Fig 2: Albumin overload promoted exosomes secretion and Rab27a expression in a dose-dependent manner.a, b Quantification of purified exosomes released from TECs exosomes treated with different doses of BSA (0, 5, 10 and 20 mg/ml). Increased, secretion of exosomes was observed shown as representative western blotting of exosome markers (Alix, CD63, and CD81) (a) and quantification using EXOCET assay kit (b). c Expression of Hrs, nSMase2A, and Rab27a mRNA in TECs exposed to different doses of BSA detected by RT-PCR. The mRNA expression is compared to cells without BSA treatment (represented by 1-fold). d, e Rab27a protein in TECs with albumin treatment. Rab27a protein was upregulated by BSA treatment in a dose-dependent manner, as shown by western blotting and cellular immunofluorescence staining. The bar graph shows fluorescence intensity from 20 mg/ml-BSA-treated TECs compared with Ctrl group (represented by 1-fold). Scale bar: 20 μm. f, g HE staining and immunofluorescence staining of Rab27a expression in renal biopsies from controls (n = 4) (normal kidney tissue from patients of kidney cancer) and patients with CKD (n = 8). Severe tubular injury, protein casts (f) and higher expression of Rab27a in the tubules was observed in CKD patients compared with controls (g). Scale bars: 50 μm. Ctrl, control. *p < 0.05 compared to Ctrl. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs TECs without BSA exposure. #p < 0.05 vs TECs with 5 mg/ml BSA treatment. BSA, bovine serum albumin. Data presented as mean ± S.E.M. of three independent experiments.

Fig 3: Particle population characteristics, zeta potential, and western blot analysis of cell-derived EVs. (A) Categorization of EV populations by size distribution by TRPS. The majority of EVs belong to a size range of 100–250 nm. t-test was performed between their counterparts. (B) pH affects the zeta potential of EVs: Compared to physiological pH (7.4), cyst microenvironmental pH (6.0) showed a drastic change in the zeta potential (measured by TRPS) of EVs derived from T1G cells. (C) Western blot analysis of EVs. Classical EV markers such as Alix, TSG 101, ARL13b, CD63, CD81, and CD9 were present in EVs derived from mIMCD3 and T1G cells. Data (n = 3) were represented as mean ± SD. Significant difference analyzed by t-test (*p < 0.05, **p < 0.01, ***p < 0.001).

Fig 4: A schematic image illustrating CD81 expression patterns during β-cell maturation and dedifferentiation. This schematic was designed using BioRender.com.

Fig 5: Cd81 expression levels correlated with the maturation state of β-cells. (A) Heat map representing the gene expression of the top 30 genes differentially regulated in P16 Cd81high and Cd81low/- β-cells. (B) Pathway enrichment analysis obtained from processing the differentially regulated genes of P16 Cd81high and Cd81low/- β-cells. (C) Violin plots of pivotal genes playing a crucial role during the maturation process of β-cells. Ucn3, Slc2a2 (Glut2), and Mafa are upregulated in Cd81low/- β-cells and Mafb and Cgha are upregulated in Cd81high β-cells. (D) Venn diagrams representing the comparison between differentially expressed genes of P16 Cd81high and Cd81low/- β-cells with highly upregulated genes from embryonic Fev+ (left) and endocrine cells (right) described in Bastidas-Ponce et al., 2019. (E) Venn diagrams representing the comparison between differentially expressed genes of P16 Cd81high and Cd81low/- β-cells with highly upregulated genes from β1 (left) and β2 (right) β-cells as described in Sachs et al., 2020. (F) Targeted analysis of specific signaling pathways differentially regulated between P16 Cd81high and Cd81low/- β-cells.