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

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.

Keywords: hematopoietic stem cell transplantation; immunology and pathology; kidney biopsy; membranous nephropathy; nephrotic syndrome.

Graphical abstract

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.

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.

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×.

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.

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).