Hyperuricaemia (HUA) is an independent risk factor for chronic kidney disease. Urate crystals are deposited in the kidney and can cause renal tubular interstitial fibrosis, leading to renal dysfunction. Chicory extract (hereafter referred to as chicory) clearly reduced serum uric acid levels in rats with HUA induced by 10% fructose. This is the first study to observe the effect of chicory on serum uric acid levels and renal function in rats with HUA and renal injury. In vivo studies using hyperuricaemic rats with renal injury induced by yeast and adenine demonstrated that chicory decreased serum uric acid level, and its effect of delaying the progression of kidney injury was better than that of benzbromarone. In vitro cell experiments showed that this effect is related to the inhibition of GLUT9 protein expression in renal tubules and that lowering blood uric acid concentrations is one of the factors that alleviates renal damage. The results of this study indicate that chicory can be used as an alternative for alleviating renal dysfunction in hyperuricaemia.
Hyperuricaemia (HUA) is a metabolic disorder associated with an abnormally high uric acid level in blood, and HUA is an independent risk factor for chronic kidney disease [
Approximately, ninety percent of hyperuricaemia cases are caused by insufficient excretion of uric acid [
Antihyperuricaemia drugs inhibit or activate these transporters, thereby increasing uric acid excretion in the kidneys to lower blood uric acid levels. Although their pharmacological effect of reducing serum uric acid is clear, these drugs do not effectively delay the progression of renal injury caused by high blood uric acid [
In a previous experiment, we found that chicory could decrease serum uric acid levels by inhibiting the activity of xanthine oxidase (XOD), the enzyme that catalyses the generation of uric acid from hypoxanthine and xanthine, and by promoting uric acid excretion by upregulating the mRNA expression of OAT3 in hyperuricaemic rats [
Therefore, the objective of this study was to examine the effect of chicory on serum uric acid and renal function in hyperuricaemic rats with renal injury through renal urate excretion and GLUT9 expression based on our previous research. In vitro cell experiments were used to verify the effect of GLUT9 expression on uric acid transport.
Yeast was purchased from OXOID (UK) and dissolved in purified water (1 g:1 ml) before being given rats. Adenine was purchased from Sigma-Aldrich (USA) and suspended in purified water (80 mg:1 ml) before being given rats. Benzbromarone tablets were purchased from Heumann Pharma GmbH (Germany). The chicory used in the study was authenticated by Professor Yan (Traditional Chinese Medicine Appraisal Teaching and Research Section of Beijing University of Chinese Medicine). Chicory was crushed and ground into powder. The powder was extracted with distilled water (1 g:10 ml) by heating to reflux for 1 h twice. The solution was concentrated by a rotary evaporator after filtering and diluted to different volumes with purified water [
Sixty male Sprague-Dawley (SD) rats (240-260 g body weight) were purchased from Beijing SPF Laboratory Animal Technology Co., Ltd. (Certificate of Quality: SCXK-2016-0002). The animals were housed in an air-conditioned room where the temperature and humidity were 20-24°C and 45-55%, respectively, with 12 h light-dark cycles. Food and water were provided ad libitum. After 5 days of acclimation, the rats were randomly divided into 5 groups (n=12): control group (CG), hyperuricaemia with renal injury group (MG), benzbromarone group (BEN), high dosage of chicory group (HD-C), and low dosage of chicory group (LD-C). Normal rats were used for the CG, and hyperuricaemia with renal injury was induced in the rats of the other groups by intragastric administration of yeast (15 g·kg−1·d−1) and adenine (80 mg·kg−1·d−1). After 8 h, the treatment groups were intragastrically administered benzbromarone (20 mg·kg−1·d−1) or chicory at a high dosage (13.2 g·kg−1·d−1) or low dosage (6.6 g·kg−1·d−1), and the CG and MG rats were intragastrically administered an equal volume purified water at the same time. The animal experiments lasted 5 weeks. The animal study protocol was approved by the Animal Care and Ethics Committee of Beijing University of Chinese Medicine.
Blood samples were collected from the tail tips after 12 h of fasting at weeks 1, 3, and 5. The serum was separated and stored at -20°C. The 24-h urine of ten rats per group was collected by the metabolic cage method at weeks 1, 3 and 5. The urine volume was recorded, and then the supernatant was taken and stored at -20°C. Serum uric acid (SUA) and serum creatinine (SCr) levels were measured according to the instructions of a uric acid assay kit (BioSino, China) and CRE assay kit (Nanjing Jiancheng, China), respectively. Additionally, urinary uric acid (UUA) and urinary creatinine (UCr) were measured according to instructions of the same kits. Urinary microalbumin (UMA) was measured according to the microalbumin assay kit (Nanjing Jiancheng, China). The 24-h UUA, 24-h urinary microalbumin and creatinine clearance (CrCl) were calculated by the following equation: 24-h UUA (mg/d) = [UUA ( 24-h UMA (mg/d) = [UMA (mg/L)]×[24-h urine volume (ml/d)]×10−3; CrCl (ml/min) = [UCr (
After 5 weeks of treatment, all animals were anaesthetized with 10% chloral hydrate solution (3.5 ml/kg), and blood samples were collected from the abdominal aorta after 12 h of fasting. The blood samples were analysed as described above. The upper half of the left kidney was fixed with 4% paraformaldehyde for histological evaluation, and the lower half was preserved at -80°C for western blot and qPCR assays.
The left kidneys were fixed with 4% buffered paraformaldehyde, dehydrated with 50-100% ethanol and embedded in paraffin. Samples were cut into 3
Total RNA from the kidneys was extracted by Trizol reagent (Thermo Scientific, USA). The concentration of total RNA was measured with a UV-Vis Spectrophotometer Q5000 (Quawell, USA), and the integrity was evaluated by electrophoresis in a 1% agarose gel. The RNA was reverse-transcribed following the manufacturer’s protocol of the Revert Aid First Strand cDNA Synthesis Kit (Thermo Scientific, USA). Quantitative real-time PCR was performed using iTaq™ Universal SYBR Green Supermix (Bio-Rad, USA) and CFX96™ (Bio-Rad, USA). The forward and reverse primers for GLUT9/SLC2A9 were designed according to the mRNA sequences shown in Table
Primers used for the quantitative RT-PCR gene expression studies.
Gene | Forward primer | Reverse primer |
---|---|---|
GLUT9 | 5′-TGCA TTGG CGTG TTTT CTGG-3′ | 5′-GTTT GGAA GGCT TTCG TGGC-3′ |
GAPDH | 5′-GGTG GACC TCAT GGCC TACA-3′ | 5′-ATTG TGAG GGAG ATCC TCAG TGT-3′ |
The proteins were extracted by lysing the kidney tissue with RIPA (Solarbio, China). The concentration of the proteins was measured by a BCA protein assay kit (Solarbio, China). The proteins were mixed with 4×SDS buffer (Solarbio, China), heated for 10 min and then separated on 10% SDS-PAGE gels. Proteins were transferred to PVDF membranes (Millipore, USA), and the membranes were blocked with TBST (containing 5% skim milk) at room temperature for 1 h. Then, the membranes were incubated with rabbit anti-GLUT9 antibody (1:2500, Millipore, USA) and rabbit anti-
The HKC cell line was purchased from the National Infrastructure of Cell Line Resource & Peking Union Medical College Cell Resource Center. The HKC cells were grown in complete medium consisting of DMEM/F12 (Corning, USA) with 5% fetal bovine serum (FBS;
HKC cells (1.0 × 104 cells/ml) in the logarithmic growth phase were seeded in 96-well plates (200
The HKC cells were incubated in complete medium containing UA, BEN, and Chi for 24 h, and then the cells were collected and lysed by RIPA (Solarbio, China). The total protein was extracted and denatured by heating in a water bath for 10 min. The proteins (5
HKC cells (1.0 × 105 cells/ml) in the logarithmic growth phase were added to the upper chamber (500
First, the culture medium in both the upper and lower chambers was removed, and the HKC cell monolayer was washed twice with 37°C HBSS. Complete medium (1.5 ml) containing the specified concentrations of BEN, Chi, and UA was added to the lower chambers, and the complete medium of the CG contained UA only. Then, 0.5 ml of HBSS was added to the upper chambers. After 30, 60, 90, and 120 min, 300
The liquid samples were filtered through a 0.45
Statistical analyses were conducted by SPSS 20.0 software. All data were expressed as the mean ± standard deviation (SD). The statistical analysis was performed using analysis of variance (ANOVA) followed by Dunnett’s multiple range tests to determine levels of significance. AP-value < 0.05 was considered statistically significant.
As shown in Figure
Effect of chicory on SUA in hyperuricaemic rats with renal injury.
As shown in Figure
Effect of chicory on SCr in hyperuricaemic rats with renal injury.
As shown in Figure
Effect of chicory on 24-h urine volume in hyperuricaemic rats with renal injury.
As shown in Figure
Effect of chicory on 24-h UUA excretion in hyperuricaemic rats with renal injury.
As shown in Figure
Effect of chicory on CrCl in hyperuricaemic rats with renal injury.
As shown in Figure
Effect of chicory on 24-h UMA in hyperuricaemic rats with renal injury.
As shown in Figures
Glomerulus (HE, ×10 objective lens). (a) Control group; (b) hyperuricaemia with renal injury group; (c) group treated with benzbromarone; (d) group treated with the high dosage of chicory; (e) group treated with the low dosage of chicory.
Renal tubule (HE, ×10 objective lens). (f) Control group; (g) hyperuricaemia with renal injury group; (h) group treated with benzbromarone; (i) group treated with the high dosage of chicory; (j) group treated with the low dosage of chicory.
Figure
Effect of chicory on kidneys GLUT9 mRNA expression in hyperuricaemic rats with renal injury examined by RT-PCR.
As shown in Figure
Effect of chicory on kidneys GLUT9 protein expression in hyperuricaemic rats with renal injury examined by western blotting.
As shown in Tables
Comparison of HKC cells proliferation ability at 24 h and 48 h with various concentrations of UA (
UA ( | 24 h | 48 h |
---|---|---|
0 (control group) | 0.2959 ± 0.0267 | 0.4022 ± 0.0333 |
100 | 0.3121 ± 0.0242 | 0.4223 ± 0.0203 |
200 | 0.3257 ± 0.0367 | 0.4406 ± 0.0345 |
400 | 0.3389 ± 0.0245 | 0.4522 ± 0.0273 |
600 | 0.3528 ± 0.0394 | 0.4851 ± 0.0375 |
800 | 0.3742 ± 0.0263 | 0.4963 ± 0.0294 |
Comparison of HKC cells proliferation ability at 24 h and 48 h with various concentrations of benzbromarone (
Benzbromarone ( | 24h | 48h |
---|---|---|
0 (control group) | 0.3065 ± 0.0216 | 0.4209 ± 0.0267 |
12.5 | 0.2992 ± 0.0297 | 0.4092 ± 0.0360 |
25 | 0.3043 ± 0.0305 | 0.3768 ± 0.0364 |
50 | 0.2790 ± 0.0237 | 0.3370 ± 0.0381 |
100 | 0.2756 ± 0.0347 | 0.3377 ± 0.0352 |
200 | 0.2551 ± 0.0384 | 0.3177 ± 0.0426 |
Comparison of HKC cells proliferation ability at 24 h and 48 h with various concentrations of chicory (
Chicory ( | 24 h | 48 h |
---|---|---|
0 (control group) | 0.2859 ± 0.0230 | 0.4097 ± 0.0433 |
100 | 0.2858 ± 0.0188 | 0.3783 ± 0.0464 |
200 | 0.2657 ± 0.0240 | 0.3724 ± 0.0501 |
400 | 0.2644 ± 0.0354 | 0.3611 ± 0.0459 |
600 | 0.2726 ± 0.0221 | 0.3408 ± 0.0317 |
800 | 0.2602 ± 0.0475 | 0.3421 ± 0.0483 |
As shown in Figure
GLUT9 protein expression in HKC by western blotting.
As shown in Figure
(a) The changes of TEER values in HKC cell monolayer; (b) the amount of UA transmembrane transport per 30 min; (c) the peak of UA from standard solution at 3.7 min; (d) the peak of UA from control group sample at 3.7 min.
The results showed in Figure
Figure
In this study, the effect of chicory on serum uric acid (SUA), renal function and GLUT9 expression in the pathological state of hyperuricaemia rats with renal injury was investigated. Hyperuricaemia with renal injury was induced in rats by intragastric administration of yeast (15 g·kg−1·d−1) and adenine (80 mg·kg−1·d−1). The purine in yeast generates uric acid (UA) by the action of XOD and other enzymes in vivo. However, most mammals have uricase, which can catalyse UA oxidation to produce allantoin. Allantoin can dissolve in water and can be excreted from the kidney without being absorbed by renal tubules. Therefore, yeast is commonly used in combination with other agents, such as ethambutol and potassium oxonate, to induce hyperuricaemia in rats and mice [
As expected, the model group (MG) displayed hyperuricaemia and renal function injury from week 3 to week 5. The levels of SUA and creatinine increased in the MG, and the 24-h urine volume also increased.
Creatinine clearance (CrCl) is an indicator that can reflect renal function more accurately and sensitively than serum creatinine [
As shown with haematoxylin and eosin (HE) staining, the numbers of glomeruli in the high dosage and low dosage of chicory groups were greater than those in the model and benzbromarone groups, and the renal tubular expansion was less than that in the model and benzbromarone groups. These changes support the protective effect of chicory on renal function.
Excretion of UA is mainly determined by the balance between renal reabsorption and secretion, and the role of various urate transporters has been debated [
In addition, GLUT9 (SLC2A9) is known to transport glucose or fructose until Doblado and Moley reported that GLUT9 may transport not only fructose but also urate [
As shown in the qPCR and western blot results, there was no significant change in GLUT9 mRNA expression in each group of kidney tissues. However, GLUT9 protein expression in the kidney increased markedly to 128.3% in hyperuricaemic rats with renal injury, and the expression levels decreased to 77.8%, 81.2%, and 84.3%, respectively, when the animals were treated with benzbromarone, high dosage chicory and low dosage chicory. This result indicated that chicory could inhibit the expression of GLUT9 protein in the kidneys of hyperuricaemic rats with renal injury. As a result, the UA reabsorption in the renal tubules is reduced, and the UA excretion by renal tubules is increased. Therefore, chicory plays a role in reducing SUA levels.
Cell experiments were performed in vitro to further verify that the effect of chicory on reducing UA is related to the regulation of GLUT9 protein expression. Benzbromarone was used as an inhibitor to inhibit GLUT9 in HKC cells, and the effects of chicory on protein expression and the ability of monolayer HKC cells to transport UA were evaluated to verify that chicory lowers SUA by regulating GLUT9 protein expression.
HKC cells are a group of cells that are mostly composed of normal human proximal tubular epithelial cells. HKC cells are widely used in pharmacological and toxicological experiments [
The effect of chicory on the expression of GLUT9 protein has been clarified. To further observe the effect of chicory on GLUT9 function, a Transwell assay was used for UA transport experiments. Literature reports indicate that Tranilast can inhibit urate transport through URAT1 and GLUT9 in a reversible, noncompetitive manner [
Finally, knockout mice are used in the research of urate transporters at present. It is reported that OAT1 and OAT3 knockout mice without obvious histological or anatomic abnormalities, and they are similar to their wild-type counterparts with respect to metabolic parameters. Thus the knockout mice could use assessment of renal transport of urate and other organic anions [
The study indicated that chicory lowered serum uric acid levels and alleviated renal function in hyperuricaemic rats with renal injury. In vitro and in vivo biochemical experiments showed that chicory acts on the renal uric acid transporter GLUT9 and down regulates its protein expression level as a mechanism for reducing uric acid. The experiment showed that the chicory had a better effect than benzbromarone in delaying the development of renal function damage caused by adenine in model animals, indicating the possibility of using chicory as an alternative to alleviating renal damage in hyperuricaemia.
The data used to support the findings of this study are included within the article.
The authors declare that they have no conflicts of interest.
The authors thank the Animal Experimental Center of Beijing University of Chinese Medicine for its support to this project. This research is funded by financially supported by the National Natural Science Foundation of China (no. 81673618), Beijing Natural Science Foundation (no. 7162117), and National Science and Technology Major Projects for “Major New Drugs Innovation and Development” (no. 2017ZX09301024).
Table S1: The effect of chicory on SUA in hyperuricaemic rats with renal injury. Table S2: The effect of chicory on SCr in hyperuricaemic rats with renal injury. Table S3: The effect of chicory on 24-h urine volume in hyperuricaemic rats with renal injury. Table S4: The effect of chicory on 24-h UUA excretion in hyperuricaemic rats with renal injury. Table S5: The effect of chicory on CrCl in hyperuricaemic rats with renal injury. Table S6: The effect of chicory on 24-h UMA in hyperuricaemic rats with renal injury. Table S7: The effect of chicory on kidneys GLUT9 mRNA expression in hyperuricaemic rats with renal injury. Table S8: The effect of chicory on kidneys GLUT9 protein expression in hyperuricaemic rats with renal injury. Table S9: The effect of chicory on GLUT9 protein expression in HKC cells. Table S10: The changes of TEER values in HKC cell monolayer (Supplementary Materials).