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. 2025 Dec 3;75(2):202–213. doi: 10.1538/expanim.25-0127

A nonsense mutation in the Mocos gene induces xanthinuria, obstructive nephropathy, and anemia in rats

Mao Urasaki 1, Kana Nagasaka 1, Minori Kido 1, Kenta Hayashi 1, Ayumi Watanabe 1, Kosuke Hattori 2, Takahiro Sekiguchi 3, Mitsuru Kuwamura 3, Miyuu Tanaka 3, Tomoji Mashimo 2, Takashi Kuramoto 1
PMCID: PMC13100697  PMID: 41338991

Abstract

Xanthinuria type II is a rare hereditary disorder caused by mutations in the MOCOS gene, leading to dual deficiency of xanthine dehydrogenase and aldehyde oxidase. To establish a robust animal model for this condition, we generated Mocos knock-in (KI) rats carrying the Arg419Ter nonsense mutation identified in Japanese patients. Homozygous KI rats exhibited severe growth retardation, anemia, and reduced survival, with all individuals dying by 14 weeks of age. Biochemical analyses revealed elevated levels of hypoxanthine and xanthine, along with decreased uric acid in both serum and urine, confirming xanthinuria. Homozygous KI rats also showed increased blood creatinine (CRE) and urea nitrogen (UN), and decreased urinary CRE and UN, indicating renal dysfunction. Histopathological examination showed obstructive nephropathy characterized by tubular atrophy, crystal deposition, and inflammation. Compared to existing mouse models, Mocos KI rats demonstrated extended lifespan, enabling more detailed investigation of disease mechanisms. This rat model provides a valuable tool for studying the pathogenesis of xanthinuria type II and exploring potential therapeutic strategies.

Keywords: anemia, Mocos, nephropathy, rats, xanthinuria

Introduction

Purines are important components of nucleotides. During nucleotide catabolism, phosphate and ribose are recycled, while purines are broken down by oxidization and finally excreted as uric acid. In this metabolic process, xanthine dehydrogenase (XDH, EC:1.17.1.4) has a crucial role in the oxidation of hypoxanthine to xanthine and xanthine to uric acid. Defects in XDH lead to the accumulation of hypoxanthine/xanthine and depletion of uric acid in urine, resulting in xanthinuria.

Xanthinuria is a rare hereditary disorder with unknown prevalence, but about 150 cases have been described so far [1]. Xanthinuria is divided into two forms: type I and type II. The xanthinuria type I is caused by XDH deficiency alone due to mutations in the XDH gene. Type II is caused by a dual deficiency of XDH and aldehyde oxidase (AOX1, EC: EC:1.2.3.1). The deficiency results from mutations in a single gene, MOCOS [2], because MOCOS (molybdenum cofactor sulfatase, EC:2.8.1.9) catalyzes the sulfurization of molybdenum cofactor of XDH and AOX1, which is required for their activities.

Both forms of xanthinuria lead to an identical clinical phenotype: accumulation of xanthine and depletion of uric acid in blood and urine. Although about two-thirds of affected individuals are asymptomatic, the remaining third form xanthine stones in the kidney, which result in varying degrees of nephrolithiasis, hydronephrosis, and renal failure [3]. There is no curative treatment, but a low purine diet and a high fluid intake are recommended.

To date, four variants associated with xanthinuria type II have been identified in human MOCOS gene. An Arg419Ter nonsense variant has been identified in two unrelated Japanese patients [2]. An Ala57Pro missense variant has been identified in a Japanese family [4]. An Arg776Cys missense variant has been identified in a Bedouin Arab family with xanthinuria [5]. This variant has also been found in an unrelated woman of Jewish descent who harbors a frameshift mutation (Gln347fsTer379), suggesting she is compound heterozygous for both mutations.

In addition, MOCOS gene variants associated with xanthinuria have been identified in several domestic animal species. A frameshift mutation was first described in the Tyrolean Grey breed of cattle [6], and the same mutation was later detected in Brown Swiss cattle [7], suggesting that this variant is prevalent among certain European breeds. A homozygous missense variant (Asp303Gly) has also been reported in goats [8]. In dogs, multiple variants have been identified: a splice variant leading to exon 2 skipping in Manchester Terriers, a missense variant (Leu46Pro) in Dachshunds, and a frameshift variant (Ala128Glyfs*30) in Cavalier King Charles Spaniels and English Cocker Spaniels [9]. All of these mutations have been detected in homozygous form in affected animals or as compound heterozygotes, supporting an idea that MOCOS variants causing xanthinuria result in loss of function.

To test this hypothesis and develop a model for xanthinuria type II, Mocos knockout (KO) mice have been generated [10]. They exhibit xanthinuria and obstructive nephropathy, with a significantly shortened lifespan of less than 4 weeks. Although the Mocos KO mice are potential models of xanthinuria, their short lifespan limits their availability in experiments with intervention.

Similarly to mice, rats are widely used in biomedical research, but their physiological characteristics differ from those of mice. Aiming to extend the lifespan and establish a better model of xanthinuria type II, we attempted to generate Mocos gene-edited rats with CRISPR/Cas9 system. To mimic a human case, we focused on the Arg419Ter nonsense variant, which has been identified in Japanese patients with xanthinuria type II [2]. We introduced a knock-in (KI) allele, AGG (Arg) to TGA (Ter), using CRISPR/Cas9 with long single-stranded oligonucleotide (lssODN). The Mocos KI rats exhibited growth retardation, anemia, and xanthinuria. Obstructive nephropathy was also observed. The Mocos KI rats survived up to 14 weeks of age, which enhances their utility for investigating the pathogenesis and developing curative treatments for xanthinuria.

Materials and Methods

Animal experiment

All animal experiments were approved by the Animal Research Committee of Tokyo University of Agriculture (Approval number: 2022130) and The University of Tokyo (Approval number: A23-34), and were conducted in accordance with their regulations on animal experimentation.

Generation of Mocos KI rats

Mocos KI rats were generated using CRISPR/Cas9 system as previously described [11]. Guide RNAs (gRNAs) were designed by Optimized CRISPR Design (crispr.mit.edu) and synthesized by Integrated DNA Technologies, Inc. (Coralville, IA, USA). LssODNs were prepared using a LsODN Preparation Kit (Biodynamics Laboratory Inc., Tokyo, Japan). Cas9 protein was purchased from Integrated DNA Technologies. Pronuclear-stage embryos of F344/Jcl rats were obtained through natural mating. Female rats with vaginal plugs were euthanized by CO2 inhalation followed by cervical dislocation, and their oviducts were collected. Embryos were flushed out from the ampullae using culture medium. Cas9 protein, lssODNs, and gRNAs were introduced into the embryos using a super electroporator NEPA 21 (NEPA GENE Co., Ltd., Ichikawa, Chiba, Japan). The sequences and positions of the induced mutation, lssODNs and gRNAs are presented in Figs. 1A and B. Embryos that developed to the two-cell stage were transferred into the oviducts of pseudopregnant female rats anesthetized with isoflurane. Genotyping of the offspring was performed using the following primer set: 5′-GTGGCCAATGTGAAGGAGTT-3′ and 5′-AATGCCTCCTACCAACAACG-3′ (Fig. 1B). Founder rats were mated with F344/Jcl rats and F1 heterozygous rats were intercrossed to obtain F2 progeny.

Fig. 1.

Fig. 1.

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Generation of Mocos KI rats. A, Exon-intron structure of the rat Mocos gene. The position of the nonsense mutation (AGG to TGA) in exon 7 is indicated by a vertical bar. B, Positions of guide RNAs (gRNAs) and the genomic region targeted by the long single-stranded oligonucleotide (lssODN) used to generate Mocos KI rats are shown. Exon 7 of the rat Mocos gene is indicated in bold. gRNA and PAM sequences are highlighted in yellow and light blue, respectively. The lssODN carrying the Arg419Ter nonsense mutation (highlighted in red) was 786 bp in length and included 250-bp and 100-bp homology arms (underlined). Forward and reverse primers (highlighted in grey) were used to screen founder rats. Solid triangles indicate breakpoints identified in the deletion line, F344-Mocosem2/Tua. C, Relative expression levels of Mocos mRNA in the liver. Transcript levels normalized to Ppia were significantly decreased in Mocos KI rats compared to wild-type rats. *: P<0.05.

RNA preparation and quantitative PCR

Total RNA was isolated using ISOGEN II (NIPPON GENE Co., Ltd., Chiyoda, Tokyo, Japan) according to the manufacturer’s instructions. Complementary DNA (cDNA) was synthesized using FastGene Scriptase II (NIPPON Genetics Co., Ltd.). Real-time PCR was performed using the Thermal Cycler Dice Real Time System (Takara Bio, Inc.) with the KAPA SYBR Fast qPCR Kit (Roche Diagnostics K.K., Minato, Tokyo, Japan). Primer sequences for quantification of Mocos were 5′-CAGTAGCATCCAGCCGATCC-3′ and 5′-GTCACCTCAAACGCAGCACAG-3′.

The number of target molecules in each test sample was analyzed by monitoring the amplification curves of both test and reference samples, each containing 101 to 106 molecules of the gene of interest, as previously described [12]. The number of target molecules was normalized to that of peptidylprolyl isomerase A (Ppia), which served as an internal control.

Genotyping of the Arg419Ter mutation

The CycleavePCR method was used to genotype the Arg419Ter mutation. This method detects fluorescent signals from allele-specific DNA-RNA chimera probes that are digested by thermostable RNaseH following hybridization with amplicon [13]. The primer sequences to obtain amplicon are 5′-CACAACCAGACGTTTCTGAC-3′ and 5′-ACTACAGGGTCTTGCACTGA-3′. The probe for detecting the wild-type allele was 5′-CACCTG(A)GG-3′ with 3′ end labelled with FAM. The probe for detecting the mutant allele was 5′- CAGTTC(A)CAG-3′ with 3′ end labelled with ROX. RNA bases in the probes are indicated in parentheses. Real-time PCR was performed with the CycleavePCR Reaction Mix (Takara Bio, Inc., Kusatsu, Shiga, Japan).

Body weight measurement and survival rate

Body weights of homozygous (male: n=8, female: n=5), heterozygous (male: n=6, female: n=6), and wild-type (male: n=6, female: n=5) rats were measured weekly from 4 to 16 weeks of age. Rats were provided with standard chow (CE-2, Japan CLEA, Tokyo, Japan) and tap water ad libitum. Survival rates were calculated by recording the number of rats that died during the experimental period. Kaplan–Meier curves and log-rank tests were generated using the EZR (Easy R) plugin for R Commander [14].

Hematology and Urine analysis

Blood samples were collected in EDTA-2K tubes (Capiject II, Terumo Corporation, Tokyo, Japan) and analyzed using an automated veterinary hematology analyzer, XN-1000V (Sysmex Corporation, Kobe, Japan). To collect urine and feces, rats at 7 weeks of age were individually housed in metabolic cages (Natsume Seisakusho Co., Ltd., Tokyo, Japan) for three nights, with free access to standard chow and tap water. Urine and feces were collected every morning, and their volumes were measured. Food and water intake were also recorded. Urine biochemical analysis was performed at the Nagahama Institute of Bioscience (OrientalBio Co., Ltd., Nagahama, Japan). Xanthine and hypoxanthine levels were determined using the Xanthine/Hypoxanthine Assay Kit (ab155900; Abcam, Cambridge, UK), following the manufacturer’s instructions.

Serum biochemistry

Blood samples were collected from the abdominal vein of male rats (9–12 weeks of age) under isoflurane anesthesia and were coagulated in Venoject II tubes (Terumo Corporation, Tokyo, Japan). Serum was collected after centrifuge at 10,000 × g for 5 min. Blood urea nitrogen (BUN) and creatinine (CRE) were measured at Nagahama Institute of Bioscience (OrientalBio Co., Ltd.).

Histological and immunohistochemical analysis

Rats (7–8 weeks of age) were euthanized under isoflurane anesthesia. Tissue samples harvested from the kidney, liver, adrenal glands, spleen, and brain were fixed in 10% neutral buffered formalin and embedded in paraffin. Sections 4 µm thick were cut and stained with hematoxylin and eosin (HE). After pretreatment by autoclaving in citrate buffer (pH 6.0), immunohistochemistry for ionized calcium-binding adapter molecule 1 (IbaI) was performed on 4 µm paraffin sections using a HISTOSTAINER 36A (Nichirei Biosciences, Tokyo, Japan). Sections were incubated for 1 h at room temperature with a rabbit anti-Iba1 polyclonal antibody (1:750; Code No. 019-19741, FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). Following incubation in 3% H2O2 for 15 min at room temperature, bound antibodies were detected using Histofine Simplestain Rat MAX-PO(R) (Nichirei Biosciences) and 3,3′-diaminobenzidine (DAB Substrate Kit; Nichirei Biosciences). Images were acquired using an Olympus VS-120 virtual slide scanner (Olympus, Tokyo, Japan).

Statistical analysis

Statistical comparisons between experimental groups were performed using one-way or two-way ANOVA. The post-hoc testing was performed by the Mann-Whitney U test. Survival analyses were conducted using the log-rank test. All statistical tests were conducted using the EZR package [14]. A P value <0.05 was considered significant.

Results

Development of Mocos KI rats harboring the Arg419Ter nonsense mutation

To develop a rat model of xanthinuria and investigate the underlying mechanisms, we generated Mocos KI rats harboring the Arg419Ter nonsense mutation using the CRISPR/Cas9 system (Fig. 1A). The mutation (AGG>TGA) was introduced by transferring lssODNs along with gRNAs to embryos (Fig. 1B). We obtained two Mocos-mutated rat strains. One strain, F344-Mocosem1Tua, harbored the Arg419Ter mutation. The other strain, F344-Mocosem2Tua, carried a 434 bp deletion that included the target site. For further investigation, we used the F344-Mocosem1Tua strain, as it harbored the Arg419Ter mutation identical to the variant found in Japanese patients [2]. Mocos mRNA was significantly reduced in the liver of the KI line. The relative expression levels compared to wild-type F344 rats was 0.56 ± 0.08 (P<0.01) in F344-Mocosem1Tua (Fig. 1C). The significant reduction in mRNA levels is likely due to nonsense-mediated decay.

Growth retardation and reduced survival in Mocos KI rats

Rats homozygous for the nonsense mutation exhibited reduced body size from the time of weaning (Fig. 2A). Their body weight was significantly lower than that of heterozygous or wild-type rats in both sexes. Notably, homozygous rats showed impaired weight gain after 5 weeks of age, indicating severe growth retardation (Fig. 2B). All homozygous rats died between 7 and 14 weeks of age, with an average lifespan of 12.6 ± 2.0 weeks. In contrast, heterozygous rats did not differ in body weight or lifespan from wild-type rats (Figs. 2B and C). There was no apparent phenotypic difference observed between males and females.

Fig. 2.

Fig. 2.

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Clinical appearance, growth and survival of Mocos KI rats. A, Appearance of homozygous Mocos KI rat (right), heterozygous (center), and wild-type littermate (left). Homozygous rats exhibited small body size and dull, unhealthy fur. B, Growth curves of homozygous, heterozygous and wild-type rats. Body weights of heterozygous rats of both sexes were comparable to wild-type controls throughout the observation period. In contrast, homozygous rats showed significantly lower body weights than heterozygous and wild-type rats from 5 to 13 weeks of age, with growth arrest evident after 5 weeks of age. A two-way ANOVA was performed to assess intergroup effect. The degrees of freedom numerator and denominator were 2 and 33 in male, and 2 and 32 in female, respectively. In males (F [2,33]=16.53, P<0.001) and in females (F [2,32]=24.48, P<0.001), the effect was significant. C, Survival rate of wild-type (n=18), heterozygous (n=34) and homozygous (n=21) Mocos KI rats over time. *: P<0.01.

Polydipsia and polyuria in homozygous Mocos KI rats

Obstructive nephropathy has been reported in Mocos-deficient mice [10]. Impaired renal reabsorption under such conditions can lead to polyuria and dehydration. To assess these symptoms, we measured water and food intake, as well as urine and feces output. No significant differences were observed in food intake or feces output among homozygous, heterozygous, and wild-type rats (Figs. 3A and B). In contrast, water intake and urine volume normalized to body weight were markedly elevated in homozygous rats compared to heterozygous and wild-type controls (Figs. 3C and D). These findings indicate that homozygous Mocos KI rats exhibit polydipsia and polyuria.

Fig. 3.

Fig. 3.

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Water and food intake, and urine and feces output in Mocos KI rats. A–D. Food intake (A), feces output (B), drinking water volume (C), and urine volume (D) of male homozygous (n=6), heterozygous (n=6), and wild-type (n=6) rats at 6–7 weeks of age. All values are normalized to body weight. Drinking water and urine volumes were significantly higher in homozygous rats compared to heterozygous and wild-type controls. A one-way ANOVA was performed to assess intergroup differences. The degrees of freedom numerator and denominator were 2 and 15, respectively. (A: F [2,15]=3.504, P=0.056; B: F [2,15]=0.586, P=0.569; C: F [2,15]=299.1, P<0.001; D: F [2,15]=307.9, P<0.001). Mann-Whitney U test was performed for post-hoc testing. *: P<0.001.

Biochemical evidence of xanthinuria in homozygous Mocos KI rats

Urine hypoxanthine/xanthine concentrations were significantly higher in homozygous Mocos KI rats compared to heterozygous and wild-type rats (Fig. 4A). Consistently, uric acid–the final metabolite of purine metabolism–was significantly lower in homozygous rats than in heterozygous or wild-type rats (Fig. 4B). We also observed significantly elevated hypoxanthine/xanthine and reduced uric acid levels in the serum of homozygous rats compared to heterozygous and wild-type controls (Figs. 4C and D). These findings indicate that homozygous Mocos KI rats developed xanthinuria.

Fig. 4.

Fig. 4.

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Serum and urinary purine metabolites in Mocos KI rats. A, B. Hypoxanthine/xanthine (A) and uric acid (UA) (B) concentrations in urine of homozygous, heterozygous, and wild-type rats. C, D. Hypoxanthine/xanthine (C) and UA (D) concentrations in serum of homozygous, heterozygous, and wild-type rats. A one-way ANOVA was performed to assess intergroup differences. The degrees of freedom numerator and denominator were 2 and 15, respectively. (A: F [2,15]=16.29, P<0.001; B: F [2,15]=3.964, P=0.041; C: F [2,15]=390.5, P<0.001; D: F [2,15]=18.11, P<0.001). Mann-Whitney U test was performed for post-hoc testing. *: P<0.001.

Hematological abnormalities in Mocos KI rats

As anemia is common in patients with chronic kidney disease (CKD) [15] and Mocos KO mice have been reported to exhibit anemia [10], we performed hematological analysis of Mocos KI rats. Red blood cell (RBC) count, hemoglobin (HGB), and hematocrit (HCT) values were significantly decreased in Mocos KI homozygous rats compared to heterozygous and wild-type rats (Fig. 5). Interestingly, RBC, HGB, and HCT values in heterozygous rats were also significantly lower than wild-type controls.

Fig. 5.

Fig. 5.

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Hematological parameters in Mocos KI rats. A–C. Red blood cell (RBC) count (A), hemoglobin (HGB) concentration (B), and hematocrit (HCT) (C) in male homozygous (n=6), heterozygous (n=6), and wild-type (n=6) rats at 6–7 weeks of age. Heterozygous rats exhibited significantly lower RBC, HGB, and HCT values compared to wild-type rats, while homozygous rats showed markedly reduced levels compared to both heterozygous and wild-type rats. A one-way ANOVA was performed to assess intergroup differences. The degrees of freedom numerator and denominator were 2 and 15, respectively. (A: F [2,15]=42.13, P<0.001; B: F [2,15]=63.59, P<0.001; C: F [2,15]=85.28, P<0.001). Mann-Whitney U test was performed for post-hoc testing. *: P<0.05, **: P<0.01.

White blood cell (WBC) count was significantly increased in homozygous KI rats. Notably, neutrophil (NEUT) counts were elevated, suggesting systemic inflammation in homozygous rats (Supplementary Fig. 1).

Obstructive nephropathy in homozygous Mocos KI rats

Urine biochemistry demonstrated significantly higher albumin concentration and significantly lower levels of CRE and urea nitrogen in homozygous Mocos KI rats compared to heterozygous and wild-type rats, indicating impaired glomerulus function (Figs. 6A–C). Additionally, homozygous rats exhibited significantly lower concentrations of electrolytes, including sodium, potassium, chloride, calcium, and phosphate (Figs. 6D–H), suggesting impaired reabsorption of water and electrolytes in the proximal tubule. In addition to urine biochemistry, serum biochemistry demonstrated significantly higher levels of BUN and CRE in homozygous rats compared to heterozygous and wild-type controls, indicating the nephropathy in Mocos KI homozygous rats (Supplementary Fig. 2).

Fig. 6.

Fig. 6.

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Urine biochemistry in Mocos KI rats. Urine biochemistry of albumin (ALB), creatinine (CRE), nitrogen (UN), sodium, potassium, chloride, calcium, and phosphor (IP) in homozygous (n=6), heterozygous (n=6), and wild-type (n=6) rats at 6–7 weeks of age. A one-way ANOVA was performed to assess intergroup differences. The degrees of freedom numerator and denominator were 2 and 15, respectively. (A: F [2,15]=8.311, P=0.004; B: F [2,15]=173.1, P<0.001; C: F [2,15]=34.20, P<0.001; D: F [2,15]=101.0, P<0.001; E: F [2,15]=129.1, P<0.001; F: F [2,15]=121.1, P<0.001; G: F [2,15]=15.73, P<0.001; H: F [2,15]=66.01, P<0.001). Mann-Whitney U test was performed for post-hoc testing. *: P<0.01.

Consistent with urine and serum biochemistry, we observed significant renal abnormalities in homozygous rats. At necropsy, the kidney of wild-type rats appeared normal (Figs. 7A and C), whereas those of homozygous rats showed a rough and pale surface and appeared atrophic, with thinner cortex (Figs. 7B and D). Histopathological examination revealed numerous renal tubules exhibiting atrophy or cystic dilation in the renal parenchyma of homozygous rats, accompanied by interstitial fibrosis and infiltration of lymphocytes, plasma cells, and macrophages (Figs. 7E and F). From the cortex to the medulla, the dilated tubular lumens contained characteristic deposits of various sizes, including round to irregularly shaped lamellated eosinophilic materials with a basophilic rim and/or unstained crystal-shaped clefts (Figs. 7E and F). These findings were associated with tubular disruption, detachment or loss of tubular epithelium, accumulation of cellular debris with neutrophils, and formation of urinary casts (Figs. 7E and F). In addition, infiltration of foreign body-type multinucleated giant cells phagocytizing deposits or crystals was frequently observed (Fig. 7F), which was rarely seen in other rat nephropathy models, including Alport syndrome and cystic fibrosis [16, 17]. The number and size of glomeruli were reduced, with expansion of Bowman’s space and thickening of Bowman’s capsule, although sclerotic glomeruli were rarely observed. Other organs appeared normal in homozygous rats. No significant histopathological changes were observed in any organs of heterozygous rats. Taken together, these findings indicate that homozygous Mocos KI rats suffered from obstructive nephropathy.

Fig. 7.

Fig. 7.

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Macroscopic and histopathological findings in the kidneys of Mocos KI rats. A, B. Macroscopic sagittal sections of kidneys from wild-type (A) and homozygous Mocos KI rats (B) at 8 weeks of age. Kidneys from homozygous rats appear pale, reduced in size, and exhibit a rough, granular surface. C, D. Low-magnification microscopic images of sagittal kidney sections from wild-type (C) and homozygous (D) rats. Homozygous kidneys show cortical thinning, irregular contours, and signs of atrophy. E. Magnified view of the boxed area in panel D (renal cortex). The atrophic renal parenchyma contains numerous tubules with atrophy or dilation, accompanied by interstitial fibrosis and mononuclear cell infiltration. F. Higher magnification of the renal medulla from a homozygous rat. Intratubular deposits (black arrows) vary in size and exhibit a lamellated appearance, consisting of eosinophilic material with a basophilic rim and unstained crystal-shaped clefts. Foreign body-type multinucleated giant cells (yellow arrowheads) phagocytosing the deposits or crystals (asterisks) are also observed. These multinucleated giant cells and macrophages are immunopositive for Iba1 (inset). Scale bars: 2 mm (A–D), 200 µm (E), 100 µm (F).

Discussion

In the present study, we generated Mocos KI rats carrying Arg419Ter nonsense mutation which was identified in Japanese patients with xanthinuria. The gene-edited homozygous rats exhibited severe growth retardation and failed to survive beyond 14 weeks of age. They showed clear biochemical features of xanthinuria, including elevated hypoxanthine/xanthine and reduced uric acid levels in both serum and urine. Additionally, lamellate deposits were observed in the renal tubules. These findings demonstrate that the Mocos KI rats recapitulate the phenotype of xanthinuria type II.

The Arg419Ter nonsense variant was identified in two unrelated Japanese xanthinuria type II patients [2]. Both patients exhibited hypouricemia and undetectable urinary uric acid. Their serum xanthine levels were 2.1 mg/dl and 0.17 mg/dl, respectively, compared to 0.077 ± 0.032 mg/dl in healthy individuals. Oxypurinol was not detected in the serum or urine of either patient following allopurinol loading, leading to a diagnosis of xanthinuria type II. Although our Mocos KI rats were not subjected to an alloprinol loading test, they mimicked the serum and urinary phenotypes of the patients, supporting that the Arg419Ter variant was causative and validating the homozygous KI rats as a model of the xanthinuria type II.

Nonetheless, the clinical symptoms in Mocos KI rats were more severe than those observed in human patients. Homozygous rats showed marked growth retardation after weaning and did not survive beyond 16 weeks. Similarly, mouse models of xanthinuria, including Xdh KO (type I) and Mocos KO (type II) mice, exhibited stunted growth and die before 6 weeks of age [10, 18]. In constant, human patients with xanthinuria are often asymptomatic and rarely progress to renal failure [19]. These phenotypic differences suggest that the development and severity of xanthinuria may vary among species. Indeed, purine metabolism has been known to differ between human and rodents. Hypoxanthine phosphoribosyl transferase (HPRT), which converts hypoxanthine into inosine monophosphate and reuses it in the purine salvage pathway, exhibits higher activity in humans than in mice [20, 21]. Therefore, hypoxanthine may accumulate more readily in rodents. Since hypoxanthine inhibits the synthesis of nicotinamide mononucleotide, a precursor of NAD+, low HPRT activity may lead to NAD+ depletion [22] and potentially contributing to the shortened lifespan observed in xanthinuria model mice such as Xdh and Mocos KO mice [18]. We hypothesize that differences in lifespan among mouse, rat, and human MOCOS mutants may reflect species-specific variations in HPRT activity.

Mocos KI rats displayed significantly reduced RBC, HGB, and HCT values compared to heterozygous and wild-type rats. These erythrocyte-related values were outside the normal range established across approximately 200 rat strains [23, 24], indicating that homozygous Mocos KI rats were anemic. Anemia is common in patients with CKD and is typically caused by reduced erythropoietin (EPO) production, inflammation, and oxidative stress in the kidney. It is likely that renal dysfunction in homozygous rats led to decreased EPO production, resulting in anemia.

Interestingly, heterozygous Mocos KI rats also showed significantly lower RBC, HGB, and HCT values than wild-type rats, despite not exhibiting xanthinuria. MOCOS deficiency inactivates XDH, which plays a key role in regulating reactive oxygen species (ROS) [25], and ROS are critical mediators of hematopoiesis [26]. We hypothesize that ROS activity may affect hematopoiesis in heterozygous rats, or that EPO production may be impaired despite the absence of overt histopathological changes in the kidney. Further investigations, including measurement of ROS and EPO levels, are needed to elucidate the mechanisms underlying the reduced erythrocyte parameters in both heterozygous and homozygous rats.

We observed various deposits in the renal tubules of homozygous rats. The deposits were surrounded by the lamellated eosinophilic material with a basophilic rim, which closely resemble those reported in Mocos KO mice [10]. Given that elevated hypoxanthine/xanthine concentrations in the urine of Mocos KI rats, these deposits are likely xanthine crystal precipitates.

Crystal deposition in the kidney can cause injury when formed or retained within the renal structures, leading to acute and chronic kidney disorders referred to as crystalline nephropathies [27]. Crystal nephropathies have been classified based on the location of crystal deposition: renal parenchyma (type 1), nephron (type 2), or urinary tract (type 3) [28]. The type 2 crystalline nephropathy involves endogenous metabolites such as xanthin and it is characterized by acute necroinflammation and tubular obstruction, often accompanied by multinucleated giant cells. Our morphological data showed that the deposits were located within the tubules, appeared to obstruct them, and were associated with multinucleated giant cells and widespread renal inflammation. These findings suggest that Mocos KI rats are classified as type 2 crystal nephropathy.

In summary, we generated Mocos KI rats harboring the Arg419Ter nonsense variant. These rats exhibited severe growth retardation, anemia, and xanthinuria. In addition, Mocos KI rats exhibited obstructive nephropathy, in which xanthine crystals were often phagocytized by multinucleated macrophages. Notably, Mocos KI rats demonstrated extended survival compared to the mouse model, providing a valuable platform for more precise and comprehensive investigations into the molecular mechanisms underlying xanthinuria and obstructive nephropathy.

Supplementary

Supplementary Materials
expanim-75-2-202-s001.pdf (330.2KB, pdf)

Acknowledgments

Authors are grateful to Kouya Murata, Aigo Kamikawa, and Akari Yoshida for their technical assistance. This work was supported by JSPS KAKENHI Grant Number JP22H04922 (AdAMS). F344-Mocosem1/Tua (NBRP-Rat #1063) and F344-Mocosem2/Tua (NBRP-Rat #1064) were deposited at the NBRP-Rat Project in Japan and distributed through the Project.

Funding Statement

This work was supported by JSPS KAKENHI Grant Number JP22H04922 (AdAMS).

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