) activity was assayed spectrophotometrically by observing the decrease in the absorbance of NADPH at 340 nm using DL-glyceraldehyde as a substrate as described by Wei-hua et al. (2008). The assay mixture contained 30 mM potassium phosphate buffer (pH 6.5), 5 mM DL-glyceraldehyde,

X 3+

AL

X LDS

2+ LDS

DS

L-

1+

AL

AL

X

nt

C o LX A nt L A +D X LX S A + LL D 1 A X+ SL L D -2 A X+ SL L D -3 A X+ SL LX D -4 +DSLSL 5 -6 A

A

LX

C on t + A D AL LX S X A + L-2 L D A X+ SL 0 LX D +D SL 40 SL -80 -1 20

Co

0

X

0

i

AL

Blood glucose level (mg/dl)

400

19

Fig. 3. Time dependent preventive effects of DSL on blood glucose level of ALX-induced diabetic rats. Cont: Blood glucose level in normal rats, ALX: Blood glucose level in ALX treated rats, DSL-1 + ALX, DSL-2 + ALX, DSL-3 + ALX: Blood glucose level in diabetic rats that were treated with DSL for 1, 2 and 3 weeks respectively prior to ALX injection. Each column represents mean± SD, n= 6. “a” indicates the significant difference between the normal control and diabetic control groups and “b” indicates the significant difference between the diabetic control and the diabetic rats pretreated with DSL (Pa b 0.05, Pb b 0.05).

0.2 M ammonium sulfate, and 1.0 mM NADPH. The results were presented in unit of μmol NADPH per minute per gram protein. Assay of reduced glutathione. Reduced glutathione levels were measured by the method of Hissin and Hilf (1976) using o-phthalaldehyde. The method takes advantage of the reaction of GSH with OPT at pH 8 and the fluorescence was determined at excitation wavelength of 360 nm and emission wavelength of 460 nm. Measurement of hydroxyproline and fibronectin levels. The hydroxyproline levels of the kidneys were estimated according to Woessner's method (1961). One ml of tissue homogenates were sealed in small Pyrex test tubes and hydrolyzed for 12 h at 110 °C by adding 5 mL of 6 N HCl. The hydrolysates were neutralized by 2.5 N NaOH and 0.25 mL of the mixture was used for analyses. Hydroxyproline oxidation was initiated by adding chloramine T and the tube contents were

Table 1 Effect of alloxan (ALX) and DSL on body weight (BW), serum glucose, plasma insulin, kidney weight (KW), KW/BW and blood urea nitrogen of the normal and experimental animals. Parameters

Normal control

DSL treated

ALX treated

ALX+ DSL

ALX+ INSULIN

Body weight (BW) (g) Serum glucose (mg/dl) Plasma insulin (μU/ml) Kidney weight (KW) (g) KW/BW (×10−3) Blood urea nitrogen (BUN) (mg/dL)

192.35. ± 7.52

190.67 ± 7.27

149.24 ± 5.36a

177.43 ± 6.78b

175.67 ± 6.23b

118.32 ± 6.56

121.45 ± 7.2

367.58 ± 17.79a

153.43 ± 8.3b

149.25 ± 8.2b

12.43 ± 0.62

7.12 ± 0.32a

10.27 ± 0.54b

0.98 ± 0.04

0.96 ± 0.04

1.27 ± 0.06a

1.03 ± 0.05b

0.99 ± 0.05b

6.4 ± 0.3

6.5 ± 0.32

11.6 ± 0.55a

7.4 ± 0.35b

6.8 ± 0.36b

22.56 ± 1.12

79.56 ± 3.87a

28.32 ± 1.84b

26.35 ± 1.56b

11.85 ± 0.6

21.79 ± 1.07

11.57 ± 0.7b

Values are expressed as mean ± SD, for 6 animals in each group. “a” indicates the significant difference between the normal control and ALX treated groups, “b” indicates the significant difference between the ALX treated and DSL or insulin treated groups, (Pa b 0.05, Pb b 0.05).

20

A

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1.2

Plasma creatinine (mg/dL)

a b

0.8

b

b

0.4

i

B

X+

-1 20 SL

AL

AL

X+ D

DS

L80

L40 DS

L20

X+

AL

AL

X+

DS

AL

X

co nt

0.0

1.2

Plasma creatinine (mg/dL)

a b

0.8

b

b

b

0.4 i

6 L-

5 DS

AL

X+

L-

4 LX+ AL

DS

DS

3 AL

X+

L-

2 LX+ AL

DS

DS

-1 X+

DS L

AL

X AL

X+

AL

co

nt

0.0

Fig. 4. A. Dose dependent effect of DSL on plasma creatinine level of ALX-induced diabetic rats. Cont: Plasma creatinine level in normal rats, ALX: Plasma creatinine level in ALX treated rats, ALX+DSL-20, ALX+DSL-40, ALX+DSL-80, ALX+DSL-120: Plasma creatinine level in DSL treated diabetic rats for 6 weeks at a dose of 20, 40, 80 and 120 mg/kg body weight. Each column represents mean±SD, n=6. “a” indicates the significant difference between the normal control and diabetic control groups and “b” indicates the significant difference between the diabetic control and DSL treated diabetic groups. (Pa b 0.05, Pb b 0.05). B. Time dependent effect of DSL on plasma creatinine level of ALX-induced diabetic rats. Cont: Plasma creatinine level in normal rats, ALX: Plasma creatinine level in ALX treated rats, ALX+DSL-1, ALX+DSL-2, ALX+DSL-3, ALX+DSL-4, ALX+DSL-5, ALX+DSL-6: Plasma creatinine level in DSL treated diabetic rats at a dose of 80 mg/kg body weight for 1, 2, 3, 4, 5, and 6 weeks. Each column represents mean±SD, n=6. “a” indicates the significant difference between the normal control and diabetic control groups and “b” indicates the significant difference between the diabetic control and DSL treated diabetic groups. (Pa b 0.05, Pb b 0.05).

kept at room temperature for 5 min. Chloramine T was then removed by adding 3.15 M perchloric acid. After 5 min, 3.25 mL of Ehrlich's reagent was finally added; the mixture was shaken, and the tubes were placed in a 60 °C water bath for 25 min. They were then cooled in tap water for 5 min. The absorbance values of the solutions were

Fig. 5. Hematotoxylin and eosin stained kidney sections. A, B: renal section of normal rat kidney, A (× 200), B (× 400); C, D: renal section from animals treated with DSL only, C (× 200), D (× 400); E, F: renal section from the diabetic group, E (× 200), F (× 400); G, H: renal section from the animal treated with DSL after diabetic induction, G (× 200), H (× 400); and I, J: renal section from animal treated with insulin after diabetic induction, I (× 200), J (× 400). The arrows indicate basement membrane thickening in the renal tissue of diabetic rats. BC — Bowman's capsule; G — glomerulus; P — proximal convoluted tubule.

determined at 558 nm. The hydroxyproline values were calculated from L-hydroxyproline standard curve. The fibronectin levels were determined by ELISA following the method of Drel et al. (2009). Assay of inflammatory cytokines. IL-1β, IL-6, IL-18 and TNF-α concentrations in the renal tissue samples were analyzed using enzyme linked immune sorbent assay (ELISA). Optical density was measured

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100 ± 4.8

102.32 ± 5.1

318.54 ± 14.92a 159.67 ± 7.88b

7.79 ± 0.38

8.62 ± 0.42

16.24 ± 0.8a

9.74 ± 0.47b

4.39 ± 0.2

4.36 ± 0.19

11.18 ± 0.5a

7.29 ± 0.35b

Values are expressed as mean ± SD, for 6 animals in each group. “a” indicates the significant difference between the normal control and ALX treated groups, “b” indicates the significant difference between the ALX treated and DSL treated groups, (Pa b 0.05, Pb b 0.05).

with a Labsystems integrated EIA Management System iEMS and the DeltaSoft 3 2.22 EMS software (Dr. E Bechthold and Biometallics). DNA fragmentation assay. Genomic DNA was isolated from renal tissues of normal as well as experimental rats by phenol-chloroform method and was then electrophoresed on 1% agarose gel in presence of ethydium bromide (Das and Sil, 2012). The fragmented DNA (DNA ladder) was visualized by UV light.

AR activity (μ μmol NADPH/min/g protein)

A

TUNEL staining to confirm DNA fragmentation. Paraffin embedded renal tissue sections (5 μm) were warmed 30 min (64 °C), deparaffinized and rehydrated. Terminal transferase mediated dUTP nick end-labeling of nuclei was performed by using APO-BrdU TUNEL Assay kit (A-23210; Molecular Probes, Eugene, OR) following the manufacturer's protocol.

DSL treated

ALX treated

ALX+ DSL

8.45 ± 0.42

8.2 ± 0.4

2.09 ± 0.1a

6.34 ± 0.3b

87.79 ± 4.38

86.99 ± 3.99

16.24 ± 0.75a

68.74 ± 3.43b

b

0.5

nt

AL

Co

10

b 8

6

a 4

2

SL +D

LX

0.39 ± 0.019

0.36 ± 0.016

19.67 ± 0.98

18.72 ± 0.85

38.34 ± 1.87

37.28 ± 1.8

0.18 ± 0.011a

6.84 ± 0.32a

18.38 ± 0.8a

0.29 ± 0.015b

16.76 ± 0.8b

28.32 ± 1.4b

SOD — superoxide dismutase, CAT — catalase, GST — glutathione-S-transferase, GR — glutathione reductase and GPx — glutathione peroxidase. Values are expressed as mean±SD, for 6 animals in each group. “a” indicates the significant difference between the normal control and ALX treated groups, “b” indicates the significant difference between the ALX treated and DSL treated groups, (Pa b 0.05, Pb b 0.05).

A

LX

A

SL D

t

0 on

SOD (unit/mg protein) CAT (μmol/min/mg protein) GST (μmol/min/mg protein) GR (nmol/min/mg protein) GPx (nmol/min/ mgprotein)

Normal control

1.0

C

Parameters

GSH level (nmol/mg protein)

Table 3 Effect of alloxan (ALX) and DSL on antioxidant status in the renal tissue of the normal and experimental animals.

a

0.0

B Isolation of mitochondria from renal tissue. Mitochondria were isolated from the renal tissue following the method of Kayal et al. (2004). Following homogenization in an ice cold solution containing 140 mM KCl and 20 mM HEPES (pH 7.4), kidney tissue homogenates were centrifuged at 1000 ×g for 10 min at 4 °C. Supernatants were taken and centrifuged at 10,000 ×g for 15 min at 4 °C. Pellets were washed twice in 140 mM KCl and 20 mM HEPES (pH 7.4), and respun

1.5

L

ROS (%) MDA (nmole/mg protein) Protein carbonyl content (nmol/mg protein)

Determination of mitochondrial membrane potential (Δψm). Mitochondrial membrane potential (Δψm) was estimated on the basis of cell retention of the fluorescent cationic probe rhodamine 123 (Zamzami and Kroemer, 2004). The fluorescence of rhodamine 123 was determined using BD-LSR flow cytometer. Cell debris,

DS

ALX+ DSL

X+

ALX treated

X

DSL treated

AL

Normal control

L

Parameters

at 10,000 ×g. Following the final wash, mitochondria were resuspended in 0.02 M phosphate buffer (pH 7.4).

DS

Table 2 Effect of alloxan (ALX) and DSL on ROS production, lipid peroxidation, and protein carbonylation in the renal tissue of the normal and experimental animals.

21

Fig. 6. A. Effect of DSL on AR activity in the renal tissue of the normal and experimental rats. Cont: normal rats, DSL: normal rats treated with DSL only, ALX: diabetic control and ALX + DSL: diabetic rats treated with DSL. The measurements were made in six times. Data represent the average ± SD of 6 separate experiments in each group. “a” indicates the significant difference between the normal control and diabetic control groups and “b” indicates the significant difference between the diabetic control and DSL treated diabetic groups (Pa b 0.05, Pb b 0.05). B. Effect of DSL on GSH level in the renal tissue of the normal and experimental rats. Cont: normal rats, DSL: normal rats treated with DSL only, ALX: diabetic control and ALX + DSL: diabetic rats treated with DSL. The measurements were made in six times. Data represent the average ± SD of 6 separate experiments in each group. “a” indicates the significant difference between the normal control and diabetic control groups and “b” indicates the significant difference between the diabetic control and DSL treated diabetic groups (Pa b 0.05, Pb b 0.05).

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stored at −80 °C. The protein contents were measured by the method of Bradford using crystalline BSA as standard (Bradford, 1976).

Assay of cytochrome c release from mitochondria to the cytosol. The concentration of cytosolic cytochrome c was measured with the cytochrome c enzyme immunometric assay kit (R&D Systems, Minneapolis, USA). Firstly, the renal tissue homogenate of the experimental rats was subjected to centrifugation at 100,000 ×g for 60 min at 4 °C to obtain the cytosolic fraction (the supernatant). The cytosolic fraction was then incubated with a monoclonal antibody specific for rat cytochrome c immobilized on a microtiter plate for 1 h. Then the sample in excess was washed out and a horseradish peroxidase conjugated monoclonal antibody specific for rat cytochrome c was added. After 1 h of incubation, excess antibody was washed out and the substrate solution (a mixture of H2O2 and tetramethylbenzidine) was added and further incubated for 30 min at room temperature. Finally, HCl was added as stop solution and the optical density was determined at 450 nm using ELISA Microplate Reader (Bio-Rad, USA).

Immunoblotting. Samples containing 50 μg proteins were subjected to 10% SDS-PAGES and transferred to a nitrocellulose membrane. Membranes were blocked at room temperature for 2 h in blocking buffer containing 5% non-fat dry milk to prevent non specific binding and then incubated with primary antibodies overnight at 4°C. The primary antibodies used in the present study were anti Bax (1:500), anti Bcl-2 (1:500), anti caspase-3 (1:1000), anti-PARP (1:1000), anti PKCα (1:500), anti PKCβ (1:500), anti PKCδ (1:500), anti PKCε (1:500), anti NF-κb p65 (1:1000), anti lamin B1 (1:500) and anti β-actin (1:1000) antibodies. The membranes were washed in TBST (50 mM Tris–HCl, pH 7.6, 150 mM NaCl, 0.1% Tween 20) for 30 min and incubated with appropriate HRP conjugated secondary antibody (1:2000) for 2 h at room temperature and developed by the HRP substrate 3,3′-diaminobenzidine tetrahydrochloride (DAB) system (Bangalore, India).

Preparation of nuclear extract from renal tissue. A nuclear extract was prepared from renal cortex as described by Iwamoto et al. (2005). In brief, frozen renal cortex was minced and suspended in 1 ml of ice-cold TBS buffer (25 mM Tris–HCl [pH 7.4], 130 mM NaCl, and 5 mM KCl) and homogenized. The homogenate was centrifuged at 7000 ×g for 2 min at 4 °C, the pellet was lysed in 1 ml of ice-cold buffer A (10 mM HEPES [pH 7.9], 10 mM KCl, 0.1 mM EDTA [pH 7.5], 0.1 mM EGTA [pH 7.5], 1 mM PMSF, and 1 mM dithiothreitol [DTT]) and incubated on ice for 20 min. Next, 100 μl of 10% Nonidet P-40 was added and vigorously vortexed, and the extract was centrifuged at 12,000 ×g for 7 min at 4 °C. The nuclei then were extracted with 100 μl of ice-cold buffer C (20 mM HEPES [pH 7.9], 400 mM NaCl, 1 mM EDTA [pH 7.5], 1 mM EGTA [pH 7.5], 1 mM PMSF, and 1 mM DTT), incubated on ice for 2 h, and centrifuged at 12,000 ×g for 7 min at 4 °C. The supernatant fraction was

Statistical analysis. All the values are expressed as mean±S.D. (n=6). Significant differences between the groups were determined with SPSS 10.0 software (SPSS Inc., Chicago, IL, USA) for Windows using one-way analysis of variance (ANOVA) and the group means were compared by Duncan's Multiple Range Test (DMRT). A difference was considered significant at the Pb 0.05 level.

2

b

SL

A a

2

b

SL +D

LX

LX

A

SL D

C

on t

1

A

A

Relative band intensity of PKC-ε (arbitrary unit)

SL +D

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ALX

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DSL

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Cont

1

SL

42 kDa

b

D

β-actin

2

on t

84 kDa

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PKC-ε

Relative band intensity of PKC-δ (arbitrary unit)

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81 kDa

+D

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SL D

on t

1

C

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+D SL

1

Relative band intensity of PKC-β (arbitrary unit)

b

a

LX

PKC-δ

2

A

77 kDa

a

SL

PKC-β

Reduction in body weight, increase in blood glucose level and decrease in plasma insulin level are the markers for the development of diabetes. This pathophysiology was observed in ALX-treated rats

D

80 kDa

Evaluation of induced diabetes and effect of DSL

on t

PKC-α α

Results

C

Relative band intensity of PKC-α (arbitrary unit)

characterized by a low FSC/SSC was excluded from analysis. The data was analysed by Cell Quest software.

Fig. 7. Western blot analysis of PKC-α, PKC-β, PKC-δ and PKC-ε in the renal tissue of diabetic and DSL treated animals. The relative intensities of the bands were determined using NIH- image software and the control band was given an arbitrary value of 1. Data represent the average ± SD of 6 separate experiments in each group. “a” indicates the significant difference between the normal control and diabetic control groups and “b” indicates the significant difference between the diabetic control and DSL treated diabetic groups (Pa b 0.05, Pb b 0.05).

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(Fig. 2 & Table 1), suggesting their diabetic nature. DSL supplements, however, lowered the blood glucose level in both dose and time dependent manners (Fig. 2), reaching almost the control values. Moreover, DSL was also found to prevent the onset of hyperglycemia as is evident from the pre-treatment study (Fig. 3). It also attenuated the reduction in insulin level as well as loss in body weight compared to the diabetic control group (Table 1) and the results are comparable to that of the positive control, insulin.

23

Effect of DSL on AR activity in diabetic kidney AR, the first and rate-limiting enzyme in the polyol pathway, is activated by hyperglycemia. AR activation is involved in the pathogenesis of diabetic complications, including diabetic nephropathy. Fig. 6A shows that the activities of AR in the diabetic group were increased by 120% compared to the normal group and this increase was, however, inhibited by DSL treatment.

Effect of DSL on kidney weight and renal function related parameters

A Hydroxyproline level (μ μg/ 100mg tissue)

In the present study, we measured the kidney weight and the serum specific renal function related parameters (creatinine and BUN) in order to elucidate whether ALX administration induced renal injury. Table 1 represents the kidney weights, KW/BW ratio and BUN levels of normal and experimental diabetic rats. The kidney weight and BUN levels were increased by about 29.5% and 265% respectively in alloxan induced diabetic rats compared with those in the control group. Administration of DSL at a dose of 80 mg/kg body weight to diabetic rats significantly reversed these changes to near normal levels and this effect was almost equal to that of insulin (positive control). However, no noteworthy changes were observed in the normal rats treated with DSL. Plasma creatinine assay was used to determine the optimum dose and time necessary for DSL for the protection of rat kidney under hyperglycemic condition. It has been observed that hyperglycemia increased the plasma creatinine level by about 2 folds and that could be attenuated by treatment with DSL at a dose of 80 mg/kg body weight for 6 weeks (Figs. 4A and B).

80 70

a

60

b

50 40 30 20 10

SL +D

SL

LX

Effect of DSL on the activities of antioxidant enzymes in diabetic kidney For studying the effect of DSL on antioxidant status, the activities of the antioxidant enzymes SOD, CAT, GPx, GST and GR were measured (Table 3). Reduced activities of the renal antioxidant enzymes were observed in diabetic animals. Treatment with DSL at a dose of 80 mg/ml for six weeks resulted in the attenuation of ALX-induced decrease in the activities of antioxidant enzymes (Table 3).

a 20

b

10

SL +D A LX

A LX

D

SL

0

t

It is well established that ALX results in the overproduction of ROS that leads to the diabetic complications (Hashemi et al., 2009). These free radicals also damage the protein molecules and degrade the membrane bound phospholipids through the process of lipid peroxidation (Baynes, 1991). Table 2 shows a significant elevation in the level of ROS (about 3 folds) in ALX-induced diabetic rats compared to normal control. The levels of MDA and protein carbonylation in diabetic rats were also significantly higher by 2 folds and 3 folds respectively than their control counterparts. DSL treatment, post to the ALX exposure, has found to be effective in reducing the ALX-induced renal oxidative stress under hyperglycemic condition (Table 2).

30

C on

Suppressive effect of DSL on ROS generation, lipid peroxidation and protein carbonylation

B Fibronectin level (μg/g protein)

Histological assessments of various renal segments of the normal and experimental rats are presented in Fig. 5. Results showed typical renal pathological changes in diabetic rats, including degenerated glomeruli, basement membrane thickening, marked proliferation of mesanglial cells, and mesanglial matrix accumulation. Treatment with DSL attenuated these changes and it was comparable to that of insulin.

A

LX

A

C

D

on

t

0 Effect of DSL on renal morphology of diabetic rats

Fig. 8. A. Study on the hydroxyproline level of the renal tissue of the normal and experimental rats. Cont: normal rats, DSL: normal rats treated with DSL only, ALX: diabetic control and ALX+DSL: diabetic rats treated with DSL. The measurements were made in six times. Data represent the average±SD of 6 separate experiments in each group. “a” indicates the significant difference between the normal control and diabetic control groups and “b” indicates the significant difference between the diabetic control and DSL treated diabetic groups (Pa b 0.05, Pb b 0.05). B. Study on the fibronectin level of the renal tissue of the normal and experimental rats. Cont: normal rats, DSL: normal rats treated with DSL only, ALX: diabetic control and ALX+DSL: diabetic rats treated with DSL. The measurements were made in six times. Data represent the average±SD of 6 separate experiments in each group. “a” indicates the significant difference between the normal control and diabetic control groups and “b” indicates the significant difference between the diabetic control and DSL treated diabetic groups (Pa b 0.05, Pb b 0.05).

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The cellular metabolite GSH is a vital intracellular protective antioxidant against oxidative stress. Fig. 6B shows that the level of reduced glutathione was significantly reduced in the renal tissue of diabetic rats compared to the normal ones. Administration of DSL to the diabetic rats ameliorated this change in GSH content and thus maintaining the antioxidant status close to normal.

Reports suggest that of the different isoforms of PKC, PKC-α, PKC-β, PKC-δ and PKC-ε are mainly involved in the pathogenesis of renal complications in diabetes mellitus (Meier et al., 2007). These are sensitively activated by hyperglycemia-induced oxidative stress in rat kidney. In our study, immunoblot analysis showed that ALX administration stimulated the expression of PKC-α, PKC-β, PKC-δ and PKC-ε in the renal tissue. Treatment with DSL reduced the protein level of these isoforms (Fig. 7).

Effect of DSL on the activation of PKCs

Effect of DSL on hydroxyproline and fibronectin levels in the kidney of DN rats

Increased AR activity and oxidative stress due to overproduction of ROS by heperglycemia stimulate the activation of PKC within cells leading to some of the pathophysiological changes associated with DN (Inoguchi et al., 2000; Ramana et al., 2006). However, PKC is not a single entity but consists of a number of different serine/threonine kinases.

Collagen and fibronectin are considered as markers of fibrosis. Collagen is usually determined by estimating the hydroxyproline content. Figs. 8A and B show that renal hydroxyproline and fibronectin levels were significantly higher in diabetic rats than in normal control rats. These reflect the severity of kidney fibrosis of diabetic

A

+D

LX

SL

0

b

0.5 a

SL

0.0 +D

ALX+ DSL

A LX

ALX

LX

DSL

A

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1.0

D SL

42 kDa

on t

β-actin

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65 kDa

Relative band intensity of cytosolic NF-κBα α (arbitrary unit)

A

Cytosolic NF-κB p65

1

LX

48 kDa

b

SL

Lamin B1

2

D

65 kDa

a

t

Nuclear NF-κB p65

3

on

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C

Relative band intensity of cytosolic NF-κB p65 (arbitrary unit)

Effect of DSL on renal level of reduced glutathione (GSH)

a

phospho I κ B α

ALX

ALX+ DSL

SL

DSL

A

Cont

1

LX +D

42 kDa

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A

35 kDa

2

SL

total I κ B α

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35 kDa

C on t

phospho κBα total IκBα (arbitrary unit)

B

Fig. 9. A, B. Western blot analysis of NF-κB p65 (Fig. 7A) and IκBα (Fig. 7B) in the renal tissue of diabetic and DSL treated animals. The relative intensities of bands were determined using NIH-image software and the control band was given an arbitrary value of 1. Data represent the average ± SD of 6 separate experiments in each group. “a” indicates the significant difference between the normal control and diabetic control groups and “b” indicates the significant difference between the diabetic control and DSL treated diabetic groups (Pa b 0.05, Pb b 0.05).

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A

B a

40

100 75

b

50 25 i

IL-6 (pg/mg protein)

0

a

30 b

20 10

C

A LX A LX +D SL

D SL

C on t

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D SL

C on t

0

D a

a

350

b

16

8

0

TNF-α (pg/mg protein)

24

300 250 b

200 150 100 50

SL

A

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+D

LX A

SL

t on C

SL

A

LX

+D

LX A

SL D

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on

t

0

D

IL-1 (β β pg/mg protein)

125

IL-18 (pg/mg protein)

25

Fig. 10. Study on the concentration of the cytokines IL-1 (Panel A), IL-6 (Panel B), IL-18 (Panel C), TNF-α (Panel D). Cont: normal rats, DSL: normal rats treated with DSL only, ALX: diabetic control and ALX + DSL: diabetic rats treated with DSL. The measurements were made in six times. Data represent the average ± SD of 6 separate experiments in each group. “a” indicates the significant difference between the normal control and diabetic control groups and “b” indicates the significant difference between the diabetic control and DSL treated diabetic groups (Pa b 0.05, Pb b 0.05).

rats. However, hydroxyproline and fibronectin levels in the kidney were significantly reduced by DSL. Effect of DSL on NF-κB activation in diabetic kidney Because NF-κB activation requires the translocation of the p65 subunit NF-κB to the nucleus, we examined the protein levels of

p65 subunit of NF-κB in both the nuclear and cytosolic fraction of the renal tissue of diabetic rats by Western blot. Fig. 9A clearly illustrates that there were low levels of NF-κB in the nuclear fraction of the renal tissue in absence and presence of DSL although high levels were observed in the cytosolic fraction. It was also observed that the levels of p65 in the cytosolic fraction decreased whereas its level increased in the nuclear fraction of ALX-induced diabetic rat

Fig. 11. A. DNA fragmentation on agarose/ethydium bromide gel. DNA isolated from renal tissues of experimental rats was loaded onto 1% (w/v) agarose gels. Lane 1: Marker (1 kb DNA ladder); Lane 2: DNA isolated from kidney of normal rats; Lane 3: DNA isolated from the renal tissue of animals treated with DSL only; Lane 4: DNA isolated from the renal tissue of the diabetic animals (arrows indicate the DNA laddering); Lane 5 and DNA isolated from the renal tissue of the animals treated with DSL after diabetic induction. B. Tunel staining in renal tissue sections in experimental animals. Cont: renal section of normal rat kidney (× 100); DSL: renal section from animals treated with DSL only (× 100); ALX: renal section from the diabetic group (× 100); and ALX+DSL: renal section from the animal treated with DSL after diabetic induction (× 100).

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kidneys. Interestingly, diabetic rats fed with DSL showed decrease in the level of nuclear NF-κB p65 (Fig. 7A). Also, as shown in Fig. 9B, the levels of IκBα protein in cytosolic fraction decreased in diabetic rats, but diabetic DSL fed rats showed higher levels of this protein. Thus, these findings strongly indicate that enhanced nuclear translocation of NF-κB during diabetic renal injury is associated with increased IκBα degradation and that these can be attenuated by DSL treatment. Effect of DSL on the levels of inflammatory cytokines Recent evidences indicate that inflammatory cytokines play a significant role in the development and progression of diabetic nephropathy (Navarro and Mora, 2005). Among them, IL-1β, IL-6, IL-18 and TNF-α are relevant to the development of diabetic nephropathy (Navarro-Gonzalez and Mora-Fernandez, 2008). Fig. 10 shows an increase in the concentration of these inflammatory cytokines in ALXinduced diabetic rats whereas treatment with DSL reduces their concentration. Effect of DSL on ALX-induced apoptosis in kidney ALX-induced renal cell death in diabetic rats was investigated by Western blot analyses, DNA gel electrophoresis and tunnel assay. Administration of ALX induced a clear apoptotic response as indicated by the ladder pattern obtained in DNA gel electrophoresis (Fig. 11A) and TUNEL positive nuclei (Fig. 11B) in the renal tissue that diminished significantly after 6 weeks of DSL treatment. Further, we have investigated the involvement of mitochondria in kidney apoptosis and observed that ALX significantly decreased the expression of Bcl-2 and increased the level of Bax (Fig. 12A), causing a decrease of Bcl-2/Bax ratio. In addition, ALX also reduced the mitochondrial membrane potential (Fig. 12B) and increased the translocation of mitochondrial cytochrome c into cytosol (Fig. 12C). This release of cytochrome c can induce apoptosis by activation of downstream caspases which subsequently modulated PARP cleavage from its full length form (116 kDa) to the cleaved form (84 kDa). In our present study, immunoblot analysis showed an increase in the level of caspase-3 (Fig. 12D) in ALX-induced diabetic rats. Western blot analysis also revealed that ALX exposure caused the degradation of 116 kDa PARP to the characteristic 84 kDa PARP fragment (Fig. 12E). However, DSL effectively reduced the ALXinduced translocation of cytochrome c into cytosol, normalized the Bcl-2/Bax ratio and the mitochondrial membrane potential in the renal tissues of ALX-exposed rats. All these results clearly indicated the anti-apoptotic role of DSL in renal tissue under ALX-induced diabetic conditions. Discussion Diabetes mellitus (DM) is the most common endocrine disease that is associated with chronic hyperglycemia (Baynes, 1991). The chronic hyperglycemia brings about a rise in oxidative stress due to overproduction of reactive oxygen species (ROS), leading to diabetic

complications and tissue damage including renal injury (Brownlee, 2001; Wolff and Dean, 1987). In the present study we demonstrated the potential benefits of DSL in ameliorating the renal injury in ALX-induced diabetes mellitus through its antioxidant, anti-apoptotic and anti-inflammatory effects. Alloxan, a classical diabetogen, is often used to induce diabetes in animals. It is a potent generator of ROS which can mediate pancreatic beta cell toxicity resulting in significant increase in blood glucose level and leading to diabetic complications (Verma et al., 2010). In this study, diabetes was induced in Swiss albino rats by intraperitoneal injection of 120 mg/kg b.w. of alloxan. We observed that DSL significantly decreased the blood glucose level for both post and pre treatment studies (Figs. 2 & 3) and attenuated the plasma insulin level (Table 1) in the diabetic rats. In addition, the kidney weight/body weight, BUN, creatinine and glomerular hypertrophy were significantly increased in ALX-induced diabetic rats compared with the normal group, indicating glomerular injury and renal dysfunction (Table 1). Treatment with DSL at an appropriate dose (80 mg/kg BW/day for 6 weeks) was useful in the attenuation of renal injury in the diabetic animals and the effect was comparable to that of insulin. The findings described herein provide the first evidence that DSL effectively prevents the progression and development of diabetic nephropathy in animal models. Recent literature suggests that diabetic subjects show an increase in ROS generation with an accompanying disturbance in the red-ox status (Bloch-Damti and Bashan, 2005; Jain, 1989; Rains and Jain, 2011). Our study also showed that kidney dysfunction resulting from diabetes was accompanied with an alteration in the antioxidant status of the renal tissue (Tables 2 and 3). The inexorable generation of ROS and lipid peroxides during diabetes-mediated oxidative stress could be correlated to the increase in the levels of MDA and protein carbonylation (Table 2).and the decline in the activities of antioxidant enzymes (SOD, CAT, GST, GR and GPx) (Table 3). This could be successfully reversed by the DSL treatment suggesting its protective action in the oxidative stress induced renal dysfunction of diabetic rats. An alternative route of glucose metabolism is the polyol pathway. Aldose reductase (AR), the first and rate-limiting enzyme in the polyol pathway, is activated by hyperglycemia and catalyzes the reduction of glucose to sorbitol using NADPH as a cofactor (Jang et al., 2010). It is likely that, during hyperglycemia, consumption of NADPH by this reaction inhibits replenishment of reduced glutathione, ultimately decreasing cellular antioxidant status. Subsequently, sorbitol is oxidized to fructose by sorbitol dehydrogenase, with NAD+ reduced to NADH providing increased substrate to complex I of the mitochondrial respiratory chain. Since the mitochondrial respiratory chain is thought to be a major source of excess ROS in diabetes, provision of additional electrons for transfer to oxygen-forming superoxide would augment mitochondrial ROS production. In addition, since sorbitol does not cross cell membranes, its intracellular accumulation results osmotic stress and decreased activity of enzymes including ATPase leading to dysfunction of renal tubular reabsorption (Forbes et al., 2008). Thus inhibition of sorbitol accumulation with aldose reductase blockade has been shown to ameliorate renal injury in diabetic complications (Manna et al., 2009;

Fig. 12. A. Western blot analysis of Bcl-2 and Bax in the renal tissue of diabetic and DSL treated animals. The ratios of the relative intensities of the bands were determined using NIH- image software and the control band was given an arbitrary value of 1. Data represent the average±SD of 6 separate experiments in each group. “a” indicates the significant difference between the normal control and diabetic control groups and “b” indicates the significant difference between the diabetic control and DSL treated diabetic groups (Pa b 0.05, Pb b 0.05). B. Study on the mitochondrial membrane potential by flow cytometric analysis. Cont: mitochondria isolated from the renal tissue of normal animals; DSL: mitochondria isolated from the renal tissue of animals treated with DSL only; ALX: mitochondria isolated from the renal tissue of the diabetic animals and ALX+DSL: mitochondria isolated from the renal tissue of the animals treated with DSL after diabetic induction. The measurements were made in six times. C. Study on the concentration of cytosolic cytochrome c. Cont: normal rats, DSL: normal rats treated with DSL only, ALX: diabetic control and ALX+DSL: diabetic rats treated with DSL. The measurements were made in six times. Data represent the average±SD of 6 separate experiments in each group. “a” indicates the significant difference between the normal control and diabetic control groups and “b” indicates the significant difference between the diabetic control and DSL treated diabetic groups (Pa b 0.05, Pb b 0.05). D. Western blot analysis of caspases 3 in the renal tissue of diabetic and DSL treated animals. The relative intensities of bands were determined using NIHimage software and the control band was given an arbitrary value of 1. Data represent the average±SD of 6 separate experiments in each group. “a” indicates the significant difference between the normal control and diabetic control groups and “b” indicates the significant difference between the diabetic control and DSL treated diabetic groups (Pa b 0.05, Pb b 0.05). E. Western blot analysis of PARP in the renal tissue of diabetic and DSL treated animals. The ratios of the relative intensities of the bands were determined using NIH- image software and the control band was given an arbitrary value of 1. Data represent the average±SD of 6 separate experiments in each group. “a” indicates the significant difference between the normal control and diabetic control groups and “b” indicates the significant difference between the diabetic control and DSL treated diabetic groups (Pa b 0.05, Pb b 0.05).

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A

27

Bcl-2

1.0

Bcl-2/Bax ratio (arbitrary unit)

26 kDa

Bax 23 kDa

β-actin

b

0.5

a

42 kDa

C 100

B

80

Concentration of cytosolic cytochrome c (pg/ml)

a

DSL

ALX+DSL

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60

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20

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101

102

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A

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on t

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0.0 SL

ALX

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A

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42 kDa

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D SL

β-actin

b

t

84 kDa

1.0

on

116 kDa

C

PARP

116 KDa/84 KDa ratio (arbitrary unit)

E

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Fig. 13. Schematic diagram of the ALX induced kidney damage in diabetes and its attenuation by DSL.

Wei-hua et al., 2008). In our study we observed an increase in the activity of AR in the renal tissue of ALX-induced diabetic rats and that could be reduced by DSL treatment suggesting that DSL might possess an inhibitory effect on aldose reductase (Fig. 6A). It has also been observed that with a decrease in AR activity in DSL fed diabetic rats there is also an increase in the level of the cellular antioxidant GSH restoring the antioxidant status (Fig. 6B). Hyperglycemia can also activate PKC by the influx of the polyol pathway (Ahmad et al., 2005), and activated PKC contributes directly to the oxidative stress environment by activating NF-κB and various membrane associated NADPH oxidase, resulting in excessive ROS production (Brownlee, 2001). It has been reported that the PKC isoforms α, β, δ and ε are activated by hyperglycemia-induced oxidative stress in diabetic rat kidney (Meier et al., 2007). In our study we, therefore, investigated the protein levels of PKC isoforms α, β, ε and δ in the renal tissue of the experimental animals and observed that ALXexposure upregulated the expression of PKCα, PKCβ, PKCε and PKCδ (Fig. 7). PKC, in turn, contributes to matrix protein accumulation by inducing the expression of TGF-β 1, fibronectin and type IV collagen which are considered as the markers for fibrosis (Rains and Jain, 2011). The results of the present study also showed an increase in the level of collagen (as indicated by increased hydroxyproline content) (Fig. 8A) and fibronectin (Fig. 8B) and in the renal tissue of ALX-induced diabetic kidney. Treatment with DSL, however, could ameliorate the ALX-induced upregulation of all these PKC isoforms as well as the accumulation of fibronectin and collagen. Oxidative stress is now well known to induce alterations in gene expression of several redox sensitive transcription factors. One potential target of ROS activation is the nuclear transcription factor, NF-κB. It appears to play a major role in regulating immune and inflammatory responses (Schreck et al., 1991). Extracellular stimuli such as ROS signal the degradation and release of the inhibitory unit IκBα of NF-κB through the direct action of protein kinases, IKKs. The free NF-κB unit (p65) is now able to translocate into the nucleus, bind to DNA and modulate the expression of several genes (Baeuerle and Henkel, 1994; Baldwin, 1996). In this study, we observed the translocation of NF-κB in the nuclear fraction of hyperglycemic renal tissue

with the simultaneous increased phosphorylation of IκBα in the cytosol (Fig. 9). An increase in NF-κB activation is also followed by an increase in the concentration of inflammatory cytokines like TNF-α, IL-6 and IL-8 in diabetic rats (Fig. 10) which are in good agreement with other studies (Elmarakby and Sullivan, 2012). However, increased NF-κB activity and the concentration of inflammatory cytokines in the kidneys of diabetic rats were inhibited by treatment with DSL, indicating that DSL effectively inhibits renal inflammation in diabetic rats. Finally, we investigated the involvement of ALX-mediated intrinsic apoptotic cell death pathway in renal tissues. The DNA ladder pattern in agarose gel (Fig. 11A) and TUNEL positive nuclei (Fig. 11B) confirms the apoptotic cell death in the renal tissues of diabetic rats. Oxidative stress seems to play a major role in mitochondrial dysfunction which is an important early event in the intrinsic pathway of apoptosis. Kroemer et al. (1997) reported that proteins of the Bcl-2 family act on the mitochondria to regulate the release of cytochrome c and initiates the caspase dependent cell death pathway. In our study, we observed that ALX treatment activates Bax and inactivates Bcl-2 (Fig. 12A) causing reduction in mitochondrial membrane potential (Fig. 12B), release of cytochrome c (Fig. 12C) followed by the activation of caspase-3 (Fig. 12D) and PARP (Fig. 12E) leading to tissue damage. Treatment with DSL against ALX exposure could attenuate the apoptotic cell death by regulating the activation of Bcl-2 family proteins and their effects on mitochondria dependent cell death pathway (Fig. 12). In conclusion, by using alloxan-induced diabetic renal injury model, our present study first demonstrated that DSL effectively inhibited oxidative stress and related inflammatory response of the kidneys and provided renoprotection in diabetic rats (Fig. 13). Although DSL does not possess radical scavenging activities, it can reduce the oxidative stress by maintaining the intracellular antioxidant machineries close to normal. Moreover, in the present study we showed that DSL could inhibit the activation of aldose reductase, PKC, NF-κB and mitochondria dependent apoptotic signaling cascades. With this benefit and absence of any adverse effect known till date, the use of DSL could be of prophylactic value in reducing renal complications usually resulting from oxidative stress in diabetes mellitus.

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Conflict of interest The authors have declared that no conflict of interest exists.

Acknowledgment The authors are grateful to Mr. Prasanta Pal for technical assistance for the study.

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