Serum Levels of BAFF and APRIL Predict Clinical Response in Anti-PLA2R-Positive Primary Membranous Nephropathy

Abstract

Primary membranous nephropathy (PMN) is a renal-specific autoimmune disease caused by circulating autoantibodies that target glomerular podocyte antigens (PLA2R/THSD7A). However, very little is known on the molecular mechanisms controlling B cell response in this nephropathy. The present study was aimed at correlating the serum levels of B cell activators BAFF/BLyS and APRIL with the presence of anti-PLA2R antibodies in PMN patients and with long-term clinical outcome. To this aim, 51 patients with anti-PLA2R-positive biopsy-proven PMN and nephrotic range proteinuria (>3.5 g/24 hours) were enrolled between January 2009 and December 2015 and treated with conventional 6-month immunosuppressive therapy. After 6 months, 29 patients (56.9%) cleared circulating anti-PLA2R, while in remaining 22 (43.1%), they persisted. Intriguingly, in the first group, baseline serum levels of BAFF/BLyS and APRIL were significantly lower than those in the second one. Moreover, after 6 months of immunosuppressive therapy, an overall reduction in both cytokine serum levels was observed. However, in PMN patients with anti-PLA2R clearance, this reduction was more prominent, as compared with those with anti-PLA2R persistence. When related to clinical outcome, lower baseline BAFF/BLyS (<6.05 ng/mL) and APRIL (<4.20 ng/mL) serum levels were associated with significantly higher probability to achieve complete or partial remission after 24-month follow-up. After dividing the entire study cohort into three groups depending on both cytokine baseline serum levels, patients with both BAFF/BLyS and APRIL below the cut-off showed a significantly higher rate of complete or partial remission as compared with patients with only one cytokine above the cut-off, while the composite endpoint was achieved in a very low rate of patients with both cytokines above the cut-off. Taken together, these results provide new insights into the role of BAFF/BLyS and APRIL in both the pathogenesis of anti-PLA2R-positive PMN and the response to immunosuppressive therapy.

Figure 1

Algorithm of the study. CYC: cyclophosphamide; CR: complete remission; LR: limited response; MP: methylprednisolone; NR: nonresponder; PMN: primary membranous nephropathy; PR: partial remission.

Figure 2

BAFF/BLyS and APRIL baseline serum levels in PMN patients and in controls. (a) BAFF/BLyS baseline serum levels in PMN patients with anti-PLA2R antibodies are significantly higher than those in patients without anti-PLA2R antibodies (5.64 ± 0.97 vs. 1.08 ± 0.33 ng/mL, p < 0.001; white and gray histogram, respectively), while no significant difference was observed with those from patients with lupus nephritis (6.01 ± 2.68 ng/mL, p = 0.09; dark gray histogram). Finally, in healthy volunteers, BAFF/BLyS was detected at very low concentration (0.31 ± 0.21 ng/mL, p = 0.12 vs. PLA2R-negative patients; black histogram). (b) APRIL baseline serum levels in PMN patients with anti-PLA2R antibodies are significantly higher than those in patients without anti-PLA2R antibodies (3.62 ± 0.84 vs. 1.08 ± 0.33 ng/mL, p < 0.001; white and gray histogram, respectively), while no significant difference was observed with those from patients with lupus nephritis (4.09 ± 2.24 ng/mL, p = 0.18; dark gray histogram). Finally, in healthy volunteers, APRIL was detected at very low concentration (0.52 ± 0.32 ng/mL, p = 0.27 vs. PLA2R-negative patients; black histogram). Mann-Whitney U test for nonparametric data. Data in the graphs are expressed as mean ± standard deviation.

Figure 3

BAFF/BLyS and APRIL serum levels before and after 6-month immunosuppressive therapy among groups. (a) Baseline serum levels of BAFF/BLyS were significantly lower in group 1 (anti-PLA2R clearance after 6-month therapy, n = 29) as compared to group 2 (anti-PLA2R persistence after 6-month therapy, n = 22) (5.03 ± 0.48 vs. 6.44 ± 0.86 ng/mL, p < 0.001). If compared with BAFF/BLyS serum levels after 6-month therapy, really a significant reduction was observed mainly in group 1 (5.03 ± 0.48 vs. 1.64 ± 0.82 ng/mL, p < 0.001, white histograms), while in group 2, this reduction, though significant, was less pronounced (6.44 ± 0.86 vs. 5.39 ± 0.73 ng/mL, p = 0.012, gray histograms). (b) Baseline serum levels of APRIL were significantly lower in group 1 (anti-PLA2R clearance after 6-month therapy, n = 29) as compared to group 2 (anti-PLA2R persistence after 6-month therapy, n = 22) (3.12 ± 0.61 vs. 4.26 ± 0.63 ng/mL, p = 0.001). If compared with APRIL serum levels after 6-month therapy, really a significant reduction was observed mainly in group 1 (3.12 ± 0.61 vs. 0.67 ± 0.50 ng/mL, p < 0.001, white histograms), while in group 2, this reduction, though significant, was less pronounced (4.26 ± 0.63 vs. 3.72 ± 0.63 ng/mL, p = 0.032, gray histograms). (c) Baseline serum levels of BAFF/BLyS were significantly lower in PMN patients who achieved complete or partial remission (CR/PR, n = 41) after 24-month follow-up, as compared with those with limited response or nonresponders (LR/NR, n = 10) (5.41 ± 0.92 vs. 6.56 ± 0.52 ng/mL, p < 0.001). Moreover, if the BAFF/BLyS baseline serum levels were compared to those assessed after 6-month follow-up, the cytokine reduction was more marked in patients who achieved the best clinical outcome (CR/PR) after 24-month follow-up (5.41 ± 0.92 vs. 2.81 ± 1.82 ng/mL, p < 0.001, white histograms), as compared with the other group (LR/NR) (6.56 ± 0.52 vs. 5.11 ± 1.87 ng/mL, p = 0.013, gray histograms). (d) Baseline serum levels of APRIL were significantly lower in PMN patients who achieved complete or partial remission (CR/PR, n = 41) after 24-month follow-up, as compared with those with limited response or nonresponders (LR/NR, n = 10) (3.43 ± 0.80 vs. 4.41 ± 0.39 ng/mL, p = 0.001). Moreover, if the APRIL baseline serum levels were compared to those assessed after 6-month follow-up, the cytokine reduction was more marked in patients who achieved the best clinical outcome (CR/PR) after 24-month follow-up (3.43 ± 0.80 vs. 1.69 ± 1.58 ng/mL, p = 0.002, white histograms), as compared with the other group (LR/NR) (4.41 ± 0.39 vs. 3.23 ± 1.25 ng/mL for APRIL, p = 0.001, gray histograms). Mann-Whitney U test for nonparametric data. Data in the graphs are expressed as mean ± standard deviation.

Figure 4

ROC curves for BAFF, APRIL, and anti-PLA2R serum levels and 24-month clinical outcome. (a) ROC curve analysis to validate the association of BAFF/BLyS serum levels at baseline with the probability to obtain CR/PR after 24-month follow-up (AUC = 0.884, CI 95% 0.792-0.976, p < 0.001). (b) ROC curve analysis to validate the association of APRIL serum levels at baseline with the probability to obtain CR/PR after 24-month follow-up (AUC = 0.880, CI 95% 0.786-0.975, p < 0.001). (c) ROC curve analysis to validate the association of anti-PLA2R serum levels at baseline with the probability to obtain CR/PR after 24-month follow-up (AUC = 0.737, CI 95% 0.598-0.875, p = 0.021).

Figure 5

Cumulative incidence of complete/partial remission after immunosuppressive therapy among patients stratified for BAFF and APRIL serum levels. (a) Anti-PLA2R-positive PMN patients with BAFF/BLyS < 6.05 ng/mL at the time of renal biopsy achieved the composite endpoint with a significantly higher percentage, as compared with the patients with BAFF/BLyS above the cut-off (97.1% vs. 43.7%, log-rank test, p < 0.001). (b) Anti-PLA2R-positive PMN patients with APRIL < 4.20 ng/mL at the time of renal biopsy achieved the composite endpoint with a significantly higher percentage, as compared with the patients with APRIL above the cut-off (94.4% vs. 46.7%, log-rank test, p < 0.001). (c) If the entire study cohort was assigned to three groups depending on both cytokine baseline serum levels, patients with both BAFF/BLyS and APRIL below the cut-off (continuous line) showed a significantly higher complete or partial remission rate as compared with patients with only one cytokine above the cut-off (dashed line) or with patients with both cytokines above the cut-off (dotted line) (100% vs. 80.% vs. 17.3%, Kaplan-Meier lifetime analysis and log-rank test, p < 0.001).