Keywords: membranous nephropathy, PLA2R1, pathogenecity, complement, pilot projects, miniature swine
Abstract
Significance Statement
Membranous nephropathy (MN) is an autoimmune kidney disease characterized by immune deposits in the glomerular basement membrane. Circulating anti-phospholipase A2 receptor 1 (PLA2R1) antibodies are detectable in 70%–80% of patients with MN, but experimental evidence of pathogenicity has been lacking. This study demonstrates the pathogenicity of human anti-PLA2R1 antibodies in minipigs, a model for MN that intrinsically expresses PLA2R1 on podocytes. After passive transfer of human anti-PLA2R1 antibody-containing plasma from patients with PLA2R1-associated MN to minipigs, antibodies were detected in the minipig glomeruli, but not in response to plasma from healthy controls. The minipigs developed histomorphological characteristics of MN, local complement activation in the glomeruli, and low-level proteinuria within 7 days, showing that human anti-PLA2R1 antibodies are pathogenic.
Background
Primary membranous nephropathy (MN) is an autoimmune kidney disease in which immune complexes are deposited beneath the epithelium in the glomeruli. The condition introduces a high risk for end-stage kidney disease. Seventy percent to 80% of patients with MN have circulating antibodies against phospholipase A2 receptor 1 (PLA2R1), and levels correlate with treatment response and prognosis. However, experimental evidence that human anti-PLA2R1 antibodies induce MN has been elusive.
Methods
In passive transfer experiments, minipigs received plasma or purified IgG from patients with PLA2R1-associated MN or from healthy controls. Anti-PLA2R1 antibodies and proteinuria were monitored using Western blot, ELISA, and Coomassie staining. Kidney tissues were analyzed using immunohistochemistry, immunofluorescence, electron microscopy, and proteomic analyses.
Results
Minipigs, like humans, express PLA2R1 on podocytes. Human anti-PLA2R1 antibodies bound to minipig PLA2R1 in vitro and in vivo. Passive transfer of human anti-PLA2R1 antibodies from patients with PLA2R1-associated MN to minipigs led to histological characteristics of human early-stage MN, activation of components of the complement cascade, and low levels of proteinuria. We observed development of an autologous, later phase of disease.
Conclusions
A translational approach from humans to minipigs showed that human anti-PLA2R1 antibodies are pathogenic in MN, although in the heterologous phase of disease only low-level proteinuria developed.
Primary autoimmune membranous nephropathy (MN) is initiated by binding of autoantibodies to antigens expressed on podocytes.1 Antibodies against PLA2R1 and Thrombospondin type-1 domain-containing protein 7A (THSD7A) are found in 70%–80% and 2%–3% of patients with MN, respectively.2,3 In patients with PLA2R1- or THSD7A-associated MN, subepithelial immune deposits in glomeruli contain antibodies against the respective molecule, suggesting that PLA2R1 and THSD7A may serve as endogenous antigens.2,3 Transfer of human anti-THSD7A antibodies to mice resulted in a disease, which mimics human MN and demonstrated the pathogenicity of human anti-THSD7A antibodies.4 In contrast to THSD7A, PLA2R1 is not expressed on rodent podocytes and the pathogenicity of human anti-PLA2R1 antibodies has so far not been shown. We used minipigs, which show a PLA2R1 expression pattern on podocytes identical to that found in human glomeruli, to study the pathogenicity of human anti-PLA2R1 antibodies.
Methods
Experiments were performed on healthy, full-grown female Göttingen minipigs (government body approval number N108/2018). Details about the animal housing conditions and all experimental procedures are outlined in the Supplemental Methods.
Anonymized samples from patients with biopsy-proven PLA2R1-associated MN or healthy controls were used for the transfer experiments. Human material was collected as part of a prospective study, which was approved by the local ethics committee of the chamber of physicians in Hamburg and conducted in accordance with the ethical principles stated by the Declaration of Helsinki. Written informed consent was received from all participants before inclusion in the study. The Supplemental Methods describe the selection criteria and preparation of samples in detail. This section also describes the anti-PLA2R1 antibody measurements, Western blot experiments, and the immunohistochemical, immunofluorescence, and electron microscopy investigations.
The mass spectrometry proteomics data have been deposited in the ProteomeXchange Consortium using the Proteomics Identifications Database partner repository with the dataset identifier PXD039024. Details of the sample preparation and mass spectrometric analysis are available as Supplemental Methods.
Results and Discussion
Sera from patients with PLA2R1-associated MN showed an IgG4-dominant reactivity to conformation-dependent epitopes of the porcine PLA2R1 (pPLA2R1), as typically shown for the human PLA2R1 (hPLA2R1) antigen (Supplemental Figures 1 and 2). PLA2R1 antibody–positive plasma from patients with PLA2R1-associated MN was transferred into minipig A at four time points (Supplemental Figure 3). As a negative control, plasma from healthy donors was transferred into the control minipig B. Seven days before the first plasma transfer, the renal mass of each minipig was reduced by removal of one kidney to increase the exposure of the remaining kidney to human antibodies.
The transfer of anti-PLA2R1 antibody–positive patient plasma into minipig A resulted in the presence of circulating anti-PLA2R1 antibodies and hIgG during the entire course of the experiment (Figure 1, A and B). Interestingly, anti-PLA2R1 antibodies specific for the pPLA2R1 were only detected for short times after infusions 3 and 4 (Figure 1A), supporting a mechanism congruent to the “kidney as a sink” hypothesis.5 At later time points (days 29 and 42), very low pig anti-hIgG antibodies were detectable in the circulation, suggesting the initiation of an autologous phase of the disease (Supplemental Figure 4). At no time point during the experiment was pig IgG specific for PLA2R1 detected in the serum of the animals.
Figure 1.
Serologic and morphologic analyses after passive transfer of human plasma from anti-PLA2R1 antibody–positive patients into minipigs. (A) Characterization of minipig A serum samples by Western blot analyses. Reactivity of minipig A serum samples (1:100) used as primary antibody with hPLA2R1 (top; 4 µg HGE/lane) and pPLA2R1 (bottom; 4 µg PGE/lane) immediately before serum transfer (pre), 5 minutes after serum transfer (post), or at the specific day of blood collection. *A longer exposure time was chosen to make the band depicting the anti-pPLA2R1 antibody at “day 29, post” more clear. (B) Detection of hIgG and pAlb in minipig A serum samples (4 µl of 1:50 serum dilution per lane) loaded as antigens in Western blot analysis. (C) Immunofluorescence staining for human IgG (hIgG) and porcine C3 (pC3) (both in green), collagen type IV (in red) and nuclei (in blue) in minipig A and the control minipig B (both day 42). The scale bars indicate 5 µm. (D) Electron microscopy depicting subepithelial electron dense immune deposits (white arrows) in the glomeruli of minipig A, but not in the control minipig B. (E) Proteomic analysis of glomeruli showed an increase of proteins of the complement system as determined by mass spectrometry. Fold changes of label-free quantitation data (minipig A versus control minipig B) were log2-transformed and z-score–transformed. (F) Reactivity of hIgG4 eluted from minipig A glomeruli as primary antibody (1:2) with recombinant hrPLA2R1 as antigen (3.7 µg hrPLA2R1-expressing human embryonic kidney cell lysate per lane) in Western blot analysis. As a positive control, serum from a patient with PLA2R1-associated MN (1:100) was used. No reactivity to PLA2R1 was seen when IgG from glomeruli of the control minipig B was eluted. (G) Development of proteinuria in minipig A, but not in the control minipig B during the course of the experiment. Analyzes of all urine samples from minipig A and minipig B using Coomassie blue staining showed dominant albumin and IgG bands, starting already at day 7, in the urine of minipig A, but not in the urine of minipig B. CL, capillary lumen; HGE, human glomerular extract; hrPLA2R1, human recombinant PLA2R1; pAlb, minipig albumin; PGE, minipig glomerular extract; PLA2R1, phospholipase A2 receptor 1; UR, urinary space.
The transfer of anti-PLA2R1 antibody–positive plasma resulted in a segmental, distinct granular positivity for hIgG and porcine complement component 3 on the urinary side of the glomerular basement membrane of minipig A (Figure 1C). Electron microscopy showed subepithelial electron dense deposits associated with podocyte foot process effacement in a pattern reflecting stage I MN in patient biopsies (Figure 1D, Supplemental Figure 5). The glomeruli of minipig B showed none of these characteristics (Figure 1, C and D). Mass spectrometry analyses of sieved glomeruli confirmed a strong accumulation of complement proteins in minipig A in comparison with control minipig B (Figure 1E). These included proteins involved in the activation of the classical pathway of complement (C1q), alternative pathway (complement factor B), and the terminal complement cascade (C5–C9).
IgG eluted from glomeruli of minipig A reacted with human PLA2R1, while no anti-PLA2R1 antibodies were found in IgG eluted from glomeruli of the control minipig B (Figure 1F). Noteworthy, the anti-PLA2R1 antibodies eluted from the glomeruli of minipig A were of the IgG4 subclass, which is the dominant subclass of anti-PLA2R1 antibodies in patients with PLA2R1-associated MN. Moreover, immunofluorescence staining confirmed that human IgG4 colocalized with pPLA2R1 in the glomeruli of minipig A, but not in minipig B (Supplemental Figure 6). Pig anti-human IgG antibodies were detectable in the glomerular eluates of minipig A, but not of minipig B (Supplemental Figure 4). Minipig A developed proteinuria starting at day 7, suggesting that this was due to the direct binding of the human antibodies to pPLA2R1, before relevant titers of pig anti-hIgG developed. By contrast, the control animal showed no proteinuria over the course of the experiment (Figure 1G and Supplemental Figure 7). The development of pig anti-human IgG antibodies in the later phase of the experiment may have played a role in the later increased albuminuria. A similar observation can also be made concerning the mechanism of complement activation in our model because it does not allow reliable differentiation between complement activation in the glomerular deposits directly induced by human anti-PLA2R1 antibody binding to pPLA2R1 or by pig anti-human IgG binding to human anti-PLA2R1 antibodies. Because both immunofluorescence staining of renal tissue and mass spectrometry analyses of the glomeruli were performed at the end of the experiment when the pig anti-human IgG had already developed, kidney biopsies taken at the early disease stages (day 7) would be needed to answer this question.
In a second passive transfer approach, hIgG purified from patients with PLA2R1-associated MN or healthy donors was transferred into minipigs C and D, respectively. Serological and histomorphological findings were similar but less pronounced compared with the passive transfer model using whole plasma preparations (Supplemental Results).
Our study has a number of limitations. The amount of human anti-PLA2R1 antibody transferred to the minipig was not sufficient to induce nephrotic-range proteinuria within the first days of disease. Owing to the limited amount of available anti-PLA2R1 antibody, we were only able to study single animals. Thus, our experiments were proof-of-principle studies in a pilot setup.
In summary, the serological, histomorphologic, and urinary findings show that the transfer of PLA2R1 antibody–positive plasma from patients with PLA2R1-associated MN to minipigs induces characteristics of MN in the minipig, as well as low-level proteinuria, showing the pathogenicity of human anti-PLA2R1 antibodies for development of MN. At the same time, our experiments do not exclude the possibility that other factors in the plasma of the patients may also contribute to the development of MN.
Supplementary Material
ACKNOWLEDGMENTS
We highly acknowledge the excellent technical support at all experimental steps by Céline Brauer, Katharina Schulz, and Mariola Reszka. We thank Maximilian Webendörfer and Lothar Weiß for their contribution in the glomeruli isolation of minipigs. We thank Ann-Katrin Ziehe, Sonia Wulf, Janine Eisenmann, Anja Obser, Eva Maria Kuhr, Jule Smolka, and Diana Teich for additional technical support. We thank Dr. Gunther Zahner for providing the anti-human PLA2R1 antibody used in immunofluorescence costainings. Purified, recombinant PLA2R1 extracellular region was kindly provided by Euroimmun.
Footnotes
R.A.K.S. and E.H. contributed equally to this work.
Disclosures
T. Wiech reports honoraria: Alexion, Bayer, GlaxoSmithKline GmbH, Novartis and advisory or leadership role: Novartis and Retrophin. M. Machalitza reports other interests or relationships: Stipend of the Else Kröner-Promotionskolleg Hamburg, translationale Euroimmun. S. Peine reports consultancy: Cerus Cooperation. R. Stahl reports patents or royalties: EUROIMMUN, Germany. E. Hoxha reports consultancy: Morphosys AG, Novartis and research funding: Deutsche Forschungsgemeinschaft. M. Rinschen reports honoraria: JASN, Physiological Genomics and advisory or leadership role: Editorial board American Physiology—Renal Physiology, Editorial board JASN, and Editorial board Physiological Genomics. Because M. Rinschen is an editor of the Journal of the American Society of Nephrology, he was not involved in the peer review process for this manuscript. Another editor oversaw the peer review and decision-making process for this manuscript. All remaining authors have nothing to disclose.
Funding
The project was funded by the DFG SFB-1192 to E. Hoxha (project B1, C1) to R.A.K. Stahl (project B1), to T. Wiech (project B6, C1), to C. Meyer-Schwesinger (project B3), and by the Heisenberg Program to E. Hoxha. M. Machalitza was supported by “Else Kröner-Promotionskolleg Hamburg—Translationale Entzündungsforschung” (iPRIME). Funding was also received from Novo Nordisk Fonden (NNF19OC0056043) to M.M. Rinschen.
Author Contributions
E. Hoxha, L. Reinhard, R.A.K. Stahl conceptualized the study; E. Hoxha, M. Lassé, L. Reinhard, and R.A.K. Stahl were responsible for data curation; E. Hoxha, Meyer-Schwesinger, R.A.K. Stahl, and T. Wiech were responsible for funding acquisition; N. Ferru, H.-J. Gröne, A. Heumann, E. Hoxha, A. Hutzfeldt, M. Lassé, D. Loreth, M. Machalitza, C. Meyer-Schwesinger, L. Reinhard, A. Reitmeier, M. Rinschen, M.-L. Schröder, F.R. Stahl, R.A.K. Stahl, and T. Wiech were responsible for investigation; N. Ferru, A. Heumann, E. Hoxha, A. Hutzfeldt, M. Lassé, M. Machalitza, L. Reinhard, A. Reitmeier, M.M. Rinschen, F.R. Stahl, and T. Wiech were responsible for methodology; E. Hoxha and R.A.K. Stahl R. Stahl were responsible for project administration; E. Hoxha, S. Peine, A. Reitmeier, M.M. Rinschen, M.-L. Schröder, and R.A.K. Stahl were responsible for resources; A. Hutzfeldt, M. Lassé, and M.M. Rinschen were responsible for software; E. Hoxha, C. Meyer-Schwesinger, L. Reinhard, M.M. Rinschen, and R.A.K. Stahl provided supervision; H.-J. Gröne, L. Reinhard, and T. Wiech were responsible for validation; E. Hoxha, A. Hutzfeldt, M. Lassé, C. Meyer-Schwesinger, L. Reinhard, M.M. Rinschen, R.A.K. Stahl, and T. Wiech were responsible for visualization; E. Hoxha, L. Reinhard, and R.A.K. Stahl wrote the original draft; and N. Ferru, H.-J. Gröne, A. Heumann, E. Hoxha, A. Hutzfeldt, M. Lassé, D. Loreth, M. Machalitza, C. Meyer-Schwesinger, S. Peine, L. Reinhard, A. Reitmeier, M.M. Rinschen, M.-L. Schröder, F.R. Stahl, R.A.K. Stahl, and T. Wiech reviewed and edited the manuscript
Data Sharing Statement
All data used in this study are available in this article.
Supplemental Material
This article contains the following supplemental material online at http://links.lww.com/JSN/D634.
Supplemental Figure 1. Porcine glomerular PLA2R1 and THSD7A expression and reactivity of human sera with human or minipig glomerular extracts.
Supplemental Figure 2. Evaluation of cross-reactivity of human anti-PLA2R1 and anti-THSD7A antibodies to the respective human and porcine antigen.
Supplemental Figure 3. Overview of the experimental timeline of experiments.
Supplemental Figure 4. Detection of pig anti-human IgG antibodies in the serum and glomeruli of minipigs after transfer of human plasma.
Supplemental Figure 5. Electron microscopic evaluation of the kidney of minipig A after passive transfer of human plasma from anti-PLA2R1 antibody–positive patients.
Supplemental Figure 6. Colocalization of human IgG4 with pPLA2R1 in the glomeruli of minipig A after passive transfer of anti-PLA2R1 antibody–positive human plasma.
Supplemental Figure 7. Urinary findings in minipigs after the passive transfer of human plasma.
Supplemental Figure 8. Validation and quality control of the IgG purification from serum of patients with PLA2R1-associated MN.
Supplemental Figure 9. PLA2R1 recognition of the infusion mixes used in passive model of anti-PLA2R1 antibody–induced MN using whole plasma.
Supplemental Figure 10. Circulating anti-PLA2R1 antibodies and hIgG in control minipig B.
Supplemental Figure 11. Species-specific mass spectrometry analyses of complement molecules deposited in the glomeruli of the minipigs after passive transfer of anti-PLA2R1 antibody–positive or healthy human plasma.
Supplemental Figure 12. Serologic and morphologic analyses after passive transfer of purified human IgG from anti-PLA2R1 antibody–positive patients into minipigs.
Supplemental Figure 13. Circulating anti-PLA2R1 antibodies and hIgG in control minipig D.
Supplemental Figure 14. Urine analyses after passive transfer of purified human IgG from anti-PLA2R1 antibody–positive patients into minipigs.
Supplemental Table 1. Anti-PLA2R1 antibody levels of the infusion samples and serum samples in the passive model of PLA2R1-associated MN after transfer of whole plasma samples.
Supplemental Table 2. Anti-PLA2R1 antibody levels of the infusion samples and serum samples in the passive model of PLA2R1-associated MN after transfer of purified IgG.
Supplemental Table 3. Protein sequence identity of human and porcine PLA2R1 domains.
Supplemental Table 4. Data of the mass spectrometry analysis of sieved glomeruli.
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Associated Data
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Data Availability Statement
All data used in this study are available in this article.