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Abstract: TH-PO063

Brevin R-SNARES Provide a Cellular Address to Localize APOL1 to Podocyte Endosomal Compartments

Session Information

Category: Glomerular

  • 1002 Glomerular: Basic/Experimental Pathology

Authors

  • Madhavan, Sethu M., MetroHealth Medical Center, Cleveland, Ohio, United States
  • O'Toole, John F., Case Western Reserve University, Cleveland, Ohio, United States
  • Konieczkowski, Martha, Case Western Reserve University, Cleveland, Ohio, United States
  • Bruggeman, Leslie A., Case Western Reserve University, Cleveland, Ohio, United States
  • Sedor, John R., Case Western Reserve University, Cleveland, Ohio, United States
Background

Genetic variants in APOL1 (G1 and G2) associate with non-diabetic kidney diseases in individuals with African ancestry. Kidney-expressed, but not circulating APOL1 variants, confer risk for CKD. Most subjects with high risk APOL1 genotypes do not develop kidney disease, suggesting a second hit is necessary. Sites of APOL1 synthesis in kidney, its subcellular localization and the identity of its cognate binding partners as well as mechanisms by which variant APOL1s promote nephropathy remain unclear.

Methods

In situ hybridization (ISH) was performed in normal human kidney tissue to detect APOL1 transcripts. APOL1 subcellular localization was examined by immunoelectron (IEM) and confocal immunofluorescence microscopy. Co-immunoprecipitation (Co-IP) experiments to study APOL1-SNARE protein interaction was performed in 293T cells ectopically expressing tagged proteins. Bacterially expressed proteins purified by metal affinity and size-exclusion chromatography were used to study protein interaction by surface plasmon resonance (SPR).

Results

ISH demonstrated that APOL1 is synthesized in podocytes of human kidney and less so in tubules. In kidneys of mice transgenic for APOL1 and human nephrectomy samples, IEM localized the protein to double membrane vesicles and endocytic compartments in podocytes. APOL1 did not localize to plasma membrane in podocytes. By immunofluorescence, podocyte APOL1 did not localize with either a mitochondrial marker, beta-subunit of ATP synthase, or a lysosomal marker, LAMP1. APOL1 interacted with the R-SNAREs, VAMP8 and VAMP1 in Co-IPs. APOL1 colocalized with VAMP8 in podocytes of normal human kidney and Co-IP and SPR experiments confirmed that APOL1-G1 and -G2 interact with VAMP8 with lower affinity than -G0.

Conclusion

Reference APOL1 is expressed in the podocyte, localizes with vesicular structures and directly interacts with VAMP8, consistent with regulation of podocyte vesicular trafficking. APOL1 variants attenuate interaction with VAMP8. We propose that reference APOL1, by interacting with VAMP8 or other R-SNAREs, identifies vesicles containing cargo capable of mediating cellular damage, and activates a cellular response, which mitigates the pathogenic potential of these cargos. Variant APOL1 proteins fail to do so, disrupting vesicular trafficking and permitting kidney disease progression.

Funding

  • NIDDK Support