Renal proximal tubular and liver cells share many of the enzymes and metabolic pathways important for ammoniagenesis. However, the two organs differ markedly in their response to systemic acidosis in that the kidney increases production of ammonia from glutamine and glutamate whereas the liver shunts urea from ammonia production to glutamine synthesis. Our major hypothesis accounting for the differential metabolic behavior of kidney and liver subjected to acidosis is that alpha-ketoglutarate dehydrogenase, one of the key enzymes involved in ammoniagenesis, is regulated dissimilarly by calcium and/or pH. Alternatively, there may be differences in the pH gradient across the inner mitochondrial membrane (delta pHmito) of renal and hepatic cells under acidotic conditions. For example, an increase in delta pHmito in renal but not hepatic cells may stimulate substrate flux into the mitochondria, thereby enhancing alpha- KGDH activity. We will test the validity of these hypotheses by two approaches. First, we will measure ammonia production by primary cultures of rat proximal renal tubular cells and hepatocytes as a function of intracellular Ca++, pH and delta pHmito. Intracellular Ca++ and pH will be measured suing Fura-2 and BCECF, respectively. THe ?pHmito will be calculated from DMO distribution. Intracellular Ca++ and pH will be varied by changed in media and by treating cells with glucagon, PTH and norepinephrine. Second, mitochondria will be isolated from kidney and liver cells and ammonia production studied as a function of intramitochondrial Ca++ and pH in a similar manner as for intact cells. Additionally, we will measure flux through alpha-ketoglutarate dehydrogenase in order to determine Km and Vmax for this enzyme as a function of Ca++ and pH. Measurement of cytosolic Ca++ and pH is possible both in suspensions of cells and on a single cell basis with the fluorescence microscopy coupled digital imaging system present in the Division of Nephrology.
|Smith, B C; Clotfelter, L A; Cheung, J Y et al. (1992) Differences in 2-oxoglutarate dehydrogenase regulation in liver and kidney. Biochem J 284 ( Pt 3):819-26|