The general aims of this project have been to improve understanding of the cellular pathophysiology of the renal proximal tubule cell injury which occurs during ischemic acute renal failure. Suspensions of proximal tubules are prepared by collagenase treatment of rabbit kidneys and their metabolic characteristics, particularly respiration, cell cation homeostasis and cell adenine nucleotide homeostasis, are studied in models of oxygen deprivation injury which have now been well defined. We have found that several maneuvers strikingly alter the sensitivity of the isolated tubules to oxygen deprivation-induced injury. These are: incubating tubules at high density, lowering the pH of the incubation medium from 7.4 to 7.0 or less, treating the tubules with high concentrations of exogenous adenine nucleotides and treating the tubules with glycine or gluthatione. Further delineation of the characteristics and mechanisms of these effects can provide the tools for critically assessing which of many possible pathophysiologic processes and subcellular sites of damage are actually important in the pathogenesis of oxygen deprivation-induces tubule cell injury and can help guide the development of methods for ameliorating tubule cell injury in vivo. During the next period of funding we will concentrate our work on three areas. 1) The potent, cytoprotective effects of glycine will be studied to more completely define the oxygen deprivation and reoxygenation conditions under which glycine is protective, the sites within the tubule cell of glycine's activity, the specificity of its protective effects for the isolated tubule preparation and oxygen deprivation-induced injury and the pathways of glycine metabolism which account for protection. 2) We will extend our studies of the protective effects of high doses of exogenous nucleotides to clarify the relative degrees of protection provided by different purines. 3) We will analyze the major protective effect of reducing pH by direct measurements of cytosolic pH and independent manipulations of medium and cytosolic pH. The interplay of multiple processes over a rapid time fram during the development of oxygen deprivation-induced tubule cell injury makes it important to be able to directly assess and manipulate them if reliable insights into pathophysiology are to be obtained. The studies in this proposal provide an established and versatile approach for achieving this.
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