Clinical Research
Open Access

Hematopoietic Stem Cell Transplant-Membranous Nephropathy Is Associated with Protocadherin FAT1

Sanjeev Sethi, Benjamin Madden, Marta Casal Moura, Samih H. Nasr, Nattawat Klomjit, LouAnn Gross, Vivian Negron, M. Cristine Charlesworth, Mariam P. Alexander, Nelson Leung, Ulrich Specks, Fernando C. Fervenza and Mark Haas
JASN May 2022, 33 (5) 1033-1044; DOI: https://doi.org/10.1681/ASN.2021111488

Significance Statement

Hematopoietic stem cell transplant (HSCT) is a treatment for certain hematologic malignancies and immune disorders. A complication of HSCT is membranous nephropathy (MN), which results from antibodies targeting an antigen in the glomerular basement membrane (GBM). The antigen in most cases of HSCT-associated MN is not known. Laser microdissection and mass spectrometry identified a novel protein, protocadherin FAT1 (FAT1), in HSCT-associated MN. Kidney biopsy specimens showed granular staining for FAT1 along the GBM in HSCT-associated MN. Antibodies to FAT1 were detected in serum and in kidney biopsy tissue in HSCT-associated MN, but not in controls. FAT1-associated MN appears to be a unique type of MN associated with HSCT. FAT1-associated MN represents the majority of MN associated with HSCT.

Abstract

Background Membranous nephropathy (MN) is a common cause of proteinuria in patients receiving a hematopoietic stem cell transplant (HSCT). The target antigen in HSCT-associated MN is unknown.

Methods We performed laser microdissection and tandem mass spectrometry (MS/MS) of glomeruli from 250 patients with PLA2R-negative MN to detect novel antigens in MN. This was followed by immunohistochemical (IHC)/immunofluorescence (IF) microscopy studies to localize the novel antigen. Western blot analyses using serum and IgG eluted from frozen biopsy specimen to detect binding of IgG to new 'antigen'.

Results MS/MS detected a novel protein, protocadherin FAT1 (FAT1), in nine patients with PLA2R-negative MN. In all nine patients, MN developed after allogeneic HSCT (Mayo Clinic discovery cohort). Next, we performed MS/MS in five patients known to have allogeneic HSCT-associated MN (Cedar Sinai validation cohort). FAT1 was detected in all five patients by MS/MS. The total spectral counts for FAT1 ranged from 8 to 39 (mean±SD, 20.9±10.1). All 14 patients were negative for known antigens of MN, including PLA2R, THSD7A, NELL1, PCDH7, NCAM1, SEMA3B, and HTRA1. Kidney biopsy specimens showed IgG (2 to 3+) with mild C3 (0 to 1+) along the GBM; IgG4 was the dominant IgG subclass. IHC after protease digestion and confocal IF confirmed granular FAT1 deposits along the GBM. Lastly, Western blot analyses detected anti-FAT1 IgG and IgG4 in the eluate obtained from pooled frozen kidney biopsy tissue and in the serum of those with FAT1-asssociated MN, but not from those with PLA2R-associated MN.

Conclusions FAT1-associated MN appears to be a unique type of MN associated with HSCT. FAT1-associated MN represents a majority of MN associated with HSCT.

Membranous nephropathy (MN) results from subepithelial deposition of immune complexes along the glomerular basement membranes (GBMs).1⇓–3 MN is often classified into primary MN, where there is an indentifiable target antigen, and secondary MN, where MN may be associated with an autoimmune disease, infection, malignancy, hematopoietic stem cell transplant (HSCT), etc.1⇓–3 The target antigen has been identified as M-type phospholipase A2 receptor (PLA2R) and thrombospondin type 1 domain-containing 7A (THSD7A) in approximately 70% and 1%–5% of primary MN, respectively.4,5 Recently, using laser microdissection and mass spectrometry, novel/putative antigens of MN have been identified in many of the remaining cases of MN. These include neural tissue encoding protein with EGF-like repeats (NELL1), semaphorin 3B (SEMA3B), protocadherin 7 (PCDH7), and serine protease HTRA1 in primary MN, and exostosin 1/exostosin 2 (EXT1/EXT2) and neural cell adhesion molecule (NCAM1) in secondary (autoimmune) MN.6⇓⇓⇓⇓⇓–12

HSCT can cause graft versus host disease (GVHD). One of the complications of chronic GVHD is MN developing in the setting of HSCT.13,14 The antigen in HSCT-associated MN is unknown, although a recent study showed NELL1 in two out of nine patients. 15 We now show that a novel protein—protocadherin FAT1 (FAT1)—is the target antigen in most cases of HSCT-associated MN.

Methods

Patients and Sample Collection

We evaluated biopsy specimens received in the Renal Pathology Laboratory, Department of Laboratory Medicine and Pathology, Mayo Clinic, for diagnosis and interpretation between January 2000 and October 2021. The diagnosis of MN was confirmed by light microscopy, immunofluorescence (IF) microscopy including PLA2R studies, and electron microscopy. The clinical information was obtained from the accompanying charts. The study was approved by the Mayo Clinic Institutional Review Board. For detection of novel proteins, we performed tandem mass spectrometry (MS/MS) in 250 patients with PLA2R-negative MN, which included the patients used for identification of EXT1/EXT2, NELL1, SEMA3B, and PCDH7.7⇓⇓–10 We detected nine patients with FAT1-positive MN by MS/MS (Mayo Clinic cohort; patients 1–5 and 11–14; Figure 1). Patient 11 was a recent case for which serum was available. All nine patients were negative for spectral counts for THSD7A, EXT1/EXT2, NELL1, SEMA3B, and PCDH7, whereas baseline spectral counts for PLA2R were present in three of the nine patients. Clinical information revealed that all nine patients with FAT1-associated MN developed MN after allogeneic HSCT.

Figure 1.
Figure 1.

Discovery and validation cohorts of FAT1-associated MN. In the discovery cohort, MS/MS was performed in 250 patients to look for novel proteins in PLA2R-negative MN. Six patients (patients 1–5 and 11) were positive for a unique protein FAT1. All patients had history of HSCT. Additional patients were discovered when three more patients with HSCT-associated MN were studied, and all (patients 12, 13, and 14) were positive for FAT1. Patients 2, 3, 12, 13, and 14 were Mayo Clinic patients. Kidney biopsy tissue from patients 1, 4, 5, and 11 were received at the Mayo Clinic renal biopsy laboratory. IHC was performed for the seven of nine patients and showed granular GBM staining for FAT1. In the validation cohorts (Cedars Sinai cohort, patients 6–10), MS/MS was performed in five known patients with HSCT-associated MN. All patients were PLA2R negative. All five patients were positive for a unique protein FAT1 by MS/MS. IHC was performed for all five patients and showed granular GBM staining for FAT1.

We then approached M.H. (Cedar Sinai Medical Center) for validation studies and performed MS/MS in a validation cohort of eight patients, of which five had a history of HSCT and three had non-HSCT, PLA2R-negative MN. All five patients with HSCT-associated MN showed spectral counts for FAT1 (Cedar Sinai cohort, patients 6–10; Figure 1). The remaining three patients with non-HSCT MN were negative for FAT1. It is important to note that the Mayo Clinic cohort kidney biopsy tissue was fixed in formalin, whereas the Cedar Sinai cohort tissue was fixed in Bouin fixative.

For control patients, we performed MS/MS on samples from 116 patients that included 15 patients who underwent zero-time kidney transplant biopsies, 17 patients with minimal change disease, 44 patients with FSGS, seven patients with diabetic glomerulosclerosis, five patients with IgA nephropathy, and 28 patients with PLA2R-associated MN. The patients with PLA2R-negative MN and the controls were the same patients that were used for MS/MS studies in the detection of EXT1/EXT2, NELL1, SEMA3B, and PCDH7.7⇓⇓–10 None of the controls showed any spectral counts for FAT1.

Protein Identification by Laser Capture Microdissection, Trypsin Digestion, Nano-LC Orbitrap MS/MS

For each patient, 10-μm-thick formalin-fixed paraffin-embedded (FFPE) tissue sections were obtained and mounted on a special polyethylene naphthalate membrane laser microdissection slide (Thermo Fisher Scientific, Waltham, MA) and, using a Zeiss Palm Microbean microscope, the glomeruli were microdissected to reach approximately 250–550,000 μm2 per case. Resulting FFPE fragments were digested with trypsin and collected for MS/MS analysis. The trypsin-digested peptides were identified by nanoflow liquid chromatography electrospray ionization MS/MS using a Thermo Scientific Q-Exactive Mass Spectrometer (Thermo Fisher Scientific, Bremen, Germany) coupled to a Thermo Ultimate 3000 RSLCnano HPLC system. All MS/MS samples were analyzed using Mascot and X! Tandem set up to search a Swiss-Prot human database. Scaffold (version 4.8.3, Proteome Software Inc., Portland, OR) was used to validate MS/MS-based peptide and protein identifications. Peptide identifications were accepted at >95.0% probability by the Scaffold Local FDR algorithm, with protein identifications requiring a two-peptide minimum and a 95% probability using Protein Prophet.16

The glomerular area dissected for each FAT1-positive case was as follows: patient 1, 116,553 μm2; patient 2, 454,701 μm2; patient 3, 277,338 μm2; patient 4, 564,474 μm2; patient 5, 1,463,913 μm2; patient 6, 51,246 μm2; patient 7, 96,333 μm2; patient 8, 126,334 μm2; patient 9, 547,169 μm2; patient 10, 610,962 μm2; patient 11, 338,810 μm2; patient 12, 100,480 μm2; patient 13, 39,2136 μm2; and patient 14, 652,180 μm2.

Immunohistochemical Staining

Immunohistochemical (IHC) staining was performed at the Pathology Research Core (Mayo Clinic, Rochester, MN) using the Leica Bond RX stainer (Leica). FFPE tissues were sectioned at 5 μm and IHC staining was performed online. Slides for the FAT1 stain were pretreated for 5 minutes using Enzyme 2 (AR9551; Leica) and incubated in Protein Block (Dako) for 5 minutes. The FAT1 primary antibody (rabbit polyclonal; Abcam) was diluted to 1:200 in Background Reducing Diluent (Dako) and incubated for 15 minutes.

The detection system used was the Polymer Refine Detection System (Leica). This system includes the hydrogen peroxidase block, post primary and polymer reagent, diaminobenzidine (DAB), and hematoxylin. Immunostaining visualization was achieved by incubating slides for 10 minutes in DAB and DAB buffer (1:19 mixture) from the Bond Polymer Refine Detection System. To this point, slides were rinsed between steps with 1× Bond Wash Buffer (Leica). Slides were counterstained for 5 minutes using Schmidt hematoxylin and molecular biology grade water (1:1 mixture), followed by several rinses in 1× Bond Wash Buffer and distilled water—this is not the hematoxylin provided with the Refine kit. Once the immunochemistry process was completed, slides were removed from the stainer and rinsed in tap water for 5 minutes. Slides were dehydrated in increasing concentrations of ethyl alcohol and cleared in three changes of xylene before permanent coverslipping in xylene-based medium.

In addition to FAT1-positive MN on MS/MS, 25 controls for IHC included six patients with minimal change disease, five patients with FSGS, six patients with PLA2R-positive MN, two patients with PLA2R-negative MN, three patients who underwent a zero-time transplant biopsy, and three patients with lupus nephritis.

Confocal Immunostaining

IF staining was performed on 8-μm sections cut from formalin-fixed (patient 5) and Bouin-fixed (patient 8) biopsy samples. Sections from two specimens of PLA2R-associated MN were also stained. Slides were deparaffinized and antigen retrieval was performed with Proteinase-K treatment for 5 minutes at room temperature. After quenching in 100 mM ammonium chloride for 20 minutes and blocking (5% normal donkey serum, 5% BSA in 0.1% Triton X-100, PBS) for 2 hours, slides were incubated with primary antibody at 4°C overnight. The following day, the secondary antibody incubation was performed at room temperature for 2 hours. Washes using PBS with 0.05% Triton X-100 were performed between the incubations. Primary antibody against FAT-1 (catalog number ab198892; Abcam) and secondary antibody donkey anti-rabbit AF555N (Thermo Scientific) were used. Microscope images (Zeiss LSM780) were acquired using a 20× lens (numerical aperture 0.3) and images were prepared using Photoshop. All exposure levels were identical within the groups.

Elution of IgG from Kidney Biopsy Specimens

IgG was acid eluted from frozen (tissue remaining after IF microscopy) kidney biopsy specimens.17 The eluate containing anti-FAT1 IgG was obtained from four patients with FAT1-associated MN that were pooled together. The eluate containing control IgG was obtained from tissue pooled together from six patients with PLA2R-associated MN. The kidney biopsy specimens from each group were frozen together in a single block, and serial cryostat sections of 4-μm thickness were mounted side by side on a glass slide. The slides were thawed, fixed with prechilled 100% acetone for 10 minutes at room temperature, and then washed for 5 minutes with PBS (0.01 M, phosphate, pH 7.2). The slide sections were covered with 0.2 ml of a 0.02 M citrate buffer (pH 3.2) and incubated overnight in a humid chamber at 4°C. The eluate was extracted with a calibrated syringe and neutralized with 0.4 M sodium hydroxide to a pH of 7.2.

Western Blot Analysis

A recombinant protein corresponding to antigenic determinants in human FAT1 (Novus Biologicals) was used under nonreducing and reducing conditions. The target molecular mass and dominant band is expected at 33 kD.18 The protein (400 ng) was diluted with nonreduced and reduced (with β-mercaptoethanol) Laemmli sample buffer (BioRad) and boiled for 5 minutes. The samples were loaded into Criterion 4%–15% TGX gels (BioRad) and electrophoresed in Tris-glycine-SDS running buffer. Proteins were transferred to nitrocellulose membrane (0.20-µm pore size) according to standard protocols. Membranes were incubated overnight at 4°C with rabbit anti-human FAT1 (0.2 µg/ml, recommended dilution range of 0.04–0.4 µg/ml; Novus Biologicals), eluates from FAT1-associated MN and PLA2R-associated MN, and patient serum from FAT1-associated MN (1:50 and 1:100 dilutions, patient 11). Serum from patients with PLA2R-associated MN, PLA2R-negative MN, IgA nephropathy, myeloperoxidase-ANCA–associated vasculitis, and one healthy control was probed against reduced FAT1 as negative controls. Subsequently, blots were washed and incubated for 1 hour at room temperature with goat anti-rabbit and goat anti-human IgG Fc (1:15000 and 1:5000, respectively; LI-COR Biosciences, Lincoln, NE ) and with mouse anti-human IgG4 Fc horseradish peroxidase (1:500; Thermo Fischer Scientific). We specifically used anti-human IgG4 for detection of anti-PLA2R IgG because anti-PLA2R antibodies belong to the IgG4 subclass, as has been shown previously.4 Near-infrared fluorescence was detected at the 700 and 800 nm channel, and by chemiluminescence in the Odyssey Infrared Imaging System (LI-COR Biosciences).

Results

Mayo Clinic discovery cohort (nine patients, patients 1–5 and 11–14): A total of 1487 allogeneic HSCTs were done at Mayo Clinic (2000–2019). Nine (0.6%) patients developed MN. Of the nine patients, tissue was available for typing by MS/MS and IHC for six patients. One (16.7%) patient was positive for PLA2R. The remaining five (83.3%) patients were positive for FAT1-associated MN.

An additional four patients with HSCT-associated MN were received at Renal Pathology Laboratory, Mayo Clinic, from other hospitals for diagnosis and interpretation. These patients were initially diagnosed as having PLA2R-negative MN. MS/MS showed that these patients were positive for FAT1, bringing the total of the discovery (Mayo Clinic) cohort to nine.

Cedars Sinai validation cohort (5 patients, patients 6–10): This cohort included five patients with HSCT-associated MN. All five (100%) patients were positive for FAT1 on MS/MS and IHC. The Cedar Sinai cohort included all patients with HSCT-associated MN received in the renal biopsy service dating to 2006.

All 14 patients developed MN after allogeneic HSCT. None of the MN cases developed after autologous HSCT. The consolidated results are described below.

Laser Dissection and MS/MS Detection of FAT1 in PLA2R-Negative MN Biopsy Specimens

We detected a unique protein, FAT1, by MS/MS in the glomeruli of 14 patients with MN (Figure 2). The counts ranged from 8 to 39, with an average±SD total spectral count of 20.9±10.1. The average±SD spectral counts of FAT1 were lower than that of PLA2R (86.1±27.5), EXT1/EXT2 (EXT1, 65.3±34.6; EXT2, 83.4±38.4), and NELL1 (63.1±21.6) in PLA2R-, EXT1/EXT2-, and NELL1-associated MN, respectively. On the other hand, the counts were comparable to that of SEMA3B (23.7±16.5), and higher than that of PCDH7 (13.2±6.6).7⇓⇓–10 In addition, the finding of FAT1 was unique in this subset of patients with PLA2R-negative MN and, importantly, all controls, including 15 who underwent zero-time transplant biopsies, 73 with other glomerulopathies, and 28 with PLA2R-positive MN, were negative for FAT1.

Figure 2.
Figure 2.

Proteomic identification of FAT1 in HSCT-associated PLA2R-negative MN. Glomeruli were microdissected and analyzed using mass spectrometry as described in Methods. (A) Detection of FAT1 in 14 patients with PLA2R-negative MN (top row). Numbers in green boxes represent total spectral counts of MS/MS matches to a respective protein. All 14 patients show moderate total spectral counts for FAT1 and Igs, baseline spectral counts of PLA2R were detected in four of ten patients. For comparison, the pooled total spectral counts from six controls (zero-time protocol transplant biopsies) are also shown, FAT1 is not present in the controls. (B) Representative sequence coverage map of FAT1 from one patient. Amino acids highlighted in bold letters over yellow background are the amino acids detected. (C) An example of MS/MS spectra match to a sequence from FAT1. Example MS/MS spectra of ion 707.36 [M+2H]2+ matched to the FAT1 peptide sequence FSAAGEYDILSIK. Ctrl, control.

The spectral counts of all 14 patients positive for FAT1, along with a representative sequence coverage map of FAT1, are shown in Figure 2, A and B. The MS/MS spectra match from one patient is shown in Figure 2C. It is important to point out that none of the patients positive for FAT1 show any spectral counts for EXT1/EXT2, THSD7A, NELL1, SEMA3B, or PCDH7, whereas baseline PLA2R counts were detected in four of the 14 patients, but the counts were much lower than that for FAT1 and appear similar to the baseline PLA2R counts seen in EXT1/EXT2-, NELL1-, and SEMA3B-associated MN.

All four subclasses of IgG were detected in FAT1-associated MN, with average±SD spectral counts of 20.9±10.1 for IgG1, 19.1±7.3 for IgG2, 21.5±9.1 for IgG3, and 17.4±7.6 for IgG4.

IHC and Confocal Staining for FAT1 in PLA2R-Negative MN Biopsy Specimens

Fourteen patients were positive for FAT1 by MS/MS. Tissue was available in 12 of the 14 patients for IHC staining. All 12 patients showed granular (1 to 3+/3) staining for FAT1 along the GBM (Figure 3A). The positive FAT1 granular staining mirrored the granular IgG along the GBM seen on IF in each patient. Patient 13 had very early MN with mild IgG on IF, only a few subepithelial deposits on electron microscopy (stage 1), and showed only a few loops with mild granular staining for FAT1. Also, FAT1 staining along the GBM was only seen after protease digestion, indicating FAT1 epitopes recognized by the commercial anti-FAT1 antibody were likely masked. FAT1 staining without protease digestion was negative in patients positive for FAT1 (bottom left panel, Figure 3A). Bowman’s capsule and mesangial staining was negative. Tubular basement membrane (TBM) staining was negative. Interestingly, the tubular epithelial cell cytoplasm was strongly positive for FAT1—the staining is likely nonspecific. Confocal microscopy studies also showed that glomeruli are positive for FAT1 along the capillary walls in two patients with HSCT-associated MN (one each from discovery cohort and validation cohort) and negative for FAT1 in two patients with PLA2R-associated MN (Figure 3B).

Figure 3.
Figure 3.

IHC and confocal IF microscopy for FAT1 in patients with HSCT-associated MN and controls. (A) IHC is positive for FAT1 in FAT1-associated MN. Twelve patients show granular capillary wall staining for FAT1 along the GBMs. Each case number of FAT-1 associated MN is shown. Note positive FAT1 staining in tubular epithelial cells. Protease treatment was required for IHC staining. One patient (patient 8) without protease treatment is shown that is negative for FAT1, and a high power is shown to highlight the FAT1 deposits. In two patients, tissue was not available for IHC. Original magnification, all 40× except 60× for bottom middle and 100× for bottom right. (B) Confocal IF microscopy analysis: Detection of FAT1 in glomerular immune deposits. Glomeruli are positive for FAT1 (red, top panel) along the capillary walls in two patients with HSCT-associated MN (one each from discovery cohort and validation cohort) and negative for FAT1 in patients with PLA2R-associated MN (40x). (C) IHC of controls shows no glomerular capillary wall FAT1 staining after protease treatment in a normal kidney, minimal change disease, PLA2R-associated MN, and a zero-time transplant biopsy. Note the positive FAT1 staining of the tubules. Original magnification, 40×.

Controls were negative for FAT1 staining along the GBM. Negative staining for FAT1 in representative patients with a normal kidney (nephrectomy specimen), minimal change disease, PLA2R-associated MN, and who underwent a zero-time kidney transplant is shown in Figure 3C. IHC of additional controls, including patients who underwent a zero-time transplant and those with proteinuric diseases such as minimal change disease, frequently-relapsing minimal change disease, secondary FSGS (obesity), primary FSGS, steroid-resistant FSGS, recurrent FSGS, and PLA2R-positive MN, are shown in the Supplemental Figure 1.

IgG Elution and Western Blot Studies

Western blot analyses were performed using recombinant human FAT1 to determine the presence of anti-FAT1 antibodies in the eluate obtained from pooled kidney biopsy specimens and serum from a recent patient with FAT1-associated MN (patient 11; Figure 4). A recombinant 33-kD human FAT1 protein corresponding to antigenic determinants (Novus Biologicals) was used in Western blot analysis. FAT1 was detected by rabbit anti-human FAT1 under both nonreducing (Figure 4A) and reducing (Figure 4B) conditions, with a dominant band at approximately 33 kD, as expected (Figure 4, A and B, lane 1). The same band was detected after exposure of the recombinant human FAT1 to IgG obtained from the eluate of FAT1-associated MN using anti-human IgG (Figure 4, A and B, lane 2). More specifically, the band was seen after exposure of recombinant human FAT1 to eluate from FAT1-associated MN using anti-human IgG4 (Figure 4A, lane 3, and Figure 4B, lane 4) but not after exposure to the eluate from PLA2R-associated MN (Figure 4A, lane 4, and Figure 4B, lanes 3 and 5). Finally, the same band was seen using serum from patient 11 using anti-human IgG4 (Figure 4B, lane 6) and anti-human IgG (Figure 4B, lane 7). Anti-FAT1 antibodies were not detected in serum from patients with PLA2R-associated-MN, PLA2R-negative MN, IgA nephropathy, and myeloperoxidase–ANCA-associated vasculitis, or in serum from a healthy control (Figure 4C). Additional controls are shown in Supplemental Figure 2.

Figure 4.
Figure 4.

Western blot analysis showing IgG from eluate and from serum of FAT1-associated MN bind to reduced FAT1 (400 ng loaded in each lane). (A) (1) Nonreduced FAT1 is detected by rabbit anti-human FAT1 (0.2 µg/ml) at approximately 33 kD (arrow). (2) Nonreduced FAT1 (arrow) is detected using eluate from FAT-1–associated MN using a secondary anti-human IgG (1:5000) and, (3) more specifically, using a secondary anti-human IgG4 (1:500). (4) The binding is not detected using eluate from PLA2R-associated MN. (B) (1) Reduced FAT1 is detected by rabbit anti-human FAT1 (0.2 µg/ml) at approximately 33 kD (arrow). (2) Reduced FAT1 (arrow) is detected using eluate from FAT-1–associated MN using a secondary anti-human IgG (1:5000) and, (3) more specifically, using a secondary anti-human IgG4 (1:500). (3 and 5) The binding is not detected using eluate from PLA2R-associated MN. (6) Reduced FAT1 was also detected using serum from a recent patient (1:100 and 1:50) and detected using anti-human IgG4 (1:500) and (7) using anti-human IgG (1:5000). (C) (1) Reduced FAT1 is detected by anti-human IgG4 (1:500) in serum from a patient with FAT1-associated MN (patient 11; 1:100 dilution), but not in serum from patients with (2) PLA2R-associated MN, (3) PLA2R-negative MN, (4) IgA nephropathy, (5) myeloperoxidase–ANCA-associated vasculitis (MPO-AAV), or (6) in serum from a healthy control. hu, human; IgAN, IgA nephropathy; neg, negative.

Clinical and Kidney Biopsy Findings of FAT1-Associated MN

The mean±SD age of those with FAT1-associated MN was 60.3±8.6 years (Table 1). MN occurred 2.4±0.8 years after HSCT. There were nine men and five women. The mean±SD serum creatinine and proteinuria at kidney biopsy was 1.4±0.5 mg/dl and 7.8±6.0 g/d, respectively. HSCT was done for the treatment of eight patients with acute myelogenous leukemia, two patients with myelodysplastic syndrome, and one patient each with chronic lymphocytic leukemia, essential thrombocytopenia, myelofibrosis, and lymphoplasmacytic disease. Kidney biopsy findings showed granular IgG (2 to 3+), κ light chains (1 to 3+), and λ light chains (1–3+) in all cases (Table 2). Subtyping of IgG performed for six patients with available tissue showed dominant IgG4 (3+) for all patients; one patient also had IgG2 (2+) present. There was only mild C3 present (0 to 1+). Other Igs and C1q were absent. Interestingly, three patients also showed TBM deposits that were positive for IgG. Two of these three cases were recently reported.19 Mass spectrometry of the tubular deposits was negative for FAT1. Chronic changes in 12 of the 14 patients were mild, including focal global glomerulosclerosis (median, 10%) and tubular atrophy and interstitial fibrosis (median, 10%–20%).20 In two of the ten patients there was extensive (>50%) tubular atrophy and interstitial fibrosis present. A representative case with kidney biopsy findings (patient 1) is shown in Figure 5. Treatment details were available for the five Mayo Clinic patients. Patient 2 was treated with prednisone and rituximab and achieved partial remission. She died 3 months after diagnosis of MN due to congestive heart failure. Patient 3 was treated with rituximab and achieved complete remission. Patient 12 achieved complete remission with prednisone and cyclosporine, but died 13 years later of probable sepsis. Patient 13 achieved complete remission with prednisone. Patient 14 achieved partial remission with prednisone and cyclosporine, but died 4 months later from sepsis. Overall, follow-up of the 11 patients showed complete remission in six patients (proteinuria of <0.3 g per 24 hours), three patients continued to have significant proteinuria (nephrotic range in one patient), one patient was a recent case, and six patients died (Tables 1 and 2). Data available in four of the six patients that died showed the cause of death as congestive heart failure in one patient and sepsis as the cause of death in three of the patients.

View this table:
Table 1.

Clinical findings in FAT1-associated MN

View this table:
Table 2.

Pathologic findings in FAT1-associated MN

Figure 5.
Figure 5.

Biopsy finding of a representative patient (patient 1) with FAT1-associated MN. (A) Light microscopy showing focally thickened GBM (Periodic acid–Schiff stain), (B and C) IF microscopy showing bright 3+ capillary wall staining for (B) IgG and (C) IgG4 along the capillary walls (20x), (D) electron microscopy showing subepithelial electron dense deposits (7140×), and (E and F) granular capillary wall staining for FAT1. Original magnification, 60× in (A), 40x in (B), 20x in (C), 7140× in (D), 40× in (E), and 100× in (F).

Discussion

The understanding of MN has undergone impressive advancements in the last 5 years, particularly due to identification of novel antigenic targets. In the first step, laser microdissection of glomeruli followed by mass spectrometry of the digested glomerular proteins has helped identify novel proteins.21 This is followed by IHC/IF to localize the novel proteins along the GBM in a granular membranous pattern. Lastly, to determine whether the protein might truly be an antigenic target, Western blot analyses of serum/frozen tissue IgG eluate demonstrates binding of extracted IgG to the newly identified protein. Using this framework, we have been successful in identifying novel/putative antigens, including EXT1/EXT2, NELL1, SEMA3B, and PCDH7, although antibodies to EXT1/EXT2 have not yet been detected.7⇓⇓–10 On reviewing the clinical data associated with each of the new antigens, we found distinguishing clinicopathologic findings for each antigen. Thus, EXT1/EXT2 was detected in MN associated with autoimmune diseases, such as lupus; NELL1 was mostly present in primary MN, but a smaller subset was associated with malignancy; SEMA3B was unique in that it was present predominantly in MN in children; and PCDH7 was seen in MN in older patients and showed minimal complement activation.

We have now identified a novel protein, FAT1, as the likely target antigen in another specific subset of MN. This subset of FAT1-associated MN develops after HSCT. HSCT is the treatment of choice for some patients with hematologic malignancies and other immune disorders. One of the complications of allogeneic HSCT is GVHD. GVHD often involves the skin, gastrointestinal system, lungs, and the kidney. MN is the most common glomerular disease in patients with allogeneic HSCT and is considered a manifestation of chronic GVHD.22⇓⇓⇓⇓–27 MN develops after a mean of 22 months post-transplant.22 In most cases, the target antigen in HSCT-associated MN is not known, although PLA2R- and NELL1-associated MN have been reported in a small number of patients.15,19,28

Using laser microdissection and MS/MS of PLA2R-negative MN, we identified the unique protein FAT1 in nine patients. Moderate FAT1 total spectral counts were present in all patients. The protein appeared unique in that it was not present in either patients with PLA2R-postive MN or in the remaining patients with PLA2R-negative MN. Clinical data then revealed that MN developed after allogeneic HSCT in all nine patients in which FAT1 was identified. Furthermore, FAT1 was not identified in any patient with nontransplant-associated MN. To confirm that FAT1 was present in HSCT-associated MN, validation studies were performed on five additional patients from a second institution (Mark Haas/Cedars-Sinai Medical Center). MS/MS in all five patients showed moderate total spectral counts of FAT1. The spectral counts of FAT1 are higher than those seen in PCDH7-, comparable with SEMA3B-, but lower than spectral counts seen in EXT1/EXT2- and NELL1-associated MN. Like PCDH7, FAT1 is a large, heavily glycosylated cadherin and, during the preparation of tissue for MS/MS, the heavy glycosylation can interfere with trypsin’s access and inhibit its binding and cleavage of the arginine and lysine residues in the glycosylated region, which likely accounts for the lower spectral counts. In fact, FAT1 is the largest protein identified to be associated with MN. The heavy glycosylation may also explain why IHC without protease digestion of the paraffin-embedded material was inconclusive. On the other hand, IHC after protease digestion showed bright anti-FAT1 granular staining along the GBMs mirroring the GBM deposits. The IHC staining was sharper in paraffin-embedded tissue that was fixed in Bouin fixative compared with tissue fixed in formalin. Interestingly, the proximal tubular epithelial cells stained for FAT1, although there was no staining along the Bowman’s capsule or TBM. Three patients also had TBM deposits on the kidney biopsy specimens, but the TBM was negative for FAT1 on IHC and mass spectrometry studies (data not shown). This finding appears similar to SEMA3B-associated MN where TBM deposits may be present, but do not stain for SEMA3B on IHC. Finally, pooled IgG eluates from frozen biopsy tissue from FAT1-associated MN also showed binding to both nonreduced and reduced recombinant FAT1. Most importantly, serum from a patient with a recent diagnosis of FAT1-associated MN also showed binding to recombinant FAT1. On the other hand, under similar conditions, IgG eluate and serum from PLA2R-positve MN showed no binding to recombinant FAT1. Lastly, typing for IgG subclasses performed for six patients showed dominant IgG4, which was also confirmed by Western blot analysis of both the eluate of pooled frozen tissue of FAT1-associated MN and in the serum of the recent patient with FAT1-associated MN (Figure 4). Taken together, these findings confirm the presence of anti-FAT1 IgG in the kidney biopsy specimens and serum of those with FAT1-associated MN.

Patient 11 is a recent and interesting patient because the suspicion of FAT1-associated MN was initially made on detection of anti-FAT1 antibodies. We received the serum from this patient to test for PLA2R and THSD7A, which were negative. On learning that the patient had developed MN after HSCT, we performed Western blot analysis and determined that the patient had anti-FAT1 antibodies. Subsequently, we obtained the biopsy material and performed both MS/MS and IHC to confirm the diagnosis of FAT1-associated MN. Thus, the diagnosis was first made on the finding of anti-FAT1 antibodies in the serum, suggesting serum testing for anti-FAT1 antibodies in patients with HSCT may be diagnostic for FAT1-associated MN, similar to PLA2R antibodies in cases of PLA2R-associated MN.29⇓–31 These findings needs to be confirmed in a larger cohort of FAT1-associated MN.

In our series of FAT-1 associated MN, the clinicopathologic findings of all 14 patients were consistent with known findings of HSCT-associated MN. Kidney biopsy tissues showed bright IgG with mild or no C3 deposition. This is in keeping with the PCHD7, another cadherin, that also showed only minimal C3 deposition. Subtyping of IgG performed for seven patients showed IgG4 (Figure 5, Table 2). The lack of C3 on the kidney biopsy specimen is in keeping with lower potential of complement activation by IgG4 compared with other IgG subclasses.32⇓–34

Our data also show that FAT1 is the most common antigen detected in HSCT-associated MN. In the Mayo Clinic discovery cohort and Cedars Sinai validation cohort, FAT1 was identified in 83.3% and 100% of patients with HSCT-associated MN, respectively. Although both PLA2R and NELL1 have been reported in HSCT-associated MN, these are rare and, in our study, we detected only one patient with PLA2R-positive MN after HSCT.

Cadherins are a large group of transmembrane proteins on the cell surface that mediate calcium-dependent cell-cell recognition and adhesion.35 The cadherins are characterized by the presence of extracellular cadherin domains composed of multiple repeats of the cadherin-specific motifs.36 Thus, cadherins are further classified into subfamilies on the basis of the extracellular domain and include the classic cadherins, desmosomal cadherins, protocadherins (including PCDH7), and the Drosophila fat protein and its mammalian homolog FAT.37 The FAT cadherins comprise four members, FAT1–FAT4, and all are large transmembrane proteins of 500–600 kD.38 The biologic function of FAT cadherins is not well understood. FAT1 is characterized by its 34 cadherin repeats in the extracellular domain, two to six endothelial growth factor repeats, and one laminin G domain. In situ hybridization studies showed widespread expression of FAT in many rat fetal tissues, including the central nervous system and kidney. In contrast to fetal tissue, FAT expression is minimal or disappears in most tissues in the adult rat. However, FAT is still expressed in the kidney, in particular in podocytes, and was first identified along the rat glomerular capillary wall as a component of the glomerular slit diaphragm.39 Subsequently, it was shown that FAT1 is located at sites of cell-cell contact in podocytes and localizes to the podocyte intercellular junctions.40 Furthermore, transgenic mice lacking FAT1 die perinatally within 48 hours with loss of glomerular slit junction and massive effacement of foot processes.41 More recently, FAT1 recessive mutations in humans have been associated with glomerulotubular nephropathy exhibiting extensive podocyte foot process effacement and resulting in steroid-resistant nephrotic syndrome.42,43 Although these studies show that both FAT1-deficient mice and mutations in FAT1 in humans results in podocyte injury, to the best of our knowledge, an alloimmune response to FAT1 has not been reported in any kidney disease. Taken together with our current understanding of podocyte localization of FAT1 and foot process effacement in patients with FAT1 mutation, we can hypothesize that severe podocyte injury would also likely result in the context of autoantibodies to FAT1. Clearly, further studies are required to determine the cause of the alloimmune response to FAT1 and subsequent development of FAT1-associated MN in patients with HSCT-associated MN. Somatic mutations in FAT1 have been described in normal tissues, such as skin, esophagus, and bronchus,44 and it is intriguing to hypothesize that a somatic/previously hidden FAT1 mutation may cause an immune response as a consequence of GVHD. Further studies are also required to confirm this finding and determine why chronic GVHD results in the development of antibodies to FAT1.

We would again like to point out that the detection of FAT1 in the very specific scenario of HSCT-associated MN argues for classification of MN on the basis of the antigen and antibody detected.6,45 To conclude, our studies show that FAT1 is a novel protein that likely represents the antigenic target in most cases of allogeneic HSCT-associated MN.

Disclosures

F.C. Fervenza reports having consultancy agreements with Alexion Pharmaceuticals, Alnylam, ByoCrystal, Novartis, and Takeda; receiving research funding from Chemocentryx, Genentech, Janssen Pharmaceutical, Questcor/Mallinckrodt, and Retrophin; serving in an advisory or leadership role for JASN, Kidney International, Nephrology, Nephrology Dialysis and Transplantation, and UpToDate; and receiving honoraria from UpToDate. M. Haas reports having consultancy agreements with Argenx, AstraZeneca, CareDx, Novartis, Retrophin, and Shire Viropharma Inc.; serving in an advisory or leadership role for Argenx, CareDx, Novartis, and Retrophin; and receiving honoraria from, and serving on a speakers bureau for, CareDx. N. Leung reports having consultancy agreements with AbbVie, Lilly, and Omeros; receiving research funding from Alnylam and Omeros; having other interests in, or relationships with, Amyloidosis Research Consortium; having ownership interest in Checkpoint Therapeutics; and serving in an advisory or leadership role for Journal of Nephrology. S. Sethi reports receiving honoraria for teaching, conducting grand rounds, giving lectures, reviewing slides for a study for Novartis, and from UpToDate. U. Specks reports having ownership interest in AbbVie, Johnson & Johnson, Pfizer, and Viatris; receiving research funding from AstraZeneca, Boehringer Ingelheim, Bristol Myer Squibb, Genentech, and GlaxoSmithKline; having consultancy agreements with AstraZeneca and ChemoCentryx; and having patents with, or receiving royalties from, Mayo Foundation for Education and Research and UpToDate. All remaining authors have nothing to disclose.

Funding

None.

Acknowledgments

We would like to thank Megan Maxwell and Jamie Altamirano-Alonso in the Renal Pathology Laboratory for help with histology. We would like to thank Amber Hummel in Dr. Ulrich Specks laboratory, and Virginia Van Keulen in the Immunology Department. We would like to thank the Mayo Clinic Genome Facility Proteomics Core (a shared resource of the Mayo Clinic Cancer Center [NCI P30 CA15083], Department of Laboratory Medicine and Pathology, and the Pathology Research Core, Mayo Clinic).

Author Contributions

M.P. Alexander, N. Klomjit, N. Leung, S.H. Nasr, and S. Sethi were responsible for data curation; M. Casal Moura, M.C. Charlesworth, L. Gross, M. Haas, B. Madden, V. Negron, S. Sethi, and U. Specks were responsible for methodology; M. Casal Moura, F.C. Fervenza, and S. Sethi were responsible for formal analysis; F.C. Fervenza, M. Haas, S. Sethi, and U. Specks reviewed and edited the manuscript and were responsible for resources; F.C. Fervenza and S. Sethi conceptualized the study and were responsible for funding acquisition; M. Haas, B. Madden, and S. Sethi were responsible for investigation; M. Haas and S. Sethi were responsible for validation; S. Sethi wrote the original draft and was responsible for visualization and project administration; and S. Sethi and U. Specks provided supervision.

Supplemental Material

This article contains the following supplemental material online at http://jasn.asnjournals.org/lookup/suppl/doi:10.1681/ASN.2021111488/-/DCSupplemental.

Supplemental Figure 1. Immunohistochemistry.

Supplemental Figure 2. Western blot.

Footnotes

  • Published online ahead of print. Publication date available at www.jasn.org.

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Hematopoietic Stem Cell Transplant-Membranous Nephropathy Is Associated with Protocadherin FAT1
Sanjeev Sethi, Benjamin Madden, Marta Casal Moura, Samih H. Nasr, Nattawat Klomjit, LouAnn Gross, Vivian Negron, M. Cristine Charlesworth, Mariam P. Alexander, Nelson Leung, Ulrich Specks, Fernando C. Fervenza, Mark Haas
JASN May 2022, 33 (5) 1033-1044; DOI: 10.1681/ASN.2021111488
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