Global Analysis of Gene Expression in Renal Ischemia–Reperfusion in the Mouse

Biochemical and Biophysical Research Communications 291, 787–794 (2002)

doi:10.1006/bbrc.2002.6535, available online at http://www.idealibrary.com on

Global Analysis of Gene Expression in RenalIschemia–Reperfusion in the Mouse

Takumi Yoshida,*,† Shiow-Shih Tang,† Li-Li Hsiao,* Roderick V. Jensen,‡Julie R. Ingelfinger,† and Steven R. Gullans*,1

*Renal Division, Department of Medicine, Brigham and Women’s Hospital, Cambridge, Massachusetts 02139;†Pediatric Nephrology Unit, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts;and ‡Department of Physics, Wesleyan University, Middletown, Connecticut 06459

Received January 22, 2002

Ischemia-induced acute renal failure (ARF) is a rel-atively common disorder with major morbidity andmortality. To study global gene expression duringARF, 6-week-old C57BL/6 male mice underwent 30 minof bilateral renal ischemia followed by reperfusion[I/R] or sham operation. Oligonucleotide microarrays[Affymetrix] with approximately 10,000 genes, 6,643 ofwhich were present in mouse kidney, were used toanalyze mRNA expression for up to 4 days followingI/R. Fifty-two genes at day 1 and 40 at day 4 wereup-regulated more than 4-fold [400%]. Seventy genes atday 1 and 30 genes at day 4 were down-regulated tounder 0.25-fold from baseline [25%]. Real-time quanti-tative RT-PCR confirmed changes in expression for 8genes of interest. Most of the induced transcripts areinvolved in cell structure, extracellular matrix, intra-cellular calcium binding, and cell division/differen-tiation. Our data identified several novel genes thatmay be important in renal repair after ischemia. © 2002

Elsevier Science (USA)

Key Words: microarray; ischemia reperfusion; renalfailure; extracellular matrix; tissue repair.

A major cause of acute renal failure (ARF) is isch-emia, which accounts for approximately 50% of allcases (1). Many clinical conditions, such as hemor-rhagic shock, sepsis, or hepatorenal syndrome lead toischemic ARF. However, the molecular basis andpathogenic progression of ARF remain incompletelyunderstood. In renal ischemia–reperfusion [I/R] dy-namic changes in gene expression have been previ-ously reported for specific genes involved in alterationsin cell polarity (2–5), signaling transduction (6–11),

1 To whom correspondence and reprint requests should be ad-dressed at Brigham and Women’s Hospital, 65 Landsdowne Street,Cambridge, MA 02139. Fax: 617-768-8595. E-mail: [email protected].

15). Ischemic injury results in disruption of the corti-cal cytoskeleton and actin microfilament network (16).ATP depletion, which is induced by ischemia, results inoxidant injury, increase in cytosolic free calcium, andapoptosis (17). Changes in extracellular matrix (ECM)genes and proteins, such as integrin, laminin, and col-lagen, known as important to the development andmaintenance of cell structure, participate in polariza-tion of renal epithelial cells, and contribute to loss ofpolarization and to cell exfoliation (12, 18). Extracellu-lar signals are transduced through the various cas-cades, one of which is the mitogen-activated protein(MAP) kinase cascade. This family, which includesp38MAPK(10), JNK, ERK (11), and AP-1 (9), has beenpreviously reported to be involved in ARF. Accompa-nying inflammation occurs in ARF, marked by infil-tration of monocytes, which induces certain adhesionmolecules within the injured region. Such moleculesinvolved in cytokine-adhesion cascade include inte-grins (12), E-selectin (13), P-selectin (14), intercellularadhesion molecule-1 (8), and neural cell adhesion mol-ecule (15). However, it is highly likely that additionalfactors yet to be appreciated are important in the evo-lution of ARF. Discovering such factors may lead toimproved strategies for preventing ARF and treatingthis serious disorder.

DNA microarray analysis followed by powerful dataanalysis has the potential to uncover previously unde-scribed genes participating in ARF and lead to in-creased understanding of ARF. High-density DNA mi-croarrays that examine expression of many genessimultaneously not only can provide new informationabout gene function, but also generate insights intotranscriptional processes, and identify new mecha-nisms of pathogenesis (19).

In the present study, we provide the first reportof large-scale changes in gene expression inducedby ischemia–reperfusion (I/R) injury. The Affymetrix

extracellular matrix, and adhesion molecules (8, 12–

787 0006-291X/02 $35.00© 2002 Elsevier Science (USA)
All rights reserved.

GeneChip probe array was used for profiling gene ex-pression. As expected, we found that many of thechanges in expression occur in genes that encode pro-teins already implicated in ischemia–reperfusion injury.Functional annotation revealed that genes involved incell structure, tissue repair, cell division/differentialwere increased at day 1, whereas immunoglobulin andmetabolism-related transcripts were decreased. Onday 4 prominent increases in ECM, neutrophil, macro-phage related genes were observed. In addition, weidentified changes in a number of genes not previouslyimplicated in ARF that may be important in tissuerepair after ischemia.

MATERIALS AND METHODS

Animals and RNA extraction. 6-week-old C57BL/6 male mice (60to 100 g) were housed in a 12-h light/dark cycle, and allowed freeaccess to food and water. Control and treated animals were weightand age-matched at time of initiation of bilateral ischemic injury,and body weights were recorded at initiation and completion ofexperiments. A total of 15 animals were assigned to two-studygroups: ischemia or sham. In both groups, RNA was harvested priorto ischemia (day 0), immediately after clamping (ischemia, 30 min),and subsequently on days 1, 2, 3, and 4. Animals were anesthetizedintraperitoneally with pentobarbital sodium, 65 mg/kg. Both renalarteries were exposed using midline incisions and then clamped for30 min followed by reperfusion. After ischemia or sham treatment,clamps were removed, muscle layer incisions sutured, and skin in-cisions closed with Michel clips. At sacrifice the kidneys from eachanimal were removed and snap-frozen in liquid nitrogen. The sam-ples were stored at �80°C until further study. Total RNA wasisolated from each animal of all five-study groups by Trizol (GIBCO)extraction. Three individual RNA samples from day 0 and ischemiadays 1 and 4 were subjected to microarray study; samples were notpooled. Real time PCR studies were performed on independent sam-ples collected from sham and ischemic animals at all time points.

DNA microarray analysis. Affymetrix Gene Chip technology wasused as previously described (20) as follows: cDNA was synthesizedfrom total RNA using Superscript Choice kit (GibcoBRL, Rockville,MD) and T7 polymerase (Mega Script T7 kit; Ambion, Austin, TX).Total RNA (8.0 �g) was annealed to T7-(dt)24 primer (100 pmol/�l) at70°C for 10 min. Reverse transcription was carried out at 42°C for 1 hin a mixture with final concentrations of 1� First Strand Buffer, 10mM dithiothreitol, 500 �M each dATP, dCTP, dGTP, and dTTP, and20,000 U of Superscript II reverse transcriptase per ml, and thereaction was terminated by placing the tube on ice. Second-strandsynthesis was carried out in 150 �l, incorporating the entire 20-�lfirst-strand reaction mixture and a 130-�l second-strand reactionmixture containing final concentrations of 1� Second Strand Buffer,250 �M each dATP, dCTP, dGTP, and dTTP, 1.2 mM dithiothreitol,65 U of DNA ligase per ml, 250 U of DNA polymerase I per ml, and13 U of RNase H per ml. The mixture was incubated at 16°C for 2 h,whereupon 2 �l of T4 DNA polymerase at 5 U/�l were added andincubation at 16°C was prolonged for 5 min. To terminate the reac-tion, 10 �l of 0.5 M EDTA was added. After purification, the cDNAwas precipitated with 5 M ammonium acetate and absolute ethanolat �20°C for 20 min. The pellet was resuspended in 1.5 �l of RNase-free water. Synthesis of biotin-labeled cRNA was carried out by invitro transcription using the MEGAscript T7 In Vitro TranscriptionKit (Ambion, Inc., Austin, TX). The reaction was carried out at 37°Cfor 5 h in a mixture with 7.5 mM ATP, 7.5 mM GTP, 5.6 mM UTP,1.9 mM biotinylated UTP, 5.6 mM CTP, 1.9 mM biotinylated CTP,1� T7-Transcription Buffer, and 1� T7-Enzyme Mix (Enzo Diagnos-tics, Farmingdale, NY). The amplified cRNA was purified with an

affinity resin column (RNeasy, Qiagen, Valencia CA). The cRNA wasfragmented by incubation at 94°C for 35 min in the presence of 40mM Tris-acetate (pH 8.1), 100 mM potassium acetate, and 30 mMmagnesium acetate. 20 �g of the fragmented cRNA was hybridized tothe GeneChip Murine U74A Array Set (Affymetrix, Santa Clara,CA), allowing us to monitor the abundance of about 10,000 mRNAtranscripts representing known genes and ESTs; 6,643 genes werefound to be present in mouse kidney. A 220-�l hybridization solutionof 1 M NaCl, 10 mM Tris (pH 7.6), 0.005% Triton X-100, 50 pMcontrol oligonucleotide B2 (Affymetrix), control cRNA (Bio B 150 pM,Bio C 500 pM, Bio D 2.5 nM, and Cre � 10 nM) (American TypeTissue Collection, Manassas, VA, and Lofstrand Labs, Gaithersburg,MD), 0.1 mg of herring sperm DNA per ml, and 0.05 �g of thefragmented cRNA per �l was heated to 95°C, cooled to 40°C, andclarified by centrifugation before being applied to chip. Hybridizationwas at 45°C in a rotisserie hybridization oven at 60 rpm for 16 h.Subsequent washing and staining of the arrays was carried out usingthe GeneChip fluidics station protocol EukGE-WS2. Briefly, theGeneChip probe arrays were washed 10 times at 25°C with NonStringent Wash Buffer (6� SSPE, 0.01% Tween20, 0.005% antifoam).The second wash consisted of 4 cycles of 15 mixes per cycle withStringent Wash Buffer (100 mM Mes (2-N-morpholinoethanesulfonicacid, Sigma, St. Louis, MO), 0.1 M NaCl, 0.01% Tween20) at 50°C.The arrays were stained for 10 min in streptavidin-phycoerythrin(SAPE) solution 1� Mes solution, 0.005% antifoam, 10 �g/ml SAPE(Molecular Probes, Eugene, OR), 2 �g/�l acetylated BSA (Sigma, St.Louis, MO) at 25°C. The post stain wash consisted of 10 cycles at25°C in the fluidics station. The probe arrays were treated for 10mins in antibody solution (1� Mes solution, 0.005% antifoam, 2�g/�l acetylated BSA, 0.1 �g/�l normal goat IgG (Sigma, St. Louis,MO), 3 �g/�l goat-anti-streptavidin, biotinylated antibody (VectorLaboratories, Burlingame, CA)) at 25°C. The final wash consisted of15 cycles of 4 mixes per cycle at 30°C in the fluidics station. Followingwashing and staining, probe arrays were scanned twice at 3 �m reso-lution using the GeneChip System confocal scanner (Hewlett–Packard,Santa Clara, CA), controlled by GeneChip 3.1 software (Affymetrix).

Quality control of samples. The quality of each total RNA samplewas checked on a 1% agarose gel. In addition, expression levels of 3�and 5� for both �-actin and GAPDH were evaluated such that the3�/5� ratio was always less than 3, according to the manufacturer’sinstructions. To assess the integrity of hybridization each probearray was spiked with several prokaryotic genes (e.g., bioB, bioC,and bioD are genes of the biotin synthesis pathway from the bacteriaE. coli, Cre is the recombinase gene from P1 bacteriophage) werechosen to serve as hybridization controls.

Data analysis. Data analysis was performed using AffymetrixGeneChip 3.1 software. All chip data were normalized to a targetintensity of 100. We decided to use conservative criteria to identifydifferentially expression transcripts. Accordingly, four-fold or greaterchange in average difference values (i.e., gene expression levels) forthe 6,643 genes present in mouse kidney on the microarrays wasdefined as signifying differentially expressed genes, as stated in themanufacturer’s instructions (Affymetrix) and confirmed by our ex-tensive experience with the system. For each gene the “AbsoluteCalls” for significant fold-changes were determined according toAffymetrix algorithms and procedures (21, 22). The calculation of theratio between perfect match (PM) to mismatch (MM) (PM/MM ratio)was used to define transcripts as present (P), marginal (M), or absent(A; undetected). We used the conservative default settings providedby Affymetrix for this determination.

Quantitative real time RT PCR. We confirmed changes in eighttranscripts using real-time one-step quantitative RT-PCR. We se-lected the following eight genes, which appeared dramatically orunexpectedly altered: calcyclin, calpactin, MAT-1, SSeCKS, An-nexin, lysozyme, KIM-1, and ribosome L24 (PLUS GAPDH, A house-keeping gene). Relative quantitation with real-time, one-stepreverse-transcriptase polymerase chain reaction (RT-PCR) was per-

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formed with SYBR Green PCR Reagents and an ABI PRISM 7700Sequence Detection System (PE Applied Biosystems, Foster City,CA), according to the manufacturer’s instructions. Reactions wereperformed using 1.0 �l of RNA at a concentration of 100 ng/�l, in areaction volume of 50 �l. An RT reaction was performed at 48°C for30 min, followed by PCR consisting of AmpliTaq activation for 10min at 95°C, then 40 cycles with heating to 95°C for 15 sec andcooling to 60°C for 1 min mRNA levels were normalized to levels ofGAPDH. The PCR primers used were as follows: glyceraldehyde-3-phosphate dehydrogenase (GAPDH): forward primer, 5�-CATGGCC-TCCAAGGAGTAAG-3�, reverse primer, 5�-CCTAGGCCCCTCCT-GTTATT-3�; calcyclin: forward primer, 5�-AGCAAGAAGGAGCT-GAAGGA-3�, reverse primer, 5�-AAGGCGACATACTCCTGGAA-3�;calpactin I heavy chain: forward primer, 5�-ACCTGGAGACGGTG-ATTTTG-3�, reverse primer, 5�-ATCTCTTGCAGCTCCTGGTT-3�,lysozyme P: forward primer, 5�-AGAATGCCTGTGGGATCAAT-3�,reverse primer, 5�-CTGGGACAGATCTCGGTTTT-3�. annexin III:forward primer, 5�-TGTGATCTCGGCTTGAGAGA-3�, reverse primer,5�-TCTCGTCAGTCCCAAGTCCT-3�. SSeCKS: forward primer,5�-CCGAGAAGAGAAAGGAGCAA-3�, reverse primer, 5�-AAGGCA-ACTCCACCTTCTCA-3�. MAT1: forward primer, 5�-CTCCACCTG-GTAAGGGACAA-3�, reverse primer, 5�-GACCAGGAAAGGATCGA-GTG-3�. KIM1: forward primer, 5�-TGTGTGCCTGGTCCATAGTC-3�,reverse primer, 5�-ATGTCACTTCCCCCATCTTG-3�. Ribosome L24:forward primer, 5�-CAACCTTGACACCACCATTG-3�, reverse primer,5�-AGGGTGCTCCTGTTTCAAGA-3�.

All primer sets were designed according to manufacturer’s recom-mendations, and when feasible, to span one or more introns. Elec-trophoretic analysis of expected product sizes was performed for allprimer sets prior to one step, real time RT-PCR, to confirm thefidelity of the reaction.

RESULTS AND DISCUSSION

Alteration of Genes after Ischemia Reperfusion

Using oligonucleotide microarrays, we monitoredgene expression levels at days 0, 1, and 4 of ischemia–reperfusion injury. Of 10,043 genes analyzed, 3,400genes were absent, i.e., not expressed, in every sample.Of the 6,643 present [or expressed] genes, 52 genes(0.78%) at day 1 and 40 genes (0.60%) at day 4 wereup-regulated more than 4-fold. Seventy genes (1.05%)at day 1 and 30 genes (0.45%) at day 4 were down-regulated to less than 0.25-fold of baseline. Genes thatwere up-regulated more than 4-fold or down-regulatedto under 0.25-fold are shown in Table 1. The data inthis table summarize genes that appear to be impor-tant in the general biological processes involved inischemia reperfusion. Most of the up-regulated genesbelong to groups that are important in maintaining cellstructure, extracellular matrix [ECM], calcium, immu-nologic processes, cell division/differentiation and tis-sue repair. No genes in the TCA [Tricarboxylic acid]cycle or ribosome related classes appeared changed.(All data are available on the internet (http://www.hugeindex.org).

Proteolysis, Extracellular Matrix, and Tissue Repair

Genes involved in cell structure, ECM, and tissuerepair increased, mainly on day 1. Of particular note,five different serine protease inhibitors increased more

than 5-fold on day 1, which were not previously re-ported. Proteases, i.e., matrix metalloproteinase (23),cathepsin G (24), elastase (25) are considered to beinvolved in ischemic injury. Therefore, increases inprotease inhibitors are likely involved in conferringprotection from ischemia reperfusion injury. In supportof this concept, von Dobschuetz et al. showed thattreatment with serine protease inhibitor, Lex032, re-sulted in significant preservation of capillary perfu-sion. Further Tsuda et al. suggested that SPI-3, encod-ing serine protease inhibitor, may protect degenerat-ing neurons (26). Bonventre reported that ischemia–reperfusion injury increases proteolysis of the tubularcells (27). Therefore, it seems likely that protease in-hibitors may be expressed to protect renal tubular cellsfrom proteolysis in response to renal ischemia reperfu-sion injury.

Walker et al. (28) showed ECM-related genes in-crease on day 1 and continue to increase 4 days afterischemia-induced ARF. They demonstrated that im-munofluorescent staining of laminin and fibronectinwas enhanced within 3–5 days post injury, suggestinga shift from an ECM-degrading environment to onethat promotes ECM synthesis. We also observed sev-eral novel changes in ECM genes, particularly procol-lagens. ECM may modulate cell function as a fragmentof collagen was shown to be a potent anti-angiogenicprotein in renal tubular cells (29, 30). Thus, proteinsand proteolytic fragment of ECM, such as procollagenand laminin appear to play roles in ischemia–reper-fusion injury and repair.

Calcium Binding Proteins and Related Genes

Calcium [Ca2�] binding proteins and related genes,such as members of the S100 family (e.g., calcyclin) andthe annexin family, were up-regulated dramatically atday 1 and were maintained at high levels on day 4.Of note, the change of calpactin 1 and annexin A3 havenot been reported previously. These three genesshowed large increases with similar time courses ofgene expression, suggesting that these Ca2� bindingproteins may be regulated by similar mechanisms. Aspreviously reported, depletion of cellular ATP is knownto lead to an increase in the cytosolic calcium concen-tration in cells (32, 33). ATP depletion also inhibitscalcium ATPase and Na-K-ATPase. The inhibition ofthese enzymes leads to increased cytoplasmic calciumdirectly and indirectly (34, 27). This high cytoplasmiccalcium concentration may increase the expression ofCa binding proteins. Andrew et al. (31) reported thatcalcyclin is increased 10-fold by day 1 after renalischemia–reperfusion injury and suggested that calcy-clin could play a role in the regulation of renal cellproliferation and shift into the G1 phase of the cellcycle. It is possible that calpactin 1 and annexin A3may potentially involve in the mechanism of cell cycle


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TABLE 1


TABLE 1—Continued

* Genes selected were those present in any of the three study groups (day 0,1 and 4) as well as having more than four-fold changes when compared withthose from day 0.

** Average difference indicates expression level of the gene.*** Fold changes were calculated using Affymetrx software algolithm.


arrest in ischemia injury. In addition, we also con-firmed the coexpression of Calcyclin, calpactin 1 andannexin A3 by real-time PCR (Fig. 1).

Cell Division and Differentiation

Evaluation of genes involved in cell cycle revealed apattern of growth arrest. p21 (also called WAF1) (35)and SSeCKS (novel finding) were up-regulated (Table1), and cyclin D (0.28 fold, data not shown in table) andcyclin E were down-regulated. It is reported that p21 isincreased in kidney with IR (35). Further, p21 inhibitscyclin E, and SSeCKS inhibits cyclin D (36). Thesealterations cause cells to shift into the G1 phase of thecell cycle. Therefore, proliferation seems to be inhibi-ted during ischemia reperfusion injury. Interestingly,MAT1, which works as a trimeric cdk7-cyclin H-MAT1complex, activates the cyclin-dependent kinases, whichadvances the cell cycle (37), was up-regulated approx-

imately 8-fold. This increase was confirmed by real-time PCR (Fig. 1). This unexpected finding suggeststhat the MAT 1 gene may play a different role in cellcycle in ischemia reperfusion injury from previous re-ports. It will require further investigation to determineits role for the pathogenesis of ARF. These genes havenot previously been identified as playing a role in isch-emic ARF and may have importance in this setting.

Inflammation

Enhanced expression of genes involved in the im-mune response included neutrophil- and macrophagerelated genes, such as lysozyme P, lysozyme S, chemo-kines orphan receptor 1 and chemokines (c-c) receptor2. In particular lysozyme P increased approximately27-fold by microarray analysis and 18-fold by real-timePCR measurements at day 4. This dramatic increase ingene expression has been not reported and likely re-

FIG. 1. Real-time PCR and microarray analysis show comparable changes in expression. Open circles (E) show real-time PCR and closedcircles (F) show microarray data. The values are expressed as fold change compared to day 0. Data for real time PCR are expressed as mean �SEM (n � 4). Ischemia � 30 min post-renal artery clamp.


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sults from infiltrating neutrophils. Ischemia reperfu-sion injury causes accumulations of neutrophils, start-ing at about 2 h (38). On the other hand, immuno-globulins, such as Ig kappa heavy chain, IgA, and IgG2b, showed a decrease at day 1. These observationsimplicate to a role for inflammation in ischemia–reperfusion injury though the nature of this processneeds further evaluation.

Cell Signaling and Growth Factors

Many genes involved in signaling were down-regu-lated. Insulin-like growth factor binding protein(IGFBP 3), epidermal growth factors (EGF), and pros-taglandin E receptor 3 were decreased, as previouslyreported (39, 40). It is also reported that the decreaseexpression of IGFBP 3 in contrast induced nephropa-thy and EGF in ischemia injury correspond to thetissue repair process (39, 40). In this study, we re-ported that the expression level of EGF decreasedmainly on day 1 whereas IGFBP 3 on day 4. Thisobservation may suggest that EGF and IGFBP 3 in-volve in different stage of tissue repair process duringthe acute renal failure. The decrease of transcripts fortransthyren and thyroid hormone receptor alpha maybe correlated with altered thyroid functions, as previ-ously reported to decrease in ischemic ARF(41).

Other Genes

Many genes involved in metabolism (hydroxysteroiddehydrogenase-2, carbonic anhydrase 14, arylsulfataseA etc) were decreased on day 1. We speculate thatperhaps these decreases are associated with growtharrest by ischemia reperfusion injury.

Several genes involved in solute transport such as aK� voltage-gated channel, aldo-keto reductase, andsolute carrier family 16, not previously reported asaltered in ARF, were also decreased. This transportersuppression is coincident with a previous report thattubular reabsorption of sodium and water is impaired(42). These data amplify prior reports that the abun-dance of major Na� transporter (i.e., Na�-K�-ATPase,type 3 Na�/H� exchanger, type II Na-Pi cotranspor-ter, type 1 bumetanide-sensitive cotransporter, andthiazide-sensitive cotransporter), are decreased by re-nal ischemia injury (42).

KIM-1 (kidney injury molecule-1) was increased, andwe confirmed this change by real-time PCR (Fig. 1).KIM-1, a gene of unknown function, was reported to beup-regulated post ischemia reperfusion rat kidney tu-bule, and it has been suggested as a new biomarkerfor renal proximal tubule injury (43). Carbonyl reduc-tase 1 (Crb1) was dramatically up-regulated (approxi-mately 28-fold) at day 1, which is a novel finding. Thisenzyme catalyzes the reduction of endogenous prosta-glandins, steroids, and other aliphatic aldehydes andketones (44). Therefore Crb1 may play an important

role in oxidative stress status, an important event inischemia reperfusion.

CONCLUSION

When ischemia reperfusion injury occurs, genes in-volved in ECM–cell interactions and cell–cell interac-tions are up-regulated based on our observations. Spe-cifically, we found that five different serine proteaseinhibitors, not reported previously, were increased sig-nificantly. This supports the notion that increasinggene expression of these protease inhibitors may pro-tect renal cells from proteolysis in response to renalischemia injury (27). In addition, it has been well doc-umented previously that oxidative stress can causeintracellular ATP depletion leading to increase of cy-toplasmic calcium concentration (32, 33). In this study,we observed three calcium-related genes, calcyclin, cal-pactin1 and annexin A3, were up-regulated dramati-cally at day 1 and remained high at day 4. Calcyclin isknown to be involved in G1 arrest (31). Although therewere no previous reports regarding functions of calpac-tin 1 and annexin A3 in ischemia injury, we proposehere that both calpactin 1 and annexin A3 may playroles in cell cycle arrest in ischemia induced acuterenal failure. Furthermore, oxidant injury and an in-crease in cytoplasmic calcium favor both apoptosis andnecrosis. Interestingly, we found differential expres-sion of only a few genes that are related to the apopto-sis such as p21, and the apoptotic cell clearance recep-tor. Our data confirmed that ischemic injury arrestscell cycle, i.e., up-regulation of p21 and SSeCKS, anddown-regulation of cyclin D and cyclin E (36). We alsoreport that MAT1, which advances the cell cycle viaactivation of cyclin-dependent kinases, was unexpect-edly up-regulated. This observation suggests thatMAT1 may play a different role in cell cycle related toischemia ARF. It will require further investigation todetermine its role for the pathogenesis of ARF. Inconclusion, microarrays enabled us to identify multiplebiological processes that are markedly altered byischemia–reperfusion. It also revealed many genes in-volved in IR injury, some of which were previouslyunknown to participate in this process and were dra-matically elevated.

ACKNOWLEDGMENTS

This work was supported by the NIH Grants DK36031 andDK58849 (S.R.G.), DK50835 (S.S.T.), and HL-48455 and DK58950(J.R.I.). The data and supplements described in the paper are avail-able at www.hugeindex.org.

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Global Analysis of Gene Expression in Renal Ischemia–Reperfusion in the Mouse

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