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Kidney Week

Abstract: PO1986

The African APOL1 E150 SNP and Cell Surface Expression Are Required for Kidney Risk-Variant (G1/G2)-Mediated Cytotoxicity in Podocytes

Session Information

  • Podocyte Biology
    October 22, 2020 | Location: On-Demand
    Abstract Time: 10:00 AM - 12:00 PM

Category: Glomerular Diseases

  • 1204 Podocyte Biology

Authors

  • Gupta, Nidhi, Genentech Inc, South San Francisco, California, United States
  • Scales, Suzie J., Genentech Inc, South San Francisco, California, United States
Background

Apolipoprotein L1 (APOL1) variants G1 and G2 protect against trypanosome infection, but homozygosity greatly increases the risk of chronic kidney diseases, purportedly by acting as surface cation channels in kidney podocytes. “Wild type” APOL1-G0 exhibits various single nucleotide polymorphisms (SNPs), most commonly haplotype E150K, M228I and R255K (“KIK”, where the Reference Sequence is “EMR”), whereas G1 and G2 are only found in a single African haplotype background (“EIK”), also seen in some G0 Africans. Lannon et al (Kidney Int. 96: 1303) recently documented that differential cytotoxicity of APOL1 G1 and G2 variants versus G0 in HEK-293 cells depended on the haplotype. However, HEK-293 cells are unusually sensitive to APOL1, and podocytes are a more relevant cell type. Furthermore, only the small fraction of APOL1 that is transported to the cell surface (from its major expression site in the ER) is responsible for cytotoxicity and cell surface levels were not shown in that study. Since APOL1 residue 150 can differ in G0 Africans, we focused on comparing the cytotoxicities of E150 vs K150 SNPs in podocytes expressing equal surface levels of APOL1.

Methods

We generated podocytes stably expressing APOL1 G0, G1 or G2 under a doxycycline-inducible promoter and compared the effect of the African E150 (EIK) vs K150 (KIK) SNPs on cytotoxicity (by the Cyto-Tox GloTM assay). Surface and total APOL1 were measured by FACS and Western blotting at increasing doxycycline levels. Brefeldin A was used to prevent APOL1 transport to the cell surface.

Results

Cell surface APOL1 levels increased in a doxycycline dose-dependent manner, but only the E150 G1 and G2 variants caused toxicity to podocytes as compared to E150 G0 at equal surface expression levels; K150 G0, G1 and G2 were not toxic. E150 G1 and G2 cytotoxicity was dose-dependent and required exit of APOL1 from the ER.

Conclusion

Using a physiologically relevant podocyte cell line, we confirmed that the African haplotype (EIK) is required for APOL1 G1 and G2 to exert cytotoxicity. Non-natural KIK versions were not toxic. Additionally, APOL1 G0 was not toxic in either the KIK or EIK background. Furthermore, African (E150) G1 and G2 cytotoxicity required ER exit, supporting the surface cation channel hypothesis. Our data thus suggest two potential therapeutic avenues for APOL1 nephropathies.

Funding

  • Commercial Support