How Xanthine Oxidoreductase Activity Affects Aristolochic Acid Nephropathy
Mar 25, 2022
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Takeo Ishii， Tomohiro Kumagae， Hiromichi Wakui， Shingo Urate， Shohei Tanaka & et al.
Introduction
Chronic kidney disease (CKD) is prevalent worldwide, and various associated diseases including fibrotic disorders pose a challenge in kidney tissues [1]. The increase in CKD has led to an increased number of patients on dialysis, and an increased financial burden, owing to dialysis costs [2]. Therefore, there is an urgent need to reduce and prevent renal dysfunction, which leads to end-stage renal failure. To study CKD, we focused on an animal model of aristolochic acid (AA) induced nephropathy, which is characterized by progressive fibrosis. AA (aristolochic acid) nephropathy is a problem on the Balkan Peninsula[3,4] and in China where the Mu Tong renal disorder [5] is endemic, leading to fibrosis and irreversible end-stage renal failure [6]. Additionally, modern lifestyle leads to hypertension, diabetes, hyperuricemia, and gout, which are posing problems globally. In addition to causing gout, hyperuricemia was recently reported as a risk factor for the development of nephrosclerosis and hypertension [7-9].
Uric acid causes the formation of crystal-associated disease networks through the deposition of monosodium urate crystals [10], resulting from purine metabolism. This causes oxidative stress-related tissue damage and tissue inflammation due to the generation of superoxides by xanthine oxidoreductase (XOR)[11,12]. XOR(xanthine oxidoreductase) was discovered approximately 100 years ago, and xanthine dehydrogenase(XDH) and xanthine oxidase (XO) con-version were discovered approximately 30 years ago [13]. Under physiological conditions, XO exists in milk and plays a role in physiological sterilization [14,15]. In vivo, XDH preferentially reacts with NAD, whereas XO cannot, but produces the superoxide anion (O2) and hydrogen peroxide (H2O2)[16-19]. It is well established that O2 and H2O2 cause tissue damage [1]. XOR(xanthine oxidoreductase) expression is upregulated by ATP degradation caused by certain pathological conditions such as ischemia, hypoxia, and oxidative stress, triggered by cellular stress. Under these pathological conditions, ATP is metabolized to hypoxanthine, xanthine, and uric acid via reactions catalyzed by XOR(xanthine oxidoreductase) [12]. XOR(xanthine oxidoreductase) produces reactive oxygen species (ROS)such as superoxides, hydroxyl radicals, hydrogen peroxide,and peroxynitrite [20]. XOR(xanthine oxidoreductase) also induces the renin-angiotensin system in tissues, which promotes organ damage and renal sclerosis through the activation of a positive feedback loop [12].
We previously reported that treatment with XOR(xanthine oxidoreductase) inhibitors can improve the survival of patients on dialysis [21]. XOR(xanthine oxidoreductase) inhibition may potentially reduce the oxidative stress associated with organ damage due to renal failure. In this study, we analyzed XOR(xanthine oxidoreductase) activity in the kidneys, plasma, heart, liver, and muscle in this AA(aristolochic acid)-induced nephropathy.AA(aristolochic acid) enters renal tubular epithelial cells through the OAT1 channel on the basement membrane. It stops the cell cycle and forces the cell to undergo apoptosis, causing irreversible fibrosis. AA(aristolochic acid) can do this in kidneys without damage to other organs. We chose AA(aristolochic acid) because it shows the fibrotic mechanism to end-stage renal failure, functioning as a model of acute kidney injury that can progress toward chronic kidney disease. The aim of this study was to investigate the relevance of XOR(xanthine oxidoreductase) activity in terms of tissue damage. The AA(aristolochic acid) model is a model of acute kidney injury with the potential to progress toward chronic kidney disease. We investigated tissue damage in this model based on time-dependent XOR(xanthine oxidoreductase) activity.
Materials and methods
Animals
Male 12 week old C57BL/6 mice(purchased from Charles River Laboratories)were assigned to either an AA(aristolochic acid) treatment group or the vehicle control group. Each group was divided into three subgroups depending on whether they were sacrificed at 0, 2, or 4 weeks after the fourth injection. AA(aristolochic acid)(2.5 mg·kgl) was administered via intraperitoneal injection once a week for four consecutive weeks as previously described [22]. This dosing regimen was used in order to induce chronic fibrosis with a low rate of death. XOR(xanthine oxidoreductase) activity was evaluated following AA(aristolochic acid) administration, and tissues were examined for XOR(xanthine oxidoreductase) related damage. Before sacrifice(24 h), urine was collected in metabolic cages as described previously [23](0 week group n=4,2 week group n=3,4 week group n=4,control n=4)(Fig. 1). Mice were subjected to a 12h light/12h dark cycle at 25 and were provided a standard diet(0.3 % NaCl,3.6 kcal gl,13.3 %energy as fat (Oriental MF;Oriental Yeast Co.,Ltd, And over, MA, USA))with free access to water. This study was approved by the Yokohama City University Laboratory Animal Committee and was conducted as per the Ministry of Health, Labor, and Welfare Laboratory Animal Guidelines. Serum albumin was measured using bromocresol green(BCG); urine albumin was measured using a turbidimetric immunoassay; and serum and urine creatinine(Cr)were measured using enzymatic methods.
Metabolic cage analysis
Metabolic cage analysis was performed as previously described (Techniplast,Paola,Malta)[23,24].Daily food and water intake were measured.
Histological analysis
Histological analysis was performed as described previously [25,26]. The kidney was fixed with 4%PFA and embedded in paraffin. Sections(4Im thick) were stained with Masson's trichrome. For analysis of renal structures, renal fibrotic areas were measured digitally using a fluorescence microscope(BZ-9000; Keyence, Osaka, Japan). The obtained image was red/blue stained, and the blue was extracted.After binarization considering the overall balance, the tubular epithelium and the blue stained part of the lumen were found to not be fibrotic and were trimmed and excluded in order to quantify the fibrotic area.Perivascular fibrosis was included in the quantification. Tubulo interstitial damage was defined as the ratio between the stained area and the area of the whole specimen. Further, we estimated simple regression between tissue XOR(xanthine oxidoreductase) activity and tubulo interstitial damage (%).
Fig.1 Experimental procedure and timeline for the mouse model. Male C57BL/6 mice were assigned to either an aristolochic acid I(AA) treatment group or the vehicle control group.Each group was divided into three subgroups depending on whether they were sacrificed at 0,2, or 4 weeks after their fourth injection.AA (2.5 mg.kg-1) was administered intraperitoneally(ip) once a week for four consecutive weeks. For 24 h before sacrifice (Sx), urine was collected in metabolic cages as described previously. For the 0-week AA group n=4, 2-week group n= 3,4-week group n=4,and control n=4. Serum creatinine clearance (Ccr) levels were measured immediately after the last AA injection.
Real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR)
Total RNA was extracted from renal tissues using ISOGEN (Nippon Gene, Tokyo, Japan), and cDNA was synthesized using the SuperScript III first strand synthesis system (Invitrogen). RT-qPCR was performed using a TaqMan PCR master mix and a designed TaqMan probe(Applied Biosystems, Foster City,CA,USA)on an ABIPRISM 7000 sequence detection system. Expression levels of target mRNAs were normalized to the 18S rRNA. The TaqMan probes used for PCR were as follows: transforming growth factor beta 1(TGF beta 1), MM01178820_ml;collagenla, Mm00801666_gl;hypoxia inducible factor 1 alpha subunit inhibitor (HIF-1a), Mm01198376_ml; and angiotensinogen (AGT), Mm00599662_m1;RAS related C3 botulinum toxin substrate 1(RAC 1)Mm01201656_ml;NADPH Oxidase 1 (NOX 1)Mm00549170_m1; CCAAT/enhancer binding protein, alpha C/EBPa Mm00514283_sl; peroxisome proliferator activated receptor gamma (PPARy) Mm00440940_ml;and sterol regulatory element binding transcription factor 1 (SREBF1), Mm00550338_m1.
XOR(xanthine oxidoreductase) activity measurement
Xanthine oxidoreductase activity was measured three times (0 week,2 weeks, 4 weeks after AA(aristolochic acid) administration)(n=4 in each group) in the heart, liver, kidney, and muscle tissues according to a previously described method [27]. In brief, the kidney, liver, heart,or muscle homogenates,or plasma was added to a mixture of [1C2.15N2] xanthine, NAD+, and oxonate in Tris buffer (pH 8.5) and was incubated at 37 for 30 min. Then, methanol containing [1C2,15N2jUA was added, and the mixture was centrifuged at 3000×g at 4 for 15min. The amount of [13C2,15N]UA produced in the supernatant was measured using LC/TQMS (Nexera/QTRAP4500, SHIMADZU/SCIEX).XOR(xanthine oxidoreductase) activity was expressed as[1C2,1NJUA nmol/min/mg protein [27].
Statistical analysis
The results of the AA(aristolochic acid) and control groups were compared using an unpaired t-test. P-values<0.05 were considered statistically significant. All analyses were performed using Prism 8 Ver.8.3.1(Yokohama, Japan).
MEDICINAL HERB CISTANCHE FOR PATIENTS WITH KIDNEY DISEASES
Results
For the mice sacrificed at 4 weeks after their fourth injection, the bodyweights in the vehicle and AA(aristolochic acid) groups were 31.1 g±0.4g and 31.1 g±0.4 g,respectively, at the first injection. Two weeks later (after the fourth injection and during the observation period), the bodyweight was 31.9 g±0.4 g in the vehicle group, whereas it reached a minimum of 27.8 g±0.7g (P<0.05)in the AA(aristolochic acid) group (Fig.2). Serum Cr levels were measured immediately after the last AA(aristolochic acid) injection. The Cr level was 0.11lg±0.02 mg·dL-1 in the vehicle group and was significantly higher in the AA (aristolochic acid)groups (P<0.05). The levels continued to increase in the experimental group at 2 and 4 weeks after the end of AA(aristolochic acid) administration.After the final AA(aristolochic acid) administration, serum blood urea nitrogen(BUN)was 25.7 mg.dL-1±0.2 mg·dL-1 and 53.8mg·dL-1±5.1 mg·dL-l in the vehicle and AA(aristolochic acid) groups, respectively(P<0.05). BUN continued to increase in the AA(aristolochic acid) group during the follow-up observation period. First Cr clearance(Ccr), which was measured at the end of the AA(aristolochic acid) administration， was 690.1 μLmin-1± 71.4 μLmin-1 in the vehicle group and 183.8 μLmin-'± 12.9 μLmin l in the AA(aristolochic acid) groups. The Ccr levels in AA(aristolochic acid) groups remained low at 2 weeks and 4 weeks after AA(aristolochic acid) administration (P<0.05;Table 1 and Fig.3A,B).
Fig.2 Bodyweight changes during the observation period. The mice were followed up 4 weeks after the final AA injection. The bodyweights in the vehicle and aristolochic acid I(AA) groups were 31.1±0.4 g and 31.1 g±0.4, respectively, at the first injection. By week 4, the control group bodyweights were 31.2 ± 0.6g, but the AA group bodyweights were 29.3± 1.4 g. Two weeks later (during the observation period), the bodyweights were 31.9± 0.4 g in the vehicle group, whereas those in the AA group reached a minimum of 27.8± 0.7 g(P<0.05).0-week group n=4,2-week group n= 3,4-week group n=4,control n=4. Error bars represent SEM.
Changes in the urinary albumin excretion
The urinary albumin excretion was measured immediately after four AA(aristolochic acid) injections. The values were Cr in 26.0±0.9 μg·mg1 and 240.8±100.0 μg·mg1 the vehicle and AA(aristolochic acid) groups, respectively. At 4 weeks, the urinary albumin excretion was 18.3±0.6 ug mg1 and 194.3± 59.8 μg·mg1Cr in the vehicle and AA(aristolochic acid) groups, respectively (P<0.05; Fig.3c).
Table 1. Activities of xanthine oxidoreductase (XOR) in the aristolochic acid (AA) nephropathy model
XOR(xanthine oxidoreductase) activity in plasma and tissues
The plasma XOR(xanthine oxidoreductase) activity was 73.3± 8.0 pmol·min'mg1 and 77.8±4.2 pmol·min mg in the vehicle and AA(aristolochic acid) groups, respectively,at the end of AA(aristolochic acid) administration. No significant difference was observed in plasma XOR(xanthine oxidoreductase) activities between the two groups at any time point (Fig. 4A).
Fig.3. Changes in serum creatinine (Cr), Cr clearance (Ccr), and urinary albumin excretion. (A) Serum Cr levels were measured immediately after the last aristolochic acid I(AA) injection. The Cr level was 0.11 ± 0.02 mg-dL-'in the vehicle group and was significantly higher in the AA group (0.30±0.02 mg.dL-1;P<0.05). The levels continued to increase in the experimental group, at 2 weeks and 4 weeks after the end of AA administration.(B) First Ccr, which was measured at the end of the AA administration, was 690.1 ± 71.4 uL-min-'in the vehicle group and 183.8± 12.9 μL·min-1 in the AA groups. The levels in the AA groups remained low at 2 weeks and 4 weeks after AA administration (P<0.05; Table 1 and Fig.3a, 3b).(C) Urinary albumin excretion was measured immediately after four AA injections. The values were 26.0 ± 0.9 and 240.8± 100 ug-mg-1 Cr in the vehicle and AA groups, respectively. Two weeks later, the urinary albumin excretion was 18.8± 1.7 ug-mg-1 and 78.3± 10.6 ug.mg-1 Cr in the vehicle and AA groups, respectively(P<0.05).At 4 weeks, the urinary albumin excretion was 18.3± 0.6 ug·mg-'and 194.3± 59.8 ug·mg-' Cr in the vehicle and AA groups, respectively (P<0.05;Fig.3C).0-week group n=4, 2-week group n=3, 4-week group n=4, control n=4.Error bars represent SEM. Statistical analysis was performed using multiple t-test in each week. *Represents P< 0.05, and ** represents P<0.001.
Except for the kidney, XOR(xanthine oxidoreductase) activities in the examined tissues did not differ between the groups. The AA(aristolochic acid) groups did not show any differences in XOR(xanthine oxidoreductase) activity in the heart, liver,and muscle compared to the control group at any time point(Fig.4BD). However, in renal tissue, the XOR(xanthine oxidoreductase) activities in the AA(aristolochic acid) groups were elevated during the observation period. At week 0, XOR(xanthine oxidoreductase) activities were 254.3± 19.9 pmolmin 1.mg1 and 556.8±52.9 pmol·min1mg1 TP in the vehicle and AA(aristolochic acid) groups, respectively(P<0.0001).Two weeks later, the XOR(xanthine oxidoreductase) activity was 231.5±20.4 pmolmin一mg1 and 430±55.6 pmolmin'mg1TP in the vehicle and AA(aristolochic acid) groups, respectively(P<0.05).At week 4, the activities were 237.5±9.9 pmolmin1mg1a and 410.5 ± 30.5 pmol:min1mg g1 TP in the vehicle and AA(aristolochic acid) groups,respectively(P<0.05;Fig. 4e).
Expression of fibrosis markers in the renal tissue
The mRNA expression levels of renal fibrosis related genes were examined. Renal expression of collagen1 (Col1) was elevated in the AA(aristolochic acid) groups compared to that in the vehicle group at all time points(P<0.05; Fig. 5A). Additionally, the renal expression of transforming growth factor β(TGF-β) was increased in the AA(aristolochic acid) groups compared to that in the vehicle group at the end of AA(aristolochic acid) administration and after 2 weeks (P<0.05), and showed a tendency to increase after 4 weeks (Fig. 5B).
Fig.4. Xanthine oxidoreductase activity in plasma and tissues.(A) The plasma xanthine oxidoreductase(XOR) activity was 73.3± 8.0 pmol·min-1.mg-1and 77.8± 4.2 pmol-min-1.mg-1 in the vehicle and aristolochic acid I(AA) groups, respectively, at the end of AA administration. No significant difference was observed in the plasma XOR activities between the two groups at any time point. (B-D)Except for the kidney,XOR activities in the examined tissues did not differ between the groups. The AA groups did not show any differences in XOR activity in the heart, liver, and muscle compared to the control group at any time point.(E)However, in renal tissue, the XOR activities in the AA groups were elevated during the observation period. At week 0, the XOR activities were 254.3± 19.9 pmol.min-1.mg-1 and 556.8± 52.9 pmol·min-1.mg-1 TP in the vehicle and AA groups, respectively (P<0.0001). Two weeks later, XOR activity was 231.5± 20.4 pmol-min-1.mg-1 and 430± 55.6 pmol-min-1.mg-1 TP in the vehicle and AA groups, respectively (P<0.05). At week 4, the activity was 237.5± 9.9 pmol.min-1.mg-1 and 410.5 ± 30.5 pmol-min-1.mg-1 TP in the vehicle and AA groups, respectively(P< 0.05). O-week group n=4, 2-week group n=3, 4-week group n=4, control n=4.Error bars represent SEM. Statistical analysis was performed using multiple t-test in each week. *Represents P< 0.05, and **Represents P<0.001.
Expression of hypoxia-inducible factor(HIF-1a)in the renal tissue
The mRNA expression levels of HIF-1a were also examined. Renal expression of HIF-1o was significantly higher in the AA(aristolochic acid) groups compared with that in the vehicle group(P<0.001) at the end of AA(aristolochic acid) administration, at 2 weeks and 4 weeks(P<0.05;Fig.5C).
Expression of NADPH components in the renal tissue
RACI and NOXI gene expression in the AA(aristolochic acid) group were elevated continuously compared with the vehicle group (P<0.05;Fig.5D,E).
Expression of adipogenesis markers in the renal tissue
Adipogenesis markers C/EBPα and PPARγ gene expression, and lipogenesis marker SREBF1 gene expression in the AA(aristolochic acid) group were elevated continuously compared with the vehicle group(P<0.05;Fig. 5F, G,H).
Fig.5. Gene expression in the renal tissue. (A) The mRNA levels of renal fibrosis-related genes were examined. Renal expression of collagen-1 (Col-1)was elevated in the aristolochic acid I(AA) groups compared to the vehicle group at all time points(P<0.05).(B)Additionally, renal expression of transforming growth factor-β(TGF-B)was increased in the AA groups compared to the vehicle group at the end of AA administration and 2 weeks later (P<0.05), and showed a tendency to increase after 4 weeks. (C) The mRNA levels of hypoxia-inducible factor-1 alpha (HIF-1α) were examined. Renal expression of HIF-1a was significantly higher in the AA groups compared with the vehicle group (P< 0.001) at the end of AA administration,at 2 weeks, and 4 weeks(P< 0.05).(D, E)The mRNA levels of NADPH components were examined. Renal expression of (RAC1) and (NOX1)was elevated in the aristolochic acid I(AA) groups compared to the vehicle group (P<0.05).(F-H) The mRNA levels of adipogenesis and lipogenesis markers were examined. Renal expression of (C/EBPa), (PPARy), and (SREBF1)was elevated in the aristolochic acid I(AA) aroups compared to the vehicle aroup(P<0.05). 0-week groun n = 4 2-week group n=3, 4-week group n = 4, control n = 4.Error bars represent SEM. Statistical analysis was performed using multiple ttest in each week.* represents p < 0.05, and **Represents P< 0.001.
Renal tissue fibrosis
The interstitial renal fibrosis areas were 1.8±0.7%and 9.1± 1.9%(P<0.05)in the vehicle and AA(aristolochic acid) groups, respectively, at the end of AA(aristolochic acid) administration. A wide range of tubular epithelial cells was lost via apoptosis. Two weeks later, the fibrotic areas were 2.1 ±0.4%and 8.1±4.7%in the vehicle and AA(aristolochic acid) groups, respectively.At 4 weeks, the interstitial renal fibrosis areas were 3.6±1.1% and 15.3±2.6%(P<0.05)in the vehicle and AA(aristolochic acid) groups, respectively. The tubular apoptotic area at O weeks was replaced with the fibrotic area via tissue remodeling (Fig.6A, B). Microscopic evaluation of Masson trichrome stain showed that kidney tissue XOR(xanthine oxidoreductase) activity was significantly correlated with the interstitial fibrotic area(%o)（P<0.0012，r²= 0.40）（Fig.6C).
Fig.6.Renal tissue fibrosis.(A, B) The interstitial renal fibrosis areas were 1.8± 0.7% and 9.1 ± 1.9%(P<0.05) in the vehicle and AA groups, respectively,at the end of AA administration. Two weeks later, the areas were 2.1+0.4% and 8.1 + 4.7% in the vehicle and AA groups, respectively. At 4 weeks, the interstitial renal fibrosis areas were 3.6± 1.1 and 15.3 ±2.6%(P<0.05) in the vehicle and AA groups, respectively, Scale bar indicated 200 um,(c) Kidney tissue XOR activity was significantly correlated with interstitial fibrotic area(%)using microscopic evaluation of Masson trichrome stain （P=0.0012 产=0.40）. This suggests that XOR activity contributed to tissue damage leading to end-stage renal disease. 0-week group n=4, 2-week group n=3,4-week group n=4,control n=4.Error bars represent SEM. Statistical analysis was performed using multiple t-test in each week.*represents P< 0.05, and ** represents P<0.001.
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