Anti at1r

The unfrozen hippocampal samples were homogenized with lysis buffer containing a cocktail of phosphatase and proteinase inhibitors and PMSF (Beyotime, Shanghai, China). After denaturation, the lysates were separated on a 10% SDS-PAGE gel and transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad, USA). The membranes were blocked with 5% non-fat powdered milk in TBST (containing 0.1% Tween 20) for 1 h at 25°C and incubated for 15−18 h at 4°C with a monoclonal rabbit anti-MasR (1 : 2000, Proteintech Group), anti-AT1R (1 : 500, Proteintech Group), or anti-GAPDH (1 : 5000, Proteintech Group) primary antibody. After TBST cleaning, the membrane was incubated for 1 h at 25°C with a horseradish peroxidase- (HRP-) conjugated goat anti-rabbit antibody (1 : 5000, Jackson ImmunoResearch Laboratories, USA) and the protein bands were visualized using an ECL system (Immun-Star™ HRP Chemiluminescence Kit, Bio-Rad). The band images were recorded using the ImageQuant LAS 4000 system (GE Healthcare, Japan), and the band intensities were quantified using ImageQuant TL software (version 7.0, GE Healthcare, Japan). Five rats from each group were randomly selected for western blotting.

Calcium oxalate monohydrate (COM) crystals were purchased from Macklin (Shanghai, China). COM crystals were weighed and suspended in sterile phosphate buffer solution at 1 mM and then were diluted into different concentrations. Ang II was purchased from Sangon Biotech (Shanghai, China). Anti-AT1R, anti-NADPH oxidase 2 (Nox2), anti-p50, and anti-p65 antibodies were purchased from Proteintech (Wuhan, China). Losartan and apocynin were purchased from Sigma Aldrich (St. Louis, MO, USA). 2′,7′-Dichlorofluorescein diacetate (DCF-DA) and Fluo-3AM were purchased from Beyotime Institute of Biotechnology (Shanghai, China). Anti-NADPH oxidase 4 (Nox4), anti-OPN, and anti-MCP-1 antibodies were purchased from Abcam (Cambridge, MA, USA). An anti-CD44 antibody was purchased from Cell Signaling Technology (Boston, MA, USA). Malondialdehyde (MDA), superoxide dismutase (SOD), and catalase (CAT) assay kits were purchased from Nanjing Jiancheng Bioengineering Institute (Jiangsu, China). An 8-OHdG assay kit was purchased from Elabscience (Wuhan, China). The transfection reagent Lipofectamine™ 2000 was purchased from Invitrogen (Carlsbad, CA, USA).