There is mounting evidence that free radicals are involved in the pathogenesis of the ischemia-reflow, certain toxin- and drug- induced nephropathies. The data accumulate in the applicant's laboratory have indicated that oxidative stress causes profound de- rangements in cellular calcium and acid-base metabolism. In view of a potential role of these changes as mediators of cell injury or as modulators of recovery/regeneration processes, it is important to study in depth problems of renal cell physiology, as they pertain to oxidative insult. To address these problems, the following techniques will be used: cultures of renal tubular cells, microfluorometric monitoring of cytosolic free calcium, cell pH, the rate of formation of free radicals, the mitochondrial membrane potential (all parameters in a single cell), studies of inositol phosphate and diacylglycerol metabolism, studies of various determinants of cell injury/regeneration (uptake of propidium iodide, release of 51Cr, formation of lipid peroxides, incorporation of 3H-thymidine and 3H-leucine), microinjection of antioxidants or promoters of oxidative stress into the cells. Anoxia-reoxygenation, hydrogen peroxide and tert-butyl peroxide- induced changes in the above parameters of cell calcium and acid- base metabolism, as we as indeces of cell viability will be used in construction of the three-dimensional correlation matrix. Various possible sources of Ca2+ mobilization and cytosolic acidification in response to oxidative stress will be examined. The possible role of the rate, amplitude, and duration of changes in cell calcium and acid-base metabolism as mediator of injury vs. adaptive accelerators of recovery will be assessed using Ca2+ and pH-clamp techniques. Experiments utilizing manipulation of the redox state of the cell and microinjection of antioxidants or substances enhancing oxidative stress (manipulation of the level of glutathione, chelated or non-chelated iron, active or blocked xanthine oxidase) with the monitoring the subsequent oxidant- induced changes in cell calcium and acid-base metabolism, together with the parameters of cell injury will provide information on the nature (causative or coincidental) of the relationships between the insult, metabolic parameters, and cell outcome. The results of these studies may shed light on the pathogenesis of some toxic and ischemic nephropathies, and help to plan rational strategies to alleviate injury.
Showing the most recent 10 out of 26 publications
