Date of Award

January 2018

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biomedical Sciences

First Advisor

Seema Somji

Abstract

The proximal tubules of the kidney are target sites of injury by various toxicants. Cadmium (Cd2+), an environmental nephrotoxicant can cause adverse effects and overt renal damage. To decipher the mechanisms involved in nephrotoxicity, an in-vitro model system is required. Mortal cultures of human proximal tubule (HPT) cells isolated from the renal cortex are used as models, but are difficult to acquire and have limited passage number. The immortalized HK-2 cell line, has served as a model but it lacks vectorial active transport and shows signs of lost epithelial features. Recently a new proximal tubule cell line, the RPTEC/TERT1, was developed. For this study we performed global gene expression analysis of this cell line in comparison to the HK-2 and HPT cells showed that the RPTEC/TERT1 cells had gene expression patterns similar to HPT cells when compared to the HK-2 cells. The HPT and the RPTEC/TERT1 cell lines showed higher expression of renal stem/progenitor like cell population, CD133+CD24+ when compared to the HK-2 cells. The level of expression of tight junctional molecules was also similar between the RPTEC/TERT1 and the HPT cells. Acute exposure to Cd2+ resulted in necrosis of the RPTEC/TERT1 cells whereas the HK-2 cells died by apoptosis. Thus, we verified that the RPTEC/TERT1 are similar to HPT cells and can serve as a good model system to study mechanisms involved in Cd2+ induced renal damage. Recent studies suggest that tubular regeneration after toxic insult may involve progenitor/ stem like cells expressing CD133 as well as CD24 markers that resides among the renal tubular cells. Our previous study shows that the RPTEC/TERT1 cells comprise of 25-30% of CD24+ cells whereas 70-75% of the CD133+CD24+ cells. To determine the response of these populations of cells to renal insult, the two populations of cells were sorted from the RPTEC/TERT1 cells, following which they were cultured and treated with 4.5μM and 9μM Cd2+ for approximately 34 days. The results demonstrate that the CD133+CD24+ cells are more resistant to cadmium exposure as there was no change in the number of double positive cells in response to cadmium treatment whereas the number of CD24+ cells significantly decrease. Both populations of cells form domes in culture indicative of vectorial active transport. The CD133+CD24+ cells show faster growth rate when compared to the CD24+ cells. When injected subcutaneously into the nude mice, these cell populations form tubule structures and CD133+CD24+ cells show positive staining for the distal as well as the proximal tubule markers suggesting their heterogeneous characteristics whereas CD24+ cells show positive staining for only the proximal tubule markers. In conclusion, the data suggests that the CD133+CD24+ cells are likely the progenitor cells in the proximal tubule involved in tubular regeneration after toxic insult whereas the role of CD24+ cells is still unclear.

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