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Abstract: SA-PO731

APOL1 Extracellular Translocation: A Possible Role of Microvesicles

Session Information

Category: Glomerular Diseases

  • 1304 Glomerular Diseases: Podocyte Biology

Authors

  • Johal, Prabhjot Kaur, Post Graduate Institute of Medical Education and Research, Chandigarh, Chandigarh, India
  • Kumar, Vinod, Post Graduate Institute of Medical Education and Research, Chandigarh, Chandigarh, India
  • Malhotra, Ashwani, Northwell Health Feinstein Institutes for Medical Research, Manhasset, New York, United States
  • Singhal, Pravin C., Northwell Health Feinstein Institutes for Medical Research, Manhasset, New York, United States
Background

Podocyte expressed APOL1 (pAPOL1) is not secretory compared to the one expressed by liver cells. However, the plasma membrane integration potential of pAPOL1 is well documented, where G0 has maximum membrane penetration than G1 & G2. Presence of pAPOL1 protein in the culture medium and biological fluids is still unclear. Here, we hypothesized that APOL1 secretion in podocyte cells adopt microvesicles (MVs) secretion pathway rather than direct secretion to the medium

Methods

APOL1 and its risk variants were over-expressed in human embryonic kidney (HEK) cells. After 48hr of incubation, the culture medium was collected and processed for MVs isolation by ultracentrifugation and MVs isolation kit. MVs were also isolated from podocytes expressing APOLG0, G1 & G2. Isolated MVs were characterized for the presence of HSP70, CD81, CD63 and absence of Calnexin by Western Blot (WB) and FACS. Size was measured using Nanosite system and Scanning Electron Microscopy. ELISA was used to detect circulating APOL1, while WB used for MVs APOL1. MVs were further incubated with non APOL1 expressing HEK cells and after 48hr HEK cell lysate was analyzed for APOL1 presence by WB.

Results

Nanosite & SEM measured size of isolated MVs was in between 90-125nm. These MVs stained positive for the expression of CD63, CD81, and HSP70, but were negative for the expression of calnexin. Thereby, confirming that there was no cytosolic contamination. We observed the presence of APOL1 protein only in the lysed MVs. However, the level of APOL1-G0 (intensity: 1.92±0.01) is much higher compared to Vector (0.11±0.01) G1 (0.12±0.01) and G2 (0.17±0.02) Figure1

Conclusion

This preliminary study shows that Podocytes secrete APOL1 through MVs pathway

Funding

  • Government Support – Non-U.S.