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

Abstract

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-1β 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.

Keywords: exercise-induced acute kidney injury (EIAKI); hypoxanthine phosphoribosyltransferase (HPRT); inborn errors; knockout; mice; renal hypouricemia (RHUC); renal tubular transport; urate transporter 1 (URAT1); urolithiasis; xanthine oxidoreductase inhibitor (XOI).

Graphical abstract

Figure 1.

Establishment of male high HPRT activity Urat1-Uox DKO mice. (A) Expression of URAT1 protein in the kidney plasma membrane of male WT (C57BL/6J), high HPRT activity Uox-KO, and Urat1-Uox DKO mice (n=3 per group). (B) HPRT activity of mouse erythrocytes in male WT, high HPRT activity Uox-KO, and Urat1-Uox DKO mice (n=5 per group). (C) Body weight, (D) plasma UA, (E) urinary UA/Cr ratio, (F) urinary AL/Cr ratio, (G) FE_UA, (H) plasma Cr, and (I) BUN in male WT (B6, n=9), high HPRT activity Uox-KO (n=8), and Urat1-Uox DKO mice (n=9). Data represent means±SEM; data were analyzed using 1-way ANOVA with Tukey’s post hoc test in (B–I). Significantly different from WT (*P<0.05, **P<0.01, ***P<0.001) and Uox-KO (^##P<0.01). N.D., not detected; NS, not significant (P>0.05).

Figure 2.

EIAKI of high HPRT activity Urat1-Uox DKO mice. (A) Plasma Cr, (B) BUN, (C) CL_Cr, (D) plasma UA, (E) urinary UA/Cr ratio, (F) FE_UA, and (G) CL_UA in WT (B6, n=8), high HPRT activity Uox-KO (n=8), and Urat1-Uox DKO mice (n=9) pre- and post-exercise. Data represent means±SEM; data were analyzed using 2-way ANOVA with Tukey’s post hoc test in (A–G). Significantly different from pre-exercise group (**P<0.01, ***P<0.001) and change in Uox-KO group (^#P<0.05, ^##P<0.01, ^###P<0.001). NS, not significant (P>0.05).

Figure 3.

Renal histology and UA crystalluria of high HPRT activity Urat1-Uox DKO mice. (A) Urine was collected from WT, Uox-KO, and Urat1-Uox DKO mice pre- and post-exercise. Bright signals in the lower images are urinary deposits. Urinary crystals were observed under polarized light (scale bar=50 μm). (B) A peak corresponding to UA was detected in solution after redissolving urinary crystals in HPLC buffer (refer to Supplemental Methods for details). (C) Urinary pH in WT (B6, n=8), Uox-KO (n=8), and Urat1-Uox DKO mice (n=9) pre- and post-exercise. (D) Periodic acid-Schiff staining of the kidney of WT, Uox-KO, and Urat1-Uox DKO mice pre- and post-exercise (scale bar =100 μm). Data represent means±SEM; data were analyzed using 2-way ANOVA with Tukey’s post hoc test in (C). Significantly different from pre-exercise group (*P<0.05, **P<0.01, ***P<0.001) and WT mice group (^###P<0.001).

Figure 4.

Expression of NLRP3 inflammasome related proteins and NKA alternated in kidney of high HPRT activity Urat1-Uox DKO mice pre- and post-exercise. (A) Representative Western blots of NLRP3, ASC, Caspase-1, IL-1β, β-actin, and NKA α1 in pre- and post-exercise. Protein expression of (B) NLRP3, (C) ASC, (D) Caspase-1, and (E) IL-1β in kidney of WT (pre, n=5; post, n=8), Uox-KO (pre, n=5; post, n=8), and Urat1-Uox DKO mice (pre, n=5; post, n=9) pre- and post-exercise as determined by Western blot analysis. Protein expression of (F) NKA α1 in the kidney plasma membrane-enriched (PM) protein of WT (pre, n=5; post, n=8), Uox-KO (pre, n=5; post, n=8), and Urat1-Uox DKO mice (pre, n=5; post, n=9) pre- and post-exercise as determined by Western blot analysis. (G) The correlation of IL-1β with NKA PM protein in kidney of WT, Uox-KO, and Urat1-Uox DKO mice after exercise. Dot plot graphs show the results of densitometric quantitation. Data represent means±SEM; data were analyzed using 2-way ANOVA with Tukey’s post hoc test in (B–F). Significantly different from pre-exercise group (**P<0.01, ***P<0.001), WT mice group (^##P<0.01, ^###P<0.001), and Uox-KO mice group (^†P<0.05, ^††P<0.01, ^†††P<0.001). NS, not significant (P>0.05). (H) Schematic representation of the pathophysiologic features of model mice.

Figure 5.

Effects of topiroxostat, a nonpurine-type XOI, on purine metabolism of high HPRT activity Urat1-Uox DKO mice. (A) Plasma UA, (B) plasma Cr, (C) urinary UA/Cr ratio, (D) urinary HX+XA/Cr ratio, (E) urinary OP/Cr ratio, (F) FE_UA, (G) FE_HX+XA, and (H) FE_OP in high HPRT activity Uox-KO (n=6) and Urat1-Uox DKO mice (DKO control, n=5; topiroxostat 0.3 mg/kg, n=6; topiroxostat 1 mg/kg, n=5). Data represent means±SEM; data were analyzed using unpaired t test (Uox-KO versus DKO control) and 1-way ANOVA with Dunnett’s post hoc test (DKO control versus topiroxostat-treated groups) in (A–H). Significantly different from Uox-KO group (*P<0.05, **P<0.01) and Urat1-Uox DKO group (^#P<0.05, ^##P<0.01, ^###P<0.001). NS, not significant (P>0.05).

Figure 6.

Effects of topiroxostat and allopurinol, XOIs, on EIAKI of high HPRT activity Urat1-Uox DKO mice. (A) Plasma Cr, (B) CL_Cr, (C) plasma UA, (D) urinary UA/Cr ratio, (E) urinary HX+XA/Cr ratio, (F) urinary OP/Cr ratio, (G) FE_UA, (H) FE_HX+XA, and (I) FE_OP in Urat1-Uox DKO control group (DKO, n=10) and topiroxostat-treated Urat1-Uox DKO group (DKO+TPR, n=10) or Urat1-Uox DKO control group (DKO, n=6) and allopurinol-treated Urat1-Uox DKO group (DKO+ALP, n=6) pre- and post-exercise. (J) BUN in Urat1-Uox DKO control group (DKO, n=10) and topiroxostat-treated Urat1-Uox DKO group (DKO+TPR, n=10) pre- and post-exercise. (K) IL-1β in the kidney of the Urat1-Uox DKO control group (DKO, n=6) and topiroxostat-treated Urat1-Uox DKO group (DKO+TPR, n=6) pre- and post-exercise as determined by Western blot analysis. Data represent means±SEM; data were analyzed using 2-way ANOVA with Tukey’s post hoc test in (A–K). Significantly different from pre-exercise groups (*P<0.05, **P<0.01, ***P<0.001) and Urat1-Uox DKO control group (^#P<0.05, ^##P<0.01, ^###P<0.001). NS, not significant (P>0.05). ALP, allopurinol; TPR, topiroxostat.

Figure 7.

Schematic representation of the mechanism of EIAKI on RHUC1 together with the therapeutic action of XOIs.