Predominant role for C5b-9 in renal ischemia/reperfusion injury

Abstract

Previous work has indicated that complement is a mediator of ischemia/reperfusion (I/R) injury. To investigate the components of complement responsible for this effect, we examined a model of renal I/R injury in C3-, C4-, C5-, and C6-deficient mice. We occluded the renal arteries and veins (40-58 minutes) and, after reperfusion (0-72 hours), assessed renal structural and functional injury. C3-, C5-, and C6-deficient mice were protected from renal I/R injury, whereas C4-deficient mice were not protected. C6-deficient mice treated with antibody to block C5a generation showed no additional protection from I/R injury. Reconstitution with C6 alone restored the I/R injury in C6-deficient mice. Tubular epithelial cells were the main structures damaged by complement-mediated attack, and, in contrast, the renal vessels were spared. Neutrophil infiltration and myeloperoxidase activity were reduced in C-deficient mouse kidney, but by a similar extent in C3-deficient and C6-deficient mice. We conclude that the membrane attack complex of complement (in which C5 and C6 participate) may account for the effect of complement on mouse renal I/R injury. Neither C5a-mediated neutrophil infiltration nor the classic pathway, in which C4 participates, appears to contribute to I/R injury in this model. By contrast with other organs, such as the heart, the primary effect of complement in the ischemic area is on the parenchymal cell rather than the vascular endothelial cell. The membrane attack complex of complement is a potential target for prevention of I/R injury in this model.

Figures

Figure 1

Effect of renal ischemia and reperfusion on renal function in C-def and WT mice. Mice underwent renal ischemia for 58 minutes (C3-, C4-, C6-def, and WT mice) or for 40 minutes (C5-def and WT mice). Serum urea nitrogen (BUN) was measured at 6–72 hours after removal of the clamps or sham surgery. (a) C3-def and WT mice; (b) C4-def and WT mice; (c) C5-def and WT mice; (d) C6-def and WT mice; and (e) B6 C3-def and WT mice. Values shown are means ± SEM. P values are for comparisons between values in deficient and WT mice. Dashed line represents the BUN level in sham-treated mice.

Figure 2

Effect of renal ischemia and reperfusion on renal morphology in C3-def and WT mice. Light microscopy of kidney corticomedullary junction showing tubular damage at 24, 48, and 72 hours of reperfusion in C3-def mice (a, c, and e) and WT mice (b, d, and f). ×80.

Figure 3

Effect of renal ischemia and reperfusion on renal morphology in C4-, C5-, and C6-def and WT mice. Light microscopy of kidney corticomedullary junction showing tubular damage at 48 hours of reperfusion in C4-def (a) and WT (b) mice; C5-def (c) and WT (d) mice; and C6-def (e) and WT mice (f). ×80.

Figure 4

Inhibition of mouse C activity by anti-C5 mAb. WT C3H/He mice received a single intraperitoneal injection of anti-C5 (BB5.1) or control (135.1) mAb (50 mg/kg), 16 hours before induction of renal ischemia. Sera were collected for analysis at 72 hours of reperfusion. (a) Hemolytic assay. Values shown are means ± SEM of triplicate determination for each of three mice. (b) Chemotactic assay. Anti-C5 mAb inhibited ZAS-induced neutrophil chemotaxis under agarose versus control mAb. Chemotactic index of nonactivated mouse serum was subtracted from the chemotactic index of ZAS in each treatment group to normalize data. Values shown are means ± SEM. AP < 0.05 versus anti-C5 mAb treated sera.

Figure 5

Effects of anti-C5 mAb on I/R injury in C6-def mice. C6-def mice received an injection of anti-C5 or control mAb (50 mg/kg) 16 hours before induction of renal ischemia. BUN was measured at 24, 48, and 72 hours of reperfusion or after sham surgery. Values shown are means ± SEM. P values are shown for comparisons between values in deficient and WT mice. Dashed line represents the BUN level in sham-treated mice.

Figure 6

Reconstitution of C6-def mice with C6 and its effect on I/R injury. C6-def mice received two injections of purified C6 (75 μg/mouse) at 16 hours before and 1 hour after the period of ischemia. BUN was measured at 24 and 48 hours of reperfusion. Values shown are means ± SEM. Numbers in parentheses represent the number of animals studied at each group. P values are for comparisons between C6-def and restored mice and WT controls. Dashed line represents the BUN level in sham mice.

Figure 7

Staining for product of complement activation, neutrophils, and fibrin on kidney tissue. C3-def and WT mice were induced with 58-minute renal ischemia followed by 24-hour reperfusion. (a and b) Immunochemical staining of C3d at kidney corticomedullary junction in C3-def (a) and WT (b) mice. Arrows point to the deposition of C3d on the basolateral surface of renal tubular epithelium; asterisks show locations of peritubular capillaries. ×128. (c and d) MSB staining at damaged tubular area in C3-def (c) and WT (d) mice. ×80. Nuclei, erythrocytes, fibrin, and connective tissue should be stained as black, yellow, red, and blue, respectively. Necrotic tissue tubular epithelial cells are stained gray-blue (d). Arrows point at peritubular capillaries showing no fibrin formation. (e and f) Immunochemical staining of neutrophils at kidney corticomedullary junction in C3-def (e) and WT (f) mice. ×50.