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Aerobic exercise training prevents kidney lipid deposition in mice fed a cafeteria diet
Life Sciences 211 (2018) 140–146
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Aerobic exercise training prevents kidney lipid deposition in mice fed a cafeteria diet C.R. Mullera, A.L.V. Américoa, P. Fiorinob, F.S. Evangelistac,
T
⁎
a
Experimental Pathophysiology Dept., Faculty of Medicine, University of São Paulo, Brazil Health and Biological Science Center (CCBS), Mackenzie University, Sao Paulo, Brazil c School of Arts, Science and Humanities, University of Sao Paulo, Brazil b
A R T I C LE I N FO
A B S T R A C T
Keywords: Kidney lipid deposition Aerobic exercise training Oxidative metabolism
Aim: The objective of this study was to investigate the potential of aerobic exercise training (AET) to prevent kidney lipid accumulation and the contribution of renal metabolism to mediate this response. Main methods: Male C57BL/6J mice were assigned into groups CHOW-SED (chow diet, sedentary; n = 13), CHOW-TR (chow diet, trained; n = 13), CAF-SED (cafeteria diet, sedentary; n = 13) and CAF-TR (cafeteria diet, trained; n = 13). AET consisted in running sessions of 60 min at 60% of maximal speed conducted five days per week for eight weeks. Key findings: AET prevented weight gain in both trained groups. Food intake was not different among groups, however water intake, urine output, urine potassium and osmolarity were reduced in CAF-SED and CAF-TR groups. Kidney lipid deposition increased in CAF-SED (4.12 ± 0.5%/area) compared with CHOW-SED (1.7 ± 0.54%/area), and the AET prevented this increase in the CAF-TR group (2.1 ± 0.5%/area). The Bowman's capsule area decreased in CAF-SED and CAF-TR groups while the Bowman' space reduced in CAF-SED compared to CHOW-SED group, which was prevented by AET in the CAF-TF group. We observed a 27% increase in the p-AMPK expression in CAF-TR compared to CHOW-SED group without differences in the SIRT-1, PGC1-α, ACC and p-ACC. β-HAD activity increased in CAF-SED (43.9 ± 4.57 nmol·min−1·ug−1) and CAF-TR (44.7 ± 2.6 nmol·min−1·ug−1) groups compared to CHOW-SED (35.1 ± 2.9 nmol·min−1·ug−1) e CHOW-TR (36.6 ± 2.7 nmol·min−1·ug−1). Significance: AET prevented kidney lipid accumulation induced by cafeteria diet and this response was not associated with changes in the renal metabolic activity that favors lipid oxidation.
1. Introduction Changes in lifestyle and dietary habits have led to several health problems, such as obesity, dyslipidemia, Diabetes Mellitus type 2 (DM2) and hypertension. The excess of lipids has been indicated as the main factor triggering pathologies associated with this lifestyle, because they accumulate in non-adipose tissue leading to lipotoxicity. In the kidney, lipid accumulation can cause proteinuria, urinary podocyte loss, insulin resistance, oxidative stress, fibrosis, apoptosis and hypertrophy culminating in chronic kidney disease (CKD) [15]. Thus, excessive lipid deposition is a common mechanism involved in the CKD development in most of pathologies such as obesity, DM2 and metabolic syndrome [3]. Studies have been conducted to better understand the effects of lifestyle change and the ectopically accumulated lipids. In a previous report, it was demonstrated that a high-fat diet induced weigh gain,
increased adiposity and renal lipid accumulation, which were associated with inflammation, glomerulosclerosis, mesangial matrix expansion and mitochondrial damage [34]. Our group previously demonstrated that a cafeteria diet increased body weight gain, adiposity, insulin resistance and total cholesterol in mice. Also, we observed that a cafeteria diet increased white adipose lipolysis and circulating free fatty acid (FFA), which may lead to lipotoxicity in peripheral tissues [12]. Thus, mice fed a cafeteria diet can be a good model to study ectopically accumulated lipids in the kidney. Impairments in the regulation of lipid metabolic activity such as lipogenesis (triacylglycerol biosynthesis and accumulation in the intracellular lipid droplet), lipolysis (triacylglycerol hydrolysis) and fatty acid oxidation may lead to an increase in the renal lipid accumulation. Kume et al. [18] showed that the renal lipid accumulation in a high-fat diet model occurs due to an altered balance between lipolysis and
⁎ Corresponding author at: School of Arts, Science and Humanities, Sao Paulo University, Av. Arlindo Bettio, 1000, Ermelino Mattarazzo, São Paulo, SP CEP 03828000, Brazil. E-mail address: [email protected] (F.S. Evangelista).
https://doi.org/10.1016/j.lfs.2018.09.017 Received 13 April 2018; Received in revised form 29 August 2018; Accepted 7 September 2018 Available online 12 September 2018 0024-3205/ © 2018 Elsevier Inc. All rights reserved.
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2.3. Running capacity test
lipogenesis, and this modification in renal lipid metabolism can be associated, at least in part, with decrease in AMP-activated protein kinase (AMPK) and increase in acetyl-CoA carboxylase (ACC) activity. Both proteins are crucial for lipid metabolism because while AMPK protein induces pathways that increase ATP (adenosine triphosphate) synthesis by providing more glucose and fatty acid for oxidation [19], the ACC modulates oxidative metabolism by inhibiting the activity of carnitine palmitoyl transferase 1 (CPT-1), an important fatty acid carrier protein in the mitochondria [14]. The aerobic exercise training (AET) has been used to treat and prevent metabolic damage associated with obesity and DM2 [12,21] because it promotes reduction in body weight and white adipose mass [12,23,25], and increases free fatty acid (FFA) oxidation in the skeletal muscle associated with better mitochondrial function [16]. Our previous study demonstrated that AET was able to prevent obesity, glucose intolerance and insulin resistance in mice fed a cafeteria diet by improving white adipose metabolism that favors fat oxidation (higher AMPK and lower ACC expression protein) instead of fat storage [12]. These data show that AET is a good non-pharmacological tool to increase lipid oxidation, however, this effect still needs to be investigated in the kidney. Evidence in the literature has confirmed that AET provides improvement in renal function and morphology. In a previous study, Silva et al. [31] showed that AET decreased proteinuria in animals with DM induced by streptozotocin. Agarwal et al. [1] have shown that AET has beneficial morphofunctional effects on the kidney. Considering the role of lipid accumulation in the development and progression of kidney damage, and the effect of AET to improve lipid metabolism by increasing fatty acid oxidation, the objective of this study was to investigate the potential of AET to prevent kidney lipid accumulation and the contribution of renal metabolism to mediate this response. Our hypothesis is that the AET prevents kidney lipid accumulation induced by a cafeteria diet and this response is associated with the improvement of renal metabolic activity that favors lipid oxidation.
Running capacity was assessed before, in the fourth and eighth weeks of AET using a progressive test with a 0% incline on a treadmill as described by Ferreira et al. [7]. The initial speed was 0.4 km/h and the speed was increased by 0.2 km/h every 3 min until exhaustion of the animal, which was characterized by the impossibility of maintaining the standard rate. The test variable was quantified as maximum time to exhaustion (min). 2.4. Body weight control Body weight was measured at the beginning and at the end of the AET protocol using a digital balance (Gehaka, Model BK4001, Brazil). Body weight gain was calculated as the difference between beginning body weight and final body weight. 2.5. Metabolic cages In the 6th week of the protocol, the animals were housed individually in metabolic cages (Tecniplast, Buguggiate, VA, Italy) for 48 h. The first 24-h were used to adapt the mice to the environment and the following 24-h were used to collect urine. Food consumption and water intake were also monitored. The water balance was calculated through the water to urine ratio. Urine samples collected during 24-h period were used to determine urine output, creatinine, sodium, potassium chloride, protein excretion and osmolarity. Creatinine and total protein were quantified in spectrophotometer using colorimetric method (LABTEST Biochemical Kit, Brazil). In addition, electrolytes (Na +, K+ and Cl−) and osmolarity were measured in a gas analyzer (ABL800 FLEX, Radiometer Copenhagen) and osmometer (Vapor Pressure Osmometer 5520 USA), respectively. The creatinine clearance for assessing the Glomerular Filtration Rate (GFR) was calculated using the formula [(Urine (Creatinine) × Urine Vol) / Serum (Creatinine)]. The calculation of the water balance was performed using the formula Water intake / Urine output (mL/24-h).
2. Materials and methods
2.6. Tissue and blood collection
2.1. Animals
Forty-eight hours after the end of the last training session, the animals were killed with an intraperitoneal injection of sodium pentobarbital (4 mg/100 g body weight). Kidneys were harvested, weighed and stored at −80 °C (right kidney) or fixed in 10% neutral-buffered formalin (left kidney) for subsequent histological analyses. The vena cava blood was collected and centrifuged at 4 °C (10.000g for 10 min) and serum was sent for creatinine analysis as described above.
Eight-week-old male C57BL/6J mice were assigned in groups CHOW-SED (chow diet, sedentary; n = 13), CHOW-TR (chow diet, trained; n = 13), CAF-SED (cafeteria diet, sedentary; n = 13) and CAFTR (cafeteria diet, trained; n = 13). Animals were maintained under the same housing conditions (12-h light/12-h dark cycle, temperature 22 ± 2 °C) with free access to tap water and food ad libitum. All procedures were performed in accordance with the guidelines of the Brazilian College for Animal Experimentation and were approved by the Ethics Committee of the Faculty of Medicine of University of Sao Paulo (#18/2014).
2.7. Histological analysis Glomerular injury was measured in paraffin sections of kidney (5 μm) stained with Picrossirius Red (Sigma). Digital images from thirty glomeruli per animal were obtained using a light microscope (Leica) at 200× magnification. After digitalization, Bowman's capsule area, glomerular tuft area, Bowman's space area and glomerular diameter were traced and calculated using a computerized morphometric analysis system (Image Pro-Plus 4.1; Media Cybernetics, Silver Spring, MD, USA). Lipid content was measured using quantitative histochemistry of Oil Red O (Sigma-Aldrich) staining of kidney. Tissue sections (thickness 10 μm) obtained in a cryostat were examined by light microscopy at 200× magnification and analyzed by a computerized morphometric analysis system (Image Pro-Plus 4.1; Media Cybernetics, Silver Spring, MD, USA). The slides were counterstained with hematoxylin to visualize the nuclei. Lipid accumulation was determined in 20 images per animal based on the percentage of area occupied by lipid droplets. Histological analyses were conducted by CR Muller, blinded to mice
2.2. Diets and aerobic exercise training The standard chow diet contained 4% of kilocalories from fat, 55% from carbohydrate and 22% from proteins (Nuvilab®, Paraná, Brazil). The cafeteria diet contained 18.8% of kilocalories from fat, 55% from carbohydrate and 14.8% from proteins [11]. Diet and AET were started simultaneously. CHOW-TR and CAF-TR animals were submitted to AET as described by Ferreira et al. [7]. Animals were trained during the dark cycle (i.e., during their active period) on a motorized treadmill for 1 h/ day at 60% of maximal velocity achieved in the running capacity test, five times per week for eight weeks. AET intensity was progressively increased and adjusted after the running capacity test done in the fourth week. To minimize the influence of the treadmill stress, sedentary mice were placed on the treadmill for 5 min twice weekly at 0.3 km/h during the experimental protocol. 141
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identities.
Table 1 Metabolic parameter evaluated in 24-h. Data presented as mean ± SE. CHOW-SED (n = 13); CHOW-TR (n = 13); CAF-SED (n = 13); CAF-TR (n = 13).
2.8. Western blot analysis Samples of frozen right kidney were homogenized in an ice-cold lysis buffer (pH: 7.2): 0.1 M sodium phosphate monobasic, 0.34 M sucrose, 0.3 M sodium chloride, protease inhibitor cocktail tablets mini complete EDTA-free (Roche®, Germany). Samples were centrifuged for 25 min at 14.000 rpm at 4 °C. Protein concentrations of the homogenates were measured by the BCA method with a protein assay kit (PIERCE Biotechnology®, Rockford, IL, USA) using bovine serum albumin as a standard. Aliquots (30 μg) of protein were subjected to SDSPAGE. The membranes were incubated overnight at 4 °C with the following primary antibodies: p-AMPK (Thr172) (1:1000), AMPK (1:1000), p-ACC (Ser79) (1:1000), ACC (1:1000) (Cell Signaling®, Beverly, MA), SIRT-1(1:1000), PGC1-α (1:1000) and Beta-Actin (1:1000) (Abcam®, Cambridge, USA). The signal on the membrane was detected via the peroxidase reaction in the ECL solution using an Image Quant LAS 4000 mini system (GE Healthcare Life Sciences®). Band intensities were quantified based on optical densitometry measurements using the Image J program (version 1.43 for Windows).
Food intake (g/24 h) Initial body weight (g) Final body weight (g) Weight gain (g) Water intake (mL/ 24 h) Urine output (mL/ 24 h) Water balance (mL/ 24 h) ⁎ #
CHOW-SED
CHOW-TR
CAF-SED
CAF-TR
4.2 ± 0.3 22.2 ± 0.9 29.2 ± 0.7 7.0 ± 0.8 5.4 ± 0.5
4.9 ± 0.3 22.3 ± 0.8 27.3 ± 0.8# 5.0 ± 0.4# 4.8 ± 0.3
4.1 ± 0.3 22.4 ± 0.7 28.8 ± 0.3 6.4 ± 0.6 3.5 ± 0.3⁎
4.3 ± 0.3 21.8 ± 0.8 26.4 ± 0.4# 4.6 ± 0.7# 4.2 ± 0.4⁎
1.6 ± 0.2
1.9 ± 0.2
1.2 ± 0.2⁎
1.2 ± 0.2⁎
4.1 ± 0.8
3.2 ± 0.5
4.5 ± 1.3
4.2 ± 0.7
p ≤ 0.05 vs. CHOW-SED and CHOW-TR. ≤0.05 vs. CHOW-SED and CAF-SED.
groups (CHOW-SED = 19.4 ± 1.7 min; CHOW-TR = 21.4 ± 0.7 min; CAF-SED = 21.8 ± 1.5 min; CAF-TR = 20.8 ± 0.8 min). However, after AET, the running capacity was increased in both trained groups (CHOW-TR = 31.3 ± 1.8 min and CAF-TR = 31.2 ± 3.1 min) compared with sedentary groups (CHOW-SED = 21.5 ± 0.7 min and CAFSED = 19.9 ± 1.0 min). These results confirm the aerobic adaptation induced by AET.
2.9. β-HAD activity For the determination β-HAD (β-hydroxyacyl-CoA dehydrogenase), approximately 0.75 g of the kidney was suspended in 750 μL of extraction buffer containing Tris-base (50 mM) and EDTA (1 mM), pH = 7.4. The material was homogenized in Precellys® 24 (Bertin Corp, USA) for 2 cycles of 20 s. The homogenate was centrifuged at 3000 rpm for 15 min at 4 °C in a 5417 C/R (Eppendorf) centrifuge. β-HAD activity was measured by the determination of the amount of oxidized NADPH considering the NADPH molar extinction coefficient. The buffer used in the assay contained triethanolamine (0.1 M), EDTA (5 mM) and NADH (0.45 mM) pH = 7.0. The reaction was started with the addition of 15 μL AcetoAcetil CoA (0.1 mM) to the enzyme extract and followed for 10 min (37 °C). Absorbance was monitored at 340 nm [2]. The proteins were quantified by the BCA™ method (PIERCE Biotechnology). The results were expressed as μg/μl of protein present in the extract.
3.2. Metabolic and urinary parameters There were no changes in the initial body weight, however the CHOW-TR and CAF-TR had smaller final weight and weight gain compared with CHOW-SED and CAF-SED (Table 1). No difference in the food intake was present among groups. On the other hand, the water intake was decreased in both cafeteria diet groups compared to chow diet groups. Similarly, the urine output was lower in both cafeteria diet groups compared with chow diet groups (CHOW-SED and CHOW-TR). Water balance was not changed (Table 1). Although non-significant, the GFR was 88% increased in CAF-SED group (0.15 ± 0.03 mL/min) compared with CHOW-SED (0.08 ± 0.022 mL/min) CHOW-TR (0.10 ± 0.02 mL/min) and CAFTR (0.11 ± 0.02 mL/min). The urinary excretion of protein, sodium and chloride were not different among groups, however the potassium excretion and the osmolarity were decreased in cafeteria groups (CAFSED and CAF-TR) compared with CHOW-SED and CHOW-TR groups (Table 2).
2.10. Statistical analyses All values are expressed as mean ± SE. The results were compared among groups using two-way analyses of variance (ANOVA). The Bonferroni post hoc test was used to determine differences between means when a significant change was observed using ANOVA. A p value equal to or < 0.05 was considered to be statistically significant (GraphPad Prism®, v.6.0).
3.3. Lipid deposition and glomerular morphometry The renal lipid deposition was significantly 2.5-fold higher in the CAF-SED (4.1 ± 0.5% area) than CHOW-SED group (1.7 ± 0.5% area) and the AET prevented this increase in the CAF-TR (2.1 ± 0.5% area). No difference was observed between CHOW-SED and CHOW-TR (2.2 ± 0.2% area) groups (Fig. 1A). Fig. 1B represents kidneys sections stained with Oil Red O.
3. Results 3.1. Aerobic performance Before AET, no differences in running capacity were present among
Table 2 GFR and urinary parameters. Data presented as mean ± SE. GFR = Glomerular Filtration Rate. CHOW-SED (n = 9); CHOW-TR (n = 8); CAF-SED (n = 11); CAF-TR (n = 12).
Protein (mg/24 h) Potassium (mMol/24 h) Sodium (mMol/24 h) Chloride (mMol/24 h) Osmolarity (mmol/kg) GFR (mL/min) ⁎
CHOW-SED
CHOW-TR
CAF-SED
CAF-TR
0.0207 ± 0.0018 0.372 ± 0.045 0.259 ± 0.032 0.920 ± 0.144 2564.3 ± 141.2 0.08 ± 0.022
0.0155 ± 0.0022 0.364 ± 0.051 0.251 ± 0.043 0.834 ± 0.136 2515.0 ± 175.3 0,10 ± 0.02
0.0184 ± 0.0023 0.227 ± 0.047⁎ 0.222 ± 0.036 0.724 ± 0.141 2038.1 ± 162.4⁎ 0.15 ± 0.03
0.0140 ± 0.0015 0.198 ± 0.022⁎ 0.185 ± 0.031 0.766 ± 0.137 1931.8 ± 176.9⁎ 0.11 ± 0.02
p ≤ 0.05 vs. CHOW-SED and CHOW-TR. 142
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B
L ip id D e p o s itio m ( % /A r e a )
A
*
5
SED TR
4
CHOW-SED
3
CHOW-TR
2
1
0 CHOW
C AF
CAF-SED
CAF-TR
Fig. 1. Kidney lipid deposition. CHOW-SED (n = 4), CHOW-TR (n = 5), CAF-SED (n = 6) and CAF-TR (n = 5). Error bars indicate SE (A). Arrows shows lipids droplets (B). Data are shown as the percentage of marked area. p ≤ 0.05, *CAF-SED vs. CHOW-SED, CHOW-TR and CAF-TR.
and maximum oxygen uptake (VO2 max) in rats and mice, revealing that higher physical capacity is followed by better aerobic capacity. This observation is relevant because higher aerobic capacity is associated with reduced cardiovascular and metabolic disease risk [26]. On the other hand, low levels of cardiorespiratory fitness (VO2 max) have been associated with a higher risk of morbidity and mortality due to chronic-degenerative diseases, including coronary artery disease, systemic arterial hypertension, DM2 and some types of cancer [26]. The cafeteria diet has been used for the development of obesity and DM2 experimental models, contributing to the investigation of mechanisms involved in the development and progression of these diseases. In our study, the cafeteria diet did not induce greater body weight gain in the CAF-SED group compared to the CHOW-SED, however, for both trained groups (CHOW-TR and CAF-TR) AET was efficient in reducing body weight gain. It is important to note that, although the CAF-SED group did not present a greater body weight gain, the weight of fat depot were higher compared with the other groups (data not showed), which is typical in rodent obesity [27]. We observed that dietary intake was not different among the groups. Although it is known that AET can induce increased food intake, our trained groups maintained the same eating pattern as previously demonstrated by Higa et al. [12]. These data corroborate the Eguchi et al. [6] study, which found no difference in the food consumption in swimming trained groups fed a cafeteria or control diet. Gollisch et al. [8] demonstrated an increase in the food consumption of trained animals, independent of the diet (normocaloric or hyperlipidic). These contradictory data can be explained by methodological differences between the studies, such as type of diet, AET protocol, duration and intensity. Considering that body weight control occurs through the balance between caloric intake and energy expenditure, it is possible that the lower body weight gain observed in the CHOW-TR and CAF-TR groups is due to the increase in energy expenditure promoted by AET, since in the present study, trained groups did not present an increase in caloric intake. There was a reduction in the water intake and urine output in both groups fed a cafeteria diet without changes in the water balance. In the same direction, both cafeteria groups had decreased potassium excretion and osmolarity. Lower renal potassium excretion can be associated with reduced sodium delivery to the distal nephron, decreased mineralocorticoid level or activity, or abnormalities in the cortical collecting duct [29]. Unfortunately, we did not measure electrolytes clearance and activity, neither the level of aldosterone to understand these changes. Although non-significant, the CAF-SED group showed high GFR
Table 3 Glomerular morphological parameters. Data presented as mean ± SE. p ≤ 0.05.
Bowman's capsule area (μm2) Bowman's space area (μm2) Glomerular tuft area (μm2) Glomerular diameter (μm)
CHOW-SED
CHOW-TR
CAF-SED
CAF-TR
470 ± 16 154 ± 15 316 ± 9 21 ± 0.4
442. ± 17 126. ± 11 312 ± 12 23. ± 1.8
405 ± 21⁎ 104 ± 5# 298 ± 18 21 ± 0.6
414. ± 13⁎ 117 ± 5 297 ± 10 21 ± 0.4
⁎ CAF-SED (n = 7) and CAF-TR (n = 9) vs. CHOW-SED (n = 9) and CHOWTR (n = 9). p ≤ 0.05. # CAF-SED vs. CHOW-SED.
The animals fed a cafeteria diet (CAF-SED and CAF-TR) have reduced the Bowman's capsule area. There were no differences in the glomerular tuft area and glomerular diameter among groups. In the other hand, the Bowman's space was reduced in the CAF-SED compared with CHOW-SED and the AET prevented this reduction in the CAF-TR group (Table 3). 3.4. Kidney lipid metabolism As shown in the Fig. 2A, the expression of p-AMPK was 27% increased in the CAF-TR (1.27 ± 0.007% vs. CHOW-SED) group compared with CHOW-SED group (1.0 ± 0.008% vs. CHOW-SED). There were no changes in the expression levels of AMPK (Fig. 2B), p-ACC (Fig. 2C), ACC (Fig. 2D), PGC1-α (Fig. 2E), SIRT-1 (Fig. 2F). The β-HAD enzyme activity was increased in CAF-SED and CAF-TR group compared with CHOW-SED and CHOW-TR (Fig. 3). 4. Discussion In the present study we investigated if AET could prevent kidney lipid accumulation and if adaptations in renal metabolic activity could explain this response in physically trained mice fed a cafeteria diet. Our results confirmed the efficiency of AET to prevent ectopically accumulated lipids in the kidney and despite the increase in p-AMPK protein expression, the lower lipid content in the CAF-TR group cannot be associated with lipid oxidation metabolism improvement. We showed through running capacity test that the animals responded adequately to the AET, since both trained groups presented better performance compared to sedentary groups. This response confirms the AET efficacy for aerobic adaptations and corroborate previous studies conducted by Ferreira et al. [7] and Higa et al. [12]. Hoydal et al. [13] demonstrated a direct relationship between running velocity 143
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Fig. 2. Expression of AMPK (A), p-AMPK Thr172 (B), ACC (C), p-ACC Ser79 (D), PGC1-α (E) and SIRT-1(F) in the kidney. CHOW-SED (n = 9), CHOW-TR (n = 9), CAF-SED (n = 9) and CAF-TR (n = 9). Error bars indicate SE. Immunoblotting data are shown as the percentage of CHOWSED (set to 1). p ≤ 0.05, *CAF-TR vs. CHOW-SED, CHOW-TR.
and CKD development [17]. In fact, Henegar et al. [10] demonstrated that dog fed a high-fat diet had higher GFR [10]. However, Wicks et al. [36] demonstrated that C57BL/J6 fed a high-fat diet are resistant to develop kidney injury. Another study with C57BL/J6 showed that high fructose and high-fat diet developed minimal injury with mild hyperfiltration (1.5 fold higher than the control group) [5]. Studies have suggested the association between lipid accumulation and the development of kidney injury, however little is known about
which was prevented by AET in the CAF-TR group. Glomerular hyperfiltration has been considered an inadequate adaptive response presented by the kidneys, which can be observed early in the course of renal disease, and may cause irreversible damage to the nephron, leading to the development of more advanced stages of CKD [30]. Hyperfiltration occurs to meet the heightened metabolic demands of the increased body weight, and this alteration can increase intraglomerular pressure and increase the risk of kidney structure damage 144
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60
.u g
- 1
*
SED TR
40
-1
( n m o l.m in
- H A D a c t iv ity
)
*
20
0 CHOW
C AF
Fig. 3. β-HAD enzyme activity in the kidney. CHOW-SED (n = 5), CHOW-TR (n = 5), CAF-SED (n = 5) and CAF-TR (n = 5). Error bars indicate SE. p ≤ 0.05, * CAF-SED and CAF-TR vs. CHOW-SED, CHOW-TR.
accumulation [20,32,33]. However, here we have shown that AET prevents renal lipid accumulation independent of the activation of the AMPK pathway. This result suggests that AET may prevent lipid deposition in the CAF-TR animals with no changes in renal metabolic activity. In this case, a reduced flow of fatty acids to the kidneys could explain the lower content of lipids in the CAF-TR group. This may occur since we have previously demonstrated the reduction of circulating FFA in the CAF-TR, which was associated with the white adipose metabolism improvement by AET [12]. Furthermore, considering that the skeletal muscle is the main tissue to oxide fatty acid and that AET induces important adaptations in the skeletal muscle to increase oxidative metabolism, it is possible that AET promotes a cooperation between adipose tissue and skeletal muscle to improve the organism ability to deal with excess lipids, which is essential to reduce the availability of fatty acids to the kidneys and, therefore, the accumulation induced by the cafeteria diet. It is important to emphasize that we cannot exclude the reduction of renal lipogenic pathways for the prevention of lipid accumulation in our experimental model, since they were not investigated in the present study. Moreover, evidence supporting the metabolic actions of AMPK independent of PGC1-α protein downstream is clear in the literature and must be considered. According to Hardie [9], at least 60 AMPK target proteins have been described. For example, AMPK can activate cellular catabolism resulting in improved mitochondrial performance and increased lipid oxidation mediated by increased expression of CD36 (differentiation cluster 36) and consequently, increased fatty acid absorption; increased expression of mitochondrial fission-related proteins (MFF-mitochondrial fission factor), and autophagy (ULK ½-Unc-51 autophagy activating kinase). Conversely, AMPK can inhibit lipogenesis, for example, by inhibiting the HMGR (HMG-CoA reductase) protein responsible for the cholesterol synthesis or GPAT (glycerol phosphate acetyl transferase), which is responsible triacylglycerol phospholipid synthesis [9]. All these AMPK actions could culminate with lower lipid deposition in the kidney, however future studies are necessary to understand this issue.
this process, especially when compared to the number of studies about the lipid deleterious effects in other organs such heart, liver and skeletal muscle [35]. In our study, the mice fed a cafeteria diet had accumulated lipids in the kidney, but AET was able to prevent this lipid excess. Zheng et al. [37] have showed that mice fed a high-fat diet accumulate renal lipids, as well as Bobulescu et al. [3] showed in humans an association between body mass index and kidney lipid deposition. The accumulation of lipid caused subtle renal morphological changes. There was a reduction in the Bowman's space in the CAF-SED group compared to CHOW-SED group. It is known that renal morphological changes can happen during the progression of CKD to the end stage, including microvascular damages with hyalinosis in the preglomerular vessel walls and thickening of the intima, and reduplication of the internal elastic lamina of the arcuate and interlobar arteries. These changes can lead to glomerular injury, glomerulosclerosis, irregular tubular atrophy and fibrosis. Usually, hyalilized glomeruli are smaller than the normal due to the loss of cellular elements [24,28]. Although some studies have shown that a hyperlipidic or hypercaloric diet may cause renal morphological alterations [4,10], these results are not well established in the literature. Dissard et al. [5] showed that animals fed a high-fat and high fructose diet did not present mesangial matrix expansion or fibrosis but only some minor podocyte effacement. The differences showed by the authors may be due to the characteristics of the studied animal model, the diet (type and duration) or another specificity of the experimental protocol. Considering that lipid deposition is determined by the balance between oxidation and storage, we decided to investigate the pathway of AMPK protein, which plays a central role in the modulation of oxidative metabolism. There was an increase in the p-AMPK levels in the kidney of CAF-TR group. The p-AMPK is the active form of AMPK, that can lead to an activation of PGC-1α, which is responsible to promote mitochondrial biogenesis and increase the activity of oxidative enzymes such β-HAD [19]. SIRT-1 exerts similar AMPK actions, activating proteins involved in energy metabolism such as PGC-1α and is also capable of inhibiting the SREBP-1 transcription factor, which is responsible for the synthesis of fatty acids [22]. The p-AMPK can also improve the availability of fatty acids for oxidation in mitochondria by phosphorylating the ACC protein (inactive form). In the present study, the increased p-AMPK did not have repercussions on changes in protein expression of SIRT-1, PGC-1α, ACC and p-ACC in the CAF-TR group. Furthermore, β-HAD activity increased in CAF-SED and CAF-TR suggesting that this enzyme response was more dependent on cafeteria diet than on AET and p-AMPK. Previous studies that used pharmacological strategies have described the role of AMPK, SIRT-1 and PGC-1α activation to prevent metabolic disorders and renal diseases associated with lipid
5. Conclusion Collectively, our study provides evidence that AET prevented kidney lipid accumulation induced by cafeteria diet and this response was not associated with changes in the renal metabolic activity that favors lipid oxidation. Future studies are necessary to investigate the fatty acid flow to the kidney and the role of renal lipogenesis, which can contribute to understand the effect of AET to prevent lipid accumulation in the kidney. 145
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Conflict of interest statement
Sci. Med. Sci. 56 (5) (2001) M298–M303. [17] C.P. Kovesdy, K. Matsushita, et al., Serum potassium and adverse outcomes across the range of kidney function: a CKD Prognosis Consortium meta-analysis, Eur. Heart J. 39 (17) (2018) 1535–1542. [18] S. Kume, T. Uzu, et al., Role of altered renal lipid metabolism in the development of renal injury induced by a high-fat diet, J. Am. Soc. Nephrol. 18 (10) (2007) 2715–2723. [19] C.T. Lim, B. Kola, et al., AMPK as a mediator of hormonal signalling, J. Mol. Endocrinol. 44 (2) (2010) 87–97. [20] J.P. Little, A. Safdar, et al., A practical model of low-volume high-intensity interval training induces mitochondrial biogenesis in human skeletal muscle: potential mechanisms, J. Physiol. 588 (Pt 6) (2010) 1011–1022. [21] K.M. Madden, Evidence for the benefit of exercise therapy in patients with type 2 diabetes, Diabetes Metab. Syndr. Obes. 6 (2013) 233–239. [22] B.B. Madison, Srebp2: a master regulator of sterol and fatty acid synthesis, J. Lipid Res. 57 (3) (2016) 333–335. [23] S.K. Malin, J.P. Kirwan, Fasting hyperglycaemia blunts the reversal of impaired glucose tolerance after exercise training in obese older adults, Diabetes Obes. Metab. 14 (9) (2012) 835–841. [24] R. Marin, M. Gorostidi, et al., Systemic and glomerular hypertension and progression of chronic renal disease: the dilemma of nephrosclerosis, Kidney Int. Suppl. (99) (2005) S52–S56. [25] T.H. Marwick, M.D. Hordern, et al., Exercise training for type 2 diabetes mellitus: impact on cardiovascular risk: a scientific statement from the American Heart Association, Circulation 119 (25) (2009) 3244–3262. [26] M. Neto, G. Albuquerque, et al., Equações de predição da aptidão cardiorrespiratória sem testes de exercício e sua aplicabilidade em estudos epidemiológicos: revisão descritiva e análise dos estudos, Rev. Bras. Med. Esporte 9 (5) (2003) 304–314. [27] K. Ohshima, M. Mogi, et al., Possible role of angiotensin-converting enzyme 2 and activation of angiotensin II type 2 receptor by angiotensin-(1-7) in improvement of vascular remodeling by angiotensin II type 1 receptor blockade, Hypertension 63 (3) (2014) e53–e59. [28] H. Ono, Y. Ono, Nephrosclerosis and hypertension, Med. Clin. North Am. 81 (6) (1997) 1273–1288. [29] B.F. Palmer, D.J. Clegg, Diagnosis and treatment of hyperkalemia, Cleve. Clin. J. Med. 84 (12) (2017) 934–942. [30] E. Premaratne, S. Verma, et al., The impact of hyperfiltration on the diabetic kidney, Diabete Metab. 41 (1) (2015) 5–17. [31] K.A. Silva, S. Luiz Rda, et al., Previous exercise training has a beneficial effect on renal and cardiovascular function in a model of diabetes, PLoS One 7 (11) (2012) e48826. [32] V. Soetikno, F.R. Sari, et al., Curcumin decreases renal triglyceride accumulation through AMPK-SREBP signaling pathway in streptozotocin-induced type 1 diabetic rats, J. Nutr. Biochem. 24 (5) (2013) 796–802. [33] A. Sriwijitkamol, D.K. Coletta, et al., Effect of acute exercise on AMPK signaling in skeletal muscle of subjects with type 2 diabetes: a time-course and dose-response study, Diabetes 56 (3) (2007) 836–848. [34] H.H. Szeto, S. Liu, et al., Protection of mitochondria prevents high-fat diet-induced glomerulopathy and proximal tubular injury, Kidney Int. 90 (5) (2016) 997–1011. [35] N. Tsuboi, Y. Okabayashi, et al., The renal pathology of obesity, Kidney Int. Rep. 2 (2) (2017) 251–260. [36] S.E. Wicks, T.T. Nguyen, et al., Diet-induced obesity and kidney disease - in search of a susceptible mouse model, Biochimie 124 (2016) 65–73. [37] Y. Zheng, L. Tang, et al., Anti-inflammatory effects of ang-(1-7) in ameliorating HFD-induced renal injury through LDLr-SREBP2-SCAP pathway, PLoS One 10 (8) (2015) e0136187.
The authors declare that there are no conflicts of interest. Acknowledgments We thank Prof. José E. Krieger for providing laboratory support and Nancy Pirro for the careful review of the English in this manuscript. This study was supported by grants from the São Paulo Research Foundation (FAPESP #2015/04948-4) to F.S. Evangelista and from CAPES to C.R. Muller. References [1] D. Agarwal, C.M. Elks, et al., Chronic exercise preserves renal structure and hemodynamics in spontaneously hypertensive rats, Antioxid. Redox Signal. 16 (2) (2012) 139–152. [2] A. Bass, D. Brdiczka, et al., Metabolic differentiation of distinct muscle types at the level of enzymatic organization, Eur. J. Biochem. 10 (2) (1969) 198–206. [3] I.A. Bobulescu, Y. Lotan, et al., Triglycerides in the human kidney cortex: relationship with body size, PLoS One 9 (8) (2014) e101285. [4] U.G. de Castro, R.A. dos Santos, et al., Age-dependent effect of high-fructose and high-fat diets on lipid metabolism and lipid accumulation in liver and kidney of rats, Lipids Health Dis. 12 (2013) 136. [5] R. Dissard, J. Klein, et al., Long term metabolic syndrome induced by a high fat high fructose diet leads to minimal renal injury in C57BL/6 mice, PLoS One 8 (10) (2013) e76703. [6] R. Eguchi, N.C. Cheik, et al., Efeitos do exercício crônico sobre a concentração circulante da leptina e grelina em ratos com obesidade induzida por dieta, Rev. Bras. Med. Esporte 14 (3) (2008). [7] J.C. Ferreira, N.P. Rolim, et al., Maximal lactate steady state in running mice: effect of exercise training, Clin. Exp. Pharmacol. Physiol. 34 (8) (2007) 760–765. [8] K.S. Gollisch, J. Brandauer, et al., Effects of exercise training on subcutaneous and visceral adipose tissue in normal- and high-fat diet-fed rats, Am. J. Physiol. Endocrinol. Metab. 297 (2) (2009) E495–E504. [9] D.G. Hardie, Keeping the home fires burning: AMP-activated protein kinase, J. R. Soc. Interface 15 (138) (2018). [10] J.R. Henegar, S.A. Bigler, et al., Functional and structural changes in the kidney in the early stages of obesity, J. Am. Soc. Nephrol. 12 (6) (2001) 1211–1217. [11] T.S. Higa, A.V. Spinola, et al., Comparison between cafeteria and high-fat diets in the induction of metabolic dysfunction in mice, Int. J. Physiol. Pathophysiol. Pharmacol. 6 (1) (2014) 47–54. [12] T.S. Higa, A.V. Spinola, et al., Remodeling of white adipose tissue metabolism by physical training prevents insulin resistance, Life Sci. 103 (1) (2014) 41–48. [13] M.A. Hoydal, U. Wisloff, et al., Running speed and maximal oxygen uptake in rats and mice: practical implications for exercise training, Eur. J. Cardiovasc. Prev. Rehabil. 14 (6) (2007) 753–760. [14] J.H. Ix, K. Sharma, Mechanisms linking obesity, chronic kidney disease, and fatty liver disease: the roles of fetuin-A, adiponectin, and AMPK, J. Am. Soc. Nephrol. 21 (3) (2010) 406–412. [15] A. Izquierdo-Lahuerta, C. Martinez-Garcia, et al., Lipotoxicity as a trigger factor of renal disease, J. Nephrol. 29 (5) (2016) 603–610. [16] G.A. Kelley, K. Sharpe Kelley, Aerobic exercise and resting blood pressure in older adults: a meta-analytic review of randomized controlled trials, J. Gerontol. A Biol.
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Aerobic exercise training prevents kidney lipid deposition in mice fed a cafeteria diet C.R. Mullera, A.L.V. Américoa, P. Fiorinob, F.S. Evangelistac,
T
⁎
a
Experimental Pathophysiology Dept., Faculty of Medicine, University of São Paulo, Brazil Health and Biological Science Center (CCBS), Mackenzie University, Sao Paulo, Brazil c School of Arts, Science and Humanities, University of Sao Paulo, Brazil b
A R T I C LE I N FO
A B S T R A C T
Keywords: Kidney lipid deposition Aerobic exercise training Oxidative metabolism
Aim: The objective of this study was to investigate the potential of aerobic exercise training (AET) to prevent kidney lipid accumulation and the contribution of renal metabolism to mediate this response. Main methods: Male C57BL/6J mice were assigned into groups CHOW-SED (chow diet, sedentary; n = 13), CHOW-TR (chow diet, trained; n = 13), CAF-SED (cafeteria diet, sedentary; n = 13) and CAF-TR (cafeteria diet, trained; n = 13). AET consisted in running sessions of 60 min at 60% of maximal speed conducted five days per week for eight weeks. Key findings: AET prevented weight gain in both trained groups. Food intake was not different among groups, however water intake, urine output, urine potassium and osmolarity were reduced in CAF-SED and CAF-TR groups. Kidney lipid deposition increased in CAF-SED (4.12 ± 0.5%/area) compared with CHOW-SED (1.7 ± 0.54%/area), and the AET prevented this increase in the CAF-TR group (2.1 ± 0.5%/area). The Bowman's capsule area decreased in CAF-SED and CAF-TR groups while the Bowman' space reduced in CAF-SED compared to CHOW-SED group, which was prevented by AET in the CAF-TF group. We observed a 27% increase in the p-AMPK expression in CAF-TR compared to CHOW-SED group without differences in the SIRT-1, PGC1-α, ACC and p-ACC. β-HAD activity increased in CAF-SED (43.9 ± 4.57 nmol·min−1·ug−1) and CAF-TR (44.7 ± 2.6 nmol·min−1·ug−1) groups compared to CHOW-SED (35.1 ± 2.9 nmol·min−1·ug−1) e CHOW-TR (36.6 ± 2.7 nmol·min−1·ug−1). Significance: AET prevented kidney lipid accumulation induced by cafeteria diet and this response was not associated with changes in the renal metabolic activity that favors lipid oxidation.
1. Introduction Changes in lifestyle and dietary habits have led to several health problems, such as obesity, dyslipidemia, Diabetes Mellitus type 2 (DM2) and hypertension. The excess of lipids has been indicated as the main factor triggering pathologies associated with this lifestyle, because they accumulate in non-adipose tissue leading to lipotoxicity. In the kidney, lipid accumulation can cause proteinuria, urinary podocyte loss, insulin resistance, oxidative stress, fibrosis, apoptosis and hypertrophy culminating in chronic kidney disease (CKD) [15]. Thus, excessive lipid deposition is a common mechanism involved in the CKD development in most of pathologies such as obesity, DM2 and metabolic syndrome [3]. Studies have been conducted to better understand the effects of lifestyle change and the ectopically accumulated lipids. In a previous report, it was demonstrated that a high-fat diet induced weigh gain,
increased adiposity and renal lipid accumulation, which were associated with inflammation, glomerulosclerosis, mesangial matrix expansion and mitochondrial damage [34]. Our group previously demonstrated that a cafeteria diet increased body weight gain, adiposity, insulin resistance and total cholesterol in mice. Also, we observed that a cafeteria diet increased white adipose lipolysis and circulating free fatty acid (FFA), which may lead to lipotoxicity in peripheral tissues [12]. Thus, mice fed a cafeteria diet can be a good model to study ectopically accumulated lipids in the kidney. Impairments in the regulation of lipid metabolic activity such as lipogenesis (triacylglycerol biosynthesis and accumulation in the intracellular lipid droplet), lipolysis (triacylglycerol hydrolysis) and fatty acid oxidation may lead to an increase in the renal lipid accumulation. Kume et al. [18] showed that the renal lipid accumulation in a high-fat diet model occurs due to an altered balance between lipolysis and
⁎ Corresponding author at: School of Arts, Science and Humanities, Sao Paulo University, Av. Arlindo Bettio, 1000, Ermelino Mattarazzo, São Paulo, SP CEP 03828000, Brazil. E-mail address: [email protected] (F.S. Evangelista).
https://doi.org/10.1016/j.lfs.2018.09.017 Received 13 April 2018; Received in revised form 29 August 2018; Accepted 7 September 2018 Available online 12 September 2018 0024-3205/ © 2018 Elsevier Inc. All rights reserved.
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2.3. Running capacity test
lipogenesis, and this modification in renal lipid metabolism can be associated, at least in part, with decrease in AMP-activated protein kinase (AMPK) and increase in acetyl-CoA carboxylase (ACC) activity. Both proteins are crucial for lipid metabolism because while AMPK protein induces pathways that increase ATP (adenosine triphosphate) synthesis by providing more glucose and fatty acid for oxidation [19], the ACC modulates oxidative metabolism by inhibiting the activity of carnitine palmitoyl transferase 1 (CPT-1), an important fatty acid carrier protein in the mitochondria [14]. The aerobic exercise training (AET) has been used to treat and prevent metabolic damage associated with obesity and DM2 [12,21] because it promotes reduction in body weight and white adipose mass [12,23,25], and increases free fatty acid (FFA) oxidation in the skeletal muscle associated with better mitochondrial function [16]. Our previous study demonstrated that AET was able to prevent obesity, glucose intolerance and insulin resistance in mice fed a cafeteria diet by improving white adipose metabolism that favors fat oxidation (higher AMPK and lower ACC expression protein) instead of fat storage [12]. These data show that AET is a good non-pharmacological tool to increase lipid oxidation, however, this effect still needs to be investigated in the kidney. Evidence in the literature has confirmed that AET provides improvement in renal function and morphology. In a previous study, Silva et al. [31] showed that AET decreased proteinuria in animals with DM induced by streptozotocin. Agarwal et al. [1] have shown that AET has beneficial morphofunctional effects on the kidney. Considering the role of lipid accumulation in the development and progression of kidney damage, and the effect of AET to improve lipid metabolism by increasing fatty acid oxidation, the objective of this study was to investigate the potential of AET to prevent kidney lipid accumulation and the contribution of renal metabolism to mediate this response. Our hypothesis is that the AET prevents kidney lipid accumulation induced by a cafeteria diet and this response is associated with the improvement of renal metabolic activity that favors lipid oxidation.
Running capacity was assessed before, in the fourth and eighth weeks of AET using a progressive test with a 0% incline on a treadmill as described by Ferreira et al. [7]. The initial speed was 0.4 km/h and the speed was increased by 0.2 km/h every 3 min until exhaustion of the animal, which was characterized by the impossibility of maintaining the standard rate. The test variable was quantified as maximum time to exhaustion (min). 2.4. Body weight control Body weight was measured at the beginning and at the end of the AET protocol using a digital balance (Gehaka, Model BK4001, Brazil). Body weight gain was calculated as the difference between beginning body weight and final body weight. 2.5. Metabolic cages In the 6th week of the protocol, the animals were housed individually in metabolic cages (Tecniplast, Buguggiate, VA, Italy) for 48 h. The first 24-h were used to adapt the mice to the environment and the following 24-h were used to collect urine. Food consumption and water intake were also monitored. The water balance was calculated through the water to urine ratio. Urine samples collected during 24-h period were used to determine urine output, creatinine, sodium, potassium chloride, protein excretion and osmolarity. Creatinine and total protein were quantified in spectrophotometer using colorimetric method (LABTEST Biochemical Kit, Brazil). In addition, electrolytes (Na +, K+ and Cl−) and osmolarity were measured in a gas analyzer (ABL800 FLEX, Radiometer Copenhagen) and osmometer (Vapor Pressure Osmometer 5520 USA), respectively. The creatinine clearance for assessing the Glomerular Filtration Rate (GFR) was calculated using the formula [(Urine (Creatinine) × Urine Vol) / Serum (Creatinine)]. The calculation of the water balance was performed using the formula Water intake / Urine output (mL/24-h).
2. Materials and methods
2.6. Tissue and blood collection
2.1. Animals
Forty-eight hours after the end of the last training session, the animals were killed with an intraperitoneal injection of sodium pentobarbital (4 mg/100 g body weight). Kidneys were harvested, weighed and stored at −80 °C (right kidney) or fixed in 10% neutral-buffered formalin (left kidney) for subsequent histological analyses. The vena cava blood was collected and centrifuged at 4 °C (10.000g for 10 min) and serum was sent for creatinine analysis as described above.
Eight-week-old male C57BL/6J mice were assigned in groups CHOW-SED (chow diet, sedentary; n = 13), CHOW-TR (chow diet, trained; n = 13), CAF-SED (cafeteria diet, sedentary; n = 13) and CAFTR (cafeteria diet, trained; n = 13). Animals were maintained under the same housing conditions (12-h light/12-h dark cycle, temperature 22 ± 2 °C) with free access to tap water and food ad libitum. All procedures were performed in accordance with the guidelines of the Brazilian College for Animal Experimentation and were approved by the Ethics Committee of the Faculty of Medicine of University of Sao Paulo (#18/2014).
2.7. Histological analysis Glomerular injury was measured in paraffin sections of kidney (5 μm) stained with Picrossirius Red (Sigma). Digital images from thirty glomeruli per animal were obtained using a light microscope (Leica) at 200× magnification. After digitalization, Bowman's capsule area, glomerular tuft area, Bowman's space area and glomerular diameter were traced and calculated using a computerized morphometric analysis system (Image Pro-Plus 4.1; Media Cybernetics, Silver Spring, MD, USA). Lipid content was measured using quantitative histochemistry of Oil Red O (Sigma-Aldrich) staining of kidney. Tissue sections (thickness 10 μm) obtained in a cryostat were examined by light microscopy at 200× magnification and analyzed by a computerized morphometric analysis system (Image Pro-Plus 4.1; Media Cybernetics, Silver Spring, MD, USA). The slides were counterstained with hematoxylin to visualize the nuclei. Lipid accumulation was determined in 20 images per animal based on the percentage of area occupied by lipid droplets. Histological analyses were conducted by CR Muller, blinded to mice
2.2. Diets and aerobic exercise training The standard chow diet contained 4% of kilocalories from fat, 55% from carbohydrate and 22% from proteins (Nuvilab®, Paraná, Brazil). The cafeteria diet contained 18.8% of kilocalories from fat, 55% from carbohydrate and 14.8% from proteins [11]. Diet and AET were started simultaneously. CHOW-TR and CAF-TR animals were submitted to AET as described by Ferreira et al. [7]. Animals were trained during the dark cycle (i.e., during their active period) on a motorized treadmill for 1 h/ day at 60% of maximal velocity achieved in the running capacity test, five times per week for eight weeks. AET intensity was progressively increased and adjusted after the running capacity test done in the fourth week. To minimize the influence of the treadmill stress, sedentary mice were placed on the treadmill for 5 min twice weekly at 0.3 km/h during the experimental protocol. 141
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identities.
Table 1 Metabolic parameter evaluated in 24-h. Data presented as mean ± SE. CHOW-SED (n = 13); CHOW-TR (n = 13); CAF-SED (n = 13); CAF-TR (n = 13).
2.8. Western blot analysis Samples of frozen right kidney were homogenized in an ice-cold lysis buffer (pH: 7.2): 0.1 M sodium phosphate monobasic, 0.34 M sucrose, 0.3 M sodium chloride, protease inhibitor cocktail tablets mini complete EDTA-free (Roche®, Germany). Samples were centrifuged for 25 min at 14.000 rpm at 4 °C. Protein concentrations of the homogenates were measured by the BCA method with a protein assay kit (PIERCE Biotechnology®, Rockford, IL, USA) using bovine serum albumin as a standard. Aliquots (30 μg) of protein were subjected to SDSPAGE. The membranes were incubated overnight at 4 °C with the following primary antibodies: p-AMPK (Thr172) (1:1000), AMPK (1:1000), p-ACC (Ser79) (1:1000), ACC (1:1000) (Cell Signaling®, Beverly, MA), SIRT-1(1:1000), PGC1-α (1:1000) and Beta-Actin (1:1000) (Abcam®, Cambridge, USA). The signal on the membrane was detected via the peroxidase reaction in the ECL solution using an Image Quant LAS 4000 mini system (GE Healthcare Life Sciences®). Band intensities were quantified based on optical densitometry measurements using the Image J program (version 1.43 for Windows).
Food intake (g/24 h) Initial body weight (g) Final body weight (g) Weight gain (g) Water intake (mL/ 24 h) Urine output (mL/ 24 h) Water balance (mL/ 24 h) ⁎ #
CHOW-SED
CHOW-TR
CAF-SED
CAF-TR
4.2 ± 0.3 22.2 ± 0.9 29.2 ± 0.7 7.0 ± 0.8 5.4 ± 0.5
4.9 ± 0.3 22.3 ± 0.8 27.3 ± 0.8# 5.0 ± 0.4# 4.8 ± 0.3
4.1 ± 0.3 22.4 ± 0.7 28.8 ± 0.3 6.4 ± 0.6 3.5 ± 0.3⁎
4.3 ± 0.3 21.8 ± 0.8 26.4 ± 0.4# 4.6 ± 0.7# 4.2 ± 0.4⁎
1.6 ± 0.2
1.9 ± 0.2
1.2 ± 0.2⁎
1.2 ± 0.2⁎
4.1 ± 0.8
3.2 ± 0.5
4.5 ± 1.3
4.2 ± 0.7
p ≤ 0.05 vs. CHOW-SED and CHOW-TR. ≤0.05 vs. CHOW-SED and CAF-SED.
groups (CHOW-SED = 19.4 ± 1.7 min; CHOW-TR = 21.4 ± 0.7 min; CAF-SED = 21.8 ± 1.5 min; CAF-TR = 20.8 ± 0.8 min). However, after AET, the running capacity was increased in both trained groups (CHOW-TR = 31.3 ± 1.8 min and CAF-TR = 31.2 ± 3.1 min) compared with sedentary groups (CHOW-SED = 21.5 ± 0.7 min and CAFSED = 19.9 ± 1.0 min). These results confirm the aerobic adaptation induced by AET.
2.9. β-HAD activity For the determination β-HAD (β-hydroxyacyl-CoA dehydrogenase), approximately 0.75 g of the kidney was suspended in 750 μL of extraction buffer containing Tris-base (50 mM) and EDTA (1 mM), pH = 7.4. The material was homogenized in Precellys® 24 (Bertin Corp, USA) for 2 cycles of 20 s. The homogenate was centrifuged at 3000 rpm for 15 min at 4 °C in a 5417 C/R (Eppendorf) centrifuge. β-HAD activity was measured by the determination of the amount of oxidized NADPH considering the NADPH molar extinction coefficient. The buffer used in the assay contained triethanolamine (0.1 M), EDTA (5 mM) and NADH (0.45 mM) pH = 7.0. The reaction was started with the addition of 15 μL AcetoAcetil CoA (0.1 mM) to the enzyme extract and followed for 10 min (37 °C). Absorbance was monitored at 340 nm [2]. The proteins were quantified by the BCA™ method (PIERCE Biotechnology). The results were expressed as μg/μl of protein present in the extract.
3.2. Metabolic and urinary parameters There were no changes in the initial body weight, however the CHOW-TR and CAF-TR had smaller final weight and weight gain compared with CHOW-SED and CAF-SED (Table 1). No difference in the food intake was present among groups. On the other hand, the water intake was decreased in both cafeteria diet groups compared to chow diet groups. Similarly, the urine output was lower in both cafeteria diet groups compared with chow diet groups (CHOW-SED and CHOW-TR). Water balance was not changed (Table 1). Although non-significant, the GFR was 88% increased in CAF-SED group (0.15 ± 0.03 mL/min) compared with CHOW-SED (0.08 ± 0.022 mL/min) CHOW-TR (0.10 ± 0.02 mL/min) and CAFTR (0.11 ± 0.02 mL/min). The urinary excretion of protein, sodium and chloride were not different among groups, however the potassium excretion and the osmolarity were decreased in cafeteria groups (CAFSED and CAF-TR) compared with CHOW-SED and CHOW-TR groups (Table 2).
2.10. Statistical analyses All values are expressed as mean ± SE. The results were compared among groups using two-way analyses of variance (ANOVA). The Bonferroni post hoc test was used to determine differences between means when a significant change was observed using ANOVA. A p value equal to or < 0.05 was considered to be statistically significant (GraphPad Prism®, v.6.0).
3.3. Lipid deposition and glomerular morphometry The renal lipid deposition was significantly 2.5-fold higher in the CAF-SED (4.1 ± 0.5% area) than CHOW-SED group (1.7 ± 0.5% area) and the AET prevented this increase in the CAF-TR (2.1 ± 0.5% area). No difference was observed between CHOW-SED and CHOW-TR (2.2 ± 0.2% area) groups (Fig. 1A). Fig. 1B represents kidneys sections stained with Oil Red O.
3. Results 3.1. Aerobic performance Before AET, no differences in running capacity were present among
Table 2 GFR and urinary parameters. Data presented as mean ± SE. GFR = Glomerular Filtration Rate. CHOW-SED (n = 9); CHOW-TR (n = 8); CAF-SED (n = 11); CAF-TR (n = 12).
Protein (mg/24 h) Potassium (mMol/24 h) Sodium (mMol/24 h) Chloride (mMol/24 h) Osmolarity (mmol/kg) GFR (mL/min) ⁎
CHOW-SED
CHOW-TR
CAF-SED
CAF-TR
0.0207 ± 0.0018 0.372 ± 0.045 0.259 ± 0.032 0.920 ± 0.144 2564.3 ± 141.2 0.08 ± 0.022
0.0155 ± 0.0022 0.364 ± 0.051 0.251 ± 0.043 0.834 ± 0.136 2515.0 ± 175.3 0,10 ± 0.02
0.0184 ± 0.0023 0.227 ± 0.047⁎ 0.222 ± 0.036 0.724 ± 0.141 2038.1 ± 162.4⁎ 0.15 ± 0.03
0.0140 ± 0.0015 0.198 ± 0.022⁎ 0.185 ± 0.031 0.766 ± 0.137 1931.8 ± 176.9⁎ 0.11 ± 0.02
p ≤ 0.05 vs. CHOW-SED and CHOW-TR. 142
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B
L ip id D e p o s itio m ( % /A r e a )
A
*
5
SED TR
4
CHOW-SED
3
CHOW-TR
2
1
0 CHOW
C AF
CAF-SED
CAF-TR
Fig. 1. Kidney lipid deposition. CHOW-SED (n = 4), CHOW-TR (n = 5), CAF-SED (n = 6) and CAF-TR (n = 5). Error bars indicate SE (A). Arrows shows lipids droplets (B). Data are shown as the percentage of marked area. p ≤ 0.05, *CAF-SED vs. CHOW-SED, CHOW-TR and CAF-TR.
and maximum oxygen uptake (VO2 max) in rats and mice, revealing that higher physical capacity is followed by better aerobic capacity. This observation is relevant because higher aerobic capacity is associated with reduced cardiovascular and metabolic disease risk [26]. On the other hand, low levels of cardiorespiratory fitness (VO2 max) have been associated with a higher risk of morbidity and mortality due to chronic-degenerative diseases, including coronary artery disease, systemic arterial hypertension, DM2 and some types of cancer [26]. The cafeteria diet has been used for the development of obesity and DM2 experimental models, contributing to the investigation of mechanisms involved in the development and progression of these diseases. In our study, the cafeteria diet did not induce greater body weight gain in the CAF-SED group compared to the CHOW-SED, however, for both trained groups (CHOW-TR and CAF-TR) AET was efficient in reducing body weight gain. It is important to note that, although the CAF-SED group did not present a greater body weight gain, the weight of fat depot were higher compared with the other groups (data not showed), which is typical in rodent obesity [27]. We observed that dietary intake was not different among the groups. Although it is known that AET can induce increased food intake, our trained groups maintained the same eating pattern as previously demonstrated by Higa et al. [12]. These data corroborate the Eguchi et al. [6] study, which found no difference in the food consumption in swimming trained groups fed a cafeteria or control diet. Gollisch et al. [8] demonstrated an increase in the food consumption of trained animals, independent of the diet (normocaloric or hyperlipidic). These contradictory data can be explained by methodological differences between the studies, such as type of diet, AET protocol, duration and intensity. Considering that body weight control occurs through the balance between caloric intake and energy expenditure, it is possible that the lower body weight gain observed in the CHOW-TR and CAF-TR groups is due to the increase in energy expenditure promoted by AET, since in the present study, trained groups did not present an increase in caloric intake. There was a reduction in the water intake and urine output in both groups fed a cafeteria diet without changes in the water balance. In the same direction, both cafeteria groups had decreased potassium excretion and osmolarity. Lower renal potassium excretion can be associated with reduced sodium delivery to the distal nephron, decreased mineralocorticoid level or activity, or abnormalities in the cortical collecting duct [29]. Unfortunately, we did not measure electrolytes clearance and activity, neither the level of aldosterone to understand these changes. Although non-significant, the CAF-SED group showed high GFR
Table 3 Glomerular morphological parameters. Data presented as mean ± SE. p ≤ 0.05.
Bowman's capsule area (μm2) Bowman's space area (μm2) Glomerular tuft area (μm2) Glomerular diameter (μm)
CHOW-SED
CHOW-TR
CAF-SED
CAF-TR
470 ± 16 154 ± 15 316 ± 9 21 ± 0.4
442. ± 17 126. ± 11 312 ± 12 23. ± 1.8
405 ± 21⁎ 104 ± 5# 298 ± 18 21 ± 0.6
414. ± 13⁎ 117 ± 5 297 ± 10 21 ± 0.4
⁎ CAF-SED (n = 7) and CAF-TR (n = 9) vs. CHOW-SED (n = 9) and CHOWTR (n = 9). p ≤ 0.05. # CAF-SED vs. CHOW-SED.
The animals fed a cafeteria diet (CAF-SED and CAF-TR) have reduced the Bowman's capsule area. There were no differences in the glomerular tuft area and glomerular diameter among groups. In the other hand, the Bowman's space was reduced in the CAF-SED compared with CHOW-SED and the AET prevented this reduction in the CAF-TR group (Table 3). 3.4. Kidney lipid metabolism As shown in the Fig. 2A, the expression of p-AMPK was 27% increased in the CAF-TR (1.27 ± 0.007% vs. CHOW-SED) group compared with CHOW-SED group (1.0 ± 0.008% vs. CHOW-SED). There were no changes in the expression levels of AMPK (Fig. 2B), p-ACC (Fig. 2C), ACC (Fig. 2D), PGC1-α (Fig. 2E), SIRT-1 (Fig. 2F). The β-HAD enzyme activity was increased in CAF-SED and CAF-TR group compared with CHOW-SED and CHOW-TR (Fig. 3). 4. Discussion In the present study we investigated if AET could prevent kidney lipid accumulation and if adaptations in renal metabolic activity could explain this response in physically trained mice fed a cafeteria diet. Our results confirmed the efficiency of AET to prevent ectopically accumulated lipids in the kidney and despite the increase in p-AMPK protein expression, the lower lipid content in the CAF-TR group cannot be associated with lipid oxidation metabolism improvement. We showed through running capacity test that the animals responded adequately to the AET, since both trained groups presented better performance compared to sedentary groups. This response confirms the AET efficacy for aerobic adaptations and corroborate previous studies conducted by Ferreira et al. [7] and Higa et al. [12]. Hoydal et al. [13] demonstrated a direct relationship between running velocity 143
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Fig. 2. Expression of AMPK (A), p-AMPK Thr172 (B), ACC (C), p-ACC Ser79 (D), PGC1-α (E) and SIRT-1(F) in the kidney. CHOW-SED (n = 9), CHOW-TR (n = 9), CAF-SED (n = 9) and CAF-TR (n = 9). Error bars indicate SE. Immunoblotting data are shown as the percentage of CHOWSED (set to 1). p ≤ 0.05, *CAF-TR vs. CHOW-SED, CHOW-TR.
and CKD development [17]. In fact, Henegar et al. [10] demonstrated that dog fed a high-fat diet had higher GFR [10]. However, Wicks et al. [36] demonstrated that C57BL/J6 fed a high-fat diet are resistant to develop kidney injury. Another study with C57BL/J6 showed that high fructose and high-fat diet developed minimal injury with mild hyperfiltration (1.5 fold higher than the control group) [5]. Studies have suggested the association between lipid accumulation and the development of kidney injury, however little is known about
which was prevented by AET in the CAF-TR group. Glomerular hyperfiltration has been considered an inadequate adaptive response presented by the kidneys, which can be observed early in the course of renal disease, and may cause irreversible damage to the nephron, leading to the development of more advanced stages of CKD [30]. Hyperfiltration occurs to meet the heightened metabolic demands of the increased body weight, and this alteration can increase intraglomerular pressure and increase the risk of kidney structure damage 144
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60
.u g
- 1
*
SED TR
40
-1
( n m o l.m in
- H A D a c t iv ity
)
*
20
0 CHOW
C AF
Fig. 3. β-HAD enzyme activity in the kidney. CHOW-SED (n = 5), CHOW-TR (n = 5), CAF-SED (n = 5) and CAF-TR (n = 5). Error bars indicate SE. p ≤ 0.05, * CAF-SED and CAF-TR vs. CHOW-SED, CHOW-TR.
accumulation [20,32,33]. However, here we have shown that AET prevents renal lipid accumulation independent of the activation of the AMPK pathway. This result suggests that AET may prevent lipid deposition in the CAF-TR animals with no changes in renal metabolic activity. In this case, a reduced flow of fatty acids to the kidneys could explain the lower content of lipids in the CAF-TR group. This may occur since we have previously demonstrated the reduction of circulating FFA in the CAF-TR, which was associated with the white adipose metabolism improvement by AET [12]. Furthermore, considering that the skeletal muscle is the main tissue to oxide fatty acid and that AET induces important adaptations in the skeletal muscle to increase oxidative metabolism, it is possible that AET promotes a cooperation between adipose tissue and skeletal muscle to improve the organism ability to deal with excess lipids, which is essential to reduce the availability of fatty acids to the kidneys and, therefore, the accumulation induced by the cafeteria diet. It is important to emphasize that we cannot exclude the reduction of renal lipogenic pathways for the prevention of lipid accumulation in our experimental model, since they were not investigated in the present study. Moreover, evidence supporting the metabolic actions of AMPK independent of PGC1-α protein downstream is clear in the literature and must be considered. According to Hardie [9], at least 60 AMPK target proteins have been described. For example, AMPK can activate cellular catabolism resulting in improved mitochondrial performance and increased lipid oxidation mediated by increased expression of CD36 (differentiation cluster 36) and consequently, increased fatty acid absorption; increased expression of mitochondrial fission-related proteins (MFF-mitochondrial fission factor), and autophagy (ULK ½-Unc-51 autophagy activating kinase). Conversely, AMPK can inhibit lipogenesis, for example, by inhibiting the HMGR (HMG-CoA reductase) protein responsible for the cholesterol synthesis or GPAT (glycerol phosphate acetyl transferase), which is responsible triacylglycerol phospholipid synthesis [9]. All these AMPK actions could culminate with lower lipid deposition in the kidney, however future studies are necessary to understand this issue.
this process, especially when compared to the number of studies about the lipid deleterious effects in other organs such heart, liver and skeletal muscle [35]. In our study, the mice fed a cafeteria diet had accumulated lipids in the kidney, but AET was able to prevent this lipid excess. Zheng et al. [37] have showed that mice fed a high-fat diet accumulate renal lipids, as well as Bobulescu et al. [3] showed in humans an association between body mass index and kidney lipid deposition. The accumulation of lipid caused subtle renal morphological changes. There was a reduction in the Bowman's space in the CAF-SED group compared to CHOW-SED group. It is known that renal morphological changes can happen during the progression of CKD to the end stage, including microvascular damages with hyalinosis in the preglomerular vessel walls and thickening of the intima, and reduplication of the internal elastic lamina of the arcuate and interlobar arteries. These changes can lead to glomerular injury, glomerulosclerosis, irregular tubular atrophy and fibrosis. Usually, hyalilized glomeruli are smaller than the normal due to the loss of cellular elements [24,28]. Although some studies have shown that a hyperlipidic or hypercaloric diet may cause renal morphological alterations [4,10], these results are not well established in the literature. Dissard et al. [5] showed that animals fed a high-fat and high fructose diet did not present mesangial matrix expansion or fibrosis but only some minor podocyte effacement. The differences showed by the authors may be due to the characteristics of the studied animal model, the diet (type and duration) or another specificity of the experimental protocol. Considering that lipid deposition is determined by the balance between oxidation and storage, we decided to investigate the pathway of AMPK protein, which plays a central role in the modulation of oxidative metabolism. There was an increase in the p-AMPK levels in the kidney of CAF-TR group. The p-AMPK is the active form of AMPK, that can lead to an activation of PGC-1α, which is responsible to promote mitochondrial biogenesis and increase the activity of oxidative enzymes such β-HAD [19]. SIRT-1 exerts similar AMPK actions, activating proteins involved in energy metabolism such as PGC-1α and is also capable of inhibiting the SREBP-1 transcription factor, which is responsible for the synthesis of fatty acids [22]. The p-AMPK can also improve the availability of fatty acids for oxidation in mitochondria by phosphorylating the ACC protein (inactive form). In the present study, the increased p-AMPK did not have repercussions on changes in protein expression of SIRT-1, PGC-1α, ACC and p-ACC in the CAF-TR group. Furthermore, β-HAD activity increased in CAF-SED and CAF-TR suggesting that this enzyme response was more dependent on cafeteria diet than on AET and p-AMPK. Previous studies that used pharmacological strategies have described the role of AMPK, SIRT-1 and PGC-1α activation to prevent metabolic disorders and renal diseases associated with lipid
5. Conclusion Collectively, our study provides evidence that AET prevented kidney lipid accumulation induced by cafeteria diet and this response was not associated with changes in the renal metabolic activity that favors lipid oxidation. Future studies are necessary to investigate the fatty acid flow to the kidney and the role of renal lipogenesis, which can contribute to understand the effect of AET to prevent lipid accumulation in the kidney. 145
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Conflict of interest statement
Sci. Med. Sci. 56 (5) (2001) M298–M303. [17] C.P. Kovesdy, K. Matsushita, et al., Serum potassium and adverse outcomes across the range of kidney function: a CKD Prognosis Consortium meta-analysis, Eur. Heart J. 39 (17) (2018) 1535–1542. [18] S. Kume, T. Uzu, et al., Role of altered renal lipid metabolism in the development of renal injury induced by a high-fat diet, J. Am. Soc. Nephrol. 18 (10) (2007) 2715–2723. [19] C.T. Lim, B. Kola, et al., AMPK as a mediator of hormonal signalling, J. Mol. Endocrinol. 44 (2) (2010) 87–97. [20] J.P. Little, A. Safdar, et al., A practical model of low-volume high-intensity interval training induces mitochondrial biogenesis in human skeletal muscle: potential mechanisms, J. Physiol. 588 (Pt 6) (2010) 1011–1022. [21] K.M. Madden, Evidence for the benefit of exercise therapy in patients with type 2 diabetes, Diabetes Metab. Syndr. Obes. 6 (2013) 233–239. [22] B.B. Madison, Srebp2: a master regulator of sterol and fatty acid synthesis, J. Lipid Res. 57 (3) (2016) 333–335. [23] S.K. Malin, J.P. Kirwan, Fasting hyperglycaemia blunts the reversal of impaired glucose tolerance after exercise training in obese older adults, Diabetes Obes. Metab. 14 (9) (2012) 835–841. [24] R. Marin, M. Gorostidi, et al., Systemic and glomerular hypertension and progression of chronic renal disease: the dilemma of nephrosclerosis, Kidney Int. Suppl. (99) (2005) S52–S56. [25] T.H. Marwick, M.D. Hordern, et al., Exercise training for type 2 diabetes mellitus: impact on cardiovascular risk: a scientific statement from the American Heart Association, Circulation 119 (25) (2009) 3244–3262. [26] M. Neto, G. Albuquerque, et al., Equações de predição da aptidão cardiorrespiratória sem testes de exercício e sua aplicabilidade em estudos epidemiológicos: revisão descritiva e análise dos estudos, Rev. Bras. Med. Esporte 9 (5) (2003) 304–314. [27] K. Ohshima, M. Mogi, et al., Possible role of angiotensin-converting enzyme 2 and activation of angiotensin II type 2 receptor by angiotensin-(1-7) in improvement of vascular remodeling by angiotensin II type 1 receptor blockade, Hypertension 63 (3) (2014) e53–e59. [28] H. Ono, Y. Ono, Nephrosclerosis and hypertension, Med. Clin. North Am. 81 (6) (1997) 1273–1288. [29] B.F. Palmer, D.J. Clegg, Diagnosis and treatment of hyperkalemia, Cleve. Clin. J. Med. 84 (12) (2017) 934–942. [30] E. Premaratne, S. Verma, et al., The impact of hyperfiltration on the diabetic kidney, Diabete Metab. 41 (1) (2015) 5–17. [31] K.A. Silva, S. Luiz Rda, et al., Previous exercise training has a beneficial effect on renal and cardiovascular function in a model of diabetes, PLoS One 7 (11) (2012) e48826. [32] V. Soetikno, F.R. Sari, et al., Curcumin decreases renal triglyceride accumulation through AMPK-SREBP signaling pathway in streptozotocin-induced type 1 diabetic rats, J. Nutr. Biochem. 24 (5) (2013) 796–802. [33] A. Sriwijitkamol, D.K. Coletta, et al., Effect of acute exercise on AMPK signaling in skeletal muscle of subjects with type 2 diabetes: a time-course and dose-response study, Diabetes 56 (3) (2007) 836–848. [34] H.H. Szeto, S. Liu, et al., Protection of mitochondria prevents high-fat diet-induced glomerulopathy and proximal tubular injury, Kidney Int. 90 (5) (2016) 997–1011. [35] N. Tsuboi, Y. Okabayashi, et al., The renal pathology of obesity, Kidney Int. Rep. 2 (2) (2017) 251–260. [36] S.E. Wicks, T.T. Nguyen, et al., Diet-induced obesity and kidney disease - in search of a susceptible mouse model, Biochimie 124 (2016) 65–73. [37] Y. Zheng, L. Tang, et al., Anti-inflammatory effects of ang-(1-7) in ameliorating HFD-induced renal injury through LDLr-SREBP2-SCAP pathway, PLoS One 10 (8) (2015) e0136187.
The authors declare that there are no conflicts of interest. Acknowledgments We thank Prof. José E. Krieger for providing laboratory support and Nancy Pirro for the careful review of the English in this manuscript. This study was supported by grants from the São Paulo Research Foundation (FAPESP #2015/04948-4) to F.S. Evangelista and from CAPES to C.R. Muller. References [1] D. Agarwal, C.M. Elks, et al., Chronic exercise preserves renal structure and hemodynamics in spontaneously hypertensive rats, Antioxid. Redox Signal. 16 (2) (2012) 139–152. [2] A. Bass, D. Brdiczka, et al., Metabolic differentiation of distinct muscle types at the level of enzymatic organization, Eur. J. Biochem. 10 (2) (1969) 198–206. [3] I.A. Bobulescu, Y. Lotan, et al., Triglycerides in the human kidney cortex: relationship with body size, PLoS One 9 (8) (2014) e101285. [4] U.G. de Castro, R.A. dos Santos, et al., Age-dependent effect of high-fructose and high-fat diets on lipid metabolism and lipid accumulation in liver and kidney of rats, Lipids Health Dis. 12 (2013) 136. [5] R. Dissard, J. Klein, et al., Long term metabolic syndrome induced by a high fat high fructose diet leads to minimal renal injury in C57BL/6 mice, PLoS One 8 (10) (2013) e76703. [6] R. Eguchi, N.C. Cheik, et al., Efeitos do exercício crônico sobre a concentração circulante da leptina e grelina em ratos com obesidade induzida por dieta, Rev. Bras. Med. Esporte 14 (3) (2008). [7] J.C. Ferreira, N.P. Rolim, et al., Maximal lactate steady state in running mice: effect of exercise training, Clin. Exp. Pharmacol. Physiol. 34 (8) (2007) 760–765. [8] K.S. Gollisch, J. Brandauer, et al., Effects of exercise training on subcutaneous and visceral adipose tissue in normal- and high-fat diet-fed rats, Am. J. Physiol. Endocrinol. Metab. 297 (2) (2009) E495–E504. [9] D.G. Hardie, Keeping the home fires burning: AMP-activated protein kinase, J. R. Soc. Interface 15 (138) (2018). [10] J.R. Henegar, S.A. Bigler, et al., Functional and structural changes in the kidney in the early stages of obesity, J. Am. Soc. Nephrol. 12 (6) (2001) 1211–1217. [11] T.S. Higa, A.V. Spinola, et al., Comparison between cafeteria and high-fat diets in the induction of metabolic dysfunction in mice, Int. J. Physiol. Pathophysiol. Pharmacol. 6 (1) (2014) 47–54. [12] T.S. Higa, A.V. Spinola, et al., Remodeling of white adipose tissue metabolism by physical training prevents insulin resistance, Life Sci. 103 (1) (2014) 41–48. [13] M.A. Hoydal, U. Wisloff, et al., Running speed and maximal oxygen uptake in rats and mice: practical implications for exercise training, Eur. J. Cardiovasc. Prev. Rehabil. 14 (6) (2007) 753–760. [14] J.H. Ix, K. Sharma, Mechanisms linking obesity, chronic kidney disease, and fatty liver disease: the roles of fetuin-A, adiponectin, and AMPK, J. Am. Soc. Nephrol. 21 (3) (2010) 406–412. [15] A. Izquierdo-Lahuerta, C. Martinez-Garcia, et al., Lipotoxicity as a trigger factor of renal disease, J. Nephrol. 29 (5) (2016) 603–610. [16] G.A. Kelley, K. Sharpe Kelley, Aerobic exercise and resting blood pressure in older adults: a meta-analytic review of randomized controlled trials, J. Gerontol. A Biol.
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