Research Paper Volume 16, Issue 4 pp 3302—3331

Figure 5. ADSC-derived exosomal miR-204 alleviates HG-induced injury in HK-2 cells. (A) Determination of ADSC-derived Exos taken up by HK-2 cells (human proximal renal tubular epithelial cell line) using PKH67 staining. ADSCs were pre-treated with GW4869 (an Exo inhibitor; 2.5 μM), followed by Exo isolation. HK-2 cells were then co-cultured with PKH67-labeled Exos to evaluate their capacity to take up Exos. (B) The expression of miR-204 in HK-2 cells using qRT-PCR. (C, D) HK-2 cell viability (at 24 h) and apoptosis assessed using CCK-8 and flow cytometry, respectively. (E) The levels of MDA, SOD, GPX, and CAT in HK-2 cells using ELISA. (F) The expression of Drp1, Fis1, OPA1, and Mfn1 in HK2 cells using western blotting. (B–F) HK-2 cells were exposed to HG (30 mM) and cultured for 24 h to construct an in vitro model of DN. To determine the function of exosomal miR-204 in DN, ADSCs were transfected with miR-204 mimic or miR NC (negative control) for 48 h. Then, Exos were separated from the transfected ADSCs and co-cultured with HK-2 cells for 12 h at the concentration of 100 μg/mL. Data were expressed as mean ± standard deviation. ^**p < 0.01 vs. Control group; ^#p < 0.05 and ^##p < 0.01 vs. HG group; ^&p < 0.05 and ^&&p < 0.01 vs. HG + Exo-miR NC group. Abbreviations: ADSC: adipose-derived stem cell; miR-204: microRNA-204; HG: high glucose; Exos: exosomes; qRT-PCR: quantitative real-time polymerase chain reaction; CCK-8: cell counting kit-8; MDA: malondialdehyde; SOD: superoxide dismutase; GPX: glutathione peroxidase; CAT: catalase; ELISA: enzyme-linked immunosorbent assay.