This application proposes to examine the phenomenon of antibody-induced glomerular injury that can occur in the absence of complement fixation. The investigator has previously determined that sheep nephrotoxic serum induces proteinuria in rats and mice in the absence of complement fixation in the glomerulus, and has further demonstrated that a major target of this polyclonal antibody is beta1 integrins in glomerular epithelial cells. The goal of the present studies is to determine whether the anti-beta1 integrin activity of this nephrotoxic serum is necessary and sufficient to produce the observed proteinuria, or whether there are additional or alternative antigens responsible for this effect. The first specific aim will examine whether the anti beta1 integrin antibody activity is necessary and sufficient to induce the glomerular injury. This will be purified using immunoabsorption techniques and in addition, antibodies will be produced to fusion proteins of external domains of beta1 integrin and alpha3 integrin (the likely alpha partner to the beta1 integrin in glomerular epithelial cells), and the nephrotoxicity of these antibodies will be tested in vivo, and will also be important in initial attempts to map the pathogenic epitope of the nephrotoxic serum, if indeed the beta1 integrin is determined to be the major or only antigenic site. The second specific aim examines the possibility that anti-integrin properties of nephrotoxic serum may be necessary but insufficient to cause renal injury, or is an epiphenomenon. These experiments are designed to determine how other as yet undefined glomerular antigens contribute to the nephrogenic effect. Immunopurification and molecular biological techniques will be used to identify other possible glomerular cell membrane proteins that may contribute to the effects. In addition, the possibility that the noncollagenous domain of the alpha3 chain of type IV collagen will be examined as a possible antigen. The third specific aim will examine the hypothesis that the binding of nephrotoxic serum to glomerular epithelial cells activates events that disrupt glomerular permselectivity by altering the relationship between the cells and basement membrane.
This specific aim will be important if the experiments in the first specific aim determine that the anti-integrin effect of nephrotoxic serum is primarily responsible for the glomerular alterations. In this revised specific aim, studies will be performed in a cultured system of rat glomerular epithelial cells grown on permeable supports. Cells exposed to antibodies will be examined for alterations in distribution and phosphorylation status of proteins that are involved in integrin mediated formation of cell and cell extracellular matrix adhesion. The morphology of cytoskeletal elements and the proteins that link them to integrins will also be examined in relation to the altered macromolecular permeability induced by nephrotoxic serum. Integrin-related signaling will be analyzed by phospholipid hydrolysis and second messenger production. Finally, the investigator will examine whether there is antigen shedding after exposure to antibody, or whether the antigen is endocytosed in response to antibody. These studies are designed to investigate further, the role of the glomerular epithelial cell as a primary target of injury in proteinuric renal diseases, and to define the role of integrins in the maintenance of normal glomerular structure and function.
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