The Relationship Between Xanthine Oxidoreductase Activity And Aristolochic Acid Induced Nephropathy

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PART Ⅱ: Tissue xanthine oxidoreductase activity in a mouse model of aristolochic acid nephropathy

Takeo Ishii， Tomohiro Kumagae， Hiromichi Wakui， Shingo Urate， Shohei Tanaka & et al.

Xanthine oxidoreductase (XOR) is a critical enzyme in purine metabolism and uric acid production, and its levels are reported to increase during stress, thereby promoting organ damage. Herein, we investigated the activity of XOR(Xanthine oxidoreductase) in a mouse model of aristolochic acid (AA) induced nephropathy, a type of nephrotoxic chronic kidney disease(CKD). A persistent decrease in renal function was observed in mice up to 4 weeks after 4 weeks of AA(aristolochic acid)(2.5 mg kg')administration. Renal histology revealed an increase in tubular interstitial fibrosis over time. Although AA(aristolochic acid) administration did not change XOR(Xanthine oxidoreductase) activity in the plasma, heart, liver, or muscle, XOR(Xanthine oxidoreductase) activity was persistently increased in renal tissue. Our results suggest that the renal tissue-specific increase in XOR(Xanthine oxidoreductase) activity is involved in the progression of tubulointerstitial disorders, specifically fibrosis.

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Discussion

The results of the present study showed that renal XOR(Xanthine oxidoreductase) activity was enhanced, concomitant with the development of renal dysfunction and fibrosis in an AA(aristolochic acid) nephropathy mouse model. Intriguingly, renal XOR(Xanthine oxidoreductase) activity was sustainably enhanced even after AA(aristolochic acid) injection, which may be involved in the formation of long-term renal fibrosis in AA(aristolochic acid) nephropathy. AA(aristolochic acid) is incorporated into tubular epithelial cells via the organic anion transporter 1 (OAT1)channel expressed on the renal tubular basement membrane [28,29]. After AA(aristolochic acid) administration, cellular cyclin B1 levels increase, reflective of the cell cycle arrest of tubular epithelial cells before mitosis, and apoptosis occurs intermittently [30]. This mechanism is the reason why this AA(aristolochic acid) nephropathy model is kidney-specific and useful for investigating the mechanism of renal fibrosis. We have not tested mice younger than 12 weeks; nevertheless, these mice would likely have exhibited similar patterns of renal tubular epithelial cell apoptosis and basement membrane drop. However, it is not known whether epithelial cell turnover is activated more rapidly in younger mice. Tubular epithelial cell apoptosis results in cell cycle arrest of the peritubular capillary (PTC)network, which causes the entire nephron to become hypoxic, thus triggering the expression of HIF-1α. The OAT1 channel mainly exists on the tubular epithelial basement membrane of the kidney but is also present in small quantities in liver cells [28]. Our results showed that, following AA(aristolochic acid) administration, XOR(Xanthine oxidoreductase) activation mainly occurred in the kidneys and was not observed in other tissues. Sun et al.[31]evaluated the PTC density as well as HIF-1a and vascular endothelial growth factor (VEGF)expression in female rats that were administered dietary AA(aristolochic acid) twice daily for eight weeks. They showed decreased PTC density over time, increased HIF-1a and VEGF, and no improvement in VEGF expression.Sun et al. concluded that the AA(aristolochic acid) mediated decrease in the PTC network resulted in higher HIF-1o expression levels.

In the present study, AA(aristolochic acid) administration significantly caused bodyweight loss,as well as renal function decline. A lower body mass index is associated with greater mortality in patients with CKD. Uremic cachexia is known to be the cause of weight loss in CKD, being associated with acidosis and inflammation.In addition, patients with CKD often have sarcopenia, which is related to increased risks of mortality and cardiovascular complications. Although we did not examine mouse food intake and skeletal muscle profiles in the present study,AA(aristolochic acid) induced weight loss may have been caused by cachexia and sarcopenia. Cachexia caused by uremia was suggested to reduce food intake and body weight. On the other hand, the increase in adipogenesis and lipogenesis that occurred concomitantly with the increase in XOR(Xanthine oxidoreductase) activity, related to C/EBPα or PPARy and SREBF1 gene expression progression, suggested that this reaction is a protective mechanism against weight loss.

We also observed that AA(aristolochic acid) administration consistently increased the levels of HIF-1α expression, likely due to tissue breakdown. Collectively, these results suggest that nephron hypoxia and increase in XOR(Xanthine oxidoreductase) may coincide. Persistently elevated expression levels of XOR(Xanthine oxidoreductase) in renal tissue were caused by AA(aristolochic acid) induced apoptosis of the tubular epithelium. Tissue hypoxia led to the break-down of ATP, thereby activating XOR(Xanthine oxidoreductase). Elevated XOR(Xanthine oxidoreductase) activity was observed not only along with ATP break-down, but during tissue damage caused by ROS production. Further investigation is required to determine whether XOR(Xanthine oxidoreductase) provoked tssue damage through production of oxidative stress. XOR(Xanthine oxidoreductase) is the rate-limiting enzyme in purine metabolism. It produces uric acid and ROS, the latter of which generates oxidative stress.

Through ROS production, proinflammatory cytokines in the NLR family pyrin domain containing 3 (NLRP3) inflammasome/IL-1β pathway are stimulated in macrophages [32].Meanwhile, the NRLP3/IL-1βpathway is also activated by monosodium urate, which exacerbates renal impairment through tissue deposition of uric acid crystals [32-34]. Yisireyili et al.[35]reported that in stress burdened C57BL/6 mice, XOR(Xanthine oxidoreductase), MCP-1, and TNF-α expression levels were elevated in the visceral adipose tissue, with an increase in tissue F4/80 and CD68 staining, which could be reversed with febuxostat. Page et al.[36] evaluated the XOR(Xanthine oxidoreductase) activity induced by inflammatory cytokines in human mammary epithelial cells. XOR(Xanthine oxidoreductase) activity was mainly increased upon treatment with IFN-y, but no reaction was observed with IL-6. Fibrosis is presumed to progress through enhanced stimulation of the TGF-β pathway by transglutaminase type 2 (TG2), which promotes collagen I-IV expression in the nuclear factor kappa light chain enhancer of activated B cells (NF-kB)[37-40]. When studying AA(aristolochic acid) nephropathy in mice with a C57BL/6 background, Scarpellini et al. [41 reported that syndecan-4,which promotes fibrosis, externalizes TG2 to the cell membrane. This results in the formation of a complex with TGF-β and leads to the progression of kidney fibrosis. This effect is significantly attenuated in syndecan-4 knockout mice. The proteasome inhibitor bortezomib was reported to inhibit fibrosis through TGF-β inhibition [30].TGF-βplays an important role in fibroblast transformation in AA(aristolochic acid) nephropathy through a fibrotic mechanism [4]]. In our AA(aristolochic acid) study,TGF-β and XOR(Xanthine oxidoreductase) were expressed simul-taneously, but this fibrotic mechanism was most likely induced through TGF-βexpression via activated XOR(Xanthine oxidoreductase). The expression of TGF-β and Col I genes was promoted in response to treatment with AA(aristolochic acid), which suggested a fibrotic mechanism of action. Moreover, the expression of HIF-la, a component of the hypoxia inducible factor, increased concurrently with XOR(Xanthine oxidoreductase) activation in the fibrotic pathway, which consequently increased oxidative stress in the tissues.

The findings of this experiment indicate that the production of reactive oxygen species (ROS) upon activation of the NADPH oxidase occurred concurrently with XOR(Xanthine oxidoreductase) activation in response to tissue necrosis caused by tissue hypoxia, followed by progression to the fibrotic pathway.Additionally, the microscopic observations showed that XOR(Xanthine oxidoreductase) activity and the fibrotic area were significantly correlated (Fig. 6C), suggesting that XOR(Xanthine oxidoreductase) might have caused partial tssue damage. However, further examination is needed.

It has been pointed out that XOR(Xanthine oxidoreductase) may regulate adipogenesis and mesenchymal transformation in the renal tubule epithelium [42]. However, it has not been clarified whether the XOR(Xanthine oxidoreductase) gene negatively regulates the adipose droplet deposition and mesenchymal transformation, or they result from xanthin deposition in kidney tssue when XOR(Xanthine oxidoreductase) is depleted. We did not investigate the serum lipid mechanism, but our study showed that the primary changes in renal tissue were apoptosis,a drop in tubular epithelial cells, and fibrosis. Lipid droplet deposition was not indicated. In this AA(aristolochic acid) model, adipogenesis and lipogenesis increased concomitantly with XOR(Xanthine oxidoreductase) activity, contrarily to the XOR(Xanthine oxidoreductase) model. This result suggested that the adipogenesis and lipogenesis that occur with C/EBPα or PPARy and SREBF1 gene progression was the result of cachexia or inflammation caused by ROS, and not the work of XOR(Xanthine oxidoreductase) directly.

Chen et al.[43] reported that XOR(Xanthine oxidoreductase) activity was attenuated in C/EBP silent cells; therefore, C/EBP gene lies upstream of XOR(Xanthine oxidoreductase) gene. Additionally, in the ob/ob mouse model, XOR(Xanthine oxidoreductase) gene depletion was observed to inhibit adipogenesis; based on this, XOR(Xanthine oxidoreductase) was considered to lie upstream of the PPAR? gene. XOR(Xanthine oxidoreductase) is suggested as a therapeutic target of metabolic syndrome with hyper uricemia. In our AA(aristolochic acid) model, PPARy, C/EBP α,and SREBFl gene expression were elevated concurrently with XOR(Xanthine oxidoreductase) activity. This result suggested that XOR(Xanthine oxidoreductase) activated adipogenesis or lipogenesis, or, conversely, that cachexia or oxidative stress caused by AA(aristolochic acid) resulted in the activation of adipogenetic or lipogenetic genes as a protective mechanism;further investigation is required on this point. Administering AA(aristolochic acid) caused apoptosis and nephron hypoxia with elevated HIF-1α expression, and induced progression to the fibrotic pathway through TGF-β and Col 1. XOR(Xanthine oxidoreductase) was induced mainly by hypoxia with oxidative stress, further suspected to produce ROS through elevated levels of NADPH oxidase,suggesting that XOR(Xanthine oxidoreductase) participated in the fibrotic mechanism. Further investigation on this mechanism is required to clarify whether adipogenesis and lipogenesis were induced by XOR(Xanthine oxidoreductase) or elevated as a consequence of tissue damage. The main limitation of this study is the small group sizes. However, we observed clear differences in the markers examined, indicating that further investigation of therapeutic interventions is warranted. Further studies should evaluate the effect of XOR(Xanthine oxidoreductase) inhibition concerning the suppression of fibrosis in progressive renal diseases. Another limitation is that we did not investigate the serum lipid mechanism thoroughly and could not measure the serum lipid metabolic marker. However, the major changes observed in the renal tissues were apoptosis, a decline in the number of tubular epithelial cells, and fibrosis, while lipid droplet deposition was not shown. Further, we assessed adipogenesis and lipogenesis renal gene expression, which allowed us to evaluate lipid metabolism under AA(aristolochic acid) administration. In this AA(aristolochic acid) model, adipogenesis progressed concomitantly with increasing XOR(Xanthine oxidoreductase) activity,in contrast to what was observed in the XOR(Xanthine oxidoreductase) model. This result suggested that XOR(Xanthine oxidoreductase) did not exert a direct protective function on renal tissues, and the fact that the adipogenesis that occurred with C/EBPo or PPARY and SREBF1 gene expression progression resulted from inflammation induced by ROS suggested this adipogenetic or lipogenetic reaction is a protective mechanism.

In conclusion, the results of the present study showed that tissue XOR(Xanthine oxidoreductase) activity, caused by tissue hypoxia induced catabolism, was consistently activated in an AA(aristolochic acid) induced nephropathy CKD model. Furthermore, kidney specific XOR(Xanthine oxidoreductase) activation was associated with organ damage and renal fibrotic lesion development. Thus, this AA(aristolochic acid) nephropathy model could be useful to further investigate a preventive mechanism against the XOR(Xanthine oxidoreductase) mediated renal fibrotic changes leading to end-stage renal disease.

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