Cytokine cooperation in renal tubular cell injury: The role of TWEAK
2006; Elsevier BV; Volume: 70; Issue: 10 Linguagem: Inglês
10.1038/sj.ki.5001866
ISSN1523-1755
AutoresPilar Justo, Ana B. Sanz, María Dolores Sánchez-Niño, Jeffrey A. Winkles, Corina Lorz, Jesús Egido, Alberto Ortíz,
Tópico(s)Trace Elements in Health
ResumoTumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK, TNFSF12) is a member of the TNF superfamily. TWEAK activates the Fn14 receptor, and may regulate apoptosis, proliferation, and inflammation, processes that play a significant role in pathological conditions. However, there is little information on the function and regulation of this system in the kidney. Therefore, TWEAK and Fn14 expression were studied in cultured murine tubular epithelial MCT cells and in mice in vivo. The effect of TWEAK on cell death was determined. We found that TWEAK and Fn14 expression was increased in experimental acute renal failure induced by folic acid. Cultured tubular cells express both TWEAK and the Fn14 receptor. TWEAK did not induce cell death in non-stimulated tubular cells. However, in cells costimulated with TNFα/interferon-gamma, TWEAK induced apoptosis through the activation of the Fn14 receptor. Apoptosis was associated with activation of caspase-8, caspase-9, and caspase-3, Bid cleavage, and evidence of mitochondrial injury. There was no evidence of endoplasmic reticulum stress. A pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-DL-Asp prevented TWEAK-induced apoptosis, but it sensitized cells to necrosis via generation of reactive oxygen species. In conclusion, cooperation between inflammatory cytokines results in tubular cell death. TWEAK and Fn14 may play a role in renal tubular cell injury. Tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK, TNFSF12) is a member of the TNF superfamily. TWEAK activates the Fn14 receptor, and may regulate apoptosis, proliferation, and inflammation, processes that play a significant role in pathological conditions. However, there is little information on the function and regulation of this system in the kidney. Therefore, TWEAK and Fn14 expression were studied in cultured murine tubular epithelial MCT cells and in mice in vivo. The effect of TWEAK on cell death was determined. We found that TWEAK and Fn14 expression was increased in experimental acute renal failure induced by folic acid. Cultured tubular cells express both TWEAK and the Fn14 receptor. TWEAK did not induce cell death in non-stimulated tubular cells. However, in cells costimulated with TNFα/interferon-gamma, TWEAK induced apoptosis through the activation of the Fn14 receptor. Apoptosis was associated with activation of caspase-8, caspase-9, and caspase-3, Bid cleavage, and evidence of mitochondrial injury. There was no evidence of endoplasmic reticulum stress. A pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-DL-Asp prevented TWEAK-induced apoptosis, but it sensitized cells to necrosis via generation of reactive oxygen species. In conclusion, cooperation between inflammatory cytokines results in tubular cell death. TWEAK and Fn14 may play a role in renal tubular cell injury. Tubular cells compose most of the mass of the functioning kidney. Loss of renal tubular cells characterizes both acute and chronic renal failure.1.Ortiz A. Apoptotic regulatory proteins in renal injury.kidney Int. 2000; 58: 467-485Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar Acute tubular necrosis is the most common form of acute renal failure.2.Liano F. Pascual J. the Madrid Acute Renal Failure Study Group Epidemiology of acute renal failure: a prospective, multicenter, community-based study.Kidney Int. 1996; 50: 811-818Abstract Full Text PDF PubMed Scopus (766) Google Scholar In humans, tubular cell death was the best histopathological correlate of renal dysfunction in acute tubular necrosis.3.Solez K. Morel-Maroger L. Sraer J.D. 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TWEAK, a member of the TNF superfamily, is a multifunctional cytokine that binds the TweakR/Fn14 receptor.Cytokine Growth Factor Rev. 2003; 14: 241-249Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar TWEAK functions as a both type II transmembrane protein and as a cleaved soluble molecule. TWEAK may promote cell death, but it also modulates cell proliferation, inflammation, and angiogenesis.15.Chicheportiche Y. Bourdon P.R. Xu H. et al.TWEAK, a new secreted ligand in the tumor necrosis factor family that weakly induces apoptosis.J Biol Chem. 1997; 272: 32401-32410Crossref PubMed Scopus (535) Google Scholar, 17.Campbell S. Michaelson J. Burkly L. et al.The role of TWEAK/Fn14 IN the pathogenesis of inflammation and systemic autoimmunity.Front Biosci. 2004; 9: 2273-2284Crossref PubMed Scopus (88) Google Scholar, 18.Chicheportiche Y. Chicheportiche R. Sizing I. et al.Proinflammatory activity of TWEAK on human dermal fibroblasts and synoviocytes: blocking and enhancing effects of anti-TWEAK monoclonal antibodies.Arthritis Res. 2002; 4: 126-133Crossref PubMed Scopus (141) Google Scholar, 19.Lynch C.N. Wang Y.C. Lund J.K. et al.TWEAK induces angiogenesis and proliferation of endothelial cells.J Biol Chem. 1999; 274: 8455-8459Crossref PubMed Scopus (210) Google Scholar, 20.Wiley S.R. Cassiano L. Lofton T. et al.A novel TNF receptor family member binds TWEAK and is implicated in angiogenesis.Immunity. 2001; 15: 837-846Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar Fibroblast growth factor-inducible 14 (Fn14) is a cell membrane TWEAK receptor.20.Wiley S.R. Cassiano L. Lofton T. et al.A novel TNF receptor family member binds TWEAK and is implicated in angiogenesis.Immunity. 2001; 15: 837-846Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar, 21.Meighan-Mantha R.L. Hsu D.K. Guo Y. et al.The mitogen-inducible Fn14 gene encodes a type I transmembrane protein that modulates fibroblast adhesion and migration.J Biol Chem. 1999; 274: 33166-33176Crossref PubMed Scopus (172) Google Scholar, 22.Nakayama M. Ishidoh K. Kojima Y. et al.Fibroblast growth factor-inducible 14 mediates multiple pathways of TWEAK-induced cell death.J Immunol. 2003; 170: 341-348Crossref PubMed Scopus (115) Google Scholar Both TWEAK and its receptor, Fn14, are present in the healthy adult kidney.15.Chicheportiche Y. Bourdon P.R. Xu H. et al.TWEAK, a new secreted ligand in the tumor necrosis factor family that weakly induces apoptosis.J Biol Chem. 1997; 272: 32401-32410Crossref PubMed Scopus (535) Google Scholar,21.Meighan-Mantha R.L. Hsu D.K. Guo Y. et al.The mitogen-inducible Fn14 gene encodes a type I transmembrane protein that modulates fibroblast adhesion and migration.J Biol Chem. 1999; 274: 33166-33176Crossref PubMed Scopus (172) Google Scholar However, there is little information on the regulation of the expression and the role of TWEAK and its receptor in kidney disease. We now report that TWEAK induces tubular cell apoptosis in the presence of proinflammatory cytokines and that these findings may be relevant to the pathogenesis of acute renal failure. In an experimental model of acute tubular injury characterized by tubular cell apoptosis,10.Ortiz A. Lorz C. Catalán M.P. et al.Expression of apoptosis regulatory proteins in tubular epithelium stressed in culture or following acute renal failure.Kidney Int. 2000; 57: 969-981Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar expression of both TWEAK (Figure 1a and b) and its receptor (Figure 1c and d) increased both at the messenger RNA (mRNA) and protein level. Fn14 was localized to injured tubules (Figure 1e). Based on this observation we studied the contribution of TWEAK to cultured tubular cell death. Renal tubular murine cortical tubular cell line (MCT) cells constitutively express TWEAK and Fn14 (Figure 2a–c). Constitutive Fn14 expression is low, but it was upregulated by proinflammatory cytokines (TNFα, interferon-gamma (INFγ)) alone or in combination (Figure 2c). Increased Fn14 expression was also observed when the ligand, TWEAK, was combined with TNFα/INFγ. This combination increased Fn14 in a time-dependent manner at the mRNA (Figure 2b) and protein (Figure 2c) levels. Quantitative polymerase chain reaction (PCR) confirmed a fourfold increase of Fn14 mRNA at 3 h, with return to baseline by 24 h (data not shown). We next evaluated a potential lethal activity of TWEAK in tubular cells. TWEAK did not induce apoptosis in non-stimulated tubular cells (Figure 3a). By contrast, costimulation with TNFα and INFγ resulted in sensitization to TWEAK-induced apoptosis (Figure 3b). Neither INFγ nor TNFα sensitized, by themselves, to death induced by TWEAK (Figure 3c). Features of apoptosis included the presence of hypodiploid cells and morphology (nuclear shrinkage, condensation, and fragmentation as well as decreased cell size) (Figure 4a and b).Figure 4TWEAK-induced tubular cell death in a proinflammatory environment has features of apoptosis. (a) Flow cytometry of DNA content. Note hypodiploid, apoptotic cells (A0) among those treated with TWEAK/TNFα/INFγ for 24 h. (b) Characteristic shrunk, pyknotic, fragmented nuclei are present among 4′,6′-diamidino-2-phenylindole-stained, TWEAK/TNFα/INFγ-treated cells, but not among control or TNFα/INFγ-treated cells (original magnification × 200).View Large Image Figure ViewerDownload (PPT) Apoptosis increased with concentration of TWEAK (Figure 3b). For further experiments we chose a concentration of 100 ng/ml of TWEAK, which induces around 30% of the cells to undergo apoptosis in 24 h (Figure 3b). Under these conditions, TWEAK-induced apoptosis was time-dependent and was apparent following 18 h of incubation (Figure 5). TNFα/INFγ induced delayed apoptosis in tubular cells (Figure 5). This delayed apoptosis maybe explained by the lethal effect of TNF in MCT cells.23.Justo P. Sanz A. Lorz C. et al.Expression of Smac/Diablo in tubular epithelial cells and during acute renal failure.Kidney Int. 2003; 64: 52-56Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar That is, TWEAK-induced apoptosis in a proinflammatory milieu is accelerated when compared to that induced by TNFα alone. MCT cells constitutively express FasL and Fas, and Fas expression is increased by TNFα/INFγ.12.Lorz C. Ortiz A. Justo P. et al.Proapoptotic Fas ligand is expressed by normal kidney tubular epithelium and injured glomeruli.J Am Soc Nephrol. 2000; 11: 1266-1277PubMed Google Scholar To rule out an autocrine role for FasL in cell death, cells were cultured with a decoy receptor which antagonizes TWEAK or with FasL blocking antibodies, in the presence of TWEAK/TNFα/INFγ. The decoy receptor protected against apoptosis, whereas blocking FasL was only marginally protective (Figure 6a). However, the decoy receptor did not significantly protect against apoptosis induced by the TNFα/INFγ combination at 48 h (TNFα/INFγ 40±7.8%, TNFα/INFγ/Fn14:Fc 33.5±8.4%, P=NS). The combination of TWEAK/TNFα/INFγ increased the expression of Fn14 receptor (Figure 2b and c). ITEM-4, an anti-Fn14 neutralizing antibody, prevented TWEAK/TNFα/INFγ-induced apoptosis (Figure 6b). These results suggest that in a proinflammatory environment TWEAK induces apoptosis in the tubular epithelium through Fn14 activation. We next investigated caspase involvement in apoptosis induced by TWEAK in the presence of TNFα/INFγ. The combination of cytokines activated caspase-3, -8, and -9 (Figure 7a–c). TWEAK/TNFα/INFγ rapidly induced the appearance of truncated Bid, which preceded caspase-9 and -3 activation (Figure 7c). Bid is a caspase-8 target in death receptor-initiated apoptosis. Caspase-9 processing started at 1 h (Figure 7c). Processing of caspase-3 started at 3 h, as shown by the appearance of caspase-3 cleavage product in Western blots (Figure 7c). Caspase-3 is mainly activated by caspase-8 or caspase-9.24.Thornberry N.A. Lazebnik Y. Caspases: enemies within.Science. 1998; 281: 1312-1316Crossref PubMed Scopus (6018) Google Scholar Truncated Bid translocates to the mitochondria and elicits cytochrome c release to the cytosol, where the latter activates caspase-9.25.Li H. Zhu H. Xu C.J. et al.Cleavage of BID by caspase 8 mediates the mitochondrial damage in the Fas pathway of apoptosis.Cell. 1998; 94: 491-501Abstract Full Text Full Text PDF PubMed Scopus (3664) Google Scholar Cytochrome c was released from the mitochondria of cells treated with TWEAK/TNFα/INFγ (Figure 7d and e). Growth-arrest and DNA damage (GADD)153 is induced during cell death triggered by endoplasmic reticulum stress.26.Zinszner H. Kuroda M. Wang X.Z. et al.CHOP is implicated in programmed cell death in response to impaired function of the endoplasmic reticulum.Gene Dev. 1998; 12: 982-995Crossref PubMed Scopus (1610) Google Scholar GADD153 expression did not change in tubular cells treated with TWEAK/TNFα/INFγ, unlike in cells treated with the endoplasmic reticulum stress or tunicamycin (Figure 7f). Moreover, cleavage of endoplasmic reticulum-specific caspase-12 was not detected in cells treated with TWEAK/TNFα/INFγ (Figure 7f). We next studied the effect of caspase inhibition on apoptosis (Figure 8). Benzyloxycarbonyl-Val-Ala-DL-Asp(zVAD)-fluoromethylketone is a broad-spectrum inhibitor of caspases.27.Garcia-Calvo M. Peterson E.P. Leiting B. et al.Inhibition of human caspases by peptide-based and macromolecular inhibitors.J Biol Chem. 1998; 273: 32608-32613Crossref PubMed Scopus (826) Google Scholar zVAD-fluoromethylketone inhibited caspase-8 and caspase-3 activity (Figure 7a), caspase-3 processing (Figure 7b), and apoptosis, efficiently blocking DNA hypoploidy, but it did not prevent cell death (Figure 8d). Notably, cells stimulated with TWEAK in the presence of zVAD and cytokines exhibited a necrotic morphology, which was characterized by trypan blue staining (79% of the cells). Similar results were found in cells treated with TWEAK/TNFα/INFγ and caspase-8 inhibitor (data not shown). A role for TWEAK in necrotic death in MCT cells in the presence of caspase inhibition was confirmed, because a TWEAK antagonist prevented the cell death (Figure 8e). In addition, zVAD did not promote cell death in the presence of TNFα/INFγ (Figure 8h). These results indicate that in a proinflammatory environment TWEAK primarily induces apoptosis in MCT cells, but it can also induce necrosis when caspases are inactivated. Recently, it has been reported that mitochondrial stress-induced reactive oxygen species were responsible for TNFα or anti-Fas monoclonal antibody-induced necrosis, which was abrogated by antioxidants such as butylated hydroxyanisole (BHA).28.Vercammen D. Brouckaert G. Denecker G. et al.Dual signaling of the Fas receptor: initiation of both apoptotic and necrotic cell death pathways.J Exp Med. 1998; 188: 919-930Crossref PubMed Scopus (443) Google Scholar,29.Khwaja A. Tatton L. Resistance to the cytotoxic effects of tumor necrosis factor alpha can be overcome by inhibition of a FADD/caspase-dependent signaling pathway.J Biol Chem. 1999; 274: 36817-36823Crossref PubMed Scopus (83) Google Scholar Thus, we tested whether BHA could inhibit TWEAK/TNFα/INFγ-induced necrosis in zVAD-treated cells. The necrotic morphology was completely abrogated by BHA (Figure 8f). These results suggest that caspase inhibition could sensitize MCT cells to TWEAK-induced necrosis via reactive oxygen species-dependent pathway. TWEAK and Fn14 expression has been detected in the normal kidney.15.Chicheportiche Y. Bourdon P.R. Xu H. et al.TWEAK, a new secreted ligand in the tumor necrosis factor family that weakly induces apoptosis.J Biol Chem. 1997; 272: 32401-32410Crossref PubMed Scopus (535) Google Scholar,21.Meighan-Mantha R.L. Hsu D.K. Guo Y. et al.The mitogen-inducible Fn14 gene encodes a type I transmembrane protein that modulates fibroblast adhesion and migration.J Biol Chem. 1999; 274: 33166-33176Crossref PubMed Scopus (172) Google Scholar We now show that both proteins are upregulated in acute tubular injury, suggesting that they may play a role in kidney disease. In the presence of proinflammatory cytokines, activation of Fn14 leads to tubular epithelial cell injury. Cell death is associated with caspase-8 activation and recruitment of the mitochondrial pathway of apoptosis. Caspase inhibitors prevent apoptosis but promote oxidative stress and a necrotic form of cell death. In different cell types TWEAK may promote cell proliferation or cell death.15.Chicheportiche Y. Bourdon P.R. Xu H. et al.TWEAK, a new secreted ligand in the tumor necrosis factor family that weakly induces apoptosis.J Biol Chem. 1997; 272: 32401-32410Crossref PubMed Scopus (535) Google Scholar, 16.Wiley S.R. Winkles J.A. TWEAK, a member of the TNF superfamily, is a multifunctional cytokine that binds the TweakR/Fn14 receptor.Cytokine Growth Factor Rev. 2003; 14: 241-249Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, 17.Campbell S. Michaelson J. Burkly L. et al.The role of TWEAK/Fn14 IN the pathogenesis of inflammation and systemic autoimmunity.Front Biosci. 2004; 9: 2273-2284Crossref PubMed Scopus (88) Google Scholar, 19.Lynch C.N. Wang Y.C. Lund J.K. et al.TWEAK induces angiogenesis and proliferation of endothelial cells.J Biol Chem. 1999; 274: 8455-8459Crossref PubMed Scopus (210) Google Scholar, 20.Wiley S.R. Cassiano L. Lofton T. et al.A novel TNF receptor family member binds TWEAK and is implicated in angiogenesis.Immunity. 2001; 15: 837-846Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar, 22.Nakayama M. Ishidoh K. Kojima Y. et al.Fibroblast growth factor-inducible 14 mediates multiple pathways of TWEAK-induced cell death.J Immunol. 2003; 170: 341-348Crossref PubMed Scopus (115) Google Scholar, 30.Potrovita I. Zhang W. Burkly L. et al.Tumor necrosis factor-like weak inducer of apoptosis-induced neurodegeneration.J Neurosci. 2004; 24: 8237-8244Crossref PubMed Scopus (117) Google Scholar, 31.Kaplan M.J. Lewis E.E. 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Burkly L. et al.Tumor necrosis factor-like weak inducer of apoptosis-induced neurodegeneration.J Neurosci. 2004; 24: 8237-8244Crossref PubMed Scopus (117) Google Scholar, 31.Kaplan M.J. Lewis E.E. Shelden E.A. et al.The apoptotic ligands TRAIL, TWEAK, and Fas ligand mediate monocyte death induced by autologous lupus T cells.J Immunol. 2002; 169: 6020-6029Crossref PubMed Scopus (138) Google Scholar, 32.Nakayama M. Ishidoh K. Kayagaki N. et al.Multiple pathways of TWEAK-induced cell death.J Immunol. 2002; 168: 734-743Crossref PubMed Scopus (145) Google Scholar, 33.Campbell S. Burkly L.C. Gao H.X. et al.Proinflammatory effects of TWEAK/Fn14 interactions in glomerular mesangial cells.J Immunol. 2006; 176: 1889-1898Crossref PubMed Scopus (138) Google Scholar By contrast, TWEAK does not induce apoptosis in non-stimulated tubular cells. Nevertheless, the lethal activity of TWEAK may require the coincubation with other agents. TWEAK promotes apoptosis when combined with INFγ in some tumor cell lines, whereas others remain resistant even in the presence of this cytokine.15.Chicheportiche Y. Bourdon P.R. Xu H. et al.TWEAK, a new secreted ligand in the tumor necrosis factor family that weakly induces apoptosis.J Biol Chem. 1997; 272: 32401-32410Crossref PubMed Scopus (535) Google Scholar,22.Nakayama M. Ishidoh K. Kojima Y. et al.Fibroblast growth factor-inducible 14 mediates multiple pathways of TWEAK-induced cell death.J Immunol. 2003; 170: 341-348Crossref PubMed Scopus (115) Google Scholar In tubular epithelial cells, the coincubation with TNFα and INFγ sensitizes to apoptosis induced by TWEAK. This effect was not observed with either cytokine alone. This requirement for both INFγ and TNF is novel. TNFα/INFγ increased Fn14 expression in tubular cells. The early peak of Fn14 expression (2 h) is consistent with previous reports, as Fn14 is an immediate-early response gene.21.Meighan-Mantha R.L. Hsu D.K. Guo Y. et al.The mitogen-inducible Fn14 gene encodes a type I transmembrane protein that modulates fibroblast adhesion and migration.J Biol Chem. 1999; 274: 33166-33176Crossref PubMed Scopus (172) Google Scholar Upregulation of Fn14 expression may underlie the sensitization to apoptosis. In fact, Fn14 transfectants become sensitive to TWEAK-induced death.22.Nakayama M. Ishidoh K. Kojima Y. et al.Fibroblast growth factor-inducible 14 mediates multiple pathways of TWEAK-induced cell death.J Immunol. 2003; 170: 341-348Crossref PubMed Scopus (115) Google Scholar Alternatively, it has been suggested that certain functions of TWEAK are mediated by a second, not yet characterized receptor.35.Polek T.C. Talpaz M. Darnay B.G. et al.TWEAK mediates signal transduction and differentiation of RAW264.7 cells in the absence of Fn14/TWEAKR. Evidence for a second TWEAK receptor.J Biol Chem. 2003; 278: 32317-32323Crossref PubMed Scopus (133) Google Scholar However, a Fn14 blocker prevented the cytotoxicity of the cytokine combination on tubular epithelium. This indicates that Fn14 mediates the lethal effect of TWEAK in tubular cells. An autocrine activation of the FasL/Fas system was also considered as a potential cause of cell death. Our group had previously described that proinflammatory cytokines increase Fas expression in tubular cells.10.Ortiz A. Lorz C. Catalán M.P. et al.Expression of apoptosis regulatory proteins in tubular epithelium stressed in culture or following acute renal failure.Kidney Int. 2000; 57: 969-981Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar The Fas receptor could theoretically be activated by autocrine FasL, as tubular epithelium constitutively expresses FasL.10.Ortiz A. Lorz C. Catalán M.P. et al.Expression of apoptosis regulatory proteins in tubular epithelium stressed in culture or following acute renal failure.Kidney Int. 2000; 57: 969-981Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar FasL blocking antibody minimally decreased the rate of cell death induced by TWEAK/TNFα/INFγ, suggesting that the mechanism is indeed active, albeit of little significance in this system. Next we investigated the intracellular pathways for apoptosis induced by TWEAK in the presence of TNFα/INFγ. We observed activation of caspase-8, proteolysis of Bid, release of cytochrome c to the cytosol and activation of caspase-9 and caspase-3. Proteolysis of Bid pre-dated caspase 9, which in turn, pre-dated caspase-3 activation. This suggests that, as it is the case for some cell types in response to activation of death receptors, caspase-8-activated Bid mediated an amplification loop through the mitochondria.25.Li H. Zhu H. Xu C.J. et al.Cleavage of BID by caspase 8 mediates the mitochondrial damage in the Fas pathway of apoptosis.Cell. 1998; 94: 491-501Abstract Full Text Full Text PDF PubMed Scopus (3664) Google Scholar Activation of caspases-3, -8, and -9 had previously been observed in some, but not all, tumor cell lines undergoing apoptosis induced by TWEAK.22.Nakayama M. Ishidoh K. Kojima Y. et al.Fibroblast growth factor-inducible 14 mediates multiple pathways of TWEAK-induced cell death.J Immunol. 2003; 170: 341-348Crossref PubMed Scopus (115) Google Scholar,32.Nakayama M. Ishidoh K. Kayagaki N. et al.Multiple pathways of TWEAK-induced cell death.J Immunol. 2002; 168: 734-743Crossref PubMed Scopus (145) Google Scholar However, it is still unclear what adaptors mediate this effect, as Fn14 lacks a death domain.22.Nakayama M. Ishidoh K. Kojima Y. et al.Fibroblast growth factor-inducible 14 mediates multiple pathways of TWEAK-induced cell death.J Immunol. 2003; 170: 341-348Crossref PubMed Scopus (115) Google Scholar On the other hand, we found no evidence supporting the participation of endoplasmic reticulum stress in the process. The apoptotic endoplasmic reticulum response is preserved in MCT cells exposed to paracetamol or tunicamycin.36.Lorz C. Justo P. Sanz A. et al.Paracetamol-induced renal tubular injury: a role for endoplasmic reticulum stress.J Am Soc Nephrol. 2004; 15: 380-389Crossref PubMed Scopus (130) Google Scholar TNF-α alone may induce a delayed apoptosis in MCT cells, but the time course differs from that of TWEAK, as there is a lag period of 48 h, which was confirmed in cells treated with TNFα/INFγ.23.Justo P. Sanz A. Lorz C. et al.Expression of Smac/Diablo in tubular epithelial cells and during acute renal failure.Kidney Int. 2003; 64: 52-56Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar In addition, the intracellular molecular mechanisms differ, as cleavage of Bid was not observed in the absence of TWEAK. Most extracellular inputs are not processed in isolation, rather, multiple inputs are perceived by cells in a proinflammatory milie
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