Michael Fay, Ph.D.

Cadmium is a major industrial environmental pollutant that is known to cause nephrotoxicity at the level of the proximal tubule. MicroRNAs are small (18-24 nucleotides) non-coding RNAs that regulate gene expression at the post-transcriptional level. In collaboration with Drs. Walter Prozialeck and Joshua Edwards, in the Department of Pharmacology, we are investigating the role of microRNAs in cadmium-induced nephrotoxicity and their usefulness as urinary biomarkers of nephrotoxicity.

Project I: The environmental pollutant cadmium (Cd) is currently ranked seventh on the EPA/ATSDR Priority List of Hazardous Substances. Human Cd exposure causes damage to various organs, and chronic low-level exposure results in Cd accumulation in the renal proximal tubule epithelial cells resulting in kidney toxicity characterized by a generalized reabsorptive dysfunction. Since Cd is a known renal toxicant, there is great interest in elucidating the molecular mechanisms of Cd-induced kidney injury, and a need to identify effective early noninvasive biomarkers to monitor Cd-induced kidney injury. MicroRNAs (miRNAs) are small (20-25 nt) non-coding RNAs that inhibit gene expression at the post-transcriptional level. Preliminary data, generated using a 12-week subchronic rat model of Cd-induced nephrotoxicity and miRNA microarray analysis, indicates that Cd causes significant dysregulation of miRNA expression in the renal cortex at week 12 when kidney injury is evident. The miRNAs that exhibited a significant ([?] 2-fold) increased expression with Cd-treatment included: miR-3084a- 3p, -34a-5p, -149-3p -1949, -224-5p, -146b-5p, -3084c-3p, -21-5p, -451-5p; and the miRNAs that exhibited a significant ([?] 2-fold) decreased expression with Cd treatment included: miR-193b-3p, miR-455-3p, miR-342-3p. The overall goal of the this research project is to evaluate the expression and function of miRNAs during Cd-induced nephrotoxicity, and to evaluate their utility as non-invasive biomarkers of Cd-induced kidney injury. The first goal is to evaluate the expression of these dysregulated miRNAs in the rat renal cortex of Cd-treated rats versus saline control rats at earlier time points in the treatment protocol prior to overt kidney injury (i.e. 3, 6, 9 weeks) using real-time PCR and in situ hybridization. Exosomes are small (30-160 nm) vesicles of endosomal origin that are secreted by cells, and their cargo (e.g. miRNAs) are representative of the cells from which they originated. There is great interest in examining exosomal miRNAs for identification of novel disease biomarkers since exosomal miRNAs are protected from degradation and exosomal shedding increases with disease. As a second goal for this research project, increased urinary exosome shedding throughout (i.e. 3, 6, 9, 12 weeks) the 12-week Cd-treated subchronic rat model will be assessed with NanoSight(r) nanoparticle tracking analysis. The expression of exosomal miRNAs, found to be increased ([?] 2-fold) by Cd in the renal cortex, will be evaluated by real-time PCR to determine their utility as biomarkers of Cd-induced injury. Finally, the function of the Cd-dysregulated miRNAs discussed above will be evaluated by transfecting NRK-52E rat proximal tubule epithelial cells with miRCURY LNATM miRNA mimics and miRCURY LNATM miRNA inhibitors and examining the effects on cell adhesion molecule expression (E- and N-cadherin, b-catenin), transepithelial electrical resistance, cell viability, autophagy, and apoptosis. Overall, these studies will determine the role of Cd-dysregulated miRNAs in Cd-induced kidney injury, and determine the utility of these dysregulated miRNAs as non-invasive biomarkers.
Oncology
Toxicology