The mammalian nephron is composed of several cell populations that possess distinctive biochemical and physiologic properties and each cell population responds differently to chemical toxicants or to pathologic conditions such as hypoxia or ischemia. To understand what properties are responsible for cell type-specific susceptibility to chemical and pathologic injury, we have focussed on two salient cellular factors, glutathione (GSH) status and mitochondrial function. GSH is a key component of cellular defense against reactive electrophiles and oxidants, two of the most common producers of cellular injury in both toxicity and disease. The kidney is unusual with respect to GSH in that it possesses both synthetic and transport capabilities. Cell type-specific differences in regulation of GSH status may be important in determining cellular susceptibility to injury. Proper mitochondrial function is critical to maintain a constant and high energy input for renal function. Consequently, renal cells are vulnerable to interruptions in energy supply due to mitochondrial dysfunction. Since mitochondria are frequent target sites in toxic or pathologic injury, cell type-specific differences in properties of mitochondria may also contribute to cellular susceptibility to injury. Research proposed in these studies will focus specifically on mechanisms of GSH transport across cellular plasma membranes and across mitochondrial inner membranes. Freshly isolated cells derived from rat kidney proximal tubule (PT) and distal tubule (DT), mitochondria derived from renal cortex, and mitochondria purified from renal PT and DT cells by a novel fractionation technique will be the biological systems used. The fractionation technique is novel because it is the only method available to isolate functionally viable mitochondria from single cells in quantities sufficient for biochemical studies.
The specific aims of this research will be: (l) To determine the mechanism of GSH transport across plasma membranes in different renal cell populations and to investigate the role of these processes in regulation of intracellular GSH status; (2) to determine the mechanisms of GSH and glutathione disulfide (GSSG) transport in renal cortical mitochondria; and (3) to characterize biochemical properties of mitochondria purified from specific renal cell populations and to determine the role of mitochondrial GSH transport and energetics in differential susceptibility to toxicants. By completion of the above aims, we will delineate mechanisms of GSH transport across cellular plasma membranes and mitochondrial inner membranes, we will identify the protein responsible for mitochondrial GSH transport, we will describe the interaction of mitochondrial GSH transport activity with mitochondrial metabolism and cellular energetics, and we will use these results to gain information on the role of mitochondrial and cellular heterogeneity in susceptibility of renal PT and DT cells to chemical and pathologic injury. These results will ultimately help in development of procedures to maintain or improve kidney function.
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