DNA fragmentation has been described in both necrotic and apoptotic forms of renal tubular epithelial cell death, however, the endonuclease responsible for the DNA fragmentation prior to the kidney cell death is unknown. The central hypothesis of this proposal is the DNase I induces DNA strand breaks and is important for cell death of kidney tubular epithelium during ischemic/hypoxic injury.
Our specific aims are: (1) To examine the role DNase I in DNA fragmentation and cell death in kidney epithelial cells during hypoxia/reoxygenation in vitro. Proposed studies will utilize TKPTS mouse proximal tubule epithelial cell lines that are different in their expression of DNase I, and tubules isolated from DNase I wild type and knockout mice. (2) To study role of DNase I in mouse kidney in vivo during ischemic injury. These studies will draw the temporal relations between the endonuclease activation, DNA fragmentation, and cell death (both apoptosis and necrosis) in the kidney during ischemia/reperfusion. The experiments will be performed using DNase I wild type and knockout mice. The effect of anti-DNase I antisense treatment in vivo will be examined in a model of ischemia/reperfusion-induced acute renal failure. A portion of DNA breaks induced in vivo by the DNase I vs. other endonucleases during ischemia/reperfusion will be detected using single-base resolution """"""""fingerprints"""""""" of in vivo DNA breaks. (3) To study regulation of DNase I during ischemic/hypoxic injury at three levels: (a) at the mRNA level, through alternative pre-mRNA splicing, (b) at the protein level, including DNase synthesis, post-translational modification, and nuclear import, and (c) at the activity level, including regulation by protein inhibitor. We believe that understanding of the mechanisms of acute renal failure and their regulation may lead to better therapeutic modalities in the future.
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