Autophagy in proximal tubules protects against acute kidney injury
2012; Elsevier BV; Volume: 82; Issue: 12 Linguagem: Inglês
10.1038/ki.2012.261
ISSN1523-1755
AutoresMan Jiang, Qingqing Wei, Guie Dong, Masaaki Komatsu, Yunchao Su, Zheng Dong,
Tópico(s)Acute Kidney Injury Research
ResumoAutophagy is induced in renal tubular cells during acute kidney injury; however, whether this is protective or injurious remains controversial. We address this question by pharmacologic and genetic blockade of autophagy using mouse models of cisplatin- and ischemia–reperfusion-induced acute kidney injury. Chloroquine, a pharmacological inhibitor of autophagy, blocked autophagic flux and enhanced acute kidney injury in both models. Rapamycin, however, activated autophagy and protected against cisplatin-induced acute kidney injury. We also established a renal proximal tubule–specific autophagy-related gene 7–knockout mouse model shown to be defective in both basal and cisplatin-induced autophagy in kidneys. Compared with wild-type littermates, these knockout mice were markedly more sensitive to cisplatin-induced acute kidney injury as indicated by renal functional loss, tissue damage, and apoptosis. Mechanistically, these knockout mice had heightened activation of p53 and c-Jun N terminal kinase, the signaling pathways contributing to cisplatin acute kidney injury. Proximal tubular cells isolated from the knockout mice were more sensitive to cisplatin-induced apoptosis than cells from wild-type mice. In addition, the knockout mice were more sensitive to renal ischemia–reperfusion injury than their wild-type littermates. Thus, our results establish a renoprotective role of tubular cell autophagy in acute kidney injury where it may interfere with cell killing mechanisms. Autophagy is induced in renal tubular cells during acute kidney injury; however, whether this is protective or injurious remains controversial. We address this question by pharmacologic and genetic blockade of autophagy using mouse models of cisplatin- and ischemia–reperfusion-induced acute kidney injury. Chloroquine, a pharmacological inhibitor of autophagy, blocked autophagic flux and enhanced acute kidney injury in both models. Rapamycin, however, activated autophagy and protected against cisplatin-induced acute kidney injury. We also established a renal proximal tubule–specific autophagy-related gene 7–knockout mouse model shown to be defective in both basal and cisplatin-induced autophagy in kidneys. Compared with wild-type littermates, these knockout mice were markedly more sensitive to cisplatin-induced acute kidney injury as indicated by renal functional loss, tissue damage, and apoptosis. Mechanistically, these knockout mice had heightened activation of p53 and c-Jun N terminal kinase, the signaling pathways contributing to cisplatin acute kidney injury. Proximal tubular cells isolated from the knockout mice were more sensitive to cisplatin-induced apoptosis than cells from wild-type mice. In addition, the knockout mice were more sensitive to renal ischemia–reperfusion injury than their wild-type littermates. Thus, our results establish a renoprotective role of tubular cell autophagy in acute kidney injury where it may interfere with cell killing mechanisms. Acute kidney injury (AKI) induced by renal ischemia–reperfusion, sepsis, and nephrotoxins is a major kidney disease characterized by rapid loss of renal function, resulting in accumulation of metabolic wastes and imbalance of electrolytes and body fluid. Despite advances in basic research and medical care during the past several decades, clinical outcomes of AKI remain poor, with steadily increasing incidence, unacceptably high mortality, and unsatisfactory therapeutic approaches.1.Waikar S.S. Curhan G.C. Wald R. et al.Declining mortality in patients with acute renal failure, 1988 to 2002.J Am Soc Nephrol. 2006; 17: 1143-1150Crossref PubMed Scopus (468) Google Scholar, 2.Xue J.L. Daniels F. Star R.A. et al.Incidence and mortality of acute renal failure in Medicare beneficiaries, 1992 to 2001.J Am Soc Nephrol. 2006; 17: 1135-1142Crossref PubMed Scopus (621) Google Scholar The pathogenesis of AKI is multiphasic and multifactorial, involving a complex interplay between renal tubules, vasculature, and inflammatory factors.3.Bonventre J.V. Yang L. Cellular pathophysiology of ischemic acute kidney injury.J Clin Invest. 2011; 121: 4210-4221Crossref PubMed Scopus (1326) Google Scholar, 4.Pabla N. Dong Z. Cisplatin nephrotoxicity: mechanisms and renoprotective strategies.Kidney Int. 2008; 73: 994-1007Abstract Full Text Full Text PDF PubMed Scopus (1345) Google Scholar, 5.Price P.M. Safirstein R.L. Megyesi J. The cell cycle and acute kidney injury.Kidney Int. 2009; 76: 604-613Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 6.Sharfuddin A.A. Molitoris B.A. Pathophysiology of ischemic acute kidney injury.Nat Rev Nephrol. 2011; 7: 189-200Crossref PubMed Scopus (525) Google Scholar Pathologically, AKI is characterized by sublethal and lethal damage of renal tubules, resulting in tubular dysfunction and cell death in the forms of necrosis and apoptosis. In this regard, the tubular segments located within the outer stripe of out medulla, including proximal tubules, are especially susceptible to injury.3.Bonventre J.V. Yang L. Cellular pathophysiology of ischemic acute kidney injury.J Clin Invest. 2011; 121: 4210-4221Crossref PubMed Scopus (1326) Google Scholar, 4.Pabla N. Dong Z. Cisplatin nephrotoxicity: mechanisms and renoprotective strategies.Kidney Int. 2008; 73: 994-1007Abstract Full Text Full Text PDF PubMed Scopus (1345) Google Scholar, 5.Price P.M. Safirstein R.L. Megyesi J. 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Molecular chaperones in the kidney.Annu Rev Physiol. 2002; 64: 503-527Crossref PubMed Scopus (48) Google Scholar p21, a conventional cell cycle regulator, is upregulated in AKI to antagonize the cell death–promoting action of cyclin-dependent kinase 2.5.Price P.M. Safirstein R.L. Megyesi J. The cell cycle and acute kidney injury.Kidney Int. 2009; 76: 604-613Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar More recently, macroautophagy (referred to as autophagy hereafter) has been demonstrated as a stress response of renal tubular cells to acute injury in vitro and in vivo. Whether autophagy is renoprotective or injurious remains very controversial.10.Lieberthal W. Macroautophagy: a mechanism for mediating cell death or for promoting cell survival?.Kidney Int. 2008; 74: 555-557Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 11.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-F256Crossref PubMed Scopus (126) Google Scholar Autophagy is a cellular process of ‘self-eating’ whereby cytoplasmic components are sequestered into autophagic vesicles or vacuoles (called autophagosomes) and then delivered to lysosomes for degradation.12.Levine B. Klionsky D.J. 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, 13.Mizushima N. Yoshimori T. Ohsumi Y. The role of atg proteins in autophagosome formation.Annu Rev Cell Dev Biol. 2011; 27: 107-132Crossref PubMed Scopus (2153) Google Scholar Recent research has delineated an evolutionarily conserved molecular machinery of autophagy, which includes a specific class of genes or proteins called autophagy-related genes, Atgs.12.Levine B. Klionsky D.J. 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, 13.Mizushima N. Yoshimori T. Ohsumi Y. The role of atg proteins in autophagosome formation.Annu Rev Cell Dev Biol. 2011; 27: 107-132Crossref PubMed Scopus (2153) Google Scholar Atgs interact with each other and other regulatory proteins to form various protein complexes for the initiation, expansion, and final maturation of autophagosomes. Deficiency of a specific Atg leads to inhibition of relevant autophagic events. For example, Atg7 is a critical regulator of the Atg5–Atg12 and Atg8–phosphotidylethanolamine conjugation systems in autophagy. Accordingly, these conjugations are attenuated in Atg7-null cells and tissues, resulting in complete blockade of autophagy.14.Komatsu M. Waguri S. Ueno T. et al.Impairment of starvation-induced and constitutive autophagy in Atg7-deficient mice.J Cell Biol. 2005; 169: 425-434Crossref PubMed Scopus (1911) Google Scholar Functionally, basal or physiological autophagy contributes to the maintenance of cellular homeostasis and quality control of proteins and subcellular organelles. In pathological conditions or cell stress, autophagy is induced and may serve as an adaptive and protective mechanism for cell survival; however, dysregulated autophagy may also contribute to cell death.15.Baehrecke E.H. Autophagy: dual roles in life and death?.Nat Rev. 2005; 6: 505-510Crossref Scopus (842) Google Scholar, 16.Maiuri M.C. Zalckvar E. Kimchi A. et al.Self-eating and self-killing: crosstalk between autophagy and apoptosis.Nat Rev. 2007; 8: 741-752Crossref Scopus (2821) Google Scholar As a result, autophagy and its deregulation has been implicated in the pathogenesis of a variety of diseases such as cancer, neurodegeneration, and heart failure.17.Mizushima N. Levine B. Cuervo A.M. et al.Autophagy fights disease through cellular self-digestion.Nature. 2008; 451: 1069-1075Crossref PubMed Scopus (5144) Google Scholar In kidneys, autophagy is essential to the homeostasis and physiological function of podocytes.18.Hartleben B. Godel M. Meyer-Schwesinger C. et al.Autophagy influences glomerular disease susceptibility and maintains podocyte homeostasis in aging mice.J Clin Invest. 2010; 120: 1084-1096Crossref PubMed Scopus (538) Google Scholar Notably, autophagy induction has been demonstrated in renal tubular cells in experimental models of AKI induced by ischemia–reperfusion and nephrotoxicants such as cisplatin and cyclosporine.19.Chien C.T. Shyue S.K. Lai M.K. 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, 20.Suzuki C. Isaka Y. Takabatake Y. et al.Participation of autophagy in renal ischemia/reperfusion injury.Biochem Biophys Res Commun. 2008; 368: 100-106Crossref PubMed Scopus (153) Google Scholar, 21.Inoue K. Kuwana H. Shimamura Y. et al.Cisplatin-induced macroautophagy occurs prior to apoptosis in proximal tubules in vivo.Clin Exp Nephrol. 2010; 14: 112-122Crossref PubMed Scopus (75) Google Scholar, 22.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: 1181-1192Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar, 23.Pallet N. Bouvier N. Legendre C. et al.Autophagy protects renal tubular cells against cyclosporine toxicity.Autophagy. 2008; 4: 783-791Crossref PubMed Scopus (138) Google Scholar, 24.Periyasamy-Thandavan S. Jiang M. Wei Q. et al.Autophagy is cytoprotective during cisplatin injury of renal proximal tubular cells.Kidney Int. 2008; 74: 631-640Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar, 25.Yang C. Kaushal V. Shah S.V. et al.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, 26.Bolisetty S. Traylor A.M. Kim J. et al.Heme oxygenase-1 inhibits renal tubular macroautophagy in acute kidney injury.J Am Soc Nephrol. 2011; 21: 1702-1712Crossref Scopus (136) Google Scholar Tubular autophagy was also detected following unilateral ureteral obstruction in mice.27.Li L. Zepeda-Orozco D. Black R. et al.Autophagy is a component of epithelial cell fate in obstructive uropathy.Am J Pathol. 2010; 176: 1767-1778Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar Despite these reports, the mechanism of autophagy induction during AKI is unclear. Moreover, the role of autophagy in the pathogenesis of AKI remains to be debated. Although Chien et al.,19.Chien C.T. Shyue S.K. Lai M.K. 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 Suzuki et al.,20.Suzuki C. Isaka Y. Takabatake Y. et al.Participation of autophagy in renal ischemia/reperfusion injury.Biochem Biophys Res Commun. 2008; 368: 100-106Crossref PubMed Scopus (153) Google Scholar and Inoue et al.,21.Inoue K. Kuwana H. Shimamura Y. et al.Cisplatin-induced macroautophagy occurs prior to apoptosis in proximal tubules in vivo.Clin Exp Nephrol. 2010; 14: 112-122Crossref PubMed Scopus (75) Google Scholar suggested a role of autophagy in tubular cell death,5.Price P.M. Safirstein R.L. Megyesi J. The cell cycle and acute kidney injury.Kidney Int. 2009; 76: 604-613Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar we and others provided evidence for autophagy as a prosurvival, renoprotective mechanism in AKI.22.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: 1181-1192Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar, 23.Pallet N. Bouvier N. Legendre C. et al.Autophagy protects renal tubular cells against cyclosporine toxicity.Autophagy. 2008; 4: 783-791Crossref PubMed Scopus (138) Google Scholar, 24.Periyasamy-Thandavan S. Jiang M. Wei Q. et al.Autophagy is cytoprotective during cisplatin injury of renal proximal tubular cells.Kidney Int. 2008; 74: 631-640Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar, 25.Yang C. Kaushal V. Shah S.V. et al.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 Although the cause of the discrepancy is currently unknown, those studies concluded mainly based on the use of pharmacological inhibitors of autophagy. Nevertheless, the latest work by Kimura et al.28.Kimura T. Takabatake Y. Takahashi A. et al.Autophagy protects the proximal tubule from degeneration and acute ischemic injury.J Am Soc Nephrol. 2011; 22: 902-913Crossref PubMed Scopus (343) Google Scholar demonstrated heightened renal ischemia–reperfusion injury in proximal tubule–specific Atg5-knockout mice, providing the first genetic evidence for a renoprotective role in an AKI model. In the present study, we first examined the effect of chloroquine, a pharmacological inhibitor of autophagy, on cisplatin-induced AKI. We further established a renal proximal tubule–specific Atg7-knockout mouse model and demonstrated the high sensitivity of these mice to both cisplatin-induced AKI and renal ischemia–reperfusion-induced AKI. Mechanistically, Atg7-knockout kidney tissues showed heightened activation of p53 and c-Jun N-terminal kinase (JNK) during cisplatin treatment, the signaling pathways contributing to tubular cell death and AKI. In contrast to the aggravating effects of autophagy inhibition, activation of autophagy by rapamycin significantly attenuated cisplatin-induced renal injury. Together, these studies establish a renoprotective role of tubular cell autophagy in AKI. We first confirmed the occurrence of autophagy in proximal tubular cells during cisplatin nephrotoxicity in C57BL/6 mice. As shown by immunoblot analysis of LC3 in Figure 1a, cisplatin led to a notable increase of LC3-II in renal cortical and outer medulla tissues, particularly at days 2 and 3. The LC3-II accumulation went down at day 4, possibly due to massive lysosomal degradation of the proteins in autophagosomes (autophagic flux). Morphologically, the formation of autophagosomes in kidneys was visualized by immunofluorescence staining of LC3. In control tissues, LC3 was diffusely distributed throughout the cells without punctated staining. Upon cisplatin treatment, intense dot-like LC3 staining puncta appeared in some renal tubular cells of renal cortex and outer medulla, indicating the formation of autophagosomes (Figure 1b, LC3). The localization of LC3 puncta was further determined by costaining with either fluorescein isothiocyanate (FITC)–labeled phaseolus vulgaris agglutinin (PHA) or peanut agglutinin, the two lectins that bind proximal and distal tubular cells, respectively.29.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 (188) Google Scholar Most of the LC3 puncta colocalized with the FITC-PHA-positive renal tubules, suggesting that autophagy was induced mainly in proximal tubular cells by cisplatin (Figure 1b, FITC-PHA and merge). Quantitatively, on average, approximately three LC3 dots per proximal tubule were found in the cisplatin-treated mice, whereas no LC3 dot was observed in the control mice (Figure 1c). By electron microscopy, our previous work detected the formation of autophagic vacuoles in proximal tubule cells during cisplatin-induced AKI in mice. Together, these results demonstrate compelling evidence for the occurrence of autophagy in this AKI model. The role of autophagy in the pathogenesis of AKI remains controversial.19.Chien C.T. Shyue S.K. Lai M.K. 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, 20.Suzuki C. Isaka Y. Takabatake Y. et al.Participation of autophagy in renal ischemia/reperfusion injury.Biochem Biophys Res Commun. 2008; 368: 100-106Crossref PubMed Scopus (153) Google Scholar, 21.Inoue K. Kuwana H. Shimamura Y. et al.Cisplatin-induced macroautophagy occurs prior to apoptosis in proximal tubules in vivo.Clin Exp Nephrol. 2010; 14: 112-122Crossref PubMed Scopus (75) Google Scholar, 22.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: 1181-1192Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar, 23.Pallet N. Bouvier N. Legendre C. et al.Autophagy protects renal tubular cells against cyclosporine toxicity.Autophagy. 2008; 4: 783-791Crossref PubMed Scopus (138) Google Scholar, 24.Periyasamy-Thandavan S. Jiang M. Wei Q. et al.Autophagy is cytoprotective during cisplatin injury of renal proximal tubular cells.Kidney Int. 2008; 74: 631-640Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar, 25.Yang C. Kaushal V. Shah S.V. et al.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 To address this question, we initially tested the effects of chloroquine, a pharmacological inhibitor of autophagy, on cisplatin-induced AKI in C57BL/6 mice. Suppression of autophagic flux by chloroquine was first confirmed by immunoblot analysis of LC3 and p62. As shown in Figure 2a and b, at day 4 of cisplatin treatment, LC3-II induction was not as obvious as the earlier time points of days 2 and 3, probably owing to autolysosomal degradation. Chloroquine blocked the lysosomal degradation of LC3 in autophagosomes, leading to a marked accumulation of LC3-II following cisplatin treatment. As a selective substrate of autophagy, p62 degradation was also inhibited by chloroquine (Figure 2a and b), further confirming the inhibitory effect of chloroquine on autophagy in kidney tissues. We then examined cisplatin-induced AKI in the absence or presence of chloroquine. Cisplatin at 25mg/kg induced moderate AKI within 3 days as indicated by blood urea nitrogen (BUN) measurement, which was not affected by chloroquine. At day 4 of cisplatin treatment, BUN level increased to 90mg/dl, which was aggravated to 181mg/dl by chloroquine (Figure 2c). Similarly, serum creatinine was increased from 0.84 to 1.54mg/dl by chloroquine at day 4 of cisplatin treatment (Figure 2d). Consistent with the functional data, cisplatin-induced tissue damage in renal cortex and outer medulla was aggravated by chloroquine (Figure 2e). Cisplatin treatment led to the loss of brush border, tubular dilation and distortion, and cell lysis in some proximal tubules. In the presence of chloroquine, the majority of the tubules were injured and the severity of the injury was also increased, showing tubular disruption and massive tubular lysis with sloughed debris in the lumen. Semiquantification confirmed that chloroquine significantly increased kidney tissue damage during cisplatin treatment (Figure 2f: 1.3 for cisplatin vs. 2.9 for cisplatin+chloroquine). Further examination of renal tissues by TUNEL (TdT-mediated dUTP nick-end labeling) assay showed that cisplatin-induced tubular cell apoptosis was increased by chloroquine (Figure 2g and h). As a control, chloroquine alone did not induce kidney injury in C57BL/6 mice (data not shown). Collectively, these results demonstrate the inhibitory effect of chloroquine on cisplatin-induced autophagy and the aggravating effect of chloroquine on AKI, suggesting a renoprotective role of autophagy in this disease model. To further define the role of tubular cell autophagy in AKI, we established a conditional knockout mouse model, in which Atg7 was deleted specifically from renal proximal tubules in kidneys. Atg7 is critical for the conjugation events in autophagy.30.Ohsumi Y. Mizushima N. Two ubiquitin-like conjugation systems essential for autophagy.Semin Cell Dev Biol. 2004; 15: 231-236Crossref PubMed Scopus (256) Google Scholar Mice bearing the floxed Atg7 alleles (Atg7flox/flox) were generated by inserting two loxP sites around exon 14 that encodes the active site cysteine essential for activation of the conjugation substrates.14.Komatsu M. Waguri S. Ueno T. et al.Impairment of starvation-induced and constitutive autophagy in Atg7-deficient mice.J Cell Biol. 2005; 169: 425-434Crossref PubMed Scopus (1911) Google Scholar Atg7flox/flox mice were bred to transgenic mice expressing the Cre recombinase under the control of a modified PEPCK promoter (PEPCK-Cre) that directs Cre expression predominantly in kidney proximal tubular cells and marginally in hepatocytes.31.Rankin E.B. Tomaszewski J.E. Haase V.H. Renal cyst development in mice with conditional inactivation of the von Hippel-Lindau tumor suppressor.Cancer Res. 2006; 66: 2576-2583Crossref PubMed Scopus (285) Google Scholar After the first round of breeding, heterozygous female progenies (Atg7flox/+XcreX) were obtained and then crossed with Atg7flox/flox males to generate Atg7flox/floxXcreY mice with PEPCK-Cre-mediated Atg7 deletion from renal proximal tubules (PT-Atg7-KO, Figure 3a). As PEPCK-Cre is linked to X chromosome,31.Rankin E.B. Tomaszewski J.E. Haase V.H. Renal cyst development in mice with conditional inactivation of the von Hippel-Lindau tumor suppressor.Cancer Res. 2006; 66: 2576-2583Crossref PubMed Scopus (285) Google Scholar we only used male mice for the study to ensure correct genotypes. To verify the genotypes, three sets of PCR were conducted for the genomic DNA sample of each mouse. The genotype of PT-Atg7-KO mice was indicated by the amplification of the ∼500-bp fragment of the floxed allele, the nonamplification of the ∼600-bp fragment of the wild-type allele, and the amplification of the ∼370-bp fragment of the Cre gene (Figure 3b, lanes 3 and 5). The absence of the Cre gene ensured the genotype of wild-type (PT-Atg7-WT) mice (Figure 3b, lanes 1, 2, 4, and 6). By immunoblot analysis of the tissues from kidney cortex and outer medulla that consist mainly of proximal tubules, we confirmed that Atg7 expression was indeed reduced in PT-Atg7-KO mice, compared with their wild-type littermates (Figure 3c for littermate tissue comparison: lanes 2 vs. 1, 4 vs. 3, and 6 vs. 5). We further verified the PT-Atg7-KO model functionally. To this end, we first analyzed the Atg7-mediated autophagic conjugations in collected kidney tissues. In wild-type tissues, LC3-II, the phosphatidylethanolamine-conjugated form of LC3, was mainly detected (Figure 3c, lanes 1, 3, and 5); however, in PT-Atg7-KO tissues, large amount of LC3-I accumulated, whereas LC3-II was barely detectable (Figure 3c, lanes 2, 4, and 6). Furthermore, wild-type kidney tissues showed high levels of Atg5–Atg12 conjugation, which was markedly reduced in PT-Atg7-KO tissues (Figure 3c, lanes 1, 3, and 5 vs. lanes 2, 4, and 6). In addition, we examined kidney tissues for the expression and localization of p62, a selective substrate of autophagy. Immunoblot analysis demonstrated markedly higher p62 in PT-Atg7-KO kidney tissues than wild type (Figure 3c, lanes 2, 4, and 6 vs. lanes 1, 3, and 5). By immunohistochemistry, positive p62 staining was detected in PT-Atg7-KO kidney tissues, and not in wild-type tissues (Figure 3d). Notably, p62 staining in PT-Atg7-KO tissues appeared as unevenly distributed cytoplasmic puncta of various sizes, characteristic of the inclusion bodies seen in autophagy-deficient cells.32.Komatsu M. Waguri S. Koike M. et al.Homeostatic levels of p62 control cytoplasmic inclusion body formation in autophagy-deficient mice.Cell. 2007; 131: 1149-1163Abstract Full Text Full Text PDF PubMed Scopus (1709) Google Scholar By morphology, the p62 staining appeared exclusively in proximal tubules. It is noteworthy that p62 staining did not show a general increase in all proximal tubular cells, suggesting that the basal level of autophagy varies in different tubular cells. Together, these studies validate the PT-Atg7-KO model, in which Atg7 is specifically deleted from proximal tubular cells in kidneys. After verifying the autophagic defects in proximal tubules of PT-Atg7-KO mice under unchallenged conditions, we further examined autophagy during cisplatin treatment. As shown in Figure 4a, in response to cisplatin treatment, wild-type kidney tissues showed LC3-II accumulation, whereas the conversion of LC3-I to LC3-II was completely abrogated in PT-Atg7-KO kidneys. Atg5–Atg12 conjugation was also reduced in PT-Atg7-KO tissues. Consistent with Figure 3, the basal level of p62 was significantly higher in PT-Atg7-KO tissues than wild-type tissues, and following cisplatin treatment p62 increased markedly (Figure 4a, PT-Atg7-KO). The effect of Atg7 deficiency on cisplatin-induced autophagy in proximal tubules was then determined morphologically by coimmunostaining of LC3 and FITC-labeled PHA. As shown in Figure 4b, punctuate or granular LC3 staining was detected in some proximal tubular cells of wild-type kidneys during cisplatin treatment, which was barely seen in PT-Atg7-KO tissues. The number of LC3 puncta per proximal tubule was significantly reduced from 3.2 in wild-type mice to 0.1 in PT-Atg7-KO tissues (Figure 4c). By immunohistochemistry, we found that, compared with the control, cisplatin induced massive p62 accumulation in proximal tubules in PT-Atg7-KO mice, as indicated by remarkably increased amount and size of cytoplasmic p62 inclusion bodies (Figure 4d, high-magnification inserts). In wild-type tissues, there was an increase in p62 staining in some proximal tubular cells after cisplatin treatment, but in general the staining was diffuse and not indicative of inclusion bodies (Figure 4d). Together, these results suggest that cisplatin-induced autophagy is suppressed in renal proximal tubular cells in PT-Atg7-KO mice. By using the PT-Atg7-KO model, we then determined the role of autophagy on cisplatin-induced AKI. Without treatment, both PT-Atg7-KO mice and their wild-type littermates showed similarly low levels of BUN and serum creatinine, indicating normal renal function. At 4 days after cisplatin injection, wild-type mice developed moderate renal failure, with BUN and serum creatinine levels increased to 151 and 0.74mg/dl, respectively. In the same experiments, PT-Atg7-KO mice had more severe loss of renal function, showing BUN level of 198mg/dl and serum creatinine level of 1.21mg/dl (Figure 5a and b). Notably, the progression of AKI was accelerated thereafter in PT-Atg7-KO mice. At 5 days after cisplatin treatment, these mice had 351mg/dl BUN and 3.24mg/dl serum creatinine levels, whereas their wild-type littermates had 244mg/dl BUN and 1.15mg/dl serum creatinine levels (Figure 5a and b). Histological analysis confirmed that, compared with wild type, cisplatin induced much more severe kidney tissue damage in PT-Atg7-KO mice, which showed widespread, extensively damaged proximal tubules in the renal cortex and outer medulla (Figure 5c). These tissues had a tubular damage score of 2.2, whereas the score was 1.3 for wild-type tissues (Figure 5d). Further examination by TUNEL assay indicated that cisplatin induced significantly more apoptosis in kidney tissues of PT-Atg7-KO mice than wild-type littermates (Figure 5e and f). Together with t
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