Usefulness of the cytokines expression of Th1/Th2/Th17 and urinary CD80 excretion in adult-onset minimal change disease

Patient selection

Adult patients with biopsy proven MCD and healthy volunteers were studied (Table 1). None of the healthy control subjects involved in the study had any underlying immunologic disease in the study. Patients with renal dysfunction were excluded (glomerular filtration rate<60 ml/min or serum creatinine>1.5 mg/dl). MCD was defined according to the established pathology criteria (Vivarelli et al., 2017). Relapse of MCD was defined as proteinuria (≥3+ using the tetrabromophenol-citrate buffer colorimetric qualitative dipstick test or urinary protein/creatinine ratio >3.0 mg/mg), edema and hypoalbuminemia (of<30 g/l). Remission of MCD was defined as a urinary protein/creatinine ratio<0.3 mg/mg or no proteinuria using the colorimetric qualitative test. Our study was approved by the Medical Ethics Committee of Ningbo First Hospital (No. 2017-R033). A written informed consent was obtained from all participants.

Clinical samples collection and cytokine antibody array measure

We randomly selected six patients with MCD in relapse, three patients with MCD in remission and three healthy controls. From January 2017 to January 2018, 10ml peripheral blood samples were collected from patients with MCD and healthy controls in Ningbo First Hospital, Medical College of Ningbo University. The total 34 cytokines of Th1/Th2/Th17 in MCD patients and healthy controls were detected by cytokine antibody array using a RayBio^® human cytokine antibody array (RayBiotech, Inc, Norcross, GA, USA, and AAH-TH17-G1). Membranes were incubated with diluted antibodies at room temperature for 2 h. The detections were accomplished according to the manufacturer’s manual and previous research (Huang, 2001).

Lymphocyte subsets assessment

We used flow cytometry analysis to detect lymphocyte subsets in whole peripheral blood in ten patients with MCD in relapse, nine patients with MCD in remission and nine healthy controls. Briefly, peripheral blood was incubated with marked monoclonal antibodies for 20 min at room temperature in the dark. Then lysing reagent was added and incubated for 15 min at room temperature in the dark. The lymphocyte subsets analysis was performed by using a BD flow cytometer (BD Bioscience).

CD80 and CTLA-4 measurements

A commercially available ELISA kit (Bender Med-Systems, Burlingame, CA, USA) was used for measuring CD80 in blood and urine. We detected CTLA-4 in blood and urine according to previous study (Oaks & Hallett, 2000). We adjusted the results of CD80 and CTLA-4 with urinary creatinine excretion. Urinary creatinine and protein and serum albumin were detected by an auto-analyzer.

Immunohistochemistry

We used immunofluorescence technique to test the expression of CD80 in glomeruli of MCD patient in relapse. Snap-frozen renal specimens were incubated with monoclonal synaptopodin or WT-1 antibody (1:50; Santa Cruz, CA) for 2 h at room temperature to reveal podocyte. Then, we washed the sections three times with PBS and incubated specimens with anti-CD80 antibody (1:100) (goat; R&D Systems, Minneapolis, MN) 2 h at room temperature. After washing three times with PBS, we incubated sections with anti-goat 488 and chicken anti-mouse 594 Alexa Fluor antibodies (1:1500, Invitrogen, Carlsbad, CA) for 1 h at room temperature.

Statistical analysis

Graph Pad Instat version 5.0 was used to perform statistical analysis and data graphics. Non-parametric ANOVA (Kruskal-Wallis test) was conducted for statistical analysis. We used Mann–Whitney U test or Wilcoxon signed rank test (when applicable) to determine the differences between means. Spearman correlation coefficient was used to calculate the correlation between urinary CD80 and proteinuria. Values were expressed as means ± standard error of mean (SEM). Results were considered statistically significant if P < 0.05.

Clinical characteristics

The clinical characteristics and laboratory results of patients with MCD in relapse (n = 10), MCD in remission (n = 9) and healthy controls (n = 9) are summarized in Table 1. A total of 28 cases were reported, 15 males and 13 females, with an average age of 34.1 ± 2.1 years (age range: 18–56 years). Patients with MCD in relapse were analyzed at onset of illness. Eight of the relapsed patients were on a tapering dose of immunosuppressive treatment while the remaining two were after drug withdrawal. All patients with MCD in remission were getting immunosuppressive treatment at the time of measuring.

Lymphocyte subsets in the peripheral blood of MCD patients and healthy controls

We analyzed lymphocyte subsets, including CD3⁺T cells, CD3⁺ CD4⁺ T cells, CD3⁺CD8⁺T cells, CD3⁻CD19⁺B cells and CD3⁻CD16⁺/CD56⁺ NK cells. The distribution of lymphocyte subsets in the peripheral blood of MCD patients and healthy controls are showed in Table 2. There were no statistically significant differences in the proportions of CD3⁺ and CD3⁺CD4⁺ cells among patients with MCD in relapse, MCD in remission and healthy controls (P = 0.445 and P = 0.802). MCD patients in relapse had significant higher proportions of CD3⁺CD8⁺ cells compared to MCD in remission (42.7 ± 2.29% vs. 27.42 ± 1.51%, P = 0.008) and healthy controls (42.7 ± 2.29% vs. 27.97 ± 2.34%, P<0.001). Furthermore, CD4⁺/CD8⁺ ratio was lower in MCD in relapse compared with MCD in remission (0.84 ± 0.09 vs. 1.45 ± 0.14, P = 0.014) and healthy controls (0.84 ± 0.09 vs. 1.4 ± 0.12, P = 0.005). The CD3⁻CD16⁺/CD56⁺ NK cell and CD3⁻CD19⁺B cell populations revealed no significant differences among patients with MCD in relapse, MCD in remission and healthy controls (P = 0.199 and P = 0.445).

Cytokine profiles in serum of MCD patients and controls

We investigated Th1/Th2/Th17 cytokines in the serum of the randomly selected MCD patients and controls by cytokine antibody array (Figs. 1A–1C and Table 3). The characteristics of the randomly selected MCD patients and healthy controls are presented in Table 4. Each cytokine antibody array included 34 cytokines (Fig. 1D). The concentrations that increased by ≥1.5-fold or decreased by ≤0.65-fold were considered as significant. Our results in Table 3 indicate that the levels of GM-CSF and tumor necrosis factor (TNF)-related activation-induced cytokine (TRANCE) in patients with MCD in relapse increased 1.5 times higher than in patients in remission. GM-CSF, IL-10, IL-22 and TNF beta levels increased significantly in patients with MCD in relapse compared to healthy controls. The expression of CD40 was found to have decreased in MCD in relapse compared to healthy controls.

Measurement of CD80 and CTLA-4 expression

We explored urinary CD80 expression adjusted by urinary creatinine in patients with MCD in relapse (n = 10), MCD in remission (n = 9) and healthy controls (n = 9) (Fig. 2A). A significant increase in the excretion of urinary CD80 was observed in MCD patients in relapse when compared with patients in remission (598.4 ± 115.8 ng/g vs. 81.78 ± 7.04 ng/g creatinine, P<0.001) and healthy controls (598.4 ± 115.8 ng/g vs. 67.44 ± 8.94 ng/g creatinine, P <0.001). The excretion of urinary CD80 showed no significant difference between MCD patients in remission and healthy controls (P = 0.269, Fig. 2A). There was no correlation between urinary CD80 and proteinuria in MCD patients in relapse (r =  − 0.32, P = 0.366, Fig. 2B).

In contrast to the urinary results, no significant differences were found in serum CD80 concentrations among patients with MCD in relapse, MCD in remission, and healthy control subjects (379.9 ± 32.3 vs. 287.6 ± 19.48 vs. 342.4 ± 28.43 pg/ml, P = 0.081). Urinary CTLA-4 levels were not significantly increased in MCD patients in relapse when compared with patients in remission (173.7 ± 8.73 vs. 155.0 ± 8.54 ng/g creatinine, P = 0.098) and healthy control subjects (173.7 ± 8.73 vs. 160.0 ± 10.85 ng/g creatinine, P = 0.253, Fig. 2C). There was no correlation between the concentrations of urinary CTLA-4 and proteinuria in MCD patients in relapse (r = 0.18, P = 0.632, Fig. 2D). There was also no inverse correlation between urinary CTLA-4 and urinary CD80 in MCD patients in relapse (r = 0.15, P = 0.682, Fig. 2E) nor in remission (r = 0.3, P = 0.437, Fig. 2F).

Compared with MCD patients in remission, the urinary CD80 to CTLA-4 ratio was elevated significantly in MCD in relapse (P = 0.004, Fig. 3A). No significant differences of serum CD80/CTLA-4 ratio (P = 0.278, Fig. 3B) and CTLA-4 concentrations (P = 0.949, Fig. 3C) were found among patients with MCD in relapse, MCD in remission and healthy controls.

The expression of CD80 in podocyte in patient with relapsed MCD

We used immunofluorescence technique to test the expression of CD80 in glomeruli of patient 5 as listed in Table 1. Two glomeruli from patient 5 with relapsed MCD were stained for CD80 in green (Figs. 4A and 4D), synaptopodin in red (Fig. 4B) and WT-1 in red (Fig. 4E). The double immunostaining for CD80 and synaptopodin in the glomerulus of MCD patient in relapse showed colocalization (Fig. 4C). CD80 and WT-1 co-localized in the glomerulus of MCD patient in relapse (Fig. 4F).