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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV150104	1/4	Homo sapiens	Urine	DC (d)(U)C	Pocsfalvi G	2015	44%
Study summary Full title Urinary extracellular vesicles as reservoirs of altered proteins during the pathogenesis of polycystic kidney disease. All authors Pocsfalvi G, Raj DA, Fiume I, Vilasi A, Trepiccione F, Capasso G. Journal Proteomics Clin Appl Abstract PURPOSE: Recent findings indicate that urinary extracellular vesicles (EVs) might reflect the pathop (show more...)PURPOSE: Recent findings indicate that urinary extracellular vesicles (EVs) might reflect the pathophysiological state of urinary system; and that EVs-induced ciliary signaling is a possible mechanism of intercellular communication within the tract. Here, we aimed to analyze the protein expression of urinary EVs during autosomal dominant polycystic kidney disease (ADPKD). EXPERIMENTAL DESIGN: EVs were isolated from pooled urine samples of healthy control and ADPKD patients at two different stages of the disease and under tolvaptan treatment using the double-cushion ultracentrifugation method. Proteins were identified and quantified by iTRAQ and multidimensional protein identification technology (MudPIT)-based quantitative proteomics. RESULTS: Quantitative analyses identified 83 differentially expressed EV proteins. Many of these have apical membrane origin and are involved in signal transduction pathways of primary cilia, Ca(2+) -activated signaling, cell-cycle regulation, and cell differentiation. CONCLUSIONS AND CLINICAL RELEVANCE: The reduced AQP-2 and the increased APO-A1 levels observed in all ADPKD-affected groups may reflects the impaired renal concentrating capability of these patients and correlated with estimated glomerular filtration rate decline. The levels of some upregulated proteins involved in Ca(2+) -activated signaling declined upon tolvaptan treatment. The results obtained suggest that the quantitative proteomics of urinary EVs might be useful to monitor proteins difficult to access noninvasively, and thus advance our understanding of urinary tract physiology and pathology. (hide) EV-METRIC 44% (80th 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 Control condition 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 DC (d)(U)C Protein markers EV: TSG101/ Alix/ AQP2/ PKD22/ NHE3/ CD9/ PKD11 non-EV: / Uromodulin/ Albumin Proteomics yes Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Urine Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Pelleting: rotor type Not specified Pelleting: speed (g) 200000 Density cushion Density medium Sucrose Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Detected EV-associated proteins CD9/ NHE3/ AQP2/ PKD11/ PKD22/ TSG101/ Alix Detected contaminants Not detected contaminants Albumin/ Uromodulin Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 95 EM EM-type Transmission-EM Image type Wide-field

	EV150104	2/4	Homo sapiens	Urine	DC (d)(U)C	Pocsfalvi G	2015	33%
Study summary Full title Urinary extracellular vesicles as reservoirs of altered proteins during the pathogenesis of polycystic kidney disease. All authors Pocsfalvi G, Raj DA, Fiume I, Vilasi A, Trepiccione F, Capasso G. Journal Proteomics Clin Appl Abstract PURPOSE: Recent findings indicate that urinary extracellular vesicles (EVs) might reflect the pathop (show more...)PURPOSE: Recent findings indicate that urinary extracellular vesicles (EVs) might reflect the pathophysiological state of urinary system; and that EVs-induced ciliary signaling is a possible mechanism of intercellular communication within the tract. Here, we aimed to analyze the protein expression of urinary EVs during autosomal dominant polycystic kidney disease (ADPKD). EXPERIMENTAL DESIGN: EVs were isolated from pooled urine samples of healthy control and ADPKD patients at two different stages of the disease and under tolvaptan treatment using the double-cushion ultracentrifugation method. Proteins were identified and quantified by iTRAQ and multidimensional protein identification technology (MudPIT)-based quantitative proteomics. RESULTS: Quantitative analyses identified 83 differentially expressed EV proteins. Many of these have apical membrane origin and are involved in signal transduction pathways of primary cilia, Ca(2+) -activated signaling, cell-cycle regulation, and cell differentiation. CONCLUSIONS AND CLINICAL RELEVANCE: The reduced AQP-2 and the increased APO-A1 levels observed in all ADPKD-affected groups may reflects the impaired renal concentrating capability of these patients and correlated with estimated glomerular filtration rate decline. The levels of some upregulated proteins involved in Ca(2+) -activated signaling declined upon tolvaptan treatment. The results obtained suggest that the quantitative proteomics of urinary EVs might be useful to monitor proteins difficult to access noninvasively, and thus advance our understanding of urinary tract physiology and pathology. (hide) EV-METRIC 33% (64th 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 autosomal dominant polycystic kidney disease (late stage) 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 DC (d)(U)C Protein markers EV: TSG101/ AQP2/ PKD2/ PKD1/ Alix/ CD9/ NHE3 non-EV: / Uromodulin/ Albumin Proteomics yes EV density (g/ml) Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Urine Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Pelleting: rotor type Not specified Pelleting: speed (g) 200000 Density gradient Only used for validation of main results Yes Type Number of initial discontinuous layers Lowest density fraction Highest density fraction Total gradient volume, incl. sample (mL) Sample volume (mL) Orientation Rotor type Speed (g) Duration (min) Fraction volume (mL) Fraction processing Pelleting: volume per fraction Pelleting: duration (min) Pelleting: rotor type Pelleting: speed (g) Pelleting-wash: volume per pellet (mL) Pelleting-wash: duration (min) Pelleting-wash: speed (g) Density cushion Density medium Sucrose Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Detected EV-associated proteins Alix/ NHE3/ AQP2/ CD9/ TSG101 Not detected EV-associated proteins PKD1/ PKD2 Detected contaminants Not detected contaminants Albumin/ Uromodulin Proteomics database No Characterization: Lipid analysis No EM EM-type EV concentration

	EV150104	3/4	Homo sapiens	Urine	DC (d)(U)C	Pocsfalvi G	2015	33%
Study summary Full title Urinary extracellular vesicles as reservoirs of altered proteins during the pathogenesis of polycystic kidney disease. All authors Pocsfalvi G, Raj DA, Fiume I, Vilasi A, Trepiccione F, Capasso G. Journal Proteomics Clin Appl Abstract PURPOSE: Recent findings indicate that urinary extracellular vesicles (EVs) might reflect the pathop (show more...)PURPOSE: Recent findings indicate that urinary extracellular vesicles (EVs) might reflect the pathophysiological state of urinary system; and that EVs-induced ciliary signaling is a possible mechanism of intercellular communication within the tract. Here, we aimed to analyze the protein expression of urinary EVs during autosomal dominant polycystic kidney disease (ADPKD). EXPERIMENTAL DESIGN: EVs were isolated from pooled urine samples of healthy control and ADPKD patients at two different stages of the disease and under tolvaptan treatment using the double-cushion ultracentrifugation method. Proteins were identified and quantified by iTRAQ and multidimensional protein identification technology (MudPIT)-based quantitative proteomics. RESULTS: Quantitative analyses identified 83 differentially expressed EV proteins. Many of these have apical membrane origin and are involved in signal transduction pathways of primary cilia, Ca(2+) -activated signaling, cell-cycle regulation, and cell differentiation. CONCLUSIONS AND CLINICAL RELEVANCE: The reduced AQP-2 and the increased APO-A1 levels observed in all ADPKD-affected groups may reflects the impaired renal concentrating capability of these patients and correlated with estimated glomerular filtration rate decline. The levels of some upregulated proteins involved in Ca(2+) -activated signaling declined upon tolvaptan treatment. The results obtained suggest that the quantitative proteomics of urinary EVs might be useful to monitor proteins difficult to access noninvasively, and thus advance our understanding of urinary tract physiology and pathology. (hide) EV-METRIC 33% (64th 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 autosomal dominant polycystic kidney disease (early stage) 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 DC (d)(U)C Protein markers EV: TSG101/ AQP2/ PKD2/ PKD1/ Alix/ CD9/ NHE3 non-EV: / Uromodulin/ Albumin Proteomics yes EV density (g/ml) Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Urine Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Pelleting: rotor type Not specified Pelleting: speed (g) 200000 Density gradient Only used for validation of main results Yes Type Number of initial discontinuous layers Lowest density fraction Highest density fraction Total gradient volume, incl. sample (mL) Sample volume (mL) Orientation Rotor type Speed (g) Duration (min) Fraction volume (mL) Fraction processing Pelleting: volume per fraction Pelleting: duration (min) Pelleting: rotor type Pelleting: speed (g) Pelleting-wash: volume per pellet (mL) Pelleting-wash: duration (min) Pelleting-wash: speed (g) Density cushion Density medium Sucrose Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Detected EV-associated proteins Alix/ NHE3/ AQP2/ CD9/ TSG101 Not detected EV-associated proteins PKD1/ PKD2 Detected contaminants Not detected contaminants Albumin/ Uromodulin Proteomics database No Characterization: Lipid analysis No EM EM-type EV concentration

	EV150104	4/4	Homo sapiens	Urine	DC (d)(U)C	Pocsfalvi G	2015	33%
Study summary Full title Urinary extracellular vesicles as reservoirs of altered proteins during the pathogenesis of polycystic kidney disease. All authors Pocsfalvi G, Raj DA, Fiume I, Vilasi A, Trepiccione F, Capasso G. Journal Proteomics Clin Appl Abstract PURPOSE: Recent findings indicate that urinary extracellular vesicles (EVs) might reflect the pathop (show more...)PURPOSE: Recent findings indicate that urinary extracellular vesicles (EVs) might reflect the pathophysiological state of urinary system; and that EVs-induced ciliary signaling is a possible mechanism of intercellular communication within the tract. Here, we aimed to analyze the protein expression of urinary EVs during autosomal dominant polycystic kidney disease (ADPKD). EXPERIMENTAL DESIGN: EVs were isolated from pooled urine samples of healthy control and ADPKD patients at two different stages of the disease and under tolvaptan treatment using the double-cushion ultracentrifugation method. Proteins were identified and quantified by iTRAQ and multidimensional protein identification technology (MudPIT)-based quantitative proteomics. RESULTS: Quantitative analyses identified 83 differentially expressed EV proteins. Many of these have apical membrane origin and are involved in signal transduction pathways of primary cilia, Ca(2+) -activated signaling, cell-cycle regulation, and cell differentiation. CONCLUSIONS AND CLINICAL RELEVANCE: The reduced AQP-2 and the increased APO-A1 levels observed in all ADPKD-affected groups may reflects the impaired renal concentrating capability of these patients and correlated with estimated glomerular filtration rate decline. The levels of some upregulated proteins involved in Ca(2+) -activated signaling declined upon tolvaptan treatment. The results obtained suggest that the quantitative proteomics of urinary EVs might be useful to monitor proteins difficult to access noninvasively, and thus advance our understanding of urinary tract physiology and pathology. (hide) EV-METRIC 33% (64th 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 autosomal dominant polycystic kidney disease (tolvaptan treatment) 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 DC (d)(U)C Protein markers EV: TSG101/ AQP2/ PKD2/ PKD1/ Alix/ CD9/ NHE3 non-EV: / Uromodulin/ Albumin Proteomics yes EV density (g/ml) Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Urine Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Pelleting: rotor type Not specified Pelleting: speed (g) 200000 Density gradient Only used for validation of main results Yes Type Number of initial discontinuous layers Lowest density fraction Highest density fraction Total gradient volume, incl. sample (mL) Sample volume (mL) Orientation Rotor type Speed (g) Duration (min) Fraction volume (mL) Fraction processing Pelleting: volume per fraction Pelleting: duration (min) Pelleting: rotor type Pelleting: speed (g) Pelleting-wash: volume per pellet (mL) Pelleting-wash: duration (min) Pelleting-wash: speed (g) Density cushion Density medium Sucrose Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Detected EV-associated proteins Alix/ NHE3/ AQP2/ CD9/ TSG101 Not detected EV-associated proteins PKD1/ PKD2 Detected contaminants Not detected contaminants Albumin/ Uromodulin Proteomics database No Characterization: Lipid analysis No EM EM-type EV concentration

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EV-TRACK ID	EV150104
species	Homo sapiens
sample type	Urine
condition	Control condition	autosomal dominant polycystic kidney disease (late stage)	autosomal dominant polycystic kidney disease (early stage)	autosomal dominant polycystic kidney disease (tolvaptan treatment)
separation protocol	DC (d)(U)C	DC (d)(U)C	DC (d)(U)C	DC (d)(U)C
Exp. nr.	1	2	3	4
EV-METRIC %	44	33	33	33