P53 Mediates the Apoptotic Response to GTP Depletion after Renal Ischemia-Reperfusion: Protective Role of a p53 Inhibitor
K. J. Kelly,
Zoya Plotkin,
Stacey L. Vulgamott and
Pierre C. Dagher
Indiana Center for Biological Microscopy, Department of Medicine, Division of Nephrology, Indiana University, Indianapolis, Indiana.
Correspondence to Dr. Pierre Dagher, Department of Medicine, Division of Nephrology, 1120 South Drive, FH 115, Indianapolis, Indiana, 46202. Phone: 317-278-2867; Fax: 317-274-8575;
ABSTRACT. Ischemic injury to the kidney is characterized inpart by nucleotide depletion and tubular cell death in the formof necrosis or apoptosis. GTP depletion was recently identifiedas an important inducer of apoptosis during chemical anoxiain vitro and ischemic injury in vivo. It has also been shownthat GTP salvage with guanosine prevented apoptosis and protectedfunction. This study investigates the role of p53 in mediatingthe apoptotic response to GTP depletion. Male Sprague-Dawleyrats underwent bilateral renal artery clamp for 30 min followedby reperfusion. p53 protein levels increased significantly inthe medulla over 24 h post-ischemia. The provision of guanosineinhibited the increase in p53. Pifithrin-, a specific inhibitorof p53, mimicked the effects of guanosine. It had no effecton necrosis, yet it prevented apoptosis and protected renalfunction. Pifithrin- was protective when given up to 14 h afterthe ischemic insult. The effects of pifithrin- on p53 includedinhibition of transcriptional activation of downstream p53 targetslike p21 and Bax and inhibition of p53 translocation to themitochondria. Similar results were obtained in cultured renaltubular cells. It is concluded that p53 is an important mediatorof apoptosis during states of GTP depletion. Inhibitors of p53should be considered in the treatment of ischemic renal injury.
Renal ischemia-reperfusion (I-R) injury is characterized histologicallyby inflammation and tubular cell death in the form of necrosisand/or apoptosis (1,2). The relative importance of these twoforms of cell death in determining the functional outcome ofinjury remains poorly understood. Nevertheless, agents thatmodulate or prevent apoptosis have been shown to be protectiveafter I-R in various tissues and organs. Such agents includeantioxidants, -MSH, caspase inhibitors, and growth factors (3–6).Despite diverse mechanisms of action, all these agents ultimatelyshow potent antiapoptotic properties that account, at leastin part, for their protective effects.
We recently identified GTP depletion as an important stimulusfor apoptosis after renal I-R in both mice and rats (7,8). Wealso showed that salvage of GTP levels with guanosine resultedin markedly reduced tubular cell apoptosis and protection fromacute renal failure. These studies extended our earlier observationsin renal tubular cells in culture that specifically linked theapoptotic phenotype to GTP depletion (9). We now investigatethe mechanism by which GTP depletion induces apoptosis.
We hypothesize that the apoptotic response to GTP depletionduring I-R is mediated by p53. Indeed, p53 plays a central roleas the initiator of the intrinsic apoptotic cascade triggeredby a wide variety of insults (10). These include UV irradiation,chemotherapeutic agents, free radicals, hypoxia, and nucleotidedepletion (11,12). In addition, a role for p53 in regulatingthe extrinsic receptor-mediated apoptotic pathway has also beenreported (13,14). Thus, p53 is poised as an ideal candidatefor mediating apoptosis after I-R, a setting where many of theabove insults coexist.
We also investigated the effects of the newly described potentand specific inhibitor of p53, pifithrin- (PIF). This syntheticcompound was recently shown to protect against intestinal epithelialapoptosis after radiation or chemotherapy and was proposed astherapy for the devastating diarrhea that results from theseagents (15,16). Our results show that we are able to directlyinhibit p53 with PIF after I-R. In fact, PIF had an overallprotective profile identical to that we described with guanosine(7). That is, inhibition of p53 protected against apoptosisand resulted in improved renal function without significantlyaffecting necrosis. Our studies further probe the complex interactionbetween PIF and p53 by examining the transcriptional activityand cytoplasmic-mitochondrial translocation of p53.
Renal Ischemia
All animal experimentation was conducted in conformity withthe "Guiding Principles for Research Involving Animals and HumanBeings." PIF (2.2 mg/kg; Calbiochem, San Diego, CA) in 0.9%NaCl or 0.0005%DMSO/0.9% NaCl or an equal volume of 0.9% NaClor DMSO/0.9% NaCl (placebo) was administered via intraperitonealinjection. The dose of PIF was based on the in vivo and in vitroanti-apoptotic efficacy reported in the literature (16). MaleSprague-Dawley rats weighing 180 to 220 g (Harlan, Indianapolis,IN) were anesthetized with intraperitoneal sodium pentobarbital(50 to 70 mg/kg) and placed on a homeothermic table to maintaincore body temperature at 37°C. Both renal pedicles wereoccluded via a midline incision for 30 or 45 min followed byreperfusion (7,17). Sham surgery consisted of an identical procedurewith the exception of application of the microaneurysm clamps.Creatinine was determined by standard picric acid reaction inserum obtained from the tail vein or via cardiac puncture.
Light Microscopy
Twenty-four, 48, or 72 h after surgery, kidneys were perfusion-fixedin situ with 4% paraformaldehyde, paraffin-embedded, sectionedat 4 microns, and stained using hematoxylin and eosin.
Fluorescence Microscopy
A Zeiss confocal microscope (LSM 510) equipped with UV, argon,and helium lasers was used. Pieces from the in situ fixed kidneyswere preserved in 20% sucrose before 10-µm frozen sectionswere obtained. Some sections were stained for p53 with a primarysheep polyclonal anti-p53 antibody (Ab-7; Oncogene, Boston,MA). This was followed with a secondary unlabeled rabbit anti-sheepIgG (Zymed Lab Inc., San Francisco, CA) and a tertiary TexasRed-labeled donkey anti-rabbit IgG (Jackson Lab., West Grove,PA). Mitochondria were stained with anti-cytochrome c oxidase(anti-cox) mouse monoclonal antibody (Molecular Probes, Eugene,OR) and a secondary goat anti-mouse IgG, FITC-conjugated (JacksonLab.). Finally, nuclei were stained with the nuclear dye To-Pro-3iodide (Molecular Probes). Images were collected and analyzedwith Zeiss LSM software and MetaMorph (Universal Imaging Corporation).In addition, we used Corr3D, a software program developed inour division by Chris Constantine. It allows the quantitationof overlap between pixels from different channels and thus yieldsan accurate estimate of colocalization (values ranging from0 [no colocalization] to 1 [100% colocalization]). It can beapplied to any field (cytoplasm alone, nucleus alone, etc.)by applying a mask to exclude surrounding areas.
Separate sections were stained with TUNEL reagent (Promega,Madison, WI) and DAPI for in situ apoptosis detection. In brief,10-µm frozen sections were treated with 20 µg/mlproteinase K and then incubated in a nucleotide mixture containingfluorescein-12-dUTP and TdT (terminal deoxynucleotidyl transferase).Positive controls were pretreated with 1 U/ml Dnase, and negativecontrols were incubated without TdT. TUNEL-positive nuclei wereexpressed as a percent of total nuclei (DAPI-positive) per field.Six to eight fields per section and 2 to 3 sections per kidneywere examined in each experiment (7).
Western Blots
In some experiments, tissue harvested form cortex or medullawas obtained at specified time points before or after I-R. Proteinswere extracted with standard techniques and measured by Coomassieblue assay (Pierce Chemical, Rockford, IL). They were then resolvedon a 15% Tris-HCl gel by electrophoresis, along with MW markers.An identical amount was loaded in each lane for a given experiment.
After electrophoresis, proteins were transferred to a PVDF filtermembrane and probed for p53 (Ab-1, mouse monoclonal), p21 (Ab-5,rabbit polyclonal), or Bax (Ab-1, rabbit polyclonal); all fromOncogene, Boston, MA. Appropriate HRP-conjugated secondary antibodieswere used along with ECL labels. Densitometry was performedusing Quantity One software from Bio-Rad.
Studies with LLC-PK1 Cells
A4.8 clones of LLC-PK1 porcine proximal tubule cells were grownin 5% CO2 at 37°C in DMEM with 10% FBS (Sigma Chemical,St. Louis, MO) and 5 mM glucose. For chemical anoxia, 0.1 µMantimycin A was used in depleted media (DMEM without amino acids,glucose, or serum). Apoptosis was detected on the basis of nuclearmorphology using a Zeiss confocal microscope. Both adherentand floating cells were visualized after staining with Hoechst33342 and propidium iodide as described previously. Cell viabilitywas based on the criteria of trypan blue exclusion and celladherence (7,9). Apoptosis was also detected with DNA electrophoresison a 1.2% agarose gel after phenol-chloroform extraction. Immunofluorescenceas well as Western blots for p53, p21, and Bax were performedas described above for renal tissues.
Effects of I-R and Guanosine on Renal Cortical and Medullary p53 Protein
As shown in Figure 1A, p53, which was not detectable in kidneysfrom sham animals, increased significantly in the medulla 24h after I-R. This increase in medullary p53 was reduced sixfoldwith guanosine, a treatment we have previously shown to restoreGTP to control levels one hour after I-R. Guanosine was alsoeffective in inhibiting the increase in p53 in animals subjectedto 45 min renal artery clamp instead of the usual 30 min. Thesedata suggest a causal relationship between GTP depletion afterI-R and the increase in medullary p53. Figure 1B shows the timecourse of p53 increase after I-R. p53 levels were detectableas early as 1.5 h after ischemia and peaked at 24 h. p53 levelstended to normalize back to baseline by 48 h after I-R (datanot shown).
Figure 1. Effects of ischemia-reperfusion (I-R) and guanosine on renal cortical and medullary p53 protein. Western blot of p53 from cortical (c) and medullary (m) tissues of control kidneys and kidneys subjected to 30 or 45 min of ischemia and different periods of reperfusion (I-R). (A) Tissues were obtained at 24 h after I-R, and rats received placebo (normal saline) or guanosine (G) intraperitoneally as detailed in Materials and Methods. pc, positive control (p53 standard). (B) p53 Western blot was performed on cortical (c) and medullary (m) tissues harvested from kidneys 1.5, 6, and 24 h after I-R (30 min ischemia time). Blots representative of n = 4.
Effects of the p53 Inhibitor PIF on Renal Histology and Morphology after I-R
Histologic evidence of injury in kidney sections removed 24h after bilateral renal ischemia was no different in the PIF-treatedrats as compared with the saline-treated group. Figure 2 showsrepresentative sections from the outer medulla of sham rats(upper panels) demonstrating normal morphology. Comparable sectionsfrom saline-treated (middle panels) or PIF-treated (lower panels)rats 24 h after renal ischemia show marked disruption of normaltubular morphology with debris and casts in most tubules. However,many small, condensed, and fragmented nuclei (arrows) characteristicof apoptosis were present only in the saline-treated group andnot in the PIF-treated group. One such nucleus from each middlesection (arrowhead) is shown at a higher magnification in theinsets. The apoptotic cells were predominantly tubular and manywere found shed in the lumen. Finally, to determine whetheradministration of PIF at the time of ischemia resulted in morerapid recovery of morphologic evidence of injury, H+E sectionswere also examined 72 h post-ischemia. The extent of tubulardebris, casts, and mitoses in sections from the I-R and I-R+ PIF groups was comparable (data not shown).
Figure 2. Effects of the p53 inhibitor pifithrin- (PIF) on histology after renal I-R. Representative hematoxylin and eosin–stained sections of renal medulla of the sham (upper panels), I-R (middle panels), and I-R + PIF (lower panels) groups 24 h after surgery are presented. The arrows indicate condensed, fragmented nuclei characteristic of apoptosis. The insets show enlargement of the nuclei identified by the arrowheads.
Effects of the p53 Inhibitor PIF on Apoptosis after Renal I-R
To quantify the extent of apoptosis after ischemia and reperfusion,the TUNEL reaction was performed. We have previously validatedthe specificity of the TUNEL assay for apoptosis in this modelby light, fluorescence, and electron microscopic criteria (7).Representative sections of outer medulla are shown in Figure 3.Virtually no TUNEL-positive nuclei were seen in kidney sectionsfrom sham rats (upper panel). After bilateral renal ischemiaand 24 h of reperfusion, large numbers of TUNEL-positive nucleiwere seen in the medulla (middle panels). Nuclear morphologywas further evaluated by examining the DAPI channel only (withoutthe FITC channel) at higher magnification. Nuclear morphology(insets) of the TUNEL-positive nuclei demonstrated condensed,fragmented nuclei consistent with apoptosis. In contrast, fewTUNEL-positive nuclei were seen in the PIF-treated group afterI-R (lower panels). The nuclei in these sections were enlargedand showed faint DAPI staining (insets). Quantification of TUNEL-positivecells showed that treatment with PIF at the time of ischemiaresulted in a decrease in TUNEL-positive cells in sections ofrenal medulla from 27.2 ± 4.5% in the I-R group to 3.9± 2.1% in the I-R + PIF group (Figure 4A). These resultsare similar to those observed after the administration of guanosinebefore renal ischemia (7). To examine the effect of PIF on apoptosisat later time points, the TUNEL reaction was performed on tissuesremoved 48 and 72 h after ischemia. Very few TUNEL-positivecells were found in any of these sections (data not shown).
Figure 3. Effects of the p53 inhibitor PIF on apoptosis after renal I-R. Representative sections of renal medulla from the sham (upper panel), I-R (middle panels), and I-R + PIF (lower panels) groups 24 h after surgery are presented. nc, negative control (incubation without terminal deoxynucleotidyl transferase); pc, positive control (incubation with DNAse). TUNEL-positive nuclei are yellow-green. All nuclei are co-stained with DAPI (blue), and the autofluorescence of tubules is brown. No TUNEL-positive nuclei are seen in the sham group. After renal ischemia and 24 h of reperfusion, many TUNEL-positive nuclei are seen in the renal medulla in all tubule segments. Few TUNEL-positive nuclei are seen post-ischemia after treatment with PIF. The insets show only the DAPI staining of the nuclei identified by the arrows.
Figure 4. Effects of the p53 inhibitor PIF on the frequency of TUNEL-positive nuclei and on renal function after renal I-R. TUNEL-positive (FITC-labeled) nuclei were counted on multiple, coded sections from 7 to 12 animals in each group. The mean number of TUNEL-positive nuclei as a percent of total (DAPI-positive) nuclei seen in each field is presented in panel A. Mean serum creatinine ± 1 SEM in the sham and I-R groups as well as that in the groups that received PIF at the time of ischemia (0 h) and 2, 8, and 14 h after renal ischemia is presented in panel B. *P < 0.01 versus I-R group.
Effects of the p53 Inhibitor PIF on Renal Function after I-R
To assess the functional significance of the differences inTUNEL-positive nuclei seen, serum urea nitrogen and creatininewere measured 24 h after renal ischemia (Figure 4B). Treatmentwith PIF at the time of renal ischemia resulted in a mean serumcreatinine that was lower than that in the I-R group and wasnot statistically different from that in the sham group. Infact, treatment with PIF as long as 14 h after renal ischemiaresulted in functional protection from renal ischemia. Meanserum creatinine post-ischemia in the group treated with PIF14 h after ischemia was 1.3 ± 0.1 mg/dl as compared with2.4 ± 0.3 mg/dl in the I-R group (P < 0.05). Meanserum urea nitrogen values after ischemia showed a similar patternof protection (data not shown). We also measured function at48 or 72 h post-ischemia. Mean serum creatinine in the I-R +PIF group was 0.3 ± 0.1 mg/dl 48 h post-ischemia (versus1.8 ± 0.3 in the I-R group) and 0.4 ± 0.72 mg/dl72 h post-ischemia (versus 1.6 ± 0.3) in the I-R group.Thus, the protective effect of PIF was sustained over a periodof at least 72 h.
Effect of the p53 Inhibitor PIF on p53 and its Transcriptional Targets p21 and Bax
PIF is known to bind p53 presumably in the cytoplasm and thusinhibit its transcriptional potential. As shown in Figure 5A,PIF did not reduce p53 levels. Rather, it caused an increase,which likely represents mobilization of otherwise poorly extractablep53.
Figure 5. Effect of PIF on levels of p53 and its transcriptional targets after I-R. Western blot of medullary p53 and cortical and medullary p21 and Bax 24 h post-ischemia (I-R, ischemia time = 30 min) are presented. Rats received placebo (normal saline) or pifithrin- (PIF) intraperitoneally before ischemia as detailed in Materials and Methods. pc, positive control; c, cortex; m, medulla. Blot are representative of n = 3.
PIF did not directly reduce p53 protein levels but rather inhibitedits action; we therefore examined its efficacy in downregulatingtwo classical transcriptional targets of p53, namely p21 andBax, which are involved in cell cycle control and apoptosis,respectively. Figure 5B shows the effects of I-R and PIF onp21. The changes in p21 after I-R were modest (1.2- to 1.5-foldincrease) in both the cortex and medulla. PIF was very effectivein reducing p21 protein after I-R to levels lower than baselinein both cortex and medulla. As shown in Figure 5C, there wasno change in Bax protein levels in the cortex and a small (butreproducible) 1.4-fold increase in the medulla after I-R. PIFsignificantly reduced Bax levels in the medulla (but not thecortex) to below baseline levels.
Effects of I-R and PIF on the Cellular Localization of p53
Whereas the changes in p53 protein levels after I-R were significant,those of Bax were comparatively modest. This suggested thatp53 might be involved in the apoptotic response to I-R via anadditional non-transcriptional effect. We therefore examinedthe cellular localization of p53 with specific emphasis on nuclear,cytoplasmic, and mitochondrial compartments. As shown in Figure 6,there was no detectable p53 in medullary tubular cells fromcontrol animals. This was expected from the Western blot resultsshown in Figure 1. After I-R, there was a remarkable increasein immunoreactive p53 across all medullary tubules examined.That is, p53 was not restricted to any particular tubular segment.Furthermore, the p53 signal was predominantly (but not exclusively)cytoplasmic and co-localized strongly with the mitochondrialsignal. Using Corr3D software, the correlation between p53 andmitochondrial signals was 0.8 ± 0.2 and that betweenp53 and nuclear signals 0.3 ± 0.1. In the presence ofPIF, the p53 signals became more finely granular and co-localizedless strongly with the mitochondrial signal (Corr3D score decreasedto 0.3 ± 0.2). PIF also modestly reduced nuclear p53colocalization with a decrease in Corr3D score to 0.2 ±0.1. These results suggest that the increase in p53 after I-Ris mostly cytoplasmic and more specifically mitochondrial. PIFproved very potent in inhibiting this mitochondrial localizationof p53.
Figure 6. Effects of I-R and PIF on the cellular localization of p53. Tissues were obtained 24 h after surgery from sham rats and rats subjected to ischemia-reperfusion (I-R, 30 min ischemia time) that received placebo or PIF intraperitoneally as detailed in Materials and Methods. Tissues were fixed and immunostained with antibodies to p53 (Texas red–label, red) and the mitochondrial (mito) marker cytochrome c oxidase (FITC-label, green). Nuclei were stained with To-Pro-3 iodide (blue).
Studies with LLC-PK Cells
Because of the architectural complexity of the kidney and thepresence of multiple cell types within any given section, weexamined the reproducibility of our results in LLC-PK renalproximal tubular cells in culture. We have previously shownthat cell death and apoptosis induced after chemical anoxia/recoveryis significantly reduced by the provision of guanosine to restoreGTP levels (7). We now show that antimycin A treatment followedby recovery upregulated p53 protein levels as early as 4 h afterrecovery (Figure 7B). In addition, Figure 7A shows that 50 µMguanosine significantly reduced the increase in p53 by 2.5-fold.Of note is the presence of significant amount of p53 in restingcontrol cells unlike renal tissues in which p53 is undetectableunder sham conditions. This is a known characteristic of manyimmortalized cell lines. Figure 7C shows that, like in renaltissues, PIF does not inhibit the increase in p53 after chemicalanoxia. Similarly, modest increases in p21 and Bax were notedafter chemical anoxia/recovery and these increases were significantlyinhibited by PIF (Figure 7D and 7E).
Figure 7. Effect of antimycin/recovery, guanosine (G), or PIF on expression of p53 and its transcriptional targets p21 and Bax in cultured renal tubular cells. Western blots of p53, p21, and Bax from control LLC-PK1 cells and LLC-PK1 cells subjected to antimycin/recovery are presented. In panel A, cells were harvested 24 h after exposure to antimycin A (0.1 µM for 45 min) with or without guanosine (G; 50 µM) as detailed in Materials and Methods. In panel B, the time course of the increase in p53 after antimycin is presented. In panels C, D, and E, the effect of the p53 inhibitor PIF with and without antimycin on the expression of p53, p21, and Bax is shown. pc, positive control. Blots are representative of n = 3.
Figure 8 shows that nuclear condensation and fragmentation typicalof apoptosis induced by chemical anoxia (panel B) were totallyinhibited by PIF (panel C). Indeed, PIF reduced the number ofapoptotic cells after chemical anoxia from 30 ± 5 perfield to 4 ± 2 per field (ten fields counted per conditionwith an average of 50 cells per field). PIF alone had no effecton nuclear morphology (panel D). We confirmed these resultswith DNA electrophoresis, showing typical laddering after chemicalanoxia that is inhibited by PIF or guanosine. Overall, PIF increasedcell viability at 24 h from 35 ± 6% (chemical anoxiagroup) to 88 ± 6% (chemical anoxia + PIF group). Theseare identical results to those seen with guanosine (7) and similarto the inhibition of apoptosis with PIF in vivo shown in Figure 3.
Figure 8. Effect of antimycin/recovery and treatment with the p53 inhibitor PIF or guanosine (G) on apoptosis in cultured renal tubular cells. A representative fluorescent micrograph of LLC-PK1 cells demonstrating normal nuclear morphology is shown in panel A. Condensed, fragmented nuclei characteristic of apoptosis are seen after antimycin/recovery (0.1 µM antimycin A for 45 min and 24 h recovery; panel B). Apoptosis was not apparent in the cells treated with PIF (10 µM) before antimycin/recovery, as shown by the normal nuclear morphology in panel C. PIF alone did not alter nuclear morphology (panel D). Apoptosis after antimycin/recovery was also evident by characteristic laddering on DNA gel electrophoresis (panel E). Prevention of laddering was seen after antimycin/recovery in the presence of PIF (1 or 10 µM) or guanosine (G; 50 µM; panel E). m, DNA size markers.
Finally, we examined the effects of chemical anoxia and PIFon the cellular localization of p53. As shown in Figure 9, p53(red) in control cells is predominantly nuclear. Chemical anoxialeads to a remarkable translocation of p53 to the cytoplasm,where it strongly colocalizes with mitochondria (green), resultingin a yellow merge (Corr3D correlation 0.9 ± 0.1). Furthermore,when chemical anoxia was performed in the presence of PIF, p53remained in the cytoplasm, but the colocalization with mitochondriadecreased significantly (Corr3D correlation 0.2 ± 0.1)as shown by distinct red and green rather than yellow fluorescence.These results are very similar to the in vivo observations shownin Figure 6.
Figure 9. Effects of antimycin/recovery and PIF on the cellular localization of p53 in cultured renal tubular cells. Cultured LLC-PK1 renal tubular epithelial cells were harvested 24 h after exposure to antimycin A (0.1 µM for 45 min) with or without pifithrin- (PIF; 10 µM) as detailed in Materials and Methods. Cells were fixed and immunostained with antibodies to p53 (Texas red–label, red) and the mitochondrial marker cytochrome c oxidase (FITC-label, green). Nuclei were stained with To-Pro-3 iodide (blue). Control cells were maintained in standard media.
There are many mediators of cell death after renal I-R, andthey likely interact in a complex array of signaling pathwaysthat result in tubular cell demise. Recently, we identifiedthe depletion of GTP pools as one such mediator and linked itspecifically to the apoptotic phenotype (9). We further showedthat enhanced GTP recovery had a profound beneficial effecton cell survival and renal function after chemical anoxia andI-R (7). This beneficial effect strictly correlated with inhibitionof apoptosis. In this article, we present strong evidence thatp53 is the downstream mediator of apoptosis in the setting ofGTP depletion and I-R injury in vivo. All the results were furtherreproduced in LLC-PK renal tubular cells.
Known as the "the guardian of the genome," p53 is the most frequentlymutated tumor suppressor in many forms of neoplasia. This underscoresits importance as the master regulator of cell proliferationand cell death. p53 primarily eliminates unwanted or damagedcells and thus insures the overall integrity of the genome.Its regulation is highly complex and involves interactions withmdm2, alterations in 53 protein levels, and also direct phosphorylationor acetylation (11,12). Once activated, p53 induces cell cyclearrest or apoptosis under many conditions where DNA damage posesthe risk of malignant transformation.
The role of p53 in mediating cell death during I-R is more controversial.Such a role is generally accepted for neuronal ischemia butnot for cardiac I-R injury (18–21). Indeed, recent reportsshow that apoptosis after cardiac I-R is p53-independent (22,23).Thus, the involvement of p53 could be tissue or organ-specific.The data on p53 after renal I-R is even more scarce. Althoughsome reports propose a potential role for p53, others presentdata showing that cell death after I-R is p53-independent (24–26).Possible reasons for these discrepancies are discussed below.
In this article, we show a significant increase in p53 proteinin the medulla after I-R. The medulla is the primary site oftubular cell apoptosis after I-R. The increase in p53 was preventedby guanosine, a treatment we have previously shown to selectivelyreplete GTP stores and inhibit apoptosis (7). Guanosine wascapable of inhibiting this increase in p53, even after 45-minrenal artery clamp, a procedure that invariably results in extensivedamage and acute renal failure. Although these data supporta role for p53 in mediating GTP depletion-induced apoptosis,it still could be simply an association rather than a causalrelationship. Our studies with PIF provide more direct prooffor a causal role of p53 in apoptosis after I-R.
PIF, a synthetic, potent, and highly specific inhibitor of p53,not only inhibited apoptosis but also reproduced our previousfindings with guanosine. That is, the inhibition of apoptosiswith PIF was accompanied by an impressive functional protectionand by the same lack of effect on necrotic death. Taken together,these findings strongly argue for an important role of p53 inmediating apoptotic cell death after I-R and GTP depletion.They further underscore the importance of apoptosis as a determinantof functional outcome after ischemia, independent of necrosis.The exact mechanisms by which inhibition of apoptosis impactrenal function post-ischemia are largely unknown. Possible mechanismsinclude preservation of tubular integrity and thus preservationof tubuloglomerular feedback. Furthermore, shed apoptotic cellscould contribute both to tubular obstruction and the formationof gaps in the tubular epithelium resulting in backleak. Recently,a protective effect of PIF was shown after neuronal ischemia(27). To our knowledge, this is the first report showing a beneficialeffect of p53 inhibition after renal I-R.
The effects of PIF on p53 activity were complex. First, thedownregulation of p21 and Bax was expected, as PIF is thoughtto bind and mobilize p53 in the cytoplasm and prevent its translocationinto the nucleus (16). Although downregulation of the pro-apoptoticBax fits well with the inhibition of apoptosis, the decreasein p21 was more surprising because this cell cycle regulatorgenerally has anti-apoptotic effects (28). However, the impactof p53 activation on cell fate depends on the balance betweenthe anti-apoptotic p21 and the pro-apoptotic Bax (29). Our datasuggest that the Bax effects dominate after renal I-R and aninhibition of p53 and Bax with PIF has an overall beneficialeffect despite the reduction in p21. Megyesi et al. (25) haveshown that p21 activation is p53-independent. However, theirstudies were performed in p53 null mice. In these geneticallyaltered mice, other p53-independent pathways that activate p21could have been induced when p53 is absent. Rapid inhibitionof p53 with PIF in wild-type animals might not permit sufficienttime for these other pathways to become activated and to stimulatep21.
The other surprising finding in this article is that the increasein p53 protein after I-R is, to a large extent, cytoplasmic.This might explain the lack of increase in p53 reported by Megyesiet al. (25), as they measured only nuclear p53. Our data furthershow that cytoplasmic p53 colocalizes strongly with mitochondria.This mitochondrial increase in p53 was recently reported incell culture after hypoxia (10,30). These authors proposed anovel non-transcriptional mechanism by which p53 causes apoptosis.This mechanism is initiated upon direct translocation of p53to mitochondria, where it localizes predominantly to the membranouscompartment. To our knowledge, our data provide the first demonstrationof mitochondrial p53 translocation in vivo after renal I-R.Our data also suggest that such a mechanism is important afterrenal I-R. Indeed, we show that the protective and anti-apoptoticproperties of PIF correlate well with its ability to preventthe colocalization of p53 with mitochondria in addition to inhibitingits transcriptional potential.
In summary, the data in this article and our previous reportsupport a model of apoptotic cell death after I-R in which GTPdepletion and p53 activation occur sequentially, leading tocell death. We also show that direct inhibition of p53 withPIF, like GTP salvage with guanosine, confers functional protectionin addition to inhibition of apoptosis. However, unlike guanosine,the therapeutic window with PIF is much wider (up to 14 h post-ischemia)due to the incremental accumulation of p53. In fact this therapeuticwindow is wider than that of most other agents proposed to preventor treat renal I-R. Therefore, agents that modulate the transcriptionalactivity of p53 or its mitochondrial translocation should bestrongly considered for the treatment of ischemic renal injury.
Acknowledgments
This work was supported by National Institute of Diabetes andDigestive and Kidney Diseases grants 1P50 DK61594–01 (PCD)and 1RO1 DK60495–01A1 (PCD) and a grant INGEN from theLilly Endowment to Indiana University School of Medicine. Dr.Kelly is the recipient of the National Kidney Foundation ClinicalScientist Award. The authors are grateful to Bruce Molitorisfor support and advice throughout this project.
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Received for publication July 18, 2002.
Accepted for publication September 16, 2002.
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Top
Abstract
Introduction
, a specific inhibitor of p53, mimicked the effects of guanosine. It had no effect on necrosis, yet it prevented apoptosis and protected renal function. Pifithrin-
B in p53-mediated programmed cell death. Nature 404: 892–897, 2000[CrossRef][Medline]
