Ischemia in vivo and ATP in vitro have been shown to result in extensive alterations of the actin cytoskeleton and surface membrane of proximal tubule cells. We hypothesize that correction of these alterations is essential for the cellular restitution process. Furthermore, we postulate the injured epithelial cell has the ability to reutilize, with high efficiency and fidelity, the internalized surface membrane components and disrupted and highly polymerized actin cytoskeletal constituents during cellular ATP repletion. Finally, we hypothesize that reorganization of the cortical actin cytoskeleton occurs prior to and is essential for the efficient and polarized reutilization of internalized surface membrane components during the restitution process. To test these different but interrelated hypotheses we have developed and extensively characterized a reversible model of ATP depletion in LLCPK1 cells. Furthermore, we have developed, characterized and are presently using a myriad of immunofluorescent, fluorescent analog, digital confocal, videoimaging, microinjection immunocytochemical, immunogold and quantitative biochemical techniques to study cellular repair at the single cell level. Specifically, we will use selective surface membrane domain specific biotinylation and avidin Texas-red fluorescent techniques in combination with digital confocal, immunogold an quantitative biochemical- spectrofluorimetric techniques to follow surface membrane internalization, intracellular trafficking and reutilization during ATP depletion and repletion. We will then use fluorescent analog, microinjection, digital confocal and videoimaging techniques to evaluate both G and F-actin during ATP depletion and repletion. Finally, we will combine these two approaches to observe morphologically and quantify biochemically and spectrofluorometrically the interaction between the actin cytoskeleton and apical and basolateral surface membrane domains.

R01DK041126

1990-05-01

2003-03-31