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Kidney Week

Abstract: FR-PO1022

Elucidating Transport Kinetics of Risk-Variant APOL1 Through the Secretory Pathway Helps to Understand Molecular Mechanism of Kidney Cell Toxicity

Session Information

Category: Genetic Diseases of the Kidney

  • 1002 Genetic Diseases of the Kidney: Non-Cystic

Authors

  • Daruwala, Zayd, CUNY Hunter College, New York City, New York, United States
  • Raper, Jayne, City University of New york, New York, New York, United States
  • Giovinazzo, Joseph A., Hunter College, New York, New York, United States
  • Friedman, Alejandro J., Hunter College, New York, New York, United States
Background

APOL1 is an innate immunity factor circulating on HDL particles, forming cation specific channels in African trypanosomes that lead to ionic imbalance and osmotic lysis. When endocytosed by the parasite, APOL1 undergoes a necessary acidification followed by neutralization upon recycling to the plasma membrane. G1 and G2 variants of APOL1 protect against human-infective trypanosomes but increase the risk of kidney disease. Within a human cell, APOL1 traffics along the secretory pathway, which is acidified at the golgi followed by neutralization at the plasma membrane. We show that the trafficking kinetics of G2 and non-risk G0 are similar as they travel along the secretory pathway. We also find that APOL1 is membrane-bound within the cell and is not secreted out.

Methods

Stably transfected FlpIn Trex HEK 293 cells were generated expressing the cDNA of APOL1 variants under a tet-inducible promoter. Soluble proteins were separated from membrane-bound proteins through subcellular fractionation. Cells were collected at different time points to analyze intracellular APOL1 localization. Organelles were separated using mechanical lysis and sucrose gradient ultracentrifugation. Protein co-localization with organelles was analyzed via Western blot. Toxicity was measured by quantifying LDH release. Culture media was immunoprecipitated to pull down APOL1.

Results

Subcellular fractionation showed APOL1 co-localizing in membrane-bound fractions. Sucrose gradient separation of organelles revealed that the majority of G0 and G2 APOL1 localized to the plasma membrane within 10 hours of induction of expression. Unlike G0, the G2 variant leads to cell swelling followed by cell lysis. No APOL1 secretion was detected in the media of cells expressing any of the variants.

Conclusion

We report that there is no secretion of G0 or G2 APOL1 into the media, indicating that the toxicity is related to intracellular localization of G2. There appears to be no distinction between G0 and G2 APOL1 in relation to their trafficking through the secretory pathway to the plasma membrane. However while both associate with the membrane fraction, G2 is toxic to cells but G0 is not, hinting to a hypothetical chaperone or regulatory mechanism that G2 evades, allowing for ion channel formation.