Autoantibodies against thrombospondin type 1 domain-containing 7A induce membranous nephropathy

Abstract

Membranous nephropathy (MN) is the most common cause of nephrotic syndrome in adults, and one-third of patients develop end-stage renal disease (ESRD). Circulating autoantibodies against the podocyte surface antigens phospholipase A2 receptor 1 (PLA2R1) and the recently identified thrombospondin type 1 domain-containing 7A (THSD7A) are assumed to cause the disease in the majority of patients. The pathogenicity of these antibodies, however, has not been directly proven. Here, we have reported the analysis and characterization of a male patient with THSD7A-associated MN who progressed to ESRD and subsequently underwent renal transplantation. MN rapidly recurred after transplantation. Enhanced staining for THSD7A was observed in the kidney allograft, and detectable anti-THSD7A antibodies were present in the serum before and after transplantation, suggesting that these antibodies induced a recurrence of MN in the renal transplant. In contrast to PLA2R1, THSD7A was expressed on both human and murine podocytes, enabling the evaluation of whether anti-THSD7A antibodies cause MN in mice. We demonstrated that human anti-THSD7A antibodies specifically bind to murine THSD7A on podocyte foot processes, induce proteinuria, and initiate a histopathological pattern that is typical of MN. Furthermore, anti-THSD7A antibodies induced marked cytoskeletal rearrangement in primary murine glomerular epithelial cells as well as in human embryonic kidney 293 cells. Our findings support a causative role of anti-THSD7A antibodies in the development of MN.

Figure 1. Serum anti-THSD7A antibody positivity and recurrence of MN after renal transplantation.

(A) Immunohistochemical staining for THSD7A and complement C4d in biopsies from a patient with circulating anti-THSD7A antibodies before and after renal transplantation. Scale bars: 50 μm. (B) Clinical course of the same patient showing 24-hour proteinuria and serum anti-THSD7A antibody titers when measured with an IFT. Serum anti-THSD7A antibody levels were high at the time of transplantation and at the time of MN recurrence in the renal allograft. CsA, cyclosporine A; MMF, mycophenolate mofetil.

Figure 2. Human anti-THSD7A antibodies bind to mouse THSD7A in vitro and in vivo.

(A) Sera from 2 patients with anti-THSD7A antibody–positive MN, but not serum from a healthy individual, immunoprecipitated THSD7A from MGEs. (B) Indirect immunofluorescence analysis using serum from a patient with THSD7A-associated MN or control serum in frozen sections of mouse kidneys. Original magnification, ×1,000, 4× zoom (enlargement of the boxed area). (C) Immunofluorescence staining for huIgG4 and nephrin 2 hours after i.v. injection of 100 μl serum from a patient with THSD7A-associated MN or a from healthy individual into male BALB/c mice (weight, 30 g). Original magnification, ×1,000, 4× zoom (enlargement of the boxed area). (B and C) Asterisks indicate podocyte nuclei. Scale bars: 50 μm.

Figure 3. Anti-THSD7A antibodies induce the histological features of MN in mice.

(A) Immunofluorescence staining (paraffin sections) for huIgG and laminin in mice injected with anti-THSD7A antibody–containing or control serum at different time points. b, blood side of GBM; u, urinary side of GBM. Scale bars: 10 μm. Enlargements are of boxed areas (original magnification, ×4) of the glomerular filtration barrier. (B) Immunofluorescence staining (frozen sections) for huIgG and THSD7A in mice injected with anti-THSD7A antibody–containing serum or control serum. Scale bars: 10 μm. (C) Immunoblots of HGEs, MGEs, and recombinant mouse THSD7A with eluates from frozen kidney sections from mice that either received anti-THSD7A antibody–containing serum or control serum.

Figure 4. Mouse IgG and complement C3 are deposited in the glomeruli of mice that were injected with anti-THSD7A–containing serum.

(A and B) Immunofluorescence staining (paraffin sections) for mIgG and laminin (A) and complement component C3 and laminin (B) on day 70. Scale bars: 10 μm.

Figure 5. Anti-THSD7A antibodies induce ultrastructural features of MN and proteinuria in mice.

(A) Immunogold electron microscopic images of THSD7A expression in mice after injection of anti-THSD7A antibody–containing or control serum using a specific anti-THSD7A antibody and 12-nm gold-coupled anti-rabbit antibodies on day 70. Scale bars: 200 nm. (B) Immunohistochemical staining for huIgG on paraffin-embedded tissue sections from a mouse 70 days after injection of anti-THSD7A antibody–containing serum. Scale bar: 20 μm. (C) Electron microscopic image from the same mouse 70 days after injection of anti-THSD7A antibody–containing serum. Arrows indicate subepithelial electron-dense deposits. Original magnification, ×12,500. (D and E) Immunofluorescence staining for SOD2 (D) and nephrin (E) on paraffin sections of kidney samples from mice that received either anti-THSD7A antibody–containing serum or control serum on day 70. Scale bars: 10 μm (D) and 5 μm (E). (F) Albumin-to-creatinine ratios measured in urine samples from mice injected with either anti-THSD7A antibody–containing serum or control serum. Error bars indicate the mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001, by 2-tailed, nonparametric Mann-Whitney U test of MN 1 or MN 2 versus control serum.

Figure 6. Affinity-purified anti-THSD7A antibodies induce MN in mice.

(A) Serum from a patient with THSD7A-associated MN was run through chromatographic columns loaded with beaded agarose that was coupled to purified recombinant human THSD7A. The serum was passed 3 times over the columns (FL1–FL3) with 2 acid elutions per round (E1–E6). Immunoblots show the reactivity of purified anti-THSD7A antibodies and serum flow-through with HGEs and recombinant human THSD7A. (B–D) Immunofluorescence staining for huIgG and laminin (paraffin sections) (B), huIgG and THSD7A (frozen sections) (C), and mIgG and laminin (paraffin sections) (D) 28 days after injection of purified anti-THSD7A antibodies or of serum depleted of anti-THSD7A antibodies. Images are representative of analyses of 4 animals per group. Scale bars: 50 μm. Enlargements of boxed areas in B–D are shown in the far right upper and lower panels (original magnification, ×1,000; 4× zoom). (E) Albumin-to-creatinine ratios measured in urine samples from mice injected with either purified anti-THSD7A antibodies or depleted serum (n = 4 per group). Error bars indicate the mean ± SEM. *P < 0.05, by 2-tailed, nonparametric Mann-Whitney U test.

Figure 7. Anti-THSD7A antibodies cause cytoskeletal rearrangement in THSD7A- expressing primary cultured GECs.

(A) Western blot analysis shows expression of THSD7A in GECs. Rec. THSD7A, recombinant human THSD7A. (B) Immunofluorescence staining for huIgG and α-actinin-4 in GECs following a 40-minute exposure to affinity-purified anti-THSD7A antibodies or to serum that was depleted of anti-THSD7A antibodies. Arrows indicate cells that did not express α-actinin-4 and that showed no huIgG binding. Asterisks indicate the glomerulus from which the cells grew. Scale bars: 50 μm. Enlargements of boxed areas are shown in the far right upper and lower panels (original magnification, ×1,000, 4× zoom). (C) Immunofluorescence staining for huIgG, F-actin (phalloidin), and p-paxillin in GECs following a 40-minute exposure to affinity-purified anti-THSD7A antibodies or to serum that was depleted of anti-THSD7A antibodies. Arrows indicate a cell that had no huIgG bound to the membrane and that did not exhibit enhanced F-actin staining. Asterisks indicate the glomerulus from which the cells grew. Scale bars: 50 μm. (D) Quantification of stress fiber formation after treatment of GECs with purified anti-THSD7A antibodies or serum depleted of anti-THSD7A antibodies. A total of 30 images from 3 independent experiments were analyzed. Data indicate F-actin OD of individually circled cells normalized to the control condition (depleted serum). Error bars represent the mean ± SEM.***P < 0.001, by 2-tailed, nonparametric Mann-Whitney U test.

Figure 8. Anti-THSD7A antibodies induce cell detachment and cytoskeletal rearrangement in THSD7A-expressing HEK293 cells.

(A) Western blot analysis shows reactivity of a specific anti-THSD7A antibody in HEK293 cells that were transfected with THSD7A cDNA or empty vector. (B and C) Quantification of detached cells by measurement of total protein concentration in cell pellets after centrifugation of cell culture supernatants following a 60-minute incubation with (B) anti-THSD7A antibody–containing or control serum (results are from at least 5 independent experiments with 2 anti-THSD7 antibody–positive sera, 5 anti-PLA₂R1 antibody–positive sera, 5 sera with no antibodies against THSD7A or PLA₂R1, and 1 control serum) or with (C) affinity-purified anti-THSD7A antibodies, depleted serum, and affinity-purified IgG from a healthy individual (results are from 5 independent experiments with antibodies purified from 1 patient, the corresponding depleted serum, and purified control IgG). Data indicate the fold change in protein content in the cell culture supernatant in comparison with the cells that were transfected with empty vector and treated with serum from a healthy donor (B) or with purified control IgG (C). Error bars indicate the mean ± SEM. ***P < 0.001, by 1-way ANOVA with Bonferroni’s post test; statistical significance is shown for all conditions versus THSD7A-transfected cells that were treated with anti-THSD7A antibody–containing serum (B) or affinity-purified anti-THSD7A antibodies (C). (D) Immunofluorescence staining for huIgG and F-actin (phalloidin) in empty vector– and THSD7A-transfected HEK293 cells after a 30-minute exposure to anti-THSD7A antibody–containing serum or control serum. Arrows show accentuated cortical F-actin rings; asterisks indicate detaching cells. Scale bars: 20 μm.