Anti aqp1

After pretreatment with benzo[a]pyrene (0,5,10,50,100 and 200 µM) for various lengths of time, C6 cells were lysed in RIPA lysis buffer (Beyotime Institute of Biotechnology) and centrifuged at 12,000 × g for 10 min at 4°C. Protein concentration was measured using a BCA assay (Pierce; Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions. A total of 30 µg protein from each sample was resolved via 10% SDS-PAGE. The proteins were then transferred to a nitrocellulose membrane at 100 V for 120 min. After blocking with 5% non-fat dry milk for 1 h at room temperature, the membranes were incubated with anti-AQP1 (cat no. ab122821; 1:100; Abcam, Cambridge, MA, USA) and anti-GAPDH (cat no. ab8245; 1:1,000; Abcam) antibodies overnight at 4°C. After 3 washes with PBS, the membranes were incubated with goat anti-rabbit (cat no. 32460; 1:1,000; Invitrogen; Thermo Fisher Scientific, Inc.) or goat anti-mouse IgG (cat no. A0216; 1:1,000; Beyotime Institute of Biotechnology) secondary antibodies conjugated with horseradish peroxidase for 1 h at room temperature. Detection was performed using the ChemiDoc™ XRS+ enhanced chemiluminescent system (Bio-Rad Laboratories, Inc.).

Kidney tissue samples were fixed in 10% formalin for 24–48 h and embedded in paraffin after standard processing procedures for immunohistochemical analysis. After washing with PBS and cell permeabilization with 0.3% Triton, the slides were blocked with 10% goat serum and incubated overnight at 2–8 °C with the primary anti-Human Nuclear Antigen antibodies (anti-HNA, 1:100, Abcam, USA), anti-AQP1 (1:100, Abcam, USA), and anti-AQP2 (1:100, Abcam, USA). The slides were then incubated for 1 h with secondary antibodies. Finally, all slides were counterstained with DAPI and analyzed using fluorescent microscopy (Olympus) after adding glycerol and PBS solutions.

For this research, human ESCs (Royan H5) at passages 40–60 and CD133+CD24⁺ renal progenitor cells were purchased from Royan Institute. while Minimum Essential Medium were obtained from Thermo Fisher (Thermo Fisher Scientific, Massachusetts, USA). In this study we used fetal bovine serum (FBS, Gibco Life Technologies, USA), penicillin and streptomycin (Gibco Life Technologies, USA), osteogenic differentiation medium (Gibco Life Technologies, Germany) and alkaline phosphatase (Sigma-Aldrich, USA), alizarin red dye (Sigma-Aldrich, USA), oil red dye (Sigma-Aldrich, USA), knock-out serum replacement (Gibco), non-essential amino acids (Gibco Life Technologies, USA), glutamine (Gibco Life Technologies, USA), β-mercaptoethanol (Sigma-Aldrich, USA), basic fibroblast growth factor (bFGF, Sigma- Aldrich, USA), Accumax (Sigma-Aldrich, USA), bovine serum albumin (BSA, Sigma- Aldrich, USA), propidium iodide (PE) -conjugated mouse anti-human CD133 (Miltenyi Biotec, USA) and fluorescein isothiocyanate (FITC) -conjugated mouse anti-human CD24 (Abcam, USA) antibodies, primary anti-Human Nuclear Antigen antibodies (anti-HNA, Abcam, USA), anti-AQP1 (Abcam, USA), and anti-AQP2 (Abcam, USA). Additionally, TNF α, IL-1, IL-2, IL-6, IL-10, TGF-β, and IFN γ were obtained from Sigma Aldrich, USA.