Post-Transplant Thrombotic Microangiopathy due to a Pathogenic Mutation in Complement Factor I in a Patient With Membranous Nephropathy: Case Report and Review of Literature

Abstract

Thrombotic microangiopathy (TMA) is characterized by microangiopathic hemolytic anemia, thrombocytopenia and organ injury occurring due to endothelial cell damage and microthrombi formation in small vessels. TMA is primary when a genetic or acquired defect is identified, as in atypical hemolytic uremic syndrome (aHUS) or secondary when occurring in the context of another disease process such as infection, autoimmune disease, malignancy or drugs. Differentiating between a primary complement-mediated process and one triggered by secondary factors is critical to initiate timely treatment but can be challenging for clinicians, especially after a kidney transplant due to presence of multiple confounding factors. Similarly, primary membranous nephropathy is an immune-mediated glomerular disease associated with circulating autoantibodies (directed against the M-type phospholipase A2 receptor (PLA2R) in 70% cases) while secondary membranous nephropathy is associated with infections, drugs, cancer, or other autoimmune diseases. Complement activation has also been proposed as a possible mechanism in the etiopathogenesis of primary membranous nephropathy; however, despite complement being a potentially common link, aHUS and primary membranous nephropathy have not been reported together. Herein we describe a case of aHUS due to a pathogenic mutation in complement factor I that developed after a kidney transplant in a patient with an underlying diagnosis of PLA2R antibody associated-membranous nephropathy. We highlight how a systematic and comprehensive analysis helped to define the etiology of aHUS, establish mechanism of disease, and facilitated timely treatment with eculizumab that led to recovery of his kidney function. Nonetheless, ongoing anti-complement therapy did not prevent recurrence of membranous nephropathy in the allograft. To our knowledge, this is the first report of a patient with primary membranous nephropathy and aHUS after a kidney transplant.

Keywords: atypical hemolytic uremic syndrome; complement factor I; complement functional analysis; kidney transplantation; membranous nephropathy; thrombotic microangiopathy.

Figure 1

Timeline of hospital course of patient after a living unrelated kidney transplantation. C3, complement 3; HD, hemodialysis; LDH, lactate dehydrogenase; OR, operative room; thymo, thymoglobulin; MPA, mycophenolic acid.

Figure 2

Biopsy image from patient. Fibrin thrombi seen in glomeruli (black arrow). Small arteries and arterioles demonstrated focal fibrinoid necrosis of the arterial wall (not shown).

Figure 3

Functional evaluation of Factor I (FI) variant Ile357Met: proteolytic activity. The fluid-phase C3b proteolytic activity of the variant factor I (357Met) with its cofactor proteins (Factor H [FH], membrane cofactor protein [MCP], or complement receptor 1 [CR1]) was assessed by cleavage of purified C3b to iC3b and compared to wild type (WT). For these assays, purified WT and FI variant proteins were diluted in physiologic salt (150 mM NaCl) buffer with C3b (10 ng; Complement Technologies, Inc, Tyler, TX USA) at 37°C. Concentrations of WT or variant FI used with the individual cofactors were 10 ng with MCP, 20 ng with FH and 15 ng with CR1. Concentration of cofactor used in the reactions were 100 ng MCP, 200 ng FH or 150 ng CR1. Reactions were carried out in a total volume of 15 µl/reaction at 37°C. Kinetic analysis of the WT and variants was achieved through collection of sample at 0, 10, 20 and 30 min. At each time point 7 µl of 3x Laemmli reducing sample buffer was added to individual reactions to stop the reaction and then heated at 95°C for 5 min. The samples were electrophoresed on 10% Tris-glycine gel and then transferred to nitrocellulose for WB analysis. Membranes were rinsed with TBS-T (0.05%Tween-20) for 5 min and blocked overnight with 5% nonfat dry milk in PBS. Blots were probed with a 1:5,000 dilution of goat anti-human C3 (Complement Technologies, Inc, Tyler, TX, USA) followed by HRP-conjugated rabbit anti-goat IgG and developed with SuperSignal substrate (Thermo Fisher Scientific, Waltham, MA, USA). The signal detected on radiographic films was scanned using a laser densitometer (Pharmacia LKB Biotechnology, Piscataway, NJ, USA). Multiple exposures were used to establish linearity. (A, B). The percentage of α’ chain remaining and generation of α41 fragment indicates cleavage of C3b to iC3b. Cleavage rate was measured by densitometric analysis of the α’ chain remaining as well as generation of α41 relative to the β chain. Data represent 2 separate experiments with bars corresponding to the standard error of mean (SEM). Upon comparison to WT FI, the proteolytic activity of variant 357Met was defective with FH. The P value for the difference in the percentage of α’chain remaining between WT and variant was 0.05 and for the difference in the percentage of α41 generation was <0.05. (C). Representative WB demonstrating cofactor activity of Factor H with the variant (357Met) compared with wild type FI as well as purified FI (D) No defect was observed with MCP or CR1 as the cofactor protein. I, isoleucine; M, methionine. Structural evaluation of Ile357Met (E) Mapping of Ile357 on the structure of FI shows that it is located in the serine protease domain of FI which harbors the catalytic activity. Although the variant is away from the catalytic serine (S525)), the substitution of I to a bulkier amino acid M likely alters the position of the disulfide bond 365-381, thus affecting the FI activity. (F) Mapping of the Ile357 on the triple complex of Factor H and C3b shows that the variant is away from the binding surface of FH, therefore we speculate that it likely leads to a conformational change resulting in low functional activity.