Autophagy is cytoprotective during cisplatin injury of renal proximal tubular cells
2008; Elsevier BV; Volume: 74; Issue: 5 Linguagem: Inglês
10.1038/ki.2008.214
ISSN1523-1755
AutoresSudharsan Periyasamy‐Thandavan, Man Jiang, Qingqing Wei, Robert G. Smith, Xiao-Ming Yin, Zheng Dong,
Tópico(s)Biomedical Research and Pathophysiology
ResumoAutophagy is a cellular process of bulk degradation of damaged organelles, protein aggregates and other macromolecules in the cytoplasm. It is thought to be a general response to stress contributing to cell death; alternatively it might act as a cytoprotective mechanism. Here we found that administration of cisplatin induced the formation of autophagic vesicles and autophagosomes in mouse kidneys. In cultured proximal tubular cells, the nephrotoxin caused autophagy in a dose- and time-dependent manner prior to apoptosis. Notably, autophagy occurred within hours of cisplatin administration but this was partially suppressed by the p53 inhibitor pifithrin-α, suggesting that p53 is involved in autophagic signaling. This cisplatin-induced autophagy was attenuated in renal cells stably transfected with Bcl-2, suggesting an anti-autophagic role for this well-known anti-apoptotic protein. Blockade of autophagy with pharmacological inhibitors (3-methyladenine or bafilomycin) or shRNA knockdown of the autophagic gene Beclin increased tubular cell apoptosis during cisplatin treatment. Our study has found that autophagy occurs in acute kidney injury and this may be an important protective mechanism for cell survival. Autophagy is a cellular process of bulk degradation of damaged organelles, protein aggregates and other macromolecules in the cytoplasm. It is thought to be a general response to stress contributing to cell death; alternatively it might act as a cytoprotective mechanism. Here we found that administration of cisplatin induced the formation of autophagic vesicles and autophagosomes in mouse kidneys. In cultured proximal tubular cells, the nephrotoxin caused autophagy in a dose- and time-dependent manner prior to apoptosis. Notably, autophagy occurred within hours of cisplatin administration but this was partially suppressed by the p53 inhibitor pifithrin-α, suggesting that p53 is involved in autophagic signaling. This cisplatin-induced autophagy was attenuated in renal cells stably transfected with Bcl-2, suggesting an anti-autophagic role for this well-known anti-apoptotic protein. Blockade of autophagy with pharmacological inhibitors (3-methyladenine or bafilomycin) or shRNA knockdown of the autophagic gene Beclin increased tubular cell apoptosis during cisplatin treatment. Our study has found that autophagy occurs in acute kidney injury and this may be an important protective mechanism for cell survival. Autophagy is a process of bulk degradation of damaged organelles, protein aggregates, and other macromolecules in the cytoplasm.1.Klionsky D.J. Autophagy: from phenomenology to molecular understanding in less than a decade.Nat Rev Mol Cell Biol. 2007; 8: 931-937Crossref PubMed Scopus (1405) Google Scholar,2.Levine B. Yuan J. Autophagy in cell death: an innocent convict?.J Clin Invest. 2005; 115: 2679-2688Crossref PubMed Scopus (1402) Google Scholar,3.Suzuki K. Ohsumi Y. Molecular machinery of autophagosome formation in yeast, Saccharomyces cerevisiae.FEBS Lett. 2007; 581: 2156-2161Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar,4.Shintani T. Klionsky D.J. Autophagy in health and disease: a double-edged sword.Science. 2004; 306: 990-995Crossref PubMed Scopus (2025) Google Scholar At the core of autophagy is a specific family of genes or proteins called autophagy-related gene (Atg). The Atg proteins are responsible for initiation, formation, and maturation of autophagosomes, which subsequently fuses with lysosomes for hydrolysis or degradation of enwrapped materials.1.Klionsky D.J. Autophagy: from phenomenology to molecular understanding in less than a decade.Nat Rev Mol Cell Biol. 2007; 8: 931-937Crossref PubMed Scopus (1405) Google Scholar,2.Levine B. Yuan J. Autophagy in cell death: an innocent convict?.J Clin Invest. 2005; 115: 2679-2688Crossref PubMed Scopus (1402) Google Scholar,3.Suzuki K. Ohsumi Y. Molecular machinery of autophagosome formation in yeast, Saccharomyces cerevisiae.FEBS Lett. 2007; 581: 2156-2161Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar,4.Shintani T. Klionsky D.J. Autophagy in health and disease: a double-edged sword.Science. 2004; 306: 990-995Crossref PubMed Scopus (2025) Google Scholar Atgs were originally identified in yeast, but their mammalian orthologs are now being discovered and shown to play critical roles in autophagy in mammalian cells and animals.1.Klionsky D.J. Autophagy: from phenomenology to molecular understanding in less than a decade.Nat Rev Mol Cell Biol. 2007; 8: 931-937Crossref PubMed Scopus (1405) Google Scholar,2.Levine B. Yuan J. Autophagy in cell death: an innocent convict?.J Clin Invest. 2005; 115: 2679-2688Crossref PubMed Scopus (1402) Google Scholar,3.Suzuki K. Ohsumi Y. Molecular machinery of autophagosome formation in yeast, Saccharomyces cerevisiae.FEBS Lett. 2007; 581: 2156-2161Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar,4.Shintani T. Klionsky D.J. Autophagy in health and disease: a double-edged sword.Science. 2004; 306: 990-995Crossref PubMed Scopus (2025) Google Scholar For example, Beclin-1, the mammalian ortholog of yeast Atg6, contributes to vesicle nucleation, an early event for autophagosome formation.5.Liang X.H. Jackson S. Seaman M. et al.Induction of autophagy and inhibition of tumorigenesis by beclin 1.Nature. 1999; 402: 672-676Crossref PubMed Scopus (2533) Google Scholar LC3, the mammalian ortholog of Atg8, is proteolytically processed and conjugated with phosphotidylethanolamine to form LC3-II, which is then localized on the autophagic vesicle for its elongation and expansion.6.Kabeya Y. Mizushima N. Ueno T. et al.LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing.EMBO J. 2000; 19: 5720-5728Crossref PubMed Scopus (5048) Google Scholar Traditionally, autophagy is recognized as a cellular response to nutrient deprivation or starvation, whereby cells digest a portion of cytoplasm to recycle nutrients for survival. However, recent studies have suggested that autophagy might be a general cellular response to stress.1.Klionsky D.J. 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Acute kidney injury by ischemia, sepsis, or nephrotoxins is a typical condition of renal stress, leading to cell death, tissue damage, and loss of renal function or renal failure.7.Bonventre J.V. Weinberg J.M. Recent advances in the pathophysiology of ischemic acute renal failure.J Am Soc Nephrol. 2003; 14: 2199-2210Crossref PubMed Scopus (608) Google Scholar,8.Devarajan P. Update on mechanisms of ischemic acute kidney injury.J Am Soc Nephrol. 2006; 17: 1503-1520Crossref PubMed Scopus (732) Google Scholar,9.Molitoris B.A. Sutton T.A. Endothelial injury and dysfunction: role in the extension phase of acute renal failure.Kidney Int. 2004; 66: 496-499Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar,10.Vaidya V.S. Ferguson M.A. Bonventre J.V. Biomarkers of acute kidney injury.Annu Rev Pharmacol Toxicol. 2008; 48: 463-493Crossref PubMed Scopus (458) Google Scholar,11.Goligorsky M.S. Whispers and shouts in the pathogenesis of acute renal ischaemia.Nephrol Dial Transplant. 2005; 20: 261-266Crossref PubMed Scopus (28) Google Scholar Is autophagy induced during acute kidney injury? Does autophagy contribute to renal cell injury and death, or survival under this pathological condition? This study was designed to address these questions by using experimental models of cisplatin nephrotoxicity. Cisplatin is a widely used chemotherapy drug, with major side effects in kidneys, inducing acute kidney injury.12.Arany I. Safirstein R.L. Cisplatin nephrotoxicity.Semin Nephrol. 2003; 23: 460-464Abstract Full Text Full Text PDF PubMed Scopus (741) Google Scholar,13.Pabla N. Dong Z. Cisplatin nephrotoxicity: mechanisms and renoprotective strategies.Kidney Int. 2008; 73: 994-1007Abstract Full Text Full Text PDF PubMed Scopus (1140) Google Scholar While multiple mechanisms have been documented to contribute to cisplatin nephrotoxicity,14.Arany I. Megyesi J.K. 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Mi Q.S. et al.ATR-Chk2 signaling in p53 activation and DNA damage response during cisplatin-induced apoptosis.J Biol Chem. 2008; 283: 6572-6583Crossref PubMed Scopus (212) Google Scholar how these signaling pathways are integrated to induce renal pathology is largely unknown.13.Pabla N. Dong Z. Cisplatin nephrotoxicity: mechanisms and renoprotective strategies.Kidney Int. 2008; 73: 994-1007Abstract Full Text Full Text PDF PubMed Scopus (1140) Google Scholar Here we demonstrate early occurrence of autophagy during cisplatin treatment of renal tubular cells and tissues. Importantly, autophagy appears to be a cytoprotective mechanism for cell survival. To determine the occurrence of autophagy during cisplatin nephrotoxicity, we examined an in vitro model of cultured proximal tubular cells (RPTC). This model has been characterized in previous studies.16.Jiang M. Pabla N. Murphy R.F. et al.Nutlin-3 protects kidney cells during cisplatin therapy by suppressing Bax/Bak activation.J Biol Chem. 2007; 282: 2636-2645Crossref PubMed Scopus (84) Google Scholar,17.Jiang M. Wei Q. Wang J. et al.Regulation of PUMA-alpha by p53 in cisplatin-induced renal cell apoptosis.Oncogene. 2006; 25: 4056-4066Crossref PubMed Scopus (167) Google Scholar,18.Jiang M. Yi X. Hsu S. et al.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 (129) Google Scholar Our initial analysis was focused on subcellular localization and redistribution of LC3, because redistribution of LC3 from cytosol to a punctate autophagosome staining is an indication of autophagy.34.Bampton E.T. Goemans C.G. Niranjan D. et al.The dynamics of autophagy visualized in live cells: from autophagosome formation to fusion with endo/lysosomes.Autophagy. 2005; 1: 23-36Crossref PubMed Scopus (299) Google Scholar To this end, cells were transfected with a green fluorescent protein (GFP)-LC3 fusion plasmid and then treated with 20 μM cisplatin for 0–16 h, followed by examination by fluorescence microscopy. In the control group, 27% of transfected (GFP-labeled) cells had punctate LC3 staining. Six hours of cisplatin treatment increased the percentage of LC3 punctate cells to ∼50% (Figure 1a). Further incubation with cisplatin to 12 or 16 h led to gradual disappearance of LC3 puncta, an observation that was consistent with the degradation of LC3 in matured autophagosomes (Figure 1a). Representative LC3 staining in control and cisplatin-treated cells is shown in Figure 1b. By analyzing LC3 redistribution, we further showed that induction of autophagy was dependent on cisplatin dose (Figure 1b). Significant increases of autophagic (LC3 punctate) cells were induced by 5–20 μM, but not 1 or 2 μM, cisplatin (Figure 1c). To further analyze autophagy, we examined formation of LC3-II, the autophagic form of LC3.35.Mizushima N. Yoshimori T. How to interpret LC3 immunoblotting.Autophagy. 2007; 3: 542-545Crossref PubMed Scopus (1736) Google Scholar RPTCs were incubated with 20 μM cisplatin for 0–16 h to collect whole-cell lysates for immunoblot analysis. As shown in Figure 1d, cisplatin induced an obvious accumulation of LC3. Of note, LC3-II, the autophagic form, showed highest accumulation at 6 h of cisplatin treatment and then decreased toward basal levels (Figure 1d), which correlated with the time course of punctate LC3 formation (Figure 1a). As shown in our previous work,16.Jiang M. Pabla N. Murphy R.F. et al.Nutlin-3 protects kidney cells during cisplatin therapy by suppressing Bax/Bak activation.J Biol Chem. 2007; 282: 2636-2645Crossref PubMed Scopus (84) Google Scholar,17.Jiang M. Wei Q. Wang J. et al.Regulation of PUMA-alpha by p53 in cisplatin-induced renal cell apoptosis.Oncogene. 2006; 25: 4056-4066Crossref PubMed Scopus (167) Google Scholar,18.Jiang M. Yi X. Hsu S. et al.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 (129) Google Scholar cisplatin at 20 μM induced significant amounts of apoptosis in RPTCs in a time-dependent manner (Figure 1e). It is noteworthy that occurrence of autophagy was hours earlier than apoptosis (Figures 1a and e). The results suggest that cisplatin-induced autophagy is not caused by or secondary to apoptosis. We further verified cisplatin-induced autophagy in RPTCs by electron microscopy. As shown by representative micrographs, autophagosomes with characteristic double or multiple membranes were identified in the cells after 6 h of cisplatin treatment (Figure 2a, arrows). In addition, there were many autophagic vacuoles or vesicles (Figure 2a, arrowheads). Counting autophagic vesicles in the micrographs also indicated higher autophagy in cisplatin-treated cells (Figure 2b). Interestingly, these cells also showed autophagic vesicles at different stages of autophagosome apparently from initiation, elongation, complete assembly, to maturation (Figure 2c–g). To gain initial evidence for autophagy during cisplatin nephrotoxicity in vivo, C57BL/6 mice were injected with a single dose of 30 mg/kg cisplatin.28.Faubel S. Ljubanovic D. Reznikov L. et al.Caspase-1-deficient mice are protected against cisplatin-induced apoptosis and acute tubular necrosis.Kidney Int. 2004; 66: 2202-2213Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar,36.Wei Q. Dong G. Franklin J. et al.The pathological role of Bax in cisplatin nephrotoxicity.Kidney Int. 2007; 72: 53-62Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar,37.Wei Q. Wang M.H. Dong Z. Differential gender differences in ischemic and nephrotoxic acute renal failure.Am J Nephrol. 2005; 25: 491-499Crossref PubMed Scopus (76) Google Scholar We first examined autophagy by electron microscopy. As shown in Figure 3a, 2 days after cisplatin injection, numerous vacuoles appeared in some proximal tubular cells and some of the vacuoles contained cytosolic materials, becoming autophagic vesicles (arrow). At day 3, the numbers of empty vacuoles decreased, but their sizes increased. In addition, more vesicles had taken up cytosolic materials to become autophagosomes (arrow). Counting cells containing autophagic vacuoles or vesicles showed progressive increase of autophagy during cisplatin nephrotoxicity (Figure 3b). By the end of 2–3 days of cisplatin treatment, over 30% of renal tubular cells were autophagic. To further confirm autophagy, we collected renal tissue lysates for immunoblot analysis of LC3. As shown in Figure 3c, there was a notable increase of LC3-II in renal tissues following cisplatin injection, particularly at day 2 and 3. Together, these results have demonstrated evidence for the occurrence of autophagy during cisplatin-induced renal cell injury. We and others have demonstrated a role for p53 in cisplatin-induced renal cell apoptosis in vitro and nephrotoxicity in vivo.13.Pabla N. Dong Z. Cisplatin nephrotoxicity: mechanisms and renoprotective strategies.Kidney Int. 2008; 73: 994-1007Abstract Full Text Full Text PDF PubMed Scopus (1140) Google Scholar,15.Cummings B.S. Schnellmann R.G. Cisplatin-induced renal cell apoptosis: caspase 3-dependent and -independent pathways.J Pharmacol Exp Ther. 2002; 302: 8-17Crossref PubMed Scopus (287) Google Scholar,17.Jiang M. Wei Q. Wang J. et al.Regulation of PUMA-alpha by p53 in cisplatin-induced renal cell apoptosis.Oncogene. 2006; 25: 4056-4066Crossref PubMed Scopus (167) Google Scholar,18.Jiang M. Yi X. Hsu S. et al.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 (129) Google Scholar,24.Seth R. Yang C. Kaushal V. et al.p53-Dependent caspase-2 activation in mitochondrial release of apoptosis-inducing factor and its role in renal tubular epithelial cell injury.J Biol Chem. 2005; 280: 31230-31239Crossref PubMed Scopus (151) Google Scholar,36.Wei Q. Dong G. Franklin J. et al.The pathological role of Bax in cisplatin nephrotoxicity.Kidney Int. 2007; 72: 53-62Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar,38.Wei Q. Dong G. Yang T. et al.Activation and involvement of p53 in cisplatin-induced nephrotoxicity.Am J Physiol Renal Physiol. 2007; 293: F1282-F1291Crossref PubMed Scopus (171) Google Scholar Interestingly, a recent study by Feng et al.39.Feng Z. Zhang H. Levine A.J. et al.The coordinate regulation of the p53 and mTOR pathways in cells.Proc Natl Acad Sci USA. 2005; 102: 8204-8209Crossref PubMed Scopus (909) Google Scholar suggests that p53 may also be involved in the regulation of autophagy. We, therefore, tested the effects of pifithrin-α (PF), a pharmacological inhibitor of p53, on cisplatin-induced autophagy in RPTCs. For this purpose, cells were transfected with GFP-LC3 and then incubated with 20 μM cisplatin in the presence of 20 μM PF. As shown in Figure 4a, cisplatin incubation for 6 h induced punctate GFP-LC3 in 56% of the cells, which was suppressed to 36% by PF. In addition, PF induced an interesting distribution pattern of punctate LC-GFP in the autophagic cells. As shown in Figure 4b, the autophagic cells in the cisplatin+PF group had many big empty vacuoles surrounded by punctate GFP-LC3 staining. We further collected cell lysates for immunoblot analysis of LC3 (Figure 4c). Consistently, PF had partial inhibitory effects on LC3-II formation during cisplatin treatment (Figure 4c and d). Together, these results suggest the involvement of p53 in triggering autophagy during cisplatin treatment of renal tubular cells. Bcl-2 is well known for its antiapoptotic activity. Interestingly, recent studies have shown that Bcl-2 is also an anti-autophagic protein,40.Pattingre S. Levine B. Bcl-2 inhibition of autophagy: a new route to cancer?.Cancer Res. 2006; 66: 2885-2888Crossref PubMed Scopus (233) Google Scholar which binds and sequesters Beclin-1 to prevent vesicle nucleation, an early step of autophagosome formation. To determine the regulation of cisplatin-induced autophagy by Bcl-2, we used an RPTC line stably transfected with Bcl-2 that was established in our previous work.41.Saikumar P. Dong Z. Patel Y. et al.Role of hypoxia-induced Bax translocation and cytochrome c release in reoxygenation injury.Oncogene. 1998; 17: 3401-3415Crossref PubMed Scopus (248) Google Scholar Wild-type RPTCs and Bcl-2-transfected cells were incubated with 20 μM cisplatin for 6 h. In wild-type RPTCs, cisplatin treatment increased autophagic cells to 57%, while no increase was shown in Bcl-2 cells (Figure 5a). Examination of GFP-LC3 signal at later time points did not reveal significant amounts of autophagic cells in the Bcl-2 group either (Figure 5b). We further analyzed LC3 by immunoblots. Consistent with earlier results (Figure 1), cisplatin induced LC3-II with a maximal accumulation at 6 h; such induction was not shown in Bcl-2 cells (Figure 5c and d). Together, the results demonstrate an impressive inhibitory effects of Bcl-2 on cisplatin-induced autophagy. Depending on the experimental conditions, autophagy can be cell killing or cytoprotective.2.Levine B. Yuan J. Autophagy in cell death: an innocent convict?.J Clin Invest. 2005; 115: 2679-2688Crossref PubMed Scopus (1402) Google Scholar,42.Maiuri M.C. Zalckvar E. Kimchi A. et al.Self-eating and self-killing: crosstalk between autophagy and apoptosis.Nat Rev Mol Cell Biol. 2007; 8: 741-752Crossref PubMed Scopus (2533) Google Scholar What is the pathophysiological role of autophagy during cisplatin-induced tubular cell injury? To address this question, we determined the effects of 3-methyladenine (3-MA) and bafilomycin (BAF), two pharmacological inhibitors of autophagy. We first verified their effects on autophagy. As shown in Figures 6a, 3-MA blocked GFP-LC3 redistribution into punctate form. In stark contrast, BAF enhanced formation of punctate LC3. The results were further supported by cell count (Figure 6b). The opposite effects of 3-MA and BAF on LC3 distribution are not surprising, as these two inhibitors block autophagy at different levels: while 3-MA inhibits the initiation of autophagosome, BAF attenuates fusion of autophagosome with lysosome.42.Maiuri M.C. Zalckvar E. Kimchi A. et al.Self-eating and self-killing: crosstalk between autophagy and apoptosis.Nat Rev Mol Cell Biol. 2007; 8: 741-752Crossref PubMed Scopus (2533) Google Scholar As a result, 3-MA blocked autophagosome formation from the beginning, whereas BAF prevented degradation of GFP-LC3 in autophagolysosomes and in turn increased LC3 punctate. This notion was also supported by our immunoblot analysis of LC3 (Figure 6c and d). 3-MA blocked the formation LC3-II in cisplatin-treated as well as in control cells, while BAF preserved LC3-II during cisplatin treatment even at 24 h. After confirming the effects of 3-MA and BAF on autophagy, we examined their effects on apoptosis during cisplatin treatment. Apoptosis was evaluated based on cellular and nuclear morphology. As shown in Figure 7a, control cells without cisplatin exposure appeared healthy. Incubation with 20 μM cisplatin for 12 h induced apoptosis in some cells, which had a shrunken cell body with condensed and fragmented nuclei. Many of these cells also showed formation of apoptotic bodies or blebs. Importantly, addition of either 3-MA or BAF significantly increased apoptosis during cisplatin incubation (Figure 7). Quantification by cell count indicated that 3-MA and BAF increased apoptosis from 27% to 47 and 55%, respectively (Figure 7b). We further examined the effects of 3-MA on apoptosis induced by lower concentrations of cisplatin. As shown in Figure 7c, 5 μM cisplatin incubation for 16 h induced ∼3% apoptosis, which was increased to ∼20% by 3-MA. Together, the pharmacological results suggest that autophagy might be a cytoprotective mechanism against apoptosis during cisplatin treatment. To further determine the role of autophagy in cell death or survival during cisplatin treatment, we used short-hairpin RNA (shRNA) to knock down Beclin-1, a critical protein involved in initial autophagic vesicle formation.5.Liang X.H. Jackson S. Seaman M. et al.Induction of autophagy and inhibition of tumorigenesis by beclin 1.Nature. 1999; 402: 672-676Crossref PubMed Scopus (2533) Google Scholar,42.Maiuri M.C. Zalckvar E. Kimchi A. et al.Self-eating and self-killing: crosstalk between autophagy and apoptosis.Nat Rev Mol Cell Biol. 2007; 8: 741-752Crossref PubMed Scopus (2533) Google Scholar The transfection efficacy was relatively low for RPTCs; thus, human embryonic kidney (HEK) cells were initially used for the shRNA study. Four Beclin-1 shRNAs and one non-targeting control shRNA was first tested for their effects on Beclin-1 expression. As shown in Figure 8a, both #3 and #4 Beclin shRNA significantly attenuated Beclin-1 expression. We then examined the effects of Beclin shRNA#4 on cisplatin-induced autophagy. As expected, transfection with #4, but not the non-targeting control shRNA, blocked autophagic GFP-LC3 distribution during cisplatin treatment (Figure 8b). Importantly, Beclin shRNA sensitized the cells to cisplatin-induced apoptosis. As shown in Figure 8c, cisplatin induced 20% apoptosis, which was increased to 53% by shRNA knockdown of Beclin-1. As a control, the non-targeting shRNA did not have significant effects. We further examined the effects of Beclin knockdown in RPTCs. The shRNA constructs used in our study also had GFP sequence and expressed the fluorescent protein. Thus, RPTCs were transfected with control or Beclin shRNA and then treated with cisplatin. The transfected cells with GFP were specifically analyzed for apoptosis. The results are shown in Figure 8d. While the group transfected with control shRNA had 32% apoptosis, the cells transfected with Beclin shRNA had 52%. Together with the pharmacological results of 3-MA and BAF, the shRNA study has provided strong evidence that autophagy during cisplatin-induced renal injury is an important mechanism for cytoprotection and cell survival. Autophagy has been intensely studied during the past a few years.1.Klionsky D.J. Autophagy: from phenomenology to molecular understanding in less than a decade.Nat Rev Mol Cell Biol. 2007; 8: 931-937Crossref PubMed Scopus (1405) Google Scholar,2.Levine B. Yuan J. Autophagy in cell death: an innocent convict?.J Clin Invest. 2005; 115: 2679-2688Crossref PubMed Scopus (1402) Google Scholar,3.Suzuki K. Ohsumi Y. Molecular machinery of autophagosome formation in yeast, Saccharomyces cerevisiae.FEBS Lett. 2007; 581: 2156-2161Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar,4.Shintani T. Klionsky D.J. Autophagy in health and disease: a double-edged sword.Science. 2004; 306: 9
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