Neutrophil-independent mechanisms of caspase-1- and IL-18-mediated ischemic acute tubular necrosis in mice

Abstract

Having recently described the injurious role of caspase-1-mediated production of the proinflammatory cytokine IL-18 in ischemic acute renal failure (ARF), we report here on the effect of the newly developed caspase inhibitor Quinoline-Val-Asp(Ome)-CH(2)-OPH (OPH-001) on caspase-1, IL-18, neutrophil infiltration, and renal function in ischemic ARF. C57BL/6 mice with ischemic ARF treated with OPH-001 had a marked (100%) reduction in blood urea nitrogen (BUN) and serum creatinine and a highly significant reduction in morphological acute tubular necrosis (ATN) score compared with vehicle-treated mice. OPH-001 significantly reduced the increase in caspase-1 activity and IL-18 and prevented neutrophil infiltration in the kidney during ischemic ARF. To evaluate whether this lack of neutrophil infiltration was contributing to the protection against ischemic ARF, a model of neutrophil depletion was developed. Neutrophil-depleted mice had a small (18%) reduction in serum creatinine during ischemic ARF but no reduction in ATN score despite a lack of neutrophil infiltration in the kidney. Remarkably, caspase-1 activity and IL-18 were significantly increased in the kidney in neutrophil-depleted mice with ARF. In addition, IL-18 antiserum-treated neutrophil-depleted mice with ischemic ARF had a significant (75%) reduction in serum creatinine and a significant reduction in ATN score compared with vehicle-treated neutrophil-depleted mice. These results suggest a novel neutrophil-independent mechanism of IL-18-mediated ischemic ARF.

Figure 1

Mice treated with OPH-001 are functionally protected against ischemic ARF. In vehicle-treated mice with ischemic ARF, serum creatinine was significantly increased at 24 hours and 48 hours of reperfusion compared with sham-operated controls. In mice treated with OPH-001 before induction of ischemic ARF, the serum creatinine was normalized at 24 hours and 48 hours of reperfu-sion compared with sham-operated controls. *P < 0.01 vs. sham; **P < 0.01 vs. vehicle-treated ARF, NS vs. sham; n = 8 at 24 hours. ⁺P < 0.05 vs. sham; ⁺⁺P < 0.05 vs. vehicle-treated ARF, NS vs. sham; n = 6 at 48 and 72 hours. Veh, vehicle.

Figure 2

Mice treated with OPH-001 are histologically protected against ischemic ARF. Mice treated with OPH-001 before induction of ischemia had a significant reduction in ATN score compared with vehicle-treated mice. *P < 0.001 vs. sham; **P < 0.001 vs. vehicle-treated ARF; n = 6.

Figure 3

Renal histopathology (comparable sections and representative pictures of at least three experiments). (a) In vehicle-treated mice with ischemic ARF, proximal tubules in the outer stripe of the outer medulla show extensive damage, with epithelial cell necrosis and sloughing with focal denudation. There are occasional apoptotic cells (arrow). There are numerous clumps of neutrophils in the interstitium surrounding necrotic tubules (arrowheads). (b) In OPH-001–treated mice with ischemic ARF, tubules are largely intact, with only focal sloughing of tubular cytoplasm and minimal loss of brush border. Neutrophils are inconspicuous. (c) In neutrophil-depleted mice with ischemic ARF, proximal tubules in the outer stripe of the outer medulla show extensive damage, with epithelial cell necrosis and sloughing. Neutrophils are inconspicuous, yet there is still extensive tubular necrosis. (d) In neutrophil-depleted mice treated with IL-18–neutralizing antiserum, there is less tubular necrosis. Tubules are largely intact, with only focal sloughing of tubular cytoplasm. Neutrophils are inconspicuous.

Figure 4

Mice treated with OPH-001 have decreased caspase-1 activity (a), IL-18 protein (b), and neutrophil infiltration (c) in the kidney in mice with ischemic ARF. (a) OPH-001 resulted in a normalization of caspase-1 activity compared with that of vehicle-treated mice. *P < 0.05 vs. sham; **P < 0.05 vs. vehicle-treated ARF, NS vs. sham; n = 4. (b) IL-18 protein (ECL assay) was increased in ischemic ARF. OPH-001 resulted in a normalization of IL-18 compared with that of vehicle-treated mice. *P < 0.01 vs. sham; **P < 0.01 vs. vehicle-treated ARF, NS vs. sham; n = 4. (c) Neutrophil infiltration (neutrophils/mm²) was increased in vehicle-treated mice with ischemic ARF and prevented in OPH-001–treated mice with ischemic ARF. *P < 0.01 vs. sham; **P < 0.01 vs. vehicle-treated ARF, NS vs. sham; n = 5.

Figure 5

Neutrophil-depleted mice have slight functional protection against ischemic ARF (a) and are not histologically protected against ischemic ARF (b). (a) There was an increase in serum creatinine in vehicle-treated mice with ischemic ARF (neutro⁺). Neutrophil-depleted mice with ischemic ARF (neutro^–) had a slight decrease in serum creatinine compared with neutro⁺ mice. *P < 0.001 vs. sham; **P < 0.05 vs. neutro⁺ ARF, P < 0.001 vs. sham; n = 8. (b) Histological scoring of ATN in the outer medulla was the same in neutro⁺ as in neutro^– mice. *P < 0.001 vs. sham; **P < 0.001 vs. sham, NS vs. neutro⁺; n = 7. The lack of protection occurs despite the prevention of neutrophil infiltration in the kidney in mice with ischemic ARF, shown in Figure 6c.

Figure 6

Caspase-1 activity (a), IL-18 protein (b), neutrophil infiltration (c), and active form of IL-18 (d) in the kidney in neutrophil-depleted mice with ischemic ARF. (a) Caspase-1 activity increased in both vehicle-treated (neutro⁺) and neutrophil-depleted (neutro^–) mice with ARF. *P < 0.001 vs. sham; **P < 0.001 vs. sham, NS vs. neutro⁺; n = 5. (b) IL-18 protein was measured by the ECL assay that detects both pro–IL-18 and active IL-18. IL-18 was increased in ischemic ARF in neutro⁺ as well as neutro^– mice with ischemic ARF compared with sham-operated controls. IL-18 was higher in neutro⁺ mice than in neutro^– mice. *P < 0.001 vs. sham; **P < 0.01 vs. sham, P < 0.01 vs. neutro⁺; n = 11. (c) Neutrophil infiltration (neutrophils/mm²) was increased in ischemic ARF in neutro⁺ and prevented in neutro^– mice with ischemic ARF. *P < 0.01 vs. sham; **P < 0.01 vs. vehicle-treated ARF, NS vs. sham; n = 7. (d) There was no difference in the amount of active IL-18 protein (18 kDa) on immunoblot analysis in whole-kidney homogenates in neutro⁺ versus neutro^– mice with ischemic ARF. Recombinant murine IL-18 (PeproTech Inc., Rocky Hill, New Jersey, USA) was used as a positive control (Pos). A representative picture of three separate experiments is shown.

Figure 7

Neutrophil-depleted mice treated with IL-18–neutralizing antiserum are both functionally (a) and histologically (b) protected against ischemic ARF. Mice were injected with the neutrophil-depleting antibody RB6-8C5 24 hours before renal pedicle clamp (neutro^–), followed by anti–IL-18 antiserum (AS) or vehicle 40 minutes before renal pedicle clamp and just before clamp release. (a) In vehicle-treated neutro^– mice with ischemic ARF, there was a significant increase in serum creatinine compared with sham-operated controls. In neutro^– mice treated with AS, the serum creatinine was significantly decreased compared with vehicle-treated mice with ARF. *P < 0.01 vs. sham; **P < 0.01 vs. vehicle-treated ARF, NS vs. sham; n = 8. (b) In vehicle-treated neutro^– mice with ischemic ARF, there was a significant increase in ATN score compared with sham-operated controls. In neutro^– mice treated with AS before induction of ischemic ARF, the ATN score was significantly decreased compared with vehicle-treated neutro^– mice with ARF. *P < 0.001 vs. sham; **P < 0.01 vs. vehicle-treated ARF; n = 4.