Ameliorative effect of caffeic acid phenethyl ester on histopathological and biochemical changes induced by cigarette smoke in rat kidney.

Abstract

It was aimed to investigate the histopathological and biochemical changes in kidney tissues of rats exposed to cigarette smoke and possible protective effects of caffeic acid phenethyl ester (CAPE) on these changes. Twenty one male Wistar albino rats were divided into three equal groups. Animals in group I were used as control. Rats in group II were exposed to cigarette smoke and rats in group III were exposed to cigarette smoke and daily administration of CAPE. At the end of the 60-day experimental period, all the animals were sacrificed by decapitation. The serum samples obtained from the animals were studied for uric acid, creatinine and blood urine nitrogen (BUN) levels. Following routine histological procedures, kidney tissue specimens were examined under a light microscope. In addition, dismutase (SOD) and glutathione peroxidase (GSH-Px) enzyme activities and malondialdehyde (MDA) and nitric oxide (NO) contents were determined spectrophotometrically in tissue samples. It was found that serum uric acid and BUN levels of the rats exposed to cigarette smoke alone were elevated, although serum creatinine levels did not significantly change. Furthermore, renal SOD, GSH-Px, NO and MDA levels were significantly increased. These increases in serum BUN, and renal SOD, GSH-Px, NO and MDA levels were significantly inhibited by CAPE treatment. In light microscopic observations of tissues from rats exposed to smoke, mesangial cell proliferation in the renal corpuscles, dilatation and congestion in the peritubular capillaries and degenerative alterations in the proximal tubules were noted. There were also atrophic renal corpuscles. However, these histopathological changes were partially disappeared in the rats exposed to cigarette smoke plus CAPE. The present findings indicate that cigarette smoke causes impairment in renal structure and function, which can be prevented by CAPE administration.

Full text

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Ameliorative effect of caffeic acid
phenethyl ester on histopathological
and biochemical changes induced by
cigarette smoke in rat kidney
Hidir Pekmez1, Murat Ogeturk2, Huseyin Ozyurt3,
Mehmet Fatih Sonmez4, Neriman Colakoglu5and
Ilter Kus2
Abstract
It was aimed to investigate the histopathological and biochemical changes in kidney tissues of rats exposed to
cigarette smoke and possible protective effects of caffeic acid phenethyl ester (CAPE) on these changes.
Twenty one male Wistar albino rats were divided into three equal groups. Animals in group I were used as
control. Rats in group II were exposed to cigarette smoke and rats in group III were exposed to cigarette
smoke and daily administration of CAPE. At the end of the 60-day experimental period, all the animals were
sacrificed by decapitation. The serum samples obtained from the animals were studied for uric acid, creatinine
and blood urine nitrogen (BUN) levels. Following routine histological procedures, kidney tissue specimens
were examined under a light microscope. In addition, dismutase (SOD) and glutathione peroxidase (GSH-
Px) enzyme activities and malondialdehyde (MDA) and nitric oxide (NO) contents were determined spectro-
photometrically in tissue samples. It was found that serum uric acid and BUN levels of the rats exposed to
cigarette smoke alone were elevated, although serum creatinine levels did not significantly change. Further-
more, renal SOD, GSH-Px, NO and MDA levels were significantly increased. These increases in serum BUN,
and renal SOD, GSH-Px, NO and MDA levels were significantly inhibited by CAPE treatment. In light micro-
scopic observations of tissues from rats exposed to smoke, mesangial cell proliferation in the renal corpuscles,
dilatation and congestion in the peritubular capillaries and degenerative alterations in the proximal tubules
were noted. There were also atrophic renal corpuscles. However, these histopathological changes were par-
tially disappeared in therats exposed to cigarette smoke plus CAPE. The present findings indicate that cigarette
smoke causes impairment in renal structure and function, which can be prevented by CAPE administration.
Keywords
cigarette, caffeic acid phenethyl ester, kidney, histopathology, biochemistry
Introduction
Cigarettes contain numerous carcinogenic substances.
Smokers are exposed to various chemical compounds
of nicotine, cadmium, lead, benzene, acetone and
mercury (Rahman and MacNee, 1996; S ¸is ¸man et al.,
2003). Nicotine, one of the toxic ingredients of
cigarettes, is known as a toxic alkaloid with many
effects on the central and peripheral nervous, cardio-
vascular and endocrine systems (Kyerematen and
Vesell, 1991; Shah and Helfant, 1988; Zavos, 1989).
Smoking also has negative effects on renal struc-
ture and function. Tobacco compounds are discharged
1Elazig School of Health Sciences, Firat University, Elazig, Turkey
2Department of Anatomy, Faculty of Medicine, Firat University,
Elazig, Turkey
3DepartmentofBiochemistry,
Gaziosmanpasa University, Tokat, Turkey
4Department of Histology and Embryology, Faculty of Medicine,
Erciyes University, Kayseri, Turkey
5Department of Histology and Embryology, Faculty of Medicine,
Firat University, Elazig, Turkey
Faculty ofMedicine,
Corresponding author:
Murat Ogeturk, Department of Anatomy, Faculty of Medicine,
Firat University, 23119 Elazig, Turkey.
Email: mogeturk@firat.edu.tr
Toxicology and Industrial Health
26(3) 175–182
ª The Author(s) 2010
Reprints and permission:
sagepub.co.uk/journalsPermissions.nav
DOI: 10.1177/0748233710362380
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through the kidneys after metabolizing in the liver,
which plays an important role in the development of
renal diseases, and their incidence and progression
(Briganti et al., 2002; Chiu et al., 2001; Klahr and
Morrissey, 2003). Since smokers are constantly
exposed to chemical substances such as cadmium,
lead and mercury, the effects of these chemicals
aggravate nephrotoxicity in presence of renal diseases
(Mortada et al., 2004). A relationship between smok-
ing and renal disease is supported by previous studies.
Glomerulosclerosis has been demonstrated to be a
progressive vasculopathic lesion linked to hyperten-
sion and smoking in earlier studies (Ward and
Studenski, 1992; Markowitz et al., 2002). Smoking
has also been shown to be an independent risk factor
for end-stage renal failure in patients with inflamma-
tory and noninflammatory renal disease (Orth, 2000).
Furthermore, it has been reported that smoking
increases the risk of renal-cell carcinoma directly cor-
related with the number of cigarettes smoked per day
(Yuan et al., 1998). Similarly, in experimental studies,
smoking has been shown to decrease renal blood flow,
increase plasma endothelin-1 concentration, and as a
result, lead to impairment of renal function (Gambaro
etal.,1998).Inaddition, a positivecorrelationhasbeen
found between exposure to cigarette smoke during
pregnancy and renal malformations (Ka ¨lle ´n, 1997;
Nelson et al., 1999). Another study on hemodialysis
patients has shown the negative effects of smoking
on plasma levels of lipid peroxidation products, which
increases the level of oxidative damage (Wang et al.,
2002).
Caffeic acid phenethyl ester (CAPE) is one of the
active components of propolis found in the extract
collected by bees from the plants. It has a sharp and
aromatic smell (Koltuksuz et al., 1999). Studies to
date have described propolis as an antioxidant, anti-
microbic, anti-inflammatory, immunomodulatory and
antimutagenic substance. All of these properties have
been demonstrated to be associated with CAPE
(Dimov et al., 1992; Dobrowolski et al., 1991; Irmak
et al., 2001). Recently, it has been shown that CAPE
exhibits protective effects against testicular oxidative
damage caused by cigarette smoking in rats (Ozyurt
et al., 2006). Additionally, we have previously
reported that hepatic injury had occurred in rats
exposed to cigarette smoke, and that this was partly
improved, presumably, by the antioxidant activity of
CAPE (Pekmez et al., 2007).
The aim of the present studywas therefore to inves-
tigate whether CAPE treatment exerts ameliorative
effects on nephrotoxicity caused by cigarette smoking.
Forthispurpose,wehavehistopathologicallyexamined
deleterious effects of smoking and possible protective
effect of CAPE on tissue damage of rat kidney. We
havealsoexaminedtissuesuperoxidedismutase(SOD),
glutathioneperoxidase(GSH-Px),nitricoxide(NO)and
malondialdehyde (MDA) levels in order to evaluate
the oxidative/antioxidative status and serum uric acid,
creatinine and blood urine nitrogen (BUN) levels in
order to evaluate renal function.
Materials and methods
Preparation of CAPE
CAPE applied in this study was synthesized in the
Physico-Chemistry Laboratory according to the stan-
dard method described by Grunberger et al. (1988).
Animals and treatments
Adult male Wistar albino rats (weighing 200–250 g,
n ¼ 21) obtained from Firat University Experimental
Research Unit were randomly divided into three
groups, of seven animals per group. All animals
received humane care in compliance with the Eur-
opean Community Guidelines on the care and use of
laboratory animals (86/609/EEC). The rats were kept
in plexiglas cages (4 rats/cage) where they received
standard chow (supplied from Elazig Feed Plant,
Elazig, Turkey) and water ad libitum in an air-
conditionedroomwith
temperature (22 + 1?C) and lighting (07.00–19.00
hours). All rats were allowed to acclimatize for a
week prior to experimentation. Animals in group I,
which inhaled fresh air, were used as control. Rats
in group II were exposed to cigarette smoke and rats
in group III were exposed to cigarette smoke and
injected daily with CAPE (10 mmol/kg body weight,
intraperitoneally). A glass cabin was prepared and
insulated with silicone (dimension: 100 ? 50 ? 20
cm, thickness 5 mm). A short plastic pipe, with one
end left outside, was inserted into the cabin. A cigar-
ette lit during the experimental period was placed to
the end of this pipe and the entire cigarette was puffed
by aquarium motor. In this study, cigarettes without a
filter tip (Birinci Cigarettes, Tekel A.S ¸., Elazig, Tur-
key) were used. Two cigarettes lit for 30 min in each
period were placed in cages four times a day during
the study period (group II and III). At the end of the
60-day experimental period, all the rats were
sacrificed.
automaticallyregulated
176Toxicology and Industrial Health 26(3)

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Determination of biochemical parameters in
serum
Blood samples were collected in routine biochemical
test tubes, allowed to clot and the serum was removed
by centrifugation at 2000g for 10 min. All serum sam-
ples were sterile, hemolysis-free and were kept at
þ4?C. Creatinin, BUN, and uric acid were measured
by autoanalyzer using Dade Behring Dimension Clin-
ical chemistry system (Dade Behring Inc., NE, USA)
according to the manufacturer’s instructions.
Preparation of tissues
The kidneys of all animals were excised immediately,
decapsulated and divided longitudinally into two
equal sections. One section was fixed in 10% formal-
dehyde solution for routine histopathological exami-
nation by light microscopy. The other section was
washed in ice-cold saline, placed into glass bottles,
labeled and stored at ?70?C until processed.
Biochemical analysis of kidney tissues
After weighing the kidney tissues, they were homoge-
nized in five volumes of ice-cold Tris-HCl buffer
(50 mM, pH 7.4) containing 0.05% Triton X-100. The
homogenization procedure (IKA Ultra-Turrax t 25
Basic, Germany) was carried out for 2 min at
13,000 rpm. All procedures were performed at 4?C.
Homogenate, supernatant and extracted samples were
prepared and the following determinations were made
on the samples using commercial chemicals (Sigma,
St. Louis, MO, USA). Protein measurements were
made in the samples according to the method
explained by Lowry et al. (1951).
Total (Cu-Zn and Mn) superoxide dismutase (SOD;
EC 1.15.1.1) activity was determined according to the
method of Sun et al. (1988). The principle of the
method is based on inhibition of nitroblue tetrazolium
(NBT) reduction by the xanthine-xanthine oxidase sys-
tem asa superoxidegenerator.Activity was assessed in
the ethanol phase of the supernatant after 1.0 mL of
ethanol-chloroform mixture (5:3, v/v) was added to the
same volume of sample and centrifuged. One unit of
SOD was defined as the amount causing 50% inhibi-
tion in the NBT reduction rate. The SOD activity is
expressed as U/mg protein (Durak et al., 1993).
Glutathione peroxidase (GSH-Px) activity was
measured by the method of Paglia and Valentine
(1967). The enzymatic reaction in the tube (contain-
ing NADPH, reduced glutathione, sodium azide and
glutathione reductase) was initiated by addition of
H2O2and the change in absorbance at 340 nm was
monitored by a spectrophotometer. Activity is
expressed as U/g protein.
The tissue MDA was determined by a method,
based on reaction with thiobarbituric acid (TBA) at
90–100?C (Esterbauer and Cheeseman, 1990). In the
TBA test reaction, MDA or MDA-like substances and
TBA react to produce a pink pigment with an absorp-
tion maximum at 532 nm. The reaction was performed
at pH 2–3 and 90?C for 15 min. The sample was mixed
with 2 vol. of cold 10% (w/v) trichloroacetic acid to
precipitate the protein. The precipitate was pelleted
by centrifugation and an aliquot of the supernatant was
reacted with an equal volume of 0.67% (w/v) TBA in a
boiling water-bath for 10 min. After cooling, the absor-
bance was read at 532 nm. Results were expressed as
nanomoles per gram wet tissue, according to the
standard graphic prepared from measurements with a
standard solution (1,1,3,3-tetramethoxypropane).
NO measurement is very difficult in biological speci-
mens;therefore,tissuenitrite(NO2?)andnitrate(NO3?)
were estimated as an index of NO production. Samples
wereinitiallydeproteinizedwithSomogyireagent.Total
nitrite (nitrite þ nitrate) was measured after conversion
of nitrate to nitrite by copperized cadmium granules by
a spectrophotometer at 545 nm. A standard curve was
established with a set of serial dilutions (10?8–
10?3mol/L) of sodium nitrite. Linear regression was
carried out using the peak area from the nitrite standard.
The resulting equation was then used to calculate the
unknownsampleconcentrations.Resultswereexpressed
as mmol/g wet tissue (Cortas and Wakid, 1990).
Microscopic examination of kidney tissue
specimens
The kidney tissue specimens were fixed in formalde-
hydesolution(10%).
embedded in paraffin wax and sectioned (thickness,
5 mm). Paraffin sections were used for light micro-
scopic examination. For light microscopic evaluation,
paraffin sections were stained with hematoxylin-eosin
(H&E) and examined with Olympus BH2 photomi-
croscope. All sections were evaluated by an experi-
enced observer unaware of the treatment.
Tissuespecimenswere
Statistical analyses
Quantitative data are expressed as mean + standard
deviation (SD). All analyses were performed with
Pekmez et al. 177

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SPSS 15.0 for windows software (SPSS Inc., Chicago,
IL, USA). Distribution of the groups was analyzed
with the Kolmogorov-Smirnov one-sample test.
One-way analysis of variance (ANOVA) test was
performed and post hoc multiple comparisons were
done with least-squares differences (LSD). Differ-
ences were considered to be statistically significant
at p < 0.05.
Results
All animals survived until the scheduled sacrifice.
Serum biochemical parameter levels
Serum uric acid, urine nitrogen and creatinine levels
of all groups are shown in Table 1. Cigarette smoke
exposure for 2 months significantly increased both
serum uric acid and urine nitrogen levels compared
with control values (p < 0.05 vs controls), which was
more prominent in the uric acid level; however, crea-
tinine level did not significantly change. CAPE treat-
ment reduced all serum levels, but a statistical
equality with the control group was provided in only
BUN level (p > 0.05).
Status of lipid peroxidation and antioxidant
enzymes
As shown in Table 1, there were marked increases in
kidney SOD, GSH-Px, MDA and NO levels in cigar-
ette smoking group, emphasizing the generation of
increased oxidative stress. CAPE treatment signifi-
cantly reduced all elevations in tissue levels (p <
0.05 vs exposed to smoke alone), and it achieved sta-
tistically equal levels with controls with the exception
of NO (p > 0.05).
Renal histopathology
In light microscopic observations of the kidney
sections obtained from control animals, the renal
corpuscle structure, glomerular tuft, Bowman’s space
and mesangial cell count were normal. Likewise,
proximal and distal tubules of the same group were
also shown in normal structural and architectural
integrity (Figure 1).
The evaluation of the renal specimens from rats
exposed to cigarette smoke for 2 months revealed
mesangial cell proliferation in the renal corpuscles,
dilatation and congestion in the peritubular capil-
laries, and degeneration in the proximal tubules. In
this group, Bowman’s spaces in the renal corpuscles
could not be distinguished. In addition, a few atrophic
renal corpuscles were detected (Figures 2 and 3).
In the evaluation of the renal samples of the rats
exposed to cigarette smoke and administered CAPE,
mesangial cell proliferation as well as dilatation and
Table 1. Uric acid, blood urea nitrogen (BUN), creatinin, SOD, GSH-Px, MDA and NO values of the study groups
Parameters ControlCigaretteCigaretteþCAPE
20.20 + 5.14a
17.60 + 2.60b
4.80 + 0.83
0.049 + 0.002b
2.360 + 0.544b
19.230 + 4.179b
0.369 + 0.089a,b
Uric Acid (mg/dL)
BUN (mg/dL)
Creatinin (mg/dL)
SOD (U/mg protein)
GSH-Px (U/g protein)
MDA (nmol/g wet tissue)
NO (mmol/g wet tissue)
9.33 + 4.08
17.83 + 3.31
5.16 + 0.40
0.050 + 0.003
2.378 + 1.048
24.348 + 4.768
0.309 + 0.062
21.60 + 6.26a
24.40 + 3.43a
5.40 + 0.54
0.074 + 0.008a
3.701 + 0.855a
40.448 + 6.295a
0.413 + 0.078a
GSH-Px, glutathione peroxidase; MDA, malondialdehyde; NO, nitric oxide; SOD, superoxide dismutase.
Values are expressed as mean + SD. n ¼ 7 per group.
ap < 0.05 significant differences compared to control.
bp < 0.05 significant differences compared to cigarette group.
Figure 1. Section of kidney from a control rat showing
normal architecture (hematoxylin and eosin ?20).
178Toxicology and Industrial Health 26(3)

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congestion
detected. However, in this group, tubular structure
was better preserved than that of rats exposed smoke
alone (Figure 4). Thus, the structural changes in this
group were relatively fewer.
in the peritubularcapillarieswere
Discussion
The negative effects of smoking on the respiratory,
digestive, urogenital, endocrine and cardiovascular
systems have been shown in many studies (Doll,
1996; Hoffmann and Hoffmann, 1997; Kyerematen
and Vesell, 1991; Nordlund et al., 1997; Shah and
Helfant, 1988; Zavos, 1989). The effects of smoking
on the histological structure and functions of the
kidneys have also been reported. Some of these
effects are renal hemodynamic and electrolyte extrac-
tion disorders, diuresis and impairment of proximal
tubules (Chiu et al., 2001; EL-Safty et al., 2003;
McLaughlin et al., 1990; Yu et al., 1997).
In ourstudy, the light microscopic evaluation ofthe
tissue samples from the rats exposed to cigarette
smoke revealed mesangial cell proliferation in the
renal corpuscle, dilatation and congestion in the peri-
tubular capillaries, degeneration in the proximal
tubules and atrophic renal corpuscle. Earlier studies
have shown that smoking leads to glomerular and
proximal tubular damage. In addition, smoking has
been held responsible for renal hemodynamic impair-
ment, diuresis and electrolyte extraction (EL-Safty
et al., 2003; Czekaj et al., 2002; Nakadaira and
Nishi, 2003; Odoni et al., 2002; Schiffl et al., 2002).
Smoking has also been found to affect the glomerular
structure and function in patients of type 2 diabetes,
which has been reported to play an important role in
nephropathy development and progression (Baggio
et al., 2002). In their electron microscopic study,
Thophon et al. (2004) used cadmium, an ingredient
of cigarette with carcinogenic effects, and detected
hydropic swelling in both nuclei and cytoplasm of the
tubular cells, destruction in the brush border,
dilatation in the granular endoplasmic reticulum and
condensations in the mitochondria. In another study,
cigarette smoking was found to cause severe hydropic
Figure 2. In the rats exposed to cigarette smoke (group II),
mesangial cell proliferation (m) in the renal corpuscles,
dilatation and congestion (*) in peritubular capillaries and
proximal tubular degeneration (d) were noted. Bowman’s
space (arrows) was not visible (hematoxylin and eosin ?20).
Figure 3. The renal preparation of the rats exposed to
cigarette smoke (group II). Atrophic renal corpuscles
(arrows) and proximal tubular degeneration (d) were
observed (hematoxylin and eosin ?20).
Figure 4. The appearance of the renal tissue of the rats
exposed to cigarette smoke and administered caffeic acid
phenethyl ester (CAPE; group III) exhibiting mesangial cell
proliferation (m) in the renal corpuscle with disappearance
of Bowman’s space (arrow), dilatation and congestion (*)
in the peritubular capillaries (hematoxylin and eosin ?20).
Pekmez et al.179

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degeneration in the renal tubules, and ethanol added
to smoking was found to cause intense inflammatory
cell infiltration in the renal cortex and calcification
focuses in the cortico-medullary junction (Cigremis
et al., 2004). Lhotta et al. (2002) detected higher rates
of myointimal hyperplasia in the small arteries, glo-
merular sclerosis and arteriolar hyalinosis in the renal
samples of smokers than in the samples of non-
smokers.
Cadmium is a nephrotoxic substance of wide use in
industry and is an ingredient of cigarette. S ¸is ¸man
et al. (2003) have reported high levels of blood cad-
mium and serum uric acid in tobacco workers exposed
to cigarette smoke. Langlois et al. (2003), however,
reported that smoking effected no changes in the level
of uric acid of hypertensive patients. Chan-Yeung
et al. (1981) found decreased level of blood urine
nitrogen with exposure to cigarette smoke and no
change in creatinine level of smoking subjects.
Similarly, Greenberg et al. (1986) also reported
normal levels of creatinine in industrial workers
exposed to cadmium. In our study, the serum uric acid
and nitrogen levels of the rats exposed to cigarette
smoke were elevated. However, there was no change
in the creatinine level.
MDA is the end product of lipid peroxidation and
an indicator of oxidative stress (Kamal et al., 1989).
Our results were compatible with the results of
Cigremis et al. (2004). As in our study, Cigremis
et al. (2004) have reported increased MDA level in
renal tissue samples. Similarly, Agarwal (2005)
detected increased MDA level in nephritic patients.
In the study of Husain et al. (2001), ethanol-
administered rats that were also exposed to cigarette
smoke had increased MDA level.
Antioxidant enzymes such as superoxide dismutase
(SOD) and glutathione peroxidase (GSH-Px) are
essential for preservation of cellular balance and elim-
ination of free radicals (McLaughlin et al., 1990).
Husain et al. (2001) have reported reduced levels of
SOD. Moreover, in the ethanol-administered group
along with exposure to cigarette smoke, they have
reported reduced level of GSH-Px. In our study,
another indicator of oxidative stress, NO, was signif-
icantly decreased in cigarette smoke-exposed animals
compared with healthy controls, while the SOD and
GSH-Px levels were elevated. Increase in the SOD
activity may have been a response of increased oxida-
tive stress in the renal tissue. GSH-Px is an important
antioxidant enzyme acting in H2O2elimination and
lipid peroxidation. Increased activity of GSH-Px
reflects increase in the products of H2O2. The
antioxidant enzyme activities of the rats exposed to
cigarette smoke (group II) were significantly higher
than the rats in the control group, which indicates that
cigarette smoke increases the rate of free radical
production. The increase in the MDA level, which is a
markerofthedamageinflictedonthelipidsbyfreeradi-
cals, is supportive of this hypothesis because the MDA
level of the rats exposed to cigarette smoke (group II)
was significantly higher than the control group.
CAPE, an active component of propolis extract,
hasbeenreportedtobeantimicrobial,anti-inflammatory,
immunomodulating and
(Dobrowolski et al., 1991; Dimov et al., 1992; Irmak
et al., 2001). Recent studies have shown that in addi-
tion to such properties, CAPE is also a potent antiox-
idant compound (Ozyurt et al., 2006; Pekmez et al.,
2007). In addition to these well-known properties,
CAPE has been reported to exert neuroprotective,
nephroprotectiveandlearning-
improving abilities in several studies (Ogeturk
et al., 2005; Ozdem Turkoglu et al., 2007; Ozdem
Turkoglu et al., 2008).
In the present study, we revealed that serum urine
nitrogen, tissue SOD, GSH-Px, NO and MDA levels
decreased in rats exposed to cigarette smoke and
treated with CAPE. Furthermore, the histopathologi-
cal alterations inflicted by cigarette smoke in this
group were substantially reduced and renal tissues
taken from these animals also showed similar
histological appearances to controls.
In conclusion, therenal functions of the rats exposed
to cigarette smoke were reduced and the renal structure
was impaired. These were partially prevented by
CAPE treatment with its antioxidant properties. We
therefore propose that antioxidant supplements in
proper dose and duration may help to protect the
kidneys against the harmful effects of smoking.
antimutagenic agent
and memory-
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