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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV190003	1/1	Homo sapiens	Urine	(d)(U)C	Sabaratnam R	2019	50%
Study summary Full title In human nephrectomy specimens, the kidney level of tubular transport proteins does not correlate with their abundance in urinary extracellular vesicles. All authors Sabaratnam R, Geertsen L, Skjødt K, Hojlund K, Dimke H, Lund L, Svenningsen P. Journal Am J Physiol Renal Physiol Abstract Human urinary extracellular vesicles (uEVs) contain proteins from all nephron segments. An assumptio (show more...)Human urinary extracellular vesicles (uEVs) contain proteins from all nephron segments. An assumption for years has been that uEVs might provide a non-invasive liquid biopsy that reflect physiological regulation of transporter protein expression in human. We hypothesized that protein abundance in human kidney tissue and uEV are directly related and tested this in paired collections of nephrectomy tissue and urine sample from 12 patients. Kidney tissue was fractioned into total kidney protein, crude membrane (plasma membrane and large intracellular vesicles) and intracellular vesicle enriched fractions, as well as sections for immunolabelling. uEVs were isolated from spot urine samples. Antibodies were used to quantify 6 segment-specific proteins (proximal tubular expressed Na/Phosphate cotransporter NaPi-2a, thick ascending limb expressed Tamm-Horsfall protein and renal-outer-medullary K+channel ROMK, distal convoluted tubular expressed NaCl cotransporter NCC, intercalated cell expressed proton-pump subunit ATP6V1G3 and principal cell expressed aquaporin 2 (AQP2)) and 3 uEV markers (exosomal CD63, microvesicle marker VAMP3 and β-actin) in each fractions. By western blotting and immunofluorescence labelling, we found significant positive correlations between abundance of CD63, NCC, AQP2 and ATP6V1G3, respectively, within the different kidney-derived fractions. We detected all 9 proteins in uEVs, but their level did not correlate with kidney tissue protein abundance. The uEV protein levels showed higher inter-patient variability than the kidney-derived fractions, indicating that factors, besides kidney protein abundance, contribute to the uEV protein level. Our data suggest that, in a random sample of nephrectomy patients, uEV protein level is not a predictor of kidney protein abundance. (hide) EV-METRIC 50% (86th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Urine Sample origin pre-nephrectomy Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: SLC34A1/ VAMP3/ CD63/ beta-actin/ ROMK non-EV: Tamm-Horsfall protein Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Urine Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed No Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ VAMP3/ beta-actin/ SLC34A1/ ROMK Detected contaminants Tamm-Horsfall protein Characterization: Lipid analysis No

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EV-TRACK ID	EV190003
species	Homo sapiens
sample type	Urine
condition	pre-nephrectomy
separation protocol	(d)(U)C
Exp. nr.	1
EV-METRIC %	50