Dr Monica Suet Ying Ng1,2,3,4,5, KURT TK GIULIANI1,3,4, XIANGJU WANG1,4, ANDREW J KASSIANOS1,3,4, HELEN G HEALY1,3,4
1Kidney Health Service, Royal Brisbane and Women’s Hospital, Brisbane, Australia, 2Institute of Molecular Biosciences, University of Queensland, Brisbane, Australia, 3Faculty of Medicine, University Of Queensland, Brisbane, Australia, 4Conjoint Internal Medicine Laboratory, Chemical Pathology, Pathology Queensland, Brisbane, Australia, 5Nephrology Department, Princess Alexandra Hospital, Brisbane, Australia
Aim
To develop a workflow for purifying and profiling single urinary extracellular vesicles (uEVs) using spectral flow cytometry.
Background
uEVs contain molecules which participate in paracrine communication between tubules, immune function and fibrotic responses. Previous analyses of uEVs primarily examined bulk populations using labour-intensive methods (i.e. proteomics and transcriptomics). Here, we present a novel, high-throughput flow cytometric method of profiling single uEVs.
Methods
50ml urine from four healthy adults were centrifuged at 650xg for 10mins to remove cells. Supernatants were treated with protease inhibitors followed by centrifugation at 2000xg for 20mins to remove cellular debris. uEVs were concentrated to 500µl using serial centrifugation at 3500xg for up to 40mins in an Amicon Centrifugal Filter unit, followed by centrifugation at 20,000xg for 30mins. Flow cytometry quantitation was completed using 400nm beads, with antibodies against markers of nephron source: podocin (podocyte), CD45 (leukocyte), CD31 (vascular), CD10 (tubular), CD13 (tubular) and MUC1 (tubular). FCMpass was used to calculate uEV size.
Results
Mean uEV concentration was 4.86 x 10⁵ particles/µl. uEVs ranged in size from 250nm to 770nm, with mean diameter of 354nm. uEVs stained positive for proximal tubular markers CD10 (mean: 3.57 x 10⁴ particles/μl) and CD13 (mean: 4.82 x 10⁴ particles/μl) and distal tubular marker MUC1 (mean: 1.05 x 10⁵ particles/μl). Comparatively, there were minimal uEVs positive for podocin (mean: 2.60 x 10³ particles/μl), CD45 (mean: 1.99 x 10² particles/μl) and CD31 (mean: 1.96 x 10² particles/μl).
Conclusions
uEVs from healthy individuals are predominantly from the tubular compartment, with minimal uEVs from podocyte, leukocyte and vascular compartments. We aim to continue developing this novel diagnostic platform for translation to kidney disease patients.
Biography:
Monica Ng is a renal registrar at Princess Alexandra Hospital and, post-doctoral researcher at Conjoint Internal Medicine Laboratory, Royal Brisbane and Women’s Hospital and Institute of Molecular Biosciences. Her research interests include registry data analysis and developing molecular workflows for kidney disease biomarker discovery. Currently, this involves optimising spatial transcriptomics and flow cytometry protocols for biosample analysis.