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. Author manuscript; available in PMC: 2018 May 9.
Published in final edited form as: Nat Rev Nephrol. 2017 Oct 30;13(12):731–749. doi: 10.1038/nrneph.2017.148

Table 3.

Proteomic analysis of extracellular vesicles in human urine

Study Sample origin Sampling method Isolation* (force, time and temperature) uEV proteome analysis
First step Second step; third step
Thongboonkerd et al. (2002)18 Healthy Spot urine UC* (2 h, 4 °C) NA; NA Discovery
Pisitkun et al. (2004)19 Healthy Spot urine UC* (1 h, 4 °C) NA; NA Discovery
Zhou et al. (2006)121 Healthy Spot urine UC* (1 h, 4 °C) NA; NA Discovery
Zhou et al. (2006)122 AKI with sepsis, AKI without sepsis Spot urine UC* (1 h, 4 °C) 1.3 M DTT then UC (1 h, 4 °C); NA Discovery
Cheruvanky et al. (2007)83 Healthy, FSGS Spot urine

  • P1: NF (Vivaspin 500, Vivaspin 4, or Vivaspin 20 concentrators)

  • P2: UC (1 h, 4 °C)

 NA; NA Targeted immunoblot
Zhou et al. (2008)123 Healthy, AKI, FSGS Spot urine Timed UC* (1 h, 4 °C) 200 mg/ml DTT then UC (1 h, 4 °C); NA Targeted immunoblot
Smalley et al. (2008)112 Healthy, bladder cancer Random urine sample UC (1 h) NA; NA Discovery
Gonzales et al. (2009)71 Healthy Spot urine UC (1 h, 25 °C) 1.3 M DTT then UC (1 h, 25 °C); NA Discovery
Hogan et al. (2009)76 Healthy, ADPKD Spot urine (1st morning void) UC (150,000 g, 1 h, 4 °C) Sucrose/D2O density gradient UC (24 h, 4 °C); UC (150,000 g, 1 h, 4 °C) for each of 14 sucrose/D2O fractions Targeted immunoblot
Gonzales et al. (2009)20 Healthy, Bartter syndrome type 1 Spot urine UC (1 h, 25 °C) 1.3 M DTT then UC (1 h, 25 °C); NA Discovery, phosphoproteomics
Merchant et al. (2010)72 Healthy Spot urine

  • P1: MF using 0.1 μm diafiltration membranes (RT)

  • P2: UC (2 h, 4 °C)

 NA; NA Discovery
Rood et al. (2010)73 Healthy, iMN and iFSGS Spot urine

  • P1: UC (2 h, 4 °C)

  • P2: UC (2 hr, 4 °C)

  • P1: NA; NA

  • P2: SEC-HPLC; UC (2 h, 4 °C); NA

 Discovery
Moon et al. (2011)114 Healthy, IgA nephropathy, thin basement membrane nephropathy Spot urine (mid-morning void) UC* (1 h, 4 °C) 60 mg/ml DTT then UC (10 min, 60 °C), then sucrose density gradient UC (270,000 g, 16 h, 4 °C); UC (200,000 g, 1 h) of sucrose fractions Discovery
Stamer et al. (2011)113 Healthy Random urine sample UC (100,000 g, 1 h, 4 °C) NA; NA Discovery
Wang et al. (2012)124 Healthy Spot urine UC (1 h, 25 °C) 1.3 M DTT then UC (1 h, 25 °C); NA Discovery
Raj et al. (2012)115 Healthy Spot urine (2nd morning void) UC (1 h, 4 °C) to isolate crude uEV preparation

  • P1: Underlay pellet with 1M sucrose/D2O density gradient and UC (110,000 g, 3 h, 4 °C)

  • P2: Underlay pellet between two sucrose layers (1 M sucrose/PBS/D2O and 2M sucrose/PBS/D2O) and UC (110,000 g, 3 h, 4 °C)

  • Third step for P1 + P2: UC (110,000 g, 90 min, 4 °C), sucrose layer diluted to recover uEVs

 Discovery
Fraser et al. (2013)117 Healthy, unknown Random urine sample UC (unknown g, time, temperature) NA; NA Discovery
Zhou et al. (2008)123 Healthy, FSGS, SSNS Spot urine UC* (1 h, 4 °C) NA; NA Targeted immunoblot
Hogan et al. (2013)77 Membranous GN Spot urine UC (150,000 g, 1 h, 4 °C) Sucrose/D2O density gradient UC (24 h, 4 °C); NA Discovery
Prunotto et al. (2013)79 Healthy Spot urine (1st morning void)

  • Centrifugation (3,500 g, 30 min) and NF (10,000 NMWCO membrane)

  • Unknown final volume

 Immunoaffinity enrichment using immobilized anti-CR1 antibody; NA Targeted immunoblot or immunfluorescence
Gerlach et al. (2013)125 Healthy, ADPKD Spot urine (1st morning void)

  • P1: UC (110,000 g, 2.5 h, 17 °C)

  • P2: UF (Vivaspin 20 with 100 kDa MWCO membranes (20 °C)

  • P3: UC (150,000 g, 1 h, unknown temperature)

  • P1: UF pellet recovery with Vivaspin concentrator (100 kDa MWCO membranes); NA

  • P2: NA; NA

  • P3: UC (24 h) separation of pellet on 5–30% sucrose/D2O gradient; NA

 Flow cytometry and lectin microarrays
Principe et al. (2013)116 Healthy, low grade, organ-confined, Gleason 6 prostate cancer Spot urine UC* (100,000 g, 5 h) NA; NA Discovery
Zubiri et al. (2014)84 Healthy, DN with CKD stage 3–5 Spot urine

  • P1: UC (110,000 g, 2.5 h, 17 °C)

  • P2: UF (Vivaspin 20 with 100 kDa MWCO membranes (20 °C))

  • P3: UC (150,000 g spin, 1 h, unknown temperature)

 NA; NA Discovery
Hogan et al. (2014)77 ADPKD Spot urine (1st morning void) Sucrose/D2O density gradient UC (1 h, 4 °C) NA; NA Discovery
Corbetta et al. (2014)126 Gitelman syndrome, Bartter syndrome Spot urine (2nd morning void) UC (1 h, 4 °C) NA; NA Targeted immunoblot
Hiemstra et al. (2014)74 Healthy Spot urine Centrifugation (17,000 g, 20 min, 0.22 μm sterile filtration) UC (234,000g, 135 min, 4 °C); NA Discovery
Esteva-Font et al. (2014)127 Kidney transplant with or without ciclosporin A treatment Timed urine collection (24 h) UC (2 h, 4 °C) NA; NA Targeted immunoblot
Saraswat et al. (2014)128 Healthy Spot urine UC (2 h, room temperature) NA; NA Discovery
Kosanovic & Jankovic (2014)129 Healthy Spot urine (1st morning void)

  • P1: salt-based precipitation of urinary mucoproteins

  • P2: no salt-based precipitation of urinary mucoproteins

  • P1: UC (100,000 g, 2 h, 20 °C); NA

  • P2: UC (100,000 g, 2 h, 20 °C); NA

 Lectin-binding assays, SEM, TEM
Bourderioux et al. (2015)119 Healthy, cystinuria Spot urine (1st morning void) UC (1 h, 20 °C) 200 mg/ml DTT then UC (1 h, room temperature); NA Discovery
Hogan et al. (2015)118 ADPKD–PKD1 Spot urine (1st morning void) Sucrose/D2O density gradient UC (1 h, 4 °C) NA; NA Discovery, targeted immunoblot
Rood et al. (2015)120 Healthy, iMN, iFSGS Spot urine (2nd morning void) UC* (2 h, 4 °C) SEC-HPLC (RT); UC (2 h, 4 °C) Discovery, targeted immunoblot
Salih et al. (2016)81 Healthy, ADPKD, non-ADPKD CKD Spot urine (2nd morning void) UC* (200,000 g, 2 h, 4 °C) NA; NA Discovery, targeted immunoblot
Turco et al. (2016)130 Before and after kidney transplant Spot urine Low-speed centrifugation to clarify cells and debris NA; NA Fluorescence-activated cell sorting
Panich et al. (2017)131 AKI Spot urine UC (1 h, temperature unknown) NA; NA Targeted immunoblot
Wolley et al. (2017)132 Primary aldosteronism Spot urine (2nd morning void) UC (1 h, 20 °C) 200 mg/ml DTT then UC (1 h, 4 °C); NA Targeted immunoblot
Lytvyn et al. (2017)133 Healthy, normotensive type 1 diabetics Biorepository spot urine sample Differential centrifugation (20,000 g) NA; NA Fluorescence-activated cell sorting

ADPKD, autosomal dominant polycystic kidney disease; AKI, acute kidney injury; CKD, chronic kidney disease; CR1, complement receptor type 1; DN, diabetic nephropathy; DTT, dithiothreitol; FSGS, focal segmental glomerulosclerosis; GN, glomerulonephritis; iFSGS, idiopathic FSGS; iMN, idiopathic membranous nephropathy; LN, lupus nephritis; MF, microfiltration; MWCO, molecular weight cut-off; NA, not applicable; NF, nanofiltration; P1, procedure 1; PBS, phosphate-buffered saline; PKD1, polycystin 1; RT, room temperature; SEC–HPLC, size-exclusion chromatography–high performance liquid chromatography; SEM, scanning electron microscopy; SSNS, steroid-sensitive nephrotic syndrome; TEM, transmission electron microscopy; UC, ultracentrifugation; uEV, urinary extracellular vesicle; UF, ultrafiltration.

*

Urine precleared of large particulate or flocculate material, usually by two sequential centrifugation steps, a short (5–30 min), low speed (1,500–3,000 g) step and a longer (20–60min), higher speed (14,000–17,500 g) step.