Borra 1

Effects of Kefir on Antioxidant Enzymes in Rat Kidneys subjected to

Doxorubicin
Nimisha Borra
Frontiers of Science Institute, University of Northern Colorado
July 22nd, 2016

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Table of Contents

Abstract
..3

Introduction..
..3-4
Literature
Review.4-7
Methods & Materials...
..7-10

Results..
.10-12

Discussion..
.13-14

Acknowledgements...
15

References.

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..16-17

Abstract
Doxorubicin is a very widely used chemotherapy drug, though it is limited due
to its cardiotoxicity and nephrotoxicity that is present. Kefir is a form of fermented
milk made from kefir grains and is used as a way to combat the negative effects of
doxorubicin. Both of these substances have their benefits and their ways of
targeting cancer cells. Nephrotoxicity is the harmful intake into the kidneys, due to
the chemotherapy drug. The oxidative stress would be measured through the four
antioxidant enzymes that are studying and the proteins identified through this
procedure. The overall goal of this project was to measure the oxidative stress of

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the rat kidneys through the use of kefir paired with doxorubicin. The rats that we
sampled were placed into four groups: Doxorubicin+Kefir, Doxorubicin+Milk,
Saline+Kefir and Saline+Milk. The Western Blot procedure was in effect once the
homogenization and Bradford Protocol was complete. This study was important in
understanding how the antioxidant enzymes were affected through the use of kefir
on animals exposed to the doxorubicin drug. The results, however, were
inconclusive and the project was unable to be completed.
Introduction
The purpose of this research is to determine if the use of kefir can reverse the
effects of Doxorubicin (DOX) in rat kidneys through the oxidative stress pathway. We
are evaluating the oxidative stress of the rats that were fed DOX, DOX+Kefir,
Control, and Control+Kefir. The aim is to study the antioxidative enzymes shown in
the unhealthy rats compared to the healthy rats.
Doxorubicin is a chemotherapy drug that is used to decrease the amount of
cancer cells present in the body. It was first discovered in the 1950s and was named
Adriamycin but later renamed Doxorubicin (1). The use of doxorubicin is restricted
due to the cardiotoxicity that comes with the drug as it damages many parts of the
body (2). If you give patients a higher dose of DOX, it causes more damage to the
body. Doxorubicin leads to membrane damage, tissue damage, DNA damage and
congestive heart failure (2). If the blood is not pumping and circulating properly,
then the blood cannot reach the kidneys. This would directly affect the rats that are
being studied in the way that they kidneys would no longer be able to function
properly without blood supply.

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Kefir is a probiotic, discovered in Russia/Eastern Europe and Southwest Asia
and is known to be very beneficial for the body. Kefir is made from kefir grains
which are then converted to sugar, or Kefirin, in the milk, fermenting them (3). Kefir
contains many different proteins and digestive enzymes and the kefir grains
themselves inhibit tumor growth and reduce inflammation. A study that was
performed in China in 2007 showed that after 6 days of drinking Kefir, there was a
reduction of cancerous cells in the human mammary cells (4). Past studies like
these show how Kefir halts the spreading of most cancerous cells, while still
protecting the healthy cells.
Oxidative stress refers to the inability to activate antioxidant enzymes that
prevent the harmful effects from taking place. The antioxidant enzymes that we will
be analyzing are SOD1, SOD2, Catalase and GPx. The enzymes that we will be
analyzing are SOD1 and SOD2. SOD1 stands for Superoxide Dismutase 1 which is
made by the SOD1 gene and it attaches with other molecules to break down toxicity
(5). SOD2 encodes a specific protein to convert hydrogen peroxide and diatomic
oxygen (6). The purpose of these two enzymes is to be able to identify if the
oxidative stress observed is similar to the oxidative stress on a normal healthy rat.
The kefir and doxorubicin will work to reduce the amount of cancerous cells,
more so the kefir will further prevent the the healthy cells from also being affected.
It was hypothesized that the intake of kefir along with the doxorubicin drug should
decrease the negative effects implemented from doxorubicin shown through the
oxidative stress measured through the experiment.

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Literature Review
Doxorubicin (DOX) is a chemotherapy drug and is known to be an effective
drug that decreases the amount of cancer cells. This classifies doxorubicin as an
anthracycline antibiotic due to these factors (7). DOX is used to treat different types
of cancers such as leukemia, bladder, breast, stomach, lung, ovarian, thyroid and
others (1). It is made from a soil fungus known as Streptomyces (7). Doxorubicin
was first used as a form of cancer treatment but was named Adriamycin which was
the brand name until it was revised to the chemical name. Its origins trace back to
the 1950s when a strain of Streptomyces was mutated, producing a red colored
antibiotic (1). Doxorubicin blocks an enzyme called topoisomerase 2, which is an
enzyme that stops the growth of cancer cells and prevents cell division (8).
However, doxorubicin is not that widely used due to the cardiotoxicity and
nephrotoxicity that accompanies it. DOX releases reactive oxygen species (ROS),
which may lead to DNA damage and membrane damage. (2) Other negative side
effects from this drug include neutropenia, anemia, and alopecia. Using a large dose
may even increase the risk of congestive heart failure and death (1). This study is
important in order to determine the effectiveness of kefir diet in order to reverse the
effects of doxorubicin.
Doxorubicin cardiotoxicity refers to the damaging of the heart, making it
unable to pump blood or circulate properly. When doxorubicin is injected into the
animal, there is a higher chance of cardiomyopathy to occur, depending on the
amount given to the subject. If a higher dose were given, that would lead to a
higher risk of damage. A dilated cardiomyopathy is observed leading to myofibrillar
loss, fibroplasia and apoptosis (9). A more efficient drug is preferred, one that

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follows the same success rate with the destruction of cancer cells but does not
contain the same level of toxicity as doxorubicin. Doxorubicin related nephropathy is
also observed because if the heart isnt pumping blood as it should, it might alter
the circulation that is delivered to the kidneys. This drug is injected through the
central line or peripheral venous line, and one must be carefully trained to
administer it so that you do not let it escape the vein. Doing so can cause extensive
tissue damage and encourage a lot of pain (7). Relating directly with the kidneys,
taking doxorubicin increases your chances of developing Tumor Lysis Syndrome,
which occurs when a mass amount of neoplastic cells are killed at a very rapid rate.
Being diagnosed with this may lead to kidney failure as a result from the
doxorubicin drug (10). In the form of a chemotherapy drug, doxorubicin is an
effective drug in terms of fighting off cancerous cells, however it cannot distinguish
between cancerous cells and normal cells (7).
Kefir is a probiotic used to enhance immunity and strengthen the body (13).
During the fermentation process, many beneficial bacteria are produced, including
digestive enzymes and amino acids (4). Kefir is essentially fermented milk made
from kefir grains, originating from Russia/Eastern Europe and Southwest Asia (3).
Since it is a fermented beverage, it enhances the microbes in our bodies to make
their own natural antibodies. Kefiran is the sugar that is produced from the bacteria
and contributes to the benefits (11). Kefir can inhibit the growth and appearance of
harmful bacteria and cleanse harmful toxins (3). Kefir contains many
microorganisms that improve lactose digestion, very similar to yogurt and has many
nutrients that are very useful (12). Kefir grains fight against cancer by inhibiting
tumor growth and reducing inflammation (4). Past studies have shown that this
product has the ability to target cancerous cells directly, without damaging the

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healthy cells (3). According to a study performed in China in 2007, the use of kefir
for six days stopped the cancer cells from growing on human mammary epithelial
cells (4). Kefir is also known to be a anti-inflammatory product so that creates a
safer foundation for the beverage to be administered (13).
Oxidative Stress is measured through the imbalance and inability to activate
antioxidants to detoxify harmful effects. The use of kefir to attenuate oxidative
stress is indicated by a previous study in which the amount of control over oxidative
stress increased. An increased amount of ROS may lead to a high level of oxidative
stress and malondialdehyde, which is a very toxic molecule (13). Doxorubicin
cardiotoxicity and nephrotoxicity will most likely lead to an increase in oxidative
stress so the kefir diet may be a contribution to the reduction of oxidative stress.
(13, 14). Antioxidant enzymes such as SOD1 and SOD2 would affect the results
under doxorubicin and kefir influence as well. Superoxide Dismutase 1 is an enzyme
made by the SOD1 gene and attaches with other molecules attempting to break
down the harmful effects from superoxide radicals. Mutations of that gene can lead
to damaging of the spinal cord, muscle weakness and a decrease in control of
muscles (5). SOD2 encodes a specific protein that leads to the conversion to
hydrogen peroxide and diatomic oxygen and is a mitochondrial isozyme (6).
Doxorubicin decreases SOD2 and the SODs convert superoxide to hydrogen
peroxide. Tsa1p regulates the oxidative stress within the cell and is considered an
enzymatic defense. In terms of a nonenzymatic defense, melanin is an example of
one that reduces oxidants and mannitol (15). The oxidative stress from the use of
doxorubicin is shown through the decrease of SOD2 and weakness from the
mutation of SOD1.

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It was hypothesized that the intake of kefir along with the chemotherapy
drug, doxorubicin, should decrease the negative effects implemented from
doxorubicin and decrease the amount of oxidative stress in the kidneys of the rats.
The lower the dose of doxorubicin intake on the rats, the more positive of a
response should occur and the use of kefir should half the damaging factors from
the drug. The beneficial progressions of Kefir would prevent the spread of the
cancer without attacking other cells and provide a more positive response.

Methods and Materials

Homogenization: The process began with the homogenization of ten kidney samples
from rats that were previously excised. The samples were removed from -80
degrees Celcius and left to thaw at room temperature. One set of 2 mL Eppendorf
microcentrifuge tubes were labeled for each kidney sample and a small piece of the
each tissue sample was cut off and placed in each tube. 500 L of RIPA lysis buffer
(Santa Cruz Biotechnology) and 10 L of protease inhibitor cocktail (Santa Cruz
Biotechnology) was added to each tube and the samples were minced using a
Scilogex D160 (Rocky Hill, CT) homogenizer. Each tube was sonicated for nuclear
bound proteins and the values were recorded. A Mini Spin microcentrifuge
(Eppendorff) was used to centrifuge the tubes for 10 minutes at 10,000 g and 13.1
RPM. The supernatant was separated and transferred into a second set of
Eppendorff tubes.

Bradford Protocol: 10 L of premade solutions (Thermo Fisher Scientific) of protein

concentration, 125 L/mL, 250 L/mL, 500 L/mL, 750 L/mL, 1000 L/mL, 1500
L/mL and 2000 L/mL were added to cuvettes containing with 1 mL of Bradford

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Reagent. A parafilm cover was used to mix the solutions and each cuvette was
wiped clean using a Kimwipe. A Genesys 20 spectrophotometer (Thermo Electron
Corporation) was used to read the absorbancy at 595 nm. From the standard
absorbancies, a graph was created using Excel including each protein concentration
and the corresponding absorbency. A trendline was selected and an R^2 value of
greater than 0.99 appeared, showing that the equation was appropriate to use.
Another set of cuvettes were used and 1mL of Bradford Reagent was added as well
as 10 L of the homogenized kidney sample set. The samples were then diluted to
4mg/mL by using 100 L of RIPA buffer. The equation that was used was X=(protein
concentration x 100)/4. An equal volume of Laemelli buffer was added to each
sample to double the amount of substance. All of the samples were then stored in
the freezer at a temperature of -80 degrees Celsius.

Western Blotting: The samples were removed from the freezer and allowed to thaw.
Then they were placed in a beaker of boiling water for 2 minutes and then set in a
tub of ice for 5 minutes. To make the running buffer, 100 mL of 10X (Invitrogen)
running buffer was added to 900 mL of deionized water. The pre cast gels
(Invitrogen by Thermo Fisher Scientific) were removed from the wrapping and the
combs were taken off to expose the wells in the gel. The gel cassettes were added
to the Xcell Surelock blot module (Invitrogen by Thermo Fisher Scientific) and it was
filled with the running buffer. About 15 L of each sample, 10 L of MagicMark
Protein Ladder (Invitrogen) and SeaBlue (Invitrogen) was added to each well. The
samples ran at 135 V and 60 mA for about 2-3 hours. The modules and cassettes
were removed and blotted dry. To transfer the buffer, 40 mL of 25x transfer buffer
(Novex) was added to 200 mL of methanol and 760 mL of deionized water. The pads

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and filter paper used in this process were soaked in the transfer buffer before being
placed on the membranes. The PVDF membranes (Novex, Life Technologies) were
placed in a dish containing methanol (Thermo Fischer Scientific) for 30 seconds then
soaked in deionized water before it was placed in the transfer buffer. For the actual
transfer, the cassettes were broken open and the gels were removed. A piece of
filter paper was placed directly on top of the gel and the foot was trimmed off of
each gel. The membrane was placed on the gel and the exposed side of the gel was
covered with filter paper. A glass Pasteur pipette was used to roll out all of the
bubbles in order to create a good transfer. The gel filter paper - membrane
sandwich was placed into the transfer module and added to the Xcell Surelock blot
module (Invitrogen by Thermo Fisher Scientific). The transfer buffer was added to
the inside of the module and this ran for 90 minutes at 25 V and 100 mA. Once that
was complete, the membrane was placed in a dish with ultrapure water and was
shaken for 5 minutes to remove the loosely bound proteins. The wash was repeated,
three times. The blocking solution was added and the membranes were left to rock
for 1 hour. Once the membrane was rinsed three times with TBSt, the primary
antibody was added and was incubated on the rocker overnight. The primary
antibody was removed and the wash TBSt three times on five minutes on the rocker.
This procedure was repeated 3 more times and the secondary antibody was added
and incubated for 1 hour. Afterwards, the wash solution was added 4 more times,
preparing it for imaging.

Antibody Probing: The membranes were blocked using 10 mL of the blocking

solution made using 1.7g of nonfat dry milk and 40 mL of TBSt. They were placed on
the rocker for one hour, washed with TBSt four times of five minutes. The primary

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antibodies were then diluted in 3 mL (A) and 2 mL (Diluent B) (Invitrogen) and 7 mL
of water. Incubation of the membranes took place overnight in 10 mL of the primary
antibody solutions. After about 18 hours on the rocker, the primary antibody
solution was rinsed out and each membrane was then washed with TBST three more
times. The secondary antibody solution was then added and incubated for one hour
on the rocker. Once the second incubation was complete, the membranes were
washed with TBST three more times.

Image Collection: The device that was being used to collect the images (Li-Cor)
warmed up for about 15 minutes. After that, chemiluminescent solution (Li-Cor) was
prepared with peroxide buffer and luminol substrate. This solution was placed into a
2mL microcentrifuge tube and poured onto the middle of the platform on the image
collector. The membranes were placed upside down on the platform and the lid was
closed. To capture the images of the protein bands, Image Studio software (Li-Cor)
was used and once the image appeared the clearest one was selected, ImageJ
(Wayne Rasband National Institute of Health, USA) was used to quantify protein
concentration of specific antibodies. All of the proteins that were identified during
the study were converted to values relative to the -Actin. Once all of the images
were collected and recorded, the membranes were stripped using a stripping buffer
(Thermo Fischer Scientific) for 20 minutes. TBST was used to wash the membranes
and this process was repeated for the remaining three proteins, CAT, GPx and SOD1.

Results

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The results from this project came out to be inconclusive. The proteins were
apparent on the membranes however, when the membranes were taken into
imaging, there was was no clear structure. Dark bands were expected to appear,
labeling the different proteins, but only black spots and black backgrounds were
visible after imaging. The imaging process was performed three times, each time
with inconclusive results.
The graphs displayed here show the constants and values we recorded and
used to create the total volume needed during the Bradford Protocol.

Figure 1 - Absorbancies of Premade Protein Concentration Solutions

Protein

Absorbancy

0.125

0.036

0.25

0.045

0.5

0.099

0.75

0.128

0.168

1.5

0.264

0.375

Figure 2 - Graph of all premade absorbancies of Bradford Reagent

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Figure 3 - Values for each kidney sample during Bradford Protocol

Animal

Absorbancy

[Protein]
Total Volume RIPA Needed
7.16536748
43.3073496

K-17

1.289

3
143.3073497
7
7.65534521
53.1069042

K-39

1.377

2
153.1069042
3
7.25445434
45.0890868

K-16

1.305

3
145.0890869
6
8.00612472
60.1224944

4
5

K-45
K-43

1.44
1.323

2
160.1224944
3
7.35467706 147.0935412 47.0935412
7.16536748
43.3073496

K-3

1.289

3
143.3073497
7
8.54621380
70.9242761

K-24

1.537

8
170.9242762
7
6.17427616
23.4855233

K-19

1.111

9
123.4855234
9
5.46158129
9.23162583

K-37

0.983

2
109.2316258
5
5.09409799
1.88195991

10

K-9

0.917

101.8819599

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This picture shows one of the membranes that was soaked in red dye. This is
important because it shows that the proteins are present on the membrane.

Discussion
The results obtained are considered inconclusive because we were unable to
identify the proteins and analyze the antioxidant enzymes. The hypothesis that was
presented was that the use of kefir to would be helpful in reducing the harmful
effects of doxorubicin in rat kidneys and remove the markers of oxidative stress.
We are unable to report whether our hypothesis was true or not due to the
lack of results. Possible reasons for this may have lead to this may be that the
primary and secondary antibodies were not compatible with each other, as well as
excessive washing of the membrane (16). In the first trial, the error that we
recognized was that the primary and secondary antibodies that we used had
expired. However, for the second trial, brand new antibodies had been utilized but

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there was still no success. For the third trial, the earlier procedure was repeated and
the new antibodies were used. The transfer of the proteins from the gel cassettes
was successful as the proteins were present.
The imaging was also attempted using another imaging device from the
Biology department in case the problem was within the imaging device. The results
from the other device was the same, leading us to believe that our error lies
between the transfer and imaging. Coming to the conclusion that the proteins were
present, a red color dye was used to soak each membrane and darken the proteins.
If this study was to be repeated, it could be successful by monitoring the
membranes more closely and to ensure that every solution used is the correct
solution as well as not expired. Having the solutions at the exact amount in terms of
ingredients and use of the solution would ensure that each step performed was
correct. Alternate methodologies would include performing the experiment from the
very beginning, starting with the homogenization using a different set of rat
kidneys. Doing so would eliminate all of the contamination and tampering that may
have taken place during the constant stripping and refilling of antibodies.
Contamination could be easily avoided by making sure to dispense every pipette tip
whenever multiple solutions are being added and by not mixing all of the chemicals
onto one membrane. This would allow the fresh, new membranes to be taken into
imaging using new antibodies leading to some form of results. If this experiment
had been successful, we would have been able to determine how much oxidative
stress the rat contains through the use of doxorubicin and kefir.

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Acknowledgements
First of all, I would like to thank Lori Ball and the entire Frontiers of Science
staff for providing me with this opportunity to be a part of this program. This
program has changed me in many ways and was definitely one of the best
beneficial things that I have done. Thank you to Jessamyn for always being on top of
things, even though we never really see you we know how much you contribute to
the program. A huge thank you to my RAs, Reece, Sergio and Sophia, for being so
incredibly helpful and for dealing with us for six weeks. They were not only our RAs,
they were our friends and I am really glad that I had the chance to meet them as we
had some amazing times. Also a thank you to our instructors, Alissa, Dean and
Nicole, for being so sweet to us and for volunteering to spend your time with us
during summer to teach us and help us better our knowledge. The classes were
very entertaining and I absolutely loved them.

Thank you to my sponsor, Brian Davidson, for contributing to the program

and for making my stay possible. You gave me an amazing opportunity and I really
appreciated it. Also, a thank you to my research sponsor, XCel Energy, for making
my research project attainable. Thank you to my mentor, Raquel Besekrus, for being

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so sweet and for helping us learn along the process and my advisor, Nicole Wood,
for always helping me out with my paper and being successful.

Thank you to my family for always being so encouraging and for supporting
me throughout everything, especially for my passion for the STEM field. Thank you
to my fellow scholars for helping me have an amazing summer, I will miss everyone
in this program and I owe my success to you all.

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Klein, T. E., & Altman, R. B. (2011). Doxorubicin pathways: Pharmacodynamics
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14. Chatterjee, K., Zhang, J., Honbo, N., & Karliner, J. S. (2009, December 11).
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