Journal: Advanced Science

Article Title: Renal Endothelial Cell‐Targeted Extracellular Vesicles Protect the Kidney from Ischemic Injury

doi: 10.1002/advs.202204626

Figure Lengend Snippet: Expression of P‐selectin in the kidneys post AKI. a) Gene expression of Selp , Sele , Icam1 , Vcam1 , and Pecam1 in renal tissues at 6, 12, and 24 h postsevere IRI. The renal tissues subjected to the sham operation served as the control, n = 3. ns, not significant versus sham, ∗ P < 0.05 versus sham. b) Western blot analysis of P‐selectin and Kim1 in renal tissues at 6, 12, and 24 h postsevere IRI (normalized to Gapdh). c) P‐selectin (red) expression in kidneys (FITC‐labeled Lotus tetragonolobus lectin (LTL), green, proximal tubules) at 0, 1, 3, 6, 12, and 24 h postsevere IRI was quantified by immunofluorescence, n = 5. ∗ P < 0.05. d) Immunofluorescence of P‐selectin (red) in kidneys (FITC‐labeled LTL, green, proximal tubules) at 12 h postmild, moderate, and severe IRI. e) Quantification of the P‐selectin‐positive area in renal sections, n = 5. * P < 0.05 versus sham. f) Average fluorescence intensity of P‐selectin in HUVECs post H/R, n = 3. * P < 0.05. g) P‐selectin (red) expression in HUVECs (Alexa Fluor 488‐labeled phalloidin, green, actin) post H/R was revealed by immunofluorescence. All data are expressed as the mean ± s.d. For a), c), and e), statistical analysis was performed using one‐way ANOVA with Tukey's multiple comparison tests. For f), statistical analysis was performed using a two‐tailed unpaired Student's t ‐test. The nuclei were counterstained with DAPI (blue). Scale bars, 100 µm.

Article Snippet: EVs or PBP‐EVs (100 μg mL −1 ) were added to the upper chamber postreoxygenation and cultured for another 24 h. Single kidney cells were isolated at day 3 postsevere IRI using the multitissue dissociation kit 2 (#130‐110‐203, Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Expressing, Gene Expression, Control, Western Blot, Labeling, Immunofluorescence, Fluorescence, Comparison, Two Tailed Test

Journal: Advanced Science

Article Title: Renal Endothelial Cell‐Targeted Extracellular Vesicles Protect the Kidney from Ischemic Injury

doi: 10.1002/advs.202204626

Figure Lengend Snippet: PBP‐EVs regulate endothelial cell functions. a) Scanning electron microscopy (SEM) images of human monocyte THP1 binding to H/R‐injured HUVECs with or without PBP‐EV blockade. Scale bar, 50 µm, n = 3. b) Myeloperoxidase (MPO) activity in renal tissue homogenate from different groups at day 3 postsevere IRI, n = 5. c) Immunohistochemistry and quantification of CD45 (brown) in renal tissues from different groups on day 3 postsevere IRI. Scale bar, 100 µm, n = 5. d) Representative images and quantification (numbers of meshes and branches per field) of the networks formed by HUVECs treated with EVs or PBP‐EVs for 6 h. Scale bar, 200 µm, n = 3. e) Vegfr2 expression in injured kidneys was monitored in real time by Fluc imaging in Vegfr2‐Fluc KI mice. f) Angiogenic tendencies of injured kidneys were quantitatively analyzed by Fluc radiance. The average radiance of Fluc was expressed as photons/s/cm 2 /steradian, n = 3. g) Quantitative analysis of CD31 + capillaries of injured renal tissues in immunofluorescence images, n = 5. h) The capillaries of the injured renal tissue were counted by immunofluorescence with CD31 (red) on day 7 postsevere IRI. The nuclei were counterstained with DAPI (blue). Scale bar, 200 µm. i) Angiogenesis‐related gene expression ( Vegfa , Vegfr2 , Ang1 , Ang2 , Plgf , and Egf ) in renal tissues was assessed by real‐time qPCR on day 7 postsevere IRI. The renal tissues subjected to the sham operation served as the control, n = 3. All data are expressed as the mean ± s.d. For a–d), g), and i), statistical analysis was performed using one‐way ANOVA with Tukey's multiple comparison tests. For f), statistical analysis was performed using two‐way ANOVA with Tukey's multiple comparison tests. * P < 0.05 versus PBS, # P < 0.05 versus EVs.

Article Snippet: EVs or PBP‐EVs (100 μg mL −1 ) were added to the upper chamber postreoxygenation and cultured for another 24 h. Single kidney cells were isolated at day 3 postsevere IRI using the multitissue dissociation kit 2 (#130‐110‐203, Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Electron Microscopy, Binding Assay, Activity Assay, Immunohistochemistry, Expressing, Imaging, Immunofluorescence, Gene Expression, Control, Comparison

Journal: Advanced Science

Article Title: Renal Endothelial Cell‐Targeted Extracellular Vesicles Protect the Kidney from Ischemic Injury

doi: 10.1002/advs.202204626

Figure Lengend Snippet: PBP‐EVs ameliorate maladaptive repair of tubular epithelial cells. a) The cell cycle of H/R‐injured HK2 cells was assessed by PI staining after administration of EVs or PBP‐EVs for 24 h. Normal HK2 cells served as a control, n = 3. b) Immunofluorescence of Ki67 (red) and p‐H3 (green) in H/R‐injured HK2 cells treated with EVs or PBP‐EVs for 24 h. Scale bar, 50 µm. c) Percentage of proliferating HK2 (Ki67 + ) cells that were in the G2/M phase (p‐H3 + ) of the cell cycle after H/R injury with administration of EVs or PBP‐EVs, n = 3. d) Cell cycles of TECs isolated from injured kidneys on day 3 postsevere IRI were analyzed by PI staining. Cells isolated from kidneys with sham operation served as a control. e) Percentage of TECs isolated from kidneys according to the cell cycle distribution, n = 3. f) Immunofluorescence of Ki67 (red) in renal tissues (FITC‐labeled LTL, green, proximal tubules) on day 3 postsevere IRI. Scale bar, 100 µm. g) Quantification of Ki67 + cells in renal immunofluorescence images, n = 5. h) Immunofluorescence of Kim1 (red, top row) and caspase3 (brown, bottom row) in renal tissues at day 3 postsevere IRI. Scale bar, 100 µm. i) Quantitative analysis of Kim1 + renal tubules and caspase3 + renal tubules in immunofluorescence images. n = 5. j) Expression of apoptosis‐related genes ( Fasl , Fas , Bad , and Bax ) in renal tissues was detected by real‐time qPCR on day 3 postsevere IRI. The renal tissues subjected to the sham operation served as the control, n = 3. All data are expressed as the mean ± s.d. Statistical analysis was performed using one‐way ANOVA with Tukey's multiple comparison tests. * P < 0.05 versus PBS, # P < 0.05 versus EVs. The nuclei were counterstained with DAPI (blue).

Article Snippet: EVs or PBP‐EVs (100 μg mL −1 ) were added to the upper chamber postreoxygenation and cultured for another 24 h. Single kidney cells were isolated at day 3 postsevere IRI using the multitissue dissociation kit 2 (#130‐110‐203, Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Staining, Control, Immunofluorescence, Isolation, Labeling, Expressing, Comparison

Journal: Advanced Science

Article Title: Renal Endothelial Cell‐Targeted Extracellular Vesicles Protect the Kidney from Ischemic Injury

doi: 10.1002/advs.202204626

Figure Lengend Snippet: PBP‐EVs facilitated renal recovery and alleviated fibrosis. a) Serum creatinine (SCr) and blood urea nitrogen (BUN) were evaluated on days 3 and 7 postsevere IRI, n = 3. b) Hematoxylin and eosin (H&E) staining of renal tissues 3 days postsevere IRI. c) Quantification of cast formation and loss of brush border in H&E staining image, n = 5. d) α ‐SMA (red) expression in renal tissues (FITC‐labeled LTL, green, proximal tubules) at day 28 postsevere IRI was quantified by immunofluorescence, n = 5. e) Collagen fibrils in renal tissues 28 days after severe IRI were visualized and quantified by Masson's trichrome staining, n = 5. f) Collagen IV (red) expression in renal tissues (FITC‐labeled LTL, green, proximal tubules) at day 28 postsevere IRI was quantified by immunofluorescence, n = 5. All data are expressed as the mean ± s.d. For a), statistical analysis was performed using two‐way ANOVA with Tukey's multiple comparison tests. For c– e), and f), statistical analysis was performed using one‐way ANOVA with Tukey's multiple comparison tests. * P < 0.05 versus PBS, # P < 0.05 versus EVs. The nuclei were counterstained with DAPI (blue). Scale bar, 100 µm.

Article Snippet: EVs or PBP‐EVs (100 μg mL −1 ) were added to the upper chamber postreoxygenation and cultured for another 24 h. Single kidney cells were isolated at day 3 postsevere IRI using the multitissue dissociation kit 2 (#130‐110‐203, Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Staining, Expressing, Labeling, Immunofluorescence, Comparison