Role of Caspases in Hypoxia-Induced Necrosis of Rat Renal Proximal Tubules

Reagents

Hepes, 3-[(cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), digitonin, hyaluronidase type III from sheep testes, N-succinyl-Leu-Tyr-7-amido-4-methyl coumarin (N-succinyl-Leu-Tyr-AMC), and DMSO were purchased from Sigma (St. Louis, MO). N-succinyl-Leu-Leu-Val-Tyr-AMC, and AMC were made by Peptide Institute (Osaka, Japan) and obtained from Peptides International (Louisville, KY). Ac-Tyr-Val-Ala-Asp-7-amido-4-methyl coumarin (Ac-YVAD-AMC), Ac-Asp-Glu-Val-Asp-7-amido-4-methyl coumarin (Ac-DEVD-AMC), Z-Asp-2,6-dichloro benzoyl oxymethylketone (Z-D-DCB), and recombinant IL-1β (murine) were obtained from Bachem (King of Prussia, PA). Purified μ-calpain (from porcine erythrocytes), PD150606 [(2)-3-(4-iodophenyl)-2-mercapto-2-propenoic acid], and acetyl-Leu-Leu-norleucinol (Calpain inhibitor 1) were purchased from Calbiochem (San Diego, CA). Collagenase type B (lot no. 0FAA133) was from Boehringer Mannheim (Indianapolis, IN). Fatty acid-free bovine albumin was from ICN Biochemical (Cleveland, OH), and interleukin-1 receptor antagonist (IL-1Ra) was provided by Dr. Vann Parker (Amgen, Boulder, CO). Recombinant human ICE (caspase 1) purified to homogeneity from Escherichia coli was obtained from Dr. Nancy Thornberry (Merck Research Laboratories, Rahway, NJ) (24).

Preparation of Tubules

Proximal tubules were isolated from kidney cortex of male Sprague Dawley rats (200 to 300 g body wt), using collagenase digestion and Percoll centrifugation (25). Six-milliliter aliquots of tubule suspension (approximately 1 to 2 mg/ml) were placed in siliconized 25-ml Erlenmeyer flasks for a “recovery” period, which included gassing with 95% O₂/5% CO₂ for 5 min on ice. Flasks were then capped with rubber stoppers and kept at room temperature for 5 min, then placed in a shaking water bath at 37°C for 10 min. To create hypoxia, the tubule suspension was regassed for 5 min with 95% N₂/5% CO₂ at a rate of 3 L/min (26). After regassing, the flasks were closed and kept in the shaking water bath for the period of hypoxia studied. At the end of the hypoxic period, 1 ml of tubule suspension was sampled for measurement of lactate dehydrogenase (LDH) and 2 ml for caspase activity and calpain activity.

Caspase Inhibition Studies

A stock solution of 50 mM of the inhibitor Z-D-DCB in 100% ethanol was freshly prepared before each experiment. After the recovery period, the tubules were preincubated with the inhibitor or vehicle (ethanol) for 10 min at 37°C in the shaking water bath before induction of hypoxia.

Measurement of LDH Release and Protein

LDH release was measured to evaluate cell damage, as described previously (25,27). The percentage of LDH released from tubules was calculated by determining the ratio of LDH in the supernatant compared to that in the lysed tubule pellet plus the supernatant. Tubule protein was measured by the Lowry method, using bovine serum albumin as standard (28).

In Vitro Calpain Assay

The calpain assay used in this study is based on that described by Sasaki et al. for purified porcine kidney calpain (29). N-succinyl-Leu-Leu-Val-Tyr-AMC was used as a susceptible substrate for calpain (29). A stock solution of 10 mM was prepared in DMSO and stored at -20°C between use. The preparation of cytosolic extracts from proximal tubules and the calpain assay in freshly isolated proximal tubules has recently been described in detail by us (4,5,6).

Briefly, the calpain assay was performed as follows. After exposure to normoxia or hypoxia, the tubule pellet was separated from 2 ml of tubule suspension by centrifugation. The imidazole-HCl buffer used contained (in mM): 63.2 imidazole, 10 mercaptoethanol (pH 7.3). The tubule pellet was immediately resuspended in a calcium-free imidazole-HCl buffer containing in addition 1 mM ethylenediaminetetraacetic acid (EDTA) and 10 mM ethyleneglycol-bis(β-aminoethyl ether)-N,N′-tetra-acetic acid (pH 7.3). The suspension was then incubated with digitonin (10 μM) at 37°C in a shaking water bath for 5 min. Digitonin (10 μM) selectively permeabilizes the plasma membrane but does not destroy lysosomal or mitochondrial membranes of hepatocytes (30) or mitochondrial membranes of rat proximal tubules (31). After incubation with digitonin, the tubule pellet and supernatant containing released calpain were again separated by centrifugation. Calpain activity in the tubule pellet after digitonin incubation was zero, confirming that all of the calpain had been released into the supernatant. The calpain assay was then performed on this supernatant as follows: 0.5 ml supernatant (0.5 to 1 mg tubule protein) and 10 μl of the substrate N-succinyl-Leu-Leu-Val-Tyr-AMC (final concentration, 50 μM) was added to the imidazole buffer with or without 5 mM CaCl₂. The total volume of the assay solution was made up to 2 ml with the imidazole buffer. The solution was incubated for 30 min at 37°C in a shaking water bath. In the assay performed without CaCl₂, the imidazole-HCl buffer containing 1 mM EDTA and 10 mM ethyleneglycol-bis(β-aminoethyl ether)-N,N′-tetra-acetic acid was used. After the 30 min incubation, fluorescence at 380 nm excitation and 460 nm emission was determined using a Hitachi F2000 spectrophotometer. Calpain activity was determined as the difference between the calcium-dependent fluorescence and the non-calcium-dependent fluorescence. An AMC standard curve was determined for each experiment. Calpain activity was expressed in pmol AMC released per minute of incubation time per mg of tubule protein.

Rat Lung Extract

Partially purified m-calpain controls were obtained from normal nonperfused rat lung tissue by a technique modified from Waxman and Krebs (32).

Caspase Assay

The caspase assay developed in rat proximal tubules is based on the biochemical characteristics of caspases (33) and the caspase assay in apoptotic cerebellar granule neurons described by Nath et al. (34). Ac-YVAD-AMC and Ac-DEVD-AMC were used as susceptible substrates for caspases (34). A stock solution of 10 mM was prepared in DMSO and stored at -20°C between use.

Proximal tubule extracts for the caspase assay were prepared as follows. After exposure to normoxia or to hypoxia, the tubule pellet was separated from 2 ml of tubule suspension by centrifugation. The tubule pellet was mixed with 1 ml of lysis buffer on ice for 10 min. The lysis buffer used contained 20 mM Hepes, 100 mM NaCl, 10 mM dithiothreitol, 1 mM EDTA, 0.1% CHAPS, 10% sucrose, and 1% Triton X-100, pH 7.2. The lysate was then centrifuged at 5000 × g for 10 min. The supernatant was mixed with an equal quantity of 50% glycerol. The caspase assay was then performed on this supernatant as follows: 200 μl of supernatant (200 to 400 μg protein) and 10 μl of the substrate (final concentration, 50 μM) were added to the caspase assay buffer with and without 100 μM of the caspase inhibitor Z-D-DCB. The total volume of the assay solution was adjusted to 2 ml using the assay buffer. The assay buffer contained 100 mM Hepes, 100 mM NaCl, 10 mM dithiothreitol, 1 mM EDTA, 10% sucrose, 0.1% CHAPS, and 10% glycerol, pH 7.4. The solution was incubated for 30 min at 37°C in a shaking water bath. After the 30-min incubation, fluorescence at 380 nm excitation and 460 nm emission was determined using a Hitachi F2000 spectrophotometer. Both total fluorescence and Z-D-DCB-inhibitable fluorescence were determined. Caspase activity was determined as the Z-D-DCB-inhibitable fluorescence. An AMC standard curve was determined for each experiment. Caspase activity was expressed in pmol AMC released per minute of incubation time per mg of lysate protein. Lysate protein was measured by the Bradford method as described in the Bio-Rad protein assay kit with bovine serum albumin as standards.

The caspase selectivity of the assay was determined by the following:

1. Purified human recombinant ICE (Caspase 1) (14 to 228 ng) (24) was added to the caspase assay using the substrate Ac-YVAD-AMC. There was a linear dose-dependent increase in caspase activity from 2.3 pmol AMC/min for 14 ng ICE to 28 pmol AMC/min for 228 ng recombinant ICE. Z-D-DCB (100 μM) added to the assay resulted in a 95% decrease in the caspase activity of the higher dose of recombinant ICE.

2. Purified μ-calpain (10 to 40 μg) and rat lung extract (260 to 520 μg), which is rich in m-calpain, were added to the caspase substrate Ac-YVAD-AMC in the presence of 5 mM CaCl₂ to activate the purified calpain (n = 3). There was no increase in fluorescence activity of the Ac-YVAD-AMC, indicating that calpain could not cleave the caspase substrate.

3. Caspase activity in the assay was defined as activity that was inhibited by the specific caspase inhibitor Z-D-DCB. In this regard, it has been demonstrated that Z-D-DCB has no effect on cathepsin activity in vitro (33).

4. In proximal tubules in culture, the activity of caspases measured with the substrate Ac-YVAD-AMC is not inhibited by inhibitors of serine proteases (phenylmethylsulfonylfluoride, aprotonin), aspartic proteases (pepstatin A), metalloprotease (EDTA, 1,10-phenanthroline), and cysteine proteases (E-64-d, leupeptin) (15).

Statistical Analyses

Multiple group comparisons were done using ANOVA with post-test according to Newman-Keuls. A P value <0.05 was considered statistically significant. Values are expressed as means ± SEM.

Mechanism of Cell Death

The presence of necrosis rather than apoptosis during 5 to 30 min of hypoxia in this model of freshly isolated rat proximal tubules has been demonstrated (35). In this study, there was endonuclease activation without the morphologic features of apoptosis.

The following study was performed to confirm that the mechanism of cell death is necrosis rather than apoptosis. Freshly isolated rat proximal tubules were exposed to 15 min of hypoxia. The proximal tubules were then stained with the DNA-specific dyes Hoechst 33342 and propidium iodide (PI). Hoechst 33342 is cell membrane-permeable. PI is membrane-impermeable and stains nuclei only in cells with a damaged cell membrane (31). Individual proximal tubules were visualized with video imaging fluorescence microscopy as we have described previously (36). Normoxic control tubules showed a normal structure of nuclei stained mostly by Hoechst 33342 (Figure 1A). These normoxic tubules effectively excluded PI thereby demonstrating maintenance of membrane integrity. In contrast, the hypoxic tubules displayed extensive necrosis characterized by loss of membrane integrity and PI staining (Figure 1B). The nuclei of these tubules were still normal with light staining in the center. Proximal tubules treated with 0.1 mM hydrogen peroxide for 1 h were used as a positive control for apoptosis. These tubules excluded PI, but demonstrated apoptotic bodies (arrow) and highly condensed nuclei with Hoechst stain (Figure 1C). These studies demonstrate that proximal tubules exposed to 15 min of hypoxia undergo necrotic rather than apoptotic cell death.

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Figure 1.

Freshly isolated rat proximal tubules exposed to 15 min of hypoxia undergo necrotic rather than apoptotic cell death. Proximal tubules were exposed to 15 min of hypoxia. The proximal tubules were then stained with the DNA-specific dyes Hoechst 33342 and propidium iodide (PI). Individual proximal tubules were visualized with video imaging fluorescence microscopy. Normoxic control tubules showed a normal structure of nuclei stained mostly by Hoechst 33342 (A). These normoxic tubules effectively excluded PI thereby demonstrating maintenance of membrane integrity. Hypoxic tubules displayed extensive necrosis characterized by loss of membrane integrity and PI staining (B). The nuclei of these tubules were still normal with light staining in the center. Proximal tubules treated with 0.1 mM hydrogen peroxide for 1 h were used as a positive control for apoptosis. These tubules excluded PI, but demonstrated apoptotic bodies (arrow) and highly condensed nuclei with Hoechst stain (C).

Caspase Activity and LDH Release after 15 Min of Hypoxia

Caspase activity was 10.7 ± 1.5 pmol/min per mg in normoxic controls and increased significantly to 17.7 ± 1.5 pmol/min per mg after 15 min of hypoxia (P < 0.01 versus hypoxia, n = 5). There was a dose-dependent decrease in caspase activity with the caspase inhibitor Z-D-DCB. Caspase activity was 15.5 ± 2.1 with 50 μM Z-D-DCB (NS versus hypoxia, n = 5), 12.7 ± 2.5 with 200 μM Z-D-DCB (P < 0.01 versus hypoxia, n = 5), and 9.6 ± 0.8 with 500 μM Z-D-DCB (P < 0.01 versus hypoxia, n = 5) (Figure 2). This dose-dependent decrease in hypoxia-induced caspase activity with Z-D-DCB was accompanied by a dose-dependent decrease in cell membrane damage as assessed by LDH release. LDH release was 12 ± 1% in normoxic controls, 38 ± 2% after 15 min of hypoxia (P < 0.001 versus hypoxia, n = 7), 36 ± 2% with 50 μM Z-D-DCB (NS versus hypoxia, n = 7), 29 ± 2% with 200 μM Z-D-DCB (P < 0.01 versus hypoxia, n = 7), and 21 ± 2% with 500 μM Z-D-DCB (P < 0.001 versus hypoxia, n = 7) (Figure 2).

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Figure 2.

Effect of the specific caspase inhibitor Z-Asp-2,6-dichloro-benzoyloxymethylketone (Z-D-DCB) on caspase activity (top panel) and lactate dehydrogenase (LDH) release (bottom panel) after 15 min in normoxic (N) and hypoxic (H) proximal tubules. Proximal tubules were preincubated with the caspase inhibitor Z-D-DCB for 10 min before being exposed to hypoxia (95% N₂/5% CO₂). After hypoxia, tubules were sampled for determination of both LDH release and caspase activity. Caspase activity was determined using the fluorescent substrate Ac-Tyr-Val-Ala-Asp-7-amido-4-methyl coumarin (Ac-YVAD-AMC), as described in Materials and Methods. ^*P < 0.001 versus normoxia; ^**P < 0.01 versus normoxia; ^***P < 0.01 versus hypoxia; ^****P < 0.001 versus hypoxia (n = 5).

Caspase Activity after 25 Min of Hypoxia

Caspase activity was 12.6 ± 3.5 pmol/min per mg in normoxic controls and increased significantly to 22.9 ± 4.3 pmol/min per mg after 25 min of hypoxia (P < 0.01 versus hypoxia, n = 4). These data demonstrate that caspase activity remains elevated after a longer period of hypoxia.

Calpain Activity in Normoxic, Hypoxic, and Z-D-DCB-Treated Proximal Tubules

It is known that calpain activity is increased during hypoxic injury in rat proximal tubules (4,5,6,37,38). The caspase inhibitor Z-D-DCB inhibited the hypoxia-induced increase in calpain activity in a dose-dependent manner. Calpain activity in proximal tubules was 24 ± 12 pmol/min per mg in normoxic controls, 73 ± 16 pmol/min per mg after 15 min of hypoxia (P < 0.001 versus hypoxia, n = 4), 67 ± 16 pmol/min per mg with 50 μM Z-D-DCB (NS versus hypoxia, n = 4), 14 ± 10 pmol/min per mg with 200 μM Z-D-DCB (P < 0.001 versus hypoxia, n = 4), and 4 ± 3 pmol/min per mg with 500 μM Z-D-DCB (P < 0.001 versus hypoxia, n = 4) (Figure 3).

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Figure 3.

Effect of the specific caspase inhibitor Z-D-DCB on calpain activity after 15 min in normoxic (N) and hypoxic (H) proximal tubules. Proximal tubules were preincubated with the caspase inhibitor Z-D-DCB for 10 min before being exposed to hypoxia (95% N₂/5% CO₂). After hypoxia, tubules were sampled for determination of calpain activity using the fluorescent substrate N-succinyl-Leu-Leu-Val-Tyr-AMC, as described in Materials and Methods. ^*P < 0.001 versus normoxia; ^**P < 0.001 versus hypoxia (n = 4).

Effect of Caspase Inhibitor Z-D-DCB on Calpain Activity in Vitro

To ascertain whether Z-D-DCB directly inhibits calpain activity in vitro, we measured the in vitro activity of purified calpains in the absence and presence of Z-D-DCB and the calpain inhibitors PD150606 and calpain inhibitor 1. The substrate N-succinyl-Leu-Tyr-AMC, which we have found to be more sensitive than N-succinyl-Leu-Leu-Val-Tyr-AMC for detecting activity of purified calpains, was used. The calpain activity of purified calpains was not affected by 100 μM Z-D-DCB but was almost completely inhibited by the calpain inhibitors PD150606 and calpain inhibitor 1 (Figure 4). The calpain activity (pmol/min per mg) of purified μ-calpain (20 μg) was 329 ± 46 with no added Z-D-DCB and 283 ± 36 after addition of 100 μM of the caspase inhibitor Z-D-DCB (NS versus purified calpain alone, n = 4). However, after addition of 100 μM of the calpain inhibitor PD150606, calpain activity decreased to 58 ± 29 (P < 0.001 versus purified calpain alone, n = 4) and was 0 after addition of 100 μM of calpain inhibitor 1 (P < 0.001 versus purified calpain alone, n = 4) (Figure 4).

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Figure 4.

Effect of the specific caspase inhibitor Z-D-DCB and the specific calpain inhibitors PD150606 and calpain inhibitor 1 (CI1) on the activity of purified calpain in vitro. Purified μ-calpain (20 μg) was preincubated with the inhibitors or the vehicle in the calpain assay buffer in the presence of 5 mM CaCl₂ for 10 min at 37°C. The fluorescent substrate N-succinyl-Leu-Tyr-AMC (final concentration, 50 μM), was then added. After an additional 30-min incubation, calpain activity was determined as described in Materials and Methods. ^*P < 0.001 versus purified calpain alone (n = 4).

Effect of Calpain Inhibitor PD150606 on Caspase Activity in Proximal Tubules

The effect of calpain inhibition on hypoxia-induced caspase activity was determined. The calpain inhibitor PD150606 was used (39). We have demonstrated previously that PD150606 inhibits calpain activity and protects against hypoxic injury in proximal tubules (5). Caspase activity was 6 ± 0.5 pmol/min per mg in normoxic controls and increased to 13 ± 1.4 pmol/min per mg after 15 min of hypoxia (P < 0.01 versus normoxia, n = 5). There was a decrease in caspase activity with the calpain inhibitor PD150606. Caspase activity was 6.4 ± 0.5 with 100 μM of PD150606 (P < 0.01 versus hypoxia, n = 5) (Figure 5).

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Figure 5.

Effect of the specific calpain inhibitor PD150606 (PD) on caspase activity after 15 min in normoxic (N) and hypoxic (H) proximal tubules. Proximal tubules were preincubated with the calpain inhibitor PD150606 for 10 min before being exposed to hypoxia (95% N₂/5% CO₂). After hypoxia, tubules were sampled for determination of caspase activity using the fluorescent substrate Ac-YVAD-AMC, as described in Materials and Methods. ^*P < 0.01 versus normoxia; ^**P < 0.01 versus hypoxia (n = 5).

Effect of Calpain Inhibition on Caspase Activity in Vitro

To ascertain whether PD150606 directly inhibits caspase activity in vitro, we measured the in vitro activity of purified caspase 1 with Z-D-DCB and in the absence and presence of the calpain inhibitors PD150606 and calpain inhibitor 1. The caspase activity of purified caspase 1 was not affected by the calpain inhibitors PD150606 and calpain inhibitor 1, but was almost completely inhibited by the caspase inhibitor Z-D-DCB. The caspase activity (pmol/min per μg) of purified caspase 1 (57 ng) was 390 ± 10 with no added inhibitors, 415 ± 55 after addition of 100 μM of the calpain inhibitor PD150606 (NS versus purified caspase alone, n = 3), and 410 ± 10 after addition of 100 μM of calpain inhibitor 1 (NS versus purified caspase alone, n = 3). After addition of both 10 and 100 μM of the caspase inhibitor Z-D-DCB, caspase activity was reduced to zero (P < 0.001 versus purified caspase alone, n = 3) (Figure 6).

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Figure 6.

Effect of the calpain inhibitors PD150606 and calpain inhibitor 1 (CI1) on the activity of purified caspase 1 in vitro. Purified caspase 1 (57 ng) was preincubated with the inhibitors or the vehicle for the inhibitors. The fluorescent substrate Ac-YVAD-AMC was then added. After an additional 30-min incubation, caspase activity was determined as described in Materials and Methods. The caspase inhibitor Z-D-DCB was used as a positive control. ^*P < 0.001 versus purified caspase alone (n = 3).

Effect of Combined Caspase and Calpain Inhibition on Proximal Tubule Cell Membrane Damage

The effect of a combination of the calpain inhibitor PD150606 (100 μM) and the caspase inhibitor Z-D-DCB (100 μM) on cell membrane damage as assessed by LDH release was determined. A total of 100 μM of each inhibitor was used. In this regard, 200 μM of either inhibitor alone does not give complete protection against LDH release (Figure 2) (5). LDH release was 12.3 ± 1% in normoxic controls, 42 ± 4% after 15 min of hypoxia (P < 0.01 versus hypoxia, n = 4), 34 ± 3% with 100 μM Z-D-DCB (P < 0.05 versus hypoxia, n = 4), 27 ± 1% with 100 μM PD150606 (P < 0.01 versus hypoxia, n = 4), and 15 ± 2% with a combination of 100 μM Z-D-DCB and 100 μM PD150606 (P < 0.001 versus hypoxia, NS versus controls, n = 4) (Figure 7). Thus, the combination of caspase and calpain inhibition gave additive and complete protection against hypoxic proximal tubular injury.

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Figure 7.

Effect of the combined caspase and calpain inhibition on LDH release after 15 min in normoxic (N) and hypoxic (H) proximal tubules. Proximal tubules were preincubated with either the caspase inhibitor Z-D-DCB (100 μM) or the calpain inhibitor PD150606 (100 μM), or a combination of both Z-D-DCB (100 μM) and PD150606 (100 μM) for 10 min before being exposed to hypoxia (95% N₂/5% CO₂). After hypoxia, tubules were sampled for determination of LDH release as described in Materials and Methods. ^*P < 0.001 versus normoxia; ^**P < 0.05 versus hypoxia; ^***P < 0.01 versus hypoxia; ^****P < 0.001 versus hypoxia (n = 5).

Caspase Activity using a Different Substrate

The caspase assay was developed using the fluorescence substrate Ac-YVAD-AMC, which is preferentially cleaved by caspases 1, 4, and 5. The fluorescence substrate Ac-DEVD-AMC, which is cleaved by caspases 2, 3, and 7, was also used. Both total fluorescence and Z-D-DCB-inhibitable fluorescence (caspase activity) were measured in normoxic and hypoxic tubules with both substrates. Significant fluorescence activity was detected with Ac-YVAD-AMC but not with Ac-DEVD-AMC (Figure 8), suggesting that caspase 1 is involved in hypoxic injury.

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Figure 8.

Caspase activity was measured in proximal tubules using the fluorescent substrate Ac-DEVD-AMC (DEVD) in addition to Ac-YVAD-AMC (YVAD). The caspase assay was performed as described in Materials and Methods. Both total fluorescence and Z-D-DCB-inhibitable fluorescence (caspase activity) was measured in normoxic (N) and hypoxic (H) tubules. The fluorescent substrate Ac-YVAD-AMC (YVAD) is preferentially cleaved by caspases 1, 4, and 5, and Ac-DEVD-AMC (DEVD) is cleaved by caspases 2, 3, and 7 and CED-3. ^**P < 0.01 versus normoxia (n = 4).

A positive control for the substrate Ac-DEVD-AMC was performed. Thymus, which is rich in thymocytes that undergo caspase 3-mediated apoptosis (40), was prepared for the caspase assay in the same manner as the proximal tubules. The thymus extract was assayed using the substrates Ac-YVAD-AMC and Ac-DEVD-AMC. Significant caspase activity was only detected with Ac-DEVD-AMC, and minimal activity was detected with Ac-YVAD-AMC (Figure 9).

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Figure 9.

Caspase activity was measured in the rat thymus using the fluorescent substrates Ac-DEVD-AMC (DEVD) and Ac-YVAD-AMC (YVAD). Thymus, which is rich in thymocytes that undergo caspase 3-mediated apoptosis (40), was prepared for the caspase assay as described in Materials and Methods. Significant caspase activity was only detected with Ac-DEVD-AMC (which is cleaved by caspases 2, 3, and 7). Minimal caspase activity was detected with Ac-YVAD-AMC (which is cleaved by caspases 1, 4, and 5). ^*P < 0.001 versus YVAD (n = 4).

Effect of Interleukin-1β on Hypoxic Injury

Pro-interleukin-1β is cleaved at the aspartic acid-alanine position (amino acids 116 to 117) to mature IL-1β by caspase 1 (ICE) (41). It is possible that caspase 1 (ICE) activation during hypoxic injury produces IL-1B, which mediates the cell membrane damage. Thus, the effect of IL-1Ra on hypoxic injury was determined. The IL-1Ra had no protective effect on hypoxic injury. LDH release (%) was 14.5 ± 1 in normoxic controls, 36 ± 3 in hypoxia (P < 0.01 versus normoxia, n = 3), 34 ± 4 in hypoxic tubules preincubated with 20 μg/ml IL-1Ra (NS versus hypoxia alone, n = 3), and 33 ± 1 in hypoxic tubules preincubated with 40 μg/ml IL-1Ra (NS versus hypoxia alone, n = 3). To further determine whether IL-1β mediates cell injury, the additive effect of recombinant IL-1β on hypoxic injury was determined. A shorter time of hypoxia with less cell membrane damage was used so that the additive effect of recombinant IL-1β on a mild degree of cell membrane damage could be determined. LDH release (%) was 10 ± 1 in normoxic controls, 19 ± 4 after 10 min of hypoxia (P < 0.05 versus normoxia, n = 4), and 18 ± 3 in hypoxic tubules preincubated for 10 min with 20 ng/ml recombinant IL-1β (NS versus hypoxia alone, n = 4).