Antinephrin, Antipodocin, and Anti-Kirrel1 Autoantibodies in Posttransplant Recurrent FSGS

Introduction

The discovery of antinephrin autoantibodies as a possible circulating factor in diffuse podocytopathies has led to a paradigm shift in our understanding of the pathogenic mechanisms in diffuse podocytopathies.¹ Antinephrin autoantibodies have been implicated in both native kidney diffuse podocytopathies and posttransplant recurrent focal segmental glomerulosclerosis (FSGS, rFSGS), and this emerging framework is reshaping the traditional classification of nephrotic syndrome and has important implications for diagnosis.2, 3, 4

Very recently, additional autoantibodies directed against slit diaphragm molecules other than nephrin—such as podocin, Kirrel1 and NEPH3—have been reported in native kidneys, expanding the spectrum of autoimmune-mediated diffuse podocytopathies.^5,6 We reported on a case of rFSGS in which graft biopsy specimens demonstrated simultaneous IgG colocalization with nephrin, podocin, and Kirrel1.⁷ Based on this case report, we examined the positivity rates of plasma or serum antipodocin and anti-Kirrel1 autoantibodies in rFSGS using enzyme-linked immunosorbent assay.

Results

Patients’ Clinical Characteristics

We retrospectively analyzed 19 Japanese pediatric patients with rFSGS who had stored graft biopsy samples obtained during posttransplant recurrence. The biopsies were performed at a median (interquartile range) of postoperative day 30 (20–54). The median (interquartile range) age of the 19 patients at the time of kidney transplantation was 12.0 (8.6–15.1) years (Supplementary Table S1). Prophylactic plasma exchange was performed in 13 patients, and 4 of them received rituximab before transplantation. All patients experienced recurrence of proteinuria 1 (1–1) days after transplantation.S1 Twelve patients achieved remission, whereas the remaining 7, classified as treatment nonresponders, developed graft failure despite intensive treatment including plasma exchange, methylprednisolone pulse therapy, and rituximab (Supplementary Table S1).

IgG Colocalization With Nephrin, Podocin, or Kirrel1

IgG depositions on podocytes were observed in 18 of 19 cases (95%) (Figure 1a). Seventeen cases (89%) showed IgG depositions that colocalized with ≥1 of the 3 slit diaphragm molecules (Figure 1a). One case (5%) showed IgG deposition, but without colocalization of IgG with any of the 3 slit diaphragm molecules (Figure 1a, Supplementary Table S2). IgG was colocalized with nephrin, podocin, and Kirrel1 in 15 (79%), 8 (42%), and 11 (58%) cases, respectively (Figure 1b, Supplementary Table S2).

Figure 1.

(a) Number of patients with IgG deposition in allograft biopsies. Among the 19 patients with stocked kidney biopsy specimens, IgG depositions on podocytes were observed in 18 cases (95%). One case (5%) showed IgG deposition without colocalization with nephrin, podocin, or Kirrel1. (b) Venn diagram showing the number of patients with single-, double-, and triple-positive for IgG colocalized with nephrin, podocin, or Kirrel1. IgG was colocalized with nephrin, podocin, or Kirrel1 in 15 (79%), 8 (42%), and 11 (58%) cases, respectively. Colocalization of IgG with 2 or 3 slit diaphragm molecules, suggesting the presence of multiple autoantibodies, was observed in 11 cases. Of these, 6 were triple-positive for IgG colocalizing with nephrin, podocin, and Kirrel1. (c) Venn diagram showing the number of patients with single-, double-, and triple-positive for circulating anti-nephrin, anti-podocin, or anti-Kirrel1 autoantibodies detected by enzyme-linked immunosorbent assay. Circulating antinephrin, antipodocin, and anti-Kirrel1 autoantibodies were detected in 9 (100%), 5 (56%), and 5 (56%) cases with available plasma or serum samples during recurrence, respectively. Multiple autoantibodies were identified in 6 cases, including 4 cases that were positive for all 3 autoantibodies. rFSGS, recurrent focal segmental glomerulosclerosis.

Colocalization of IgG with 2 or 3 slit diaphragm molecules was observed in 11 cases. Of these, 6 were triple-positive for IgG colocalizing with nephrin, podocin, and Kirrel1 (Figures 1b and 2, Supplementary Table S2). Notably, nephrin colocalized with IgG exhibited altered localization to the intracellular areas of podocytes, whereas podocin and Kirrel1 remained localized along the basement membrane despite IgG colocalization (Figure 2, Supplementary Figure S1). Prophylactic maneuvers were performed in 13 of 19 cases. IgG colocalization with these 3 slit diaphragm molecules in biopsy specimens during recurrence was frequently observed, irrespective of prophylactic maneuvers (Supplementary Table S3). None of the control cases showed IgG deposition colocalizing with any of the 3 molecules (Supplementary Figure S2).

Figure 2.

Representative immunofluorescence images from a case with triple-positive for anti-nephrin, anti-podocin, and anti-Kirrel1 autoantibodies. IgG showed colocalization with all 3 slit diaphragm molecules. Nephrin colocalized with IgG exhibited altered localization. In contrast, both podocin and Kirrel1 retained their localization along the basement membrane and individually colocalized with IgG. The allograft biopsy specimen and plasma sample were obtained during recurrence on postoperative day 22. Scale bar = 20 μm.

Circulating Antinephrin, Antipodocin, and Anti-Kirrel1 Autoantibodies in Plasma or Serum Detected Using Enzyme-Linked Immunosorbent Assay

Plasma or serum autoantibodies against nephrin, podocin, and Kirrel1 were measured using enzyme-linked immunosorbent assay in 9 patients with available samples during posttransplant recurrence (Supplementary Methods).S2 Among the 9 cases who had available plasma samples obtained during recurrence at a median (interquartile range) of postoperative day 21 (7–28), biopsy and plasma or serum findings were consistent in most cases; however, a discrepancy was observed in 1 case each for antipodocin and anti-Kirrel1 autoantibodies. Circulating antinephrin, antipodocin, and anti-Kirrel1 autoantibodies were detected in 9, 5, and 5 cases, respectively (Figure 1c, Supplementary Figure S3, and Supplementary Tables S2 and S4).

Longitudinal assessment of circulating antislit diaphragm autoantibodies demonstrated that in responders with available plasma samples after remission, antinephrin autoantibodies became negative in all 3 cases (Supplementary Figure S4). Similarly, antipodocin and anti-Kirrel1 autoantibodies in positive cases during recurrence showed reduction after remission or after initiation of therapeutic plasma exchanges. After graft failure, several cases, including both responders and nonresponders, tested positive for these autoantibodies (Supplementary Figure S4).

Discussion

Recent studies have shown that antinephrin autoantibodies may be involved in the pathogenesis of both minimal change disease and FSGS.1, 2, 3, 4, 5 In addition, Raglianti et al.⁶ demonstrated that some cases with minimal change disease and FSGS showed colocalization of IgG with podocin or Kirrel1 in native kidney biopsies; and reported that, in a subset of these cases, both antinephrin and antipodocin or anti-Kirrel1 antibodies were double-positive. The identification of multiple autoantibodies suggests that various slit diaphragm proteins can serve as autoantigens. Remarkably, in the present study, among the 19 cases with rFSGS, 11 exhibited IgG deposits that colocalized with ≥ 2 of these molecules—nephrin, podocin, and Kirrel1—and 6 demonstrated IgG colocalization with all 3.

In a previous report by Raglianti et al.,⁶ stimulated emission depletion microscopy demonstrated IgG colocalization with podocin in 11 of 116 patients (9%) and with Kirrel1 in 3 of 116 patients (3%) among patients with minimal change disease or FSGS, respectively. The prevalence of antipodocin and anti-Kirrel1 autoantibodies in their study, which included both children and adults with native kidney minimal change disease or FSGS,⁶ was lower than that observed in our cohort of rFSGS. This difference may reflect variations in the study population, because our cohort consisted exclusively of Japanese pediatric kidney transplant recipients with rFSGS. Another possible explanation is variation in the recombinant proteins used for enzyme-linked immunosorbent assay. In the present study, the recombinant podocin antigen consisted of the full-length protein, whereas the antigen used by Raglianti et al.⁶ included only partial sequences. Similarly, for Kirrel1, we used a recombinant protein encompassing a broader region, including the entire N-terminal portion. As a result, different epitopes may have been assayed compared with previous study.⁶

The production of multiple autoantigens has been reported in several autoimmune diseases.⁸ Tissue destruction caused by a primary immune response against a specific epitope can lead to phagocytosis of tissue debris by antigen-presenting cells and the subsequent induction of secondary immune responses against neo-epitopes.⁸ This phenomenon, known as epitope spreading, can occur within a single antigen or across distinct antigens, a process referred to as intermolecular epitope spreading.⁹ Our findings suggest that an initial immune response against slit diaphragm antigen may lead to podocyte injury, resulting in the exposure of additional slit diaphragm antigens.

In conclusion, antinephrin, antipodocin, and anti-Kirrel1 autoantibodies are frequently detected in rFSGS. Despite its retrospective, single-center design, small sample size, and the absence of adult cases, this study showed that most patients with posttransplant rFSGS exhibit IgG reactivity against multiple slit diaphragm proteins, suggesting a broader and potentially polyclonal humoral response.

Disclosure

YS reported grant funding for the Development of Human Resources in Science and Technology, Initiative for Realizing Diversity in the Research Environment from the Ministry of Education, Culture, Sports, Science and Technology of Japan; Grants-in-Aid Scientific Research (C) (JP 24K11031) from the Ministry of Education, Culture, Sports, Science and Technology of Japan and The Japan Foundation for Pediatric Research. KM reports grant funding from Grants-in-Aid Scientific Research (C) (JP 24K10960) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. All the other authors declared no competing interests.

Data Availability Statement

The data underlying this article will not be made publicly available because of the privacy of the study participants. These data will be shared upon reasonable request to the corresponding author.

Supplementary Material

Supplementary File (PDF)

Supplementary Methods. Supplementary References. Figure S1. Representative immunofluorescence images of dual staining for IgG and nephrin, podocin, or Kirrel1 in recurrent focal segmental glomerulosclerosis. Figure S2. Representative immunofluorescence images on control cases. Figure S3. The ELISA results measuring plasma or serum autoantibodies against nephrin, podocin, and Kirrel1. Figure S4. Longitudinal changes in plasma or serum antislit autoantibody levels.Table S1. Detailed clinical characteristics of patients. Table S2. Results of immunofluorescence staining and ELISA for antinephrin, antipodocin, and anti-Kirrel1 autoantibodies in each patient during recurrence. Table S3. The relationship between prophylactic maneuvers and autoantibody positivity. Table S4. Summary of ELISA and immunofluorescence results in patients with available plasma or serum samples during recurrence (n = 9). STROBE Checklist.