The broad, long-term objectives of the proposed research are both pragmatic and heuristic. The pragmatic objective is to ameliorate the nephrotoxic side effects of certain classes of drugs by blocking their access to the site(s) of injury. The heuristic objective is to delineate the similar spatial and temporal relationships that follow exposure to dissimilar noxious agents. The experimental approach to achieving these objectives will be to establish an in vitro model employing tissue culture cells and isolated plasma membrane vesicles: thereby integrating information obtained with subcellular fractions with that obtained with intact cells.
The specific aims are to establish the validity of the concept that renal cells can be protected from injury by specifically blocking transport, and to determine if perturbation of calcium is a common, early event in pathogenesis regardless of the initiating agent. The tissue culture cells to be used are the following: LLC-PK1 cells, an established cell line of pig kidney origin, and dog primary cultures. The LLC-PK1 cells have been thoroughly characterized by others, and therefore, we will seize this opportunity and use them as a model. Additionally, they will be useful for establishing the parameters to be measured in the primary cultures as our characterization of the latter develops. The primary cultures provide two unique advantages. One, we have extensive experience using membrane vesicles isolated from dog kidney for studying transport phenomena; thereby providing the opportunity for correlating information obtained from different methodologies. Two, the primary cultures can be grown on porous filters which in effect separates the basal and apical compartments; thereby providing the opportunity for studying events occurring at either face of the cell independently. Kidney damage caused by exposure to drugs or environmental toxins is an important public health problem. A successful outcome of the proposed research would ensue the development of this approach by protecting against xenobiotic-induced nephrotoxicity, and would shed light on the molecular events underlying cell injury.
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