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Abstract: FR-PO107

Inhibiting ApoL1 Translocation to Mitochondrial Matrix Can Protect Against Risk Variant Mediated Cytotoxicity

Session Information

Category: CKD (Non-Dialysis)

  • 1903 CKD (Non-Dialysis): Mechanisms

Authors

  • Shah, Shrijal, Beth Israel Deaconess Medical Center, Boston, Massachusetts, United States
  • Lannon, Herbert F., Beth Israel Deaconess Medical Center, Boston, Massachusetts, United States
  • Dias, Leny, Beth Israel Deaconess Medical Center, Boston, Massachusetts, United States
  • Pollak, Martin R., Beth Israel Deaconess Medical Center/Harvard Medical School, Boston, Massachusetts, United States
  • Friedman, David J., Beth Israel Deaconess Medical Center/Harvard Medical School, Boston, Massachusetts, United States
Background

African Americans with two copies of ApoL1 risk variants (G1 and G2) are at high risk of developing chronic kidney disease. The subcellular localization of ApoL1 based on imaging studies has been a subject of conflicting reports.

Methods

HEK293 stable cells that express G0, G1, G2 in the presence of tetracycline were made using the T-Rex system. Cells were transfected with siRNA for specific outer mitochondrial membrane (OMM) and inner mitochondrial membrane (IMM) translocase machinery components and then ApoL1 expression was induced with tetracycline. After ApoL1 induction, mitochondrial and cytosolic fractions were prepared by differential centrifugation and presence of ApoL1 in each fraction was determined by immunoblotting. We also measured oxygen consumption rate using a Seahorse assay and measured cytotoxicity after knocking down specific OMM and IMM components.

Results

Using a biochemical approach to study ApoL1 localization, we found a large fraction of G0 as well as G1 and G2 in the mitochondria. Protease digestions of isolated mitochondria demonstrated that ApoL1 protein had been translocated into the mitochondrial matrix. siRNA knock-down of OMM protein TOMM20, which is involved in recognizing matrix-targeting presequences on nuclear-encoded proteins, reduces ApoL1 levels in mitochondria. TOMM20 knockout also rescues G1 and G2 mediated mitochondrial dysfunction as measured by oxygen consumption rate and reduces cell death. Knock-down of TIMM23/TIMM17 complex components, which are essential for translocating matrix-targeted proteins, rescues G1- and G2-mediated cytotoxicity whereas knock-down of TIMM22, which is essential for protein insertion into the IMM, has no effect.

Conclusion

G1 and G2 need to translocate to mitochondrial matrix to induce toxicity. Since both G0 and risk variants are translocated to the mitochondrial matrix, elucidating different behavior of these variants after mitochondrial translocation will be important for understanding why only the risk variants cause kidney injury.

Funding

  • Other NIH Support