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• W2119210248 abstract "C5b-9-induced glomerular epithelial cell (GEC) injury in vivo (in passive Heymann nephritis) and in culture is associated with damage to the endoplasmic reticulum (ER) and increased expression of ER stress proteins. Induction of ER stress proteins is enhanced via cytosolic phospholipase A2 (cPLA2) and limits complement-dependent cytotoxicity. The present study addresses another aspect of the ER unfolded protein response, i.e. activation of protein kinase R-like ER kinase (PERK or pancreatic ER kinase), which phosphorylates eukaryotic translation initiation factor 2-α (eIF2α), thereby generally suppressing translation and decreasing the protein load on a damaged ER. Phosphorylation of eIF2α was enhanced significantly in glomeruli of proteinuric rats with passive Heymann nephritis, compared with control. In cultured GECs, complement induced phosphorylation of eIF2α and reduced protein synthesis, and complement-stimulated phosphorylation of eIF2α was enhanced by overexpression of cPLA2. Ischemia-reperfusion in vitro (deoxyglucose plus antimycin A followed by glucose re-exposure) also stimulated eIF2α phosphorylation and reduced protein synthesis. Complement and ischemia-reperfusion induced phosphorylation of PERK (which correlates with activation), and fibroblasts from PERK knock-out mice were more susceptible to complement- and ischemia-reperfusion-mediated cytotoxicity, as compared with wild type fibroblasts. The GEC protein, nephrin, plays a key role in maintaining glomerular permselectivity. In contrast to a general reduction in protein synthesis, translation regulated by the 5′-end of mouse nephrin mRNA during ER stress was paradoxically maintained, probably due to the presence of short open reading frames in this mRNA segment. Thus, phosphorylation of eIF2α and consequent general reduction in protein synthesis may be a novel mechanism for limiting complement- or ischemia-reperfusion-dependent GEC injury. C5b-9-induced glomerular epithelial cell (GEC) injury in vivo (in passive Heymann nephritis) and in culture is associated with damage to the endoplasmic reticulum (ER) and increased expression of ER stress proteins. Induction of ER stress proteins is enhanced via cytosolic phospholipase A2 (cPLA2) and limits complement-dependent cytotoxicity. The present study addresses another aspect of the ER unfolded protein response, i.e. activation of protein kinase R-like ER kinase (PERK or pancreatic ER kinase), which phosphorylates eukaryotic translation initiation factor 2-α (eIF2α), thereby generally suppressing translation and decreasing the protein load on a damaged ER. Phosphorylation of eIF2α was enhanced significantly in glomeruli of proteinuric rats with passive Heymann nephritis, compared with control. In cultured GECs, complement induced phosphorylation of eIF2α and reduced protein synthesis, and complement-stimulated phosphorylation of eIF2α was enhanced by overexpression of cPLA2. Ischemia-reperfusion in vitro (deoxyglucose plus antimycin A followed by glucose re-exposure) also stimulated eIF2α phosphorylation and reduced protein synthesis. Complement and ischemia-reperfusion induced phosphorylation of PERK (which correlates with activation), and fibroblasts from PERK knock-out mice were more susceptible to complement- and ischemia-reperfusion-mediated cytotoxicity, as compared with wild type fibroblasts. The GEC protein, nephrin, plays a key role in maintaining glomerular permselectivity. In contrast to a general reduction in protein synthesis, translation regulated by the 5′-end of mouse nephrin mRNA during ER stress was paradoxically maintained, probably due to the presence of short open reading frames in this mRNA segment. Thus, phosphorylation of eIF2α and consequent general reduction in protein synthesis may be a novel mechanism for limiting complement- or ischemia-reperfusion-dependent GEC injury. The unfolded protein response (UPR) 1The abbreviations used are: UPR, unfolded protein response; AA, arachidonic acid; CMV, cytomegalovirus; cPLA2, cytosolic phospholipase A2-α; eIF2α, eukaryotic translation initiation factor 2-α; ER, endoplasmic reticulum; GEC, glomerular epithelial cell; GFP, green fluorescent protein; HIS, heat-inactivated human serum; LDH, lactate dehydrogenase; NS, normal human serum; PAN, puromycin aminonucleoside nephrosis; PHN, passive Heymann nephritis; PERK, protein kinase R-like ER kinase or pancreatic ER kinase; grp, glucose-related protein. is one of several cell stress responses, which in part consists of up-regulating the capacity of the endoplasmic reticulum (ER) to process abnormal proteins (1Kaufman R.J. Scheuner D. Schroder M. Shen X. Lee K. Liu C.Y. Arnold S.M. Nat. Rev. Mol. Cell. Biol. 2002; 3: 411-421Crossref PubMed Scopus (506) Google Scholar, 2Ron D. J. Clin. Invest. 2002; 110: 1383-1388Crossref PubMed Scopus (745) Google Scholar, 3Kaufman R.J. J. Clin. Invest. 2002; 110: 1389-1398Crossref PubMed Scopus (1105) Google Scholar, 4Zhang K. Kaufman R.J. J. Biol. Chem. 2004; 279: 25935-25938Abstract Full Text Full Text PDF PubMed Scopus (491) Google Scholar). Following accumulation of unfolded proteins in the ER, or depletion of ER Ca2+ stores, activating transcription factor-6 moves to the Golgi, where it is cleaved by site-1 and site-2 proteases to yield a cytosolic fragment. The fragment migrates to the nucleus to activate transcription of ER stress proteins, e.g. the glucose-related proteins (grp), grp94 and bip (grp78), and others. In parallel, inositol requiring-1 dimerizes and activates its endoribonuclease activity. IRE1 cleaves X-box-binding protein-1 mRNA and changes the reading frame to yield a potent transcriptional activator. Under normal conditions, ER stress proteins may serve as protein chaperones for exocytosis from the ER, and they may complex with defective proteins and target them for degradation. During stress, the induction of ER stress proteins may limit accumulation of abnormal proteins in cells. A third aspect of the UPR involves PERK (protein kinase R-like ER kinase or pancreatic ER kinase), which is activated to phosphorylate the α subunit of eukaryotic translation initiation factor-2 (eIF2α). This process reduces AUG codon recognition, and consequently, the general rate of translation is reduced (which aims at decreasing the protein load on a damaged ER), but selective mRNAs can be preferentially translated under these conditions. Current literature supports the view that bip serves as a master UPR regulator, being involved in the activation of activating transcription factor-6, inositol requiring-1, and PERK. Induction of the UPR may allow cells to recover from ER stress and may be protective to additional insults, at least in part via cross-talk with the extracellular signal-regulated kinase (5Hung C.C. Ichimura T. Stevens J.L. Bonventre J.V. J. Biol. Chem. 2003; 278: 29317-29326Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). However, prolonged or more substantial ER stress may lead to cell death via apoptosis (2Ron D. J. Clin. Invest. 2002; 110: 1383-1388Crossref PubMed Scopus (745) Google Scholar, 4Zhang K. Kaufman R.J. J. Biol. Chem. 2004; 279: 25935-25938Abstract Full Text Full Text PDF PubMed Scopus (491) Google Scholar). Upon activation of the complement cascade near a cell surface, there is assembly of terminal components, exposure of hydrophobic domains, and insertion of the C5b-9 membrane attack complex into the lipid bilayer of the plasma membrane (6Morgan B.P. Curr. Top Microbiol. Immunol. 1992; 178: 115-140PubMed Google Scholar, 7Nicholson-Weller A. Halperin J.A. Immunol. Res. 1993; 12: 244-257Crossref PubMed Scopus (131) Google Scholar). C5b-9 assembly results in formation of transmembrane channels or rearrangement of membrane lipids with loss of membrane integrity. In nucleated cells, multiple C5b-9 complexes are required for lysis, but at lower doses, C5b-9 induces sublethal (sublytic) injury (6Morgan B.P. Curr. Top Microbiol. Immunol. 1992; 178: 115-140PubMed Google Scholar, 7Nicholson-Weller A. Halperin J.A. Immunol. Res. 1993; 12: 244-257Crossref PubMed Scopus (131) Google Scholar, 8Shin M.L. Rus H.G. Niculescu F.I. Lee A.G. Biomembranes. JAI Press, Greenwich, CT1996: 119-146Google Scholar, 9Rus H.G. Niculescu F.I. Shin M.L. Immunol. Rev. 2001; 180: 49-55Crossref PubMed Scopus (108) Google Scholar). At the same time, complement attack may result in activation of various pathways, including those that restrict injury or facilitate recovery (6Morgan B.P. Curr. Top Microbiol. Immunol. 1992; 178: 115-140PubMed Google Scholar, 7Nicholson-Weller A. Halperin J.A. Immunol. Res. 1993; 12: 244-257Crossref PubMed Scopus (131) Google Scholar, 8Shin M.L. Rus H.G. Niculescu F.I. Lee A.G. Biomembranes. JAI Press, Greenwich, CT1996: 119-146Google Scholar). Recently we demonstrated that induction of the UPR is a mechanism of protection from complement attack (10Cybulsky A.V. Takano T. Papillon J. Khadir A. Liu J. Peng H. J. Biol. Chem. 2002; 277: 41342-41351Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). An example of sublytic C5b-9-mediated cell injury in vivo is passive Heymann nephritis (PHN) in the rat, a widely accepted model of human membranous nephropathy (11Heymann W. Hackel D.B. Harwood S. Wilson S.G. Hunter J.L. J. Am. Soc. Nephrol. 2000; 11: 183-188PubMed Google Scholar, 12Cybulsky A.V. Foster M.H. Quigg R.J. Salant D.J. Seldin D.W. Giebisch G. The Kidney: Physiology and Pathophysiology. 3rd Ed. Lippincott-Raven, Philadelphia2000: 2645-2697Google Scholar). Injury involves the visceral glomerular epithelial cell (GEC), a highly specialized cell type that is involved in the maintenance of glomerular permselectivity (13Pavenstadt H. Kriz W. Kretzler M. Physiol. Rev. 2003; 83: 253-307Crossref PubMed Scopus (1215) Google Scholar). Expression of unique cell surface molecules by GECs (e.g. nephrin) appears to be key to maintaining normal ultrastructure and permselective properties (14Kestila M. Lenkkeri U. Mannikko M. Lamerdin J. McCready P. Putaala H. Ruotsalainen V. Morita T. Nissinen M. Herva R. Kashtan C.E. Peltonen L. Holmberg C. Olsen A. Tryggvason K. Mol. Cell. 1998; 1: 575-582Abstract Full Text Full Text PDF PubMed Scopus (1574) Google Scholar, 15Wartiovaara J. Ofverstedt L.G. Khoshnoodi J. Zhang J. Makela E. Sandin S. Ruotsalainen V. Cheng R.H. Jalanko H. Skoglund U. Tryggvason K. J. Clin. Invest. 2004; 114: 1475-1483Crossref PubMed Scopus (244) Google Scholar). In PHN, antibody binds to GEC antigens, and leads to the in situ formation of subepithelial immune complexes (11Heymann W. Hackel D.B. Harwood S. Wilson S.G. Hunter J.L. J. Am. Soc. Nephrol. 2000; 11: 183-188PubMed Google Scholar, 12Cybulsky A.V. Foster M.H. Quigg R.J. Salant D.J. Seldin D.W. Giebisch G. The Kidney: Physiology and Pathophysiology. 3rd Ed. Lippincott-Raven, Philadelphia2000: 2645-2697Google Scholar). C5b-9 assembles in GEC plasma membranes, “activates” GECs, and leads to proteinuria and sublytic GEC injury (11Heymann W. Hackel D.B. Harwood S. Wilson S.G. Hunter J.L. J. Am. Soc. Nephrol. 2000; 11: 183-188PubMed Google Scholar, 12Cybulsky A.V. Foster M.H. Quigg R.J. Salant D.J. Seldin D.W. Giebisch G. The Kidney: Physiology and Pathophysiology. 3rd Ed. Lippincott-Raven, Philadelphia2000: 2645-2697Google Scholar). Based on studies in GEC culture and in vivo, C5b-9 assembly induces transactivation of receptor tyrosine kinases (16Cybulsky A.V. Takano T. Papillon J. McTavish A.J. Am. J. Pathol. 1999; 155: 1701-1711Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar), an increase in cytosolic free Ca2+ concentration, and activation of cytosolic phospholipase A2-α (cPLA2) (17Cybulsky A.V. Monge J.C. Papillon J. McTavish A.J. Am. J. Physiol. 1995; 269: F739-F749Crossref PubMed Google Scholar, 18Panesar M. Papillon J. McTavish A.J. Cybulsky A.V. J. Immunol. 1997; 159: 3584-3594PubMed Google Scholar, 19Cybulsky A.V. Papillon J. McTavish A.J. Kidney Int. 1998; 54: 360-372Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 20Cybulsky A.V. Takano T. Papillon J. McTavish A.J. Kidney Int. 2000; 57: 1052-1062Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 21Leslie C.C. J. Biol. Chem. 1997; 272: 16709-16712Abstract Full Text Full Text PDF PubMed Scopus (743) Google Scholar, 22Hirabayashi T. Shimizu T. Biochim. Biophys. Acta. 2000; 1488: 124-138Crossref PubMed Scopus (177) Google Scholar). cPLA2 is an important mediator of C5b-9-dependent GEC injury. First, arachidonic acid (AA) released by cPLA2 is metabolized to prostaglandin E2 and thromboxane A2, and inhibition of prostanoid production reduces proteinuria in PHN and in human membranous nephropathy (12Cybulsky A.V. Foster M.H. Quigg R.J. Salant D.J. Seldin D.W. Giebisch G. The Kidney: Physiology and Pathophysiology. 3rd Ed. Lippincott-Raven, Philadelphia2000: 2645-2697Google Scholar). Second, cPLA2 may mediate GEC injury more directly, probably by inducing damage to the membrane of the ER, and by participating in the ER stress response (10Cybulsky A.V. Takano T. Papillon J. Khadir A. Liu J. Peng H. J. Biol. Chem. 2002; 277: 41342-41351Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Thus, assembly of C5b-9 in GEC in culture and in vivo increased expression of the ER stress proteins, bip and grp94, in a cPLA2-dependent manner, and induction of these proteins restricted complement-mediated GEC injury. In addition to complement, other types of stress may injure GEC. For example, during or after renal ischemia-reperfusion injury there is up-regulation of glomerular heat shock and ER stress proteins, activation of nitric-oxide synthases, glomerular infiltration with leukocytes, and development of sclerosis (23Klausner J.M. Paterson I.S. Goldman G. Kobzik L. Rodzen C. Lawrence R. Valeri C.R. Shepro D. Hechtman H.B. Am. J. Physiol. 1989; 256: F794-F802PubMed Google Scholar, 24Azuma H. Nadeau K. Takada M. Mackenzie H.S. Tilney N.L. Transplantation. 1997; 64: 190-197Crossref PubMed Scopus (199) Google Scholar, 25Pagtalunan M.E. Olson J.L. Tilney N.L. Meyer T.W. J. Am. Soc. Nephrol. 1999; 10: 366-373Crossref PubMed Google Scholar, 26Smoyer W.E. Ransom R. Harris R.C. Welsh M.J. Lutsch G. Benndorf R. J. Am. Soc. Nephrol. 2000; 11: 211-221Crossref PubMed Google Scholar, 27Valdivielso J.M. Crespo C. Alonso J.R. Martinez-Salgado C. Eleno N. Arevalo M. Perez-Barriocanal F. Lopez-Novoa J.M. Am. J. Physiol. 2001; 280: R771-R779PubMed Google Scholar). The aim of the present study was to determine if induction of ER stress in GECs activates the PERK pathway and suppresses protein synthesis. We demonstrate that complement, as well as ischemia-reperfusion, induce phosphorylation of PERK and eIF2α, and suppress general protein synthesis. Furthermore, activation of the PERK pathway is functionally important, because it limits complement- and ischemia-reperfusion-dependent cytotoxicity. Translation regulated by the 5′-end of nephrin mRNA was, however, maintained following ischemia-reperfusion injury. Materials and Plasmid Construction—Tissue culture reagents were obtained from Invitrogen. Tunicamycin, 2-deoxyglucose, antimycin A, and puromycin aminonucleoside (PA) were purchased from Sigma-Aldrich. Electrophoresis and immunoblotting reagents were from Bio-Rad Laboratories (Mississauga, Ontario, Canada). [3H]AA (100 Ci/mmol) and [α-32P]dCTP (3000 Ci/mmol) were purchased from PerkinElmer Life Sciences. [35S]Methionine/cysteine (1000 Ci/mmol) was purchased from Amersham Biosciences. Rabbit anti-phospho-eIF2α serine 51 and rabbit anti-phospho-PERK threonine 980 antibodies were purchased from New England Biolabs (Mississauga, Ontario, Canada). Rabbit anti-PERK antibody was from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit anti-bip antibody was from Stressgen (Vancouver, BC). pEGFP-N3 vector was from BD Biosciences (Mississauga, Ontario, Canada). Pwo polymerase was purchased from Roche Diagnostics. Restriction enzymes and other molecular biology reagents were from Invitrogen or New England Biolabs. PERK knock-out and wild type mouse embryonic fibroblasts (28Harding H.P. Zeng H. Zhang Y. Jungries R. Chung P. Plesken H. Sabatini D.D. Ron D. Mol. Cell. 2001; 7: 1153-1163Abstract Full Text Full Text PDF PubMed Scopus (1014) Google Scholar) were kindly provided by Drs. Heather Harding and David Ron (New York University, New York, NY). An ∼8.5-kb DNA fragment of the 5′-flanking region of mouse nephrin (29Moeller M.J. Kovari I.A. Holzman L.B. J. Am. Soc. Nephrol. 2000; 11: 2306-2314Crossref PubMed Google Scholar, 30Moeller M.J. Sanden S.K. Soofi A. Wiggins R.C. Holzman L.B. J. Am. Soc. Nephrol. 2002; 13: 1561-1567Crossref PubMed Scopus (92) Google Scholar, 31Michaud J.L. Lemieux L.I. Dube M. Vanderhyden B.C. Robertson S.J. Kennedy C.R. J. Am. Soc. Nephrol. 2003; 14: 1200-1211Crossref PubMed Scopus (128) Google Scholar) was kindly provided by Dr. Chris Kennedy (University of Ottawa, Ottawa, Ontario, Canada). Male Sprague-Dawley rats (150 g) were purchased from Charles River. The transcription start site of the nephrin mRNA is reported to be either at nucleotide –415 or –381, as counted from the nephrin translation initiation ATG codon in the cDNA (30Moeller M.J. Sanden S.K. Soofi A. Wiggins R.C. Holzman L.B. J. Am. Soc. Nephrol. 2002; 13: 1561-1567Crossref PubMed Scopus (92) Google Scholar, 32Beltcheva O. Kontusaari S. Fetissov S. Putaala H. Kilpelainen P. Hokfelt T. Tryggvason K. J. Am. Soc. Nephrol. 2003; 14: 352-358Crossref PubMed Scopus (40) Google Scholar). To construct pEGFP-N5′(391bp)-enhanced green fluorescent protein (GFP), a 391-nucleotide fragment corresponding to the mouse nephrin 5′-flanking region (nucleotides –391 to 1) was produced using PCR. The primers were 5′-CTGGGCTCGAGCAATGCTCAGTGCTG-3′ (forward-1) and 5′-CGCGGATCCCATCACCAGCAGCTTGTTGT-3′ (reverse-1). The ∼8.5-kb nephrin 5′-flanking DNA served as template. The PCR product was digested with XhoI and BamHI restriction enzymes, and was subcloned into the XhoI and BamHI sites of pEGFP-N3, between the 589-nucleotide cytomegalovirus (CMV) promoter region (contained within the vector), and GFP. To mutate three ATG sequences in the 391-bp nephrin 5′-flanking fragment, two PCRs were performed using the 5′-end of nephrin DNA as template. In the first reaction, the forward primer was the same as above, except that the ATG sequence was changed to ATA (forward-1A), and the reverse primer was 5′-TGACTGTCGCAGTCTTTCTGTCCCGGGATCGCCTTT-3′ (reverse-2). In the second reaction, the primers were 5′-CAGAAAGACTGCGACAGTCACAGACATTGGTAGGAA-3′ (forward-2), and reverse-1. Single base substitutions were introduced into primers forward-2 and reverse-2 to remove ATG sequences (bold letters). Products of the two PCRs were allowed to anneal (as the forward-2 and reverse-2 primers contain 20 overlapping nucleotides), and then the nephrin 5′-flanking fragment containing three mutated ATG sequences was produced using PCR and the primers forward-1A and reverse-1. An additional construct that contained a 1.25-kb fragment of the 5′-end of nephrin, subcloned into pEGFP (at XhoI and BamHI sites), was produced as above (using PCR and the ∼8.5-kb nephrin 5′-flanking DNA as template). Primers were 5′-AAGCACTCGAGAGGTGAGAGGTTTGTAG-3′ and reverse-1. Cell Culture and Transfection—Rat GEC culture and characterization has been published previously (17Cybulsky A.V. Monge J.C. Papillon J. McTavish A.J. Am. J. Physiol. 1995; 269: F739-F749Crossref PubMed Google Scholar, 18Panesar M. Papillon J. McTavish A.J. Cybulsky A.V. J. Immunol. 1997; 159: 3584-3594PubMed Google Scholar, 33Coers W. Reivinen J. Miettinen A. Huitema S. Vos J.T. Salant D.J. Weening J.J. Exp. Nephrol. 1996; 4: 184-192PubMed Google Scholar). GECs were cultured in K1 medium, and studies were done with cells between passages 8 and 60. Production and characterization of the GECs that stably overexpress cPLA2 or only the neomycin resistance gene (neo) was described previously (17Cybulsky A.V. Monge J.C. Papillon J. McTavish A.J. Am. J. Physiol. 1995; 269: F739-F749Crossref PubMed Google Scholar, 18Panesar M. Papillon J. McTavish A.J. Cybulsky A.V. J. Immunol. 1997; 159: 3584-3594PubMed Google Scholar). Compared with neo GECs, PLA2 activity is increased ∼5-fold in GECs overexpressing cPLA2, but is nevertheless within a physiological range. PERK knock-out and wild type fibroblasts, and COS-1 cells were cultured in Dulbecco's modified Eagle's medium with 10% fetal calf serum. The protocol for transient transfection of COS-1 cells was described previously (18Panesar M. Papillon J. McTavish A.J. Cybulsky A.V. J. Immunol. 1997; 159: 3584-3594PubMed Google Scholar, 20Cybulsky A.V. Takano T. Papillon J. McTavish A.J. Kidney Int. 2000; 57: 1052-1062Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Incubation with Complement and in Vitro Ischemia-reperfusion— The standard protocol involved incubation of GECs in monolayer culture with rabbit anti-GEC antiserum (5% v/v) in modified Krebs-Henseleit buffer, containing 145 mm NaCl, 5 mm KCl, 0.5 mm MgSO4, 1 mm Na2HPO4, 0.5 mm CaCl2, 5 mm glucose, and 20 mm Hepes, pH 7.4, for 40 min at 22 °C (10Cybulsky A.V. Takano T. Papillon J. Khadir A. Liu J. Peng H. J. Biol. Chem. 2002; 277: 41342-41351Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 17Cybulsky A.V. Monge J.C. Papillon J. McTavish A.J. Am. J. Physiol. 1995; 269: F739-F749Crossref PubMed Google Scholar, 18Panesar M. Papillon J. McTavish A.J. Cybulsky A.V. J. Immunol. 1997; 159: 3584-3594PubMed Google Scholar). GECs were then incubated with normal human serum (NS; diluted in Krebs-Henseleit buffer), or heat-inactivated (decomplemented) human serum (HIS; 56 °C, 30 min) in controls, for 40 min at 37 °C. Except for studies of cytolysis, experiments were carried out at concentrations of complement that induced minimal or no lysis (NS at 2.5–4.0% v/v). As in previous studies, we have generally used heterologous complement to minimize possible signaling via complement-regulatory proteins, although we have demonstrated that homologous complement can also induce activation of analogous pathways (17Cybulsky A.V. Monge J.C. Papillon J. McTavish A.J. Am. J. Physiol. 1995; 269: F739-F749Crossref PubMed Google Scholar, 18Panesar M. Papillon J. McTavish A.J. Cybulsky A.V. J. Immunol. 1997; 159: 3584-3594PubMed Google Scholar). Previous studies have shown that in GECs, complement is not activated in the absence of antibody (17Cybulsky A.V. Monge J.C. Papillon J. McTavish A.J. Am. J. Physiol. 1995; 269: F739-F749Crossref PubMed Google Scholar, 18Panesar M. Papillon J. McTavish A.J. Cybulsky A.V. J. Immunol. 1997; 159: 3584-3594PubMed Google Scholar). In vitro ischemia-reperfusion or chemical anoxia/recovery was induced by incubating cells in glucose-free medium with 2-deoxyglucose (10 mm) plus antimycin A (10 μm) for 90 min (anoxia). Then, cells were incubated in glucose-replete medium for 30 min, 4 h, or 24 h (recovery) (10Cybulsky A.V. Takano T. Papillon J. Khadir A. Liu J. Peng H. J. Biol. Chem. 2002; 277: 41342-41351Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Induction of PHN and Puromycin Aminonucleoside Nephrosis in Rats—PHN was induced by a single intravenous injection of 0.4 ml of sheep anti-Fx1A antiserum, as described previously (10Cybulsky A.V. Takano T. Papillon J. Khadir A. Liu J. Peng H. J. Biol. Chem. 2002; 277: 41342-41351Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 19Cybulsky A.V. Papillon J. McTavish A.J. Kidney Int. 1998; 54: 360-372Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). PAN was induced by a single intravenous injection of PA (80 mg/kg) (10Cybulsky A.V. Takano T. Papillon J. Khadir A. Liu J. Peng H. J. Biol. Chem. 2002; 277: 41342-41351Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Urine was collected on day 14, and rats were then sacrificed and glomeruli were isolated by differential sieving. All rats with PHN and PAN had heavy proteinuria (urine protein > 100 mg/day; normal < 20) All studies were approved by the McGill University Animal Care Committee. Measurement of free [3H]AA—GEC phospholipids were labeled to isotopic equilibrium with [3H]AA for 48–72 h, as detailed previously (17Cybulsky A.V. Monge J.C. Papillon J. McTavish A.J. Am. J. Physiol. 1995; 269: F739-F749Crossref PubMed Google Scholar, 18Panesar M. Papillon J. McTavish A.J. Cybulsky A.V. J. Immunol. 1997; 159: 3584-3594PubMed Google Scholar). Lipids were extracted from ∼1 × 106 cells and cell supernatants. Methods for extraction and separation of radiolabeled lipids (e.g. [3H]AA) by thin layer chromatography are published (17Cybulsky A.V. Monge J.C. Papillon J. McTavish A.J. Am. J. Physiol. 1995; 269: F739-F749Crossref PubMed Google Scholar, 18Panesar M. Papillon J. McTavish A.J. Cybulsky A.V. J. Immunol. 1997; 159: 3584-3594PubMed Google Scholar). Immunoprecipitation, Immunoblotting, and Northern Hybridization—Preparation of GEC and glomerular lysates and cell fractions was described previously (16Cybulsky A.V. Takano T. Papillon J. McTavish A.J. Am. J. Pathol. 1999; 155: 1701-1711Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 19Cybulsky A.V. Papillon J. McTavish A.J. Kidney Int. 1998; 54: 360-372Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). After incubation, ∼6 × 106 GECs were lysed, and proteins were immunoprecipitated with primary antiserum. Immune complexes were incubated with agarose-coupled protein A. Complexes were boiled in Laemmli sample buffer, and subjected to SDS-PAGE under reducing conditions. Proteins were then electrophoretically transferred onto nitrocellulose paper, blocked with 5% milk, and incubated with primary antibody in 3% bovine serum albumin, and then with horseradish peroxidase-conjugated secondary antibody. The blots were developed using the enhanced chemiluminescence technique (ECL, Amersham Biosciences). Protein content was quantified by scanning densitometry, using National Institutes of Health Image software. Preliminary studies demonstrated that there was a linear relationship between densitometric measurements and the amounts of protein loaded onto gels. Northern hybridization was performed as described previously (10Cybulsky A.V. Takano T. Papillon J. Khadir A. Liu J. Peng H. J. Biol. Chem. 2002; 277: 41342-41351Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Briefly, total RNA was extracted from cells using TRIzol reagent. RNA was separated by gel electrophoresis (1% agarose containing 1.9% formaldehyde) and was transferred to an Immobilon-Ny+ membrane (Millipore). The coding region of GFP cDNA was radiolabeled with [α-32P]dCTP using the random primer method. Membranes were hybridized in buffer containing 7% SDS, 0.5 m sodium phosphate, pH 7.1, 2 mm EDTA, 4 mm sodium pyrophosphate, and 1–2 × 106 cpm/ml radiolabeled probe for 16 h at 42 °C. Membranes were washed in 1× SSPE buffer (180 mm NaCl, 1 mm EDTA, 10 mm sodium phosphate, pH 6.8) with 0.5% SDS twice for 5 min at 22 °C, and then 0.2× SSPE buffer with 0.2% SDS, twice for 15 min at 68 °C. Membranes were exposed to x-ray film with an intensifying screen at –70 °C for 48–72 h. Measurement of Cytotoxicity—Complement-mediated cytolysis was determined by measuring release of lactate dehydrogenase (LDH), similarly to the method described previously (10Cybulsky A.V. Takano T. Papillon J. Khadir A. Liu J. Peng H. J. Biol. Chem. 2002; 277: 41342-41351Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Specific release of LDH was calculated as [NS – HIS]/[100 – HIS], where NS represents the percent total LDH released into cell supernatants in incubations with NS, and HIS is the percent total LDH released into cell supernatants in incubations with HIS. By analogy, cytolysis after anoxia/recovery was calculated as [A – U]/[100 – U], where A represents the percent total LDH released into cell supernatants in cells subjected to anoxia/recovery, and U represents the percent total LDH released in untreated cells. Statistics—Data are presented as mean ± S.E. The t statistic was used to determine significant differences between two groups. One-way analysis of variance was used to determine significant differences among groups. Where significant differences were found, individual comparisons were made between groups using the t statistic, and adjusting the critical value according to the Bonferroni method. Two-way analysis of variance was used to determine significant differences in multiple measurements among groups. Complement Induces Phosphorylation of eIF2α and a Reduction in Protein Synthesis—In an earlier study, we demonstrated that expression of ER stress proteins (bip and grp94) was increased in PHN, an in vivo model of C5b-9-induced GEC injury (10Cybulsky A.V. Takano T. Papillon J. Khadir A. Liu J. Peng H. J. Biol. Chem. 2002; 277: 41342-41351Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). To determine if other aspects of the UPR were activated by C5b-9, we examined phosphorylation of eIF2α on serine 51 in rats with PHN at day 14, a time point when these rats show heavy proteinuria. There was significantly greater phosphorylation of eIF2α in glomeruli isolated from rats with PHN, as compared with control glomeruli (Fig. 1). Further studies to delineate the mechanisms of eIF2α phosphorylation were carried out in cultured GECs. Previously, we showed that complement induced expression of ER stress proteins in cultured GECs and that the induction was dependent on C5b-9 assembly (10Cybulsky A.V. Takano T. Papillon J. Khadir A. Liu J. Peng H. J. Biol. Chem. 2002; 277: 41342-41351Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Incubation of GECs with antibody and sublytic doses of complement (NS) to assemble C5b-9" @default.
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• W2119210248 title "Role of the Endoplasmic Reticulum Unfolded Protein Response in Glomerular Epithelial Cell Injury" @default.
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