An experimental model of acute humoral rejection of renal allografts associated with concomitant cellular rejection

Abstract

Acute humoral rejection (AHR), which occurs in up to 8% of kidney transplant recipients, is a significant cause of renal allograft dysfunction and loss. More efficacious treatment modalities are needed to eliminate or curtail alloantibody production and its deleterious effects on the kidney. The availability of animal models mimicking human AHR is essential to understand its pathophysiology and develop new treatment strategies. Using a mouse kidney transplant model, we demonstrate that presensitization of recipients with donor skin grafts results in rejection of subsequent renal allografts. All presensitized mice developed renal failure 8.6 +/- 4.3 days after engraftment, with serum creatinine values near 100 micromol/dl. Graft histology revealed mild, diffuse, interstitial, mononuclear cell infiltrates; prominent peritubular capillary inflammatory cell margination; patchy interstitial hemorrhage; interstitial edema; and focal glomerular fibrin deposition. Complement (C3d) deposition was diffuse and prominent in peritubular capillaries. Serum analysis demonstrated high levels of circulating alloantibodies with broad cross-reactivity to many MHC haplotypes. The clinical setting and histological findings of our model strongly resemble AHR, which is frequently associated with cellular rejection, a situation commonly encountered in human renal allograft recipients. This animal model provides a valuable tool to study the pathogenesis of AHR, its relationship to cellular alloimmunity, its contribution to graft injury, and the effects of various potential therapeutic interventions.

Figure 1

Renal allograft rejection by skin-presensitized C57BL/6 recipients. A: C57BL/6 mice were either presensitized by DBA/2 skin graft rejection (▴, n = 24) or left nonsensitized (▪, n = 12) and then received DBA/2 renal allografts. B: Presensitized recipients were either left untreated (▴, n = 4) or were depleted of CD8 T cells (•, n = 4) at the time of renal allograft transplantation. Renal allograft rejection was considered at the time animals displayed signs of ill health and creatinine levels greater than 70 μmol/L.

Figure 2

Creatinine levels in skin-presensitized versus nonsensitized renal allograft recipients. Whole blood was collected and analyzed for creatinine levels in presensitized mice (n = 24) at the time of rejection and in nonsensitized mice (n = 10) 10 days after transplant. The mean creatinine level in the serum of presensitized mice was 127.8 ± 48.6 compared to 36.7 ± 7.6 in nonsensitized mice.

Figure 3

Histological characteristics of rejected renal allografts in presensitized C57BL/6 recipients. Renal allografts were harvested at the time of rejection. A: Low magnification shows mild diffuse interstitial inflammation (H&E). B: Most of the inflammatory cells appear to localize to the PTCs. Note the absence of tubulitis (H&E). C: Higher magnification reveals that the inflammatory cells are not infiltrating tubules. Many of the inflammatory cells are in the PTCs (arrows) (periodic acid-Schiff). Left arrows point to interstitial edema, which was a common finding (H&E). D: Glomerular capillaries filled by fuchsinophilc (red) amorphous material, probably representing fibrin (Masson’s trichrome). Original magnifications: ×40 (A); ×400 (B); ×600 (C, D).

Figure 4

Characterization of inflammatory infiltrate of rejected renal allografts in presensitized C57BL/6 recipients. A: Most of the infiltrating inflammatory cells were T cells (anti-CD3, immunoperoxidase). B: Only rare B cells were seen in the interstitium; B cells were mainly present in perivascular clusters (B220 antibody, immunoperoxidase). C: Macrophages/monocytes were present in the PTCs and interstitium (F4/80 antibody, immunoperoxidase). D: Large numbers of monocytes/macrophages marginating along the endothelium of a vein (F4/80 antibody, immunoperoxidase). Original magnifications: ×400 (A, C, D); ×40 (B).

Figure 5

Immunofluorescence for C3d. A: Diffuse strong PTC C3d staining within rejected renal allografts of presensitized recipients. Arrows point to the positive staining in the PTCs. B: PTC C3d staining is absent in renal allografts of nonsensitized recipients collected at day 10 after transplant. There is tubular basement membrane C3d staining, which is probably a common nonspecific finding and was seen in most animals. Indirect immunofluorescence. Original magnifications, ×400.

Figure 6

Ultrastructural findings. A: A glomerular capillary with swollen endothelium (E). Note the platelets in the lumen (arrows). B: A swollen PTC endothelial cell is lifting off from the underlying basement membrane (arrows). C: Platelets, admixed with fibrin (asterisk) and inflammatory cells fill the lumen of this small artery. Note that the smooth muscle cells (sm) are intact. E, endothelia; m, monocyte. D: A small artery with necrotic smooth muscle cells. Note that RBCs (arrows) are present in the necrotic/degenerated smooth muscle layer. Uranyl acetate-lead citrate (all images). Original magnifications: ×4,400 (A, B); ×3,000 (C, D).

Figure 7

Alloantibody development in presensitized renal allograft recipients. A: Sera were collected from four individual presensitized recipients on the day of skin transplantation (d-14 relative to kidney transplantation), after 5 days (d-9), after 10 days (d-4), on the day of kidney transplant (day 0), and at the time of severe renal allograft dysfunction (rejection), diluted 1:16 and then tested for reactivity to donor splenocytes. B: Sera from presensitized recipients (n = 8) collected at the time of rejection and nonsensitized recipients (n = 5) collected at day 10 after transplant were diluted 1:16 and assessed for DBA/2-reactive IgG, IgG1, IgG2a, and IgG2b antibodies by flow cytometry. Antibody binding was measured as the mean fluorescence intensity (linear values). C: Sera from presensitized recipients (n = 5) collected at the time of rejection was diluted 1:20 and assessed for reactivity to DBA/2 (donor, H-2^d), BALB/c (H-2^d), FVB/N (H-2^q), CBA/J (H-2^k), BALB.B (H-2^b), and C57BL/6 (recipient, H-2^b) splenocytes by flow cytometry.