Xanthine Oxidoreductase Inhibitors Suppress the Onset of Exercise-Induced AKI in High HPRT Activity Urat1-Uox Double Knockout Mice.

BACKGROUND: Hereditary renal hypouricemia type 1 (RHUC1) is caused by URAT1/SLC22A12 dysfunction, resulting in urolithiasis and exercise-induced AKI (EIAKI). However, because there is no useful experimental RHUC1 animal model, the precise pathophysiologic mechanisms underlying EIAKI have yet to be elucidated. We established a high HPRT activity Urat1-Uox double knockout (DKO) mouse as a novel RHUC1 animal model for investigating the cause of EIAKI and the potential therapeutic effect of xanthine oxidoreductase inhibitors (XOIs). METHODS: The novel Urat1-Uox DKO mice were used in a forced swimming test as loading exercise to explore the onset mechanism of EIAKI and evaluate related purine metabolism and renal injury parameters. RESULTS: Urat1-Uox DKO mice had uricosuric effects and elevated levels of plasma creatinine and BUN as renal injury markers, and decreased creatinine clearance observed in a forced swimming test. In addition, Urat1-Uox DKO mice had increased NLRP3 inflammasome activity and downregulated levels of Na+-K+-ATPase protein in the kidney, as Western blot analysis showed. Finally, we demonstrated that topiroxostat and allopurinol, XOIs, improved renal injury and functional parameters of EIAKI. CONCLUSIONS: Urat1-Uox DKO mice are a useful experimental animal model for human RHUC1. The pathogenic mechanism of EIAKI was found to be due to increased levels of IL-1ß via NLRP3 inflammasome signaling and Na+-K+-ATPase dysfunction associated with excessive urinary urate excretion. In addition, XOIs appear to be a promising therapeutic agent for the treatment of EIAKI.

Uricase deficiency causes mild and multiple organ injuries in rats.

Uricase-deficient rats could be one of the optimal model animals to study hyperuricemia. The present study aimed to find the biological differences between uricase-deficient (Kunming-DY rats) and wild-type male rats. Uricase-deficient rats and wild-type rats were commonly bred. Their body weight, water and food consumption, 24-h urine and feces, uric acid in serum and organs, and serum indexes were recorded or assayed. Organs, including the heart, liver, spleen, lung, kidney, thymus, stomach, duodenum, and ileum, were examined using a routine hematoxylin-eosin staining assay. We found that the growth of male uricase-deficient rats was retarded. These rats excreted more urine than the wild-type rats. Their organ indexes (organ weight body weight ratio), of the heart, liver, kidney, and thymus significantly increased, while those of the stomach and small intestine significantly decreased. The uricase-deficient rats had a significantly higher level of serum uric acid and excreted more uric acid via urine at a higher concentration. Except for the liver, uric acid increased in organs and intestinal juice of uricase-deficient rats. Histological examination of the uricase-deficient rats showed mild injuries to the heart, liver, spleen, lung, kidney, thymus, stomach, duodenum, and ileum. Our results suggest that uricase-deficient rats have a different biological pattern from the wild-type rats. Uricase deficiency causes growth retardation of young male rats and the subsequent increase in serum uric acid results in mild organs injuries, especially in the kidney and liver.

Hyperuricemia causes kidney damage by promoting autophagy and NLRP3-mediated inflammation in rats with urate oxidase deficiency.

Epidemiological research has shown that elevated serum urate concentration is a risk factor for the development of kidney disease; however, the mechanisms underlying this process have not yet been elucidated. To examine the role of urate in the kidney, we used Wistar rats to functionally disrupt expression of urate oxidase (UOX) by using the CRISPR/Cas9 system. In comparison to wild-type (WT) rats, serum urate levels spontaneously and persistently increased in UOX-KO rats, without showing a significant decrease in survival rate. Architecture and function of the kidneys in UOX-KO rats were impaired. Injury to the kidney resulted in increased interstitial fibrosis, macrophage infiltration, increased expression of NLRP3 and IL-1ß, and activation of multiple cell-signaling pathways associated with autophagy, such as AMPK, p38 MAPK, ERK and JNK pathways. Inhibition of autophagy with the PI3K inhibitor 3-MA abrogated the development of kidney damage and attenuated renal fibrosis, macrophage infiltration, and expression of NLRP3 and IL-1ß in injured kidneys. In conclusion, the UOX-KO rat is a great model to study hyperuricemia-related diseases. Hyperuricemia-induced autophagy and NLRP3-dependent inflammation are critically involved in the development of renal damage and, therefore, highlight the inhibition of autophagy and inflammation in search of therapeutic strategies to treat uric acid nephropathy.

Impaired intestinal barrier function in a mouse model of hyperuricemia.

Previous studies have demonstrated the effects of hyperuricemia on the damage to target organs, including the kidneys, joints and the heart. However, it is unclear whether hyperuricemia results in damage to the intestines. The aim of the present study was to investigate intestinal barrier dysfunction in a mouse model of hyperuricemia constructed by knocking out the urate oxidase (Uox) gene. The morphology of the intestine was assessed via hematoxylin and eosin, and alcian blue staining. The serum and intestinal tissue levels of uric acid, tumor necrosis factor (TNF)­α and interleukin (IL)­6, in addition to the presence of uremic toxins in the serum, were assessed. The levels of diamine oxidase (DAO), D­lactate (D­LAC) and endotoxins in the serum, which are markers of the intestinal permeability, were measured using ELISA. The expression of the intestinal tight junction proteins zona occludens­1 (ZO­1) and occludin were detected by reverse transcription­quantitative polymerase chain reaction, western blotting and immunohistochemical analysis. The Uox­knockout mice spontaneously developed hyperuricemia. Histopathological analysis indicated notable intestinal defects including sparse villi, mucosal edema and a declining mucus layer in hyperuricemic mice. The expression levels of ZO­1 and occludin in the intestines were downregulated, and the serum levels of DAO, D­LAC and endotoxins were higher in the hyperuricemic mice, compared with control mice. The serum and intestinal tissue levels of IL­6 and TNF­α were significantly increased. Additionally, the expression levels of the serum uremic toxins, serum creatinine, blood urea nitrogen were significantly increased in hyperuricemic mice compared with the control mice, while only a marked increase in indoxyl sulfate (IS) and p­cresol sulfate was reported. Collectively, the results of the present study suggested that intestinal barrier dysfunction and subsequent enhanced intestinal permeability may occur as a result of hyperuricemia in mice. Furthermore, we proposed that the loss of intestinal epithelium barrier function may be associated with uric acid­induced inflammatory responses; however, further investigation is required.

Knockout of the urate oxidase gene provides a stable mouse model of hyperuricemia associated with metabolic disorders.

The urate oxidase (Uox) gene encodes uricase that in the rodent liver degrades uric acid into allantoin, forming an obstacle for establishing stable mouse models of hyperuricemia. The loss of uricase in humans during primate evolution causes their vulnerability to hyperuricemia. Thus, we generated a Uox-knockout mouse model on a pure C57BL/6J background using the transcription activator-like effector nuclease (TALEN) technique. These Uox-knockout mice spontaneously developed hyperuricemia (over 420 µmol/l) with about 40% survival up to 62 weeks. Renal dysfunction (elevated serum creatinine and blood urea nitrogen) and glomerular/tubular lesions were observed in these Uox-knockout mice. Male Uox-knockout mice developed glycol-metabolic disorders associated with compromised insulin secretion and elevated vulnerability to streptozotocin-induced diabetes, whereas female mice developed hypertension accompanied by aberrant lipo-metabolism. Urate-lowering drugs reduced serum uric acid and improved hyperuricemia-induced disorders. Thus, uricase knockout provides a suitable mouse model to investigate hyperuricemia and associated disorders mimicking the human condition, suggesting that hyperuricemia has a causal role in the development of metabolic disorders and hypertension.

Disruption, but not overexpression of urate oxidase alters susceptibility to pentylenetetrazole- and pilocarpine-induced seizures in mice.

There is a continuous drive to find new, improved therapies that have a different mechanism of action in order to help diminish the sizable percentage of persons with pharmacoresistant epilepsy. Uric acid is increasingly recognized as contributing to the pathophysiology of multiple disorders, and there are indications that uric acid might play a role in epileptic mechanisms. Nevertheless, studies that directly investigate its involvement are lacking. In this study we assessed the susceptibility to pentylenetetrazole- and pilocarpine-induced seizures in mice with genetically altered uric acid levels by targeting urate oxidase, which is the enzyme responsible for uric acid breakdown. We found that although disruption of urate oxidase resulted in a decreased susceptibility to all behavioral end points in both seizure models, overexpression did not result in any alterations when compared to their wild-type littermates. Our results suggest that a chronic increase in uric acid levels may result in decreased brain excitability.

Uric acid is released in the brain during seizure activity and increases severity of seizures in a mouse model for acute limbic seizures.

Recent evidence points at an important role of endogenous cell-damage induced pro-inflammatory molecules in the generation of epileptic seizures. Uric acid, under the form of monosodium urate crystals, has shown to have pro-inflammatory properties in the body, but less is known about its role in seizure generation. This study aimed to unravel the contribution of uric acid to seizure generation in a mouse model for acute limbic seizures. We measured extracellular levels of uric acid in the brain and modulated them using complementary pharmacological and genetic tools. Local extracellular uric acid levels increased three to four times during acute limbic seizures and peaked between 50 and 100 min after kainic acid infusion. Manipulating uric acid levels through administration of allopurinol or knock-out of urate oxidase significantly altered the number of generalized seizures, decreasing and increasing them by a twofold respectively. Taken together, our results consistently show that uric acid is released during limbic seizures and suggest that uric acid facilitates seizure generalization.

Impulsivity is associated with uric acid: evidence from humans and mice.

BACKGROUND: The ability to control impulses varies greatly, and difficulty with impulse control can have severe consequences; in the extreme, it is the defining feature of many psychiatric disorders. Evidence from disparate lines of research suggests that uric acid is elevated in psychiatric disorders characterized by high impulsivity, such as attention-deficit/hyperactivity disorder and bipolar disorder. The present research tests the hypothesis that impulsivity is associated with higher uric acid in humans and mice. METHODS: Using two longitudinal, nonclinical community samples (total n = 6883), we tested whether there is an association between uric acid and normal variation in trait impulsivity measured with the Revised NEO Personality Inventory. We also examined the effect of uric acid on behavior by comparing wild-type mice, which naturally have low levels of uric acid, with mice genetically modified to accumulate high levels of uric acid. RESULTS: In both human samples, the emotional aspects of trait impulsivity, specifically impulsiveness and excitement seeking, were associated with higher levels of uric acid concurrently and when uric acid was measured 3 to 5 years later. Consistent with the human data, the genetically modified mice displayed significantly more exploratory and novelty-seeking behavior than the wild-type mice. CONCLUSIONS: Higher uric acid was associated with impulsivity in both humans and mice. The identification of biological markers of impulsivity may lead to a better understanding of the physiological mechanisms involved in impulsivity and may suggest potential targets for therapeutic intervention.

Diabetes insipidus in uricase-deficient mice: a model for evaluating therapy with poly(ethylene glycol)-modified uricase.

Uricase-deficient mice develop uric acid nephropathy, with high mortality rates before weaning. Urate excretion was quantitated and renal function was better defined in this study, to facilitate the use of these mice as a model for evaluating poly(ethylene glycol)-modified recombinant mammalian uricases (PEG-uricase) as a potential therapy for gout and uric acid nephropathy. The uric acid/creatinine ratio in the urine of uricase-deficient mice ranges from 10 to >30; on a weight basis, these mice excrete 20- to 40-fold more urate than do human subjects. These mice consistently develop a severe defect in renal concentrating ability, resulting in an approximately sixfold greater urine volume and a fivefold greater fluid requirement, compared with normal mice. This nephrogenic diabetes insipidus leads to dehydration and death of nursing mice but, with adequate water replacement, high urine flow protects adults from progressive renal damage. Treatment of uricase-deficient mice with PEG-uricase markedly reduced urate levels and, when initiated before weaning, preserved the renal architecture (as evaluated by magnetic resonance micros-copy) and prevented the loss of renal concentrating function. PEG-uricase was far more effective and less immunogenic than unmodified uricase. Retention of uricase in most mammals and its loss in humans and some other primates may reflect the evolution of renal function under different environmental conditions. PEG-uricase could provide an effective therapy for uric acid nephropathy and refractory gout in human patients.

Hyperuricemia and urate nephropathy in urate oxidase-deficient mice.

Urate oxidase, or uricase (EC 1.7.3.3), is a purine metabolic enzyme that catalyzes the conversion of uric acid to allantoin in most mammals except humans and certain other primates. The loss of urate oxidase in the human during primate evolution predisposes man to hyperuricemia, a metabolic disturbance that can lead to gouty arthritis and renal stones. To create a mouse model for hyperuricemia and gout, and to address the question of whether urate oxidase is essential in lower mammalian species, we have disrupted the urate oxidase gene in the mouse by homologous recombination in embryonic stem cells. Unlike the human situation, urate oxidase deficiency in mice causes pronounced hyperuricemia and urate nephropathy. More than half of the mutant mice died before 4 weeks of age, indicating that urate oxidase is essential in mice. These mutant mice may also serve as animal models for hyperuricemia and its related nephropathy in humans.

Uric acid catabolism in the woolly monkey.

The degradation and excretion of 2-14C-uric acid were examined in three adult woolly monkeys (Lagothrix lagothrichia) to determine the basis for the relatively high serum and urinary uric acid concentrations previously reported in this species. Like man and the great apes which lack uricase, but in distinction to most other mammals, these animals converted very little urate to allantoin. Uric acid turnover, as has been reported for other New World monkeys, was several times that of normal man. Renal urate excretion as well as disposition by extrarenal mechanisms may protect Lagothrix vrom hyperuricemia. The capacity to convert urate to allantoin appears to have been lost late in the evolution of New World monkeys. The woolly monkey deserves further study as a primate model for investigations of enzyme replacement strategies.