We propose to test two hypotheses: One, in the rat proximal tubule, Na+/Ca2+ exchange (Na/Ca) regulates, in part, the concentration of cytosolic Ca2+ (Ca/i). Two, in the rat proximal tubule, anoxia alters the homeostatic interdependence of Ca2+ and Na+. This alteration reverses Na/o-dependent Ca2+ efflux to Ca/o-dependent Ca2+ influx. To achieve this purpose, four aims are outlined: One, to investigate the modulation of Ca/i by fluctuations in the electrochemical gradients of Na+ and Ca2+ delta uNa+ and delta uCa2+). Two, to examine the direction of the fluxes of Na+ and Ca2+ evoked by fluctuations in delta uNa+ and delta uCa2+ in the intact rat proximal tubule. Three, to examine the contribution from intracellular organelles to the regulation of Ca/i by Na+. Four, to define the alterations in Na/Ca exchange in the anoxic rat proximal tubule. The basis for these investigations are theoretical and empirical. Based on a thermodynamic analysis, it is proposed that delta uNa+ and delta uCa2+ are forces oriented from the outside of the cell to the inside of the cell. These two forces drive Na+ influx in exchange for Ca2+ efflux and Ca2+ influx in exchange for Na+ efflux, respectively (Na/CA exchange). The orientation of Na/Ca exchange may change in pathological states, caused by changes in the magnitude of one force relative to the magnitude of the other force. These changes may have profound effects on Ca/i homeostasis. This notion has been confirmed, in part, in our laboratory. We showed that fluctuations of delta uNa+ and delta uCa2+ change Ca/i in rat proximal tubules in accord with the thermodynamic model. These investigations support the idea that Ca/i is regulated by Na/Ca exchange. We propose to extend this work, by measuring Ca/i, using aequorin and the fluxes of 45Ca and 22Na in rat proximal tubules. In addition, we propose to study the effect of anoxia on Na/Ca exchange. It is proposed that in anoxia, alterations in Ca/i may be caused, in part, by the prevalence of the force delta uCa2+ over delta uNa+. Thus far, the role of Na/Ca exchange in Ca/i and Na/i homeostasis has been ignored, in part because of the assumption that Na/Ca exchange may not operate in nonexcitable cells. In this proposal we provide evidence to the contrary. The proposed work, should also improve our understanding of the pathogenesis of alterations in Ca/i homeostasis in the proximal tubule in anoxia. These alterations may participate in ischemic renal injury.
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