Project description:Recurrence of focal segmental glomerulosclerosis (rFSGS) after kidney transplantation is a cause of early and accelerated graft loss. Immuneadsorption can alleviate renal dysfunction and suggests that circulating antibodies (Ab) are likely implicated in disease pathogenesis. To evaluate pathogenic Ab in rFSGS, we processed 141 unique serum samples from patients with and without primary rFSGS (n=64) and 34 non-FSGS control, transplanted at five (US and EU) hospitals. 9000 antigens were screened in pre-transplant sera by protein arrays and 10 Ab targeting glomerular antigens were selected for ELISA validation. A panel of 7 Ab (CD40, PTPRO, CGB-5, FAS, P2RY11, SNRPB2 and APOL2) could predict post-transplant FSGS recurrence with 92% accuracy. Pre-transplant elevation of anti-CD40 Ab levels alone had a substantial impact (78% accuracy) on the identification of rFSGS risk after transplantation. Epitope mapping of CD40 with customized peptide arrays and rFSGS sera demonstrated altered immunogenicity of the extracellular CD40 domain in rFSGS. Immunohistochemistry of CD40 demonstrated a differential expression of these antigens in FSGS compared to non-FSGS. Anti-CD40 Ab purified from rFSGS patients were uniquely pathogenic in human podocyte cultures; injection of these Ab resulted in heightened proteinuria, independently and in combination with suPAR in a rodent model, abrogated by injection of monoclonal Ab to CD40. In conclusion, a panel of 7 Ab can identify primary FSGS patients at high risk of recurrence prior to transplantation, allowing for customized therapies and improved patient selection for transplant. Intra-renal CD40 is an important axis of disease pathogenesis, and human trials of anti-CD40 therapies are warranted to evaluate their efficacy in preventing rFSGS and improving graft survival. The purpose of the study was to identify potential auto-Abs associated with rFSGS. We used a discovery set of pre-transplant sera from 20 unique patients with biopsy confirmed diagnosis of primary FSGS as their cause of ESRD, of which 10 had progressed to rFSGS within the first post-transplant year and 10 did not have recurrence of proteinuria or histological disease after transplantation (nrFSGS).

Project description:Background: Studies recently support that non-HLA antigens could be additional targets of injury in organ transplant recipients, and MICA was associated with an increased risk of graft loss. Methods: A ProtoArray platform was used to study 37 serum samples from 22 unique patients (15 renal recipients and 7 healthy controls). Thirty paired pre- and post-transplant serum samples were analyzed for detection of de novo post-transplant antibody responses in the 15 patients (10 acute rejection (AR), 5 Stable). Probes on ProtoArray and cDNA platforms (GSE: 3931) were re-annotated and compartment specific gene lists were analyzed using the integrated genomics method. Normal and transplant kidney IHC were performed for MICA antigen localization. Results: Mean MICA-Ab (antibody) signal intensity was significantly higher in transplant patients compared with healthy controls and de novo MICA-Ab were detected in 73% transplant patients. The mean post-transplant signal intensity of MICA-Ab was the highest in C4d+AR. Detection of MICA-Ab responses did not correlate with time post-transplantation, but significantly correlated with decline in graft function over the subsequent year. Integrative genomics predicted localization of the MICA antigen to the glomerulus. IHC confirmed cytoplasmic MICA staining solely in glomerular podocytes in normal kidney. In the transplant kidney, infiltrating mononuclear lymphocytes (T, B and NK) in AR had additional MICA staining. Conclusions: MICA can be highly detected regardless of graft dysfunction or AR. The intensity signal of the MICA antibody correlates with subsequent decline in graft function. The MICA antigen localizes to the glomerulus and infiltrating mononuclear cells in AR. Pre- and post-transplant serum antibodies were profiled for each patient, using the Invitrogen ProtoArrayÂ® Human Protein Microarray v3.0 platform (Invitrogen, Carlsbad, CA). This platform contains 5,056 non-redundant human proteins expressed in a baculovirus system, purified from insect cells and printed in duplicate onto a nitrocellulose-coated glass slide. Each protein is spotted twice on each array, to measure the quality of the signal intensity. Details for experiment processing and analysis follow the previous publication from our group (13). Prospector software was used to retrieve the expression based on immune response profiling of the .gal files.

Project description:C3 glomerulopathy (C3G), which encompasses C3 glomerulonephritis (C3GN) and dense deposit disease (DDD), results from dysregulation of the alternative complement pathway. Data on disease recurrence after kidney transplantation is limited, and details on histologic features of recurrent C3G are scarce. We aimed to evaluate C3G recurrence in the allograft, with a focus on histologic presentation and progression. We retrospectively analyzed 18 patients with native kidney failure attributed to C3G (12 C3GN and 6 DDD) who received a kidney transplant from January 2016 to January 2023. Demographic, genetic, clinical, and histologic data were studied. The Nanostring 770 genes immune profiling panel was used for transcriptomic analysis. Disease recurrence was the primary outcome. During a median follow-up period of 37 (18, 56) months, C3G recurrence occurred in 16 (89%) of patients (11 with C3GN and 5 with DDD), at a median of 33 (13, 141) days post-transplantation. Over a third (38%) of recurrent cases were detected in protocol biopsies, and only 31% of patients presented with >300 mg/g of proteinuria. Recurrence in index biopsies was mainly established through a combination of immunofluorescence and electron microscopy findings, while it showed only subtle histologic alterations and no characteristic transcriptomic signals. Over time, histologic chronicity indices increased, but all allografts were functioning at the end of follow-up. Patients with recurrence of C3GN and DDD showed overlapping immunofluorescence and electron microscopy findings and had similar recurrence rate and time to recurrence. The majority of patients with native kidney failure attributed to C3G developed disease recurrence very early after kidney transplantation, usually with minimal proteinuria, mild histologic alterations, and favorable short-term allograft survival. Immunofluorescence and electron microscopy played crucial role in detecting early, sub-clinical recurrence of C3GN and DDD, which showed significant overlapping features. To identify transcriptomic signals that may distinguish recurrent C3G, we utilized the 770 genes PanCancer Immune Profiling Panel in a discovery cohort of native kidney biopsies followed by a testing cohort of allograft biopsies. For the discovery cohort, we residual tissue from formalin-fixed paraffin-embedded samples (FFPE) was used from 18 native kidney biopsies with C3G (11 C3GN and 7 DDD) and 36 native biopsies with other glomerular diseases (GD controls: 15 IgA nephropathy, 14 membranous nephropathy, 7 diffuse podocytopathy). For the testing cohort, we ass essedresidual FFPE tissue was used from 13 index allograft biopsies with recurrent C3G (combining 10 from our study with enough residual tissue and 3 additional cases that were added to increase sample size) and 41 allograft glomerular disease (GD) controls. Since recurrent GD is overall more commonly encountered than de novo GD although the frequency of latter might be underestimated20, the GD controls were selected to roughly reflect this notion [15 de novo GD (5 membranous nephropathy, 6 immune complex-mediated glomerulonephritis not otherwise specified, 3 IgA nephropathy, 1 HCV-associated glomerulonephritis) and 26 recurrent GD (10 membranous nephropathy, 8 IgA nephropathy, 7 diffuse podocytopathy, 1 HCV-associated glomerulonephritis)].

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