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Autophagy Is a Renoprotective Mechanism During in Vitro Hypoxia and in Vivo Ischemia-Reperfusion Injury

2010; Elsevier BV; Volume: 176; Issue: 3 Linguagem: Inglês

10.2353/ajpath.2010.090594

1525-2191

Man Jiang, Kebin Liu, Jia Luo, Zheng Dong,

Cannabis and Cannabinoid Research

Autophagy mediates bulk degradation and recycling of cytoplasmic constituents to maintain cellular homeostasis. In response to stress, autophagy is induced and may either contribute to cell death or serve as a cell survival mechanism. Very little is known about autophagy in renal pathophysiology. This study examined autophagy and its pathological role in renal cell injury using in vitro and in vivo models of ischemia−reperfusion. We found that hypoxia (1% O2) induced autophagy in cultured renal proximal tubular cells. Blockade of autophagy by 3-methyladenine or small-interfering RNA knockdown of Beclin-1 and ATG5 (two key autophagic genes) sensitized the tubular cells to hypoxia-induced apoptosis. In an in vitro model of ischemia−reperfusion, autophagy was not induced by anoxic (0% O2) incubation in glucose-free buffer, but was induced during subsequent recovery/reperfusion period. In this model, suppression of autophagy also enhanced apoptosis. In vivo, autophagy was induced in kidney tissues during renal ischemia−reperfusion in mice. Autophagy was not obvious during the ischemia period, but was significantly enhanced during reperfusion. Inhibition of autophagy by chloroquine and 3-methyladenine worsened renal ischemia/reperfusion injury, as indicated by renal function, histology, and tubular apoptosis. Together, the results demonstrated autophagy induction during hypoxic and ischemic renal injury. Under these pathological conditions, autophagy may provide a protective mechanism for cell survival. Autophagy mediates bulk degradation and recycling of cytoplasmic constituents to maintain cellular homeostasis. In response to stress, autophagy is induced and may either contribute to cell death or serve as a cell survival mechanism. Very little is known about autophagy in renal pathophysiology. This study examined autophagy and its pathological role in renal cell injury using in vitro and in vivo models of ischemia−reperfusion. We found that hypoxia (1% O2) induced autophagy in cultured renal proximal tubular cells. Blockade of autophagy by 3-methyladenine or small-interfering RNA knockdown of Beclin-1 and ATG5 (two key autophagic genes) sensitized the tubular cells to hypoxia-induced apoptosis. In an in vitro model of ischemia−reperfusion, autophagy was not induced by anoxic (0% O2) incubation in glucose-free buffer, but was induced during subsequent recovery/reperfusion period. In this model, suppression of autophagy also enhanced apoptosis. In vivo, autophagy was induced in kidney tissues during renal ischemia−reperfusion in mice. Autophagy was not obvious during the ischemia period, but was significantly enhanced during reperfusion. Inhibition of autophagy by chloroquine and 3-methyladenine worsened renal ischemia/reperfusion injury, as indicated by renal function, histology, and tubular apoptosis. Together, the results demonstrated autophagy induction during hypoxic and ischemic renal injury. Under these pathological conditions, autophagy may provide a protective mechanism for cell survival. Autophagy is a cellular process of “self-eating” wherein various cytoplasmic constituents are broken down and recycled through the lysosomal degradation pathway.1Levine B Klionsky DJ Development by self-digestion: molecular mechanisms and biological functions of autophagy.Dev Cell. 2004; 6: 463-477Abstract Full Text Full Text PDF PubMed Scopus (3189) Google Scholar This process consists of several sequential steps, including sequestration of cytoplasmic portions by isolation membrane to form autophagosome, fusion of the autophagosome with lysosome to create an autolysosome, and degradation of the engulfed material to generate monomeric units such as amino acids.2Levine B Kroemer G Autophagy in the pathogenesis of disease.Cell. 2008; 132: 27-42Abstract Full Text Full Text PDF PubMed Scopus (5542) Google Scholar Identification of the autophagy-related genes (ATG) in yeast and their orthologs in other organisms including mammals demonstrates that autophagy is evolutionarily conserved in all eukaryotic cells. The ATG genes constitute the core molecular machinery of autophagy and function at the different levels to regulate autophagy induction, progression, and completion.1Levine B Klionsky DJ Development by self-digestion: molecular mechanisms and biological functions of autophagy.Dev Cell. 2004; 6: 463-477Abstract Full Text Full Text PDF PubMed Scopus (3189) Google Scholar Autophagy occurs at basal level in most cells and contributes to the turnover of long-lived proteins and organelles to maintain intracellular homeostasis. In response to cellular stress, autophagy is up-regulated and can provide an adaptive strategy for cell survival, but may also directly or indirectly lead to cell demise.3Baehrecke EH Autophagy: dual roles in life and death?.Nat Rev Mol Cell Biol. 2005; 6: 505-510Crossref PubMed Scopus (842) Google Scholar, 4Levine B Yuan J Autophagy in cell death: an innocent convict?.J Clin Invest. 2005; 115: 2679-2688Crossref PubMed Scopus (1471) Google Scholar, 5Maiuri MC Zalckvar E Kimchi A Kroemer G Self-eating and self-killing: crosstalk between autophagy and apoptosis.Nat Rev Mol Cell Biol. 2007; 8: 741-752Crossref PubMed Scopus (2821) Google Scholar, 6Shintani T Klionsky DJ Autophagy in health and disease: a double-edged sword.Science. 2004; 306: 990-995Crossref PubMed Scopus (2163) Google Scholar With the dual role in life and death, autophagy is involved in various physiological processes, and more importantly, linked to the pathogenesis of a wide array of diseases, such as neurodegeneration, cancer, heart disease, aging, and infections.1Levine B Klionsky DJ Development by self-digestion: molecular mechanisms and biological functions of autophagy.Dev Cell. 2004; 6: 463-477Abstract Full Text Full Text PDF PubMed Scopus (3189) Google Scholar, 2Levine B Kroemer G Autophagy in the pathogenesis of disease.Cell. 2008; 132: 27-42Abstract Full Text Full Text PDF PubMed Scopus (5542) Google Scholar, 6Shintani T Klionsky DJ Autophagy in health and disease: a double-edged sword.Science. 2004; 306: 990-995Crossref PubMed Scopus (2163) Google Scholar, 7Mizushima N Levine B Cuervo AM Klionsky DJ Autophagy fights disease through cellular self-digestion.Nature. 2008; 451: 1069-1075Crossref PubMed Scopus (5144) Google Scholar However, it remains largely unknown how autophagy makes the life and death decisions of a stressed cell. Moreover, the conundrum is further complicated by the cross talk and coordinated regulation between autophagy and apoptosis.4Levine B Yuan J Autophagy in cell death: an innocent convict?.J Clin Invest. 2005; 115: 2679-2688Crossref PubMed Scopus (1471) Google Scholar, 5Maiuri MC Zalckvar E Kimchi A Kroemer G Self-eating and self-killing: crosstalk between autophagy and apoptosis.Nat Rev Mol Cell Biol. 2007; 8: 741-752Crossref PubMed Scopus (2821) Google Scholar, 8Thorburn A Apoptosis and autophagy: regulatory connections between two supposedly different processes.Apoptosis. 2008; 13: 1-9Crossref PubMed Scopus (429) Google Scholar Despite rapid progress of autophagy research in other organ systems, the role of autophagy in the pathogenesis of renal diseases was not recognized until very recently. In 2007, Chien et al9Chien CT Shyue SK Lai MK Bcl-xL augmentation potentially reduces ischemia/reperfusion induced proximal and distal tubular apoptosis and autophagy.Transplantation. 2007; 84: 1183-1190Crossref PubMed Scopus (125) Google Scholar suggested the first evidence of autophagy during renal ischemia−reperfusion in rats. Subsequent work by Suzuki et al10Suzuki C Isaka Y Takabatake Y Tanaka H Koike M Shibata M Uchiyama Y Takahara S Imai E Participation of autophagy in renal ischemia/reperfusion injury.Biochem Biophys Res Commun. 2008; 368: 100-106Crossref PubMed Scopus (153) Google Scholar further showed autophagy in ischemic mouse kidneys and notably, in transplanted human kidneys. In nephrotoxic models of acute kidney injury, we and others have demonstrated autophagy during cisplatin nephrotoxicity and have suggested a role for autophagy in renoprotection.11Periyasamy-Thandavan S Jiang M Wei Q Smith R Yin XM Dong Z Autophagy is cytoprotective during cisplatin injury of renal proximal tubular cells.Kidney Int. 2008; 74: 631-640Crossref PubMed Scopus (263) Google Scholar, 12Yang C Kaushal V Shah SV Kaushal GP Autophagy is associated with apoptosis in cisplatin injury to renal tubular epithelial cells.Am J Physiol Renal Physiol. 2008; 294: F777-F787Crossref PubMed Scopus (229) Google Scholar A prosurvival role of autophagy was also shown in tubular cells during cyclosporine A nephrotoxicity.13Pallet N Bouvier N Legendre C Gilleron J Codogno P Beaune P Thervet E Anglicheau D Autophagy protects renal tubular cells against cyclosporine toxicity.Autophagy. 2008; 4: 783-791PubMed Google Scholar In contrast, Gozuacik et al14Gozuacik D Bialik S Raveh T Mitou G Shohat G Sabanay H Mizushima N Yoshimori T Kimchi A DAP-kinase is a mediator of endoplasmic reticulum stress-induced caspase activation and autophagic cell death.Cell Death Differ. 2008; 15: 1875-1886Crossref PubMed Scopus (211) Google Scholar suggested that autophagy may serve as a second cell killing mechanism that acts in concert with apoptosis to trigger kidney damage in tunicamycin-treated mice. A cell killing role for autophagy was also suggested by Suzuki et al10Suzuki C Isaka Y Takabatake Y Tanaka H Koike M Shibata M Uchiyama Y Takahara S Imai E Participation of autophagy in renal ischemia/reperfusion injury.Biochem Biophys Res Commun. 2008; 368: 100-106Crossref PubMed Scopus (153) Google Scholar during H2O2-induced renal tubular cell injury. As a result, whether autophagy is a mechanism of cell death or survival in renal pathology remains unclear. In this study, we have determined the role of autophagy in renal tubular cell injury using in vitro and in vivo models of renal ischemia−reperfusion. We show that autophagy is induced in these models. Importantly, blockade of autophagy sensitizes renal cells and tissues to injury by hypoxia and ischemia−reperfusion, suggesting a prosurvival role for autophagy. Immortalized rat kidney proximal tubular cell line (RPTC) was originally obtained from Dr. Ulrich Hopfer (Case Western Reserve University, Cleveland, OH) and maintained for experiments as described previously.15Woost PG Orosz DE Jin W Frisa PS Jacobberger JW Douglas JG Hopfer U Immortalization and characterization of proximal tubule cells derived from kidneys of spontaneously hypertensive and normotensive rats.Kidney Int. 1996; 50: 125-134Crossref PubMed Scopus (116) Google Scholar, 16Jiang M Yi X Hsu S Wang CY Dong Z Role of p53 in cisplatin-induced tubular cell apoptosis: dependence on p53 transcriptional activity.Am J Physiol Renal Physiol. 2004; 287: F1140-F1147Crossref PubMed Scopus (143) Google Scholar, 17Jiang M Wei Q Wang J Du Q Yu J Zhang L Dong Z Regulation of PUMA-alpha by p53 in cisplatin-induced renal cell apoptosis.Oncogene. 2006; 25: 4056-4066Crossref PubMed Scopus (177) Google Scholar Isolation and primary culture of proximal tubular cells from mice were described in our recent work.18Jiang M Wang CY Huang S Yang T Dong Z Cisplatin-induced apoptosis in p53-deficient renal cells via the intrinsic mitochondrial pathway.Am J Physiol Renal Physiol. 2009; 296: F983-F993Crossref PubMed Scopus (87) Google Scholar, 19Wei Q Dong G Franklin J Dong Z The pathological role of Bax in cisplatin nephrotoxicity.Kidney Int. 2007; 72: 53-62Crossref PubMed Scopus (188) Google Scholar, 20Wei Q Dong G Yang T Megyesi J Price PM Dong Z Activation and involvement of p53 in cisplatin-induced nephrotoxicity.Am J Physiol Renal Physiol. 2007; 293: F1282-F1291Crossref PubMed Scopus (191) Google Scholar Antibodies in the study were from the following sources: anti-LC3 from Dr. Tamotsu Yoshimori and Dr. Noboru Mizushima,21Kabeya Y Mizushima N Ueno T Yamamoto A Kirisako T Noda T Kominami E Ohsumi Y Yoshimori T LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing.EMBO J. 2000; 19: 5720-5728Crossref PubMed Scopus (5433) Google Scholar anti-Beclin-1 from Santa Cruz Biotechnology (Santa Cruz, CA), anti-ATG5 and anti-β-actin from Sigma (St. Louis, MO), all secondary antibodies from Jackson ImmunoResearch Laboratories Inc (West Grove, PA). Carbobenzoxy-Asp-Glu-Val-Asp-7-amino-4-trifluoromethyl coumarin (DEVD.AFC) and 7-amino-4-trifluoromethyl coumarin (AFC) were from Enzyme Systems Products (Livermore, CA). Lipofectamine transfection reagents were from Invitrogen (Carlsbad, CA). Unless indicated, other reagents including 3-methyladenine (3-MA) and chloroquine were from Sigma (St. Louis, MO). The GFP-LC3 fusion plasmid was generously provided by Dr. Tamotsu Yoshimori and Dr. Noboru Mizushima.21Kabeya Y Mizushima N Ueno T Yamamoto A Kirisako T Noda T Kominami E Ohsumi Y Yoshimori T LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing.EMBO J. 2000; 19: 5720-5728Crossref PubMed Scopus (5433) Google Scholar Green fluorescent protein (GFP)-tagged plasmids for the short hairpin RNA (shRNA) of Beclin-1, ATG5 and their negative control shRNA were purchased from SuperArray (Frederick, MD). Transient transfection of RPTC cells and primary proximal tubular cells was described in our recent work.22Brooks C Wei Q Cho SG Dong Z Regulation of mitochondrial dynamics in acute kidney injury in cell culture and rodent models.J Clin Invest. 2009; 119: 1275-1285Crossref PubMed Scopus (558) Google Scholar Briefly, cells were plated on a coverslip at approximately 50% confluence and then transfected with 1.0 μg plasmid DNA using Lipofectamine PLUS reagents for RPTC cells or Lipofectamine 2000 reagents for primary cells. After incubation in serum-free medium for 4 to 5 hours, the cells were transferred into full culture medium and incubated for ∼24 hours to reach 80 to 90% confluence before experiment. The transfection efficiency for both RPTC and primary cells was around 20%. Cells were plated in 35-mm dishes at a density of ∼1.0 × 106 cells/dish for RPTC cells or ∼0.3 × 106 cells/dish for primary tubular cells and reached ∼90% confluence by next day for experiment. Hypoxia treatment was conducted in a hypoxia chamber as before.23Wang J Biju MP Wang MH Haase VH Dong Z Cytoprotective effects of hypoxia against cisplatin-induced tubular cell apoptosis: involvement of mitochondrial inhibition and p53 suppression.J Am Soc Nephrol. 2006; 17: 1875-1885Crossref PubMed Scopus (60) Google Scholar Briefly, cells were incubated in a hypoxia chamber (COY Laboratory Products, Ann Arbor, MI) with a compact gas oxygen controller to maintain oxygen concentration at 1% by injecting a gas mixture of 95% N2 and 5% CO2. For in vitro ischemia, RPTC cells were washed with phosphate-buffered saline and incubated for 2 hours in a glucose-free Krebs-Ringer bicarbonate buffer in an anaerobic chamber equilibrated with 5% CO2, 5% H2 and 90% N2. After ischemic treatment, the cells were transferred back to full culture medium with oxygen for reperfusion. The incubation medium used for hypoxia or ischemia treatment was pre-equilibrated overnight in the respective chambers. Control cells were incubated in a regular cell culture incubator with 21% oxygen. At the end of treatment, cells were monitored morphologically or harvested with indicated buffers to collect cell lysates for biochemical analyses. For cell lysis, both floating and adherent cells were collected. The two commonly used methods for autophagy analysis were described in our recent study.11Periyasamy-Thandavan S Jiang M Wei Q Smith R Yin XM Dong Z Autophagy is cytoprotective during cisplatin injury of renal proximal tubular cells.Kidney Int. 2008; 74: 631-640Crossref PubMed Scopus (263) Google Scholar To monitor the formation of GFP-LC3 puncta, RPTC or primary tubular cells were transiently transfected with 1.0 μg GFP-LC3 plasmid and then treated with hypoxia as described above. After treatment, the cells were fixed with 4% paraformaldehyde for fluorescence microscopic examination. Twenty fields of ×600 magnification with 20 to 30 GFP-labeled green cells per field were counted in each condition. The following criteria were used to determine the cells with punctuate GFP-LC3 (positive cells): 1, with uneven, ring-shaped dots in the cytoplasm; 2, with more than 10 dots per cell. The percentage of such positive cells was recorded for quantification. For LC3 immunoblot analysis, whole cell or tissue lysates were extracted in 2% SDS buffer and protein concentration was determined with bicinchoninic acid reagent from Pierce (Rockford, IL). Equal amounts of protein were loaded in each lane and resolved in 12% SDS-polyacrylamide electrophoresis gel. After transferred onto polyvinylidene difluoride membrane, the blots were subsequently incubated with 5% milk, anti-LC3 primary antibody and horseradish peroxidase-conjugated anti-rabbit secondary antibody. Antigens on the blots were revealed using the enhanced chemiluminescence kit from Pierce (Rockford, IL). Same blots were also probed with anti-β-actin to monitor protein loading and transferring. Apoptosis was determined by morphological and biochemical methods as described in our previous work.16Jiang M Yi X Hsu S Wang CY Dong Z Role of p53 in cisplatin-induced tubular cell apoptosis: dependence on p53 transcriptional activity.Am J Physiol Renal Physiol. 2004; 287: F1140-F1147Crossref PubMed Scopus (143) Google Scholar, 17Jiang M Wei Q Wang J Du Q Yu J Zhang L Dong Z Regulation of PUMA-alpha by p53 in cisplatin-induced renal cell apoptosis.Oncogene. 2006; 25: 4056-4066Crossref PubMed Scopus (177) Google Scholar, 18Jiang M Wang CY Huang S Yang T Dong Z Cisplatin-induced apoptosis in p53-deficient renal cells via the intrinsic mitochondrial pathway.Am J Physiol Renal Physiol. 2009; 296: F983-F993Crossref PubMed Scopus (87) Google Scholar Morphologically, after treatment, cells were stained with 10 μg/ml Hoechst 33342. Cellular and nuclear morphology was examined by phase contrast and fluorescence microscopy, respectively. Typical apoptotic cells were identified by their morphology including cellular shrinkage, nuclear condensation and fragmentation, and formation of apoptotic bodies. Four fields with ∼200 cells per field were examined in each condition to estimate the apoptosis percentage. Biochemically, the enzymatic activity of caspases was measured using DEVD.AFC, a fluorogenic peptide substrate. Briefly, cells were extracted with 1% Triton X-100. The lysates of 25 μg protein were added to enzymatic reactions containing 50 μmol/L DEVD.AFC. After 1 hour incubation at 37°C, fluorescence was measured at excitation 360 nm/emission 530 nm. For each measurement, a standard curve was constructed using free AFC. Based on the standard curve, the fluorescence reading from each enzymatic reaction was converted into the nanomolar amount of liberated AFC per mg protein to indicate caspase activity. C57BL/6 mice were originally purchased from Jackson Laboratory and maintained in the animal facility of Charlie Norwood VA Medical Center under a 12-hour light/12-hour dark pattern with free access to food and water. All animal experiments were performed according to a protocol approved by the Institutional Animal Care and Use Committee of Charlie Norwood VA Medical Center. Both littermate and age/sex-matched nonlittermate male mice of 8 to 10 weeks were used for renal ischemia− reperfusion surgery as described in our recent studies.22Brooks C Wei Q Cho SG Dong Z Regulation of mitochondrial dynamics in acute kidney injury in cell culture and rodent models.J Clin Invest. 2009; 119: 1275-1285Crossref PubMed Scopus (558) Google Scholar, 24Wei Q Yin XM Wang MH Dong Z Bid deficiency ameliorates ischemic renal failure and delays animal death in C57BL/6 mice.Am J Physiol Renal Physiol. 2006; 290: F35-F42Crossref PubMed Scopus (56) Google Scholar Briefly, after anesthetized with pentobarbital (50 mg/kg, i.p.), the mice were kept on a Homeothermic Blanket Control Unit (Harvard Apparatus Ltd, UK) with a rectal probe to monitor and maintain body temperature at ∼36.4°C. Flank incisions were made to expose both renal pedicles for bilateral clamping to induce 30 or 28 minutes of renal ischemia. The clamps were then released for reperfusion. Kidneys and blood were collected after indicated durations of reperfusion for the following examinations. Color changes of kidneys during the initiation of clamping and after removal of clamps were monitored to indicate sufficient renal ischemia and reperfusion. Control animals were subjected to sham operation without renal pedicle clamping. To detect the effects of chloroquine, the mice were treated with chloroquine (60 mg/kg, i.p.) 1 hour before renal ischemia, and were continuously subjected to daily chloroquine injection for up to 2 days through reperfusion. To examine the effects of 3-MA, the mice were injected with one dose (30 mg/kg) of 3-MA 1 hour before ischemia−reperfusion. Renal tissue electron microscopy assay was described in our recent work.11Periyasamy-Thandavan S Jiang M Wei Q Smith R Yin XM Dong Z Autophagy is cytoprotective during cisplatin injury of renal proximal tubular cells.Kidney Int. 2008; 74: 631-640Crossref PubMed Scopus (263) Google Scholar, 22Brooks C Wei Q Cho SG Dong Z Regulation of mitochondrial dynamics in acute kidney injury in cell culture and rodent models.J Clin Invest. 2009; 119: 1275-1285Crossref PubMed Scopus (558) Google Scholar Briefly, after indicated treatment, the mice were sacrificed and perfused with 10 ml (10 units/ml) heparin, followed by 50 ml fixative (100 mmol/L sodium cacodylate, 2 mmol/L CaCl2, 4 mmol/L MgSO4, 4% paraformaldehyde, and 2.5% glutaraldehyde). Kidneys were then harvested and postfixed in the same fixative. An approximately 1 mm3 of tissue cube was collected from each kidney, including a portion of renal cortex and outer medulla for standard electron microscopy processing. According to their morphology, various autophagic structures including phagophore, autophagosome, and autolysosome in proximal tubular cells were revealed at high magnification (×10,000). For quantification, 20 to 30 fields of low magnification (×1000) were randomly selected from each kidney and digital images with scale bars were taken. Using AxioVision 4 software, the amount of autophagic vacuoles per unit cytoplasmic area of 100 μm was evaluated. Renal function was monitored by blood urea nitrogen (BUN) and serum creatinine as described before.19Wei Q Dong G Franklin J Dong Z The pathological role of Bax in cisplatin nephrotoxicity.Kidney Int. 2007; 72: 53-62Crossref PubMed Scopus (188) Google Scholar, 24Wei Q Yin XM Wang MH Dong Z Bid deficiency ameliorates ischemic renal failure and delays animal death in C57BL/6 mice.Am J Physiol Renal Physiol. 2006; 290: F35-F42Crossref PubMed Scopus (56) Google Scholar Briefly, blood samples were collected and coagulated at room temperature, followed by centrifugation to have serum. BUN was measured with a kit from Biotron Diagnostics Inc (Hemet, CA) and absorbance at 540 nm was recorded at the end of reaction. Serum creatinine was determined using a kit from Stanbio Laboratory (Boerne, TX) and kinetic absorbance at 510 nm was monitored at 20 and 80 second of reaction. BUN and creatinine levels (mg/dl) were then calculated based on standard curves. For histology, kidneys were fixed with 4% paraformaldehyde and embedded in paraffin. The tissues were then sectioned at 4 μm for H&E staining. As described previously,19Wei Q Dong G Franklin J Dong Z The pathological role of Bax in cisplatin nephrotoxicity.Kidney Int. 2007; 72: 53-62Crossref PubMed Scopus (188) Google Scholar, 20Wei Q Dong G Yang T Megyesi J Price PM Dong Z Activation and involvement of p53 in cisplatin-induced nephrotoxicity.Am J Physiol Renal Physiol. 2007; 293: F1282-F1291Crossref PubMed Scopus (191) Google Scholar, 22Brooks C Wei Q Cho SG Dong Z Regulation of mitochondrial dynamics in acute kidney injury in cell culture and rodent models.J Clin Invest. 2009; 119: 1275-1285Crossref PubMed Scopus (558) Google Scholar histopathological changes, including loss of brush border, tubular dilation, cast formation, and cell lysis, were evaluated. Tissue damage was examined in a blind manner and scored according to the percentage of damaged tubules: 0, no damage; 1, 75%. As shown in our recent studies,19Wei Q Dong G Franklin J Dong Z The pathological role of Bax in cisplatin nephrotoxicity.Kidney Int. 2007; 72: 53-62Crossref PubMed Scopus (188) Google Scholar, 20Wei Q Dong G Yang T Megyesi J Price PM Dong Z Activation and involvement of p53 in cisplatin-induced nephrotoxicity.Am J Physiol Renal Physiol. 2007; 293: F1282-F1291Crossref PubMed Scopus (191) Google Scholar, 22Brooks C Wei Q Cho SG Dong Z Regulation of mitochondrial dynamics in acute kidney injury in cell culture and rodent models.J Clin Invest. 2009; 119: 1275-1285Crossref PubMed Scopus (558) Google Scholar, 24Wei Q Yin XM Wang MH Dong Z Bid deficiency ameliorates ischemic renal failure and delays animal death in C57BL/6 mice.Am J Physiol Renal Physiol. 2006; 290: F35-F42Crossref PubMed Scopus (56) Google Scholar apoptosis in renal tissue was identified by TdT-mediated dUTP nick-end labeling (TUNEL) assay using an in situ cell death detection kit (Roche Applied Science, Indianapolis, IN). Briefly, paraffin-embedded renal tissue sections of 4 μm were deparaffinized and permeabilized with 0.1 mol/L sodium citrate, PH6.0 at 65°C for 2 hours. The sections were then exposed to a TUNEL reaction mixture containing terminal deoxynucleotidyl transferase and nucleotides including tetramethylrhodamine-labeled dUTP. After 1 hour incubation at 37°C in a humidified atmosphere, positive staining with nuclear DNA fragmentation was detected by fluorescence microscopy. For quantification, 10 representative fields were selected from each tissue section and the amount of TUNEL-positive cells per 100 mm2 was evaluated. Qualitative data including immunoblots and cell images are representatives of at least three experiments. Quantitative data were expressed as means ± SD. Statistical analysis was conducted using the GraphPad Prism software. Statistical differences in multiple groups were determined by multiple comparisons with analysis of variance followed by Tukey's post-tests. Statistical differences between two groups were determined by two-tailed unpaired Student's t-test. P < 0.05 was considered significantly different. Accumulation of LC3 (also called ATG8) in autophagosomes and lipidation of LC3 to form LC3-II are two hallmarks of autophagy and are commonly used for autophagy detection.25Klionsky DJ Abeliovich H Agostinis P Agrawal DK Aliev G Askew DS Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes.Autophagy. 2008; 4: 151-175PubMed Google Scholar, 26Mizushima N Yoshimori T How to interpret LC3 immunoblotting.Autophagy. 2007; 3: 542-545PubMed Google Scholar Thus we initially examined autophagy by analyzing the formation of fluorescent puncta or autophagosomes in GFP-LC3-transfected cells. As shown in Figure 1A, most control RPTC cells had an even and diffused GFP-LC3 staining with occasional puncta. On hypoxic incubation, some cells showed numerous unevenly distributed, cup- or ring-shaped green dots of various sizes. Cell counting indicated that 6 to 12 hours of hypoxia increased GFP-LC3 punctuate cells from the basal level of 15 to 34%, which decreased thereafter to 23% at the end of 24 hours (Figure 1B). We further examined LC3-II formation by immunoblot analysis. As shown in Figure 1C, hypoxic incubation induced a time-dependent accumulation of LC3-II in RPTC cells, starting at 6 hours and increasing markedly after 12 to 24 hours of treatment. The results were confirmed by densitometry of immunoblots from separate experiments (Figure 1D). Of note, the formation of GFP-LC3 puncta seemed to occur earlier than LC3-II (Figure 1, B and D), suggesting that LC3 may first accumulate to autophagic vesicles and then undergo lipidation. Autophagy is a dynamic, multistep process, and an accumulation of autophagosome content may reflect either increased autophagic activity or reduced autophagic flux and lysosomal degradation.25Klionsky DJ Abeliovich H Agostinis P Agrawal DK Aliev G Askew DS Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes.Autophagy. 2008; 4: 151-175PubMed Google Scholar, 26Mizushima N Yoshimori T How to interpret LC3 immunoblotting.Autophagy. 2007; 3: 542-545PubMed Google Scholar Did hypoxia induce autophagy or block autophagic flux to lysosomal degradation? To address this question, we tested the effects of E64d and pepstatin A, two lysosomal protease inhibitors used to study autophagic flux.27Tanida I Minematsu-Ikeguchi N Ueno T Kominami E Lysosomal turnover, but not a cellular level, of endogenous LC3 is a marker for autophagy.Autophagy. 2005; 1: 84-91Crossref PubMed Scopus (936) Google Scholar As shown in Figure 1E, the lysosomal inhibitors significantly increased LC3-II accumulation during hypoxic incubation of RPTC cells at each time point (lanes 6–9 vs. 2–5). The results suggest that hypoxia did not block autophagic flux; rather the autophagic activity was induced in these cells. Of note, hypoxia did not induce significant apoptosis in RPTC until 24 hours of incubation (Figure 1F). We further showed autophagy during hypoxic incubation of primary proximal tubular cells that were isolated from C57BL/6 mice (data not shown). In these cells, apoptosis or cell death was not induced even after 72 hours of hypoxic incubation (data not shown), further suggesting that autophagy is an early response to hypoxic stress whereas apoptosis is a late outcome. Autophagy induction under cellular stress