Alzet osmotic minipump

All animal procedures were approved by the Animal Care and Use Committee of Fudan University and were in compliance with the Guidelines for the Care and Use of Laboratory Animals published by the National Academy Press (NIH Publication No. 85‐23, revised in 1996). In brief, C57BL/6 male mice, aged 8–10 weeks, were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). Ang II (1000 ng/kg/min.; Sigma‐Aldrich, St. Louis, MO, USA), Ang‐(1‐7) (500 μg/kg/day; Sigma‐Aldrich) and A779 (500 μg/kg/day; Sigma‐Aldrich) were continuously administered using Alzet osmotic minipumps (Model 2002; DURECT, Cupertino, CA, USA) implanted subcutaneously into the experimental animals. Four weeks later, all mice were killed and hearts were excised for further examination.

Nicotine Bitartrate (Sigma-Aldrich, Saint-Louis, MO, USA) was administered to mice as described previously [16 ]. Briefly, a 100 mg/mL solution in PBS or a solution of PBS alone was freshly prepared 24h before pump implantation and loaded into Alzet osmotic minipumps (model 1007D, Durect Corporation). The pumps were implanted subcutaneously on the right side of the back of the mouse while animals were kept under anesthesia using 2–3% of isoflurane, and continuously delivered either PBS or nicotine salt at ~13 mg of nicotine free base/kg/d for the duration of the experiment. After 4 days of nicotine treatment, LPS (1 mg/kg, Sigma-Aldrich) or an equivalent amount of PBS was injected in the intraperitoneal cavity. Mice were then euthanized 1, 6 or 24 hours after the LPS injection.

All experiments were approved by and carried out in accordance with the guidelines of the Norwegian Committee for Experiments on Animals (Forsøksdyrutvalget, FDU: ID 2015-7661). Female outbred Sprague-Dawley rats (Mollegaard) were kept under standard conditions with an artificial 12:12-hour light/dark cycle (lights on: 08:00) and constant 48% humidity. Animals were housed 5 per cage and allowed access to tap water and free (ad libitum) access to standard laboratory chow (Special Diets Services) during the whole experimental period. Care was taken to ensure minimal suffering of the animals at all stages of the experiment. The rats were anesthetized with 2.5% isoflurane gas (Isoba vet; Schering-Plough, Denmark) and Alzet osmotic minipumps (model 2ML4; DURECT Corporation) were implanted according to the manufacturer’s instructions. Rats received either vehicle (n = 9) or 2 mmol/kg/day (84.8 mg/kg/day) of lithium chloride (n = 8) for 4 days. After deep anesthetization and sacrifice by decapitation, truncal blood was collected in EDTA tubes, and brains were rapidly removed from the skull, briefly washed in ice-cold phosphate buffered saline (PBS), and placed on ice. The whole CC was carefully dissected. Plasma concentrations of lithium were photometrically measured on a Cobas 8000 C702 module (Roche Diagnostics).

Early-onset preeclampsia was induced using Alzet^® osmotic mini-pumps (DURECT Corporation, Cupertino, CA, USA, mods. 2002, 1003D) loaded with sFlt1 (recombinant mouse VEGFR type 1/Flt1 Fc chimera; R&D Systems, Minneapolis, MN, USA, cat # 7756-FL). Mini-pumps were implanted intraperitoneally in animals that were anesthetized by controlled isoflurane inhalation vapour (3%) on gestation day (GD) 7, and maintained until GD19. During this period, sFlt1 was infused at a rate of 3.7 µg/kg/day. Samples were processed at GD19 (PE-19 group, n = 8), except for specific experiments performed in an additional group of animals (n = 6) that were sacrificed at GD11 (PE-11 group, i.e., sFlt1 infused from GD7 to GD11) to study potential signs of early renal damage.