Division of Renal Diseases and Hypertension, University of Colorado Health Sciences Center, Denver, Colorado 80262
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ABSTRACT |
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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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INTRODUCTION |
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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.
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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.
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.
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% -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
-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.
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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RESULTS |
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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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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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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 · min1 · 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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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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DISCUSSION |
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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, -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)-1
levels in the plasma but also have a decrease in IL-1
(27). Whereas caspase 1 is the pro-IL-1
processing
enzyme, calpain is the preferred pro-IL-1
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.
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ACKNOWLEDGEMENTS |
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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).
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FOOTNOTES |
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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.
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