Rat glomerular cells do not express podocytic markers when cultured in vitro

Abstract

A panel of cell-type specific monoclonal and polyclonal antibodies and lectins was used to examine the early, morphologically epithelial outgrowth of rat renal glomerular cells in culture. The cell type-specific reactivity of the monoclonal antibodies has been previously verified on tissue sections of rat kidneys at light and electron microscopic levels. Morphologically distinct epithelial cells grew out from the isolated glomeruli within 3 days in culture, followed by the growth of morphologically typical stellate mesangial-like cells. Endothelial and mesangial cells were positively identified from the early cultures (up to 10 days) with antibodies to a 350 kD protein, dipeptidyl peptidase IV, podocalyxin, factor VIII, OX-43 and with Bandeiraea simplicifolia (BS-I B4) lectin, and with antibodies to smooth muscle actin, desmin, Thy1.1 antigens and with Ricinus communis (RCA-1) lectin, respectively. The antibodies recognizing podocytes in vivo (antipodocalyxin, anti-O-acetyl GD3 ganglioside, anti-gp330, anti-C3b complement receptor, anti-vimentin and anti-CALLA) consistently failed to bind to the predominant epithelial cells in early cultures, although these antibodies readily bound to the cells of the intact glomeruli remaining in culture. The attempts to augment the expression of cell-type specific epitopes by culturing glomeruli on various matrices or by enriching the medium with various growth factors, failed to induce podocytic epitopes on the growing epithelial cells. Glomeruli from newborn rats cultured in vitro, but were also constantly negative for the markers of podocytes. In addition, we cultured glomerular-like bodies from in vitro were induced metanephric mesenchymes but failed to obtain evidence of growing podocytes. However, the epithelial cells reacted with antibodies to thrombospondin and cytokeratin that react with the parietal epithelium of glomeruli on tissue sections. The results show that early glomerular cultures consist of mesangial, endothelial and presumably parietal epithelial cells readily identifiable by immunocytochemical methods. No podocytes could be grown under the various growth conditions tested. This suggests that glomerular podocytes are effectively growth arrested and call for new approaches to obtain these cells in culture.