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Abstract: SA-PO985

APOL1 G1-Mediated Cation Transport Inhibits Amino Acid Transport and Increases Endoplasmic Reticulum Calcium Release, Causing Podocytopathy

Session Information

Category: Glomerular Diseases

  • 1403 Podocyte Biology

Authors

  • Olabisi, Opeyemi A., Duke University, Durham, North Carolina, United States
  • Datta, Somenath, Duke University, Durham, North Carolina, United States
  • Antonio, Brett M., Ligand Pharmaceuticals Incorporated Icagen Ion Channel Technology Center, Durham, North Carolina, United States
  • Zahler, Nathan, Ligand Pharmaceuticals Incorporated Icagen Ion Channel Technology Center, Durham, North Carolina, United States
  • Krafte, Douglas, Ligand Pharmaceuticals Incorporated Icagen Ion Channel Technology Center, Durham, North Carolina, United States
  • Hohmeier, Hans-Ewald, Duke University, Durham, North Carolina, United States
  • Chaves, Alec, Duke University, Durham, North Carolina, United States
  • Nystrom, Sarah, Duke University, Durham, North Carolina, United States
  • Zhang, Guofang, Duke University, Durham, North Carolina, United States
  • Ilkayeva, Olga, Duke University, Durham, North Carolina, United States
  • Silas, Daniel Philip, Duke University, Durham, North Carolina, United States
  • Theile, Jonathan W., Ligand Pharmaceuticals Incorporated Icagen Ion Channel Technology Center, Durham, North Carolina, United States
  • Muehlbauer, Michael, Duke University, Durham, North Carolina, United States
  • Becker, Thomas C., Duke University, Durham, North Carolina, United States
  • Li, Guojie, Duke University, Durham, North Carolina, United States
  • Bain, James R., Duke University, Durham, North Carolina, United States
  • Soldano, Karen, Duke University, Durham, North Carolina, United States
  • Newgard, Christopher B., Duke University, Durham, North Carolina, United States
Background

How two coding variants of APOL1 gene (G1 and G2) cause kidney disease is poorly understood. While experimental models have shown that these APOL1 renal risk variants induce cytotoxicity, the causal mechanism underlying these effects is unknown. Previously, we reported that RRVs form cation pores that transport Na+ and K+ across the plasma membrane of mammalian cells.

Methods

We blocked APOL1 G1-cation channel function with a new small molecule inhibitor, VX-147 in inducible HEK293 cells and human-derived podocytes and measured cytotoxic phenotypes. We used Real-time cation sensors including genetically-encoded Ca2+ sensor to measure levels of cations in the cytoplasm and the endoplasmic reticulum (ER). We used inhibitor of IP3R and CRISPR-Cas9-mediated IP3R knockout to determine the impact of IP3R on APOL1-G1-induced increases in cytoplasmic Ca2+ and cell viability. Finally, using metabolomics and related methods, we measured APOL1-GI-mediated changes in amino acid transport, protein synthesis and ATP production, in the presence and absence of VX-147 treatment.

Results

We demonstrate for the first time that APOL1 G1-mediated transport of Na+ and K+ induces Ca2+ release from the ER via IP3R and ryanodine receptor (RyR), and that the liberated Ca2+ triggers a sequence of downstream cytotoxic events including mitochondrial dysfunction and inhibition of protein synthesis via AMPK-TSC2-mTORC1 and eIF2a signaling. We also discovered for the first time that APOL1 G1 cation function impedes amino acid uptake in kidney cells. All observed cytotoxic phenotypes are reversed by VX-147.

Conclusion

These findings established APOL1-mediated Na+/K+ transport as the proximal driver of podocyte injury, and that Ca2+ signaling and protein synthesis are potential therapeutic targets for APOL1 nephropathy.

Funding

  • Other NIH Support