Creatine as an Antioxidant to Attenuate Kidney Damage Caused by Doxorubicin

Stated by the World Health Organization in their key facts about cancer, the second cause of death in the world is cancer, being mostly made up by the low- and middle-income countries (World Health Orginazation, 2018). While there are treatments for cancer such as chemotherapy, it can take a major toll on the patient’s body while treating the cancer.

Doxorubicin (DOX), also known as Adriamycin, is a chemotherapy drug that is commonly used to treat a wide range of cancers (Gale, 2015). In the article by Ayla, et al., 2011, it states how doxorubicin has shown to cause nephrotoxicity in the renal system. This is specifically oxidative stress, an imbalance between free radical species and antioxidants. Free radical species are defined as molecular species that are unstable and reactive that cause damage to biologically necessary molecules, chemicals that are free radicals are hydrogen peroxide, hydroxyl radical and more (Lobo, Patil, Phantak, & Chandra, 2010). DOX mainly plays a role in Nitric oxide production, another free radical, by direct or indirect stimulation, otherwise it’s not completely understood the mechanism behind DOX induced nephrotoxicity (Ayla, et al., 2011).

Oxidative stress is an imbalance between free radicals and antioxidants. Antioxidants, also called free radical scavengers, are what prevent oxidation reactions. Antioxidants can range from many vitamins, foods, and supplements having no identified side effects (Jones, Newton, Odle, & Schenker, 2015). Creatine (Cr) has gained attention recently for studies such as within the article by Sestili in 2011, its reviews studies on Cr to find antioxidant properties, giving cites to studies that show Cr has been able to decrease formation of free radical species and tissue oxidative damage (Sestili, et al., 2011). Cr is more commonly known and used as a physical enhancement, Cr is also common in the body made as a product of the arginine biosynthesis pathway, being stored in the skeletal muscle and a large amount in the brain (Riesberg, Weed, McDonald, Eckerson, & Drescher, 2016).

The research that is being conducted is to correct DOX induced nephrotoxicity in a rat model. They were distributed between four groups, control and saline, control and DOX, creatine and saline, and a group with Cr and DOX. The collected information incudes, body mas before and after treatments, optical density of GPx (glutathione peroxidase) and catalase in tissue samples of the kidneys, and kidney mass relative to the body mass after treatments. Using the information collected, it was used to determine activity levels of an antioxidant, catalase, finding higher amounts says that free radicals was to be lower, decreasing oxidative stress. It is being hypothesized that creatine as an antioxidant can help protect against nephrotoxicity from an imbalance of reactive oxygen spices and antioxidants due to doxorubicin.

METHODS AND MATERIALS

The Institutional Animal Care and Use Committee at the University of Northern Colorado approved all procedures which were followed within the Animal Welfare Act. Twenty-eight Sprague-Dawley rats kept in a controlled environment being fed regular food as a control or regular food with a 3% addition of Cr. Body mass was collected before introduction of food and three times after for two and half weeks of treatment. At two weeks the rats were injected with equivalent volumes of DOX (Control + Dox, n=7 and Cr + DOX, n=7) or saline (C + Sal, n=7 and Cr + Sal, n=7). Five days after the injections the rats were euthanized, and the kidneys were removed.

The kidneys were homogenized. From the solution, proteins concentrations were prepared for Western blotting. PVDF membranes were blocked with a milk solution overnight with antibody for β – actin (Santa Cruz Biotechnology). Within morning the primary antibody was removed to add a secondary mouse antibody for another period, after to have the antibodies removed and the membranes washed three time for five minutes.

Images of the membrane was collected using a scanning device, C-DiGit blot scanner, and the image studio by Li-COR (Li-COR, Inc.). The buffer used for the scanner to detect the blots was a 1:1 ratio using peroxide buffer and luminal substrate in a 2mL centrifuge container. The buffer being used in the center of the device, the membrane was placed face down and using the image software by Li-COR, the image was developed giving a small change to the picture for best quality. The membrane was then removed from the device to be stripped using a buffer and having the procedure repeated for catalase using a second antibody with β – actin as loading control. Software ImageJ (National Institute of Health) the proteins concentrations were quantified using optical density.

The data was collected and complied into a Microsoft Excel worksheet, gathering average and standard deviations. Statistical analysis was performed using Prism Graphpad (Graphpad software) that allowed comparison between groups to be determined by a one-way analysis of variance (ANOVA), significance was also used to collect the P<0.05.

RESULTS

With all data, significant difference was reported when P < 0.05. There was no significant difference between kidneys masses, as shown in figure 1. Also, in figure 2 the before and after injections of DOX, the rats body mass significantly decreased, this is due to the treatment involving the removal of chow, to better model how DOX causes nausea and unwillingness to eat. There is also average difference in body mass and kidney mass (Table 1) with standard deviation between the four groups. There is a ± difference that can show Cr + Dox could possibly be higher than C + DOX. Catalase (CAT) expression was significantly lower in the Cr + Dox group than in the Control (C) + Saline (Sal) group as shown in figure 3. This shows that while the rats were treated with doxorubicin and had been getting Cr to help counteract the oxidative stress there was a significant decrease in the CAT. The average of the protein expressions with each group on the enzymes GPx and CAT and standard deviations (Table 2).