PHOSPHORYLATION DEPENDENT SIGNALING IN HYPOXIC TUBULE INJURY

University Of Michigan At Ann Arbor, Ann Arbor MI

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Abstract

Reversibly injured proximal tubule cells undergo major remodeling of both their apical and basolateral surfaces that is critical for the functional recovery of the tissue during ischemic and related types of acute renal failure and there is substantial evidence that this form of injury rather than cell killing predominates in many settings. Phosphorylation/dephosphorylation events involving both proteins and lipids are well recognized as critical regulators of cellular protein localization and cellular ATP depletion followed by recovery induces profound alterations in phosphorylation states of both types of molecules. however, the specific implications of these changes and their interactions in the setting of tissue injury have been large unexplored, in part because of the lack of models that accurately recapitulate the events in fully differentiated cells that can be studied in vitro. Recent models that accurate recapitulate the vents in fully differentiated cells that can be studied in vitro. Recent advances in the understanding of factors that account for the normal in vivo resistance to lytic membrane damage have now made it possible to study these issues in freshly isolated proximal tubules. Among cellular phospholipids, the phosphoinositides are the most rapidly and completely lost during ATP deletion. Concomitantly, multiple tyrosine-phosphorylated proteins, including phosphoinositide 3- kinase (PI(3)K), are completely dephosphorylated and focal adhesions disassemble. During recovery under favorable metabolic conditions these structures rapidly reassemble. The expression of this behavior in isolated tubules will allow a unique test of the hypothesis that the phosphoinositides play a critical structural role interfacing the plasma membrane with cytoskeletal proteins and components of signaling complexes that co-assemble there. We will use freshly isolated rabbit proximal tubules to address three specific aims: 1) Quantitate the extent of decrease of phosphoinositide mass that occurs during hypoxia and determine whether Ca2+ and phospholipase C-dependent diacylglycerol accumulation during hypoxia is large derived from phosphoinositides and is limited because of their prior depletion of CA2+-independent processes. 2) Characterize the restoration of phosphoinositide mass during reoxygenation. Assess the extent of concomitant recovery of ATP and the associated perturbations of diacylglycerol. Assess the extent of concomitant recovery of ATP and the associated perturbations of diacylglycerol. Assess the contribution of these events to restoration of focal adhesion structure. 3) Determine whether further metabolism of phosphoinositides of PI(3)K contributes to the reassembly of focal adhesions during reoxygenation and investigate involvement of the enzyme in other signaling molecule complexes which also reassemble at that time.