Advances in Research on Pathological and Molecular Mechanism of Hyperuricemic Nephropathy Based on Animal Models / 实验动物与比较医学

Uric acid (UA), the final product of human purine metabolism, can cause hyperuricemia (HUA) when excessively accumulated. HUA is closely linked to chronic kidney diseases (CKD) and is considered an independent risk factor. Hyperuricemic nephropathy, a form of CKD induced by HUA, has seen significant advances in understanding through research into the pathogenic roles of uric acid and the development of HUA animal models. Although progress has been made in understanding the pathophysiological mechanisms by which UA induces CKD, much remains to be learned about its pathological molecular mechanisms. New approaches in animal modeling or the selection of model animals may potentially lead to significant breakthroughs in research on hyperuricemia as well as related CKD. This paper reviews the research progress on the molecular mechanisms of hyperuricemic nephropathy, focusing on oxidative stress, inflammation, autophagy, fibrosis, and gut microbiota. Oxidative stress is induced by uric acid intracellularly through xanthine oxidase, NADPH oxidases, and mitochondria, leading to cellular damage. In terms of inflammation, uric acid crystals can activate the NLRP3 inflammasome, triggering an inflammatory cascade. The role of free uric acid as a pro-inflammatory agent, however, remains controversial. Depending on the study conducted, autophagy has been found to either alleviate or exacerbate inflammation induced by uric acid. Fibrosis, particularly through epithelial-mesenchymal transition (EMT), is a major mechanism by which uric acid causes glomerulosclerosis and tubulointerstitial fibrosis. Extensive research has explored various signaling pathways involved in uric acid-induced EMT. Beneficial gut microbiota protect the kidneys by synthesizing short-chain fatty acids, reducing urea’s enterohepatic circulation, and decreasing uric acid production. This paper aims to enhance understanding of the complex relationships between HUA and CKD, serving as a reference for further research and new drug development.

Effects of compound active tea of Lithocarpus litseifolius on uric acid and renal function in mice with hyperuricemia nephropathy / 中国比较医学杂志

Objective To explore the effect of compound active tea of Lithocarpus litseifolius on uric acid levels and kidney function of mice with hyperuricemia nephropathy and to provide an experimental basis for the development of hyperuricemia nephropathy drugs and functional food.Methods A mouse model of hyperuricemia nephropathy was established by administering potassium oxazinate with adenine.Mice were randomly divided into common,model,positive drug(10 mg/(kg·d))and compound active tea of Lithocarpus litseifolius high-,middle-and low-dose groups(10 g/(kg ·d),3.33 g/(kg·d)and 1.11g/(kg·d),respectively).One hour after the last gavage,urine protein(UP)was measured by CBB method,urea nitrogen(UUN)was measured by urease method.Orbital blood pampling,blood was collected for uric acid(UA)analysis by enzyme ratio method,urea nitrogen(BUN)was measured by urease method.The serum contents of interleukin 6(IL-6)and tumor necrosis factor(TNF-α)were measured by ELISA.Take kidney tissue,levels of urate transporter 1(URAT1)and glucose transporter 9(GLUT9)were measured by quantitative fluorescence,kidney histopathological changes were observed by HE stainning.Results Compared with the control group,the model group's levels of UP,UUN,UA,BUN,IL-6,URAT1,ULUT9 and TNF-α were significantly increased(P＜0.01,P＜0.05),and the renal tissue structure was normal.Compared with the model group,the positive group's levels of UP,UUN,UA,BUN,IL-6 and TNF-α were significantly decreased(P＜0.01,P＜0.05),there was little glomerular atrophy or deformation in the kidneys,kidney tubular dilatation was occasionally seen,but there was no inflammatory cell infiltration.Compared with the model group,the high-dose compound active tea of Lithocarpus litseifolius group's UP,UUN,UA,BUN,IL-6,URAT1,TNF-α and GLUT9 levels were significantly decreased(P＜0.01,P＜0.05).The middle-dose compound active tea of Lithocarpus litseifolius group's UP,UUN,UA content,IL-6,URAT 1,GLUT9,BUN and TNF-αwere significantly decreased(P＜0.01,P＜0.05).The low-dose compound active tea of Lithocarpus litseifolius group's UP,UUN,UA,IL-6,URAT1,BUN,TNF-α and GLUT9 levels were significantly decreased(P＜0.01,P＜0.05).Conclusions Compound active tea of Lithocarpus litseifolius can reduce uric acid in mice with hyperuricemia nephropathy and has a certain protective effect on the kidneys.The mechanism may be related to the inhibition of uric acid reabsorption,and the specific mechanistic details should be further investigated.

Establishment and optimization of a hyperuricemic nephropathy mouse model / 药学学报

The aim of this study was to establish an efficient and stable mouse model of hyperuricemic nephropathy (HN) by testing different modes of administration of potassium oxonate (PO) combined with hypoxanthine (Hx). Animal welfare and experimental procedures were in accordance with the regulations of the Animal Ethics Committee of Guangdong Pharmaceutical University. Male C57BL/6 mice were randomly divided into a control group, a PO+Hx group (i.g.; 100 mg·kg-1·d-1 and 500 mg·kg-1·d-1, respectively), and a PO+Hx group (i.p.; 100 mg·kg-1·d-1, and 500 mg·kg-1·d-1). This HN model was induced by combination of PO and Hx administration once daily for 21 days. The results of serum biochemistry showed that the levels of serum creatinine and 24 h albuminuria were increased compared with the normal group in intragastric administration of PO combined with Hx (P < 0.05), but there was no significant difference in serum uric acid and hepatic levels of xanthine oxidase. The maximum value of serum uric acid and creatinine was 349.3 μmol·L-1 and 26.4 μmol·L-1, respectively, in mice injected with PO combined with Hx. The levels of liver xanthine oxidase and 24 h albuminuria were significantly increased in mice injected with PO combined with Hx (P < 0.01). Pathological data showed that renal tubules were dilated, the epithelial cells of renal tubules were disordered, and the production of collagen fibers, reactive oxygen species (ROS) and lipid peroxidase 4-hydroxynonenal (4-HNE) were slightly increased after intragastric administration of PO combined with Hx mice. Obvious infiltration of inflammatory cells and large area of collagen deposition, with a large amount of ROS and the lipid peroxide 4-HNE were produced in mice injected with PO combined with Hx. Western blot analysis showed that the expression of fibronectin (FN) and urate transporter 1 (URAT1) was increased after intragastric administration of PO combined with Hx in mice and further increased in mice injected with PO combined with Hx. This study demonstrates that injection with 100 mg·kg-1 potassium oxonate combined with 500 mg·kg-1 hypoxanthine establishes a stable and efficient mouse HN model.

Role of 3-methyladenine in uric acid-induced apoptosis of renal tubular epithelial cells / 中华肾脏病杂志

Objective:To investigate the effect of autophagy inhibitor 3-methyladenine (3-MA) on uric acid (UA)-induced apoptosis of renal tubular epithelial cells.Methods:(1) Totally 24 SD rats were randomly divided into 4 groups: control group, treatment with 3-MA group, hyperuricemic nephropathy (HN) group, and HN+3-MA group, with 6 rats in each group. According to the body weight of the rats, adenine (100 mg/kg) and potassium oxonate (1 500 mg/kg) were mixed with distilled water to make a suspension, and the rats were given intragastrically once daily for consecutive 21 days to establish HN rat model. The control group and the 3-MA treatment group were fed an equivalent amount of distilled water. At the same time, the 3-MA treatment group and the HN+3-MA group were intraperitoneally injected with 3-MA (15 mg/kg), and the control group and HN group were intraperitoneally injected with an equal volume of saline once daily for 21 consecutive days. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay (TUNEL) was used to observe renal cell apoptosis. Western blotting was used to detect the expression levels of cleaved caspase-3 and Bax, and immunofluorescence staining was used to detect the expression and localization of cleaved caspase-3 in renal tissue. (2) Human renal tubular epithelial cells (HK-2) were stimulated with UA (800 μmol/L), and cells were administrated with different concentrations of 3-MA or Beclin-1 small interfering RNA (siRNA). The apoptosis of renal tubular epithelial cells was detected by Western blotting and immunofluorescence staining.Results:Compared with the normal rats, the apoptosis of renal tubular epithelial cells in the HN group was significantly increased ( P<0.01), and the expression levels of cleaved caspase-3 and Bax were significantly up-regulated (both P<0.05). Compared with the HN group, the apoptosis of renal tubular epithelial cells in the HN+3-MA group was significantly decreased ( P<0.01). In addition, high level of uric acid could significantly increase the levels of apoptosis associated proteins in HK-2 cells (all P<0.05), and using different concentrations of 3-MA or transfecting with Beclin-1 siRNA could significantly reduce the expression of cleaved caspase-3 and Bax (all P<0.05). Conclusion:Autophagy plays an important role in uric acid-induced apoptosis of renal tubular epithelial cells. Inhibiting the excessive activation of autophagy may be a new strategy to prevent the progression of HN.

Dioscin attenuates inflammatory injury in uric acid-induced renal tubular epithelial cells by suppression of NF-κB signaling pathway / 中国临床药理学与治疗学

AIM: To observe the effect of Dioscin treatment on NF-κB signaling pathway and cellular inflammatory injury and explore its potential mechanism in uric acid-induced mouse tubular epithelial cells (mTECs). METHODS: After 1.2 mol/L uric acid induced mTECs, Dioscin and NF-κB P65 inhibitor BAY11-7082 were given to intervene respectively. IκB-α, NF-κB P65, PP65, NLRP3, IL-1β and β-actin were detected by Western Blot, immunofluorescence staining and real-time PCR. RESULTS: Western Blot, immunofluorescence staining and real-time PCR analysis showed that expression levels of PP65, NLRP3 and IL-1β were significantly downregulated in the uric acid-induced mTECs with Dioscin and BAY11-7082 treatment. CONCLUSION: Dioscin attenuates uric acid-induced cellular inflammatory damage by suppression NF-κB signaling pathway.

Anti-hyperuricemic and anti-inflammatory actions of vaticaffinol isolated from Dipterocarpus alatus in hyperuricemic mice / 中国天然药物

The present study was designed to examine the anti-hyperuricemic and anti-inflammatory effects and possible mechanisms of vaticaffinol, a resveratrol tetramer isolated from ethanol extracts of Dipterocarpus alatus, in oxonate-induced hyperuricemic mice. At 1 h after 250 mg·kg potassium oxonate was given, vaticaffinol at 20, 40, and 60 mg·kg was intragastrically administered to hyperuricemic mice once daily for seven consecutive days. Vaticaffinol significantly decreased serum uric acid levels and improved kidney function in hyperuricemic mice. It inhibited hepatic activity of xanthine dehydrogenase (XDH) and xanthine oxidase (XOD), regulated renal mRNA and protein levels of urate transporter 1 (URAT1), glucose transporter 9 (GLUT9), organic anion transporter 1 (OAT1), organic cation transporter 1 (OCT1), OCT2, organic cation/carnitine transporter 1 (OCTN1), and OCTN2 in hyperuricemic mice. Moreover, vaticaffinol markedly down-regulated renal protein levels of NOD-like receptor 3 (NLRP3), apoptosis-associated speck-like (ASC), and Caspase-1, resulting in the reduction of interleukin (IL)-1β, IL-18, IL-6 and tumor necrosis factor-α (TNF-α) levels in this animal model. Additionally, HPLC and LC-MS analyses clearly testified the presence of vaticaffinol in the crude extract. These results suggest that vaticaffinol may be useful for the prevention and treatment of hyperuricemia with kidney inflammation.

Relationship between oxidative stress and inflammation in peripheral and cerebral system of oxonate-induced hyperuricemic rats

To study what kind of role uric acid play on the relationship between oxidative Stress and inflammation in peripheral and cerebral system of oxonate-induced hyperuricemic rats. Twenty-six eight male Wistar rats were divided into two groups randomly. Potassium oxonate was used to establish hyperuricemic model for four weeks. In 2nd and 4th week, uric acid (UA) level, total superoxide dismutase (T-SOD), Gu,Zn-SOD activity and interleukin-1beta (IL-1ß) concentration in serum were determined respectively. In 4th week, one hour after last PO treatment, five rats of every group were given Evans Blue to test blood-brain barrier (BBB) permeability. Other brains were obtained to analysis T-SOD, Gu,Zn-SOD activity and IL-1ß concentration in cerebral system. Meanwhile, brain and kidney were stained with hematoxylin and eosin (H&E) to observe pathological change. In 2nd week, both of T-SOD and Gu,Zn-SOD activity in serum increased obviously (P<0.05) in hyperuricemia rats. However, IL-1ß content didn't change remarkably. In the 4th week, T-SOD activity in model group had become similar with control group, and at the same time IL-1ß content in serum increased significantly (P<0.05). Pathological section showed the structural and functional unit of the kidney had been damaged. On the contrary, both of T-SOD and Gu,Zn-SOD activity in brain increased obviously (P<0.05), but IL-1ß concentration was no significant difference between two groups. In addition, the results of Evans Blue and H&E suggested the integrity of BBB and structure of brain were not changed after PO treatment. The permeability of BBB and form of UA would be potential factors to decide what kind role UA play on keeping balance between anti-oxidative stress and induction of inflammatory response.

Effects of 3,5,2 ’,4 ’-tetrahydroxychalcone on urate excretion in hyperuricemic mice / 中国药理学通报

Aim To investigate the effects of 3 ,5 ,2 ’ , 4’-tetrahydroxychalcone (P40) on urate excretion, as well as mRNA and protein expressions of renal URAT1 and GLUT9 in hyperuricemic mice. Methods Sixty Kunming mice were randomly divided into six groups:normal control group, hyperuricemic group ( model group), P40 2. 0, 4. 0, 8. 0 mg·kg-1 groups and positive control group. All drugs were administered in-tragastrically to mice for 5 doses. Hyperuricemic mice were induced by intraperitoneal injection of uric acid (0. 15 g·kg-1 body weight) for 3 times. The urate levels were assayed with the phosphotungstic acid method. The mRNA and protein expressions of GLUT9 and URAT1 were determined by RT-PCR and Western blot. Results P40 at a dose of 4. 0 and 8. 0 mg · kg-1 significantly reduced the serum urate levels in a dose-dependent manner, when compared with untreat-ed hyperuricemic mice ( P<0. 05 or 0. 01 ) . The he-patic urate contents decreased in untreated-and treated-hyperuricemic mice as compared with normal mice ( P<0. 01 ) . Furthermore, P40 had no influence on the renal URAT1 mRNA and protein expression levels, while it could down-regulate renal GLUT9 protein ex-pression but not mRNA expression in hyperuricemic mice. Conclusion P40 possesses potent uricosuric effects associated with urate reabsorption by down-regu-lating the protein expression of GLUT9 in kidney.

A Novel UMOD Mutation (c.187T>C) in a Korean Family with Juvenile Hyperuricemic Nephropathy

Familial juvenile hyperuricemic nephropathy (FJHN; OMIM 162000) is an autosomal dominant disorder characterized by hyperuricemia and gouty arthritis due to reduced kidney excretion of uric acid and progressive renal failure. Gradual progressive interstitial renal disease, with basement membrane thickening and glomerulosclerosis resulting from fibrosis, starts in early life. In most cases of FJHN, uromodulin gene (UMOD) is responsible for the disease; however, there has been only one report of a genetically confirmed FJHN family in Korea. Here we report another Korean family with FJHN, in which three male members. a father and 2 sons.developed gout and progressive renal insufficiency. The clinical, laboratory, and radiological findings were consistent with FJHN, and renal biopsy showed chronic parenchymal damage, which can be found in FJHN but is not specific to this disease. In order to confirm the diagnosis, sequence analysis of the UMOD was performed, and a novel heterozygous missense variant (c.187T>C; p.Cys63Arg) in exon 3 was identified. We assume that this variant is likely to be the causative mutation in this family, as the variant segregated with the disease. In addition, approximately two-thirds of the known mutations lead to a cysteine amino acid change in uromodulin, and all such variants have been shown to cause UMOD-associated kidney disease. In summary, we report a Korean FJHN family with three affected members by genetic analysis of the UMOD, and provide the first report of a novel heterozygous missense mutation.