This experiment was performed on 35 male Sprague–Dawley rats (180–220 g) obtained from the Experimental Animal Centre of Fasa University of Medical Sciences. All procedures have been carried out in accordance with relevant guidelines and regulations regarding the care and the use of animals for the experimental procedures and were approved by the Committee of Animal Care of the Fasa University of Medical Sciences (IR.FUMS.REC.1397.014) in compliance with the ARRIVE guidelines. All Animals were acclimatized under the controlled standard conditions of 12 h light/12 h dark cycles, at temperature 20-22 ° C, standard pellet diets, and water ad libitum for one week before the start of experiments. Alamandine, and DOX were obtained from Phoenix Pharmaceuticals Inc., CA, USA and Tocris Bioscience, respectively
Experimental Group Design
After one week of acclimatization to the cages, the rats were randomly divided into five groups:
2-Sham group that, received normal saline as a vehicle for 42 days via mini-osmotic pumps (model 2006; ALZET Osmotic Pumps, CA, USA) and was surgically placed subcutaneously between the scapulae. This group also received normal saline intraperitoneal (i.p) on day(s) 14, 21, 28 and 35.
3-DOX group: Received DOX (3.750 mg/kg) i.p on day (s) 14, 21, 28 and 35 to reach total cumulative dose (15 mg/kg).
4- Alamandine group: Received alamandine for 42 days by mini-osmotic pumps with an infusion rate of 0.15 μl/h (50 μg alamandine/kg/day).
5- DOX+Alamandine group: Received alamandine by mini-osmotic pumps for 42 days (50 μg alamandine/kg/day) and also DOX (3.750 mg/kg) i.p on day (s) 14, 21, 28 and 35 to reach total cumulative dose (15 mg/kg).
On 41th day, rats were housed in the individual metabolic cages. The 24-hour urine sample from each animal was collected for measuring the creatinine clearance, creatinine, albumin, and TGF-B levels. At the end of the experiment, the animals were euthanized with pentobarbital sodium (150 mg/kg, intraperitoneal, IP), the blood samples were taken, and the two kidneys were removed. The blood samples were centrifuged at 4000 rpm for ten minutes. Serums for evaluation of biochemical parameters were taken and maintained at -80 0C. Creatinine clearance as an estimate of the glomerular filtration rate (GFR) was determined based on the 24-hour urine samples obtained under the following formula and expressed as mL/min (1).: Creatinine clearance (ml/min) = mg creatinine/dl urine× ml urine 24 h /mg creatinine/dl serum×1440
The right kidneys were dissected and washed with PBS (10 mM PO43−, 137 mM NaCl, and 2.7 mM KCl; pH = 7.4) and then dried on filter paper and weighed. Then were homogenized in the PBS and centrifuged at 10,000×g for 20 minutes at 4 ° C. The supernatant was stored at −20 ° C until the oxidative stress parameters, antioxidants, and TGF-β were further analyzed using Eliza kits.
Assessment of Water Intake and Urine Volume
It was achieved by using metabolic cages. Water intake was calculated by the difference between the last measured and the remaining volume over 24 hours. This measurement was considered as the daily water intake for every animal in each experimental group.
Both volumes for water intake and urine output were measured by measuring cylinders.
Assessment of inflammatory cytokines in serum:
IL-1β, IL-6 and NF-κB levels in the serum samples were determined using ELISA kits (Cloud-Clone Crop Technology co., Ltd., Wuhan, China), according to the manufacturer's protocol. All measurements were performed in duplicate.
Assessment of oxidative stress markers and TGF-β in kidney tissue and urine:
TGF-β levels in the urine and kidney tissue were determined using ELISA kits (R&D Systems, Minneapolis, MN), according to the manufacturer's protocol. All measurements were performed in duplicate. Samples of urine were collected in the metabolic cages and stored at 200C. Before refrigeration, 10 mL of commercial protease inhibitor cocktail were added to the urine samples.
The markers of oxidative stress, malondialdehyde (MDA), superoxide dismutase (SOD), and Glutathione Peroxidase (GPx), were determined in kidney tissue homogenates using available kits according to the manufacturer's protocol
IL-6, IL-1, P53, and NF-κB expressions were evaluated on paraffin-embedded tissues by a standard immunostaining assay. Briefly, Xylene and a graded alcohol series were used for deparaffinization and rehydration, respectively. Then the slides were incubated for 30 minutes in a blocking reagent containing 1.5% hydrogen peroxide in methanol. Antigen retrieval was performed on slides using microwave protocol and then incubated in serum for 30 minutes and immunostained with IL-6 (cat. no. sc-28343; Santa Cruz Biotechnology, Inc.), IL-1 (cat. no. sc-32294; Santa Cruz Biotechnology, Inc.), P53 (cat. no. sc-81168; Santa Cruz Biotechnology, Inc.) and NF-κB (cat. no. sc-48366; Santa Cruz Biotechnology, Inc.) primary antibodies, for one hour at room temperature. The slides were washed with PBS three times and then incubated with secondary antibodies for 30 minutes. The sections were stained using 3,30- diaminobenzidine (Dako liquid DAB color solution), and the slides were then counterstained with hematoxylin. An Olympus BX51 microscope was used to visualize the results (Olympus, Tokyo, Japan). Five different microscopic fields were selected randomly in each slide, and positive staining within each slide was measured by Image Pro Plus 6.1. Quantitative analysis was performed in a blinded manner.
Left kidneys of male rats were harvested and fixed immediately in 10% buffered formalin phosphate (pH 7.4) for histological tests. The tissue samples were then dehydrated by passing through graded concentrations of alcohol, cleaned to remove alcohol by xylene, incorporated in paraffin, and allowed to harden. Subsequently, 5 μm sections of the paraffin blocks were prepared by microtome and remained floating in the water bath. Floating kidney sections were then mounted on microscopical slides, placed into the drying oven at 60 °C, stained with Harris’ hematoxylin and 1% eosin, and histological examination was carried out with light microscopy.
Data and statistical analyses
Statistical analyses were performed using GraphPad Prism software (GraphPad Prism software v6 Inc., La Jolla, CA, USA). All data were expressed as mean ± standard deviation (SD). A one-way Analysis of Variance (ANOVA) was used to compare all groups, followed by Tukey post-hoc test.