Zebrafish model of hyperuricemia

Hyperuricemia (HUA) is a common metabolic disorder that seriously affects human health. It is characterized by high levels of uric acid in the blood due to the excessive production or impaired excretion of uric acid.1 According to the 2019 Chinese Guidelines for the Diagnosis and Treatment of Hyperuricemia and Gout, published by the Chinese Medical Association, HUA is diagnosed in adults with non-same-day 2 fasting blood uric acid levels greater than 420 μmol/L. The body maintains a dynamic balance between the production, absorption, excretion, and breakdown of uric acid. However, certain factors can disturb this balance and cause abnormalities in uric acid levels, leading to HUA. HUA can lead to several complications, including gout and joint injury, caused by the deposition of urate crystals. Another common complication is kidney damage, which results from an inflammatory response in the kidney tissue due to the continuous deposition of urate crystals in the renal tubules.2 Animal models are indispensable tools for studying the pathological changes and therapeutic drug mechanisms of HUA as well as drug development for patients with HUA. Currently, avian species and rodents are the preferred choices for constructing animal models of HUA because of their similarities to humans in uric acid metabolism, physiology, and biochemistry. Modeling methods are categorized based on the purpose of the study, such as increasing the source of uric acid, inhibiting uricase activity, inhibiting uric acid excretion, and gene knockout. However, in addition to the fact that avian species are not mammals, their specific pathogen-free (SPF) grade feeding conditions and intake of inducers via the transoral route are difficulties faced at present. Rodents retain the uricase gene and lack homology to the human HUA model. Although the process of human uric acid metabolism can be mimicked using a uric acid oxidase (UOX) knockout technique, this is technically difficult and not applicable to high-volume operations. In contrast, zebrafish are genetically more homologous to humans and their small size (3–4 cm) saves space, making them easy to maintain. Additionally, they have a short reproductive cycle, no seasonal limitations on mating, a large number of eggs, and a fast embryonic development rate. Embryos are transparent and easy to observe, allowing real-time visualization and tracking of cells, organs, and tissues. Many of the organs and cell types of typical vertebrates are similar to those of mammals. Major organs develop in larvae within 5–6 days post-fertilization (dpf).3 In terms of socioeconomic benefits, zebrafish have lower maintenance costs than mammals. Therefore, zebrafish could replace rodents as an excellent model for studying gene function and metabolic diseases. UOX is a crucial enzyme involved in purine metabolism. It catalyzes the oxidation of uric acid to 5-hydroxyisouric acid, which is the first rate-limiting step in uric acid catabolism. This process converts urate into a more soluble allantoin. Owing to the loss of pseudogenes during primate evolution, humans are unable to convert uric acid to soluble allantoins in the body.4 This results in the inactivation of the gene encoding UOX and elevated levels of urates in the blood. Additionally, an efficient reabsorption system for this compound by the kidneys can lead to sensitivity to consequences that may follow the concomitant elevation of uric acid. Bhargava et al.5 discovered that zebrafish larvae begin to express Uox at 4 dpf and reach the highest levels of uric acid at 5 dpf, which correlates with the expression of this gene, mainly in the liver. Before liver formation, uric acid accumulates throughout the body of zebrafish in the absence of Uox expression. After 5 dpf, Uox is expressed in the progressively forming liver, resulting in a dramatic decrease in systemic uric acid levels. Therefore, 5 dpf zebrafish embryos were used to model HUA. Additionally, Uox expression in zebrafish liver maintains uric acid oxidase activity similar to that observed in mice. Zhang et al.6 first successfully constructed a zebrafish HUA model by co-treating 5 dpf wild-type zebrafish AB strain larvae with 200 μM potassium oxonate (PO) and 10 μM xanthine sodium salt (XSS). The uric acid level of zebrafish in the model group was significantly higher than that of the normal group after treatment. PO is an inhibitor of uric acid oxidase, and by inhibiting the activity of xanthine oxidase, it can increase uric acid levels for a short period and maintain the increased levels for a long time. XSS is a precursor of uric acid, and the exogenous intake of purines leads to increased uric acid production. The current modeling approach used to screen for uric acid-lowering drugs in zebrafish followed that of combining PO and XSS developed by Zhang et al.6 (Table 1). To model HUA development, Hall et al.13 developed a monosodium urate (MSU) crystal-driven model of acutely inflamed zebrafish larvae by microinjecting MSU crystals into the posterior ventricle of zebrafish larvae at 2 dpf. By imaging the behavior of macrophages and neutrophils in response to the MSU crystals in real time, the molecular mechanism of macrophage activation in an intact animal model was identified for the first time, revealing a mechanism of the immuno-metabolic action of macrophage activation during acute gouty inflammation. In 2022, Hall et al.14 further constructed a zebrafish model with a deletion of the uricase gene (Uox−/−) and the first zebrafish mutant with a genetically induced HUA phenotype using CRISPR/Cas9 gene editing technology to successfully inactivate the target gene, thus enabling targeted manipulation of the zebrafish genome. The Uox−/− zebrafish larval livers showed significantly reduced Uox expression and elevated urate levels. In addition, Uox−/− zebrafish showed no signs of embryonic death, in addition to being fertile. Moreover, zebrafish larvae have been reported to have a strong regenerative capacity.15 The ability of zebrafish to rapidly repair damaged organs and tissues can be used as a reference for studying several complications of HUA caused by urate crystal deposition, such as local inflammation, vascular injury, and/or impaired organ function, or as a promising direction for developing a zebrafish HUA model. For the construction and application of HUA models in zebrafish, depending on the purpose of the study, it is currently possible to construct a high-throughput drug screening model using UOX inhibitors. Because of the small size of zebrafish, the aquatic environment, and the permeability of small molecules, this model is ideal for high-throughput drug screening, but there are certain limitations. For example, few zebrafish tissues are available for experimental analyses, which, in turn, constrains in-depth studies of drug mechanisms. Microinjections of MSU crystals can mimic the development of HUA but cannot accurately replicate the elevated urate levels that often occur superimposed on the inflammatory response to MSU crystals in humans. Gene editing technology can address this obstacle but is technically challenging. Meanwhile, the current lack of effective techniques or equipment to automatically observe and analyze a large number of morphologies in zebrafish and the different modes of drug delivery means that the screening of some compounds in zebrafish still needs to be validated in other animal models. However, the development of new technologies, such as genomic analysis, in vivo imaging, and high-throughput small-molecule screening, suggests that zebrafish will continue to play a significant role in the analysis and identification of the regulation of embryonic development and genes, as well as in drug screening and development. All authors wrote and critically revised the manuscript for important intellectual content. All authors have read and approved the submitted manuscript. None. This study was supported by grants from Yunnan Clinical Research Center for Rheumatism in Traditional Chinese Medicine (202102AA310006), the Construction Project of the National Traditional Chinese Medicine Clinical Research Base (2018No.131), Scientific Research Fund Project of Yunnan Provincial Education Department (2023Y0463 and 2024Y380), The High level Key Discipline of TCM Construction Project, Rheumatology, of National Administration of Traditional Chinese Medicine (2023NO.85) and the National Natural Science Foundation of China (81960863 and 82160901). The authors have no conflict of interest relevant to this study. The data are not publicly available due to privacy or ethical restrictions.