Abstract: FR-PO238

Exogenous ApoL1 Alters Acidification and Trafficking of Endocytic Compartments in Human Podocytes

Session Information

Category: Cell Biology

  • 202 Apoptosis, Proliferation, Autophagy, Cell Senescence, Cell Transformation

Author

  • Edwards, John C., St. Louis University, Saint Louis, Missouri, United States
Background

Variants in ApoL1 confer increased risk of certain types of chronic kidney disease in people of African ancestry. We assessed effects of exogenous wild type ApoL1 on immortalized human podocytes.

Methods

6 His/T7-tagged recombinant ApoL1 was prepared by Ni affinity and gel filtration. Immortalized human podocytes were differentiated by growth at restrictive temperature for 2 weeks. Purified protein was added to serum-free culture medium at 5 μg/ml. Localization of exogenous ApoL1 was determined with confocal microscopy. Rates of endocytosis were determined by uptake of fluorescently labeled dextran or transferrin as assessed by flow cytometry. Endosomal acidification was assayed using confocal ratiometric fluorescence microscopy of living podocytes that had been loaded with dual labeled FITC/TRITC dextran.

Results

UPTAKE: Endocytic compartments of podocytes exposed to ApoL1 were labeled with fluorescently-tagged dextran or transferrin, then stained for ApoL1. ApoL1 infrequently colocalized with both dextran and transferrin in small endosomes. Transferrin also accumulated in large peripheral structures that stained for ApoL1 and were not present in cells not exposed to ApoL1. ENDOCYTIC KINETICS: Differentiated podocytes were exposed to FITC-labeled dextran or transferrin in the presence or absence of ApoL1 and uptake/cell determined. Dextran uptake was not significantly altered by ApoL1, but uptake of transferrin was significantly increased (P<0.00001 at 80 minutes of uptake). ENDOSOMAL ACIDIFICATION: Cells were exposed to ApoL1 for 1 hour prior to pulse loading with dual-labeled dextran. 24 minutes after endocytic loading, the fraction of endosomes with pH above 7 was 11% in the control cells and 56% in the ApoL1-treated cells ( P<0.0001).

Conclusion

Exogenous ApoL1 is endocytosed by podocytes and accumulates in structures that colocalize with a recycling pathway marker. ApoL1 has little effect on endocytic kinetics of the fluid phase pathway, but increases accumulation of a marker of the recycling pathway. Exogenous ApoL1 induces accumulation of endosomes which fail to acidify. Taken together, the data are consistent with the hypothesis that endocytosed ApoL1 alters endosomal trafficking and acidification with selective effects on kinetics of the recycling pathway. Whether these properties vary among the disease associated variants remains to be determined.