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