FIGURE 8.

Podocytes overexpressing Neph1 are resistant to PAN-induced injury. A, schematic representation of the construction of Cherry-Neph1 construct (where Cherry was introduced after the transmembrane domain and before any potential tyrosine phosphorylation sites in Neph1) is shown. B, expression pattern of Cherry-Neph1 was similar to endogenous Neph1 in podocytes, where both proteins localized at the cell-cell junctions (arrows). C, podocytes without and with stable expression of Cherry-Neph1 were created and subjected to PAN treatment for 48 h. Unlike the control cells transfected with empty vector, the Cherry-Neph1-transfected cells were resistant to PAN-induced cytoskeletal damage, and their cell-cell junctions were well maintained. D, BSA permeability assay was performed with vector-transfected (control) and Cherry-Neph1-overexpressing podocytes that were cultured as a monolayer on Transwell filters and treated with PAN. The passage of albumin across the podocytes monolayer was assessed by a paracellular albumin flux assay using Texas red-labeled albumin. The Cherry-Neph1-overexpressing cells showed increased resistance to PAN-induced albumin leakage as compared with control podocytes. ns, nonsignificant. Scale bar, 20 μm (B); 10 μm (C).