Recent in vitro and in vivo studies lend strong support to the hypothesis that reactive oxygen metabolites (ROM) play an important role in the functional and morphological changes observed in glomerular diseases. Passive Heymann nephritis (PHN), induced by a injection of anti-Fx1A, is a complement-dependent and neutrophil-independent model of glomerular disease that resembles membranous nephropathy in humans. We have recently shown marked reduction in proteinuria in PHN with hydroxyl radical scavengers and an iron chelator. Using the PHN model of membranous nephropathy, the major objective of the present proposal is to examine those aspects of ROM in which there is virtually no information as it relates to glomerular disease.
Specific Aim : A. To examine intracellular generation of hydrogen peroxide by normal glomeruli and by glomeruli in response to anti-Fx1A antibody. Based on the ability of 3-amino-1,2,4-triazole to irreversibly inactivate endogenous catalase only in the presence of hydrogen peroxide, the purpose of these studies is to examine in vitro and in vivo the intracellular generation of hydrogen peroxide by glomeruli during normal metabolism and in response to anti-Fx1A. In addition the effect of complement depletion of the generation of hydrogen peroxide by glomeruli in PHN will be examined. B. To examine the effect of anti-Fx1A antibody on glomerular GSH, GSSG levels, GSSG/GSH ratios and lipid peroxidation. The detection of enhanced lipid peroxidation and changes in glutathione (GSH) """"""""oxidized"""""""" glutathione, (GSSG) and the glutathione redox ratio (GSSG/GSH serve as an important marker of oxygen-mediated injury in a particular tissue, 1. In vitro studies. The effect of enzymatically generated ROM, and the effect in vitro of anti-Fx1A (with and without complement) on glomerular on GSH, GSSG and glutathione redox ratio and lipid peroxidation will be examined. 2. In vivo studies. The effect of PHN on glomerular GSH,GSSG and glutathione redox ratio and lipid peroxidation will be examined. C. To examine the role of intracellularly generated superoxide anion and hydrogen peroxide in PHN. Using liposome entrapped SOD and catalase (which results in increased intracellular levels) on anti-Fx1A- induced proteinuria will be examined. D. To examine the role of glomerular enzymatic defenses against ROM in PHN. Teh tissue injury by ROM is determined not only by enhanced generation, but also by the enzymatic and non-enzymatic defenses of ROM. I plan to examine: 1. The specific activity of SOD, catalase and glutathione peroxidase in glomeruli, tubules and renal cortex in normal and in anti-Fx1A-treated rats. ii. The effect of reducing endogenous glomerular defenses against hydrogen peroxide (catalase and glutathione peroxidase) on proteinuria in PHN. E. To examine the role of glutathione in PHN. GSH has been postulated to be an important defense against free radical injury. I propose to examine the effect of decreasing glomerular GSH levels and the effect of increasing glomerular non-protein sulfhydryl content on anti-Fx1A-induced nephrotic syndrome. F. To examine the role of iron in PHN. Since iron is critical in the generation of hydroxyl radical, I propose to examine: 1. In vitro studies. The effect of enzymatically generated ROM and the effect of anti-Fx1A antibody (with and without complement) on the mobilization of iron in glomeruli. 2. In vivo studies. The effects of anti-Fx1A on the glomerular total and non-heme iron, low molecular wight iron and bleomycin detectable iron (which detects low molecular wight iron available for participation in the Haber-Weiss reaction). ii. Based on recent studies that iron deficiency is protective and that iron-loading enhances tissue injury, the effect of iron deficiency and the effect of iron loading on anti-Fx1A-induced proteinuria will be examined.
|Ueda, N; Kaushal, G P; Hong, X et al. (1998) Role of enhanced ceramide generation in DNA damage and cell death in chemical hypoxic injury to LLC-PK1 cells. Kidney Int 54:399-406|
|Hagar, H; Ueda, N; Shah, S V (1997) Tyrosine phosphorylation in DNA damage and cell death in hypoxic injury to LLC-PK1 cells. Kidney Int 51:1747-53|
|Ueda, N; Walker, P D; Hsu, S M et al. (1995) Activation of a 15-kDa endonuclease in hypoxia/reoxygenation injury without morphologic features of apoptosis. Proc Natl Acad Sci U S A 92:7202-6|
|Ueda, N; Guidet, B; Shah, S V (1994) Measurement of intracellular generation of hydrogen peroxide by rat glomeruli in vitro. Kidney Int 45:788-93|
|Kaushal, G P; Walker, P D; Shah, S V (1994) An old enzyme with a new function: purification and characterization of a distinct matrix-degrading metalloproteinase in rat kidney cortex and its identification as meprin. J Cell Biol 126:1319-27|
|Mayeux, P R; Shah, S V (1993) Glomerular thromboxane A2/prostaglandin H2 receptors: characterization and effect of adriamycin-induced nephrotic syndrome. Biochim Biophys Acta 1181:148-54|
|Golconda, M S; Ueda, N; Shah, S V (1993) Evidence suggesting that iron and calcium are interrelated in oxidant-induced DNA damage. Kidney Int 44:1228-34|
|Ueda, N; Guidet, B; Shah, S V (1993) Gentamicin-induced mobilization of iron from renal cortical mitochondria. Am J Physiol 265:F435-9|
|Ueda, N; Shah, S V (1992) Role of intracellular calcium in hydrogen peroxide-induced renal tubular cell injury. Am J Physiol 263:F214-21|
|Ueda, N; Shah, S V (1992) Endonuclease-induced DNA damage and cell death in oxidant injury to renal tubular epithelial cells. J Clin Invest 90:2593-7|
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