In our study, 266 patients (> 18 years old) were recruited according to the 2010 ACR/EULAR criteria for definite RA at The Second Affiliated Hospital of Nanjing Medical University between January 2018 and December 2020. We excluded patients suffering from other autoimmune diseases, such as systemic lupus erythematosus (SLE) and dermatomyositis (DM), acute infectious disease, gout, and severe hepatic and renal dysfunction, or those taking uric acid-lowering drugs. Current or former smokers were also excluded. General condition, past medical history, smoking status and duration of RA were obtained from medical clinical records. The ILD of all patients collected in this study was defined according to HRCT evidence. Meanwhile, the patients were evaluated with pulmonary function tests (PFTs) and bronchoalveolar lavage (BAL). Additionally, 138 individuals matched by age and sex were also included as healthy controls (HCs).
All subjects who participated in this study voluntarily and provided written informed consent. The study protocol was approved by the medical ethics committee of The Second Affiliated Hospital of Nanjing Medical University.
Clinical and laboratory assessments
We registered the patients’ general condition, including age, sex, duration of RA and comorbidities. Laboratory indexes such as C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), presence and plasma level of rheumatoid factor (RF) and anti-cyclic peptide containing citrulline (anti-CCP) were collected. Serum samples of patients were analyzed for UA level and the concentration of Krebs von den Lungen-6 (KL-6). All basic demographic and clinical characteristics of the participants are shown in Table.
Bronchoalveolar lavage (BAL)
Based on current evidence, BAL cellular analysis might be a supplementary means for the diagnostic evaluation of ILD. All RA patients included in this study underwent BAL, bronchoalveolar lavage fluid (BALF) was collected and then UA concentrations were measured as described previously.
Measurement of Krebs von den Lungen-6 (KL-6)
The concentrations of KL-6 in serum were measured by an enzyme-linked immunosorbent assay (ELISA) kit according to previously described procedures.
According to the American Thoracic Society/European Respiratory Society (ATS/ERS) classification of idiopathic interstitial pneumonias (IIPs)[18, 19], the HRCT patterns of RA-ILD were categorized into two major subgroups, namely, serum UAl interstitial pneumonia (UIP) and nonspecific interstitial pneumonia (NSIP). Separately, the UIP pattern was characterized by traction bronchiectasis/bronchiectasis, reticular opacity and honeycombing, while the HRCT characteristic of NSIP was extensive ground glass opacity. HRCT scans of each patient were independently reviewed by two trained thoracic radiologists who were blinded to all clinical information such as demographics and general state. Disagreements over the diagnosis were resolved by consensus.
Pulmonary function tests (PFTs)
Patients with RA-ILD who underwent PFTs fulfilled the American Thoracic Society (ATS)/European Respiratory Society (ERS) guidelines[21–23], including forced vital capacity (FVC) and forced expiratory volume in 1 s (FEV1). All the data are presented as percentages of the normal predicted values. Moreover, we calculated the values of FEV1/FVC and FEV1% predicted.
Cell culture, treatment and analysis
A549 cell line was used to resemble type II alveolar epithelial cells. A549 cells were cultured in vitro. Before UA treatment, A549 cells were maintained in serum-free media for 12 h prior to stimulation and then changed to complete media with increasing concentrations of uric acid (UA, 0, 200 and 400µmol/L) for 24 h. Supernatants were then collected and IL-1, IL-6, and TGF-β concentrations were determined by ELISA.
Real time Reverse transcription semiquantitative polymerase chain reaction (Real-Time RT-PCR).
Total RNA was extracted from A549 cells by TRIzol® RNA isolation (Gibco; Thermo Fisher Scientific, Inc.) and purified with DNase I (Invitrogen; Thermo Fisher Scientific, Inc.). according to the manufacturer's protocol. The primer sequences are available upon request. RNAs were reverse-transcribed into cDNA using SuperScriptTMII (Invitrogen Life Technology). Real-time quantitative PCR was performed by fluorescent dye SYBR Green methodology using SYBR Green PCR Master Mix (Applied Biosystems) and the ABI Prism 7000 apparatus (Perkin-Elmer, Foster City, CA). Gene expression was normalized to the corresponding β-actin level and is presented as the fold change relative to that of the control.
A549 cells were seeded in 6‑well plates. After the indicated treatment, the cells were fixed and incubated with primary antibodies against α-SMA (Santa Cruz Biotechnology, Inc.) and E‑cadherin (Santa Cruz Biotechnology, Inc.) overnight at 4°C, after which the cells were washed three times with PBS. The cells were stained as described in the online Supplementary Information.
All statistical analyses were carried out using GraphPad Prism version 8.0.2. Differences between groups were analyzed by the Student’s t-test. Comparisons of categorical variables were conducted using Pearson chi-square tests. For nonparametric data, the results were expressed as median (range) values, and the differences between groups were analyzed by the Mann-Whitney U test. Spearman correlation analysis was performed to analyze the association of UA with clinical and laboratory indexes in RA patients. Univariate logistic regression analysis was performed to determine the factors associated with the presence of ILD. Multivariate logistic regression analysis was performed by including the confounding factors that were found to be significantly associated with the univariate analyses. The receiver operating characteristic (ROC) curve and area under the curve (AUC) were used to assess the validity and value of UA for RA-ILD. Any difference with a p value < 0.05 was regarded as statistically significant.