N 4 hydroxyphenyl retinamide 4 hpr

Male Wistar rats (120–150 g) were purchased from the Vital River Laboratories (Beijing, China). All rats were raised in an environment with a 12-h light/dark cycle at a temperature of 22 ± 1°C with free access to food and water. Animal care and all experiments were performed according to the guidelines established by the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Animal Care Committee of Peking Union Medical College & Chinese Academy of Medical Sciences (PUMC&CAMS). Porcine serum was purchased from Solarbio Science & Technology (Beijing, China). Lipopolysaccharide (LPS), D-galactosamine (D-gal), and N-(4-hydroxyphenyl) retinamide (4-HPR) were purchased from Sigma-Aldrich (St Louis, MO, USA). Cytochrome C primary antibody was purchased from Santa Cruz (Dallas, Texas, USA). Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) kits were purchased from Biosino (Beijing, China). Prothrombin times (PTs) determination kit and plasma ammonia kit were purchased from the Nanjing Institute of Jiancheng Bioengineering (Nanjing, China).

Reactive oxygen species (ROS) production in HK-2 cells in response to HG treatment was detected as described previously.11 (link) The HK-2 cells were plated on six-well plates at 5×10⁴ cells per well, cultured overnight, and then pretreated with or without NHE1/PKG inhibitor before being incubated with 30 mM HG. DMSO and N-(4-hydroxyphenyl) retinamide (4HPR) (Sigma-Aldrich, St. Louis, MO, USA) were used as controls. After 48 h of incubation, the cells were harvested, washed in PBS, and then loaded with 50 µM 2',7'-dichlorofluorescein-diacetate (DCFH-DA) at 37℃ for 10 min. Fluorescence was detected using a FACSCanto cytometer (BD) on the FL1 channel and analyzed with FACSDiva 6.0 software.