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Originally published In Press as doi:10.1074/jbc.M500729200 on April 29, 2005

J. Biol. Chem., Vol. 280, Issue 26, 24396-24403, July 1, 2005
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Role of the Endoplasmic Reticulum Unfolded Protein Response in Glomerular Epithelial Cell Injury*

Andrey V. Cybulsky{ddagger}§, Tomoko Takano{ddagger}, Joan Papillon, and Krikor Bijian

From the Department of Medicine, McGill University Health Centre, McGill University, Montreal, Quebec H3A 1A1, Canada

Received for publication, January 20, 2005 , and in revised form, April 27, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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-{alpha} (eIF2{alpha}), thereby generally suppressing translation and decreasing the protein load on a damaged ER. Phosphorylation of eIF2{alpha} was enhanced significantly in glomeruli of proteinuric rats with passive Heymann nephritis, compared with control. In cultured GECs, complement induced phosphorylation of eIF2{alpha} and reduced protein synthesis, and complement-stimulated phosphorylation of eIF2{alpha} was enhanced by overexpression of cPLA2. Ischemia-reperfusion in vitro (deoxyglucose plus antimycin A followed by glucose re-exposure) also stimulated eIF2{alpha} 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 eIF2{alpha} and consequent general reduction in protein synthesis may be a novel mechanism for limiting complement- or ischemia-reperfusion-dependent GEC injury.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The unfolded protein response (UPR)1 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 (1–4). 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 {alpha} subunit of eukaryotic translation initiation factor-2 (eIF2{alpha}). 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 (5). However, prolonged or more substantial ER stress may lead to cell death via apoptosis (2, 4).

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 (6, 7). 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 (6–9). At the same time, complement attack may result in activation of various pathways, including those that restrict injury or facilitate recovery (6–8). Recently we demonstrated that induction of the UPR is a mechanism of protection from complement attack (10).

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 (11, 12). Injury involves the visceral glomerular epithelial cell (GEC), a highly specialized cell type that is involved in the maintenance of glomerular permselectivity (13). Expression of unique cell surface molecules by GECs (e.g. nephrin) appears to be key to maintaining normal ultrastructure and permselective properties (14, 15). In PHN, antibody binds to GEC antigens, and leads to the in situ formation of subepithelial immune complexes (11, 12). C5b-9 assembles in GEC plasma membranes, "activates" GECs, and leads to proteinuria and sublytic GEC injury (11, 12). Based on studies in GEC culture and in vivo, C5b-9 assembly induces transactivation of receptor tyrosine kinases (16), an increase in cytosolic free Ca2+ concentration, and activation of cytosolic phospholipase A2-{alpha} (cPLA2) (17–22). 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 (12). 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 (10). 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 (23–27).

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 eIF2{alpha}, 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.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
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 [{alpha}-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-eIF2{alpha} 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 (28) 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 (29–31) 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 (30, 32). 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 (17, 18, 33). 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 (17, 18). 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 (18, 20).

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 (10, 17, 18). 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 (17, 18). Previous studies have shown that in GECs, complement is not activated in the absence of antibody (17, 18). 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) (10).

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 (10, 19). PAN was induced by a single intravenous injection of PA (80 mg/kg) (10). 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 (17, 18). Lipids were extracted from ~1 x 106 cells and cell supernatants. Methods for extraction and separation of radiolabeled lipids (e.g. [3H]AA) by thin layer chromatography are published (17, 18).

Immunoprecipitation, Immunoblotting, and Northern Hybridization—Preparation of GEC and glomerular lysates and cell fractions was described previously (16, 19). After incubation, ~6 x 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.



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  		FIG. 1.
Phosphorylation of eIF2{alpha} is increased in PHN. Glomeruli were isolated from proteinuric rats with PHN (day 14), or from normal (control) rats (Ctrl). Lysates were immunoblotted with antibody to phospho-eIF2{alpha} serine 51 (p-eIF2{alpha}) (A, representative immunoblot of p-eIF2{alpha}, ~40 kDa; B, densitometric quantification, arbitrary units). *, p < 0.02 PHN versus control. PHN: 7 rats, control: 5 rats.

 
Northern hybridization was performed as described previously (10). 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 [{alpha}-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 x 106 cpm/ml radiolabeled probe for 16 h at 42 °C. Membranes were washed in 1x 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 0.2x 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 (10). 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.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Complement Induces Phosphorylation of eIF2{alpha} 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 (10). To determine if other aspects of the UPR were activated by C5b-9, we examined phosphorylation of eIF2{alpha} 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 eIF2{alpha} in glomeruli isolated from rats with PHN, as compared with control glomeruli (Fig. 1).

Further studies to delineate the mechanisms of eIF2{alpha} 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 (10). Incubation of GECs with antibody and sublytic doses of complement (NS) to assemble C5b-9 induced phosphorylation of eIF2{alpha} (Fig. 2, A and B). Phosphorylation increased significantly above control after 40 min of exposure to complement and returned to basal levels by 6 h. Induction of eIF2{alpha} phosphorylation was associated with a decrease in [35S]methionine/cysteine incorporation into protein, indicating a reduction in protein synthesis (Fig. 2C). For comparison, we examined the effects on eIF2{alpha} phosphorylation of other stimuli previously shown to induce ER stress (10). Chemical anoxia followed by re-exposure to glucose (recovery) is an in vitro model of ischemia-reperfusion injury, shown to induce expression of ER stress proteins in GECs. Exposure to anoxia/recovery induced eIF2{alpha} phosphorylation within 30 min (Fig. 3, A and B). Phosphorylation was increased, but at a lower level at 4 h, and returned to basal levels by 24 h. Another stimulus, UV light, also induced eIF2{alpha} phosphorylation within 30 min (Fig. 3, A and C), but in contrast to complement and anoxia/recovery, phosphorylation persisted for at least 24 h after UV irradiation. Anoxia/recovery reduced [35S]methionine/cysteine incorporation into protein, indicating a decrease in global protein synthesis (Fig. 3D). Tunicamycin is a nucleoside antibiotic that blocks N-linked glycosylation and is believed to cause an accumulation of unfolded proteins in the ER. Interestingly, incubation of GECs with tunicamycin had no significant effect on eIF2{alpha} phosphorylation (Fig. 3E), even though tunicamycin was a potent inducer of ER stress proteins at 24 h (10). The Ca2+ ionophore, ionomycin, can induce ER stress proteins via depletion of Ca2+ from intracellular stores (10). By analogy to tunicamycin, ionomycin was a potent inducer of ER stress proteins in GECs but did not affect eIF2{alpha} phosphorylation (results not shown).



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  		FIG. 2.
Complement induces phosphorylation of eIF2{alpha} and reduction in protein synthesis in GEC. A and B, GECs were incubated with antibody and complement (NS) for 40 min, 6 h or 24 h (HIS in control incubations). Lysates were immunoblotted with antibody to phospho-eIF2{alpha} (A, representative immunoblot; B, densitometric quantification). *, p < 0.02; **, p < 0.0001 versus HIS; 6 experiments. C, GECs were incubated with antibody and complement for 40 min. Then, cells were pulsed with [35S]methionine/cysteine for 20 min. *, p < 0.02 versus HIS; 6 experiments.

 
Overexpression of cPLA2 Enhances Complement-mediated Phosphorylation of eIF2{alpha}—In an earlier study, we demonstrated that overexpression of cPLA2 in GEC augmented complement-dependent induction of the ER stress proteins, bip and grp94, and that this enhancement was a direct effect of cPLA2 on phospholipid hydrolysis and not due to AA or its metabolites (10). Moreover, inhibition of endogenous cPLA2 reduced ER stress protein induction by complement. To determine the potential role of cPLA2 in regulating translation during ER stress, eIF2{alpha} phosphorylation was compared in GECs stably transfected with cPLA2, and neo (control) GECs, which express cPLA2 at a lower level (17, 18). In keeping with earlier results (17, 18), overexpression of cPLA2 enhanced complement-mediated release of [3H]AA (Fig. 4A). For comparison, we also examined [3H]AA release after exposure to UV light or anoxia/recovery. UV light did not induce any significant increase in free [3H]AA, whereas in response to anoxia/recovery, free [3H]AA increased to a similar extent in both cPLA2-overexpressing and neo GEC (Fig. 4A), suggesting that release of [3H]AA occurred independently of cPLA2. In keeping with the dependence of C5b-9-mediated [3H]AA release on cPLA2, complement-induced phosphorylation of eIF2{alpha} was further enhanced in cPLA2-overexpressing GECs, as compared with neo (Fig. 4B). Anoxia/recovery- or UV light-induced phosphorylation of eIF2{alpha} occurred independently of cPLA2, as increases in eIF2{alpha} phosphorylation were similar in cPLA2-overexpressing and neo GEC (Fig. 4C).



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  		FIG. 3.
Anoxia/recovery (A/R) or UV light induce phosphorylation of eIF2{alpha}. A and B, GECs were incubated with antimycin A, 10 µM plus 2-deoxyglucose, 10 mM, for 90 min (anoxia). Then, cells were incubated in glucose-replete medium for 30 min, 4 h, or 24 h (recovery). In other experiments, GECs were exposed to UV light for 2 min, and were then incubated in medium for 30 min, 4 h, or 24 h (A and C). Lysates were immunoblotted with antibody to phospho-eIF2{alpha}. In B: *, p < 0.0001; **, p < 0.005 versus control (Ctrl); 6 experiments. In C: *, p = 0.05; **, p < 0.001; +, p < 0.005 versus control; 3 experiments). D, anoxia/recovery reduces protein synthesis (protocol as above). Cells were pulsed with [35S]methionine/cysteine for 20 min. *, p < 0.0001 anoxia/recovery versus control; 5 experiments. E, incubation with tunicamycin (Tunic; 40 min, 4 h, or 24 h) did not induce phosphorylation of eIF2{alpha} in GECs.

 
Complement and Anoxia/Recovery Induce Phosphorylation of PERK—During ER stress, phosphorylation of eIF2{alpha} is mediated via PERK. Activation of PERK is associated with dimerization and phosphorylation on threonine 980 (2) and was assessed by immunoblotting with a phospho-PERK-specific antibody. Incubation of GECs with antibody and complement induced PERK phosphorylation (Fig. 5A). Furthermore, immunoblotting with antibody to PERK showed that in complement-treated GECs, a portion of PERK underwent a gel-mobility shift (i.e. migration of PERK on SDS-PAGE was retarded), in keeping with phosphorylation. Exposure of GECs to anoxia/recovery also induced PERK phosphorylation. Phosphorylation of PERK was faint, but detectable in glomeruli of two of three rats with PHN, but not in any of three control rats (Fig. 5D). These results confirm that phosphorylation of eIF2{alpha} and reduction in protein synthesis following exposure to complement or anoxia/recovery are due to ER stress. In contrast, exposure of cultured GECs to UV light for 4 or 24 h did not result in PERK phosphorylation consistently (data not shown). Thus, UV light-induced phosphorylation of eIF2{alpha} may be mediated via a distinct kinase.



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  		FIG. 4.
Overexpression of cPLA2 enhances complement-mediated release of [3H]AA and phosphorylation of eIF2{alpha}. A, [3H]AA-labeled GEC, stably transfected with cPLA2, or neo (control) GECs (which express cPLA2 at a lower level) were incubated with antibody and complement (2.5% NS) for 40 min (HIS in control incubations). For comparison, GECs were exposed to UV light for 2 min, and then incubated in medium for 30 min, or were subjected to anoxia/recovery (30 min), as in Fig. 3. Complement-induced [3H]AA release was significantly greater in GEC that overexpress cPLA2 as compared with neo (*, p < 0.0001; 5 experiments). UV light did not stimulate [3H]AA release (4 experiments). After anoxia/recovery, free [3H]AA increased in both GEC lines, but there were no significant differences between the two cell lines (3–6 experiments). B, GECs, stably transfected with cPLA2, or neo GECs were incubated with antibody and complement (2.5% or 4.0% NS), as above. Lysates were immunoblotted with antibody to phospho-eIF2{alpha} (representative immunoblot and densitometric quantification). *, p < 0.001; **, p < 0.025 versus HIS; 4 experiments. C, GECs stably transfected with cPLA2, or neo (control) GECs were subjected to anoxia/recovery (A/R) or UV light treatment as described above. Lysates were immunoblotted with antibody to phospho-eIF2{alpha}. Anoxia/recovery- or UV-induced phosphorylation of eIF2{alpha} occurred independently of cPLA2. The band partially visible at the top of the gel is nonspecific.

 
Complement and Anoxia/Recovery-induced Cell Injury Is Enhanced in PERK Knock-out Fibroblasts—To determine if activation of PERK by complement or anoxia/recovery is functionally important, we assessed cell injury in PERK knock-out fibroblasts (28). The protocol allows for complement to increase eIF2{alpha} phosphorylation, but with increasing incubation time and complement dose, a portion of the cells will undergo lysis. After 40 min of incubation, complement lysis (LDH release) was consistently greater in PERK knock-out (–/–) fibroblasts, as compared with wild type (+/+) (Fig. 6A). Thus, inability to activate PERK results in enhanced cytolysis, indicating that PERK plays a functionally important role in limiting the amount of complement-dependent injury.



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  		FIG. 5.
Complement and anoxia/recovery (A/R) induce phosphorylation of PERK threonine 980. A and B, GECs were incubated with antibody and complement (NS) for 40 min (HIS in control incubations). Lysates were immunoprecipitated with antibody to PERK, and then immunoblotted with antibody to phospho-PERK (p-PERK; A) or PERK (B). PERK migrates at ~150 kDa; phosphorylation induces a slight gel mobility shift. C, GECs were subjected to anoxia/recovery (30 min), as in Fig. 3. Lysates were immunoblotted with antibody to phospho-PERK. D, glomeruli were isolated from rats with PHN or control (Ctrl). Lysates were immunoprecipitated with antibody to PERK, and then immunoblotted with antibody to phospho-PERK.

 
In the next series of experiments, we examined the role of PERK in cytotoxicity following exposure to anoxia/recovery. Similar to complement, cytolysis after anoxia/recovery was significantly greater in the PERK knock-out fibroblasts, as compared with wild type (Fig. 6B). Thus, PERK also plays a functionally important role in limiting the amount of cell injury after anoxia/recovery.

ER Stress in PAN—GECs in culture are particularly sensitive to the cytotoxic effect of PA, and injection of this compound into rats induces GEC injury and proteinuria in vivo. Previously, we showed that incubation of cultured GECs with PA increased expression of bip at 2 and 4 h and that preincubation with PA attenuated complement-dependent cytotoxicity (10). In the present study, we show that, unlike complement, PA did not acutely stimulate eIF2{alpha} phosphorylation in cultured GECs (Fig. 7A). By analogy, eIF2{alpha} phosphorylation was not detected consistently in glomeruli from rats with PAN at day 14, a time point at which rats showed heavy proteinuria (Fig. 7B). In contrast, bip expression was clearly increased in the majority of rats with PAN (Fig. 7B). These results suggest that in GECs, PA is selective in activating one aspect of the UPR, but not another, which resembles the effects of tunicamycin and ionomycin. In addition, rats with PHN and with PAN showed heavy proteinuria. Thus, the presence of eIF2{alpha} phosphorylation in PHN, but absence in PAN, suggests that eIF2{alpha} phosphorylation in PHN was not due to an effect of proteinuria, but was due to complement.



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  		FIG. 6.
Complement and anoxia/recovery-induced cell injury is enhanced in PERK knock-out fibroblasts. A, wild type (+/+) and PERK knock-out (–/–) fibroblasts were incubated with antibody and complement (NS) for 40 min (HIS in control incubations). B, fibroblasts were subjected to anoxia/recovery (A/R; 30 min). The protocol was similar to GECs, as described in Fig. 3. Cell injury was monitored as specific release of LDH. *, p < 0.015; **, p < 0.04 PERK –/– versus PERK +/+; 4 experiments (A), 8 experiments (B).

 



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  		FIG. 7.
ER stress in PAN. A, cultured GECs were incubated with or without PA (50 µg/ml) for 30 min, and lysates were immunoblotted with antibody to phospho-eIF2{alpha}. PA did not induce eIF2{alpha} phosphorylation. B, glomeruli were isolated from two normal rats (Ctrl), and four rats with PAN (day 14), and lysates were immunoblotted with antibodies to phospho-eIF2{alpha} (upper panel), or bip (lower panel). eIF2{alpha} phosphorylation was not detected consistently in PAN. Bip expression was increased in the majority of rats with PAN.

 
Effects of ER Stress on Regulation of Translation from the Nephrin 5'-Flanking Region—Activation of PERK and phosphorylation of eIF2{alpha} reduces AUG codon recognition, and consequently, the general rate of translation is reduced. However, translation of certain mRNAs, containing short open reading frames upstream of the principal ATG, may be paradoxically increased during ER stress (34). Nephrin is a component of the GEC filtration slit diaphragm and plays an important role in maintaining glomerular permselectivity. The transcription start site of the mouse nephrin mRNA is ~400 nucleotides upstream of the nephrin translation initiation ATG codon (30, 32). There are three other ATGs within this fragment of the nephrin 5'-flanking region, which potentially encode for short open reading frames (26, 13, and 3 amino acids; Fig. 7A, construct 2). Cultured GECs do not typically express nephrin. To test the potential effect of ER stress on nephrin mRNA translation, we employed DNA constructs consisting of a CMV promoter region (contained within the pEGFP vector) linked with a fragment of mouse nephrin 5'-flanking region and the cDNA encoding enhanced GFP (Fig. 8A, constructs 1–3). The constitutive CMV promoter region contains a transcription start site, and it would drive transcription of ~40 nucleotides of CMV and vector DNA sequence, plus the entire nephrin 5'-flanking fragment and GFP reporter, while translation of GFP would be regulated by the nephrin 5'-flanking region (34). In control experiments (CMV promoter without the nephrin 5'-flanking region; Fig. 8A, construct 4), both transcription and translation of GFP would depend on the CMV promoter region.



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  		FIG. 8.
Effect of ER stress on the regulation of translation by the nephrin 5'-flanking region. A, DNA constructs employed in these experiments consisted of the CMV promoter (CMV P; contained within the pEGFP vector) with a 1.25-kb fragment of the 5'-end of nephrin (N5'(1.25kb); –1.25 kb 5' to the nephrin translation initiation ATG codon), and the cDNA encoding enhanced GFP (construct 1). Construct 2 included the CMV promoter (CMV P) with a fragment of nephrin 5'-flanking region beginning at –391 bp 5' to the nephrin translation initiation ATG codon (N5'(391bp)), and the cDNA encoding enhanced GFP ("1" denotes the translation initiation ATG codon). The three short open reading frames within N5'(391bp) are indicated with arrows. Construct 3 was similar to the second, except that the three ATG sequences were mutated (N5'(391bp)*), abolishing the three short open reading frames. Construct 4 consisted of the CMV promoter and GFP, without N5'(391bp). The solid circle denotes the transcription start site. B and C, COS cells were transiently transfected with CMV promoter-1.25 kb 5'-end nephrin fragment-GFP (N5'(1.25kb)-GFP), or CMV promoter-GFP (GFP) DNAs. At 48 h, lysates were subjected to Northern analysis or immunoblotting. Transfection of the GFP DNA resulted in production of GFP mRNA (B) and GFP polypeptide (C). Transfection of the N5'(1.25kb)-GFP DNA resulted in production of a longer mRNA, consistent with GFP plus the nephrin 5'-flanking fragment (B), but GFP polypeptide was absent (C). D–H, COS cells were transiently transfected with CMV promoter-N5'(391bp)-GFP (N5'(391bp)-GFP), CMV promoter-mutated N5'(391bp)-GFP (N5'(391bp)*-GFP), or CMV promoter-GFP (GFP) DNAs. After 24 h, COS cells were incubated with antimycin A, 10 µM plus 2-deoxyglucose, 10 mM for 90 min (anoxia; A). Then, cells were incubated in glucose-replete medium for 4 h (recovery; R). Control (Ctrl) cells were untreated. Lysates were immunoblotted with anti-GFP antibody. The panels show representative immunoblots and densitometric quantification of the changes in GFP expression in A/R-treated cells/control. D and F, A/R increased GFP expression in N5'(391bp)-GFP transfections, but GFP expression decreased in the absence of N5'(391bp). *, p < 0.04 N5'(391bp)-GFP versus GFP; 6 experiments. E and G, A/R decreased GFP expression in N5'(391bp)*-GFP transfections; 5 experiments. H, in untreated COS cells, basal GFP expression in N5'(391bp)-GFP transfections normalized for GFP transfections was significantly lower (*, p < 0.01) than N5'(391bp)*-GFP transfections normalized for GFP transfections.

 
Preliminary experiments involved transient transfection of COS cells with a plasmid containing the CMV promoter, a 1.25-kb fragment of nephrin 5'-flanking region, and GFP reporter (Fig. 8A, construct 1). Control transfections included the CMV promoter and GFP (Fig. 8A, construct 4). We observed that, in control transfections, synthesis of GFP polypeptide increased steadily for at least the first 48 h after transfection. Fig. 8 (B and C) demonstrates GFP mRNA and GFP polypeptide at 48 h after transfection of the CMV promoter-GFP DNA (construct 4). Transfection of the CMV promoter-nephrin 5'-end 1.25-kb fragment-GFP DNA (construct 1) resulted in production of a longer mRNA, consistent with GFP plus the nephrin 5'-end 1.25-kb fragment, but there was no production of GFP polypeptide (Fig. 8, B and C). Such transcription of the 1.25-kb nephrin 5'-flanking region is unphysiological, because transcription would normally start at around nucleotide –400 in GECs in vivo, but this result indicates that the CMV promoter can indeed induce transcription of both 1.25-kb nephrin 5'-flanking region and GFP mRNAs. There was, however, inefficient translation of GFP, perhaps because the nephrin 5' 1.25-kb fragment may contain structural features that affect translation adversely (see below).

In the next series of experiments, COS cells were transiently transfected with CMV promoter linked to a 391-nucleotide fragment of nephrin 5'-flanking region (beginning near the nephrin transcription start site to the nephrin initiation ATG codon), and GFP reporter (Fig. 8A, construct 2), or with CMV promoter-GFP DNAs (Fig. 8A, construct 4). At 24 h post-transfection (a time point at which GFP polypeptide production is increasing), cells were untreated or were subjected to anoxia/recovery, and translation regulated by nephrin 5'-flanking region was assessed by immunoblotting for GFP, similar to a protocol described by Harding et al. (35). When CMV promoter-GFP was transfected without the nephrin 5'-flanking region (construct 4), anoxia/recovery reduced GFP expression significantly, as compared with untreated control (Fig. 8, D and F), consistent with reduced general protein synthesis (Fig. 3D). In contrast, in the CMV promoter-nephrin 5'-flanking region-GFP transfections (construct 2), anoxia/recovery induced a small but significant increase in GFP expression (Fig. 8, D and F). Thus, ER stress, while inducing a general reduction in protein synthesis (Fig. 3D), appears to preserve synthesis of GFP reporter when the latter is regulated by the nephrin 5'-flanking region. In another set of experiments, we employed the same nephrin 5'-end fragment, except that the three ATG sequences were mutated, preventing any translation of the short open reading frames upstream of the nephrin ATG codon (Fig. 8A, construct 3). In these CMV promoter-mutant nephrin 5'-flanking region-GFP transfections (construct 3), anoxia/recovery reduced GFP production, similar to CMV promoter-GFP (construct 4; Fig. 8, E and G). It should also be noted that, in untreated cells, the amount of GFP polypeptide was substantially lower in CMV promoter-nephrin 5'-flanking region-GFP transfections (construct 2), as compared with CMV promoter-GFP transfection (construct 4; Fig. 8H). However, relative GFP expression increased when the mutant nephrin 5'-flanking region (construct 3) was transfected instead of the wild type (Fig. 8H). The results are thus consistent with the view that basal translation regulated by the nephrin 5'-flanking region appears to be less efficient, as compared with translation regulated by the CMV mRNA sequence, but can be improved by mutating the three nephrin 5'-end ATG sequences. We could not carry out analogous GFP translation experiments using complement to induce ER stress because our nephritogenic antibodies were not able to activate complement in COS-1 cells.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Increased amounts of ER stress proteins have been characterized in experimental models of glomerulonephritis (10) and kidney ischemia-reperfusion injury (36–39). The present study focuses on another aspect of the ER unfolded protein response, i.e. activation of PERK, which leads to phosphorylation of eIF2{alpha}, and suppression of global translation initiation and protein synthesis. This response potentially decreases the protein load on a damaged ER, conserves amino acid pools for essential functions, and facilitates recovery of cells from injury. The present study demonstrates that phosphorylation of PERK and eIF2{alpha}, and consequent general reduction in protein synthesis may be a novel mechanism for limiting complement- or ischemia-reperfusion-dependent GEC injury. First, we demonstrated that phosphorylation of eIF2{alpha} and PERK was enhanced in GEC injury in vivo, i.e. in proteinuric rats with PHN (Fig. 1). In cultured GECs, complement induced phosphorylation of PERK and eIF2{alpha}, which was associated with a reduction in protein synthesis (Figs. 2 and 5). Fibroblasts from PERK-knock-out mice were more susceptible to complement-mediated cytotoxicity, as compared with wild type fibroblasts (Fig. 6), indicating that activation of PERK and suppression of protein synthesis plays an important role in limiting complement-induced injury. Finally, the complement-stimulated phosphorylation of eIF2{alpha} was enhanced by overexpression of cPLA2 (Fig. 4).

In a previous study, we showed that increases in ER stress proteins (bip and grp94) occurred in the PHN model of GEC injury (10). Furthermore, pretreatment of rats with subnephritogenic stimuli that increased ER stress protein expression reduced C5b-9-mediated GEC injury, manifested by proteinuria. In cultured GECs, complement-induced activation of cPLA2 resulted acutely in phospholipid hydrolysis at the membrane of the ER (10) and was associated with leakage of luminal ER stress proteins (bip and grp94) into the cytosol, suggesting that cPLA2-induced phospholipid hydrolysis resulted in impairment of ER membrane integrity. Such damage to the ER membrane could potentially cause leakage of Ca2+ and other ER luminal components, as well as impair ER Ca2+ uptake. Chronic incubation of GEC with complement increased expression of the ER stress proteins, bip and grp94, and these increases were enhanced with cPLA2 activation. Thus, complement is able to activate distinct aspects of the UPR, although with different kinetics. In GECs, eIF2{alpha} phosphorylation occurred acutely, whereas increased expression of bip and grp94 required chronic exposure, which is in keeping with results in other systems.

Similar to complement, ischemia-reperfusion in vitro (chemical anoxia induced by deoxyglucose plus antimycin A, followed by recovery during glucose re-exposure) stimulated PERK and eIF2{alpha} phosphorylation, and reduced protein synthesis (Figs. 3 and 5). Fibroblasts from PERK knock-out mice were more susceptible to anoxia/recovery-mediated cytotoxicity, as compared with wild type fibroblasts (Fig. 6). Unlike complement, the effect of anoxia/recovery eIF2{alpha} phosphorylation was not augmented by overexpression of cPLA2, even though anoxia/recovery stimulated release of AA in GECs (Fig. 4). This result suggests that anoxia/recovery may have stimulated another PLA2 in GECs, such as the Ca2+-independent PLA2 (40), but that activation of this other PLA2 was not coupled to ER stress. Phosphorylation of PERK and eIF2{alpha} and reduction in protein synthesis during ischemia-reperfusion in GECs are in keeping with the up-regulation of bip and grp94 expression demonstrated previously (10). Similar to complement, eIF2{alpha} phosphorylation occurred early, whereas bip and grp94 expression was evident at 24 h. Our results in GECs are in keeping with previous studies (41–43), which showed that PERK and eIF2{alpha} became phosphorylated after experimental brain ischemia. However, unlike GECs, another study reported that brain ischemia is associated with activation of cPLA2 (44).

Exposure of GECs to UV light induced phosphorylation of eIF2{alpha}, but not PERK. Similar to complement and anoxia/recovery, eIF2{alpha} phosphorylation was evident early after UV exposure, but unlike the other two stimuli, phosphorylation persisted for several hours. Three earlier studies demonstrated that UV light can induce eIF2{alpha} phosphorylation, however, in one study this phosphorylation was shown to be dependent on PERK, whereas the two other studies implicated another eIF2{alpha} kinase, GCN2 (45–47). Thus, it is possible that the prolonged kinetics of eIF2{alpha} phosphorylation in GECs may be due to activation of non-UPR pathway(s). Interestingly, incubation of GECs with tunicamycin, Ca2+ ionophore, or with PA did not induce eIF2{alpha} phosphorylation. However, at similar doses, these stimuli (particularly tunicamycin) were potent inducers of bip and grp94 expression (10). Thus, our results suggest that different stimuli may preferentially activate distinct aspects of the UPR. The possibility that different stress conditions can selectively activate only one or two of the ER sensors has been recognized (3), and the reason for this discrepancy will require further study. Also, it is now appreciated that some stimuli at low doses will induce ER stress that is cytoprotective, whereas at higher doses ER stress becomes overwhelming and additional pathways become activated, leading to cell death (1–4). It should be noted that complement did not increase glomerular expression of the transcription factor, CHOP (results not shown), which is typically associated with induction of ER stress-mediated apoptosis (48), and actually, apoptosis is not a feature of PHN.

Nephrin is a protein expressed almost exclusively in GECs in vivo and is a component of the filtration slit diaphragm (14, 15). Because nephrin plays a key role in maintaining glomerular permselectivity, it is important to understand the potential effects of GEC injury on nephrin expression. Analysis of the mouse nephrin DNA sequence upstream of the nephrin ATG codon revealed that there are three other ATGs within the transcribed nephrin mRNA, each of which potentially encodes for a short open reading frame. In unstressed cells, low levels of eIF2{alpha} phosphorylation may direct ribosomes to such upstream open reading frames, such that translation from the principal initiating AUG becomes inefficient. In stressed cells, high levels of eIF2{alpha} phosphorylation may impair interaction of ribosomes with upstream AUGs, which would favor initiation of translation at the principal AUG (2, 4). Thus, it was reasonable to propose that nephrin translation may be differentially regulated during ER stress. Cultured GEC lines do not, however, typically express nephrin, and to study the effect of ER stress on nephrin translation, we established a transient transfection model in COS cells, where a 391-nucleotide fragment of the nephrin 5'-flanking region was fused between the CMV promoter and a GFP reporter (Fig. 8A). Thus, the CMV promoter induced transcription of the nephrin mRNA fragment plus GFP mRNA, whereas translation of GFP would be regulated by the transcribed nephrin mRNA fragment. Under basal conditions, expression of GFP was lower in the presence of the nephrin 5'-flanking region, as compared with CMV promoter alone (Fig. 8). Following exposure of COS cells to ER stress (anoxia/recovery), expression of GFP in the presence of the nephrin 5'-flanking region was increased slightly, whereas expression of GFP in the absence of the nephrin 5'-flanking region was decreased, the latter consistent with a global decrease in protein synthesis (Fig. 3). These results would predict that mutation of the ATG codons within the fragment of nephrin 5'-end should improve basal GFP expression and suppress GFP expression during ER stress, and indeed our data are consistent with this prediction (Fig. 8). Our result with nephrin is analogous to activating transcription factor-4, whose translation is increased during ER stress, whereas global protein synthesis is suppressed; although activating transcription factor-4 translation appears to be markedly up-regulated, whereas the increase in nephrin was modest (34). It should be noted that regulation of translation in genes with short upstream open reading frames is complex, and the 5'-end of nephrin will require further study. For example, upstream open reading frames may regulate translation through various mechanisms, which may depend in part on the number, position, and length of the open reading frames, as well as nucleotide sequence (49). Moreover, translation of certain genes with short upstream open reading frames (e.g. CHOP and GADD34) is basally suppressed, but there does not appear to be translational up-regulation in response to eIF2{alpha} phosphorylation (50).

Activation of PERK and the UPR is important in pancreatic {beta} cell function and glucose homeostasis, as well as in normal osteoclast function (2, 28). Together with our previous study showing up-regulation of ER stress proteins following assembly of C5b-9, the present study indicates that activation of PERK and reduction in protein synthesis is a mechanism that limits complement-induced damage, or facilitates recovery. Furthermore, the UPR restricts ischemia-reperfusion injury. During ER stress, the general reduction in protein synthesis may help to sustain physiological functions and viability of GECs, whereas preservation of nephrin synthesis may facilitate maintenance of glomerular permselectivity. The present study provides further rationale for developing non-toxic methods to modulate the UPR in vivo, which may eventually have applications to therapy of glomerular disease. The importance of these results extends beyond GEC injury. For example, preinduction of the UPR prior to xenotransplantation may potentially be a means of reducing hyperacute xenograft rejection, which is complement-dependent (51), or may offer novel opportunities for the treatment of insulin resistance and type 2 diabetes associated with obesity (52).


   FOOTNOTES
 
* This work was supported in part by Research Grants from the Canadian Institutes of Health Research and the Kidney Foundation of Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} Both authors hold scholarships from the Fonds de la Recherche en Santé du Québec. Back

Awarded a fellowship from the McGill University Health Centre Research Institute. Back

§ To whom correspondence should be addressed: Division of Nephrology, Royal Victoria Hospital, 687 Pine Ave. West, Montreal, Quebec H3A 1A1, Canada. Tel.: 514-398-8148; Fax: 514-843-2815; E-mail: andrey.cybulsky{at}mcgill.ca.

1 The abbreviations used are: UPR, unfolded protein response; AA, arachidonic acid; CMV, cytomegalovirus; cPLA2, cytosolic phospholipase A2-{alpha}; eIF2{alpha}, eukaryotic translation initiation factor 2-{alpha}; 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. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Kaufman, R. J., Scheuner, D., Schroder, M., Shen, X., Lee, K., Liu, C. Y., and Arnold, S. M. (2002) Nat. Rev. Mol. Cell. Biol. 3, 411–421[CrossRef][Medline] [Order article via Infotrieve]
  2. Ron, D. (2002) J. Clin. Invest. 110, 1383–1388[CrossRef][Medline] [Order article via Infotrieve]
  3. Kaufman, R. J. (2002) J. Clin. Invest. 110, 1389–1398[CrossRef][Medline] [Order article via Infotrieve]
  4. Zhang, K., and Kaufman, R. J. (2004) J. Biol. Chem. 279, 25935–25938[Free Full Text]
  5. Hung, C. C., Ichimura, T., Stevens, J. L., and Bonventre, J. V. (2003) J. Biol. Chem. 278, 29317–29326[Abstract/Free Full Text]
  6. Morgan, B. P. (1992) Curr. Top Microbiol. Immunol. 178, 115–140[Medline] [Order article via Infotrieve]
  7. Nicholson-Weller, A., and Halperin, J. A. (1993) Immunol. Res. 12, 244–257[Medline] [Order article via Infotrieve]
  8. Shin, M. L., Rus, H. G., and Niculescu, F. I. (1996) in Biomembranes (Lee, A. G., ed) pp. 119–146, JAI Press, Greenwich, CT
  9. Rus, H. G., Niculescu, F. I., and Shin, M. L. (2001) Immunol. Rev. 180, 49–55[CrossRef][Medline] [Order article via Infotrieve]
  10. Cybulsky, A. V., Takano, T., Papillon, J., Khadir, A., Liu, J., and Peng, H. (2002) J. Biol. Chem. 277, 41342–41351[Abstract/Free Full Text]
  11. Heymann, W., Hackel, D. B., Harwood, S., Wilson, S. G., and Hunter, J. L. (2000) J. Am. Soc. Nephrol. 11, 183–188[Medline] [Order article via Infotrieve]
  12. Cybulsky, A. V., Foster, M. H., Quigg, R. J., and Salant, D. J. (2000) in The Kidney: Physiology and Pathophysiology (Seldin, D. W., and Giebisch, G., eds) 3rd Ed., pp. 2645–2697, Lippincott-Raven, Philadelphia
  13. Pavenstadt, H., Kriz, W., and Kretzler, M. (2003) Physiol. Rev. 83, 253–307[Abstract/Free Full Text]
  14. Kestila, 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., and Tryggvason, K. (1998) Mol. Cell 1, 575–582[CrossRef][Medline] [Order article via Infotrieve]
  15. Wartiovaara, J., Ofverstedt, L. G., Khoshnoodi, J., Zhang, J., Makela, E., Sandin, S., Ruotsalainen, V., Cheng, R. H., Jalanko, H., Skoglund, U., and Tryggvason, K. (2004) J. Clin. Invest. 114, 1475–1483[CrossRef][Medline] [Order article via Infotrieve]
  16. Cybulsky, A. V., Takano, T., Papillon, J., and McTavish, A. J. (1999) Am. J. Pathol. 155, 1701–1711[Abstract/Free Full Text]
  17. Cybulsky, A. V., Monge, J. C., Papillon, J., and McTavish, A. J. (1995) Am. J. Physiol. 269, F739–F749
  18. Panesar, M., Papillon, J., McTavish, A. J., and Cybulsky, A. V. (1997) J. Immunol. 159, 3584–3594[Abstract]
  19. Cybulsky, A. V., Papillon, J., and McTavish, A. J. (1998) Kidney Int. 54, 360–372[CrossRef][Medline] [Order article via Infotrieve]
  20. Cybulsky, A. V., Takano, T., Papillon, J., and McTavish, A. J. (2000) Kidney Int. 57, 1052–1062[CrossRef][Medline] [Order article via Infotrieve]
  21. Leslie, C. C. (1997) J. Biol. Chem. 272, 16709–16712[Free Full Text]
  22. Hirabayashi, T., and Shimizu, T. (2000) Biochim. Biophys. Acta 1488, 124–138[Medline] [Order article via Infotrieve]
  23. Klausner, J. M., Paterson, I. S., Goldman, G., Kobzik, L., Rodzen, C., Lawrence, R., Valeri, C. R., Shepro, D., and Hechtman, H. B. (1989) Am. J. Physiol. 256, F794–F802
  24. Azuma, H., Nadeau, K., Takada, M., Mackenzie, H. S., and Tilney, N. L. (1997) Transplantation 64, 190–197[CrossRef][Medline] [Order article via Infotrieve]
  25. Pagtalunan, M. E., Olson, J. L., Tilney, N. L., and Meyer, T. W. (1999) J. Am. Soc. Nephrol. 10, 366–373[Abstract/Free Full Text]
  26. Smoyer, W. E., Ransom, R., Harris, R. C., Welsh, M. J., Lutsch, G., and Benndorf, R. (2000) J. Am. Soc. Nephrol. 11, 211–221[Abstract/Free Full Text]
  27. Valdivielso, J. M., Crespo, C., Alonso, J. R., Martinez-Salgado, C., Eleno, N., Arevalo, M., Perez-Barriocanal, F., and Lopez-Novoa, J. M. (2001) Am. J. Physiol. 280, R771–R779
  28. Harding, H. P., Zeng, H., Zhang, Y., Jungries, R., Chung, P., Plesken, H., Sabatini, D. D., and Ron, D. (2001) Mol. Cell 7, 1153–1163[CrossRef][Medline] [Order article via Infotrieve]
  29. Moeller, M. J., Kovari, I. A., and Holzman, L. B. (2000) J. Am. Soc. Nephrol. 11, 2306–2314[Abstract/Free Full Text]
  30. Moeller, M. J., Sanden, S. K., Soofi, A., Wiggins, R. C., and Holzman, L. B. (2002) J. Am. Soc. Nephrol. 13, 1561–1567[Abstract/Free Full Text]
  31. Michaud, J. L., Lemieux, L. I., Dube, M., Vanderhyden, B. C., Robertson, S. J., and Kennedy, C. R. (2003) J. Am. Soc. Nephrol. 14, 1200–1211[Abstract/Free Full Text]
  32. Beltcheva, O., Kontusaari, S., Fetissov, S., Putaala, H., Kilpelainen, P., Hokfelt, T., and Tryggvason, K. (2003) J. Am. Soc. Nephrol. 14, 352–358[Abstract/Free Full Text]
  33. Coers, W., Reivinen, J., Miettinen, A., Huitema, S., Vos, J. T., Salant, D. J., and Weening, J. J. (1996) Exp. Nephrol. 4, 184–192[Medline] [Order article via Infotrieve]
  34. Lu, P. D., Harding, H. P., and Ron, D. (2004) J. Cell Biol. 167, 27–33[Abstract/Free Full Text]
  35. Harding, H. P., Zhang, Y., and Ron, D. (1999) Nature 397, 271–274[CrossRef][Medline] [Order article via Infotrieve]
  36. Kuznetsov, G., Bush, K. T., Zhang, P. L., and Nigam, S. K. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 8584–8589[Abstract/Free Full Text]
  37. Benjamin, I. J., and McMillan, D. R. (1998) Circ. Res. 83, 117–132[Abstract/Free Full Text]
  38. Bush, K. T., Keller, S. H., and Nigam, S. K. (2000) J. Clin. Invest. 106, 621–626[Medline] [Order article via Infotrieve]
  39. Paschen, W. (2001) Cell Calcium 29, 1–11[CrossRef][Medline] [Order article via Infotrieve]
  40. Six, D. A., and Dennis, E. A. (2000) Biochim. Biophys. Acta 1488, 1–19[Medline] [Order article via Infotrieve]
  41. Kumar, R., Azam, S., Sullivan, J. M., Owen, C., Cavener, D. R., Zhang, P., Ron, D., Harding, H. P., Chen, J. J., Han, A., White, B. C., Krause, G. S., and DeGracia, D. J. (2001) J. Neurochem. 77, 1418–1421[CrossRef][Medline] [Order article via Infotrieve]
  42. Little, E., Tocco, G., Baudry, M., Lee, A. S., and Schreiber, S. S. (1996) Neuroscience 75, 209–219[CrossRef][Medline] [Order article via Infotrieve]
  43. DeGracia, D. J., Kumar, R., Owen, C. R., Krause, G. S., and White, B. C. (2002) J. Cereb. Blood Flow Metab. 22, 127–141[CrossRef][Medline] [Order article via Infotrieve]
  44. Bonventre, J. V., Huang, Z., Taheri, M. R., O'Leary, E., Li, E., Moskowitz, M. A., and Sapirstein, A. (1997) Nature 390, 622–625[CrossRef][Medline] [Order article via Infotrieve]
  45. Wu, S., Hu, Y., Wang, J. L., Chatterjee, M., Shi, Y., and Kaufman, R. J. (2002) J. Biol. Chem. 277, 18077–18083[Abstract/Free Full Text]
  46. Deng, J., Harding, H. P., Raught, B., Gingras, A. C., Berlanga, J. J., Scheuner, D., Kaufman, R. J., Ron, D., and Sonenberg, N. (2002) Curr. Biol. 12, 1279–1286[CrossRef][Medline] [Order article via Infotrieve]
  47. Jiang, H. Y., and Wek, R. C. (2005) Biochem. J. 385, 371–380[CrossRef][Medline] [Order article via Infotrieve]
  48. Zinszner, H., Kuroda, M., Wang, X., Batchvarova, N., Lightfoot, R. T., Remotti, H., Stevens, J. L., and Ron, D. (1998) Genes Dev. 12, 982–995[Abstract/Free Full Text]
  49. Morris, D. R., and Geballe, A. P. (2000) Mol. Cell. Biol. 20, 8635–8642[Free Full Text]
  50. Jousse, C., Bruhat, A., Carraro, V., Urano, F., Ferrara, M., Ron, D., and Fafournoux, P. (2001) Nucleic Acids Res. 29, 4341–4351[Abstract/Free Full Text]
  51. Samstein, B., and Platt, J. L. (2001) J. Am. Soc. Nephrol. 12, 182–193[Abstract/Free Full Text]
  52. Ozcan, U., Cao, Q., Yilmaz, E., Lee, A. H., Iwakoshi, N. N., Ozdelen, E., Tuncman, G., Gorgun, C., Glimcher, L. H., and Hotamisligil, G. S. (2004) Science 306, 457–461[Abstract/Free Full Text]

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X. Zheng, X. Zhang, B. Feng, H. Sun, M. Suzuki, T. Ichim, N. Kubo, A. Wong, L. R. Min, M. E. Budohn, et al.
Gene Silencing of Complement C5a Receptor Using siRNA for Preventing Ischemia/Reperfusion Injury
Am. J. Pathol., October 1, 2008; 173(4): 973 - 980.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Renal Physiol.Home page
M. Kitamura
Endoplasmic reticulum stress and unfolded protein response in renal pathophysiology: Janus faces
Am J Physiol Renal Physiol, August 1, 2008; 295(2): F323 - F334.
[Abstract] [Full Text] [PDF]

Home page
 J. Am. Soc. Nephrol.Home page
T. Marumo, K. Hishikawa, M. Yoshikawa, and T. Fujita
Epigenetic Regulation of BMP7 in the Regenerative Response to Ischemia
J. Am. Soc. Nephrol., July 1, 2008; 19(7): 1311 - 1320.
[Abstract] [Full Text] [PDF]

Home page
 J. Am. Soc. Nephrol.Home page
R. Inagi, T. Kumagai, H. Nishi, T. Kawakami, T. Miyata, T. Fujita, and M. Nangaku
Preconditioning with Endoplasmic Reticulum Stress Ameliorates Mesangioproliferative Glomerulonephritis
J. Am. Soc. Nephrol., May 1, 2008; 19(5): 915 - 922.
[Abstract] [Full Text] [PDF]

Home page
 J. Am. Soc. Nephrol.Home page
A. Nakajo, J. Khoshnoodi, H. Takenaka, E. Hagiwara, T. Watanabe, H. Kawakami, R. Kurayama, Y. Sekine, F. Bessho, S. Takahashi, et al.
Mizoribine Corrects Defective Nephrin Biogenesis by Restoring Intracellular Energy Balance
J. Am. Soc. Nephrol., September 1, 2007; 18(9): 2554 - 2564.
[Full Text] [PDF]

Home page
 Hum Mol GenetHome page
Z. Zhang, Y.-C. Lee, S.-J. Kim, M. S. Choi, P.-C. Tsai, A. Saha, H. Wei, Y. Xu, Y.-J. Xiao, P. Zhang, et al.
Production of lysophosphatidylcholine by cPLA2 in the brain of mice lacking PPT1 is a signal for phagocyte infiltration
Hum. Mol. Genet., April 1, 2007; 16(7): 837 - 847.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Renal Physiol.Home page
A. V. Cybulsky, T. Takano, J. Papillon, W. Hao, A. Mancini, J. A. Di Battista, and M. I. Cybulsky
The 3'-untranslated region of the Ste20-like kinase SLK regulates SLK expression
Am J Physiol Renal Physiol, February 1, 2007; 292(2): F845 - F852.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Renal Physiol.Home page
K.-H. Han, U-Y. Lee, Y.-S. Jang, Y. M. Cho, Y. M. Jang, I.-A Hwang, J. Y. Ghee, S.-W. Lim, W.-Y. Kim, C. W. Yang, et al.
Differential regulation of B/K protein expression in proximal and distal tubules of rat kidneys with ischemia-reperfusion injury
Am J Physiol Renal Physiol, January 1, 2007; 292(1): F100 - F106.
[Abstract] [Full Text] [PDF]

Home page
 J. Am. Soc. Nephrol.Home page
B. S. Kasinath, M. M. Mariappan, K. Sataranatarajan, M. J. Lee, and D. Feliers
mRNA Translation: Unexplored Territory in Renal Science
J. Am. Soc. Nephrol., December 1, 2006; 17(12): 3281 - 3292.
[Abstract] [Full Text] [PDF]

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