Olsalazine Sodium Increases Renal Urate Excretion by Modulating Urate Transporters in Hyperuricemic Animals

Abstract

Hyperuricemia is mainly the result of relative underexcretion of urate. Urate is mainly eliminated by kidney and several important transporters expressed on the membrane of renal tubular cells involved in urate excretion. Olsalazine sodium was screened from 3167 authorized small compounds/drugs, targeting xanthine oxidoreductase. In previous study, we reported that olsalazine sodium significantly reduced the serum urate levels, and the anti-hyperuricemic activity linked with inhibiting urate formation by reducing the activity of xanthine oxidoreductase. The current research aimed to assess olsalazine sodium renal urate excretion and likely molecular mechanism. The results showed that administration of olsalazine sodium 5.0 mg/kg decreased the levels of serum urate in hyperuricemic rats, and noticeably improved the fractional excretion of urate and urate clearance, exhibiting an uricosuric action. Moreover, olsalazine sodium (2.5, 5.0, 10.0 mg/kg) reduced the level of blood urea nitrogen in rats. Further study showed that olsalazine sodium reduced the mRNA expression of urate reabsorptive transporter glucose transporter 9 (GLUT9), increased the mRNA expression of urate secretory transporters, organic anion transporter 1 (OAT1), OAT3 and type 1 sodium-dependent phosphate transporter (NPT1) as well as the protein expression of OAT3 in the kidney in hyperuricemic mice. In conclusion, olsalazine sodium exhibited a promotion of urate excretion in kidney by increasing the expression of OAT3.

INTRODUCTION

Excessive production and/or underexcretion of urate can lead to hyperuricemia. Due to the global prevalence of hyperuricemia in the last decades, hyperuricemia has received more attention. It is reported that the prevalence of gout is about 0.1–10% all over the world,¹⁾ and it is increasing in developing countries. The prevalence of gout and hyperuricemia in China was 1.1 and 13.3%, respectively.²⁾ Furthermore, hyperuricemia is not only a major risk factor of gout, but also leades to hypertension, metabolic syndromes, and cardiovascular diseases.^3–5)

The homeostasis of urate depends on the balance of its production and excretion in the body. Underexcretion is the prominent feature of hyperuricemia in patients with gout.⁶⁾ It is usually believed that the kidney is responsible for over 70% of urate excretion, and that hyperuricemia is mostly the result of relative underexcretion of urate by the kidney. Diamond and Paolino⁷⁾ proposed that urate homeostasis in blood or urine is accomplished in the form of a complex interaction, which involves glomerular filtration, reabsorptive and secretory in the renal proximal tubule cells, with approximately 10% of urate that is primarily filtered finally being excreted.⁸⁾ Reabsorption and secretion coexist along the proximal renal tubule. As a result, research on the treatment of renal excretion and the mechanism of hyperuricemia has been extensively carried out. Recent studies have found that several vital transporters are involved in urinary excretion of the renal tubules,⁹⁾ these transporters can be classified into reabsorptive urate-anion exchangers and secretory organic anion-exchange transporters. Reabsorptive urate-anion exchangers involve urate transporter 1 (URAT1, encoded by SLC22A12), glucose transporter 9 (GLUT 9, encoded by SLC2A9)¹⁰⁾ and organic anion transporter 4 (OAT4, encoded by SLC22A11). The secretory organic anion transporters include organic anion transporter 1 (OAT1, encoded by SLC22A6), organic anion transporter 3 (OAT3, encoded by SLC22A8),¹¹⁾ breast cancer resistance protein (BCRP/ABCG2, encoded by Abcg2),¹²⁾ the type 1 sodium-dependent phosphate transporter (NPT1, encoded by SLC17A1)¹³⁾ and so on. Among them, URAT1 has become a key target of drugs that promote the excretion of urate, such as probenecid, benzbromarone and lesinurad.^14,15) Therefore, serum urate homeostasis is closely related to the urate transport system in renal proximal tubulesthe.¹⁶⁾ Hyperuricemia is suggested to be associated with the altered expression and function of the urate transporters.¹⁷⁾

Olsalazine sodium (Fig. 1), an anti-ulcerative-colitis drug in clinic, is a xanthine oxidoreductase inhibitor screened by using the protein-ligand docking software from 3167 authorized small compounds/drugs. In a previous study, we reported for the first time that the serum urate levels in hyperuricemic mice can be significantly decreased by olsalazine sodium and the anti-hyperuricemic effects are associated with decrease the formation of urate by inhibiting the activity of xanthine oxidoreductase in the liver.¹⁸⁾ However, whether the hypouricemic effects linked with urate elimination and underling mechanism are unclear. Therefore, this study evaluated the effects of olsalazine sodium on the excretion of urate in the kidney, and suggested that its possible molecular mechanism is related to the renal urate transporter.

Fig. 1. The Chemical Structure of Olsalazine Sodium

MATERIALS AND METHODS

Materials

Olsalazine sodium was purchased from Selleck Chemicals (U.S.A.). Potassium oxonate and benzbromarone were products of Sigma and Aldrich Chemical Co. (St. Louis, MO, U.S.A.). The biochemical kits were gained from Nanjing Jiancheng Bioengineering Institute. All other reagents are commercially available and of analytical grade.

The primer sequences were synthesized by Sangon Biotech Co., Ltd. Mem-PER Plus membrane protein extraction kit was purchased from Thermo Fisher Scientific Inc. (U.S.A.). Radio immunoprecipitation assay (RIPA) lysis buffer kit was get from Beijing Com Win Biotech Co., Ltd. (China). Enhanced chemiluminescence (ECL) detection reagent is the production of Bio-Rad (U.S.A.).

URAT1 and ABCG2 antibody are the products of Proteintech. GLUT9 antibody was purchased from Sigma. OAT1, OAT3 and β-actin antibody was gained from Origene. Na, K-ATPase antibody was purchased from Cell Signaling Technology (U.S.A.).

Animals

Sprague-Dawley rats, ♂, body weight of 180–200 g were obtained from the Chengdu Dashuo Bioengineering Company, Sichuan, China (Certificate No. SCXK 2015-030) and Kun-Ming mice, ♂, weighing 18–22 g were purchased from Beijing HFK Bioscience Co., Ltd. (Certificate No. SCXK 2014-0004). The animals were kept in a temperature and humidity-controlled room with a constant cycle of 12 h light and dark. During the study, animals were freely given food and water. Before the experiment, the animals were reared one week to adapt to the environment. All experiments were conducted in accordance with the Institute Ethical Committee for Experimental Use of Animals of Kunming Medical University.

Effects of Olsalazine Sodium on Urate Excretion in Potassium Oxonate-Induced Hyperuricemic Rats

As described previously,¹⁹⁾ rats used in this study were induced hyperuricemia with potassium oxonate. Then, rats were randomly divided into six groups (n = 10). The normal control group and the hyperuricemic control group were received vehicle only, whereas other hyperuricemic rats were administrated with olsalazine sodium (2.5, 5.0, 10.0 mg/kg) and benzbromarone (25.0 mg/kg), respectively. All trial compounds were suspended in 0.5% sodium carboxymethylcellulose (CMC-Na) and intraperitoneal injected twice a day for five doses. Potassium oxonate (200 mg/kg) were intraperitoneally injected 1 h before the last dose was given. Then, urine of all rats was collected for 3 h in the metabolic cages. Urine supernatant was obtained by centrifuging at 2000 × g for 10 min, then urate and creatinine were analyzed. At the end of trial, the rats were anesthetized by intraperitoneal injection of pentobarbital (30 mg/kg), and blood was obtained by abdominal aortic puncture. Centrifugation of 3600 × g blood for 10 min to obtain serum for the determination of uric acid and creatinine. In order to assess the capability of urate handling by kidney, fractional excretion of urate (FEUA) and the clearance of urate (C_ur) were calculated as follows²⁰⁾: FEUA = (urinary urate × serum creatinine)/(serum urate × urinary creatinine) × 100. C_ur = urine volume × urinary urate/(serum urate ×180).

Effects of Olsalazine Sodium on Renal Urate Transporters in Potassium Oxonate-Induced Hyperuricemic Mice

Hyperuricemic mice were intragastric administration 500 mg/kg potassium oxonate at 9:00 and 17:00 for 3.5 consecutive days and were further divided into five subgroups (n = 10) intraperitoneal injection 0.5% CMC-Na, olsalazine sodium (5.0, 10.0 and 20.0 mg/kg), benzbromarone (25.0 mg/kg) began at 10:00 a.m. on the day after potassium oxonate was given, respectively. Normal control group was given 0.5% CMC-Na. Serum was collected at 1 h after the last dosing and the serum urate levels was determinated. Renal cortex tissues were dissected immediately on the ice, frozen in liquid nitrogen, and then stored at −80°C for real time fluorescence quantitative PCR (q-PCR) and Western blotting analysis, respectively.

Real-Time q-PCR Analysis

q-PCR was used to analyze the gene expression of urate transporter in the renal cortex of mice. Add 30 mg of renal cortex tissue to 0.5 mL of Trizol reagent and extract total RNA according to the manufacturer’s protocol. An equal amount of total RNA (2 µg) was used for cDNA synthesis according to the manufacturer’s instruction. The reverse transcription reaction products were kept at −20°C for the following amplification. The primer pairs used were as follows: 5′-GGT GCT GAC CTG GAG CTA TC-3′ and 5′-CAG CAG GAA ACG AAA CAG GC-3′ for URAT1; 5′-TTG CCA AGA GGA ACA AGG CT-3′ and 5′-GTG GAG GAT GAA TCG GGC TC-3′ for GLUT9; 5′-CTG CAT T TTC CGG CTC CTC T-3′ and 5′-CTG GCC CAA GCT GTA GAC AT-3′ for OAT1; 5′-TCT GGC CTG GTT TGC TAC TG-3′ and 5′-TGT CAA CCC CAC CGA AGA TG-3′ for OAT3; 5′-CCT CAC CTT ACT GGC TTC CG-3′ and 5′-ATC CGC AGG GTT GTT GTA G G-3′ for ABCG2; 5′-GTA TGG TCG TGG TTC AGG CT-3′ and 5′-GTG GGA GGA G CAA GGA CAT C-3′ for NPT1; 5′-AGC TGA GAG GGA AAT CGT GC-3′ and 5′-ACG GAT GTC AAC GTC ACA CTT-3′ for β-actin.

Western Blotting Analysis

Extract the protein of renal cortex tissue according to the manufacturer’s instructions for transporters assay. The total proteins contents were determined by bicinchoninic acid (BCA) method. The proteins were incubated with 1/5 volume 5 × sodium dodecyl sulfate (SDS) loading buffers in the boiling water for 10 min. Equivalent proteins were isolated on 10% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene difluoride (PVDF) membrane. At room temperature, membranes were sealed in TBST (Tris-buffered saline (TBS) containing 0.5% Tween-20) containing 5% non-fat milk powder for 1 h. Primary antibodies included rabbit polyclonal antibodies against URAT1 (1 : 800), GLUT9 (1 : 1000), OAT1 (1 : 2000), OAT3 (1 : 1000), ABCG2 (1 : 800), Na⁺, K⁺-ATPase (1 : 1000), mouse-anti-β-actin antibody (1 : 2000) were used. HRP conjugated goat anti-mouse immunoglobulin G (IgG) or goat anti-rabbit IgG were used to detect the immunoreactive bands. ECL was used to visualize the protein and ChemiScope was used to quantify the densitometry density.

Statistical Analysis

The results were expressed as mean ± standard deviation (S.D.). The experimental data were analyzed by one-way ANOVA, and then Tukey’s Multiple Comparison Test was carried out with GraphPad Prism version 5.0 software. Statistical significance was accepted at the level of p < 0.05.

RESULTS

Effects of Olsalazine Sodium on Urate Excretion in Potassium Oxonate-Induced Hyperuricemic Rats

As shown in Table 1, the serum urate level of rats treated with potassium oxonate was 260.9 ± 33.6 µmol/L at the end of the experiment, which was significantly increased compared with that of normal control group (110.1 ± 17.9 µmol/L, p < 0.01). The treatment of olsalazine sodium markedly decreased the levels of serum urate at the dose of 5.0 mg/kg (206.1 ± 34.4 µmol/L). Urine urate levels were much higher in the hyperuricemic rats (3.1 ± 0.3 mmol/L) than in normal controls (1.2 ± 0.8 mmol/L), (p < 0.01). Compared with control group, administration of potassium oxonate increased the blood urea nitrogen (BUN) levels of rats from 7.5 ± 0.9 to 8.1 ± 1.6 µmol/L, olsalazine sodium lowered the BUN levels to 6.6 ± 0.9, 6.3 ± 0.9, 6.5 ± 1.0 µmol/L in rats with hyperuricemia, respectively (p < 0.05). In addition, FEUA and C_ur are the sensitive indicators of kidney urate excretion, therefore we detected FEUA and C_ur to evaluate the kidney urate excretion of rats. As shown in Table 1, compared with model group, olsalazine sodium effectively increased FEUA and C_ur in hyperuricemic rats at the dose of 5.0 mg/kg. However, benzbromarone did not significantly affect the level of serum urate, urine urate, BUN, FEUA and Ucr in model group rats. These data demonstrated that olsalazine sodium might increase kidney urate excretion to decrease serum urate levels in hyperuricemic rats.

Table 1. Effects of Olsalazine Sodium on Urate Excretion in Potassium Oxonate-Treated Rat

Group	Con	Model	Olsalazine sodium			Benzbromarone 25.0 (mg/kg)
			2.5	5.0	10.0
Serum urate (µmol/L)	110.1 ± 17.9	260.9 ± 33.6d	241.1 ± 34.4d	206.1 ± 34.4ad	254.3 ± 43.7d	257.9 ± 42.7d
Urine urate (mmol/L)	1.2 ± 0.8	3.1 ± 0.3d	2.9 ± 0.7d	2.8 ± 0.7d	2.7 ± 0.5d	2.9 ± 0.7d
Serum creatinine (mmol/L)	49.7 ± 2.6	44.0 ± 5.2c	44.4 ± 3.5d	42.6 ± 3.3d	49.9 ± 10.3	46.1 ± 5.5
Urine creatinine (mmol/L)	1.9 ± 0.8	1.0 ± 0.3c	0.9 ± 0.3d	0.9 ± 0.4d	1.2 ± 0.4c	0.8 ± 0.4d
BUN (µmol/L)	7.5 ± 0.9	8.1 ± 1.6	6.6 ± 0.9a	6.3 ± 0.9ac	6.5 ± 1.0ac	7.2 ± 0.5
3 h-Urinary volume (mL)	1.6 ± 0.9	4.9 ± 1.0d	5.4 ± 1.4 d	5.5 ± 1.2d	5.8 ± 1.7d	5.1 ± 0.8d
FEUA (%)	30.3 ±17.7	49.4 ± 9.3c	63.0 ± 24.1c	86.7 ± 34.7ad	56.1 ± 22.5c	76.8 ± 44.0c
Cur (mL/min)	0.06 ± 0.03	0.32 ± 0.07d	0.35 ± 0.08d	0.41 ± 0.08ad	0.39 ± 0.13d	0.28 ± 0.04d

Con-normal control, Model-hyperuricemic control. The data are expressed as the mean ± S.D. for ten mice per group. ^ap < 0.05, ^bp < 0.01 vs. the model group; ^cp < 0.05, ^dp < 0.01 vs. the control group (Tukey’s Multiple Comparison Test). FEUA, fraction excretion of uric acid; Cur, clearance of uric acid; BUN, blood urea nitrogen.

Effects of Olsalazine Sodium on the Serum Urate Level of in Potassium Oxonate-Induced Hyperuricemic Mice

As reported in Fig. 2, Potassium oxonate gavaging in mice caused a marked increase in serum urate (p < 0.05), compared with that of normal control group, suggesting that the mouse model of hyperuricemia was successfully established. The olsalazine sodium (5.0 and 10.0 mg/kg) and the positive drug of benzbromaron (25.0 mg/kg) significantly reduced the levels of serum urate.

Fig. 2. Effects of Olsalazine Sodium on Serum Urate Levels in Potassium Oxonate-Treated Mice

Con-normal control, Model-hyperuricemic control. The data are expressed as the mean ± S.D. for ten mice per group. ^ap < 0.05, ^bp < 0.01 vs. the model group; ^cp < 0.05, ^dp < 0.01 vs. the normal control group (Tukey’s Multiple Comparison Test).

Effects of Olsalazine Sodium on the mRNA Expression of Renal Urate Transporters in Potassium Oxonate-Induced Hyperuricemic Mice

As shown in Fig. 3, compared with the normal control group, the mRNA expression of renal urate transporters URAT1, GLUT9 and ABCG2 in model mice increased significantly (p < 0.01), but there was no significant change in OAT1, OAT3 and NPT1 (p > 0.05). Olsalazine sodium resulted in a decrease in GLUT9 mRNA expression at the dose of 10 mg/kg and an increase in the mRNA expression of OAT3, OAT1 and NPT1 at the dose of 5.0, 10, 20 mg/kg, respectively, compared with model mice (p < 0.05, 0.01). However, there was no significant changes in the mRNA expression of urate reabsorptive URAT1. As a positive control, benzbromarone had no effects on the mRNA expression of URAT1, GLUT9, OAT1, OAT3, ABCG2 and NPT1.

Fig. 3. Effects of Olsalazine Sodium on the mRNA Expression of URAT1/GLUT9/OAT1/OAT3/ABCG2 and NPT1 in Potassium Oxonate-Treated Mice

Con-normal control, Model-hyperuricemic control. The data are expressed as the mean ± S.D. for ten mice per group. ^ap < 0.05, ^bp < 0.01 vs. the model group; ^cp < 0.05, ^dp < 0.01 vs. the normal control group (Tukey’s Multiple Comparison Test).

Effects of Olsalazine Sodium on the Protein Expression of Renal Urate Transporters in Potassium Oxonate-Induced Hyperuricemic Mice

Effects of olsalazine sodium on protein level of renal urate transporters are reported in Fig. 4, from which it can be seen that olsalazine sodium had no effects on the protein expression of URAT1, GLUT9, ABCG2, OAT1 and NPT1 in hyperuricemic mice compared with model mice. Administration of olsalazine sodium remarkably elevated the protein expression of OAT3 at doses of 5.0 and 10.0 mg/kg. However, benzbromarone had no significant alteration in the protein levels of URAT1, GLUT9, ABCG2, OAT1, OAT3 and NPT1 in the hyperuricemic mice.

Fig. 4. Effects of Olsalazine Sodium on the Protein Expression of URAT1/GLUT9/OAT1/OAT3/ABCG2 and NPT1 in Potassium Oxonate-Treated Mice

Con-normal control, Model-hyperuricemic control. The data are expressed as the mean ± S.D. for ten mice per group. ^ap < 0.05, ^bp < 0.01 vs. the model group; p > 0.05 vs. the normal control group (Tukey’s Multiple Comparison Test).

DISCUSSION

Two-thirds of urate is excreted through the kidneys, so the homeostasis of urate was mainly dependent on the normal renal function.²¹⁾ Insufficient excretion of urate by kidney, such as reduced glomerular filtration, increased resorption or decreased secretion in renal tubules, is the primary cause of hyperuricemia. Therefore, enhancement of urate excretion by kidney is an effective therapeutic approach. In the present study, we demonstrated that olsalazine sodium has potent uricosuric action. Treatment of olsalazine sodium at the dose of 5.0 mg/kg significantly lowered the serum urate levels although the treatment at a dose of 10 mg/kg had no significant effect on the serum urate level. It may be that p-aminosalicylate, a metabolite of high-dose olsalazine sodium, competitively inhibits uric acid excretion. Olsalazine sodium at the dose of 5.0 mg/kg increased the FEUA and C_ur in the hyperuricemic rats induced by potassium oxonate. FEUA represents the percentage of filtered urate that is excreted in the final urine. FEUA and C_ur are the sensitive indicators of urate excretion by kidney. The data suggest that olsalazine sodium is a potent uricosuric agent as the observed urate lowering effect is related to its ability of promoting renal urate excretion.

Urate exists in the form of organic anion (pK_a value 5.75) in vivo, which requires membrane transporter to penetrate into cells through plasma membrane. In kidney, urate freely filtered by glomerulus is almost completely reabsorpted in proximal tubules and then secreted back into the filtrate. This process is mediated by several urate transporters, mainly including URAT1, GLUT9, OAT1, OAT3, ABCG2 and NPT1, expressed on apical membrane and basement membrane of proximal tubular cells, respectively. Reabsorptive URAT1 and GLUT9 are responsible for transmembrane transport of urate from lumen into cells and from intracellular to interstitial space in the blood, respectively. Secretory OAT1 and OAT3 are responsible for the first step of urate secretion, transporting urate from the interstitial space into tubular epithelial cells. Then, ABCG2 and NPT1 mediate the second step of urate secretion, transporting urate from intracellular to lumen. In this study, to elucidate the underlying molecular mechanisms of uricosuric effects of olsalazine sodium, we detected the mRNA and protein expression of transporters. The results showed that olsalazine sodium suppressed the mRNA expression of reabsorptive GLUT9 and enhanced the mRNA expressions of OAT1, OAT3 and NPT1 in kidney of the hyperuricemic mice. Most noteworthy, olsalazine sodium significantly increased the protein expression of renal OAT3 in the current condition, which was different from the approved uricosuric drugs targeting URAT1, such as benzbromarone and lesinurad. OAT3 in the basolateral membrane of renal proximal tubules contributes to urate secretion rather than reabsorption. In human, OAT3 is believed to be the first step for the kidneys to secrete urate.²²⁾ There is evidence that the expression of OAT3 mRNA in human kidney is three times than that of OAT1 and more than ten times higher compared to OAT2 or OAT4, which indicates that the expression of OAT3 is the most abundant.²³⁾ Therefore, OAT3 is likely to be a potential target for drugs affecting urate homoeostasis. Our study demonstrates for the first time that olsalazine sodium exerts uricosuric actions might be associated with increasing renal OAT3 expression.

It is well known that the urate crystallization can be deposited in body regions and induce acute arthritis that marks the onset of gout, when serum urate level is higher than the threshold of 6.0 mg/dL.²⁴⁾ Current management guidelines recommend that serum urate should be maintenanced at <6.0 mg/dL in patients with gout. This is seldom achieved with either a xanthin oxidase (XOD) inhibitor or a uricosuric agent. Allopurinol, XOD inhibitor as a first-line urate-lowering drug, has demonstrated that more than half of patients do not reach the serum urate target of below 6.0 mg/dL at doses below 300 mg daily^25,26) and induces serious side effects including renal failure, impaired hepatic function and hypersensitivity.²⁷⁾ Therefore, it is clinically desirable to combine a compound with allopurinol therapy to potentiate the effects of allopurinol and to reduce the required high-dose of the drug for better treatment safety.²⁸⁾ Lesinurad is a new selective URAT1 inhibitor approved by the U.S. Food and Drug Administration and European Medicines Agency for the treatment of gout patients with hyperuricemia. It can inhibit the urate reabsorption via targeting URAT1, thus increasing the urate excretion, resulting in the reduction of serum urate. But lesinurad 400 or 600 mg/d reduced serum urate by 44 and 47%, respectively, and did not reduce the target serum urate level of <6 mg/dL. Lesinurad added to allopurinol reduced serum urate by 60 and 72%, respectively and a 100% response rate of serum urate level <6 mg/dL was achieved.^25,29,30) This provides a dual mechanism for the decrease of serum urate—increasing the excretion of urate and reducing the production of urate. Therefore, researching for dual inhibitors is important for future development of gout drugs. RLBN1001 and KUX-1151 are dual inhibitors under study for gout.³¹⁾ Although dual inhibitors are still under study, they provide the valuable example for the study of gout drug. Previously, we have reported that olsalazine sodium decreased urate generation by inhibiting the activity of xanthine oxidoreductase, resulting in the reduction of serum urate.¹⁸⁾ Currently, we found that in a certain dose range, sodium oxalozatin may promote urate excretion by increasing renal urate transporter OAT3 expression, resulting in the reduction of serum urate levels.

Acknowledgments

This work was supported by National Natural Science Foundation of China (81760666). Applied Basic Research Project of Yunnan (2018FB149, 2018FE001(-166)) and the Innovation Fund Designated for Graduate Students of Kunming Medical University (2018S009), and Yunnan Provincial academician workstation of X.F. Kong.

Conflict of Interest

The authors declare no conflict of interest.

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