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Published as doi: 10.1096/fj.07-8882com.
 (The FASEB Journal. 2008;22:510-521.)
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(The FASEB Journal. 2008;22:510-521.)
© 2008 FASEB

Protective role of the inhibitor of apoptosis protein, survivin, in toxin-induced acute renal failure

Nele Kindt, Axel Menzebach, Marlies Van de Wouwer, Inge Betz, Astrid De Vriese and Edward M. Conway

VIB Department of Transgene Technology and Gene Therapy, K.U. Leuven, Leuven, Belgium

1Correspondence: Center for Transgene Technology and Gene Therapy, Flanders Institute for Biotechnology, University of Leuven, Herestraat 49, B-3000 Leuven, Belgium. E-mail: ed.conway{at}med.kuleuven.be


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Acute renal failure (ARF) is a major worldwide cause of morbidity and mortality, lacking specific targeted, effective therapies. Renal tubular cell apoptosis has been recognized to play a critical role in the pathogenesis of ARF, yet few studies have evaluated whether intervention in apoptotic pathways can ameliorate the deterioration in renal function associated with ARF. Using transgenic mice with diminished levels of the inhibitor of apoptosis protein, survivin, we show that survivin is required to protect the kidney from apoptosis, to suppress renal expression of p53, and to ameliorate renal dysfunction in two models of ARF. Gene delivery of survivin to wild-type mice and mice with 50% levels of survivin, prior to or at the time of induction of ARF, interferes with the deterioration of renal function and preserves the integrity of the kidneys and the renal tubular cells by inhibiting activation of apoptotic pathways in the kidneys and suppressing expression of p53. These results encourage further evaluation of survivin, its active structural domains, and other inhibitors of apoptosis proteins, for preventing and/or treating acute renal failure.—Kindt, N., Menzebach, A., Van de Wouwer, M., Betz, I., De Vriese, A., Conway, E. M. Protective role of the inhibitor of apoptosis protein, survivin, in toxin-induced acute renal failure.


Key Words: caspase • cisplatin • kidney • transgenic mice • p53 • hydrodynamic gene therapy


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
ACUTE RENAL FAILURE (ARF) LEADING TO renal insufficiency is a common disorder, estimated to occur in at least 5% of all hospitalized patients, and in 30–50% of those admitted to the intensive care unit. Morbidity and mortality from ARF remain unacceptably high, and indeed, despite advances in supportive care, outcomes have not improved in the past four decades (1) . The most common cause of ARF—acute tubular necrosis (ATN)—is most frequently observed in the setting of sepsis, postrenal transplant, postmyocardial infarct, in the elderly with diminished fluid intake, and as a consequence of exposure to a wide range of toxins, including cisplatin (cis-diammine-dichloro-platinumII), aminoglycosides, amphotericin B, acyclovir, and radio-contrast agents (2 3 4) . The notion that only severe renal failure impacts on long-term morbidity is dispelled by the fact that even modest degrees of renal insufficiency significantly increase the risk of death for critically ill patients (5) . Despite intensive investigation into the pathophysiology of ARF, effective therapeutic strategies remain elusive.

While the pathogenesis of renal tubular cell death in ATN is complex and varies depending on the etiology, severity, and stage of the illness, strong evidence supports the concept that apoptosis plays a central role (3 , 6 7 8 9) . Ischemia-reperfusion-induced ARF is associated with activation of caspases and prominent increases in renal tubular expression of several proapoptotic genes and/or proteins, including Fas-associated death domain (FADD), p53, Bad, Fas, Smac/Diablo, and prostate apoptosis response-4 (Par-4) (9 10 11 12) (reviewed in ref. 13 ). In mouse models, Bid deficiency ameliorates ischemia-induced renal failure and renal tubular apoptosis (14) . At doses used clinically, cisplatin induces ARF that is associated with caspase activation and histological changes consistent with renal tubular cell apoptosis (2 , 15) . Postrenal transplant, apoptosis of donor kidney tubular epithelial cells, and low Bcl-xL and Bcl-2 expression are associated with a high incidence of ARF (16 , 17) . Overall, the long-held view that cellular necrosis is the sole mechanism responsible for tubular epithelial cell death in ARF has thus been supplanted by a paradigm in which apoptosis plays a key role. Interfering with one or more proapoptotic pathways at crucial times during progression of ARF is therefore likely to be protective.

Major gains have been made in elucidating the molecular mechanisms regulating apoptosis, and consequently, several molecular steps may be targeted to interfere with downstream activation of caspases. Survivin is a unique member of the inhibitor of apoptosis protein (IAP) family (reviewed in ref. 18 , 19 ). It is minimally expressed in adult tissues but abundant in most proliferating cells (20) . Overexpression of survivin can protect cells from Fas- and injury-induced apoptosis (21) , in part by interfering with effector caspases and stabilizing mitochondrial function (22) , whereas suppression of survivin expression by antisense, ribozymes, or transgenic inactivation in mice leads to spontaneous apoptosis, and increased sensitivity to Fas and ischemia/hypoxia (23 24 25 26 27 28) . In contrast to other IAPs, survivin also plays a role in facilitating cell cycle progression, and, furthermore, is a chromosome passenger protein that is critical for regulation of mitosis and cytokinesis (28 , 29) .

The versatility of survivin in modulating apoptosis and cell division suggests that this unique IAP might be suitable for prevention and/or treatment of renal tubular cell damage in the setting of ATN. We used two well-established murine models of toxin-induced ARF (30 , 31) in wild-type mice and in transgenic mice that have diminished levels of survivin (27) in order to elucidate the role of survivin in the pathophysiology of ARF. Our results indicate that survivin, when delivered as a single dose prior to the onset of ARF induced by folic acid or cisplatin, effectively reduces renal tubular cellular damage and preserves renal function.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Transgenic mice
Generation of survivin+/– mice by homologous recombination in embryonic stem cells has been reported (27) . Transgenic mice were maintained on a Swiss:129s (50:50) genetic background and housed in a specific pathogen-free environment. The survivin+/– mice express ~50% levels of survivin mRNA, and under nonstress conditions, have no phenotypic abnormalities (27 , 32) . Experiments were performed with 10- to 12-wk-old, 25–30-g male mice for the folic acid–induced ARF model and with 25–35 g, 7-wk-old female mice for the cisplatin-induced ARF model. Sex-matched survivin+/+ littermates were used as controls for experiments on survivin+/– mice, thereby excluding strain-related differences. Studies were approved by the animal ethics committee at the University of Leuven.

Induction of acute renal failure
ARF was induced by either a single i.p. injection of folic acid, 250 mg/kg body weight, dissolved in 150 µl sodium bicarbonate (NaHCO3) (30 , 33) , or a single i.p. injection of Platosin® (cisplatin 1 mg/ml; Teva Pharma Belgium, Wilrijk-Antwerpen, Belgium), 20 mg/kg body weight, dissolved in saline (34 , 35) . In pilot studies to induce ARF in wild-type mice, the doses of folic acid and cisplatin were established, such that serum creatinine levels rose significantly but without the agents causing severe illness or death when monitored over 7 and 4 days, respectively. Control animals were administered an equivalent volume of the carrier i.p., that is, sodium bicarbonate for the folic acid model, and saline for the cisplatin model.

Plasmid preparation and hydrodynamic gene delivery
The cDNAs encoding full-length murine survivin (survivin140), survivin121, and survivin40 (36) were each cloned into the expression vector pcDNA3 (Invitrogen, San Diego, CA, USA), resulting in the vectors survivin140/pcDNA3, survivin121/pcDNA3, and survivin40/pcDNA3. For in vivo gene delivery, 25 or 50 µg plasmid DNA in 2 ml of saline or Ringer solution was injected in 5–6 s via the tail vein of mice (37 , 38) . Empty vector was injected for nontreatment controls.

Renal function and preparation of kidneys for histopathological analyses
At different time points after the induction of ARF, mice were anesthetized (ketamine 10 mg/ml, xylazine 1 mg/ml, atropine 3 µg/ml in NaCl 0.9%). The chest wall and abdomen were surgically exposed, and blood was drawn from the inferior vena cava (IVC). Mice were perfused transcardially with saline, after which the left kidney was removed, frozen in liquid nitrogen, and stored at –80°C. Perfusion was resumed with zinc-buffered formalin (Z-fix; Anatech, Battlecreek, MI, USA), after which the right kidney was removed for histology. For the cisplatin experiments, mice were anesthetized, the abdomen was surgically exposed, and blood was drawn from the IVC, after which the kidneys were removed for further analysis, without perfusion.

Kidneys were incubated overnight in Z-fix, dehydrated through increasing ethanol concentrations, embedded in paraffin wax, and prepared for histological sectioning. Seven-micrometer sections were stained with hematoxylin and eosin (H&E) or incubated after antigen retrieval with specific antibodies, followed by addition of appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies and visualization by immunoperoxidase staining. Control primary antibodies were used to exclude nonspecific staining. Serum creatinine levels were quantified with a commercial kit (Roche Pharmaceuticals, Brussels, Belgium) in the University of Leuven Hospital clinical laboratory.

Detection of apoptosis
Apoptosis was detected in situ by staining deparaffinized sections using the ApopTag peroxidase in situ apoptosis detection kit (Chemicon, Hofheim, Germany), according to the manufacturer’s instructions. Quantification of apoptosis by an investigator blinded to experimental conditions and/or mouse genotype was accomplished by microscopically counting the number of peroxidase-positive nuclei relative to the total number of nucleated cells in randomly selected nonadjacent cortical regions in five x400 microscopic fields per section. For the folic acid–induced ARF model, the absolute number of peroxidase-positive nuclei per x200 microscopic field was counted (39) . Results are expressed as the means ± SE.

Detection of active caspase-3 and caspase-9
To detect caspase-3 or caspase-9 activity in kidney lysates, the Caspase-Glo 3/7 or Caspase-Glo 9 assays (Promega; Leiden, The Netherlands) were used, respectively, according to the manufacturer’s instructions. Briefly, 50 µg of total protein (kidney lysate) was added to each well of a 96-well plate (performed in duplicate for each sample) and incubated for 1 h with the appropriate caspase reagent, after which luminescence was measured. Luminescence is proportional to the amount of specific caspase activity in the sample and is measured in relative light units (RLU).

SDS-PAGE, Western immunoblots, and antibodies
Kidneys were lysed and homogenized on ice in a solution containing 1% Triton X-100; 150 mM NaCl; 0.1 mM ethylenediaminetetraacetate; 20 mM HEPES, pH 7.5; 20% glycerol; and 1 mM MgCl2 in the presence of protease inhibitors. For detection of cytochrome c, cytosolic fractions were prepared as previously reported (27) . Protein content of cleared lysates was quantified with the BCA kit (Promega) or the Bradford kit (Bio-Rad, Hercules, CA, USA). One hundred micrograms of each sample was separated by SDS-PAGE under reducing conditions and transferred to a nitrocellulose membrane, which was blocked with 5% nonfat dried milk powder in PBS with 0.1% Tween 20 and incubated for 2 to 24 h with the primary antibody. The following primary antibodies were used: hamster anti-Bad and rabbit anticaspase-3 from Pharmingen Europe (Aalst, Belgium); rabbit antisurvivin (NB500-201) from Novus Biologicals (Hiddenhausen, Germany); rabbit anticaspase-9 from Eurobiochem (Bierges, Belgium); rabbit anticytochrome c, murine monoclonal antip53 (DO-1), and rabbit anti-Bcl-xL (H-62) from Santa Cruz Biotechnology (Santa Cruz, CA, USA). After washing and incubation of the membrane with the appropriate HRP-conjugated secondary antibody, detection was accomplished using the enhanced chemiluminescence method (Amersham-Biosciences, Freiburg, Germany). Equal loading was confirmed either by running parallel gels for detection of β-actin, or by stripping and reblotting the membranes for expression of β-actin. In addition, densitometry was used to quantify protein expression, in which case, control lanes, relative to the corresponding expression of actin, were arbitrarily set at 1 in each blot, and the signals of other conditions on the same blot were normalized with the control to yield their protein expression levels.

Statistical analyses
Statistics were performed with InStat® software (MacKiev Company, Cupertino, CA, USA). Data were tested using one-way ANOVA, followed by Tukey-Kramer multiple comparisons test. For comparisons between two groups, the unpaired two-tailed t test was used, with Welch correction if necessary. Results are reported as means ± SE, unless otherwise indicated. Values of P < 0.05 were considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Survivin+/– mice are more susceptible to folic acid–induced ARF
Folic acid was administered to induce ARF with tubular epithelial cell death, as reported (33) . Survivin+/+ and survivin+/– mice were evaluated at 6 h, 24 h, and 7 days (Fig. 1 ). Under baseline conditions, there were no discernible differences between the survivin+/+ and the survivin+/– mice in terms of renal function (serum creatinine), or histological appearance of the kidneys, as assessed by H&E staining and evidence of apoptosis (Figs.1 , 2 ). At 6 h, the serum creatinine level of survivin+/+ mice had not changed from baseline. By 24 h, the serum creatinine level became notably elevated, but by 7 days, renal function, as measured by serum creatinine, had decreased in the survivin+/+ mice, a pattern of recovery that is typical for folic acid–induced ARF in mice (38) .


Figure 1
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  		Figure 1. Survivin-dependent response to folic acid induced ARF. Acute renal failure was induced with folic acid in survivin+/+ and survivin+/– mice. Serum creatinine (A) and the number of apoptotic cells detected by TUNEL staining of kidney sections (B) were quantified. Survivin+/– mice (+/–) were more sensitive than survivin+/+ mice (+/+) to induction of acute renal failure. Results reflect measures on a minimum of 3–5 mice. *P < 0.05.


Figure 2
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  		Figure 2. Histological changes in kidneys after folic acid. Representative kidney sections from survivin+/+ mice (A–C) and survivin+/– mice (D–F) were stained with hematoxylin and eosin (H&E). Prior to folic acid administration (A, D), no detectable differences were noted between the two genotypes. 6 h (B, E) and 24 h (E, F) after folic acid, histological changes were notably worse in kidneys of survivin+/– mice, with clear loss of brush borders, and greater frequency of condensed nuclei of renal tubular cells (arrows).

The response to folic acid in the survivin+/– mice was very different (Fig. 1) . A more rapid onset of ARF was observed, as the serum creatinine became significantly elevated at 6 h. By 24 h, renal function had deteriorated to a similar extent to that observed with the survivin+/+ mice. But at 7 days, rather than a full recovery in renal function, the serum creatinine remained significantly elevated in the survivin+/– mice. Overall, the results indicate that diminished levels of survivin in the survivin+/– mice confer increased sensitivity to folic acid–induced ARF.

Low levels of survivin render renal tubular cells sensitive to apoptosis induced by folic acid
The mechanisms by which low levels of survivin result in increased sensitivity to folic acid–induced ARF were evaluated. In keeping with the absence of early changes in serum creatinine in the survivin+/+ mice, the kidneys exhibited no histological evidence of damage by H&E staining 6 h after folic acid injection (Fig. 2) . Nonetheless, TUNEL staining revealed a 20-fold increase in the number of apoptotic renal tubular epithelial cells as compared with baseline (Figs. 1B and 3 ), highlighting the early onset of apoptosis following this toxic injury, as well as the limited sensitivity of the serum creatinine (40) . By 24 h, when the serum creatinine was elevated, the number of apoptotic cells increased further. Otherwise, the most prominent histological finding in the kidneys was that the collecting ducts appeared dilated (Fig. 2) . A minority of the renal tubular epithelial cells exhibited loss of brush borders and condensed nuclei. By 7 days, when renal function had returned to normal, the kidneys appeared histologically normal by H&E staining (not shown), although the number of apoptotic cells was still somewhat elevated, but near normal (Fig. 1B ).


Figure 3
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  		Figure 3. TUNEL staining of kidneys after folic acid. Representative kidney sections from survivin+/+ mice (A–C) and survivin+/– mice (D–F) were stained for detection of apoptotic cells (arrows). Before folic acid administration (A, D), no detectable differences were noted between the two genotypes. Six hours (B, E) after folic acid, apoptotic cells were detected in sections from survivin+/– mice, but not from survivin+/+ mice. Twenty-four hours (C, F) after folic acid injection, apoptosis was more prominent in kidneys from survivin+/– mice.

Histological changes were much more dramatic in the survivin+/– mice throughout the study period (Fig. 2) . Six hours after folic acid administration, H&E staining revealed more evidence of renal tubular cell damage, with condensed nuclei and loss of brush borders. There was a 47-fold increase in the number of apoptotic cells, as compared to baseline, and this was significantly more than that which was observed with the survivin+/+ kidneys at the same time point (P<0.05) (Figs. 1B and 3) . By 24 h, the kidneys of survivin+/– mice exhibited diffuse and major histological changes, reflecting extensive tubular epithelial cell damage, with evidence of both necrosis and apoptosis, as is commonly seen with this model (38) (Fig. 2) . The majority of renal tubular cells were swollen, with the accumulation of apical cytoplasmic vacuoles and loss of brush borders. Tubular epithelial cell nuclei were condensed, with evidence of apoptotic bodies, accompanied by shedding of cells and cellular debris and/or casts into the collecting ducts. TUNEL staining of kidney sections from survivin+/– mice confirmed the significant increase in renal tubular cell death relative to that observed in the kidneys of survivin+/+ mice at 24 h. These findings persisted until at least 7 days after folic acid was administered (Fig. 1B ), at which time there was still a significant increase in apoptotic cells.

To further confirm activation of apoptotic pathways, we performed Western immunoblots of kidney lysates. Under baseline conditions, caspase-3 activation was not detectable in the kidneys of either genotype mice (Fig. 4 ). There was minimal evidence of caspase-9 activation (appreciated by detection of a caspase-9 specific 10-kDa activation cleavage fragment), and this was only observed from kidney lysates of survivin+/– mice (2/4 mice). Twenty-four hours after folic acid administration, there was readily detectable activation of caspase-9 in all the kidney lysates from survivin+/+ and survivin+/– mice (n=4/group). Activation appeared more evident (but not to a statistically significant level) in survivin+/– lysates, with 2.1 ± 0.4-fold more cleavage product, measured by densitometry (n=4, P>0.05). However, the 17-kDa caspase-3 activation cleavage product was only detectable from the survivin+/– lysates (n=4/4), and cytochrome c release into the cytosol was consistently higher (1.8±0.2-fold) in survivin+/– vs. survivin+/+ kidney lysates after folic acid exposure, confirming more activation of apoptosis in the survivin+/– mice in response to the nephrotoxin. Further supporting this conclusion was the notable increase, 24 h following induction of ARF, of the proapoptotic tumor suppressor p53 to a significantly greater extent in the survivin+/– kidney lysates (Fig. 4) (densitometry measures from Western blots: 3.0±0.5-fold, n=4, P=0.015) as compared to survivin+/+ lysates. In this respect, ARF has been associated with augmented expression of p53 (41) , and there are several reports—consistent with our findings—of an inverse relationship between p53 and survivin (29 , 42 , 43) . Overall, the data demonstrate that survivin plays an important role in protecting renal tubular cells against apoptosis associated with folic acid–induced ARF, that heterozygous deficiency of survivin enhances the sensitivity to toxin-induced, caspase-mediated cell death, and that apoptosis in the setting of low survivin is likely linked to increased expression of p53.


Figure 4
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  		Figure 4. Activation of apoptosis after folic acid. Lysates of kidneys from survivin+/+ and survivin+/– mice 24 h after administration of folic acid or saline were separated by SDS-PAGE and Western immunoblotted for detection of active fragments of caspase-3 (17 kDa), caspase-9 (10 kDa), release of cytochrome c into the cytosol (27) , and expression of p53. Representative blots are shown, accompanied by corresponding blots to detect actin. Activation of caspase-3 is only detected in lysates from survivin+/– mice exposed to folic acid, whereas the caspase-9 activation fragment is detectable in folic acid exposed to either genotype. Under baseline conditions, small amounts of caspase-9 activation fragment are detected in lysates from survivin+/– mice, which is increased in ARF. Cytochrome c release is more prominent in lysates from survivin+/– mice with or without ARF. Expression of p53 is dramatically increased in response to folic acid–induced ARF, and the level is significantly higher in the survivin+/– kidneys as compared to the survivin+/+ kidneys (n=4, P<0.05).

Gene therapy with survivin140 or survivin121 prevents folic acid–induced acute tubular necrosis
On the basis of the preceding results, we predicted that delivery of supraphysiological levels of survivin might be protective against ARF. To overexpress survivin in mice, we used hydrodynamic gene delivery, in which an expression plasmid vector is rapidly infused in a large volume intravenously into the tail vein. This method has been successfully utilized to deliver a variety of genes in mice, with persistence in elevated gene expression for more than 5–7 days (38) , and up to 4 months (37) . In pilot studies, we used hydrodynamic gene delivery of a vector with a CMV promoter driving lacZ to assess tissue distribution. At 6, 24, and 48 h, beta-galactosidase was detected in several tissues, with diffuse and prominent expression in the tubular epithelial cells of proximal and distal collecting ducts of the kidneys (not shown). A transient 1.2- to 1.4-fold increase in serum levels of the liver enzyme, alanine aminotransferase, was observed 24 h after gene delivery, but this normalized at 48 h.

We have previously reported that, in the mouse, there are three distinct survivin mRNAs that encode functionally distinct proteins (36) . Both survivin140 (the full-length form) and survivin121 retain the BIR domain that is crucial for interfering with caspase activation, whereas survivin121 lacks the C-terminal coiled-coil domain, which functionally links survivin to the cell cycle (44) . Survivin40 lacks both the coiled-coil domain and the BIR domain and thus has no known independent antiapoptotic function (36) .

Hydrodynamic gene delivery was first used to treat survivin+/+ mice with survivin140 or control vector 24 h before folic acid injection. A further 24 h and 7 days later, renal function and histopathology were assessed (Figs. 5 7 ). Augmented expression of survivin in renal tubular cells in survivin-treated mice, relative to vector-alone-treated mice, 24 h after folic acid injection was confirmed first by Western immunoblotting kidney lysates (Fig. 5) and by immunostaining kidney sections with specific antisurvivin antibodies (Fig. 7) . Several parameters indicated a beneficial response to the treatment with survivin140 24 h after folic acid administration. Most critically, mice that were pretreated with survivin140 had significantly lower levels of serum creatinine (0.7 mg/dl treated vs. 1.5 mg/dl control, P<0.05, n=4 in each group) (Fig. 6A ). Survivin140 significantly reduced quantitive activity levels in kidney lysates of caspase-3 at 6 h (8.1x104±4.5x103 vs. 5.6x104±4.2x103 RLU, untreated vs. treated, respectively, n=4, P=0.0062), and caspase-9 at 6 h (1.95x106±2.2x105 vs. 1.1x106±1.1x105 RLU, untreated vs. treated, respectively, n=4, P<0.05) and at 24 h (1.9x106±1.1x105 vs. 15x106±0.6x105 RLU, untreated vs. treated, respectively, n=4, P=0.0064). By Western blot analysis, expression of the antiapoptotic Bcl-2 member, Bcl-xL, was significantly enhanced by treatment (Fig. 5) . At 24 h, there were also fewer renal tubular cells that stained positive for the proapoptotic Bad (132±38 cells/x200 microscopic field (mf) treated vs. 250±25 cells/x200 mf control, P<0.05), and a significantly reduced number of apoptotic cortical renal tubular cells by TUNEL staining (129±11 cells/x200 mf vs. 1030±10 cells/x200 mf control, P<0.05) (Fig. 6B ). Furthermore, survivin140 pretreatment prevented dilatation of the collecting ducts, renal tubular cell swelling, and brush border changes, and it decreased the number of cells with condensed nuclei.


Figure 5
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  		Figure 5. Expression of survivin after gene delivery and its effect on Bcl-xL expression in folic acid induced ARF. Survivin+/+ or survivin+/– mice were treated with either survivin140 cDNA (+) or with vector alone (–) via hydrodynamic gene delivery. Twenty-four hours later, folic acid was administered, and 24 h after that, kidneys were harvested and Western blots were prepared from the lysates (n=4). Representative blots are shown. Gene delivery of survivin140 increased levels of survivin significantly over the respective vector-alone controls (n=4). Densitometry measures of survivin expression for survivin+/– mice: 2.04 ± 0.13 with survivin140 vs. 1.0 ± 0.24 with control cDNA, P = 0.0089; and for survivin+/+ mice: 1.82 ± 0.24 with survivin140 vs. 0.59 ±.016 with control cDNA, P = 0.0053. For both genotypes, survivin140 induced a significant increase in Bcl-xL expression (2.1±0.2-fold and 2.3±0.3-fold increase in survivin+/+ and survivin+/– kidneys, relative to respective vector-alone-treated control, P<0.05).


Figure 6
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  		Figure 6. Protection from folate-induced ARF after treatment with survivin140. Mice were treated with either survivin140 cDNA or vector alone (control) via hydrodynamic gene delivery. Twenty-four hours later, folic acid was administered to induce ARF. A further 24 h or 7 days later, serum creatinine (A) and the number of apoptotic cells detected by TUNEL staining of kidney sections (B) were quantified. In mice of either genotype, treatment with survivin140 results in significant improvement in both renal function as measured by serum creatinine, and in the number of apoptotic renal tubular cells. *P < 0.05.


Figure 7
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  		Figure 7. Hydrodynamic gene delivery of survivin140 to kidneys during ARF. Survivin+/+ mice (A, B, E, F), and survivin+/– mice (C, D, G, H) were pretreated with either survivin140 (B, D, F, H) or with vector alone (control) (A, C, E, G) via hydrodynamic gene delivery. Twenty-four hours later, mice were exposed to folic acid, and after a further 24 h, mice were sacrificed, and kidneys were TUNEL stained for apoptosis (A–D) or for survivin expression (E–H). As noted before, kidneys from vector-alone-treated survivin+/– mice exhibited more apoptosis and histological changes than vector-alone-treated survivin+/+ mice, while survivin140 decreased the number of apoptotic cells in both genotypes. Survivin is barely detectable in vector-alone-treated survivin+/– kidneys (G), but easily seen in vector-alone survivin+/+ kidneys (E), and prominent in all mice treated with survivin140 (F, H).

The effect of overexpressing survivin140 in survivin+/– mice was also evaluated. Similar to the response in survivin+/+ mice, pretreatment with survivin140 protected the mice as assessed 24 h after folic acid, from ARF, maintaining serum creatinine levels in the normal range, enhancing expression of Bcl-xL (Fig. 5) , significantly diminishing the number of Bad-positive cells (203±62 cells/x200 mf control vs. 133±15 cells/x200 mf), and apoptotic renal tubular cells, and notably ameliorating the extent of renal tubular cell damage (Figs. 6 and 7) . Because the renal function of survivin+/– mice remained disturbed until at least 7 days postfolic acid injection (in contrast to survivin+/+ mice, which had recovered by then), we could also evaluate the response to pretreatment with survivin140 over a longer interval. After 7 days, serum creatinine levels decreased from 0.45 to 0.24 mg/dl in treated vs. control mice, respectively (P<0.05), and the number of apoptotic renal tubular cells were significantly diminished, showing that the protective effects of survivin administration are sustainable (Fig. 6) . When survivin+/– mice were treated with survivin140 at the same time as folic acid was administered, serum creatinine levels were significantly improved as compared with treatment with vector alone (0.36±0.2 mg/ml vs. 1.81±0.3 mg/ml, respectively, n=4, P=0.0069).

These results indicate that prior to or at the onset of induction of ARF with folic acid, treatment with survivin140 is beneficial. We also assessed the effects of other isoforms of survivin. Administration of survivin121 cDNA to survivin+/– mice 24 h prior to folic acid injection also protected them against ARF as assessed 24 h after the induction procedure, preventing a rise in serum creatinine (0.65±0.2 mg/dl treated vs. 1.5±0.2 mg/dl control, P<0.05, n=3 in each group), and significantly diminishing the number of apoptotic renal tubular cells (from 1310±29 apoptotic cells/x200 mf in controls to 440±30 apoptotic cells/x200 mf in survivin121 treated mice, n=3, P<0.05). Thus, there did not appear to be a discernible difference in response to therapy with survivin140 and survivin121. In contrast, pretreatment of survivin+/– mice with survivin40 provided no protection against folic acid–induced ARF. Twenty-four hours after folic acid injection, and with survivin40 pretreatment, the serum creatinine remained elevated at 1.4 ± 0.2 mg/dl, as did the number of apoptotic renal tubular cells (1210±38 cells/x200 mf) (n=3, P>0.5 as compared with untreated control group above).

Gene therapy with survivin140 protects against cisplatin induced ARF
On the basis of the promising results in the folic acid model of ARF, we proceeded to evaluate the therapeutic potential of survivin in the clinically more relevant model of cisplatin induced ARF (45) . Inbred female Swiss wild-type mice were administered a single i.p. dose of cisplatin, after which, renal function was quantified with serum creatinine measurements at different time points. Pilot studies revealed that following a dose of 20 mg/kg body weight of cisplatin, the chemotherapeutic agent reproducibly induced a progressive deterioration in renal function, such that by 4 days, most animals became ill and were sacrificed. At 24 h, cisplatin caused serum creatinine levels in wild-type mice to increase ~1.6-fold—not quite to a significant level—over saline-treated controls (serum creatinines: 0.25±0.02 mg/dl (n=5) and 0.40±0.06 mg/dl (n=8) at 0 and 24 h, respectively). At 48 and 72 h, serum creatinine levels rose significantly over the controls to 0.62 ± 0.05 mg/dl (n=5) and 1.53 ± 0.4 mg/dl (n=8), respectively, P<0.01). This peak in renal dysfunction at 3 days postcisplatin is consistent with that previously reported (34 , 45) .

The therapeutic efficacy of survivin140, delivered by hydrodynamic gene delivery 24 h prior to cisplatin, was first tested. Delivery of survivin to the kidney was confirmed by Western blot analysis of SDS-PAGE-separated lysates (Fig. 8 ): by densitometry, there was a significant increase in survivin, relative to vector alone (densitometric analyses, n=4 per group) at 6 and 24 h (1.7±0.1-fold and 1.8±0.1-fold, respectively, P < 0.05). Elevated expression of survivin in the kidney persisted (density of bands on blots from survivin-treated mice 1.6- to 1.8-fold higher than with vector alone, n=4), but not to a statistically significant extent. Renal function and histological evidence of renal tubular cell apoptosis were evaluated 24, 48, and 72 h after cisplatin injection. In all wild-type mice exposed to the cisplatin, serum creatinine levels progressively rose, but mice pretreated with survivin140 cDNA as compared with vector alone had significantly lower serum creatinine levels at 48 and 72 h (0.48±0.01 mg/dl vs. 0.60±0.04 mg/dl at 48 h, P=0.041; and 1.22±0.12 mg/dl vs. 1.59±0.3 mg/dl at 72 h, P=0.032; for survivin140 cDNA and vector alone, respectively, n=5–11) (Fig. 9 A). This protective response to survivin140 occurred in concert with a significantly more rapid decrease in the number of apoptotic renal tubular epithelial cells in the cortex, as measured by TUNEL staining (Figs. 9 B, 10 F–J). At 24 h after cisplatin administration, more than 30% of the nucleated cells underwent apoptosis, both in the kidneys of mice treated with survivin140 and in those treated with vector-alone (controls). At that interval and even earlier in the course (6 h), Western blot analysis revealed that p53 levels were significantly reduced, while Bcl-xL was enhanced (Fig. 8) . Consistent with the pattern of response previously reported using the same cisplatin model (34) , the number of apoptotic cells abruptly decreased at 48 h, remaining relatively low at 72 h. However, treatment with survivin140 yielded significantly fewer apoptotic cells at both of the latter time points (at 48 h, % apoptosis=7.2±1.0 vs. 10.5±1.0, for survivin140 cDNA and vector alone, respectively, P=0.038; at 72 h, % apoptosis=5.1±0.5 vs. 11.1±0.8, P<0.001, for survivin140 cDNA and vector alone, respectively) (Fig. 9B ). The beneficial effects of survivin140 were also revealed by H&E staining of kidney sections (Fig. 10) . In the vector-alone-treated mice (Fig. 10B , C, G, H), cisplatin induced extensive renal tubular cell swelling, loss of brush borders, and, at 72 h, extensive accumulation of renal tubular casts. In contrast, administration of survivin140 provided obvious evidence of protection against the cisplatin, both at 48 and 72 h (Fig. 10D , E, I, J), in parallel with improved renal function.


Figure 8
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  		Figure 8. Expression of survivin after gene delivery and its effect on Bcl-xL and p53 in cisplatin induced ARF. Wild-type mice were treated with either survivin140 cDNA (+) or vector alone (–), and 24 h later, cisplatin was administered. Western blots of kidney lysates were prepared 6 and 24 h later (n=4/group). Representative blots are shown. Gene delivery of survivin140 caused a significant rise in survivin levels in the kidneys of mice (see text), concomitant with a significant increase in Bcl-xL levels (1.6±0.2-fold and 1.8±1.2-fold, at 6 and 24 h, respectively, P<0.05) over the control. p53 levels were markedly suppressed by administration of survivin140 at both 6 and 24 h after cisplatin.


Figure 9
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  		Figure 9. Protection from cisplatin-induced ARF after treatment with survivin140. Wild-type mice were treated with either survivin140 cDNA or vector alone (control) via hydrodynamic gene delivery. Twenty-four hours later, cisplatin was administered to induce ARF. A further 24, 48, or 72 h later, serum creatinine (A) and the number of apoptotic cells detected by TUNEL staining of kidney sections (B) were quantified. Treatment with survivin140 results in significant improvement in both renal function as measured by serum creatinine and in the number of apoptotic renal tubular cells. *P < 0.05; **P < 0.01.


Figure 10
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  		Figure 10. Histological changes after cisplatin-induced ARF and treatment with survivin140. Wild-type mice were treated with either survivin140 cDNA (D, E, I, J) or vector alone (control) (C, D, G, H) via hydrodynamic gene delivery. Twenty-four hours later, cisplatin was administered to induce ARF. A further 48 h (B, D, G, I) or 72 h later (C, E, H, J), mice were sacrificed, and kidney sections were stained with H&E (A–E) or by TUNEL (F–J). Images A and B are representative kidney sections from a wild-type mouse unexposed to cisplatin or hydrodynamic gene delivery. Cisplatin preceded by vector-alone treatment causes profound histological changes (B, C) with accumulation of tubular casts (C), and apoptotic renal tubular cells (G, H). Treatment with survivin140 results in significant improvement by H&E staining (D, E), and TUNEL (I, J).

We also assessed whether the delivery of survivin140 closer to the time of administration of cisplatin would be protective against ARF. Therefore, survivin140 cDNA or vector alone was delivered via hydrodynamic gene delivery 3 h before cisplatin, and renal function was evaluated at 72 h. Serum creatinine levels were 1.86 ± 0.19 and 0.83 ± 0.21 mg/dl for vector-alone and survivin140 cDNA, respectively (P=0.007, n=5 in each group). At the same time point, caspase-3 activity in kidney lysates, measured by the Caspase-Glo 3/7 assay (Promega) was significantly suppressed by survivin140 cDNA (1.0x106±5.9x104 RLU for vector alone vs. 0.83x106±5.5x104 RLU for survivin140 cDNA, n=5, P=0.038), as was the number of apoptotic renal tubular cells, quantified after Apoptag staining of kidney sections (at 72 h, % apoptosis=4.5±1.1, n=6, vs. 9.9±1.0, n=5, for survivin140 cDNA and vector alone, respectively; P=0.0069). Thus, with this preventive strategy, survivin provided significant protection against cisplatin-induced ARF.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The results of this study highlight the importance of the IAP survivin in protecting the kidney against toxin-induced ARF, and furthermore, they demonstrate that gene therapy with forms of survivin in which the BIR domain is intact may have therapeutic utility.

Despite observations that quiescent adult cells express little or no survivin, many tissues require >50% levels of survivin, i.e., both survivin alleles, to maintain homeostasis in the face of a variety of pathophysiologic stresses. For example, survivin+/– mice, which under baseline conditions appear normal, are more sensitive than their wild-type counterparts to fasL-induced hepatocellular necrosis (27) . Survivin+/– mice also have a dampened angiogenic response to stroke (32) , and their neutrophils exhibit a poor survival response to interleukin-3 (46) . Given the evidence that apoptosis is a critical factor in the pathogenesis and progression of ARF (reviewed in refs. 1 , 7 ) and the prominent role of survivin in cell and tissue homeostasis, we predicted that renal tubular epithelial cells of survivin+/– mice would exhibit enhanced sensitivity to toxin-induced acute tubular necrosis and renal failure, and finally that survivin may thus be a therapeutic target for intervention.

Folic acid–induced ARF resulted in a worse outcome in survivin+/– mice as compared to wild-type sibling controls in terms of renal histology and renal function. Onset of ARF was more rapid in the survivin+/– mice, and recovery was delayed. Although serum creatinine levels were similarly elevated in survivin+/+ and survivin+/– mice at 24 h, the morphology of the kidneys of the survivin+/– mice was notably worse, with more evidence of renal tubular epithelial cell necrosis and apoptosis. By 7 days after injection of folic acid, renal function and morphology of the survivin+/+ mice returned almost to normal, whereas the recovery time of the survivin+/– mice was delayed. The contribution of apoptosis to the more rapid and severe progression of renal failure in the survivin+/– mice was substantiated by TUNEL staining kidney sections and was also documented biochemically by greater increases in caspase-3 and caspase-9 activation, release of cytochrome c, and accumulation of p53.

In view of the profound effect that low levels of survivin had on renal function when exposed to folic acid, we tested the hypothesis that administration of survivin might be protective. The relatively simple naked DNA gene therapy approach, referred to as "hydrodynamic gene delivery" (47) , has been successfully used to evaluate the efficacy of hepatocyte growth factor for ARF in mice (38) . By this method, we first demonstrated that the CMV promoter driving a cDNA encoding β-galactosidase results in high expression in renal tubular cells. We then showed that after a single injection of survivin140 cDNA, the renal failure induced by folic acid could be almost entirely prevented in both survivin+/+ and survivin+/– mice and that the effect was sustainable for at least 7 days. Serum creatinine levels were decreased, morphology was improved, and the number of apoptotic cells was suppressed. Similar protective effects of survivin140 were obtained in the clinically more relevant model of cisplatin-induced ARF. A sensitive assay to detect tissue levels of murine survivin is lacking. However, our data confirmed increased kidney expression after induction of ARF for at least 24 h, and likely beyond 72 h. Nonetheless, it remains to be determined whether high tissue levels of survivin must be maintained to achieve optimal therapeutic benefit, or whether secondary protective effects proceed after its initial administration.

The apparent inverse relationship of survivin with p53 was particularly intriguing. ARF may be initiated by depletion of intracellular GTP (48) , which is associated with rapid induction of p53 (41) , which may in turn promote cell death. Also, several studies support a functional and opposing relationship between p53 and survivin (29 , 42 , 43) . Our results, in which renal expression of p53 was suppressed after survivin140 gene therapy, suggest that suppression of p53 expression may be one mechanism by which administration of survivin prevents renal tubular epithelial cell death in some forms of toxin-induced ARF.

A beneficial effect on toxin-induced ARF was also observed when we pretreated the mice with cDNA encoding survivin121, an isoform that retains the BIR domain that is critical for antiapoptotic functional. In contrast, and not surprisingly, survivin40, which lacks the BIR domain, had no effect in the ARF model. The therapeutic efficacy of survivin121, which lacks the C-terminus coiled-coil structure, may be relevant in the design of safe therapies. Survivin is highly expressed in essentially all tumors (reviewed in ref. 18 ), and there are concerns that treatment with survivin might induce tumor growth, even though transplanted pancreatic islet cells that overexpress full-length survivin did not show evidence of malignant transformation (49) . Nonetheless, in vitro studies show that full-length survivin promotes cell proliferation in hepatocellular carcinoma (50) , and in vivo, survivin may oppose the elimination of cancerous cells by p53 (42) . This concern might be mitigated if one could identify those domains of survivin that do not induce tumor formation. We are currently evaluating which isoforms or domains of survivin retain their renal-protective properties yet do not promote tumor growth.

Although we have shown that administration of survivin protects against toxin-induced ARF, we have not yet determined the cellular target. The hydrodynamic gene delivery method caused survivin expression in the renal tubular epithelial cells to be augmented, and thus it is likely that survivin at that location provided protection. However, survivin has downstream effects and interacts with several protein partners, and the mechanisms underlying ARF are complex and involve multiple cells. For example, inflammation has been implicated in playing a major role in ARF (51 52 53) , and it is possible that overexpression of survivin in different leukocyte populations may modulate the disease. Evaluation of the role of these different cells in ARF, using transgenic mice in which the survivin gene has been inactivated in a cell-specific manner, is ongoing. We are furthermore cognizant of the danger implicit in extrapolating between the various models of human disease. ARF that is precipitated by, for example, ischemia-reperfusion, undoubtedly involves the recruitment of distinctly different molecular mechanisms from those that are involved in, for example, cisplatin-induced kidney damage. Thus, a strategy using a single antiapoptotic agent for therapy is not expected to be sufficiently robust to widely apply it to all causes of ARF. Further study with a variety of models will ultimately facilitate the design of more targeted approaches to prevent and/or treat the different causes of ARF with appropriately selected antiapoptotic agents.

In the past 5–10 yr, several therapies have been evaluated for treatment and/or prevention of ARF, none of which have been shown to be effective in humans. These include, for example, insulin growth factor I (54) , lysophosphatidic acid (55) , minocycline (56) , interleukin-10 (45) , antioxidants (57 , 58) , parathyroid hormone-related protein, hepatocyte growth factor (HGF) (59) , and atorvastatin (60) . There is clearly an urgent need for new agents that may be used singly or in combination, and these must be safe and efficacious. The described studies suggest that survivin may be an effective therapeutic target to treat and/or prevent acute renal failure.


   ACKNOWLEDGMENTS
 
Funding for research by E.M.C. and N.K. was provided by the Fonds voor Wetenschappelijk Onderzoek, Belgium, and the Dutch Kidney Foundation, the Netherlands. M.V. was supported by a fellowship from the Instituut voor Innovatie door Wetenschap en Technologie, Belgium. A.M. was supported by a grant from the Deutsche Forschungsgemeinschaft, Bonn, Germany.

Received for publication April 30, 2007. Accepted for publication August 9, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Liu, K. D. (2003) Molecular mechanisms of recovery from acute renal failure. Crit. Care Med. 31,S572-S581[CrossRef][Medline]
  2. Sheikh-Hamad, D., Cacini, W., Buckley, A. R., Isaac, J., Truong, L. D., Tsao, C. C., Kishore, B. K. (2004) Cellular and molecular studies on cisplatin-induced apoptotic cell death in rat kidney. Arch. Toxicol. 78,147-155[CrossRef][Medline]
  3. Wan, L., Bellomo, R., Di Giantomasso, D., Ronco, C. (2003) The pathogenesis of septic acute renal failure. Curr. Opin. Crit. Care 9,496-502[CrossRef][Medline]
  4. Evenepoel, P. (2004) Acute toxic renal failure. Best Pract. Res. Clin. Anaesthesiol. 18,37-52[CrossRef][Medline]
  5. Silvester, W., Bellomo, R., Cole, L. (2001) Epidemiology, management, and outcome of severe acute renal failure of critical illness in Australia. Crit. Care Med. 29,1910-1915[CrossRef][Medline]
  6. Ortiz, A., Lorz, C., Catalan, M. P., Danoff, T. M., Yamasaki, Y., Egido, J., Neilson, E. G. (2000) Expression of apoptosis regulatory proteins in tubular epithelium stressed in culture or following acute renal failure. Kidney Int. 57,969-981[CrossRef][Medline]
  7. Ortiz, A., Justo, P., Catalan, M. P., Sanz, A. B., Lorz, C., Egido, J. (2002) Apoptotic cell death in renal injury: the rationale for intervention. Curr. Drug Targets Immune. Endocr. Metabol. Disord. 2,181-192[CrossRef][Medline]
  8. Padanilam, B. J. (2003) Cell death induced by acute renal injury: a perspective on the contributions of apoptosis and necrosis. Am. J. Physiol. Renal. Physiol. 284,F608-F627[Abstract/Free Full Text]
  9. Devarajan, P., Mishra, J., Supavekin, S., Patterson, L. T., Steven Potter, S. (2003) Gene expression in early ischemic renal injury: clues towards pathogenesis, biomarker discovery, and novel therapeutics. Mol. Genet. Metab. 80,365-376[CrossRef][Medline]
  10. Nogae, S., Miyazaki, M., Kobayashi, N., Saito, T., Abe, K., Saito, H., Nakane, P. K., Nakanishi, Y., Koji, T. (1998) Induction of apoptosis in ischemia-reperfusion model of mouse kidney: possible involvement of Fas. J. Am. Soc. Nephrol. 9,620-631[Abstract]
  11. Justo, P., Sanz, A., Lorz, C., Gomez-Garre, D., Mezzano, S., Egido, J., Ortiz, A. (2003) Expression of Smac/Diablo in tubular epithelial cells and during acute renal failure. Kidney Int. (Suppl),S52-S56
  12. Xie, J., Guo, Q. (2007) Par-4 is a novel mediator of renal tubule cell death in models of ischemia-reperfusion injury. Am. J. Physiol. Renal. Physiol. 292,F107-F115[Abstract/Free Full Text]
  13. Bonegio, R., Lieberthal, W. (2002) Role of apoptosis in the pathogenesis of acute renal failure. Curr. Opin. Nephrol. Hypertens. 11,301-308[CrossRef][Medline]
  14. Wei, Q., Yin, X. M., Wang, M. H., Dong, Z. (2006) Bid deficiency ameliorates ischemic renal failure and delays animal death in C57BL/6 mice. Am. J. Physiol. Renal. Physiol. 290,F35-F42[Abstract/Free Full Text]
  15. Kaushal, G. P. (2003) Role of caspases in renal tubular epithelial cell injury. Semin. Nephrol. 23,425-431[CrossRef][Medline]
  16. Oberbauer, R., Rohrmoser, M., Regele, H., Muhlbacher, F., Mayer, G. (1999) Apoptosis of tubular epithelial cells in donor kidney biopsies predicts early renal allograft function. J. Am. Soc. Nephrol. 10,2006-2013[Abstract/Free Full Text]
  17. Schwarz, C., Hauser, P., Steininger, R., Regele, H., Heinze, G., Mayer, G., Oberbauer, R. (2002) Failure of BCL-2 up-regulation in proximal tubular epithelial cells of donor kidney biopsy specimens is associated with apoptosis and delayed graft function. Lab. Invest. 82,941-948[Medline]
  18. Li, F. (2003) Survivin study: what is the next wave?. J. Cell Physiol. 197,8-29[CrossRef][Medline]
  19. Li, F., Ling, X. (2006) Survivin study: an update of "what is the next wave"?. J. Cell Physiol. 208,476-486[CrossRef][Medline]
  20. Altieri, D. C. (2003) Survivin, versatile modulation of cell division and apoptosis in cancer. Oncogene 22,8581-8589[CrossRef][Medline]
  21. Tamm, I., Wang, Y., Sausville, E., Scudiero, D. A., Vigna, N., Oltersdorf, T., Reed, J. C. (1998) IAP-family protein survivin inhibits caspase activity and apoptosis induced by Fas (CD95), Bax, caspases, and anticancer drugs. Cancer Res. 58,5315-5320[Abstract/Free Full Text]
  22. Dohi, T., Altieri, D. C. (2005) Mitochondrial dynamics of survivin and "four dimensional" control of tumor cell apoptosis. Cell Cycle 4,21-23[Medline]
  23. Chen, J., Wu, W., Tahir, S. K., Kroeger, P. E., Rosenberg, S. H., Cowsert, L. M., Bennett, F., Krajewski, S., Krajewska, M., Welsh, K., Reed, J. C., Ng, S. C. (2000) Down-regulation of survivin by antisense oligonucleotides increases apoptosis, inhibits cytokinesis and anchorage-independent growth. Neoplasia 2,235-241[Medline]
  24. Olie, R. A., Simoes-Wust, A. P., Baumann, B., Leech, S. H., Fabbro, D., Stahel, R. A., Zangemeister-Wittke, U. (2000) A novel antisense oligonucleotide targeting survivin expression induces apoptosis and sensitizes lung cancer cells to chemotherapy. Cancer Res. 60,2805-2809[Abstract/Free Full Text]
  25. Grossman, D., McNiff, J. M., Li, F., Altieri, D. C. (1999) Expression and targeting of the apoptosis inhibitor, survivin, in human melanoma. J. Invest. Dermatol. 113,1076-1081[CrossRef][Medline]
  26. Xia, C., Xu, Z., Yuan, X., Uematsu, K., You, L., Li, K., Li, L., McCormick, F., Jablons, D. M. (2002) Induction of apoptosis in mesothelioma cells by antisurvivin oligonucleotides. Mol. Cancer Ther. 1,687-694[Abstract/Free Full Text]
  27. Conway, E. M., Pollefeyt, S., Steiner-Mosonyi, M., Luo, W., DeVriese, A., Lupu, F., Bono, F., Leducq, N., Dol, F., Schaeffer, P., Collen, D., Herbert, J.-M. (2002) Deficiency of survivin in transgenic mice exacerbates Fas-induced apoptosis via mitochondrial pathways. Gastroenterology 123,619-631[CrossRef][Medline]
  28. Uren, A. G., Wong, L., Pakusch, M., Fowler, K. J., Burrows, F. J., Vaux, D. L., Choo, K. H. (2000) Survivin and the inner centromere protein INCENP show similar cell-cycle localization and gene knockout phenotype. Curr. Biol. 10,1319-1328[CrossRef][Medline]
  29. Beltrami, E., Plescia, J., Wilkinson, J. C., Duckett, C. S., Altieri, D. C. (2004) Acute ablation of survivin uncovers p53-dependent mitotic checkpoint functions and control of mitochondrial apoptosis. J. Biol. Chem. 279,2077-2084[Abstract/Free Full Text]
  30. Klingler, E. L., Jr, Evan, A. P., Anderson, R. E. (1980) Folic acid-induced renal injury and repair. Correlation of structural and functional abnormalities. Arch. Pathol. Lab. Med. 104,87-93[Medline]
  31. Tsuruya, K., Ninomiya, T., Tokumoto, M., Hirakawa, M., Masutani, K., Taniguchi, M., Fukuda, K., Kanai, H., Kishihara, K., Hirakata, H., Iida, M. (2003) Direct involvement of the receptor-mediated apoptotic pathways in cisplatin-induced renal tubular cell death. Kidney Int. 63,72-82[Medline]
  32. Conway, E. M., Zwerts, F., Van Eygen, V., DeVries, A., Nagai, N., Luo, W., Collen, D. (2003) Survivin-dependent angiogenesis in ischemic brain: Molecular mechanisms of hypoxia-induced upregulation. Am. J. Pathol. 163,935-946[Abstract/Free Full Text]
  33. Justo, P., Sanz, A. B., Sanchez-Nino, M. D., Winkles, J. A., Lorz, C., Egido, J., Ortiz, A. (2006) Cytokine cooperation in renal tubular cell injury: the role of TWEAK. Kidney Int. 70,1750-1758[CrossRef][Medline]
  34. Faubel, S., Ljubanovic, D., Reznikov, L., Somerset, H., Dinarello, C. A., Edelstein, C. L. (2004) Caspase-1-deficient mice are protected against cisplatin-induced apoptosis and acute tubular necrosis. Kidney Int. 66,2202-2213[CrossRef][Medline]
  35. Jiang, M., Wei, Q., Wang, J., Du, Q., Yu, J., Zhang, L., Dong, Z. (2006) Regulation of PUMA-alpha by p53 in cisplatin-induced renal cell apoptosis. Oncogene 25,4056-4066[CrossRef][Medline]
  36. Conway, E. M., Pollefeyt, S., Cornelissen, J., DeBaere, I., Steiner-Mosonyi, M., Ong, K., Baens, M., Collen, D., Schuh, A. C. (2000) Three differentially expressed survivin cDNA variants encode proteins with distinct antiapoptotic functions. Blood 95,1435-1442[Abstract/Free Full Text]
  37. Alino, S. F., Crespo, A., Dasi, F. (2003) Long-term therapeutic levels of human alpha-1 antitrypsin in plasma after hydrodynamic injection of nonviral DNA. Gene. Ther. 10,1672-1679[CrossRef][Medline]
  38. Dai, C., Yang, J., Liu, Y. (2002) Single injection of naked plasmid encoding hepatocyte growth factor prevents cell death and ameliorates acute renal failure in mice. J. Am. Soc. Nephrol. 13,411-422[Abstract/Free Full Text]
  39. Wan, B., Hao, L., Qiu, Y., Sun, Z., Cao, Q., Zhang, Y., Zhu, T., Wang, H., Zhang, Y. (2006) Blocking tumor necrosis factor-alpha inhibits folic acid-induced acute renal failure. Exp. Mol. Pathol. 81,211-216[CrossRef][Medline]
  40. Perco, P., Pleban, C., Kainz, A., Lukas, A., Mayer, G., Mayer, B., Oberbauer, R. (2006) Protein biomarkers associated with acute renal failure and chronic kidney disease. Eur. J. Clin. Invest. 36,753-763[CrossRef][Medline]
  41. Kelly, K. J., Plotkin, Z., Vulgamott, S. L., Dagher, P. C. (2003) P53 mediates the apoptotic response to GTP depletion after renal ischemia-reperfusion: protective role of a p53 inhibitor. J. Am. Soc. Nephrol. 14,128-138[Abstract/Free Full Text]
  42. Grossman, D., Kim, P. J., Blanc-Brude, O. P., Brash, D. E., Tognin, S., Marchisio, P. C., Altieri, D. C. (2001) Transgenic expression of survivin in keratinocytes counteracts UVB-induced apoptosis and cooperates with loss of p53. J. Clin. Invest. 108,991-999[CrossRef][Medline]
  43. Hoffman, W. H., Biade, S., Zilfou, J. T., Chen, J., Murphy, M. (2002) Transcriptional repression of the anti-apoptotic survivin gene by wild-type p53. J. Biol. Chem. 277,3247-3257[Abstract/Free Full Text]
  44. Li, F., Ambrosini, G., Chu, E. Y., Plescia, J., Tognin, S., Marchisio, P. C., Altieri, D. C. (1998) Control of apoptosis and mitotic spindle checkpoint by survivin. Nature 396,580-584[CrossRef][Medline]
  45. Deng, J., Kohda, Y., Chiao, H., Wang, Y., Hu, X., Hewitt, S. M., Miyaji, T., McLeroy, P., Nibhanupudy, B., Li, S., Star, R. A. (2001) Interleukin-10 inhibits ischemic and cisplatin-induced acute renal injury. Kidney Int. 60,2118-2128[CrossRef][Medline]
  46. Altznauer, F., Martinelli, S., Yousefi, S., Thurig, C., Schmid, I., Conway, E., Schoni, M., Vogt, P., Mueller, C., Fey, M., Zangemeister-Wittke, U., Simon, H.-U. (2004) Inflammation-associated cell cycle-independent block of apoptosis by survivin in terminally differentiated neutrophils. J. Exp. Med. 199,1343-1354[Abstract/Free Full Text]
  47. Liu, F., Song, Y., Liu, D. (1999) Hydrodynamics-based transfection in animals by systemic administration of plasmid DNA. Gene Ther. 6,1258-1266[CrossRef][Medline]
  48. Kelly, K. J., Plotkin, Z., Dagher, P. C. (2001) Guanosine supplementation reduces apoptosis and protects renal function in the setting of ischemic injury. J. Clin. Invest. 108,1291-1298[CrossRef][Medline]
  49. Dohi, T., Salz, W., Costa, M., Ariyan, C., Basadonna, G. P., Altieri, D. C. (2006) Inhibition of apoptosis by survivin improves transplantation of pancreatic islets for treatment of diabetes in mice. EMBO Rep. 7,438-443[Medline]
  50. Ito, T., Shiraki, K., Sugimoto, K., Yamanaka, T., Fujikawa, K., Ito, M., Takase, K., Moriyama, M., Kawano, H., Hayashida, M., Nakano, T., Suzuki, A. (2000) Survivin promotes cell proliferation in human hepatocellular carcinoma. Hepatology 31,1080-1085[CrossRef][Medline]
  51. Liu, M., Chien, C. C., Burne-Taney, M., Molls, R. R., Racusen, L. C., Colvin, R. B., Rabb, H. (2006) A pathophysiologic role for T lymphocytes in murine acute cisplatin nephrotoxicity. J. Am. Soc. Nephrol. 17,765-774[Abstract/Free Full Text]
  52. Li, S., Gokden, N., Okusa, M. D., Bhatt, R., Portilla, D. (2005) Anti-inflammatory effect of fibrate protects from cisplatin-induced ARF. Am. J. Physiol. Renal. Physiol. 289,F469-F480[Abstract/Free Full Text]
  53. Kawai, H., Sato, W., Yuzawa, Y., Kosugi, T., Matsuo, S., Takei, Y., Kadomatsu, K., Muramatsu, T. (2004) Lack of the growth factor midkine enhances survival against cisplatin-induced renal damage. Am. J. Pathol. 165,1603-1612[Abstract/Free Full Text]
  54. Daemen, M. A., van’t Veer, C., Denecker, G., Heemskerk, V. H., Wolfs, T. G., Clauss, M., Vandenabeele, P., Buurman, W. A. (1999) Inhibition of apoptosis induced by ischemia-reperfusion prevents inflammation. J. Clin. Invest. 104,541-549[Medline]
  55. De Vries, B., Matthijsen, R. A., van Bijnen, A. A., Wolfs, T. G., Buurman, W. A. (2003) Lysophosphatidic acid prevents renal ischemia-reperfusion injury by inhibition of apoptosis and complement activation. Am. J. Pathol. 163,47-56[Abstract/Free Full Text]
  56. Kelly, K. J., Sutton, T. A., Weathered, N., Ray, N., Caldwell, E. J., Plotkin, Z., Dagher, P. C. (2004) Minocycline inhibits apoptosis and inflammation in a rat model of ischemic renal injury. Am. J. Physiol. Renal. Physiol. 287,F760-F766[Abstract/Free Full Text]
  57. Tsuruya, K., Tokumoto, M., Ninomiya, T., Hirakawa, M., Masutani, K., Taniguchi, M., Fukuda, K., Kanai, H., Hirakata, H., Iida, M. (2003) Antioxidant ameliorates cisplatin-induced renal tubular cell death through inhibition of death receptor-mediated pathways. Am. J. Physiol. Renal. Physiol. 285,F208-F218[Abstract/Free Full Text]
  58. Santos, N. A., Bezerra, C. S., Martins, N. M., Curti, C., Bianchi, M. L., and Santos, A. C. (2007) Hydroxyl radical scavenger ameliorates cisplatin-induced nephrotoxicity by preventing oxidative stress, redox state unbalance, impairment of energetic metabolism and apoptosis in rat kidney mitochondria. Cancer Chemother. Pharmacol. doi: 10.1007/s00280-007-0459-y
  59. Fiaschi-Taesch, N. M., Santos, S., Reddy, V., Van Why, S. K., Philbrick, W. F., Ortega, A., Esbrit, P., Orloff, J. J., Garcia-Ocana, A. (2004) Prevention of acute ischemic renal failure by targeted delivery of growth factors to the proximal tubule in transgenic mice: the efficacy of parathyroid hormone-related protein and hepatocyte growth factor. J. Am. Soc. Nephrol. 15,112-125[Abstract/Free Full Text]
  60. Sabbatini, M., Pisani, A., Uccello, F., Serio, V., Seru, R., Paterno, R., Cianciaruso, B., Fuiano, G., Andreucci, M. (2004) Atorvastatin improves the course of ischemic acute renal failure in aging rats. J. Am. Soc. Nephrol. 15,901-909[Abstract/Free Full Text]



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