Neutrophil-independent mechanisms of caspase-1– and IL-18–mediated ischemic acute tubular necrosis in mice (original) (raw)
Mice treated with OPH-001 do not develop functional ischemic ARF. Mice were treated with vehicle (DMSO) or OPH-001 (120 mg/kg) before induction of ischemic ARF. Mice treated with OPH-001 before induction of ARF had a 100% reduction in serum creatinine at 24 hours of reperfusion compared with vehicle-treated mice with ischemic ARF, and a serum creatinine level similar to that of sham-operated controls (Figure 1). At 48 hours of reperfusion, OPH-001–treated mice remained completely protected against ischemic ARF (Figure 1). By 72 hours of reperfusion, renal function returned to normal in vehicle-treated mice, confirming the reversible nature of this model of ischemic ARF (Figure 1). BUN demonstrated a similar pattern at 24 hours of reperfusion. BUN (mg/dl) was 24 ± 0.7 in sham-operated control mice, 102.4 ± 13 in mice with vehicle-treated ischemic ARF (P < 0.001 vs. sham, n = 6–8), and 36 ± 4 in OPH-001–treated mice with ischemic ARF (P < 0.01 vs. vehicle-treated ischemic ARF, not significant [NS] vs. sham, n = 6–8). A dose of 120 mg/kg OPH-001 was chosen, as it gave the best functional protection compared with 40 mg/kg and 80 mg/kg.
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.
Sham-operated mice had an ATN score of 0, representing no tubular necrosis (Figure 2). Vehicle-treated mice with ARF had a mean ATN score of 4.7, representing necrosis in 46–75% of tubules (Figure 2). Mice treated with OPH-001 before induction of ARF had a mean ATN score of 1.8, representing tubular necrosis in 11–25% of tubules (Figure 2). Renal histopathology is shown in Figures 3, a and b.
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.
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.
OPH-001 inhibits caspase-1 activity, IL-18 protein expression, and neutrophil infiltration during ischemic ARF. OPH-001 inhibited the increase in caspase-1 activity in whole kidney homogenates during ischemic ARF (Figure 4a). IL-18 protein was determined by the ECL assay in kidney homogenates. IL-18 protein was increased in vehicle-treated mice with ischemic ARF compared with sham-operated control mice (Figure 4b). There was a large reduction in IL-18 protein in OPH-001–treated mice with ischemic ARF compared with vehicle-treated mice with ischemic ARF (Figure 4b).
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.
Because OPH-001 inhibited both caspase-1 and IL-18, both of which are proinflammatory, its effect on inflammation during ischemic ARF was determined. MPO activity in ischemic ARF was decreased in OPH-001–treated mice. MPO activity (OD/min/mg) was 0.003 ± 0.2 in sham-operated control mice, 0.056 ± 0.012 in vehicle-treated mice with ischemic ARF (P < 0.5 vs. sham, n = 4), and 0.018 ± 0.005 in OPH-001–treated mice with ischemic ARF (P < 0.05 vs. vehicle-treated ischemic ARF, n = 4).
Because MPO activity identifies the activity of monocytes and macrophages as well as neutrophils, neutrophil infiltration in the kidney was quantified (12). Neutrophil infiltration (neutrophils/mm2) in the outer medulla was 4.3 ± 0.9 in sham-operated animals, 594 ± 199 in vehicle-treated mice with ischemic ARF (P < 0.01 vs. sham), and 10.8 ± 4 in OPH-001–treated mice with ischemic ARF (P < 0.01 vs. vehicle-treated ischemic ARF, NS vs. sham) (Figure 4c). Thus, neutrophil infiltration was increased more than 100-fold in mice with ischemic ARF, and OPH-001 completely prevented the increase in neutrophil infiltration in the kidney during ischemic ARF.
Mouse model of neutrophil depletion. Mice were injected with 0.1 mg of the rat IgG2b mAb RB6-8C5 (BD Pharmingen) or vehicle (sterile water) intraperitoneally 24 hours before renal pedicle clamp (8). The mAb results in depletion of neutrophils in the peripheral blood within 24 hours. Differential peripheral blood neutrophil count was 77% ± 1.9% in vehicle-treated mice and 0.8% ± 0.3% in mice treated with the mAb RB6-8C5 (P < 0.0001 vs. untreated, n = 12).
Ischemic ARF in neutrophil-depleted mice. Ischemic ARF was induced in neutrophil-depleted mice. Serum creatinine (mg/dl)was 0.27 ± 0.03 in sham-operated control mice, 2.2 ± 0.07 in vehicle-treated mice with ischemic ARF (P < 0.001 vs. sham), and 1.8 ± 0.12 in neutrophil-depleted mice with ischemic ARF (P < 0.05 vs. wild-type ischemic ARF, P < 0.001 vs. sham) (Figure 5a).
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.
Both vehicle-treated mice with ischemic ARF and neutrophil-depleted mice with ischemic ARF had a high ATN score (Figure 5b). Renal histopathology demonstrating extensive tubular necrosis in the outer medulla in the absence of neutrophils is shown in Figure 3c.
Caspase-1 activity, IL-18 protein expression, and neutrophil infiltration during ischemic ARF in neutrophil-depleted mice. Caspase-1 activity was increased in whole-kidney homogenates during ischemic ARF (Figure 6a). Caspase-1 activity in ischemic ARF was the same in neutrophil-depleted mice as in vehicle-treated mice (Figure 6a). IL-18 protein was determined by the ECL assay. IL-18 protein was increased in vehicle-treated mice with ischemic ARF compared with sham-operated control mice (Figure 6b). Neutrophil-depleted mice with ARF had significantly more IL-18 than sham-operated mice and less IL-18 than vehicle-treated mice with ARF (Figure 6b).
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.
The ECL assay detects both pro–IL-18 and active IL-18. Thus, immunoblots for the active form of IL-18 (18 kDa) were performed in whole-kidney homogenates. There was no difference in the amount of active IL-18 protein in neutrophil-depleted versus normal kidneys with ischemic ARF, confirming a neutrophil-independent source of active IL-18 (Figure 6d).
Neutrophil infiltration (neutrophils/mm2) in the outer medulla was 5 ± 0.2 in sham-operated mice, 273 ± 34 in vehicle-treated mice with ischemic ARF (P < 0.01 vs. sham), and 6 ± 1.9 in neutrophil-depleted mice with ischemic ARF (P < 0.01 vs. ischemic ARF, NS vs. sham) (Figure 6c). Thus neutrophil depletion, like OPH-001, prevented the increase in neutrophil infiltration in the kidney during ischemic ARF.
IL-18–neutralizing antiserum protects neutrophil-depleted mice against ischemic ARF. To investigate neutrophil-independent mechanisms of IL-18–mediated injury, neutrophil-depleted mice were treated with IL-18–neutralizing antiserum. Neutrophil-depleted mice with ischemic ARF treated with IL-18–neutralizing antiserum had a 75% reduction in serum creatinine at 24 hours of reperfusion compared with vehicle-treated neutrophil-depleted mice with ARF (Figure 7a). Histological scoring of ATN in the outer medulla was significantly less in neutrophil-depleted mice with ischemic ARF that were treated with IL-18–neutralizing antiserum compared with vehicle-treated neutrophil-depleted mice with ischemic ARF (Figure 7b). Representative renal histopathology is shown in Figure 3d.
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.
In additional experiments, serum creatinine was determined at 48 hours of reperfusion. Mice treated with IL-18–neutralizing antiserum before renal pedicle clamp release as well as mice treated with an additional dose of 300 μl intraperitoneally at 24 hours of reperfusion were not functionally protected against ischemic ARF at 48 hours of reperfusion compared with vehicle-treated mice with ischemic ARF.
IL-18 and proximal tubules. We have previously demonstrated an increase in IL-18 in the urine of mice with ischemic ARF (7). Thus, we investigated proximal tubules as the possible source and target of IL-18. Studies were performed on freshly isolated proximal tubules from C57BL/6 mice. OPH-001 protected against 25 minutes of hypoxic injury in these tubules. LDH release was 11% ± 1% in normoxic tubules, 38% ± 6% in hypoxic tubules preincubated with vehicle (DMSO) (P < 0.001 vs. normoxia, n = 6), and 22% ± 1% in hypoxic tubules preincubated with OPH-001 (100 μM) (P < 0.01 vs. hypoxia, n = 6). Immunoblotting (n = 6) of normoxic proximal tubules demonstrated the presence of pro–IL-18 (24 kDa). Exogenous recombinant IL-18 (1 μg per 6 ml of tubule suspension) exacerbated sublethal (12 minutes) hypoxic proximal tubular injury. LDH release was 10% ± 1% in normoxic tubules, 13% ± 1% in hypoxic tubules preincubated with vehicle (saline) (NS vs. normoxia, n = 6), and 18% ± 1% in hypoxic tubules preincubated with recombinant IL-18 before induction of hypoxia (P < 0.01 vs. hypoxia, n = 6). It will be interesting to further study the role of IL-18 in hypoxic injury in proximal tubules.
OPH-001 inhibits both caspase-1 and caspase-3. The IC50 of OPH-001 for recombinant caspase-1 was determined to be 6.5 μM. The IC50 of OPH-001 for recombinant caspase-3 was determined to be 1.0 μM. Thus OPH-001 is a caspase inhibitor that inhibits both caspase-1 and caspase-3 in vitro.
To confirm that OPH-001 also inhibits proapoptotic caspase-3 in vivo, both caspase-3 activity and apoptosis were determined. Caspase-3 activity was increased during ischemic ARF. OPH-001 inhibited the increase in caspase-3 activity. Caspase-3 activity (nmol/min/mg) was 9.6 ± 1.7 in sham-operated control mice, 31.2 ± 4.6 in vehicle-treated mice with ischemic ARF (P < 0.01 vs. sham, n = 4), and 16.5 ± 4.2 in OPH-001–treated mice with ischemic ARF (P < 0.01 vs. vehicle-treated ischemic ARF, NS vs. sham, n = 4). The number of apoptotic tubular epithelial cells in the outer stripe of the outer medulla per ten high-power fields was 0 in sham-operated control mice, 10.6 ± 3.6 in vehicle-treated mice with ischemic ARF (P < 0.05 vs. sham, n = 4), and 2.7 ± 2.1 in OPH-001–treated mice with ischemic ARF (P < 0.05 vs. vehicle-treated ischemic ARF, n = 6).
Specificity of OPH-001. We have determined the specificity of OPH-001 for caspases versus two other major cysteine proteases, calpain and cathepsin B, both of which are mediators of cell death. The IC50 of OPH-001 for purified calpain was 727 μM. The IC50 for purified cathepsin B was 218 μM. These values are considerably higher than the IC50 for caspase-1 and caspase-3 reported above. We also measured cathepsin B activity in the kidney during ischemic ARF. There was no increase in cathepsin B activity in the kidney in mice with ischemic ARF compared with sham-operated mice, and OPH-001 had no effect on cathepsin B activity. Cathepsin B activity (μmol/min/mg) was 51.2 ± 6 in sham-operated control mice, 52 ± 2 in vehicle-treated mice with ischemic ARF (NS vs. sham, n = 4), and 53.6 ± 3 in OPH-001–treated mice with ischemic ARF (NS vs. vehicle-treated ischemic ARF, n = 4).