MEDIATION OF ANTIBODY-INDUCED GLOMERULAR INJURY

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The long-term goals of this program are to define the humoral mechanisms of glomerular damage. Attention is presently focused on the visceral glomerular epithelial cell (GEC) as the target of antibody-directed, complement-mediated injury. The proposal utilizes passive Heymann nephritis (PHN) in rats, a model that faithfully reproduces many features of human membranous nephropathy. Specific aim 1 is based on two observations. Fish oil feeding ameliorates glomerular injury in PHN, and complement causes membrane phospholipid hydrolysis and intracellular calcium mobilization in cultured GECs . Three groups of experiments are proposed to examine the beneficial effects of fish oil. Cell membrane phospholipids of rat GECs in culture will be enriched with either omega-3 (n-3) or omega-6 (n-6) fatty acids and the response to complement attack will be compared in terms of: their susceptibility to sublethal cytotoxicity; alterations in phospholipid hydrolysis and intracellular calcium levels; and changes in cell membrane fluidity. The functional effects of n-3 fatty acid substitution of target GECs, independent of systemic influences, will be examined in the isolated perfused rat kidney. Glomerular hemodynamic effects will be studied in vivo by micropuncture and macromolecular sieving techniques. Specific aim 2 is designed to determine if the GEC cytoskeleton plays a role in the morphological and functional changes that accompany complement-mediated injury in PHN. Morphological, labelling and functional techniques, and agents that either disrupt or stabilize microfilaments and microtubules will be used to study the effect of complement on the GEC cytoskeleton in cell culture and in the isolated kidney. Specific aim 3 examines the role of antibody and complement on extracellular matrix production in PHN. The work will extend preliminary findings in which cells occupying the periphery of PHN glomeruli selectively expressed mRNA transcripts that hybridize in situ to a probe for the a1 chain of type I collagen. Two groups of studies are planned. First, studies will be done in vivo to define the time course of type I collagen mRNA expression and relate it to the expression of type IV collagen and laminin by in situ hybridization. RNA from PHN and control glomeruli will be analyzed by Northern blot and ribonuclease protection assays and compared to RNA from rat fibroblasts to determine if hybridization is to genuine type I collagen mRNA. Immunodetection techniques will be used to determine if type I collagen is intercalated into the basement membrane or secreted into the urinary space. Second, rat GECs in culture will be used to develop an in vitro model to study signalling pathways by which complement may influence matrix synthesis.