Abstract: FR-OR108

RNA of APOL1 Risk Alleles Causes Cellular Toxicity through the PKR Pathway

Session Information

Category: Cell Biology

  • 202 Apoptosis, Proliferation, Autophagy, Cell Senescence, Cell Transformation

Authors

  • Han, Zhe, Children''s National Health System, Washington, District of Columbia, United States
  • Fu, Yulong, Children''s National Health System, Washington, District of Columbia, United States
  • Zhu, Jun-yi, Children''s National Health System, Washington, District of Columbia, United States
Background

African Americans are at higher risk for developing chronic kidney diseases due to APOL1 risk alleles (RA), but the mechanism of the cellular toxicity remains unclear. We generated Drosophila models of APOL1 nephropathy by expressing APOL1-RA in nephrocytes, which shares striking similarities with podocytes. Using fly genetic screen, we identified genes that interact with APOL1-RA. One of these genes is Pyk, homolog of human protein kinase R (PKR). PKR was suggested to interact with APOL1-RA RNA. Here we test the hypothesis that the APOL1-RA RNA induce toxicity through activating the PKR pathway.

Methods

A series of transgenic flies carrying APOL1-RA with different mutations were generated. Some carry an early stop codon so that these APOL1 proteins are gone but RNA remains same (STOP mutations), some carry synonymous mutations so that the APOL1 RNA no longer interacts with the Pyk protein, but proteins are same (Synonymous mutations). These transgenic lines, together with controls, were tested in different cell types. We also tested how Pyk knocking down affect the phenotype of these transgenes in different cell types, and whether the PKR inhibitor is beneficial for fly models of APOL1 nephropathy.

Results

We found that expression of either STOP or Synonymous mutations of APOL1-RA lead to cellular toxicity. In the fly wing, phenotypes caused by APOL1 RNA and protein appear different, suggesting that APOL1 RNA and protein have different molecular mechanism of cellular toxicity. In the nephrocytes, however, APOL1 STOP and Synonymous mutations cause similar phenotype, characterized as nephrocyte hypertrophy followed by functional decline and cell death. Knocking down of Pyk could rescue the toxicity caused by APOL1-RA with STOP mutations but not by APOL1-RA with Synonymous mutations, suggesting that PKR interaction is uniquely required for the APOL1 RNA toxicity. Feeding with PKR inhibitor reduced the overall toxicity of APOL1-G1 and G2, suggesting that inhibiting the PKR pathway is beneficial for reducing the cellular toxicity caused by the RNA of APOL1-RA.

Conclusion

Our findings demonstrated that both the RNA and the protein of APOL1-RA contribute to the overall cellular toxicity. We provided the in vivo evidence for the mechanism of APOL1 RNA toxicity through the PKR pathway, and showed that PKR inhibitor could be used as a potential therapeutic treatment to reduce APOL1 RNA toxicity.

Funding

  • NIDDK Support