Fig 2: Fibulin-1 (FBLN1) induced epithelial–mesenchymal transition (EMT) in proximal tubular epithelial cells (PTECs). (A) EMT markers, including E-cadherin, N-cadherin, and vimentin, and extracellular matrix marker (ECM; fibronectin) levels were assessed in HK-2 cells treated with normal control (NC) and FBLN1 protein (300 ng/ml). (B) Cells were transfected with FBLN1 siRNA (20 nM) or control siRNA for 24 h. After transfection, the cells were treated with normal glucose (NG) or high glucose (HG) for 48 h to examine EMT and fibronectin expression using Western blotting. *p < 0.05, **p < 0.01, and ***p < 0.001 by Student’s t-test or ANOVA followed by the post-hoc test adjusted with Tukey’s correction.

Fig 3: Identification of differentially exosomal proteins derived from proximal tubular epithelial cells (PTECs) in diabetic nephropathy (DN). (A) Detection of normal glucose (NG; 5.5 mM)- or high glucose (HG; 25 mM)-treated HK-2 cell-derived exosomes uptake by HK-2 cells using immunofluorescence stain. (B) Examination of the markers of exosomes derived from HK-2 cells treated with NG or HG for 48 h using Western blotting. (C) The morphology of HK-2 cells treated with NG or exosomes derived from HK-2 cells under NG or HG for 48 h (exosomes: HK-2 cell = 5:1) was examined using a light microscope. (D) Epithelial–mesenchymal transition (EMT) markers were assessed in HK-2 cells treated with NG, HG, or exosomes derived from HK-2 cells under NG and HG conditions for 48 h using Western blotting. (E) Flowchart of identification of potential exosomal proteins derived from HK-2 cells treated with NG and HG for 48 h using liquid chromatography–tandem mass spectrometry (LC-MS/MS) and following bioinformatics analysis. (F,G) Gene ontology of cellular component and biological process of exosomal proteins derived from NG- and HG-treated HK-2 cells. The pie chart indicates the-Log10 (false discovery rate (FDR)) of each term, and the numbers that are shown at the outside of each pie segment indicate the number of genes involved in each term. (H) The protein–protein interaction network analysis of genes associated with extracellular matrix organization. STRING database (version 10) was used in the bioinformatics analysis. (I,J) Fibulin-1 (FBLN1) expression in HK-2 cells treated with NG and HG for 48 h, and with exosomes derived from HK-2 cells under NG and HG conditions for 48 h. (K) The FBLN1 protein level in HK-2 cells treated with NG or exosomes derived from HK-2 cells under NG and HG conditions for 48 h (exosomes: HK-2 cells = 5:1). (L) The expression of FBLN1 in the proximal tubules of kidneys in mice is shown. The kidney sections of nondiabetic db/m mice (n = 3) and diabetic db/db mice (n = 3) were stained with FBLN1 (brown). The bar graph represents the mean ± SEM of at least three independent experiments. * p < 0.05, *** p < 0.001 by Student’s t-test.

Fig 4: Urinary exosomal miR-1269b level and urinary Fibulin-1 (FBLN1) level as potential biomarkers of kidney injury in humans. (A) Urinary exosomal miR-1269b level was measured in normal individuals (N = 49) and type 2 diabetes mellitus (DM) patients (N = 59). (B) The correlation between urinary exosomal miR-1269b level and albumin–creatinine ratio (ACR) was assessed. (C) The difference of urinary exosomal miR-1269b levels across the severity of albuminuria in humans. (D–G) The association of urinary exosomal miR-1269b levels with kidney injury molecule 1/creatinine (KIM-1/Cr), neutrophil gelatinase-associated lipocalin/creatinine (NGAL/Cr), and estimated glomerular infiltration rate (eGFR) were examined. (G) Urinary FBLN1 level was examined in study subjects. (H,I) The correlations between urinary FBLN1 levels and urinary exosomal miR-1269b levels and ACR were assessed. (J) The difference of urinary FBLN1 level across the severity of albuminuria in humans. (K–M) The associations of urinary FBLN1 level with KIM-1/Cr, NGAL/Cr, and eGFR were investigated. Exosomal miR-1269b in the urine of humans was isolated and then assessed by qRT-PCR. Urine albumin was measured using the immunoturbidimetric assay, and urine creatinine was determined using the enzymatic method. The concentrations of FBLN1, NGAL, and KIM-1 in urine were measured using ELISA. Serum creatinine was measured using the compensated Jaffé (kinetic alkaline picrate) method. eGFR was calculated using the equation eGFR = 186 × Serum creatinine−1.154 × Age−0.203 × 0.742 (if female). The bar graph represents the mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001 by Student’s t-test or ANOVA followed by the post-hoc test with Tukey’s correction, and p-value of correlation was analyzed by Spearman’s analysis.

Fig 5: Illustration of the mechanism by which high glucose (HG) induced epithelial–mesenchymal transition (EMT) in proximal tubular epithelial cells (PTECs) through PTEC-derived exosomal Fibulin-1 (FBLN1) in diabetic nephropathy (DN). HG suppressed miR-1269b expression in PTECs, leading to increased expression of FBLN1. FBLN1 further promoted EMT in PTECs through autocrine PTEC-derived exosome delivery.