Our long term goals are to understand the relation between hydration and polymerization in the sickle red cell and to predict its behavior as it moves through the hypertonic environment of the renal medulla. (Severe sickling commonly occurs in the kidney, probably because of the strong concentration dependence of HbS polymerization.) We will investigate and characterize the sources of membrane lesions that are responsible for perturbations of membrane transport that lead to different hydration and polymerization states of these cells. We will investigate differences between osmotic equilibrium properties of normal and sickle cells and the extent that these differences account for volume regulation problems encountered by sickle cells in various states. This will include measurements of how the osmotic coefficient of sickle cell Hb and fixed charge changes with concentration, pH and 02 tension, and how these results are altered as the Hb polymerizes. We will also examine the extent that hydration, polymerization, and transport properties of sickle cells are perturbed by the hypertonic and high urea environments characteristic of the renal medullary circulation. This will include measurements of how the sickle cell permeabilities of Na+, K+, Ca++, urea and glutathione depend on the hypertonic salt and urea concentrations in their environment and how these factors are modified by pH and hypoxia. Further, the kinetics of Hb polymerization in response to concentration jumps under simulated renal medulla environments will be studied, and particular attention will be paid to inhibition of polymerization by intracellular urea. Data obtained in the above projects will be assembled in a model designed to predict the fate of a sickle red cell as it enters the renal medulla. Finally we will investigate biochemical mechanisms responsible for these membrane perturbations. We will test the hypothesis that the membrane defects are a result of oxidative damage that accrues when the membrane is deformed by osmotic stress.
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