Gamma Oryzanol Treats Obesity-Induced Kidney Injuries by Modulating the Adiponectin Receptor 2/PPAR- α Axis

Abstract

The kidney is an important organ in the maintenance of body homeostasis. Dietary compounds, reactive metabolites, obesity, and metabolic syndrome (MetS) can affect renal filtration and whole body homeostasis, increasing the risk of chronic kidney disease (CKD) development. Gamma oryzanol ( γ Oz) is a compound with antioxidant and anti-inflammatory activity that has shown a positive action in the treatment of obesity and metabolic diseases. Aim . To evaluate the effect of γ Oz to recover renal function in obese animals by high sugar-fat diet by modulation of adiponectin receptor 2/PPAR- α axis Methods . Male Wistar rats were initially randomly divided into 2 experimental groups: control and high sugar-fat diet (HSF) for 20 weeks. When proteinuria was detected, HSF animals were allocated to receive γ Oz or maintain HSF for more than 10 weeks. The following were analyzed: nutritional and biochemical parameters, systolic blood pressure, and renal function. In the kidney, the following were evaluated: inflammation, oxidative stress, and protein expression by Western blot. Results . After 10 weeks of γ Oz treatment, γ Oz was effective to improve inflammation, increase antioxidant enzyme activities, increase the protein expression of adiponectin receptor 2 and PPAR- α , and recover renal function. Conclusion . These results permit us to confirm that γ Oz is able to modulate PPAR- α expression, inflammation, and oxidative stress pathways improving obesity-induced renal disease.

Full text

Research Article
Gamma Oryzanol Treats Obesity-Induced Kidney Injuries by
Modulating the Adiponectin Receptor 2/PPAR-αAxis
Fabiane Valentini Francisqueti ,
1
Artur Junio Togneri Ferron,
1
Fabiana Kurokawa Hasimoto,
2
Pedro Henrique Rizzi Alves,
2
Jéssica Leite Garcia,
1
Klinsmann Carolo dos Santos ,
1
Fernando Moreto,
1
Vanessa dos Santos Silva ,
1
Ana Lúcia A. Ferreira,
1
Igor Otávio Minatel,
2
and Camila Renata Corrêa
1
1
Medical School, São Paulo State University (UNESP), Botucatu, SP, Brazil
2
Institute of Biosciences, São Paulo State University (UNESP), Botucatu, SP, Brazil
Correspondence should be addressed to Fabiane Valentini Francisqueti; fabiane_vf@yahoo.com.br
Received 17 May 2018; Accepted 22 July 2018; Published 9 September 2018
Academic Editor: Germán Gil
Copyright © 2018 Fabiane Valentini Francisqueti et al. This is an open access article distributed under the Creative Commons
Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work
is properly cited.
The kidney is an important organ in the maintenance of body homeostasis. Dietary compounds, reactive metabolites, obesity, and
metabolic syndrome (MetS) can affect renal filtration and whole body homeostasis, increasing the risk of chronic kidney disease
(CKD) development. Gamma oryzanol (γOz) is a compound with antioxidant and anti-inflammatory activity that has shown a
positive action in the treatment of obesity and metabolic diseases. Aim. To evaluate the effect of γOz to recover renal function in
obese animals by high sugar-fat diet by modulation of adiponectin receptor 2/PPAR-αaxis Methods. Male Wistar rats were
initially randomly divided into 2 experimental groups: control and high sugar-fat diet (HSF) for 20 weeks. When proteinuria
was detected, HSF animals were allocated to receive γOz or maintain HSF for more than 10 weeks. The following were analyzed:
nutritional and biochemical parameters, systolic blood pressure, and renal function. In the kidney, the following were evaluated:
inflammation, oxidative stress, and protein expression by Western blot. Results. After 10 weeks of γOz treatment, γOz was
effective to improve inflammation, increase antioxidant enzyme activities, increase the protein expression of adiponectin
receptor 2 and PPAR-α, and recover renal function. Conclusion. These results permit us to confirm that γOz is able to modulate
PPAR-αexpression, inflammation, and oxidative stress pathways improving obesity-induced renal disease.
1. Introduction
Kidneys exert a central role in the maintenance of body
homeostasis by regulating electrolyte concentrations, blood
pressure, degradation of hormones, lipid metabolism, and
excretion of waste metabolites [1]. Despite many factors
leading to kidney disease, such as age, gender, smoking
status, alcohol use, physical inactivity, diabetes mellitus,
and hypertension, studies reveal that obesity is an inde-
pendent risk factor for development of CKD [1–3].
The pathways activated by obesity to induce kidney
disease are not fully understood. Studies have identified
several new injurious pathways in the kidney led by insulin
resistance (IR), chronic inflammation (a major contributor
to microvascular remodeling), dyslipidemia and excessive
nutrient availability (both may induce mitochondrial dys-
function and oxidative stress), and adipokine production
unbalance [4–6].
Adiponectin is an adipocyte-derived protein hormone
which plays a role in the suppression of inflammation-
associated metabolic disorders. Adiponectin receptor 1
(Adipo-R1) and adiponectin receptor 2 (Adipo-R2) are the
two major receptors for adiponectin and appear to be integral
membrane proteins [7], expressed in different tissues, among
them the kidney [8]. Kadowaki et al. [7] reported previously
that the receptor expression levels are reduced in obesity,
apparently in correlation with reduced adiponectin sensitiv-
ity. Moreover, the authors relate that Adipo-R1 may be more
Hindawi
Oxidative Medicine and Cellular Longevity
Volume 2018, Article ID 1278392, 9 pages
https://doi.org/10.1155/2018/1278392

tightly linked to activation of AMPK pathways, whereas
Adipo-R2 seems to be associated with the activation of
PPAR-αpathways and the inhibition of inflammation. So,
the modulation of these pathways could be important to treat
renal injuries.
Considering this situation, natural compounds have
received attention as a promising pool of substances to treat
diseases [9]. Rice bran is rich in gamma oryzanol (γOz), a
natural compound with antioxidant and anti-inflammatory
activities that showed a positive action in the treatment of
hyperlipidemia, hyperglycemia, insulin resistance, and
increased levels of adiponectin [10–14]. So, considering that
obesity, inflammation, and oxidative stress are able to induce
renal disease and there are no studies that evaluate the effect
of γOz in renal disease, the aim of this study was to evaluate
the effect of γOz in the recovery of renal function in obese
animals by high sugar-fat diet by modulation of the adipo-
nectin receptor 2/PPAR-αaxis.
2. Methods
2.1. Experimental Protocol. All of the experiments and proce-
dures were approved by the Animal Ethics Committee of
Botucatu Medical School (1150/2015) and were performed
in accordance with the National Institute of Health’s Guide
for the Care and Use of Laboratory Animals. Male Wistar
rats (±187 g) were kept in an environmental controlled
room (22
°
C±3
°
C, 12 h light-dark cycle, and relative humid-
ity of 60 ±5%) and initially randomly divided into 2 experi-
mental groups (control, n=15, and high sugar-fat diet
(HSF), n=30) for 20 weeks. HSF groups also received
water + sucrose (25%). The diets and water were provided
ad libitum. The HSF diet contained soybean meal, sorghum,
soybean peel, dextrin, sucrose, fructose, lard, vitamins, and
minerals, plus 25% sucrose in drinking water; the control diet
contained soybean meal, sorghum, soybean peel, dextrin, soy
oil, vitamins, and minerals. The nutrients and nutritional
composition of each diet was described in our previous study
[15]. At week 20 of this study, when proteinuria was detected
in the HSF groups, animals were divided to begin the treat-
ment with γOz or continue receiving HSF for 10 more weeks
as described below.
2.2. Group Characterization. After 20 weeks of experimental
protocol, a 95% confidence interval (CI) was built for the
protein/creatinine ratio from the HSF and control groups
and was adopted as the separation point (SP) between the
groups, the midpoint between the upper limit of the control
group and the lower limit of the HSF group. The protein/cre-
atinine ratio was adopted since it reflects proteinuria and is
considered a marker of kidney function [16]. From this point,
the control animals with a protein/creatinine ratio above of
SP and the HSF animals with a protein/creatinine ratio below
the SP were excluded from the control and HSF groups,
respectively, ensuring the homogeneity of the treated and
control groups. About the remaining animals in the HSF
group, they were randomly divided to receive γOz or only
diet. This criterion was adopted because animals submitted
to different diet models do not always present the expected
response. This fact can lead to erroneous animal classifica-
tion and, consequently, false conclusions. The values for pro-
tein/creatinine ratio on the 20th week were 2.5 for the control
group and 3.3 for the HSF group (p=0 0006).
2.3. Treatment with Gamma Oryzanol. After the characteri-
zation on the 20th week, the groups were the following: con-
trol diet (control, n=8), high sugar-fat diet (HSF, n=8), and
HSF/HSF + gamma oryzanol (HSF/HSF + γOz, n=8). The
treatment duration was 10 weeks, totaling 30 weeks of exper-
iment. The γOz dose used in this study was added in the
chow (0.5 w/w) according to our previous study [15].
2.4. Body Composition and Caloric Ingestion. The nutritional
profile was evaluated according to the following parameters:
caloric intake, body weight, and adiposity index. Caloric
intake was determined by multiplying the energy value of
each diet (g ×kcal) by the daily food consumption. For the
HSF group, caloric intake also included calories from water
(0.25 ×4×mL consumed). Body weight was measured
weekly. After euthanasia, fat deposits (visceral (VAT), epi-
didymal (EAT), and retroperitoneal (RAT)) were used to cal-
culate the adiposity index (AI) by the following formula:
VAT + EAT + RAT /FBW × 100.
2.5. Metabolic and Hormonal Analysis. After 12 h fasting,
blood was collected and the plasma was used to measure
insulin and biochemical parameters. Glucose concentration
was determined by using a glucometer (Accu-Chek Per-
forma, Roche Diagnostics Brazil Limited); triglycerides
were measured with an automatic enzymatic analyzer system
(Chemistry Analyzer BS-200, Mindray Medical International
Limited, Shenzhen, China). The insulin and adiponectin
levels were measured using enzyme-linked immunosorbent
assay (ELISA) methods using commercial kits (EMD Milli-
pore Corporation, Billerica, MA, USA). The homeostatic
model of insulin resistance (HOMA-IR) was used as an
insulin resistance index, calculated according to the follow-
ing formula: HOMA-IR = (fasting glucose (mmol/L) ×fast-
ing insulin (μU/mL))/22.5.
2.6. Systolic Blood Pressure. Systolic blood pressure (SBP)
evaluation was assessed in conscious rats by the noninvasive
tail-cuffmethod with a Narco Bio-Systems® electrosphygmo-
manometer (International Biomedical, Austin, TX, USA).
The animals were kept in a wooden box (50 ×40 cm) between
38 and 40
°
C for 4-5 minutes to stimulate arterial vasodilation
[17]. After this procedure, a cuffwith a pneumatic pulse sen-
sor was attached to the tail of each animal. The cuffwas
inflated to 200 mmHg pressure and subsequently deflated.
The blood pressure values were recorded on a Gould RS
3200 polygraph (Gould Instrumental Valley View, Ohio,
USA). The average of three pressure readings was recorded
for each animal.
2.7. Renal Function. Renal function was evaluated by mea-
surements of plasma and urine. At twenty-four hours,
urine was collected from the metabolic cages to measure
the excretion of creatinine and the total protein. The urea
and creatinine content of the plasma were measured. All
2 Oxidative Medicine and Cellular Longevity

analyses were performed with an automatic enzymatic
analyzer system (biochemical analyzer BS-200, Mindray,
China). The glomerular filtration rate (GFR = (urine crea-
tinine ×flux)/plasma creatinine) and proteinuria were also
calculated.
2.8. Renal Tissue Analysis
2.8.1. Inflammatory Parameters. Renal tissue (±150 mg) was
homogenized (ULTRA-TURRAX® T 25 basic IKA® Werke,
Staufen, Germany) in 1.0 mL of phosphate-buffered saline
70
80
90
100
110
120
Calories/day
(kcal/d)
HSFControl HSF/HSF + Oz
(a)
⁎
⁎
0
5
10
15
Adiposity index (%)
Control HSF/HSF + Oz
HSF
(b)
⁎
0
50
100
150
Glucose (mg/dl)
HSF/HSF + Oz
HSFControl
(c)
⁎
⁎
0
20
40
60
80
HOMA-IR
HSF HSF/HSF + Oz
Control
(d)
⁎
⁎
0
50
100
150
Triglycerides (mg/dL)
Control HSF/HSF + Oz
HSF
(e)
⁎
⁎
100
120
140
160
180
Systolic blood pressure
(mmHg)
HSF HSF/HSF + Oz
Control
(f)
Figure 1: Nutritional, metabolic, and cardiovascular parameters: (a) caloric intake (kcal/day); (b) adiposity index (%); (c) glucose (mg/dL);
(d) HOMA-IR; (e) triglycerides (mg/dL); (f ) systolic blood pressure (mmHg). Data expressed in mean ±standard deviation or median.
Comparison by one-way ANOVA with Tukey post hoc. HSF: high sugar-fat diet; γOz: gamma oryzanol. ∗indicates p<005;n=8
animals/group.
3Oxidative Medicine and Cellular Longevity

(PBS) pH 7.4 cold solution and centrifuged at 800gat 4
°
C for
10 min. The supernatant (100 μL) was used in analysis.
Tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6),
and monocyte chemoattractant protein-1 (MCP-1) levels
were measured using the enzyme-linked immunosorbent
assay (ELISA) method using commercial kits from R&D
System, Minneapolis, USA. The supernatant (100 μL)
was used for analysis, and the results were corrected by the
protein amount.
2.8.2. Hydrophilic Antioxidant Capacity. The hydrophilic
antioxidant capacity in the kidney was in the prepared
supernatant as described in the previous item. It was
determined fluorometrically, using a VICTOR X2 reader
(PerkinElmer, Boston, MA). The antioxidant activity was
quantitated by comparing the area under the curve relating
to the oxidation kinetics of the suspension phosphatidylcho-
line (PC), which was used as the reference biological matrix.
The peroxyl radical 2′,2′-azobis-(2-amidinopropane) dihy-
drochloride (AAPH) was used as an initiator of the reaction.
The results represent the percent inhibition (4,4-difluoro-5-
(4-phenyl 1-3 butadiene)-4-bora-3,4-diaza-s-indacene)
(BODIPY) 581/591 plasma with respect to the control
sample of BODIPY 581/591 PC liposome. All analyses were
performed in triplicate. The results are reported as a percent-
age of protection [18].
2.8.3. Antioxidant Enzyme Activity. For these analyses, a
100 mg kidney was homogenized (1 : 10 v/v)inKH
2
PO
4
(10 mmol/L)/KCl (120 mmol/L), pH 7.4, and centrifuged at
2.000 ×g for 20 min. Superoxide dismutase (SOD) activity
was measured based on the inhibition of a superoxide radical
reaction with pyrogallol, and the absorbance values were
measured at 420 nm [19]. Catalase activity was evaluated by
following the decrease in the levels of hydrogen peroxide in
240 nm [20]. The activity is expressed as pmol of H
2
O
2
reduced/min/mg protein. Glutathione peroxidase (GP)
activity was measured by following β-nicotinamide ade-
nine dinucleotide phosphate (NADPH) oxidation at 340 nm
as described by Flohé and Günzler [21]. The results were
expressed as μmol hydroperoxide-reduced/min/mg protein.
Protein was quantified based on Lowry et al.’s method [22]
using bovine serum albumin as the standard. The absor-
bance values for all analyses were measured in a UV/VIS spec-
trophotometer (Pharmacia Biotech, Houston, Texas, USA),
and the values are expressed as units per milligram of protein.
⁎
⁎
0
20
40
60
80
100
Plasma urea (mg/dL)
HSFControl HSF/HSF + Oz
(a)
HSFControl HSF/HSF + Oz
0.0
0.2
0.4
0.6
0.8
Plasma creatinin
(mg/dL)
(b)
⁎⁎
0
1
2
3
4
Glomerular ltration rate (GFR)
(mL/min)
HSF/HSF + Oz
HSFControl
(c)
⁎⁎
HSFControl HSF/HSF + Oz
0
1
2
3
4
5
Protein/creatinine ratio
(d)
Figure 2: Renal function parameters: (a) plasma urea (mg/dL); (b) plasma creatinine (mg/dL); (c) glomerular filtration rate (GFR) (mL/min);
(d) protein/creatinine ratio. Data expressed in mean ±standard deviation or median. Comparison by one-way ANOVA with Tukey post hoc.
∗indicates p<005;n=8 animals/group.
4 Oxidative Medicine and Cellular Longevity

2.8.4. Western Blot. Renal samples were homogenized in
RIPA buffer with a protease and phosphatase cocktail inhib-
itor. After determination of protein concentration by the
Bradford method [23], samples were diluted in Laemmli
buffer and loaded (50 μg of protein) into a 10% SDS–poly-
acrylamide gel. Transfer to a nitrocellulose membrane was
carried out using Trans-Blot Turbo-Transfer System
(BioRad). Incubation with the primary antibodies was per-
formed overnight at 4
°
C in Tris-buffered saline solution con-
taining Tween 20 (TBS-T) and 3% bovine serum albumin.
Antibody dilutions were 1 : 1000 for Adipo-R1 (ABCAM
ab126611), 1 : 1000 for Adipo-R2 (ABCAM ab77612),
1 : 500 for PPAR-α(ABCAM ab8934), 1 : 1000 for total
AMPK (Cell Signaling #2532), 1 : 1000 for phospho-AMPH
(Thr172) (Cell Signaling #2531), and 1 : 1000 for beta-actin
(ABCAM ab8227). After incubation overnight at 4
°
Cin
TBS-T containing 1% nonfat dried milk with the Abcam
secondary antibodies (dilution 1 : 3000 for anti-goat and
1 : 1000 for anti-rabbit). Protein was revealed using the
chemiluminescence method according to the manufacturer’s
instructions (ECL SuperSignal® West Pico Chemilumines-
cent Substrate, Thermo Scientific). Band intensities were
evaluated using ImageQuant TL 1D Version 8.1 (GE Health-
care Life Sciences).
2.9. Statistical Analysis. Data are presented as means ±stan-
dard deviation (SD) or median (interquartile range). Differ-
ences among the groups were determined by one-way
analysis of variance. Statistically significant variables were
subjected to the Tukey post hoc test to compare all the
groups. Statistical analyses were performed using Sigma
Stat for Windows Version 3.5 (Systat Software Inc., San
Jose, CA, USA). A pvalue of 0.05 was considered statisti-
cally significant.
3. Results
Figure 1 shows caloric intake, adiposity index, and cardio-
metabolic risk factors for kidney disease (glucose, HOMA-
IR, triglycerides, and systolic blood pressure). It is possible
to verify that both HSF groups presented higher values for
all the parameters. There was no difference for caloric intake.
Figure 2 shows renal function parameters. Gamma oryza-
nol was effective for recovery of renal function of the HSF/
HSF + γOz group, characterized by lower proteinuria and
high glomerular filtration rate compared to the HSF group.
Figure 3 shows inflammatory parameters in kidney tis-
sue. γOz was effective to reduce the inflammatory response
for levels similar to those observed in the control group.
⁎
HSFControl HSF/HSF + Oz
0
200
400
600
Renal TNF-
(pg/gprotein)
⁎
(a)
⁎⁎
HSFControl HSF/HSF + Oz
0
500
1000
1500
2000
2500
Renal IL-6
(pg/gprotein)
(b)
⁎⁎
0
50
100
150
200
Renal MCP-1
(pg/gprotein)
HSFControl HSF/HSF + Oz
(c)
Figure 3: Inflammatory parameters in kidney tissue: (a) tumor necrosis factor-alpha (TNF-α; pg/g protein); (b) interleukin-6 (IL-6; pg/g
protein); (c) monocyte chemoattractant protein-1 (MCP-1; pg/g protein). Data expressed in mean ±standard deviation or median.
Comparison by one-way ANOVA with Tukey post hoc. ∗indicates p<005;n=8 animals/group.
5Oxidative Medicine and Cellular Longevity

Figure 4 shows redox state parameters in the kidney. It
is possible to verify a positive action of γOz on the HSF/
HSF + γOz group to increase hydrophilic antioxidant protec-
tion, catalase, and superoxide dismutase levels compared
to HSF.
Figure 5 presents plasma adiponectin levels. The HSF
group presented higher levels while the treatment with
gamma oryzanol was able to reduce the levels.
Figure 6 shows protein expression of Adipo-R1, Adipo-
R2, phosphorylated and total AMPK, and PPAR-αin the kid-
ney. It is possible to note the effect of γOz which increased
the expression of Adipo-R2 and PPAR-αwhen compared
to HSF.
4. Discussion
The aim of this study was to evaluate the potential of γOz to
recover renal function in obese animals by high sugar-fat
diet consumption. In this study, the animals feeding on a
HSF diet developed obesity and signals of kidney injury,
characterized by proteinuria and decreased glomerular
filtration rate. Obesity, insulin resistance, hypertension,
chronic inflammation, dyslipidemia, and oxidative stress are
considered the major risk factors for renal disease [1, 4, 6].
The HSF group developed all these risk factors, which were
expected considering the diet used in this study, rich in sugar
and fat [15], but the noneffect of γOz on these parameters
was observed. In opposition to our results, Wang et al. and
⁎⁎
0
20
40
60
0
8
Hydrophilic antioxidant capacity
(% protection/g protein)
HSF HSF/HSF + Oz
Control
(a)
⁎⁎
0
2
4
6
Catalase
(pmol/mg protein/min)
HSF HSF/HSF + Oz
Control
(b)
5
6
7
8
9
10
Glutathione peroxidase
(mol/mg protein/min)
HSF HSF/HSF + Oz
Control
(c)
⁎⁎
0
2
4
6
8
Superoxide dismutase
(U/mg protein/min)
HSF HSF/HSF + Oz
Control
(d)
Figure 4: Redox state parameters in the kidney: (a) hydrophilic antioxidant capacity (% protection/g protein); (b) catalase (pmol/mg protein/
min); (c) glutathione peroxidase (μmol/mg protein/min); (d) superoxide dismutase (U/mg protein/min). Data expressed in mean ±standard
deviation. Comparison by one-way ANOVA with Tukey post hoc. ∗indicates p<005;n=6animals/group.
⁎⁎
0
10,000
20,000
30,000
40,000
Plasma adiponectin (ng/ml)
HSFControl HSF/HSF + Oz
Figure 5: Plasma adiponectin levels (ng/mL). Data expressed in
mean ±standard deviation. Comparison by one-way ANOVA with
Tukey post hoc. ∗indicates p<005;n=8animals/group.
6 Oxidative Medicine and Cellular Longevity

Justo et al. found in their studies improvement in some param-
eters after treatment with γOz [10, 24]. It is important to
emphasize that in these studies, animal models and the
dose of γOz were different from ours, which can explain
these opposite results.
Once metabolic disorders are risk factors for renal dis-
ease, it would be expected that both HSF groups presented
renal function impairment. However, analyzing the clinical
signals of renal disease (proteinuria, most conveniently per-
formed by estimation of the protein/creatinine ratio and glo-
merular filtration rate) [25], we can note an improvement in
the treatment group with γOz characterized by lower pro-
teinuria and higher GFR. Therefore, better understanding
of the mechanisms by which γOz acted in this group is very
important to enable novel therapeutic target development.
Oxidative stress is one condition associated with
impaired renal function [8, 26]. Kidney disease progression
is related with a significant increase of ROS, which influences
cell function and damages proteins, lipids, and nucleic acids,
and can also inhibit enzymatic activities of the cellular respi-
ratory chairs. On the other hand, endogenous enzymatic and
nonenzymatic antioxidant mechanisms protect against dam-
aging effects of oxidative products [27]. The first line of enzy-
matic antioxidant defense is SOD, which accelerates the
dismutation rate of oxygen to H
2
O
2
, but the catalase reduces
H
2
O
2
to water. Glutathione peroxidase reduces H
2
O
2
and
other organic peroxides to water and oxygen and requires
glutathione as a hydrogen donor which is a scavenger for
H
2
O
2
, hydroxyl radicals, and chlorinated oxidants [27]. Usu-
ally, patients suffering from renal insufficiency have dimin-
ished antioxidant defense when compared to healthy
controls [28]. In the case of this study, the results showed
an increase of antioxidant capacity, SOD, and catalase activ-
ities after treatment with gamma oryzanol, confirming the
potential of the compound to improve the antioxidant
system. But some authors relate difficulty in establishing a
pattern of antioxidant status in kidney disease due to assess-
ment by different measurement techniques [28]. In this case,
Adipo-R1

-Actin
Control HSF
HSF/HSF
+ Oz
0.5 1.0 1.50.0
Control
HSF
HSF/HSF + Oz
Adipo-R1/-actin
(relative expression)
(a)
Control HSF
Adipo-R2

-Actin
HSF/HSF
+ Oz
123450
⁎
⁎
Control
HSF
HSF/HSF + Oz
Adipo-R2/

-actin
(relative expression)
(b)
Phospho-
AMPK
Total AMPK
Control HSF HSF/HSF
+ Oz
0.5 1.0 1.50.0
Control
HSF
HSF/HSF + Oz
phospho-AMPK total AMPK
(relative expression)
(c)
PPAR-


-Actin
Control HSF
HSF/HSF
+ Oz
0.5 1.0 1.50.0
⁎
Control
HSF
HSF/HSF + Oz
PPAR-

/

-actin
(relative expression)
(d)
Figure 6: Relative protein expression in kidney tissue: (a) Adipo-R1; (b) Adipo-R2; (c) phospho-AMPK; (d) PPAR-α. Data expressed in
mean ±standard deviation. Comparison by one-way ANOVA with Tukey post hoc. ∗indicates p<005;n=6animals/group.
7Oxidative Medicine and Cellular Longevity

information associating various parameters can give a better
representation of a patient’s current antioxidant status.
The literature reports that the renoprotection can also
be related to some mechanisms involving improvement
of the endothelial dysfunction, reduction of oxidative stress,
and upregulation of endothelial nitric oxide synthase expres-
sion, all effects dependent on adiponectin receptor activation
[29]. In contrast, the dysfunction regulation of adiponectin
and its receptors has been observed in the development of
various diseases, including obesity, insulin resistance, type 1
and type 2 diabetes, and chronic kidney disease [29].
Adiponectin is secreted primarily by adipose tissue and
plays a key role in kidney disease. In obesity, reduced adipo-
nectin levels are also associated with insulin resistance and
cardiovascular disease. However, in conditions of established
chronic kidney disease, adiponectin levels are elevated and
positively predict progression of disease [30, 31]. Corroborat-
ing these findings, the HSF group presented higher levels of
adiponectin associated with reduced GFR which confirms
kidney disease. In opposition, the HSF group that received
the compound showed reduction in the levels, which can be
explained by the amelioration of glomerular filtration rate
by γOz in these animals, since adiponectin is excreted via
kidney glomerular filtration [32].
Adipo-R1 and Adipo-R2 are expressed in many tissues
[8], but in the specific case of the kidneys, no studies evaluated
the effect of γOz in this pathway and its role on renal function.
The compound showed capacity to upregulate the Adipo-R2/
PPAR-αaxis. PPAR-αis highly expressed in tissues that pos-
sess high mitochondrial and β-oxidation activity, as the kid-
ney. Decreased renal PPAR-αexpression might contribute
to the pathogenesis of kidney injuries [33], whereas its high
expression is associated with metabolic control in the organ
[34]. Moreover, PPAR-αactivation can attenuate or inhibit
several mediators of vascular injury involved in renal damage,
such as lipotoxicity, reactive species oxygen (ROS) genera-
tion, and inflammation [34, 35]. Corroborating this informa-
tion, our animals of the HSF/HSF + γOz group did not
present inflammation in the kidney, showing lower levels of
TNF-α, IL-6, and MCP-1 compared to the HSF group.
In summary, this study introduces very important find-
ings since γOz was effective in ameliorating renal dysfunc-
tion by acting on the Adipo-R2/PPAR-αaxis and also by
improving the antioxidant response in the organ. γOz could
be a therapeutic alternative for restoring/ameliorating meta-
bolic dysfunctions, in special renal injuries that are developed
in an obese individual. These results permit us to confirm
that γOz is able to modulate PPAR-αexpression, inflamma-
tion, and oxidative stress pathways improving obesity-
induced renal disease.
Data Availability
The data used to support the findings of this study are
available from the corresponding author upon request.
Conflicts of Interest
The authors declare no conflict of interest.
Acknowledgments
This work was supported by Fundação de Amparo à Pesquisa
do Estado de São Paulo (FAPESP) (2015/10626-0 and 2018/
15288-3).
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