Markers of Oxidative Stress and Mature Red Blood Cell Mirnome in Cats with Diabetes Mellitus
Abstract
Despite previously accepted dogma, several recently published studies in humans and mice have shown that mature red blood cells (RBCs) contain a pool of microRNAs. Their role is currently uncertain; however, it has been suggested that microRNAs may play a role in cellular communications as they can be transferred from the RBC to endothelial cells or other cells. This thesis investigated the set of differentially abundant microRNAs found in mature RBCs of felines with oxidative stress, using diabetes mellitus as an oxidant stressor. We postulated that individual microRNAs identified in this study, might be valuable targets for future studies, investigating the role specific microRNAs play in the development or progression of diabetes and in the oxidative damage inflicted on the red cell and other cells by this disease. The first specific objective of this thesis was to document oxidative stress in diabetic cats. In the absence of validated assays to document the presence of oxidative protein damage in felines, we first evaluated the performance of a commonly used colorimetric assay for measurement of protein carbonyls (PC) in serum and plasma. Although within run variation was acceptable and performed well over a wide range of PC content values, there were severe limitations related to excessive between run-variation, hemoglobin interference, and difficulty of assay performance. Therefore, we developed and validated a new method, using a fluorescent probe. This new assay had good within and between-run variations, a broad analytical range, and was easy and rapid to run. Hemoglobin and triglyceride only affected the results when present at moderate to higher levels. To further evaluate their redox status, free-radical production and oxidative stress were measured in diabetic and healthy, control cats. The presence of oxidative stress was assessed by measurement of the resulting damage to biomolecules, and detection of antioxidant levels. Our data indicated the presence of protein and membrane lipid oxidation in diabetic individuals and suggest that the redox status of the mature RBC was shifted toward an oxidation state. In the final chapter of this thesis, we document the presence of an abundant and diverse set of microRNAs in the mature erythrocytes of healthy and diabetic cats. While their function in the mature erythrocyte remains unknown, a difference was found in the microRNA expression patterns of diabetic and healthy cats. Our data uncovered severe bias in the microRNA sequencing such that the expression levels of some microRNAs appeared to be artificially increased and other diminished. The library construction kit used, appeared to be the cause of this bias. Among the 899 erythrocyte microRNAs sequenced, 12 differentially abundant microRNAs were identified in diabetic cats, however only 6 were differentially abundant by RT-qPCR. Let-7b, miR-1692, miR339, miR-486 and a feline specific microRNA were increased in mature RBCs of diabetic cats, while miR-451 was decreased. In conclusion, we have shown that diabetic cats have evidence of significant systemic protein and lipid oxidation as well as erythrocytic oxidative stress. The new, fluorescent PC content assay developed and validated herein could serve as useful tool to better understand the role and consequence of oxidative stress in feline diabetes or other diseases and to monitor antioxidant treatment. Further, this test could be readily adapted for use in other domestic species.
Degree
Ph.D.
Advisors
Messick, Purdue University.
Subject Area
Biochemistry|Pharmaceutical sciences
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