Immune-mediated membranous nephropathy: Long term fluconazole usage caused podocyte autophagy.

The primary consequences of membranous nephropathy (MN) are the development of nephrotic syndrome including hypogammaglobulinemia, the increased infectious risk, the loss of protein-bound vitamin D, and, above all, an elevated thromboembolic incidence of up to 50% in severe proteinuria patients. Membrane nephropathy may be either idiopathic or primary, not recognized (70%-80%) or secondary (20%-30%) to pathological sicknesses such as hepatitis B, systemic lupus erythematosus, malignancies, and side-effects of medicines. The immunological responses in MN involve multiple components: immunoglobulin G (IgG), long-escaped antigens, and the membrane attachment complex, formed by the supplement to form C5b-9. In general, IgG4 is the most significant IgG subclass deposited in idiopathic membranous nephropathic disease but fluctuating IgG1 levels also are linked with immunological deposits. In contrast, IgG1, IgG2, and IgG3 deposition are greater than IgG4 deposition in secondary nephropathy. Fluconazole is a synthetic antifungal triazole that is often used. It is well tolerated in general and has never been identified as a cause of nephropathies. We report on the development of MN caused by fluconazole therapy that could potentiate podocyte autophagy.

A Novel Insight into the Role of PLA2R and THSD7A in Membranous Nephropathy.

Membranous nephropathy (MN) is an organ-restricted autoimmune disease mainly caused by circulating autoantibodies against podocyte antigens, including the M-type phospholipase A2 receptor (PLA2R) and thrombospondin domain-containing 7A (THSD7A). Antibodies against PLA2R are present in 70%-80% and against THSD7A in 2% of adult patients, which provides a paradigm shift in molecular diagnosis and management monitoring. Both antigens share some similar characteristics: they are expressed by podocytes and have wide tissue distributions; they are bound by autoantibodies only under nonreducing conditions, and the subtype of most autoantibodies is IgG4. However, the factors triggering autoantibody production as well as the association among air pollution, malignancy, and the pathogenesis of MN remain unclear. In this review, we discuss the similarity between the pathological mechanisms triggered by disparate antigens and their associated diseases. Furthermore, we demonstrated the possibility that PM2.5, malignancy, and gene expression specifically induce exposure of these antigens through conformational changes, molecular mimicry, or increased expression eliciting autoimmune responses. Thus, this review provides novel insights into the pathological mechanism of MN.

Ferret Podoplanin Is Detected by PMab-241 in Immunohistochemistry.

Podoplanin (PDPN) plays an important role in the development of many normal tissues and is expressed in various cancers. We have previously developed multiple monoclonal antibodies (mAbs) against PDPNs from a variety of animal species and characterized each of these PDPNs using the anti-PDPN mAbs. In this study, we evaluated whether these anti-PDPN mAbs possess cross-reactivity with ferret PDPN (ferPDPN) using flow cytometry. Comprehensive analysis using 17 differing anti-PDPN mAbs available for immunohistochemistry use, demonstrated that the anti-bear PDPN mAb (clone PMab-241) strongly cross-reacts with ferPDPN-overexpressed Chinese hamster ovary-K1 (CHO/ferPDPN) cells. Immunohistochemistry analysis demonstrated intense PMab-241 staining within Bowman's capsules and glomeruli of the ferret kidney, and lymphatic endothelial cells of the ferret lung. These results demonstrate that PMab-241 is suitable for the detection of PDPN in ferret tissues.

Development of Monoclonal Antibody PMab-269 Against California Sea Lion Podoplanin.

The development of protein-specific antibodies is essential for understanding a wide variety of biological phenomena. Parasitic and viral infections and cancers are known to occur within California sea lion (Zalophus californianus) populations. However, sensitive and specific monoclonal antibodies (mAbs) for the pathophysiological analysis of California sea lion tissues have not yet been developed. A type I transmembrane glycoprotein, podoplanin (PDPN), is a known diagnostic marker of lymphatic endothelial cells. We have previously developed several anti-PDPN mAbs in various mammalian species, with applications in flow cytometry, Western blotting, and immunohistochemistry. In this study, we established a novel mAb against California sea lion PDPN (seaPDPN), clone PMab-269 (mouse IgG1, kappa), using a Cell-Based Immunization and Screening method. PMab-269 is specifically detected in seaPDPN-overexpressed Chinese hamster ovary (CHO)-K1 cells using flow cytometry and Western blotting. Moreover, PMab-269 clearly identified pulmonary type I alveolar cells, renal podocytes, and colon lymphatic endothelial cells in California sea lion tissues using immunohistochemistry. These findings demonstrate the usefulness of PMab-269 for the pathophysiological analysis of lung, kidney, and lymphatic tissues of the California sea lion.

Autoantibodies in the Diagnosis, Monitoring, and Treatment of Membranous Nephropathy.

The discovery of anti-podocyte antibodies in primary membranous nephropathy (MN) has revolutionized our approach toward the diagnosis and treatment of this disease. Evaluation of serum levels of anti-podocyte antibodies paved the way for non-invasive diagnosis and helped distinguish between primary and secondary MN although the relationship between anti-podocyte antibodies and cancer remains to be elucidated. Serum levels of anti-PLA2R antibodies directed against the major podocyte autoantigen are related to MN activity and the decrease in serum levels of anti-PLA2R antibodies in response to treatment (immunologic remission) also serves as an early indicator of the later putative proteinuric remission, enabling personalization of the treatment. The serum levels of anti-podocyte antibodies also enable the prediction of renal outcomes in terms of both remission and the risk of progression to end-stage renal disease. The positivity of anti-PLA2R antibodies before renal transplantation is associated with the risk of recurrence of MN. It remains to be established if all these relations observed in patients with anti-PLA2R antibodies are also valid for expanding spectrum of antibodies directed against recently discovered minor antigens (e.g., THSD7A, NELL-1, semaphorin 3B).

Expression of immunoglobulin G in human proximal tubular epithelial cells.

Proximal tubular epithelial cells (PTECs) have innate immune characteristics, and produce proinflammatory factors, chemokines and complement components that drive epithelial­mesenchymal transition (EMT). Our previous studies revealed that human mesangial cells and podocytes were able to synthesize and secrete immunoglobulin (Ig)A and IgG, respectively. The aim of the present study was to evaluate the expression of Igs in PTECs. Firstly, IgG was detected in the cytoplasm, the cell membrane and the lumen of PTECs in the normal renal cortex by immunohistochemistry. Secondly, Igγ gene transcription and V(D)J recombination were detected in single PTECs by nested PCR and Sanger sequencing. Thirdly, Igγ, Igκ and Igλ were clearly detected in an immortalized PTEC line (HK­2) by immunostaining and western blotting, in which RP215 (an antibody that predominantly binds to non­B cell­derived IgG) was used. In addition, Igγ, Igκ and Igλ gene transcripts, conservative V(D)J recombination in the Igγ variable region, recombination activating gene 1/2 and activation­induced cytidine deaminase were all detected in HK­2 cells. These data suggested that PTECs may express IgG in a similar manner to B cells. Furthermore, IgG expression was upregulated by TGF­ß1 and may be involved in EMT.

Collapsing Focal Segmental Glomerulosclerosis in Viral Infections.

Collapsing glomerulopathy represents a special variant of the proteinuric kidney disease focal segmental glomerulosclerosis (FSGS). Histologically, the collapsing form of FSGS (cFSGS) is characterized by segmental or global condensation and obliteration of glomerular capillaries, the appearance of hyperplastic and hypertrophic podocytes and severe tubulointerstitial damage. Clinically, cFSGS patients present with acute kidney injury, nephrotic-range proteinuria and are at a high risk of rapid progression to irreversible kidney failure. cFSGS can be attributed to numerous etiologies, namely, viral infections like HIV, cytomegalovirus, Epstein-Barr-Virus, and parvovirus B19 and also drugs and severe ischemia. Risk variants of the APOL1 gene, predominantly found in people of African descent, increase the risk of developing cFSGS. Patients infected with the new Corona-Virus SARS-CoV-2 display an increased rate of acute kidney injury (AKI) in severe cases of COVID-19. Besides hemodynamic instability, cytokine mediated injury and direct viral entry and infection of renal epithelial cells contributing to AKI, there are emerging reports of cFSGS associated with SARS-CoV-2 infection in patients of mainly African ethnicity. The pathogenesis of cFSGS is proposed to be linked with direct viral infection of podocytes, as described for HIV-associated glomerulopathy. Nevertheless, there is growing evidence that the systemic inflammatory cascade, activated in acute viral infections like COVID-19, is a major contributor to the impairment of basic cellular functions in podocytes. This mini review will summarize the current knowledge on cFSGS associated with viral infections with a special focus on the influence of systemic immune responses and potential mechanisms propagating the development of cFSGS.

A case of immune complex mediated tubulointerstitial disease and nephrotic syndrome: anti LRP-2 Nephropathy with diffuse podocyte effacement.

Anti-LDL Receptor-Related Protein 2 (Anti-LRP2) nephropathy is a rare form of kidney disease that affects the older patients and is characterized with acute kidney injury (AKI) and progressive renal tubular injury associated with IgG immune complex deposits along the basement membrane of proximal tubules, and circulating autoantibodies to the proximal tubule brush border protein LRP2 (megalin). We present the case of a 79-year-old man who was hospitalized for worsening malaise, abdominal distention and bilateral lower extremity edema, diagnosed with AKI and had nephrotic range proteinuria. Percutaneous kidney biopsy revealed tubulointerstitial nephritis with IgG immune complex deposits along the basement membrane of proximal tubules and brush borders. Immunofluorescence staining for LRP2 (megalin) showed similar granular tubular basement membrane deposits along the proximal tubules and proximal tubule brush borders. Electron microscopy revealed global podocyte foot process effacement. The patient was started on oral prednisolone 1 mg/kg and rituximab at a dose of 375 mg/m2 once weekly for 4 weeks with gradual tapering of prednisone. This case with AKI and nephrotic syndrome highlights the significant morphologic overlap with minimal change disease and anti-LRP2 nephropathy, which is associated with autoantibodies to the tubular brush border protein LRP2/megalin.

Altered glycosylation of IgG4 promotes lectin complement pathway activation in anti-PLA2R1-associated membranous nephropathy.

Primary membranous nephropathy (pMN) is a leading cause of nephrotic syndrome in adults. In most cases, this autoimmune kidney disease is associated with autoantibodies against the M-type phospholipase A2 receptor (PLA2R1) expressed on kidney podocytes, but the mechanisms leading to glomerular damage remain elusive. Here, we developed a cell culture model using human podocytes and found that anti-PLA2R1-positive pMN patient sera or isolated IgG4, but not IgG4-depleted sera, induced proteolysis of the 2 essential podocyte proteins synaptopodin and NEPH1 in the presence of complement, resulting in perturbations of the podocyte cytoskeleton. Specific blockade of the lectin pathway prevented degradation of synaptopodin and NEPH1. Anti-PLA2R1 IgG4 directly bound mannose-binding lectin in a glycosylation-dependent manner. In a cohort of pMN patients, we identified increased levels of galactose-deficient IgG4, which correlated with anti-PLA2R1 titers and podocyte damage induced by patient sera. Assembly of the terminal C5b-9 complement complex and activation of the complement receptors C3aR1 or C5aR1 were required to induce proteolysis of synaptopodin and NEPH1 by 2 distinct proteolytic pathways mediated by cysteine and aspartic proteinases, respectively. Together, these results demonstrated a mechanism by which aberrantly glycosylated IgG4 activated the lectin pathway and induced podocyte injury in primary membranous nephropathy.

Is primary membranous nephropathy a complement mediated disease?

Membranous nephropathy (MN) is an immune complex mediated disease. Although limited to the kidney, in up to 20% of patients MN is associated with other autoimmune, infectious or malignant diseases. The initial pathogenetic event in what is still considered "primary" MN is the binding of circulating autoantibodies to proteins (autoantigens) expressed in glomerular podocytes. This antibody binding leads to the formation of immune complexes in the glomerular basement membrane. There is clinical and experimental evidence that these immune deposits lead to the activation of the complement system. Experimental studies in the MN model of Heymann's nephritis show that the terminal membrane attack complex (MAC) of the complement system induces a disturbance of the glomerular filtration barrier and leads to proteinuria, the clinical hallmark of MN. After the discovery of the phospholipase A2 receptor 1 and thrombospondin type 1 domain containing protein 7A as endogenous antigens, it is assumed that IgG4 antibodies directed against these proteins induce MN in over 85% of patients with primary MN. As a result, the role of complement in the pathogenesis of MN needs to be defined in light of these developments. In this review we describe the current knowledge on the function of the complement system in primary MN and discuss the open questions, which have to be solved for a better understanding of the potential role of complement in the pathophysiology of primary MN.

Podocytopathies.

Podocytopathies are kidney diseases in which direct or indirect podocyte injury drives proteinuria or nephrotic syndrome. In children and young adults, genetic variants in >50 podocyte-expressed genes, syndromal non-podocyte-specific genes and phenocopies with other underlying genetic abnormalities cause podocytopathies associated with steroid-resistant nephrotic syndrome or severe proteinuria. A variety of genetic variants likely contribute to disease development. Among genes with non-Mendelian inheritance, variants in APOL1 have the largest effect size. In addition to genetic variants, environmental triggers such as immune-related, infection-related, toxic and haemodynamic factors and obesity are also important causes of podocyte injury and frequently combine to cause various degrees of proteinuria in children and adults. Typical manifestations on kidney biopsy are minimal change lesions and focal segmental glomerulosclerosis lesions. Standard treatment for primary podocytopathies manifesting with focal segmental glomerulosclerosis lesions includes glucocorticoids and other immunosuppressive drugs; individuals not responding with a resolution of proteinuria have a poor renal prognosis. Renin-angiotensin system antagonists help to control proteinuria and slow the progression of fibrosis. Symptomatic management may include the use of diuretics, statins, infection prophylaxis and anticoagulation. This Primer discusses a shift in paradigm from patient stratification based on kidney biopsy findings towards personalized management based on clinical, morphological and genetic data as well as pathophysiological understanding.

Podocyte Antigen Staining to Identify Distinct Phenotypes and Outcomes in Membranous Nephropathy: A Retrospective Multicenter Cohort Study.

RATIONALE & OBJECTIVE: Membranous nephropathy (MN) is characterized by the deposition of immune complexes along glomerular basement membranes. M-Type phospholipase A2 receptor (PLA2R), thrombospondin type 1 domain-containing 7A (THSD7A), exostosin 1 and 2 (EXT1/2), and neural epidermal growth factor-like 1 protein (NELL-1) have been identified as established or potential podocyte antigens in MN. We investigated the association of podocyte antigen staining with MN clinical phenotype and outcomes. STUDY DESIGN: Multicenter retrospective cohort study. SETTING & PARTICIPANTS: 177 consecutive patients with MN unrelated to lupus erythematosus, identified after screening of 3,875 native kidney biopsies performed in the Belgian UCLouvain Kidney Disease Network from 2000 through 2018. PREDICTOR: Positive immunostaining for podocyte antigens on archived kidney biopsy samples. OUTCOMES: Association with different phenotypes (baseline characteristics of patients and pathologic findings on kidney biopsy), time to cancer and to kidney failure. ANALYTICAL APPROACH: Kaplan-Meier estimates and Cox regression analyses to assess time to cancer and kidney failure. RESULTS: 177 patients were followed up for a median of 4.0 (IQR, 1.3-8.0) years. Diagnosis of PLA2R-positive (PLA2R+), THSD7A+, and double-negative (PLA2R-/THSD7A-) MN was made in 117 (66.1%), 6 (3.4%), and 54 (30.5%) patients, respectively. Progression to kidney failure was similar in all groups. Although the number of patients with THSD7A+MN was small, they showed a higher incidence (50%) and increased risk for developing cancer during follow-up (adjusted HR, 5.0 [95% CI, 1.4-17.9]; P=0.01). 8% and 5% of patients with double-negative MN stained positively for EXT1/2 and NELL-1, respectively. Most patients with EXT1/2+MN were women, had features of systemic autoimmunity, and showed glomerular C1q deposits. LIMITATIONS: Retrospective design; small number of patients in the THSD7A group; lack of evaluation of immunoglobulin G subclasses deposition. CONCLUSIONS: Our real-world data describe the relative prevalence of subgroups of MN and support the hypothesis that a novel classification of MN based on podocyte antigen staining may be clinically relevant.

MiR-770-5p facilitates podocyte apoptosis and inflammation in diabetic nephropathy by targeting TIMP3.

OBJECTIVE: Diabetic nephropathy (DN) is one of the most severe and frequent diabetic complications. MicroRNAs (miRNAs) have been reported to play a vital role in DN pathogenesis. The present study aimed to investigate the molecular mechanism of miR-770-5p in DN. METHODS: Podocyte injury model was established by treating mouse podocytes with high glucose (HG, 33 mM) for 24 h. The levels of miR-770-5p and TIMP3 were examined in kidney tissues and podocytes using quantitative real-time PCR (qRT-PCR). Flow cytometry analysis was applied to detect apoptosis in podocytes. Western blot assay was used to measure the protein levels of B-cell lymphoma 2 (Bcl-2), Bcl-2 associated X (Bax) and tissue inhibitors of metalloproteinase 3 (TIMP3). Enzyme-linked immunosorbent assay (ELISA) was conducted to measure the levels of inflammatory factors. The interaction between miR-770-5p and TIMP3 was determined by MicroT-CDS and luciferase reporter assay. RESULTS: MiR-770-5p was up-regulated and TIMP3 was down-regulated in DN kidney tissues and HG-stimulated podocytes. Depletion of miR-770-5p suppressed cell apoptosis and the release of pro-inflammatory factors in HG-treated podocytes. Additionally, TIMP3 was a target of miR-770-5p in HG-treated podocytes. TIMP3 inhibited cell apoptosis and inflammation in HG-treated podocytes. Moreover, TIMP3 knockdown alleviated the inhibitory effect of miR-770-5p silencing on podocyte apoptosis and inflammatory response. CONCLUSION: Knockdown of miR-770-5p suppressed podocyte apoptosis and inflammatory response by targeting TIMP3 in HG-treated podocytes, indicating that miR-770-5p may be a potential therapeutic target for DN therapy.

Further Evidence That the Soluble Urokinase Plasminogen Activator Receptor Does Not Directly Injure Mice or Human Podocytes.

BACKGROUND: The role of the soluble urokinase plasminogen activator receptor (suPAR) in focal segmental glomerulosclerosis (FSGS) as the circulating factor or as a predictor of recurrence after transplantation remains controversial. Previously published studies in mice and isolated podocytes produced conflicting results on the effect of suPAR on podocyte injury, effacement of foot processes, and proteinuria. These discordant results were in part due to diverse experimental designs and different strains of mice. The aim of our study was to determine the reasons for the inconsistencies of the previous studies results with suPAR by using uniform methods and studies in different strains of mice. METHODS: We utilized a primary culture of human podocytes and 2 mouse models, the wild type (WT) and the urokinase plasminogen activator receptor (uPAR) KO (uPAR), in an attempt to resolve the reported conflicting results. RESULTS: In both WT and uPAR mouse models, injection of recombinant uPAR, even at a high dose (100 µg), did not induce proteinuria, effacement of podocytes, or disruption of the cytoskeleton. Injection of suPAR resulted in its deposition exclusively in the glomerular endothelial cells and not in the podocytes of WT mice and was not detected at the uPAR KO mice. Kidneys from patients with recurrent FSGS had negative immunostaining for uPAR. We also evaluated the effect of recombinant uPAR on primary culture of human podocytes. uPAR did not result in podocytes damage. CONCLUSIONS: suPAR by itself is not the cause for direct podocyte injury, in vitro or in vivo. These findings suggest a more complex and still poorly understood role of suPAR in FSGS.

Association of vascular endothelial growth factor and renal thrombotic microangiopathy-like lesions in patients with Castleman's disease.

AIM: Renal thrombotic microangiopathy (TMA) is a common pathological manifestation of Castleman's disease (CD)-associated renal lesions. Increased level of plasma vascular endothelial growth factor (VEGF) has been shown in single-case reports. We aimed to investigate the dysregulation of VEGF in the pathogenesis of CD-associated TMA-like lesions (CD-TMA) in a larger cohort. METHODS: Nineteen patients with clinico-pathologically diagnosed CD with renal involvement were enrolled. Ten patients with pregnancy TMA or TMA of unknown reasons were enrolled as TMA control group. The plasma levels of VEGF, soluble Flt-1 and interleukin-6 (IL-6) were detected using enzyme-linked immunosorbent assay kits. The expression of VEGF in the kidney biopsied tissue sections and the lymph node specimens were detected by immunostaining. RESULTS: The plasma levels of VEGF and IL-6 levels were the highest in CD-TMA group compared to TMA control group and healthy controls. The levels of plasma VEGF was positively correlated with that of IL-6, and increased expression of VEGF and IL-6 was also observed in the lymph nodes from CD-TMA patients. However, the expression of VEGF in the glomerular podocytes was significantly decreased in CD-TMA group as well as in the TMA control. CONCLUSION: Our findings suggest that renal VEGF expression might be important in the pathogenetic mechanism of CD-associated TMA-like lesions.

Role of A20/TNFAIP3 deficiency in lupus nephritis in MRL/lpr mice.

BACKGROUND: The activation of the nuclear factor-κB (NF-κB) signaling pathway gives rise to inflammation in the pathogenesis of lupus nephritis (LN), with A20 serving as a negative feedback regulator and ubiquitin C­terminal hydrolase L1 (UCH-L1) acting as a downstream target protein. However, their roles in the mechanism of LN remain undetermined. METHODS: In the present study, the expression of A20 and UCH-L1, the activity of NF-κB and ubiquitin-proteasome system (UPS) were measured in MRL/lpr mice and A20 gene silenced podocytes. The severity of podocyte injury and immune complex deposits were detected by transmission electron microscopy. RESULTS: The in vivo experiments revealed that A20 failed to terminate the activation of NF-κB, which was accompanied by UCH-L1 overexpression, ubiquitin accumulation, and glomerular injury in LN mice. Immunosuppression therapy did improve LN progression by attenuating A20 deficiency. In vitro experiments confirmed that tumor necrosis factor-α induced NF-κB activation, which led to UCH-L1 overexpression, UPS impairment, the upregulation of desmin and the downregulation of synaptopodin in A20 gene silenced podocytes. CONCLUSION: Thus, the results of the present study suggest that A20 regulates UCH-L1 expression via the NF-κB signaling pathway and A20 deficiency might play an important role in LN pathogenesis. Therefore, the A20 protein may serve as a promising therapeutic target for LN.

Autophagy in Immune-Related Renal Disease.

Autophagy is an important biology process, central to the maintenance of biology process in both physiological and pathological situations. It is regarded as a "double-edged sword"-exerting both protective and/or detrimental effects. These two-way effects are observed in immune cells as well as renal resident cells, including podocytes, mesangial cells, tubular epithelial cells, and endothelial cells of the glomerular capillaries. Mounting evidence suggests that autophagy is implicated in the pathological process of various immune-related renal diseases (IRRDs) as well as the kidney that underwent transplantation. Here, we provide an overview of the pathological role of autophagy in IRRDs, including lupus nephritis, IgA nephropathy, membrane nephropathy, ANCA-associated nephritis, and diabetic nephropathy. The understanding of the pathogenesis and regulatory mechanisms of autophagy in these renal diseases may lead to the identification of new diagnostic targets and refined therapeutic modulation.

The Epitope of PMab-210 Is Located in Platelet Aggregation-Stimulating Domain-3 of Pig Podoplanin.

Podoplanin (PDPN)/T1alpha/Aggrus, a small mucin-type transmembrane glycoprotein, has been shown to be expressed on lymphatic endothelial cells and epithelial cells of many organs. PDPN is also upregulated in many cancers, and is involved in cancer metastasis and malignant progression. Human PDPN possesses three platelet aggregation-stimulating (PLAG) domains and the PLAG-like domain, which bind to C-type lectin-like receptor-2 (CLEC-2). Previously, we reported a novel antipig PDPN (pPDPN) monoclonal antibody (PMab-210) using Cell-Based Immunization and Screening (CBIS) method. PMab-210 specifically detected pPDPN-overexpressed Chinese hamster ovary (CHO)-K1 cells by flow cytometry and Western blot analysis. Immunohistochemical analyses demonstrated that PMab-210 stained pulmonary type I alveolar cells strongly and renal corpuscles weakly in pig or microminipig. However, the specific binding epitope of PMab-210 for pPDPN could not be determined by enzyme-linked immunosorbent assay using a series of pPDPN peptides. In this study, deletion mutants or point mutants of pPDPN were produced for analyzing the PMab-210 epitope using flow cytometry. The analysis of deletion mutants showed that N-terminus of PMab-210 epitope exists between 45th amino acid (aa) and 50th aa of pPDPN. In addition, the analysis of point mutants demonstrated that the critical epitope of PMab-210 could include Glu47, Asp48, Tyr49, Thr50, and Val51 of pPDPN, indicating that PMab-210 epitope is located in PLAG3 domain of pPDPN.