Chemico-Biological Interactions 245 (2016) 1e11

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Chemico-Biological Interactions
journal homepage: www.elsevier.com/locate/chembioint

Protective effect of thymol on high fat diet induced diabetic

nephropathy in C57BL/6J mice
Settu Saravanan, Leelevinothan Pari*
Department of Biochemistry and Biotechnology, Annamalai University, Annamalai Nagar, 608 002, Tamil Nadu, India

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 29 June 2015
Received in revised form
23 November 2015
Accepted 30 November 2015
Available online 8 December 2015

Obesity is one of several factors implicated in chronic kidney disease (CKD). Thymol, a monoterpene
phenolic compound found in the oils of thyme with multiple biological properties especially antidiabetic
activity. The present study was undertaken to evaluate the thymol against diabetic nephropathy by high
fat diet (HFD)-induced diabetic C57BL/6J mice. After 10 weeks of continuous dietary intervention, HFD
(fat- 35.2%) to mice presented characteristic features of progressive nephropathy by signicant increased
in kidney weight, blood, and urinary parameters, glomerulosclerosis, oxidative stress, hyperlipidemia
and subsequent renal injuries. After intragastric administration of thymol (40 mg/kg BW) daily for the
subsequent 5 weeks signicantly decreased the blood, urinary parameters and kidney weight. Thymol
inhibited the activation of transforming growth factor-b1 (TGF-b1) and vascular endothelial growth
factor (VEGF). Also, signicantly increased the antioxidants and suppresses the lipid peroxidation
markers in erythrocytes and kidney tissue compared to the diabetic mice. Thymol downregulated the
expression level of sterol regulatory element binding protein-1c (SREBP-1c) and reduced the lipid
accumulation in renal. Histopathological study of kidney tissues showed that extracellular mesangial
matrix expansion, glomerulosclerosis in diabetic mice were suppressed by thymol. Further, our results
indicate that administration of thymol afforded remarkable protection against HFD-induced diabetic
nephropathy.
2015 Published by Elsevier Ireland Ltd.

Keywords:
Thymol
High fat diet
Diabetic nephropathy
Glomerulosclerosis
Lipids

1. Introduction
Diabetes mellitus is a major health problem affecting people
worldwide. Obesity is all most an important risk factor for diabetes
mellitus, hypertension, metabolic disease and chronic renal disease. Diabetic nephropathy (DN) is a serious and progressive
complication of diabetes and occurs in 30%e40% of diabetic patients [1]. For a long time, the renal diseases have been considered a
victim of metabolic syndrome, abnormalities of glucose and lipid
metabolism related to insulin resistance [2]. Excess consumption of
fat induces hypersecretion of insulin leads to insulin resistance,
increases nutrient uptake and subsequently increases the electron
ow in the mitochondrial respiratory chain, resulting in an amplied reactive oxygen species (ROS) generation, thereby establishing
oxidative stress [3]. This may induce lipotoxicity, morphological
changes and tissue injury in the kidney of high fat diet (HFD)induced mice. Under these conditions, the activation of

* Corresponding author.
E-mail address: jayampari@gmail.com (L. Pari).
http://dx.doi.org/10.1016/j.cbi.2015.11.033
0009-2797/ 2015 Published by Elsevier Ireland Ltd.

endogenous enzymatic and non-enzymatic antioxidants may play

crucial role in the early defense of the body from the HFD-induced
oxidative stress [4]. Insulin resistance, a key pathophysiological
feature of metabolic syndrome, is characterized by generation of
higher levels of ROS, oxidative stress and organ injuries [5].
Injuries in the structures of the kidney, leading to changes in
renal structure, including glomerular hypertrophy, mesangial
expansion, abnormalities of the podocyte foot processes, increased
glomerular capillary wall tension and vascular abnormalities are
observed in obese humans and animals. These changes decrease
the glomerular ltration rate and increase proteinuria or albuminuria and worsening of renal function [6].
Once obvious DN occurs, it progresses slowly or rapidly to the
most advanced stage of chronic kidney disease (CKD), the progression to end-stage renal disease is irreversible. So, which necessitates dialysis or transplantation for treatment. Although the
positive effects on the development and progression of DN through
strict control of blood glucose [7]. Hyperglycemia initiates, sustains
and promotes the progression of DN. Cautious maintenance of
controlled glycemia is the most effective means to delay the onset

S. Saravanan, L. Pari / Chemico-Biological Interactions 245 (2016) 1e11

of DN. Along with the control of blood glucose other factors such as
inammation, hypoxia, and hyperlipidemia may all play important
roles. However, each of these factors is initially triggered by higher
glucose levels [8].
Mice are vital to biomedical research due to almost unlimited
changes that can be made to their genome. Mouse and human
kidneys were similar in physiology, structure and cell types [9,10].
In our study, C57BL/6J mice are provoked by high-fat diet and it is
associated with obesity, hyperglycemia and hyperinsulinemia
which results in type 2 diabetes along with DN [11]. HFD-induced
hyperglycemia and hyperlipidemia initiates loss of kidney function and injury, which stimulates mesangial and tubulointerstitial
cells that produce cytokines and chemokines. The production of
cytokines and chemokines cause mesangial cell proliferation via an
increased production of transforming growth factor-b1 (TGF-b1)
and vascular endothelial growth factor (VEGF) which promotes
deposition of extracellular matrix components in the mesangium
and the tubulointerstitium, thereby causing renal failure. Diabetic
renal diseases also supported by upregulated renal expression of
transcriptional factor sterol regulatory element binding protein
(SREBP)-1c, that causes increased synthesis and accumulation of
triglycerides and this is correlated with renal sclerosis and proteinuria [12]. However, a full understanding of the mechanisms
involved in progressive renal disease is not yet evaluated. Thus, we
investigated the renal alterations and accompanying systemic abnormalities in experimental C57BL/6 mice. We demonstrated that
HFD-induced mice developed the core features of metabolic syndrome and subsequently developed albuminuria. In addition, we
attempted to investigate several pathophysiological alterations
occurring in the kidney, which may be potent therapeutic targets
for the prevention of renal diseases.
While a number of oral medicines are available for treating
diabetes mellitus, these drugs have several side effects, including
weight gain and gastrointestinal distress [13]. So investigating
potential natural products to prevent the DN induced by HFD has
increasingly attracted the interest of the academic community.
Thymol is a dietary monoterpene phenol, which is found in the oils
of thyme and plants such as Thymus vulgaris, Thymbra spicata,
Thymus ciliates, Trachyspermum ammi, Monarda stulosa and Nigella
sativa seeds. It exhibits multiple biological activities such as antibacterial [14], anti-fungal [15], anti-inammatory [16], radioprotective [17], antioxidant and anti-myocardial infarction [18]. US
Food and Drug Administration (US-FDA) have reported thymol as a
food additive in generally recognized as safe (GRAS) and it is
nontoxic [19]. Previously we reported the antidiabetic effects of
thymol on HFD-induced diabetic mice. In continuation of our
research work on thymol, we made an attempt to evaluate the
protective effects of thymol on the pathophysiological and histological alterations in the kidney of HFD-induced diabetic C57BL/6J
mice.

(temperature 23 2  C, humidity 65e70% and 12 h light/dark cycle)

in the Central Animal House, Department of Experimental Medicine, Rajah Muthiah Medical College, Annamalai University and
were fed with standard pellet diet and water ad libitum. They were
maintained under standard conditions with a 12 h light/dark cycle
and were provided free access to food and water. All studies were
conducted in accordance with the National Institute of Health,
guide for the care and use of laboratory animals and CPCSEA
guidelines. The study protocols were approved by the Institutional
Animal Ethics Committee of Rajah Muthiah Medical College and
Hospital (Reg No. 160/1999/CPCSEA, Proposal number: 1001),
Annamalainagar.

2.3. Diet and experimental design

The standard diet, commercially obtained from Sai Enterprises,
Chennai. It was composed of 4.2 g fat. The beef tallow based high fat
diet was composed of protein 17.7 g, fat 35.2 g, carbohydrate 34.5 g,
ber 3.4 g, minerals 6.8 g and vitamins 1.8 g [20]. The experimental
design consisted of four groups of mice. (Group IIeIV) were fed high
fat diet for a period of 15 weeks. At the end of the 10th week, blood
was collected by tail bleeding into heparin-coated tubes after a 4 h
fast; blood glucose was checked using a glucometer (OneTouch;
LifeScan, Inc, U.K). Animals with blood glucose more than 180 mg/
dl were considered to be diabetic and used for the experiment.
Thymol dissolved in 0.5% dimethyl sulfoxide (DMSO) was given
orally by an intragastric tube. Group I: Normal mice fed with a
standard diet for 15 weeks. Group II: Normal mice fed with standard diet for 15 weeks and administered with thymol (40 mg/kg
BW) by gavage for last 5 weeks. Group III: HFD diabetic mice fed
with high fat diet for a period of 15 weeks. Group IV: HFD-induced
diabetic mice administered with thymol (40 mg/kg BW) by gavage
for last 5 weeks.

2.4. Sample collection

At the end of the experiment, all the mice were sacriced and
blood samples were obtained and centrifuged at 4000 rpm/min for
10 min to separate serum which was then frozen at 70  C for the
determination of serum biochemistry parameters. During the end
of the experimental period the animals were placed in individual
metabolic cages before decapitation, urine samples were collected
for the measurement of urea, creatinine, total protein and creatinine clearance. After decapitation, kidneys were immediately
removed, weighed, minced and homogenized (10%, w/v) separately
in ice cold 1.15% KCl-0.01 M sodium, potassium phosphate buffer
(pH 7.4) in a Potter-Elvehjem type homogenizer. The homogenate
was centrifuged at 18,000  g for 20 min at 4  C, and the resultant
supernatant was used for subsequent biochemical analyses.

2. Materials and methods

2.1. Chemicals and reagents

2.5. Biochemical parameters

Thymol (HPLC purity
99.0%) was purchased from Sigma

Chemical Co., St. Louis, MO, USA. All the other chemicals used in this
study were analytical grade and were obtained from HIMEDIA,
Mumbai, India.

Glucose was estimated by the method of Trinder [21]. The insulin in the plasma was measured by the method of Burgi [22].
Nephropathy was evaluated by estimating blood urea nitrogen
(BUN) and urinary protein. Further creatinine clearance was also
determined as a measure of glomerular ltration rate (GFR) [23].
Creatinine clearance was assessed from the urinary and serum
creatinine. Blood and urinary urea were estimated by the diacetyl
monooxime method of Netlson [24], serum and urinary creatinine
were measured by the alkaline picrate method of Jaffe [25]. Urinary
protein was quantied by Lowry's method of Lowry [26].

2.2. Experimental animals

Healthy adult male C57BL/6J mice 3 weeks of age was obtained
from NIN Hyderabad and housed in polypropylene cages. The animals were housed in well-ventilated polypropylene cages

S. Saravanan, L. Pari / Chemico-Biological Interactions 245 (2016) 1e11

2.6. Estimation of lipid peroxidation products

Thiobarbituric acid reactive substances (TBARS) and lipid hydroperoxides (LOOH) in plasma and tissues were estimated by the
methods of Niehaus and Samuelsson [27] and Jiang et al. [28].

enhanced chemiluminescence method using an ECL kit. Bands were

scanned using a scanner and quantitative by Image-J, a public
domain Java image processing software, Wayne Rasband, NIH,
Bethesda, MD, USA, which of control was set to 1.
2.10. Histopathological studies

2.7. Evaluation of antioxidant enzymes

Superoxide dismutase (SOD) was assayed according to Misra
and Fridovich [29]. Catalase (CAT) activity was assayed by the
method of Aebi [30]. Reduced glutathione (GSH) content was
measured using the method of Ellman [31]. Glutathione S-transferase (GST) activity was determined according to Habig et al. [32].
The activity of glutathione peroxidase (GPx) in the erythrocytes and
tissues were measured by the method of Rotruck et al. [33].
Glutathione reductase (GR) in the erythrocytes and tissues were
measured by the method of Pinto and Bartley [34]. Vitamin C and
vitamin E in the plasma and tissues were estimated by the methods
of Roe and Kuether [35] and Baker et al. [36].
2.8. Measurement of lipids
Tissue lipids were extracted with chloroform/methanol mixture
(2v/1v) according to the method of Folch et al. [37]. The contents of
total lipids in the kidney were quantied gravimetrically by evaporating off the solvents using a rotary evaporator (Heidolph,
Laborota 4010 digital, Germany). Triglyceride (TG), total cholesterol
(TC), free fatty acid (FFA) and phospholipids (PL) were enzymatically
analyzed with a commercial kit (Agappe Diagnostics, Kerala, India).
2.9. SDS-PAGE and western blot analysis
Western blotting was performed to analyze the expression
patterns of SREBP-1c, TGF-b1, and VEGF by using the method of
Laemmli [38]. The kidney tissue samples were homogenized in an
ice-cold RIPA buffer (1% Triton, 0.1% SDS, 0.5% deoxycholate,
1 mmol/l EDTA, 20 mmol/l Tris (pH 7.4), 150 mmol/l NaCl, 10 mmol/l
NaF, and 0.1 mmol/l phenylmethylsulfonyl uoride (PMSF). The
homogenate was centrifuged at 12,000 g for 30 min at 4  C to
remove debris. Protein concentration was measured in nanodrop.
Samples containing 50 mg of total cellular proteins were loaded and
separated using 10% SDS polyacrylamide gel electrophoresis. The
resolved proteins were blot transferred onto a PVDF membrane
(Millipore). The membranes were incubated with the blocking
buffer containing BSA for 2 h to reduce non-specic binding sites
and then incubated with anti b-actin (rabbit polyclonal); 1:500
dilution in 5% BSA in Tris buffered saline and 0.05% Tween-20
(TBST), anti-rabbit SREBP-1c (monoclonal; 1:1000), anti rabbit
TGF-b1 (monoclonal; 1:1000) and anti-mouse VEGF (monoclonal;
1:1000) with gentle shaking overnight at 4  C. After this, membranes were incubated with their corresponding secondary antibodies (anti-goat or anti-mouse IgG conjugated to horseradish
peroxidase) for 2 h at room temperature. Membranes were washed
thrice with TBST for 30 min. Protein bands were visualized by an

4% paraformaldehyde-xed kidneys were embedded in parafn,

sectioned at 5 mm, and stained with hematoxylineeosin (H&E) for
histological examination under light microscopy. For renal ber
staining quantied with Masson's trichrome (MT) was used. Renal
tissue sections were stained by deparafnized with xyline followed
by the MT staining kit (Yucca Diagnostics, India). MT stain was
graded for the presence of interstitial brosis. Each section was rst
viewed at low power (40x magnication).
Kidney samples were analyzed for lipid and fat depots by
staining with a fat-specic dye, Oil Red O. Frozen samples were cut
into 30 mm-thick sections using a sliding microtome and xated
with 10% formol calcium. Sections were washed with distilled
water and rinsed with 60% isopropanol. Then, sections were stained
with freshly prepared Oil Red O working solution for 20 min (Oil
Red O stock stain: 0.5 g of Oil Red O and 100 mL of isopropanol;
Oil Red O working solution: 30 mL of the stock stain and 20 mL of
distilled water). Sections were rinsed with 60% isopropanol, lightly
stained with Mayer's hematoxylin, rinsed with tap water and
mounted in aqueous mountant. The sections were examined by
light microscopy (10x magnication).
2.11. Statistical analysis
The results were expressed as a mean standard deviation (S.D.)
for 6 mice (n 6) in each group. Data were analyzed by one-way
analysis of variance followed by Duncan's Multiple Range Test
(DMRT) using SPSS version 17 (SPSS, Chicago, IL). Post hoc testing
was performed for intergroup comparisons using the least signicant difference (L.S.D.) test. P values < 0.05 were considered as
signicantly altered.
3. Results
3.1. Effect of thymol on glucose, insulin, BUN, serum creatinine and
serum protein in C57BL/6J mice
The blood glucose and insulin levels of the HFD-Induced diabetic group were signicantly (p < 0.05) higher compared to other
groups. Thymol (40 mg/kg BW) treated to diabetic group had
signicantly (p < 0.05) lower blood glucose and plasma insulin
levels. BUN and serum creatinine level were signicantly increased
and serum protein level was signicantly decreased in HFDinduced diabetic mice. After the treatment with thymol (40 mg/
kg BW) to HFD-induced diabetic mice, BUN and serum creatinine
level were signicantly decreased and serum protein level was
signicantly increased as shown in Table 1.

Table 1
Effect of thymol supplementation on glucose, insulin, BUN, serum creatinine and serum protein in normal and experimental mice.
Groups

Normal

Glucose (mg/dL)
Insulin (mU/l)
BUN (mg/dl)
Serum creatinine (mg/dl)
Serum protein (mg/dl)

94.45
22.42
20.23
0.35
9.56

Normal thymol (40 mg/kg)

7.32a
1.71a
1.54a
0.03a
0.73a

93.44
22.25
20.21
0.34
9.57

7.74a
1.70a
1.55a
0.03a
0.73a

HFD thymol (40 mg/kg)

HFD
245.55
153.62
62.56
2.22
5.32

21.13b
13.87b
4.76b
0.17b
0.41b

135.51
39.68
26.63
0.47
8.22

11.72c
2.74c
2.04c
0.04 ac
0.63c

Values were means SD for six samples from six mice in each group. Values not sharing a common superscript differ signicantly at p < 0.05. Duncan's multiple range test
(DMRT). BUN e blood urea nitrogen; ap < 0.05 vs HFD thymol, bp < 0.05 vs normal, cp < 0.05 vs HFD.

S. Saravanan, L. Pari / Chemico-Biological Interactions 245 (2016) 1e11

3.2. Effect of thymol on kidney weight of normal and experimental

mice
HFD-induced diabetic mice revealed increased kidney weight
compared to the normal control mice. Treatment with thymol to
HFD-induced diabetic mice reduced signicantly (p < 0.05) the
kidney weight as shown in Fig. 1.

There was a signicant excretion in the urine glucose, urinary

urea and urinary protein and reduced level of urinary creatinine
and creatinine clearance in HFD-induced diabetic mice. After the
treatment with the thymol to HFD-induced diabetic mice, the level
of urinary glucose, urinary urea and urinary protein were signicantly (p < 0.05) reduced and urinary creatinine and creatinine
clearance were increased signicantly compared to the HFDinduced diabetic mice were shown in (Fig. 2aee).
3.4. Effect of thymol on the levels of TBARS and LOOH in the plasma
and kidney
There was a signicant (p < 0.05) elevation in the levels of lipid
peroxidation markers (TBARS and LOOH) on the plasma and kidney
in HFD-induced diabetic mice when compared to normal control
mice. A remarkable signicant (p < 0.05) decrease in the levels of
TBARS and LOOH were also observed in thymol treated mice to
HFD-induced diabetic mice (Table 2).
3.5. Effect of thymol on erythrocyte antioxidant activity
The levels of enzymatic antioxidants such as (SOD, catalase, GPx,
GST and GR) and non enzymatic antioxidants such as (GSH, vitamin
C and vitamin E) were signicant (p < 0.05) decreased in HFDinduced diabetic mice. Administration of thymol to diabetic mice
signicantly (p < 0.05) elevated the level of antioxidants compared
to the HFD-induced diabetic mice (Tables 3 and 4).
3.6. Effect of thymol on renal antioxidant status
The levels of enzymatic antioxidants such as (SOD, catalase, GPx,
GST and GR) and non enzymatic antioxidants such as (GSH, vitamin C
and vitamin E) were signicantly (p < 0.05) decreased in HFDinduced diabetic mice. After treatment with thymol to diabetic
mice signicantly increased the level of antioxidants (Tables 5 and 6).

Kidney weight
b
a

(mg)

200
150
100
50
0

NC

NC+THY 40
mg/kg

HFD

HFD-induced diabetic mice group showed signicantly

(p < 0.05) increased levels of TG, TC, FFA, and PL (Table 7). Treatment with thymol signicantly (p < 0.05) decreased the levels of
these renal lipids in the diabetic mice group.
3.8. Effect of thymol on SREBP-1c, TGF-b and VEGF protein
expression in kidney of normal and experimental mice

3.3. Effects of thymol on urine biochemistry in normal and

experimental mice

250

3.7. Effect of thymol on renal lipid proles

HFD+THY 40
mg/kg

Groups

Fig. 1. Effect of thymol on kidney weight of normal and experimental mice. Values
were means SD for six samples from six mice in each group. Values not sharing a
common superscript differ signicantly at p < 0.05. Duncan's multiple range test
(DMRT). THY e Thymol; NCeNormal control; HFD e High fat diet. ap < 0.05 vs HFD,
b
p < 0.05 vs NC.

The western blot protein expressions of SREBP-1c, TGF-b and

VEGF in the kidney of normal and experimental mice were
described in (Fig. 3a). Densitometric analyses of western blots were
given in (Fig. 3bed). The expressions of SREBP-1c, TGF-b and VEGF
proteins were up-regulated in HFD-induced diabetic mice and
treatment with thymol to diabetic mice resulted in a downregulation of SREBP-1c, TGF-b and VEGF proteins compared to the
diabetic mice.
3.9. Effect of thymol on histopathology in kidney of normal and
experimental mice
Histological sections of kidney (H&E) stain shown in Fig. 4. HFDinduced mice showed signicant vacuolar degeneration of renal
tubular. Thymol treated diabetic mice shown normal renal
glomeruli and mild fatty inltration. Fig. 5 shows the Masson's
trichrome stain of the kidney. HFD-induced mice showed degeneration of renal tubular and glomeruli with deposition of collagen
were the thymol treated diabetic mice showed moderate reduction
in collagen.
Fig. 6 shows Oil Red O staining in the kidney. HFD-induced
mice shows increased red lipid droplet accumulation. Treatment
of thymol signicantly reduced the lipid accumulation. For all the
above mentioned biochemical, molecular and histological parameters studied, HFD-induced diabetic mice treated with thymol
group (Group-IV) was compared with HFD-induced diabetic mice
group (Group-III).
4. Discussion
An emerging body of evidence suggests that obesity is the most
important risk factor for the development of hypertension, metabolic disease, diabetes mellitus and CKD [39]. Diabetic nephropathy
is characterized by hyperltration, microalbuminuria, renal and
glomerular hypertrophy, thickening of glomerular basement
membrane and reduced renal function [40]. Hyperglycemia,
hyperinsulinemia, insulin resistance, hyperlipidemia, oxidative
stress and proteinuria itself, contribute to progression of renal
damage. As stated by Soler et al. [41], in our study, HFD-induced
C57BL/6J mice cause major systemic alterations of metabolic syndrome which leads to diabetic nephropathy. However, each of these
factors is initially triggered by high glucose [8]. Similar to the
ndings of this study, HFD-induced mice also elevated the insulin
resistance by increased level of plasma glucose and insulin. We
previously reported that thymol (40 mg/kg BW) treatment led to
plasma glucose and plasma insulin levels to the normal values [42].
Diabetes mellitus is also grossly reected by profound changes in
protein metabolism and by loss of nitrogen from most organs.
Hyperglycemia triggers elevated levels of urea and creatinine,
which are considered as signicant markers of renal dysfunction
[43]. The HFD-induced diabetic mice markedly decreased creatinine clearance, increased BUN, creatinine, urinary urea, creatinine
and urinary albumin excretion with increased kidney weight,
which are consistent with a previous study [44]. Albumin is used as
the marker of protein leakage, which is an indicator of kidney

S. Saravanan, L. Pari / Chemico-Biological Interactions 245 (2016) 1e11

Fig. 2. Effects of thymol on urine biochemistry in normal and experimental mice. (a) Urinary glucose, (b) urinary urea, (c) urinary creatinine, (d) urinary protein, (e) creatinine
clearance. Values were means SD for six samples from six mice in each group. Values not sharing a common superscript differ signicantly at p < 0.05. Duncan's multiple range
test (DMRT). THY - Thymol; NCeNormal control; HFD e High fat diet. ap < 0.05 vs HFD THY, bp < 0.05 vs normal, cp < 0.05 vs HFD.

Table 2
Effect of thymol on the levels of TBARS and LOOH in the plasma and kidney tissues.
Groups

Plasma
TBARS (mmol/dL)

Normal
Normal Thymol (40 mg/kg)
HFD
HFD Thymol (40 mg/kg)

0.96
0.94
4.23
1.50

0.07a
0.07a
0.39b
0.09c

Kidney
LOOH (mmol/dL)
10.34
10.93
36.32
14.43

0.95a
0.91a
2.36b
0.95c

TBARS (mmol/100 g
wet tissue)
0.63
0.61
2.13
0.96

0.05a
0.05a
0.18b
0.08c

LOOH (mmol/100 g
wet tissue)
66.00
67.03
146.35
83.66

4.79a
5.39a
13.65b
7.53c

Values were means SD for six samples from six mice in each group. Values not sharing a common superscript differ signicantly at p < 0.05. Duncan's multiple range test
(DMRT).
a
p < 0.05 vs HFD thymol, bp < 0.05 vs normal, cp < 0.05 vs HFD.

damage [45]. The elevated levels of serum creatinine and urea

indicate diminished ability of the kidneys to lter these waste
products from the blood and excrete them in the urine. An association between hyperuricemia and renal injury has been reported
by Feig et al. [46], where uric acid-mediated arteriopathy and

interstitial inammation suggest mechanisms that would exacerbate or potentiate progressive renal functional decline after injury.
In this study, elevation of uric acid together with the histological
alterations clearly indicate the induction of renal dysfunction in
HFD-induced diabetic mice. The therapeutic property of thymol

S. Saravanan, L. Pari / Chemico-Biological Interactions 245 (2016) 1e11

Table 3
Effect of thymol on the activity of enzymatic antioxidant in the erythrocyte of normal and experimental mice.
Groups

Normal

SOD (U1/mg dL)

Catalase (U2/mg dL)
GPx (U3/mg dL)
GST (U4/mg dL)
GR (U5/mg dL)

7.65
133.24
21.62
2.18
28.65

Normal thymol (40 mg/kg BW)

0.71a
10.66a
1.83a
0.17a
2.18a

7.21
134.63
22.06
2.17
28.64

0.68a
11.53a
1.96a
0.17a
2.19a

HFD thymol (40 mg/kg BW)

HFD
2.64
65.52
8.36
1.02
17.66

0.17b
5.90b
0.72b
0.08b
1.34b

6.11
118.73
19.42
1.58
24.45

0.35c
11.1c
1.73c
0.12c
1.87c

Values were means SD for six samples from six mice in each group. Values not sharing a common superscript differ signicantly at p < 0.05. Duncan's multiple range test
(DMRT).
U1Enzyme concentration required for 50% inhibition of NBT reduction/min.
U2 mmole of hydrogen peroxide consumed/min.
U3 mmole of reduced glutathione consumed/min.
U4 mg of CDNB conjugate formed/min.
U5 mg of reduced glutathione formed/min.
a
p < 0.05 vs HFD thymol, bp < 0.05 vs normal, cp < 0.05 vs HFD.

Table 4
Effect of thymol on the activity of non enzymatic antioxidant in the erythrocyte of normal and experimental mice.
Groups
GSH (mg/dL)
Vitamin C (mg/dL)
Vitamin E (mg/dL)

Normal thymol (40 mg/kg BW)

Normal
a

47.21 4.04
3.03 0.21a
2.93 0.17a

47.03 3.55
3.12 0.21a
2.87 0.16a

HFD thymol (40 mg/kg BW)

HFD
b

21.72 1.96
0.93 0.06b
0.77 0.06b

41.87 3.80c
2.97 0.18c
2.55 0.17c

Values were means SD for six samples from six mice in each group. Values not sharing a common superscript differ signicantly at p < 0.05. Duncan's multiple range test
(DMRT).
a
p < 0.05 vs HFD thymol, bp < 0.05 vs normal, cp < 0.05 vs HFD.

Table 5
Effect of thymol on the activity of enzymatic antioxidant in the kidney of normal and experimental mice.
Groups

Normal

SOD (U1/mg protein)

Catalase (U2/mg protein)
GPx (U3/mg protein)
GST (U4/mg protein)
GR (U5/mg protein)

19.78
39.74
16.03
1.81
11.45

Normal thymol (40 mg/kg BW)

1.82a
3.56a
1.39a
0.14a
0.87a

19.01
40.50
16.78
1.80
11.43

1.60a
3.94a
1.55a
0.14a
0.88a

HFD thymol (40 g/kg BW)

HFD
9.36
19.40
9.52
1.17
6.87

0.84b
1.36b
0.75b
0.09b
0.52b

17.73
34.67
13.63
1.58
9.66

1.42 ac
3.44c
1.24c
0.12c
0.74c

Values were means SD for six samples from six mice in each group. Values not sharing a common superscript differ signicantly at p < 0.05. Duncan's multiple range test
(DMRT).
U1Enzyme concentration required for 50% inhibition of NBT reduction/min.
U2 mmole of hydrogen peroxide consumed/min.
U3 mmole of reduced glutathione consumed/min.
U4 mg of CDNB conjugate formed/min.
U5 mg of reduced glutathione formed/min.
a
p < 0.05 vs HFD thymol, bp < 0.05 vs normal, cp < 0.05 vs HFD.

Table 6
Effect of thymol on the activity of non enzymatic antioxidant in the kidney of normal and experimental mice.
Groups

Normal

Normal thymol (40 mg/kg BW)

HFD

HFD thymol (40 mg/kg BW)

GSH (mg/mg protein)

Vitamin C (mg/mg protein)
Vitamin E (mg/mg protein)

21.64 1.85a
1.06 0.09a
4.76 0.35a

22.10 1.94a
1.11 0.09a
4.75 0.39a

9.60 0.86b
0.54 0.04b
1.99 0.11b

18.43 1.34c
0.89 0.07c
3.78 0.32c

Values were means SD for six samples from six mice in each group. Values not sharing a common superscript differ signicantly at p < 0.05. Duncan's multiple range test
(DMRT).
a
p < 0.05 vs HFD thymol, bp < 0.05 vs normal, cp < 0.05 vs HFD.

Table 7
Effect of thymol on the concentration of lipids in the kidney of normal and experimental mice.
Groups

Normal

TC (mg/g tissue)
TGs (mg/g tissue)
FFAs (mg/g tissue)
PLs (mg/g tissue)

4.34
5.64
20.22
3.21

Normal thymol (40 mg/kg BW)

0.39a
0.49a
1.96a
0.25a

4.31
5.21
19.45
3.11

0.38a
0.46a
1.78a
0.25a

HFD thymol (40 mg/kg BW)

HFD
14.63
12.27
48.52
9.64

1.23b
1.11b
4.65b
0.85b

5.34
7.42
25.74
4.01

0.48c
0.69c
2.34c
0.35c

Values were means SD for six samples from six mice in each group. Values not sharing a common superscript differ signicantly at p < 0.05. Duncan's multiple range test
(DMRT).
a
p < 0.05 vs HFD thymol, bp < 0.05 vs normal, cp < 0.05 vs HFD.

S. Saravanan, L. Pari / Chemico-Biological Interactions 245 (2016) 1e11

Fig. 3. (A) Effect of thymol on SREBP-1c, TGF-b1 and VEGF protein expression in the kidney of normal and experimental mice by western blot. THY e Thymol; NCeNormal control;
HFD e High fat diet; Lane 1. Normal control, Lane 2. Normal THY (40 mg/kg b/w), Lane 3. HFD, Lane 4. HFD THY (40 mg/kg b/w). (B) Effect of thymol on SREBP-1c, (C) Effect of
thymol on TGF-b1 and (D) Effect of thymol on VEGF protein band intensities scanned by densitometer histogram depicts quantization of three independent experiments
(means S.D.), with data normalized by dening the normal group, with SREBP-1c, TGF-b1and VEGF protein, as 1 unit. TGF-b1- Transforming growth factor-b1; VEGF e Vascular
endothelial growth factor; SREBP-1c- Sterol regulatory element binding protein-1c. ap < 0.05 vs HFD THY, bp < 0.05 vs normal, cp < 0.05 vs HFD.

seems propitious in improving kidney function by signicantly

increased serum and urine biochemical parameters. Moreover, in
molecular mechanism, renal injuries were connected with over
expression of proteins. Mesangial cell expansion, extracellular
matrix accumulation, proteinuria and glomerulosclerosis are all
associated with over expression of TGF-b1 and VEGF [47]. As we
observed in the present study, HFD-induced diabetic mice exhibited over expression of TGF-b1 and VEGF as well as the extracellular
matrix proteins observed in histology which increases the mesangial matrix thickening of the glomerular basement membrane
(GBM) and growing vascular permeability in the nephron which is
mainly due to elevated levels of proteins that are normally present
in these structures ultimately to progress the diabetic nephropathy.
However, ve weeks of treatment to diabetic mice with thymol

reduced the extracellular matrix proteins on GBM and down

regulated the expression of TGF-b1 and VEGF, thereby preventing
the above mentioned kidney function and alterations in
morphology.
Insulin resistance increases oxidative stress, which has also been
implicated in the renal progression of glycoxidation and lipid
peroxidation [48]. When the production of ROS exceeds cellular
defense, these unstable ROS will interact with essential biological
cellular macromolecules and lead to tissue damage as well as
functional abnormalities [49]. The increased levels of oxidative
stress are not only associated with the functional changes, but
also decreased serum antioxidant enzyme activities, indicating
cellular leakage and loss of functional integrity of renal membrane
which lead to an increased glomerular ltration rate and

S. Saravanan, L. Pari / Chemico-Biological Interactions 245 (2016) 1e11

Fig. 4. Illustrates H&E stain on the kidney tissues section. (A) Normal: Normal renal tubular architecture and glomeruli, (B) Normal thymol: showing normal architecture and
glomeruli, (C) HFD: renal parenchyma showing signicant vacuolar degeneration of renal tubular, (/) shows severe degeneration along with necrosis of tubular vacuolization and
dilation of cells, (D) HFD thymol: normal renal glomeruli maintaining structure similar to the observed normal (/) and mild fatty inltration. (G) Indicates normal glomeruli.

Fig. 5. Illustrates Masson's trichrome stain on the kidney tissue section. (A) Normal: Normal mice kidney showing normal distribution of collagen with normal architecture and
glomeruli, (B) Normal thymol: Showing with normal architecture and glomeruli, (C) HFD: showing degeneration of renal tubular and glomeruli with deposition of collagen (blue
color), (/) demonstrates acellular nodular sclerosis as well as periglomerular and interstitialbrosis, (D) HFD thymol: shows normal glomeruli with moderate reduction in
collagen. (For interpretation of the references to colour in this gure legend, the reader is referred to the web version of this article.)

glomerulosclerosis. Antioxidant enzymes present an inspiring array

of defense mechanisms that are efcient in maintaining ROS under
adequate concentrations. Antioxidant enzymes such as SOD, CAT,
GPx, GST and GR that balance with non-enzymatic antioxidants

such as vitamin E, vitamin C and GSH to neutralize free radicals and

counteract the oxidative cellular damage. Any small deviations in
the physiological concentrations of these antioxidant enzymes
have a dramatic effect on the resistance of cellular lipids, proteins

S. Saravanan, L. Pari / Chemico-Biological Interactions 245 (2016) 1e11

Fig. 6. Histopathological changes in kidney Oil RedO'staining. (A) Normal: showing the vacuoles are negative for Oil Red O Staining, (B) Normal thymol: showing the vacuoles are
negative for Oil Red O Staining, (C) HFD: (/) showing increase the red droplet lipid accumulation, (D) HFD thymol: (/) showing moderate lipid droplet accumulation.

and DNA to oxidative damage [50]. The function of SOD is to

neutralize superoxide, one of the most damaging free radicals. CAT
transforms toxic hydrogen peroxide to harmless byproducts such as
water and molecular oxygen. GPx reduces lipid hydroperoxides to
their corresponding alcohols and free hydrogen peroxide to water.
The decreased activities of CAT and GPx activity might be due to
glycation of these enzymes [51]. The decrease in GSH content
represents increased utilization due to oxidative stress. The
depletion of GSH level also decreases GST activity as GSH is required
as a substrate for GST activity.
Nonenzymic antioxidants play an excellent role in preventing
the cells from oxidative threats. Vitamin-E is a well-known physiological antioxidant and membrane stabilizer. It interrupts the
chain reaction of lipid peroxidation by reacting with lipid peroxy
radicals, thus protecting the cell structures against damage.
Vitamin-C is a hydrophilic antioxidant because it disappears faster
than other antioxidants when erythrocyte are exposed to ROS [43].
In the present study, HFD-induced mice cause insulin resistance
and increased the levels of lipid peroxidation products and
decreased the levels of antioxidants, which inhibit mesangial cell
activation that results in diabetic nephropathy [52]. The increased
levels of lipid peroxidation products could be due to the increased
levels of ROS and decreased levels of non-enzymatic antioxidants
as reported earlier by Rizvi [53]. In our study, thymol improved
erythrocytes and kidney antioxidants in HFD-induced diabetic
nephropathy. It also suppresses the lipid peroxidation markers,
thereby decreasing the production of ROS and reactive nitrogen
species implicated in cell injury by virtue of its antilipid peroxidation effect. In this context, Meeran and Stanely Mainzen Prince
[18] also noted an increase in the levels of antioxidants by treatment with thymol, by virtue of its antioxidant effects.
Insulin resistance and a defective insulin action on the metabolism of lipoproteins are the main mechanisms of altered lipid
prole in patients with diabetic nephropathy [54]. Thus, there is a

possibility of lipid deposition occurs in the kidney. The altered renal

lipid metabolism may enhance lipotoxicity and inammation in the
kidney and subsequently cause renal abnormalities. It has been
found that hyperlipidemia induced by HFD could lead to increased
renal lipid accumulation and glomerulosclerosis in mice via a
SREBP-1c dependent pathway [55]. SREBP-1c regulates the lipogenic process by activating genes involved in fatty acid and triglyceride biosynthesis [56]. HFD-induced mice increased SREBP-1c
protein in the kidney that might contribute to the development of
renal tubular lipid accumulation. In the present study, we observed
increased levels of lipids in HFD-induced diabetic mice. In addition,
scientic reports revealed an increase in neutral lipids as determined by increase of Oil Red O staining in the kidney after HFD
induction [57,58]. Our present ndings also conrmed the Oil Red
O positive for the deposition of phospholipids and cholesteryl
esters in the kidney. Treatment with thymol to diabetic nephropathic mice showed reduced triglycerides, cholesterol, phospholipids and free fatty acids, which is due to an increased insulin
secretion and down regulation of SREBP-1c that ultimately resulting in the prevention of glomerulosclerosis [59]. Our previous study
also revealed the lipid lowering effect of thymol [42]. Histopathological observations of the kidney also conrmed the nephroprotective role of thymol.
5. Conclusion
The data present in this study demonstrated the protective effect of thymol on HFD-induced diabetic nephropathy. The bioactive
natural product thymol's antioxidant property lowered lipid peroxidation products. It also improved glucose homeostasis,
decreased kidney weight, biochemical parameters in serum and
urine and restored the TGF-b and VEGF proteins in HFD-induced
diabetic mice. Further, thymol signicantly reduced the lipid prole by downregulating SREBP-1c protein expression and also

10

S. Saravanan, L. Pari / Chemico-Biological Interactions 245 (2016) 1e11

preserved renal architecture and decreased renal brosis. Thus,

thymol is a potent nephroprotectant in HFD-induced diabetic
nephropathy.
Conict of interest
The authors declare that they have no conicts of interest concerning this article.
Acknowledgment
We thank the University Grants Commission (UGC), New Delhi,
India for funding support in the form of research fellow under
Research Fellowship in Science for Meritorious Students (RFSMS)
Scheme (F4-1/2006 (BSR)/7e10/2007 (BSR)) to Mr. S. Saravanan.
Transparency document
Transparency document related to this article can be found
online at http://dx.doi.org/10.1016/j.cbi.2015.11.033.
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