Valsartan

Islets from 10-12 week old lean C57BL/6J male and female mice were isolated by collagenase digestion and recovered overnight in RPMI 1640 media. Islets were then pre-incubated for 90 minutes in Krebs buffer containing 2.8 mmol/l glucose, with valsartan (1 μmol/l; Sigma-Aldrich, St Louis, MO, USA) being added during the last 15 minutes. Triplicate batches of islets were then transferred to Krebs buffer containing 2.8 or 20 mmol/l glucose alone, or plus 100 nmol/l angiotensin II (AnaSpec Inc, Freemont, CA, USA), 1 μmol/l valsartan and/or 1 μmol/l sacubitrilat (Sigma-Aldrich, St. Louis, MO, USA). After 60 minutes, supernatant was collected for insulin assay (Alpco, Salem, NH, USA). Doses of angiotensin II and valsartan were based on prior literature (31 (link), 32 (link)). The dose of sacubitrilat, the active metabolite of sacubitril, was chosen to achieve a 1:1 molar ratio with valsartan, as administered in the mouse diet.

HEK293/GC-A⁺, HEK293/GC-A⁺/AT₁⁺, and HEK293/GC-A⁺/AT₂⁺ cells were plated in a 24-well plate and cultured to reach 80–90% confluency before treatment. The treatment buffer included 0.5 mM 3-isobutyl-1-methylxanthine (IBMX, Sigma-Aldrich, St. Louis, MO, USA) to prevent cGMP degradation. Cells were then treated with a treatment buffer (vehicle), 10⁻⁸, 10⁻⁷, or 10⁻⁶ M ANP with and without 10⁻⁸ M ANGII for 10 min before being harvested. Cells were then lysed, sonicated, centrifuged, and the supernatants were extracted and reconstituted in 300 μL 0.1 M HCl for cGMP assay. The samples were then assayed using a cGMP ELISA (Enzo Life Sciences, Farmingdale, NY, USA). To study the effect of valsartan or Go6983 (protein kinase C inhibitor) on cGMP, 1 µM valsartan (Sigma-Aldrich, St. Louis, MO, USA) or 5 µM Go6983 (Bio-Techne Corporation, Minneapolis, MN, USA) were also added together with treatment buffer and peptides as described above.
Human renal proximal tubular epithelial cells (HRPTCs) (ScienCell Research Laboratories, Carlsbad, CA, USA) were maintained and subcultured according to the manufacturer’s protocols. For cGMP generation by ANP with or without ANGII, HRPTCs were cultured in 6-well plate and the same procedure was followed as in HEK293 cells.

Human umbilical vascular endothelial cell (HUVEC) lines (CRL-1730) were obtained from the American Type Culture Collection (Manassas, VA). Cells were cultured in a humidified incubator under 5% CO2 at 37°C using Dulbecco's modified Eagle's medium (DMEM)-F12 media (Corning) supplemented with 10% fetal bovine serum (FBS, Quacell Biotechnology).
The HUVECs were seeded on 6-well plates (Thermo Fisher Scientific) at a density of 2 × 10⁵ cells/well for 24 hours and were serum-starved for 12 hours. The medium was changed to fresh serum-free DMEM/F12 before treatment. To examine the effects of AT1 receptor blockade, HUVECs were pretreated with either 1 μM valsartan (Sigma, USA) (protocol 1) or 1 mM Hcy (Sigma, USA) (protocol 2) for 30 min and then treated with Hcy or valsartan for 24 h, respectively. To determine whether PKC-GSK3β-mTOR pathway is involved in Hcy-induced HUVEC injury, HUVECs were pretreated with 1 μM PKC inhibitor RO318820 (Tocris, UK) or 1 μM GSK3β inhibitor TDZD-8 (MCE, China) for 30 min and then treated with 1 mM Hcy for 24 h (protocol 3).
To determine the effects of autophagy inhibition or ER stress inhibition, HUVECs were pretreated with 10 μM 3-methyladenine (3-MA, MCE, China) or 1 mM 4-phenylbutyric acid (4-PBA, MCE, China) for 30 min and then treated with 1 mM Hcy for 24 h (protocol 4).