Aliskiren

About 1 week after the diabetes was established with the confirmation of significantly increased blood glucose level, either Aliskiren (5 or 25 mg/kg/day) diluted in PBS (n = 12 in each group), olmesartan (10 mg/kg/day) (n = 6), Candesartan (20 mg/kg/day) (n = 6) or PBS alone (n = 12) were administered to the diabetic mice via an osmotic minipump (Alzet model 2006) for 6 weeks. hydralazine (2 or 10 mg/kg/day) (n = 12 in each group) was given in the drinking water. In the hydralazine-treated group, we measured the amount of the water they drank daily. The blood pressures were also measured weekly to make sure the accurate drug delivery. The doses of the drugs were chosen according to the reference [10 (link),17 (link)–20 (link)]. Aliskiren and olmesartan were kindly given from Novartis and Pfizer respectively. Candesartan and hydralazine were purchesed from Sigma.
Some diabetic mice received both Aliskiren (25 mg/kg/day) and intraperitoneal injection of anti-SDF-1 neutralizing monoclonal antibody (mAb, 50 μg; R&D) 3 times per week up to 2 weeks. Mouse IgG1 isotype was administrated as a control. The protocol of animal study was approved by the Institutional Animal Care and Use Committee (IACUC) of National Yang-Ming University, Taipei, Taiwan, ROC. The study was conducted according to European Commission guidelines. (Fig 1).

Osmotic minipump installation was conducted according to our previous studies [6 (link), 7 (link), 13 (link)]. An osmotic minipump (No. 2002, 14 days of active life, Alza Corp) was filled with aliskiren dissolved in deionized water (Novartis Pharmaceuticals Co., NJ, USA), which was implanted subcutaneously in the rats under brief anesthesia with sodium pentobarbital (40 mg/kg BW, intraperitoneal injection).

Elabscience ELISA (Enzyme-Linked Immunosorbent Assay) rat kits were used for TSH, renin, angiotensin I, angiotensin II, and aldosterone analysis. Fosinopril sodium (Monopril) 20 mg manufactured by Deva was purchased from a licensed pharmacy in Istanbul, Turkey. Aliskiren (Rasilez) 150 mg manufactured by Novartis was purchased from Germany. Propylthiouracil (Prouracil) 50 mg manufactured by Iran Hormone was used for induction of hypothyroidism. Levothyroxine (Eltroxin) 100 mcg manufactured by aspen was used for hyperthyroid induction.

Aliskiren (Novartis Pharma Stein AG, Stein) was dissolved in 0.5 ml of normal saline and administered by gavage immediately after preparation as a single daily dose of 60 mg/kg.
Sacubitril (MuseChem) was suspended in 2 ml of 0.5% carboxymethylcellulose in distilled water and 30 mg/kg was administered twice daily by gavage immediately after preparation. Aliskiren was given at least 1 h after the first sacubitril dose to avoid any possible drug interactions. Sham‐operated animals received similar volumes of the vehicles at timings similar to the treated groups.
Treatment of sacubitril and Aliskiren was commenced 6 and 4 days, before the IRI, respectively. The treatment continued daily for 2 days thereafter until the sacrifice of the animals and kidney collection (Figure 1).
As shown in Figure 1, blood was collected from the tail vein to measure serum creatinine and urea at three stages: before starting any medication (basal values), just before the IRI (Pre‐IRI values), and 48 h (Post‐IRI values). Rats were placed in metabolic cages at the same stages to collect urine for measurement of urine volume and levels of albumin and creatinine.

For the blood pressure analysis, mice were anesthetized with intraperitoneal chloral hydrate (300 mg/kg; an anesthetic with limiting effects on blood pressure) and then placed in 25° C or 7° C (plastic plates on ice) environments. Because of the surgical operation, the mice became weak during the experimental course of blood pressure measurements; thus, a relatively milder cold exposure at 7° C was employed, as compared with 4° C that was used in other cold exposure experiments without surgical operation. During anesthesia, the femoral artery was cannulated and connected to a pressure transducer (MEMSCAP, Skoppum, Norway) to record arterial pressure on a polygraph recorder (eDAQ Pty Ltd., New South Wales, Australia). The operation was completed within 10 min, leaving a small wound (<0.5 cm²), and each mouse was placed on a cardboard. The mice were exposed to cold by placing a reusable cold pack (7° C; 3 M, Saint Paul, Minnesota, USA) under half of the cardboard under a mouse's body (head side), and a gauze pad was placed under the opposite side. Cold exposure was maintained for up to 7 h. To analyze the involvement of the RAAS, aliskiren (5 mg/kg; Novartis, Basel, Switzerland) and losartan (3 mg/kg; Merck & Co., Inc., Kenilworth, NJ, USA) were intraperitoneally injected 2 h after cold exposure.