Impact of EGCG Supplementation on the Progression of Diabetic


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IMPACT OF EGCG SUPPLEMENTATION ON THE PROGRESSION OF DIABETIC NEPHROPATHY IN RATS: AN INSIGHT INTO FIBROSIS AND APOPTOSIS Kalaiselvi Periandavan, Thangarajeswari Mohan, Prema Velusamy, Lakshmi Narasimhan Chakrapani, Ashok Kumar Srinivasan, Abhilasha Singh, and Thanka Johnson J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 21 Aug 2017 Downloaded from http://pubs.acs.org on August 21, 2017

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Journal of Agricultural and Food Chemistry

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IMPACT OF EGCG SUPPLEMENTATION ON THE PROGRESSION OF

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DIABETIC NEPHROPATHY IN RATS: AN INSIGHT INTO FIBROSIS AND

3

APOPTOSIS

4 5 6 7 8 9

Thangarajeswari Mohan1, Prema Velusamy1, Lakshmi Narasimhan Chakrapani1, Ashok Kumar Srinivasan1, Abhilasha Singh1, Thanka Johnson2, Kalaiselvi Periandavan1*

10 11

1

Department of Medical Biochemistry, Dr.ALMPGIBMS, University of Madras, Taramani Campus, Chennai. 2 Department of Pathology, Sri Ramachandra University, Chennai.

*-Corresponding author Abstract

12

Apoptosis is an active response of cells to altered microenvironments which is

13

characterized by cell shrinkage, chromatin condensation and DNA fragmentation, in variety

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of cell types such as renal epithelial cells, endothelial cells, mesangial cells and podocytes.

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Hyperglycemia is among the micro environmental factors that may facilitate apoptosis which

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plays a decisive role in the initiation of diabetic nephropathy. Transforming growth factor-β,

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emerges as a powerful fibrogenic factor in the development of renal hypertrophy. Although, a

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number of potential treatment strategies exist for diabetic nephropathy, considering the ease

19

of use and bioavailability, phytochemicals stands distinct as the preeminent option. EGCG, a

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green tea catechin is one such phytochemical which possess hypoglycemic and anti-fibrotic

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activity. The present study aims to explore the potential of EGCG to prevent apoptosis in

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high-fat diet and STZ induced diabetic nephropathy rats by assessing renal function, pro-

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fibrotic marker, and the expression of apoptotic and anti-apoptotic proteins. Our results

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validate EGCG as a potential anti-apoptotic agent evidently by improving renal function via

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down regulating TGF-β, consequently ameliorating diabetic nephropathy. In accordance,

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EGCG might be regarded as a prospective therapeutic candidate in modulating diabetic

27

nephropathy, thus being a promising treatment.

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Key words: Diabetic nephropathy, EGCG, High-fat diet, STZ, TGF-β, Apoptosis.

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Introduction

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Apoptosis contributes to the development of diabetic nephropathy leading to the

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functional deterioration of the kidney. Epithelial cells, endothelial cells, mesangial cells and

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podocytes play a crucial role in the maintenance of structure and function of the glomerulus.

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Injury to the glomerulus is usually characterized by effacement of foot processes leading to

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proteinuria,1 accumulation of extracellular matrix, mesangial matrix expansion, and tubular

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fibrosis.

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Transforming growth factor-β (TGF-β) is shown to induce podocyte loss via apoptosis

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in diabetic nephropathy contributing to the development of glomerulosclerosis2 and regulate

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urinary albumin excretion by increasing both glomerular permeability and decreasing

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reabsorption in the proximal tubules.3 Even though, a number of potential treatment strategies

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have shown benefit in improving surrogate markers like albuminuria, but the translation to

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preserve GFR and prevent ESRD has not always been followed.4

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Therefore, any phytochemical that addresses glycemic control, podocytes loss by

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preventing apoptosis and regulate TGF-β levels will be a promising therapeutic agent that

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targets diabetic nephropathy. EGCG (Epigallocatechin-3-gallate) is shown to possess anti-

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hyperglycemic effect5,6 and anti-fibrotic effect,7,8 also has an afforded protective effect

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against ROS production in diabetic nephropathy induced mice kidneys.9 Therefore, we

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intended to investigate the role of EGCG in ameliorating apoptosis in the kidneys of rats

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induced with diabetic nephropathy.

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Materials and Methods

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Chemicals

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Epigallocatechin-3-gallate and Bovine Serum Albumin were procured from Sigma–

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Aldrich, USA. Streptozotocin (STZ) was purchased from Cayman chemicals, Europe. All

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other chemicals used were of analytical grade and were obtained from SRL (Sisco Research

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Laboratories Pvt. Ltd) and CDH (Central Drug House Pvt. Ltd., Mumbai, India). Enhanced

55

chemiluminescence (ECL) kit was purchased from BioRad, USA.

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Laboratory animals

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Male albino rats of Wistar strain (100-110g) were obtained from Central Animal

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House facility, University of Madras, Taramani campus, and experiments were conducted in

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accordance with guidelines approved by the Institutional Animal Ethical Committee (IAEC

60

No.

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conditions of controlled temperature (25±2 °C) with 12/12 h light/dark cycle and were given

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food and water ad libitum. The animals were maintained on a commercial rat feed

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manufactured by Hindustan Lever Ltd., Mumbai under the trade name “GOLD MOHUR

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RAT FEED”. The feed contained 5% fat, 21% protein, 55% nitrogen free extract, 4% fibre

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(w/w) with adequate vitamins and minerals.

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Experimental design

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01/07/2016). The animals were housed two per cage in large spacious cages under

Diabetic nephropathy model was established by High-fat Diet (HFD) comprising of

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the normal rat chow

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30% coconut oil and 30% fructose water. After sixty days of dietary manipulation, HFD fed

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rats was injected intraperitoneally (i.p.) with low dose of streptozotocin (STZ, 30 mg/kg,

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Cayman chemicals, Europe) dissolved in 0.1 M citrate buffer, pH 4.3. The fasting blood

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glucose was measured 3 days after the STZ injection. Rats with the fasting blood glucose

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16.7 mmol/L and above were considered as diabetic rats. Then these rats are left for fortnight

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to establish diabetic nephropathy and allowed to continue to feed on their respective diets

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until the end of the study.

supplemented with 4% cholesterol and 1.5% cholic acid along with

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Group 1: Normal control rats were given saline orally for 28 days. Group 2:

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Diabetic nephropathy induced HFD fed rats. Group 3: Diabetic nephropathy induced HFD

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fed rats treated with EGCG (100mg/kg body weight) through oral gavage for 28 days. Group

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4: Rats were treated with EGCG (100mg/kg body weight) through oral gavage for 28 days.

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Urine was collected from the rats housed in metabolic cages for 24hr. (Based on the literature

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survey, bioavailability of EGCG is about 1.6% accounting to approximately 320µg for a rat

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weighing 200 g).

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At the end of the experimental period, rats were anesthetized with ketamine (22

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mg/kg body weight, i/p) and kidneys were excised immediately, immersed in ice-cold

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physiological saline and 10% cortex of the kidney tissue homogenate were prepared using

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Tris-HCl buffer (0.01 M, pH 7.4), followed by centrifugation at 12,000 rpm for 10 min and

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the supernatant was used for the analysis of various parameters. Rest of the tissue was stored

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at -80oC for protein expression studies. For histological examination, the kidney was

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collected and fixed in 10 % neutral buffered formalin, embedded in paraffin. Standard

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sections of 5 μm thickness were cut, which were then stained with hematoxylin and eosin

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(H&E) and Masson Trichrome Stains.

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Fasting blood glucose measurements

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Fasting blood glucose levels were measured weekly on lateral tail vein blood samples TM

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using an One touch select simple

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treatment period.

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Determination of glycosylated hemoglobin (HbA1c)

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Glycosylated

hemoglobin

meter and system (Life scan, Scotland) throughout the

was

assessed

using

the

Hemoglobin

Analyzer

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(D-10 - Biorad) based on chromatographic separation of the analytes by ion exchange high

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performance liquid chromatography (HPLC) and the changes in the absorbance measured at

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415 nm.

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Urinary parameter

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The 24 h urine sample collected from each rat was centrifuged and the supernatant

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was used for further analysis. Creatinine levels were determined using Kit method

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(Medsource Ozane Biomedicals). Glomerular Filtration Rate (GFR) was calculated on the

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basis of urinary creatinine, serum creatinine and urine volume using the following equation:

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GFR (mL/min) = [urinary creatinine (mg/dl × urine volume (mL)/serum creatinine (mg/dl)]

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×1/1440 (min)]. Albumin creatinine ratio (ACR) was calculated from the urinary creatinine

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and urinary albumin values.

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Assay of urinary albumin by ELISA

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Urinary proteins were extracted using 10% TCA and High affinity 96 well microtitre

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plates were coated with 10 µg of protein in 50µl of 0.2 M sodium carbonate/bicarbonate

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buffer (pH 9.6) per well and incubated at 4°C for overnight. After that plates were washed

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thrice with 100 µl of 0.01M phosphate buffered saline (pH 7.4) and then blocked for 2 hours

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with 200 µl of 1% BSA in PBS at 37°C. Following four washes as above in PBS, appropriate

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primary antibody diluted in PBS were added and incubated at 4°C for overnight. Further,

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plates were washed thrice with phosphate buffered saline (pH 7.4) in 0.2 % Tween 20 (PBS-

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T) and incubated with 100 µl of streptavidin-horse radish peroxidase for 1 hour.

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Subsequently after washing, the reaction was visualized by the addition of 100µl/well 2,2’

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Azinobis [3-ethylbenzothiazoline-6sulfonic acid] diammonium salts (ABTS) substrate for

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20min at room temperature. The reaction was stopped by the addition of 100µl/well 2N

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H2SO4 optical densities were determined at 415 nm using iMark™ Microplate Absorbance

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Reader (Biorad). The urinary albumin was expressed as ng/mL. 10

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Analysis of serum lipid profile

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Total Cholesterol, Triglycerides and High density lipoproteins (HDL) in serum were

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analyzed using commercial kits from Spin react in the auto analyzer (Rx Monza, Randox,

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U.K). The values of total cholesterol, triglycerides, LDL (Low Density Lipoprotein), VLDL

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(Very Low Density Lipoprotein) and HDL are expressed as mg/dl.

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Biochemical analysis of protein carbonyl and lipid peroxidation in kidney cortex tissue

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The estimation of protein carbonyl in kidney cortex tissue were determined

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using 2, 4-dinitrophenyl hydrazine (DNPH) with slight modifications of the method of

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Levine et al.,11 and expressed as nmoles of protein carbonyl/mg protein/ml.

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The level of lipid peroxides was assayed by the method of Devasagayam and

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Tarachand, 12 and expressed as nmoles of MDA released/g of tissue.

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Western blot analysis

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Cortical kidney tissue samples were homogenized in Tris-HCl buffer (0.01 M, pH

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7.4) and protein concentrations were determined.13 For immunoblotting, aliquots of lysate

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were mixed with loading buffer containing dithiothreitol and maintained at 95oC for 10 min

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before loading on 10% SDS-PAGE. Following SDS-PAGE separation, proteins were

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transferred to polyvinyl difluoride membrane (PVDF). Membranes were blocked in Tris-

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buffered saline with Tween-20 containing 5% (w/v) non-fat milk for 1 h at room temperature.

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Membrane strips were incubated with primary antibodies (TGF-β1, Bax, Bcl-2 and cleaved

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caspase-3) overnight at 4oC. Following extensive washing, membrane strips were incubated

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with horseradish peroxidase conjugated secondary antibodies for 1 h. Protein bands were

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detected by the detection reagent mixture (an equal volume of luminol with H2O2) using

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standard enhanced chemiluminescence method in Bio-rad Chemidoc XRS imaging system.

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Statistical analysis

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Data are presented as mean ± Standard Error of Mean (SEM) of the results obtained

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from the average of at least three or six independent experiments. Differences between

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groups were analyzed by one-way analysis of variance (ANOVA) using the SPSS software

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package for Windows (Version: SPSS 20.0). Post hoc testing was performed for inter-group

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comparisons using Least Significant Difference (LSD) test. Values are statistically significant

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at the level of p<0.05.

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Results

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Influence of EGCG on body weight in the high-fat diet and streptozotocin induced

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diabetic rats

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Rats fed with high-fat diet and induced with streptozotocin had higher fasting blood

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glucose levels and decreased body weight compared with the control rats (p < 0.05).

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Treatment with EGCG significantly decreased the blood glucose levels and increased the

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body weight of diabetes induced rats (Figure 1). These results demonstrate that EGCG

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treatment ameliorates hyperglycemia and increases the body weight in rats with diabetes.

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Impact of EGCG on HbA1c

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High Fat diet and STZ administration augments the formation of glycosylated

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hemoglobin by 30.5% (Figure 2). The anti-glycation effect of EGCG was evident in the

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treated rats with their HbA1c levels decreased by 12.98% than that of the diabetic rats.

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Effect of EGCG on renal functions markers in rats with early diabetic nephropathy

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Urinary creatinine level, GFR and urinary albumin/creatinine ratio (ACR) were

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assessed as markers of renal function. Urinary creatinine (2.5 fold), GFR (3.5 fold) and ACR

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(24.9 fold) levels were significantly elevated in the diabetic nephropathy rats, wherein with

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EGCG treatment, the levels of the markers were near normalcy, signifying the renal function

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restoration (Figure 3).

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Effect of EGCG on the lipid profile of rats with early diabetic nephropathy

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Table 1 depicts the role of EGCG on the lipid profile of diabetic rats. The present

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study observed increased serum levels of total cholesterol, triglyceride, LDL, VLDL in

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diabetic rats with the concomitant diminution in the HDL as compared to control rats. EGCG

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treatment significantly improved the lipid profile in the diabetic rats.

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Influence of EGCG on the oxidative stress markers in rats with early diabetic

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nephropathy

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Oxidative stress was assessed by measuring renal protein carbonyl and

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malondialdehyde levels. Both the levels were significantly (p<0.05) elevated (51.89% and

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33.89%) in diabetic nephropathy rats compared with control rats. Administration of EGCG

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curtails the macro molecular damage in hyperglycemic condition by declining renal protein

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carbonyl (21.17%) and malondialdehyde (15.89%) respectively (Figure 4).

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Effect of EGCG on kidney histoarchitecture of high-fat diet and streptozotocin-induced

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early diabetic nephropathy in rats (H&E 40X)

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Figure 5 shows the histoarchitecture of diabetic kidney. The rats of control group (G1) shows

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thin and delicate glomerular capillary loops. The endothelial cells, mesangial cells and

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surrounding tubules were normal in number. The early diabetic nephropathy group (G2)

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shows mesangial matrix expansion (indicated by red arrow), loss of separation between

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glomerular capillaries and Bowman’s capsules (indicated by green arrow), and occlusion of

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the capillary network (indicated by black arrow). Diabetic nephropathy induced rats treated

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with EGCG (G3) shows slight mesangial cell hyperplasia, and adhesion between glomerular

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capillaries and Bowman’s capsules with intact walls (indicated by green arrow). EGCG alone

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treated rats (G4) shows no pathological changes.

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Effect of EGCG on kidney histopathology of high-fat diet and streptozotocin-induced

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early diabetic nephropathy in rats (Masson trichrome staining 40X).

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Figure 6 shows the masson trichrome staining of the kidney tissue. The control group (G1)

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shows normal glomerular structure. The early diabetic nephropathy group (G2) shows lighter

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blue stain (indicates collagen deposition), reduced visibility of capillary lumen, thicken

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glomerular basement membrane, mesangial matrix expansion, tubular atrophy, extensive

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glomerular and interstitial fibrosis. While diabetic nephropathy induced rats treated with

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EGCG (G3) shows mild blue staining, visible capillary lumen and restoring occlusion of the

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capillary network. Whereas the tissue sections from EGCG alone treated rats (G4) shows no

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pathological changes.

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Effect of EGCG on protein expression of TGF-β in high-fat diet and streptozotocin

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induced diabetic rats

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The upregulation of TGF-β is proposed to play a pivotal role in aiding matrix gene

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activation and subsequent glomerulosclerosis in diabetic glomerular injury. Hence we studied

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the protein expression of TGF-β in diabetic kidney (Figure 7), which exhibited

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approximately 1.95 fold increase when compared with non-diabetic controls (P < 0.05).

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Treatment with EGCG significantly attenuated the upregulation of TGF-β1 (2.25 fold).

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The anti-apoptotic effect of EGCG in high-fat diet and streptozotocin induced diabetic

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rats

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In hyperglycemia mediated oxidative stress, proapoptotic (Bax) and anti-apoptotic

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(Bcl-2) proteins are involved in response to apoptosis. Oxidative stress induced apoptosis is

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initiated through the activation of caspase 3 cleavage. Immunoblot analysis shows that the

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expression of Bax (2.3 fold) and cleaved caspase-3 (1.38 fold) were up regulated in the HFD

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and STZ-induced diabetic renal tissue (Figure 8). However, supplementation with EGCG

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significantly altered the expression of these proteins by 1.81 fold and 1.64 fold respectively.

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Our results showed that HFD and STZ-induced diabetic condition significantly

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decreased the expression of Bcl-2 by about 27.37% (Figure 9). Nevertheless, treatment with

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EGCG effectively increased Bcl-2 (23.14%). This explicit, EGCG could act as an effective

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anti-apoptotic agent by increasing the expression of anti-apoptotic proteins in the diabetic

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kidney.

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Discussion

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To elucidate the pathophysiology of diabetic nephropathy, animal models mimicking

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the pathology of human type II diabetes are of great value. One such type II diabetic animal

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model is the high-fat diet and streptozotocin induced rat model.14-16 The present model

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involves a combination of a high-fat diet and fructose, to bring about hyperinsulinemia and

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insulin resistance and/or glucose intolerance followed by treatment with the β‐cell toxin STZ,

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which results in a severe reduction in functional β‐cell mass. We confirmed that consumption

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of 4% cholesterol and 1.5% cholic acid along with coconut oil and 30% fructose water in the

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diet and a single dose of STZ (30 mg/kg body weight) led to the rapid induction of diabetes in

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eight weeks and instigates diabetic nephropathy in two weeks.

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Hyperglycemia is a key factor in mediating the development and progression of

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diabetic nephropathy.17 In the present study, we found that animals fed with high-fat diet and

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low dose of STZ (30 mg/kg, ip) exhibited fairly high fasting blood glucose levels which is

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supported by Li et al.,18 .Wu et al.,19 also reported that Sprague-Dawley rats with diabetes

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induced by HFD and STZ show a marked increase in the fasting glucose, glycosylated

239

hemoglobin and urinary excretion of proteins. In the present study, a drastic reduction in the

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body weight was observed in the diabetic group which corroborates with the studies done by

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Xu et al.,20 who have reported that STZ induced diabetic type I mice exhibit increased

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glomerular area and extracellular matrix accumulation. Our observations are consistent with

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the findings of Yoon et al.,21 who have reported that EGCG treatment (100mg/kg b.wt) to

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STZ induced diabetic rats exhibit a decrease in the fasting blood glucose with a concomitant

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increase in the body weight.

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During diabetes, advanced glycation end products are formed, when glucose reacts

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with various proteins namely hemoglobin, albumin, collagen, LDL, or crystalline proteins to

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form labile Schiff bases, which then go through further modification to form Amadori

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products.22 The rate of glycation of hemoglobin is proportional to the blood glucose

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concentration and glycosylated hemoglobin was found to increase in the patients with

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uncontrolled diabetes mellitus.23 HbA1c drastically increases over time simultaneously with a

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pronounced urinary flow and urinary glucose excretion in the STZ induced diabetic rats.24 In

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the current study, oral administration of EGCG significantly decreased the levels of

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glycosylated hemoglobin. These results indicate the beneficial effects of EGCG in preventing

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the pathogenesis of diabetic nephropathy caused by impaired glucose metabolism.

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To check whether diabetic nephropathy has been established, we assessed the levels

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of albumin creatinine ratio in the urine of the experimental animals. The present study shows

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a significant elevation in the levels of urinary creatinine, glomerular filtration rate and

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increased albumin: creatinine ratio indicating a progressive nephrotoxicity in the diabetic

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animals. Fu et al.,25 have reported a substantial increase in the urinary albumin: creatinine

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ratio in the STZ-induced diabetic nephropathy. Moreover, a decline in urinary creatinine and

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albumin in diabetic nephropathy is associated with the renal protection. EGCG, effectively

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reversed this pathophysiology by lowering urinary creatinine, GFR and albumin/ creatinine

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ratio. These results are consistent with the findings of Thangapandiyan and Miltonprabu,26

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who have reported that EGCG has the ability to lessen the serum uric acid and urinary

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creatinine in rats with fluoride intoxication.

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Urinary albumin has been suggested as a predictive indicator for prognosis of diabetic

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nephropathy.27 The renal podocyte foot processes and associated slit diaphragms ensure the

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integrity of the renal basement membrane that forms the barrier to urinary protein loss.28 The

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charge for glomerular basement membrane is imparted by the sulfated glycosaminoglycan

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side chains of proteoglycans, and to a lesser extent by carboxyl and sialyl groups of

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glycoproteins in glomerular basement membrane and mesangial matrix where they serve as

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charge-selective barrier of the filtration process.29 It has been hypothesized that decreased

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glycosaminoglycan levels in glomerular basement membrane of kidney results in its leakiness

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leading to albuminuria.30 The declining levels of heparan sulfate, which is responsible for the

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maintenance of the selective permeability of the glomerular basement membrane during

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diabetes due to decreased synthesis31 or increased degradation by heparanase activity32 might

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be the cause for albuminuria. On EGCG administration, the urinary albumin levels were

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reduced signifying its renoprotective effect. Tsai et al.,33 have reported that EGCG prevents

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proteinuria, renal function impairment, and severe renal lesions in lupus nephritis

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development in mice. Previous studies done by Yamabe et al.,34 demonstrated that EGCG

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treated groups showed suppressed hyperglycemia and proteinuria in STZ induced diabetic

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rats.

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The resultant of urinary protein loss is hypoalbuminemia, which leads to an

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upregulation of 3-hydroxy-3-methylglutaryl CoA reductase that stimulates LDL synthesis in

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the liver with a consequent hypercholesterolemia.35 Impaired clearance of chylomicrons and

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VLDL has emerged as the leading factor for the amplified concentration of triglyceride in

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serum. Mooradian,36 stated that under the diabetic environment, hormone sensitive lipase is

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activated, which results in the release of free fatty acid from the adipose tissue and it

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promotes hepatic triglyceride production, leading to excessive apoB and VLDL synthesis.

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These changes result in augmented serum levels of TG and remnant particles. Apolipoprotein

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(apoB 48), a major component of chylomicron, is also increased in diabetes, suggesting an

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accumulation of chylomicron remnants. 37

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In the present study also, high-fat diet and STZ induced diabetic rats show abnormal

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lipid profile as reflected by raised total cholesterol, LDL, VLDL, TG, and a concomitant

296

decrease in the HDL levels. Wu et al.,19 also stated that Sprague-Dawley rats with diabetes

297

induced by HFD and STZ showed a marked increase in the total cholesterol and triglyceride.

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EGCG treated rats show a significant decline in the total cholesterol, LDL, VLDL, TG with a

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concomitant increase in the HDL levels. It has been proposed that the galloyl moiety of

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EGCG may play a critical role in its lipid lowering effect.38

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Ruiz-Hurtado et al.,39 stated that albuminuria is accompanied by an amplified

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oxidative damage in patients in early stages of chronic kidney disease. Glomerular mesangial

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cells, retinal capillary endothelial cells, and neuronal cells are particularly susceptible to

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hyperglycemic assault since they are unable to avert intracellular high glucose ambience in

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states of the systemic rise in blood glucose levels. As a result of high glucose in diabetic

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condition accelerated oxidative phosphorylation and excessive leakage of O2.−, mitochondria

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are subjected to extraordinary ROS-mediated oxidative stress.40 Protein carbonylation may be

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related to elevated oxygen free radicals causing glucose auto-oxidation, non-enzymatic

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glycation of proteins leading to advanced glycation end products, and enhanced glucose flux

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through polyol pathway.41 Highly reactive oxygen metabolites, especially hydroxyl radicals,

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act on unsaturated fatty acids of phospholipid components of membranes to produce

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malondialdehyde. Reports state that high-fat diet and low dose of streptozotocin-induced type

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II diabetes rats show increased lipid peroxidation14 and increased protein carbonyl42 in the

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kidney tissue. Similarly, the current study also evidenced an increased level of MDA, an

315

index of endogenous lipid peroxidation and protein carbonyl content in the diabetic group

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reflecting the oxidative milieu of the cell. Furthermore, EGCG could decrease the levels of

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protein carbonyl and malondialdehyde formation in the treated group. El-Mowafy et al.,43

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have stated that EGCG scavenges ROS, thereby preventing the lipid peroxidation, protein

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carbonylation and act as renal protective agent.

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Altunkaynak et al.,44 suggested that there is a significant association between fatty

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diet intake and structural changes in the kidney such as, decrease in the density of glomeruli,

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tubular deformations, glomerular necrosis and basement membrane thickening. Moreover, the

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meta-analysis of clinical studies have shown that control of hyperlipidemia is extra beneficial

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for prevention of progression of albuminuria45 as hyperlipidemia, due to high-fat intake

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causes macrophage infiltration and foam cell formation, leading to glomerulosclerosis.46

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To authenticate the effect of EGCG in diabetic nephropathy, we assessed the

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histopathology of the diabetic kidney tissue. The histopathology observation showed

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mesangial matrix expansion, loss of separation between glomerular capillaries and Bowman’s

329

capsules, and occlusion of the capillary network in the diabetic nephropathy group. This

330

could be due to the accumulation of reactive oxygen species as a consequence of increased

331

lipid peroxidation and protein carbonyls leading to loss of membrane integrity and other

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pathological alterations in the diabetic kidney. Treatment with EGCG prominently

333

diminished the significant pathological changes triggered by hyperglycemia. This can be

334

accredited to the antioxidant and anti-inflammatory efficacy of EGCG.

335

The severity of diabetic nephropathy is correlated with the accumulation of extra

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cellular matrix especially collagen, which was determined using Masson Trichrome staining.

337

Current study observed lighter blue staining indicating collagen deposition, visible capillary

338

lumen, tubular atrophy and extensive glomerular and interstitial fibrosis in the diabetic

339

nephropathy group. Our results also corroborate with the work done by Zhou et al.,47 who

340

have reported pathological alterations in diabetic kidney including glomerular basement

341

membrane thickening, mesangial matrix expansion, and renal interstitial fibrosis.

342

Increased oxidative stress leads to the up regulation of TGF-β, which is recognized as

343

a crucial player involved in the development of glomerulosclerosis and interstitial fibrosis.48,

344

49

345

oxidase.50 Jin et al.,51 stated that the STZ induced diabetic rats had an up regulation of TGF-

346

β1 protein expression. Our results are consistent with the above findings wherein we found an

347

increased expression of TGF-β1 in the diabetic animals. Iglesias-De et al.,52 have reported

348

that TGF-β1upregulation aggravates accumulation of extra cellular matrix in human

349

mesangial cells. Similarly, we have also observed increased collagen deposition in the kidney

350

of rats with diabetic nephropathy as revealed by Masson Trichrome staining. Nevertheless

On the other hand, TGF-β itself could cause ROS production via NADH/NADPH

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351

treatment with EGCG significantly down regulates the expression of the pro-fibrotic factor,

352

which was coherent with the results of previous studies carried out by Park et al.,7 who have

353

also reported that EGCG prevents the up regulation of TGF-β1 in high glucose stimulated

354

glomerular epithelial cells.

355

Adding to that, oxidative stress also disturbs the proapoptotic-antiapoptotic (Bax and

356

Bcl-2) balance and activates mitochondrial-dependent apoptosis via caspase 9 and caspase-

357

3.53 Translocation of Bax from cytosol to mitochondria in hyperglycemia induces oxidative

358

stress and causes damages to the outer mitochondrial membrane; which in turn, initiates the

359

mitochondrial dependent apoptotic pathway.54 According to Lee,

360

podocyte loss via apoptosis in diabetic nephropathy contributing to the development of

361

glomerulosclerosis.

55

TGF-β1 may induce

362

Chen et al.,56 reported that activation of caspase-3 is associated with TGF-β1 induced

363

apoptosis in the human hepatoma cell line (Hep3B). Caspase-3 is a central mediator of

364

apoptosis and is normally present in the cell in an inactive form and its activation occurs

365

through proteolytic cleavage of a prodomain.57 Cao et al.,58 have stated that caspase-3 is a

366

downstream target of TGF-β/Smad3 signaling in RIE-1/Smad3 cells as it activates caspase-9

367

by inducing protein cleavage, mitochondrial membrane depolarization, and cytochrome c

368

release. Similar to the above findings, hyperglycemia induced TGF-β mediated activation of

369

mitochondrial apoptosis pathway is evident by the prevalent high levels of cleaved caspase-3

370

in the kidneys of rats with diabetic nephropathy. Activation of apoptotic pathway is partially

371

reduced by EGCG dictating its protective role in safeguarding the renal cells against

372

hyperglycemic assault. Hence, the observed increase in apoptotic activity might be due to rise

373

in TGF-β levels.

374

High glucose induced apoptosis in the mouse mesangial cell is linked to the

375

expression of Bax, Bcl-2 and cleaved caspase-3.59 According to Aktuğ et al.,60 decreased

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376

Bcl-2 expression in the renal tissues of STZ induced diabetic rats demonstrates that the

377

degree of apoptotic activity is higher under diabetic conditions. Correspondingly, down

378

regulation of anti-apoptotic marker Bcl-2 has been reported in the high-fat diet and

379

streptozotocin induced diabetic nephropathy.61 Ying et al.,62 conveyed that both bax and

380

cleaved caspase-3 levels were increased in the HFD and STZ induced diabetic rats. Increased

381

Bax expression along with decreased Bcl2 expression and caspase-3 in its activated form in

382

the kidneys of diabetic nephropathy rats observed in the current study demonstrates the

383

greater extent of apoptotic activity prevailing in the kidney. In the present study, EGCG

384

supplementation up regulates the protein expression of Bcl-2 and decreases the levels of

385

cleaved caspase-3 and Bax substantiating EGCG to be a renoprotective agent.

386

Conclusion

387

To conclude, this study shows that EGCG is more effective in diminishing ROS

388

generation, ROS mediated TGF-β up regulation and apoptosis through the down regulation of

389

bax and cleaved caspase-3, as well as it bolsters the Bcl-2 protein expression in the kidneys

390

of high-fat diet and STZ induced diabetic rats. EGCG might be a potential candidate in

391

preventing the progression of diabetic nephropathy and thus may endorse a promising

392

treatment. However, the molecular mechanism behind the ability of EGCG to protect the

393

renal cells from hyperglycemia induced oxidative stress is to be explored.

394

List of abbreviations

395

DN

-

Diabetic nephropathy

396

HFD

-

High-Fat Diet

397

STZ

-

Streptozotocin

398

EGCG -

Epigallocatechin-3-gallate

399

TGF-β -

Transforming growth factor-β

400

Acknowledgements

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This work was supported by DST PURSE Phase II programme/UGC SAP.

402

Competing interests

403

The authors declare that they have no competing interests.

404

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Figures and legends:

579

Figure 1 Influence of EGCG on fasting blood glucose levels and body weight

580

Group 1- control rats; Group 2- Diabetic rats; Group 3 -Diabetic + EGCG treated rats; Group

581

4 -EGCG alone treated rats. Values are expressed as mean ± SEM for six animals in each

582

group. Values are statistically significant at the level of p<0.05, where ‘a’ -compared with

583

Group 1, ‘b’ -compared with Group 2.

584

Figure 2 Effect of EGCG on serum glycosylated Hb

585

Group 1- control rats; Group 2- Diabetic rats; Group 3 -Diabetic + EGCG treated rats; Group

586

4 -EGCG alone treated rats. Values are expressed as mean ± SEM for six animals in each

587

group. Values are statistically significant at the level of p<0.05, where ‘a’ -compared with

588

Group 1, ‘b’ -compared with Group 2.

589

Figure 3 Effect of EGCG on the kidney function markers

590

Group 1- control rats; Group 2- Diabetic rats; Group 3 -Diabetic + EGCG treated rats; Group

591

4 -EGCG alone treated rats. Values are expressed as mean ± SEM for six animals in each

592

group. Values are statistically significant at the level of p<0.05, where ‘a’ -compared with

593

Group 1, ‘b’ -compared with Group 2.

594

Table 1 Effect of EGCG on lipid profile

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Journal of Agricultural and Food Chemistry

Parameter

Group 1

Group 2

Group 3

Group 4

Total cholesterol (mg/dl)

52.99±1.49

136.98±4.65a

64.99±3.11b 55.99±1.53

Triglycerides(mg/dl)

65.99±3.52

124.03±4.51a

76.01±3.61b 63.99±8.74

HDL (mg/dl)

22.96±1.95

12.01±0.54 a

18.99±1.41b 20.01±1.89

LDL(mg/dl)

18.39±0.86

60.93±2.07 a

28.36±1.75b 20.09±0.96

VLDL (mg/dl)

13.59±0.67

32.01±0.91 a

15.58±0.77b 12.77±0.67

595 596

Group 1- control rats; Group 2- Diabetic rats; Group 3 -Diabetic + EGCG treated rats; Group

597

4 -EGCG alone treated rats. Values are expressed as mean ± SEM for six animals in each

598

group. Values are statistically significant at the level of p<0.05, where ‘a’ -compared with

599

Group 1, ‘b’ -compared with Group 2.

600

Figure 4 Effect of EGCG on oxidative stress markers

601

Group 1- control rats; Group 2- Diabetic rats; Group 3 -Diabetic + EGCG treated rats; Group

602

4 -EGCG alone treated rats. Values are expressed as mean ± SEM for six animals in each

603

group. Values are statistically significant at the level of p<0.05, where ‘a’ -compared with

604

Group 1, ‘b’ -compared with Group 2.

605

Figure 5 Effect of EGCG on kidney histoarchitecture of high-fat diet and

606

streptozotocin-induced early diabetic nephropathy in rats (H&E 40X)

607

G1- Control rats; G2- DN rats; G3- Diabetic + EGCG treated rats; G4- EGCG alone treated

608

rats.

609

G1- Sections from control rat kidney shows thin and delicate glomerular capillary loops.

610

Endothelial cells, mesangial cells and surrounding tubules are normal in number. G2- shows

611

mesangial matrix expansion (indicated by red arrow), loss of separation between glomerular

612

capillaries and Bowman’s capsules (indicated by green arrow), and occlusion of the capillary

613

network (indicated by black arrow). G3- shows slight mesangial cell hyperplasia, and

614

adhesion between glomerular capillaries and Bowman’s capsules with intact walls (indicated

615

by green arrow). G4- shows no pathological changes.

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616

Figure 6 Effect of EGCG on kidney histopathological picture of high-fat diet and

617

streptozotocin-induced early diabetic nephropathy in rats (Masson trichrome 40X)

618

G1- Control rats; G2- DN rats; G3- Diabetic + EGCG treated rats; G4- EGCG alone treated

619

rats.

620

G1- Control group shows normal structures of glomeruli. G2-Early diabetic nephropathy

621

group shows lighter blue stain (indicates collagen deposition), reduced visibility of capillary

622

lumen, thicken glomerular basement, mesangial matrix expansion, tubular atrophy, extensive

623

glomerular and interstitial fibrosis. G3- Diabetic nephropathy pathology rats treated with

624

EGCG shows mild blue staining, visible capillary lumen and restoring occlusion of the

625

capillary network. G4- Sections from EGCG alone treated rat shows no pathological changes.

626

Figure 7 Effect of EGCG on the expression of pro fibrotic marker TGF-β in diabetic

627

nephropathy induced rats

628

Group 1- control rats; Group 2- Diabetic rats; Group 3 -Diabetic + EGCG treated rats; Group

629

4 -EGCG alone treated rats. Values are expressed as mean ± SEM for three independent

630

observations. Values are statistically significant at the level of p<0.05, where ‘a’ -compared

631

with Group 1, ‘b’ -compared with Group 2.

632

Figure 8 Impact of EGCG on apoptotic protein

633

Group 1- control rats; Group 2- Diabetic rats; Group 3 -Diabetic + EGCG treated rats; Group

634

4 -EGCG alone treated rats. Values are expressed as mean ± SEM for three independent

635

observations. Values are statistically significant at the level of p<0.05, where ‘a’ -compared

636

with Group 1, ‘b’ -compared with Group 2.

637

Figure 9 Effect of EGCG on protein expression of anti-apoptotic protein (Bcl-2)

638

Group 1- control rats; Group 2- Diabetic rats; Group 3 -Diabetic + EGCG treated rats; Group

639

4 -EGCG alone treated rats. Values are expressed as mean ± SEM for three independent

640

observations. Values are statistically significant at the level of p<0.05, where ‘a’ -compared

641

with Group 1, ‘b’ -compared with Group 2.

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Figure 1

FASTING BLOOD GLUCOSE 35

BODY WEIGHT 300

a

mmole/L

Body weight in grams

a

25 20

a

a

a

a

30

15

ab ab

10 5 0

ab

250

b

200

b a

b

150 100 50 0

1

2

3

4

1

GROUPS

2

3

GROUPS

Day 1 of EGCG administration

Day 1 of EGCG administration

Day 14 of EGCG administration

Day 14 of EGCG administration

Day 28 of EGCG administration

Day 28 of EGCG administration

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4

a

Journal of Agricultural and Food Chemistry

Figure 2

Glycosylated Hb 9

a

%of glycated Hb

8

b

7 6 5 4 3 2 1 0 1

2

3

4

GROUPS

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Journal of Agricultural and Food Chemistry

Figure 3 A

B

URINARY CREATININE

GFR 7

a

12

6

10

5

8

ml/min

mg/day

14

b

6 4

a

4 b

3 2 1

2

0

0 1

2

3

1

4

2

D

12

a

µg/ml of urine

8 6

b

4 2 0

1

2

3

ALBUMIN/CREATININE RATIO albumin creatinine ratio ((mg/g)

URINARY ALBUMIN

10

4

GROUPS

GROUPS

C

3

25

a

20 15 10 5

b

0

4

GROUPS

ACS Paragon Plus Environment

1

2

3 GROUPS

4

Journal of Agricultural and Food Chemistry

Page 28 of 34

Figure 4

B

nmoles of protein carbonyl/mg protein/ml

PROTEIN CARBONYL 20 18 16 14 12 10 8 6 4 2 0

a b

1

2

3

4

LPO nmoles of MDA released/g of tissue

A

100 a

90 80

b

70 60 50 40 30 20 10 0

GROUPS

1

2

3

GROUPS

ACS Paragon Plus Environment

4

Page 29 of 34

Journal of Agricultural and Food Chemistry

Figure 5 G1

G2

G3

G4

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Figure 6 G1

G3

G2

G4

ACS Paragon Plus Environment

Page 30 of 34

Page 31 of 34

Journal of Agricultural and Food Chemistry

Figure 7 TGF-β 25 kDa

β- actin 45 kDa

TGF-β 9000000

a

Relative intensity TGF-β / β-actin

8000000 7000000 6000000 5000000

b

4000000 3000000 2000000 1000000 0 1

2

3 GROUPS

ACS Paragon Plus Environment

4

Journal of Agricultural and Food Chemistry

Page 32 of 34

Figure 8

Bax 21 kDa

Cleaved caspase-3 19&17 kDa

β- actin 45 kDa

β- actin 45 kDa

Cleaved Caspase-3

6000000

6000000 5000000

a

Relative intensity Cleaved Caspase 3/ β-actin

Relative intensity Bax / β-actin

Bax

4000000 3000000

b

2000000 1000000 0 1

2

3

a

5000000 4000000 b 3000000 2000000 1000000

4

GROUPS

0 1

2

3 GROUPS

ACS Paragon Plus Environment

4

Page 33 of 34

Journal of Agricultural and Food Chemistry

Figure 9 Bcl-2 26 kDa

β- actin 45 kDa

BCL-2 14000000

Relative intensity BCl2/ β-actin

12000000 b

10000000 a

8000000 6000000 4000000 2000000 0 1

2

3 GROUPS

ACS Paragon Plus Environment

4

Graphical abstract

Journal of Agricultural and Food Chemistry

Page 34 of 34

High-fat diet and Streptozotocin Hyperglycemia

Hyperlipidemia

Reactive oxygen species

Bcl2

Bax Up regulation of TGF-β

Activation of caspase-3 Collagen deposition Apoptosis

Progression of ACS Paragon Plus Environment diabetic nephropathy

Sites blocked by EGCG