Date of Award

January 2015

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biomedical Sciences

First Advisor

Scott H. Garrett

Abstract

The proximal tubule of the kidney is particularly susceptible to toxicant-induced damage and cell cultures of human proximal tubule cells are widely utilized to study the role of epithelial-mesenchymal transition (EMT) in renal disease. Cadmium is a toxic metal ion that is known to produce renal tubular necrosis and accumulate in the proximal tubule. This metal binds to a family of cysteine rich metal binding proteins known as metallothioneins (MT) that are found in abundance in the kidney. Previous studies from our laboratory have shown that the third isoform of metallothionein (MT-3) is expressed in the epithelial cells of the human kidney, including those of the proximal tubule. An immortalized proximal tubule cell line does not express MT-3 and does not demonstrate vectorial active transport. Transfection of the MT-3 gene into the HK-2 cells restores vectorial active transport as evidenced by dome formation. This suggests that MT-3 is involved in mesenchymal to epithelial transition (MET), the reverse of EMT, and promotes and epithelial phenotype. The goals of the present study were to examine the role of growth media composition on classic EMT responses, quantitatively evaluate the expression levels of E- and N-cadherin, define the functional epitope of MT-3 that mediates MET in HK-2 cells, and identify proteins that interact with MT-3 to promote epithelial features in the proximal tubule. It was shown that both E- and N-cadherin mRNA and protein are expressed in the human renal proximal tubule. Based on the pattern of cadherin expression, vectorial active transport, and transepithelial resistance, it seems that the HK-2 cell line has already undergone many of the early features associated with EMT. Our data indicates the unique, six amino acid C-terminal sequence of MT-3 is required to induce MET in HK-2 cells. A combination of co-immunoprecipitation and western blotting indicate that MT-3 interacts with myosin-IIa, β-actin, enolase-1, tropomyosin-3, and aldolase-a in vitro. Together, the data suggests the HK-2 cell line can be an effective model to study later stages in the conversion of the renal epithelial cell to a mesenchymal cell and when transfected with MT-3 it may be an effective model to study the process of MET. MT-3 protein-protein interactions provide insight into the potential mechanism by which MT-3 promotes cytoskeletal organization in non-diseased epithelial proximal tubule cells and offers the opportunity to investigate these interactions under pathological conditions.

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