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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV210305	2/10	Homo sapiens	urine	Lectin affinity capture (Lotus tetragonolobus) PEG precipitation	Isaksson, Gustaf L.	2021	50%
Study summary Full title Proteinuria is accompanied by intratubular complement activation and apical membrane deposition of C3dg and C5b-9 in kidney transplant recipients All authors Gustaf Lissel Isaksson, Marie Bodilsen Nielsen, Gitte Rye Hinrichs, Nicoline Valentina Krogstrup, Rikke Zachar, Heidi Stubmark, Per Svenningsen, Kirsten Madsen, Claus Bistrup, Bente Jespersen, CONTEXT Study Group; Henrik Birn, Yaseelan Palarasah, Boye L Jensen Journal Am J Physiol Renal Physiol Abstract Proteinuria predicts accelerated decline in kidney function in kidney transplant recipients (KTRs). (show more...)Proteinuria predicts accelerated decline in kidney function in kidney transplant recipients (KTRs). We hypothesized that aberrant filtration of complement factors causes intraluminal activation, apical membrane attack on tubular cells and progressive injury. Biobanked samples from two previous studies in albuminuric KTRs were used. Complement activation split products C3c, C3dg and sC5b-9 associated C9 neoantigen were analyzed by ELISA in urine and plasma using neoepitope-specific antibodies. Urinary extracellular vesicles (uEV) were enriched by lectin- and immunoaffinity-isolation and analyzed by immunoblotting. Urine complement excretion increased significantly in KTRs with albumin/creatinine ratio ≥ 300 mg/g compared to < 30 mg/g. Urine C3dg and C9 neoantigen excretion correlated significantly to changes in albumin excretion from 3 to 12 months after transplantation. The fractional excretion of C9 neoantigen was significantly higher than for albumin indicating post-filtration generation. C9 neoantigen was detected in uEVs in six of nine of albuminuric KTRs but was absent in non-albuminuric controls (n = 8). In C9 neoantigen positive KTRs, lectin-affinity enrichment of uEVs from the proximal tubules yielded signal for iC3b, C3dg, C9 neoantigen and SGLT2 but only weakly for AQP2. Co-isolation of podocyte markers and Tamm-Horsfall protein was minimal. Our findings show that albuminuria is associated with aberrant filtration and intratubular activation of complement with deposition of C3 activation split products and C5b-9 associated C9 neoantigen on uEVs from the proximal tubular apical membrane. Intratubular complement activation may contribute to progressive kidney injury in proteinuric kidney grafts. (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 Kidney transplant recipient controls 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 Lectin affinity capture (Lotus tetragonolobus) PEG precipitation Protein markers EV: CD9/ CD63/ TSG101/ AQP2/ C9 neoantigen/ iC3b/C3dg/ CR1/ Podocalyxin/ SGLT2 non-EV: Tamm-Horsfall protein Proteomics no Show all info Study aim Biomarker/New methodological development Sample Species Homo sapiens Sample Type urine Separation Method Other Name other separation method Lectin affinity capture (Lotus tetragonolobus) Other Name other separation method PEG precipitation Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9/ CD63/ TSG101/ C9 neoantigen/ iC3b/C3dg/ SGLT2 Not detected EV-associated proteins AQP2/ CR1/ Podocalyxin Not detected contaminants Tamm-Horsfall protein Characterization: Lipid analysis No

	EV210305	3/10	Homo sapiens	urine	Lectin affinity capture (Dolichus biflorus) PEG precipitation	Isaksson, Gustaf L.	2021	50%
Study summary Full title Proteinuria is accompanied by intratubular complement activation and apical membrane deposition of C3dg and C5b-9 in kidney transplant recipients All authors Gustaf Lissel Isaksson, Marie Bodilsen Nielsen, Gitte Rye Hinrichs, Nicoline Valentina Krogstrup, Rikke Zachar, Heidi Stubmark, Per Svenningsen, Kirsten Madsen, Claus Bistrup, Bente Jespersen, CONTEXT Study Group; Henrik Birn, Yaseelan Palarasah, Boye L Jensen Journal Am J Physiol Renal Physiol Abstract Proteinuria predicts accelerated decline in kidney function in kidney transplant recipients (KTRs). (show more...)Proteinuria predicts accelerated decline in kidney function in kidney transplant recipients (KTRs). We hypothesized that aberrant filtration of complement factors causes intraluminal activation, apical membrane attack on tubular cells and progressive injury. Biobanked samples from two previous studies in albuminuric KTRs were used. Complement activation split products C3c, C3dg and sC5b-9 associated C9 neoantigen were analyzed by ELISA in urine and plasma using neoepitope-specific antibodies. Urinary extracellular vesicles (uEV) were enriched by lectin- and immunoaffinity-isolation and analyzed by immunoblotting. Urine complement excretion increased significantly in KTRs with albumin/creatinine ratio ≥ 300 mg/g compared to < 30 mg/g. Urine C3dg and C9 neoantigen excretion correlated significantly to changes in albumin excretion from 3 to 12 months after transplantation. The fractional excretion of C9 neoantigen was significantly higher than for albumin indicating post-filtration generation. C9 neoantigen was detected in uEVs in six of nine of albuminuric KTRs but was absent in non-albuminuric controls (n = 8). In C9 neoantigen positive KTRs, lectin-affinity enrichment of uEVs from the proximal tubules yielded signal for iC3b, C3dg, C9 neoantigen and SGLT2 but only weakly for AQP2. Co-isolation of podocyte markers and Tamm-Horsfall protein was minimal. Our findings show that albuminuria is associated with aberrant filtration and intratubular activation of complement with deposition of C3 activation split products and C5b-9 associated C9 neoantigen on uEVs from the proximal tubular apical membrane. Intratubular complement activation may contribute to progressive kidney injury in proteinuric kidney grafts. (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 Kidney transplant recipient controls 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 Lectin affinity capture (Dolichus biflorus) PEG precipitation Protein markers EV: CD9/ CD63/ TSG101/ AQP2/ C9 neoantigen/ iC3b/C3dg/ CR1/ Podocalyxin/ SGLT2 non-EV: Tamm-Horsfall protein Proteomics no Show all info Study aim Biomarker/New methodological development Sample Species Homo sapiens Sample Type urine Separation Method Other Name other separation method Lectin affinity capture (Dolichus biflorus) Other Name other separation method PEG precipitation Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9/ CD63/ TSG101/ AQP2/ C9 neoantigen/ iC3b/C3dg/ SGLT2 Not detected EV-associated proteins CR1/ Podocalyxin Detected contaminants Tamm-Horsfall protein Characterization: Lipid analysis No

	EV210305	7/10	Homo sapiens	urine	Lectin affinity capture (Lotus tetragonolobus) PEG precipitation	Isaksson, Gustaf L.	2021	50%
Study summary Full title Proteinuria is accompanied by intratubular complement activation and apical membrane deposition of C3dg and C5b-9 in kidney transplant recipients All authors Gustaf Lissel Isaksson, Marie Bodilsen Nielsen, Gitte Rye Hinrichs, Nicoline Valentina Krogstrup, Rikke Zachar, Heidi Stubmark, Per Svenningsen, Kirsten Madsen, Claus Bistrup, Bente Jespersen, CONTEXT Study Group; Henrik Birn, Yaseelan Palarasah, Boye L Jensen Journal Am J Physiol Renal Physiol Abstract Proteinuria predicts accelerated decline in kidney function in kidney transplant recipients (KTRs). (show more...)Proteinuria predicts accelerated decline in kidney function in kidney transplant recipients (KTRs). We hypothesized that aberrant filtration of complement factors causes intraluminal activation, apical membrane attack on tubular cells and progressive injury. Biobanked samples from two previous studies in albuminuric KTRs were used. Complement activation split products C3c, C3dg and sC5b-9 associated C9 neoantigen were analyzed by ELISA in urine and plasma using neoepitope-specific antibodies. Urinary extracellular vesicles (uEV) were enriched by lectin- and immunoaffinity-isolation and analyzed by immunoblotting. Urine complement excretion increased significantly in KTRs with albumin/creatinine ratio ≥ 300 mg/g compared to < 30 mg/g. Urine C3dg and C9 neoantigen excretion correlated significantly to changes in albumin excretion from 3 to 12 months after transplantation. The fractional excretion of C9 neoantigen was significantly higher than for albumin indicating post-filtration generation. C9 neoantigen was detected in uEVs in six of nine of albuminuric KTRs but was absent in non-albuminuric controls (n = 8). In C9 neoantigen positive KTRs, lectin-affinity enrichment of uEVs from the proximal tubules yielded signal for iC3b, C3dg, C9 neoantigen and SGLT2 but only weakly for AQP2. Co-isolation of podocyte markers and Tamm-Horsfall protein was minimal. Our findings show that albuminuria is associated with aberrant filtration and intratubular activation of complement with deposition of C3 activation split products and C5b-9 associated C9 neoantigen on uEVs from the proximal tubular apical membrane. Intratubular complement activation may contribute to progressive kidney injury in proteinuric kidney grafts. (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 Kidney transplant albuminuria 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 Lectin affinity capture (Lotus tetragonolobus) PEG precipitation Protein markers EV: CD9/ CD63/ TSG101/ AQP2/ C9 neoantigen/ iC3b/C3dg/ CR1/ Podocalyxin/ SGLT2 non-EV: Tamm-Horsfall protein Proteomics no Show all info Study aim Biomarker/New methodological development Sample Species Homo sapiens Sample Type urine Separation Method Other Name other separation method Lectin affinity capture (Lotus tetragonolobus) Other Name other separation method PEG precipitation Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9/ CD63/ TSG101/ C9 neoantigen/ iC3b/C3dg/ SGLT2 Not detected EV-associated proteins AQP2/ CR1/ Podocalyxin Not detected contaminants Tamm-Horsfall protein Characterization: Lipid analysis No

	EV210305	8/10	Homo sapiens	urine	Lectin affinity capture (Dolichus biflorus) PEG precipitation	Isaksson, Gustaf L.	2021	50%
Study summary Full title Proteinuria is accompanied by intratubular complement activation and apical membrane deposition of C3dg and C5b-9 in kidney transplant recipients All authors Gustaf Lissel Isaksson, Marie Bodilsen Nielsen, Gitte Rye Hinrichs, Nicoline Valentina Krogstrup, Rikke Zachar, Heidi Stubmark, Per Svenningsen, Kirsten Madsen, Claus Bistrup, Bente Jespersen, CONTEXT Study Group; Henrik Birn, Yaseelan Palarasah, Boye L Jensen Journal Am J Physiol Renal Physiol Abstract Proteinuria predicts accelerated decline in kidney function in kidney transplant recipients (KTRs). (show more...)Proteinuria predicts accelerated decline in kidney function in kidney transplant recipients (KTRs). We hypothesized that aberrant filtration of complement factors causes intraluminal activation, apical membrane attack on tubular cells and progressive injury. Biobanked samples from two previous studies in albuminuric KTRs were used. Complement activation split products C3c, C3dg and sC5b-9 associated C9 neoantigen were analyzed by ELISA in urine and plasma using neoepitope-specific antibodies. Urinary extracellular vesicles (uEV) were enriched by lectin- and immunoaffinity-isolation and analyzed by immunoblotting. Urine complement excretion increased significantly in KTRs with albumin/creatinine ratio ≥ 300 mg/g compared to < 30 mg/g. Urine C3dg and C9 neoantigen excretion correlated significantly to changes in albumin excretion from 3 to 12 months after transplantation. The fractional excretion of C9 neoantigen was significantly higher than for albumin indicating post-filtration generation. C9 neoantigen was detected in uEVs in six of nine of albuminuric KTRs but was absent in non-albuminuric controls (n = 8). In C9 neoantigen positive KTRs, lectin-affinity enrichment of uEVs from the proximal tubules yielded signal for iC3b, C3dg, C9 neoantigen and SGLT2 but only weakly for AQP2. Co-isolation of podocyte markers and Tamm-Horsfall protein was minimal. Our findings show that albuminuria is associated with aberrant filtration and intratubular activation of complement with deposition of C3 activation split products and C5b-9 associated C9 neoantigen on uEVs from the proximal tubular apical membrane. Intratubular complement activation may contribute to progressive kidney injury in proteinuric kidney grafts. (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 Kidney transplant albuminuria 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 Lectin affinity capture (Dolichus biflorus) PEG precipitation Protein markers EV: CD9/ CD63/ TSG101/ AQP2/ C9 neoantigen/ iC3b/C3dg/ CR1/ Podocalyxin/ SGLT2 non-EV: Tamm-Horsfall protein Proteomics no Show all info Study aim Biomarker/New methodological development Sample Species Homo sapiens Sample Type urine Separation Method Other Name other separation method Lectin affinity capture (Dolichus biflorus) Other Name other separation method PEG precipitation Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9/ CD63/ TSG101/ AQP2/ C9 neoantigen/ iC3b/C3dg/ SGLT2 Not detected EV-associated proteins CR1/ Podocalyxin Detected contaminants Tamm-Horsfall protein Characterization: Lipid analysis No

	EV210305	9/10	Homo sapiens	urine	IAF PEG precipitation	Isaksson, Gustaf L.	2021	50%
Study summary Full title Proteinuria is accompanied by intratubular complement activation and apical membrane deposition of C3dg and C5b-9 in kidney transplant recipients All authors Gustaf Lissel Isaksson, Marie Bodilsen Nielsen, Gitte Rye Hinrichs, Nicoline Valentina Krogstrup, Rikke Zachar, Heidi Stubmark, Per Svenningsen, Kirsten Madsen, Claus Bistrup, Bente Jespersen, CONTEXT Study Group; Henrik Birn, Yaseelan Palarasah, Boye L Jensen Journal Am J Physiol Renal Physiol Abstract Proteinuria predicts accelerated decline in kidney function in kidney transplant recipients (KTRs). (show more...)Proteinuria predicts accelerated decline in kidney function in kidney transplant recipients (KTRs). We hypothesized that aberrant filtration of complement factors causes intraluminal activation, apical membrane attack on tubular cells and progressive injury. Biobanked samples from two previous studies in albuminuric KTRs were used. Complement activation split products C3c, C3dg and sC5b-9 associated C9 neoantigen were analyzed by ELISA in urine and plasma using neoepitope-specific antibodies. Urinary extracellular vesicles (uEV) were enriched by lectin- and immunoaffinity-isolation and analyzed by immunoblotting. Urine complement excretion increased significantly in KTRs with albumin/creatinine ratio ≥ 300 mg/g compared to < 30 mg/g. Urine C3dg and C9 neoantigen excretion correlated significantly to changes in albumin excretion from 3 to 12 months after transplantation. The fractional excretion of C9 neoantigen was significantly higher than for albumin indicating post-filtration generation. C9 neoantigen was detected in uEVs in six of nine of albuminuric KTRs but was absent in non-albuminuric controls (n = 8). In C9 neoantigen positive KTRs, lectin-affinity enrichment of uEVs from the proximal tubules yielded signal for iC3b, C3dg, C9 neoantigen and SGLT2 but only weakly for AQP2. Co-isolation of podocyte markers and Tamm-Horsfall protein was minimal. Our findings show that albuminuria is associated with aberrant filtration and intratubular activation of complement with deposition of C3 activation split products and C5b-9 associated C9 neoantigen on uEVs from the proximal tubular apical membrane. Intratubular complement activation may contribute to progressive kidney injury in proteinuric kidney grafts. (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 Kidney transplant albuminuria 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 Immunoaffinity capture (non-commercial) PEG precipitation Protein markers EV: C9 neoantigen/ iC3b/C3dg/ CD63/ TSG101/ SGLT2 non-EV: None Proteomics no Show all info Study aim Biomarker/New methodological development Sample Species Homo sapiens Sample Type urine Separation Method Immunoaffinity capture Selected surface protein(s) Complement factor C3dg Other Name other separation method PEG precipitation Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins C9 neoantigen/ iC3b/C3dg/ SGLT2 Not detected EV-associated proteins CD63/ TSG101 Characterization: Lipid analysis No

	EV210305	1/10	Homo sapiens	urine	(d)(U)C	Isaksson, Gustaf L.	2021	44%
Study summary Full title Proteinuria is accompanied by intratubular complement activation and apical membrane deposition of C3dg and C5b-9 in kidney transplant recipients All authors Gustaf Lissel Isaksson, Marie Bodilsen Nielsen, Gitte Rye Hinrichs, Nicoline Valentina Krogstrup, Rikke Zachar, Heidi Stubmark, Per Svenningsen, Kirsten Madsen, Claus Bistrup, Bente Jespersen, CONTEXT Study Group; Henrik Birn, Yaseelan Palarasah, Boye L Jensen Journal Am J Physiol Renal Physiol Abstract Proteinuria predicts accelerated decline in kidney function in kidney transplant recipients (KTRs). (show more...)Proteinuria predicts accelerated decline in kidney function in kidney transplant recipients (KTRs). We hypothesized that aberrant filtration of complement factors causes intraluminal activation, apical membrane attack on tubular cells and progressive injury. Biobanked samples from two previous studies in albuminuric KTRs were used. Complement activation split products C3c, C3dg and sC5b-9 associated C9 neoantigen were analyzed by ELISA in urine and plasma using neoepitope-specific antibodies. Urinary extracellular vesicles (uEV) were enriched by lectin- and immunoaffinity-isolation and analyzed by immunoblotting. Urine complement excretion increased significantly in KTRs with albumin/creatinine ratio ≥ 300 mg/g compared to < 30 mg/g. Urine C3dg and C9 neoantigen excretion correlated significantly to changes in albumin excretion from 3 to 12 months after transplantation. The fractional excretion of C9 neoantigen was significantly higher than for albumin indicating post-filtration generation. C9 neoantigen was detected in uEVs in six of nine of albuminuric KTRs but was absent in non-albuminuric controls (n = 8). In C9 neoantigen positive KTRs, lectin-affinity enrichment of uEVs from the proximal tubules yielded signal for iC3b, C3dg, C9 neoantigen and SGLT2 but only weakly for AQP2. Co-isolation of podocyte markers and Tamm-Horsfall protein was minimal. Our findings show that albuminuria is associated with aberrant filtration and intratubular activation of complement with deposition of C3 activation split products and C5b-9 associated C9 neoantigen on uEVs from the proximal tubular apical membrane. Intratubular complement activation may contribute to progressive kidney injury in proteinuric kidney grafts. (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 Kidney transplant recipient controls 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 (Differential) (ultra)centrifugation Protein markers EV: CD63/ C9 neoantigen non-EV: None Proteomics no Show all info Study aim Biomarker/New methodological development Sample Species Homo sapiens Sample Type urine Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 100 Pelleting: rotor type Type 70-Ti Pelleting: speed (g) 220,000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63/ C9 neoantigen Characterization: Lipid analysis No

	EV210305	6/10	Homo sapiens	urine	(d)(U)C	Isaksson, Gustaf L.	2021	44%
Study summary Full title Proteinuria is accompanied by intratubular complement activation and apical membrane deposition of C3dg and C5b-9 in kidney transplant recipients All authors Gustaf Lissel Isaksson, Marie Bodilsen Nielsen, Gitte Rye Hinrichs, Nicoline Valentina Krogstrup, Rikke Zachar, Heidi Stubmark, Per Svenningsen, Kirsten Madsen, Claus Bistrup, Bente Jespersen, CONTEXT Study Group; Henrik Birn, Yaseelan Palarasah, Boye L Jensen Journal Am J Physiol Renal Physiol Abstract Proteinuria predicts accelerated decline in kidney function in kidney transplant recipients (KTRs). (show more...)Proteinuria predicts accelerated decline in kidney function in kidney transplant recipients (KTRs). We hypothesized that aberrant filtration of complement factors causes intraluminal activation, apical membrane attack on tubular cells and progressive injury. Biobanked samples from two previous studies in albuminuric KTRs were used. Complement activation split products C3c, C3dg and sC5b-9 associated C9 neoantigen were analyzed by ELISA in urine and plasma using neoepitope-specific antibodies. Urinary extracellular vesicles (uEV) were enriched by lectin- and immunoaffinity-isolation and analyzed by immunoblotting. Urine complement excretion increased significantly in KTRs with albumin/creatinine ratio ≥ 300 mg/g compared to < 30 mg/g. Urine C3dg and C9 neoantigen excretion correlated significantly to changes in albumin excretion from 3 to 12 months after transplantation. The fractional excretion of C9 neoantigen was significantly higher than for albumin indicating post-filtration generation. C9 neoantigen was detected in uEVs in six of nine of albuminuric KTRs but was absent in non-albuminuric controls (n = 8). In C9 neoantigen positive KTRs, lectin-affinity enrichment of uEVs from the proximal tubules yielded signal for iC3b, C3dg, C9 neoantigen and SGLT2 but only weakly for AQP2. Co-isolation of podocyte markers and Tamm-Horsfall protein was minimal. Our findings show that albuminuria is associated with aberrant filtration and intratubular activation of complement with deposition of C3 activation split products and C5b-9 associated C9 neoantigen on uEVs from the proximal tubular apical membrane. Intratubular complement activation may contribute to progressive kidney injury in proteinuric kidney grafts. (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 Kidney transplant albuminuria 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 (Differential) (ultra)centrifugation Protein markers EV: CD63/ C9 neoantigen non-EV: None Proteomics no Show all info Study aim Biomarker/New methodological development Sample Species Homo sapiens Sample Type urine Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 100 Pelleting: rotor type Type 70-Ti Pelleting: speed (g) 220000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63/ C9 neoantigen Characterization: Lipid analysis No

	EV210305	4/10	Homo sapiens	urine	IAF PEG precipitation	Isaksson, Gustaf L.	2021	38%
Study summary Full title Proteinuria is accompanied by intratubular complement activation and apical membrane deposition of C3dg and C5b-9 in kidney transplant recipients All authors Gustaf Lissel Isaksson, Marie Bodilsen Nielsen, Gitte Rye Hinrichs, Nicoline Valentina Krogstrup, Rikke Zachar, Heidi Stubmark, Per Svenningsen, Kirsten Madsen, Claus Bistrup, Bente Jespersen, CONTEXT Study Group; Henrik Birn, Yaseelan Palarasah, Boye L Jensen Journal Am J Physiol Renal Physiol Abstract Proteinuria predicts accelerated decline in kidney function in kidney transplant recipients (KTRs). (show more...)Proteinuria predicts accelerated decline in kidney function in kidney transplant recipients (KTRs). We hypothesized that aberrant filtration of complement factors causes intraluminal activation, apical membrane attack on tubular cells and progressive injury. Biobanked samples from two previous studies in albuminuric KTRs were used. Complement activation split products C3c, C3dg and sC5b-9 associated C9 neoantigen were analyzed by ELISA in urine and plasma using neoepitope-specific antibodies. Urinary extracellular vesicles (uEV) were enriched by lectin- and immunoaffinity-isolation and analyzed by immunoblotting. Urine complement excretion increased significantly in KTRs with albumin/creatinine ratio ≥ 300 mg/g compared to < 30 mg/g. Urine C3dg and C9 neoantigen excretion correlated significantly to changes in albumin excretion from 3 to 12 months after transplantation. The fractional excretion of C9 neoantigen was significantly higher than for albumin indicating post-filtration generation. C9 neoantigen was detected in uEVs in six of nine of albuminuric KTRs but was absent in non-albuminuric controls (n = 8). In C9 neoantigen positive KTRs, lectin-affinity enrichment of uEVs from the proximal tubules yielded signal for iC3b, C3dg, C9 neoantigen and SGLT2 but only weakly for AQP2. Co-isolation of podocyte markers and Tamm-Horsfall protein was minimal. Our findings show that albuminuria is associated with aberrant filtration and intratubular activation of complement with deposition of C3 activation split products and C5b-9 associated C9 neoantigen on uEVs from the proximal tubular apical membrane. Intratubular complement activation may contribute to progressive kidney injury in proteinuric kidney grafts. (hide) EV-METRIC 38% (73rd 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 Kidney transplant recipients controls 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 Immunoaffinity capture (non-commercial) PEG precipitation Protein markers EV: C9 neoantigen/ iC3b/C3dg/ CD63/ TSG101/ SGLT2 non-EV: None Proteomics no Show all info Study aim Biomarker/New methodological development Sample Species Homo sapiens Sample Type urine Separation Method Immunoaffinity capture Selected surface protein(s) Complement factor C3dg Other Name other separation method PEG precipitation Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins C9 neoantigen/ iC3b/C3dg/ SGLT2 Not detected EV-associated proteins CD63/ TSG101 Characterization: Lipid analysis No

	EV210305	5/10	Homo sapiens	urine	IAF PEG precipitation	Isaksson, Gustaf L.	2021	38%
Study summary Full title Proteinuria is accompanied by intratubular complement activation and apical membrane deposition of C3dg and C5b-9 in kidney transplant recipients All authors Gustaf Lissel Isaksson, Marie Bodilsen Nielsen, Gitte Rye Hinrichs, Nicoline Valentina Krogstrup, Rikke Zachar, Heidi Stubmark, Per Svenningsen, Kirsten Madsen, Claus Bistrup, Bente Jespersen, CONTEXT Study Group; Henrik Birn, Yaseelan Palarasah, Boye L Jensen Journal Am J Physiol Renal Physiol Abstract Proteinuria predicts accelerated decline in kidney function in kidney transplant recipients (KTRs). (show more...)Proteinuria predicts accelerated decline in kidney function in kidney transplant recipients (KTRs). We hypothesized that aberrant filtration of complement factors causes intraluminal activation, apical membrane attack on tubular cells and progressive injury. Biobanked samples from two previous studies in albuminuric KTRs were used. Complement activation split products C3c, C3dg and sC5b-9 associated C9 neoantigen were analyzed by ELISA in urine and plasma using neoepitope-specific antibodies. Urinary extracellular vesicles (uEV) were enriched by lectin- and immunoaffinity-isolation and analyzed by immunoblotting. Urine complement excretion increased significantly in KTRs with albumin/creatinine ratio ≥ 300 mg/g compared to < 30 mg/g. Urine C3dg and C9 neoantigen excretion correlated significantly to changes in albumin excretion from 3 to 12 months after transplantation. The fractional excretion of C9 neoantigen was significantly higher than for albumin indicating post-filtration generation. C9 neoantigen was detected in uEVs in six of nine of albuminuric KTRs but was absent in non-albuminuric controls (n = 8). In C9 neoantigen positive KTRs, lectin-affinity enrichment of uEVs from the proximal tubules yielded signal for iC3b, C3dg, C9 neoantigen and SGLT2 but only weakly for AQP2. Co-isolation of podocyte markers and Tamm-Horsfall protein was minimal. Our findings show that albuminuria is associated with aberrant filtration and intratubular activation of complement with deposition of C3 activation split products and C5b-9 associated C9 neoantigen on uEVs from the proximal tubular apical membrane. Intratubular complement activation may contribute to progressive kidney injury in proteinuric kidney grafts. (hide) EV-METRIC 38% (73rd 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 Kidney transplant recipients controls 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 Immunoaffinity capture (non-commercial) PEG precipitation Protein markers EV: C9 neoantigen/ CD63/ TSG101/ SGLT2 non-EV: None Proteomics no Show all info Study aim Biomarker/New methodological development Sample Species Homo sapiens Sample Type urine Separation Method Immunoaffinity capture Selected surface protein(s) Complement factor C9 Other Name other separation method PEG precipitation Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins C9 neoantigen/ CD63/ SGLT2 Not detected EV-associated proteins TSG101 Characterization: Lipid analysis No

	EV210305	10/10	Homo sapiens	urine	IAF PEG precipitation	Isaksson, Gustaf L.	2021	38%
Study summary Full title Proteinuria is accompanied by intratubular complement activation and apical membrane deposition of C3dg and C5b-9 in kidney transplant recipients All authors Gustaf Lissel Isaksson, Marie Bodilsen Nielsen, Gitte Rye Hinrichs, Nicoline Valentina Krogstrup, Rikke Zachar, Heidi Stubmark, Per Svenningsen, Kirsten Madsen, Claus Bistrup, Bente Jespersen, CONTEXT Study Group; Henrik Birn, Yaseelan Palarasah, Boye L Jensen Journal Am J Physiol Renal Physiol Abstract Proteinuria predicts accelerated decline in kidney function in kidney transplant recipients (KTRs). (show more...)Proteinuria predicts accelerated decline in kidney function in kidney transplant recipients (KTRs). We hypothesized that aberrant filtration of complement factors causes intraluminal activation, apical membrane attack on tubular cells and progressive injury. Biobanked samples from two previous studies in albuminuric KTRs were used. Complement activation split products C3c, C3dg and sC5b-9 associated C9 neoantigen were analyzed by ELISA in urine and plasma using neoepitope-specific antibodies. Urinary extracellular vesicles (uEV) were enriched by lectin- and immunoaffinity-isolation and analyzed by immunoblotting. Urine complement excretion increased significantly in KTRs with albumin/creatinine ratio ≥ 300 mg/g compared to < 30 mg/g. Urine C3dg and C9 neoantigen excretion correlated significantly to changes in albumin excretion from 3 to 12 months after transplantation. The fractional excretion of C9 neoantigen was significantly higher than for albumin indicating post-filtration generation. C9 neoantigen was detected in uEVs in six of nine of albuminuric KTRs but was absent in non-albuminuric controls (n = 8). In C9 neoantigen positive KTRs, lectin-affinity enrichment of uEVs from the proximal tubules yielded signal for iC3b, C3dg, C9 neoantigen and SGLT2 but only weakly for AQP2. Co-isolation of podocyte markers and Tamm-Horsfall protein was minimal. Our findings show that albuminuria is associated with aberrant filtration and intratubular activation of complement with deposition of C3 activation split products and C5b-9 associated C9 neoantigen on uEVs from the proximal tubular apical membrane. Intratubular complement activation may contribute to progressive kidney injury in proteinuric kidney grafts. (hide) EV-METRIC 38% (73rd 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 Kidney transplant albuminuria 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 Immunoaffinity capture (non-commercial) PEG precipitation Protein markers EV: C9 neoantigen/ CD63/ TSG101/ SGLT2 non-EV: None Proteomics no Show all info Study aim Biomarker/New methodological development Sample Species Homo sapiens Sample Type urine Separation Method Immunoaffinity capture Selected surface protein(s) Complement factor C9 Other Name other separation method PEG precipitation Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins C9 neoantigen/ CD63/ SGLT2 Not detected EV-associated proteins TSG101 Characterization: Lipid analysis No

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EV-TRACK ID	EV210305
species	Homo sapiens
sample type	urine
condition	Kidney transplant recipient controls	Kidney transplant recipient controls	Kidney transplant albuminuria	Kidney transplant albuminuria	Kidney transplant albuminuria	Kidney transplant recipient controls	Kidney transplant albuminuria	Kidney transplant recipients controls	Kidney transplant recipients controls	Kidney transplant albuminuria
separation protocol	Lectin affinity capture (Lotus tetragonolobus)/ PEG precipitation	Lectin affinity capture (Dolichus biflorus)/ PEG precipitation	Lectin affinity capture (Lotus tetragonolobus)/ PEG precipitation	Lectin affinity capture (Dolichus biflorus)/ PEG precipitation	IAF capture (non-commercial)/ PEG precipitation	dUC	dUC	IAF capture (non-commercial)/ PEG precipitation	IAF capture (non-commercial)/ PEG precipitation	IAF capture (non-commercial)/ PEG precipitation
Exp. nr.	2	3	7	8	9	1	6	4	5	10
EV-METRIC %	50	50	50	50	50	44	44	38	38	38

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