Genistein ameliorates hyperuricemia-associated nephropathy in hyperuricemic mice

ABSTRACT

Hyperuricemia is a metabolic disease caused by excessive production of uric acid (UA), which can cause gout, hypertension, hyperlipidemia and other diseases. This study aimed to explore the effect of genistein on hyperuricemia and renal protection in hyperuricemic mice induced by potassium oxonate. Furthermore, genistein significantly downregulated the xanthine oxidase (XOD), adenosine deaminase levels, renal gene expressions of glucose transporter type 9 (mGLUT9) and uric acid transporter 1 (mURAT1) and upregulated organic anion transporters (mOAT1 and mOAT3) and organic cation transporters (mOCT1 and mOCT2). Additionally, genistein improved renal function, renal histopathological features and antioxidant activities in hyperuricemic mice. Genistein also attenuated renal fibrosis by suppressing the JAK2/STAT3 and Wnt/β-catenin signalling pathways in hyperuricemic mice. Overall, this study suggested that both doses of genistein have strong potential against hyperuricemia and associated disorders and may be used as natural supplements for the treatment of UA-related disorders.

KEYWORDS:

• Hyperuricemia
• polyphenols
• urate transporters
• oxidative stress
• genistein

1. Introduction

Hyperuricemia is a disease caused by excessive secretion or reduced excretion of uric acid (UA), which can lead to gout, hypertension, hyperlipidemia and other diseases (Guo et al., 2020). Abnormal high-serum UA levels can cause the accumulation of UA and acute inflammatory reaction in the kidneys and joints, leading to joint deformity, UA nephropathy (Pan et al., 2019) and acute gouty arthritis (Robinson, 2018). Reduced excretion of UA is the most dominant cause of hyperuricemia, with approximately 90% of patients presenting with these symptoms (Chen et al., 2019). Changes in lifestyle such as consumption of a high-purine diet are also leading to hyperuricemia (Wang et al., 2010). In China, the occurrence of hyperuricemia has reached 13.3% (men 19.4% and women 7.9%), and worldwide, the incidence of hyperuricemia ranges from 2.6% to 36% in different populations (Liu et al., 2015).

Metabolic cleavage of purine nucleotides results in the production of UA. Xanthine oxidase (XOD) and adenosine deaminase (ADA) catalyzes the oxidation of xanthine or hypoxanthine to UA. The product UA is excreted mainly through the kidneys and intestines. The kidney plays an important role in UA excretion. Excretion of UA in the kidney includes secretion and reabsorption of proximal tubular cells and uric acid transport systems such as glomerular filtration. There are many urate transporters that play vital roles in UA reabsorption and excretion, such as GLUT9, URAT1, ABCG2, OAT1, OAT2, OCT1 and OCT2 (Dinour et al., 2010; Ichida et al., 2004; Zeng et al., 2012).

Excessive UA levels result in the over production of reactive oxygen species (ROS), which further promotes the activation of inflammasomes (Cui et al., 2020). Inflammasomes and cytokines such as cysteine-containing aspartate-specific protease-1 (caspase-1), inflammatory cytokine interleukin 1β (IL-1β), TNF-alpha and inflammasomes including NOD-like receptor family pyrin domain containing 3 (NLRP3) are released extracellular, accelerating the process of kidney disease (Tan et al., 2019). Furthermore, higher UA is also the main cause of hyperuricemia nephropathy (Li et al., 2021).

Nowadays, hyperuricemia and other UA-related disorders are being treated with hypouricemic drugs, including renal urate reabsorption and XOD inhibitors. However, long-term use of these drugs, including benzbromarone, allopurinol and febuxostat, has adverse effects, such as severe hepatotoxicity (Li et al., 2021), gastrointestinal and renal toxicity and severe hypersensitivity (Zhou et al., 2017). Therefore, there is a need to develop effective, safe and improved therapies to treat UA-related disorders, and natural products could lead to an innovative source of such treatments. Presently, natural products are used to treat and prevent hyperuricemia due to their advantages of high compliance, remarkable efficacy and few adverse reactions (Guo et al., 2020; Chen et al., 2019; Cui et al., 2020; Mehmood et al., 2020a, 2020b, 2020c, 2020d, 2020e, 2019a, 2019b; Ishaq et al., 2020; Chen et al., 2013; Müller et al., 2019; Pan et al., 2021).

Genistein is an isoflavone compound naturally found in soybeans and soybean products. Previously, it was documented that genistein can be used for the prevention of various metabolic diseases linked with cardiovascular disease (CVD), diabetes, cancer and obesity (Pavese et al., 2010; Rehman et al., 2019). Moreover, it may also protect against acute kidney injury (AKI)/chronic kidney injury (CKI), such as cisplatin, unilateral ureteral obstruction (UUO)-induced renal fibrosis, radiation, ischemia/reperfusion (I/R)-induced renal injury, morphine and diabetic nephrotoxicity (Stephenson et al., 2005; Sung et al., 2008; Canyilmaz et al., 2016; Ning et al., 2020; Jalili et al., 2020; Javani et al., 2019; Jia et al., 2019).

In this study, we analysed the antihyperuricemic activities of genistein in an animal model of hyperuricemia induced by potassium oxonate (PO), and underlying mechanism was further explored.

2. Materials and methods

2.1. Chemicals

Genistein, allopurinol, benzbromarone, PO and carboxymethyl cellulose (CMC) were purchased from Sigma-Aldrich Reagent (Shanghai, China). The total protein (A045-2-2), ADA (A048-2-1), XOD (A002-1-1), UA (C02-2-1), creatinine (C011-2-1), blood urea nitrogen (BUN), malondialdehyde (MDA) (A003-1-2), superoxide dismutase (SOD) (A001-3-2), catalase (CAT) (A007-1-1), glutathione (GSH) (A006-2-1), total antioxidant capacity (T-AOC) (A015-2-1), tumor necrosis factor alpha (TNF-α) (H052-1), interleukin-1β (IL-1β) (H002), monocyte chemoattractant protein-1 (MCP-1) (H115), Caspase-1 (H074), prostaglandin E₂ (PGE₂) (H099-1) and interleukin-6 (IL-6) (H007-1-1) were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). All other chemicals used in this study were of analytical grade.

2.2. Animals

Animal experiments were conducted in accordance with the Institutional Guidelines for the Care of Laboratory Animals of the Chinese Academy of Medical Science, and approved by the Institutional Ethics Committee for Research on Laboratory Animal Use. 50 male Kunming mice (SPF) were procured from Beijing Vital River Laboratory Animal Technology Co., Ltd. (approval number SCXK (BJ) 2012-0001). The animals were housed in a control room maintaining the room relative humidity 60%, temperature at 25 ± 5°C on 12-h light/dark cycle along with free access to water and food for 1 week.

2.3. Induction of hyperuricemia

After one week of environmental adjustment, mice were divided into five groups named as G1: normal control group, G2: model group, G3: positive group, G4: low dose genistein group and G5: high dose genistein group (detail presented in Table 1). The hyperuricemia model was established by the oral gavage of PO (0.25% CMC solution) at the dosage of 250 mg/kg bw for 15 days (Ishaq et al., 2020). CMC solution (0.25%) was also given to mice in the normal control group for 15 days. The allopurinol was used as a positive control and 10 mg/kg bw was administrated to mice. G4 and G5 groups received genistein at the dosage of 10 and 20 mg/kg bw for 15 days (suspension was made in 0.25% CMC solution).

Table 1. Animal study plan.

Treatments	Dosage Information
G1	Normal diet + 0.5% (w/v) CMC-Na
G2	Normal diet + potassium oxonate (250 mg/kg bw)
G3	Normal diet + potassium oxonate (250 mg/kg bw) + allopurinol (10 mg/kg bw)
G4	Normal diet + potassium oxonate (250 mg/kg bw) + low dosage of genistein (10 mg/kg bw)
G5	Normal diet + potassium oxonate (250 mg/kg bw) + High dosage of genistein (20 mg/kg bw)

Notes: G1: normal group, G2: Model control group, G3: positive control group, G4: low dose genistein, G5: high dose of genistein.

2.4 . Collection of urine, blood and tissues

On the last day, mice were transferred to metabolic cages for urine collection. The collected urine samples were centrifuged (2000g for 10 min) and stored at −80°C for further analysis. The blood samples from each mouse were collected after the administration of the final dosage via cardiopuncture. The serum was obtained after centrifugation at 10,000g for 5 min and stored at −80°C. The tissues such as kidneys, liver and heart were quickly and carefully dissected and rinsed with ice-cold PBS and some parts were fixed for H&E staining and others stored at −80°C for various biochemical and PCR assay.

2.5. Determination of serum and urine UA levels and renal function tests

The UA levels in the serum and urine samples were measured using the Nanjing Institute of Bioengineering Co. Ltd (Nanjing, China) kit. The renal function tests such as serum Cr and serum BUN levels were also determined using Nanjing Institute of Bioengineering Co. Ltd assays kits and kits’ catalogue numbers are presented in section 2.1.

2.6. Measurement of XOD and ADA activity

The XOD and ADA activities in the serum and liver tissues were also determined. The liver tissue (0.1 g) added into ice-cold PBS was prepared by centrifuging at 8000g at 4°C for 10 min and the supernatant was used for the analysis of XOD and ADA activities as per the manufacturers’ instruction provided by the Nanjing Institute of Bioengineering Co. Ltd (Nanjing, China).

2.7. Determination of oxidative stress indicators

The Oxidative stress (OS) indicators such as malondialdehyde (MDA) (A003-1-2), SOD (A001-3-2), catalase (CAT) (A007-1-1), glutathione (GSH) (A006-2-1) and T-AOC (A015-2-1) levels in the serum and renal tissues (homogenize in PBS) were determined using kits. The kits were purchased from the Nanjing Jiancheng Bioengineering Institute, and assays were performed according to the method provided by the manufacturer.

2.8. Determination of inflammatory indicators

The inflammatory indicators such as TNF-α, IL-6, MCP-1, Caspase-1 and PGE₂ were determined in serum and renal tissues using the enzyme-linked immunosorbent assay (ELISA) kits as per the instructions provided by the manufacturer.

2.9. Histopathological examination

The fixed renal tissues (paraformaldehyde, 4%) were embedded in paraffin, cut, sectioned (5 μm) and stained with hematoxylin and eosin (H&E). Later, images were obtained using microscope BX53 (Olympus, Tokyo, Japan).

2.10. Effects of genistein on mRNA expressions

The renal urate transporters such as UART1, GLUT9, OAT1, OAT3, OCT1, OCT2, and inflammatory indicators NF-κβ, JAK2/STAT3 and Wnt/β-Catenin downstream genes expressions were determined using RT- qPCR analysis. In detail, total RNA from renal cortex samples (1 mg) were isolated using Takara, Japan. After that, 500 mg of RNA was reverse transcribed into cDNA using PrimeScript™ RT reagent kit (TaKaRa, Japan) and subjected to PCR amplification using SYBR Premix Plus and specific primers (Table S1). RT-PCR was performed in a CFX96 Real-Time PCR Detection system (Bio-Rad, USA).

2.11. In silico study

2.11.1. Homology modeling and evaluation of URAT1

The amino acid sequence of URAT1 was obtained from the UNIPROTKB database with accession numbers Q96S37. Then, the 3D structure of URAT1 was built using SWISS-MODEL server (https://swissmodel.expasy.org/). Finally, the quality of 3D structures of URAT1 was evaluated using ERRAT and PROCHECK method, and the results are shown in Figure S1. The ERRAT value was 96.084, and the PROCHECK results showed 93.3% of the amino acid residues in favored regions, 6.1% in allowed regions, 0.6% in generous regions and 0.0% in disallowed regions, indicating the reliability of URAT1 model prediction. Moreover, the quality of URAT1 model established in this study was better than that of the study by Yong et al. (2016).

2.11.2. Molecular docking study of URAT1 and genistein

The 3D structure of genistein was obtained from PubChem (https://pubchem.ncbi.nlm.nih.gov/) with CID numbers 5280961, and structure optimization was performed using the steepest descent method with MMFF94 in the Avogadro program (Hanwell et al., 2012). AutoDock Vina package was used for genistein and URAT1 docking (Trott and Olson). AutoDock Tools 1.5.6 software was performed to edit the 3D structure of genistein and URAT1, Gasteiger charges were added. The active pocket positions of URAT1 were set as x = −11.6, y = −13.3, z = 8.0, the grid box was 40 × 40 × 40. After docking, the optimal model was selected according to the binding free energy and binding mode for interaction analysis. Open-Source PyMOL and Discovery Studio 2016 Client were used to visualize the analysis.

2.12. Statistical analysis

The obtained data were analysed using one-way ANOVA followed by multiple comparisons test using GraphPad Prism software. The significance was set at a P < .05, and the results are expressed as mean ± standard error.

3. Results

3.1. Genistein attenuated hyperuricemia via inhibiting key enzymes, reducing UA level and improving renal function

As depicted in Figure 1(E), PO administration significantly elevated the serum UA levels and decreased urine UA levels in the model group (G2) compared to the normal control group (G1), which indicated that hyperuricemic animal model was successfully developed. Allopurinol (G3) and genistein (G4 and G5) treatment markedly (p < .05) decreased the serum UA and increased urine uric acid (UUA) in hyperuricemia mice compared to model control group (G2). Both genistein doses (10 and 20 mg/kg bw) were effective in reducing the UA level. As presented in Figure 1(A–E), serum and hepatic XOD (p < .05) and ADA (p < .05) activities were significantly (p < .05) elevated in model group (G2) compared to the normal control group (G1), which indicated the increase in UA level. Allopurinol (G3) and genistein (G4 and G5) at the dosage of 10 and 20 mg/kg simultaneously inhibited XOD (p < .05) and ADA (p < .05) enzymes compared to the PO group. Serum BUN and Cr (Figure 2(A,B)) levels in the model group were significantly (p < .05) elevated compared to the normal control group. Higher UA level could result in the damage of renal tissues and inflammation. The genistein treatments significantly (p < .05) restored the serum BUN and Cr levels, suggesting that genistein alleviated UA-mediated renal damage.

Figure 1. Effect of genistein on enzymatic activities and UA levels in mice, (A) SXOD, (B) LXOD, (C) SADA, (D) LADA, (E) SUA, (F) UUA, (G) UA synthesis pathway and effect of genistein on targeted enzyme SXOD: Serum xanthine oxidase, LXOD: liver xanthine oxidase, SADA: Serum adenosine deaminase, LADA: Liver adenosine deaminase, SUA: Serum uric acid, UUA: Urine uric acid,#G1: normal group, G2: Model control group, G3: positive control group, G4: low dose genistein treated group, G5: High dose genistein treated group, *p < .05, **p < .01, ***p < .001 compare with G1, ^#p < .05, ^##p < .01, ^###p < .001 compare with G2.

Figure 2. Effect of genistein on renal function tests (A) Serum urea nitrogen, and (B) Serum creatinine,#G1: normal group, G2: Model control group, G3: positive control group, G4: low dose genistein treated group, G5: High dose genistein treated group, *p < .05, **p < .01, ***p < .001 compare with G1, ^#p < .05, ^##p < .01, ^###p < .001 compare with G2.

3.2. Genistein restore UA-mediated urate transporters in PO-induced hyperuricemic mice

The docking analysis results of genistein and URAT1 are shown in Figure 3. As seen in Figure 3(A–F), genistein binded with the surrounding amino acids (Arg, Phe, Pro and Ala) of URAT1 via hydrogen bond and pi−pi interaction. In addition, in the animal experiment, PO supplementation increased the URAT1 gene expression in model group compared to the normal control group (Figure 4(A)). The genistein (G4 and G5) markedly (p < .05) downregulated the renal gene expression of mURAT1 with comparison to the model group (G2). Similarly, gene expression of mGLUT9 was also upregulated by PO in model group (G2) compared with the normal group, which was further restored by allopurinol (G3) and genistein (G3 and G4) treatments (Figure 4(B)).

Figure 3. Molecular docking results of genistein and URAT1. (A) Conformation of complexes URAT1-genistein. (B) Profile map of complexes URAT1-genistein. (C) Genistein could bind to the hydrophobic region of URAT1. (D) Vertical view of complexes URAT1-genistein. (E) and (F) represent 3D and 2D interactions between genistein and URAT1, respectively. Open-Source PyMOL and Discovery Studio 2016 Client software were used to create the image.

Figure 4. Effect of genistein on renal genes expression of urate transporters and underlying mechanism. (A) URAT1, (B) GLUT9, (C) OCT1, (D) OAT1, (E) OAT3, (F) OCT2, (G) potential mechanism of uric acid lowering via genistein,#G1: normal group, G2: Model control group, G3: positive control group, G4: low dose genistein treated group, G5: High dose genistein treated group, *p < .05, **p < .01, ***p < .001 compare with G1, ^#p < .05, ^##p < .01, ^###p < .001 compare with G2.

The effects of genistein on organic anion and cation transporters are presented in Figure 4(C–F). The PO administration significantly downregulated the gene expression of mOAT1, mOAT3, mOCT1 and mOCT2 compared to G1. The allopurinol (G3) and genistein (G3 and G4) treatments markedly (p < .05) upregulated these organic anions and cations transporters compared to the G2 group. These results suggested that genistein has dual function like inhibiting key enzymes and aiding UA secretion via interacting with urate transporters (Figures 1(G) and 3(G)).

3.3. Genistein restored UA-mediated OS in PO-induced hyperuricemic mice

The results of serum and renal OS indicators, such as MDA, SOD, T-AOC, CAT and GSH levels are shown in Table 2. Our results showed that compared with the NC group, SOD, T-AOC, CAT, GSH and other OS indexes in serum and kidney tissues of G2 group were downregulated, while MDA content was upregulated (Table 2). Interestingly, allopurinol (G3) and genistein (G3 and G4) markedly (p < .05) restored antioxidant status in hyperuricemic mice via increasing SOD, T-AOC, CAT, GSH levels and decreasing MDA content in serum and renal tissues (Table 2). These results confirmed that genistein has a strong potential to improve antioxidant activities in hyperuricemic mice.

Table 2. Effect of genistein on serum and renal antioxidant activities in PO-induced hyperuricemic mice (n = 10, mean ± SD).

	Antioxidant parameters	G1	G2	G3	G4	G5
Serum	SOD (U/ml)	159.30 ± 8.23	86.11 ± 5.36***	121.6 ± 6.60^###	142.30 ± 6.12^###	149.1 ± 7.49^###
	GSH (U/ml)	101.40 ± 5.56	33.49 ± 2.26***	75.19 ± 7.46^###	58.69 ± 5.00^#	99.56 ± 4.49^###
	T-AOC (U/ml)	5.77 ± 0.35	2.71 ± 0.12***	4.96 ± 0.25^##	4.63 ± 0.12^#	5.98 ± 0.36^###
	CAT (U/ml)	17.45 ± 1.62	7.44 ± 0.66***	12.57 ± 0.77^###	11.21 ± 1.08^###	14.95 ± 1.33^###
	MDA (nmole/ml)	8.45 ± 0.89	16.79 ± 1.33***	9.87 ± 0.70^###	9.12 ± 0.97^###	7.95 ± 0.75^###
Kidney	SOD (U/mg protein)	59.41 ± 4.67	18.15 ± 2.29***	40.93 ± 2.94^###	55.76 ± 4.71^###	56.90 ± 5.27^###
	GSH (U/mg protein)	29.03 ± 2.01	8.67 ± 0.08***	26.24 ± 2.37^###	25.32 ± .04^##	2.63 ± 1.08^###
	T-AOC (U/mg protein)	2.92 ± 0.20	0.75 ± 99***	1.68 ± 0.81^##	2.77 ± 0.24^###	2.79 ± 0.38^###
	CAT (U/mg protein)	78.31 ± 4.58	20.13 ± 2.77***	62.19 ± 5.44^##	53.60 ± 2.90^#	68.09 ± 4.19^###
	MDA (nmole/mg protein)	2.97 ± 0.34	6.98 ± 0.09***	3.22 ± 0.10^###	3.39 ± 0.09^##	2.33 ± 0.02^###

Notes: G1: normal group, G2: Model control group, G3: positive control group, G4: low dose genistein treated group, G5: High dose genistein treated group, SOD: Superoxide dismutase, T-AOC: Total antioxidant activities, CAT: Catalase, MDA: Malondialdehyde.

*p < .05, **p < .01, ***p < .001 compare with G1, ^#p < .05, ^##p < .01, ^###p < .001 compare with G2.

3.4. Genistein protect renal fibrosis via inhibiting Wnt/β-catenin signalling pathway in hyperuricemic mice

To further confirm the protective effect of genistein on hyperuricemia-associated renal fibrosis, we determined the expression of downstream renal genes involved in Wnt/β-catenin signalling pathway. We noticed that compared with the normal control group (G1), PO significantly downregulated the mAxin (p < .05), mGSK (p < .05) and mAPC (p < .05), and upregulated mβ-catenin (p < .05), mWnt3 (p < .05), mWnt4 (p < .05), mLRP5 (p < .05), mLRP6 (p < .05) and mFirizzled4 (p < .05) gene expression. The allopurinol (G3) and genistein (G4 and G5) administration restored these gene expressions compared with the G2 group (Figure 6(A–I)).

3.5. Genistein protected renal fibrosis via inhibiting JAK2/STAT3 signalling pathway in hyperuricemic mice

As shown in Figure 7(A–C), compared with the normal group, PO (G1) markedly elevated the gene expressions of mJAK2 (p < .05), mSTAT3 (p < .05) and mSOCS3 (p < .05). The genistein treated groups (G4 and G5) and allopurinol (G3) treatments significantly (p < .05) downregulated the renal mJAK2 (p < .05), mSTAT3 (p < .05) and mSOCS3 (p < .05) gene expression compared with the model group (PO).

3.6. Genistein protected renal morphological and inflammation in hyperuricemic mice

As presented in Figure 5, results of H&E staining clearly showed severe tubular injury, swelling, atrophy and dilation in the renal tissues of the G2 group with high UA. Furthermore, glomerular cells proliferated, and inflammatory cells infiltrated into the interstitium in the hyperuric acid model group. The genistein treatment (G3 and G4) markedly (p < .05) restored the UA-mediated renal injury compared to the model group. The G2 also showed protection in a similar way. These morphological features of the kidney also provided supporting factors for biochemical analysis.

Figure 5. Effect of genistein on renal#histopathology features (magnification at 300×). G1: normal group, G2: Model control group, G3: positive control group, G4: low dose genistein treated group, G5: High dose genistein treated group.

The protective effect of genistein on hyperuricemia-associated renal inflammation was also investigated by analyzing the levels of inflammatory cytokines such as (TNF-α, PGE₂, IL-1β, COX-2, MCP-1 and IL-6) in serum and renal tissues. As shown in Figure 8(A–G), PO significantly (p < .05) elevated the serum (TNF-α, IL-1β, and IL-6) and renal (PGE₂, COX-2, and MCP-1) levels in model group (G2) compared with the normal control group (G1). The supplementation of genistein (G4 and G5) and allopurinol (G3) significantly (p < .05) decreased the serum and renal TNF-α, PGE2, IL-1β, COX-2 and IL-6 levels compared with the model group G2. Furthermore, renal gene expression of mNF-κβ was also upregulated in the G2 group compared with the G1 group, which was restored by the allopurinol (G3) and genistein (G3 and G4) treatments (Figure 8).

4. Discussion

The overproduction of UA results to hyperuricemia, and per long time persist might trigger renal inflammation and fibrosis which positively contribute to hyperuricemia nephropathy (Li et al., 2021). That is why a new safe and effective UA-lowering strategy is urgently needed to cope hyperuricemia and associated renal nephropathy. Hyperuricemia in the animal can be induced by several ways such as administration of high fructose, yeast extract, potassium oxonate (PO) and adenine. Among all, the model of hyperuricemia induced by PO is most commonly used. PO selectively inhibits uricase of UA metabolism, which causes hyperuricemia in rodents and blocks the activity of hepatic uricase (Tang et al., 2017). Effectiveness of therapeutic agents and pathophysiological mechanism of hyperuricemia can be assessed using PO-induced hyperuricemia disease model in animals (Lee et al., 2018). In this study, we induced hyperuricemia by oral gavage of PO (0.25% CMC solution) at the dosage of 250 mg/kg bw for 15 days. The PO administration significantly alter various biochemical parameters such UA, XOD, ADA, BUN and SCr levels.

XOD is an important enzyme responsible for UA production via catalyzing xanthine to hypoxanthine to UA (Fukunari et al., 2004). Furthermore, ADA is also an important enzyme involved in the UA synthesis. ADA is a catalytic enzyme responsible for the production of hypoxanthine nucleoside from adenosine (Pang et al., 2017). The PO administration increases the serum and hepatic XOD and ADA levels in hyperuricemic mice, which were further restored by genistein treatment (Figure 1). Many authors reported that polyphenolic compounds potently inhibited XOD and ADA both in vitro and in vivo experiments. Authors proposed that polyphenolic compounds bind closely to enzymes (XOD and ADA) via hydrogen bonds or pi−pi interactions with the surrounding amino acid, thus occupying the active site of enzymes and slowing down the catalysis of UA (Figure 1(G)) (Guo et al., 2020; Chen et al., 2019; Cui et al., 2020; Mehmood et al., 2020a, 2020b, 2020c, 2020d, 2020e, 2019a, 2019b; Ishaq et al., 2020; Chen et al., 2013; Müller et al., 2019; Pan et al., 2021; Li et al., 2021).

The BUN and creatinine (Cr) are the key indicators of renal function. The higher these values, the greater the kidney damage. The PO administration (G2) significantly increases the serum BUN and creatinine levels which were further restored by genistein (G4 and G5) (Figure 2). Mehmood et al., 2020c also observed that the polyphenolic compounds enrich stevia residue extract downregulated serum Cr and BUN in hyperuricemic mice. Ishaq et al. (2020) also reported that sinapic acid could improve renal function via decreasing the serum Cr and BUN in PO-induced hyperuricemic mice. Recently, sunflower head, apigenin, and other bioactive compounds have also been reported to improve renal function in hyperuricemic mice (Tan et al., 2019; Mehmood et al., 2018, 2020b, 2020c, 2020e, 2020d; Tang et al., 2017; Lee et al., 2018; Pang et al., 2017).

It has been reported that approximately 90% of hyperuricemia patients cannot adequately excrete UA (Ichida 2009). More than 70−75% UA excreted via the renal pathway, whereas 20−25% UA excreted by intestinal tract. Notably, UA is a polar molecule and cannot easily pass through the membrane. Thus, it depended on ion channels to finish secretion and reabsorption in the renal proximal convoluted tubules. Therefore, renal tubular secretion and reabsorption processes are the key mechanisms of UA excretion. There are many urate transporters (URAT1, GLUT9, OCT1, and OCT2, OAT1, and OAT3) involved in excreting UA levels (Mandal & Mount, 2015). URAT1 is mainly involved in the reabsorption of UA in the renal proximal convoluted tubules and is encoded by the SLC22A12 gene. It is a key target of reducing UA drugs such as benzbromarone, losartan and fenofibrate (Ichida et al., 2004). GLUT9 (encoded by the SLC2A9 gene) is also responsible for the reabsorption of UA (Nigam et al., 2007). Organic anions transporters (OAT1 and OAT3) and organic cations transporters (OCT1 and OCT2) were involved in the renal secretion of urate. Reduced urate secretion was observed in OAT1-knockout mice, which confirmed the importance of organic anions transporters in the pathogenesis of hyperuricemia (Nigam et al., 2007). The results of our study concluded that genistein (G4 and G5) treatment markedly rebalance urate transporters (URAT1, GLUT9, OCT1, OCT2, OAT1 and OAT3) (Figures 3 and 4). Natural products and isolated compounds have been reported to regulate urate transporters such as sunflower head, stevia residue extract, grapefruit juice, Cordyceps militaris, Camellia japonica bee pollen, curcumin, phloretin and mangiferin (Wang et al., 2010; Chen et al., 2019; Xu et al., 2021; Pan et al., 2019; Yang et al., 2018; Mehmood et al., 2018, 2020b, 2020c, 2020d; Cui et al., 2020).

OS is defined as the higher production of ROS. The oxidant producing enzyme (XO) is released from the liver and linked to glycosaminoglycans and endothelial cells. . During the production of UA, XO generates ROS (oxygen radicals or hydrogen peroxide) around endothelial cells, further leading to the production of OH or ONOO⁻ (Aslan et al., 2001; Osarogiagbon et al., 2000; Schmidt et al., 2018). It is well-documented that the OS of asymptomatic young patients with primary hyperuricemia is significantly higher than that of healthy subjects (Zhou et al., 2018). Moreover, it also reported that people with hyperuricemia and gout are at higher risk of developing cardiovascular diseases (Hayyan et al., 2016; Cristóbal-García et al., 2015). Abnormal UA levels lead to OS, decrease endogenous antioxidant defenses, increase ROS production, lipid peroxidation and initiate a cascade of reactions. MDA, SOD, CAT, T-AOC and GSH play an important role in UA-mediated OS (Lan et al., 2012). Therefore, this study investigated the protective effect of genistein on UA-mediated OS in hyperuricemia mice. We observed that genistein restored UA-mediated OS via modulating serum and renal MDA, SOD, CAT, GSH and T-AOC levels (Table 2). Genistein was reported to possess strong antioxidant activities. For example, genistein can markedly decrease the renal MDA content in diabetic mice (Kim & Lim, 2013). Similar results have been reported by many authors (Kim & Lim, 2013; Mehmood et al., 2020b, 2020c, 2020d). In an hyperuricemic model study, Mehmood et al. (2020c, 2020d) documented that polyphenolic compounds-rich stevia residue extract ameliorate UA-mediated OS via restoring the activity of MDA, SOD, T-AOC, CAT, GSH-Px and NO levels in serum and renal tissues. In another study, the same research group observed that grapefruit juice markedly improved renal antioxidant activity (CAT, MDA, GSH, and SOD) in PO-induced hyperuricemic mice (Mehmood et al., 2020b). Many other bioactive compounds such as curcumin, phloretin and mangiferin were also reported to restore UA-mediated OS in animal experiments (Chen et al., 2019; Cui et al., 2020;Yang et al., 2018).

Wnt/β-catenin is an evolutionary conserved signalling pathway that controls embryogenesis, injury repair and pathogenesis of different diseases. Wnt/β-catenin is activated mainly in response to renal injury. Frequent activation of Wnt/β-catenin leads to the development of AKI to chronic kidney disease (CKD) (Xiao et al., 2016; Nusse & Clevers, 2017; Zuo & Liu, 2018). The genistein administration restored these gene expressions of Wnt/β-catenin signalling pathway compared with the G2 group (Figure 6). Our results were closely related to the previous authors’ findings. Many authors have reported that natural products/compounds ameliorate renal fibrosis via suppressing downstream genes involved in the Wnt/β-catenin signalling pathway (Li et al., 2021; Wang et al., 2018a; Wang, et al., 2018b). In detail, Zhang and Chen (2011) reported that genistein inhibited the human colon cancer cell line via Wnt signaling pathway. In another study, poricoic acid ZG and poricoic acid ZH were reported to attenuate renal fibrosis via inhibiting the activation of Wnt/β-Catenin signalling pathway (Wang et al., 2018a; Wang, et al., 2018b). Mehmood et al. (2020d) reported that polyphenolic-enrich stevia residue extract attenuates renal fibrosis via inhibiting the activation of Wnt/β-Catenin signalling pathway in mice. In addition, recently Li et al. (2021) recently documented that apigenin alleviated renal fibrosis in hyperuricemic mice via the Wnt/β-catenin pathway.

Figure 6. Genistein attenuates renal fibrosis inhibits the Wnt/β-catenin and its downstream target genes expression in PO-induced hyperuricemic mice., G1: normal group, G2: Model control group, G3: positive control group, G4: low dose genistein treated group, G5: High dose genistein treated group, *p < .05, **p < .01, ***p < .001 compare with G1, ^#p < .05, ^##p < .01, ^###p < .001 compare with G2.

Renal fibrosis is an important pathologic process of hyperuricemia nephropathy, which is mainly caused by deposition of extracellular matrix (ECM). Many mechanisms of UA-mediated renal fibrosis have been reported. Among them, JAK2/STAT3 is an important pathway of renal fibrosis. Previously, Pan et al. (2021) documented that renal interstitial fibrosis may be due to the activation of Src/STAT3 pathway in hyperuricemic renal nephropathy. We also determined renal gene expressions involved in the JAK2/STAT3 signalling pathway. The genistein treatment significantly (p < .05) downregulated the renal mJAK2 (p < .05), mSTAT3 (p < .05) and mSOCS3 (p < .05) gene expression compared with the model group (G2) (Figure 7). Many polyphenolic compounds (genistein, fisetin, caffeic acid, astilbin, etc.) were reported to suppress the JAK2-STAT3 signalling cascade (Cheng et al., 2020; Hu et al., 2021). Ethanol extract of Liriodendron chinense (Hemsl.) Sarg barks were also reported to attenuate hyperuricemic nephropathy via inhibiting JAK2/STAT3 signalling pathway in mice (Pan et al., 2021).

Figure 7. Genistein attenuates renal fibrosis via inhibiting JAK2/STAT3 pathway and its downstream target genes expression in PO-induced hyperuricemic mice, G1: normal group, G2: Model control group, G3: positive control group, G4: low dose genistein treated group, G5: High dose genistein treated group *p < .05, **p < .01, ***p < .001 compare with G1, ^#p < .05, ^##p < .01, ^###p < .001 compare with G2.

It has been documented that UA-mediated ROS activated the mitogen-activated protein kinase (MAPK) signal transduction pathway which further affected various transcription factors, including nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κβ). ROS enhances the NF-kβ translocation from cytosol into the nucleus during OS, which leads to the generation of nitric oxide (iNOS) and COX-2 inflammatory enzymes. Moreover, NF-κβ altered the gene expression encoding mediators and cytokines such as IL-1β and TNF-α (Ho & Bray, 1999; Lee & Burckart, 1998). Hence, NF-κβ caspase-1 activation, cytokines play a pivotal role in the inflammatory response during UA-mediated ROS (Qian et al., 2019;Yin et al., 2015). The genistein (G4 and G5) treatment significantly rebalances the UA-associated renal inflammation in mice (Figure 8). The polyphenolic compounds were reported to restore the UA-mediated renal inflammation via downregulating inflammatory cytokines levels and inhibiting NRLP3 pathway (Pan et al., 2019; Chen et al., 2019; Mehmood et al., 2020c, 2020d; Chen et al., 2013; Müller et al., 2019; Qian et al., 2019).

Figure 8. Effect of genistein on serum inflammatory cytokines levels in PO-induced hyperuricemic mice. (A) Renal PGE2, (B) Renal COX-2, (C) Serum TNF-α, (D) Renal genes expression of NF-κβ, (E) Serum IL-6, (F) Serum IL-1β, (G) Renal MCP-1, G1: normal group, G2: Model control group, G3: positive control group, G4: low dose genistein treated group, G5: High dose genistein treated group, *p < .05, **p < .01, ***p < .001 compare with G1, ^#p < .05, ^##p < .01, ^###p < .001 compare with G2.

5. Conclusions

In this study, we investigated the protective effect of genistein on PO-induced hyperuricemia in mice. Administration of genistein at the dosage of 10 and 20 mg/kg bw decreased the serum UA level and renal function biomarkers. Genistein decreased XOD and ADA activities in serum and liver. Moreover, genistein treatments (G4 and G5) also downregulated the renal mRNA expression of mURAT1 and mGLUT9 and upregulated mOAT1, mOAT3, mOCT1 and mOCT2 expression in hyperuricemic mice. Genistein also improved antioxidant activities and decreased renal inflammation and fibrosis in PO-induced hyperuricemia mice via JAK2/STAT3 and Wnt/β-catenin pathway. In summary, our results provide an effective treatment for hyperuricemia.

Institutional review board statement

The animal experiment was according to the protocols and Ethical number; A356-60 that approved by Ethical Committee for Scientific the Beijing Key Laboratory of Functional Food from Plant Resources.

Sample availability

Samples of the compounds available from the authors.

Acknowledgment

Bi Wei-Yun contributes to software, validation, formal analysis, writing—original draft preparation and Zhu Cailin contribute interview and editing, visualization, supervision, project administration, funding acquisition

Data availability statement

All data are presented in the figure. However, additional information can be obtained from corresponding author on request.

Disclosure statement

No potential conflict of interest was reported by the author(s).