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Apoptosis and Autophagy in Cold Preservation Ischemia

Turkmen, Kultigin1; Martin, Jessica1; Akcay, Ali1; Nguyen, Quocan1; Ravichandran, Kameswaran1; Faubel, Sarah1; Pacic, Arijana2; Ljubanović, Danica2; Edelstein, Charles L.1; Jani, Alkesh3,4

doi: 10.1097/TP.0b013e31821ab9c8
Basic and Experimental Research
Free

Background. Prolonged cold ischemia (CI) is a risk factor for the development of delayed graft function that predicts reduced 5-year kidney transplant survival. CI results in caspase-3 activation, tubular injury, and apoptosis. Autophagy, a highly conserved pathway that permits recycling of nutrients within the cell during stress, is linked to apoptosis. We hypothesized that CI during kidney preservation would induce autophagy. We sought to determine apoptosis and autophagic flux in CI.

Methods. Autophagic flux and apoptosis were examined in kidneys of wild-type and green fluorescent protein (GFP)-microtubule-associated protein1 light chain 3 (LC3) transgenic mice that were subjected to 48 hr of CI. Autophagic flux was determined by performing experiments with and without bafilomycin A1.

Results. CI alone significantly increased the number of apoptotic cells/hpf, caspase-3/7 activity, and protein expression of autophagy markers LC3 II and autophagy-related protein 5. To determine the effect of inhibiting autophagic flux on apoptosis, kidneys of wild-type and GFP-LC3 transgenic mice were subjected to 48 hr of CI in the presence of lysosomal inhibitor bafilomycin A1. The combination of CI and bafilomycin A1 suppressed autophagic flux and significantly reduced the number of apoptotic cells/hpf, caspase-3/7 activity, LC3 II (both by immunoblot and in GFP-LC3 transgenic mice), and autophagy-related protein 5 protein expression.

Conclusion. In summary, we have shown that autophagy and autophagic flux are reduced in cold ischemic kidneys treated with bafilomycin A1. Reduced autophagy and autophagic flux were associated with a significant reduction in apoptotic cell death, which may provide a therapeutic rationale for including bafilomycin A1 in University of Wisconsin solution during organ preservation.

1University of Colorado, Denver, CO.

2University Zagreb SOM, Zagreb University Hospital Dubrava, Zagreb, Croatia.

3Denver Veterans Affairs Medical Center, Denver, CO.

This work was supported by KO8DK069512 (A.J.), R01HL095363 (S.F.), and NIH grants RO1DK056851 and RO1-DK-074835 (C.L.E.), and Turkish Society of Nephrology (K.T.).

The authors declare no conflict of interest.

4Address correspondence to: Alkesh Jani, M.D., Division of Renal Diseases and Hypertension, University of Colorado Denver, 12700 East 19th Avenue, C281, Aurora, CO 80045.

E-mail: Alkesh.jani@ucdenver.edu

K.T. participated in the performance of the research; J.M., A.A., Q.N., A.P., and D.L. participated in research design and performance of the research and contributed new reagents or analytic tools; K.R. and S.F. participated in the performance of the research; and C.L.E. and A.J. participated in research design, writing of the manuscript, performance of the research, and data analysis and contributed new reagents or analytic tools.

Received 13 January 2011.

Accepted 13 March 2011.

Delayed graft function (DGF) occurs in 8% to 50% of primary deceased donor renal transplants in the United States and independently predicts reduced 1- and 5-year kidney transplant survival (1). Several studies have identified prolonged cold ischemic time as a risk factor for the development of DGF (1–3). Prolonged cold ischemic time and DGF have also been associated with increased serum creatinine at 1 year posttransplant (4). Our previous work demonstrates that cold ischemia (CI) results in caspase-3 activation, tubular injury, and apoptosis in a model of CI in mice (5). Preservation of kidneys with a pan caspase inhibitor (OPH-100) prevented caspase-3 activation and significantly reduced tubular injury and apoptosis.

Autophagy (derived from the Greek “auto” meaning “self” and “phagy” meaning “eat”) (6) is a cellular process in which cytoplasmic proteins and macromolecules are delivered by autophagosomes to lysosomes for degradation (7). The rate at which cytoplasmic proteins and macromolecules are delivered to lysosomes through the autophagic pathway is defined as the autophagic flux (8). Autophagy is a highly conserved pathway that permits recycling of nutrients within the cell and is rapidly up-regulated during starvation or cell stress (6, 9). Inhibition of autophagy during cell stress may result in apoptosis (7, 10, 11).

Because organ preservation for transplantation represents both a cell stress and nutrient deprivation, one might predict an increase in autophagic flux during CI. Alternatively, it is possible that CI, by virtue of limiting enzymatic reactions, may reduce autophagic flux. Whether autophagic flux is induced and results in apoptosis during CI of kidneys procured for transplantation is not known. We hypothesized that CI during kidney preservation would induce autophagic flux. We further sought to determine the effect of reducing autophagic flux with bafilomycin A1 on apoptosis.

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RESULTS

Effect of Cold Ischemia on Apoptosis and Caspase Activity

The number of apoptotic cells/hpf was significantly increased in cold ischemic kidneys versus controls (Fig. 1A). Treatment with bafilomycin A1 significantly reduced the number of apoptotic cells/hpf after 48 hr of CI (Fig. 1A). Caspase-3/7 activity was also significantly increased in cold ischemic kidneys versus controls (Fig. 1B). Treatment of control kidneys exposed to CI for 48 hr with bafilomycin A1 significantly reduced caspase-3/7 activity (Fig. 1B). Immunoblotting was performed to detect the protein expression of the active form of caspase-3 (20 kDa). In cold ischemic kidneys, there was 2-fold increase in active caspase-3 compared with controls (Fig. 2). Treatment with bafilomycin A1 significantly reduced active caspase-3 protein expression in cold ischemic kidneys.

FIGURE 1.

FIGURE 1.

FIGURE 2.

FIGURE 2.

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Effect of Cold Ischemia on Autophagy

To assess autophagy in whole kidney, microtubule- associated protein1 light chain 3 (LC3) I and II protein expression was examined. A significant increase in LC3 II protein expression was observed in cold ischemic kidneys versus controls (Fig. 3). Treatment of cold ischemic kidneys with bafilomycin A1 significantly reduced LC3 II protein expression. To determine autophagic flux, control mice were compared with bafilomycin A1-treated mice, and LC3 I and II protein expression was assessed (12). LC3 II protein expression was significantly increased at 0 hr in bafilomycin A1-treated mice versus vehicle-treated mice (Fig. 3), suggesting positive autophagic flux and the constitutive presence of autophagosomes (13). In cold ischemic kidneys, LC3 II protein expression was significantly decreased in bafilomycin A1-treated mice versus cold ischemic treated with vehicle alone, suggesting negative autophagic flux (Fig. 3). To confirm these findings, autophagic flux was examined in green fluorescent protein (GFP)-LC3 transgenic mice. A significant increase in GFP-LC3 puncta was observed in cold ischemic kidneys (Fig. 4), whereas treatment with bafilomycin A1 significantly reduced the number of GFP-LC3 puncta after CI. Autophagy was further assessed by examination of the protein expression of the autophagy-related protein (ATG)-5. ATG-5 increased 4-fold in cold ischemic kidneys versus controls (Fig. 5). Treatment of cold ischemic kidneys with bafilomycin A1 prevented the increase in ATG-5.

FIGURE 3.

FIGURE 3.

FIGURE 4.

FIGURE 4.

FIGURE 5.

FIGURE 5.

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DISCUSSION

Autophagy is a tightly regulated, highly conserved physiologic process that involves recycling of cytoplasmic proteins and molecules within autophagolysosomes (7, 12, 14). Autophagy occurs at low-baseline levels in all cells (9) and during embryonic development (7). Potent stimulators of autophagy, both in vitro and in vivo, include starvation, hypoxemia, and energy depletion (12). Under these conditions, autophagy is believed to enable cell survival through recycling of amino acids and essential cell metabolites (7). Three distinct types of autophagy have been recognized: macroautophagy (referred as autophagy in this article), microautophagy, and chaperone-mediated autophagy (9). Autophagy begins with the formation of a double-membraned vesicle, the autophagosome, which envelopes cytoplasmic contents (12). The autophagosome subsequently fuses with lysosomes permitting the degradation of the cytoplasmic material (14) and recycling of amino acids (12). Autophagosome generation begins with the induction of several autophagy-related genes, including LC3, ATG-5, and Beclin-1 (7, 15). LC3 II, which stably associates with the autophagosomal membrane, has been used as a marker for the presence of autophagosomes (9). The accumulation of LC3 II within a cell can indicate the presence of increased autophagy (increased autophagic flux) or an inhibition of autophagosome breakdown (decreased autophagic flux) (12). Bafilomycin A1, a macrolide antibiotic which inhibits the H+-ATPase responsible for acidification of autophagolysosomal vacuoles (16), results in reduced autophagic flux (12).

Deceased donor organ preservation involves the rapid cooling of ex vivo donor organs to 4°C. Because donor organs are subjected to nutrient deprivation and energy depletion, we hypothesized that cells would induce autophagy enabling them to withstand these stresses. Lu et al. (17) examined the effect of CI and warm reperfusion on autophagy in rat livers. Autophagy, measured by the ratio of LC3 II:LC3 I, was increased during both cold preservation and reengraftment in a model of liver transplantation. Gotoh et al. (18) examined autophagic flux using the phosphatidylinositol 3-kinase inhibitors in a rat model of orthotopic liver transplantation. After graft reperfusion, numerous autophagosomes and autolysosomes were observed in hepatocytes, which subsequently obstructed sinusoids causing massive necrosis. Inhibition of autophagy with wortmannin or LY294002 reduced both liver damage and the mortality rate of recipient rats.

There have been no previous studies examining the relationship between CI and autophagic flux in kidneys nor any studies examining the effect of downstream lysosomal inhibitors such as bafilomycin A1. We observed an increase in LC3 II protein expression in CI kidneys compared with the baseline contralateral controls, suggesting an increase in autophagy. In contrast, treatment of cold ischemic kidneys with bafilomycin A1 reduced LC3 II protein expression. This finding was confirmed by immunofluorescence (Fig. 4) and by the finding of reduced ATG-5 protein expression in bafilomycin A1-treated CI kidneys (Fig. 5). These results indicate that CI alone increased phagophore and autophagosome production, whereas the combination of CI and bafilomycin A1 suppressed phagophore and autophagosome production and reduced autophagic flux. The mechanism by which autophagic flux was reduced by the combination of CI and bafilomycin A1 will require further detailed study.

Both human and animal studies suggest that the adverse impact of CI may be associated with caspase-3 activation and apoptosis. Caspase-3 is the “executioner caspase” that is centrally important in apoptotic cell death in vivo (19). Apoptosis rate has been shown to correlate significantly with cold ischemia time in human cadaveric renal transplants (20). Biopsies of human donor kidneys, which subsequently develop postoperative acute tubular necrosis, demonstrate an increase in apoptosis in renal tubular epithelial cells, which predicts early transplant function (21). Our previous work demonstrates that CI results in caspase-3 activation, tubular injury, and apoptosis in a non-donation after cardiac death (DCD) model of CI in mice (5).

In our model, reduced autophagy and autophagic flux after 48 hr of CI in the presence of bafilomycin A1 was associated with a significant reduction in the number of apoptotic cells/hpf. The relationship between apoptosis and autophagy is complex. Yang et al. (7) demonstrated a reduction in apoptosis after inhibition of autophagy in cisplatin-treated renal tubular epithelial cells (7). Autophagy inhibition has been associated with increased apoptosis in studies of nutrient-deprived cells (11), in colon cancer cells (22), in sodium selenite–treated NB4 cells (23), and in a Myc-induced model of lymphoma (10). Conversely, inhibition of autophagy prevented apoptosis in dopaminergic neuroblastoma cells (24). Induction of autophagy in CD4 lymphocytes by HIV-infected cells triggers apoptotic CD4 T-cell death (25). The heterogeneity of possible outcomes after the induction or inhibition of autophagy may be explained by common upstream signaling events that trigger both autophagy and apoptosis concurrently or in a mutually exclusive fashion (26). Inhibition of autophagy in CI kidneys treated with bafilomycin A1 was associated with reduced apoptosis in our model, supporting the possibility of common triggering events for both processes (26).

In summary, we have shown that autophagy and autophagic flux are reduced in cold ischemic kidneys treated with bafilomycin A1. Reduced autophagy and autophagic flux were associated with a significant reduction in apoptotic cell death, which may therefore provide a therapeutic rationale for including bafilomycin A1 in University of Wisconsin (UW) solution during organ preservation.

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MATERIALS AND METHODS

Animals

Wild-type C57BL/6 male mice (The Jackson Laboratory, Bar Harbor, ME) weighing 20 to 25 g and GFP-LC3 transgenic mice (Riken Laboratories, Hirosawa, Japan) were used. The GFP-LC3 mice contain a GFP-LC3 cassette inserted between the combination of chicken beta-actin promoter and the cytomegalovirus immediate-early enhancer element (CAG promoter) and the SV40 late polyadenylation signal (27). Assessment of autophagy and autophagic flux using GFP-LC3 transgenic mice is well established (27–29). Transgene expression in GFP-LC3 mouse tail was confirmed using reverse-transcriptase polymerase chain reaction as described previously (27).

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Cold Ischemia

Wild-type or GFP-LC3 transgenic mice male mice C57BL/6 male mice (The Jackson Laboratory) weighing 20 to 25 g were used. The mice were anesthetized with an intraperitoneal injection of Avertin (2, 2, 2-tribromoethanol; Sigma-Aldrich, Milwaukee, WI). After cervical dislocation, a thoracotomy was performed, and the left ventricle of the beating heart was identified. The kidneys were perfused through puncture of the left ventricle with 5 mL of cold UW solution containing bafilomycin A1 (2 mg/kg) or the vehicle (0.1% dimethyl sulfoxide). Adequate perfusion was determined by observing the development of a pale color to both kidneys. The left and right kidneys were then removed. The right kidney was immediately cut in half longitudinally. The right kidney was immediately processed and preserved in 4% paraformaldehyde for histology, placed in optimal cutting temperature (OCT) media for immunofluorescence, or frozen in liquid nitrogen. The frozen kidneys were later prepared for the caspase-3 assay as described below. The left kidney was stored in UW solution containing bafilomycin A1 or vehicle for 48 hr at 4°C to produce cold preservation injury. After 48 hr of cold preservation, the left kidney was preserved in 4% paraformaldehyde, OCT media, or liquid nitrogen as described for the right kidney. Consequently, there were four groups of kidneys comprising: (1) control right kidney perfused with UW solution+vehicle and immediately processed (control); (2) the contralateral left kidney also perfused with UW solution+vehicle and subjected to 48 hr CI (CI kidneys); (3) right kidney perfused with bafilomycin A1 (control+ bafilomycin A1); and (4) contralateral left kidney perfused with bafilomycin A1 and subjected to 48 hr of CI (CI+bafilomycin A1).

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Renal Histology

Kidney sections were preserved in 4% paraformaldehyde or placed in OCT media for immunoflourescent examination. Paraformaldehyde-fixed (4%) and paraffin-embedded kidneys were sectioned at 4 μm and stained with hematoxylin-eosin and periodic acid-Schiff by standard methods. OCT media-embedded kidneys were sectioned at 5 μm, air dried at room temperature for 30 min, washed in phosphate-buffered saline, and mounted on glass slides. Histologic examination for apoptosis was performed by a renal pathologist in a blinded fashion. Apoptotic tubular epithelial cells were quantitatively assessed per high power field (400×). At least 10 high power fields per slide were counted. Morphologic criteria were used to count apoptotic tubular epithelial cells on hematoxylin-eosin and periodic acid-Schiff staining. These characteristics included cellular rounding and shrinkage, nuclear chromatin compaction, and formation of apoptotic bodies (30).

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Caspase Activity

The activity of caspase-3/7 was determined by using fluorescent substrates as we have previously described (31, 32). Briefly, renal cortex was mixed with a lysis buffer containing 25 mM Na HEPES, 2 mM dithiothreitol, 1 mM EDTA, 0.1% 3-[(3-cholamidopropyl) dimethylammonio]-1-pro-panesulfonate, 10% sucrose, 1 mM phenylmethylsulfonyl fluoride, and 1 μM pepstatin A, pH 7.2 and homogenized with 10 strokes in a glass-Teflon homogenizer. The lysate was then centrifuged at 4°C at 100,000g in a Beckman Ti70 rotor for 1 hr. The resultant supernatants (cytosolic extracts) were immediately aliquoted, frozen in liquid N2, and stored at −70°C. Lysate protein was measured by the Bradford method as described in the Bio-Rad protein assay kit with bovine serum albumin as standards. The caspase assay was performed as follows: 200 μg of protein extract (20–50 mL of lysate) was mixed with 10 μL of the substrate (final concentration: 50 μM). The assay volume was made up to 200 μL with assay buffer. The assay buffer for caspase-3/7 contained 250 mM K+ HEPES, 50 mM KCl, 1 mM dithiothreitol, 1 mM EDTA, and 0.1% 3-[(3-cholamidopropyl) dimethylammonio]-1-pro-panesulfonate, pH 7.4. Ac-Asp-Glu-Val-Asp-7-amido-4-methyl coumarin in 100% dimethyl sulfoxide was used as a susceptible substrate for caspase-3 and -7 (25). The reaction was then initiated by addition of substrate. Peptide cleavage was measured over 1 hr at 300°C using a Cytofluor 4000 series fluorescent plate reader (Perseptive Biosystems, Framingham, MA) at an excitation wavelength of 380 nm and an emission wavelength of 460 nm. An AMC standard curve was determined for each experiment. Caspase activity was expressed in nanomoles mido-4-methylcoumarin (AMC) released per minute of incubation time per milligram of lysate protein.

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Immunoblotting and Immunofluorescence

Immunoblotting was performed as described previously (5). Immunoblot analyses was performed with the following antibodies: a rabbit polyclonal antibody raised against a recombinant protein corresponding to amino acids 1 to 277, representing the full-length precursor form of caspase-3 of human origin (1:1000; catalogue number sc-7148, Santa Cruz Biotechnology Inc, Santa Cruz, CA). The antibody has species reactivity to human, mouse, and rat. The antibody reacts with the precursor of caspase-3 (also designated as CPP 32; 32 kDa) and the processed forms (11 and 20 kDa). Purified recombinant caspase-3 (Upstate Group Inc, Lake Placid, NY) was used as a positive control; a rabbit antibody raised against a synthetic peptide to the C-terminal of ATG-5 (Novus Biologicals, Littleton, CO; catalogue number R-111-100); anti-bis in die (Latin for “twice a day,” BID) (Cell Signaling; catalogue 2003, 22 kDa); and a rabbit polyclonal antibody with specificity for LC3 I (∼16 kDa) and LC3 II (∼14 kDa; Cell Signaling, Danvers, MA; catalogue number 2775).

For immunofluorescence, 7-μm-thick tissue sections were prepared from OCT media-embedded tissue using a cryostat. The sections were air dried at room temperature for 30 min, washed in phosphate-buffered saline, and mounted onto glass slides as described previously (27).

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REFERENCES

1. Ojo AO, Wolfe RA, Held PJ, et al. Delayed graft function: Risk factors and implications for renal allograft survival. Transplantation 1997; 63: 968.
2. Kyllonen LE, Salmela KT, Eklund BH, et al. Long-term results of 1047 cadaveric kidney transplantations with special emphasis on initial graft function and rejection. Transpl Int 2000; 13: 122.
3. Hetzel GR, Klein B, Brause M, et al. Risk factors for delayed graft function after renal transplantation and their significance for long-term clinical outcome. Transpl Int 2002; 15: 10.
4. Hariharan S, McBride MA, Cherikh WS, et al. Post-transplant renal function in the first year predicts long-term kidney transplant survival. Kidney Int 2002; 62: 311.
5. Jani A, Ljubanovic D, Faubel S, et al. Caspase inhibition prevents the increase in caspase-3, -2, -8 and -9 activity and apoptosis in the cold ischemic mouse kidney. Am J Transplant 2004; 4: 1246.
6. Periyasamy-Thandavan S, Jiang M, Schoenlein P, et al. Autophagy: Molecular machinery, regulation, and implications for renal pathophysiology. Am J Physiol Renal Physiol 2009; 297: F244.
7. Yang C, Kaushal V, Shah SV, et al. Autophagy is associated with apoptosis in cisplatin injury to renal tubular epithelial cells. Am J Physiol Renal Physiol 2008; 294: F777.
8. Bauvy C, Meijer AJ, Codogno P. Assaying of autophagic protein degradation. Methods Enzymol 2009; 452: 47.
9. Levine B, Kroemer G. Autophagy in the pathogenesis of disease. Cell 2008; 132: 27.
10. Amaravadi RK, Yu D, Lum JJ, et al. Autophagy inhibition enhances therapy-induced apoptosis in a Myc-induced model of lymphoma. J Clin Invest 2007; 117: 326.
11. Boya P, Gonzalez-Polo RA, Casares N, et al. Inhibition of macroautophagy triggers apoptosis. Mol Cell Biol 2005; 25: 1025.
12. Mizushima N, Yoshimori T, Levine B. Methods in mammalian autophagy research. Cell 2010; 140: 313.
13. Mizushima N, Yoshimori T. How to interpret LC3 immunoblotting. Autophagy 2007; 3: 542.
14. Bolisetty S, Traylor AM, Kim J, et al. Heme oxygenase-1 inhibits renal tubular macroautophagy in acute kidney injury. J Am Soc Nephrol 2010; 21: 1702.
15. Jiang M, Liu K, Luo J, et al. Autophagy is a renoprotective mechanism during in vitro hypoxia and in vivo ischemia-reperfusion injury. Am J Pathol 2010; 176: 1181.
16. Yamamoto A, Tagawa Y, Yoshimori T, et al. Bafilomycin A1 prevents maturation of autophagic vacuoles by inhibiting fusion between autophagosomes and lysosomes in rat hepatoma cell line, H-4-II-E cells. Cell Struct Funct 1998; 23: 33.
17. Lu Z, Dono K, Gotoh K, et al. Participation of autophagy in the degeneration process of rat hepatocytes after transplantation following prolonged cold preservation. Arch Histol Cytol 2005; 68: 71.
18. Gotoh K, Lu Z, Morita M, et al. Participation of autophagy in the initiation of graft dysfunction after rat liver transplantation. Autophagy 2009; 5: 351.
19. Liu X, Zou H, Slaughter C, et al. DFF, a heterodimeric protein that functions downstream of caspase-3 to trigger DNA fragmentation during apoptosis. Cell 1997; 89: 175.
20. Castaneda MP, Swiatecka-Urban A, Mitsnefes MM, et al. Activation of mitochondrial apoptotic pathways in human renal allografts after ischemiareperfusion injury. Transplantation 2003; 76: 50.
21. Oberbauer R, Rohrmoser M, Regele H, et al. Apoptosis of tubular epithelial cells in donor kidney biopsies predicts early renal allograft function. J Am Soc Nephrol 1999; 10: 2006.
22. Wu YC, Wu WK, Li Y, et al. Inhibition of macroautophagy by bafilomycin A1 lowers proliferation and induces apoptosis in colon cancer cells. Biochem Biophys Res Commun 2009; 382: 451.
23. Ren Y, Huang F, Liu Y, et al. Autophagy inhibition through PI3K/Akt increases apoptosis by sodium selenite in NB4 cells. BMB Rep 2009; 42: 599.
24. Castino R, Bellio N, Follo C, et al. Inhibition of PI3k class III-dependent autophagy prevents apoptosis and necrosis by oxidative stress in dopaminergic neuroblastoma cells. Toxicol Sci 2010; 117: 152.
25. Espert L, Denizot M, Grimaldi M, et al. Autophagy is involved in T cell death after binding of HIV-1 envelope proteins to CXCR4. J Clin Invest 2006; 116: 2161.
26. Maiuri MC, Zalckvar E, Kimchi A, et al. Self-eating and self-killing: Crosstalk between autophagy and apoptosis. Nat Rev Mol Cell Biol 2007; 8: 741.
27. Mizushima N. Methods for monitoring autophagy using GFP-LC3 transgenic mice. Methods Enzymol 2009; 452: 13.
28. Martinet W, De Meyer GR, Andries L, et al. Detection of autophagy in tissue by standard immunohistochemistry: Possibilities and limitations. Autophagy 2006; 2: 55.
29. French CJ, Taatjes DJ, Sobel BE. Autophagy in myocardium of murine hearts subjected to ischemia followed by reperfusion. Histochem Cell Biol 2010; 134: 519.
30. Gobe G, Zhang XJ, Willgoss DA, et al. Relationship between expression of Bcl-2 genes and growth factors in ischemic acute renal failure in the rat. J Am Soc Nephrol 2000; 11: 454.
31. Melnikov VY, Ecder T, Fantuzzi G, et al. Impaired IL-18 processing protects caspase-1-deficient mice from ischemic acute renal failure. J Clin Invest 2001; 107: 1145.
32. Melnikov VY, Faubel S, Siegmund B, et al. Neutrophil-independent mechanisms of caspase-1- and IL-18-mediated ischemic acute tubular necrosis in mice. J Clin Invest 2002; 110: 1083.
Keywords:

Autophagy; Apoptosis; Cold preservation ischemia

© 2011 Lippincott Williams & Wilkins, Inc.