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Abstract: SA-PO986

Characterizing Roles of Reference and Haplotype FAT10 in APOL1-Related Kidney Diseases in Human Podocyte

Session Information

Category: Glomerular Diseases

  • 1403 Podocyte Biology

Authors

  • Hti Lar Seng, Nang San, New York City Health and Hospitals Jacobi, Bronx, New York, United States
  • Karttunen, Heidi, Albert Einstein College of Medicine, Bronx, New York, United States
  • Chaudhry, Sahir K., Albert Einstein College of Medicine, Bronx, New York, United States
  • Ross, Michael J., Montefiore Medical Center, New York, New York, United States
Background

FAT10 is a ubiquitin-like modifier, which can covalently link to target proteins or non-covalently interact with proteins, thereby altering protein function and/or stability. FAT10 is highly expressed in HIV-associated nephropathy (HIVAN) and is upregulated in glomeruli of nephrotic patients with APOL1 high-risk genotypes. FAT10 was also reported to interact with APOL1 and to accelerate degradation of APOL1 risk variants in vitro. A FAT10 haplotype with 4 missense mutations, referred to here as FAT10b, has a high allele frequency in persons of African ancestry. However, it is not known whether the FAT10b variant has altered effects on APOL1 compared to the FAT10a (reference) allele. Since expression of APOL1 risk alleles in podocytes promotes glomerular injury, we compared the effects of FAT10 variants upon APOL1 G0 and G1 proteins in human podocytes.

Methods

APOL1G0/G0 (G0) and APOL1G1/G1 (G1) podocytes were transfected with GFP-FAT10 variants using a Neon Transfection System. Immunoprecipitation (IP) was performed using anti-GFP antibody to pull down GFP-FAT10 and western blotting was performed to detect FAT10 and APOL1. Proteasome inhibitor MG132 (5 µM) was added 4 hours before IP.

Results

FAT10a increased endogenous APOL1-G0 and -G1 protein abundance, whereas FAT10b suppressed them. Both APOL1-G0 and G1 co-immunoprecipitated with FAT10a and FAT10b. 70kD and 110kD bands were observed only in APOL1G1/G1 podocytes transfected with FAT10b, likely representing novel covalent FAT10 conjugates but western blotting revealed these FAT10 conjugates did not contain APOL1.

Conclusion

FAT10a and FAT10b interacted with APOL1-G0 and -G1, but FAT10a increased APOL1 G0 and G1 abundance whereas FAT10b increased APOL1 degradation. The emergence of high molecular weight FAT10 covalent conjugates only in podocytes co-expressing APOL1 G1 and FAT10b may indicate important new targets that contribute to podocyte injury in persons with high risk APOL1 and FAT10b genotypes. These data support the importance of further studies to elucidate mechanisms by which FAT10 variants modify APOL1-mediated kidney injury.