Anti-slit Antibodies against Podocin and Kirrel1 in Pediatric and Adult Podocytopathies

Podocytopathies are kidney disorders characterized by proteinuria and nephrotic syndrome, potentially progressing to CKD and kidney failure if untreated.¹ Their causes include genetic abnormalities, immune responses, toxins, and infections, complicating diagnosis and treatment.¹ Diagnosis typically relies on kidney biopsy assessed using immunofluorescence, light and electron microscopy.¹ However, immunofluorescence often yields negative results, and light microscopy shows patterns like minimal change disease and FSGS, which indicate podocytopathies but do not clarify the underlying cause of proteinuria.¹ This often leads to nonspecific treatments, risking drug resistance or unnecessary side effects.¹

Recent research has identified in some patients autoantibodies against Nephrin, which disrupt the slit diaphragm, contributing to proteinuria.^2–7 In several patients, high-resolution microscopy has proven effective in detecting IgG colocalized with Nephrin in the podocyte slit diaphragm.⁶ However, in some patients, IgG deposits did not colocalize with Nephrin, suggesting the presence of antibodies directed against other proteins within the filtration slit.^6,7

In a multicenter study involving four centers (two pediatric and two adult), we sought to identify slit proteins other than Nephrin as potential autoantigens in autoimmune podocytopathies. We analyzed 116 kidney biopsies from patients with minimal change disease or FSGS lesions using high-resolution confocal microscopy. IgG deposits in the slit diaphragm were found in 44 of 116 patients (38%), with different percentages in association with minimal change disease (50%) and FSGS (26%) lesions, but in none of the 68 kidney biopsies obtained from controls with other types of GN.

To establish the slit antigen targeted by IgG deposits, we used stimulated emission depletion (STED) microscopy, with a resolution up to 60 nm, as previously described,⁶ using the polyclonal rabbit anti-Podocin (Merck, P0372) and anti-Kirrel1 (Merck, ABS1211). STED microscopy showed IgG colocalization with Nephrin in 22 of 44 patients (50%), with a higher prevalence in pediatric (65%) compared with adult (33%) patients (Figure 1A), but not in the remaining 22 patients (Figure 1B). We then screened IgG-positive patients for colocalization with other slit diaphragm proteins, focusing on Podocin and Kirrel1, given their direct interaction with Nephrin (Figure 1, C–F). STED microscopy showed IgG colocalization with Podocin in 11 patients (25%, Figure 1, C and F). Interestingly, in five patients, IgG colocalized with both Nephrin and Podocin in the same patient (Figure 1F). Kirrel1 colocalized with IgG in three patients (7%, Figure 1, D and F). In the other patients, Kirrel1 and IgG never showed colocalization (Figure 1E).

Figure 1.

STED microscopy and ELISA assays for IgG colocalization and autoantibody detection in podocytopathy patients. (A) Representative STED microscopy image of immunofluorescence staining showing colocalization (yellow) of IgG (green) and Nephrin (red) in a kidney biopsy from a patient with podocytopathy. Bar=2 μm. Representative fluorescence intensity profile plot (inlet) showing the complete overlap between IgG (green) and Nephrin (red) signals. (B) Representative STED microscopy image from a different patient with podocytopathy showing no IgG (green) colocalization with Nephrin (red). Bar=1 μm. Representative fluorescence intensity profile plot (inlet) showing the lack of overlap between IgG (green) and Nephrin (red) signals. (C) Representative STED microscopy image of immunofluorescence staining demonstrating colocalization (yellow) of IgG (green) and Podocin (red) in a kidney biopsy from a patient with podocytopathy. Bar=1 μm. Representative fluorescence intensity profile plot (inlet) showing the complete overlap between IgG (green) and Podocin (red) signals. (D) Representative STED microscopy image of immunofluorescence staining demonstrating colocalization (yellow) of IgG (green) and Kirrel1 (red) in a kidney biopsy from a patient with podocytopathy. Bar=1 μm. Representative fluorescence intensity profile plot (inlet) showing the complete overlap between IgG (green) and Kirrel (red) signals. (E) Representative STED microscopy image of a biopsy from a patient with podocytopathy showing no IgG (green) colocalization with Kirrel1 (red). Bar=2 μm. Representative fluorescence intensity profile plot (inlet) showing the lack of overlap between IgG (green) and Kirrel (red) signals. (F). Percentage of different autoantibodies in patients with minimal change disease or FSGS lesions with IgG deposits on the slit, as assessed by STED microscopy. (G) Levels of anti-Podocin antibodies in serum samples by ELISA assay, indicating detectable serum anti-Podocin antibodies in some patients with podocytopathies, whereas none were detected in 150 nonproteinuric controls and 50 proteinuric controls. The dashed line represents the cutoff threshold for positivity determined as mean+4SD (232.5 ng/ml) of all nonproteinuric controls. (H) Levels of anti-Kirrel1 antibodies in serum samples by ELISA assay, indicating detectable serum anti-Kirrel1 antibodies in some patients with podocytopathies, whereas none were detected in the 150 nonproteinuric controls and 50 proteinuric controls. The dashed line represents the cutoff threshold for positivity determined as mean+4SD (7.1 μg/ml) of all nonproteinuric controls. (I) Age distribution of different autoantibodies in pediatric and adult patients. (J) Comparison of levels of anti-Podocin IgG in serum samples of six patients with podocytopathies during nephrotic phase and subsequent remission, according to KDIGO guidelines.⁸ The paired sample Wilcoxon signed rank test was used to compare differences between the active phase and remission samples, P = 0.03. (K) Serum levels of anti-Podocin antibodies in a patient over time. Anti-Podocin levels show a progressive decrease becoming undetectable at remission and correlating with reduced proteinuria after immunosuppressive therapy. (L) Serum levels of anti-Kirrel1 antibodies in a patient over time. Anti-Podocin levels show a progressive decrease, becoming undetectable at remission and correlating with reduced proteinuria after immunosuppressive therapy. (L) Serum levels of anti-Kirrel1 antibodies in a patient over time. Anti-Kirrel1 levels show a progressive decrease correlating with reduced proteinuria after immunosuppressive therapy. ctrl, control; KDIGO, Kidney Disease Improving Global Outcomes; MC, minimal change; RTX, rituximab; STED, stimulated emission depletion; UPCR, urine protein-creatinine ratio.

To test for circulating anti-Podocin and anti-Kirrel1 autoantibodies, we developed two ELISA assays. ELISA plates were coated with 100 ng/well of recombinant human Podocin (PO 9287, R&D Biotechne) or Kirrel1 (10165 K1, R&D Biotechne) and incubated overnight at 4°C. Plates were washed and blocked with SuperBlock (Thermo Fisher). Patient samples were diluted and incubated on the plates. After washing, plates were incubated with 0.75 μg/ml of biotin-conjugated goat anti-human IgG Fc antibody (Thermo Fisher) in SuperBlock with 0.1% Tween 20. Plates were washed, incubated with horseradish peroxidase–conjugated avidin diluted 1:2000 in SuperBlock with 0.1% Tween 20, and washed again before adding tetramethylbenzidine substrate and stop solution (BioLegend). Absorbance was measured at 450 nm. Wells with coating buffer in the absence of recombinant proteins served as controls to assess nonspecific binding, with background correction performed by subtracting their OD from that of antigen-coated wells for each patient sample.

In a cohort of 66 patients with active idiopathic nephrotic syndrome, 12 (18%) were positive for anti-Podocin IgG (Figure 1G). The anti-Podocin ELISA correctly identified all seven patients for whom biopsy results were available that had shown IgG/Podocin colocalization (Figure 1G). Conversely, 33 of 34 patients without IgG/Podocin colocalization at biopsy were negative for serum anti-Podocin autoantibodies (Figure 1G), i.e., a sensitivity of 100% and specificity of 97% for the anti-Podocin assay. In addition, other 150 controls with nonproteinuric and 50 with other proteinuric kidney diseases tested negative for anti-Podocin antibodies underlining assay specificity (Figure 1G).

We also developed an anti-Kirrel1 ELISA assay, detecting autoantibodies in three of 66 patients (5%) with active idiopathic nephrotic syndrome (Figure 1H). Among the 41 biopsy-validated patients, all patients with IgG/Kirrel1 colocalization had anti-Kirrel1 antibodies in serum, whereas those without colocalization all tested negative (Figure 1H), underlining the specificity of the assay. Similarly, none of the 150 nonproteinuric and 50 proteinuric kidney disease controls tested positive for anti-Kirrel1 antibodies (Figure 1H). Notably, anti-Nephrin antibodies were significantly more prevalent in pediatric patients compared with adults (Fisher's exact test: P = 0.006; 22/33 versus 6/22). Conversely, anti-Podocin autoantibodies showed a similar distribution between pediatric and adult patients (Fisher's exact test: P = 0.768; 9/33 versus 7/22; Figure 1I).

In six patients with anti-Podocin antibodies, serum samples were also available during remission, revealing a significant reduction in antibody levels (Figure 1J, Wilcoxon test: P = 0.03). Longitudinal tracking of serum anti-Podocin and anti-Kirrel1 antibodies in two patients showed their presence during active proteinuria and a decline, eventually disappearing during remission achieved through immunosuppressive therapy (Figure 1, K and L).

The identification of these autoantibodies, occasionally multiple in the same patient, broadens the spectrum of autoimmune podocytopathies. This finding suggests that various slit diaphragm proteins can serve as autoantigens, leading to the production of anti-slit antibodies. This would be consistent with the observation that pathogenic mutations in the slit diaphragm genes, including NPHS2 and KIRREL1, cause monogenetic podocytopathies.¹ Further studies are needed to determine whether autoimmunity against Podocin or Kirrel1 is linked to specific clinical features compared with anti-Nephrin antibodies. Measuring these autoantibodies using ELISA assays offers a potentially convenient and noninvasive diagnostic tool. This approach could help in distinguishing patients who may benefit from immunosuppressive treatments from genetic, toxic, infectious, or adaptive podocytopathies in children and adults, which typically do not respond to immunosuppressive therapy.

Disclosures

Disclosure forms, as provided by each author, are available with the online version of the article at http://links.lww.com/JSN/F52.

Funding

This project has received funding from the European Union's Horizon 2020 research and innovation programme and from Tuscany Region (for P. Romagnani and E. Lazzeri, GA n°779282), German Bundesministerium für Bildung und Forschung BMBF (for H.J. Anders, project: 01KU2204), and Health Department-Generalitat de Catalunya DS-CAT (for J.M. Cruzado, SLD078/21/000001) under the ERA-Net Cofund in Personalised Medicine ERA PerMed. F. Becherucci received funding from Call for research projects of young researchers of the University of Florence (2023–2024).

Author Contributions

Conceptualization: Hans-Joachim Anders, Maria Lucia Angelotti, Letizia De Chiara, Valentina Raglianti, Paola Romagnani.

Data curation: Marco Allinovi, Maria Lucia Angelotti, Letizia De Chiara, Benedetta Mazzinghi, Valentina Raglianti, Paola Romagnani.

Formal analysis: Maria Lucia Angelotti, Letizia De Chiara, Benedetta Mazzinghi, Valentina Raglianti.

Funding acquisition: Elena Lazzeri, Paola Romagnani.

Investigation: Giulia Antonelli, Maria Elena Melica, Valentina Raglianti.

Methodology: Carolina Conte, Benedetta Mazzinghi, Anna Julie Peired.

Resources: Marco Allinovi, Giulia Antonelli, Francesca Becherucci, Luigi Cirillo, Carolina Conte, Josep Maria Cruzado, Bärbel Lange-Sperandio, Anna Manonelles, Paola Romagnani.

Supervision: Elena Lazzeri.

Validation: Maria Lucia Angelotti, Letizia De Chiara, Valentina Raglianti.

Writing – original draft: Paola Romagnani.

Writing – review & editing: Hans-Joachim Anders, Francesca Becherucci, Luigi Cirillo, Carolina Conte, Laura Lasagni, Anna Julie Peired, Paola Romagnani.

Data Sharing Statement

Partial restrictions to the data and/or materials apply. The methods of ELISA are under patenting but can be released upon publication.