Weak Expression of Terminal Complement in Active Antibody-Mediated Rejection of the Kidney

Abstract

Background: The role of the complement system in antibody-mediated rejection (ABMR) is insufficiently understood. We aimed to investigate the role of local and systemic complement activation in active (aABMR). We quantified complement activation markers, C3, C3d, and C5b-9 in plasma of aABMR, and acute T-cell mediated rejection (aTCMR), and non-rejection kidney transplant recipients. Intra-renal complement markers were analyzed as C4d, C3d, C5b-9, and CD59 deposition. We examined in vitro complement activation and CD59 expression on renal endothelial cells upon incubation with human leukocyte antigen antibodies.

Methods: We included 50 kidney transplant recipients, who we histopathologically classified as aABMR (n=17), aTCMR (n=18), and non-rejection patients (n=15).

Results: Complement activation in plasma did not differ across groups. C3d and C4d deposition were discriminative for aABMR diagnosis. Particularly, C3d deposition was stronger in glomerular (P<0,01), and peritubular capillaries (P<0,05) comparing aABMR to aTCMR rejection and non-rejection biopsies. In contrast to C3d, C5b-9 was only mildly expressed across all groups. For C5b-9, no significant difference between aABMR and non-rejection biopsies regarding peritubular and glomerular C5b-9 deposition was evident. We replicated these findings in vitro using renal endothelial cells and found complement pathway activation with C4d and C3d, but without terminal C5b-9 deposition. Complement regulator CD59 was variably present in biopsies and constitutively expressed on renal endothelial cells in vitro.

Conclusion: Our results indicate that terminal complement might only play a minor role in late aABMR, possibly indicating the need to re-evaluate the applicability of terminal complement inhibitors as treatment for aABMR.

Keywords: C5b-9; antibody-mediated rejection; complement system; kidney transplantation; membrane-attack complex.

Figure 1

Patient Selection with Inclusion and Exclusion Criteria. Flow chart representing selection of patients based on exclusion criteria. Total amount of patients excluded and included are depicted as (n). Exclusion of patients is presented in box with dotted lines. KTR, Kidney Transplant Recipients; UMCG, University Medical Center; yr, years; non-KTR, patients without a renal transplant; aABMR, active antibody-mediated rejection; iABO, blood group incompatible; aTCMR, acute T-cell mediated rejection; NR, non-rejection. a) with blood samples directly placed on ice.

Figure 2

Complement Levels in Plasma of aABMR, aTCMR, and NR patients. Plasma levels were measured for C3 (A), C3d (B), and C5b-9 (D). Ratios were calculated from respective C3 and C3d levels (C), indicating systemic complement consumption. Circles represent individual patients with open circles indicating C4d-positive patients and filled circles symbolizing C4d-negative patients. P values depict results from Mann-Whitney U statistical analysis. Statistical significance is defined as P < 0.05. aABMR, active antibody-mediated Rejection; aTCMR, acute T-cell mediated rejection; NR, non-rejection; ns, not significant.

Figure 3

Semiquantitative scores for C3d and C5b-9 deposition in renal biopsy specimens of aABMR, aTCMR, and NR patients. Semiquantitative scores range from 0 to 4, with higher scores indicating increased positivity in biopsy. Groups are compared regarding glomerular C3d deposition (A), peritubular capillary C3d (B), glomerular C5b-9 (C), and peritubular capillary C5b-9 (D) deposition. Data represent staining results of individual patients. Error bars represent median with interquartile range. Unfilled circles symbolize biopsies of C4d-positive patients, filled circles C4d-negative patients. P-values are derived from Mann-Whitney U tests with statistical significance defined as *P < 0.05, **P < 0.001; aABMR, active antibody-mediated rejection; aTCMR, acute T-cell mediated rejection; NR, non-rejection; ns, not significant.

Figure 4

Renal biopsy staining for complement factors in a KTR diagnosed with C4d-positive, aABMR. Selected section of biopsy slide stained for C3d in glomeruli (A) and in peritubular capillaries (B). Selected section of biopsy slide stained for C5b-9 in glomeruli (C) and in peritubular capillaries (D). White arrows indicate glomeruli and black arrows indicate peritubular capillaries. Double-compound arrows indicate C5b-9 negative peritubular capillaries (D). Semi-quantitative scores (0-4) are indicated in the lower right corner of each picture. KTR, kidney transplant recipients; aABMR, active antibody-mediated rejection; s, semi-quantitative score.

Figure 5

Renal biopsy staining for complement factors in a KTR diagnosed with aTCMR. Selected section of biopsy slide stained for C3d in glomeruli (A) and in peritubular capillaries (B). Selected section of biopsy slide stained for C5b-9 in glomeruli (C) and in peritubular capillaries (D). White arrows indicate glomeruli and black arrows indicate peritubular capillaries. Semi-quantitative scores (0-4) are indicated in the lower right corner of each picture. KTR, kidney transplant recipients; aTCMR, acute T-cell-mediated rejection; s, semi-quantitative score.

Figure 6

Renal biopsy staining for complement factors in a KTR without indication for undergoing biopsy and without signs of rejection in biopsy (NR patient). Selected section of biopsy slide stained for C3d in glomeruli (A) and in peritubular capillaries (B). Selected section of biopsy slide stained for C5b-9 in glomeruli (C) and in peritubular capillaries (D). White arrows indicate glomeruli and black arrows indicate peritubular capillaries. Semi-quantitative scores (0-4) are indicated in the lower right corner of each picture. KTR, kidney transplant recipients; s, semi-quantitative score.

Figure 7

CD59 staining in vivo. Renal biopsy staining for complement regulator CD59 in glomeruli (A, C) and peritubular capillaries (B, D). Staining was performed on human kidney before transplantation as control (A, B), on biopsy specimen from a C4d-positive (C4d+) aABMR (C, D). Black arrows point to CD59-positive peritubular capillaries, double-compound arrows to CD59-negative peritubular capillaries. aABMR, active antibody-mediated rejection.

Figure 8

Complement system activation on conditionally immortalized glomerular endothelial cells in vitro in flow cytometric analysis. Complement factors C3 (activated), C4d, and C5b-9 on conditionally immortalized glomerular endothelial cells in vitro in flow cytometric analysis are depicted in (A) with the four different incubation conditions plotted on the y-axis. Deposition of complement regulator, CD59, was measured in flow-cytometry under five different incubation conditions (B). cABO, ABO-compatible; HLA, Human Leucocyte Antigen; Abs, antibodies; IgG, Immunoglobulin G; FITC, Fluorescein isothiocyanate; iABO, ABO-incompatible.