Methods
Animal treatment and experimental protocol: The male adult Sprague-Dawley rats (240-260g) were procured from the central animal facility of the institute, NIPER. They were maintained under standard environmental conditions and provided with feed (Pranaw Agro Industries, New Delhi) and water ad libitum. All the animals were fed on normal pellet diet (NPD) one week prior to the experimentation. The guidelines of committee for the purpose of control and supervision of experiments on animals (CPCSEA), Govt. of India were followed and prior permission was sought from the institutional animal ethics committee (IAEC) for conducting the study. Rats were randomly divided into two groups at the start of the experiment. In first group, diabetes was induced by a single intraperitoneal (ip) injection of streptozotocin (STZ, 55 mg/kg, dissolved in ice cold 10 mM citrate buffer, pH 4.4). Second group was considered as control. Age matched controls were injected with citrate buffer. Animals having blood glucose concentrations >250 mg/dl were considered diabetic. Rats were sacrificed at a regular interval of 2 weeks till the 8th week.
Estimation of plasma creatinine, blood urea nitrogen The blood samples (approximately 0.3 mL) were collected from rat tail vein under light ether anesthesia in heparinized centrifuge tubes. The plasma was separated by centrifugation (5000 rpm, 10 minutes) and analyzed for plasma glucose, blood urea nitrogen and plasma albumin using commercially available spectrophotometric kits.Plasma creatinine concentration was measured by the picric acid colorimetric method [1]. BUNwas determined colorimetrically using commercially available standard kits.
Materials and Apparatus For two-dimensional electrophoresis (2-DE) analyses, the IPGphor IEF system, and Bio Rad Protean XI SDS system with 13 cm ImmobilineDryStrips (pH 3-10NL and 3-11NL)were obtained from GE Healthcare. Urea,immobilized pH gradient (IPG) buffer (pH 3-10NL and 3-11NL), and dithiothreitol(DTT) were acquired from GE Healthcare. DryStrip cover fluid, CHAPS, Tris, glycine, acrylamide,SDS, and ammonium persulfate, Iodoacetamide(IAA), TEMED, glycerol, bromophenol blue (BPB), silver nitrate,thiourea, acetone, and ammonium bicarbonate were all provided by the Sigma Chemical Co. (St. Louis, MO). The protease inhibitor cocktail (PIC) was acquired from Roche (Indianapolis, IN). All chemicals used in the 2-DE and MALDI-TOF/TOF analyses were of either electrophoresis-grade or analytic-grade. All buffers were prepared using Milli-Q water.
Sample Collection: Urine samples were taken from type 1 diabetic rats and their respective controls every week. 6 rats from each control and diabetic groups were placed individually in the metabolic cages after weekly intervals of 2nd, 4th and 8th weeks. After initial acclimatization for 3 hours, total urine was collected overnight in clean and sterilized collection tubes attached to the metabolic cage assembly. These tubes were kept at 4°C by covering them with ice packs to avoid protein degradation.
Extraction of Urine Proteins for 2-D PAGE Urine proteins were extracted by ultracentrifugation of urine at 200,000g for 2 hours. This yielded the hydrophobic proteins and then the supernatant was mixed with 20% TCA followed by acetone washing for 1 hour yielding hydrophilic fraction of proteins. The protein pellets obtained were resuspended and in rehydration buffer.Protein concentration levels were measured spectrophotometrically by Bradford method.
Extraction of Renal Proteins for 2-D PAGE: Rats were terminally sacrificed at the intervals of 2nd, 4th and 8thweek of induction of diabetes by decapitation. Protein extraction of the whole kidney was performed using standard procedures. Kidneys were perfused with ice-cold PBS and then were snap frozen in liquid nitrogen; ground to powder; resuspended in a buffer containing 40 mMTris, 7.92 M urea, 0.06% SDS, 1.76% ampholytes, 120 mM DTT, and 3.2% Triton X-100. Protein concentration levels were measured spectrophotometrically by Bradford method. Nuclei were isolated and acid soluble proteins were extracted as described previously[2].
Two dimensional Electrophoresis: Equal amounts of urinary proteins (1 mg) and Renal Proteins (200ug) from Control (2nd and 8th week) and Diabetic rats at time periods of 2nd, 4th and 8th weeks were used for proteomic analysis of urinary and renal proteins respectively. Two strips and gels were run for biological replicates from each control and diabetic groups.. First Dimensional separation was carried out using IPG-IEF in the presence of thiourea in addition to urea and CHAPS as denaturants in order to improve protein recovery. Appropriate protein amounts were mixed with gel rehydration buffer containing 7 M urea, 2 M thiourea, 2% CHAPS, 20 mM DTT, 0.2% Pharmalyte 3–10 and a trace amount of bromophenol blue. The mixture was then centrifuged at 15 000 rpm for 15 min, and the supernatant was used for rehydration of IPG gels. For separation of proteins, Immobiline Dry- Strip pH 3-10 (13 cm) was rehydrated with 250 L rehydrating solution containing 150 g proteins. After rehydration for 12–16 h at 20ºC, IEF was carried out at 20ºC on an electrophoretic apparatus (EttanIGphorII, Amersham Biosciences). The voltage was increased with the upper limit of current at 50 mA/gel, and after reaching 3500 V (for 13 cm gels), the voltage was kept constant for 4–6 h. The total Vh were 18000–24000 for 13 cm gels. After the first dimensional run, gels were incubated in 5 mL of equilibration buffer (8 M urea, 50 mM DTT, 2% SDS, 50 mMTris-HCl, pH 8.8) for 30 min, the buffer was discarded, and repeated two more times and the strips were transferred to an SDS-PAGE gel, and covered with 1% agarose in 50 mMTris-HCl, pH 8.8. The second dimensional gel was manually cast, being 20cms wide 16cms height with a 12.5% acrylamide without a stacking zone. The gels were scanned on Vilbert Lourmat gel documentation system and analyzed by Image Master 2D Platinum 6 software (GE Healthcare).Figures 1A to 1E show the gel images of from control and diabetic groups.
Silver Staining and Image Analysis Proteins were stained and visualized via silver staining method as described by Chevallet et al [3].The patterns of the spots were automatically analyzed using image Master 2D Platinum 6 (GE Healthcare). Spot intensity levels were determined by calculating the volume of each spot divided by the total volume of all the spots in the gel (called total spot normalization), and was expressed in terms of relative intensity. For each of the spots, the relative intensity was averaged and expressed as means (standard error of the mean (SEM). Statistical comparisons between the intensity of control and diabetic spots were performed using Sigma Stat software.
Protein isolation and Western blotting: Renal proteins were isolated as described previously [4]. Immunoblot analysis was performed using primary antibodies viz. anti-Fibronectin (1:500, Santa Cruz), anti- Collagen type IV (1:500, Santa Cruz), anti-Calgranulin A/B (1:500, Santa Cruz), anti-alpha 2u globulin (1:500, Santa Cruz), anti-phospho (Ser14) H2B antibody (1:1000, Millipore). Secondary antibodies were HRP conjugated anti-rabbit and were procured from Calbiochem. Proteins were detected using Enhanced Chemiluminescence System (ECL) and ECL hyperfilm (GE Healthcare).
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