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

Abstract

BackgroundAPOL1 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.MethodsImmunohistochemistry, 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.ResultsBoth 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.ConclusionsAPOL1 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.

Lack of APOL1 in proximal tubules of normal human kidneys and proteinuric APOL1 transgenic mouse kidneys

“…The work presented here and other published studies have shown significant similarities between humans [8][9][10]24] and transgenic mouse APOL1 expression patterns [17,18,24]. A reproducible and consistent observation from these combined studies is expression of APOL1 in podocytes and in endothelial cells of glomerular capillaries, peritubular capillaries, and larger blood vessels.…”

“…Although monoclonal antibodies would eliminate the variation inherent in polyclonal antibody production lots, the commercial monoclonal antibodies tested had significant non-specificity or poor sensitivity for APOL1. A recent report describing the development and validation of a large number of monoclonal antibodies against human APOL1 also did not identify proximal tubule staining in human kidney [ 24 ].…”

“…Unlike the APOL1 expressed and secreted by hepatocytes, the major APOL1 transcript in podocytes does not have a complete signal peptide [ 15 ], and there is no conclusive evidence that podocytes secrete APOL1. Alternatively, some studies observed the disease-induced high levels of APOL1 can also produce rare alternatively spliced isoforms with different signal peptide sequences possibly permitting secretion [ 24 , 31 – 33 ]. These potential alternative sources of APOL1 in filtrate would have been below limits of detection in the assays we and other investigators have used in kidney tissue and urine, and would be an unlikely explanation for the robust APOL1 expression previously reported in proximal tubules.…”

APOL1 at 10 years: progress and next steps

“…Scales emphasized the critical role of APOL1 haplotypes for G1-and G2-mediated cytotoxicity in cultured podocytes stably expressing APOL1 RVs. 49,50 Specifically, the podocytotoxicity was dependent on the African APOL1 haplotype (E150/I228/K255), not simply the specific RV, even though there were no differences in localization or expression level between haplotypes. 51 She demonstrated that critical thresholds of expression at the plasma membrane must be surpassed to achieve cytotoxicity.…”

APOL1 risk variants and the development of HIV‐associated nephropathy

“…Furthermore, the administration of endoplasmic reticulum stress inhibitors to human podocytes in vitro was shown to be protective against cytoskeletal damage and cell death [53]. Although this hypothesis is compelling, however, a recent in vitro study of ApoL1 localization in podocytes using anti-ApoL1 antibodies was unable to find any ApoL1 localization in lipid droplets; further study is required to determine the in vivo localization pattern of ApoL1 and to resolve this apparent contradiction between study results [54].…”