Expression of SSAT, a novel biomarker of tubular cell damage, increases in kidney ischemia-reperfusion injury

Kamyar Zahedi1,*, Zhaohui Wang2,*, Sharon Barone2, Anne E. Prada1, Caitlin N. Kelly1, Robert A. Casero3, Naoko Yokota4, Carl W. Porter5, Hamid Rabb4, and Manoocher Soleimani2,6

1 Division of Nephrology and Hypertension, Department of Pediatrics, Children's Hospital Medical Center, 2 Division of Nephrology and Hypertension, Department of Medicine, University of Cincinnati, and 6 Veterans Affairs Medical Center, Cincinnati, Ohio 45267; 4 Division of Nephrology, Department of Medicine, and 3 Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21218; and 5 Roswell Park Cancer Institute, Buffalo, New York 14263


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Ischemia-reperfusion injury (IRI) is the major cause of acute renal failure in native and allograft kidneys. Identifying the molecules and pathways involved in the pathophysiology of renal IRI will yield valuable new diagnostic and therapeutic information. To identify differentially regulated genes in renal IRI, RNA from rat kidneys subjected to an established renal IRI protocol (bilateral occlusion of renal pedicles for 30 min followed by reperfusion) and time-matched kidneys from sham-operated animals was subjected to suppression subtractive hybridization. The level of spermidine/spermine N1-acetyltransferase (SSAT) mRNA, an essential enzyme for the catabolism of polyamines, increased in renal IRI. SSAT expression was found throughout normal kidney tubules, as detected by nephron segment RT-PCR. Northern blots demonstrated that the mRNA levels of SSAT are increased by greater than threefold in the renal cortex and by fivefold in the renal medulla at 12 h and returned to baseline at 48 h after ischemia. The increase in SSAT mRNA was paralleled by an increase in SSAT protein levels as determined by Western blot analysis. The concentration of putrescine in the kidney increased by ~4- and ~7.5-fold at 12 and 24 h of reperfusion, respectively, consistent with increased functional activity of SSAT. To assess the specificity of SSAT for tubular 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. To distinguish 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 mRNA and protein increased by ~3.5- and >10-fold, respectively, in renal tubule epithelial cells subjected to ATP depletion and metabolic poisoning (an in vitro model of kidney IRI). Our results suggest that SSAT is likely a new marker of tubular cell injury that distinguishes acute prerenal from intrarenal failure.

acute renal failure; polyamines; putrescine; spermidine/spermine N1-acetyltransferase


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

CONDITIONS ASSOCIATED WITH ischemia-reperfusion injury (IRI), such as stroke, myocardial infarction, and acute tubular necrosis, are among the major causes of morbidity and mortality. The tissue damage associated with IRI 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 and accumulation of metabolites leading to cellular acidosis as a result of switching from aerobic to anaerobic metabolism, whereas reperfusion causes the production of reactive oxygen intermediates (6, 48). The aforementioned factors contribute to tissue damage associated with IRI (6, 48).

Acute tubular necrosis resulting from IRI is characterized by the presence of necrotic and apoptotic areas in the epithelium of several nephron segments, including the proximal tubules, and formation of casts in the affected distal tubules (6, 36). At the cellular level, disruptions in the basement membrane, alteration in the cellular morphology due to disruption of the cytoskeleton and cell adhesion components, and structural changes in the microvilli and the mitochondria are characteristic of the affected areas (26, 27, 36, 38). Dedifferentiated and mitotic cells have also been detected, providing evidence for an ongoing regenerative process (36, 46). Despite significant developments in our understanding of the pathophysiology of renal IRI, there is no specific therapy for patients, except supportive care. There is a major need for the development of new therapeutic and diagnostic strategies. It is believed that harnessing new technologies and incorporating previous developments in renal IRI will be important in accelerating progress toward this goal.

Polyamines (putrescine, spermidine, and spermine) are aliphatic cations derived 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 growth and differentiation (15, 20, 21, 23). Depletion of polyamines through addition of inhibitors of their synthesis or enhancement of their catabolism leads to apoptosis (13, 31).

Spermidine/spermine N-acetyltransferase (SSAT), the rate-limiting enzyme in polyamine catabolism, is encoded by a single gene on the X chromosome and is active only in its dimeric form (8). It acetylates spermidine and spermine and regulates their catabolism (8). The transcription and enzymatic activity of SSAT increase in response to cerebral IRI (29, 47). SSAT expression can be induced in response to increased polyamine levels, reactive oxygen intermediates, interleukin-1, and hepatocyte growth factor (7, 10, 40). The increase in the levels of SSAT leads to a decrease in the cellular content of spermidine and spermine in rat models of cerebral IRI (16, 17). The enhanced catabolism of spermidine and spermine due to increased SSAT levels and a concomitant increase in putrescine, aminopropionaldehyde, and H2O2 production may account for the tissue damage associated with cerebral IRI. The following schematic diagram depicts the polyamine metabolism pathway


View larger version (14K):
[in this window]
[in a new window]
 


where SSAT is spermidine/spermine N1-acetyltransferase, PAO is polyamine oxidase, and SPMS is spermine synthase.

According to this scheme, SSAT activation results in the generation of acetylated polyamines (acetylspermidine and acetylspermine), which are converted to putrescine by PAO. Putrescine is generated along with H2O2 and various aldehydes (such as aminobutyraldehyde), which cause apoptosis and cell damage.

The biological consequences of enhanced SSAT expression have been studied in transgenic mice and rats. Animals that express high levels of SSAT show a decrease in their tissue spermidine and spermine pools and an increase in the putrescine and acetylated spermine levels (1-3, 22, 24, 30, 43). Mice overexpressing SSAT develop follicular cysts in the dermis, permanent hair loss, and excessive 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 in spermine/spermidine levels secondary to SSAT overexpression is detrimental to cell survival and growth.

Despite information on the activity of SSAT and related molecules in cerebral injury, little information is available regarding the role of these molecules in renal IRI. During a gene discovery process to identify novel therapeutic and diagnostic targets in renal IRI using suppression subtractive hybridization (SSH), we observed a major increase in SSAT mRNA levels (see RESULTS). The association of the SSAT increase with tubular injury, but not with a significant rise in uremic toxins, was confirmed in a toxic nephropathy model with cis-platinum. To assess potential diagnostic specificity to tubular injury, SSAT levels were compared in a volume depletion model of acute renal failure (ARF) without tubular injury. To demonstrate the transferability of these findings to other species, a mouse model of renal IRI that also demonstrated SSAT upregulation was used. Furthermore, to elucidate the mechanisms and cellular source underlying the increase in SSAT, hypoxia-induced ATP depletion of tubular epithelial cells in vitro defined the tubular epithelial cell as a major source of SSAT in ARF.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. [32P]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 from GIBCO 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. The blood flow to the kidneys was reestablished by removal of the clamps (reperfusion) with visual verification of blood return. Animals subjected to sham operation (identical treatment, except renal pedicles were not clamped) were used as controls. During the procedure, animals were 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, and 48 h after ischemia, animals were killed and their kidneys were harvested. Mice were killed at 2, 12, 24, and 48 h of reperfusion after 30 min of ischemia. To induce Na+ depletion, rats (80-120 g) were placed on an Na+-free diet for 5 days before euthanasia. The urine Na+ excretion decreased 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 < 0.05), indicating significant Na+ 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 was collected for blood urea nitrogen (BUN) and creatinine measurement at the 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 as described by Sacchi and Chomczynski (39). Total RNA from control rats and rats subjected to IRI was used to make driver and tester cDNA. SSH was performed by using the PCR-select cDNA subtraction kit (Clontech). Subtracted PCR products were ligated into the pGEM-T Easy vector (Promega, WI), and ligation mixtures were transformed into the DH-5alpha strain of Escherichia coli (Invitrogen Life Technologies, Gaithersburg, MD). Differentially expressed products were selected by using the PCR-select differential screening kit (Clontech). The cloned products were sequenced, and the results were compared with GenBank database sequences using the BLAST homology search program (National Institutes of Health, Bethesda, MD).

RNA isolation and Northern hybridization. Total cellular RNA was extracted from renal cortex or medulla using the Tri-reagent method (MRC, Cincinnati, OH) following the manufacturer's protocol. Total cellular RNA (30 µg/lane) was size fractionated on a 1.2% agarose-formaldehyde gel and transferred to nylon membranes by capillary transfer using 10× sodium chloride-sodium phosphate-EDTA buffer. Membranes were cross-linked by ultraviolet light or baked. Hybridization was performed according to established methods. Membranes were washed, blotted dry, exposed to PhosphorImager screens at room temperature for 24-72 h, and scanned by PhosphorImager. A 32P-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 nucleotides 98-500 of a human PAO cDNA; GenBank accession no. AY033889) was used 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.

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 ascending limb (cTAL), or cortical collecting duct (CCD)] were dissected from freshly killed rat kidney at 4-6°C. The dissection medium (in mM: 140 NaCl, 2.5 K2HPO4, 2 CaCl2, 1.2 MgSO4, 5.5 D-glucose, 1 sodium citrate, 4 sodium lactate, and 6 L-alanine, pH 7.4) was bubbled with 100% 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 were pooled in a small volume (5-10 µl) of phosphate-buffered saline (PBS) at 4°C in three or four segments per pool. After centrifugation and incubation in lysis solution, the tubules were gently agitated, and 1 µl (0.5 µg) of oligo(dT) primer, 1 µl of filtered H2O, 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 to 42°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 with each PCR vial containing 10 µl of cDNA, 5 µl of 10× PCR buffer (with 20 mM MgCl2), 1 µl of 10 mM dNTPs, 10 pmol of each primer, and 2.5 U of Taq DNA polymerase in a final volume of 50 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 size fractionated on a 1% Tris-acetic acid-EDTA gel and stained with ethidium bromide for visualization of the amplified bands.

ATP depletion in cultured renal tubule epithelial cells. Cultured Madin-Darby canine kidney (MDCK) cells that are designated Super Tube by American Type Culture Collection and were derived from a kidney of a normal adult female cocker spaniel were subjected to metabolic 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 KH2PO4, 25 NaHCO3, 1 CaCl2, 1 MgCl2, 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 buffer containing 5 mM glucose for 30 min and then washed and incubated in growth medium for the duration of the experiment. Cells subjected to in vitro IRI and time-matched controls were harvested at timed intervals (4, 12, and 24 h) and processed for RNA or cell extract preparation.

Preparation of kidney extracts. Briefly, the tissue samples (cortex or medulla) were homogenized in ice-cold isolation solution (250 mM sucrose and 10 mM triethanolamine, pH 7.6) containing protease inhibitors [phenazine methylsulfonyl fluoride (0.1 mg/ml) and leupeptin (1 µg/ml)], using a Polytron homogenizer. The homogenate was centrifuged at low speed (1,000 g) for 10 min at 4°C to remove nuclei and cell debris.

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 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM sodium orthovanadate, and 100 µg/ml phenylmethylsulfonyl fluoride containing the protease inhibitors aprotinin, leupeptin, pepstatin, and antipain). Disrupted cells were centrifuged to separate the cytosolic (supernatant) and nuclear fractions (sediment). Cytosolic fractions were harvested, and their protein contents were determined by bicinchoninic acid assay (Pierce, Rockford, IL) following the manufacturer's protocol.

Western blot analysis of SSAT expression. Western blot analyses were performed using anti-SSAT antibody previously utilized by Fogel-Petrovic et al. (11). Briefly, 150 µg of protein from each extract were loaded onto an SDS-12% polyacrylamide gel. After size fractionation, proteins were transferred to a polyvinylidene difluoride membrane in buffer containing 20 mM Tris, 192 mM glycine, 20% methanol, and 0.1% SDS (pH 8.3) using a transfer apparatus (Idea Scientific, Minneapolis, MN) at a constant power of 400 mA for 2 h. The blocking reaction was performed 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-SSAT primary antibody (diluted 1:1,000) was carried out in 5% dry milk in TBS for 4 h at room temperature. After the membrane was washed twice 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 secondary antibody (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 (ECL kit, Amersham, Piscataway, NJ) and exposed for 5-30 s to an X-ray film. This antibody is highly specific and detects the SSAT as a ~20-kDa band (11).

Measurement of putrescine concentration in the kidney. Kidneys were harvested at 12 and 24 h of reperfusion and utilized for measurement of polyamine concentration by HPLC, as described previously (35).

Statistical analyses. Values are means ± SE. The significance of difference between mean values was examined using ANOVA. P < 0.05 was considered statistically significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Identification of SSAT by SSH as a transcript that is upregulated during renal IRI. Total RNA from the cortical regions of control rat kidneys and rat kidneys subjected to IRI was used to identify differentially expressed transcripts using SSH (occurrence of tissue damage was determined by the measurement of BUN and serum creatinine levels; Table 1). A number of transcripts, including SSAT, were identified as being differentially regulated in response to IRI. Other genes that were altered include (but are not limited to) MAP17, stathmin, and mitochondrial transcripts such as those involved in oxidative reduction and electron transport chain (unpublished observations). To confirm the results of SSH, total RNA from the renal cortex 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 cDNA probe. The results are shown in Fig. 1 and indicate that SSAT mRNA expression is increased by nearly threefold and nearly fivefold in cortex and medulla, respectively, of kidneys with IRI (P < 0.05 and P < 0.05 vs. control). Further Northern blot analyses were performed to examine the time course of expression of SSAT and other enzymes involved in polyamine 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 < 0.05) and return to baseline levels at 48 h of reperfusion. To determine whether the increase in SSAT mRNA also reflects an increase in SSAT protein abundance, Western blot analysis of kidney extracts harvested at 12 and 24 h of reperfusion was performed. A representative blot shown in Fig. 2C demonstrates that SSAT protein abundance is increased significantly at 12 and 24 h of IRI.

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   BUN and serum creatinine levels in animal models of acute renal failure



View larger version (29K):
[in this window]
[in a new window]
 
Fig. 1.   Northern blot analysis of spermidine/spermine N1-acetyltransferase (SSAT) mRNA expression in cortical and medullary areas of control and ischemia-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 of reperfusion) was size fractionated and subjected to Northern blot analysis using radiolabeled SSAT cDNA probe. C: quantitative analyses of results in A and B. Equal loading was confirmed 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).



View larger version (36K):
[in this window]
[in a new window]
 
Fig. 2.   Time course of expression of enzymes involved in polyamine catabolism during IRI. Northern blot analyses were performed (30 µg/well of total RNA) to examine time course of expression of SSAT and polyamine oxidase (PAO) in IRI. A and B: mRNA levels of SSAT in renal cortex and medulla, respectively. Expression of SSAT increased significantly at 12 h of reperfusion in cortex and medulla (P < 0.05 for each zone) and 24 h of reperfusion in medulla (P < 0.05). C: representative Western blot analysis of SSAT on IRI samples at 12 and 24 h of reperfusion. Expression of SSAT is enhanced significantly in IRI compared with sham animals. D: mRNA levels of PAO in renal cortex and medulla. E: for quantitative analysis, mRNA levels for PAO were measured in 3 control and 3 IRI medulla samples. Expression of PAO increased significantly at 12 h of reperfusion (P < 0.05). Expression of PAO also increased at 24 and 48 h of reperfusion (P < 0.05 vs. control). Equal loading was confirmed by examination of 28S rRNA bands (A, B, D, and E, bottom).

Degradation of polyamines by SSAT results in increased acetylated levels of spermidine and spermine, which are excreted or oxidized by PAO to putrescine, the product that is responsible for tissue damage in several biological systems. In the next series of experiments, the expression levels of PAO in IRI were examined by Northern hybridization. As shown in a representative blot in Fig. 2D, expression of PAO in the cortex and medulla was very low at baseline but was increased considerably at 12 h of IRI and remained elevated at 48 h of IRI. The upregulation of PAO was of a higher magnitude in the medulla than in the renal cortex. Figure 2E shows the expression of PAO in kidney medulla of three control animals and three animals subjected to IRI (30 min of ischemia-12 h of reperfusion). PAO expression is increased considerably in kidneys subjected to IRI.

Measurement of polyamine levels in kidney samples and urine. To determine whether enhanced expression of SSAT is associated with increased activity of this enzyme, the concentration of polyamines in kidney samples was measured by HPLC. Putrescine concentration was significantly increased at 12 and 24 h of reperfusion compared with sham-operated animals, consistent with increased SSAT activity in IRI (Fig. 3). Putrescine concentration increased by ~4.0- and ~7.5-fold at 12 and 24 h of reperfusion, respectively, compared with sham-operated controls (P < 0.05 and P < 0.05 vs. control).


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 3.   Polyamine concentration in kidneys with IRI. Putrescine concentration ([putrescine]) was measured by HPLC in whole kidney samples at 12 and 24 h of reperfusion and compared with that in sham-operated animals.

Urine samples from animals subjected to 30 min of ischemia and 12 (or 24) h of reperfusion were collected and processed for polyamine level measurement by HPLC. The urine samples were collected at 3-h intervals. For 12 h of reperfusion, the urine samples that were collected 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 of putrescine was not significantly different between sham and IRI animals. 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 urinary spermidine concentration decreased (69 ± 8 vs. 239 ± 15 µmol/l in IRI vs. sham, respectively, P < 0.05, n = 3), but the concentration of spermine, although it was 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).

Nephron segment RT-PCR of SSAT. Previous studies indicate that SSAT mRNA in the kidney is exclusively expressed in the epithelium of the distal convoluted and straight tubules tubules (4, 5). Because the aforementioned results did not match our Northern blot analysis, site-specific expression of SSAT was examined using nephron segment RT-PCR. Figure 4 demonstrates that SSAT mRNA transcript is expressed in CCD, cTAL, mTAL, and PST, indicating widespread distribution of SSAT in kidney tubules.


View larger version (81K):
[in this window]
[in a new window]
 
Fig. 4.   Nephron segment RT-PCR of SSAT. Total RNA from various nephron segments was subjected to RT-PCR, and samples were size fractionated on a 1% Tris-acetic acid-EDTA gel and stained with ethidium bromide for visualization of amplified bands. SSAT mRNA transcript is expressed in cortical collecting duct (CCD), cortical thick ascending limb (cTAL), medullary thick ascending limb (mTAL), and proximal straight tubule (PST), indicating widespread distribution of SSAT in kidney tubules.

Expression of SSAT in a model of kidney failure without tubular injury. IRI is associated with severe cell injury and renal failure (6, 26, 27, 36, 38, 46). To determine whether enhanced expression of SSAT was due to tissue damage secondary to IRI or accumulation of nitrogenous wastes as a consequence of renal dysfunction, a model of renal failure secondary to kidney hypoperfusion was utilized. In addition, a common clinical dilemma is to distinguish tubular injury from volume depletion as the cause of renal failure, and specific markers are needed. Rats were subjected to Na+ depletion for 5 days. ARF was verified by increases in serum BUN and creatinine at the time of euthanasia (Table 1). The SSAT mRNA levels did not change in kidneys of Na+-depleted rats (1.01- vs. 1.04-fold in Na+ depletion vs. control, P > 0.05, n = 3; Fig. 5A). These results indicate that renal failure per se does not increase the expression of IRI.


View larger version (24K):
[in this window]
[in a new window]
 
Fig. 5.   Expression of SSAT in a model of kidney failure without tubular injury (Na+ depletion) and a model of tubular injury without kidney failure (cis-platinum nephrotoxicity). Total RNA (30 µg/well) from renal cortex of Na+-depleted rats (A) and rats treated with cis-platinum (cis-plat, B) was size fractionated and subjected to Northern blot analysis using radiolabeled SSAT cDNA probe. Equal loading was confirmed 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). SSAT expression remained unchanged in Na+ depletion (P > 0.05, n = 3). C: quantitative analysis of results of cis-platinum treatment.

Expression of SSAT in a model of tubular injury without kidney failure. As an alternative approach to distinguish the SSAT increase from tubular injury as opposed to renal failure and an increase in uremic toxins, we evaluated a toxic nephropathy model. In this series of experiments, rats were treated with cis-platinum and killed 1 and 3 days later. SSAT mRNA levels are increased 1 day after cis-platinum treatment (P < 0.05, n = 3) and remained elevated 3 days after treatment (P < 0.05, n = 3; see Fig. 5C for quantitative analysis of results). BUN and creatinine were normal 1 day after cis-platinum injection and were mildly increased 3 days after cis-platinum injection (Table 1). These results indicate that SSAT expression is increased before the onset of renal failure and correlates with cell injury. Sustained elevation of SSAT mRNA levels 3 days after cis-platinum injection suggests that ongoing cell injury persists at this later time point after a single injection.

Expression of SSAT in an in vitro model of kidney IRI. The purpose of the next series of experiments was to examine the mechanisms underlying the increase in SSAT, as well as to confirm its tubular epithelial cell source. Expression of SSAT in cultured renal cells subjected to metabolic poisoning (see MATERIALS AND METHODS), a model of in vitro kidney IRI, was used. MDCK cells were subjected to 30 min of ATP depletion and 2 h of metabolic poisoning "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 < 0.05, n = 3; Fig. 6A). This observation was further confirmed using Western blot analysis. Our results indicate that SSAT protein levels increase by >10-fold as early as 2 h after release from ATP depletion and metabolic poisoning. SSAT protein levels peak at ~24 h after release and by 36 h start to decline (Fig. 6B). 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 died immediately at the end of metabolic poisoning and before the beginning of reperfusion were not included in the death rate calculation).


View larger version (37K):
[in this window]
[in a new window]
 
Fig. 6.   Expression of SSAT in an in vitro model of kidney IRI. Cultured Madin-Darby canine kidney cells were subjected to 30 min of ATP 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 and time-matched control samples was subjected to Northern blot analysis using radiolabeled SSAT cDNA probe. Equal loading was confirmed by examination of 28S rRNA bands (bottom). A single ~1.3-kb band was recognized by SSAT probe (top). SSAT expression increased significantly at 2, 6, and 24 h after ATP repletion (P < 0.05). B: Western blot analysis of SSAT; 150 µg of protein were loaded in each lane. Abundance of SSAT, which appears as a ~20-kDa band, increased by >10-fold at 2 h after ATP repletion, remained elevated at 24 h, and started to decrease by 36 h.

Expression of SSAT in mice subjected to kidney IRI. In the next series of experiments, expression of SSAT was examined in a mouse model of IRI and compared with another well-characterized marker of renal cell injury, KIM-1. Mice were subjected to 30 min of kidney ischemia followed by 2, 12, 24, or 48 h of reperfusion. The results demonstrate that SSAT expression in the kidney increased as early 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 the early stages of reperfusion (Fig. 7, middle). Enhanced levels of KIM-1 were observed at 12 h and were strongly induced by 24 h after reperfusion (Fig. 7, middle). The levels of both transcripts were lower but still above background 48 h after reperfusion. These results indicate that enhanced expression of SSAT is an early event in IRI and precedes the upregulation of KIM-1.


View larger version (49K):
[in this window]
[in a new window]
 
Fig. 7.   Expression of SSAT and kidney injury molecule-1 (KIM-1) in mice subjected to kidney IRI. Mice were subjected to 30 min of kidney ischemia followed by 2, 12, 24, or 48 h of reperfusion. Representative Northern blots (top) demonstrate that SSAT expression in kidney increased as early as 2 h after reperfusion. Expression of SSAT peaked by 12 h after reperfusion (top). In contrast, KIM-1 expression was not detected at 2 h of reperfusion in the same membranes that were stripped and reprobed for KIM-1 (middle). Levels of KIM-1 were enhanced at 12 h and peaked at 24 h of reperfusion (middle). Levels of SSAT and KIM-1 transcripts were lower but still above background at 48 h after reperfusion. Equal loading was confirmed by examination of 28S rRNA bands (bottom). Expression of SSAT increased at 2, 12, 24, and 48 h of reperfusion (P < 0.05 vs. control, n = 3 for each group). Expression of KIM-1 increased at 12, 24, and 48 h of reperfusion (P < 0.05 vs. control, n = 3 for each group).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The results of the present experiments demonstrate that SSAT mRNA levels in kidney IRI increased at 12 h of reperfusion and returned to 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 putrescine concentration in kidney samples (Fig. 3), confirming increased functional activity of SSAT. Nephron segment RT-PCR was used to demonstrate 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 expressed in the kidney. Kidney SSAT mRNA levels remained unchanged in rats subjected to Na+ depletion (renal failure with no tubular injury) but increased in cis-platinum-treated animals before the onset of renal failure (tubular cell injury alone; Fig. 4). Subjecting cultured renal tubule MDCK cells to ATP depletion (an in vitro model of kidney IRI) increased the expression of SSAT (Fig. 6).

A large body of evidence demonstrates a fundamental role for cellular polyamines in cell proliferation. As a result, enzymes that alter the cellular concentration of polyamines have come under intense investigation for their role in regulating the cell growth. Enhanced expression of SSAT by a spermine analog in the breast cancer cell line L56Br-C1 resulted in the depletion of the cellular pools of polyamine within 24-48 h (18). Cell proliferation appeared to be totally inhibited, and within 48 h of treatment, there was an extensive apoptotic response. In Ehrlich ascites tumor cells, treatment with the antitumor drug 1'-acetoxychavicol acetate resulted in increased activity of SSAT with subsequent lowering of intracellular polyamines (25). Apoptosis immediately followed. Administration of exogenous polyamines prevented 1'-acetoxychavicol acetate-induced apoptosis. A similar observation has been documented in human leukemic cells, where depletion of intracellular polyamines secondary to the overexpression of SSAT resulted in decreased cell growth and caused the induction of apoptosis (12). Taken together, these studies indicate that enhanced expression of SSAT depletes the cellular polyamine pools, decreases cell growth, and leads to apoptosis.

Intracellular polyamines in the brain are very sensitive to various pathological 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 in decreased spermidine/spermine and increased putrescine concentration in the affected neurons (29, 47). SSAT expression peaked at 12-18 h of reperfusion. The SSAT expression remained unchanged on the contralateral side, indicating that SSAT upregulation is a specific and sensitive marker for ischemic injury. Restoration of spermidine level and reduction in putrescine concentration with the help of a PAO inhibitor (MDL-72527) reduced the tissue edema and infarct size in cerebral ischemia (16, 17). Taken together, these results indicate that SSAT is upregulated in ischemic brain injury and may be detrimental to cell viability.

The most salient feature of the present studies is the detection of enhanced SSAT expression and activity in kidney IRI (Figs. 1-3). The induction of SSAT at 12 h and its return to baseline levels at 48 h of reperfusion correlate with the peak onset of tubular injury and recovery from the injury, respectively. Furthermore, the induction 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 the essential enzymes in the polyamine catabolic pathway are operating in unison. Increased putrescine concentration in kidneys (Fig. 3) confirms increased functional activity of these two enzymes in IRI and is consistent with the upregulation of their proteins. With regard to the levels of polyamines in the urine, we found that spermidine concentration decreased but the levels of putrescine and spermine remained unchanged at 12 and 24 h of reperfusion. The levels of putrescine in the urine do not correlate with increased levels of this chemical in kidney homogenates in IRI. Reductions in glomerular filtration rate at 12 and 24 h of reperfusion (Table 1), as well as possible alteration in the expression of membrane-bound polyamine transporter(s), may independently affect the concentration of polyamines in the urine, making the interpretation of urine polyamine levels in IRI difficult.

A recent report identified KIM-1 as a biomarker of kidney injury (14). KIM-1 expression increased at 24 h and remained elevated at 48 h of reperfusion (19), a pattern distinct from SSAT expression. On the basis of functional and structural studies indicating improvement in kidney function and initiation of repair in damaged tubules at 48 h of reperfusion, we propose that SSAT is an early biomarker of tubule injury, whereas KIM-1 is a closer marker of the extension phase and recovery from injury in kidney IRI. In support of this conclusion, we observe that enhanced expression of SSAT precedes the upregulation of KIM-1 in renal IRI. In this regard, the upregulation of SSAT is similar to that of CYR61, an angiogenic cysteine-rich protein (28). The upregulation of kidney SSAT expression in rats treated with cis-platinum (Fig. 5) and before the onset of renal failure strongly supports the value of this enzyme in the detection of cell injury. Our present results strongly suggest that cell injury precedes the onset of renal failure in response to cis-platinum treatment.

Induction of ATP depletion in cultured renal cells has been used as an in vitro model of kidney cell injury (41, 44). However, and aside from distortions in cytoskeletal structure and alteration in the expression of stress-related genes, no specific biomarker of kidney cell injury has been identified in this model of kidney IRI. Enhanced expression of SSAT (Fig. 6) indicates that monitoring the expression of this enzyme can be used as an indicator of the state of injury in cultured kidney tubules subjected to ATP depletion. Stable overexpression of SSAT in cultured Chinese hamster ovary cells caused perturbations in polyamine homeostasis and led to a reduction in the rate of growth (9). Whether enhanced expression of SSAT in cultured renal cells similarly depletes intracellular polyamine pools and decreases growth in cells subjected to ATP depletion remains to be seen.

In conclusion, SSAT expression increases in kidneys of rats subjected to IRI. Enhanced expression of SSAT reflects the state of cell injury and is not due to accumulation of the uremic toxins that results from renal failure. We propose that SSAT is an early marker of kidney cell injury, and its level of expression could be used as a diagnostic tool in the early phase of renal IRI. Future experiments are planned to elucidate the role of SSAT at the molecular level in in vitro and in vivo models of renal IRI, as well as to characterize the diagnostic utility of measuring SSAT levels in blood and urine.


    NOTE ADDED IN PROOF

After this manuscript had been accepted, the cloning of a new PAO was reported (Vujcic S, Liang P, Diegelman P, Kramer DL, and Porter CW). Genomic identification and biochemical characterization of the mammalian PAO involved polyamine back-conversion (Biochem J 370: 19-28, 2003). The new PAO may play an important role in the generation of putrescine. Studies are underway to examine the regulation of the new PAO in IRI.


    ACKNOWLEDGEMENTS

These studies were supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-54220 (M. Soleimani) and DK-54770 (H. Rabb), a grant from the Greater Cincinnati Kidney Foundation (K. Zahedi), a Merit Review Award (M. Soleimani), a National Kidney Foundation Clinical Scientist Award (H. Rabb), and grants from Dialysis Clinic Incorporated (M. Soleimani).


    FOOTNOTES

* K. Zahedi and Z. Wang contributed equally to this work.

Address for reprint requests and other correspondence: M. Soleimani, 231 Albert Sabin Way, MSB 259G, Cincinnati, OH 45267-0585 (E-mail: Manoocher.soleimani{at}uc.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published January 28, 2003;10.1152/ajprenal.00318.2002

Received 5 September 2002; accepted in final form 3 January 2003.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Alhonen, L, Karppinen A, Uusi-Oukari M, Vujcic S, Korhonen VP, Halmekyto M, Kramer DL, Hines R, Janne J, and Porter CW. Correlation of polyamine and growth responses to N1,N11-diethylnorspermine in primary fetal fibroblasts derived from transgenic mice overexpressing spermidine/spermine N1-acetyltransferase. J Biol Chem 273: 1964-1969, 1998[Abstract/Free Full Text].

2.   Alhonen, L, Parkkinen JJ, Keinanen T, Sinervirta R, Herzig KH, and Janne J. Activation of polyamine catabolism in transgenic rats induces acute pancreatitis. Proc Natl Acad Sci USA 97: 8290-8295, 2000[Abstract/Free Full Text].

3.   Alhonen, L, Rasanen TL, Sinervirta R, Parkkinen JJ, Korhonen VP, Pietila M, and Janne J. Polyamines are required for the initiation of rat liver regeneration. Biochem J 362: 149-153, 2002[ISI][Medline].

4.   Bettuzzi, S, Astancolle S, Pinna C, Monti MG, and Moruzzi MS. Androgen responsiveness and intrarenal localization of transcripts coding for the enzymes of polyamine metabolism in the mouse. Eur J Cancer 37: 281-289, 2001[ISI][Medline].

5.   Bettuzzi, S, Marinelli M, Strocchi P, Davalli P, Cevolani D, and Corti A. Different localization of spermidine/spermine N1-acetyltransferase and ornithine decarboxylase transcripts in the rat kidney. FEBS Lett 377: 321-324, 1995[ISI][Medline].

6.   Bonventre, JV. Mechanisms of ischemic acute renal failure. Kidney Int 43: 1160-1178, 1993[ISI][Medline].

7.   Chopra, S, and Wallace HM. Induction of spermidine/spermine N1-acetyltransferase in human cancer cells in response to increased production of reactive oxygen species. Biochem Pharmacol 55: 1119-1123, 1998[ISI][Medline].

8.   Coleman, CS, Huang H, and Pegg AE. Structure and critical residues at the active site of spermidine/spermine-N1-acetyltransferase. Biochem J 316: 697-701, 1996[ISI][Medline].

9.   Coleman, CS, Pegg AE, and McCloskey DE. Properties and regulation of human spermidine/spermine N1-acetyltransferase stably expressed in Chinese hamster ovary cells. J Biol Chem 274: 6175-6182, 1999[Abstract/Free Full Text].

10.   Desiderio, MA, Pogliaghi G, and Dansi P. Regulation of spermidine/spermine N1-acetyltransferase expression by cytokines and polyamines in human hepatocarcinoma cells (HepG2). J Cell Physiol 174: 125-134, 1998[ISI][Medline].

11.   Fogel-Petrovic, M, Vujcic S, Brown PJ, Haddox MK, and Porter CW. Effects of polyamines, polyamine analogs, and inhibitors of protein synthesis on spermidine-spermine N1-acetyltransferase gene expression. Biochemistry 35: 14436-14444, 1996[ISI][Medline].

12.   Fraser, AV, Woster PM, and Wallace HM. Induction of apoptosis in human leukaemic cells by IPENSpm, a novel polyamine analogue and anti-metabolite. Biochem J 367: 307-312, 2002[ISI][Medline].

13.   Gardini, G, Cabella C, Cravanzola C, Vargiu C, Belliardo S, Testore G, Solinas SP, Toninello A, Grillo MA, and Colombatto S. Agmatine induces apoptosis in rat hepatocyte cultures. J Hepatol 35: 482-489, 2001[ISI][Medline].

14.   Han, WK, Bailly V, Abichandani R, Thadhani R, and Bonventre JV. Kidney injury molecule-1 (KIM-1): a novel biomarker for human renal proximal tubule injury. Kidney Int 62: 237-244, 2002[ISI][Medline].

15.   Hasan, R, Alam MK, and Ali R. Polyamine induced Z-conformation of native calf thymus DNA. FEBS Lett 368: 27-30, 1995[ISI][Medline].

16.   Hatcher, JF, Baskaya MK, Dempsey RJ, and Dogan A. Effects of MDL 72527, a specific inhibitor of polyamine oxidase, on brain edema, ischemic injury volume, and tissue polyamine levels in rats after temporary middle cerebral artery occlusion. Neurosci Lett 256: 65-68, 1998[ISI][Medline].

17.   Hatcher, JF, Dempsey RJ, and Rao AM. Elevated N1-acetylspermidine levels in gerbil and rat brains after CNS injury. J Neurosci Res 58: 697-705, 1999[ISI][Medline].

18.   Hegardt, C, Johannsson OT, and Oredsson SM. Rapid caspase-dependent cell death in cultured human breast cancer cells induced by the polyamine analogue N1,N11-diethylnorspermine. Eur J Biochem 269: 1033-1039, 2002[Abstract/Free Full Text].

19.   Ichimura, T, Bonventre JV, Bailly V, Wei H, Hession CA, Cate RL, and Sanicola M. Kidney injury molecule-1 (KIM-1), a putative epithelial cell adhesion molecule containing a novel immunoglobulin domain, is up-regulated in renal cells after injury. J Biol Chem 273: 4135-4142, 1998[Abstract/Free Full Text].

20.   Igarashi, K, and Kashiwagi K. Polyamines: mysterious modulators of cellular functions. Biochem Biophys Res Commun 271: 559-564, 2000[ISI][Medline].

21.   Janne, J, Alhonen L, and Leinonen P. Polyamines: from molecular biology to clinical applications. Ann Med 23: 241-259, 1991[ISI][Medline].

22.   Korhonen, VP, Niiranen K, Halmekyto M, Pietila M, Diegelman P, Parkkinen JJ, Eloranta T, Porter CW, Alhonen L, and Janne J. Spermine deficiency resulting from targeted disruption of the spermine synthase gene in embryonic stem cells leads to enhanced sensitivity to antiproliferative drugs. Mol Pharmacol 59: 231-238, 2001[Abstract/Free Full Text].

23.   Marton, LJ, and Pegg AE. Polyamines as targets for therapeutic intervention. Annu Rev Pharmacol Toxicol 35: 55-91, 1995[ISI][Medline].

24.   Min, SH, Simmen RCM, Alhonen L, Halmekyto M, Porter CW, Janne J, and Simmen FA. Altered levels of growth-related and novel gene transcripts in reproductive and other tissues of female mice overexpressing spermidine/spermine N1-acetyltransferase (SSAT). J Biol Chem 277: 3647-3657, 2002[Abstract/Free Full Text].

25.   Moffatt, J, Hashimoto M, Kojima A, Kennedy DO, Murakami A, Koshimizu K, Ohigashi H, and Matsui-Yuasa I. Apoptosis induced by 1'-acetoxychavicol acetate in Ehrlich ascites tumor cells is associated with modulation of polyamine metabolism and caspase-3 activation. Carcinogenesis 21: 2151-2157, 2000[Abstract/Free Full Text].

26.   Molitoris, BA. Putting the actin cytoskeleton into perspective: pathophysiology of ischemic alterations. Am J Physiol Renal Physiol 272: F430-F433, 1997[Abstract/Free Full Text].

27.   Molitoris, BA, Leiser J, and Wagner MC. Role of the actin cytoskeleton in ischemia-induced cell injury and repair. Pediatr Nephrol 11: 761-767, 1997[ISI][Medline].

28.   Muramatsu, Y, Tsujie M, Kohda Y, Pham B, Perantoni AO, Zhao H, Jo SK, Yuen PS, Craig L, Hu X, and Star RA. Early detection of cysteine-rich protein 61 (CYR61, CCN1) in urine following renal ischemic reperfusion injury. Kidney Int 62: 1601-1610, 2002[ISI][Medline].

29.   Nagesh Babu, G, Sailor KA, Sun D, and Dempsey RJ. Spermidine/spermine N1-acetyl transferase activity in rat brain following transient focal cerebral ischemia and reperfusion. Neurosci Lett 300: 17-20, 2001[ISI][Medline].

30.   Niiranen, K, Halmekyto M, Pietila M, Diegelman P, Parkkinen JJ, Eloranta T, Porter CW, Alhonen L, and Janne J. Relation of skin polyamines to the hairless phenotype in transgenic mice overexpressing spermidine/spermine N-acetyltransferase. Mol Pharmacol 59: 231-238, 2001[Abstract/Free Full Text].

31.   Nitta, T, Igarashi K, and Yamamoto N. Polyamine depletion induces apoptosis through mitochondria-mediated pathway. Exp Cell Res 276: 120-128, 2002[ISI][Medline].

32.   Pegg, AE. Polyamine metabolism and its importance in neoplastic growth and a target for chemotherapy. Cancer Res 48: 759-774, 1988[Abstract].

33.   Pietila, M, Alhonen L, Halmekyto M, Kanter P, Janne J, and Porter CW. Activation of polyamine catabolism profoundly alters tissue polyamine pools and affects hair growth and female fertility in transgenic mice overexpressing spermidine/spermine N1-acetyltransferase. J Biol Chem 272: 18746-18751, 1997[Abstract/Free Full Text].

34.   Pombo, CM, Tsujita T, Kyriakis JM, Bonventre JV, and Force T. Activation of the Ste20-like oxidant stress response kinase-1 during the initial stages of chemical anoxia-induced necrotic cell death. Requirement for dual inputs of oxidant stress and increased cytosolic [Ca2+]. J Biol Chem 272: 29372-29379, 1997[Abstract/Free Full Text].

35.   Porter, CW, Ganis B, Libby PR, and Bergeron RJ. Correlations between polyamine analogue-induced increases in spermidine/spermine N1-acetyltransferase activity, polyamine pool depletion, and growth inhibition in human melanoma cell lines: characterization of human spermidine/spermine N1-acetyltransferase purified from cultured melanoma cells. Cancer Res 51: 3715-3720, 1991[Abstract].

36.   Rabb, H, Haq M, Shull GE, and Soleimani M. Possible molecular basis for changes in potassium handling in acute renal failure. J Am Soc Nephrol 9: 605-613, 1998[Abstract].

37.   Rabb, H, Mendiola CC, Dietz J, Saba SR, Issekutz TB, Abanilla F, Bonventre JV, and Ramirez G. Role of CD11a and CD11b in ischemic acute renal failure in rats. Am J Physiol Renal Fluid Electrolyte Physiol 267: F1052-F1058, 1994[Abstract/Free Full Text].

38.   Rabb, H, Mendiola CC, Saba SR, Dietz JR, Smith CW, Bonventre JV, and Ramirez G. Antibodies to ICAM-1 protect kidneys in severe ischemic reperfusion injury. Biochem Biophys Res Commun 211: 67-73, 1995[ISI][Medline].

39.   Sacchi, N, and Chomczynski P. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162: 156-159, 1987[ISI][Medline].

40.   Shappell, NW, Fogel-Petrovic MF, and Porter CW. Regulation of spermidine/spermine N1-acetyltransferase by intracellular polyamine pools. Evidence for a functional role in polyamine homeostasis. FEBS Lett 321: 179-183, 1993[ISI][Medline].

41.   Shih, T, Menza SA, Lieberthal W, and Sheridan AM. Renal mouse proximal tubular cells are more susceptible than MDCK cells to chemical anoxia. J Am Soc Nephrol 10: 2297-2305, 1999[Abstract/Free Full Text].

42.   Soleimani, M, Greeley T, Petrovic S, Wang Z, Amlal H, Kopp P, and Burnham CE. Pendrin: an apical Cl-/OH-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger in the kidney cortex. Am J Physiol Renal Physiol 280: F356-F364, 2001[Abstract/Free Full Text].

43.   Suppola, S, Heikkinen S, Parkkinen JJ, Uusi-Oukari M, Korhonen VP, Keinanen T, Alhonen L, and Janne J. Concurrent overexpression of ornithine decarboxylase and spermidine/spermine N1-acetyltransferase further accelerates the catabolism of hepatic polyamines in transgenic mice. Biochem J 358: 343-348, 2001[ISI][Medline].

44.   Wang, YH, Lieberthal W, Burke PR, and Schwartz JH. Graded ATP depletion can cause necrosis or apoptosis of cultured mouse proximal tubular cells. Am J Physiol Renal Physiol 272: F347-F355, 1997[Abstract/Free Full Text].

45.   Wang, Z, Conforti L, Petrovic S, Amlal H, Burnham CE, and Soleimani M. Mouse Na+:HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter isoform NBC-3 (kNBC-3): cloning, expression, and renal distribution. Kidney Int 59: 1405-1414, 2001[ISI][Medline].

46.   Witzgall, R, Brown D, Schwarz C, and Bonventre JV. Localization of proliferating cell nuclear antigen, vimentin, c-Fos, and clusterin in the postischemic kidney. Evidence for a heterogenous genetic response among nephron segments and a large pool of mitotically active and dedifferentiated cells. J Clin Invest 93: 2175-2188, 1994[ISI][Medline].

47.   Zoli, M, Pedrazzi P, Zini I, and Agnati LF. Spermidine/spermine N1-acetyltransferase mRNA levels show marked and region-specific changes in the early phase after transient forebrain ischemia. Mol Brain Res 38: 122-134, 1996[Medline].

48.   Zweier, JL, Duilio C, Kuppusamy P, Santoro G, Elia PP, Tritto I, Cirillo P, Condorelli M, and Chiariello M. A short burst of oxygen radicals at reflow induces sustained release of oxidized glutathione from postischemic hearts. J Biol Chem 268: 18532-18541, 1993[Abstract/Free Full Text].


Am J Physiol Renal Fluid Electrolyte Physiol 284(5):F1046-F1055