D mannitol

Primary human glomerular endothelial cells (HGECs, ScienCell Company, Beijing Yuhengfeng Agency, China) were cultured in Endothelial Cell Medium containing 10% fetal bovine serum (Endothelial Cell Medium and serum were from ScienCell Company, Beijing Yuhengfeng Agency, China). The cells were used within six passages. Cells in the high insulin concentration group were treated with medium containing different concentrations of insulin: 7.14 ul, 14.28ul, 35.7ul, 71.4ul of Novolin insulin solution (Novo Nordisk, Copenhagen, Denmark) was added to 2 ml ECM medium separately(The ECM medium did not contain any insulin) to form 5 ng/ml, 10ng/ml, 25ng/ml, 50ng/ml high concentration insulin medium. Cells in the high glucose concentration group were treated with medium containing anhydrous glucose (Solarbio, Beijing, China) at 8.3, 11.1, 16.7, or 33.3 mmol/L. D-Mannitol (Solarbio, Beijing, China) was used as a control for osmolarity. HGECs were transfected with miR-21 mimics or miR-21 inhibitor (GenePharma, Shanghai, China) using the Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA). The transfection was performed under strictly aseptic conditions according to the manufacturer’s instructions. The effectiveness of miR-21 mimics and inhibitor was verified by PCR after transfection (Figures 4A, E).

P8 fibrils were prepared following the published protocol [20 ]. A concentration of 1 mM synthesized peptide P8 (NH2-PILTIITL-COOH) was dissolved in 1.5 mL of 5% D-mannitol (Solarbio, Beijing, China) and 0.01% sodium azide (pH 5.5). P8 suspensions were sonicated with bath sonicators to make clear solutions. The P8 peptide solutions were incubated for more than 8 h at a speed of 50 rpm on a rotating mixture at 37 °C. Then, the sample was observed by transmission electron microscopy (TEM). Spotting of P8 fibrils was performed on copper-coated Formvar grids, followed by washing with Milli-Q water and staining for 20 min with 0.1% uranyl formate solution. Before use, a 0.22 mm sterile syringe filter was used to filter the uranium formate solution (Merck Millipore). TEM images were taken at 120 kV on an electron microscope (FeiTecnai12 D312, Waltham, USA).