Recurrence of Anti-Semaphorin 3B–Mediated Membranous Nephropathy after Kidney Transplantation

Results

A 7-year-old boy was referred to our center in Montpellier, France by Georgian pediatricians for a refractory primary MN. No family history of kidney disease was reported. Nephrotic syndrome occurred in 2014. No treatment was then started. A kidney biopsy performed in 2015 showed MN stage III with granular IgG deposits along the glomerular capillary walls. Immunostaining for PLA2R and THSD7A was negative. Anti-PLA2R antibodies were not detected. First-line therapy included corticosteroids, cyclophosphamide, and angiotensin-converting enzyme inhibitors and was ineffective on proteinuria. Two years later, tacrolimus was started without any efficacy on the proteinuria level.

On admission in February 2020, the patient had CKD stage 4 (serum creatinine 157 μmol/L, eGFR according to Schwartz formula was 26 ml/min per 1.73m²), with serum albumin 3.3 g/dl and nephrotic range proteinuria (2.45 g/L, urine protein/creatinine ratio 345 mg/mmol) (Figure 1). All causes of secondary MN including autoimmune disease, infections, drugs, and malignancy were excluded. Search for circulating anti-PLA2R and anti-THSD7A antibodies was negative. A kidney biopsy was performed, revealing severe glomerular sclerosis (>50% glomeruli) associated with extensive interstitial fibrosis and tubular atrophy (>30%).

• Download figure
• Open in new tab
• Download powerpoint

Figure 1.

Time course of clinical and immunologic activity. (A) Time course of clinical parameters, treatment and sample collection. (B) Detection of anti-Semaphorin 3B protein antibodies in the serum by Western blot. All incubations of individual strips were performed at the same time in the same experiment at the end of follow-up when all samples were available. Note decrease of antibodies after transplantation and RTX treatment.

Due to the coronavirus disease pandemic and given the severity of kidney lesions, rituximab was not started and only conservative care was maintained. ESKD occurred within 2 months, complicated with a cardiac tamponade and a pleural effusion that required pericardiocentesis and an urgent dialysis start. On dialysis, the patient had unusually persistent diuresis with nephrotic range proteinuria, which led to a unilateral nephrectomy being performed on the day of kidney transplantation. Strong subepithelial staining for IgG was detected along glomerular capillary walls, associated with granular staining along the tubular basement membranes. Electron microscopy showed very abundant, almost contiguous subepithelial electron dense deposits (Figure 2). The nephrectomy specimen was then stained for potential antigens. Although immunostaining for PLA2R, NELL1, EXT1, and EXT2 was negative, a strong positive, granular staining for semaphorin 3B was revealed along the glomerular capillary walls. By confocal microscopy, semaphorin 3B and IgG staining showed impressive colocalization in immune deposits (Figure 2).

• Download figure
• Open in new tab
• Download powerpoint

• Download figure
• Open in new tab
• Download powerpoint

Figure 2.

Semaphorin 3B (SEMA3B) protein localized to immune deposits in glomerular membrane. (A–F) Nephrectomy specimen of native kidney. (A and B) Representative segment of the capillary wall containing immune deposits revealed as electron dense material localized in the basement membrane by electron microscopy (red arrows). (C–E) Detection of SEMA3B protein and IgG in glomerular immune deposits by confocal immunofluorescence microscopy analysis. Biopsy was double-labeled with (C) anti-SEMA3B (green) and (D) anti-human IgG (red). (E) Merged image of (C) and (D). (C′–E′) Enlarged images of the boxed areas in (C), (D), and (E) respectively. (F) Graphs showing quantitative analysis of the fluorescence recorded across sections of a representative capillary loop. Note the superimposition of the two signals, which indicates that subepithelial immune deposits are composed of SEMA3B (green) and IgG (red). (G–J) Kidney graft biopsy performed 1 month after transplantation. (G–I) Confocal microscopy showed weak, granular deposits of SEMA3B which colocalized with IgG. Biopsy was double-labeled with (G) anti-SEMA3B (green) and (H) anti-human IgG (red). (I) Merged image of (G) and (H). (G′–I′) Enlarged images of the boxed areas in (G), (H), and (I) respectively. (J) Graphs showing quantitative analysis of the fluorescence recorded across sections of a representative capillary loop. (K–Q) Kidney graft biopsy performed 5 months after transplantation. (K–M) Electron microscopy showed small subepithelial electron dense deposits, mostly located by the podocyte sole with areas of foot process effacement (red arrows). (N–P) Confocal microscopy showed persistent weak subepithelial deposits of SEMA3B which colocalized with IgG staining. (N′–P′) Enlarged images of the boxed areas in (N), (O), and (P) respectively. (Q) Graphs showing quantitative analysis of the fluorescence recorded across sections of a representative capillary loop.

Kidney transplantation from a deceased pediatric donor was successfully performed in November 2020. Immunosuppressive regimen included an induction with basiliximab 10 mg at day 1 and day 4 in association with a pulse of corticosteroids (300 mg/m²), Maintenance immunosuppressive regimen included corticosteroids (60 mg/m² per day), tacrolimus (0.3 mg/kg per day), and mycophenolate mofetil (1200 mg/m² per day). On day 14, serum creatinine, serum albumin, and proteinuria/urinary creatinine ratio levels were 56 µmol/L, 3.5 g/dl, and <20 mg/mmol, respectively.

On day 25, isolated nephrotic range proteinuria (2.45 g/L, urine protein/creatinine ratio 583 mg/mmol) appeared. A kidney transplant biopsy was then performed. Light microscopy examination was normal but immunofluorescence analysis revealed finely granular, subepithelial IgG deposits with rare C3 and no C1q deposits, posing the diagnosis of MN recurrence. Electron microscopy was not performed. Confocal microscopy showed weak, granular deposits of semaphorin 3B which colocalized with IgG (Figure 2).

Treatment with rituximab was then started (four infusions, 375 mg/m² each week) in association with renin-angiotensin-aldosterone system blockers (enalapril). B cell depletion was obtained after the first infusion. Proteinuria dramatically decreased within 2 months and complete remission was obtained within 4 months (Figure 1). The immunosuppressive regimen was not changed: corticosteroids (tapering to a daily dose of 7.5 mg), mycophenolate mofetil (target area under the concentration time curve concentration of mycophenolate acid 30–60 mg⋅ h/L), and tacrolimus (target through level of 6–8 ng/ml). A protocol kidney transplant biopsy was performed at 5 months after kidney transplant, revealing persistent subepithelial deposits of IgG1 which colocalized with semaphorin 3B staining. Other IgG subclasses, C3 and C1q, were absent. Electron microscopy showed small subepithelial electron dense deposits, mostly located by the podocyte sole with areas of foot process effacement (Figure 2).

At 6 months, eGFR according to Schwartz formula was 72 ml/min per 1.73m². The patient has sustained complete, clinical remission (urine protein/creatinine ratio 22 mg/mmol, urinary albumin/creatinine ratio 0.6 mg/mmol) with a normal serum albumin level (4.2 g/dl). Complete B cell depletion has been maintained to date.

We retrospectively asked whether anti-semaphorin 3B antibody reactivity in serum paralleled clinical activity, and thus could serve as biomarker for the monitoring of the patient. We retrieved all available sera at various time points, which we incubated with reduced recombinant semaphorin 3B. We found a strong signal until transplantation (November 27, 2020) which rapidly dropped thereafter (Figure 1).

At 1 year after transplantation (11 months after recurrence of MN), the patient had a sustained complete immunologic (absence of anti-semaphorin 3B antibodies) and clinical (urine protein/creatinine ratio 21 mg/mmol, urinary albumin/creatinine ratio 6.1 mg/mmol, and serum albumin level 4.4 g/dl) remission.