Figure 4.
PAN nephrosis induces upregulation of ShcA and nephrin endocytosis. (A) Glomerular lysates from PAN-injected rats and controls were immunoblotted as indicated. Nephrin tyrosine phosphorylation on Y1217 decreases on day 4 of the injury time course, and then both Y1193 and Y1217 increase on day 7, coincident with ShcA expression. (B) Densitometric quantitation of p46/52 ShcA levels from (A), showing a significant increase in ShcA levels on days 7 and 14 (n=6). *P<0.05 by two-tailed t test. (C) Lysates from differentiated human podocytes treated with PAN (10 mg/ml) or vehicle control for 24 hours were immunoblotted for ShcA or β-actin loading control. (D) Densitometric quantitation of p46/52 ShcA levels from (C), showing a significant increase in ShcA after PAN exposure (n=4). *P<0.05 by two-tailed t test. (E) Dual immunofluorescence staining for p-nephrinY1217 (green) and nephrin (red) or EEA1 (red) on kidney sections of control and PAN-injected rats on day 7. Phosphorylated nephrin is decreased and relocalized on day 7. Regions of intense p-nephrin signal show colocalization with the early endosome marker EEA1. Scale bar, 20 μm.