Specific pathogen-free experimental animals were obtained from Vital River Laboratories Co., Ltd (Beijing, China). The rats were caged individually under controlled temperature (23±2°C) and humidity (55±5%) with an artificial 12-h light/dark cycle, and were given free access to food and tap water. Wistar rats (40 female, 20 male; age 10–12 weeks; weight 180–220 g) were used for the present study. All experimental procedures were carried out in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (14). All protocols were approved by the Animal Care and Research Committee of Tianjin First Central Hospital (Tianjin, China; Permit number: E20130825-003A).

Estrous female rats were mated overnight with one male. The next morning, the presence of a vaginal plug indicated successful mating and was documented as day 0 of gestation. A total of 40 female pregnant rats were randomly divided into five groups: Control pregnant rats, pregnancy-induced hypertension (PIH) rats, PIH rats + icariin [10 mg/kg, low (L)], PIH rats + icariin [50 mg/kg, medium (M)] and PIH rats + icariin [100 mg/kg, high (H)]. Nitric oxide synthase inhibitor NG-nitro-L-arginine methylester (L-NAME; Cayman Chemical Company, Ann Arbor, MI, USA); 0.5 g/l drinking water) was administered from day 12 of gestation to induce PIH. Icariin was administered intragastrically from day 1 to day 18 of gestation.

Systolic blood pressure (SBP) and diastolic blood pressure (DBP) in pregnant rats were measured at day 1 and day 18 of gestation with the CODA^TM2 non-invasive single channel blood pressure measuring instrument (Shanghai Zande Medical Devices Co., Ltd., Shanghai, China).

Rats were selected randomly from each group and were placed in metabolic cages to collect urine for 24 h. The total volume of urine was recorded and used to detect urinary total protein and concentration. Urine protein concentration was measured using the Coomassie Brilliant Blue method, serum creatinine was measured using the picric acid method and blood urea nitrogen (BUN) was measured via an enzymatic kinetic method using commercial kits purchased from Nanjing Jiancheng Biological Engineering Research Institute (Nanjing, China). Plasma levels of angiotensin II (Ang II) were detected using a bioactive Ang II ELISA assay (catalog no. C506065; Sangon Biotech, Co., Ltd., Shanghai, China), according to the manufacturer's protocol.

Kidney tissues were collected at day 18 of gestation by intraperitoneal injection of sodium pentobarbital (2%; 200 mg/kg; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) and were fixed with 4% formalin at room temperature for 24 h and paraffin-embedded. Tissues were then cut into ~5 µm-thick sections, which were stained with H&E at room temperature for 1–2 min and visualized under a microscope (Leica DM 2500; Leica Microsystems GmbH, Wetzlar, Germany).

The immunohistochemical staining analysis of kidney tissue was evaluated using anti-rat Ang II (catalog no. sc-20718; 1:50; Santa Cruz Biotechnology, Inc., Dallas, TX, USA). In brief, the paraffin sections (5 µm) were heated in an oven at 65°C for 24 h, dewaxed with water and rinsed with PBS for 5 min, 3 times. Paraffin sections were then placed in EDTA buffer (Beyotime Institute of Biotechnology, Haimen, China) for microwave antigen retrieval and boiled for 10 min. Following natural cooling, the sections were washed with PBS 3 times. The sections were placed into 3% hydrogen peroxide solution and incubated at room temperature for 10 min, to block endogenous peroxidase activity, and then washed with PBS 3 times. They were then blocked with 5% bovine serum albumin (BSA; Beyotime Institute of Biotechnology) for 20 min at room temperature, following drying. Following removal of BSA liquid, each section was incubated with 50 µl diluted anti-rat Ang II primary antibody overnight at 4°C, then washed with PBS 3 times. Following the removal of PBS liquid, each slice was incubated with 50–100 µl goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (catalog no. sc-2004; 1:2,000; Santa Cruz Biotechnology, Inc.) at 4°C for 50 min, then washed with PBS 3 times. A total of 50–100 µl freshly prepared DAB solution was added to each sample. Following washing, sections were counterstained with hematoxylin at room temperature for 5 min, rinsed with tap water, dehydrated, mounted and visualized under a microscope (Leica DM 2500; Leica Microsystems GmbH).

The renal total RNA extraction was performed using TRIzol^®, according to the manufacturer's protocol (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Synthesis of cDNAs was performed by RT reactions with 2 µg total RNA using moloney murine leukemia virus reverse transcriptase (Invitrogen; Thermo Fisher Scientific, Inc.) with oligo dT 15 primers (Fermentas; Thermo Fisher Scientific, Inc.) and 4 µl Maxima 5X Reaction Mix (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. The RT temperature protocol was as follows: 37°C for 50 min and 70°C for 15 min. The first strand cDNAs served as the template for the PCR, performed using a DNA Engine (ABI 7300; Thermo Fisher Scientific, Inc.). GAPDH served as an internal control, and was used to normalize the data to determine the relative expression of the target genes using the 2^−∆∆Cq method (15). Reaction mixtures (20 µl) were prepared using the TaqMan Universal PCR Master Mix (Thermo Fisher Scientific, Inc.). The reaction conditions were set according to the manufacturer's protocol. The PCR primers used in this study were as follows: Forward, 5′-AGCTCGTGTCTCCCAGAGT-3′ and reverse, 5′-CGTTCACGTTTGCAGAGATGT-3′ for nephrin; forward, 5′-CTGGAGCTAAAGGACACACAGA-3′ and reverse, 5′-GTGAAGGGACCCAAGCTCTC-3′ for angiotensinogen (AGT) and forward, 5′-GGATTTGGTCGTATTGGG-3′ and reverse, 5′-GGAAGATGGTGATGGGATT-3′ for GAPDH.

The kidney was homogenized and protein extracted using NP-40 buffer (Beyotime Institute of Biotechnology), followed by 5–10 min boiling and centrifugation (4°C, 10 min, 12,000 × g) to obtain the supernatant. Protein concentrations were determined using the Bicinchoninic Acid kit for Protein Determination (Sigma-Aldrich; Merck KGaA). Samples containing 50 µg protein were separated on 10% SDS-PAGE gel and transferred to nitrocellulose membranes (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Following saturation with 5% (w/v) non-fat dry milk in TBS and 0.1% (w/v) Tween-20 (TBST), the membranes were incubated with the following primary antibodies, against nephrin (catalog no. sc-377246; 1:1,000; Santa Cruz Biotechnology, Inc.) and Ang II (catalog no. sc-20718; 1:500; Santa Cruz Biotechnology, Inc.) at 4°C overnight. Following 3 washes with TBST, membranes were incubated with secondary immunoglobulins (donkey anti-goat IgG; catalog no. sc-2020; 1:10,000; Santa Cruz Biotechnology, Inc.) conjugated to IRDye 800 CW Infrared Dye (LI-COR Biosciences, Lincoln, NE, USA). Following a 2 h incubation period at 37°C, membranes were washed 3 times with TBST. Blots were visualized using the Odyssey Infrared Imaging System (LI-COR Biosciences). Signals were densitometrically assessed (Odyssey Application Software, version 3.0; LI-COR Biosciences) and normalized to the GAPDH signals to correct for unequal loading (catalog no. sc-365062; 1:2,000; Santa Cruz Biotechnology, Inc.).

The data from these experiments are presented as the mean ± standard deviation for each group. All statistical analyses were performed using PRISM version 4.0 (GraphPad Software, Inc., La Jolla, CA, USA). Inter-group differences were analyzed using one-way analysis of variance followed by Tukey's multiple comparison test as a post-hoc test to compare the group means. P<0.05 was considered to indicate a statistically significant difference.

Firstly, the SBP and DBP were measured in the control, PIH and icariin treated rats, and it was demonstrated that no significant differences were present in SBP (Fig. 1A) and DBP (Fig. 1B) among the five experimental groups on day 1. However, SBP and DBP were significantly increased in the PIH group compared with control group on day 18. The significant increase in SBP in PIH rats was prevented by icariin at a high concentration on day 18. However, icariin administration had no effect on DBP compared with PIH group on day 18 (Fig. 1C and D).

The urinary protein excretion increased ~4-fold in PIH rats compared with control pregnant rats (Fig. 2A). However, icariin administration reversed increased urinary protein in PIH rats at medium and high concentrations on day 18 (Fig. 2A). Furthermore, BUN and serum creatinine were significantly elevated in PIH rats compared with control pregnant rats. BUN and serum creatinine levels in PIH rats were attenuated following treatment with icariin at medium and high concentrations on day 18 (Fig. 2B and C).

Litters from PIH rats were significantly smaller in number compared with control pregnant rats, and icariin prevented the reduction in litter number induced by PIH (Fig. 3A). Furthermore, L-NAME administration (PIH group) resulted in a reduction in the average weight of the pup, however, icariin administration did not result in any significant weight gain of the pups during pregnancy (Fig. 3B).

H&E staining demonstrated that L-NAME administration resulted in severe mesangial expansion and significant basement membrane thickening, however, kidney morphology was well preserved in control pregnant rats. Compared with the PIH group, rats with L-NAME-induced PIH treated with icariin (50 or 100 mg/kg) exhibited markedly reduced severity of glomerular lesions (Fig. 4A). The podocyte protein nephrin is essential for maintaining the filtration barrier of the kidney and preventing proteinuria (16). A previous study indicated that glomerular expression of nephrin is decreased in kidney sections from women with pre-eclampsia (17). In the present study, the expression of nephrin was measured in the kidney from control pregnant and L-NAME administered PIH rats. The results demonstrated that L-NAME administration exerted a marked decrease in the mRNA and protein expression of nephrin in the kidneys, compared with the control group. However, icariin (50 or 100 mg/kg) treatment significantly reversed L-NAME-induced downregulation of nephrin levels in the kidney (Fig. 4B and C).

Previous studies suggest that the circulating and local renin-angiotensin systems (RAS) are activated during rat pregnancy (18,19). However, the role of local RAS activity in the kidney of PIH rats remains to be elucidated. The present study investigated the renoprotective effects of icariin in PIH rats, and the association between icariin and the local renal RAS in the progression of PIH was examined. The expression of Ang II, as a key active peptide in the RAS, was measured in the kidney of control pregnant and PIH rats. The results demonstrated that circulating Ang II and AGT mRNA expression levels were significantly elevated in PIH rats compared with control pregnant rats. Rats that received icariin at the two greater doses (50 or 100 mg/kg), exhibited alleviated circulating Ang II levels (Fig. 5A) and AGT mRNA expression (Fig. 5B), compared with the PIH group. Furthermore, the protein expression of Ang II was significantly increased in the kidneys of PIH rats compared with control group. However, icariin administration significantly reversed the L-NAME-induced upregulation of Ang II levels in the kidney (Fig. 5C). These effects of icariin on Ang II expression were verified by immunohistochemical staining (Fig. 5D).