This is a proposal to study the structural biology of the glomerular visceral epithelial cell (GEC) by analyzing a GEC-specific plasma membrane protein that is the target of a nephritogenic monoclonal antibody, mAb 5- 1-6. In tissue sections, mAb 5-1-6 binds exclusively to rat GECs, especially in the vicinity of GEC slit diaphragms, and it precipitates a 51 kDa protein from normal rat glomeruli. After a single in vivo injection, mAb 5-1-6 localizes to GEC foot processes and causes immediate, massive proteinuria in the absence of complement or inflammatory cells. Simultaneously, the antibody:antigen complex appears to redistribute on the GEC surface and may be endocytosed. It is suggested that the antigen (p51) recognized by mAb 5-1-6 may be an integral transmembrane protein of the slit diaphragm and that binding of antibody to its extracellular domain alters the molecular arrangement that constitutes a normally functioning slit diaphragm. An alternative possibility is that divalent antibody binding causes p5l to shift from the basolateral surface of the podocyte to the slit diaphragm where the complex might interfere with the normal molecular arrangement between cytoplasmic and as yet unknown transmembrane proteins.
In specific aim #1, a series of complementary methods are proposed to define the primary amino acid composition and structure of p51, identify putative nephritogenic domains and seek mouse and human homologues.
In specific aim #2, anti-p51 antibodies and isolated glomerular cells in short term culture will be used to: examine the normal synthesis, cellular distribution and organization of p51; and explain, in cell biological terms, what happens when p51 is bound by antibody. An attempt will also be made to develop a p51-expressing line of rat GECs to facilitate these studies.
Specific aim #3 is designed to: define the precise location of p51 and its relationship to the slit diaphragm in the normal mature and developing GEC; and determine how this is altered by mAb 5-1-6 binding in vivo and in certain proteinuric states in which predictable and well documented changes occur in the slit diaphragm. Immunohistology will be used to define the distribution of p5l and immunoblot analysis the amount of membrane-associated p5l. Metabolic labelling studies and mRNA levels will provide evidence of new p5l synthesis. Changes in p5l oligomerization status will be studied by cell surface labeling of cells isolated from injured and recovering glomeruli. Defining the molecular structure of this protein, together with its cellular organization and response to antibody binding in vitro and in vivo, will give insight into its role in maintaining the integrity of the GEC as a permeability barrier and perhaps shed light on the molecular composition of the slit diaphragm and how it is altered in proteinuric diseases.
| Kawachi, H; Koike, H; Kurihara, H et al. (2000) Cloning of rat nephrin: expression in developing glomeruli and in proteinuric states. Kidney Int 57:1949-61 |
| Topham, P S; Kawachi, H; Haydar, S A et al. (1999) Nephritogenic mAb 5-1-6 is directed at the extracellular domain of rat nephrin. J Clin Invest 104:1559-66 |
| Kawachi, H; Kurihara, H; Topham, P S et al. (1997) Slit diaphragm-reactive nephritogenic MAb 5-1-6 alters expression of ZO-1 in rat podocytes. Am J Physiol 273:F984-93 |
| Kawachi, H; Abrahamson, D R; St John, P L et al. (1995) Developmental expression of the nephritogenic antigen of monoclonal antibody 5-1-6. Am J Pathol 147:823-33 |
| Salant, D J (1994) The structural biology of glomerular epithelial cells in proteinuric diseases. Curr Opin Nephrol Hypertens 3:569-74 |
