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Kidney Week

Abstract: PO1996

The Renal Risk Variants of Apolipoprotein L-1 Lead to an Influx of Sodium and Calcium That Drive Cytotoxicity

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

  • Giovinazzo, Joseph A., Hunter College, New York, New York, United States
  • Thomson, Russell P., Hunter College, New York, New York, United States
  • Khalizova, Nailya, Hunter College, New York, New York, United States
  • Zager, Patrick J., Weill Cornell Medicine, New York, New York, United States
  • Schreiner, Ryan, Weill Cornell Medicine, New York, New York, United States
  • Raper, Jayne, Hunter College, New York, New York, United States
Background

Apolipoprotein L-1 (APOL1) is an innate immunity gene that protects against protozoan parasites. Recently evolved variants, G1 and G2, provide increased immunity against African trypanosomes while increasing the risk of chronic kidney disease. There is little consensus on how these renal risk variants (RRVs) lead to cell death or kidney disease, or which pathways to target for drug development. As APOL1 kills trypanosomes by forming cation channels, and because many of the pathways associated with the RRVs are linked to pore-forming toxins, we hypothesize that a similar mechanism is involved in kidney disease. In this study, we performed a series of experiments to delineate the events leading up to RRV-mediated cell death.

Methods

Stable cell lines were generated to express the RRVs and non-toxic G0 variant of APOL1. We also generated constructs using the retention using selective hooks system (RUSH) to control the trafficking of APOL1. Live-cell fluorescent microscopy was performed to measure the influx of cations Ca2+ and Na+ with GCaMP6f and FliCR sensors (membrane voltage), respectively. Confocal imaging was performed to test APOL1 localization. Ion reduction experiments were performed to test the effect of each ion on cell death. Finally, planar lipid bilayers were used to test for APOL1 channel selectivity.

Results

We discovered that in addition to K+ and Na+, the APOL1 channel is permeable to Ca2+. The RRVs led to an influx of Ca2+ and Na+ that preceded cell swelling and lysis by several hours. These events required RRV trafficking out of the ER and to the plasma membrane, where they localized prior to cation flux. Reduction of Ca2+ and Na+ in the media inhibited RRV-mediated cell death. We also found that the previously reported high K+ media protects against the RRVs due to a lack of Na+.

Conclusion

We report that the earliest event in RRV-mediated cytotoxicity is localization to the plasma membrane, followed by cation flux driven by Ca2+ and Na+. Because many of the proposed models of RRV cytotoxicity and kidney disease can be activated by pore-forming toxins, we propose that the cytotoxic cation channels at the cell surface are the upstream even that links them together. Our data suggests that targeting RRV channel activity represents a promising avenue for drug development.