lschemic renal disease remains a major cause of mortality and morbidity in this country. It is an important etiologic factor in many cases of renal failure in patients with vascular disease. While the pathophysiology of ischemic tissue damage is complex and multifactorial, several mediators of injury have been identified. Among these, ATP depletion is generally viewed as the hallmark of acute ischemia in many settings. Indeed, mush has been learned using in vitro models of chemical anoxia- induced ATP depletion in cell culture systems. However, the dynamics of cellular GTP pools during ischemia/reperIusion or chemical anoxia/recovery have not been investigated systematically. Thus, there remains a big gap in our understanding of the role of guanine nucleotide depletion in ischemia. Such a role for guanine nucleotides is suggested by the importance of the cellular GTP/GDP ratio in the functioning of a variety of signaling GTPases that control trafficking, polarity, the cytoskeleton and cell death in epithelia. In particular, the small GTPases Rho, Rac and Cdc42 are emerging as central participants in the injury-repair cycles observed in ischemia/reperfusion. The central hypothesis to be tested in this proposal is that GTP depletion during ischemia/reperfusion is an important and independent variable in determining the form of cell death observed. Apoptosis is increasingly recognized as a significant mode of cell loss during ischemia. Our hypothesis postulates a major role for GTP depletion in modulating this form of cell death, possibly via an effect on Rho family GTPases. We propose to develop models of selective GTP and ATP depletion and correlate them with cell survival and apoptotic death in culture systems. We will also investigate the role of Rho GTPases in modulating apoptosis using cells transfected with constitutively active and dominant negative forms of the GTPases Rho, Rac and Cdc42. Finally, we will examine the role of GTP in renal ischemia in vivo using control and HPRT-null mice that cannot salvage guanosine to GTP. The effects of enhanced GTP levels on morphology, apoptosis and renal function will be determined. These studies will establish GTP as a key . modulator of ischemic injury and might lead to new therapeutic options for this devastating disease.
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