Respiratory dysfunction affects quality of life and facilitates the development of noncommunicable chronic lung diseases such as chronic obstructive pulmonary disease (COPD), a condition characterized by symptoms such as increased average airspace size, decreased elasticity, and decreased forced expiratory volume in one second (FEV1)/forced vital capacity (FVC) [1
]. Generally, respiratory dysfunction is associated with the accumulation of oxidative stress, a condition of imbalance between reactive oxygen species (ROS) formation and cellular antioxidant capacity due to enhanced ROS generation and/or dysfunction of the antioxidant system, indicating that decreased oxidative stress is crucial for lung homeostasis maintenance [2
]. Consistently, some epidemiological studies showed that dietary intake of antioxidants delays the decline of pulmonary function in healthy adults [4
Among the endogenous antioxidants in epithelial lining fluid (ELF), uric acid (UA) exists in higher concentrations [6
] and has the potential role of scavenging ROS (e.g., singlet oxygen and hydroxyl radicals) [7
]. Although UA is a terminal product of purine metabolism in humans and great apes, this is not the case for most mammals; human and great apes lost the enzymatic activity of urate oxidase, or uricase (Uox), which converts UA to allantoin, due to a loss-of-function mutation during evolution [9
]. In consequence, UA levels in human blood are much higher than in other mammals, such as mice. UA is known as an abundant antioxidant in human blood [10
]. In fact, it was reported that high levels of UA are beneficial for central nervous system (CNS) disorders related to oxidative damage [12
]. Paradoxically, high plasma UA levels are detrimental to the host due to the ability of UA to be pro-oxidative under certain conditions, thereby increasing not only the risk of gout, but also of many oxidative stress-related diseases [15
]. The UA paradox in respiratory disease, especially in COPD, is often discussed in epidemiological studies [18
], but the conclusions are controversial. Horsfall et al. claimed that lower blood UA levels were associated with COPD risk [19
], while others showed that higher blood UA levels were correlated with COPD exacerbation [20
]. However, whether UA directly impacts on lung function under physiological and pathological conditions remains unclear. It is also undefined which other factors, including gender, age, alcohol consumption, obesity, and urate transporter genes regulating renal absorption of UA, e.g., SLC22A12 [22
], SLC2A9/GLUT9 [23
], and ABCG2 [24
], are key factors for UA-dependent lung function.
In the present study, we utilized an experimental mouse model of COPD (C57/BL6J-βENaC-transgenic mice) [26
] and showed that pharmacological increase of plasma UA levels improved emphysematous phenotype and lung dysfunction in female but not in male mice. In vitro cellular experiments further determined that UA significantly suppressed hydrogen peroxide (H2
)-induced oxidative stress only in the absence of estrogen, implying that the benefit of higher UA is limited to the female airway in postmenopausal conditions. We also conducted a cross-sectional retrospective longitudinal study among Japanese people in a health program and found a novel association of higher blood UA levels with higher lung function in elderly female subjects. Moreover, genetic assaying with a UA-increasing single nucleotide polymorphism (SNP) of SLC2A9/GLUT9, or the rs11722228 T/T genotype, confirmed the impact of blood UA levels on lung function in elderly female subjects. Overall, these experimental and clinical observational analyses provide the first unique evidence that blood UA directly affects lung function in female mice under pathological conditions and in human females under aging-associated physiological conditions.
2. Materials and Methods
2.1. Animal Sources and Care
We used age-matched male and female wild-type (WT) C57/BL6J mice (Charles River Laboratories, Atsugi, Japan). All mice were maintained in the animal house at Kumamoto University in accordance with the guidelines of the animal facility center of Kumamoto University and were fed with normal chow (Oriental Yeast, Tokyo, Japan) ad libitum. All experiments were performed according to the protocols approved by the Animal Welfare Committee of Kumamoto University (No. I29-200) and all methods were performed in accordance with the relevant guidelines and regulations. Uox-KO (B6;129S7-Uoxtm1Bay/J) [28
], originally obtained from The Jackson Laboratory (Bar Harbor, ME, USA), was backcrossed to C57/BL6J for at least three generations. The production of C57/BL6J-βENaC-Tg mice was previously reported [26
]. C57/BL6J-Uox knockout (KO) mice were produced by the Center for Animal Resources and Development (CARD) in Kumamoto University using our own reported reproductive technique [29
2.2. Oxonate Treatment
To increase UA concentration in the blood, 500 mg/kg oxonic acid potassium salt in solution (oxonate) (Sigma-Aldrich Japan, Tokyo, Japan) was orally administered to 7–8-week-old wild-type (WT) and C57/BL6J-βENaC-Tg female/male mice per a day for 4 or 5 weeks. Oxonate was prepared by dissolving in 0.5% (w/v) carboxymethyl cellulose solution. Carboxymethyl cellulose sodium salt (Nacalai Tesque, Kyoto, Japan) was dissolved in distilled water for injection (Otsuka Pharmaceutical, Tokyo, Japan) and 0.5% (w/v) carboxymethyl cellulose solution as vehicle was orally administered. After 4–5 weeks of treatment, pulmonary function was measured using the flexiVent system (SCIREQ, Montreal, QC, Canada). Mouse lungs and blood were collected 2–3 h after the last oxonate dosing. For the healthy control group, the vehicle was administered orally.
2.3. Elastase-Induced Pulmonary Emphysema Model
WT and C57/BL6J-Uox KO female and male (12- or 13-week-old) mice were administrated intratracheally with elastase from porcine pancreas (male: 0.06 mg per mouse; female: 0.05 mg per mouse) (Sigma-Aldrich Japan) dissolved in saline (Otsuka Pharmaceutical). As control, saline was treated intratracheally. After 3 weeks, we measured the pulmonary function using the flexiVent system and then collected mouse lungs and blood.
2.4. Measurement of Pulmonary Mechanics and Function by flexiVent
Measurement of pulmonary mechanics (resistance, compliance, and elastance) was performed with a computer-controlled small animal ventilator flexiVent as previously described [26
]. Briefly, mice were anesthetized with somnopentyl (Kyoritsu, Tokyo, Japan) (1.7–1.8 mL/kg) and then an 18-gauge needle was inserted into the trachea. Mice were mechanically ventilated by the flexiVent system. Pulmonary mechanics were measured by the forced oscillation technique. The FEV0.1/FVC ratio was also measured using the flexiVent system connected to a negative pressure reservoir. Based on the data of forced expiratory volume in 0.1 second (FEV0.1) and forced vital capacity (FVC), pulmonary functional parameter FEV0.1/FVC, or forced expiratory volume % in 0.1 seconds (FEV0.1%), was determined using the flexiVent software, enabling comparison between mice and human data.
2.5. Histological Analysis and the Measurement of Mean Linear Intercepts (MLI)
After we measured the pulmonary parameters of each mouse by flexiVent, the whole lung was filled with approximately 1 mL of 10% formalin neutral buffer solution (FUJIFILM Wako Pure Chemical, Osaka, Japan) and then preserved in formalin for 12 h at room temperature. Samples were then washed with phosphate buffered saline (PBS) and embedded in paraffin using the histoprocessor Histos-5 (Milestone Medical, Sorisole, Italy). The tissue samples were separated into 3 sections, cut into sections that were 6 μm thick by a rotary microtome Leica RM2125RT (Leica Biosystems, Wetzlar, Germany), and stained with periodic acid-Schiff (PAS) (Sigma-Aldrich, Tokyo, Japan) and alcian blue (pH 2.5) (Nacalai Tesque) to observe mucus-secreting goblet cells. After PAS and alcian blue staining, these samples were subjected to hematoxylin and eosin (H&E) (Sigma-Aldrich, Japan) staining to visualize in light microscopy for lung morphology. The images of 10 lung sections (3 upper, 4 middle, and 3 lower) were captured randomly to draw 6 lines (300 μm in width) by BioRevo BZ-X710 (KEYENCE, Osaka, Japan). To determine the mean linear intercepts (MLI), the number of the intersections of 6 lines and the alveolar walls were counted and divided by the total length of the line, as previously described in detail [30
2.6. Blood Collection and Measurement of Plasma UA and BUN
Blood was collected from the inferior vena cava using heparinized syringes with a 27-gauge needle. Plasma samples were obtained by centrifuging at 3000 rpm at 4 °C for 15 min and preserved at −80 °C. UA and blood urea nitrogen (BUN) in plasma were measured using DRI-CHEM (FUJIFILM, Tokyo, Japan).
2.7. Evaluation of Plasma Antioxidant Capacity and Oxidative Stress Level
To measure plasma reactive oxygen metabolites (ROMs) as an oxidative stress marker, plasma samples from mice were subjected to d-ROMs test (Wismer Co., Tokyo, Japan), which measured total peroxides, including hydroperoxides (ROOH), i.e., metabolites produced when active oxygen and free radicals oxidize lipids, proteins, amino acids, and nucleic acids in the body. The unit of measurement was represented by Carratelli units “CARR U” (1 CARR U = 0.08 mg H2O2/dL). To evaluate plasma antioxidant capacity, we used the biological antioxidant potential (BAP) test (Wismer Co.), a photometric test to detect reduced iron from ferric (Fe3+) to ferrous (Fe2+). The reducing power was evaluated as the antioxidant power. The results of the BAP test were presented in μmol/L of reduced iron.
2.8. Cell Culture
The human bronchial 16HBE14o- cell line [31
] was previously generated and grown in fibronectin/bovine serum albumin (BSA)-coated dishes [32
] and maintained in minimum essential medium (Sigma-Aldrich Japan). The A549 cell line was maintained in Dulbecco’s modified Eagle’s medium (DMEM) (low glucose) (FUJIFILM Wako Pure Chemical). These media were added with 10% fetal bovine serum, 100 U/mL of penicillin, and 100 mg/mL of streptomycin. Primary normal (NHBE) cells from a female donor (lot: 0000543643) were purchased from LONZA (Basel, Switzerland) and maintained according to the manufacturer’s instructions. All cells were cultured in a humidified incubator at 37 °C with 5% CO2
2.9. Intracellular ROS Detection Assay
ROS production was detected using 5(-and-6)-chloromethyl-2’,7’-dichlorodihydro-fluorescein diacetate acetyl ester (CM-H2DCFDA) (Invitrogen, Carlsbad, CA, USA) dye according to the manufacturer’s instructions. 16HBE14o-, A549 (1.0 × 104 cells/well), and NHBE (3200 cells/well) cells were seeded in 96-well plates and incubated until 90% confluent. NHBE was incubated with 1 nM 17β-estradiol (FUJIFILM Wako Pure Chemical, Japan) for 24 h before reagent treatment. After washing cells with PBS, 1.7 μM CM-H2DCFDA was added and cells were incubated for 30 min in a CO2 incubator. After 30 min, the reagent was removed and 7 or 14 mg/dL of UA (Sigma-Aldrich, Japan) was added. NaOH at a concentration of 5 mM and 10 mM N-acetylcysteine (NAC) (Sigma-Aldrich Japan) were used as the negative and positive controls, respectively. After 30 min, cells were incubated with 100 or 400 μM H2O2 (FUJIFILM Wako Pure Chemical, Japan)/well for 30 min and the 2′, 7′-dichloro-fluorescein (DCF) level was measured. For longer time treatment, 16HBE14o- and NHBE cells were incubated with 7 or 14 mg/dL of UA for 48 h before cells were stained with CM-H2DCFDA. The DCF level was immediately measured using a fluorescence microplate reader (excitation/emission = 485 nm/535 nm), TECAN infinite M1000 (TECAN, Männedorf, Switzerland).
2.10. Human Subjects
We retrospectively investigated 725 health screening program participants in the Japanese Red Cross Kumamoto Health Care Center. We excluded 90 subjects with lung or airway disease (e.g., asthma, tuberculosis, lung cancer). Furthermore, to investigate the association between UA and respiratory function in postmenopausal female subjects, we excluded 82 subjects under 50 years of age. Finally, the remaining 553 subjects were enrolled in this study. The study complied with the Declaration of Helsinki and was approved by the ethics committees of the Japanese Red Cross Kumamoto Health Care Center and the Faculty of Life Sciences at Kumamoto University (Genome No. 169). All subjects provided their written informed consent prior to enrollment in the study.
2.11. Measurements in Humans
The laboratory tests were performed using the standard methods of the Japan Society of Clinical Chemistry. The information regarding smoking habits and alcohol intake was obtained via face-to-face interviews with healthcare providers. Hypertension was defined as a systolic blood pressure (BP) of ≥140 mmHg, a diastolic BP of ≥90 mmHg, or a history of hypertension [33
]. Dyslipidemia was defined as a value of triglycerides of ≥150 mg/dL, high-density lipoprotein cholesterol of <40 mg/dL, low-density lipoprotein cholesterol of ≥140 mg/dL, or a history of dyslipidemia [34
]. Hepatic ultrasonography was used to diagnose fatty liver disease. The liver was characterized as fatty liver when the liver had areas of significantly increased echogenicity relative to the renal parenchyma, the ultrasound beam was attenuated with the diaphragm indistinct, or the echogenic walls of the portal veins were less visible [35
]. The diagnosis of fatty liver disease was made by the radiographer. Genomic DNA was extracted from whole blood using a DNA purification kit (FlexiGene DNA kit; QIAGEN, Hilden, Germany). The SLC2A9/GLUT9 rs11722228 (g.9914117C > T) genotype was determined using a real-time TaqMan allelic discrimination assay (Applied Biosystems, Waltham, MA, USA) in accordance with the manufacturer’s protocol (assay no. C_1216578_10). To ensure the genotyping quality, we included DNA samples as internal controls, hidden samples of a known genotype, and negative controls (water).
2.12. Statistical Analyses
For quantitative analysis for the in vivo and in vitro studies of the mice, the data were presented as the mean ± SEM performed in indicated replicates and were analyzed by either Student’s t-test or the Tukey–Kramer test using JMP® (SAS Institute Inc., Cary, NC, USA), as indicated in each figure legend. For correlation analysis, raw data of plasma UA and the oxidative stress marker in C57/BL6J-βENaC-Tg in untreated or oxo-treated mice were subjected to Pearson’s correlation coefficient test, also analyzed by JMP. The level of significance was set at p < 0.05. In some comparisons, effect size estimates (d) were calculated with d = 0.2 considered as a small effect, d = 0.5 as a medium effect, and d = 0.8 as a large effect.
For our clinical analyses, categorical variables were compared using Fisher’s exact test. Parametric or nonparametric continuous valuables were compared using Student’s t-test or the Mann–Whitney U test, respectively. The correlation between UA and FEV1/FVC was examined using Pearson correlation analysis. The values of UA were classified into 4 groups as follows: <5 mg/dL, 5–6 mg/dL, 6–7 mg/dL, and ≥7 mg/dL for male subjects and <4 mg/dL, 4–5 mg/dL, 5–6 mg/dL, and ≥6 mg/dL for female subjects. The associations of the classified UA levels with FEV1/FVC were examined using a multiple regression analysis with calculations of the partial regression coefficients (Bs) and standard errors (SEs). The B values were adjusted for potentially confounding factors, i.e., age and body mass index (BMI) as continuous variables and the presence of hypertension, dyslipidemia, fatty liver disease, and smoking status as nominal variables. Structural equation modeling was used to perform pathway analysis to assess the effects of UA level on respiratory function. The goodness-of-fit on the structural equation modeling was evaluated based on a goodness-of-fit index (GFI) of >0.90, an adjusted GFI (AGFI) of >0.90, and a root mean square error of approximation (RMSEA) of <0.10. A value of p < 0.05 was considered to be statistically significant. In order to examine the accuracy of the parameters of the multiple regression models and the structural equation model, bootstrap analyses were performed using 1000 replicated datasets generated by random sampling with replacement. The structural equation modeling process and the other statistical analyses were performed using the SPSS Amos software program (version 23.0; IBM Japan, Inc., Tokyo, Japan) and the SPSS software package (version 23.0; IBM Japan, Inc.), respectively.
UA is one of the antioxidants that is prominent in the airway mucous membrane [6
], but its actual role, especially in the regulation of lung function, remains unspecified. In this study, we examined the impact of blood UA levels on lung dysfunction between genders by utilizing experimental mice model as well as human observational analyses. Overall, our findings demonstrated that UA is highly likely to be a protective factor against aging-associated physiological and COPD-associated pathological decline of lung function in female humans and mice. Because the negative aspects of UA (e.g., UA crystal formation, oxidative stress induction, etc.) have attracted attention in a variety of diseases [15
], this study provides a novel concept of the beneficial aspects of UA in the field of human health.
Whether UA has epidemiologically beneficial effects on physiological and pathological lung dysfunction is controversial. Song et al. reported that higher serum UA levels were positively correlated with lung function in the middle-aged healthy population, regardless of gender [41
]. Horsfall et al. showed that lower serum UA levels were related to a higher risk of COPD in a heavy smoker group in a population-based cohort study [19
]. Nicks et al. reported that lower UA levels were associated with COPD severity in the cross-sectional study [42
]. However, these reports did not clarify whether UA alteration directly contributes to protection against lung dysfunction. Our study focused on identifying whether experimental alteration of UA affected lung function under physiological and pathological conditions. Our data supported the other groups’ reports except for gender segregation; unexpectedly, a specific limited condition, i.e., female mice under COPD conditions, was uncovered whereby UA worked as a pulmonary protective factor. Serum UA levels in females are generally lower than in males, implying that females may have a lower antioxidant capacity in the airways, thereby inducing susceptibility to oxidative stress-induced damage. In fact, decreased antioxidant capacity is associated with age-related decline of pulmonary function [43
], therefore, our data suggested that high levels of UA in elderly women exert an antioxidant effect in the lungs and maintain lung function by removing increased ROS.
A significant increase in plasma UA levels due to Uox gene deficiency did not affect lung function in mice under normal conditions (Figure 1
). Furthermore, high-dose UA treatment did not affect ROS production under normal conditions (Figure 4
). On the other hand, increased UA levels improved lung function in female mice in a COPD-dependent pathological decline in lung function (Figure 2
and Figure 3
), and high-dose UA treatment protected against H2
-induced oxidative stress in human airway epithelial cells (Figure 4
). However, these results did not explain the sexual difference in the response to improved lung function. In female COPD mice, plasma UA levels were significantly negatively correlated with plasma oxidative stress, but this was not observed in male mice. In the COPD model, the pulmonary phenotype in females was more severe than in males [44
]. This may be attributed to the fact that the female model in the control group had a slightly higher level of oxidative stress than the male model mice. In an in vitro study using NHBE cells from a female donor, UA treatment with estrogen did not suppress oxidative stress. When estrogen is sufficient, the induction of oxidative stress is lower (Figure 4
). Considering these results, UA might beneficially work only under conditions with higher oxidative stress, such as aging in postmenopausal and COPD-like conditions.
The results of our human study showed that the SLC2A9/GLUT9 rs1917760 polymorphism and UA levels were associated with respiratory function among the elderly female subjects. A previous meta-analysis of population-based cohort studies indicated that female subjects had faster rates of age-related decline in respiratory function than male subjects in a smoking population [45
]. In another study, postmenopausal female subjects who used hormone replacement therapy (i.e., use of estrogen and progesterone) exhibited higher levels of FEV1 and were less likely to suffer from airway obstruction, even after adjusting for variables known to influence respiratory function [46
], suggesting that female sex hormones may protect against respiratory function decline. Combining our results and these previous findings, the antioxidative effect of UA may have an important role in protecting respiratory function in elderly female individuals.
The results of structural equation modeling suggested that high BMI was associated with a protective effect of high UA levels on respiratory function among elderly females. A previous epidemiological study showed that many Japanese COPD patients were categorized as having emphysema and being underweight [47
]. A recent cohort study conducted on 282,135 Korean subjects showed that underweight status was associated with respiratory function decline [48
]. Moreover, the cohort study also showed that subjects with low FEV1 and FVC were more likely to have low muscle mass [48
]. Since malnutritional and/or physically inactive individuals are more likely to have low muscle mass and strength, especially in elderly [49
], we suggest that lifestyle modifications, including nutritional and/or exercise management for underweight elderly females, may provide respiratory function protection partially through increased UA levels.
Fatty liver was reported to be significantly associated with not only high serum UA levels [51
] but also decreased lung function [54
]. Additionally, nonalcoholic fatty liver disease occurs at a higher rate in women after menopause [55
]. In this study, we analyzed the association between the classified UA levels and the FEV1/FVC using multiple linear regression analysis incorporating the presence or absence of fatty liver. Accordingly, the FEV1/FVC value was higher in individuals with UA ≥ 6.0 mg/dL than in those with UA < 4.0 mg/dL, even after adjusting for the presence or absence of fatty liver disease, but only in females (Table 2
). Furthermore, we could not incorporate the fatty liver into the structural equation modeling diagram of respiratory function and UA levels in females (Figure 5
E) because the parameters did not converge. As was the case with the previous studies [51
], although the prevalence of fatty liver disease was greater in subjects with high UA levels than in those with low UA levels among male and female subjects (Supplementary Tables S1 and S2
), age, but not fatty liver disease, was significantly associated with a decrease in the FEV1/FVC value (Supplementary Table S3
). In our clinical investigation, we focused on an aging-dependent decline in lung function as the respiratory dysfunction in humans. Therefore, a large portion (37.6%) of our study subjects were elderly, i.e., >70 years old, individuals, whereas most subjects seemed to be non-elderly individuals in previous studies that showed a relationship between nonalcoholic fatty liver disease and decreased lung function [54
]. Nevertheless, further studies evaluating the UA–fatty liver–sex dimorphism triangle in terms of lung function are needed in a larger population.
Although the present clinical investigations showed that UA is likely a protective factor for aging-related physiological and pathological decline in lung function among females, it should be recognized that high UA levels are related to several diseases (e.g., coronary heart disease (CHD)) and pathophysiological conditions (e.g., hypoxia). A previous meta-analysis reported that hyperuricemia appeared to increase the incidence of CHD as well as the mortality of CHD patients [56
]. In addition, epidemiological studies showed that hyperuricemia was an independent risk factor for the development of chronic kidney disease (CKD) and end-stage renal disease [57
]. Furthermore, serum UA levels were reported to be higher in individuals with other risk factors for CHD and CKD (e.g., hypertension, hyperglycemia, dyslipidemia, fatty liver, high BMI, alcohol consumption, and smoking) than in those without [58
]. Extracellular UA is known to have antioxidant activity in terms of scavenging peroxynitrite and reacting with singlet oxygen, lipid-derived radicals, and ferric iron [59
]. On the other hand, increased intracellular UA is produced by xanthine oxidoreductase (XOR) or by uptake via urate transporters, thereby activating mitogen-activated protein kinases (MAPK) and NADPH oxidase, resulting in increased mitochondrial and cytoplasmic oxidative stress, which may be implicated in metabolic diseases [59
]. Intracellular UA was also reported to accumulate by increased XOR activity under hypoxic conditions [60
]. Previous human studies showed that serum UA levels were increased in hypoxic conditions among patients with acute exacerbation of COPD [61
], obstructive sleep apnea [62
], pulmonary hypertension [63
], and chronic respiratory failure [64
]. Moreover, smoking under hypoxic conditions is a potent risk factor for various inflammatory diseases [65
], and both acute and chronic smoking exposures increased mRNA expression and promoter activity of XOR in rats in vivo [66
]. Furthermore, an epidemiological study of 8662 Japanese individuals who underwent a health checkup reported that smoking habits increased serum UA levels [67
]. Therefore, whether UA acts as a protective or harmful factor for lung function may vary depending on the conditions under the presence or absence of hypoxia and smoking habits. Based on the above-stated information, when considering the relationship between UA and lung function, careful attention should be paid not only to serum UA levels but also to the background affecting the UA level (e.g., the presence of cardiovascular and renal disease, hypoxic condition, and lifestyle).
Several limitations associated with the present human study should be noted. First, the human study was a retrospective design with a small number of subjects. In particular, there were few female smokers (n = 3), therefore, the association of UA with respiratory function in female smokers could not be examined. To address this limitation, we validated the findings using 1000 replicated datasets generated by random sampling of the original dataset (Table 2
). Since female smokers are at greater risk of respiratory dysfunction than male smokers [68
] and we could not entirely exclude the effects of other potential confounding factors on serum UA levels, such as dietary patterns including fructose consumption and amount of daily alcohol intake, further studies in larger populations including female smokers with more detailed lifestyle information are needed to verify these findings.
Despite a number of reports on the detrimental effect of UA to the host due to its ability to be pro-oxidative under certain conditions, our data unveiled a direct beneficial connection between higher blood UA levels and maintenance of lung function in female humans and mice under aging-associated and/or COPD-associated physiopathological conditions. Because the idea of sex differences in disease susceptibility and drug responses is well-accepted, our finding of a novel concept whereby UA has a beneficial role in the lungs of postmenopausal elderly women and patients/mice suffering from aging-associated and/or COPD-associated pulmonary function decline contributes to the acceleration of sex-based medical study.