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Abstract: SA-PO363

APOL1 RNA Is Differentially Spliced in Nephrotic Syndrome and in Response to Therapy

Session Information

Category: Glomerular Diseases

  • 1202 Glomerular Diseases: Immunology and Inflammation

Authors

  • Kino, Jiro, The Research Institute at Nationwide Children's Hospital, Columbus, Ohio, United States
  • Webb, Amy, Ohio State University, Dublin, Ohio, United States
  • Chanley, Melinda A., Nationwide Children's Hospital, Columbus, Ohio, United States
  • Smoyer, William E., Nationwide Children's Hospital, Columbus, Ohio, United States
  • Agrawal, Shipra, The Research Institute at Nationwide Children's Hospital, Columbus, Ohio, United States
Background

APOL1 has been a subject of intense research since the discovery of the association of its risk alleles with heightened risk for end stage kidney disease in African Americans. However, the potential role of alternate splicing of APOL1 in podocyte biology and in the regulation of kidney disease is unknown. Recent evidence suggests that the presence and/or absence of exons 2 and 4 distinguish known splice variants of APOL1, and that exon 4 contributes to cytotoxicity. We hypothesized that APOL1 RNA is differentially spliced in nephrotic syndrome (NS) and in response to glucocorticoid (GC) therapy, thus playing a potential pathophysiological role in both disease and health, regardless of APOL1 haplotype.

Methods

APOL1 RNA and splice variant expression was analyzed in podocytes injured with puromycin aminonucleoside (PAN), and treated with dexamethasone (Dex) by RT-PCR using splice variant specific primers. Splice variant analyses were also performed in circulating leukocytes of children with steroid sensitive (SSNS) and steroid resistant NS (SRNS), before and following initial GC therapy (N =8 SSNS and 8 SRNS paired samples). T-test and 2 way ANOVA statistical analyses were performed to measure relative variant expression.

Results

Podocytes expressed all 5 known splice variants of APOL1 [v.A, variant3 v.A, v.B1,v.B3 and v.C]. Higher relative expressions of exon 4 (+) [v.A, v.B1] and exon 2 (-) [v.A, v.C] forms were observed, with variant 1 [v.A] the most predominantly expressed form. Dex increased the relative expression of splice variants exon 4 (+) and exon2 (-), which in contrast was decreased with PAN-induced injury. Although RNASeq data analysis was unable to detect these variants, in-depth RT-PCR analyses revealed that both exon 4 (+) and (-) variants were expressed in the circulating leukocytes of SSNS and SRNS patients, while exon 2 (+) forms were only minimally expressed. SRNS patients had increased levels of almost all APOL1 variants [except exon 2 (+)] compared to SSNS patient prior to GC treatment. GC treatment induced expression of most of the recognized splice variants increased in SSNS, but decreased them to varying degrees in SRNS.

Conclusion

Cultured podocytes and circulating human leukocytes express distinct APOL1 splice variant patterns and differential splicing of APOL1 is associated with NS and in response to GC therapy.