The Nephroprotective effect of resveratrol in aluminum chloride
induced toxicity in rats
.
Abstract
Methods: This study was conducted from April 2015 to June 2015 at King Khalid
University, Abha, Kingdom of Saudi Arabia (KSA). The experiments were performed on 36
male Wistar rats. The rats were randomly allocated into six groups; group A: control, group
B: received 1 ml of saline solution containing of 20% hydroxypropyl cyclodextrin, group C:
received RES, group D: received aluminium chloride (20 mg/kg bwt) dissolved in 1 ml
normal saline for 40 days. group E: received 1 ml solution of ALCl 3 dissolved in normal
saline (20 mg/kg bwt) with a concomitant dose of 1 ml of saline solution containing 20%
hydroxypropyl cyclodextrin vehicle and group F: received 20 mg/kg bwt RES in a total
volume of 1 ml and a concomitant dose of ALCl3 saline solution.
After 40 days of
treatments, urine and blood were collected and histopathological assay was performed.
Results: all findings clearly showed significant alteration in kidney function and
histopathological status after AlCl3 exposure associated with increased renal oxidative stress
and inflammation suggesting strong pro-oxidant activity of AlCl3 in spite of its non-redox
status. Astonishingly, RES co-treatment with AlCl3 to the rats showed significant
improvement in all biochemical and histological parameters related to kidney function and
structure.
Conclusion, this study is the first in literature that shows a protective effect of RES against
ALCL3 induced renal damage.
Address correspondence and reprint request to:
1
INTRODUCTION:
There is an increasing concern about aluminum (Al) poisoning in humans and several
studies have highlighted that Al has the potential to be toxic for both human and
animals1. In 2007, Al was included in the priority list of hazardous substances
identified by The Agency for Toxic Substances and Disease Registry2.
Al is widely distributed in the environment and extensively used in daily life, which
causes its easy exposure to human beings3. It gets access to the human body via the
gastrointestinal and the respiratory tracts3. The sources of Al exposure including
foods especially corn, yellow cheese, grain products (flour), salt, herbs, spices, tea
leaves, many food products, vegetables, cereals, beverages cosmetics, cookware, cans
and containers. Also, Al is added to drinking water for purification purposes4. Al
compounds are also widely used in medicines, e.g., antacids, phosphate binders,
buffered aspirins, vaccines and injectable allergens4. Moreover, environmental
pollution with different Al-containing compounds, especially industrial waste water,
exposes people to higher than normal levels of Al5.
Because excretion in the urine is the primary route by which the Al is eliminated from
the body, chronic Al exposure could increase the risk of developing Al retention, and
consequently, Al induced toxicity in the kidneys6. The nephrotoxic actions of Al arise
from its accumulation in the kidneys, with the resultant degeneration of the renal
tubular cells. It has been suggested that Al generates reactive oxygen species that
cause the oxidative damage to cellular lipids, proteins, and DNAs7, 8. Also, Al
intoxication has been reported to cause a decrease in the intracellular levels of
reduced glutathione9. Also, salts of Al may inhibit enzymes like acid and alkaline
phosphatases, phosphodiesterase and phosphooxydase10 .
2
Natural antioxidants can reduce the risk of various diseases and also protect tissues
and organs from various environmental contaminants. In this regard, Resveratrol
(RES) might be a promising agent in the treatment of Al induced renal toxicity. RES,
a phenolic compound that is found in various plants, especially red grapes and their
derivatives, has demonstrated many beneficial effects, including anti-inflammatory
and antioxidant roles by enhancing the production of antioxidant enzymes
modulating nuclear factors involved in the inflammation-oxidative stress cycle
and
11, 12
.
A growing body of evidence indicates that RES may play potential therapeutic roles
by acting on a large number of target molecules mediating the abovementioned
protective effects including the endothelial nitric oxide synthase (eNOS)13, the
mitogen-activated protein kinase (MAPK)14, the hemeoxygenase-1(HO-1) and the
nuclear factor E2-related factor-2 (Nfr2) and nuclear factor-kappa B (NF-κB)15.
In spite of these studies, after searching the search engines and pubmed, no single
study has demonstrated the effect of RES on Al induced kidney toxicity in relation to
oxidative stress. Therefore, the aim of the present study is to investigate the potential
protective effect of RES on aluminum chloride (ALCl3)-induced nephrotoxicity in
rats.
MATERIALS AND METHODS
Resveratrol (RES) is only commercially available as the trans-isomer (transResveratrol), and the stable and pharmacologically active form of RES was purchased
from Sigma-Aldrich (St. Louis, MO, USA). RES was prepared by dissolving in a
saline solution (0.9% NaCl) of 20% hydroxypropyl cyclodextrin (American MaizeProducts Co., Hammond, IN, USA) to the desired final volume used in the
3
experimental procedure. Aluminium chloride (ALCl3) in crystalline form was
obtained from Sigma-Aldrich (St. Louis, MO, USA) and was dissolved in 0.9% saline
to the desired final volume used in the experimental procedure.
Assay kits for
determination of Malondialdehyde (MDA, Cat No. NWK-MDA01) were purchased
from NWLSS, USA. Assay kit for determination of superoxide Dismutase (SOD, Cat
NO.706002), glutathione peroxidase activity (GPx, Cat NO.703102) and reduced
glutathione (Cat NO.703002) were purchased from Cayman Chemical, MI, USA.
Kinetic reagents for determination of blood urea (Cat., no. 283-30) were purchased
from Diagnostic Chemicals Limited. Assay colorimetric kit for determination serum
and urinary Creatinine (Cr) concentration (Cat., no. 700460 & Cat., no. 500701;
respectively) were purchased from Cayman Company (Ann Arbor, MI, USA). All
analyses were performed in accordance with the manuals provided by the
manufacturers.
Animals
The experiments were performed on 36 male Wistar rats of eight weeks old and have
an initial body weight of 190- 200 g. They were supplied from the animal house at the
college of medicine of King Khalid University. The rats were fed with standard
laboratory diets, given water ad libitum and maintained under laboratory conditions of
temperature 22°C (±3°C), with 12 h light and 12 h dark cycle. The experimental
procedures involving the handling and treatment of animals were approved by the
research ethical committee of the medical college at King Khalid University and all
procedures were conducted in accordance with the National Institute of Health’s
Guide for the Care and Use of Laboratory Animals.
4
Experimental Procedure:
After an adaptation period of one week, the rats were randomly allocated into six
groups ( n=6) :
Group A: Control animals and they received 1 ml of normal saline solution (0.9%
NaCl).
Group B: Received 1 ml of saline solution containing of 20% hydroxypropyl
cyclodextrin.
Group C: Received RES at a dose of 20 mg/kg body weight (bwt) in a total volume of
1 ml.
The dose selected for RES was based on a previous study that showed
antioxidant, anti-inflammatroy and anti-apoptotic effects of RES16.
Group D: Received aluminium chloride (20 mg/kg bwt, 1/ 20 LD50) dissolved in 1 ml
normal saline for 40 days. The LD 50 of (ALCl3) when administered orally to rats was
reported to be (380 - 400 mg/kg bwt)17. The dose selected for ALCl3 is based on
previous dose-response studies that showed the severe renal damage at a similar dose
and time administration18.
Group E: Received 1 ml solution of ALCl3 dissolved in normal saline (20 mg/kg bwt)
with a concomitant dose of 1 ml of saline solution containing 20% hydroxypropyl
cyclodextrin vehicle.
Group F: Received 20 mg/kg bwt RES in a total volume of 1 ml and a concomitant
dose of ALCl3 saline solution (20 mg/kg bwt).
All treatments were continued for 40 days on daily basis. Treatments were given to all
groups orally i.e. oral gavage. During this period, the rats were weekly adapted to
metabolic cages (These cages offer big space to rats to move freely without any
restrained stress). At the end of day 40 and after a 12 h fasting, rats were placed in
5
their metabolic cages and the and urine samples were collected into tubes containing
20 µL of 2.5 mol/L HCl over 24 h. Then, the volume of urine were measured
individually and filtered with 0.2 µm Millipore filters and stored at -78°C to measure
the levels of urinary Creatinine (Cr) levels. After collection of urine, all rats were
anesthetized with light diethyl ether and 2 mL blood was collected directly by cardiac
puncture, placed into plain tubes which was allowed to clot and then centrifuged at
5000 rpm for 10 min at room temperature to collect serum. Serum samples were
stored at - 80°C for further analysis of urea and creatinine levels using commercial
available kits according to manufacturer’s instruction
Creatinine (Cr) Clearance
Cr clearance (Ccr) was calculated using the following equation19:
Ccr (mL/min/kg) = [urinary Cr (mg/dL) × urinary volume (mL) / serum Cr
(mg/dL)]×[1000/body weight (g)]×[1/1440 (min)]
Preparation of Tissue Homogenates
Immediately after blood collection, The animals were returned to their cages and
allowed to recover to 48 hours after which animals were weighted and then
anesthetized by diethyl ether and killed by decapitation. Both kidneys form each rat
of each group were quickly collected, weighted and washed with phosphate buffered
saline, pH 7.4, containing 0.16 mg/mL heparin to remove any red blood cells or clots.
Then one kidney from each rat was homogenized with an ultrasonic homogenizer in
cold phosphate buffer, pH 7.0 with Ethylenediaminetetraacetic Acid (EDTA), for
Malondialdehyde (MDA) measurement and with cold 20 mM N-(2-hydroxyethyl)
piperazine-N’-2-ethanesulfonic acid (HEPES) buffer, pH 7.2, containing 1 mM
ethyleneglycol-bis (2- aminoethoxy)-tetraacetic acid (EGTA), 210 mM mannitol and
6
70 mM sucrose for superoxide dismutase (SOD) activity measurements. Other parts
of the kindeys were homogenized in cold buffer consists of 50 mM trisHCl, pH 7.5, 5
mM ED-TA, 1 nM DTT for determination of Glutathione Peroxidase (GPx) activity
and the levels of reduced Glutathione (GSH). All supernatants were kept in separate
tubes and stored at - 20. However, the other kidney obtained from each rat was placed
in 10% formalin solution for histopathological evaluation.
Measurement of Malondialdehyde (MDA)
Levels of Lipid peroxidation in the kidney homogenates were measured by the
thiobarbituric acid reaction20 using the commercially available kits as per manufacture
instructions. This method was used to measure spectrophotometrically the colour
produced by the reaction of TBA with Malondialdehyde (MDA) at 532 nm. For this
purpose, MDA levels were measured using a commercial assay as the
Malondialdehyde Assay (Cat No. NWK-MDA01) supplied from NWLSS, USA. In
brief, tissue supernatant (50 µL) were added to test tubes containing 2 µL of
Butylated Hydroxytoluene (BHT) in methanol. Next, 50 µL of acid reagent (1 M
phosphoric acid) was added and finally 50 µL of TBA solution was added. The tubes
were mixed vigorously and incubated for 60 min at 60°C. The mixture was
centrifuged at 10,000× g for 3 min. The supernatant was put into wells on a
microplate in aliquots of 75 µL and its absorbance was measured with a plate reader
at 532 nm. MDA levels were expressed as nmol/mg protein.
Measurement of Superoxide Dismutase (SOD) Activity
SOD activities in the kidneys homogenates of all groups were measured using the
supplied commercially assay kit according to the manufacturer’s instructions. The
7
SOD assay consisted of a combination of the following reagents: 0.3 mM xanthine
oxidase, 0.6 mM diethylenetriaminepenta acetic acid (DETAPAC), 150 µM Nitroblue
Tetrazolium (NBT), 400 mM Sodium Carbonate (Na2CO3) and bovine serum
albumin (1 g L−1). The principle of the method is based on the inhibition of NBT
reduction by superoxide radicals produced by the xanthine/xanthine oxidase system.
For the assay, standard SOD solutions and tissue supernatant (10 µL) were added to
wells containing 200 µL of NBT solution that was diluted by adding 19.95 mL of 50
mM Tris-HCl, pH 8.0, containing 0.1 mM DETAPAC solution and 0.1 mM
hypoxanthine. Finally, 20 µL of xanthine oxidase was added to the wells at an interval
of 20 s. After incubation at 25°C for 20 min, the reaction was terminated by the
addition of 1 mL of 0.8 mM cupric chloride. The formazan was measured
spectrophotometrically by reading the absorbance at 560 nm with the help of plate
reader. One Unit (U) of SOD is defined as the amount of protein that inhibits the rate
of NBT reduction by 50%. The calculated SOD activity was expressed as U/mg
protein.
Measurement of Glutathione Peroxidase
(GPx) Activity Glutathione peroxidase activities in kidneys homogenates were
measured using the commercially supplied kit provided as per manufacture
instructions. Glutathione peroxidase catalyzes the reduction of hydroperoxides,
including hydrogen peroxide, by reduced glutathione and functions to protect the cell
from oxidative damage. With the exception of phospholipid hydroperoxide GPX, a
monomer, all of the GPX enzymes are tetramers of four identical subunits. Each
subunit contains a selenocysteine in the active site, which participates directly in the
two-electron reduction of the perox-ide substrate. The enzyme uses glutathione as the
8
ultimate electron donor to regenerate the reduced form of the selenocysteine. The
Cayman Chemical Glutathione Peroxidase Assay Kit measures GPx activity indirectly
by a coupled reaction with Glutathione Reductase (GR). Oxidized Glutathione
(GSSG) is produced upon reduction of hydroperoxide by GPX and is recycled to its
reduced state by GR and NADPH. The oxidation of NADPH to NADP+ is
accompanied by a decrease in absorbance at 340 nm. Under conditions in which the
GPX activity is rate limiting, the rate of decrease in the A340 is directly proportional
to the GPX activity in the sample. The results were presented as nmol/min/g protein.
One unit is defined as the amount of enzyme that causing the oxidation of 0.1 nmol of
NADPH to NADP+/min at 25°C.
Glutathione Assay
The levels of reduced GSH were measured using Cayman GSH assay kit which
involved an optimized enzymatic recycling method and GR. The sulfhydryl group of
GSH reacts with 5,5-ditho-bis-2-nitrobenzoic acid (DTNB) and produces yellow
colored 5-Thio-2- Nitrobenzoic Acid (TNB). A mixed disulfide, GSTNB (between
GSH and TNB), is also produced that is reduced by GR to recycle GSH, thereby
producing more TNB. The rate of TNB production is directly proportional to this
recycling reaction which is in turn directly proportional to the concentration of GSH
in the sample. The absorbance of TNB at 410 nm was used to estimate the amount of
GSH in the sample. The GSH level was expressed as µmol/mg protein.
9
Inflammatory mediators assay:
Levels of TNF-α and IL-6 in kidney homogenates were determined by ELISA (Cat
no. ab46070, Abcam, Cambridge, MA, USA and Cat No. ELR-IL6-001, RayBio, MO,
USA, respectively). As per the manufacturer’s instruction, 100 μl of homogenate
supernatant was used in the reaction and the intensity of the developed color at 450
nm was directly proportional to the concentration of TNF-α and IL-6 contained in the
samples. LV levels of TNF-α and IL-6 levels were expressed as pg/mg protein.
Histopathological Evaluation
Kidney specimens from all groups of rats were processed routinely in 10% formalin
solution and embedded in paraffin. Tissue sections of 5 µm were obtained and stained
with Hematoxylin and Eosin (H&E). All histopathological examinations were
performed under a light microscope (NIKON, Japan) by different histologists at the
college of medicine at King Khalid University who were blinded to all tissue
specimens regarding their group. A minimum of 10 fields for each kidney were
examined.
Statistical analysis:
Statistical analyses were performed by using Graphpad prism statistical software
package (version 6). The data are represented as the mean ± SD. All comparisons
were analyzed by one-way ANOVA followed by post hoc tukeys t- test and accepted
as significant at P < 0.05.
10
RESULTS
Changes in Body Weight and relative Kidneys Weight
Changes in the final body and relative kidneys weight are shown in figure 1 (A & B).
In all experimental groups under various treatments, no mortality was observed during
the period of the study. In this study, significant decrease (p<0.01) in final body
(20.4%) and relative kidneys weights (23.7%) were observed in ALCl3 intoxicated
animals when compared to control rats received NS. However, no significant changes
in both parameters were seen in the control rats received the vehicle or resveratrol
(RES) and their levels were within the control values. In contrast, ALCl3 group of
rats received RES showed significantly higher the weights of their body and relative
kidneys weight kidneys (P = 0.011, P = 0.008, respectively) than their corresponding
levels seen in ALCl3 intoxicated rats. No significant difference in these parameters
when comparing ALCl3+RES group with the control group.
Markers of Kidney Function
Indices of kidney functions are shown in Figure 2. There was no significant changes
in the levels of urea and creatinine (Cr) and urinary levels of Cr or urine volume in the
control rats received the vehicle or RES when compared to control rats. However, the
levels of serum urea and Cr significantly (P < 0.0001) increased (127.2% & 275.5,
respectively) where urine creatinine levels and urine volume were significantly (P <
0.0001) decreased (53.9% & 64.5%, respectively) in ALCl3 intoxicated rats in
comparison to control group. ALCl3 intoxication caused significant reductions (P <
0.0001) in Cr clearance in both ALCl3 intoxicated rats received NS or vehicle
(95.7% & 94.9%, respectively), Figure 3. Interestingly, the levels of all these
11
parameters showed significant improvement toward their normal levels when RES
was concomitantly administered with ALCl3.
Oxidative Stress Parameters in the Renal Homogenates:
Figure 4 showed no significant change in the levels of Malondialdehyde (MDA) and
reduced glutathione (GSH) or in the activities of total superoxide dismutase (SOD)
or glutathione peroxidase (GPx) in the groups of control rats received NS or vehicle.
Significant depression (P < 0.001) in the activities of SOD (54.6% & 64.5%), GPX
(50.44 % & 48.26%) & as well as levels of GSH (51.6% & 50.4%) with a concurrent
significant enhancement ( P < 0.0001) in levels of MDA (235% & 208.3%) were
reported in the renal homogenates of ALCl3 intoxicated animals received NS or
vehicle, respectively. Meanwhile, concomitant administration of RES with ALCl3 to
rats resulted in significant increases (P < 0.001) in the activities of SOD and GPX and
in GSH levels and resulted in a significant decrease ( P < 0.0001) in MDA levels in
their renal homogenates as compared to ALCl3 intoxicated rats.
Inflammatory cytokines:
Figure 5 showed significant elevations (P < 0.001) of three fold increases in TNF-α
and IL-6 in AlCl3 intoxicated animals as compared to control animals. However,
concomitant administration of RES with AlCl3 resulted in significant decreases (P <
0.001) in the levels of both cytokines as compared to AlCl3 intoxicated animals.
12
Histopathogical Findings of Liver and Kidney
Histology of kidney in the control, vehicle or RES treated rats showed normal
structure, Figure 6 (A- C). kidney sections of ALCl3 intoxicated group (D- F) showed
mild thickening of the basement membrane along with mild change in the density of
mesenchyme, atrophy and degeneration of glomerular capillaries with increased
Bowman’s space (urinary space) and mild tubular necrosis. Macrophage infiltration
was also dominant (D-F). However, rats administered ALCl3 and RES (G-H) showed
improvement in their histological architectures including normal glomerulus, normal
basement membrane and capillaries. Moreover, Bowman’s space (urinary space) and
tubular necrosis were improved towards the normal condition. Macrophage
infiltration was rarely seen.
13
Figure 1: Changes in the absolute total and kidneys weights in all groups (n=6) .
Values are expressed as Mean ± SD. Values were considered significantly different at
P < 0.05. aSignificantly different when compared to Control group. bSignificantly
different when compared to Control+Vehicle cSignificantly different when compared
to Control+ RES. dSignificantly different when compared to ALCl3. eSignificantly
different when compared to ALCl3+RES.
14
Figure 2: kidney functions parameters in the serum and urine of all groups (n = 6).
Values are expressed as Mean ± SD. Values were considered significantly different at
P < 0.05. aSignificantly different when compared to Control group. bSignificantly
different when compared to Control+Vehicle cSignificantly different when compared
to Control+ RES. dSignificantly different when compared to ALCl3. eSignificantly
different when compared to ALCl3+RES.
15
Figure 3: kidney Creatinine clearance values in all groups (n = 6). Values are
expressed as Mean ± SD. Values were considered significantly different at P < 0.05.
a
Significantly different when compared to Control group. bSignificantly different
when compared to Control+Vehicle cSignificantly different when compared to
Control+ RES. dSignificantly different when compared to ALCl3. eSignificantly
different when compared to ALCl3+RES.
16
Figure 4: Oxidative stress related parameter evaluation in the renal homogenates of
all groups (n = 6) . Values are expressed as Mean ± SD. Values were considered
significantly different at P < 0.05. aSignificantly different when compared to Control
group. bSignificantly different when compared to Control+Vehicle cSignificantly
different when compared to Control+ RES. dSignificantly different when compared to
ALCl3. eSignificantly different when compared to ALCl3+RES.
17
Figure 5: Levels of tumor necrosis factor-α (TNF-α) and interlukin 6 (IL-6) in the
renal homogenates of in all groups (n = 6) . Values are expressed as Mean ± SD.
Values were considered significantly different at P < 0.05. aSignificantly different
when compared to Control group.
Control+Vehicle
d
c
b
Significantly different when compared to
Significantly different when compared to Control+ RES.
Significantly different when compared to ALCl3. eSignificantly different when
compared to ALCl3+RES.
18
19
Figure 6: Photomicrographs of kidneys from A: control rat. B: Control + Vehicle
and C: Control +RES treated rats, these groups show normal architecture of kidney
with prominent Bowman’s capsule, epithelial Cells and normal tubules. D and E:
ALCl3 and F: ALCl3+Vehicle. The kidney sections of these rat shows mild thickening
of the basement membrane along with change in the density of mesenchyme, atrophy
and degeneration of glomerular capillaries with increased Bowman's space (urinary
space) and tubular necrosis. Glomerular capillaries severely damaged and shrieked in
some glumerulus. Also, there was severe infiltration (E). G and H: ALCl3 + RES
treated rat shows normal architecture of glomerular capillaries, intact epithelial cells
with the presence of some degeneration in the tubules and slight shrinkage of the
glomerular capillaries.
20
DISCUSSION:
The present study was undertaken to determine whether resveratrol (RES) can prevent
and/or reduce AlCl3 induced renal damage by examining different biochemical and
histological parameters related to kidney function of intoxicated and treated rats.
Findings clearly showed significant alteration in kidney function in histopathological
status after AlCl3 exposure associated with increased renal oxidative stress and
inflammation suggesting strong pro-oxidant activity of AlCl3 in spite of its non-redox
status
21
. However, RES co-treatment with AlCl3 to the rats showed significant
improvement in all biochemical and histological parameters related to kidney function
and structure. This data is the first evidence in the literature that shows that RES is
able to ameliorate AlCl3 induced nephrotoxicity by improving levels of endogenous
antioxidants and reduction of inflammatory biomarkers.
Aluminum (Al) accumulates in all tissues of mammals such as the kidneys, liver,
heart, blood, bones and brain22 and it was found that one of the main organs targeted
by Al exposure is the kidney which plays a major role in preventing accumulation of
Al by excreting it out through urine6. Different mechanisms of renal excretion of Al
have been suggested. These include glomerular filtration23, tubular reabsorption of
filtered Al and secretion in distal nephron24,25 and excretion in the distal tubules25.
Consequently, Al accumulates in the kidneys and induces in renal toxicity 21.
Indeed, Al accumulation in the kidney has been related to worsening renal function 21,
26
. It has been reported that the kidney may be exposed to high concentrations of Al
during the normal process of renal excretion making kidney vulnerable to Almediated toxicity which was dependent on the rout of exposure1 . In this regard, most
of the published studies have investigated the toxic effects of ALCl3 in animals after
21
intraperitoneal or parenteral administration which does not exhibit the main route of
human exposure1, 27.
It is well reported that Al enters the body via two major routes, pulmonary and oral.
Although only a small portion of Al is absorbed through the gastrointestinal tract, oral
intake is associated with the greatest toxicological implications1, 27. For this reason, in
this study, rats were treated with ALCl3 for 40 days through intra-gastric tube to
mimic chronic toxicity of Al as may occur in humans. The high dose of orally
administered ALCl3 was chosen because the intestine plays a role of a protective
barrier against aluminum toxicity since only a small fraction (0.1 to 0.5%) of ingested
aluminum is absorbed28.
This study showed that ALCl3 induced elevation in serum urea and creatinine levels
with the concomitant decrease in creatinine clearance. These findings accord to those
obtained by Katyal et al. (1997) who reported that Al has a significant role in the
pathogenesis of renal dysfunction and in many clinical disorders. Chronic exposure of
AlCl3 to rats has shown the nephrotoxicity and glomerular tuft, and renal tubules were
reported to be the primary sites of renal damage30. AlCl3 is excreted mainly by
kidneys and it causes marked degeneration of tubules. Increased serum urea and
creatinine concentration can be a consequence of critical accumulation of this metal in
kidneys and eventually resulting in renal failure31, 32 .
The kidneys are involved in the excretion of various xenobiotics, pollutants, toxins
and hence they are prone to liberate high quantities of free radicals which contribute
to high oxidative stress that is involved in the pathogenesis of kidney damage33. AlCl3
toxicity appears to be mediated, in part, by free-radical generation. Up to date, several
evidences have shown that the toxic effects associated with AlCl3 are due to the
22
generation of reactive oxygen species (ROS), which in turns results in the oxidative
deterioration of cellular lipids, proteins and DNA34, 35. It was demonstrated that Al
may alter the activity and levels of a number of components of tissue antioxidant
defense system, such as GSH, SOD leading to enhance production of free radicals
especially ROS and development of lipid peroxidation36 .
Lipid peroxidation of biological membranes leads to a loss of membrane fluidity,
changes in membrane potential, an increase in membrane permeability and alterations
in receptor functions37. In the same line, our data support these evidences; TBARS
levels as a marker of lipid peroxidation were significantly increased with a
concomitant decrease in the levels of GSH and activities of SOD and GPX in the
kidney homogenates of intoxicated rats. Although Al is not a transition metal, and
therefore, cannot initiate peroxidation, many studies have searched for mechanisms
between aluminum Al and oxidative damage in tissues37.
A previous study reported that exposure to Al could promote disruptions in the
mineral balance, resulting in Al ions replacing iron and magnesium, which would then
lead to a reduction in Fe2+ binding to ferritin38. Free iron ions released from biological
complexes by Al can catalyze hydroperoxides decomposition to hydroxyl radicals via
Fenton’s reaction. This high hydroxyl radical reactivity could initiate the peroxidation
of membrane lipids, causing membrane damage38.
ALCl3 resulted in a significant elevation in the levels of pro-inflammatory cytokines ;
including TNF-α and IL-6 which fits with many previous reports34,
39, 40
. Also, an
increase in the expression of TNFα mRNA has been reported in the cerebrum of mice
treated with aluminum via the drinking water41.
23
Kidney sections of AlCl3 treated group showed mild thickening of the basement
membrane along with mild change in the density of mesenchyme, atrophy and
degeneration of glomerular capillaries with increased Bowman’s space (urinary
space) and mild tubular necrosis with increase and dominant macrophage infiltration.
AlCl3 affects the Bowman’s capsule by increase thickening of its basement
membrane, which is the first step in the filtration of blood to form urine. AlCl3 also
resulted in atrophy of glomerulus capillaries and damaging of both proximal and
distal tubules. This finding accords with previous those findings of in vivo and in vitro
studies42, 43.
In this study, administration of RES to ALCl3 treated rats effectively improves renal
function, as concluded from (1) decreased serum urea and creatinine concentrations
and enhancing of Cr clearance (2) ameliorated oxidative stress and inflammatory
cytokines and (3) attenuated histological changes characteristic of AlCl3
nephrotoxicity. Previously, it has been reported that treatment with RES (5 mg or
10 mg/kg orally) for 2 weeks improved urinary protein excretion, renal dysfunction,
and renal oxidative stress in streptozotocin- (STZ-) induced diabetic kidneys44.
Furthermore, pretreatment with RES (25 mg/kg, intraperitoneal injection) attenuated
signs of cisplatin-induced renal injury45.
This study also suggested that the main mechanism by which RES acts in
ameliorating ALCl3 induced renal damage is due to its potent and anti-inflammatory
effect. Most of the previous studies accord with our finding as they have shown that
resveratrol can directly scavenge ROS such as superoxide and toxic hydroxyl radicals
46, 47
.
In addition to scavenging ROS, exogenously administered RES modulates the
expression and activity of antioxidant enzymes, such as
SOD, GPx, and catalase,
24
through transcriptional regulation via nuclear factor E2-related factor 2 (Nrf2),
activator proteins (AP)-1, forkhead box O (FOXO), and SP-1 or through enzymatic
modification44,
48
.
Kitada et. al. (2011) also reported that
RES treatment
(400 mg/kg, orally, administered at concentration of 0.3% resveratrol) alleviated
albuminuria and
histological mesangial expansion and
reduced the increased
levels of renal oxidative stress and inflammation in the kidneys of db/db mice through
the scavenging of ROS and normalizing manganese (Mn)-SOD function by
decreasing its levels of nitrosative modification.
Additionally, Chen et al. (2011) reported that RES treatment improved diabetesinduced glomerular hypertrophy and urinary albumin excretion;
reduced the
expression of glomerular fibronectin, collagen IV, and transforming growth factor;
reduced the thickness of the glomerular basement membrane; and reduced nephrin
expression in the kidneys of STZ-induced diabetic rats. Furthurmore, Wu et al.
(2012) demonstrated that RES has protective effects on diabetic kidneys by
modulating the SIRT1/FOXO1 pathway. As these mechanisms were proven as an act
of RES in diabetic model, they should not be neglected in our study and further
studies will be required to investigate them.
Conclusion, this study is the first in literature that shows a protective effect of RES
against ALCL3 induced renal damage. Reduction of oxidative stress and inflammation
may be considered as main pathways of action.
25
Acknowledgements
I would like to thank my collaborator Prof. Mohammed Hairdah , Professor of
Physiology and ethical committee member at KKU for his valuable advices and
support to accomplish my work. Also, I would like to thank Mr. Mohmoud Al
Khateeb, lecturer of physiology at college of medicine, KKU for all technical &
experimental support.
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References:
1. Krewski D, Yokel RA, Nieboer E, Borchelt D, Cohen J, Harry J, et al. Human
health risk assessment for aluminium, aluminium oxide, and aluminium
hydroxide. J Toxicol Environ Health B Crit Rev. 2007;10(S1):1-269.
2. Agency for Toxic Substances and Disease Registry, (ATSDR). Notice of the
revised priority list of hazardous substances that will be the subject of toxicological
profiles. Fed Regist. 2007;73:12178-9.
3. Kumar D, Idzikowski C, Wingate DL, Soffer EE, Thompson P, Siderfin C.
Relationship between enteric migrating motor complex and the sleep cycle.
Am J Physio. 1990;259:983-90.
4. Türkez H, Yousef MI, Geyikoglu F. Propolis prevents aluminium-induced genetic
and hepatic damages in rat liver. Food and Chemical Toxicology.
2010;48(10):2741-6.
5. Malekshah AK, Torabizadeh Z, Naghshwar F. Developmental toxicity of aluminum
from high doses of AlCl3 in mice. J Appl Res. 2005;5:575-9.
6. Stoehr G, Luebbers K, Wilhelm M, Hoelzer J, Ohmann C. Aluminum load in ICU
patients during stress ulcer prophylaxis. Eur J Intern Med. 2006;17(8):561-6.
7. Gonzalez MA, del Lujan Alvarez M, Pisani GB, Bernal CA, Roma MG, Carrillo
MC. Involvement of oxidative stress in the impairment in biliary secretory function
induced by intraperitoneal administration of aluminum to rats. Biological Trace
Element Research. 2007;116(3):329-48.
8. Newairy A-SA, Salama AF, Hussien HM, Yousef MI. Propolis alleviates
aluminium-induced lipid peroxidation and biochemical parameters in male rats.
Food Chem Toxicol. 2009;47(6):1093-8.
9. Gonzalez-Munoz M, Meseguer I, Sanchez-Reus M, Schultz A, Olivero R, Benedí J,
et al. Beer
consumption reduces cerebral oxidation caused by aluminum toxicity
by normalizing gene expression of tumor necrotic factor alpha and several
antioxidant enzymes. Food Chem. Toxicol. 2008;46(3):1111-8.
10. Ochmański W, Barabasz W. Aluminum--occurrence and toxicity for organisms.
Przeglad Lekarski. 2000;57(11):665-8.
11. Ghanim H, Sia CL, Abuaysheh S, Korzeniewski K, Patnaik P, Marumganti A, et
al. An antiinflammatory and reactive oxygen species suppressive effects
of an
extract of Polygonum cuspidatum containing resveratrol. J Clin
Endocrinol Metab. 2010;95(9):1-8.
12. Palsamy P, Subramanian S. Resveratrol protects diabetic kidney by attenuating
hyperglycemia-mediated oxidative stress and renal inflammatory cytokines via
Nrf2–Keap1
signaling. Biochim Biophys Acta-Molecular Basis of Disease.
2011;1812(7):719-31.
13. Wang S, Qian Y, Gong D, Zhang Y, Fan Y. Resveratrol attenuates acute hypoxic
injury
in
cardiomyocytes: Correlation with inhibition of iNOS–NO signaling pathway. Eur.
J Pharm Sci.2011;44(3):416-21.
27
14. Yu H-P, Hwang T-L, Hsieh P-W, Lau Y-T. Role of estrogen receptor-dependent
upregulation of P38 MAPK/heme oxygenase 1 in resveratrol-mediated
attenuation of intestinal injury after trauma-hemorrhage. Shock. 2011;35(5):51723.
15. Zhang W, Lin TR, Hu Y, Fan Y, Zhao L, Stuenkel EL, et al. Ghrelin stimulates
neurogenesis in the dorsal motor nucleus of the vagus. Journal of Physiology
(London). 2004;559:729-37.
16. Eleawa SM, Alkhateeb MA, Alhashem FH, Bin-Jaliah I, Sakr HF, Elrefaey HM,
et al. Resveratrol Reverses Cadmium Chloride-induced Testicular Damage and
Subfertility by Downregulating p53 and Bax and Upregulating Gonadotropins and
Bcl-2 gene Expression. J Reprod Dev. 2014;60(2):115.
17. Krasovskiĭ G, Vasukovich L, Chariev O. Experimental study of biological effects
of leads and aluminum following oral administration. Environmental Health
Perspectives. 1979;30:47.
18. Mahmoud ME, Elsoadaa SS. Protective Effect of Ascorbic Acid, Biopropolis and
Royal Jelly against Aluminum Toxicity in Rats. J Nat Sci Res. 2013;3(1):102-12.
19. Liu X, Ramirez S, Pang PT, Puryear CB, Govindarajan A, Deisseroth K, et al.
Optogenetic stimulation of a hippocampal engram activates fear memory recall.
Nature. 2012;484(7394):381-5.
20. Holtbäck U, Brismar H, DiBona GF, Fu M, Greengard P, Aperia A. Receptor
recruitment: a mechanism for interactions between G protein-coupled receptors.
Proc Natl Acad Sci USA. 1999;96:7271-5.
21. Exley C. The pro-oxidant activity of aluminum. Free Radic Biol Med.
2004;36(3):380-7.
22. Al-Kahtani MA. Renal damage mediated by oxidative stress in mice treated with
aluminium chloride: Protective effects of taurine. J Biol Sci. 2010;10(7):584-95.
23. Yokel RA, McNamare PJ. Aluminum bioavailability and disposition in adult and
immature rabbits. Toxicology and Applied Pharmacology. 1985;77(2):344-52.
24. Shirley D, Lote C. Renal handling of aluminium. Nephron Physiology.
2004;101(4):99-103.
25. Monteagudo F, Isaacson L, Wilson G, Hickman R, Folb P. Aluminium excretion
by the distal tubule of the pig kidney. Nephron. 1988;49(3):245-50.
26. Garbossa G, Gálvez G, Castro ME, Nesse A. Oral aluminum administration to rats
with normal renal function. 1. Impairment of erythropoiesis. Hum Exp Toxicol.
1998;17(6):312-7.
27. Testolin G, Erba D, Ciappellano S, Bermano G. Influence of organic acids on
aluminium absorption and storage in rat tissues. Food Addit Contam.
1996;13(1):21-7.
28. Drüeke TB. Intestinal absorption of aluminium in renal failure. Nephrol Dial
Transpla. 2002;17(suppl 2):13-6.
29. Katyal R, Desigan B, Sodhi CP, Ojha S. Oral aluminum administration and
oxidative injury. Biol Trace Elem Res. 1997;57(2):125-30.
28
30. Stacchiotti A, Borsani E, Ricci F, Lavazza A, Rezzani R, Bianchi R, et al.
Bimoclomol ameliorates mercuric chloride nephrotoxicity through recruitment of
stress proteins. Toxicol Lett. 2006;166(2):168-77.
31. Ebina Y, Okada S, Hamazaki S, Midorikawa O. Liver, kidney, and central
nervous system toxicity of aluminum given intraperitoneally to rats: a multipledose subchronic study using aluminum nitrilotriacetate. Toxicol Appl Pharmacol.
1984;75(2):211-8.
32. Graczyk A, Radomska K, Dlugaszek M. Synergizm i antagonizm miedzy
biopierwiastkami i metalami toksycznymi. Ochrona Środowiska i Zasobów
Naturalnych. 2000(18):39-45.
33. Ghosh J, Das J, Manna P, Sil PC. Acetaminophen induced renal injury via
oxidative stress and TNF-α production: Therapeutic potential of arjunolic acid.
Toxicology. 2010;268(1):8-18.
34. El-Demerdash FM. Antioxidant effect of vitamin E and selenium on lipid
peroxidation, enzyme activities and biochemical parameters in rats exposed to
aluminium. J Trace Elem Med Biol 2004;18(1):113-21.
35. Sargazi M, Shenkin A, Roberts NB. Aluminium-induced injury to kidney
proximal tubular cells: Effects on markers of oxidative damage. J Trace Elem
Med Biol. 2006;19(4):267-73.
36. Moumen R, Ait-Oukhatar N, Bureau F, Fleury C, Bouglé D, Arhan P, et al.
Aluminium increases xanthine oxidase activity and disturbs antioxidant status in
the rat. J Trace Elem Med Biol. 2001;15(2):89-93.
37. Nehru B, Anand P. Oxidative damage following chronic aluminium exposure in
adult and pup rat brains. J Trace Elem Med Biol. 2005;19(2):203-8.
38. Ward RJ, Zhang Y, Crichton RR. Aluminium toxicity and iron homeostasis. J
Inorg Biochem. 2001;87(1):9-14.
39. El-Sayed WM, Al-Kahtani MA, Abdel-Moneim AM. Prophylactic and therapeutic
effects of taurine against aluminum-induced acute hepatotoxicity in mice. J
Hazard Mater 2011;192(2):880-6.
40. Yousef MI. Aluminium-induced changes in hemato-biochemical parameters, lipid
peroxidation and enzyme activities of male rabbits: protective role of ascorbic
acid. Toxicology. 2004;199(1):47-57.
41. Tsunoda M, Sharma RP. Modulation of tumor necrosis factor α expression in
mouse brain after exposure to aluminum in drinking water. Arch Toxicol.
1999;73(8-9):419-26.
42. Stacchiotti A, Rodella L, Ricci F, Rezzani R, Lavazza A, Bianchi R. Stress
proteins expression in rat kidney and liver chronically exposed to aluminium
sulphate. Histol Histopathol. 2006;21(2):131-40.
43. Sargazi M, Shenkin A, Roberts NB. Aluminium-induced injury to kidney
proximal tubular cells: Effects on markers of oxidative damage. J Trace Elem
Med Biol. 2006;19(4):267-73.
29
44. Sharma S, Anjaneyulu M, Kulkarni S, Chopra K. Resveratrol, a polyphenolic
phytoalexin, attenuates diabetic nephropathy in rats. Pharmacology.
2005;76(2):69-75.
45. Do Amaral CL, Francescato HDC, Coimbra TM, Costa RS, Darin JDaC, Antunes
LMG, et al. Resveratrol attenuates cisplatin-induced nephrotoxicity in rats. Arch
Toxicol 2008;82(6):363-70.
46. Leonard SS, Xia C, Jiang B-H, Stinefelt B, Klandorf H, Harris GK, et al.
Resveratrol scavenges reactive oxygen species and effects radical-induced cellular
responses. Biochem Biophys Res Commun. 2003;309(4):1017-26.
47. Holthoff JH, Woodling KA, Doerge DR, Burns ST, Hinson JA, Mayeux PR.
Resveratrol, a dietary polyphenolic phytoalexin, is a functional scavenger of
peroxynitrite. Biochem Pharmacol. 2010;80(8):1260-5.
48. Pervaiz S, Holme AL. Resveratrol: its biologic targets and functional activity.
Antioxidants & redox signaling. 2009;11(11):2851-97.
49. Kitada M, Koya D. Renal protective effects of resveratrol. Oxid Med Cell Longev.
2013;2013 :568093. doi: 10.1155/2013/568093
50. Chen K-H, Hung C-C, Hsu H-H, Jing Y-H, Yang C-W, Chen J-K. Resveratrol
ameliorates early diabetic nephropathy associated with suppression of augmented
TGF-β/smad and ERK1/2 signaling in streptozotocin-induced diabetic rats.
Chemico-biological interactions. 2011;190(1):45-53.
51. Wu L, Zhang Y, Ma X, Zhang N, Qin G. The effect of resveratrol on FoxO1
expression in kidneys of diabetic nephropathy rats. Mol Biol Rep.
2012;39(9):9085-93.
30