Figure 5.

In vivo injection of 27A IgG causes apical dislocation and tyrosine phosphorylation of nephrin. A: One hour after intravenous injection of 27A IgG₃, the antibody binds to the podocytes. In contrast to the IgG₃-injected rats (data not shown), bound 27A is detected in the glomerulus by rhodamine conjugated anti-mouse IgG (A). B, C, and D: The effacement-like morphological changes at the podocyte filtration slits are shown in C and D. At these altered filtration slits, nephrin is found at the apical pole and often in clusters as demonstrated by the 15-nm gold particles (C and D). For comparison, nephrin at normal wide filtration slits of the control-injected rat is shown (B). Original magnifications, ×34,000. E: The apical dislocation is accompanied by tyrosine phosphorylation of nephrin. One hour after injection of 27A and control IgG₃, glomerular membrane fractions were co-immunoprecipitated with anti-nephrin and labeled with anti- phosphotyrosine. Tyrosine phosphorylated nephrin was detected for the 27A-injected rats but not for the control animals. Equal loading of nephrin after immunoprecipitation was shown by probing with anti-nephrin antibody (right lanes).