Recombinant human prorenin was expressed in Chinese hamster ovary (CHO) cells and purified as previously described (22). In brief, cells harboring cDNA coding for human prorenin were cultured in Dulbecco's modified Eagle's medium (DMEM; Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) supplemented with 5% fetal bovine serum (FBS; PAA Laboratories GmbH, Pasching, Austria), 0.1 mM non-essential amino acids (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), 2 mM glutamine, 100 U/ml streptomycin and 0.6 mM methotrexate. The cells were maintained at 37°C under 5% CO₂ in 25-cm² cell culture flasks (CELLSTAR^®; Greiner Bio-One GmbH, Frickenhausen, Germany) until they were 100% confluent. Human prorenin was purified from the CHO cell conditioned medium by cation-exchange chromatography on a Resource S column (GE Healthcare, Amersham, UK). The concentration of purified prorenin was measured using a human prorenin enzyme-linked immunosorbent assay (ELISA) kit (cat no. IHPRENKT-NP; Innovative Research, Inc., Novi, MI, USA). Prior to experimentation, purified prorenin was incubated at 37°C for 1 h (23) to avoid cryo-activation due to preservation (24). Recombinant sheep angiotensinogen (AOG) was also expressed in CHO cells and purified as previously described (25,26).

HUVECs (cat no.: KJB-110; DS Pharma Biomedical Co., Ltd., Osaka, Japan) were cultured in human endothelial cell medium (DS Pharma Biomedical Co., Ltd.) supplemented with 2% FBS, essential endothelial growth factors (included in the endothelial cell medium), 0.1% heparin, 1% ascorbic acid, 0.04% hydrocortisone and 0.1% gentamicin/amphotericin-1000. The cells were maintained at 37°C under 5% CO₂ in 25-cm² cell culture flasks (CELLSTAR^®) until they were 80–90% confluent.

HUVECs were seeded at a density of 150,000 cells/well in 12-well sterile plates, and incubated at 37°C. After 24 h, the medium was removed, and the cells were washed with 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES)-buffered saline [(HBS); 1.0 g/l dextrose, 5.0 g/l HEPES, 0.37 g/l KCl, 8.0 g/l NaCl and 0.14 g/l Na₂HPO₄·2H₂O, pH 7.4]. The cells were then treated with or without aliskiren (10 nM; Novartis Pharma AG, Basel, Switzerland) at 37°C for different durations (0, 8, 16, and 24 h) in FBS-free DMEM (Fig. 1). Following each completed duration of treatment: i) the cultured medium (hereafter referred to as Medium-1) was collected to determine the concentration of s(P)RR, and the cells were washed 3 times with HBS and incubated at 37°C with prorenin (1.0 nM) for 1 h; ii) the medium containing the unbound prorenin (hereafter referred to as Medium-2) was collected, and the cells were further washed 3 times with HBS and incubated at 37°C with sheep AOG (0.3 µM) for 30 min in 5 mM sodium phosphate buffer (pH 7.2) containing 5 mM diisopropyl fluorophosphates, 5 mM EDTA, 100 mM NaCl and 0.1% (w/v) bovine serum albumin fraction V (Roche Diagnostics GmbH, Mannheim, Germany); and finally iii) the medium containing the possible generated angiotensin I (AngI; hereafter referred to as Medium-3) was collected. These experimental procedures are depicted in Fig. 1. The cells treated without aliskiren served as controls.

For cell counting, HUVECs were seeded, cultured and treated with or without aliskiren, as described above. After each duration of treatment, the total number of cells per well was counted using a hemocytometer. This cell culture was performed independently of collecting the three media described above.

The concentration of prorenin contained in Medium-2 was determined using the human prorenin ELISA kit, and was considered as the concentration of unbound prorenin (C_UB). The percentage of prorenin bound to the cell membrane was calculated using the following equation: (C_In - C_UB)/C_In ×100, where C_In was the initial concentration of prorenin that was incubated with HUVECs (1.0 nM).

The concentration of AngI in Medium-3 was determined by ELISA using an assay developed and prepared in our laboratory (27). The AngI concentration was divided by the reaction time (30 min) to calculate renin activity.

The concentration of s(P)RR contained in Medium-1 was determined by sandwich ELISA using an assay developed and prepared in our laboratory (20) with a primary anti-(P)RR antibody (anti-human renin R polyclonal antibody; cat no. AF5716; R&D systems, Inc., Minneapolis, MN, USA) and a secondary anti-(P)RR antibody [biotin-labeled anti-237/250-antibody (20)]. The resulting antigen-antibody complex was tagged with a high-sensitivity streptavidin-horseradish peroxidase conjugate, and detected by a chromogenic reaction using hydrogen peroxide and 3,3′,5,5′-tetramethylbenzedine. The s(P)RR standards were prepared as previously described (28).

GraphPad Prism 7.0 software (GraphPad Software, Inc., La Jolla, CA, USA) was used to analyze the data. Data were expressed as the mean ± standard deviation of four replicates for each treatment. The propagation of error formula (29) was used to calculate the standard deviation of either of three ratio values: bound prorenin per cell, renin activity per cell and s(P)RR per cell. A two-tailed unpaired Student's t-test was performed to compare between two groups (aliskiren-treated cells and controls). P<0.05 was considered to indicate statistical significance.

HUVECs were cultured in the presence or absence of aliskiren. The cell number of aliskiren-treated HUVECs was lower than that of the controls, most notably at 16 h (P=0.0090) and 24 h (P=0.0081; Fig. 2). Thus, aliskiren appeared to reduce the growth of HUVECs.

(P)RR binds renin and its inactive precursor, prorenin (1–3). To estimate the expression of full-length (P)RR on the plasma membrane of HUVECs, recombinant prorenin (exogenous prorenin) was added to the culture medium (Fig. 1), and the amount of prorenin bound to the cell membrane was estimated. Following aliskiren treatment for different durations (0, 8, 16 and 24 h), cells were incubated with exogenous prorenin, and the quantity of bound prorenin was evaluated (Fig. 3A). Approximately 10 and 50% of the exogenous prorenin bound to control membranes after 0 and 24 h of treatment, respectively (Fig. 3A, open circles). The quantity of bound prorenin was significantly decreased with aliskiren treatment for 8–24 h (P<0.001; Fig. 3A, filled squares). Additionally, it was observed that the concentration of endogenous prorenin secreted by HUVECs was almost 1,000 times lower than that of exogenous prorenin (data not shown), indicating that endogenous prorenin would have a negligible effect on the estimation of exogenous prorenin bound to HUVEC membrane. The quantity of bound prorenin per cell with aliskiren treatment was similar to that of untreated cells except at 16 h, where significant difference was determined (P=0.0029; Fig. 3B).

To further estimate the amount of full-length (P)RR on HUVEC membranes, the renin activity of membrane-bound exogenous prorenin was measured. As reported previously (1,30,31), the receptor-bound prorenin may become activated to exert renin activity, which can be estimated by measuring the production of AngI following incubation with sheep AOG (Fig. 1). In each condition <3% total sheep AOG was utilized, which indicated that initial velocities of the renin-AOG reaction were estimated appropriately. The renin activity per cell of aliskiren-treated HUVECs was significantly lower than that of the control group by 8 h (P=0.0127), and to the greatest extent at 24 h (P=0.0025; Fig. 3C). After 8 h of treatment, both aliskiren-treated HUVECs and controls exhibited a plateau in renin activity per cell (Fig. 3C).

To examine the effect of aliskiren on the level of s(P)RR released, HUVECs were treated with or without the drug for different durations (0–24 h), and s(P)RR concentration was measured in the culture medium collected following treatment. Not only total s(P)RR concentration (Fig. 4A) but also s(P)RR concentration per cell (Fig. 4B) was significantly reduced by the treatment with aliskiren by 8 h (P<0.05) and most notably at 16–24 h (P<0.01).