Apolipoprotein L1-Specific Antibodies Detect Endogenous APOL1 inside the Endoplasmic Reticulum and on the Plasma Membrane of Podocytes

Significance Statement Specific variants of APOL1, G1 and G2, are associated with CKD in the Black population. Overexpression of these variants kills cells, through different proposed mechanisms in different subcellular compartments. The localization of endogenous APOL1 has not been conclusively established because all studies have used antibodies that crossreact with APOL2. Generation and use of APOL1-specific antibodies show that endogenous podocyte APOL1 localizes mainly inside the endoplasmic reticulum, with a few molecules on the cell surface. These findings potentially support the endoplasmic reticulum stress or cell surface cation channel models of cytotoxicity. Background APOL1 is found in human kidney podocytes and endothelia. Variants G1 and G2 of the APOL1 gene account for the high frequency of nondiabetic CKD among African Americans. Proposed mechanisms of kidney podocyte cytotoxicity resulting from APOL1 variant overexpression implicate different subcellular compartments. It is unclear where endogenous podocyte APOL1 resides, because previous immunolocalization studies utilized overexpressed protein or commercially available antibodies that crossreact with APOL2. This study describes and distinguishes the locations of both APOLs. Methods Immunohistochemistry, confocal and immunoelectron microscopy, and podocyte fractionation localized endogenous and transfected APOL1 using a large panel of novel APOL1-specific mouse and rabbit monoclonal antibodies. Results Both endogenous podocyte and transfected APOL1 isoforms vA and vB1 (and a little of isoform vC) localize to the luminal face of the endoplasmic reticulum (ER) and to the cell surface, but not to mitochondria, endosomes, or lipid droplets. In contrast, APOL2, isoform vB3, and most vC of APOL1 localize to the cytoplasmic face of the ER and are consequently absent from the cell surface. APOL1 knockout podocytes do not stain for APOL1, attesting to the APOL1-specificity of the antibodies. Stable re-transfection of knockout podocytes with inducible APOL1-G0 , -G1 , and - G2 showed no differences in localization among variants. Conclusions APOL1 is found in the ER and plasma membrane, consistent with either the ER stress or surface cation channel models of APOL1-mediated cytotoxicity. The surface localization of APOL1 variants potentially opens new therapeutic targeting avenues.