Xinyi (Cindy) Chen- Sigma Xi Presentation 2015
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Transcript of Xinyi (Cindy) Chen- Sigma Xi Presentation 2015
VITAMIN D MITIGATES DIABETIC AND
OXIDATIVE STRESSES ON KIDNEY CELLS
Xinyi (Cindy) Chen
Research in Biology
North Carolina School of Science and Math
Background: Diabetes and P-glycoprotein
• Diabetes is now the seventh leading cause of death in the
world.
• The elevated glucose levels due to diabetes yield a down
regulation of P-glycoprotein, a membrane transporter that
eliminates toxins from cells.
• P-glycoprotein plays a critical role in the absorption and
elimination of drugs and toxins, so increasing the
expression level of P-glycoprotein can delay or even
prevent the onset of diabetes.
A proposed mechanism by which
P-glycoprotein secretes substrates
2
P-gp
substrate
1
P-gp
Cell
membrane
Intracellular
space
Extracellular
space Secretion
1. Passive uptake of
substrate across
cellular membrane.
2. Formation of P-
glycoprotein channel
through which the
substrate is secreted
out into the
extracellular space.
Background: Vitamin D
• The active form of vitamin D, calcitriol, has been shown to
up regulate P-glycoprotein levels in the cell membrane.
• The effects of vitamin D on diabetes have not yet been
tested on the kidney, an organ that can be damaged by high
glucose levels.
• One of my goals was to determine if vitamin D can
beneficially elevate P-glycoprotein expression levels to
prevent diabetes by an in vitro study of canine kidney cells.
Background: Vitamin D and Hydrogen Peroxide
• Research has shown that reactive oxygen species (ROS)
including hydrogen peroxide (H2O2) significantly reduce P-
glycoprotein expression, but this reduction was later
prevented by co-treatment with various antioxidants.
• Vitamin D can act as a membrane antioxidant, and this
ability may be useful in counteracting the down regulation
of P-glycoprotein expression induced by H2O2.
• Investigating this link between vitamin D and H2O2 can
shed light on the mechanism that vitamin D takes to up
regulate P-glycoprotein levels.
Digoxin as an indicator for P-glycoprotein activity level
• Digoxin is a toxin that’s pumped out of cells via the
P-glycoprotein transporter in the cell membrane.
• Lowered activity of P-glycoprotein causes a higher
accumulation of digoxin in the cells, thus leading to greater
cell death.
2
P-gp substrate
(such as digoxin)
1
P-gp
Cell membrane
Intracellular
space
Extracellular
space
• Therefore, digoxin can be
used to quantitatively
measure P-glycoprotein
activity levels via cell
viability.
Objectives
1. Viability assay: Determine the duration of exposure to
digoxin necessary to induce MDCK-II (canine kidney) cell
death as well as the optimal concentration of digoxin that must
be used.
2. Vitamin D and Glucose experiment: Determine if vitamin D
has a beneficial effect on MDCK-II cells in different glucose
concentrations by up regulating P-glycoprotein activity.
3. Vitamin D and H2O2 experiment: Determine if vitamin D can
act as an antioxidant and reverse the down regulation of P-
glycoprotein activity induced by H2O2, a reactive oxygen
species.
General Methods
• An established cell line of canine kidney cells (MDCK-II) was
continuously cultured in Minimum Essential Media (MEM).
• For the glucose and H2O2 experiments, all cells were kept in
their respective treatments for 24 hours before they were
removed, stained with trypan blue, and counted under a light
microscope using a hemocytometer.
• The number of live cells per unit volume (mL) was counted for
each well, and the ratio of the number of live cells per mL in
each well to the number of live cells per mL in the control well
was calculated as the response variable.
Cell Viability Assays: Methods
• In my first viability assay, the duration of exposure to digoxin was
varied (6 time points) and the concentration of digoxin was kept
constant at 40 nM (from previous research). Cell death was measured
after each time point to determine which, if any, time point would
induce significant cell death.
• However, since 40 nM of digoxin wasn’t enough to induce cell death at
any time point, a second test for viability was conducted using higher
concentrations of digoxin, as shown in the next slide, and measuring
cell death after a set time of 24 hours.
• In both tests for cell viability, normal glucose (5 mM) and diabetic
glucose (25 mM) concentrations were tested.
• Photos were taken and analyzed using Fiji software to indicate live cell
density.
5 mM
(Normal)
25 mM
(Diabetic)
Glucose concentration
128 nM
Dig
oxin
concentr
ation
400 nM
700 nM
1000 nM
1280 nM
N=3
Cell Viability Assays: Experimental Design
*Control = Ethanol
Cell Viability Assays: Results
EtOH
700 nM 1000 nM
1280 nM
• Each photo represents cells
treated with a different digoxin
concentration (shown in upper
right of photos).
• As digoxin concentration
increased, cell density decreased.
• Both 700 and 1000 nM of digoxin
gave reasonable percentages of
dead cells, so 850 nM of digoxin
was used for the following two
experiments investigating glucose
and H2O2.
• In the figure, dark gray areas represent areas with live cells and light gray
areas represent areas with no cells.
Photo credit: Xinyi Chen. Magnification: 100x
Vitamin D and Glucose: Methods
• This experiment tested the effects of 5 concentrations of vitamin
D on MDCK-II cells in 3 glucose concentrations.
• Vitamin D concentrations:• 0 ng/mL = control
• 12 ng/mL = concentration associated with a vitamin D deficiency
• 20 ng/mL = lower bound for an adequate amount of vitamin D for health
• 35 ng/mL = adequate concentration for health
• 50 ng/mL = concentration above which vitamin D may yield adverse effects
• Glucose concentrations:• 5 nM = normal glucose concentration in healthy individuals
• 15 nM = concentration associated with prediabetes
• 25 nM = concentration associated with diabetes
Vitamin D and Glucose: Experimental Design
N = 3
5 25
12
20
50
35
15
Vitam
in D
concentr
ation (
ng/m
L)
Glucose concentration (mM)
0
Vitamin D and Glucose: Results
0
1
2
3
4
0 12 20 35 50
pro
po
rtio
n o
f live
ce
lls
, re
lati
ve
to
co
ntr
ol
vitamin D concentration (ng/mL)
5 mM glucose
15 mM glucose
25 mM glucose
*Control = cells treated with 5 mM of glucose and 0 ng/mL of vitamin D
Vitamin D and Glucose: Results
• There is a clear upward trend in the proportion of live cells as vitamin D
concentration increases, with a significant increase from 0 to 35 ng/mL of
vitamin D, indicating that vitamin D is beneficial to MDCK-II cells.
• A slight dip follows as vitamin D increases from 35 to 50 ng/mL, indicating
that 50 ng/mL may be nearing the range where vitamin D becomes
harmful.
• For the cells treated with 15 mM glucose, all vitamin D concentrations
brought the proportion of live cells up to or above the control, indicating
that vitamin D can help cells survive in prediabetic conditions.
• There is no upward trend among the cells treated with 25 mM glucose,
suggesting that vitamin D is probably not an effective diabetes treatment
when glucose levels are high but more so as a prevention strategy when
glucose levels are lower.
Vitamin D and H2O2: Methods
• This experiment tested the beneficial effects of vitamin D as an antioxidant by varying vitamin D and H2O2 concentrations.
• Vitamin D concentrations:• 0 ng/mL = control
• 12 ng/mL = concentration associated with a vitamin D deficiency
• 20 ng/mL = lower bound for an adequate amount of vitamin D for health
• 35 ng/mL = adequate concentration for health
• 50 ng/mL = concentration above which vitamin D may yield adverse effects
• H2O2 concentrations: • 0 μM = control
• 200 μM = concentration used in previous experiment done on MDCK-II cells
• 400 μM = double 200 μM to investigate the effects of increasing H2O2
concentrations on MDCK-II cells
Vitamin D and H2O2: Experimental Design
N = 3
0
12
20
50
35
Vita
min
D c
oncentr
ation (
ng/m
L)
Hydrogen Peroxide concentration (μM)
200 400
0
Vitamin D and H2O2: Results
0
1
2
3
4
5
0 12 20 35 50
pro
po
rtio
n o
f live c
ells.
rela
tive
to
co
ntr
ol
vitamin D concentration (ng/mL)
0 μM H2O2
200 μM H2O2
400 μM H2O2
*Control = cells treated with 0 ng/mL of vitamin D and 0 μM of H2O2
** *
Vitamin D and H2O2: Results
• Once again, increasing vitamin D concentrations yielded greater
proportions of live cells, with a clear upward trend in cell viability as
vitamin D concentration increases from 0 to 50 ng/mL.
• Higher concentrations of H2O2 yielded lower proportions of live cells,
with the harmful consequences becoming more pronounced with each
increase in vitamin D concentration.
• Increasing vitamin D concentrations counteracted the negative effects
of increasing H2O2 concentrations, as shown by the 3 asterisked bars
in the graph that have approximately the same proportion of live cells.
• According to the dotted reference line, as little as 12 ng/mL of vitamin
D is enough to offset the detrimental effects of the highest level of
H2O2, further indicating the beneficial effects of vitamin D.
Conclusions
2
P-gp substrate
(such as digoxin)
1
P-gp
Cell
membrane
Intracellular
space
Extracellular
spaceSecretion
In the presence of vitamin D:
• The expression of P-glycoprotein is up-regulated,
allowing more digoxin molecules that enter the kidney
cells to be secreted out.
• Cell mortality is
lowered because
more cells are able to
effectively secrete out
the toxic molecules.
Conclusions
• Higher vitamin D concentrations generally yielded less cell death, thus
implying increased expression of P-glycoprotein; however, there is an
optimal concentration of approximately 35 ng/mL of vitamin D.
• Vitamin D had a more beneficial effect on cells treated with 5 mM and
15 mM glucose than with 25 mM glucose.
• Vitamin D, as an antioxidant, neutralizes the down regulating effects of
H2O2, with increasing levels of vitamin D counteracting the negative
effects of increasing H2O2 concentrations.
• Vitamin D can potentially be used as a supplement to delay or prevent
the onset of diabetes by up-regulating P-glycoprotein expression
through the same mechanism that H2O2, a ROS, takes to down
regulate P-glycoprotein activity levels.
Future Work
• Measure the percentage of live or dead cells in each well rather
than just counting the number of live cells in each well and dividing
it by the number of live cells in the control well.
• Focus on a narrower range of vitamin D concentrations around 35
ng/mL in smaller gradations, since 35 ng/mL appears to be
approximately the optimal concentration.
• Study the mechanisms of vitamin D at the expression and
transcription levels using molecular biology techniques.
• Use human kidney cells, instead of canine kidney cells, to gain a
more accurate picture of how vitamin D affects P-glycoprotein
levels in humans.
Acknowledgments
• Dr. Amy Sheck, Dean of Science at NCSSM
• Ms. Korah Wiley, Instructor of Biology at NCSSM
• Dr. Floyd Bullard, Instructor of Mathematics at NCSSM
• Research in Biology Class of 2014
• Research in Biology Class of 2015
• Glaxo endowment at NCSSM
Works Cited
1. American Diabetes Association. Statistics About Diabetes. 2014.
http://www.diabetes.org/diabetes-basics/statistics/. Accessed 7/15/14.
2. Chow, E., M. Durk, C. Cummins, and K. Pang. 2011. 1α, 25-Dihydroxyvitamin D3 up-
regulates P-glycoprotein via the vitamin D receptor and not farnesoid X receptor in
both fxr(-/-) and fxr(+/+) mice and increased renal and brain efflux of digoxin in mice
in vivo. The Journal of Pharmacology and Experimental Therapeutics 337: 846-859.
3. Matheny, C., M. Lamb, K. Brouwer, and G. Pollack. 2001. Pharmacokinetic and
pharmacodynamic implications of P-glycoprotein modulation. Pharmacotherapy 21:
778-796.
4. Qiu, J., H. Gao, Y. Liang, H. Yu, and R. Zhou. 2008. Comparative proteomics analysis
reveals role of heat shock protein 60 in digoxin-induced toxicity in human endothelial
cells. Biochimica et Biophysica Acta 1784: 1857-1864.
5. Yeh, S., H. Pan, C. Lin, Y. Kao, Y. Chen, and C. Lin. 2012. Hyperglycemia induced
down regulation of renal P-glycoprotein expression. European Journal of
Pharmacology 690: 42-50.