Downregulation of the calpain inhibitor protein calpastatin by caspases during renal ischemia-reperfusion

Yuexian Shi, Vyacheslav Y. Melnikov, Robert W. Schrier, and Charles L. Edelstein

Division of Renal Diseases and Hypertension, University of Colorado Health Sciences Center, Denver, Colorado 80262


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The interaction between the cysteine proteases calpain and caspases during renal ischemia-reperfusion (I/R) was investigated. An increase in the activity of calpain, as determined by 1) the appearance of calpain-mediated spectrin breakdown products and 2) the conversion of procalpain to active calpain, was demonstrated. Because intracellular calpain activity is regulated by calpastatin, the effect of I/R on calpastatin was determined. On immunoblot of renal cortex, there was a 50-100% decrease of a low molecular weight (LMW) form of calpastatin (41 kDa) after I/R. Calpastatin activity was also significantly decreased after I/R compared with sham-operated rats, indicating that the decreased protein expression had functional significance. In rats treated with the caspase inhibitor, z-Asp-2,6-dichlorobenzoyloxymethylketone (Z-D-DCB), the decrease in both calpastatin activity and protein expression was normalized, suggesting that caspases may be proteolyzing calpastatin. Caspase 3 activity increased significantly after I/R and was attenuated in ischemic kidneys from rats treated with the caspase inhibitor. In summary, during renal I/R injury, there is 1) calpain activation associated with downregulation of calpastatin protein and decreased calpastatin activity and 2) activation of caspase 3. In addition, in vivo caspase inhibition reverses the decrease in calpastatin activity. In conclusion, proteolysis of calpastatin by caspase 3 may regulate calpain activity during I/R injury. Although the protective effect of cysteine protease inhibition against hypoxic necrosis of proximal tubules has previously been demonstrated, the functional significance in ischemic acute renal failure in vivo merits further study.

caspase 3; calpain; calpastatin; ischemia; reperfusion; kidney


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THE CYSTEINE PROTEASES are intracellular proteases that have a cysteine residue at their active site. There are three major groups of cysteine proteases, cathepsins, the calcium-dependent calpains, and the newly discovered caspases. The cathepsins are non-calcium-dependent lysosomal proteases that do not appear to play a role in lethal cell injury (4, 21, 41). Although calpain plays an injurious role in ischemic injury to liver, heart, and brain (26, 28, 60), the effect of in vivo renal ischemia-reperfusion (I/R) on calpain is not known.

Cells that contain calpain also contain a specific endogenous calpain inhibitor protein called calpastatin (7). Calpastatin, like calpain, is ubiquitously present in most cell types (31). Proteolysis and downregulation of calpastatin occurs in rat heart and brain after I/R (44, 53). This proteolysis of calpastatin may be mediated by caspases. In this regard, it has recently been demonstrated that caspase-mediated fragmentation and inhibition of calpastatin occurs during cell death (61). It is possible that caspases may activate calpain either directly or via calpastatin degradation.

The caspases are a newly discovered family of intracellular cysteine proteases. Caspases participate in two distinct signaling pathways: 1) activation of proinflammatory cytokines and 2) promotion of apoptotic cell death (47). The role of caspases in hypoxic/ischemic injury in the kidney has previously been investigated. We have recently demonstrated that caspase 1 contributes to necrotic cell death in hypoxia-induced rat renal proximal tubular injury (14). Rat kidneys subjected to I/R demonstrate an increase in both caspase 1 and caspase 3 mRNA and protein expression (23). Caspase inhibition attenuates renal I/R injury by inhibition of apoptosis (9). However, the interaction between caspases and calpain/calpastatin during renal I/R is not known. For these reasons, we investigated calpain/calpastatin as a target of caspases during renal I/R.

With this background, the aims of the present study were to determine 1) the effect of renal I/R on caspases, calpain, and calpastatin and 2) the interaction between caspases and the calpain/calpastatin system during renal I/R.


    MATERIALS AND METHODS
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Renal I/R injury model. Male Sprague-Dawley rats weighing 200-250 g were allowed free access to standard food and tap water. At the start of the experiments, the animals were anesthetized with pentobarbital sodium (50 mg/kg, ip). Ischemia was induced by bilateral renal pedicle clamping for 45 min with smooth vascular clamps through a midline abdominal incision. After clamp removal, the kidney was inspected for restoration of blood flow. The abdomen was then closed in two layers. Sham-operated rats underwent the same anesthesia and surgical procedure as the ischemic rats, except that renal clamps were not applied to the pedicles. The rats were killed at 15 min, 6 h, or 24 h of reperfusion. Both kidneys were removed, and the cortices were separated and immediately frozen in liquid nitrogen. The kidney cortices were then stored at -80°C until later analysis. Caspase inhibitor-treated animals were injected intraperitoneally with a pancaspase inhibitor z-Asp-2,6-dichlorobenzoyloxymethylketone (Z-D-DCB, Bachem, King of Prussia, PA). The rats were injected intraperitoneally three times with 16 mg/kg of Z-D-DCB 1) 30 min before clamping, 2) at clamp release, and 3) 3 h after clamp release. The dose and route of administration of Z-D-DCB was based on a study that demonstrates that intraperitoneal injection of 100 mg/kg of Z-D-DCB attenuates caspase-mediated spectrin breakdown in a neonatal rat model of N-methyl-D-aspartate (NMDA)-induced brain apoptosis (37).

Calpastatin assay. The preparation of partially purified calpastatin extracts was performed as previously described (53). Briefly, the frozen kidney cortices were homogenized in sucrose-Tris-EGTA buffer (pH 7.4) containing 20 mM Tris · HCL, 0.25 M sucrose, 1 mM EGTA, 1 mM EDTA, 5 mM 2-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 10 µM pepstatin A. Homogenization was performed with a T Line laboratory stirrer (Talboys Engineering, Emerson, NJ) for 5 min at maximum speed. The homogenate was centrifuged at 1,000 g for 15 min to remove large particles. The supernatant was then centrifuged at 100,000 g for 45 min. The supernatant (cytosolic fraction) and the pellet resuspended in sucrose-Tris-EGTA buffer (membrane fraction) were boiled for 10 min and then centrifuged for 20 min at 10,000 g to remove denatured proteins. The heat-treated sample (100 µg protein) was incubated with purified µ-Calpain from porcine erythrocytes (Calbiochem, San Diego, CA), and calpain activity was determined as described below. Calpastatin activity (inhibition rate, %) was defined as calpain activity of purified calpain alone minus calpain activity of purified calpain with calpastatin extract (unknown sample)/calpain activity of purified calpain alone, as described previously (34).

In vitro calpain assay of purified calpain. The calpain assay used in this study was based on that described by Sasaki et al. (50) for purified porcine kidney calpain. N-succinyl-Leu-Tyr-7-amido-4-methyl coumarin (Sigma, St. Louis, MO) was used as a susceptible substrate for calpain (50). A stock solution of 10 mM was prepared in DMSO and stored at -20°C between use. Briefly, the calpain assay was performed as we have previously described (12). Purified µ-calpain (10 µg) was added to the calpain assay buffer containing (in mM): Imidazole 63.2 (pH 7.3), 2-mercaptoethanol 10, cysteine 5, and CaCl2 5. Ten microliters of the calpain substrate N-succinyl-Leu-Tyr were added to achieve a final concentration of 50 µM. The suspension was then incubated at 37°C in a shaking water bath for 30 min. After the 30-min incubation, fluorescence at 380 nm excitation and 460 nm emission was determined with a Hitachi F2000 spectrophotometer. An AMC standard curve was determined for each experiment. Calpain activity was expressed in picomoles of AMC released per minute of incubation time. The assay of purified calpain was performed with and without addition of the calpastatin extract described in the previous section. The percent inhibition of calpain was determined as the calpastatin activity, as described in the previous section.

To confirm the inhibitory effect of calpastatin on calpain activity in our assay, a pilot study was performed. Increasing doses of recombinant human calpastatin (Calbiochem) were added to the assay of purified calpain. The concentration of purified calpastatin that inhibits 20 µg of purified calpain by 50% (IC50) was determined to be 25 nM.

Caspase 3 assay. The activity of caspase 3 was determined by use of a fluorescent substrate as previously described (54). Ac-Asp-Glu-Val-Asp-AMC (Ac-DEVD-AMC; Bachem) was used as a susceptible fluorescent substrate for caspase 3 (58). Briefly, the frozen kidney cortices were homogenized in a buffer containing 20 mM Tris · HCl (pH 7.4), 100 mM NaCl, 5 mM dithiothreitol (DTT), 5 mM EDTA, 5 mM EGTA, 0.1% 3-([3-cholamidopropyl]dimethylammonio)-1-propanesulfonate (CHAPS), 10% sucrose, 1% (wt/vol) Triton X-100, 500 µM 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), 20 µM bestatin, 7 µM trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane (E-64), 11 µM leupeptin, 7.5 µM pepstatin A, and 0.4 µM aprotinin. The homogenate was then centrifuged at 5,000 g for 10 min. The resulting supernatant was stored at -70°C in 50% (vol/vol) glycerol. The caspase 3 activity was then measured in this supernatant as follows. Supernatant (containing ~250 µg total protein) was incubated with 50 µM of the caspase 3 substrate Ac-DEVD-AMC in a cell-free system buffer containing 100 mM HEPES (pH 7.4), 100 mM NaCl, 10 mM DTT, 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 shaking water bath. After 30 min, fluorescence was measured with a Hitachi F2000 spectrophotometer with an excitation wavelength of 380 nm and an emission wavelength of 460 nm. Caspase activity was determined as total fluorescence. An AMC standard curve was determined for each experiment. Caspase 3 activity was expressed in nanomoles AMC released per minute of incubation time per milligram of homogenate protein. Homogenate protein was measured by the Bradford method, as described in the Bio-Rad protein assay kit.

The caspase selectivity of the assay was determined by the following. 1) Purified recombinant caspase 3 (1-100 ng) (Upstate Biotechnology, Lake Placid, NY) was added to the caspase assay by use of the substrate Ac-DEVD-AMC. There was a linear dose-dependent increase in caspase activity from 19 nmol/min for 1 ng caspase 3 to 1,800 nmol/min for 100 ng caspase 3. Addition of 100 µM of Z-D-DCB to the assay resulted in a 100% decrease in the caspase activity of the higher dose of recombinant caspase 3. 2) Purified µ-calpain (10-40 µg) was added to the caspase substrate Ac-DEVD-AMC in the presence of 5 mM CaCl2 to activate the purified calpain (n = 3). There was no increase in fluorescent activity of the Ac-DEVD-AMC, indicating that calpain could not cleave the caspase substrate. 3) The pancaspase inhibitor Z-D-DCB (100 µM) was added to the assay of the kidney cortices, and the caspase 3 activity was completely inhibited. 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 (PMSF, aprotonin), aspartic proteases (pepstatin A), metalloprotease (EDTA, 1,10-phenanthroline), and cysteine proteases (E-64-d, leupeptin) (24).

Western blot analysis. Kidney cortices were homogenized in radioimmunoprecipitation assay (RIPA) buffer containing 5 mM Na2HPO4, 5 mM NaH2PO4, 150 mM NaCl, 2 mm EDTA, 2 mm EGTA, 1% sodium deoxycholate, 1% Nonidet p-40, o.1% SDS, 50 mm NaF, 200 µM Na3VO4, 0.1% beta -mercaptoethanol, pH 7.2, 500 µM AEBSF, 20 µM bestatin, 7 µM E-64, 11 µM leupeptin, 7.5 µM pepstatin A, and 0.4 µM aprotinin. The homogenate was then centrifuged at 1,000 g for 20 min to remove large particles. Aliquots of the supernatant were kept for protein determination using a detergent-compatible modified Lowry method, as described in the Bio-Rad DC protein assay kit. The supernatant was mixed with 5× sample buffer (containing 0.35 M Tris base, 10% glycerol, 0.01% bromophenol blue, 15% SDS, 3.6 mM beta -mercaptoethanol, pH 6.5) and stored at -20°C. Samples were boiled at 100°C for 3 min before loading onto the gel. For calpastatin, calpain, and amino-terminal spectrin breakdown products (BDPn), 100-µg samples were loaded onto the gel.

Western blotting was performed with a standard protocol (5, 32). Extracted proteins were separated by electrophoresis on a denaturing 7.5% (for BDPn) or 12.5% (for caspase 3) polyacrylamide gels. A constant current of 150 mA was used for 2 h. Proteins were then transferred to a polivinylidene difluoride (PVDF) membrane by wet electroblotting (100 V for 105 min). Blots were blocked for 1 h with 5% nonfat dry milk in TTBS (20 mM Tris base, 137 mM NaCl, 0.1% Tween-20, pH 7.5). Fresh skim milk in TTBS was used for the calpain blots. Western blot analysis was performed with the following antibodies: 1) rabbit polyclonal antibodies developed to the amino-terminal of the calpain cleavage site in alpha -spectrin (BDPn) (1). The blotted proteins were then probed with BDPn antibodies (1:50) overnight at 4°C. Secondary antibody used was horseradish peroxidase-conjugated donkey anti-rabbit IgG 1:1,000. BDPn protein (147 kDa was detected by enhanced chemiluminescence according to manufacturer's instruction). Positive controls for BDPs were run with calcium-treated hippocampal slices (1). Prestained proteins markers were used for molecular mass determinations. 2) A 1:2,500 dilution of a monoclonal panspectrin antibody (Affiniti Research Products, Exeter, UK), which recognizes both calpain-mediated spectrin BDPs 5/150 kDa) and caspase-specific spectrin BDPs (120 kDa) (35, 36). Secondary antibody used was horseradish peroxidase-conjugated donkey anti-rabbit IgG 1:1,000. 3) A 1:1,000 dilution of monoclonal antibody to calpastatin (Chemicon, Temecula, CA) overnight at 4°C. Secondary antibody used was horseradish peroxidase-conjugated donkey anti-rabbit IgG 1:1,000. 4) A 1:2,000 dilution of a specific monoclonal antibody to human µ-calpain (obtained from Dr. John Elce, Queen's University, Kingston, Ontario, Canada). Secondary antibody used was a purified goat anti-mouse IgG alkaline phosphatase conjugate 1:3,000 (Bio-Rad, Hercules, CA). Procalpain (80 kDa) and calpain (76 kDa) were detected in a color reaction by the ImmunoPure nitroblue tetrazolium chloride and 5-bromo-4-chloro-3"-indolyl phosphate p-toluidine salt substrate kit (Pierce, Rockford, IL). This monoclonal antibody is highly specific for both the human procalpain large subunit (80 kDa) and the human calpain large subunit (76 kDa) and cross-reacts with rat calpain (10, 18, 49).

Caspase-mediated cleavage of homogenates of kidney cortex. Kidney cortices from control animals were homogenized in RIPA buffer, as mentioned above. The homogenate was then centrifuged at 1,000 g for 20 min and at 100,000 g for 1 h. The supernatant was incubated with 0.5 µg/ml of purified caspase 3 (Upstate Biotechnology) at 37°C for 30 min. The samples were then mixed with 5× sample buffer and stored at -20°C. Samples were boiled at 100°C for 3 min before being loaded onto the gel. The samples were then separated on SDS-PAGE and analyzed by Western blotting, as described above.

Statistical analysis. Values are expressed as means ± SE. Multiple-group comparisons were performed by ANOVA with posttest according to Newman-Keuls. A P value of <0.05 was considered statistically significant.


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Effect of renal I/R on calpain. The 80-kDa inactive proenzyme form of µ-calpain undergoes calcium-dependent autolysis to be converted to the active 76-kDa form of calpain (19, 48). A pilot experiment to demonstrate a positive control for the conversion of procalpain to calpain was performed. Purified µ-calpain (Calbiochem; 0.48 U) was added to 3 ml of a Ca2+-free HEPES buffer. The buffer was divided into 1-ml aliquots to which the following were added: no Ca2+, 5 mM Ca2+, or excess EGTA before addition of Ca2+. Samples were immunoblotted with the monoclonal antibody against µ-calpain described in MATERIALS AND METHODS (19, 48). The sample containing Ca2+ demonstrated disappearance of procalpain (80 kDa) and appearance of calpain (76 kDa) that did not occur in the Ca2+-free or EGTA samples (Fig. 1A). We also determined that the activation of purified µ-calpain (1.4 U) resulted in increased activity as measured in the calpain assay (Fig. 1B).


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Fig. 1.   Positive control for the conversion of procalpain (80 kDa) to calpain (76 kDa). Purified procalpain was incubated at 37°C for 10 min with calcium-free (-Ca), excess calcium of 5 mM (+Ca), and EGTA buffers (E). Aliquots were used for 1) immunoblotting for calpain using a monoclonal µ-calpain antibody and 2) determination of calpain activity. A: on immunoblot analysis, procalpain (80 kDa) was activated to calpain (76 kDa) in the presence of +Ca but not in the presence of -Ca or E buffers. B: the activated calpain (+Ca) but not procalpain (-Ca) demonstrated calpain activity as measured in the calpain assay.

To confirm the specificity of the µ-calpain antibody, positive and negative controls were performed. µ-Calpain (5 ng) purified from porcine erythrocytes (Calbiochem) was used as a positive control. Rat recombinant m-calpain [5 ng; obtained from Dr John Elce (18)] was used as a negative control and was not detected by the µ-calpain antibody (Fig. 2). The specificity of this µ-calpain antibody for µ-calpain, as opposed to m-calpain, has also previously been determined (49).


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Fig. 2.   Specificity of µ-calpain antibody. To confirm the specificity of the µ-calpain antibody, positive and negative controls were performed. µ-Calpain (µ, 5 ng) purified from porcine erythrocytes was used as a positive control. Rat recombinant m-calpain [m, 5 ng (18)] was used as a negative control. m-Calpain was not detected by the µ-calpain antibody.

To determine whether the activation process of calpain occurs during I/R injury, renal cortex was immunoblotted using the µ-calpain antibody described earlier. As shown in Fig. 3, the proenzyme (80 kDa) was decreased in kidney cortex after 45 min ischemia followed by 6 h reperfusion, and the activated calpain (76 kDa) was increased at that time. The results indicate that µ-calpain is activated in kidney after 45 min ischemia followed by 6 h reperfusion.


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Fig. 3.   Activation of procalpain (80 kDa) to calpain (76 kDa) during renal ischemia. Renal ischemia was induced by bilateral renal pedicle clamp for 45 min followed by 6 h reperfusion (I). Sham-operated kidney was used as a control (C). Renal cortex was immunoblotted for µ-calpain and demonstrates the conversion of procalpain (80 kDa) to calpain (76 kDa) in I vs. C kidneys (n = 3). The positive control for the conversion of procalpain to calpain is shown in Fig. 1A. The specificity of the antibody for µ-calpain as opposed to m-calpain is shown in Fig. 2.

Proteolysis of spectrin to its calpain-specific BDPs is an established measurement of calpain activation in intact cells (1, 43, 46). Renal cortex from sham-operated control kidneys and ischemic kidneys were immunoblotted for SBPn. There was an increase in SBPn in renal cortex after 45 min ischemia followed by 6 h reperfusion compared with sham-operated controls (Fig. 4).


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Fig. 4.   Increase in calpain-specific spectrin breakdown products (SBPn) during renal ischemia-reperfusion (I/R). Renal I/R was induced by bilateral renal pedicle clamp for 45 min followed by 6 h reperfusion (I). Sham-operated kidney was used as a control (C). Renal cortex was immunoblotted for SBPn and demonstrates an increase in calpain-specific SBPn (147 kDa) in I vs. C kidneys. Pos, positive controls (calcium-treated hippocampal slices) (n = 3).

This conversion of procalpain to calpain and the appearance of calpain-specific SBPn are in situ assays of calpain activity. These data demonstrate that there is increased calpain activity in the early reperfusion period after renal ischemia. The activity of calpain is regulated by its endogenous inhibitor calpastatin. For this reason, the activity of calpastatin during renal I/R was examined next.

Calpastatin protein expression and activity is decreased in rat kidney after I/R. Calpastatin protein expression during renal I/R was examined. Homogenates of renal cortex were immunoblotted for calpastatin with a monoclonal antibody (Chemicon, Temecula, CA) that recognizes both the high molecular weight (HMW) and low molecular weight (LMW) forms of calpastatin (61). A time course of calpastatin protein expression during renal I/R was performed. A LMW form of calpastatin (41 kDa) (34) was found in renal cortex (Fig. 5A). As shown in Fig. 5A, calpastatin protein expression was markedly reduced after 45 min ischemia followed by 6 h reperfusion. The effect of caspase inhibition on the decrease in calpastatin protein was determined. Rats were injected with the pancaspase inhibitor Z-D-DCB, as outlined in MATERIALS AND METHODS. In vivo caspase inhibition attenuated the decrease in calpastatin protein expression (Fig. 5B).


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Fig. 5.   Calpastatin protein expression during renal I/R. Renal I/R was induced by bilateral renal pedicle clamp for 45 min followed by 15-min (15R), 6-h (6R), and 24-h reperfusion (24R). Sham-operated kidney was used as a control. Renal cortex was immunoblotted for calpastatin protein by means of a monoclonal antibody that recognizes both the high (HMW) and low molecular weight (LMW) forms of calpastatin. A LMW calpastatin protein was detected in renal cortex. There was a decrease in the LMW calpastatin after 6 h (45I/6R) reperfusion vs. C. The decrease in LMW calpastatin protein was improved at 24 h reperfusion (45I/24R; n = 3; A). B: Rats pretreated with the caspase inhibitor z-Asp-2,6-dichlorobenzoyloxymethylketone (Z-D-DCB; 48 mg/kg ip; 6R + Z) before induction of I/R demonstrated an attenuation of the decrease in calpastatin protein.

To determine whether this decrease in calpastatin protein has functional significance, calpastatin activity in cytosolic fractions was determined. Calpastatin activity is expressed as percent calpain inhibition; thus the more the calpain inhibition, the higher the calpastatin activity (see MATERIALS AND METHODS). Calpastatin activity was 47 ± 3.9% in sham-operated kidneys, 44.6 ± 5.9% after 45 min ischemia followed by 1 h reperfusion, 24.0 ± 2.2% after 45 min ischemia followed by 6 h reperfusion (P < 0.001 vs. sham-operated), and 38.3 ± 6.6% after 45 min ischemia followed by 6 h reperfusion in rats pretreated with Z-D-DCB (48 mg/kg ip) before I/R (P < 0.05 vs. 45 min ischemia followed by 6 h reperfusion alone; Fig. 6).


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Fig. 6.   Calpastatin activity is decreased during renal I/R. To determine whether the decrease in calpastatin protein (Fig. 5) has functional significance, calpastatin activity was determined. Renal I/R was induced by bilateral renal pedicle clamp for 45 min followed by 15 min (45I/15R), 6 h (45I/6R), and 24 h reperfusion (45I/24R). Sham-operated kidney was used as a control (C). Calpastatin activity was determined as percent calpain inhibition as described in MATERIALS AND METHODS. Note that a lower percentage of calpain inhibition represents a lower amount of calpain activity. There was a decrease in calpastatin activity after 6 h reperfusion (45I/6R) vs. C. By 24 h reperfusion, calpastatin activity had increased. Rats pretreated with Z-D-DCB (48 mg/kg ip, 45I/6R + Z) before induction of I/R demonstrated an attenuation of the decrease in calpastatin activity I/R. * P < 0.001 vs. C; ** P < 0.05 vs. 45I/6R; *** P < 0.01 vs. 45I/6R (n = 5).

Calpastatin activity was also determined in the membrane fractions and found to be minimal compared with the cytosolic fractions (described above). Calpastatin activity was 0.53% ± 0.5 in sham-operated kidneys and 1.1 ± 1% after 45 min ischemia followed by 6 h reperfusion [not significant (NS) vs. sham-operated, n = 3]. These data suggest that calpastatin does not translocate to the cellular membrane during I/R injury. Thus these data demonstrate that the activation of calpain during early renal I/R is associated with decreased calpastatin protein and activity in the cytosol.

Caspase 3 activity is increased in rat kidney after I/R. The improvement in calpastatin activity and protein expression in rats pretreated with the caspase inhibitor Z-D-DCB suggested that another group of cysteine proteases, caspases, may be proteolyzing calpastatin during renal I/R. In this regard, we have previously demonstrated that the cysteine proteases calpain and caspases interact during hypoxia in isolated rat renal proximal tubules (14). Thus the activity of caspase 3 was next determined in early renal I/R. Caspase 3 activity (nmol · min-1 · mg-1) in rat kidney cortices was 3.0 ± 0.8 in sham-operated controls, 7.4 ± 0.8 after 45 min ischemia followed by 6 h reperfusion (P < 0.01 vs. control), and 4.9 ± 0.6 after 45 min ischemia followed by 6 h reperfusion in rats pretreated with Z-D-DCB (P < 0.05 vs. I/R, n = 6; Fig. 7).


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Fig. 7.   Caspase 3 activity is increased during renal I/R. Renal I/R was induced by bilateral renal pedicle clamp for 45 min followed by 15 min (45I/15R), 6 h (45I/6R), and 24 h reperfusion (45I/24R). Sham-operated kidney was used as a control (C). Caspase 3 activity was determined with the fluorescent substrate Ac-Asp-Glu-Val-Asp 7-amido-4-methyl coumarin (AC-DEVD-AMC), as described in MATERIALS AND METHODS. There was an increase in caspase 3 activity after 45I/6R vs. C. By 24 h reperfusion (I45/24R), caspase 3 activity had normalized. Rats pretreated with Z-D-DCB (48 mg/kg ip; 45I/6R + Z) before induction of I/R demonstrated attenuation of the increase in caspase 3 activity. * P < 0.001 vs. C; ** P < 0.05 vs. 45I/6R; *** P = not significant (NS) vs. C (n = 5).

To further confirm the activation of caspase 3, in situ determination of caspase-specific spectrin BDPs was determined. In cells undergoing caspase-mediated cell death, a spectrin BDP of 120 kDa was observed. The formation of this 120-kDa spectrin BDP is insensitive to calpain inhibitors and is completely blocked by the caspase inhibitor Z-D-DCB in cultured cells (35, 36). Rats underwent bilateral renal pedicle clamp for 45 min followed by 6 h of reperfusion. Control rats were sham operated. The 120-kDa caspase-specific spectrin BDP was not present in control rats and increased dramatically after renal I/R (Fig. 8A). Calpain-specific spectrin BDPs are also detectable using this antibody (36). Interestingly, the calpain-specific spectrin BDPs (145/150) were also increased during renal I/R (Fig. 8A). These data confirm the in situ calpain activation demonstrated in Fig. 4. Thus there is both calpain and caspase activation during renal I/R that is detectable on immunoblot. In rats treated with Z-D-DCB, the increase in the 120-kDa caspase-specific spectrin BDP was attenuated (Fig. 8B). In Fig. 8B, the increase in the calpain-specific 145/150-kDa spectrin BDP was also inhibited by Z-D-DCB, reinforcing the suspicion that caspase activation may be upstream of calpain.


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Fig. 8.   Increase in caspase-mediated spectrin breakdown products (120 kDa) during I. Rats underwent bilateral renal pedicle clamp for 45 min followed by 6 h reperfusion. Sham-operated kidney was used as a control (C). Renal cortex was immunoblotted with a panspectrin antibody. There was an increase in caspase-mediated spectrin breakdown products (120 kDa) during renal I/R (A). In rats pretreated with Z-D-DCB (I+Z), there was attenuation of the increase in caspase-mediated spectrin breakdown products (120 kDa; B). Of note is that calpain-specific spectrin breakdown products (145/150) are also increased during I/R, confirming data shown in Fig. 4.

In vitro proteolysis of calpastatin by caspase 3. To further confirm the potential role of caspase 3 in calpastatin cleavage, the soluble fraction of rat kidney cortex homogenate was incubated for 30 min at 37°C with recombinant caspase 3 (0.5 µg/ml) in the presence and absence of Z-D-DCB (10 mM). Calpastatin protein was decreased in the presence of recombinant caspase 3, and this decrease was prevented by Z-D-DCB (Fig. 9). To exclude a direct effect of Z-D-DCB on calpastatin activity, purified calpastatin was incubated with or without Z-D-DCB at 37°C for 30 min, and then the calpastatin activity of the sample was measured. Z-D-DCB did not show the direct effect on calpastatin activity in vitro (data not shown).


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Fig. 9.   Proteolysis of calpastatin by recombinant caspase 3 in renal cortex homogenate in vitro. The soluble fraction of rat kidney cortex homogenate was incubated for 30 min at 37°C with recombinant caspase 3 (0.5 µg/ml) in the presence and absence of Z-D-DCB (10 mM). The samples were then analyzed by immunoblot with specific monoclonal antibody for calpastatin, as described in MATERIALS AND METHODS. Calpastatin protein was decreased in the presence of recombinant caspase 3, and this decrease was prevented by Z-D-DCB (n = 3).


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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The calcium-dependent calpains have been shown to be a mediators of hypoxic/ischemic injury to brain, liver, and heart (28, 29, 52, 60). Calpain also plays an injurious role in hypoxic injury to rat renal proximal tubules in vitro (12, 15, 16, 59, 62). In the present study, we investigated whether there is an increase in calpain activity during renal I/R in vivo. Determination of calpain activity during renal I/R in vivo has not previously been performed. Activation of calpain in situ can be detected on immunoblot analysis by the conversion of procalpain to calpain and by detection of calpain-specific spectrin breakdown products.

Calpain exists in the cytosol as the inactive proenzyme, procalpain, which translocates from the cytosol to the cell membrane in the presence of micromolar concentrations of Ca2+. Autocatalytic activation of procalpain to active calpain occurs at the membrane in the presence of Ca2+ and phosphatidylinositol (56). It is thought that autocatalytic activation of calpain cannot occur until the calpain molecule attaches to a phospholipid (phosphatidylinositol) binding site on the cell membrane. This binding lowers the Ca2+ requirement for autolysis to physiological concentrations of free Ca2+. The autolyzed calpain with its reduced Ca2+ requirement is now able to proteolyze substrate proteins. In the present study, the conversion of procalpain to calpain was detected during renal I/R by means of an antibody that detects the low calcium-sensitive µ-calpain isoform.

Cytoskeletal and cell membrane organizational proteins that anchor cytoskeletal elements to the plasma membrane are excellent substrates for calpain (45). For example, calpain cleaves actin-binding proteins such as spectrin, talin, filamin, alpha -actinin, and ankyrin (33). Specifically, ischemia of hippocampal neurons triggers proteolysis of cytoskeletal spectrin, and inhibition of this calpain-mediated proteolysis protects hippocampal neurons from ischemia (28, 52). Antibodies that recognize the specific calpain cleavage products of spectrin but not native uncleaved spectrin have been developed (1, 43, 46). This is a major advance, which allows for in situ detection of evidence for calpain activation as well as study of the specific effects of calpain on spectrin in the intact cell. In the present study, calpain-mediated spectrin breakdown products were also detected during early renal I/R.

Cells that contain calpain also contain the specific endogenous inhibitor of calpain called calpastatin (7). There is a molecular diversity of calpastatin in mammalian organs (57). For example, rat brain contains 92, 29, and 13 kDa calpastatins on SDS-PAGE (34). Low molecular weight calpastatins can be produced in the cell by proteolytic degradation of a high molecular weight precursor. Despite the molecular diversity of calpastatins, their inhibitory activity is specific for calpains (31, 57). This calpastatin-calpain interaction is calcium dependent. The general structure of calpastatin contains five domains, and each repeating domain has inhibitory activity (7). Thus low molecular weight forms of calpastatin, which may contain only one domain, also have inhibitory activity. In the present study, a low molecular weight form of calpastatin was detected in renal cortex.

Decreases in calpastatin protein and activity have been reported in various injury models. Proteolysis of calpastatin in rat heart occurs after brief ischemia (53). Downregulation of calpastatin has been described in postischemic hippocampus (44). Also, calpastatin protein levels decrease in rabbit hippocampus after hypoxia or glucocorticoid treatment (40). In view of the increase in calpain activity during renal I/R and the fact that the activity of autolyzed calpain is subject to final regulation by calpastatin, we investigated calpastatin activity and protein during renal I/R. The disappearance of a low molecular weight form of calpastatin during renal I/R that was associated with decreased calpastatin activity was found.

Next, we demonstrated a role of caspases in the downregulation of calpastatin. The caspases are a newly discovered family of intracellular cysteine proteases. The term "caspase" embodies two properties of these proteases in which the "c" refers to cysteine and "aspase" refers to their specific predilection to cleave substrates after an aspartate residue (3, 17). The members of the caspase family can be divided into three subfamilies, based on substrate specificity and function (58). Caspases participate in two distinct signaling pathways: 1) activation of proinflammatory cytokines and 2) promotion of apoptotic (2, 17, 39, 47) and necrotic cell death (55). Caspase inhibition has been demonstrated to reduce ischemic and excitotoxic neuronal damage (20, 30, 51). Moreover, mice deficient in caspase 1 demonstrate reduced ischemic brain injury produced by occlusion of the middle cerebral artery (27, 51). Caspase inhibitors also protect against liver ischemia induced by vascular clamping (8) and preservation injury (38).

Caspases also play a role in renal proximal tubular injury, as evidenced by the following studies. Inhibition of caspases also protects against necrotic cell death induced by the mitochondrial inhibitor antimycin A in cultured renal tubules (24). Rat kidneys subjected to I/R demonstrate an increase in both caspase 1 and caspase 3 mRNA and protein expression (23). Activation of caspases 1 and 3 associated with apoptosis has recently been described during early renal I/R (9). We have recently demonstrated that caspase 1 is a mediator of hypoxia-induced necrosis in rat proximal tubules (14).

Thus, although there is increasing evidence for a role of caspases during renal hypoxic/ischemic injury, the target of caspases during this injury is not known. A potential target of caspases during renal I/R is calpain/calpastatin. Because there are a number of aspartate residues within the conserved regions of calpastatin molecule, calpastatin may be a possible substrate for caspases (61). An interaction between caspases and calpain/calpastatin is also suggested by the following. Caspase 1 knockout mice treated with lipopolysaccharide have the expected decrease in interleukin (IL)-1beta levels in the plasma but also have a decrease in IL-1alpha (27). Whereas caspase 1 is the pro-IL-1beta processing enzyme, calpain is the preferred pro-IL-1alpha processing enzyme (6, 25). Thus it is possible that caspase 1 may indirectly activate calpain via calpastatin degradation. The lack of caspase 1 in the caspase 1 knockout mouse, therefore, may prevent this increased calpain activity that occurs with calpastatin degradation. In this regard, we have recently demonstrated that caspase inhibitors inhibit calpain activity and protect against hypoxic injury in proximal tubules, suggesting that caspase activation may be upstream of calpain (14). Also, several recent studies have found that calpastatin can be cleaved by caspase 1 and caspase 3 during apoptosis and that caspase inhibitors blocked the proteolysis (42, 61). Wang et al. (61) demonstrated that high molecular weight calpastatin (110 kDa) is rapidly cleaved in vitro by caspase 3 to produce 75-, 35-, 20-, and 15-kDa proteolytic fragments. In this study, low molecular weight calpastatin was also degraded in vitro into smaller proteolytic fragments by caspase 1.

For these reasons, we investigated whether there was caspase activation during renal I/R and whether inhibition of caspase activity could attenuate the changes in calpastatin. Our results demonstrated an increase in caspase 3 activity during renal I/R, and caspase inhibition attenuated the decreased calpastatin activity during I/R.

The triggers for the caspase 3 activation in I/R-induced injury are not known. Calcium is an important factor in renal tubular epithelial cell injury (11, 13). The activation of caspases is not directly calcium dependent in vitro (54). However, calcium was recently demonstrated to induce caspase 3-like activity by regulating the release of cytochrome c from mitochondria (22). The role of intracellular calcium in caspase activation during I/R merits further study.

The functional significance of changes in calpain and caspase activity has been investigated in a model of hypoxia-induced necrotic injury to freshly isolated rat renal proximal tubules in vitro (14). There was a 40% increase in tubular caspase activity after 15 min of hypoxia in association with increased cell membrane damage, as indicated by a threefold increase in lactate dehydrogenase (LDH) release. The specific caspase inhibitor Z-D-DCB attenuated the increase in caspase activity during 15 min of hypoxia and markedly decreased LDH release in a dose-dependent fashion. In the proximal tubules, Z-D-DCB also inhibited the hypoxia-induced increase in calpain activity. This study in proximal tubules demonstrates that caspase inhibition protects against necrotic injury by inhibition of hypoxia-induced calpain and caspase activity. Further studies to determine whether calpain and caspase inhibition protects against the functional and morphological changes during ischemic acute renal failure in vivo would be interesting.

In summary, our study demonstrates that there is activation of both calpain and caspase 3 during early renal I/R. The activation of calpain is associated with a downregulation of a low molecular weight calpastatin protein and a decrease in calpastatin activity. Treatment of the rats with a caspase inhibitor inhibits the renal I/R-induced increase in caspase 3 activity and attenuates the downregulation of calpastatin protein and decrease in calpastatin activity. Thus proteolysis of calpastatin by caspase 3 may regulate calpain activity during I/R injury.


    ACKNOWLEDGEMENTS

This research was supported by National Institutes of Health grant DK-52599 and a Young Investigator Grant from the National Kidney Foundation (C. L. Edelstein).


    FOOTNOTES

Address for reprint requests and other correspondence: C. L. Edelstein, Division of Renal Diseases and Hypertension, Univ. of Colorado School of Medicine, Box C281, 4200 E. 9th Ave., Denver, CO 80262 (E-mail: Charles.edelstein{at}uchsc.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Received 5 January 2000; accepted in final form 23 May 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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