Preventive Effects of Green Tea ( Camellia Sinensis var. Assamica ) on Diabetic Nephropathy

Abstract

This study aimed to evaluate the preventive effects of Camellia sinensis var. assamica (CSVA) on diabetic nephropathy in in vitro and in vivo models.
MDCK cells were incubated with 1 mM of oxalate with or without different concentrations of CSVA, then MTT and malondialdehyde (MDA) assays were performed to investigate the preventive effects of CSVA on oxalate-induced cytotoxicity and oxidative stress. Thirty male db/db mice were divided into three groups. Group 1 were fed AIN-93G ad libitum; group 2 were fed AIN-93G mixed with 10% fermented CSVA ad libitum; group 3 were fed AIN-93G mixed with 10% non-fermented CSVA ad libitum. The mice were sacrificed 14 weeks later, and the serum glucose level, 24-hour urine chemistry, and morphological changes in the kidneys were examined.
As CSVA concentrations increased, viable MDCK cells increased in concentration. MDA production decreased over time in the CSVA treated group. The creatinine clearance of group 3 was lower than those of groups 1 and 2. The amount of urine microalbumin and protein in group 1 were higher than those in groups 2 and 3. Also, more glomerulus basement membrane foot processes were preserved in groups 2 and 3.
In conclusion, CSVA has beneficial preventive tendencies towards diabetic nephropathy in both in vitro and in vivo models.

Full text

Yonsei Med J http://www.eymj.org Volume 53 Number 1 January 2012
138
Original Article http://dx.doi.org/10.3349/ymj.2012.53.1.138
pISSN: 0513-5796, eISSN: 1976-2437 Yonsei Med J 53(1):138-144, 2012
Preventive Effects of Green Tea (Camellia Sinensis var. Assamica)
on Diabetic Nephropathy
Min-Yong Kang,1 Yong Hyun Park,1 Bong Sub Kim,1 Su Yeon Seo,1 Byong Chang Jeong,2
Jung-In Kim,3 and Hyeon Hoe Kim1
1Department of Urology, Seoul National University College of Medicine and Clinical Research Institute, Seoul;
2Department of Urology, Sungkyunkwan University School of Medicine, Seoul;
3School of Food and Life Science, Inje University, Gimhae, Korea
Received: September 28, 2010
Revised: April 4, 2011
Accepted: April 21, 2011
Corresponding author: Dr. Hyeon Hoe Kim,
Department of Urology, Seoul National
University College of Medicine,
101 Daehak-ro, Jongno-gu,
Seoul 110-744, Korea.
Tel: 82-2-2072-2420, Fax: 82-2-742-4665
E-mail: hhkim@snu.ac.kr
∙ The authors have no financial conflicts of
interest.
© Copyright:
Yonsei University College of Medicine 2012
This is an Open Access article distributed under the
terms of the Creative Commons Attribution Non-
Commercial License (http://creativecommons.org/
licenses/by-nc/3.0) which permits unrestricted non-
commercial use, distribution, and reproduction in any
medium, provided the original work is properly cited.
Purpose: This study aimed to evaluate the preventive effects of Camellia sinensis
var. assamica (CSVA) on diabetic nephropathy in in vitro and in vivo models. Ma-
terials and Methods: MDCK cells were incubated with 1 mM of oxalate with or
without different concentrations of CSVA, then MTT and malondialdehyde (MDA)
assays were performed to investigate the preventive effects of CSVA on oxalate-
induced cytotoxicity and oxidative stress. Thirty male db/db mice were divided
into three groups. Group 1 were fed AIN-93G ad libitum; group 2 were fed AIN-
93G mixed with 10% fermented CSVA ad libitum; group 3 were fed AIN-93G
mixed with 10% non-fermented CSVA ad libitum. The mice were sacriced 14
weeks later, and the serum glucose level, 24-hour urine chemistry, and morpholog-
ical changes in the kidneys were examined. Results: As CSVA concentrations in-
creased, viable MDCK cells increased in concentration. MDA production de-
creased over time in the CSVA treated group. The creatinine clearance of group 3
was lower than those of groups 1 and 2. The amount of urine microalbumin and
protein in group 1 were higher than those in groups 2 and 3. Also, more glomeru-
lus basement membrane foot processes were preserved in groups 2 and 3. Conclu-
sion: In conclusion, CSVA has benecial preventive tendencies towards diabetic
nephropathy in both in vitro and in vivo models.
Key Words: Camellia sinensis var. assamica, diabetic nephropathy, prevention
INTRODUCTION
Diabetic nephropathy is a microvascular complication of diabetes mellitus associat-
ed with significant morbidity and mortality.1 Because alterations to glomerular
structures are irreversible, the primary goal of management for patients with diabet-
ic nephropathy is to prevent disease progression to end stage renal disease. There-
fore, strict control of blood pressure as well as maintaining normal glucose and lipid
levels are important for preserving renal function.2 However, it is well known that
attempts to control these factors are not sufcient for the prevention of progression
of diabetic nephropathy. Thus, many investigators have started to study the effects

Effects of Green Tea Extracts on Diabetic Nephropathy
Yonsei Med J http://www.eymj.org Volume 53 Number 1 January 2012
139
added, followed by incubation for four hours at 37°C. Optical
densities were measured on a microplate reader (Becton
Dickinson Labware, Franklin Lakes, NJ, USA) at 540 nm.
Malondialdehyde (MDA) assay
MDCK cells were exposed to 1 mM of sodium oxalate for
various periods of time (30, 60, and 120 minutes) with or
without 50 μg/L of CSVA extract. The lipid peroxidation
levels were then evaluated by measuring the MDA as de-
scribed by Wong, et al.8
In vivo study
Animals and group controls
All surgical and experimental procedures were approved by
the Seoul National University Institutional Animal Care and
Use Committee (SNUIACUC, No. 08-0213) and conducted
between November 2008 and February 2009. Thirty male
C57BL/Ks db/db mice were divided into three groups by ran-
dom sampling methods after adaptation to their new environ-
ment for one week. Group 1 (n=10) were fed AIN-93G ad li-
bitum. Group 2 (n=10) were fed AIN-93G mixed with 10%
fermented CSVA ad libitum. Group 3 (n=10) were fed AIN-
93G mixed with 10% non-fermented CSVA ad libitum. Table
1 shows the composition of the basal and experimental diets
in each group.
Experimental protocols
We monitored the total amounts of food and water intake
and the body weight of the mice every week. At 6 and 12
weeks of oral intake, blood was collected from the tail-vein
and fasting blood glucose levels were measured using Accu-
Chek Performa blood glucose monitoring system (glucome-
ter) (Roche Diagnostics, Pleasanton, CA, USA). After 14
weeks of the designated oral intake, 24-hour urine was col-
lected from each mouse in their metabolic cages. We evalu-
ated several factors to assess the effects of CSVA on diabetic
nephropathy. These factors included the results of the 24-
hour urine analysis, which measured creatinine clearance
(Ccr), amount of microalbumin, and total protein. In addi-
tion, we examined the histology of the kidneys of all experi-
mental mice to determine the effects of CSVA on the mor-
phology of diabetic nephropathy. For these examinations, all
mice were sacriced using the cardiac puncture technique
after fasting for 12 hours. The kidneys were carefully re-
moved after the abdominal cavity was opened. The fatty tis-
sue was gently removed from the renal parenchyma and the
of traditional medicines, folk remedies, and herbal medicines
to identify novel agents that might be effective managing dia-
betic complications, especially diabetic nephropathy.3-7
Recently, interest in green tea as a promising agent for the
prevention or reduction of risk for many human diseases
has increased worldwide. With regard to preventing diabet-
ic complications, the antioxidant activity of green tea might
play an important role. In this study, we aimed to evaluate the
preventive effects of Camellia sinensis var. assamica (CSVA),
traditionally consumed as a health-promoting beverage in
Korea, on diabetic nephropathy in both in vitro and in vivo
models. In addition, we classied CSVA as a fermented or
non-fermented leaf in the in vivo protocol and evaluated
whether the preventive effects on diabetic nephropathy dif-
fered according to the degree of fermentation.
MATERIALS AND METHODS
In vitro study
Preparation of CSVA
Non-fermented CSVA were obtained from CSVA in Gim-
hae, Korea. Twenty grams of CSVA leaves were extracted
with 400 mL of 100% methanol in a round-bottom flask
equipped with a condenser. The temperature of extraction
was kept at boiling temperature and the extraction mixture
was constantly stirred with a magnetic bar. After two hours
of extraction, the extraction mixture was cooled, the vacu-
um ltered (0.45 μm), and the methanol evaporated under
the vacuum at 40°C.
Cell culture
MDCK cells (American Type Culture Collection, Washington
D.C, USA) were cultured in a Dulbecco’s modified Eagle
medium (Invitrogen, Carlsbad, CA, USA) supplemented with
10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO,
USA) at 37°C, in air containing 5% CO2.
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) assay
MDCK cells were subcultured in 96-well plates and grown to
70-80% conuence. The cells were then exposed to 1 mM of
sodium oxalate (Alfa Aesar, Ward Hill, MA, USA) to induce
renal cell damage and various concentrations of CSVA extract
(0, 10, and 25 μg/L) for 24 hours. Subsequently MTT (0.1
mg, 50 μL of 2 mg/mL) in phosphate-buffered saline was

Min-Yong Kang, et al.
Yonsei Med J http://www.eymj.org Volume 53 Number 1 January 2012
140
pylene oxide inltration, epoxy resin was treated for over-
night and embedded with freshly prepared resin. Finally, the
specimens were polymerized at 60°C for 48 hours. Follow-
ing the E-rm block formation, ultrathin sections (60 nm)
were obtained and subjected to electron staining, meaning
double staining with uranyl acetate for 30 min and lead ci-
trate for 10 min; the specimens were then viewed on trans-
mission electron microscopy (JEM-1200EX, JEOL, Tokyo,
Japan) operating at 120 kV, which was documented by the
digital imaging. The remnant renal tissues were then placed
in formalin for parafn block formation. The parafn blocks
were sectioned and then stained with hematoxylin and eosin
(H&E). The tissues were then carefully examined by Olym-
pus SZX7 stereomicroscope (Olympus, Tokyo, Japan) with
a plain objective.
Statistical analysis
The Statistical Package for the Social Sciences (SPSS), ver-
sion 11.0 (SPSS Inc., Chicago, IL, USA) was used for the
statistical analysis. All experimental results were analyzed
using ANOVA and Dunnett’s test and are reported as mean
values±standard deviation. p<0.05 was accepted as statisti-
cally signicant.
RESULTS
The effect of CSVA extracts on MDCK cell viability
following exposure to sodium oxalate
The number of viable MDCK cells signicantly decreased
by 40% after exposure to 1 mM of sodium oxalate as com-
pared to the control group (Fig. 1). In contrast, the number
kidneys were weighed. For the histological examination un-
der an electron microscope, we excised 1×1 mm of cortical
tissue from each kidney and immediately xed the samples
in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer
at 4°C for 4 hours and wash them twice (each time for 1
hour) with 0.1 M buffer. Then, we performed secondary x-
ation in 2% osmium tetroxide in 0.2 M buffer for 1 hour and
rinsed twice (each time for 5 min) in 0.2 M buffer and multi-
step dehydration in 70%, 90% and 100% ethanol. After pro-
Fig. 1. MDCK cell viability on the concentration of CSVA following exposure
to sodium oxalate (*p<0.05). MDCK, Madin-Darby canine kidney; CSVA,
Camellia sinensis var. assamica.
0
20
40
60
80
100
Control 0 µg/L 10 µg/L 25 µg/L
**
*
Cell survival fraction (%)
Fig. 2. MDA levels following exposure to sodium oxalate and CSVA accord-
ing to time (*p<0.05 and **p<0.01 ). MDA, malondialdehyde; CSVA, Camellia
sinensis var. assamica.
0
0.05
0.10
0.15
0.20
0.25
Control 30 minutes 60 minutes 120 minutes
Absorbance
*
*
*
** ** **
Sodium oxalate
Co-exposure to CSVA extravts
Table 1. Composition of the Basal and Experimental Diets
Ingredients (%) Group 1 Group 2 Group 3
Corn starch 39.8 39.8 39.8
Casein 20.0 17.9 18.1
Dextrinized starch 13.2 13.2 13.3
Sucrose 10.0 10.1 9.8
Alpha-cellulose 5.0 5.1 5.0
Mineral mixture 3.5 3.5 3.5
Vitamin mixture 1.0 1.0 1.0
L-cystine 0.3 0.3 0.3
Choline bitartrate 0.25 0.25 0.25
Tert-butyl hydroquinone 0.0014 0.0014 0.0014
Soybean oil 7.0 6.9 7.1
Fermented CSVA* 0 10.0 0.0
Non-fermented CSVA* 0 0.0 10.0
CSVA, Camellia sinensis var. assamica.
*Freeze-dried.

Effects of Green Tea Extracts on Diabetic Nephropathy
Yonsei Med J http://www.eymj.org Volume 53 Number 1 January 2012
141
Histopathological assessment
Fig. 3 shows the mean weight of the kidneys. Groups 2 and
3 had signicantly decreased kidney weights (the sum of
right and left; 0.45±0.04 g, 0.36±0.03 g, and 0.34±0.03 g in
groups 1, 2 and 3, respectively, p<0.05). Optic microscopy
showed no signicant differences in the architecture of the
glomerulus basement membrane (GBM). However, the
electron microscope showed that more GBM foot process-
es were preserved in groups 2 and 3, and especially group 3
had a greater number of foot processes than group 2, while
group 1 had loss of all of GBM foot processes (Fig. 4).
DISCUSSION
Hyperglycemia is strongly associated with diabetic compli-
cations. Effective control of hyperglycemia is an important
goal of treatment and prevention.9 However, there are few
effective agents that prevent the development of diabetic
nephropathy, although strict control of hyperglycemia is
possible with the use of several diabetic treatments.10,11 Re-
cently, green tea has been the focus of great interest due to
its benecial properties for the prevention and treatment of
human disease. Several epidemiology and experimental
of MDCK cells increased as the concentration of CSVA in-
creased, as noted by the fraction of viable MDCK cells:
62.9%, 66.0%, and 75.1% in order (Fig. 1) (p<0.05).
MDA levels following exposure to sodium oxalate and
CSVA extracts
The MDA production increased with time in the sodium
oxalate-treated group. In contrast, in the group with co-ex-
posure to CSVA extracts, MDA levels decreased with time
until 60 minutes, then increased slightly after 120 minutes
(Fig. 2) (p<0.05).
Changes in mean body weight
The mean body weights of the three experimental groups
were 45.7±2.3 g, 42.5±6.4 g, and 36.2±5.5 g, respectively.
The mean body weight of group 3 was signicantly lower
than the other two groups (p<0.05) (Table 2).
Serum glucose and urine chemistry
After 6 weeks of oral intake, the mean serum glucose level
of the control group was higher than that of the CSVA-
treated group (528.9±83.5 mg/dL versus 402.0±100.3 mg/
dL, p<0.05) (Table 2). However, after 12 weeks of oral in-
take, there was no statistical difference in the mean serum
glucose level between the control and CSVA-treated groups
(457.5±191.5 mg/dL versus 390.8±115.0 mg/dL, p>0.05)
(Table 2).
The Ccr of group 3 was 2.8±1.6 μL/min; this result indicat-
ed a signicant decrease in the Ccr in group 3 compared to
those in groups 1 and 2 (p<0.05) (Table 2). The amounts of
microalbumin and protein in the 24-hour urine of group 1 were
signicantly higher than those of groups 2 and 3 (20.8±5.7 μg
versus 11.6±5.6 μg and 11.5±9.6 μg in the 24-hour microalbu-
min excretion; 73.3±25.2 μg versus 21.4±9.2 μg and
24.1±14.0 μg in total protein excretion, p<0.05) (Table 2).
0
0.1
0.2
0.3
0.4
0.5
0.6
Group 1 Group 2 Group 3
Kidney weight (g)
**
**
** **
Left Right Total
Fig. 3. The mean kidney weights of the db/db mouse (*p<0.05 and **p<0.01
relative control).
Table 2. The Overall Results of In Vivo Protocol (db/db mouse)
Group 1 Group 2 Group 3
Mean body weight (g) 45.7±2.3 42.5±6.4 36.2±5.5*
Blood gloucose level (mg/dL)
At 6 wks 528.9±83.5 402.0±100.3**
At 12 wks 457.5±191.5 390.8±115.0
24 hrs urine analysis
Ccr (µL/min) 6.4±3.2 6.4±3.4 2.8±1.6*
Microalbumin (µg) 20.8±5.7 11.6±5.6* 11.5±9.6*
Total protein excretion (µg) 73.3±25.2 21.4±9.2* 24.1±14.0*
Ccr, creatinine clearance.
*p<0.05 by Kruskal-Wallis test.
**p<0.05 by Mann-Whitney U test.

Min-Yong Kang, et al.
Yonsei Med J http://www.eymj.org Volume 53 Number 1 January 2012
142
and atherosclerosis, which have been associated with free-
radical damage.4-6
We demonstrated that administration of CSVA for 6 weeks
lowered serum glucose levels, indicating the benecial ef-
fect of CSVA on glucose tolerance in the early period of in-
take. However, after 12 weeks of feeding, serum glucose
levels did not statistically differ between the control and the
CSVA treated groups. This nding might be explained by
some experiments that showed that insulin resistance im-
proved gradually and developed to control serum glucose
levels effectively with age in diabetic mice.7,15
The results of our study showed that there was less uri-
nary protein excretion in the experimental group than in the
control group. These ndings suggest that administration of
CSVA reduced the amount of proteinuria, which is consis-
tent with the ndings of several previous investigations into
the benecial effects of green tea on proteinuria.16,17
Unexpectedly, the 24-hour Ccr and the total kidney weight
were significantly higher in the control group than in the
CSVA, especially non-fermented, treated groups. However,
more GBM foot processes were preserved in the CSVA treat-
ed group. This paradoxical nding might be attributed to a
hyperfiltration phenomenon caused by loss of the GBM.
Glomerular ltration rate, kidney size, and plasma renal ow
were found to be supra-normal in young diabetes patients as
compared to the non-diabetes controls.18 It was hypothesized
that alterations to glomerular microcirculation would lead to
single nephron injury; remnant nephrons would compensate
with increases in pressures and ows; and that a self-perpetu-
ating cycle of renal injury would be established.19
In addition, we found that non-fermented CSVA was more
effective with regards to controlling body weight, Ccr, and
histological changes in diabetic nephropathy than ferment-
ed CSVA. This suggests that fermentation of CSVA might
attenuate the benecial effects on body weight control, Ccr
studies have provided evidence that green tea has antioxi-
dant activity, anticarcinogenic potential, and preventive ef-
fects towards cardiovascular disease.4-7 However, there is
still controversy regarding the benecial effects of green tea
on diabetic nephropathy.
Renno, et al.3 recently showed that green tea had long-
term benecial effects on diabetic nephropathy in an animal
model. They reported that green tea catechins improved lev-
els of serum glucose and glycosylated protein as well as the
urine parameters. Ryu, et al.12 did not nd that green tea had
benecial effects on serum glucose levels or insulin resis-
tance in their study. However, these conflicting findings
might be due to differences in doses, amounts of green tea,
time of exposure and type of green tea as well as the degree
of hyperglycemia and the methods of experimentation.
There are two basic varieties of green tea; CSVA, a large-
leaf, tall and quick-growing tree well-suited to very warm
climates, and Camellia sinensis var. sinensis, a small-leaf,
slower-growing bush that can withstand colder climates.
Green tea produced from CSVA has been known to have
higher polyphenol content (30%) than green tea from Ca-
mellia sinensis var. sinensis (20%).13 However, most of
studies have focused on Camellia sinensis var. sinensis. To
our knowledge, ours is the rst study to show the preven-
tive effect of CSVA on diabetic nephropathy.
We showed that CSVA prevented oxalate-induced cyto-
toxicity and oxidative stress in in vitro experiments. Viable
MDCK cells proliferated after the administration of CSVA
extracts, up to about 15%, under conditions of oxalate ex-
posure. In addition, the MDA levels in the CSVA extract-
treated group decreased with time until 60 minutes. Consis-
tent with these results, Jeong, et al.14 reported that green tea
administration reduced oxalate induced free-radical stress
in NRK-52E cells. Several studies have reported the anti-
oxidative effects of green tea even in Parkinson’s disease
Fig. 4. Electron microscopy (EM) of GBM (glomerulus basement membrane): (A) Group 1 is the control group. (B) Group 2 is the fermented
Camellia sinensis var. assamica (CSVA)-treated group. (C) Group 3 is the non-fermented CSVA-treated group. The black arrows show the
GBM foot process.
A B C

Effects of Green Tea Extracts on Diabetic Nephropathy
Yonsei Med J http://www.eymj.org Volume 53 Number 1 January 2012
143
estry, Ministry for Agriculture, Forestry and Fisheries, Re-
public of Korea.
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Finally, several parameters used in this study, such as
MTT assay, weight of kidneys, albuminuria, and the EM
images of foot process effacement may not be enough to
show the obvious preventive effect of CSVA on diabetic ne-
phropathy and to elicit the signicant correlation between
in vitro and in vivo study. Thus, we may not conclude that the
CSVA has denite preventive effects on diabetic nephropa-
thy; rather, we may say that CSVA has benecial tenden-
cies regarding prevention of diabetic nephropathy. We con-
sider this study to be a pilot study for the preventive effect
of CSVA on diabetic nephropathy. More denitive evidence
is needed in in vitro and in vivo models in further studies
using several parameters, such as urinary 8-OH-deoxy-
huanosine, 8-iso-PGEF2a, HbA1c levels, and EM exami-
nation of GBM thickening and mesangial volume increase.
In conclusion, the results of the present study demon-
strate that CSVA is associated with preventive effects in ox-
alate-induced cytotoxicity and production of lipid peroxida-
tion in an in vitro setting. Furthermore, CSVA has benecial
preventive tendencies with regard to early stage diabetic
nephropathy, acting by preventing glomerular hyperltra-
tion, hypertrophic changes, and subsequent protein loss in
the urine.
ACKNOWLEDGEMENTS
We acknowledge the nancial support for this study from
Technology Development Program for Agriculture and For-

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