The overall objectives of the proposed studies are to define the cellular and molecular mechanisms which are responsible for the loss of cellular organization and disassembly of the cytoskeleton following renal ATP depletion and to delineate those adaptive and reparative processes which result in restitution of cellular structure during early recovery from an ischemic insult.
Three specific aims will be investigated: 1) The pattern and sequence of disassociation and restitution of the cytoskeleton in response to ATP depletion will be studied using an integrated approach in which cellular ATP is determined by 31P NMR spectroscopy in vivo and the molecular processing of cytoskeletal proteins and integral membrane proteins is evaluated at graded levels of ATP depletion. Detached or unstable proteins will be detected by detergent solubility and Western immunoblotting. The disruption and reconstitution of the cytoskeleton will be confirmed and the intracellular localization of proteins will be defined by confocal and immunocytochemical electron microscopy. To assess the molecular processing involved in the restoration of cellular structure, the message (mRNA) and the specific transcription rate for each of the cytoskeletal proteins and for Na/K ATPase (alpha and beta subunits) will be determined at specific ATP levels and reflow intervals. 2) The mechanism of disassembly of the cytoskeleton-integral membrane protein complex will be determined by an assessment of the binding of ankyrin and fodrin to Na/K ATPase. An ankyrin binding assay will be used to asses the susceptibility of specific binding sites to graded reductions In cellular ATP. 3) The molecular process which contributes to the restitution of cellular structure and integrity during recovery from energy depletion will be determined by an assessment of stress protein responses, particularly heat shock protein 70-72 and ubiquitin. The principal focus of this group of studies will be the hypothesis that stress proteins participate in the rescue and/or disposal of disassembled cytoskeletal proteins and/or Na/K ATPase during recovery from an ischemic insult. Co- precipitation and colocalization studies will be utilized to assess the structural and functional interaction of the stress proteins with specific cytoskeletal proteins during the restitution of cellular integrity. These studies will provide important new information concerning the basic biological mechanisms by which renal epithelial cells are injured and through which recovery of sublethally injured cells is attained.
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