De novo localization of APOL1 to the renal arterial wall in FSGS. Confocal immunofluorescence imaging of medium sized renal arterioles from the normal human kidney (A–C) and from FSGS (D–F). (A & C, green) APOL1 signal is identified in cells anatomically consistent with endothelium of medium-sized renal arterioles (arrowheads) in the normal adult human kidney and adjacent tubular segments but does not colocalize with vascular smooth muscle cells of renal arterioles stained with anti-α-SMA antibody (B & C, red). APOL1-positive cells are located in the luminal wall of the blood vessel with surrounding α-SMA positive vascular smooth muscle cells, consistent with endothelial localization. (D & F, green) Representative cross-section of a renal arteriole from an FSGS biopsy demonstrates persistent endothelial APOL1 signal and de novo appearance within the vessel wall compared with the normal arteriole (A). (E & F, red) Anti-α-SMA antibody staining identifies APOL1-positive cells as vascular smooth muscle (F, green). The vascular endothelium remains positive for APOL1 staining (arrowhead). (C & F, blue) Nuclei were visualized with TOTO-3 staining. Scale bars: 50 μm (A–F).