Event Title

The Effect of Sweeteners on Nox 4 mRNA and Protein Expression in the Rat Kidney

Location

CSU Ballroom

Start Date

20-4-2015 10:00 AM

End Date

20-4-2015 11:30 AM

Student's Major

Chemistry and Geology

Student's College

Science, Engineering and Technology

Mentor's Name

Theresa Salerno

Mentor's Email Address

theresa.salerno@mnsu.edu

Mentor's Department

Chemistry and Geology

Mentor's College

Science, Engineering and Technology

Description

Dietary sugars have been documented to cause health issues such as obesity, insulin resistance, and hypertension. Because of this, it’s important to study alternative sweeteners. An increase in reactive oxygen species (ROS) can play a prominent role in kidney disease developed from hypertension. The source of the ROS has been linked to the NADPH oxidases (Nox enzymes) that create superoxide radical products. This protein exists as isoforms, with the Nox 4 form being most associated with kidney damage. The purpose of this project was to ask the question whether normotensive rats fed diets supplemented with sugar (sucrose) or sweeteners, stevia and saccharin, exhibit changes in the expression of Nox 4 at both the mRNA and protein levels. Reverse transcription was performed on previously purified RNA, and the Taqman cDNA Reverse Transcription Kit was used to make cDNA templates. Specific primers and a probe were designed to amplify the Nox 4 target and the efficiency of the method was validated. Expression data from the samples were analyzed using the ΔΔCt method to give the expression of Nox 4 relative to the endogenous control, GAPDH. The Western blot was performed by extracting the proteins, measuring total protein content using the BCA Protein Assay Kit, and loading equal amounts of protein onto a 12% Criterion TGX gel. Proteins were transferred to a nitrocellulose membrane and detected using specific primary and IR labeled secondary antibodies. Expression of the Nox 4 protein was measured relative to the amount of GAPDH using a LiCor Odyssey imager.

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Apr 20th, 10:00 AM Apr 20th, 11:30 AM

The Effect of Sweeteners on Nox 4 mRNA and Protein Expression in the Rat Kidney

CSU Ballroom

Dietary sugars have been documented to cause health issues such as obesity, insulin resistance, and hypertension. Because of this, it’s important to study alternative sweeteners. An increase in reactive oxygen species (ROS) can play a prominent role in kidney disease developed from hypertension. The source of the ROS has been linked to the NADPH oxidases (Nox enzymes) that create superoxide radical products. This protein exists as isoforms, with the Nox 4 form being most associated with kidney damage. The purpose of this project was to ask the question whether normotensive rats fed diets supplemented with sugar (sucrose) or sweeteners, stevia and saccharin, exhibit changes in the expression of Nox 4 at both the mRNA and protein levels. Reverse transcription was performed on previously purified RNA, and the Taqman cDNA Reverse Transcription Kit was used to make cDNA templates. Specific primers and a probe were designed to amplify the Nox 4 target and the efficiency of the method was validated. Expression data from the samples were analyzed using the ΔΔCt method to give the expression of Nox 4 relative to the endogenous control, GAPDH. The Western blot was performed by extracting the proteins, measuring total protein content using the BCA Protein Assay Kit, and loading equal amounts of protein onto a 12% Criterion TGX gel. Proteins were transferred to a nitrocellulose membrane and detected using specific primary and IR labeled secondary antibodies. Expression of the Nox 4 protein was measured relative to the amount of GAPDH using a LiCor Odyssey imager.

Recommended Citation

Newman, Logan. "The Effect of Sweeteners on Nox 4 mRNA and Protein Expression in the Rat Kidney." Undergraduate Research Symposium, Mankato, MN, April 20, 2015.
http://cornerstone.lib.mnsu.edu/urs/2015/poster_session_A/38