Serum levels of tumor necrosis factor alpha in patients with IgA nephropathy are closely associated with disease severity

Serum samples were collected from 147 patients with biopsy-proven primary IgAN between July 2016 and April 2017. The patients accepted renal biopsy between November 1999 and December 2016. In addition, 126 serum samples were collected from healthy volunteers who had no known kidney diseases between August 2015 and February 2016. Clinical and laboratory data were collected at the time of cytokines measurement in the clinical laboratory of Peking Union Medical College Hospital. Patients with lupus nephritis, Henoch–Schönlein purpura, liver cirrhosis and other secondary causes of IgAN were excluded from the study. Patients with concomitant systemic disease, autoimmune diseases, neoplastic diseases and infection which might cause abnormal elevation of serum TNF-α or IL-6 were also excluded. All patients gave written informed consent to the study, which had received Local Ethical Committee approval.

Full medical histories and physical findings were documented. The demographic and the clinical parameters of patients including age, gender, blood pressure (BP), urinary red blood cells count, urine protein/creatinine ratio, 24-h urine protein excretion were recorded. Blood chemistry tests included serum creatinine, Cystatin C and IgA. Estimated glomerular filtration rate (eGFR) was calculated using the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation [19]. Chronic Kidney Disease (CKD) stages 1–5 were divided by eGFR≥90 (G1), 60–89 (G2), 45–59 (G3a), 30–44 (G3b), 15–29 (G4), and < 15 (G5) mL/min/1.73 m², respectively, according to the new KDIGO (Kidney Disease: Improving Global Outcomes) classification [20]. Histologically, the updated Oxford classification was used for evaluating the pathological lesion [5].

Medication history, including the usage of cortisone, immunosuppressant and renin-angiotensin system blockers such as angiotensin-converting enzymes inhibitors (ACE-Is) and angiotensin II receptor blockers (ARBs) was also recorded.

Blood samples were obtained from each patient after an overnight fast upon admission. The blood samples were allowed to clot at room temperature for 30 mins and centrifuged for 10 minutes at 3000 rpm. The serum samples were separated as soon as possible from the clot of red cells after centrifugation to avoid TNF-α production by blood cells that falsely could increase its values. Separated sera were measured immediately. Levels of serum TNF-α were measured in the clinical laboratory at Peking Union Medical College Hospital using Tumor necrosis factor-alpha Assay Kit (chemiluminescent assay) through IMMULITE® 1000 system (Siemens Healthcare Diagnostics Inc., United Kingdom) according to the manufacturers’ instructions. Detection limit of the kits was obtained from the manufacturers, as follows: TNF-α (detection limit 1.7–1000 pg/mL). The recommended reference range was as follows: TNF-α (≤ 8.1 pg/mL).

Statistical calculations were performed using SPSS software for Windows, version 20.0 (SPSS Inc., Chicago, IL) and Graph Pad Prism 5.0 (Graph Pad Software Inc., San Diego, CA). Data are presented as medians (interquartile range) or frequency in percent according to the types of variables. The Mann–Whitney U-test was used for statistical comparisons between two groups, and the Kruskal-Wallis Test was used for statistical comparisons among more than two groups. Categorical variables were compared using chi-squared test. To set the cut-off values between IgAN patients and healthy controls (HC), we used the receiver operating characteristic (ROC) curve analyses to find the best compromise value between sensitivity and specificity; we also calculated area under the curve (AUC) with 95% confidence interval (CI) and P values. Serum levels of TNF-α were subjected to logarithmic transformation before correlation analyses. Bivariate correlation analyses were performed using Spearman’s correlation analysis. Multivariate linear regression analyses adjusted for sex, systolic BP and diastolic BP were performed to examine the correlations between serum levels of TNF-α and clinical parameters. All tests were two-sided and a P-value < 0.05 was considered statistically significant.

In our study, 147 patients with IgAN [mean age, 35 (29–45) years; male/female ratio, 71/76] and 126 healthy subjects [mean age, 40 (30–49) years; male/female ratio, 61/65] were involved. The main demographic, clinical and the pathological features of patients with IgAN were summarized in Table 1. The average levels of BP, 24-h urine protein excretion, and eGFR were 120 (110–130) / 75 (70–80) mmHg, 0.82 (0.38–1.55) g/24 h, 79.29 (60.00–104.29) mL/min•1.73m², respectively. In this study, 35 (23.8%) IgAN patients were untreated, 99 (67.3%) were treated with ACE-Is/ARBs, 71 (48.3%) were treated with oral corticosteroids, and 76 (51.7%) were treated with other immunosuppressive agents at the time of sample collection. The detailed demographic and clinical data of healthy subjects were showed in Additional file 1.

As showed in Fig. 1, serum levels of TNF-α [9.20 (7.70–10.60) pg/mL vs. 6.04 (5.11–7.23) pg/mL, P < 0.0001] were significantly higher in IgAN group than that in HC group.

ROC analysis confirmed that TNF-α had better discrimination between patients with IgAN and healthy controls (HC) than eGFR [TNF-α: (AUC, 0.87; 95% CI, 0.83–0.91; P < 0.0001) vs. eGFR: (AUC, 0.76; 95% CI, 0.71–0.82; P < 0.0001), P = 0.007] (Fig. 2a and Fig. 2b). The respective optimal derived cut-off values were 7.79 pg/mL (Sensitivity: 74.8%; Specificity: 86.5%) for TNF-α, 80.36 mL/min•1.73m² (Sensitivity: 96.8%; Specificity: 52.4%) for eGFR.

We divided the patients into two subgroups based on the reference range of TNF-α which was established by the clinical laboratory in our hospital. The recommended reference range of serum TNF-α was from nondetectable to 8.1 pg/mL. Among 147 patients with IgAN, 98 patients were with elevated serum TNF-α and 49 patients were without elevated serum TNF-α. As showed in Table 2, compared with patients without elevated serum levels of TNF-α, patients with elevated serum levels of TNF-α had older age (P = 0.03) and higher proportion of male patients (P = 0.047). Significantly higher levels of systolic BP (P = 0.001), diastolic BP (P = 0.02), mean arterial pressure (P = 0.003), 24-h urine protein excretion (P = 0.03), urinary protein to serum creatinine ratio (P = 0.009), serum creatinine (P < 0.0001), Cystatin C (P < 0.0001) and lower levels of eGFR (P < 0.0001) were showed in patients with elevated serum levels of TNF-α. Proportions of mesangial hypercellularity (P = 0.04), segmental glomerulosclerosis (P = 0.02) and tubular atrophy/interstitial fibrosis grade (P = 0.008) were significantly higher in patients with elevated TNF-α than that in patients without elevated TNF-α.

Significant higher serum levels of TNF-α were observed in male patients with IgAN than that in female patients [9.70 (8.10–11.10) pg/mL vs. 8.50 (7.20–10.38) pg/mL, P = 0.02]. Serum levels of TNF-α were significantly positively correlated with systolic BP (r = 0.30, P < 0.0001), diastolic BP (r = 0.19, P = 0.02), 24-h urine protein excretion (r = 0.22, P = 0.007), urinary protein to serum creatinine ratio (r = 0.26, P = 0.002), serum creatinine (r = 0.49, P < 0.0001) and Cystatin C (r = 0.51, P < 0.0001) in IgAN. And serum levels of TNF-α were negatively correlated with eGFR (r = − 0.47, P < 0.0001) in bivariate correlation analysis (Table 3). Serum levels of TNF-α was not significantly correlated with age, serum IgA levels or urinary red blood cells count. Multivariate linear regression analyses showed that serum levels of TNF-α were positively correlated with 24-h urine protein excretion (r = 0.33, P = 0.04), urinary protein to serum creatinine ratio (r = 0.33, P = 0.03), serum creatinine (r = 0.46, P < 0.0001) and Cystatin C (r = 0.59, P < 0.0001) in IgAN and negatively correlated with eGFR (r = − 0.49, P < 0.0001) after adjustment for sex, systolic BP and diastolic BP (Table 3).

As showed in Fig. 3, IgAN patients with higher scores in the variable mesangial hypercellularity showed significant higher serum levels of TNF-α [M1: 9.30 (7.90–10.60) pg/mL vs. M0: 6.70 (6.00–10.58) pg/mL, P = 0.03]. Patients with higher scores in the variable tubular atrophy/interstitial fibrosis grade showed higher serum levels of TNF-α [T2:10.60 (8.25–12.85) pg/mL vs. T1: 9.20 (8.00–10.20) pg/mL vs. T0: 8.20 (6.70–10.05) pg/mL, overall P = 0.001]. Serum levels of TNF-α in patients with tubular atrophy/interstitial fibrosis grade T2 were significantly higher than that in grade T0 [T2:10.60 (8.25–12.85) pg/mL vs. T0: 6.70 (6.00–10.58) pg/mL, P = 0.0005] and T1 [T2: 10.60 (8.25–12.85) pg/mL vs. T1: 9.20 (8.00–10.20) pg/mL, P = 0.02]; But there was no difference between patients with grade T1 and T0 (P = 0.05). However, serum levels of TNF-α in patients with different scores of endocapillary hypercellularity, segmental glomerulosclerosis and cellular/fibrocellular crescents show no significant difference (P > 0.05).