Animals
The present study was conducted on 72 adult female Wistar rats (180–200 g). The rats were kept under natural conditions of 12:12 h light-dark cycle at 24 ˚C in the animal center of Afzalipour faculty medicine. All the tests were performed according to ethical instructions of the Institutional Animal Care Committee of Kerman University of Medical Sciences (ethics code: IR.KMU.REC.1398.469) and the national guidelines for the care and use of animals.
The rats were divided into two groups (6/group) of sham (ovary-intact) and OVX; each group received the diets [standard diet (SD), CR, and TR] for 2 months. Afterward, the groups were divided into two subgroups of A (no AKI) and B (AKI). In subgroup A, kidney injury indicators and biochemical parameters of serum and kidney tissue were measured, and then the animals were sacrificed. In subgroup B, at the end of the second month, AKI was induced, and the factors mentioned above were measured after AKI (24). There were six animals in each subgroup (Fig. 1).
Sham groups
1- Control (sham CTL): Ovary-intact animals in this group received the SD (Pars Animal, Iran), which contained 5.7%, 72.2%, and 22.1% calories from fat, carbohydrate, and protein, respectively (the total amount of calorie was 3.1 kc/g). At the end of the second month, the animals were divided into two subgroups of A (no AKI) and B (AKI induction).
2-Calorie restriction (sham CR): This group was similar to the control group but received only 70% of the food the control group (6).
3- Time restriction (sham TR): This group was also similar to the control group but were given only 5 hours of daily free access to food and water per day for 2 months (6).
OVX groups: The animals were categorized in the same way as the sham groups (OVX-CLT, OVX-TR, and OVX-CR); however, they were ovariectomized.
Drugs
Ketamine and xylazine were purchased from Alfasan Company (Utrecht, the Netherlands) and glycerol from Kimia Gostar Pooyesh Company, Iran.
Bilateral Ovariectomy
Following anesthesia by ketamine and xylazine (Alfasan Company Utrecht, the Netherlands) 80/10 mg/kg intraperitoneally, a small longitudinal incision was made in the abdomen. The skin, fascia, and abdominal muscles were opened, and ovaries were identified and removed. Then, 1–2 ml of saline solution was poured into the abdomen, and the skin and muscles were sutured. In the sham surgery, a similar incision was made, but the ovaries were not removed. The subsequent tests were performed 2 weeks after OVX (25, 26).
Serving size calculation and using CR
The amount of food intake of one week was measured in the CTL group to determine the average serving size in the CR group, in which the animals had free access to the food, and the amount of daily intake was calculated as an average. Then, 70% of the daily intake amount in the group with free access to food was measured and provided for the CR group for 2 months (27).
AKI induction
Rats were deprived of water for 24 hours at the end of the 2 months of being diets to AKI induction, and then single intramuscular (10 ml/kg) dose of hypertonic glycerol (Kimia Gostar Pooyesh Company, Iran) solution (50% dissolved in sterile saline) was equally injected into both lower limbs of the rat. Twenty four hours after the glycerol injection rhabdomyolysis occurred which in turn caused myoglobinuria and nephropathy to develop rapidly. In this study, urea, creatinine, and GFR were measured to confirm AKI induction (24).
Measurement of serum and urinary urea and creatinine
To measure these indicators in serum and urine, 1 day before and 1 day after AKI induction, the levels of urea and creatinine in serum and urine were measured using the ELISA test according to the kit instructions (MAN, Iran). The values were reported in mg/dl. The blood samples were taken from the choroid sinus and were immediately centrifuged, and the serum was separated. Moreover, to measure these indicators in urine, 1 day before and 1 days after AKI induction, urine was collected using a metabolic cage (Behboud Tahghigh, Kerman, Iran) (28).
Measurement of 24-h urine albumin excretion
Using a metabolic cage, urine of the animals was collected before and 24 hours after AKI induction, and urine albumin concentration was measured using the ELISA test according to the kit instructions (MAN, Iran). Then, the levels of urine albumin excretion in 24 hours was measured using a 24-h urine volume, and the following equation, and the values were reported in mg/24h (29).
Albumin excretion in 24 h = (urine volume in 24 h × concentration of urine Albumin)
Measurement of GFR
The rats were kept in a metabolic cage, and their urine was collected. This was performed before and 1 day after the AKI induction. Afterward, based on the a 24-h volume of urine and concentration of creatinine in urine and serum, GFR(ml/24h) was measured based on creatinine clearance according to the following equation (1):
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)
Measurement of MDA and TAC in kidney tissue
At the end of study, kidneys were removed and placed in cold physiological saline. Then, they were homogenized and centrifuged for 10 minutes at 1000 rpm to produce a supernatant. the levels of MDA and TAC in homogenized kidney tissue were measured using the ELISA kits (Behboud Tahghigh, Kerman, Iran) according to the manufacturer’s instruction. The values were reported in nmol/mg protein (30).
Measurement of SIRT1 and TGF-β1 in kidney tissue
The supernatant of kidney tissue was separated using the same method used for MDA and TAC measurement. Subsequently, The levels of SIRT1 were measured in supernatant of kidney tissue using the ELISA test according to the kit instructions (ZellBio, Germany), and the values were expressed as ng/mg (19). The levels of TGF-β1 in kidney tissue were also measured using the ELISA kit according to the manufacturer’s instruction (KPG, Iran), and the values were reported in pg/mg (23).
Measurement of fasting blood sugar (FBS) and lipid profile
The FBS of the rats was measured in the beginning of the study and at the end of the first and second months. Blood sugar was measured using a glucometer; the data were calculated as the percentage of changes in blood sugar compared to the first day. Blood samples for lipid profile testing, were taken from the choroid sinus of the animals. the samples were centrifuged, and the serum was separated, and the lipid profile were measured in serum using a kit (Man, Iran) according to the manufacturer’s instruction. The values were expressed in mg/dl.
Measurement of body weight
Rats weight were measured in the beginning and in the end of the study using a digital scale (Grampresicion digital scale, Canada) and reported as the percentage of changes.
Statistical analyses
To compare the quantitative variables between the groups, if the assumptions are satisfied (normal distribution of data), ANOVA and then post-hoc Tukey's tests were used; otherwise, the Kruskal-Wallis test was used. Results are presented as mean ± S.E.M and P < 0.05 was considered statistically significant and statistical analyses were done using GraphPad Prism 8.