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Am J Physiol Renal Physiol 274: F587-F595, 1998;
0363-6127/98 $5.00
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Vol. 274, Issue 3, F587-F595, March 1998

Identification of gene family of caspases in rat kidney and altered expression in ischemia-reperfusion injury

Gur P. Kaushal, Amar B. Singh, and Sudhir V. Shah

Division of Nephrology, University of Arkansas for Medical Sciences, and John L. McClellan Memorial Veterans Affairs Hospital, Little Rock, Arkansas 72205

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

In the present study, we demonstrate that rat kidney contains caspase activity that was markedly inhibited by specific peptide inhibitors of caspases but not by inhibitors of Ser, Cys, Asp, or metalloproteinases. Using primers based on the nucleotide sequence of known members of Ced-3/interleukin-1beta -converting enzyme (ICE) family from human origin, we have identified by reverse-transcription (RT) polymerase chain reaction (PCR) analyses that rat kidney transcribes the genes for caspase-1 (ICE), caspase-2 (Nedd2), caspase-3 (CPP32), and caspase-6 (Mch2). RT-PCR products, when subcloned and sequenced, provided full-length cDNAs for ICE (1,209 bp) and CPP32 (786 bp) and partial cDNA products for Mch2 (561 bp) and Nedd2 (811 bp). The sequence analysis of the caspase cDNAs showed conserved catalytic site QACRG as well as Asp cleavage site. Rat kidneys subjected to ischemia-reperfusion injury revealed differential expression of caspases with marked increase in CPP32 and ICE mRNA and proteins during reperfusion, transient increase in Nedd2 mRNA and proteins during ischemia and the early period of reperfusion, and little change in Mch2 expression during the ischemia or reperfusion period. The altered expression suggests that caspases may act in concert in a cascade and may play an important role in ischemic acute renal failure.

gene expression; interleukin-1beta -converting enzyme; CPP32; Nedd2; Mch2; cysteine proteases

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

CELL DEATH due to ischemia-reperfusion in vivo and hypoxia-reoxygenation in vitro results predominantly in necrosis (12, 24, 57, 61). Recent studies have demonstrated that endonuclease-mediated DNA fragmentation, a generally considered feature of apoptosis (13, 62, 63), may also be important in cell death traditionally considered to result in a necrotic form of cell death. Thus several in vivo studies have documented DNA fragmentation in kidney (34, 41, 46) and other tissues (9, 19, 21, 23, 32, 55) subjected to ischemia-reperfusion injury. In addition, it has been shown that endonuclease activation is responsible for DNA fragmentation and cell death in rat renal proximal tubules subjected to hypoxia-reoxygenation and in LLC-PK1 cells subjected to hypoxic injury (20, 58). Similarly, endonuclease-mediated DNA fragmentation has been shown in nonrenal cells subjected to hypoxia-reoxygenation injury (51, 64).

Recent studies have documented that cell death proteases now designated as caspases and also known as interleukin-1beta -converting enzyme (ICE) and its family of proteases (2) play an essential role in the execution phase of apoptotic cell death and act upstream of DNA fragmentation (6, 18, 38, 43, 50). The term "caspase" for the cell death proteases embodies two distinct catalytic properties of these enzymes in which "c" refers to the Cys protease and "aspase" refers to their specific ability to cleave after Asp amino acid (2). The role of caspases in apoptosis was first recognized from studies that demonstrated significant sequence homology of the first caspase homolog, ICE, to the cell death gene, ced-3, which encodes a programmed cell death protein in Caenorhabditis elegans (65). The findings that overexpression of the genes encoding caspase ICE (35) and that other caspase homologs in transfected cells induced apoptosis and their inhibition prevented DNA fragmentation and cell death in a variety of cells (reviewed in Refs. 6, 18, 39, 43, 50) provided further evidence that caspases play an important role in apoptosis.

Although several members of the caspase family of enzymes have recently been characterized from a variety of cells, there is little information on their role in hypoxia-reoxygenation injury. We have recently demonstrated that, in LLC-PK1 cells, specific caspase inhibitors prevent, in a dose-dependent manner, chemical hypoxia-induced DNA strand breaks as determined by DNA unwinding assay, DNA fragmentation as determined by agarose gel electrophoresis, and in situ labeling of cell nuclei determined by the terminal deoxynucleotidyl transferase nick end labeling (TUNEL) method (27). Caspase inhibitors also prevent cell death in chemical hypoxic injury to LLC-PK1 cells in response to antimycin A (27). In addition, other recent studies in nonrenal cells have also suggested that caspases may be involved in cell death induced by hypoxia-reoxygenation in vitro (22, 47, 48).

Although these studies suggest the potential importance of caspases in ischemia-reperfusion injury to the kidney, there is no information on caspases in the kidney. We first examined the presence of the caspases activity in the kidney and then identified the genes for the caspase homologs expressed in rat kidney cortex by reverse-transcription (RT) polymerase chain reaction (PCR), using primers designed from the coding sequences of the known genes of caspase homologs. Finally, we have determined the expression of the gene, protein and enzyme activity of the caspase homologs in ischemia-reperfusion injury.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Isolation of mRNA. Total RNA from rat kidney cortex was extracted by guanidinium-thiocyanate-phenol-chloroform extraction procedure (7). Poly(A)+ RNA was enriched from total RNA by using oligo(dT) columns (Life Technologies, Gaithersburg, MD) and quantified by absorbance at 260 nm.

RT-PCR and characterization of rat kidney ICE, Nedd2, CPP32, and Mch2 cDNAs. A strategy utilizing the coding sequences of known genes of ICE/Ced-3 family showing least similarity within related genes was used to construct the primers. We also took advantage of the pentapeptide, QACRG, motif that encompasses the catalytic site Cys of ICE and is conserved among the members of ICE/Ced-3 protein family (18, 65). The catalytic motif was considered to become an internal part in the amplified products. The primers (Table 2) based on the published sequences of known members of Ced-3/ICE protein family (ICE, CPP32, Mch2, Mch3, ICErelII, and LAP6) were synthesized (National Bioscience, Plymouth, MA) and used for RT-PCR.

The rat kidney cortex poly(A)+ RNA was reverse transcribed into first-strand cDNA using oligo(dT) primer and Moloney murine leukemia virus reverse transcriptase (Perkin-Elmer, Branchburg, NJ). By use of first-strand cDNA as a template, the specific primers (as described above) for ICE/Ced-3 family were subjected to 35 cycles of PCR amplification (30-s denaturation at 94°C, 30-s annealing at 52°C, and 2-min extension at 72°C) in a Perkin-Elmer GeneAmp PCR System 9600. The amplified products were resolved by gel electrophresis on 1% agarose (FMC BioProducts). RT-PCR fragments detected by ethidium bromide staining were then extracted, purified (gel extraction kit, Qiagen, Chatsworth, CA), and subcloned into pGEMT vector (Promega, Madison, WI) for large-scale plasmid preparation and sequencing. Sequencing was performed on both strands using dideoxy DNA cycle sequencing system (Life Technologies). The nucleotide sequences for rat Mch2 and Nedd2 reported in this paper have been submitted to the Genbank/Data Bank with accession numbers AF025670 and AF025671, respectively.

Ischemia-reperfusion model. Adult male Sprague-Dawley rats used in the present study were subjected to 40 min of ischemia by occluding renal pedicles with smooth vascular clamps as previously described (4) and then allowed to recover for 1, 4, 16, and 48 h. Each group consisted of four rats. Blood samples were collected for creatinine and urea nitrogen determination, and kidneys were removed and processed (as described below) for isolation of RNA and for Western blot analysis. The control group consisted of rats undergoing surgical exposure of kidneys without clamping renal pedicles.

Assay of caspase activity. The kidney cortices from control rats and rats subjected to ischemia-reperfusion injury were homogenized with 20 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES, pH 7.5), containing 10% sucrose, 0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 2 mM dithiothreitol (DTT), 0.1% NP-40, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 µg/ml leupeptin, and 1 µg/ml pepstatin A. The supernatants obtained after centrifugation at 2,000 g were used to determine enzyme activity. The caspase activity was determined by fluorometric assay using the substrate, Ac-Tyr-Val-Ala-Asp-AMC, which is specifically cleaved by the enzyme to release the fluorescent leaving group, amino-4-methylcoumarin (AMC) (54). The appropriate amounts of enzyme extracts were incubated with 100 mM HEPES (pH 7.5), containing 10% sucrose, 0.1% CHAPS, 10 mM DTT, and 14 µM substrate in a total reaction volume of 0.5 ml. The reaction mixture was incubated for 30 min at 25°C. At the end of incubation, the liberated fluorescent group was monitored continuously using a spectrofluorometer (Perkin-Elmer) with an excitation wavelength of 380 nm and an emission wavelength of 460 nm. AMC was used as a standard. One unit of enzyme activity is defined as the amount of enzyme required to liberate 2 nM of AMC in 30 min. The data for caspase activity are expressed as units per milligram of protein.

cDNA probes and Northern blot analysis. Plasmid preparations containing RT-PCR-amplified products for ICE, Nedd2, CPP32, and Mch2 were used for the preparation of cDNA probes. Gel-purified restriction enzymes digested cDNA inserts from pGEMT plasmids were random-primed labeled (Amersham Life Science, Arlington Heights, IL) with [alpha -32P]dCTP and purified by chromatography on Nuc-Trap Push Columns (Stratagene, La Jolla, CA). The mRNA (~6 µg) isolated from control kidneys and kidneys subjected to ischemia-reperfusion were size fractionated by electrophoresis on 1% agarose gels under denaturing conditions using 6% formaldehyde and transferred to Immobilon N nylon membranes (Millipore, Bedford, MA). The membranes were baked under vacuum at 80°C for 1 h and hybridized overnight at 42°C with labeled cDNA probes under standard conditions previously described (49). The blots were washed three times (20 min each) in 2× saline-sodium citrate (SSC) buffer containing 0.05% sodium dodecyl sulfate (SDS) at room temperature, followed by three washes in 0.1% SSC containing 0.1% SDS at 60°C and exposed to Kodak XAR-5 films with an intensifying screen at -70°C for autoradiography to detect mRNA signals. The blots were then stripped by boiling in 0.5% SDS for 15 min and rehybridized with random-primed labeled glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe (Ambion, Austin, TX) under similar conditions. The intensity of the bands present on the autoradiogram was determined on a scanning densitometer. The levels of ICE, CPP32, Nedd2, and Mch2 mRNAs were normalized relative to GAPDH mRNA after scanning densitometry of hybridization signal intensities.

Polyacrylamide gel electrophoresis. SDS-polyacrylamide gel electrophoresis (PAGE) was performed as previously described (31) in 12% polyacrylamide gels. The gels were stained with 0.05% Coomassie blue in 10% acetic acid containing 10% isopropanol and destained in the same solvent without Coomassie blue.

Western blot analysis. Protein samples were subjected to reducing SDS-PAGE, and resolved proteins were electrophoretically transferred to nitrocellulose (Millipore) membrane and processed further for antibody staining as described by Towbin et al. (56). After this transfer, the membranes were washed in a buffer containing 50 mM tris(hydroxymethyl)aminomethane (Tris) · HCl (pH 7.5), 150 mM NaCl, and 0.05% Tween 20 for 5 min and then in the same buffer containing 5% nonfat dry milk for 1 h at room temperature. The membranes were then incubated for 5 h with antibodies (1:500 dilution) to ICE homologs (Upstate Biotechnology) in the same buffer with 5% nonfat dry milk as described above. The incubations were done at room temperature on a platform shaker to ensure thorough mixing. At the end of this time, the membranes were thoroughly washed (at least 4 times) with 50 mM Tris · HCl (pH 7.5) containing 0.05% Tween 20. The membrane filters were then incubated for 2 h with horseradish peroxidase-conjugated goat anti-rabbit antibody diluted at 1:3,000 times in Tris buffer containing 5% nonfat dry milk. After incubation, membrane filters were then washed in the same buffer (50 mM Tris · HCl and 150 mM NaCl only) containing 0.05% Tween 20 and developed with a solution of 1 part of 0.3% 4-chloro-1-naphthol in methanol and 5 parts of 0.01% hydrogen peroxide.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Because caspase activity has never previously been described in kidney cortex, we first assayed the caspase activity using fluorometric peptide substrate, Ac-Tyr-Val-Ala-Asp-AMC, that contains the specific Asp cleavage site for caspase proteases. The kidney cortex contains caspase activity which is directly proportional to protein concentration up to 0.5 mg and time of incubation for up to 2 h. This activity is not inhibited by inhibitors of Ser proteases [PMSF, aprotinin, and soybean trypsin inhibitor (SBTI)], Asp proteases (pepstatin A), and metalloproteinases (EDTA and 1,10-phenanthroline) (Table 1). Although caspases are classified under Cys proteases because of the presence of Cys in the catalytic site, Cys protease inhibitors including leupeptin, E64, E64d, and specific calpain peptide inhibitor did not inhibit the caspase activity (Table 1). In contrast, the specific and competitive tetrapeptide inhibitors of caspase, Ac-Tyr-Val-Ala-Asp-aldehyde (inhibitor I) and by Ac-Asp-Glu-Val-Asp-aldehyde (inhibitor II), which are based on the cleavage site of Asp and other amino acids required to the left of the cleavage site, markedly inhibited the caspase activity. The use of the specific substrate, lack of inhibition of the activity by other protease inhibitors, and marked inhibition by the caspase inhibitors confirmed the presence of the caspase activity in rat kidney cortex and validated the assay utilized for the measurement of the activity.

                              
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  		Table 1.   Effect of inhibitors of various classes of proteinases on ICE activity of kidney cortex homogenate

Isolation and identification of ICE, Nedd2, CPP32, and Mch2 cDNAs. We used the RT-PCR technique to identify and isolate members of caspase (ICE/Ced-3 family) family present in the rat kidney. The primers based on the coding sequences of ICE, CPP32, Mch2, Mch3, ICErelII, and LAP6 (Table 2) were used to examine the expression of caspases mRNAs in kidney by RT-PCR analyses. As shown in Fig. 1, A and B, RT-PCR cDNA products of expected sizes for ICE (1,209 bp), CPP32 (786 bp), Nedd2 (811 bp), and Mch2 (561 bp) were obtained, whereas RT-PCR products for Mch3, LAP6, and ICErelII could not be detected. RT-PCR provided full-length cDNAs for CPP32 and ICE when primers designed from 5'- or 3'-terminus of the known coding sequences were used (Table 2). Attempts to obtain full-length cDNA for Nedd2 and Mch2 by RT-PCR were unsuccessful when primers (including degenerate) from 5'- or 3'-terminus based on mouse and human sequences were used. However, partial cDNA products for Nedd2 and Mch2 were obtained when primers downstream to 5'-regions were used for RT-PCR, indicating that sequences for Nedd2 and Mch2 at the 5'- or 3'-terminus are different in human, mouse, and rat homologs (Fig. 1, A and B).

                              
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  		Table 2.   Oligonucleotide primers synthesized for reverse-transcription polymerase chain reaction analyses


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  		Fig. 1.   Reverse-transcription (RT) polymerase chain reaction (PCR) amplification of rat kidney poly(A)+ RNA using degenerate primers from coding sequences of caspases. A: kidney cortex poly(A)+ RNA (1 µg) was reverse transcribed and amplified by PCR using primers designed from coding sequences of caspases [interleukin-1beta -converting enzyme (ICE), CPP32, Mch2, Mch3, LAP6, and ICErelII, all of human origin, and Nedd2 of mouse origin]. Products were electrophoresed and analyzed by ethidium bromide staining. B: restriction digestion of RT-PCR products subcloned into pGEMT vector. Subcloned plasmid (1 µg) was cut with Sac I and Sac II restriction enzymes and analyzed by agarose electrophoresis.

The cDNA fragments of each of the RT-PCR-amplified products were gel purified, subcloned into pGEMT vector, and sequenced. Comparison of the rat kidney ICE, CPP32, Nedd2, and Mch2 cDNAs sequences with corresponding caspases of human origin revealed 72, 84, 87, and 85% homology, respectively, at the nucleotide level and 62, 89, 77, and 89% homology, respectively, at the amino acid level. The sequences of rat ICE (28) and CPP32 (25), but not of Mch2 and Nedd2, have previously been described. The present study describes the partial sequences of Mch2 (Fig. 2A) and Nedd2 (Fig. 2B) from rat and the comparison of their protein and nucleotide sequences with the corresponding human sequences. The difference in the amino acid sequence may reflect interspecies differences as well as functions including substrate specificity and potentially different roles in renal injury. All of these caspases contain conserved catalytic site, QACRG, found in all of the members of ICE-like proteases.


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  		Fig. 2.   Comparison of nucleotide and predicted protein sequence of RT-PCR-amplified rat kidney Mch2 (A) and Nedd2 (B) with corresponding known protein and nucleotide sequence data of human origin. Dashes indicate positions of sequence identity. Conserved catalytic sites QACRG and potential Asp cleavage sites are shown in boldface.

Caspase activity in ischemia-reperfusion injury. Caspase activity was assayed using the substrate, Ac-Tyr-Val-Ala-Asp-AMC, which is specifically cleaved by the caspase-1-like enzyme to release the fluorescent-leaving group, AMC (33, 54). This activity does not represent the sum of the activities of all of the caspases that can specifically hydrolyze the fluorescent substrate at the Asp amino acid. As shown in Fig. 3, caspase-1-like activity is slightly decreased during 40 min of ischemia and then recovered at 4, 16, and 48 h of reperfusion. It is possible that some caspase homologs are downregulated during ischemia, whereas others may be upregulated during the reperfusion period. Also, in crude cortex homogenate, the possibility of inhibitory effect of closely related truncated proteins or unidentified inhibitors expressed during ischemia-reperfusion injury cannot be excluded. Indeed, closely related short transcripts for ICE and ICH-1 that are capable of inhibiting these caspases have been described (1, 59). Availability of more specific substrates for each of the caspases will further help in exploring these activities. Thus, to know which of the caspases are altered or upregulated in ischemia-reperfusion injury, we examined mRNA expression for each of the caspase homologs by Northern blots and protein expression by Western blots . 


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  		Fig. 3.   Caspase activity in kidney cortex subjected to ischemia-reperfusion injury. Homogenates (250 µg protein) prepared from control kidneys and kidneys rendered ischemic in 50 mM HEPES buffer (pH 7.2), containing 10% sucrose, 2 mM dithiothreitol (DTT), 0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), and protease inhibitors, including leupeptin, pepstatin, phenylmethylsulfonyl fluoride (PMSF), and EDTA, were incubated in an assay mixture containing fluorometric substrate, Ac-Tyr-Val-Ala-Asp-amino-4-methylcoumarin (AMC; 14 µM), 20 mM HEPES (pH 7.5), containing 10% sucrose, 0.1% CHAPS, 2 mM DTT, 0.1% NP-40, 1 mM EDTA, 1 mM PMSF, 1 µg/ml leupeptin, and 1 µg/ml pepstatin A in a total volume of 0.5 ml for 30 min at 25°C. At end of incubation, liberated AMC was determined using a spectrofluorometer with an excitation wavelength of 380 nm and an emission wavelength of 460 nm. One unit of enzyme activity is defined as amount of enzyme required to liberate 2 nM of AMC in 30 min. Results are means ± SE; n = 4.

Western blot analysis for ICE, CPP32, and Nedd2. We examined the protein expression of ICE, CPP32, Nedd2, and Mch2 in kidneys of normal and ischemia-reperfusion injury using specific antibodies to probe ICE homologs by Western blot analysis. ICE expression was slightly increased during 40 min of ischemia and thereafter considerably increased at 4-16 h after reperfusion compared with sham-operated control kidneys (Fig. 4A). As shown in Fig. 4A, much of the ICE precursor band at 45 kDa was markedly reduced while, at the same time, the 20-kDa size was markedly increased at 16 h after reperfusion, indicating activation of ICE enzyme. Caspases are expressed as inactive precursors that are proteolytically processed to become functionally active enzymes (54). Similarly, as shown in Fig. 4B, the CPP32 precursor appears to be induced as well as cleaved to a lower molecular size (17-20 kDa) in ischemia as well as during reperfusion period. The antibody to Nedd2 peptide (mouse origin) showed cross-reactivity with rat kidney homogenate and showed increased expression as well as activation of protein during 40 min of ischemia and thereafter at early periods of reperfusion at 1 and 4 h after ischemia (Fig. 4C). The available antibody to Mch2 (from human origin) did not show cross-reactivity with rat kidney Mch2.


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  		Fig. 4.   Western blot analysis using specific polyclonal peptide antibodies to ICE (human origin, Transduction Laboratories) and ICE homologs (CPP32 and Nedd2 of human and mouse origin, Transduction Laboratories and Santa Cruz Biotechnology) of homogenates of rat kidney subjected to ischemia-reperfusion injury. A: Western blot with ICE antibody; expected molecular masses shown by arrows are 45 and 21 kDa. B: Western blot with CPP32 antibody; expected molecular masses shown by arrows are 31 and 21 kDa. C: Western blot with Nedd2 antibody. Expected molecular mass shown by an arrow is 47 kDa. A and B: lane A, control; lane B, 40 min of ischemia; lane C, 4 h of reperfusion after 40 min of ischemia; lane D, 16 h of reperfusion after 40 min of ischemia.

Gene expression of caspases in ischemia-reperfusion. To determine the gene expression of ICE-like proteases in ischemia-reperfusion injury, mRNAs from control and ischemic kidneys were analyzed by Northern blot analysis using appropriate cDNA probes (isolated from pGEMT plasmids containing RT-PCR-amplified partial cDNA inserts of ICE homologs). The expression of ICE and ICE homologs were not detectable in total RNA samples and were therefore analyzed using poly(A)+ RNA. The levels of ICE, CPP32, Nedd2, and Mch2 were normalized relative to GAPDH mRNA. As shown in a representative autoradiogram (Fig. 5), both normal and ischemic kidneys expressed 1.6-kb ICE, 2.5-kb CPP32, 4.0-kb Nedd2, and 1.6-kb Mch2 mRNAs. The expression of ICE, CPP32, and Mch2 decreased during 40 min of ischemia, whereas that of Nedd2 increased over the sham-operated control kidneys (Figs. 5 and 6). The gene expression of Nedd2 gradually increased at 1 and 4 h of reperfusion after ischemia and then declined to basal levels at 48 h. ICE mRNA expression gradually increased at 16 h of reperfusion and was most pronounced at 48 h of reperfusion. CPP32 mRNA level was increased at 16 and 48 h of reperfusion after ischemia over the sham-operated kidneys. The expression of Mch2 mRNA further declined at 1, 4, and 16 h of reperfusion but recovered slightly at 48 h of reperfusion (Figs. 5 and 6).


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  		Fig. 5.   Northern blot analysis. Restriction enzymes digested cDNA inserts from pGEMT plasmids were random-primed labeled with [alpha -32P]dCTP. mRNA (6 µg) isolated from control kidneys and kidneys subjected to ischemia-reperfusion injury was fractionated on 1% agarose gels, transferred to Immobilon N nylon membranes, hybridized with labeled cDNA probes under standard conditions, and exposed to Kodak XAR-5 films with an intensifying screen to detect mRNA signals.


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  		Fig. 6.   Quantitation by scanning densitometric analysis of ICE, CPP32, Mch2, and Nedd2 mRNAs.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Our previous study (27), which demonstrated prevention of cell death and DNA damage in chemical hypoxia-induced LLC-PK1 cells by the active site-based peptide inhibitors of caspases, suggests an important role for caspases in ischemic renal injury. However, there is no information on caspases in the kidney. In the present study, we have demonstrated that rat kidney cortex contains caspase activity which was inhibited by specific inhibitors of caspases such as Ac-Tyr-Val-Ala-Asp-aldehyde or Ac-Asp-Glu-Val-Asp-aldehyde but not by the inhibitors of Ser proteases (PMSF, aprotinin, and SBTI), Cys proteases (leupeptin, E64, and E64d), Asp proteases (pepstatin A), or metalloproteinases (EDTA and 1,10-phenanthroline). Because the caspase homologs present in the kidney cortex were not known, we first examined the mRNA expression of the caspases transcribed by the normal kidney cortex using RT-PCR analyses. The sequence analyses of the amplified cDNAs as well as Northern blots revealed mRNA expression of caspase-1 (ICE), caspase-2 (Nedd2), caspase-3 (CPP32), and caspase-6 (Mch2) by the rat kidney cortex. When degenerate primers for LAP6, Mch3, and ICErelII were used, no RT-PCR product could be amplified. At present, 11 members of the caspase family from mammalian cells have been characterized including caspase-1 (ICE) (65), caspase-2 (Nedd2/ICH-1) (30, 59), caspase-3 (Yama/CPP32/apopain) (16, 40, 52), caspase-4 (Tx/ICH-2/ICErelII) (14, 26, 36), caspase-5 (ICErelIII) (36), caspase-6 (Mch2) (17), caspase-7 (Mch3/ICE-LAP3) (10, 17a), caspase-8, (ICE-LAP6/Mch6) (11), caspase-9 (FLICE/Mach/Mch5) (37), caspase-10 (Mch4) (15), and caspase-11 (ICH-3) (60). It is likely that in addition to the caspase homologs characterized in the present study, other caspases including newly discovered caspase-7 to -11 as well as yet to be discovered caspases may also be transcribed by the rat kidney.

Our results provide the first demonstration not only of the gene expression of caspases in rat kidney but also of altered expression of their proteins and mRNAs in ischemia-reperfusion injury. Western blot analysis exhibited both induction as well as activation of caspase-1 protein during ischemia and reperfusion period. Caspase-1 (ICE) mRNA decreased during ischemia but increased during reperfusion compared with normal control. A recent study on global forebrain ischemia in gerbils reported increased mRNA and protein expression of ICE during 48 h after ischemia (5).

Caspase-2 (Nedd2) mRNA was increased during ischemia and early period of reperfusion and then returned to basal levels at longer periods of reperfusion. This transient induction of Nedd2 mRNA indicates that Nedd2 expression occurs at an early phase of ischemia-reperfusion injury. These results in renal ischemia-reperfusion injury seem consistent with the study performed in gerbil forebrain that showed significant increase in Nedd2 mRNA at 3-6 h after 10 min of ischemia and declined to the basal level by 24 h after ischemia (29). An increase in Nedd2 mRNA at 8 h after ischemic injury was also recently reported in rat brain after permanent occlusion of the middle cerebral artery (3). Western blot analysis of Nedd2 indicates that its protein expression is maximum during the period of ischemia. The protein expression of Nedd2 did not match with its mRNA expression during ischemia. The early increase in protein levels may result from rapid translation of Nedd2 message due to a posttranscriptional modification. In ischemia, the mRNA and protein levels tend to dissociate (42), probably because of the alterations in posttranscriptional machinery (45, 53).

Northern blot analysis for caspase-3 (CPP32) revealed downregulation of the message during ischemia, and thereafter moderate increase in the message was observed at reperfusion period. CPP32 mRNA expression was consistent with protein expression during the reperfusion period but not during the ischemia. It is known that differential regulation of genes may occur in response to ischemia-reperfusion injury (44). Increased induction of CPP32/Yama mRNA at 16-24 h after ischemic injury was recently reported in rat brain after permanent occlusion of the middle cerebral artery (3). In an another study, however, the expression of CPP32 did not change within 48 h after global forebrain ischemia in gerbils (5).

In conclusion, the present study demonstrates the gene expression of various caspases in kidney and altered gene and protein expression of caspase-1, -2, -3, and -6 in ischemia-reperfusion injury. The altered gene expression may result from the differential transactivation of caspase genes by transcription factors induced in response to ischemia and/or reperfusion injury. The activation of caspases observed for ICE and CPP32 but not for Nedd2 suggest proteolytic cascade of events in which one caspase may activate the other caspase via direct interaction (8). Further studies are required to understand the exact sequence of events in the mechanisms of activation as well as their potential target substrates in ischemic acute renal failure. Nevertheless, the present study provides a first step in delineating the role of caspases in ischemia-induced cell death in kidney.

    ACKNOWLEDGEMENTS

The authors thank Xiaoman Hong, Xuede Xiong, and Dainette Craig for technical assistance and Ellen Satter for secretarial assistance.

    FOOTNOTES

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant 5RO1-DK-47963, Department of Veteran Affairs Merit Review Grant, and Department of Defense, Office of the Navy.

Address for reprint requests: G. P. Kaushal, University of Arkansas for Medical Sciences, 4301 W. Markham St., Slot 501, Little Rock, AR 72205.

Received 26 September 1997; accepted in final form 12 December 1997.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.   Alnemri, E. S., T. Fernandes-Alnemri, and G. Litwack. Cloning and expression of four novel isoforms of human interleukin-1 beta converting enzyme with different apoptotic activities. J. Biol. Chem. 270: 4312-4317, 1995[Abstract/Free Full Text].

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