Defense Date

2014

Document Type

Thesis

Degree Name

Doctor of Philosophy

Department

Physiology and Biophysics

First Advisor

Vladimir Vladimirov; MD, PhD

Second Advisor

Catherine Dumur, PhD

Abstract

Kidney failure affects over 600,000 people in the United States with over 100,000 new cases of kidney failure diagnosed every year. Chronic allograft dysfunction (CAD), a disease state highlighted by a decreased eGFR and a histo-pathological diagnosis interstitial fibrosis and renal tubular atrophy, has emerged as one of the leading causes of kidney transplantation. Gene expression analysis can identify reproducible patterns of disease in ribonucleic acid (RNA) expression and may provide insights into the quality of kidney donations heretofore overlooked by current clinical analytical methods. Additionally, genetic expression profiles can be obtained from patient blood or urine samples, diminishing the necessity of costly and invasive tissue biopsies. Micro-RNA (miR) profiles have been obtained from non-invasive sources and shown to act as biomarkers of CAD. Specifically, miR-142-3p, miR-204, and miR-211 are shown to be differentially expressed in both tissue and urine samples of patients with CAD. The role of these CAD-specific miRs in disease development is not well understood, but given the nature of miR function, they may play a significant role in CAD development.

An independent validation of CAD-specific miR expression was done in 155 deceased donor transplant recipients using RT-qPCR. This new patient cohort delineated the importance of both decreased clinical renal function as measure by GFR, and diagnosed histopathology in identifying CAD. Previous work delineating a messenger RNA signature of CAD was used to produce a list of potential targets for CAD-specific miRs via in silico analysis. Briefly, targets for each miR were consolidated from 11 miR-targeting networks: PITA, miRanda, TargetScan 5.1, DNA-microT, NBmiRTar, MicroInspector, PicTar, MirTarget2, RNA22, miTarget, and RNA hybrid (all can be found at: http://c1.accurascience.com/miRecords/prediction_query.php). Targets that appeared in at least 3 of databases were considered for further study. Experimental targets were then chosen according to negative Pearson correlations of miR/mRNA pairings and potential roles in apoptosis or fibrogenesis. Urine samples showed differential expression of transmembrane protein 14A (miR-142-3p target), CD44 molecule (miR-204), basic helix-loop-helix family member e41 (miR-204), and dual specificity kinase 6 (miR-211) between patients with CAD and those with normal allografts. Human embryonic kidney (HEK) cell lines were used to validate these targets via transfection of the CAD-specific miRs and observing the modulation of their selected gene targets. Overexpression the CAD-specific miRs in HEK cells modulated their respective target gene expression. The relationship between CAD-miRs and their targets was validated using luciferase expression. This research verifies the differential expression of these CAD-specific miRs from non-invasive sources, and validates gene targets for these miRs that modulate significant biological pathways in the development of CAD.

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

12-15-2014

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