Journal: Journal of Extracellular Vesicles
Article Title: β‐catenin‐controlled tubular cell‐derived exosomes play a key role in fibroblast activation via the OPN‐CD44 axis
Figure Lengend Snippet: N‐OPN increases in kidney and urine in various clinical nephropathies and associates with CKD progression primarily through encapsulation within exosomes. (a) Representative micrographs showing the abundance and localization of N‐OPN protein in various human CKD: chronic tubulo‐interstitial nephritis (CTIN), IgA nephropathy (IgAN), membranous nephritis (MN), diabetic nephropathy (DN), focal segmental glomerulosclerosis (FSGS) and lupus nephritis (LN). (b) Normal: nontumor kidney tissue from patients with renal cell carcinoma were used as healthy subjects. Arrow indicate positive staining (scale bar: 50 μm). (c) Quantitative analysis of immunohistochemical staining of N‐OPN in patients with CKD and healthy subjects. (d) Correlation between N‐OPN and fibrosis score. (e–f) Western blot analyses show urinary N‐OPN protein in healthy subjects and patients with CKD. Representative western blot (e) and quantitative data (f) are shown. Numbers (1–10) indicate urine samples from each subject. ** p < 0.01 versus healthy subjects. (g) Pie chart shows the composition of human urine samples. (h) Diagram shows the experimental plan. Morning urine was collected, centrifuged at 3000 rpm for 5 min. The supernatant was collected and tested by ELISA; in addition, after differential centrifugation, the exosomes were extracted by ultracentrifugation. (i) Graphic presentation shows urinary N‐OPN protein levels in cohorts of patients with CKD ( n = 183) and healthy subjects ( n = 30). Urinary N‐OPN levels are presented as pmol/μmol urinary creatinine (Ucr). *** p < 0.001 versus healthy subjects. (j) Graphic presentation shows urinary N‐OPN protein levels in different stages of CKD. There was no statistical difference among different CKD stages. (k) Linear regression shows a negative correlation between urinary N‐OPN protein and kidney function (estimated glomerular filtration rate [eGFR]). (l) Linear regression shows a significant correlation between urinary N‐OPN levels and urinary albumin to creatinine ratio (ACR). (m–t) Analyses of exosomes isolated from the urine of patients with CKD and healthy subjects. (m) Transmission electron microscopy (TEM) image showing the exosomes isolated from urine from patients with CKD. (n) Representative images of Coomassie blue staining of exosomes from the urine of healthy subjects and patients with CKD. (o) Gene ontology (GO) enrichment analysis of the specific proteins from urinary exosomes of patients with CKD. (p) Representative micrographs showing two specific peptides of OPN from urinary exosomes isolated from CKD patients were identified by mass spectrometry. (q) An interaction network of OPN protein with other proteins was identified using the STRING database. (r) GO enrichment analysis of OPN‐interacted proteins shows the potential function of OPN. (s) Exosomes were prepared from the same amounts of urine from healthy subjects or patients with CKD, and were lysed and immunoblotted with antibodies against Alix, CD81, TSG101, CD63, OPN and N‐OPN, respectively. (t) Western blot analyses show N‐OPN protein expression in urine and exosome‐removed urine from patients with CKD. (u) Colloidal gold electron microscopy analysis demonstrates that N‐OPN was encapsulated in urinary exosomes from patients with CKD. N‐OPN was labelled with 10 nm colloidal gold particles. Arrows indicate positive staining; Scale bar: 100 nm
Article Snippet: The following primary antibodies were used: anti‐N‐OPN (Abcam, Cat. ab181440, 1:1000), anti‐CD63 (Abcam, Cat. ab59479, 1:1000), anti‐OPN (Boster Biotechnology, Cat. PB0589, 1:1000), anti‐CD44 (Boster Biotechnology, Cat. A00052, 1:1000), anti‐α‐tubulin (Beijing Ray Antibody Biotech, Cat. RM2007, 1:5000), anti‐fibronectin (Sigma, Cat. F3648, 1:50000), anti‐α‐SMA (Abcam, Cat. ab5648, 1:1000), anti‐PDGFR‐β (Santa Cruz, Cat. sc‐374573, 1:1000), anti‐Collagen I (Boster Biotechnology, Cat. BA0325, 1:1000), anti‐Vimentin (Abcam, Cat. ab8978, 1:1000), anti‐PCNA (Abcam, Cat. ab29; 1:1000), anti‐active‐β‐catenin (Cell Signaling, Cat. #4270s, 1:1000), anti‐c‐Myc (Cell Signaling, Cat. #5605s, 1:1000), and anti‐Foxo4 (Cell Signaling, Cat. #9472s, 1:1000), anti‐TSG101 (Abcam, Cat. Ab83; 1:1000), anti‐CD81 (Boster Biotechnology, Cat. A01281‐2, 1:1000), anti‐Alix (Boster Biotechnology, Cat. BM5496, 1:1000), anti‐Flag (Boster Biotechnology, Cat. M30971, 1:1000).
Techniques: Staining, Immunohistochemical staining, Western Blot, Enzyme-linked Immunosorbent Assay, Centrifugation, Filtration, Isolation, Transmission Assay, Electron Microscopy, Mass Spectrometry, Expressing