We propose to continue the efforts of the past few years to study a variety of aspects of the interrelationship between metabolism and transport in renal tubules. Many of our recent advances in this area were dependent on the development of a viable rabbit proximal tubule suspension with open lumens and an array of methods to study this preparation. Methods that will continue to be used include: oxygen consumption, rapid potassium uptake, fluorometry, transport in isolated perfused tubules and tubule suspensions, and chemical determinations of cellular adenine nucleotides. We propose to develop methodology for cellular fractionation to separate mitochondrial and cytoplasmic fractions, as well as the use of high performance liquid charomatography (HPLC) for complete nucleotide and specific metabolite measurements in each of these fractions. Standard biochemical assays will still be required for other metabolites. All these techniques will be used to separately probe the metabolic and transport dysfunctions that occur upon phosphate depletion and anoxia. This information is expected to be of clinical relevance, since the added basic knowledge should suggest interventions to ameliorate the dysfunctions, and these will be tested in our preparation. On a more basic level, the kinetic properties of the Na, K-ATPase will be tested as a function of cellular ATP and other nucleotides in the intact renal cell. In addition, we will continue to measure the cytosolic free calcium concentration in renal tubules using the null-point method, first, in an attempt to understand how this concentration is regulated and, ultimately, to determine how this variable affects metabolism and tranport in kidney tubules. Electron probe microanalysis will be performed on samples from kidney tubules, using a new facility being developed in our Department. Initially, the cytoplasmic and mitochondrial levels of calcium will be assayed. The long-term goal is to obtain cellular maps fot Na, K, Cl, Ca, Mg, Pi, and S. Finally, a new preparation will be developed of rabbit thick ascending limb tubules in suspension to study transport and metabolism in this segment. The substrate specificity, hormonal action, loop diuretic effects and anoxia will be particularly studied.
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