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.
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.
MATERIALS AND METHODS
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).
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).
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).
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.
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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Keywords:© 2011 Lippincott Williams & Wilkins, Inc.
Autophagy; Apoptosis; Cold preservation ischemia