MECHANISMS UNDERLYING SEGMENT-SPECIFIC NEPHROTOXICITY

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Previous in vivo and in vitro research has demonstrated that most nephrotoxic chemicals induce selective injury within the kidney effecting either renal proximal straight (PST) or proximal convoluted (PCT) tubules. Selective injury observed in vitro suggests that innate cellular differences in metabolism and/or transport exist which may explain why each of these segments is susceptible to specific nephrotoxicants. Biochemical investigations to elucidate these innate cellular mechanisms in PST and PCT segments have been difficult to study due to the lack of tissue mass or a mixed population of tubular cell types in most preparations. Recently a new method was developed to isolate PST and PCT segments from one another in bulk making it possible to now compare and contrast basic mechanistic differences which render these segments innately susceptible to nephrotoxicant injury. The major goals of this proposal are to systematically investigate segment- specific metabolism- and transport-dependent mechanisms of nephrotoxicity by measuring the differential distribution or activity of several drug metabolizing enzymes and epithelial transport systems which may predispose specific nephron segments to nephrotoxic injury. this research should provide mechanistic information needed to develop rational approaches for preventing many forms of chemically-induced renal injuries. In these studies PST and PCT segments will be respectively isolated from the outer stripe region of the renal medulla or from the outer regions of the renal cortex using bulk dissection and standard Percoll gradient separation techniques. Once isolated and the purity characterized by marker enzyme analysis and histological examination, both fractions will be examined for various metabolic (e.g. glycolytic and gluconeogenic capacities, substrate utilization preferences), biochemical (e.g. rates of oxygen consumption, glutathione metabolism, phase I and II biotransformation enzyme activities), and transport (rates of organic acid, base, sugar and amino acid transport) differences. These measurements will be used to elucidate the differences in normal functions which might render the particular tubular segment susceptible to specific toxicant induced injury. In vitro exposure of both proximal tubular fractions to toxicants which effect either PST (acetaminophen, cis-platinum, hexachlorobutadiene, mercuric chloride) or PCT (hypoxia/anoxia, ethylene dibromide, potassium dichromate) will then be conducted to determine the mechanistic role of metabolism and transport as they relate to the pathophysiology of nephron specific injury. Additional studies will be conducted to: (1) manipulate the targeting of chemical gents to specific cell types (selective segmental delivery) through complexation of metals with specific carriers (cysteine or metallothionine); (2) evaluate the toxic consequences of delivering chemicals to the apical verses the basolateral cell surfaces; and (3) evaluate the interactive role between liver metabolism and nephrotoxicity in vitro by evaluating alterations in nephrotoxic responses in the presence and absence of liver tissue (co-incubations of liver and kidney tissues).