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1
Authors
Fabiane Valentini Francisqueti-
Ferron¹
Artur Junio Togneri Ferron¹
Alessandra Altomare²
Jéssica Leite Garcia¹
Fernando Moreto¹
Ana Lúcia A. Ferreira¹
Igor Otávio Minatel³
Giancarlo Aldini²
Camila Renata Corrêa¹
1Universidade Estadual Paulista
(UNESP), Faculdade de Medicina,
Botucatu, SP, Brasil.
2Università degli Studi di Milano,
Department of Pharmaceutical
Sciences (DISFARM), Milan, Italy.
3Universidade Estadual Paulista
(UNESP), Instituto de Biociências,
Botucatu, SP, Brasil.
Gamma-oryzanol reduces renal inflammation and oxidative
stress by modulating AGEs/RAGE axis in animals submitted
to high sugar-fat diet
Gama-orizanol reduz a inflamação e o estresse oxidativo nos rins
pela modulação do eixo RAGE/AGEs em animais submetidos a uma
dieta rica em gordura e açúcar
Introdução: O receptor para AGEs (RAGE)
é um membro multiligante da superfamília
das imunoglobulinas dos receptores de
superfície celular expresso em muitos órgãos,
entre eles, os rins. Quando ativado, o RAGE
leva a uma sequência de sinalização que
resulta em inflamação e estresse oxidativo,
ambos envolvidos na patogênese de doenças
renais. O gama-orizanol (γOz) compreende
uma mistura de ésteres de ácido ferúlico (AF)
e fitoesteróis (esteróis e álcoois triterpenos)
encontrados principalmente no arroz, com
atividades antioxidantes e anti-inflamatórias.
Objetivo: Avaliar o efeito do γOz para reduzir
a inflamação renal e o estresse oxidativo pela
modulação do eixo RAGE/AGEs em animais
submetidos a uma dieta rica em gordura
e açúcar. Métodos: Ratos Wistar machos
(±187g) foram divididos aleatoriamente em
dois grupos experimentais: controle (n = 7
animais) e dieta rica em gordura e açúcar
(HSF, do inglês high sugar-fat diet, n = 14
animais) por 20 semanas. Após este período,
quando foi detectada a presença de fatores
de risco de doença renal no grupo HSF
(resistência à insulina, dislipidemia, aumento
da pressão arterial sistólica e obesidade),
os animais HSF foram divididos para
iniciar o tratamento com γOz ou continuar
recebendo apenas HSF por mais 10 semanas.
Resultados: Não foi observado nenhum
efeito do γOz na obesidade e nos parâmetros
metabólicos. No entanto, a inflamação e o
estresse oxidativo renais diminuíram assim
que os níveis de RAGE foram reduzidos em
HSF + γOz. Conclusão: É possível concluir
que o gama- orizanol foi eficaz em reduzir a
inflamação e o estresse oxidativo no rim pela
modulação do eixo RAGE/AGEs.
Resumo
Descritores: Receptor RAGE; Inflamação;
Antioxidante; Rim; Obesidade.
Introduction: The receptor for AGEs
(RAGE) is a multiligand member of the
immunoglobulin superfamily of cell
surface receptors expressed in many
organs, among them, the kidneys. When
activated, RAGE leads to a sequence of
signaling that results in inflammation and
oxidative stress, both involved in kidney
disease pathogenesis. Gamma-oryzanol
(γOz) comprises a mixture of ferulic acid
(FA) esters and phytosterols (sterols and
triterpene alcohols) mainly found in rice,
with antioxidant and anti-inflammatory
activities. Aim: To evaluate the effect of
γOz to reduce renal inflammation and
oxidative stress by modulating AGEs/
RAGE axis in animals submitted to a high
sugar-fat diet. Methods: Male Wistar
rats (±187g) were randomly divided
into two experimental groups: control
(n = 7 animals) and high sugar-fat diet
(HSF, n = 14 animals) for 20 weeks.
After this period, when the presence of
renal disease risk factors was detected
in the HSF group (insulin resistance,
dyslipidemia, increased systolic blood
pressure and obesity), the HSF animals
were divided to begin the treatment with
γOz or continue receiving only HSF for
10 more weeks. Results: No effect of γOz
on obesity and metabolic parameters was
observed. However, kidney inflammation
and oxidative stress decreased as soon as
RAGE levels were reduced in HSF + γOz.
Conclusion: It is possible to conclude
that the gamma- oryzanol was effective
in reducing inflammation and oxidative
stress in the kidney by modulating the
AGEs/RAGE axis.
AbstRAct
Keywords: AGE receptor; Inflammation;
Antioxidants; Kidney; Obesity.
Submitted on: 01/05/2021.
Approved on: 04/18/2021.
Correspondence to:
Fabiane Valentini Francisqueti-Ferron.
E-mail: fabiane_vf@yahoo.com.br
DOI: https://doi.org/10.1590/2175-
8239-JBN-2021-0002
Braz. J. Nephrol. (J. Bras. Nefrol.) 2021. Ahead of print
Oryzanol and AGE/RAGE axis
2
Intro ductIon
Protein glycation is a complex series of sequential
reactions collectively called the Maillard reaction
that result in advanced glycation end products
(AGEs) formation. Endogenous sources of AGEs
can be found in all tissues and fluids where glucose
concentration is enough to react with proteins, such
as in conditions of hyperglycaemia and diabetes1.
Moreover, the degradation of glycated proteins,
glycolytic intermediates, and degradation of aldoses
and ketoses result in the formation of reactive
carbonyl species such as glyoxal (G), methylglyoxal
(MG), and 3-deoxyglucosone, which are also able
to react with proteins to form more AGEs directly2.
AGEs are also present in ingested food, characterizing
exogenous AGEs sources. The content depends on the
nutrient composition and how the food is processed
(for example, high levels of AGEs are found in
roasted, smoked, and baked foods)1,3.
At the cellular level, the damaging effects of AGEs
have been attributed to several AGE-binding proteins,
such as RAGE (receptor for AGEs), AGEs receptor
(AGER) 1, R2, R3, and scavenger receptors such as
CD-3647 and SCR-II 4. It is important to emphasize
that these cell surface receptors can bind to advanced
glycation end products, and also can interact with
multiple ligands called multi-ligand receptor, among
them: high-mobility group protein (B)1 (HMGB1),
S-100 calcium-binding protein, amyloid-β-protein,
Mac-1, and phosphatidylserine5. Among AGEs
receptors, the RAGE is the most notable one, and it
triggers oxidative stress and inflammation in both
acute and chronic diseases. Specifically, the binding
of RAGE leads to a sequence of signaling with the
activation of the transcription factor nuclear factor
kappa-B (NFκB) resulting in proinflammatory
cytokines production, among them tumoral necrosis
factor alpha (TNF-α), interleukin-6 (IL-6), and
monocyte chemoattractant protein-1 (MCP-1)6.
RAGE activation leads to oxidative stress by
inducing nicotinamide adenine dinucleotide phosphate
(NADPH)-oxidase (NOX), especially NOX-4.
Oxidation driven by the AGEs/RAGE axis induces
protein and lipid oxidation leading to the formation
of protein carbonyls and lipid-peroxidation, being
responsible for lipid-derived reactive carbonyl species
which in turn form protein carbonyl adducts (ALEs)
which are also RAGE binders, thus sustaining the
RAGE activation7. Thus, according to the information
above described, it is possible to note that the
AGEs/RAGE axis is an interface between oxidative
stress and inflammation, which are pillars for the
development of several diseases, especially in organs
that express these receptors for AGEs, as brain, heart,
and kidneys8.
The podocyte is the main RAGE expressing cell in
the renal glomerulus1,9. It has been described in the
literature that RAGE-dependent signal transduction
in podocytes leads to apoptosis, production of
monocyte chemoattractant protein-1, inflammatory
mediators, and oxidative stress via NOX-4, causing
renal structural changes, resulting in increased
proteinuria and reduced glomerular filtration rate
(GFR)9. Once GFR is the main way to excrete
AGEs, the exacerbation of kidney injury contributes
to accumulating AGEs, characterizing a vicious
positive feedback of AGEs accumulation through
AGEs/RAGE-induced oxidative stress in the course
of CKD progression1.
In this way, the search for strategies to lower
the ligand burden (AGEs and other RAGE ligands)
and strategies to dampen RAGE activation have
received attention, such as the use of natural
compounds as a promising pool of substances
to treat diseases10,11. Gamma-oryzanol (γOz)
comprises a mixture of ferulic acid (FA) esters and
phytosterols (sterols and triterpene alcohols) mainly
found in rice, a very important grain in the human
diet. A great variety of biological effects have been
attributed to γOz, such as antidiabetic, antioxidant,
anti-inflammatory, and anti-obesity effects12. Other
studies have already demonstrated a positive effect
of γOz to prevent cardiorenal metabolic syndrome13,
improve renal disease10, and increase muscle growth
and sports performance14.
Thus, since the AGEs/RAGE ligation is able to
induce kidney inflammation and oxidative stress
and given the lack of studies that evaluate the effect
of γOz in renal AGEs/RAGE modulation, the aim
of this study was to evaluate the effect of γOz in
reducing renal inflammation and oxidative stress by
modulating AGEs/RAGE axis in animals submitted
to a high sugar-fat diet. The rationale for using the
γOz in this animal model is sustained by the recent
literature demonstrating that this class of compounds,
besides having a well-established antioxidant activity,
exerts a direct antiglycation effect15.
Braz. J. Nephrol. (J. Bras. Nefrol.) 2021. Ahead of print
Oryzanol and AGE/RAGE axis
3
MaterIal and Met hods
ExpErimEntal protocol
All the experiments and procedures 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 housed in individual
cages in an environment-controlled room (22±3 °C; 12
h light-dark cycle, and relative humidity of 60±5 %)
and randomly divided into two experimental groups:
control (n = 7 animals) and high sugar-fat diet (HSF,
n = 14 animals) for 20 weeks. At the 20th week, when
the presence of renal disease risk factors was detected
in HSF group10,13 (insulin resistance, dyslipidemia,
increased systolic blood pressure, and obesity), the
animals were divided to begin the treatment with
γOzor continue receiving only HSF for 10 more weeks:
HSF, n=7 animals and HSF + γOz, n= 7 animals. 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 study13.
Gamma- oryzanol
The compound was purchased from Tokyo Chemical
Industry Co., Ltd. (Toshima, Kita-ku, Tokyo)
(lot.5ZZYLPJ). The γOz used in this study was added
in the chow (0.5 w/w) in line with our previous study13
in order to simulate the daily consumption of rice of
an adult individual in Brazil, according to data from
the Family Budget Survey (POF) 2008–200916.
nutritional paramEtErs and obEsity- rElatEd dis-
ordErs Evaluation
The nutritional profile considered: final body weight
(FBW), adiposity index (AI), insulin resistance,
triglycerides levels, and systolic blood pressure (SBP).
Body weight was measured weekly. After euthanasia,
the fat deposits (visceral (VAT), epididymal (EAT),
and retroperitoneal (RAT)) were used to calculate
the adiposity index (AI) by the following formula:
VAT+EAT+RAT /FBW ×10017.
After 12 h fasting, blood was collected and the
plasma was used to measure insulin and biochemical
parameters. Blood from fasted animals was collected
in tubes containing EDTA and centrifuged at 3500
rpm and the plasma was collected for analysis.
Glucose concentration was determined using a
glucometer (Accu-Chek Performa, 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 levels were
measured using the enzyme-linked immunosorbent
assay (ELISA) method using commercial kits (EMD
Millipore Corporation, Billerica, MA, USA). The
homeostatic model of insulin resistance (HOMA-
IR) was used as an insulin resistance index,
calculated according to the following formula:
HOMA-IR= (fasting glucose (mmol/L) × fasting
insulin (μU/mL)) / 22.518.
Systolic blood pressure (SBP) evaluation was
assessed in conscious rats by the non-invasive
tail-cuff method with a Narco Bio-Systems®
electrosphygmomanometer (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 vasodilation19. After
this procedure, a cuff with a pneumatic pulse sensor
was attached to the tail of each animal. The cuff was
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.
raGE lEvEls
Renal tissue (±150 mg) was homogenized (ULTRA-
TURRAX® T 25 basic IKA® Werke, Staufen,
Germany) in 1.0 mL of phosphate-buffered saline
(PBS) pH 7.4 cold solution and centrifuged at 800 g
at 4 °C for 10 min. The supernatant (100 μL) was
used in analysis. Receptors for advanced glycation
end products (RAGE) levels were measured using
the enzyme-linked immunosorbent assay (ELISA)
method using commercial kits from R&D System,
Minneapolis, USA (DY- 1616; 4000- 31.3 pg/mL of
detection). The results were corrected according to
the protein amount.
aGEs lEvEls
Most AGEs have a characteristic fluorescence.
Thus, the determination of AGEs was based on
spectrofluorometric detection according to Henle et
al.(1991)20 and Münch et al. (1997)21. Plasma and
Braz. J. Nephrol. (J. Bras. Nefrol.) 2021. Ahead of print
Oryzanol and AGE/RAGE axis
4
urine were diluted 1:20 with PBS (phosphate buffer)
pH 7.4 and fluorescence intensity was recorded in
emission maximum (440 nm) upon excitation at 370
nm (spectrofluorometer Fluoromax-3, Jobin Yvon
Horiba, USA). Fluorescence intensity is expressed in
arbitrary units (UF/mg protein).
rEnal inflammatory paramEtErs
Inflammation itself is a risk factor for renal function
loss22. The activation of RAGE leads to a sequence of
signaling with activation of inflammatory response1.
Renal tissue (±150 mg) was homogenized (ULTRA-
TURRAX® T 25 basic IKA® Werke, Staufen, Germany)
in 1.0 mL of phosphate-buffered saline (PBS) pH 7.4 cold
solution and centrifuged at 800 g at 4°C for 10min. The
supernatant (100 μL) was used in the analysis. Tumoral
necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and
monocyte chemoattractant protein-1 (MCP-1) levels
were measured by ELISA method using commercial kits
from R&D Systems, Minneapolis, USA (TNF-α: DY 510;
IL-6: DY 506; MCP-1). The TNF-α limit of detection
was 4000-62.5 pg/mL, IL-6 limit of detection was 8000-
125 pg/mL, and the MCP-1 limit was 1000-15.6 pg/mL.
The supernatant (100 μL) was used for analysis, and the
results were corrected according to the protein amount.
rEnal protEin carbonylation
Carbonylation is an irreversible protein modification
induced by reactive oxygen species (ROS). It can be
produced by oxidative cleavage of the backbone of
the protein or by an attack by ROS radicals on some
specific amino acids in the side chains such as lysine,
arginine, proline, or threonine. Protein carbonyls are
the most widely used markers to measure oxidative
protein damage23.
The supernatant described above was used for
renal protein carbonylation analysis. Carbonylated
proteins were measured by an unspecific method that
uses DNPH (2,4-dinitrophenylhydrazine derivatizing
agent) and photometric detection of any modified
protein by carbonylation24. Carbonylated protein
levels are expressed in nmol of DNPH/mg of protein.
rEnal nox-4 GEnE ExprEssion
Of the NOX isoforms, Nox4 is abundantly expressed
in the kidney and is an important source of renal ROS
triggered by RAGE25. Total RNA was extracted from
renal tissue using the reagent TRIzol (Invitrogen).
The SuperScript II First-Strand Synthesis System for
RT-PCR (Invitrogen) kit was utilized for the synthesis
of 20 mL of complementary DNA from 1000 ng of
total RNA. The mRNA levels of NOX-4 (assay Rn
00585380_m1; Applied Biosystems) were determined
by real-time PCR. Quantitative measurements
were made with a commercial kit (TaqMan qPCR;
Applied Biosystems) in a detection system (StepOne
Plus; Applied Biosystems). Cycling conditions were
as follows: enzyme activation at 50 ºC for 2 min,
denaturation at 95 ºC for 10 min; complementary
DNA products were amplified for forty cycles of
denaturation at 95 ºC for 15 s and annealing/extension
at 60 ºC for 1 min. Gene expression was quantified
in relation to the values of the Control group after
normalization by an internal control (cyclophilin:
assay Rn 00690933_m1; Applied Biosystems) by the
method 22DDCT, as described previously26.
rEnal function
After the collection of 24-h urine from the metabolic
cages, the renal function was evaluated considering
the glomerular filtration rate (GFR = (urine creatinine
× flux)/plasma creatinine)10 and the protein/creatinine
ratio, since it reflects proteinuria and is considered a
marker of kidney function27.
statistical analysis
Results are reported as means ± standard deviation
(SD) or median (interquartile range). Differences
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 p value of 0.05
was considered statistically significant.
results
Figure 1 presents the nutritional parameters of groups. It
is possible to verify that both groups that received HSF
diet (HSF and HSF + γOz groups) presented increased
adiposity index, insulin resistance, dyslipidemia, and
systolic blood pressure compared to control group. No
effect of γOz on these parameters was observed.
The plasma AGEs levels were the same in the HSF
and HSF + γOz groups and higher than the control
group. The HSF group presented lower urine AGE
and increased kidney RAGE compared to the control
group. The HSF + γOz group presented increased
AGEs levels in urine and reduced RAGE levels
compared to the HSF group (Figure 2).
Braz. J. Nephrol. (J. Bras. Nefrol.) 2021. Ahead of print
Oryzanol and AGE/RAGE axis
5
Kidney oxidative stress parameters are presented
in Figure 3. The HSF group presented increased NOX-
4 gene expression compared to the control group.
The treatment with gamma-oryzanol was effective to
reduce carbonylation and NOX-4 gene expression in
the HSF + γOz compared to the HSF group.
Figure 4. Kidney inflammatory parameters. (A)
Interleukin-6 (IL-6, pg/g protein); (B) Tumoral necrosis
factor alpha (TNF-α, pg/g protein); (C) Monocyte
chemoattractant protein -1 (MCP-1, pg/g protein).
Data are reported in means ± standard deviations
or medians and interquartile ranges (n = 7 animals/
group). Comparison by one-way ANOVA with Tukey
post-hoc test. *p < 0.05. HSF- high sugar-fat diet;
HSF + γOz- high sugar-fat diet + gamma-oryzanol.
The renal function parameters are presented
in Figure 5. The HSF group presented increased
protein/creatinine ratio and lower GFR compared
to the control group. Otherwise, the HSF + γOz
group presented improvement in kidney function,
characterized by increased glomerular filtration rate.
This group also presented reduction in kidney injury,
Figure 1. Nutritional and obesity-related disorders parameters at the end of 30 weeks. (A) Final body weight (g); (B) Adiposity index (%); (C) HOMA-
IR; (D) Plasma triglycerides levels (mg/dL); (E) Systolic blood pressure (mmHg). Data are expressed in means ± standard deviations or medians
and interquartile ranges (n = 7 animals/group). Comparison by one-way ANOVA with Tukey post-hoc. *p < 0.05. HSF: high sugar-fat diet; HSF +
γOz: high sugar-fat diet + gamma-oryzanol.
Braz. J. Nephrol. (J. Bras. Nefrol.) 2021. Ahead of print
Oryzanol and AGE/RAGE axis
6
Figure 2. AGEs and RAGE levels. (A) Plasma AGEs levels (UF/mg protein); (B) Urine AGEs levels (UF/mg protein); (C) Kidney RAGE levels (pg/g
protein) (C). Data are reported in means ± standard deviations (n = 7 animals/group). Comparison by one-way ANOVA with Tukey post-hoc test.
*p < 0.05. HSF: high sugar-fat diet; HSF + γOz: high sugar-fat diet + gamma- oryzanol.
Figure 3. Kidney oxidative stress parameters. (A) Kidney protein carbonylation (nmol/mg protein); (B) NOX-4 relative gene expression. Data are
reported in means ± standard deviations (n = 7 animals/group). Comparison by one-way ANOVA with Tukey post-hoc test. *p < 0.05. HSF: high
sugar-fat diet; HSF + γOz: high sugar-fat diet + gamma-oryzanol.
with lower proteinuria (protein/ creatinine ratio)
compared to the HSF.
dIscu ssI on
The aim of this study was to evaluate the effect of
γOz in reducing renal inflammation and oxidative
stress by modulating the AGEs/RAGE axis in animals
submitted to a high sugar-fat diet. Obesity is a
condition associated with several disorders considered
risk factors for renal disease development, among
them, central obesity, increased triglyceride levels,
low high-density lipoproteins, hypertension, and
elevated fasting glucose28. Some possible pathways
for inducing kidney disease are insulin resistance
Braz. J. Nephrol. (J. Bras. Nefrol.) 2021. Ahead of print
Oryzanol and AGE/RAGE axis
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Figure 4. shows the renal inflammatory parameters. The HSF group presented increased pro-inflammatory parameters levels in comparison to the
control group while the HSF + γOz group presented lower IL-6, TNF-α, and MCP-1 levels than the HSF group.
Figure 5. Renal function parameters. (A) Urine protein/creatinine ratio; (B) Glomerular filtration rate (GFR, mL/min). Data are reported in medians
and interquartile ranges (n = 7 animals/group). Comparison by one-way ANOVA with Tukey post-hoc test. *p < 0.05. HSF: high sugar-fat diet; HSF
+ γOz: high sugar-fat diet + gamma-oryzanol.
and chronic inflammation, a major contributor to
microvascular remodeling; dyslipidemia and excessive
nutrient availability that may induce mitochondrial
dysfunction; adipokines unbalance; the renin-
angiotensin system; and oxidative stress. Our results
showed no effect of γOz on the metabolic parameters,
in contrast to the literature which shows positive
effects, especially on glucose levels and dyslipidemia.
This divergence can be explained by the use of different
animal models, dose of γOz, and time of treatment.
Our study treated male Wistar rats with 0.5% γOz
in the chow for 10 weeks, a dose correspondent to an
average consumption of 50 mg/day of γOz. Wang et
al. (2015)29. fed male Sprague Dawley rats with a high-
fat and high-fructose diet supplemented with 0.05%
of FA or 0.16% of γOz for 13 weeks and they found
Braz. J. Nephrol. (J. Bras. Nefrol.) 2021. Ahead of print
Oryzanol and AGE/RAGE axis
8
that FA and γOz exhibited similar effects in alleviating
obesity, hyperlipidemia, hyperglycemia, and insulin
resistance. Cheng et al. (2013)30 studied the effect of
a diet with 15% of palm oil with the addition of 5.25
g of gamma-oryzanol for 5 weeks in male Wistar rats
with type 2 diabetes induced by streptozotocin. No
effect of γOz was observed on body fat, glucose, and
insulin; however, insulin resistance (area under the
curve), triglycerides, and LDL cholesterol levels were
reduced with the compound.
The role of inflammation in chronic kidney
disease (CKD) pathogenesis and progression has been
recognized since the late 1990s when IL-1 levels were
associated with major complications and increased
rate of mortality in patients undergoing chronic
dialysis. Following studies have demonstrated that
persistent inflammation is able to promote adverse
consequences to kidneys31. Evidence shows that
inflammation and inflammatory reactions from any
cause can modify or interfere with the intrarenal
microcirculatory regulation and perfusion distribution
and can induce renal damage, thus enhancing kidney
disease progression31. Our results showed that the
inflammatory response in the kidney was attenuated
in the HSF group treated with γOz , corroborating the
anti-inflammatory effect of the compound.
Oxidative stress pathogenesis in CKD patients
has been well documented in the current literature32.
Excessive production of ROS results in the activation
of several enzymatic systems such as nicotinamide
adenine dinucleotide phosphate (NADPH) oxidase and
the mitochondrial respiratory chain, and, together with
impaired antioxidant defense mechanisms, are the main
factors for the oxidative stress condition that occurs in
CKD, which leads to oxidation of macromolecules, tissue
damage, and dysfunction. Therefore, excess generation
of ROS has been directly linked to disease mechanisms
and processes associated with CKD initiation and
progression, including proteinuria, arterial hypertension,
and diabetes mellitus. Moreover, the association among
oxidative stress and chronic and endothelial dysfunction
maintain and perpetuate the vicious circle where chronic
kidney damage generates more kidney injury and systemic
complications of CKD, as cardiovascular dysfunction.
Evidence shows that oxidative stress is already present
even in the early stages of CKD with increased NADPH
oxidase production, especially NADPH subunit NOX-4.
Oxidized lipoprotein particles, as carbonylated proteins,
have been shown to accumulate in CKD as renal
dysfunction progresses 33. Thus, since our results show
that the HSF + γOz group presented reduced protein
carbonylated and NOX-4 levels compared to the HSF
group, we can confirm the antioxidant effect of γOz.
The metabolic changes present in the HSF
groups are able to lead to renal inflammation and
oxidative stress activation pathways. However, even
with no effect of γOz, the treated animals showed
reduced inflammation and oxidative stress. This
can be explained by the effect of γOz on modulating
the AGEs/RAGE axis in the HSF + γOz animals.
AGEs are complex fluorescent products and they
are formed through different pathways involving a
direct glycation followed by re-arrangements or by
the reaction of reactive carbonyl species (RCS) such
as glyoxal, methylglyoxal, and 3-deoxyglucosone,
which are formed through degradation pathways of
sugars. Based on the AGEs formation mechanism,
we can consider that in the present animal model,
AGEs are formed by the increased sugars present
in the diet leading to a direct protein glycation
and then fluorescent products by re-arrangement
reactions. AGEs induce oxidative stress through
RAGE activation as demonstrated here by NOX-4
upregulation, which further sustains AGEs formation
by forming RCS species through reducing sugar
oxidation. Oxidative stress also promotes protein
carbonylation and advanced lipoxidation end products
(ALEs) formation, which result either from a direct
oxidation or by RCS from lipid peroxidation, such
as 4- hydroxynonenal (HNE), MDA, and acrolein34.
The above-mentioned events were confirmed in the
study, and in particular we found that the increase
of RAGE and NOX-4 expression in the kidney were
accompanied by an increase of protein carbonylation.
AGEs were found to be reduced in the urine of
HSF in comparison to control animals, and this can
be explained by considering a reduced excretion of
these protein adducts. The mechanism explaining this
reduction is being evaluated, and one explanation
could be that the AGEs are trapped by RAGE whose
content has increased in the kidney of HSF animals,
thus reducing the urinary content of the AGEs. Hence
in HSF animals the AGEs/RAGE axis is activated
leading to an oxidative stress response, which
promotes an inflammatory condition as observed
by finding an increase of IL-6, TNF-α, and MCP-1.
γOz was found to significantly reduce all the above-
mentioned events and in particular the AGEs/RAGE
Braz. J. Nephrol. (J. Bras. Nefrol.) 2021. Ahead of print
Oryzanol and AGE/RAGE axis
9
axis by increasing the urinary excretion of AGEs
and reducing RAGE expression, and the oxidative
damage, by reducing protein oxidation and finally the
anti-inflammatory response35. The present paper does
not indicate through which mechanisms γOz elicits
such a protective mechanism but can be speculated
based on the literature. Very recently, Sobhy et al.
(2020)15 found that oryzanols inhibit glycation and
AGEs formation by a direct anti-glycation effect and
by scavenging the free radicals generated during the
glycation reactions. Furthermore, the antioxidant
activity of γOz has been reported in both in vitro and
in vivo experiments by several groups10,36,37.
In summary, this study found that the group
treated with gamma-oryzanol showed reduced kidney
RAGE levels, inflammation, and oxidative stress and
increased renal AGEs excretion probably due the
positive effect on glomerular filtration rate. Thus, it
is possible to conclude that the gamma-oryzanol was
effective in reducing inflammation and oxidative stress
in the kidney by modulating the AGEs/RAGE axis.
It is important to report some limitations of this
study. Only general, and not specific AGEs or RAGE,
were analyzed, more oxidative stress and inflammation
markers could be included in the parameters analyzed,
the immunohistochemical analysis for RAGE could have
been performed, as well as histological analysis of the
kidneys to demonstrate possible damage to structures.
acknowled gMe nts
The authors thank to Universitá degli Studi di Milano
and the funding by Fundação de Amparo à Pesquisa
do Estado de São Paulo (FAPESP) - process
#2015/10626-0 e #2018/15294-3- and CAPES (PDSE
- 88881.132505/2016-01).
autho rs’ contrI but Ion
Conceptualization: Francisqueti- Ferron FV; Minatel
IO; Aldini G; Correa CR. Data curation: Francisqueti-
Ferron FV; Garcia JL; Ferron AJT; Altomare AA;
Moreto F. Formal analysis: Francisqueti-Ferron FV;
Minatel IO; Ferron AJT; Altomare AA; Ferreira ALA;
Aldini G; Correa CR; Funding acquisition: Correa
CR. Methodology: Francisqueti-Ferron FV; Garcia
JL; Ferron AJT; Altomare AA; Moreto F; Minatel
IO. Project administration: Francisqueti-Ferron FV;
Correa CR. Writing - original draft: Francisqueti-
Ferron FV; Minatel IO; Aldini G; Correa CR.
confl Ict of Intere st
The authors declare no conflict of interest.
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