Apolipoprotein L1-Specific Antibodies Detect Endogenous APOL1 inside the Endoplasmic Reticulum and on the Plasma Membrane of Podocytes
Scales, Suzie J; Gupta, Nidhi; De Mazière, Ann M; Posthuma, George; Chiu, Cecilia P; Pierce, Andrew A; Hötzel, Kathy; Tao, Jianhua; Foreman, Oded; Koukos, Georgios; Oltrabella, Francesca; Klumperman, Judith; Lin, WeiYu; Peterson, Andrew S
(2020) Journal of the American Society of Nephrology, volume 31, issue 9, pp. 2044 - 2064
(Article)
Abstract
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
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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.
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Keywords: Apolipoprotein L1, Apolipoprotein L2, chronic kidney disease, immunofluorescence, immunohistochemistry, podocyte, Nephrology, Journal Article
ISSN: 1046-6673
Publisher: American Society of Nephrology
Note: Funding Information: Dr. Suzie Scales reports other from Genentech and other from Roche, outside the submitted work. Ms. Nidhi Gupta reports other from Genentech, outside the submitted work. Dr. Cecilia Chiu reports other from Genentech, and other from Roche, outside the submitted work. Dr. Andrew Pierce reports other from Genentech and other from Roche, outside the submitted work. Ms. Kathy Hötzel reports other from Genentech and other from Roche, outside the submitted work. Dr. Oded Foreman reports personal fees from Genentech and personal fees from Roche, outside the submitted work. Dr. Georgios Koukos reports grants from Roche and personal fees from Genentech, outside the submitted work. Ms. WeiYu Lin reports other from Genentech and other from Roche, outside the submitted work. Dr. Andrew Peterson reports other from Genentech and other from Roche, outside the submitted work. Publisher Copyright: Copyright © 2020 by the American Society of Nephrology Copyright: Copyright 2020 Elsevier B.V., All rights reserved.
(Peer reviewed)