After obtaining ethical committee approval, 24 Wistar albino rats – 200–270 g – were randomly divided into four groups: Group C; Group DM-C; Group DM-I/R; and Group DM-I/R-D. Diabetes was induced using Streptozotocin (Sigma Chemical, St Louis. MO, USA) at a single dose of 55 mg/kg in citrate buffer (0.1 Molar, pH 4.5). Rats with a blood glucose level – determined (GlucoDr Super Sensor, Allmedicus, Korea) from blood samples drawn via tail vein – above 250 mg/dl is accepted as diabetic. No additional intervention was done in Group C and Group DM-C. After a four-week follow-up period [16 (link)], laparotomy was performed in all groups detailed below. General anesthesia induction was done using intramuscular injection of 100 mg/kg ketamine hydrochloride (Ketalar® flakon, Parke-Davis, USA). Rats were kept under a heat lamp. All procedures were performed in the supine position. After skin asepsis, midline laparotomy was performed. After removing intestines from the surgical field, the infra-renal abdominal aorta was explored. The aorta was clamped using an atraumatic microvascular clamp. The clamp was removed at the end of 120 minutes of ischemia, then reperfusion was provided for another 120 minutes. Ischemia was determined when distal aorta pulsation disappeared, while reperfusion was determined when it reappeared. In the control group, laparotomy and abdominal aorta dissection were applied during the same time period (240 minutes); however, I/R was not applied in these groups. In other groups, in order to minimize heat and fluid loss, intraperitoneal serum physiological was administered at clamping and declamping periods. Also, the abdominal incision was covered with wet gauze. In Group DM-I/R-D, 100 μg/kg dexmedetomidine was administered intraperitoneally 30 minutes before ischemia period. At the end of reperfusion period, biochemical and histopathological evaluations of renal tissue specimen were performed. Rats were decapitated at the end of experiment.
Histopathological evaluation was performed in the Kirikkkale University Medical Faculty Histology and Embryology Department. After routine fixation process, specimens were embedded in paraffin blocks, then tissue sections of 5 μ were mounted on slides for staining with hematoxylin and eosin (H&E). Histopathological evaluation under light microscopy was performed, and findings were scored using a scoring system by Bostan et al. [17 (link)]. Glomerular vacuolization (GV), tubular dilatation (TD), vascular vacuolization and hypertrophy (VVH), tubular cell degeneration and necrosis (TCDN), Bowman space dilatation (BSD), tubular hyaline cylinder (THC), leucocyte infiltration (LI), and tubular cell spillage (TCS) were scored using a scoring system: 0: no change; +1: minimal change; +2: medium; +3: severe.
Biochemical evaluation was performed in the Gazi University Medical Faculty Medical Biochemistry Department. Oxidative stress and lipid peroxidation were evaluated using Thiobarbituric acid reactive substance (TBARS) levels as Malondialdehyde (MDA) indicators in renal tissue. Also, Catalase (CAT), Glutathione s transferase (GST), Nitric oxide synthase (NOS) and Superoxide Dismutase (SOD) activities were measured.
SOD, CAT, GST, and NOS enzyme analyses were performed as described by Durak, Aebi, Habig, and Durak [respectively 18 (link)–21 (link)]. The SOD activity method is based on the measurement of absorbance increase at 560 nm due to reduction of NBT to NBTH2. One unit of SOD activity was defined as the enzyme protein amount causing 50% inhibition in NBTH2 reduction rate. The CAT activity method is based on the measurement of the absorbance decrease due to H2O2 consumption at 240 nm. The GST activity method is based on the measurement of absorbance changes at 340 nm due to formation of a GSH-CDNB complex. The NOS activity method is based on the diazotization of sulfanilic acid by nitric oxide at acid pH and subsequent coupling to N-(1-napthyl-ethylene diamine), and absorbance of the sample tube is measured against the blank tube at 540 nm. In this method, sodium nitroprusside is used as the chemical standard.
The TBARS assay was carried out to determine lipid peroxidation using the thiobarbituric acid method [22 (link)]. TBARS measurements were conducted based on the reaction of MDA with thiobarbituric acid (TBA), which form a pink pigment with an absorption maximum at 532 nm in acid pH, and 1,1,3,3-tetraethoxypropane was used as a standard MDA solution.
All procedures were performed at 4°C throughout the experiment.
Enzyme activities and TBARS levels were determined by continuously monitoring and end point change in absorbance at 25°C with a Shimadzu UV- 1601 spectrophotometer. Results were expressed IU/ mg protein for CAT and NOS, for GST and SOD mIU/mg protein and U/mg protein respectively. TBARS results were given nmol/mg protein.
Histopathological evaluation was performed in the Kirikkkale University Medical Faculty Histology and Embryology Department. After routine fixation process, specimens were embedded in paraffin blocks, then tissue sections of 5 μ were mounted on slides for staining with hematoxylin and eosin (H&E). Histopathological evaluation under light microscopy was performed, and findings were scored using a scoring system by Bostan et al. [17 (link)]. Glomerular vacuolization (GV), tubular dilatation (TD), vascular vacuolization and hypertrophy (VVH), tubular cell degeneration and necrosis (TCDN), Bowman space dilatation (BSD), tubular hyaline cylinder (THC), leucocyte infiltration (LI), and tubular cell spillage (TCS) were scored using a scoring system: 0: no change; +1: minimal change; +2: medium; +3: severe.
Biochemical evaluation was performed in the Gazi University Medical Faculty Medical Biochemistry Department. Oxidative stress and lipid peroxidation were evaluated using Thiobarbituric acid reactive substance (TBARS) levels as Malondialdehyde (MDA) indicators in renal tissue. Also, Catalase (CAT), Glutathione s transferase (GST), Nitric oxide synthase (NOS) and Superoxide Dismutase (SOD) activities were measured.
SOD, CAT, GST, and NOS enzyme analyses were performed as described by Durak, Aebi, Habig, and Durak [respectively 18 (link)–21 (link)]. The SOD activity method is based on the measurement of absorbance increase at 560 nm due to reduction of NBT to NBTH2. One unit of SOD activity was defined as the enzyme protein amount causing 50% inhibition in NBTH2 reduction rate. The CAT activity method is based on the measurement of the absorbance decrease due to H2O2 consumption at 240 nm. The GST activity method is based on the measurement of absorbance changes at 340 nm due to formation of a GSH-CDNB complex. The NOS activity method is based on the diazotization of sulfanilic acid by nitric oxide at acid pH and subsequent coupling to N-(1-napthyl-ethylene diamine), and absorbance of the sample tube is measured against the blank tube at 540 nm. In this method, sodium nitroprusside is used as the chemical standard.
The TBARS assay was carried out to determine lipid peroxidation using the thiobarbituric acid method [22 (link)]. TBARS measurements were conducted based on the reaction of MDA with thiobarbituric acid (TBA), which form a pink pigment with an absorption maximum at 532 nm in acid pH, and 1,1,3,3-tetraethoxypropane was used as a standard MDA solution.
All procedures were performed at 4°C throughout the experiment.
Enzyme activities and TBARS levels were determined by continuously monitoring and end point change in absorbance at 25°C with a Shimadzu UV- 1601 spectrophotometer. Results were expressed IU/ mg protein for CAT and NOS, for GST and SOD mIU/mg protein and U/mg protein respectively. TBARS results were given nmol/mg protein.
