Journal: iScience

Article Title: Urinary extracellular vesicles signature for diagnosis of kidney disease

doi: 10.1016/j.isci.2022.105416

Figure Lengend Snippet: ELISA platform for assessment of uEVs (A) Schematic illustration of sandwich ELISA using Tim4. (B) Expression of MGAM, MUC1, and CD9 in nephron segments. The expression pattern was visualized by kidney cell explorer and by fluorescent immunohistochemistry using human kidney tissue. The numbers above the figure are the same as in Figure 1 G. Scale bars, 100 μm. See also Figures S4 and .

Article Snippet: Antibodies used as capture reagents were as follows: anti-CD63 antibody contained in the abovementioned kit, and mouse monoclonal anti-CD9 antibody (clone12A12, Shionogi, Osaka, Japan), rabbit polyclonal anti-MGAM antibody (Cat# 22195-1-AP, Proteintech, Chicago, IL, USA), mouse monoclonal anti-MUC1 antibody (Cat# sc-7313, Santa Cruz, Dallas, TX, USA), mouse monoclonal anti-Poliovirus receptor (PVR) antibody (Cat# 66913-1-Ig, Proteintech), mouse monoclonal anti-PKD2 antibody (Cat# H00005311-M01, Abnova, Taipei, Taiwan), mouse monoclonal anti-CD133 (PROM1) antibody (Cat# 66666-1-lg, Proteintech), and rabbit monoclonal anti-CD90/Thy1 antibody (Cat# ab92574, Abcam, Cambridge, UK).

Techniques: Enzyme-linked Immunosorbent Assay, Sandwich ELISA, Expressing, Immunohistochemistry

Journal: iScience

Article Title: Urinary extracellular vesicles signature for diagnosis of kidney disease

doi: 10.1016/j.isci.2022.105416

Figure Lengend Snippet: Application of expression signature of uEVs for diagnosis of CKD (A) Comparison of MUC1 and MGAM levels (absorbance at 450 nm) measured by customized ELISA in samples from healthy controls and patients with CKD. G1 indicates CKD patients with eGFR ≥90 and G2-5 with <90 mL/min/1.73m 2 . (B and C) ROC curve for distinguishing patients with decreased renal function (eGFR <60 (B), eGFR <90 (C)) from healthy controls by logistic regression. (D) ROC curve for distinguishing patients with CKD having normal eGFR (≥90) from healthy controls by logistic regression. (E) Box and beeswarm plot of the assay value of MGAM/MUC1 (the expression of MGAM divided by that of MUC1) in patients with CKD having each renal function and healthy controls in the discovery and validation cohorts. (F) ROC curve for distinguishing patients with decreased renal function (eGFR <90) from healthy controls by logistic regression using combinations of MGAM/MUC1, urine creatinine (uCr), urine albumin (uAlb), and L-FABP. (G) The change in eGFR in follow-up period in two groups (MGAM/MUC1 below 0.35 and the others). Boxes in boxplots indicate the 25th and 75th percentiles, and the horizontal lines inside the boxes indicate the median. Bars indicate the 10th and 90th percentiles. The data were compared using the two-tailed Mann–Whitney U test. See also Figures S6–S8 .

Article Snippet: Antibodies used as capture reagents were as follows: anti-CD63 antibody contained in the abovementioned kit, and mouse monoclonal anti-CD9 antibody (clone12A12, Shionogi, Osaka, Japan), rabbit polyclonal anti-MGAM antibody (Cat# 22195-1-AP, Proteintech, Chicago, IL, USA), mouse monoclonal anti-MUC1 antibody (Cat# sc-7313, Santa Cruz, Dallas, TX, USA), mouse monoclonal anti-Poliovirus receptor (PVR) antibody (Cat# 66913-1-Ig, Proteintech), mouse monoclonal anti-PKD2 antibody (Cat# H00005311-M01, Abnova, Taipei, Taiwan), mouse monoclonal anti-CD133 (PROM1) antibody (Cat# 66666-1-lg, Proteintech), and rabbit monoclonal anti-CD90/Thy1 antibody (Cat# ab92574, Abcam, Cambridge, UK).

Techniques: Expressing, Biomarker Discovery, Comparison, Enzyme-linked Immunosorbent Assay, Two Tailed Test, MANN-WHITNEY

Journal: iScience

Article Title: Urinary extracellular vesicles signature for diagnosis of kidney disease

doi: 10.1016/j.isci.2022.105416

Figure Lengend Snippet:

Article Snippet: Antibodies used as capture reagents were as follows: anti-CD63 antibody contained in the abovementioned kit, and mouse monoclonal anti-CD9 antibody (clone12A12, Shionogi, Osaka, Japan), rabbit polyclonal anti-MGAM antibody (Cat# 22195-1-AP, Proteintech, Chicago, IL, USA), mouse monoclonal anti-MUC1 antibody (Cat# sc-7313, Santa Cruz, Dallas, TX, USA), mouse monoclonal anti-Poliovirus receptor (PVR) antibody (Cat# 66913-1-Ig, Proteintech), mouse monoclonal anti-PKD2 antibody (Cat# H00005311-M01, Abnova, Taipei, Taiwan), mouse monoclonal anti-CD133 (PROM1) antibody (Cat# 66666-1-lg, Proteintech), and rabbit monoclonal anti-CD90/Thy1 antibody (Cat# ab92574, Abcam, Cambridge, UK).

Techniques: Labeling, Enzyme-linked Immunosorbent Assay, Isolation, Software, Microplate Reader Absorbance Measurement, Mass Spectrometry, Transmission Assay, Electron Microscopy, Fluorescence, Microscopy

Journal: iScience

Article Title: Urinary extracellular vesicles signature for diagnosis of kidney disease

doi: 10.1016/j.isci.2022.105416

Figure Lengend Snippet: Characterization of uEVs from healthy controls (A) A schematic of the study outlining the discovery and validation cohort. (B) Negative stain transmission electron microscopy of uEVs. Scale bars, 200 nm. (C) Nanoparticle tracking analysis of uEVs isolated from healthy controls. (D) Venn diagram of total proteins detected in uEVs isolated from healthy controls. (E) Bar plot representing the abundance of common classical exosomal markers (CD63, CD9) and markers for classical microvesicles (Annexin A1) and arrestin-domain-containing protein 1-mediated microvesicles (TSG101) in healthy controls. The yaxis represents log10 relative abundance. (F) KEGG/Wiki pathway analysis (g:profiler) of the 1,298 common proteins in uEVs from healthy controls. The top five terms with the lowest adjusted p values were extracted. (G) Intrarenal expression of top 50 molecules in uEVs from healthy controls visualized using kidney cell explorer ( Ransick et al., 2019 ) in order of relative abundance and immunofluorescence of human kidney specimens of 5 out of 50 molecules. Glutathione hydrolase 1 proenzyme (GGT1), phosphoglycerate kinase 1 (PGK1), uromodulin (UMOD), annexin A11 (ANXA11), keratin, and type I cytoskeletal 14 (KRT14) are expressed in the proximal tubule, loop of Henle, the distal tubule, collecting duct, and deep medullary epithelium of pelvis, respectively. The numbers above the figure represent each nephron segment: 1, podocytes; 2, parietal epithelium; 3, proximal tubule; 4, the loop of Henle; 5, distal tubule; 6, nephron connecting tubule; 7, cortical collecting duct; 8, medullary collecting duct; 9, deep medullary epithelium of pelvis. Scale bars, 200 μm.

Article Snippet: Mouse monoclonal anti-GGT1 antibody (Cat# sc-166908, Santa Cruz), mouse monoclonal anti-THP (UMOD) antibody (Cat# sc-271022, Santa Cruz), rabbit polyclonal anti-PGK1 antibody (Cat# 17811-1-AP, Proteintech), rabbit polyclonal anti-KRT14 antibody (Cat# 10143-1-AP, Proteintech), rabbit polyclonal anti-ANXA11 antibody (Cat# 10479-2-AP, Proteintech) and above mentioned anti-CD9 antibody, anti-MGAM antibody, and anti-MUC1 antibody were used as primary antibodies.

Techniques: Biomarker Discovery, Staining, Transmission Assay, Electron Microscopy, Isolation, Expressing, Immunofluorescence

Journal: iScience

Article Title: Urinary extracellular vesicles signature for diagnosis of kidney disease

doi: 10.1016/j.isci.2022.105416

Figure Lengend Snippet:

Article Snippet: Mouse monoclonal anti-GGT1 antibody (Cat# sc-166908, Santa Cruz), mouse monoclonal anti-THP (UMOD) antibody (Cat# sc-271022, Santa Cruz), rabbit polyclonal anti-PGK1 antibody (Cat# 17811-1-AP, Proteintech), rabbit polyclonal anti-KRT14 antibody (Cat# 10143-1-AP, Proteintech), rabbit polyclonal anti-ANXA11 antibody (Cat# 10479-2-AP, Proteintech) and above mentioned anti-CD9 antibody, anti-MGAM antibody, and anti-MUC1 antibody were used as primary antibodies.

Techniques: Labeling, Enzyme-linked Immunosorbent Assay, Isolation, Software, Microplate Reader Absorbance Measurement, Mass Spectrometry, Transmission Assay, Electron Microscopy, Fluorescence, Microscopy

Journal: iScience

Article Title: Urinary extracellular vesicles signature for diagnosis of kidney disease

doi: 10.1016/j.isci.2022.105416

Figure Lengend Snippet: ELISA platform for assessment of uEVs (A) Schematic illustration of sandwich ELISA using Tim4. (B) Expression of MGAM, MUC1, and CD9 in nephron segments. The expression pattern was visualized by kidney cell explorer and by fluorescent immunohistochemistry using human kidney tissue. The numbers above the figure are the same as in Figure 1 G. Scale bars, 100 μm. See also Figures S4 and .

Article Snippet: Antibodies used as capture reagents were as follows: anti-CD63 antibody contained in the abovementioned kit, and mouse monoclonal anti-CD9 antibody (clone12A12, Shionogi, Osaka, Japan), rabbit polyclonal anti-MGAM antibody (Cat# 22195-1-AP, Proteintech, Chicago, IL, USA), mouse monoclonal anti-MUC1 antibody (Cat# sc-7313, Santa Cruz, Dallas, TX, USA), mouse monoclonal anti-Poliovirus receptor (PVR) antibody (Cat# 66913-1-Ig, Proteintech), mouse monoclonal anti-PKD2 antibody (Cat# H00005311-M01, Abnova, Taipei, Taiwan), mouse monoclonal anti-CD133 (PROM1) antibody (Cat# 66666-1-lg, Proteintech), and rabbit monoclonal anti-CD90/Thy1 antibody (Cat# ab92574, Abcam, Cambridge, UK).

Techniques: Enzyme-linked Immunosorbent Assay, Sandwich ELISA, Expressing, Immunohistochemistry

Journal: iScience

Article Title: Urinary extracellular vesicles signature for diagnosis of kidney disease

doi: 10.1016/j.isci.2022.105416

Figure Lengend Snippet: Application of expression signature of uEVs for diagnosis of CKD (A) Comparison of MUC1 and MGAM levels (absorbance at 450 nm) measured by customized ELISA in samples from healthy controls and patients with CKD. G1 indicates CKD patients with eGFR ≥90 and G2-5 with <90 mL/min/1.73m 2 . (B and C) ROC curve for distinguishing patients with decreased renal function (eGFR <60 (B), eGFR <90 (C)) from healthy controls by logistic regression. (D) ROC curve for distinguishing patients with CKD having normal eGFR (≥90) from healthy controls by logistic regression. (E) Box and beeswarm plot of the assay value of MGAM/MUC1 (the expression of MGAM divided by that of MUC1) in patients with CKD having each renal function and healthy controls in the discovery and validation cohorts. (F) ROC curve for distinguishing patients with decreased renal function (eGFR <90) from healthy controls by logistic regression using combinations of MGAM/MUC1, urine creatinine (uCr), urine albumin (uAlb), and L-FABP. (G) The change in eGFR in follow-up period in two groups (MGAM/MUC1 below 0.35 and the others). Boxes in boxplots indicate the 25th and 75th percentiles, and the horizontal lines inside the boxes indicate the median. Bars indicate the 10th and 90th percentiles. The data were compared using the two-tailed Mann–Whitney U test. See also Figures S6–S8 .

Article Snippet: Antibodies used as capture reagents were as follows: anti-CD63 antibody contained in the abovementioned kit, and mouse monoclonal anti-CD9 antibody (clone12A12, Shionogi, Osaka, Japan), rabbit polyclonal anti-MGAM antibody (Cat# 22195-1-AP, Proteintech, Chicago, IL, USA), mouse monoclonal anti-MUC1 antibody (Cat# sc-7313, Santa Cruz, Dallas, TX, USA), mouse monoclonal anti-Poliovirus receptor (PVR) antibody (Cat# 66913-1-Ig, Proteintech), mouse monoclonal anti-PKD2 antibody (Cat# H00005311-M01, Abnova, Taipei, Taiwan), mouse monoclonal anti-CD133 (PROM1) antibody (Cat# 66666-1-lg, Proteintech), and rabbit monoclonal anti-CD90/Thy1 antibody (Cat# ab92574, Abcam, Cambridge, UK).

Techniques: Expressing, Biomarker Discovery, Comparison, Enzyme-linked Immunosorbent Assay, Two Tailed Test, MANN-WHITNEY

Journal: iScience

Article Title: Urinary extracellular vesicles signature for diagnosis of kidney disease

doi: 10.1016/j.isci.2022.105416

Figure Lengend Snippet:

Article Snippet: Antibodies used as capture reagents were as follows: anti-CD63 antibody contained in the abovementioned kit, and mouse monoclonal anti-CD9 antibody (clone12A12, Shionogi, Osaka, Japan), rabbit polyclonal anti-MGAM antibody (Cat# 22195-1-AP, Proteintech, Chicago, IL, USA), mouse monoclonal anti-MUC1 antibody (Cat# sc-7313, Santa Cruz, Dallas, TX, USA), mouse monoclonal anti-Poliovirus receptor (PVR) antibody (Cat# 66913-1-Ig, Proteintech), mouse monoclonal anti-PKD2 antibody (Cat# H00005311-M01, Abnova, Taipei, Taiwan), mouse monoclonal anti-CD133 (PROM1) antibody (Cat# 66666-1-lg, Proteintech), and rabbit monoclonal anti-CD90/Thy1 antibody (Cat# ab92574, Abcam, Cambridge, UK).

Techniques: Labeling, Enzyme-linked Immunosorbent Assay, Isolation, Software, Microplate Reader Absorbance Measurement, Mass Spectrometry, Transmission Assay, Electron Microscopy, Fluorescence, Microscopy

Journal: iScience

Article Title: Urinary extracellular vesicles signature for diagnosis of kidney disease

doi: 10.1016/j.isci.2022.105416

Figure Lengend Snippet:

Article Snippet: Antibodies used as capture reagents were as follows: anti-CD63 antibody contained in the abovementioned kit, and mouse monoclonal anti-CD9 antibody (clone12A12, Shionogi, Osaka, Japan), rabbit polyclonal anti-MGAM antibody (Cat# 22195-1-AP, Proteintech, Chicago, IL, USA), mouse monoclonal anti-MUC1 antibody (Cat# sc-7313, Santa Cruz, Dallas, TX, USA), mouse monoclonal anti-Poliovirus receptor (PVR) antibody (Cat# 66913-1-Ig, Proteintech), mouse monoclonal anti-PKD2 antibody (Cat# H00005311-M01, Abnova, Taipei, Taiwan), mouse monoclonal anti-CD133 (PROM1) antibody (Cat# 66666-1-lg, Proteintech), and rabbit monoclonal anti-CD90/Thy1 antibody (Cat# ab92574, Abcam, Cambridge, UK).

Techniques: Labeling, Enzyme-linked Immunosorbent Assay, Isolation, Software, Microplate Reader Absorbance Measurement, Mass Spectrometry, Transmission Assay, Electron Microscopy, Fluorescence, Microscopy