A total of 40 male Sprague-Dawley rats (250.2±1.9 g; 6 weeks-old) were obtained from the Experimental Animal Center of the Sun Yat-sen University (Guangzhou, China). All rats were housed in plastic cages at 25°C under a 12-h light-dark cycle and were provided with rodent chow and water. They were housed for one week to adapt to their environment prior to the start of the study. The study was conducted 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 the Sun Yat-sen University. The current study was approved by the Committee of Biomedical Ethics of the Sun Yat-sen University [IACUC-2013-0604].

Allopurinol tablets were purchased from Guangzhou Kanghe Pharmaceutical Co., Ltd. (Guangzhou, China), diluted with distilled water to a final concentration of 5 mg/ml. Adenine tablets were purchased from Amresco LLC (Solon, OH, USA) and diluted with 0.15% sodium carboxymethylcellulose (CMC-Na) to a final concentration of 3%. Hematoxylin (DF001) was purchased from Guangzhou Dingguo Bio-technology Co., Ltd. (Guangzhou, China). TRIzol, reverse transcription buffer, SYBR green I polymerase chain reaction (PCR) buffer, dNTPs, MMLV and Taq DNA polymerase were purchased from Takara Biotechnology, Co., Ltd. (Dalian, China). Rabbit polyclonal OAT1 antibody purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). OAT1 constructs were purchased from OriGene Technologies, Inc. (Beijing, China) in wild-type form and inserted a myc tail in the C terminal by routine sub-cloning (23). The RhoA construct was also purchased from OriGene Technologies, Inc. and the constitutively active RhoA G14V, and dominant-negative RhoA T19N site mutations were produced using the QuikChange Site-Directed Mutagenesis kit (with PfuUltra High-Fidelity DNA Polymerase; Agilent Technologies, Inc., Santa Clara, CA, USA) and followed by DpnI (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) digestion of the template DNA. The surface protein extraction kit was purchased from BioVersion Life Sciences (Dehradun, India), sulfo-NHS-SS-biotin and Avidin Agarose beads were purchased from Thermo Fisher Scientific, Inc. MSU crystals were produced as previously described (24).

The 40 Sprague-Dawley rats were randomly assigned into 4 groups. All rats were treated by intra-gastric administration. Group 1 was allocated as the control and was fed 5 ml CMC-Na (adenine dissolving buffer), while in the remaining 3 groups, rats were fed with adenine (30 mg/kg body weight) for 18 days to establish the UAN model. On the 19th day, 3 rats were randomly selected, anesthetized using 10% chloral hydrate (0.4 ml/100 g; Sigma-Aldrich, St. Louis, MO, USA), sacrificed using cervical dislocation and kidney samples were collected. Hematoxylin-eosin (H&E) staining of the kidney tissue was then conducted to assess whether the UAN model was successfully established. The 4 groups of rats were treated as follows: Group 1, blank control, rats were fed with the same amount of saline buffer; group 2, disease model control, rats were administered with the same amount of saline buffer as in group 1; group 3, allopurinol group, rats were treated with allopurinol (5 mg/kg body weight/day); group 4, folic acid group, rats were treated with folic acid (2 mg/250 g body weight/day). In all groups, rats were administered with the drug or saline treatment for 23 days. During the process, no rats died. Samples were collected 18 days subsequent to model establishment, and 5 ml peripheral blood (fasting for 12 h) was collected for detection of blood urea nitrogen (BUN), serum creatinine (Scr) and serum uric acid (UA). At the end of the 42-day experimental procedure, rats were fasted for 12 h, and then sacrificed under 20% urethane anesthesia (Sigma-Aldrich). Peripheral blood was collected to detect BUN, Scr and UA, and the left kidney was resected for further investigation.

Kidney sections were stained with hematoxylin for 2 min and then eosin for 30 sec. Subsequent to washing, the slides were dehydrated and sealed with Entellan (Merck Millipore, Darmstadt, Germany) prior to examination under a light microscope (model BX43/53; Olympus Corporation, Tokyo, Japan). Quantification of cell densities was conducted using AlphaEaseFC software version 4.0 (Alpha Innotech Corporation, San Leandro, CA, USA). Results were analyzed using Image Pro Plus software, version 6.0 (Media Cybernetics, Inc., Rockville, MD, USA), and in each section 10 fields were selected (magnification, ×200).

Kidney tissues from rats in the different groups were frozen and stored at −20°C immediately subsequent to dissection. The tissues were mechanically dissociated and homogenized (model AH96; Omni International, Kennesaw, GA, USA) following the manufacturer's instructions to obtain the surface protein. For HEK cell (American Type Culture Collection, Manassas, VA, USA) surface biotinylation, OAT1-stable HEK cells were treated with monosodium urate (MSU) for 24 h and washed twice with cold phosphate-buffered saline (PBS). OAT1-stable HEK cells without MSU treatment were used as a control. The cells were then incubated with PBS supplemented with 0.5 mg/ml sulfo-NHS-SS-biotin for 1 h with gentle shaking, and excess biotin was quenched with 50 mM Tris-PBS buffer (Sigma-Aldrich) for 20 min. Cells were lysed in radioimmunoprecipitation assay (RIPA) buffer and then were subjected to streptavidin-agarose beads at 4°C for an additional 3 h. For the HEK cell internalization assay, surface biotinylated cells were incubated at 37°C for 30 min, subsequent to quenching, to allow internalization, then were washed with cold cleavage buffer [50 mM glutathione, 90 mM NaCl, 1.25 mM CaCl₂ dihydrate, 1.25 mM MgSO₄ (all from Sigma-Aldrich), and 0.2% endotoxin-free bovine serum albumin (BSA; Cell Signaling Technology, Inc., Danvers, MA, USA), pH 8.6] for 20 min with gentle shaking. Cells were lysed in RIPA buffer, then were subjected to streptavidin-agarose beads at 4°C for another 3 h. Protein samples were boiled in Laemmli buffer (Amresco LLC), separated by 12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (ProSpec, Ness-Ziona, Israel), then transferred to polyvinylidene difluoride membranes (Hybond-C-extra; GE Healthcare Life Sciences, Chalfont, UK). Membranes were blocked using 5% BSA in Tris-buffered saline with Tween-20 (TBST; Sigma-Aldrich) and then were incubated overnight at 4°C with mouse monoclonal anti-OAT1 (1:100; cat. no. ab118346; Abcam, Cambridge, MA, USA) or rabbit monoclonal anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:8,000; cat. no. ab181603; Abcam) antibodies. The membranes were then washed in TBST and incubated with peroxidase-conjugated goat anti-rabbit or anti-mouse antibodies (1:4,000; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) for 1 h. Protein bands were developed using Enhanced Chemiluminescence plus reagent (Western Lightning; PerkinElmer, Inc., Waltham, MA, USA).

Total RNA (2 µg) was extracted with TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.) and converted to cDNA (1 µg) using the reverse transcription kit (Takara Biotechnology, Co., Ltd.) according to the manufacturer's instructions. RT-qPCR was conducted according to the following procedure: 94°C for 3 min, followed by 40 cycles of 94°C for 30 sec, 55°C for 45 sec and 72°C for 1 min. OAT1, F 5′-CAGCCAAGGAGGCTGCTGTC-3′ and R 5′-AGTCAAACCTTTTAATGA TG-3′; GAPDH, F 5′-TGGTCTACATGTTCCAGTATGACT-3′ and R 5′-CCATTTGATGTTAGCGGGATCTC-3′. Data were presented as the normalized OAT1 mRNA level/GAPDH mRNA level. Results are expressed as the mean ± standard error of three independent experiments.

OAT1 stable HEK cells were treated with or without MSU for 24 h and live cells were used for staining the OAT1 extracellular region at 4°C for 1 h. Cells were then transferred back to the 37°C incubator for 30 min to allow for internalization. Cells were fixed with 4% paraformaldehyde (Sigma-Aldrich) and surface stained using the Alexa Fluor 488 donkey anti-goat polyclonal antibody (1:200; cat. no. ab150129; Abcam) for 1 h. Subsequently, the cells were permeabilized with 0.25% Triton X-100 (Sigma-Aldrich) for 5 min and incubated with the mouse monoclonal anti-Myc antibody (1:1,000; cat. no. ab23; Abcam) at 4°C overnight. On the second day, cells were stained with the Alexa Fluor 594 donkey anti-goat antibody (1:300; cat. no. ab150132; Abcam) for internalized OAT1 and Alexa Fluor 405 goat anti-mouse polyclonal antibody (1:200; cat. no. ab175660; Abcam) for total OAT1. Representative images were captured using an Olympus FV1000 confocal microscope (Olympus Corporation, Tokyo, Japan).

Quantitative data was expressed as the mean ± standard error. Comparisons of the means among multiple groups were performed using a one-way analysis of variance followed by Dunnett's or Tukey-Kramer's post hoc tests using GraphPad Prism software, version 4.0 (GraphPad Software, Inc., La Jolla, CA, USA). Quantification of images were conducted using Image J software (National Institutes of Health, Bethesda, MD, USA). Asterisks were used to indicate the following: ^*P<0.05, ^**P<0.01 and ^***P<0.001. P<0.05 was considered to indicate a statistically significant difference.

Compared with the controls, serum UA, BUN and creatinine levels in adenine-treated rats (UAN model rats) were significantly increased, suggesting that the UAN model had been successfully established. Treatment with allopurinol led to a reduction in serum UA, BUN and creatinine levels compared with the UAN model group, however, serum BUN and creatinine levels of allopurinol-treated rats remained significantly greater than those of the control group (Fig. 1A–C). Using H&E staining, it was additionally identified that UAN model rats exhibited kidney damage (14) (renal interstitial tissue was characterized by edema, inflammatory cell infiltration, granuloma hyperplasia, glomeruli exhibiting partial atrophy and tubular cells with diffuse swelling), while treatment with allopurinol attenuated these kidney impairments (Fig. 1D). The above-mentioned data indicate that the model was successful as previously reported (18).

To further investigate the role of OAT1 in UAN, the OAT1 protein expression levels were measured. As presented in Fig. 1E, no significant different in the total OAT1 level was observed between the different groups, and the mRNA levels of OAT1 were not altered (Fig. 1F). As OAT1 is a membrane protein, the total plasma membrane proteins were extracted, and the surface OAT1 levels were measured. It was identified that although total OAT1 expression was not altered, there was a significant reduction in the level of surface OAT1 in the UAN group (Fig. 1G). The results indicated that high uric acid generation may reduce the surface OAT1 levels.

To further investigate the detailed mechanism of how uric acid regulates OAT1 expression or trafficking, OAT1 stable expressing HEK cells were used and they were treated with or without MSU crystals (1 µM) for 24 h. The results demonstrated that treatment with MSU crystals resulted in a reduction in surface OAT1 levels however not in the total protein (Fig. 2A) or mRNA levels (Fig. 2B). The internalized OAT1 levels were then measured, and it was identified that following treatment with MSU crystals, OAT1 internalization was increased (Fig. 2C and D). Notably, it was observed that treatment of HEK cells exposed to MSU with folic acid led to a reversal of these effects (Fig. 2A–D). Using confocal imaging it was also confirmed that although total OAT1 was not significantly altered (Fig. 2E), the reduction in surface expression levels corresponded with an increase in OAT1 internalization, while treatment with folic acid reversed this effect (Fig. 2F).

How the OAT1 trafficking process is regulated was then investigated. Considering that the cytoskeleton is a major component that controls vesicular movement, the experiments focused upon the small guanosine triphosphatase, RhoA, which is known to regulate actin-stress fiber formation and facilitates endocytosis following activation (24,25). HEK cells were treated with uric acid crystals, and it was identified that although total RhoA levels were not significantly altered, the guanosine triphosphate bound form of RhoA (active form) was increased significantly (Fig. 3A B, C and D). Notably, treatment with folic acid led to a reversal in RhoA levels. To further investigate the effects of RhoA on OAT1 internalization, constitutively active RhoA G14V and dominant-negative RhoA T19N site mutation constructs were produced. By overexpression of these constructs in OAT1-HEK cells, RhoA V14 was identified to lead to an increase in OAT1 internalization without crystal stimulation, while RhoA N19 inhibited uric acid-induced OAT1 internalization (Fig. 3E and F).

As it has been previously reported that folic acid was able to inhibit RhoA activity in cell models (13), the current study aimed to demonstrate whether folic acid possesses benefits on high uric acid-induced kidney damage. The results demonstrated that daily supplements of folic acid were able to rescue uric acid induced kidney damage and repress serum uric acid, BUN and creatinine levels by restoring the levels of surface OAT1 back to normal levels in vivo (Fig. 1). Treatment with MSU-treated HEK cells with or without folic acid (10 µM) demonstrated that folic acid was able to rescue OAT1 membrane distribution by inhibiting RhoA activation (Figs. 2 and 3).