PMC9207246_01
Histology of the pre- and post-implantation biopsies. (A) PAS staining showing no pre-existent damage apart from <5% atrophy (arrow) seen as broadened tubular basement membranes, fibrosis, and smaller tubuli in the preimplantation biopsy. (B) Jones' methenamine silver staining showing a normal glomerulus in the preimplantation biopsy. (C) Hematoxylin and eosin staining showing hemorrhagic areas outside the vascular bed (black arrow) and inflammatory cells with destruction of endothelial cells (open arrow) in the posttransplantation biopsy. (D) Jones' methenamine silver staining showing slightly expanded glomerular capillaries with abundant inflammatory cells (open arrows) and somewhat pronounced endothelial cells (black arrow) in the posttransplantation biopsy.
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No relevant pre-existing damage was found in the routine preimplantation biopsy of the kidney (Figures 2A, B), and no surgical complications occurred (cold ischemia time, 142 min; warm ischemia time, 33 min) and renal perfusion, as determined by Doppler ultrasound, was excellent with immediate diuresis. There was extensive peritubular hemorrhage (Figures 2C, D).
Immunohistochemistry of the hyperacutely rejected allograft. (A) Staining for C4d showing no presence of C4d in the peritubular capillaries. The inset shows a positive control with acute humoral rejection. (B) C3d staining showing variable staining along the peritubular capillaries (arrows) and hilus of the glomerulus (star).
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Immunoglobulin G (IgG) and immunoglobulin M (IgM) staining by immunofluorescence was barely seen (data not shown), and the C4d staining was negative (Figure 3A). C3d staining was most pronounced in the peritubular capillaries and the glomerular hilus (Figure 3B).
CD34 and ERG stainings show loss of endothelial cells. (A) CD34 staining in the peritubular capillaries of the patient's biopsy is diminished when compared to control tissue (posttransplant biopsy without signs of rejection). (B) ERG staining also shows a loss of endothelial cell nuclei in the peritubular capillaries.
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Additional staining for endothelial cell-specific markers CD34 and ERG showed less CD34- and ERG-positive cells compared to a posttransplantation biopsy without rejection (Figures 4A,B).
Complement-dependent endothelial cytotoxicity using machine perfusion-derived primary renal endothelial cells (MP-PRECs) and pre-transplant patient serum. A representative experiment of a complement-dependent endothelial cytotoxicity crossmatch assay (CDC) using MP-PRECs (HLA typing A1, A2, B7, B8, Bw6, Cw7, DR15, DR11, DR51, DR52, DQ6, DQ7, and bloodgroup A, incubated with blood group-compatible serum, a monoclonal antibody directed against A2 IgM, and the AECA-positive patient serum. Rabbit complement was used as the complement source. Living cells are depicted in green and apoptotic cells in red.
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Crossmatching with serum taken 1 day prior to transplantation resulted in clear complement-mediated cell death; 72% of all cells appeared to be apoptotic (Figure 5).
Complement-dependent endothelial cytotoxicity and flow crossmatch assays using machine perfusion-derived primary renal endothelial cells (MP-PRECs) and post-transplant patient serum. (A) A representative experiment of a complement-dependent endothelial cytotoxicity crossmatch assay (CDC) using MP-PRECs (HLA typing A2, B62, B60, Bw6, Bw6, Cw10, DR1, DR4, DR53, DQ7, DQ5, and bloodgroup A, incubated with blood group-compatible serum, blood group- and HLA-incompatible serum (anti-A2 serum), a monoclonal antibody directed against A2 IgG, a monoclonal antibody directed against A2 IgM, and the AECA-positive patient serum. Rabbit complement was used as the complement source. Living cells are depicted in green and apoptotic cells in red. (B) Quantification of n = 3 experiments of apoptotic (red) cells. p-values are depicted in the figure. (C) Flow cytometry showing IgG, IgM, and activated C3 binding in percentages after incubation of MP-PRECs with blood group- and HLA-compatible serum, blood group- and HLA-incompatible serum (anti-A2 serum), a monoclonal antibody directed against A2 IgG, a monoclonal antibody directed against A2 IgM, and the patients' serum having AECA. A total of 20% human serum was used as the complement source. MP-PRECs that were propidium iodide negative were included in the analysis, indicating living cells. p-values are depicted in the figure. Experiments with a positive result for IgG, IgM, and activated C3 binding after incubation with anti-A2 serum were included in the analysis. Data are normalized to the negative control (blood group- and HLA-compatible serum) and are presented on a logarithmic scale. p-values depict results using the unpaired t-test. Dots represent individual values.
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When crossmatching MP-PRECs from 3 different donors with serum taken on POD 3, extensive complement-mediated cell death occurred (p = 0.002 compared to blood group- and HLA-compatible serum) (Figure 6). In comparison, control serum from patients with HLA class I directed against HLA antigens expressed on the MP-PRECs caused less complement-mediated cell death (p = 0.05) when compared to the patient's serum (Figure 6). Crossmatching with the patient's serum resulted in IgG and IgM binding and activated C3 to varying extents (Figure 6C).
Serum samples collected before and after plasmapheresis show a reduction in the binding of IgG, IgM, activated C3, C4d, and C5b-9 to MP-PRECs during the course of 4 cycles of plasmapheresis. Serum samples were collected right before every plasmapheresis (1, 2, 3, and 4) at the time points (T) before (0) and after (1) every plasmapheresis. (A) Binding of IgG to MP-PRECs after incubation with the patient's serum collected during the course of plasmapheresis. (B) Binding of IgM to MP-PRECs after incubation with the patient's serum collected during the course of plasmapheresis. (C) Binding of activated C3 to MP-PRECs after incubation with the patient's serum collected during the course of plasmapheresis and 20% NHS as a complement source. (D) Binding of C4d to MP-PRECs after incubation with the patient's serum collected during the course of plasmapheresis and 20% NHS as a complement source. (E) Binding of C5b-9 to MP-PRECs after incubation with the patients' serum collected during the course of plasmapheresis and 20% NHS as a complement source.
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When the patient serum obtained during desensitization treatment was analyzed for the presence of AECA utilizing MP-PRECs, a time-dependent decrease in IgG, IgM, C3, C4d, and C5b-9 was observed (Figure 7), suggesting an effective removal of non-HLA AECA.