High-Protein Diet Induces Hyperuricemia in a New Animal Model for Studying Human Gout

Abstract

Hyperuricemia is a central risk factor for gout and increases the risk for other chronic diseases, including cardiometabolic disease, kidney disease, and hypertension. Overproduction of urate is one of the main reasons for hyperuricemia, and dietary factors including seafoods, meats, and drinking are contributed to the development of it. However, the lack of a suitable animal model for urate metabolism is one of the main reasons for the delay and limitations of hyperuricemia research. Combining evolutionary biological studies and clinical studies, we conclude that chicken is a preferred animal model for hyperuricemia. Thus, we provided chickens a high-protein diet (HPD) to evaluate the changes in the serum urate levels in chickens. In our study, the HPD increased the serum urate level and maintained it at a long-term high level in chickens. Long-term high serum urate levels induced an abnormal chicken claw morphology and the precipitation of monosodium urate (MSU) in joint synovial fluid. In addition, a long-term HPD also decreased the glomerular filtration rate and induced mild renal injury. Most importantly, allopurinol and probenecid displayed the positive effects in decreasing serum urate and then attenuated hyperuricemia in chicken model. These findings provide a novel model for hyperuricemia and a new opportunity to further investigate the effects of long-term hyperuricemia on other metabolic diseases.

Keywords: chicken; gout; high protein diet; hyperuricemia; renal disease.

Figure 1

Purine metabolism and urate degradation in different species. Purine metabolism consists of de novo synthesis and salvage pathways. Humans, hominoid primates, birds, reptiles, and terrestrial insects exhibit urate ammonotelism. Mammals other than primates and carnivorous dipteras exhibit allantoin ammonotelism. Amphibians and teleosts exhibit urea ammonotelism. Marine invertebrates, plants, bacteria, and fungi directly excrete ammonia. The arrow from PRPP to IMP means the reactions is complexed and need about 10 steps; the arrow from IMP to PRPP represents the uric acid cycle which is particularly belong to the birds, reptiles and insects; the red dotted boxes represent the four product of urate degradation; the green arrows represent the species which put the correspondence product as end product of urate degradation; the other arrows generally represent one step reaction.

Figure 2

Chicken as a suitable hyperuricemia model among various organisms. (A) Phylogenetic tree of urate metabolism-related genes. All participate sequence alignment information was based on database in NCBI gene, the uncertain sign is represented for no related information could obtain in this database. (B) Plasma concentrations of urate and allantoin in humans, mice, and chickens. The urate and allantoin level in plasma of human and mouse is referenced from Cantor J et al. [23]; and the level of allantoin is referenced from Carro M.D. et al. [30]; the range value of plasma urate in chicken is from our studies.

Figure 3

High-protein diet (HPD) increases serum levels of urate in chickens (A) Conceptual graph of the treatment group in chickens (n = 10). (B) Body weight. (C) Representative images of the feed condition. (D) Water intake. (E) Food intake. (F) Serum urate levels during consumption for 70 days. (G) Serum urate levels at 144 h when chickens were fed HPD (n = 6) and dotted line presented the serum urate average value of CON group. Values are presented as the mean ± SEM. Differences were assessed by non-parametric t test followed by Mann–Whitney test and denoted as follows: ** p < 0.01, *** p < 0.001 and **** p < 0.0001 compared with CON.

Figure 4

HPD induces an abnormal claw morphology in chickens. (A) Representative images of chicken claws after being fed for 28, 42, and 56 days. (B) Gout incidence rate. (C) Claw circumference, (D) tarsal joint circumference, and (E) toe bone circumference of chicken. Values are presented as the mean ± SEM.

Figure 5

HPD induces monosodium urate (MSU) crystal production in synovial fluid and other tissue fluids. (A) Representative images of chicken claws in normal and X-ray conditions. (B) Representative images of MSU in synovial fluids using polarized light microscopy. (scale: 50 and 20 µm) (C) Representative images of crystal in the ureter, cecum, and jejunum fluids using polarized light microscopy (scale: 20 μm).

Figure 6

HPD increases the production of urate in the liver. (A) Liver, intestine, left kidney (LK), right kidney (RK), and spleen relative weight. (B) Serum AST and ALT content. (C) H&E staining of liver (scale: 50 μm). (D) Liver total purine content, including adenine, guanine, hypoxanthine, and xanthine. (E) Liver urate content. (F) The expression of urate-producing-related genes including Xdh, Adsl, Hprt1, Impdh2, and Ppat in the liver. Values are presented as the mean ± SEM. Differences were assessed by non-parametric t test followed by Mann–Whitney test and denoted as follows: * p < 0.05 and ** p < 0.01 compared with CON.

Figure 7

HPD induces renal injury in chickens. (A) Representative images of kidney surfaces under polarized light microscopy (scale: 100 μm and 50 μm). (B) Serum UREA and CRE content. (C) Kidney urate and (D) total purine content. (E) H&E, Masson, and Alcian Blue Periodic acid Schiff (AB-PAS) staining of kidney (scale: 50 μm). Arrows denote lesions in the kidney. Values are presented as the mean ± SEM. Differences were assessed by non-parametric t test followed by Mann–Whitney test and denoted as follows: * p < 0.05 and *** p < 0.001 compared with CON.

Figure 8

Allopurinol and probenecid treatment decreases serum urate in chickens of hyperuricemia. (A) Body weight. (B) Serum urate levels during consumption for 73 days, and allopurinol and probenecid treatment began at 57 days and remained until experiment finished. (C) Serum urate levels at the end of experiment. (D) Feces purine and urate content. (E) Liver purine and urate content. Values are presented as the mean ± SEM. n = 8–10, *** p < 0.001 and **** p < 0.0001 compared with CON; # p < 0.05 ### p < 0.001 and #### p < 0.0001 compared with HPD by one-way ANOVA.

Figure 9

Allopurinol and probenecid treatment decreases gout incidence rate in chickens. (A) Gout incidence rate (n = 8–10). (B) Tarsal joint circumference and tarsal joint circumference/BW of chicken claws. (C) Representative images of chicken claws in 73 days. (D) Representative images of chicken claws in X-ray conditions. The red arrows are the position of swell in the joint of chickens. (E) Representative images of MSU crystal in synovial fluids of chicken claws using polarized light microscopy (scale: 50 µm). Values are presented as the mean ± SEM.