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Abstract: SA-PO988

Microvesicular Passage of APOL1

Session Information

Category: Glomerular Diseases

  • 1403 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
  • Skorecki, Karl, Bar-Ilan University, Ramat Gan, Tel Aviv, Israel
  • Singhal, Pravin C., Northwell Health Feinstein Institutes for Medical Research, Manhasset, New York, United States
Background

Podocyte-APOL1 (pAPOL1) carry a signal peptide, but its secretion and paracrine/endocrine role are controversial. We propose that APOL1 secretion in podocytes use microvesicles (MVs) to get out of cells rather than direct secretion to the medium.

Methods

APOL1 (wild-type, G0) and its risk variants (G1 or G2) were over-expressed in human podocytes. After 48hr of incubation, the culture medium was collected and processed for MVs isolation by ultracentrifugation and MVs isolation kit. Isolated MVs were characterized by HSP70, CD81, and CD63 and the absence of Calnexin by Western Blot (WB) and FACS. The size was measured using the Nanosite system and Scanning Electron Microscopy (SEM) . APOL1 was detected in the culture medium and lysed MVs using ELISA & WB. The isolated MVs were further incubated with non-APOL1 expressing Human Embryonic Kidney (HEK) cells, and after 48hr, HEK cell lysate was analyzed for APOL1 presence by WB.

Results

Nanosite & SEM measured the size of isolated MVs 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) as shown in the figure.

Conclusion

This preliminary study shows that podocyte and HEK cells secrete APOL1 through the MVs pathway.

MVs size was measured using Nanosite system (A&B), and SEM (C ). The expression of MVs protein marker and APOL1 are displayed (D). ELISA of APOL1 media was performed to detect MVs (E). ***p<0.001, #p<0.001 (*G0 Vs. V, G1 or G2; #media vs. MVs)

Funding

  • NIDDK Support