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Kidney International, Vol. 61 (2002), pp. 1646–1654

CELL BIOLOGY – IMMUNOLOGY – PATHOLOGY

Monitoring changes in expression in renal ischemia-reperfusion in the rat

TAKUMI YOSHIDA,MANJULA KURELLA,FRANCISCA BEATO,HYUNSUK MIN, JULIE R. INGELFINGER,ROBIN L. STEARS,RITA D. SWINFORD,STEVEN R. GULLANS, and SHIOW-SHIH TANG

Pediatric Nephrology Unit, Massachusetts General Hospital, Renal Division, Department of Medicine, Brigham & Women’s Hospital, Harvard Medical School, Boston, Massachusetts, Division of Pediatric Nephrology Unit, University of Texas Medical Center in Houston, Texas, USA

Monitoring changes in gene expression in renal ischemia-reper- Acute renal failure (ARF), characterized by rapid de- fusion in the rat. cline in glomerular filtration rate (GFR), is potentially Background. Although acute renal failure (ARF) is a rela- reversible but is associated with major morbidity and tively common disorder with major morbidity and mortality, its molecular basis remains incompletely defined. The present mortality. Despite new insights into the pathogenesis of study examined global gene expression in the well-character- ARF, neither the incidence nor mortality has declined ized ischemia-reperfusion model of ARF using DNA micro- in decades [1]. During the induction and course of ARF array technology. many intrarenal alterations occur, including changes in Methods. Male Wistar rats underwent bilateral renal isch- emia (30 min) or sham operation, followed by reperfusion for cell polarity [2–5], levels of signaling molecules [6–11] 1, 2, 3 or 4 days. Plasma creatinine increased approximately expression of adhesion molecules [8, 12–15] and patterns fivefold over baseline, peaking on day 1. Renal total RNA was of apoptosis and necrosis [16]. Nevertheless, it is proba- used to probe cDNA microarrays. ble that additional factors participate in the evolution Results. Alterations in expression of 18 were identified and repair of ARF but are yet to be appreciated. Dis- by microarray analysis. Nine genes were up-regulated (ADAM2, HO-1, UCP-2, and thymosin ␤4 in the early phase and clusterin, covering such factors may lead to improved strategies vanin1, fibronectin, heat-responsive protein 12 and FK506 for preventing ARF and treating this serious disorder. binding protein in the established phase), whereas another nine Certain changes seen in the course of ARF involve were down-regulated (glutamine synthetase, cytochrome p450 infiltrating cells and adhesion molecules. Neutrophil in- IId6, and cyp 2d9 in the early phase and cyp 4a14, Xist gene, filtration is induced by adhesion molecules such as inter- PPAR␥, ␣-albumin, uromodulin, and ADH B2 in the estab- lished phase). The identities of these 18 genes were sequence- cellular adhesion molecule-1 (ICAM-1) and E-selectin verified. Changes in gene expression of ADAM2, cyp2d6, fi- via the mediation of tumor necrosis factor-␣ (TNF␣) bronectin, HO-1 and PPAR␥ were confirmed by quantitative and nuclear factor-␬B (NF-␬B). Infiltrating neutrophils real-time polymerase chain reaction (PCR). ADAM2, cyp2d6, induce adhesion molecules such as integrins [12], E-selec- ␥ and PPAR have not previously been known to be involved tin [13], P-selectin [14], intercellular adhesion molecule- in ARF. Conclusion. Using DNA microarray technology, we identi- 1 [8], and neural cell adhesion molecule [15]. Inhibition fied changes in expression of 18 genes during renal ischemia- of NF-␬B using a decoy oligonucleotide has been re- reperfusion injury in the rat. We confirmed changes in five genes ported to suppress the inflammatory response that fol- ␥ (fibronectin, ADAM2, cyp 2d6, HO-1 and PPAR ) by quanti- lows ischemia-reperfusion injury [17]. Animals that ex- tative real-time PCR. Several genes, not previously been identi- fied as playing a role in ischemic ARF, may have importance press aberrant adhesion molecules may be protected in this disease. from renal injury, as noted in ICAM-1–deficient mice that are resistant to ischemic renal injury [18]. Depolarization of epithelial cells occurs in the course of renal injury and repair, in part secondary to changes in adhesion molecules. For example, in ARF induction Key words: microarray, ischemia-reperfusion, gene expression. of transforming growth factor-␤ (TGF-␤), deactivation of integrin, and increased expression of adhesion mole- Received for publication August 16, 2001 and in revised form October 23, 2001 cules is associated with disruption of the cytoskeleton Accepted for publication November 19, 2001 in proximal tubular cells [19]. In this setting the actin  2002 by the International Society of Nephrology microfilament network becomes redistributed into large

1646 Yoshida et al: ARF and DNA microarray 1647 cytoplasmic aggregates [19], which leads to depolariza- tion and exfoliation of epithelial cells. Newly expressed integrin appears to restore cell polarization, accompanied by increased expression of laminin and fibronectin [19]. Ischemic injury results in apoptosis as well as necrosis. Apoptosis is mediated through several signaling cas- cades, one of which is the mitogen-activated protein (MAP) kinase cascade. Members of this family, such as p38MAPK [10], JNK and ERK [11] have been previously reported to play a role in ARF. Park, Chen and Bonven- tre suggested that activation of JNK or p38 is involved in protection against ischemic injury in the kidney [11]. TNF-␣ and integrin also have been reported to mediate Fig. 1. Serum creatinine in sham-operated (᭺) and ischemia-reperfu- sion (᭹) rats at various times. Creatinine rose to a peak at 24 hours in apoptosis [20, 21]. the ischemia-reperfusion rats. Data are expressed as mean Ϯ SEM for DNA microarray technology offers the advantage of 4 rats. analyzing thousands of genes simultaneously, with the potential to uncover new mechanisms of pathogenesis. Thus, microarray analysis coupled with bioinformation could detect changes in genes previously unknown to RNA from animals that showed more prominent creati- participate in renal injury and repair. Such an approach nine changes. Animals were then followed for 1, 2, 3, or ϭ could lead, potentially, to a new paradigm through which 4 days (at least N 3 per group per time point). ARF may be better understood. In the present study, we At sacrifice the kidneys were removed and snap-frozen Ϫ Њ examined changes in gene expression induced by ische- in liquid nitrogen. The samples were stored at 70 C mia-reperfusion injury. A total of 2,100 mouse cDNAs until further study. The serum creatinine level over time spotted onto a glass slide were used for profiling gene ex- is shown in Figure 1, and documents successful induction pression. As expected, we found that many of the changes of ARF. Total RNA was isolated from kidney by Trizol in expression occur in genes that encode proteins pre- (Gibco, Rockville, MD, USA) extraction. viously implicated in ischemia-reperfusion injury. How- High-density DNA microarray preparation ever, and importantly, ADAM2 (a disintegrin domain and ), cytochrome p450 IID6, peroxi- High-density cDNA microarrays were generated by some proliferator-activated receptor gamma (PPAR␥) standard methods [22]: Mouse expressed sequence tags were newly identified to participate in ARF. (ESTs; Research Genetics, Huntsville, AL, USA) were amplified by the polymerase chain reaction (PCR), ac- cording to the manufacturer’s recommendations. Each METHODS sample was run in a 1% agarose gel for quality verifica- Animals and RNA extraction tion (data not shown). The sizes of PCR products ranged Male Wistar rats (200 to 250 g, N ϭ 16 ischemia- from approximately 300 bp to 1.2 kb. DNA microarrays, reperfusion, N ϭ 20 sham) were housed in a 12-hour containing 2,100 distinct ESTs, were printed onto glass light/dark cycle, and allowed free access to food and slides using a pin-style arrayer (BioRobotics, Cambridge, water. Control and treated animals were weight- and UK). The identities of all 18 genes were confirmed by age-matched at the time of initiation of bilateral ischemic DNA sequencing using an ABI PRISM 377 DNA se- injury, and body weights were recorded at initiation and quencer (Applied Biosystems, Foster City, CA, USA) completion of experiments. The animals were allowed performed by the Genome Center staff at Brigham & free access to standard rodent chow and water until over- Women’s Hospital. The sequence was confirmed using night prior to surgery when food (but not water) was BLAST (http://www.ncbi.nlm.nih.gov/BLAST/). removed. Animals were anesthetized intraperitoneally To make microarrays, sialylated glass slides (CEL As- with pentobarbital sodium, 65 mg/kg. Both renal arteries sociates Inc., Houston, TX, USA) were cleaned for two were exposed using a midline incision and then clamped hours in a solution of 2 N NaOH. After rinsing in distilled for 30 minutes followed by reperfusion. Sham animals water, the slides were treated with a 1:5 dilution of poly- had an incision plus 30 minutes of waiting time without l-lysine adhesive solution (Sigma, St. Louis, MO, USA) clamping. After ischemia or sham treatment, muscle for one hour, and then dried for five minutes at 80ЊC layer incisions were sutured, and skin incisions closed in a vacuum oven. DNA samples from 100-␮L PCR with Michel clips. We gave a total of 6 mL of 0.9% saline reactions were precipitated with ethanol in 96-well mi- solution IP to the rats during and after surgery to prevent crotiter plates. The resulting precipitates were resus- dehydration. In the present study, we chose to analyze pended in 20 ␮L3ϫ standard sodium citrate (SSC; 20 ϫ 1648 Yoshida et al: ARF and DNA microarray

SSC; 3.0 mol/L NaCl and 0.3 mol/L sodium citrate) and The cut off was based on at least twofold changes for the transferred to new plates for arraying. After printing, genes of interest. The chips were scanned in a ScanArray the microarrays were rehydrated for 10 seconds in a 5000 confocal laser scanner (GSI Lumonics, Marina Del humidity chamber. The DNA was then ultraviolet-cross- Rey, CA, USA). The signal and background signals were linked to the surface by subjecting the slides to 60 mJ then quantified with ImaGene 4.0 quantification software of energy (Stratagene Stratalinker, La Jolla, CA, USA). (BioDiscovery, Billerica, MA, USA). Signals were mea- Directly after the blocking reaction, the bound DNA sured as the mean pixel intensity within each circumscribed was denatured by a two-minute incubation in distilled spot and average background was measured using the water at ϳ95ЊC. The slides were then transferred into a mean pixel intensities of blank spots in the array (without bath of Ϫ20ЊC ethanol, rinsed, and then spun dry in a cDNA) and as the mean pixel intensity outside the circum- clinical centrifuge. Slides were stored in a closed box at scribed spot diameter for calculating specific signals. room temperature until used. Quantitative real time PCR cDNA probe labeling Quantitation with real-time, one-step reverse-tran- cDNA probes were synthesized as previously reported scriptase polymerase chain reaction (RT-PCR) was per- [23] to improve signal detection on high-density micro- formed with SYBR Green PCR Reagents and an ABI arrays using a fluorescent oligonucleotide dendrimeric PRISM 7700 Sequence Detection System (PE Applied ␮ signal amplification system. Briefly, 10 g of total RNA Biosystems, Foster City, CA, USA), according to the was primed with 1.0 picomole of modified reverse tran- manufacturer’s instructions. Reactions were performed scription (RT) primer with capture sequence for Cy3 or using 1.0 ␮L of RNA at a concentration of 100 ng/␮L, Cy5 (Genisphere, Montvale, NJ, USA). The RT reaction in a reaction volume of 50 ␮L. An RT reaction was was performed according to standard protocols (Super- performed at 48ЊC for 30 minutes, followed by PCR ␮ Script RT II; Gibco), followed by the addition of 3.5 L consisting of AmpliTaq activation for 10 minutes at 95ЊC, of 0.5 mol/L NaOH/50 mmol/L ethylenediaminetetra- then 40 cycles with heating to 95ЊC for 15 seconds, and acetic acid (EDTA), and heating at 65ЊC for ten minutes cooling to 60ЊC for one min. mRNA levels were normal- to denature the DNA/RNA hybrids. This reaction was ized to levels of glyceraldehyde-3-phosphate dehydroge- then neutralized with 5 ␮L of 1 mol/L Tris, pH 7.5. For nase (GAPDH). We elected to confirm changes in five double-labeled detection, RT reactions were mixed and genes using real-time one-step quantitative RT-PCR. precipitated using 3 mol/L sodium acetate and 100% These five genes and PCR primers are as follows: ethanol, then washed once with 70% ethanol. The pellets Fibronectin. were resuspended in 20 ␮L distilled H O. 2 Forward primer, 5Ј-GTGGCTGCCTTCAACTTCTC-3Ј Hybridization Reverse primer, 5Ј-GTGGGTTGCAAACCTTCAAT-3Ј ␮ HO-1. cDNA microarrays were prehybridized with 500 Lof Ј Ј Њ Forward primer, 5 -TCTATCGTGCTCGCATGAAC-3 preheated (65 C) ExpressHyb (Clontech, Palo Alto, CA, Ј Ј USA) for an hour in a microarray hybridization chamber Reverse primer, 5 -CAGCTCCTCAAACAGCTCAA-3 (Grace Bio-Labs, Bend, OR, USA) in a rotisserie hybrid- ADAM2. Ј Ј ization oven. Probe was put into the hybridization cham- Forward primer, 5 -GGGATCTGTTTGGCACTGAT-3 Ј Ј ber and incubated overnight in a rotisserie hybridization Reverse primer, 5 -ATTAGGTGCGCAGTCCTTGT-3 ␥ oven. The next day the microarrays were washed for five PPAR . Ј Ј minutes in 2 ϫ SSC/0.2% sodium dodecyl sulfate (SDS) Forward primer, 5 -TGTGGACCTCTCTGTGATGG-3 Ј Ј at 65ЊC, then in 2 ϫ SSC and 0.2 ϫ SSC for two minutes Reverse primer, 5 -CATTGGGTCAGCTCTTGTGA-3 each. For dendrimer detection, 1.0 ␮L of the Cy3- and Cytochrome P450 IId6. Ј Ј Cy5-labeled dendrimers (Genisphere) were suspended Forward primer, 5 -AGCTTCAACACCGCTATGGT-3 Ј Ј in 15 ␮Lof4ϫ SSC/2% SDS and layered on the array. Reverse primer, 5 -CAGCAGTGTCCTCTCCATGA-3 The arrays were covered with a coverslip and then incu- GAPDH. bated at 65ЊC for eight hours in a microarray hybridiza- Forward primer, 5Ј-ACTCCCATTCCTCCACCTTT-3Ј tion cassette (Telechem International, Sunnyvale, CA, Reverse primer, 5Ј-TTACTCCTTGGAGGCCATGT-3Ј USA). The chips were then washed as above. All primer sets were designed according to manufac- turer’s recommendations, and when feasible, to span one Data collection, normalization, and analysis or more introns. All amplicons ranged between 142 and Each time point was analyzed twice with reverse color 148 base pairs. Electrophoretic analysis of expected labeling. The mean of the two measurements was used product sizes was performed for all primer sets prior to to determine the fold changes. As was stated, dye rever- one step, real time RT-PCR, to confirm the fidelity of sal was used. This analysis uses a log-normal error model. the reaction. Yoshida et al: ARF and DNA microarray 1649

RESULTS days, consistent with other reports [12, 25]. ADAM2 was cDNA microarray analysis up-regulated by 3.3 fold and gradually normalized. Representative differential expression patterns are Genes down-regulated by ischemia-reperfusion injury shown for sham (Fig. 2A) and ischemia-reperfusion sam- Figure 5 shows nine genes that were decreased in IR. ples (Fig. 2B) at day 1 on one glass slide. Ischemia-reper- Of these nine, four genes decreased acutely, including fusion (IR) samples were labeled with Cy3 and sham alpha-albumin protein, cytochrome P450 IID6 (cyp2d6), with Cy5. This figure demonstrates clear signals with cytochrome P450 IId9 (cyp2d9), and glutamine synthe- little background and several instances of clear differen- tase (Fig. 5A). Cyp2d6 was markedly decreased, to ap- tial gene expression. Every spot is well rounded and has proximately 20% of the sham value. virtually the same diameter. Figure 2C shows an overlay Five genes were chronically down-regulated, includ- picture of sham and IR such that green spots indicate ing alcohol dehydrogenase-B2, cytochrome P450 IVa14 up-regulation and red spots indicate down-regulation, (cyp4a14), PPAR␥, Xist gene, and uromodulin (Fig. 5B). and yellow spots indicate no change, respectively. The Uromodulin was decreased to approximately 20 to 25% numbers 1, 2, 3, 4, and 5 indicate clusterin, ADAM2, of the sham value. HO-1, cyp2d6, PPAR␥, respectively. Of these, clusterin, ADAM2, and HO-1 are increased and cyp2d6 and PPAR␥ Confirmation of change in expression are decreased in IR. Microarray data of five genes of interest were con- As shown in Figure 3A, a scatter plot of sham (Cy3) firmed by real-time quantitative PCR (Fig. 6); others versus sham (Cy5) fell on a line of identity. When a scat- were not tested. Fibronectin was chosen because of its ter plot of sham (Cy5) versus IR (Cy3) was performed, well-known increase in ischemic injury [12]. HO-1 was some changes in individual genes were seen (Fig. 3B). selected as increased on gene array and previously de- Red spots, (identified as Adam2, HO-1, and clusterin) scribed to be increased in ARF [24, 26–28]. ADAM2 signify up-regulated genes in IR and green spots (identi- ␥ was chosen as a gene that was increased on gene array, fied as cyp2d6 and PPAR ) signify down-regulated genes. and a previously undescribed finding in ARF. PPAR␥ To confirm these data, reverse labeling was performed. and Cyp 2d6 were chosen as they were decreased on Instead of labeling IR with Cy3 and sham with Cy5, IR gene array, previously undescribed findings in ARF. Us- was labeled with Cy5 and sham with Cy3. In Figure 3C, ing real-time PCR, HO-1 and ADAM 2 were confirmed a scatter plot of this reverse labeling is shown; now green to be transitionally increased whereas cytochrome p450 spots (identified as Adam2, HO-1, and clusterin) signify IId6 and PPAR␥ were decreased in ischemia-reperfu- genes decreased in IR and red spots (identified as cyp2d6 ␥ sion. Thus, there was excellent concordance between and PPAR ) signify transcripts increased in IR. Thus, microarray data and real time PCR, though the magni- these reverse labeling experiments demonstrated consis- tude of changes were more pronounced than those seen tent results. Furthermore, all 18 transcripts altered were with microarray analysis. sequence-verified to confirm their identities.

Genes up-regulated by ischemia-reperfusion injury DISCUSSION Figure 4 shows the nine genes increased in IR. Of these, In the present study, we demonstrated that multiple three increased acutely. These include heme oxygenase-1, gene transcripts were increased or decreased by isch- thymosin ␤4, and uncoupling protein (UCP)-2 (Fig. 4A). emia-reperfusion, and we identified several genes pre- Heme oxygenase-1 (HO-1) increased at day 1 followed viously unknown to be involved in renal injury and re- by normalization, a finding previously reported in a rat pair. For example, it has been previously reported that model of ARF [24]. Thymosin ␤4 (Tb4) increased ap- clusterin [16] and fibronectin [12, 29] are up-regulated proximately twofold. Uncoupling protein (UCP)-2 mRNA by renal ischemia-reperfusion injury, and that HO-1 is level increased by 2.8-fold at day 1 and recovered to up-regulated during ARF [24, 30, 31]. Uromodulin has baseline at day 4. previously been shown to decrease after renal ischemia- Six genes were elevated throughout the four-day time reperfusion injury [32]. course of IR (Fig. 4B). These included ADAM2, clus- The transcript with the largest observed change was terin, FK506 binding protein 1a (Fkbp1a), fibronectin, clusterin, known as a marker for cell injury in the tissues. vanin1, and heat-responsive protein 12 (hrsp12). The ex- In the present studies clusterin was increased from day 1 pression of clusterin (also known as a TRPM-2 or sul- to day 4 after ischemic injury. Clusterin mRNA is overex- fated glycoprotein 2) was increased approximately five- pressed in apoptosis-resistant cells [33] and is required fold and maintained at that level until day 4. Fibronectin for survival of cells treated with TNF␣ or subjected to increased by about 2.5 fold and remained so for four oxidative stress [34–36]. Thus, we infer from these find- 1650 Yoshida et al: ARF and DNA microarray

Fig. 2. Representative differential expression by cDNA array. Representative differential expression is shown by microarray of sham (A, Cy3) and ischemia-reperfusion (B, Cy5) at day 1 on one glass slide. (C) Overlay picture of sham and IR microarray is shown. In this picture, each red spot indicates higher signal in sham and each green spot indicates higher in IR. Numbers denote: (1) clusterin, (2) ADAM2, (3) HO-1, (4) cyp2d6, (5) PPAR␥. Note that the signal of spots numbered 1, 2 and 3 are higher on the right compared to the left, indicating an increase. In contrast, the spots numbered 4 and 5 indicate a decrease in expression.

Fig. 3. Scatter plot graph of microarray data. (A) Sham labeled by Cy3 versus sham labeled by Cy5. All data points are on a 45Њ angle, confirming the accuracy of this technique. (B) Scatter plot of sham labeled by Cy5 versus is- chemia-reperfusion (IR) labeled by Cy3 at day 1 was shown. Red spots, (typed as ADAM2, HO-1, and clusterin) signify genes that are up- regulated and green spots, (typed as cyp2d6 and PPAR␥) signify genes that are down-reg- ulated. (C) Scatter plot of IR labeled by Cy5 vs sham labeled by Cy3 as shown on day 1, as a reverse labeling of scatter plot B. Red spots, (typed as ADAM2, HO-1, and clusterin) sig- nify up-regulated and green spots, (typed as cyp2d6 and PPAR␥), down-regulated. Thus, reverse labeling confirmed the data. Abbrevi- ations are: ADAM2, a disintegrin domain and metalloproteinase; Clu, clusterin; HO1, heme oxygenase-1; Cyp2d6, cytochrome P450 2d6; PPAR␥, peroxisome proliferator activated receptor-gamma. Yoshida et al: ARF and DNA microarray 1651

Fig. 5. Time course of down-regulated genes. Relative fold change of ischemia-reperfusion/sham is shown in time-dependent manner. Abbre- viations are: IR; ischemia-reperfusion; Aalb, alpha-albumin protein; Fig. 4. Time course of up-regulated genes. The relative fold change Adhb2, alcohol dehydrogenase-B2; Cyp2d6, cytochrome P450 2d6; Cyp2d9, cytochrome P450 2d9; Cyp4a14, cytochrome P450, 4a14 of ischemia-reperfusion/sham is shown in a time-dependent manner. ␥ Abbreviations are: IR, ischemia-reperfusion; ADAM2, a disintegrin do- (Cyp4a14); PPAR , peroxisome proliferator activated receptor gamma; Glns-ps1, intronless glutamine synthetase gene; Xist, Xist gene, P0 main and metalloproteinase; Clu, clusterin; Fkbp1a, FK506 binding ᭺ ᭹ protein 1a; Fn, fibronectin; HO1, heme oxygenase-1; Hrsp12, heat- region; Umod, uromodulin. Symbols in A are: ( ) Adhb2; ( ) Cyp4a14; (ᮀ) PPAR␥;(᭿) Xist; (᭝) Umod. Symbols in B are: (᭡) Aalb; (᭛) responsive protein 12; Tmsb4, thymosin beta-4; Ucp2, UCP2; Vnn1, ᭜ ᭺ vanin 1. Symbols in A are: (᭺) HO1; (᭹) Tmsb4; (ᮀ) Ucp2. Symbols Cyp2d6; ( ) Cyp2d9; ( ) Glns-ps1. in B are: (᭿) Adam2; (᭡) Clu; (᭝) Fkbp1a; (᭜) FN; (᭛) Vnn1; (᭺) Hrsp12.

the thick ascending limb of Henle’s loop [37]. Safirstein et al and Bachmann et al reported that uromodulin mRNA ings that clusterin may play role in protection from isch- declined to undetectable levels by 24 hours after renal emia-induced apoptosis in kidney. ischemia-reperfusion [32, 37]. We confirm their observa- In the present study, HO-1 increased at day 1, de- tion on day 1 after ischemia injury. In addition, our data creased gradually at day 2 and normalized by day 4. show that uromodulin gene expression was continuously HO-1 has already been shown to be increased in the inhibited up to day 4 post-injury. kidney in various models of ARF [24, 26–28]. Shimizu The microarray data showed that ADAM2 was in- et al reported that HO-1 conferred a protective effect creased on day 1 after IR injury and gradually returned in ischemic acute renal failure, in that when HO activity to normal on day 4. The real-time PCR confirmed that was blocked by tin mesoporphyrin, increases in micro- there is an increase of ADAM2 on day 1. However, the somal heme concentration, and serum creatinine concen- PCR results further showed no changes or decrease after tration were observed, along with extensive tubular epi- day 2. We do not know the reasons for this discrepancy. thelial cell injury [31]. Nevertheless, the PCR results did confirm that ADAM2 Uromodulin (Tamm-Horsfall protein) was greatly de- was transiently up-regulated on day 1. Novel observa- creased in our model. This protein, one of the most tions included increased ADAM2 in ARF. This gene, abundant in the renal tubule, is exclusively localized to also known as fertilin beta, is a well-characterized mole- 1652 Yoshida et al: ARF and DNA microarray

in re-polarization of renal tubular epithelial cells after IR injury. UCP-2 was increased by IR injury, a previously unre- ported finding. This gene is involved in mitochondrial hydrogen peroxide generation [48]. UCP-2 mRNA has been reported to be up-regulated approximately three- fold in fatty liver, during which hydrogen peroxide pro- duction by mitochondria was increased. It has been speculated that this increase may result from chronic apoptotic stress and may play a role in adaptation to such stress [49]. Li et al reported that UCP-2 is involved in cellular defense against oxidative stress (hydrogen peroxide 200 mmol/L) in clonal beta-cells that over- express UCP-2 [50], further supporting this theory. We speculate that UCP-2 plays an analogous role in renal Fig. 6. Time course of relative fold change of mRNA by real-time quantitative polymerase chain reaction (PCR). Relative fold change of injury. ischemia-reperfusion/sham is shown in time-dependent manner. Abbre- Vanin-1 increased by about 2.7-fold and remained ele- viations are: IR, ischemia-reperfusion; ADAM2, a disintegrin domain vated throughout the study period. Vanin-1, also called and metalloproteinase; Cyp2d6, cytochrome P450 2d6; Fn, fibronectin; HO1, heme oxygenase-1; PPAR␥, peroxisome proliferator activated pantetheinase, hydrolyzes pantethein to cysteamine (a receptor gamma. Symbols are: (᭿) Adam2; (ᮀ) Cyp2d6; (᭜) FN; (᭺) powerful antioxidant) and pantothenic acid [51]. There- ᮀ ␥ HO-1; ( ) PPAR . The values are expressed by log 2. Data are ex- fore, we hypothesize that vanin-1 acts as an endogenous pressed as mean Ϯ SEM; N ϭ 4. anti-oxidant in renal repair after ischemia-reperfusion injury [52]. We noted PPAR␥ to be down-regulated in ischemia- cule that mediates cell-cell communication in sperm-egg reperfusion injury, a novel finding. PPARs (peroxisome interactions. ADAM2 has not previously been known proliferator-activated receptors), a family of transcrip- to be involved in ARF. Interestingly, this protein in- tion factors, have multiple roles that are of great interest. ␣ creases in the central nervous system in cases of Alzhei- Although etomoxir, a PPAR agonist, was reported to mer’s disease, along with alteration in cell-matrix contact protect the kidney in ischemia-reperfusion injury [53], in the brain [38]. We thus infer that this molecule plays a to our knowledge the present work is the first to find ␥ ␥ role in cell-matrix interactions within the kidney. Several changes in PPAR in this model. PPAR is a nuclear members of the ADAM family have the ability to cleave receptor that serves as a transcription factor and thus transmembrane proteins, such as growth factors, as well regulates several physiologic pathways, particularly as their receptors, cell adhesion molecules, and cytokines, those involved in lipid metabolism [54]. Fahmi reported ␥ resulting in soluble forms of those substances, a phenom- that PPAR inhibited interleukin-1 induced matrix met- alloproteinase 13 [54]. Furthermore, Yee et al reported enon that is termed ectodomain shedding [39]. For exam- that the stromelysin-1 (MMP-3) gene, which is involved ple, ADAM-17 (TNF␣ converting , TACE), a in tissue remodeling, contains a PPAR element in its member of this family, cleaves transmembrane pro- promotor [55]. Therefore, it seems likely that PPAR␥ TNF␣ to produce mature form of TNF␣. Tissue inhibitor plays a role in tissue repair, especially in control of MMP. of metalloproteases-3 (TIMP-3) inhibits these proteins Nakajima et al observed that heterozygous PPAR␥-de- that are involved in ectodomain shedding [40–43]. It may ficient mice subjected to intestinal ischemia suffered more be that ADAM2 plays a role as a shedder, during the pronounced injury than control mice [56]. Furthermore, regeneration needed during tissue repair after renal dam- when such mice were treated with a PPAR␥-activating age. Thus, we speculate that ADAM2 plays an important ligand, BRL-49653, they were partially reconstituted and role in repair after tissue injury with in the kidney. suffered less damage. This report supports our hypothesis ␤ We also noted that thymosin 4 (Tb4) was increased that PPAR␥ may act through one of its pathways related in ischemia-reperfusion injury. The beta-thymosins con- to apoptosis and/or tissue repair after ischemic injury. stitute a family of actin monomer-sequestering proteins Cytochrome p450 IId6, IId9, and IVa14, all of which widely distributed among vertebrates, and Tb4 is most are involved with oxidation, were suppressed. When these abundant protein in this family. Tb4 has been reported are active, they lead to consumption of oxygen. to bind to actin monomers and to enhance wound healing Hence, when tissue is subjected to severe ischemic injury, [44–46]. Additionally, it is up-regulated following focal these enzymes are likely suppressed in an attempt to brain ischemia [47]. Our findings lead us to hypothesize minimize ischemic injury. We speculate that these en- that Tb4 has a general role in repair of cytoskeleton and zymes are suppressed during ischemic renal injury in an Yoshida et al: ARF and DNA microarray 1653 attempt to minimize oxidation injury. Suppression of mediated inhibition of renal injury and neutrophil adhesion. Am J Physiol (Renal Physiol) 279:F809–F818, 2000 these cytochrome p450s may occur during the ischemic 9. Sakai M, Tsukada T, Harris RC: Oxidant stress activates AP-1 state to slow down oxidative metabolism, which would and heparin-binding epidermal growth factor-like growth factor lead to cytoprotection. transcription in renal epithelial cells. Exp Nephrol 9:28–39, 2001 10. Faccio L, Chen A, Fusco C, et al: Mxi2, a splice variant of p38 The present analysis of the global gene expression stress-activated kinase, is a distal nephron protein regulated with profile in ischemia-reperfusion used whole kidney. Thus, kidney ischemia. Am J Physiol (Cell Physiol) 278:C781–C790, 2000 the expression pattern within various intrarenal struc- 11. Park KM, Chen A, Bonventre JV: Prevention of kidney ischemia/ reperfusion-induced functional injury and JNK, p38, and MAPK tures is not distinguishable from the whole. It also is pos- kinase activation by remote ischemic pretreatment. J Biol Chem sible that certain of the observations could be the result 276:11870–11876, 2001 of inflammatory cell infiltration or injury to the microvas- 12. Zuk A, Bonventre JV, Brown D, et al: Polarity, integrin, and extracellular matrix dynamics in the postischemic rat kidney. Am culature. Nonetheless, given the early stage and high J Physiol (Cell Physiol) 275:C711–C731, 1998 cost of array technology, we chose not to focus on dis- 13. Singbartl K, Ley K: Protection from ischemia-reperfusion induced sected cell types (such as, glomerulus, proximal tubule, severe acute renal failure by blocking E-selectin. Crit Care Med thick ascending loop of the limb of Henle, distal tubule, 28:2507–2514, 2000 14. Singbartl K, Green SA, Ley K: Blocking P-selectin protects from collecting duct, etc.) in an initial study of renal ischemia- ischemia/reperfusion-induced acute renal failure. FASEB J 14:48– reperfusion. Once routine amplicfication technology is 54, 2000 established, pursuing such a goal will be more feasible. 15. Abbate M, Brown D, Bonventre JV: Expression of NCAM reca- pitulates tubulogenic development in kidneys recovering from In conclusion, using DNA microarray we have identi- acute ischemia. Am J Physiol 277:F454–F463, 1999 fied 18 genes that in mRNA expression lead in the course 16. Schumer M, Colombel MC, Sawczuk IS, et al: Morphologic, bio- of ischemia-reperfusion injury. The identities of these chemical, and molecular evidence of apoptosis during the reperfu- sion phase after brief periods of renal ischemia. Am J Pathol 140: 18 genes were confirmed by DNA sequencing. Changes 831–838, 1992 in five genes (fibronectin, ADAM2, cytochrome p450 IId6, 17. Vos IH, Govers R, Grone HJ, et al: NFkappaB decoy oligode- HO-1 and PPAR␥) were further confirmed by quantita- oxynucleotides reduce monocyte infiltration in renal allografts. FASEB J 14:815–822, 2000 tive real-time PCR. Several genes that have not pre- 18. Kelly KJ, Williams WW, Jr, Colvin RB, et al: Intercellular adhe- viously been identified in ischemic ARF may have impor- sion molecule-1-deficient mice are protected against ischemic renal tant implications concerning the evolution of this disease. injury. J Clin Invest 97:1056–1063, 1996 19. Kellerman PS, Clark RA, Hoilien CA, et al: Role of microfila- ments in maintenance of proximal tubule structural and functional ACKNOWLEDGMENTS integrity. Am J Physiol 259:F279–F285, 1990 This study was supported by National Institutes of Health Grants 20. Daemen MA, van de Ven MW, Heineman E, et al: Involve- DK 50836 (to S.S. Tang), HL 48455 and DK 58950 (to J.R. Ingelfinger), ment of endogenous interleukin-10 and tumor necrosis factor- DK 36031, DK 51606, and DK58849 (to S.R. Gullans). Microarray exper- alpha in renal ischemia-reperfusion injury. Transplantation 67:792– iments were performed in the BWH NIDDK DNA microarray Biotech- 800, 1999 nology Center. 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