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The Use of Cannabinoids to Stimulate an Increase of Nitric Oxide Production
as a Basis for a Novel Treatment of Chronic Renal Disease
Experimental Design
Procedure Results
Application to Biotechnology
References
Purpose: To establish a basis for a novel
pharmaceutical treatment of CRD that works
against the cause and fights against
progression of the disease rather than
decreasing the severity of the symptoms
HE: If cells are exposed to ADMA with mAEA,
then total NO production will increase.
H0: There will be no statistically significant
difference between the individual means of
the control and experimental groups.
Independent Variable: The chemicals that
the cells were exposed to (mAEA, ADMA,
both, or neither)
Dependent Variable: Total NO production of
the cells as measured by nitrite concentration
Preparation of chemical solutions
Establishment and culture of
cells
Cells exposed to chemicals
Griess assay for nitrite
determination
((
Cells were lysed
using a Potter-
Elvehjem pestle
and exposed to the
Griess reagents,
forming an azo dye
to measure nitrite
concentration
(Figure 6).1. Schmidt, R. J., & Baylis, C. (2000). Total nitric
oxide production is low in patients with chronic
renal disease. Kidney International, 58, 1261-
1266. doi:10.1046/j.1523-1755.2000.00281.x
2. Baylis, C. (2012). Nitric oxide synthase
derangements and hypertension in kidney
disease. Current Opinion in Nephrology and
Hypertension, 21(1), 1-6.
doi:10.1097/mnh.0b013e32834d54ca
3. Carney, S. T., Lloyd, M. L., MacKinnon, S. E.,
Newton, D. C., Jones, J. D., Howlett, A. C., &
Norford, D. C. (2009). Cannabinoid regulation of
nitric oxide synthase I (nNOS) in neuronal
cells. Journal of Neuroimmune
Pharmacology, 4(3), 338-349.
doi:10.1007/s11481-009-9153-7
*See additional references attached.
Acknowledgments
Conclusions
• Allison Hennings, R.N., B.S.N., M.A.T.
• Mentors: Dr. Eric Kelley, West Virginia
University; Professor Allyn Howlett, Ph.D.,
Wake Forest Unviersity
Discussion
Although the calculated p-values (TABLES 1 and
2) were greater than the alpha value of 0.05, the
results still show promise for the usage of
cannabinoids as a treatment of CRD. The
quantitative data show a difference, although
insignificant, between groups, supporting the
positive effects that come from the interactions
between mAEA and ADMA (Figure 5).
There were many obstacles that had to be
overcome during the procedure. After two initial
rounds of testing, the assay produced no
readings. To solve this, the researcher
concluded that there were not enough cells and
let them grow for an extra day. The third round
still did not work. For the next attempt the cells
were grown to full confluency, but the assay still
produced no readings. The assay was then tried
following three separate protocols, each to no
avail. Finally, the conclusion was drawn that the
phenol red in the DMEM medium was interfering
with the acidic conditions necessary for the
assay, so cells were resuspended in PBS before
the assay, and this procedure gave readings.
ADMA and fatty
acid free bovine
serum albumin (faf-
BSA) solutions
were prepared at
120µM and
5mg/mL (Figure 1).
Cells from cell line
NRK-52E were
cultured with 12mL
DMEM (10% FBS
and 1% NEAA) in
75cm2 flasks
(Figure 2).
Cells were exposed
to 1µM mAEA in faf-
BSA, 9µM ADMA,
both, or neither for
3-5 hours (Figures 3
and 4).
Figure 1. Figure 2.
Figure 3. Figure 4.
Figure 6.
• All current pharmaceutical treatments aim to
artificially induce kidney function instead of
actually fixing the disease
o They act as diuretics to minimize fluid
retention and eliminate excess electrolytes
A new pharmaceutical that increases NO
production and therefore kidney function offers a
better solution to CRD.
• Cannabinoids provide an opportunity to slow
down the progression of the disease and
extend the lives of CRD patients.
Garrett Hauck
Oak Park River Forest High School
BioGENEius 2017
Introduction
There are 31 million Americans who suffer
from chronic renal disease (CRD), and it is
agreed upon that a defining characteristic is
a lowered amount of nitric oxide (NO) in the
kidney.1 This leads to the development of
hypertension and atherosclerosis, and
contributes to the progression of the
disease.1 One of the main factors that
causes lowered NO levels is an increased
concentration of asymmetric dimethylarginine
(ADMA), a nitric oxide synthase (NOS)
inhibitor that is elevated in CRD patients due
to the lower rate of degradation and excretion
in the failing kidney.2 Cannabinoids, a class
of molecules derived from cannabis, increase
the activity of NOS and stimulate NO
production, and (R)-methanandamide
(mAEA) is one of the most potent agonists of
the cannabinoid receptors.3 In this study,
cells from the cell line NRK-52E, established
from the kidney of an adult Osborne-Mendel
rat, were treated with mAEA after exposure
to ADMA in an effort to increase NO
production and demonstrate that a
cannabinoid pharmaceutical could potentially
be used as a treatment for CRD.
0
10
20
30
40
50
60
Control mAEA ADMA Both
Avera
ge N
itrite
Conce
ntr
ation (
µM
)
Group
Average Nitrite Concentration Produced
Statistical Analyses
Figure 5.
Source of
Variation SS df MS F p-value F crit
Between
Groups 488.16 3 162.72 0.29 0.83 3.05
Within
Groups 12259.01 22 557.23
Total 12747.17 25
TABLE 1. Single factor ANOVA test
Group 1 Group 2 Variance p-value
Control mAEA Equal 0.56
Control ADMA Equal 0.41
Control Both Equal 0.63
mAEA ADMA Equal 0.80
mAEA Both Equal 0.84
ADMA Both Equal 0.62
TABLE 2. Results of t-tests