The Absence of Interleukin-6 Enhanced Arsenite-Induced Renal Injury by Promoting Autophagy of Tubular Epithelial Cells with Aberrant Extracellular Signal-Regulated Kinase Activation
2009; Elsevier BV; Volume: 176; Issue: 1 Linguagem: Inglês
10.2353/ajpath.2010.090146
ISSN1525-2191
AutoresAkihiko Kimura, Yuko Ishida, Takashi Wada, Tomoko Hisaoka, Yoshihiro Morikawa, Takeshi Sugaya, Naofumi Mukaida, Toshikazu Kondo,
Tópico(s)Paraquat toxicity studies and treatments
ResumoSodium arsenite (NaAs)-induced autophagic cell death (ACD) of a mouse renal tubular epithelial cell line (mProx24), which expresses enhanced levels of interleukin-6 (IL-6), was reduced by the suppression of autophagy by 3-methyladenine or Atg7 knockdown. The inhibition of the IL-6/signal transducer and activator of transcription 3 (STAT3) signal pathway by anti-IL-6 antibody or a Jak2 inhibitor (AG490) exaggerated ACD of mProx24 cells after NaAs challenge, attenuating STAT3 activation and reciprocally enhancing extracellular signal-regulated kinase (ERK) phosphorylation. In contrast, an ERK inhibitor, PD98059, reduced NaAs-induced ACD in mProx24 cells. Subcutaneous injection of NaAs (12.5 mg/kg) into BALB/c (wild-type) mice enhanced intrarenal expression of IL-6, mainly produced by tubular cells, and caused severe renal injury characterized by hemorrhages, acute tubular necrosis, cast formation, and brush border disappearance, with increases in serum urea nitrogen (blood urea nitrogen) and creatinine levels. In addition, IL-6-deficient (IL-6−/−) mice exhibited exaggerated histopathological changes with higher blood urea nitrogen and creatinine levels. Moreover, in IL-6−/− mice treated with NaAs, ACD in renal tubular cells was significantly augmented, along with diminished STAT3 activation and reciprocal enhancement of ERK signaling, compared with wild-type mice. Finally, the administration of exogenous IL-6 into wild-type mice significantly reduced NaAs-induced ACD along with diminished ERK activation and eventually alleviated acute renal dysfunction. Thus, IL-6/STAT3 signal pathway could inhibit ERK activation, a crucial step for ACD, eventually attenuating NaAs-induced renal dysfunction. Sodium arsenite (NaAs)-induced autophagic cell death (ACD) of a mouse renal tubular epithelial cell line (mProx24), which expresses enhanced levels of interleukin-6 (IL-6), was reduced by the suppression of autophagy by 3-methyladenine or Atg7 knockdown. The inhibition of the IL-6/signal transducer and activator of transcription 3 (STAT3) signal pathway by anti-IL-6 antibody or a Jak2 inhibitor (AG490) exaggerated ACD of mProx24 cells after NaAs challenge, attenuating STAT3 activation and reciprocally enhancing extracellular signal-regulated kinase (ERK) phosphorylation. In contrast, an ERK inhibitor, PD98059, reduced NaAs-induced ACD in mProx24 cells. Subcutaneous injection of NaAs (12.5 mg/kg) into BALB/c (wild-type) mice enhanced intrarenal expression of IL-6, mainly produced by tubular cells, and caused severe renal injury characterized by hemorrhages, acute tubular necrosis, cast formation, and brush border disappearance, with increases in serum urea nitrogen (blood urea nitrogen) and creatinine levels. In addition, IL-6-deficient (IL-6−/−) mice exhibited exaggerated histopathological changes with higher blood urea nitrogen and creatinine levels. Moreover, in IL-6−/− mice treated with NaAs, ACD in renal tubular cells was significantly augmented, along with diminished STAT3 activation and reciprocal enhancement of ERK signaling, compared with wild-type mice. Finally, the administration of exogenous IL-6 into wild-type mice significantly reduced NaAs-induced ACD along with diminished ERK activation and eventually alleviated acute renal dysfunction. Thus, IL-6/STAT3 signal pathway could inhibit ERK activation, a crucial step for ACD, eventually attenuating NaAs-induced renal dysfunction. Arsenic is ubiquitously distributed in the natural environment such as soil, water, and air and is commonly associated with the ores of metals like copper, lead, and gold.1Oremland RS Stolz JF The ecology of arsenic.Science. 2003; 300: 939-944Crossref PubMed Scopus (1217) Google Scholar Acute arsenic exposure can cause a profound injury to various organs, including kidney, liver, intestine, and brain, and can result in high mortality and morbidity,2Liu J Liu YP Goyer RA Chanzar W Waalkes MP Metallothionein-I/II null mice are more sensitive than wild-type mice to the hepatotoxic and nephrotoxic effects of oral or injected inorganic arsenicals.Toxicol Sci. 2000; 55: 460-467Crossref PubMed Scopus (144) Google Scholar and massive renal tubular necrosis is a characteristic pathological feature of renal injury caused by acute arsenic exposure.3Kimura A Ishida Y Hayashi T Wada T Yokoyama H Sugaya T Mukaida N Kondo T Interferon-γ plays protective roles in sodium arsenite-induced renal injury by up-regulating intrarenal multidrug resistance-associated protein 1 expression.Am J Pathol. 2006; 169: 1118-1128Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 4Kimura A Ishida Y Wada T Yokoyama H Mukaida N Kondo T MRP-1 expression levels determine strain-specific susceptibility to sodium arsenic-induced renal injury between C57BL/6 and BALB/c mice.Toxicol Appl Pharmacol. 2005; 203: 53-61Crossref PubMed Scopus (35) Google Scholar Moreover, the environmental pollution of arsenic sometimes causes serious health problems in several developing countries, because chronic exposure to arsenic results in the dysfunctions in renal and nervous systems5Snow E Metal carcinogenesis: mechanistic implications.Pharmacol Ther. 1992; 53: 31-65Crossref PubMed Scopus (334) Google Scholar, 6Thompson DJ A chemical hypothesis for arsenic methylation in mammals.Chem Biol Interact. 1993; 88: 89-114Crossref PubMed Scopus (198) Google Scholar and often acts as carcinogen of skin, lung, bladder, liver, and kidney.7Goering PL Aposhian HV Mass MJ Cebrian M Beck BD Waalkes MP The enigma of arsenic carcinogenesis: role of metabolism.Toxicol Sci. 1999; 49: 5-14Crossref PubMed Scopus (225) Google Scholar, 8Abernathy CO Liu Y-P Longfellow D Aposhian HV Beck B Fowler B Goyer R Menzer R Rossman T Thompson C Waalkes MP Arsenic: health effects, mechanisms of actions and research issues.Environ Health Perspect. 1999; 107: 593-597Crossref PubMed Scopus (527) Google Scholar On the other hand, arsenic trioxide (As2O3) has recently been shown to be effective for acute promyelocytic leukemia without causing bone marrow (BM) suppression,9Chen GQ Shi XG Tang W Xiong SM Zhu J Cai X Han ZG Ni JH Shi GY Jia PM Liu MM He KL Niu C Ma J Zhang TD Paul P Naoe T Kitamura K Miller W Waxman S Wang ZY Chen SJ Chen Z Use of arsenic trioxide (As2O3) in the treatment of acute promyelocytic leukaemia (APL): As2O3 exerts dose-dependent dual effects on APL cells.Blood. 1997; 89: 3345-3353Crossref PubMed Google Scholar, 10Shen ZX Chen GQ Ni JH Li XS Xiong SM Qiu QY Zhu J Tang W Sun GL Yang KQ Chen Y Zhou L Fang ZW Wang YT Ma J Zhang P Zhang TD Chen SJ Chen Z Wang ZY Use of arsenic trioxide (As2O3) in the treatment of acute promyelocytic leukaemia (APL). 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IL-6 is produced by various types of cells and exhibits various similar activities as tumor necrosis factor-α on a wide variety of cells including lymphocytes, hepatocytes, and neuronal cells.27Akira S Kishimoto T IL-6 and NF-IL6 in acute-phase response and viral infection.Immunol Rev. 1992; 127: 25-50Crossref PubMed Scopus (468) Google Scholar However, it remains to be investigated on the effects of IL-6 on autophagic cell death. We observed that sodium arsenite (NaAs) exposure caused autophagic cell death as well as IL-6 production in a murine renal tubular epithelial cell line, mProx24. Moreover, tubular cell necrosis due to autophagic cell death was observed in acute NaAs-induced renal injury. These observations prompted us to investigate the roles of IL-6 and its downstream signaling molecules in NaAs-induced autophagic death of renal tubular cells. We demonstrated that NaAs-induced autophagic cell death of mProx24 cells was augmented by anti-IL-6 antibodies (Abs) and inhibitors of Janus kinase 2 (JAK2) and that the inhibition of extracellular signal-regulated kinase (ERK)1/2 suppressed NaAs-induced autophagic cell death of mProx24 cells. These molecules that are localized downstream the IL-6/signal transducer and activator of transcription 3 (STAT3) pathway. Furthermore, genetic ablation of IL-6 gene and IL-6 administration enhanced and alleviated NaAs-induced acute renal tubular cell necrosis with autophagic cell death, respectively. Thus, IL-6-mediated signals can counteract NaAs-induced acute renal injury characterized by autophagic cell death of renal tubular epithelial cells. NaAs was purchased from Wako Chemical Industries (Osaka, Japan). 3-Methyladenine (3-MA, an inhibitor of autophagy), AG490 (a JAK2 inhibitor), and PD98059 (an ERK1/2 kinase inhibitor) were obtained from Sigma- Aldrich (Tokyo, Japan). E64d and pepstatin A (lysosomal protease inhibitors) were obtained from Calbiochem (San Diego, CA). The following polyclonal Abs (pAbs) were used in this study; goat anti-mouse IL-6 pAbs (R&D Systems, Minneapolis, MN), rabbit anti-LC3 pAbs (Sigma-Aldrich), rabbit anti-STAT3 pAbs, rabbit anti-phospholyrated-STAT3 (p-STAT3) at Tyr705 pAbs, rabbit anti-ERK1/2 pAbs, and rabbit anti-phospholyrated-ERK1/2 (p-ERK1/2) at Thr202/Tyr204 pAbs (Cell Signaling Technology, Danvers, MA). Murine recombinant IL-6 was prepared as described previously.28Yasumoto K Mukaida N Harada A Kuno K Akiyama M Nakashima E Fujioka N Mai Kasahara T Fujimoto-Ouchi K Mori K Tanaka Y Matsushima M Molecular analysis of the cytokine network involved in cachexia in colon 26 adenocarcinoma-bearing mice.Cancer Res. 1995; 55: 921-927PubMed Google Scholar Pathogen-free 8- to 10-week old male BALB/c mice were obtained from Sankyo Laboratories (Tokyo, Japan) and were designated as wild-type mice in the following experiments. Age- and sex-matched IL-6−/− mice, backcrossed to BALB/c mice for more than 10 generations, were used in this study as described previously.29Lin ZQ Kondo T Ishida Y Takayasu T Mukaida N Essential involvement of IL-6 in the skin wound-healing process as evidenced by delayed wound healing in IL-6-deficient mice.J Leukoc Biol. 2003; 73: 713-721Crossref PubMed Scopus (403) Google Scholar All mice were housed individually in cages under the specific pathogen-free conditions during the experiments. All of the experimental procedures were approved by Animal Research Committee of Wakayama Medical University (Wakayama, Japan). NaAs-induced renal injury was induced as described previously.3Kimura A Ishida Y Hayashi T Wada T Yokoyama H Sugaya T Mukaida N Kondo T Interferon-γ plays protective roles in sodium arsenite-induced renal injury by up-regulating intrarenal multidrug resistance-associated protein 1 expression.Am J Pathol. 2006; 169: 1118-1128Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 4Kimura A Ishida Y Wada T Yokoyama H Mukaida N Kondo T MRP-1 expression levels determine strain-specific susceptibility to sodium arsenic-induced renal injury between C57BL/6 and BALB/c mice.Toxicol Appl Pharmacol. 2005; 203: 53-61Crossref PubMed Scopus (35) Google Scholar Briefly, mice were administered s.c. with NaAs (12.5 mg/kg). In other series of experiments, mice received s.c. injection of recombinant IL-6 (0.5 μg/mouse) or vehicle (phosphate-buffered saline) 1 hour after NaAs challenge (13.5 mg/kg) or received i.p. injection of PD98059 (5 mg/kg in 10% DMSO) or vehicle (10% DMSO) immediately after NaAs challenge.30Lee PJ Zhang X Shan P Ma B Lee CG Homer RJ Zhu Z Rincon M Mossman BT Elias JA ERK1/2 mitogen-activated protein kinase selectively mediates IL-13-induced lung inflammation and remodeling in vivo.J Clin Invest. 2006; 116: 163-173Crossref PubMed Scopus (107) Google Scholar BM chimeric mice were prepared as described previously.31Ishida Y Kimura A Kondo T Hayashi T Ueno M Takakura N Matsushima K Mukaida N Essential roles of the CC chemokine ligand 3-CC chemokine receptor 5 axis in bleomycin-induced pulmonary fibrosis through regulation of macrophage and fibrocyte infiltration.Am J Pathol. 2007; 170: 843-854Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar Briefly, IL-6−/− BM to wild-type mice, wild-type BM to IL-6−/− mice, wild-type BM to wild-type mice, and IL-6−/− BM to IL-6−/− mice. BM cells were collected from the femurs of donor mice by aspiration and flushing. Recipient mice were irradiated to 15 Gy using an RX-650 irradiator (Faxitron X-ray, Wheeling, IL). Then, the animals i.v. received 5 × 106 BM cells from donor mice in a volume of 200 μl of sterile PBS under the anesthesia. Thereafter, the mice were housed in sterilized microisolator cages and were fed normal chow and autoclaved hyperchlorinated water for 60 days. Successful engraftment and reconstruction of the BM in the transplanted mice were confirmed by PCR analysis for wild-type or mutant IL-6 gene of peripheral blood of each chimeric mouse 30 days after BMT. After durable BM engraftment was confirmed, mice were treated with NaAs as described above. At the indicated time intervals after NaAs challenge, sera were collected to determine blood urea nitrogen (BUN) and creatinine (CRE) levels with a Fuji DRI-CHEM 5500V (Fuji Medical System, Tokyo, Japan), according to the manufacturer's instructions. Kidney tissues were obtained at the indicated time intervals after NaAs challenge. Paraffin-embedded sections (6 μm thick) were prepared and subjected to H&E staining or PAS staining. The degree of renal damage was scored with examination of hemorrhages, the disappearance of PAS-positive brush border, and cast formation in 10 randomly chosen regions in each sample at a magnification of ×100. Renal morphological alterations were graded on a scale of 0 to 3+: 0, normal; 1+, slight; 2+, moderate; and 3+, severe. The mean of all of the fields was taken as the damage score in each sample. Immunohistochemical analyses for IL-6 or LC3 were performed as described previously.3Kimura A Ishida Y Hayashi T Wada T Yokoyama H Sugaya T Mukaida N Kondo T Interferon-γ plays protective roles in sodium arsenite-induced renal injury by up-regulating intrarenal multidrug resistance-associated protein 1 expression.Am J Pathol. 2006; 169: 1118-1128Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 4Kimura A Ishida Y Wada T Yokoyama H Mukaida N Kondo T MRP-1 expression levels determine strain-specific susceptibility to sodium arsenic-induced renal injury between C57BL/6 and BALB/c mice.Toxicol Appl Pharmacol. 2005; 203: 53-61Crossref PubMed Scopus (35) Google Scholar Histopathological and immunohistochemical evaluation was conducted by an examiner without a prior knowledge on the experimental procedures. Intrarenal arsenic contents were analyzed with the use of certified arsenic standard solution (As 100: Lot. RWN9791; Wako Chemical Industries).3Kimura A Ishida Y Hayashi T Wada T Yokoyama H Sugaya T Mukaida N Kondo T Interferon-γ plays protective roles in sodium arsenite-induced renal injury by up-regulating intrarenal multidrug resistance-associated protein 1 expression.Am J Pathol. 2006; 169: 1118-1128Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 4Kimura A Ishida Y Wada T Yokoyama H Mukaida N Kondo T MRP-1 expression levels determine strain-specific susceptibility to sodium arsenic-induced renal injury between C57BL/6 and BALB/c mice.Toxicol Appl Pharmacol. 2005; 203: 53-61Crossref PubMed Scopus (35) Google Scholar Briefly, a portion of the kidney (∼100 mg) was digested in nitric acid (100 mg/ml). Total arsenic was determined using graphite furnace atomic absorption spectrometry ACF-6800 (Shimadzu, Kyoto, Japan). The data were expressed as arsenic (μg)/sample weight (g). A murine renal proximal tubular epithelial cell line (mProx24), derived from C57BL/6J adult mouse kidney,32Takaya K Koya D Isono M Sugimoto T Sugaya T Kashiwagi A Haneda M Involvement of ERK pathway in albumin-induced MCP-1 expression in mouse proximal tubular cells.Am J Physiol. 2003; 284: F1037-F1045Crossref PubMed Scopus (105) Google Scholar was maintained in Dulbecco's modified Eagle's medium with 10% fetal bovine serum at 37°C in 5% CO2. Then, the cells were incubated for the indicated time intervals in the presence of various combinations of NaAs (20 μmol/L), goat anti-IL-6 pAbs (2 μg/ml), 3-MA (2 mmol/L), AG490 (50 μmol/L), PD98059 (20 μmol/L), E64d (10 μg/ml), and pepstatin A (10 μg/ml). Cell viability was assessed with a Cell Counting Kit (Dojin Laboratories, Kumamoto, Japan) to count living cells with the combination of 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium and 1-methoxyphenazine methosulfate. mProx24 cells at 70% confluence were transfected with EGFP-LC3 plasmid33Kabeya 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 (a gift from Dr. T. Yoshimori, Osaka University, Japan) using Lipofectamine reagent (Invitrogen, Carlsbad, CA), according to the manufacturer's instructions. After 24 hours, cells were treated with NaAs, and the images were observed using a LSM5Pascal Exciter (Carl Zeiss Japan, Tokyo, Japan) laser scanning confocal microscope. Small interfering RNA (siRNA) (Accell SMART pool siRNA Reagent) for Atg7 were purchased from Thermo Fisher Scientific (Yokohama, Japan). Nontargeting siRNA (Accell Non-targting siRNA number 1; Thermo Fisher Scientific) was used as a control. mProx24 cells at ∼30% confluence were transfected with siRNA (1 μmol/L) in Accell delivery media (Thermo Fisher Scientific). After 72 hours, cells were treated with NaAs, followed by cell viability assay or Western blotting analyses. Transmission electron microscopy was performed with some modifications, as described previously.34Matsui T Kinoshita T Morikawa Y Tohya K Katsuki M Ito Y Kamiya A Miyajima A K-Ras mediates cytokine-induced formation of E-cadherin-based adherens junctions during liver development.EMBO J. 2002; 21: 1021-1030Crossref PubMed Scopus (69) Google Scholar Briefly, mProx24 cells were harvested by trypsinization and fixed with 1% glutaraldehyde in 100 mmol/L phosphate buffer (pH 7.4) at 4°C for 1 hour. After washing in 100 mmol/L PBS, the cells were postfixed in 2% osmium tetroxide at 4°C for 1 hour, dehydrated in graded ethanol, and embedded in Quetol-812 resin (Nisshin EM, Tokyo, Japan). Ultrathin sections (70 nm thick) of the cells were stained with uranyl acetate and lead citrate and observed with a transmission electron microscope (JEM-1220; JEOL, Tokyo, Japan). A semiquantitative RT-PCR was conducted as described previously.35Ishida Y Kondo T Ohshima T Fujiwara H Iwakura Y Mukaida N A pivotal involvement of IFN-γ in the pathogenesis of acetaminophen-induced acute liver injury.FASEB J. 2002; 16: 1227-1236Crossref PubMed Scopus (192) Google Scholar Total RNAs were extracted from kidney samples or mProx24 cells using ISOGEN (Nippon Gene, Toyama, Japan). Five micrograms of total RNA was reverse-transcribed at 42°C for 1 hour in 20 μl of reaction mixture containing mouse Moloney leukemia virus reverse transcriptase (Toyobo, Osaka, Japan) and oligo(dT) primers (Amersham Biosciences Japan, Tokyo, Japan). Thereafter, cDNA was amplified together with Taq polymerase (Nippon Gene) using specific primers (Table 1). The PCR products were fractionated on a 2% agarose gel and visualized by ethidium bromide staining. The band intensities were measured using NIH Image Analysis Software version 1.61 (National Institutes of Health, Bethesda, MD) and were calculated as the ratios to β-actin.Table 1Sequences of the Primers Used for RT-PCRTranscriptSequenceAnnealing temperature (°C)CycleProduct size (bp)IL-6(F) 5′-TGGCCAAAGTTCCTGACTTGTTTG-3′6436489(R) 5′-CAGGCTATTTAACCAAGTGGTGCT-3′MRP1(F) 5′-GTTCCCTCCGCATGAACTTG-3′5530550(R) 5′-CTGGCTCATGCCTGGACTCTG-3′MRP2(F) 5′-TGAACTGGCTAGTGAGGA-3′5530379(R) 5′-GTCAGTCTCCCTCGAAGG-3′β-Actin(F) 5′-TTCTACAATGAGCTGCGTGTGGC-3′6228456(R) 5′-CTCATAGCTCTTCTCCAGGGAGGA-3′F, forward primer; R, reverse primer. Open table in a new tab F, forward primer; R, reverse primer. At the indicating time intervals after NaAs challenge, kidney tissues were obtained and homogenized with a lysis buffer (10 mmol/L PBS (pH 7.4) containing 0.01% Triton X-100, 0.5% sodium deoxycholate, and 0.1% SDS) containing complete protease inhibitor mixture, and phosphatase inhibitor mixtures for serine/threonine protein phosphatases and tyrosine protein phosphatases (P2850 and P5726; Sigma-Aldrich) and were centrifuged to obtain lysates. Thereafter, equal amounts of protein were separated on 10 or 15% SDS-polyacrylamide gel electrophoresis and transferred to an Immobilon-P transfer membrane (Millipore, Billerica, MA). After the incubation of the membrane with anti-LC3 pAbs, anti-STAT3 pAbs, anti-p-STAT3 pAbs, anti-ERK1/2 pAbs, p-ERK1/2 pAbs, or anti-β-actin, immune complexes were detected using an enhanced chemiluminescence reagent (Millipore), according to the manufacturer's instructions. In another series of experiments, mProx24 cells were scraped and centrifuged. The cell pellets were dissolved with SDS sample buffer. Thereafter, Western blotting analysis was performed to detect STAT3, p-STAT3, ERK1/2, p-ERK1/2, and anti-β-actin, as mentioned above. The means and SEMs were calculated for all parameters determined in this study. Statistical significance was evaluated using analysis of variance (analysis of variance) or Mann-Whitney U-test. A value of P < 0.05 was accepted as statistically significant. NaAs-induced acute renal injury is characterized by a massive renal tubular cell necrosis.3Kimura A Ishida Y Hayashi T Wada T Yokoyama H Sugaya T Mukaida N Kondo T Interferon-γ plays protective roles in sodium arsenite-induced renal injury by up-regulating intrarenal multidrug resistance-associated protein 1 expression.Am J Pathol. 2006; 169: 1118-1128Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 4Kimura A Ishida Y Wada T Yokoyama H Mukaida N Kondo T MRP-1 expression levels determine strain-specific susceptibility to sodium arsenic-induced renal injury between C57BL/6 and BALB/c mice.Toxicol Appl Pharmacol. 2005; 203: 53-61Crossref PubMed Scopus (35) Google Scholar Because arsenic trioxide can induce autophagic cell death in several tumor cells,18Kanzawa T Kondo Y Ito H Kondo S Germano I Induction of autophagic cell death in malignant glioma cells by arsenic trioxide.Cancer Res. 2003; 63: 2103-2108PubMed Google Scholar, 19Qian W Liu J Jin J Ni W Xu W Arsenic trioxide induces not only apoptosis but also autophagic cell death in leukemia cell lines via up-regulation of Beclin-1.Leuk Res. 2007; 31: 329-339Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar we first examined whether NaAs-induced renal tubular cell necrosis was due to autophagic cell death. LC3 protein induces autophagosome formation through its conversion of LC3-I to LC3-II,33Kabeya 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, 36Tanida I Sou YS Ezaki J Minematsu-Ikeguchi N Ueno T Kominami E HsAtg4B/HsApg4B/autophagin-1 cleaves the carboxyl termini of three human Atg8 homologues and delipidates microtubule-associated protein light chain 3- and GABAA receptor-associated protein-phospholipid conjugates.J Biol Chem. 2004; 279: 36268-36276Crossref PubMed Scopus (282) Google Scholar, 37Aoki H Kondo Y Aldape K Yamamoto A Iwado E Yokoyama T Hollingsworth EF Kobayashi R Hess K Shinojima N Shingu T Tamada Y Zhang L Conrad C Bogler O Mills G Sawaya R Kondo S Monitoring autophagy in glioblastoma with Ab against isoform B of human microtubule-associated protein 1 light chain 3.Autophagy. 2008; 4: 1-9Google Scholar and therefore, the amount of LC3-II is increased as autophagic process proceeds. Hence, we examined LC3 protein in NaAs-treated mProx24 cells as a maker of autophagic cell death. NaAs challenge increased LC3-II amounts (Figure 1, A and B) and decreased the viability of mProx24 cells (Figure 1C). Ultrastructurally, NaAs-treated cells exhibited typical features of autophagic cell death, with the appearance of vacuoles engulfing bulk cytoplasm and cytoplasmic organelles (Figure 1D). An autophagy inhibitor, 3-MA, abrogated NaAs-induced increases in LC3-II and decreases in cell viability (Figure 1, A–C). Similarly, the block of autophagy by the knockdown of Atg7, a key molecul
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