The mechanisms ultimately responsible for the functional and morphological changes in glomerular disease remain unknown. My overall, long-term objective is to examine the role of reactive oxygen metabolites (ROM) in glomerular diseases. ROM affect a variety of biological processes important in glomerular diseases and their role in neutrophil-dependent glomerular diseases has been recently demonstrated. The ability of golmerular cells to generate ROM in response to a variety of stimuli has been recently well established. The objective of the present proposal is to examine the role of ROM generated by glomerular cells in glomerular diseases. A single intravenous injection of Adriamycin in rats causes nephrotic syndrome (NS) with morphological and functional changes similar to minimal change disease in humans. Based on an increasing body of evidence indicating an important role of ROM in Adriamycin's cardiotoxicity, the role of ROM in Adriamycin-induced NS will be examined. In other tissues, ROM increase vascular permeability, cause vasoconstriction and in glomeruli, increase cyclic AMP. Therefore, the direct effects of enzymatically generated ROM on renal function will be determined. I. The role of ROM in Adriamycin-induced NS. A) The effect of Adriamycin on the generation of superoxide anion, hydrogen peroxide and lipid peroxidation by mitochondria and microsomes obtained from isolated glomeruli will be determined. B) Since intracellular defenses against ROM are important determinants of toxicity, the specific activity of superoxide dismutase, catalase and glutathione peroxidase in glomeruli from normal and adriamycin-treated rats will be examined. C) Lipid peroxidation a potential mechanism of ROM-induced tissue damage will be examined in glomeruli from normal and Adriamycin-treated animals. D) For Adriamycin to stimulate the generation of ROM it must be reduced to a semiquinone. Since methylene blue, a redox dye can compete with Adriamycin for the reducing equivalents, the effect of methylene blue on Adriamycin -induced NS will be examined. E) The effects of scavengers of ROM on Adriamycin-induced NS will be examined. F) Based on the demonstrated role of reduced glutathione (GSH) in the protection of cells against the effects of ROM, the effect of increasing or decreasing glomerular GSH levels on Adriamycin- induced NS will be determined. G) The effect of Adriamycin on biological processes of potential importance in glomerular disease will be examined. i) Based on the evidence that cyclic nucleotides modulate glomerular function and that ROM markedly increased cyclic AMP content in golomeruli, the effect of Adriamycin on the cyclic AMP and cyclic GMP content in isolated rat glomeruli and the role of ROM in the observed response will be examined ii) The ability of cell-free supernatants obtained from isolated glomeruli incubated with Adriamycin to degrade GBM and the role of enzymes and ROM in the GBM degradation will be examined. II) The direct effects of intraaortic infusion (above the renal arteries) of enzymes that generate ROM on glomerular filtration rate, renal plasma flow and urinary protein will be examined using standard clearance techniques.

R01DK028452

1981-08-01

1990-07-31