Abstract: SA-PO329
ApoL1 G0, G1, and G2 Are Expressed on the Podocyte Cell Membrane with Similar Overall Topologies
Session Information
- Cellular Crosstalk in Glomerular Diseases - II
October 27, 2018 | Location: Exhibit Hall, San Diego Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: CKD (Non-Dialysis)
- 1903 CKD (Non-Dialysis): Mechanisms
Authors
- Gupta, Nidhi, Genentech, South San Francisco, California, United States
- Scales, Suzie J., Genentech, South San Francisco, California, United States
Background
Human Apolipoprotein L1 (ApoL1) is responsible for innate immunity against trypanosome infections. Two variants of ApoL1, G1 and G2, are responsible for the high rate of kidney disease in African diaspora and cause kidney podocyte damage by unclear mechanisms. ApoL1 can form pH-gated cation (K+ and Na+) channels in lipid bilayers, as well as pH-independent chloride channels in liposomes. Two papers showed overexpressed ApoL1 in HEK-293 cells forms potassium efflux channels at the cell surface, which leads to stress-activated protein kinase stimulation and cell death. However, contradictory results were obtained regarding ApoL1 G0 toxicity thereby questioning if G1 and G2 toxicity is variant specific or expression level dependent. Total expression levels (by Western Blot) may not be indicative of cell surface ApoL1 levels. It is actually unclear if endogenous ApoL1 and its variants are found at the cell surface and whether differences in trafficking, expression level or topology could account for the differential toxicity. Also, not much is known regarding toxicity in podocytes.
Methods
Using a panel of in house generated ApoL1-specific monoclonal antibodies to various domains, we performed flow cytometry on wild type podocytes. We also generated several clones of ApoL1 knockout podocytes stably re-expressing doxycycline-inducible ApoL1 G0, G1 or G2 to avoid any interactions (potential heterozygosity) with endogenous ApoL1. Cells were also tested for variant specific cell toxicity at comparable surface expression levels.
Results
By flow cytometry, endogenous ApoL1 was found at the cell surface of wild type podocytes, with most of the pore forming domain and end of the SRA-interacting domain exposed, but not the membrane-addressing domain. Surface ApoL1 was increased up to 500x in the inducible podocytes in a dose-dependent manner, but G0/G1/G2 maintained similar topology to endogenous ApoL1. We aim to clarify if cytotoxicity correlates with cell surface expression or variant genotype.
Conclusion
Endogenous ApoL1 and stably transfected G0/G1/G2 are all expressed on the podocyte plasma membrane with similar gross topology and are therefore correctly located to form potential surface ion channels. Thus, any differential ionic conductance is likely not due to gross topology differences at the plasma membrane.
Funding
- Commercial Support – Genentech