Novel 13A antigen is an integral protein of the basolateral membrane of rat glomerular podocytes

Abstract

Background: We described recently the 13A monoclonal antibody recognizing a 120 kilodalton protein located at the bases of podocyte foot processes in rat glomeruli. The antigen was extracellular, either a component of the glomerular basement membrane or an integral membrane protein. As only few markers exist for the basal domain of the podocyte membranes, we wanted to characterize the antigen further.

Experimental design: The distribution of the 13A antigen in rat tissues and cultured cells was studied by immunofluorescence and immunoelectron microscopy. Cultured cells were also used for its biochemical and functional characterization.

Results: The antigen was detected in several rat epithelial and smooth muscle tissues. In polarized epithelia, it was restricted to the basolateral membranes, and in stratified epithelia, to the basal cell layer. In contrast to its limited distribution in vivo, the antigen was detected in vitro in several cultured fibroblastoid or epithelial rat cell lines, and in cultured mesangial cells. In nonpolarized cells, it had a diffuse granular distribution at the cell surface, and at the ventral surface, it colocalized with vinculin in areas resembling focal adhesions, as shown by double immunofluorescence staining. In polarized epithelial cells, the 13A antigen was concentrated at the basolateral membranes. By immunoelectron microscopy, it was often present at the tips of cell extensions and at adhesion sites. Pretreatment of cryostat sections or cultured cells with trypsin partially inhibited antibody binding, whereas detergents abolished it totally. The antigen of cultured cells could not be identified by Western blotting or immunoprecipitation techniques. The antibodies did not seem to affect cell growth or adhesion.

Conclusions: The 13A antigen is an integral membrane protein of several rat epithelial tissues and cultured cells, and is particularly abundant in the podocyte foot processes. Although its identity and function remain unknown, the 13A protein is a valuable marker for the basal membrane domain of the podocyte.