ASN's Mission

ASN leads the fight to prevent, treat, and cure kidney diseases throughout the world by educating health professionals and scientists, advancing research and innovation, communicating new knowledge, and advocating for the highest quality care for patients.

learn more

Contact ASN

1401 H St, NW, Ste 900, Washington, DC 20005

email@asn-online.org

202-640-4660

The Latest on Twitter

Kidney Week

Abstract: SA-PO362

Characterization of Novel APOL1 G0, G1S342G and APOL1-/- Podocyte Cell Lines and Their Response to HIV Infection

Session Information

Category: Glomerular Diseases

  • 1202 Glomerular Diseases: Immunology and Inflammation

Authors

  • Karttunen, Heidi, Albert Einstein College of Medicine, Bronx, New York, United States
  • Rosales, Alan, Albert Einstein College of Medicine, Bronx, New York, United States
  • Gao, Xiaobo, Albert Einstein College of Medicine, Bronx, New York, United States
  • Ross, Michael J., Albert Einstein College of Medicine/ Montefiore Medical Center, Bronx, New York, United States
Background

Persons with APOL1 high risk genotypes are at increased risk of several forms of progressive CKD and remarkably, have a 29 to 89-fold increased risk of HIV-associated nephropathy (HIVAN). HIV gene expression in renal epithelial cells, including podocytes, is a critical mediator of HIVAN pathogenesis but previous studies have failed to detect an increase in APOL1 expression in HIVAN biopsy specimens, suggesting that changes in APOL1 function, and not expression levels, may drive pathogenesis. The APOL1 G1 risk allele consists of 2 nonsynonymous SNPs (S342G and I384M) and S342G is likely sufficient to drive the risk attributed to G1. Since overexpression models may not accurately model APOL1 function, we used CRISPR-Cas9 to generate isogenic human podocyte lines with APOL1 G0, G1S342G, and APOL1-/- and determine the effects of APOL1 genotype upon response to HIV infection.

Methods

We used CRISPR-Cas9 to modify conditionally immortalized APOL1G0/G0 podocytes. Guide RNAs and single stranded oligonucleotides were designed to target the APOL1 locus to generate the G1S342G allele or delete exons 4 and 5 (APOL1-/-). In lentiviral transduction studies, podocytes were transduced with VSV-pseudotyped NL4-3DG/P-EGFP (HIV), or HR-IRES-EGFP (control). Endogenous APOL1 was immunprecipitated after HIV or control infection and APOL1-binding proteins were identified by mass spectrometry.

Results

Clonal homozygous G1S342G and APOL1-/- podocytes lines were generated. All podocyte lines expressed normal podocyte markers, including podocin. APOL1 expression was lower in G1S342G than G0 podocytes and APOL1 expression increased as podocytes were differentiated at 37C. Proteasome inhibition increased APOL1 protein abundance, suggesting that it is degraded by the ubiquitin-proteasome system. HIV infection did not increase APOL1 expression in any podocyte cell lines. Immunoprecipitation of endogenous APOL1 after HIV or control infection and identification of APOL1 binding proteins by mass spectrometry identified numerous cellular proteins that differentially bind G1S342G in the presence of HIV infection with known roles in kidney injury.

Conclusion

We developed novel podocyte cell lines with alterations in the APOL1 coding sequence that will allow for studies to determine changes in function of the APOL1 alleles when expressed at physiologic levels.

Funding

  • NIDDK Support