Semester

Summer

Date of Graduation

2002

Document Type

Dissertation

Degree Type

PhD

College

Davis College of Agriculture, Natural Resources and Design

Department

Biochemistry

Committee Chair

Donald A. Sens

Committee Co-Chair

Joginder Nath

Abstract

The heat shock proteins (hsps) are believed to provide protection against a variety of insults such as elevated temperature, heavy metals and chemical agents. The goal of this study was to determine if the immortalized HK-2 human proximal tubule cell line could provide a model system to study the stress response of the proximal tubule cell. Heat stress, elevated temperature at 42.5°C for 1 hr, caused a marked increase only in hsp 70 mRNA and protein, but not that of hsp 27 or hsp 60 mRNA and protein. Similar results were obtained when the cells were subjected to 100 muM sodium arsenite or 53.4 muM CdCl 2 for 4 hrs. These findings were in contrast to those found previously with mortal human proximal tubule (HPT) cells, where acute stress by all three stimuli elicited marked increases in hsp 27, hsp 60 and hsp 70 mRNA and protein. The basal levels of expression of hsp 27 and hsp 60 in the HK-2 cells were elevated compared to unstressed HPT cells. These results suggest that failure of HK-2 cells to increase hsp 27 and hsp 60 levels in response to stress is because of their elevated basal levels, indicating that the genetic events that resulted in the immortalization of the HK-2 cells also elicited a stress response for hsp 27 and hsp 60, but not for hsp 70. Thus, there are differences in the regulation of the stress response between the immortal HK-2 and mortal HPT cell lines, and as long as these differences are recognized, the HK-2 cell line should be a valuable adjunct to study the stress response of the proximal tubule in general and when exposed to environmental pollutants such as cadmium.;The metallothoneins (MT) are a family of cysteine-rich, low molecular weight (6 kD), intracellular proteins that bind transitional metals. The goal of this study was to further characterize the basal expression of MT-3 in the in situ human kidney, in mortal human proximal tubule (HPT) cell cultures and the immortalized proximal tubular cell line, HK-2. MT-3 mRNA was detected in the proximal tubule of the in situ kidney with relative expression in excess to that of the ∃-actin housekeeping gene. Human proximal tubule cells also expressed both MT-3 mRNA and protein, and were able to form domes. Exposure of HPT cells to Cd+2 resulted in a transient increase in MT-3 mRNA and protein. In contrast, the HK-2 cell line did not express MT-3 and did not form domes. Stable transfection of the HK-2 cell line with the pcDNA3.1/Hygro(+) vector containing the MT-3 gene restored MT-3 expression and dome formation to the HK-2 cells. This result demonstrated that MT-3 is involved in the transport function of a human renal cell line that retains properties of the proximal tubule.

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