The objective of this project is to acquire new information about kidney cell growth and regeneration after injury that can be used to design novel therapeutic approaches to enhance recovery from acute renal failure (ARF). We found previously that infused amino acids act directly on regenerating kidney tissue to stimulate phosphatidlycholine and protein biosynthesis and ameliorate renal functional insufficiency in a rat model of ARF. Subsequent work using cultures of nontransformed monkey kidney epithelial cells (BSC-1 line) permitted us to identify other growth-stimulating agents such as adenosine diphosphate (ADP). This nucleotide is the most potent mitogen yet described for these renal epithelial cells, and suggests that provision of exogenous ADP could speed regeneration after ARF by stimulating the proliferation of non- injured or sublethally-injured cells. Our recent studies aimed at defining the molecular mechanisms by which ADP stimulates DNA synthesis in BSC-1 cells indicate that the nucleotide rapidly activates expression of several proto-ocogenes and a novel gene that is an apparent transcriptional regulator. We have also found evidence which suggests that ADP binds to a cell surface protein, and induces the appearance of a previously unidentified autocrine growth factor. To define the molecular mechanisms of ADP action, we plan to explore the hypothesis that the nucleotide initiates DNA synthesis in renal epithelial cells by binding to a protein on the cell surface, activating gene and proto-oncogene expression, and stimulating release of a novel autocrine growth factor.
Specific aims of the proposed research are to: 1) Use chromatographic techniques to isolate and purify: a) the growth factor protein in conditioned medium of BSC-1 cells exposed to ADP, and b) the cell surface ADP binding protein; 2) Partially sequence the proteins, use the information to clone the genes that encode them, and study the regulation of gene expression; and 3) Develop and characterize polyclonal nd monoclonal antibodies for use in immunoassays to study the physiology of these proteins.
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