Apoptosis is critical to the regulation of renal development, and has been implicated in a myriad of kidney disorders, including ischemia, polycystic disease, obstruction, transplant rejection, and inflammation. Many of the cellular and molecular events in apoptosis result from Fas- or TNF receptor-dependent protein- protein interactions, facilitated by intermediary molecules that possess a conserved """"""""death domain"""""""". We have recently cloned and characterized a renal-specific isoform or ankyrin, a multivalent adapter molecule that also possesses the characteristic death domain. Our preliminary studies reveal that (a) cultured kidney epithelial (MDCK and LLC-PK1) cells retain Fas- amd TNF receptor- dependent apoptotic pathways; (b) both in vitro and in vivo renal ischemia activate apoptosis; (c) kidney ankyrin expression is upregulated following both in vitro and in vivo ischemia, temporarily coinciding with the appearance of apoptotic cells; (d) transfection of kidney ankyrin cDNA induces apoptosis in renal cell lines, as well as in Jurkat cells; and (e) the death domain of kidney ankyrin interacts with Fas. These findings strongly support the hypothesis that ankyrin plays a crucial role in mediating renal cell apoptosis.
The specific aims of this proposal will rigorously test this hypothesis by identifying (SA1) the structural determinants on ankyrin that mediate its apoptotic effects on cultured renal cells; (SA2) the specific apoptotic pathways activated by over-expression of active kidney ankyrin constructs; and (SA3) the proteins that interact with ankyrin and contribute to the apoptotic cascade. For (SA1) we will employ several well-established assays for the detection of apoptosis in a variety of cells transfected with constructs encoding full-length ankyrin or a series of deletions. The results will indicate whether ankyrin's death domain is necessary and sufficient to induce programmed cell death, and will also identify motifs within this multivalent adapter molecule outside of the death domain that may cooperatively involved in apoptosis. For (SA2) we will use a variety of known inhibitors of apoptosis (including inhibitory anti-Fas antibodies, anti-TNF receptor antibodies, and caspase inhibitors) to identify specific cell death pathways activated by ankyrin over-expression. For (SA3) we will utilize a series of complementary binding assays (including co-immunoprecipitation, gel overlays, and solution binding assays) to identify other mediators that participate with ankyrin in renal cell apoptosis. The results will provide important insights into apoptotic cell death that accompanies ischemia and several other renal pathophysiologic states, and reveal critical clues for potential therapeutic interventions.
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