The objectives of the proposed research are to arrive at a better understanding of how oxidative and anaerobic energy metabolism in renal epithelial cells relate to the maintenance of cell membrane integrity, and how impairment of energy metabolism and the generation of ATP can cause cell damage by producing pathological alterations in the lipid composition of cell membranes. Experiments will be conducted on an established renal epithelial cell line with proximal tubule characteristics, primary cultured proximal tubule cells and freshly isolated proximal tubule segments. Cellular energy metabolism will be manipulated by the use of anoxia, or inhibitors of mitochondrial oxidative phosphorylation, and by graded deletion of glucose from the experimental medium. These manipulations will result in graded levels of cell ATP, derived either from oxidative metabolism or anaerobic glycolysis. The effects of these manipulations on cells will be studied by measurement of phospholipids, neutral lipids and cellular electrolytes, by morphological examination and by a variety of techniques to determine viability. After this characterization, studies will focus on the use of supplemental or alternative glycolytic substrates to sustain cell viability during injury, and the study of the effect of cellular work on the evolution of cell injury. A variety of biochemical and morphological techniques will be used to achieve these objectives. Cells with impaired energy metabolism will be metabolically characterized for oxygen consumption, cytochrome a (a3) reduction, mitochondrial membrane potential and glucose utilization. Alterations of lipid metabolism will be studied by lipid extraction, chromatographic separation and quantification of lipid mass. In addition, lipid metabolism will be studied by radiolabelling techniques. Cell electrolytes will be measured by atomic absorption spectroscopy and electron probe microanalysis. Cell morphology will be studied by light and electron microscopy. Cell viability will be studies by determining the ability of cells to recover their ATP levels, perform complex integrated functions such as protein synthesis, and by electron microscopy of the cells following restoration to a normal environment to allow recovery. These studies may help devise effective strategies for the preservation of cell integrity during injury caused by conditions characterized by impaired energy metabolism, such as ischemia or anoxia.
Showing the most recent 10 out of 50 publications
