Abstract: SA-PO330
ApoL1 Is Not Normally Internalized by Podocytes
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
- Scales, Suzie J., Genentech, South San Francisco, California, United States
- Gupta, Nidhi, Genentech, South San Francisco, California, United States
- Peterson, Andrew S., Genentech, South San Francisco, California, United States
Background
Human Apolipoprotein L1 (ApoL1) is secreted by the liver and circulates on HDL particles, where it protects from human African trypanosomiasis (sleeping sickness). This innate immunity is achieved by internalization of HDL particles to the trypanosome lysosomes, where ApoL1 forms pores leading to trypanolysis. ApoL1 G1 and G2 variants evolved in Africans to kill additional species of trypanosome, but greatly increase the risk of chronic kidney disease (ApoL1-nephropathies) by unclear mechanisms involving loss of kidney podocytes. The “internalization model” posits that circulating ApoL1 is internalized into podocytes to cause damage akin to trypanolysis, while the “intrinsic model” supposes that endogenous ApoL1 variants in podocytes are cytotoxic. Recombinant ApoL1 was reportedly internalized into podocytes, potentially supporting the internalization model, but the destination compartment was unclear. Most other studies support the intrinsic model, but have yet to agree on a cellular mechanism, ranging from ER stress, mitochondrial or lysosomal permeability, endocytic or autophagy disruption to cell surface cation channel activity. Clarification on whether intrinsic ApoL1 or ApoL1 in HDL particles is really internalized in podocytes might help to pinpoint the correct mechanism.
Methods
We evaluated internalization of recombinant ApoL1, ApoL1 in HDL particles, endogenous ApoL1 and stably expressed ApoL1 variants in cultured podocytes by immunofluorescence microscopy with ApoL1-specific monoclonal antibodies.
Results
Recombinant ApoL1 was internalized by podocytes, but only by virtue of being sticky, as it bound to other proteins in the media. Non-sticky ApoL1 in native HDL particles up to 1mg/ml or in amphipol was not visibly internalized by podocytes. Monoclonal antibodies detected endogenous ApoL1 in podocytes at the cell surface, but were not endocytosed. Overexpressed ApoL1 in podocytes likewise remained at the cell surface, unless the antibodies were aggregated.
Conclusion
Neither intrinsic ApoL1 nor HDL-bound ApoL1 is detectably internalized by podocytes in vitro, suggesting ApoL1 is not actively trafficked to endosomes or lysosomes. These data do not support the circulating model of ApoL1 activity and further suggest that intrinsic secreted ApoL1 might exert its effect at the plasma membrane rather than in the endolysosomal system.
Funding
- Commercial Support – Genentech