Expression of SSAT, a novel biomarker of tubular cell damage,increases in kidney - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-21 13:37 |只看该作者 |倒序浏览 |打印

作者：KamyarZahedi, ZhaohuiWang, SharonBarone, Anne E.Prada, Caitlin N.Kelly, Robert A.Casero, NaokoYokota, Carl W.Porter, HamidRabb,  ManoocherSoleimani,作者单位：1 Division of Nephrology and Hypertension, Department ofPediatrics, Children‘s Hospital Medical Center, Division ofNephrology and Hypertension, Department of Medicine, University ofCincinnati, and Veterans Affairs Medical Center,Cincinnati, Ohio 45267; Division of Nephrology, Departmentof Medi 3 Q1 t) B' }) N( f5 J! }
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      【摘要】+ S; @" b" C, J0 S
   Ischemia-reperfusioninjury (IRI) is the major cause of acute renal failure in native andallograft kidneys. Identifying the molecules and pathways involved inthe pathophysiology of renal IRI will yield valuable new diagnostic andtherapeutic information. To identify differentially regulated genes inrenal IRI, RNA from rat kidneys subjected to an established renal IRIprotocol (bilateral occlusion of renal pedicles for 30 min followed byreperfusion) and time-matched kidneys from sham-operated animals wassubjected to suppression subtractive hybridization. The level ofspermidine/spermine N 1 -acetyltransferase (SSAT)mRNA, an essential enzyme for the catabolism of polyamines, increasedin renal IRI. SSAT expression was found throughout normal kidneytubules, as detected by nephron segment RT-PCR. Northern blotsdemonstrated that the mRNA levels of SSAT are increased by greater thanthreefold in the renal cortex and by fivefold in the renal medulla at12 h and returned to baseline at 48 h after ischemia.The increase in SSAT mRNA was paralleled by an increase in SSAT proteinlevels as determined by Western blot analysis. The concentration ofputrescine in the kidney increased by ~4- and ~7.5-fold at 12 and24 h of reperfusion, respectively, consistent with increasedfunctional activity of SSAT. To assess the specificity of SSAT fortubular injury, a model of acute renal failure from Na depletion (without tubular injury) was studied; SSAT mRNA levels remained unchanged in rats subjected to Na depletion. Todistinguish SSAT increases from the effects of tubular injury vs.uremic toxins, SSAT was increased in cis -platinum-treated animals before the onset of renal failure. The expression of SSAT 10-fold, respectively, in renaltubule epithelial cells subjected to ATP depletion and metabolicpoisoning (an in vitro model of kidney IRI). Our results suggest thatSSAT is likely a new marker of tubular cell injury that distinguishesacute prerenal from intrarenal failure. # n- d8 W, ~2 Y% y2 b+ z1 E9 }
      【关键词】 acute renal failure polyamines putrescine spermidine/spermine N acetyltransferase
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CONDITIONS ASSOCIATED WITH ischemia-reperfusion injury (IRI), such as stroke,myocardial infarction, and acute tubular necrosis, are among the majorcauses of morbidity and mortality. The tissue damage associated withIRI is caused by a temporary cessation of blood flow (ischemia)followed by restoration of blood flow (reperfusion) to a tissue( 6 ). Ischemic conditions lead to ATP depletion andaccumulation of metabolites leading to cellular acidosis as a result ofswitching from aerobic to anaerobic metabolism, whereas reperfusioncauses the production of reactive oxygen intermediates ( 6, 48 ). The aforementioned factors contribute to tissue damageassociated with IRI ( 6, 48 ).

Acute tubular necrosis resulting from IRI is characterized bythe presence of necrotic and apoptotic areas in the epithelium ofseveral nephron segments, including the proximal tubules, and formationof casts in the affected distal tubules ( 6, 36 ). At thecellular level, disruptions in the basement membrane, alteration in thecellular morphology due to disruption of the cytoskeleton and celladhesion components, and structural changes in the microvilli and themitochondria are characteristic of the affected areas ( 26, 27, 36, 38 ). Dedifferentiated and mitotic cells have also beendetected, providing evidence for an ongoing regenerative process( 36, 46 ). Despite significant developments in our understanding of the pathophysiology of renal IRI, there is no specifictherapy for patients, except supportive care. There is a major need forthe development of new therapeutic and diagnostic strategies. It isbelieved that harnessing new technologies and incorporating previousdevelopments in renal IRI will be important in accelerating progresstoward this goal.* g% `3 F0 Q1 R7 b0 Y
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Polyamines (putrescine, spermidine, and spermine) are aliphatic cationsderived from ornithine ( 23, 32 ). They play a fundamental role in the stabilization of DNA structure, modulate gene expression, and regulate protein synthesis, signal transduction, and cell growthand differentiation ( 15, 20, 21, 23 ). Depletion ofpolyamines through addition of inhibitors of their synthesis orenhancement of their catabolism leads to apoptosis ( 13, 31 ).+ g' `  D: Z8 V
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Spermidine/spermine N -acetyltransferase (SSAT), therate-limiting enzyme in polyamine catabolism, is encoded by a singlegene on the X chromosome and is active only in its dimeric form( 8 ). It acetylates spermidine and spermine and regulatestheir catabolism ( 8 ). The transcription and enzymaticactivity of SSAT increase in response to cerebral IRI ( 29, 47 ). SSAT expression can be induced in response to increasedpolyamine levels, reactive oxygen intermediates, interleukin-1, andhepatocyte growth factor ( 7, 10, 40 ). The increase in thelevels of SSAT leads to a decrease in the cellular content ofspermidine and spermine in rat models of cerebral IRI ( 16, 17 ). The enhanced catabolism of spermidine and spermine due toincreased SSAT levels and a concomitant increase in putrescine,aminopropionaldehyde, and H 2 O 2 production mayaccount for the tissue damage associated with cerebral IRI. Thefollowing schematic diagram depicts the polyamine metabolismpathway
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where SSAT is spermidine/spermine N 1 -acetyltransferase, PAO is polyamine oxidase,and SPMS is spermine synthase.

According to this scheme, SSAT activation results in the generation ofacetylated polyamines (acetylspermidine and acetylspermine), which areconverted to putrescine by PAO. Putrescine is generated along withH 2 O 2 and various aldehydes (such asaminobutyraldehyde), which cause apoptosis and cell damage.2 R( i/ d% ^3 \6 W

The biological consequences of enhanced SSAT expression have beenstudied in transgenic mice and rats. Animals that express high levelsof SSAT show a decrease in their tissue spermidine and spermine poolsand an increase in the putrescine and acetylated spermine levels( 1-3, 22, 24, 30, 43 ). Mice overexpressing SSATdevelop follicular cysts in the dermis, permanent hair loss, andexcessive wrinkling of the skin ( 33 ). Overexpression of SSAT in transgenic rats results in acute pancreatitis and an inability to initiate hepatic regeneration after partial hepatectomy ( 2, 3 ), strongly supporting the notion that reduction inspermine/spermidine levels secondary to SSAT overexpression isdetrimental to cell survival and growth.. w! P  V; m- e  K7 M
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Despite information on the activity of SSAT and related molecules incerebral injury, little information is available regarding the role ofthese molecules in renal IRI. During a gene discovery process toidentify novel therapeutic and diagnostic targets in renal IRI usingsuppression subtractive hybridization (SSH), we observed a majorincrease in SSAT mRNA levels (see RESULTS ). The associationof the SSAT increase with tubular injury, but not with a significantrise in uremic toxins, was confirmed in a toxic nephropathy model with cis -platinum. To assess potential diagnostic specificity totubular injury, SSAT levels were compared in a volume depletion modelof acute renal failure (ARF) without tubular injury. To demonstrate thetransferability of these findings to other species, a mouse model ofrenal IRI that also demonstrated SSAT upregulation was used.Furthermore, to elucidate the mechanisms and cellular source underlyingthe increase in SSAT, hypoxia-induced ATP depletion of tubularepithelial cells in vitro defined the tubular epithelial cell as amajor source of SSAT in ARF., g, P3 R7 o6 S; ~  u* }7 T/ b6 V3 A

MATERIALS AND METHODS

Materials. [ 32 P]dCTP was purchased from New England Nuclear (Boston,MA). Nitrocellulose filters and chemicals were purchased from Sigma Chemical (St. Louis, MO). RadPrime DNA labeling kit was purchased fromGIBCO BRL.

Animal models. IRI was induced as previously described ( 37, 38 ). Briefly,bilateral IRI was induced in male Sprague-Dawley rats (200-250 g)or Swiss mice (25-30 g) by occluding the renal pedicles with microvascular clamps for 30 min under ketamine (150 µg/g)-xylazine (3 µg/g) anesthesia. Completeness of ischemia was verified by blanching of the kidneys, signifying termination of blood flow. Theblood flow to the kidneys was reestablished by removal of the clamps(reperfusion) with visual verification of blood return. Animalssubjected to sham operation (identical treatment, except renal pedicleswere not clamped) were used as controls. During the procedure, animalswere well hydrated and their body temperature was controlled at~94°F using an adjustable heating pad. After ischemia,animals were kept under veterinarian observation. At 12, 24, and48 h after ischemia, animals were killed and their kidneyswere harvested. Mice were killed at 2, 12, 24, and 48 h ofreperfusion after 30 min of ischemia. To induce Na depletion, rats (80-120 g) were placed on an Na -freediet for 5 days before euthanasia. The urine Na excretiondecreased from 0.92 ± 0.06 meq/day in normal rats to 0.014 ± 0.005 meq/day in Na -depleted animals during the 24 h before death ( P significantNa depletion. To induce cis -platinum injury,rats (80-120 g) received a single injection of cis -platinum (5 mg/kg body wt ip) and were killed at 1 and 3 days. All animals had free access to water and food. Blood wascollected for blood urea nitrogen (BUN) and creatinine measurement atthe time of death for the above-described experiments.

SSH. The cortex and medulla were separated, snap-frozen in liquid nitrogen,and processed for RNA extraction using the Tri-reagent method asdescribed by Sacchi and Chomczynski ( 39 ). Total RNA fromcontrol rats and rats subjected to IRI was used to make driver andtester cDNA. SSH was performed by using the PCR-select cDNA subtractionkit (Clontech). Subtracted PCR products were ligated into the pGEM-TEasy vector (Promega, WI), and ligation mixtures were transformed intothe DH-5 strain of Escherichia coli (Invitrogen LifeTechnologies, Gaithersburg, MD). Differentially expressed products wereselected by using the PCR-select differential screening kit (Clontech).The cloned products were sequenced, and the results were compared withGenBank database sequences using the BLAST homology search program(National Institutes of Health, Bethesda, MD).
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RNA isolation and Northern hybridization. Total cellular RNA was extracted from renal cortex or medulla using theTri-reagent method (MRC, Cincinnati, OH) following the manufacturer'sprotocol. Total cellular RNA (30 µg/lane) was size fractionated on a1.2% agarose-formaldehyde gel and transferred to nylon membranes bycapillary transfer using 10× sodium chloride-sodium phosphate-EDTAbuffer. Membranes were cross-linked by ultraviolet light or baked.Hybridization was performed according to established methods. Membraneswere washed, blotted dry, exposed to PhosphorImager screens atroom temperature for 24-72 h, and scanned by PhosphorImager. A 32 P-labeled cDNA fragment of the mRNA-encoding SSAT(corresponding to nucleotides 323-892 of a mouse SSAT cDNA;GenBank accession no. NM009121) or PAO (corresponding to nucleotides98-500 of a human PAO cDNA; GenBank accession no. AY033889 ) wasused as a specific probe. For kidney injury molecule-1 (KIM-1), a PCR fragment corresponding to nucleotides 811-1319 of rat KIM-1 cDNA (GenBank accession no. AF035963 ) was used.
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Nephron segment RT-PCR. Nephron segment RT-PCR was performed as described elsewhere ( 42, 45 ). Briefly, single nephron segments [proximal straight tubule(PST), medullary thick ascending limb (mTAL), cortical thick ascendinglimb (cTAL), or cortical collecting duct (CCD)] were dissected fromfreshly killed rat kidney at 4-6°C. The dissection medium (inmM: 140 NaCl, 2.5 K 2 HPO 4, 2 CaCl 2,1.2 MgSO 4, 5.5 D -glucose, 1 sodium citrate, 4 sodium lactate, and 6 L -alanine, pH 7.4) was bubbled with100% oxygen. Tubule length was ~0.6 mm for PST, ~0.7 mm for mTAL,~0.6 mm for cTAL, and ~0.5 mm for CCD. The nephron segments werepooled in a small volume (5-10 µl) of phosphate-buffered saline(PBS) at 4°C in three or four segments per pool. After centrifugationand incubation in lysis solution, the tubules were gently agitated, and1 µl (0.5 µg) of oligo(dT) primer, 1 µl of filteredH 2 O, 4 µl of 5× RT buffer, 2 µl of DTT (0.1 M), and 1 µl of dNTPs (10 mM each) were added. The reaction was equilibrated to42°C for 2 min, and 1 µl of SuperScript II reverse transcriptase (Life Technologies) was added, mixed, and incubated for 1 h at 42°C. Amplification of the SSAT cDNA by the PCR was performed witheach PCR vial containing 10 µl of cDNA, 5 µl of 10× PCR buffer (with 20 mM MgCl 2 ), 1 µl of 10 mM dNTPs, 10 pmol of eachprimer, and 2.5 U of Taq DNA polymerase in a final volume of50 µl. Cycling parameters were as follows: 95°C for 45 s,47°C for 45 s, and 72°C for 2 min. Samples were then sizefractionated on a 1% Tris-acetic acid-EDTA gel and stained withethidium bromide for visualization of the amplified bands.. }6 X7 N' {( C% ~9 F3 d9 ^
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ATP depletion in cultured renal tubule epithelial cells. Cultured Madin-Darby canine kidney (MDCK) cells that are designatedSuper Tube by American Type Culture Collection and were derived from akidney of a normal adult female cocker spaniel were subjected tometabolic poisoning according to established methods( 34 ). Briefly, cells were subjected to 30 min of ATP depletion in Krebs-Henseleit (KH) buffer (in mM: 115 NaCl, 1.3 KH 2 PO 4, 25 NaHCO 3, 1 CaCl 2, 1 MgCl 2, pH 7.4) followed by 2 h of metabolic poisoning in the same buffer containing deoxyglucose (5 mM)and sodium cyanide (5 mM). Cells were then washed twice with KH buffer.Cells were released from metabolic poisoning by incubation in KH buffercontaining 5 mM glucose for 30 min and then washed and incubated ingrowth medium for the duration of the experiment. Cells subjected to invitro IRI and time-matched controls were harvested at timed intervals(4, 12, and 24 h) and processed for RNA or cell extract preparation.+ \1 G9 ]- I( z$ k

Preparation of kidney extracts. Briefly, the tissue samples (cortex or medulla) were homogenized inice-cold isolation solution (250 mM sucrose and 10 mM triethanolamine,pH 7.6) containing protease inhibitors [phenazine methylsulfonylfluoride (0.1 mg/ml) and leupeptin (1 µg/ml)], using a Polytronhomogenizer. The homogenate was centrifuged at low speed (1,000 g ) for 10 min at 4°C to remove nuclei and cell debris.4 p! O$ A) C6 \6 L2 |

Preparation of cell extracts. Cell extracts were prepared by disrupting the cells in RIPA buffer (9.1 mM dibasic sodium phosphate, 1.7 mM monobasic sodium phosphate, 150 mMNaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM sodiumorthovanadate, and 100 µg/ml phenylmethylsulfonyl fluoride containingthe protease inhibitors aprotinin, leupeptin, pepstatin, and antipain).Disrupted cells were centrifuged to separate the cytosolic(supernatant) and nuclear fractions (sediment). Cytosolic fractionswere harvested, and their protein contents were determined bybicinchoninic acid assay (Pierce, Rockford, IL) following themanufacturer's protocol.

Western blot analysis of SSAT expression. Western blot analyses were performed using anti-SSAT antibodypreviously utilized by Fogel-Petrovic et al. ( 11 ).Briefly, 150 µg of protein from each extract were loaded onto anSDS-12% polyacrylamide gel. After size fractionation, proteins weretransferred to a polyvinylidene difluoride membrane in buffercontaining 20 mM Tris, 192 mM glycine, 20% methanol, and 0.1% SDS (pH8.3) using a transfer apparatus (Idea Scientific, Minneapolis, MN) at aconstant power of 400 mA for 2 h. The blocking reaction wasperformed overnight in 5% nonfat dry milk in Tris-buffered saline(TBS; 137 mM NaCl and 20 mM Tris, pH 7.4). Exposure to the anti-SSATprimary antibody (diluted 1:1,000) was carried out in 5% dry milk inTBS for 4 h at room temperature. After the membrane was washedtwice in TBS containing 2.5% dry milk and 0.1% Tween 20 (TTBS) for 10 min each and then once in TBS, it was incubated with a secondaryantibody (goat anti-rabbit IgG peroxidase conjugate diluted 1:2,000 in 2.5% dry milk in TTBS) for 1 h at room temperature. After two 10-min washes in 2.5% dry milk in TTBS and two 10-min washes in TBS,the membrane was developed using peroxidase detection reagents (ECLkit, Amersham, Piscataway, NJ) and exposed for 5-30 s to an X-rayfilm. This antibody is highly specific and detects the SSAT as a~20-kDa band ( 11 ).2 C" u9 @5 r  k3 F0 n; f, b1 d
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Measurement of putrescine concentration in the kidney. Kidneys were harvested at 12 and 24 h of reperfusion and utilizedfor measurement of polyamine concentration by HPLC, as described previously ( 35 ).

Statistical analyses. Values are means ± SE. The significance of difference betweenmean values was examined using ANOVA. P statistically significant." t' E, [& e6 S2 P
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RESULTS

Identification of SSAT by SSH as a transcript that is upregulatedduring renal IRI. Total RNA from the cortical regions of control rat kidneys and ratkidneys subjected to IRI was used to identify differentially expressedtranscripts using SSH (occurrence of tissue damage was determined bythe measurement of BUN and serum creatinine levels; Table 1 ). A number of transcripts, includingSSAT, were identified as being differentially regulated in response toIRI. Other genes that were altered include (but are not limited to)MAP17, stathmin, and mitochondrial transcripts such as those involvedin oxidative reduction and electron transport chain (unpublishedobservations). To confirm the results of SSH, total RNA from the renalcortex and medulla of three control and three animals subjected to IRI (30 min of ischemia-12 h of reperfusion) was size fractionated and subjected to Northern blot analysis using a radiolabeled SSAT cDNAprobe. The results are shown in Fig. 1 and indicate that SSAT mRNA expression is increased by nearly threefoldand nearly fivefold in cortex and medulla, respectively, of kidneyswith IRI ( P P analyses were performed to examine thetime course of expression of SSAT and other enzymes involved inpolyamine catabolism. As shown in Fig. 2, A and B, the mRNA levels of SSAT are the highest in renal cortex and medulla at 12 h of reperfusion( P and return to baseline levels at 48 hof reperfusion. To determine whether the increase in SSAT mRNA alsoreflects an increase in SSAT protein abundance, Western blot analysisof kidney extracts harvested at 12 and 24 h of reperfusion wasperformed. A representative blot shown in Fig. 2 C demonstrates that SSAT protein abundance is increased significantly at12 and 24 h of IRI." X" v$ p+ v- d: o

Table 1. BUN and serum creatinine levels in animal models of acute renal failure

Fig. 1. Northern blot analysis of spermidine/spermine N 1 -acetyltransferase (SSAT) mRNA expression incortical and medullary areas of control andischemia-reperfusion-injured (IRI) kidneys. Total RNA (30 µg/well) from renal cortex ( A ) and medulla ( B )of 3 sham-operated animals and 3 animals subjected to IRI (12 h ofreperfusion) was size fractionated and subjected to Northern blotanalysis using radiolabeled SSAT cDNA probe. C : quantitativeanalyses of results in A and B. Equal loading wasconfirmed by examination of 28S rRNA bands ( A and B,bottom ). A single ~1.3-kb band was recognized by SSAT probe( A and B, top ).5 ?" M3 b& R& P  m6 X
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Fig. 2. Time course of expression of enzymes involved in polyaminecatabolism during IRI. Northern blot analyses were performed (30 µg/well of total RNA) to examine time course of expression of SSATand polyamine oxidase (PAO) in IRI. A and B : mRNAlevels of SSAT in renal cortex and medulla, respectively. Expression ofSSAT increased significantly at 12 h of reperfusion in cortex andmedulla ( P P C :representative Western blot analysis of SSAT on IRI samples at 12 and24 h of reperfusion. Expression of SSAT is enhanced significantlyin IRI compared with sham animals. D : mRNA levels of PAO inrenal cortex and medulla. E : for quantitative analysis, mRNAlevels for PAO were measured in 3 control and 3 IRI medulla samples.Expression of PAO increased significantly at 12 h of reperfusion( P P A, B,D, and E, bottom ).- L2 _  F, E- H+ b4 B) K" ]0 F

Degradation of polyamines by SSAT results in increased acetylatedlevels of spermidine and spermine, which are excreted or oxidized byPAO to putrescine, the product that is responsible for tissue damage inseveral biological systems. In the next series of experiments, theexpression levels of PAO in IRI were examined by Northernhybridization. As shown in a representative blot in Fig. 2 D,expression of PAO in the cortex and medulla was very low at baselinebut was increased considerably at 12 h of IRI and remainedelevated at 48 h of IRI. The upregulation of PAO was of a highermagnitude in the medulla than in the renal cortex. Figure 2 E shows the expression of PAO in kidney medulla of three control animalsand three animals subjected to IRI (30 min of ischemia-12 h ofreperfusion). PAO expression is increased considerably in kidneyssubjected to IRI.
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Measurement of polyamine levels in kidney samples and urine. To determine whether enhanced expression of SSAT is associated withincreased activity of this enzyme, the concentration of polyamines inkidney samples was measured by HPLC. Putrescine concentration wassignificantly increased at 12 and 24 h of reperfusion comparedwith sham-operated animals, consistent with increased SSAT activity inIRI (Fig. 3 ). Putrescine concentrationincreased by ~4.0- and ~7.5-fold at 12 and 24 h ofreperfusion, respectively, compared with sham-operated controls( P P control).

Fig. 3. Polyamine concentration in kidneys with IRI. Putrescineconcentration () was measured by HPLC in whole kidneysamples at 12 and 24 h of reperfusion and compared with that insham-operated animals.

Urine samples from animals subjected to 30 min of ischemia and12 (or 24) h of reperfusion were collected and processed for polyaminelevel measurement by HPLC. The urine samples were collected at 3-hintervals. For 12 h of reperfusion, the urine samples that werecollected 9-12 h after reperfusion were assayed. For 24 h, the urine samples that were collected 21-24 h after reperfusion were assayed. The results indicated that the urinary concentration ofputrescine was not significantly different between sham and IRIanimals. The urine putrescine concentration was 332 ± 12 and 285 ± 23 µmol/l in sham animals and animals with IRI at 12 h of reperfusion, respectively ( P 0.05, n = 3). At 12 h of reperfusion, the urinaryspermidine concentration decreased (69 ± 8 vs. 239 ± 15 µmol/l in IRI vs. sham, respectively, P n = 3), but the concentration of spermine, although itwas lower, did not achieve statistical significance (12.1 ± 2.1 vs. 16.1 ± 2.6 µmol/l in IRI vs. sham, respectively, P 0.05, n = 3). Similar to the 12 h of reperfusion, the urinary concentration of putrescine and spermine remained unchanged, whereas the concentration of spermidine decreased at 24 h of reperfusion (data not shown).
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Nephron segment RT-PCR of SSAT. Previous studies indicate that SSAT mRNA in the kidney is exclusivelyexpressed in the epithelium of the distal convoluted and straighttubules tubules ( 4, 5 ). Because the aforementioned resultsdid not match our Northern blot analysis, site-specific expression ofSSAT was examined using nephron segment RT-PCR. Figure 4 demonstrates that SSAT mRNA transcriptis expressed in CCD, cTAL, mTAL, and PST, indicating widespreaddistribution of SSAT in kidney tubules.

Fig. 4. Nephron segment RT-PCR of SSAT. Total RNA from variousnephron segments was subjected to RT-PCR, and samples were sizefractionated on a 1% Tris-acetic acid-EDTA gel and stained withethidium bromide for visualization of amplified bands. SSAT mRNAtranscript is expressed in cortical collecting duct (CCD), corticalthick ascending limb (cTAL), medullary thick ascending limb (mTAL), andproximal straight tubule (PST), indicating widespread distribution ofSSAT in kidney tubules.

Expression of SSAT in a model of kidney failure without tubularinjury. IRI is associated with severe cell injury and renal failure ( 6, 26, 27, 36, 38, 46 ). To determine whether enhanced expression ofSSAT was due to tissue damage secondary to IRI or accumulation ofnitrogenous wastes as a consequence of renal dysfunction, a model ofrenal failure secondary to kidney hypoperfusion was utilized. Inaddition, a common clinical dilemma is to distinguish tubular injuryfrom volume depletion as the cause of renal failure, and specificmarkers are needed. Rats were subjected to Na depletionfor 5 days. ARF was verified by increases in serum BUN and creatinineat the time of euthanasia (Table 1 ). The SSAT mRNA levels did notchange in kidneys of Na -depleted rats (1.01- vs. 1.04-foldin Na depletion vs. control, P 0.05, n = 3; Fig. 5 A ). These results indicatethat renal failure per se does not increase the expression of IRI.' ^& o# [/ Q+ H. B1 m  Q+ W$ U  z

Fig. 5. Expression of SSAT in a model of kidney failure withouttubular injury (Na depletion) and a model of tubularinjury without kidney failure ( cis -platinum nephrotoxicity).Total RNA (30 µg/well) from renal cortex of Na -depletedrats ( A ) and rats treated with cis -platinum( cis -plat, B ) was size fractionated and subjectedto Northern blot analysis using radiolabeled SSAT cDNA probe. Equalloading was confirmed by examination of 28S rRNA bands ( A and B, bottom ). A single ~1.3-kb band was recognized bySSAT probe ( A and B, top ). SSAT expressionremained unchanged in Na depletion ( P 0.05, n = 3). C : quantitative analysis ofresults of cis -platinum treatment.2 K$ n8 R7 d  u6 X

Expression of SSAT in a model of tubular injury without kidneyfailure. As an alternative approach to distinguish the SSAT increase fromtubular injury as opposed to renal failure and an increase in uremictoxins, we evaluated a toxic nephropathy model. In this series ofexperiments, rats were treated with cis -platinum and killed1 and 3 days later. SSAT mRNA levels are increased 1 day after cis -platinum treatment ( P n = 3) and remained elevated 3 days after treatment( P n = 3; see Fig. 5 C for quantitative analysis of results). BUN and creatininewere normal 1 day after cis -platinum injection and weremildly increased 3 days after cis -platinum injection (Table 1 ). These results indicate that SSAT expression is increased before theonset of renal failure and correlates with cell injury. Sustainedelevation of SSAT mRNA levels 3 days after cis -platinuminjection suggests that ongoing cell injury persists at this later timepoint after a single injection." L$ a$ F7 n* ]* f1 H/ g& v

Expression of SSAT in an in vitro model of kidney IRI. The purpose of the next series of experiments was to examine themechanisms underlying the increase in SSAT, as well as to confirm itstubular epithelial cell source. Expression of SSAT in cultured renalcells subjected to metabolic poisoning (see MATERIALS AND METHODS ), a model of in vitro kidney IRI, was used. MDCK cellswere subjected to 30 min of ATP depletion and 2 h of metabolicpoisoning "ischemia" and examined 2, 6, 12, 24, and 36 h after ATP repletion "reperfusion." SSAT expression was enhanced as early as 2 h and remained above background levels up to 24 h after ATP depletion, with mRNA levels increasing by ~3.5-fold compared with control ( P n = 3; Fig. 6 A ). This observation was further confirmed using Western blot analysis. Our results 10-fold as early as 2 hafter release from ATP depletion and metabolic poisoning. SSAT proteinlevels peak at ~24 h after release and by 36 h start to decline(Fig. 6 B ). Cells subjected to metabolic poisoning showed 6, 10, and 21% death rate as determined by trypan blue exclusion 2, 6, and 24 h after metabolic poisoning, respectively (cells that diedimmediately at the end of metabolic poisoning and before the beginningof reperfusion were not included in the death rate calculation).
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Fig. 6. Expression of SSAT in an in vitro model of kidney IRI.Cultured Madin-Darby canine kidney cells were subjected to 30 min ofATP depletion and 2 h of metabolic poisoning (ischemia)and examined at 2, 6, 24, and 36 h after ATP repletion(reperfusion). A : total RNA (10 µg/well) from IRI andtime-matched control samples was subjected to Northern blot analysisusing radiolabeled SSAT cDNA probe. Equal loading was confirmed byexamination of 28S rRNA bands ( bottom ). A single ~1.3-kbband was recognized by SSAT probe ( top ). SSAT expressionincreased significantly at 2, 6, and 24 h after ATP repletion( P B 10-fold at 2 hafter ATP repletion, remained elevated at 24 h, and started todecrease by 36 h.4 `, W" B6 \4 X. g. J/ c$ I. p; u

Expression of SSAT in mice subjected to kidney IRI. In the next series of experiments, expression of SSAT was examined in amouse model of IRI and compared with another well-characterized markerof renal cell injury, KIM-1. Mice were subjected to 30 min of kidneyischemia followed by 2, 12, 24, or 48 h of reperfusion. The results demonstrate that SSAT expression in the kidney increased asearly as 2 h after reperfusion. Expression of SSAT peaked by 12 h after reperfusion (Fig. 7, top ). In contrast, KIM-1 expression was not detected in theearly stages of reperfusion (Fig. 7, middle ). Enhancedlevels of KIM-1 were observed at 12 h and were strongly induced by24 h after reperfusion (Fig. 7, middle ). The levels ofboth transcripts were lower but still above background 48 h afterreperfusion. These results indicate that enhanced expression of SSAT isan early event in IRI and precedes the upregulation of KIM-1.

Fig. 7. Expression of SSAT and kidney injury molecule-1 (KIM-1)in mice subjected to kidney IRI. Mice were subjected to 30 min ofkidney ischemia followed by 2, 12, 24, or 48 h ofreperfusion. Representative Northern blots ( top ) demonstratethat SSAT expression in kidney increased as early as 2 h afterreperfusion. Expression of SSAT peaked by 12 h after reperfusion( top ). In contrast, KIM-1 expression was not detected at2 h of reperfusion in the same membranes that were stripped andreprobed for KIM-1 ( middle ). Levels of KIM-1 were enhancedat 12 h and peaked at 24 h of reperfusion( middle ). Levels of SSAT and KIM-1 transcripts were lowerbut still above background at 48 h after reperfusion. Equalloading was confirmed by examination of 28S rRNA bands( bottom ). Expression of SSAT increased at 2, 12, 24, and48 h of reperfusion ( P n = 3 for each group). Expression of KIM-1 increased at12, 24, and 48 h of reperfusion ( P n = 3 for each group).5 u* t" W7 c6 z/ }% Z7 j
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The results of the present experiments demonstrate that SSAT mRNAlevels in kidney IRI increased at 12 h of reperfusion and returnedto baseline at 48 h of reperfusion (Figs. 1 and 2 ). Similar findings were observed in a mouse model of renal IRI (data not shown).Enhanced expression of SSAT was associated with increased putrescineconcentration in kidney samples (Fig. 3 ), confirming increasedfunctional activity of SSAT. Nephron segment RT-PCR was used todemonstrate that SSAT is expressed in the PST, mTAL, cTAL, and CCD(Fig. 4 ). Taken together with previous results by Bettuzzi et al.( 4, 5 ), our results indicate that SSAT is widely expressedin the kidney. Kidney SSAT mRNA levels remained unchanged in ratssubjected to Na depletion (renal failure with no tubularinjury) but increased in cis -platinum-treated animals beforethe onset of renal failure (tubular cell injury alone; Fig. 4 ).Subjecting cultured renal tubule MDCK cells to ATP depletion (an invitro model of kidney IRI) increased the expression of SSAT (Fig. 6 ).7 K  b3 ?4 E) @, `

A large body of evidence demonstrates a fundamental role for cellularpolyamines in cell proliferation. As a result, enzymes that alter thecellular concentration of polyamines have come under intenseinvestigation for their role in regulating the cell growth. Enhancedexpression of SSAT by a spermine analog in the breast cancer cell lineL56Br-C1 resulted in the depletion of the cellular pools of polyaminewithin 24-48 h ( 18 ). Cell proliferation appeared tobe totally inhibited, and within 48 h of treatment, there was anextensive apoptotic response. In Ehrlich ascites tumor cells,treatment with the antitumor drug 1'-acetoxychavicol acetate resultedin increased activity of SSAT with subsequent lowering of intracellularpolyamines ( 25 ). Apoptosis immediately followed.Administration of exogenous polyamines prevented 1'-acetoxychavicol acetate-induced apoptosis. A similar observation has beendocumented in human leukemic cells, where depletion of intracellularpolyamines secondary to the overexpression of SSAT resulted indecreased cell growth and caused the induction of apoptosis( 12 ). Taken together, these studies indicate that enhancedexpression of SSAT depletes the cellular polyamine pools, decreasescell growth, and leads to apoptosis.& Q/ b7 M' v" Z2 X

Intracellular polyamines in the brain are very sensitive to variouspathological states and are perturbed in central nervous system injury.Transient focal cerebral ischemia followed by reperfusion increases SSAT expression and activity in the rat, thereby resulting indecreased spermidine/spermine and increased putrescine concentration inthe affected neurons ( 29, 47 ). SSAT expression peaked at 12-18 h of reperfusion. The SSAT expression remained unchanged onthe contralateral side, indicating that SSAT upregulation is a specificand sensitive marker for ischemic injury. Restoration ofspermidine level and reduction in putrescine concentration with thehelp of a PAO inhibitor (MDL-72527) reduced the tissue edema andinfarct size in cerebral ischemia ( 16, 17 ). Taken together, these results indicate that SSAT is upregulated inischemic brain injury and may be detrimental to cell viability.) q& A% p9 v/ Z- j6 ?% k6 N. \2 O
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The most salient feature of the present studies is the detection ofenhanced SSAT expression and activity in kidney IRI (Figs. 1-3 ).The induction of SSAT at 12 h and its return to baseline levels at48 h of reperfusion correlate with the peak onset of tubular injury and recovery from the injury, respectively. Furthermore, theinduction of PAO, the final enzyme in the degradation of cellular polyamines and generation of putrescine, at 12 and 24 h of IRI (Fig. 2, C and D ) strongly suggests that theessential enzymes in the polyamine catabolic pathway are operating inunison. Increased putrescine concentration in kidneys (Fig. 3 ) confirmsincreased functional activity of these two enzymes in IRI and isconsistent with the upregulation of their proteins. With regard to thelevels of polyamines in the urine, we found that spermidineconcentration decreased but the levels of putrescine and spermineremained unchanged at 12 and 24 h of reperfusion. The levels ofputrescine in the urine do not correlate with increased levels of thischemical in kidney homogenates in IRI. Reductions in glomerularfiltration rate at 12 and 24 h of reperfusion (Table 1 ), as wellas possible alteration in the expression of membrane-bound polyaminetransporter(s), may independently affect the concentration ofpolyamines in the urine, making the interpretation of urine polyaminelevels in IRI difficult.$ ^8 E; G, @3 _

A recent report identified KIM-1 as a biomarker of kidney injury( 14 ). KIM-1 expression increased at 24 h and remainedelevated at 48 h of reperfusion ( 19 ), a patterndistinct from SSAT expression. On the basis of functional andstructural studies indicating improvement in kidney function andinitiation of repair in damaged tubules at 48 h of reperfusion, wepropose that SSAT is an early biomarker of tubule injury, whereas KIM-1is a closer marker of the extension phase and recovery from injury inkidney IRI. In support of this conclusion, we observe that enhancedexpression of SSAT precedes the upregulation of KIM-1 in renal IRI. Inthis regard, the upregulation of SSAT is similar to that of CYR61, anangiogenic cysteine-rich protein ( 28 ). The upregulation ofkidney SSAT expression in rats treated with cis -platinum(Fig. 5 ) and before the onset of renal failure strongly supports thevalue of this enzyme in the detection of cell injury. Our presentresults strongly suggest that cell injury precedes the onset of renalfailure in response to cis -platinum treatment.
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Induction of ATP depletion in cultured renal cells has been used as anin vitro model of kidney cell injury ( 41, 44 ). However,and aside from distortions in cytoskeletal structure and alteration inthe expression of stress-related genes, no specific biomarker of kidneycell injury has been identified in this model of kidney IRI. Enhancedexpression of SSAT (Fig. 6 ) indicates that monitoring the expression ofthis enzyme can be used as an indicator of the state of injury incultured kidney tubules subjected to ATP depletion. Stableoverexpression of SSAT in cultured Chinese hamster ovary cells causedperturbations in polyamine homeostasis and led to a reduction in therate of growth ( 9 ). Whether enhanced expression of SSAT incultured renal cells similarly depletes intracellular polyamine poolsand decreases growth in cells subjected to ATP depletion remains to be seen.6 p' l' N' v, O9 O8 X1 d6 O5 t) i
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In conclusion, SSAT expression increases in kidneys of rats subjectedto IRI. Enhanced expression of SSAT reflects the state of cell injuryand is not due to accumulation of the uremic toxins that results fromrenal failure. We propose that SSAT is an early marker of kidney cellinjury, and its level of expression could be used as a diagnostic toolin the early phase of renal IRI. Future experiments are planned toelucidate the role of SSAT at the molecular level in in vitro and invivo models of renal IRI, as well as to characterize the diagnosticutility of measuring SSAT levels in blood and urine.( {1 ?9 w" b$ v4 S; z" V. i
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NOTE ADDED IN PROOF
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After this manuscript had been accepted, the cloning of a newPAO was reported (Vujcic S, Liang P, Diegelman P, Kramer DL, and PorterCW). Genomic identification and biochemical characterization of themammalian PAO involved polyamine back-conversion ( Biochem J 370: 19-28, 2003). The new PAO may play an important role in thegeneration of putrescine. Studies are underway to examine theregulation of the new PAO in IRI.3 `) c! s+ W/ b
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ACKNOWLEDGEMENTS# E( c. T% d4 p' y% L, U# B

These studies were supported by National Institute of Diabetes andDigestive and Kidney Diseases Grants DK-54220 (M. Soleimani) andDK-54770 (H. Rabb), a grant from the Greater Cincinnati Kidney Foundation (K. Zahedi), a Merit Review Award (M. Soleimani), a NationalKidney Foundation Clinical Scientist Award (H. Rabb), and grants fromDialysis Clinic Incorporated (M. Soleimani).9 n" t4 f- x* o8 i
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