The goal of this project is to determine the stoichiometric relationships between O2 consumption, ATP synthesis, and Na transport by the intact kidney in vivo. The Na/O2 ratio of the kidney is greater than the theoretical maximum of 18 (assuming ATP/O = 3, ATP/O2 = 6, Na/ATP = 3). It has been proposed that the high Na/O2 is due to passive reabsorption of Na in the proximal tubule. This hypothesis will be tested by measuring ATP synthesis using 31P NMR saturation transfer and simultaneously determining O2 consumption and Na reabsorption by clearance techniques. In order to test certain assumptions which are necessary for an unambiguous interpretation of the NMR results, we will: 1) Reduce or eliminate the contribution of extracellular Pi and red cell metabolites to the intracellular Pi peak. 2) Determine if a NMR-invisible Pi pool participates in ATP synthesis by comparing the rates of synthesis and hydrolysis. 3) Determine if NMR measures ATP synthesis or Pi-ATP exchange by measuring the sensitivity of the reaction to cyanide or rotenone; ATP synthesis is inhibited by these agents while ATP-Pi exchange is not. 4) Determine if the ATP/O can be measured with reasonable precision by error analysis experiments. It is expected that these experiments will demonstrate with reasonable certainty that the ATP/O in vivo is 2-3. It has been proposed that the high Na/O2 of the kidney is due to passive Na reabsorption (Na/ATP greater than 3) in the proximal tubule. To test this we will measure: 1) the effect of inhibiting the loop of Henle with furosemide and 2) the effect of inhibiting the proximal tubule with acetazolamide, mannitol, or vanadate. The results should indicate the tubular segment responsible for the high Na/O2. Finally, to determine if the stoichiometry between O2 consumption, ATP synthesis, and Na transport varies in a regulated fashion, we will measure ATP/O and Na/ATP during the following maneuvers: 1) changes of glomerular filtration rate. 2) Hypoxia. 3) Hypo - and hyperthyroidism. The significance of this project is that: 1) Important assumptions necessary to interpret saturation transfer measurements of ATP synthesis will be tested. 2) The stoichiometry of oxidative phosphorylation in vivo will be measured. 3) The site of the high Na/ATP in the kidney will be determined. 4) Factors which may alter coupling of oxidative phosphorylation and ATP utilization for Na transport may be identified.
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