Animals: 38 male Wistar rats, age 21 days (50g) (come from the CCA-UFOP) divided randomly, into 4 groups (4 animals/box): (1) sedentary rats fed with standard diet (S-SD), n = 14; (2) T rats fed with standard diet (T-SD), n = 9; (3) sedentary rats fed with high sucrose diet (S-SUD), n = 05; and (4) T rats fed with SUD (T-SUD), n = 10; marked on the tail; allocated in a room at a temperature of 24 ± 2 °C and 12-hour light/dark cycle (7 am - 7 pm). Body weight assessments: realized at weeks 1, 4, 5, 7, and 11. Food intake assessments: realized at weeks 1, 5, and 10. Approval of the study: by the Ethics Committee for Animal Use, UFOP (Protocol 45/2014). The experimental design scheme was shown in figure S2.
Diets: SD - commercial rodent feed in pellets (Nuvilab CR1 Quimtia®) and SUD as previously published [13] administered for 12 weeks from weaning.
Exercise training and evaluation of endurance capacity: Local - collective glass tank of CCA-UFOP with warm water at 28 ± 2 ºC and 45 cm depth. Adaptation period: 15 min (first day) increased by 15min each day until reaching 60 min (fourth day) (adapted from [14]). Maximal test: at the 4th and 12th (24 h after the last training section) weeks as proposed by others [15]. Fatigue point was adapted from [16]. Training: swimming for 8 weeks. The first 4 weeks didn’t use a workload, but that was added at the fifth week (60% of that obtained in maximal test) as adapted from [15].
Water Intake, Urinary Volume and Water Balance Measurements in 24h:
Rats were allocated in metabolic cages (Tecnoplast® SPA) during the 12-week period (CCA-UFOP); weighed (SF–400 scale), individually housed for a period of 24 h with free access to water and food. Measurements of urine volume and water intake were realized. Samples of urine 1.5 mL were obtained, labeled, and frozen at −20 °C. Calculation of water balance was done according to the equation:
Equation 1:
Water Balance (mL/24h) = (Water Intake [mL/24h]− Urinary Volume [mL/24h]).
Euthanasia: 48 h after the last T session. Collected materials: Adipose tissues inguinal, retroperitoneal and epididymal (IAT, RAT and EAT, respectively), blood and kidneys. Blood was centrifuged (CENTRIBIO 80-2 B scale) at 3000 rpm for 10 min to separate the plasma and then maintained at a temperature of −20 ºC. Details: Supplementary Material (Table S2).
Determination of the LI:
LI was measured according to [17]. Animals’ body mass and the nasoanal length [17], were calculated using the formula below:
Equation 2:
Determination of the BAI:
BAI was measured according to [19]. The EAT, IAT, and RAT were removed and weighed (BEL precision scale), and used in the following equation:
Equation 3:
Evaluation of RAT:
RAT analyse provide an assessment of the risk of developing cardiovascular diseases [20].
Plasma and Urine Creatinine Concentration and Urine Protein:
Samples - plasma and urine; kit - commercially available kit (Labtest, Belo Horizonte/MG, Brazil) – by colorimetric modified Jaffé approach. Calculation of creatinine clearance (CrCl):
Equation 4:
Proteinuria was determined with the use of pyrogallol red technique using a commercially available kit (Bioclin, Belo Horizonte, Brazil), n = 38 rats. Results were expressed in mg/dL.
Renal Histology:Collected material - right kidneys; Stage and calculation - as described by others [21, 22]. Equipment - light microscope (Leica DM5000). Analysis - Analysis and Image Processing Software Leica Qwin (Germany) [23].
Data and Statistical Analysis: Normality test - Kolmogorov-Smirnov. Statistical tests used - One-way or two-way analysis of variance (ANOVA) and Tukey’s post-test for multiple comparisons following ANOVA. Software used - GraphPad Prism 7.0 for Windows (GraphPad Software, San Diego California USA). Data were expressed as mean ± standard deviation of mean and differences between pairs of means were considered significant when the probability of type I error was less than 5% (p < 0.05). The data were analyzed blindly by the researchers involved.