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Abstract: SA-PO983

ApoL1 in Podocyte-Derived Urinary Microparticles

Session Information

Category: Glomerular Diseases

  • 1403 Podocyte Biology

Authors

  • Edwards, John C., Saint Louis University, Saint Louis, Missouri, United States
  • Winkler, Rebecca L., Saint Louis University, Saint Louis, Missouri, United States
Background

ApoL1 variants contribute the the high risk of progressive kidney disease in people of African ancestry. Overexpression of ApoL1 in podocytes may drive disease in people with high risk genotypes; ability to assess podocyte ApoL1 expression in vivo could be useful. Microparticles are 100-1000 nm vesicles that represent a sample of plasma membrane of their cells of origin, and hence may reflect the level of expression of specific proteins. Cells lining the nephron shed microparticles into the urine, and may provide a non-invasive way to assess protein expression in vivo.

Methods

The study was approved by the St. Louis University IRB. Urine was collected from 10 people with proteinuric kidney disease (3 membranous GN, 4 with immune complex GN (2 SLE nephritis, 2 infection-related), 2 diabetic nephropathy, and 1 AL amyloidosis) and one control. Microparticle-enriched urine sediment from 1.4 ml of urine was prepared by differential centrifugation, resuspended in buffered saline and incubated with fluorescently labelled probes to define microparticles (Annexin V), to identify podocyte origin (antibody to podocalyxin) and with antibody to ApoL1. Microparticles were analyzed by flow cytometry. Matched negative controls for each probe were used to define signal thresholds. Size gate was determined using polystyrene reference beads. Total particle number was normalized to urine creatinine concentration.

Results

A size gate of 250 to 1000 nm was applied. Annexin V positive population identifies the urinary microparticles (UMPs). Podocalyxin positive UMPs define the podocyte-derived urinary microparticles (PDUMP), and PDUMPs which also stain for ApoL1 are the ApoL1-positive PDUMPs (A+PDUMPs). Urine from the patients had much higher total UMPs, PDUMPs and A+PDUMPs than did control urine. Number of PDUMPS and A+PDUMPs seemed to correlate with cause and activity of kidney disease: number of both PDUMPs and A+PDUMPS were greatest in two patients with active membranous GN. The number of A+PDUMPS, the ratio of PDUMPs to total UMPs, and the ratio of A+PDUMPS to total PDUMPs all correlated weakly with proteinuria.

Conclusion

We have demonstrated the presence of ApoL1 in podocyte derived urinary microparticles. In this small sample, the A+PDUMPs seem to correlate with the type of glomerular disease, with the highest level in patients who would be expected to have direct podocyte injury driving the disease.

Funding

  • NIDDK Support