(A) 3D reconstructed confocal images of representative individual human PTECs stained for rhPTX-2 (red) and clathrin, LAMP-2, or caveolin (green) showing x, y, and z axes. The left panels indicate the surface distribution of hPTX-2 following rhPTX-2 incubation for 30 minutes on ice (arrowheads). The right panels show the distribution of internalized rhPTX-2 following incubation 30 minutes at 37°C. (B and C) Time-course Western blots showing c-Jun phosphorylation (P-S63 c-Jun) and total c-Jun levels from 15 to 60 minutes after rhPTX-2 and/or plasma shock in human fetal kidney proximal tubule epithelial cells, normalized to GAPDH. Data represent 3 experiments. (D) Images and graph quantifying nuclear p–c-JUN immunofluorescence staining intensity in human PTECs in response to plasma and PTX-2. (E) Images and quantification of E-cadherin and vimentin expression by human PTECs in resting culture conditions or in response to human plasma or TGF-β1 for 24 hours. Note that 1 hour pretreatment of rhPTX-2 markedly blocked these responses. (F) Quantification of mitochondrial dysfunction by MitoSOX (left) and apoptosis by activated caspase-3 (right) detection 24 hours after human PTECs were challenged with human plasma or TGF-β1, with or without 1-hour rhPTX-2 pretreatment. (G) Inhibitory effect of rhPTX-2 on factors secreted by human PTECs in response to TGF-β1 stimulation. Fold change versus vehicle-treated PTECs and percent inhibition by rhPTX-2 are shown, and presence of c-Jun binding sites in promoters are noted. Scale bars: 25 μm. n = 3–6/group. *P < 0.05, **P < 0.01 (ANOVA with post hoc testing for multiple comparisons). VEH, vehicle.