Abstract: SA-OR052

Immunolocalization of Apolipoprotein L1 with Specific Antibodies

Session Information

Category: Chronic Kidney Disease (Non-Dialysis)

  • 301 CKD: Risk Factors for Incidence and Progression

Authors

  • Scales, Suzie J., Genentech, Inc., South San Francisco, California, United States
  • Brezski, Randall J., Genentech, Inc., South San Francisco, California, United States
  • Foreman, Oded, Genentech, Inc., South San Francisco, California, United States
  • Peterson, Andrew S., Genentech, Inc., South San Francisco, California, United States
  • Gupta, Nidhi, Genentech, Inc., South San Francisco, California, United States
  • Hotzel, Kathy J., Genentech, Inc., South San Francisco, California, United States
  • Pierce, Andrew A, Genentech, Inc., South San Francisco, California, United States
  • Koukos, Georgios, Genentech, Inc., South San Francisco, California, United States
  • Moran, Paul, Genentech, Inc., South San Francisco, California, United States
  • Lipari, Michael Terry, Genentech, Inc., South San Francisco, California, United States
  • Wang, Xinhua, Genentech, Inc., South San Francisco, California, United States
  • Kirchhofer, Daniel, Genentech, Inc., South San Francisco, California, United States
Background

Human Apolipoprotein (ApoL1) is the only secreted member of the apolipoprotein (ApoL) family (ApoL1-ApoL6), with 53% amino acid identity (63% similarity) to its closest relative, ApoL2. ApoL1 has been widely studied because of its protective role against Trypanosoma infections and the association of its variants G1 and G2 with chronic kidney disease. To better understand the role of ApoL1 in kidney disease, it is important to identify its localization within the kidney. By immunohistochemistry, ApoL1 is reportedly strongest in proximal tubules but only just detectable in podocytes, the susceptible kidney cell type in ApoL1 nephropathies. There are conflicting reports of immunofluorescent ApoL1 subcellular localization using different commercially available antibodies: in the endoplasmic reticulum, on the plasma membrane, endosomes and even mitochondria. However, these antibodies have not been well characterized for cross-reactivity with other members of the ApoL family, giving rise to misleading results.

Methods

We generated monoclonal antibodies to ApoL1 and characterized their cross-reactivity with all members of the ApoL family in parallel with the commercially available polyclonal antibodies. ApoL family expression in podocytes was determined by Taqman analysis. ApoL1-specific antibodies were used to determine the true localization of ApoL1 in kidney and liver by immunohistochemistry, and in cultured podocytes by dual immunofluorescence labeling.

Results

All the commercially available antibodies previously published for localization studies cross-react with ApoL2, as did half of our monoclonals. Human podocytes express ApoL1, 2 and 6 mRNAs. In tissues, ApoL1 was specifically and robustly detected in serum, liver hepatocytes and kidney podocytes, but not in most proximal tubules. In cells, overexpressed ApoL1 and ApoL2 were found on opposite faces of the endoplasmic reticulum. Endogenous ApoL1 was also detected inside the endoplasmic reticulum (but not endosomes or mitochondria) of wild type podocytes and was enhanced by gamma interferon. The specificity of the staining was proven by its absence from ApoL1 knockout (CRISPR) podocytes.

Conclusion

Non-ApoL2 cross-reactive antibodies are essential for determining the true localization of endogenous ApoL1.

Funding

  • Commercial Support