The long-term goal of this research is to identify the cascade of events that lead to cell injury and death in renal proximal tubules (RPT), and to identify and examine the mechanism of action of known cytoprotective agents. The role of Ca2+ in cell injury and death has been studied and debated. Calpains, non-lysosomal calcium-activated cysteine proteases, have been implicated in cell injury also. Calpain inhibitor 1 decreased RPT cell death produced by bromohydroquinone, antimycin A, T-butylhydroperoxide, and tetrafluoroethly-L-cysteine, suggesting that calpains plan an important role in cell injury produced by diverse toxicants. The plasma membrane Ca2+ channel blocker nifedipine, calpain inhibitor 1 and the extracellular Ca2+ chelator EGTA acted in the late phase of cell injury and blocked influx of extracellular Ca2+ calpain activity, Cl- influx and cell death in rabbit RPT exposed to a mitochondrial toxicant (antimycin A). The late phase of cell injury is important to study since a variety of cytoprotectants (ex. Glycine, muscimol, neurosteroids, nifedipine, Cl- channel blockers) act in the late phase of cell injury to prevent cell death/lysis. Furthermore, experiments have shown that many of these cytoprotectants allow RPT to regain mitochondrial function and active Na+ transport following the removal of the cellular insult, strongly suggesting that these compounds are true cytoprotectants. Thus, the investigators propose that during the late phase of cell injury, influx of Ca2+ through a nifedipine-sensitive channel and calpain activation play a critical role in RPT cell death/lysis.
The specific aims of this application are:
Specific aim 1 : Use biochemical and fluorescence techniques, and immunoblot analysis to define the role of cytosolic free Ca2+, extracellular Ca2+ influx and calpains in the late phase of RPT cell injury and death.
Specific aim II : Use patch clamp techniques to determine and characterize the signaling pathway for Ca2+ entry through a nifedipine-sensitive channel in the late phase of RPT cell injury.
Specific aim III : Use results from specific aims I and II to determine the mechanisms of action of a diverse group of cytoprotectants that act in the late phase of cell injury. Completion of these specific aims will add significant new information to our understanding of cell injury and death, particularly to those events that occur in the late phase. Determining the mechanisms of action of a number of known cytoprotectants from diverse chemical classes may ultimately result in the identification of new pharmacological agents that prevent cell death and organ failure.
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