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Abstract: SA-PO361

What’s the RUSH? The Role of Calcium in APOL1 Toxicity

Session Information

Category: Glomerular Diseases

  • 1202 Glomerular Diseases: Immunology and Inflammation

Authors

  • Giovinazzo, Joseph A., CUNY Hunter College, Brooklyn, New York, United States
  • Thomson, Russell P., CUNY Hunter College, Brooklyn, New York, United States
  • Khalizova, Nailya, CUNY Hunter College, Brooklyn, New York, United States
  • Malani, Nirav, Independent, Flemington, New Jersey, United States
  • Schreiner, Ryan, Weill CornellMedical College, New York, New York, United States
  • Raper, Jayne, CUNY Hunter College, Brooklyn, New York, United States
Background

APOL1 is an innate immunity protein that forms toxic ion channels in trypanosomes. Variants of APOL1, G1 and G2 but not G0, are linked to kidney disease, however the mechanism responsible remains controversial. Here we propose that the key upstream event is APOL1 localization to the plasma membrane (PM), where it forms channels that lead to the passage of ions across the membrane, with Ca2+ influx being a driver of cell death.

Methods

APOL1 was expressed in FlpIn TREX 293 cells or HEK and CHO cells via the Retention Using Selective Hooks (RUSH) system. RUSH is a streptavidin-binding method that retains tagged APOL1 in the ER until biotin addition. Toxicity was measured via LDH release. rAPOL1 was purified from E. coli and reconstituted in planar lipid bilayers to measure channel selectivity. High throughput widefield microscopy was performed to quantify cytosolic and ER Ca2+ in cells expressing indicators GCaMP6f and ER-LAR-GECO, along with viability dye DRAQ7. IF with confocal microscopy was performed to quantify APOL1 localization.

Results

When retained in the ER, G1 and G2 are not toxic, and only release from the ER leads to cell death. The toxicity of G1 is delayed relative to G2, and G0 is not toxic. A previous study in Xenopus oocytes reported that APOL1 leads to Ca2+ influx. We tested rAPOL1 selectivity and found that all variants form channels that are permeable to Ca2+. Furthermore, G1 and G2 toxicity is dependent on extracellular Ca2+, as increasing [Ca2+] led to more cell death, while chelation with EGTA rescued. Measurement of cytosolic Ca2+ revealed that G1 and G2, only after ER release, led to a significant Ca2+ influx over 2-4h that preceded cell swelling and death. IF indicated that APOL1 localizes to the PM prior to Ca2+ influx, and that G1 traffics slower than G2. Simultaneous measurement of cytosolic and ER Ca2+ demonstrated that there is no ER Ca2+ release.

Conclusion

These data demonstrate that the toxicity of G1 and G2 requires PM localization and an influx of Ca2+. We also report a novel difference between the trafficking and toxicity kinetics of G1 and G2. The ion channel at the plasma membrane is likely the key event that leads to cell death, and the sustained influx of Ca2+ may unify the disparate theories of APOL1 toxicity, from mitochondrial damage to inflammatory pathways, which can all be triggered by Ca2+.

Funding

  • Other NIH Support