A total of 30 male Sprague Dawley rats (weight, 167.2±1.9 g; age, 6–8 weeks), were randomly assigned into three experimental groups. The rats were housed in temperature-(22–25°;C) and humidity-(40–70%) controlled conditions with a 12-h light/dark cycle and ad libitum access to food and water. All rats were treated by intragastric administration. Group 2 and 3 rats were administered with 30 mg/kg adenine once a day for 18 days to establish a rat model of UAN. Group 1 was allocated as the normal control group and rats werefed an equivalent amount of carboxymethylcellulose sodium (CMC-Na; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). Rats in Group 2 were then fed with an equivalent amount of saline buffer (30 mg/kg body weight) for 18 days to establish the disease model. Rats in Group 3 were fed with a single dose of fucoidan polysaccharide sulfate (400 mg/kg body weight) to establish the fucoidan treatment group.

Rats were obtained from the Experimental Animal Center of Sun Yat-sen University (Guangzhou, China). The research was carried out in accordance with the Guidelines for Human Treatment of Animals set by the Association of Laboratory Animal Sciences and the Center for Laboratory Animal Sciences at Sun Yat-sen University. The study was approved by the Committee of Biomedical Ethics of Sun Yat-sen University [SYXK (yue): 2007-0081].

Fucoidan polysaccharide sulfate was diluted with 10% dimethyl sulfoxide in ethanol to a final concentration of 50 mg/ml. Fucoidan was purchased from South Product Co., Ltd. (Uruma, Japan). Adenine tablets were purchased from Amresco, LLC (Solon, OH, USA) and diluted with 0.15% CMC-Nato a final concentration of 3%. Cell Surface Protein Isolation kit (K295) was purchased from BioVision, Inc., (Milpitas, CA, USA). Sulfo-NHS-SS-biotin and Avidin Agarose beads were purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). COS-7 cells were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (both Gibco; Thermo Fisher Scientific, Inc.) and 100 U/ml penicillin and 100 U/ml streptomycin (Amresco, LLC). The cells were cultured in a 5% CO₂ atmosphere at 37°C. 8-bromo-cAMP (Sigma-Aldrich; Merck KGaA) was used asaPKA activator. OCT2-expressing COS-7 cells were treated with PKA activator 8-bromo-cAMP (1 µmol/l) or phosphate-buffered saline (PBS) for 12 h at 37°C.

After treatment for 18 days, rats were euthanized by CO₂ inhalation, and the renal tissues from rats in different groups were frozen and stored at −20°C immediately after dissection. Renal tissues were mechanically dissociated and homogenized according to the manufacturer's instructions for the surface protein extraction kit. For COS-7 cell surface biotinylation, after treatment with fucoidan (500 µg/ml) or control salinefor 24 h, cells were washed and incubated with PBS supplemented with 0.5 mg/ml sulfo-NHS-SS biotin for 40 min at 4°C, and excess biotin was quenched with 50 mM Tris-PBS buffer for 20 min at 4°C. Subsequently, cells were collected, lysed in radioimmunoprecipitation buffer from BioVision, Inc., and subjected to streptavidin-agarose beads at 4°C for a further 3 h. The protein samples (30 µg) obtained were boiled, subjected to 12% SDS-PAGE and transferred to polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked with Tris-buffered saline-Tween-20 containing 2% bovine serum albumin (Sigma-Aldrich; Merck KGaA) at room temperature for 2 h, and the membranes were then incubated with specific antibodies against OCT2 (sc-233; Santa Cruz Biotechnology, Inc., Dallas, TX, USA; 1:1,500), PKA 2β (sc-365615; Santa Cruz Biotechnology, Inc; 1:1,500), p-PKA 2β (sc-293036; Santa Cruz Biotechnology, Inc.; 1:1,000), GAPDH (ab8245; Abcam, Cambridge, UK; 1:8,000). Horseradish peroxidase-conjugated goat anti-rabbit secondary antibodies (20162; ProMab Biotechnologies, Inc., Richmond, CA, USA; 1:1,000) were used. For incubation, all antibodies were incubated at 4°C for 12 h. The membranes were washed inTBST (Sigma-Aldrich; Merck KGaA) at room temperature four times between the incubations with the primary and secondary antibodies. Protein bands were detected using an enhanced chemiluminescence reaction (Biological Industries, Kibbutz Beit Haemek, Israel). The intensity of each band was quantified using Quantity One software v4.62 (Bio-Rad Laboratories, Inc., Hercules, CA, USA). All experiments were repeated three times.

Renal tissues of rats were removed, fixed with 4% paraformaldehyde in PBS at room temperature for 2 h, decalcified for 10 days at room temperature with EDTA and embedded in paraffin for histological analysis. Kidney sections (5 µm thick) were stained with hematoxylin for 2 min, then eosin for 30 sec. Images of the kidney from multiple sections in each rat were observed under a light microscope with identical parameters (magnification, resolution and light intensity).

In brief, rat kidneys were harvested, cut along the short axis at the maximum area of the whole kidney, fixed in Carnoy's solution (Beyotime Institute of Biotechnology, Haimen, China) at 37°C for 2 h, embedded in paraffin, sectioned (1 µm thick), and stained with PAM. Images of the kidney from multiple sections in each rat were observed under a light microscope with identical parameters (magnification, resolution and light intensity).

Immunohistochemistry was performed by National Engineering Center for Biochip at Shanghai (China), as described previously (16,17). Immunohistochemistry was carried out with antibodies specific for PKA using anti-rat PKA (sc-365615; Santa Cruz Biotechnology, Inc.; 1:300) in blocking buffer and incubated overnight at 4°C. Negative control sections were incubated with PBS instead of primary antibodies.

Green fluorescent protein (GFP)-OCT2 and GFP-C (control) vector plasmids were purchased from OriGene Technologies, Inc., (Rockville, MD, USA) and transfected into COS-7 cells using a Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) reagent, according to the manufacturer's protocol.

Frozen renal tissue sections (3 µm thick) were cut and mounted on 3-aminopropyltriethoxysilane-treated glass slides. The TUNEL protocol was performed using the In Situ Cell Death Detection kit (Sigma-Aldrich; Merck KGaA), according to the manufacturer's instructions. Sections were dried overnight, fixed in 1% formal dehydeat room temperature for 2 h, washed with PBS four times for 5 min each, permeabilized with 0.2% Triton X-100 at 4°C for 10 min, and re-washed with PBS before application of TUNEL reagents. Images were observed under a light microscope with identical parameters (magnification, resolution and light intensity).

All data were presented as the mean ± standard error of the mean of at least three independent experiments. Student's t-test was used to evaluate differences between two groups with GraphPad Prism v5.01 (GraphPad Software, Inc., La Jolla, CA, USA). P<0.05 was considered to indicate a statistically significant result.

An experimental model of UAN was induced using uricopoiesis promoter adenine. Histopathological analysis of renal tissues in each group was performed. H&E staining of kidney tissue indicated that the control group maintained normal glomerular size, integrity and demonstrated no notable inflammatory response (Fig. 1A). In the UAN model group, cellular infiltrate in the tubules, atrophic glomeruli, tubular ectasia, granuloma hyperplasia focal fibrosis and accumulated urate crystals were observed. However, treatment with fucoidan decreased the deposition of urate crystals and inflammatory cellular influx in the tubule compared with the model group. Furthermore, tubular ectasia and interstitial fibrosis were improved by fucoidan treatment, suggesting that kidney damage was attenuated by fucoidan. The renal tissues were also processed and stained with PAM-Masson for examination under a light microscope. The PAM-Masson results revealed that renal interstitial fibrosis was less prominent among renal tissues from the fucoidan treatment group compared with the model group (Fig. 1B). In addition, the fucoidan treatment group demonstrated a lower proportion of apoptotic nuclei in the kidney compared with the model group, as assessed by TUNEL staining (Fig. 1C). These results suggested that fucoidan was able to alleviate the symptoms of UAN in a rat model.

Given that fucoidan alleviated the symptoms of UAN, further study to investigate the specific molecular mechanism was required. Thus, the protein levels of PKA 2β, p-PKA 2β, surface OCT2 and total OCT2 were evaluated in renal tissues of the different groups. As demonstrated in Fig. 2A and B, PKA 2β and p-PKA 2β protein expression levels were significantly elevated in the renal tissues of the fucoidan treatment group compared with the model group (P<0.05 and P<0.01, respectively), suggesting that increased expression of PKA occurred in kidney tissue of fucoidan treatment group rats. The upregulation of PKA protein expression was further demonstrated by immunohistochemistry staining of PKA in rat renal tissues (Fig. 2C). The level of surface OCT2 protein was significantly increased by fucoidan treatment compared with the model group (P<0.01), with no significant change in total OCT2 level (Fig. 2A and B).

To further ascertain the role of fucoidan in regulating PKA and surface OCT2 expression, COS-7 cells ectopically expressing OCT2 were established. COS-7 cells ectopically expressing OCT2 were treated with fucoidan (500 µg/ml) or PBS for 24 h. A significant increase in the proportion of surface OCT2 protein was induced by fucoidan treatment compared with the control (P<0.05; Fig. 3), with no significant difference in total OCT2 level between the two groups. A significantly elevated PKA 2β and p-PKA 2β level was observed in OCT2-expressing COS-7 cells treated with fucoidan compared with the control (both P<0.05; Fig. 3). These results demonstrated that fucoidan may increase the expression of PKA and upregulate surface OCT2 in OCT2-expressing COS-7 cells.

Previous studies have reported that PKA regulates OCT2-mediated organic cation transport (18,19). The increased expression of PKA by forskolin leads to stimulation of 4-[4-(dimethylamino) styryl]-N-methylpyridinium uptake in IHKE-1 cells (20). To study the relationship between PKA expression and surface OCT2 upregulation in COS-7 cells, surface, intracellular and total OCT2 protein levels were evaluated in OCT2-expressing COS-7 cells treated with PKA activator 8-bromo-cAMP. OCT2-expressing COS-7 cells were treated with PKA activator, 8-bromo-cAMP (1 µmol/l) or PBS. As demonstrated in Fig. 4, in the 8-bromo-cAMP-treated COS-7 cells, the surface OCT2 protein level was increased significantly compared with the control (P<0.05), while intracellular OCT2 level decreased significantly (P<0.01), with no significant change in total OCT2. The findings presented in the present study indicate that fucoidan may upregulate surface OCT2 via PKA to alleviate the symptoms of UAN.