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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV110064	2/2	Mus musculus	NAY	(d)(U)C DG	Street JM	2011	33%
Study summary Full title Exosomal transmission of functional aquaporin 2 in kidney cortical collecting duct cells All authors Street JM, Birkhoff W, Menzies RI, Webb DJ, Bailey MA, Dear JW Journal J Physiol Abstract Exosomes are vesicles released following fusion of endosomes with the plasma membrane. Urine contain (show more...)Exosomes are vesicles released following fusion of endosomes with the plasma membrane. Urine contains exosomes that are released from the entire length of the nephron and change in composition with kidney disease. Exosomes can shuttle information between non-renal cells via transfer of protein and RNA. In this study murine kidney collecting duct (mCCDC11) cells were used to demonstrate that exosomes can act as a signalling mechanism between cells. First, the release of exosomes by mCCDC11 cells was confirmed by multiple approaches. Following isopynic centrifugation, exosomal proteins flotillin-1 and TSG101 were identified in fractions consistent with exosomes. Electron microscopy demonstrated structures consistent in size and shape with exosomes. Exposure of mCCDC11 cells to the synthetic vasopressin analogue, desmopressin, did not affect exosomal flotillin-1 or TSG101 but increased aquaporin 2 (AQP2) in a dose- and time-dependent manner that was highly correlated with cellular AQP2 (exosomal AQP2 vs. cellular AQP2, Pearson correlation coefficient r = 0.93). To test whether the ratio of exosomal AQP2/flotillin-1 is under physiological control in vivo, rats were treated with desmopressin. The ratio of AQP2/flotillin-1 in the urinary exosome was significantly increased. Inter-cellular signalling by exosomes was demonstrated: exosomes from desmopressin-treated cells stimulated both AQP2 expression and water transport in untreated mCCDc11 cells (water flow across cells: control exosome treatment 52.8 ± 11 ?l cm(-2); AQP2-containing exosomes 77.4 ± 4 ?l cm(-2), P = 0.05, n = 4). In summary, the amount of AQP2 in exosomes released from collecting duct cells is physiologically regulated and exosomal AQP2 closely reflects cellular expression. Exosomes can transfer functional AQP2 between cells and this represents a novel physiological mechanism for cell-to-cell communication within the kidney. (hide) EV-METRIC 33% (75th 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 Cell culture supernatant Sample origin NAY Focus vesicles exosomes 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 DG Protein markers EV: TSG101/ Flotilin1/ AQP2 non-EV: Proteomics no EV density (g/ml) 1.1-1.15 Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Density gradient Only used for validation of main results Yes Lowest density fraction 0.25 Highest density fraction 2 Orientation Top-down Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins Flotilin1/ TSG101/ AQP2 ELISA Antibody details provided? No Detected EV-associated proteins AQP2 Characterization: Particle analysis EM EM-type transmission EM Image type Close-up

	EV110064	1/2	Rattus norvegicus/rattus	Urine	(d)(U)C	Street JM	2011	0%
Study summary Full title Exosomal transmission of functional aquaporin 2 in kidney cortical collecting duct cells All authors Street JM, Birkhoff W, Menzies RI, Webb DJ, Bailey MA, Dear JW Journal J Physiol Abstract Exosomes are vesicles released following fusion of endosomes with the plasma membrane. Urine contain (show more...)Exosomes are vesicles released following fusion of endosomes with the plasma membrane. Urine contains exosomes that are released from the entire length of the nephron and change in composition with kidney disease. Exosomes can shuttle information between non-renal cells via transfer of protein and RNA. In this study murine kidney collecting duct (mCCDC11) cells were used to demonstrate that exosomes can act as a signalling mechanism between cells. First, the release of exosomes by mCCDC11 cells was confirmed by multiple approaches. Following isopynic centrifugation, exosomal proteins flotillin-1 and TSG101 were identified in fractions consistent with exosomes. Electron microscopy demonstrated structures consistent in size and shape with exosomes. Exposure of mCCDC11 cells to the synthetic vasopressin analogue, desmopressin, did not affect exosomal flotillin-1 or TSG101 but increased aquaporin 2 (AQP2) in a dose- and time-dependent manner that was highly correlated with cellular AQP2 (exosomal AQP2 vs. cellular AQP2, Pearson correlation coefficient r = 0.93). To test whether the ratio of exosomal AQP2/flotillin-1 is under physiological control in vivo, rats were treated with desmopressin. The ratio of AQP2/flotillin-1 in the urinary exosome was significantly increased. Inter-cellular signalling by exosomes was demonstrated: exosomes from desmopressin-treated cells stimulated both AQP2 expression and water transport in untreated mCCDc11 cells (water flow across cells: control exosome treatment 52.8 ± 11 ?l cm(-2); AQP2-containing exosomes 77.4 ± 4 ?l cm(-2), P = 0.05, n = 4). In summary, the amount of AQP2 in exosomes released from collecting duct cells is physiologically regulated and exosomal AQP2 closely reflects cellular expression. Exosomes can transfer functional AQP2 between cells and this represents a novel physiological mechanism for cell-to-cell communication within the kidney. (hide) EV-METRIC 0% (median: 22% 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 NAY Focus vesicles exosomes 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: Flotilin1/ AQP2 non-EV: Proteomics no Show all info Study aim Function Sample Species Rattus norvegicus/rattus Sample Type Urine Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins Flotilin1/ AQP2 ELISA Antibody details provided? No Detected EV-associated proteins AQP2 Characterization: Particle analysis None

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EV-TRACK ID	EV110064
species	Mus musculus	Rattus norvegicus/rattus
sample type	Cell culture	Urine
cell type	NAY	NA
medium	EV Depleted
condition	NAY	NAY
separation protocol	(d)(U)C DG	(d)(U)C
Exp. nr.	2	1
EV-METRIC %	33	0