m2:04694 (revised) complement c5b-9 membrane attack

M2:04694 (Revised)

COMPLEMENT C5b-9 MEMBRANE ATTACK COMPLEX INCREASES EXPRESSION OF

ENDOPLASMIC RETICULUM STRESS PROTEINS IN GLOMERULAR EPITHELIAL CELLS*

Andrey V. Cybulsky, Tomoko Takano, Joan Papillon, Abdelkrim Khadir, Jianhong Liu and Hongwei

Peng

Department of Medicine, McGill University Health Centre, and Department of Physiology, McGill

University, Montreal, Quebec, Canada, H3A 1A1.

Running title: C5b-9 increases endoplasmic reticulum stress proteins

Address for correspondence: Andrey V. Cybulsky, MD, Division of Nephrology, Royal Victoria

Hospital, 687 Pine Avenue West, Montreal, Quebec, Canada H3A 1A1. Tel: (514) 398-8148. Fax:

(514) 982-0897. E-mail: [email protected].

Copyright 2002 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on August 20, 2002 as Manuscript M204694200 by guest on M

arch 27, 2018http://w

ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

2

SUMMARY

In the passive Heymann nephritis (PHN) model of membranous nephropathy, complement

C5b-9 induces glomerular epithelial cell (GEC) injury, proteinuria, and activation of cytosolic

phospholipase A2 (cPLA2). This study addresses the role of endoplasmic reticulum (ER) stress

proteins (bip, grp94) in GEC injury. GEC that overexpress cPLA2 (produced by transfection), and

“neo” GEC (which express cPLA2 at a lower level) were incubated with complement (40 min), and

leakage of constitutively-expressed bip and grp94 from ER into cytosol was measured to monitor

ER injury. Greater leakage of bip and grp94 occurred in complement-treated GEC that overexpress

cPLA2, as compared with neo, implying that cPLA2 activation perturbed ER membrane integrity.

After chronic incubation (4-24 h), C5b-9 increased bip and grp94 mRNAs and proteins, and the

increases were dependent on cPLA2. Expression of bip-antisense mRNA reduced stimulated bip

protein expression, and enhanced complement-dependent GEC injury. Glomerular bip and grp94

proteins were upregulated in proteinuric rats with PHN, as compared with normal control.

Pretreatment of rats with tunicamycin or adriamycin, which increase ER stress protein expression,

reduced proteinuria in PHN. Thus, C5b-9 injures the ER and enhances ER stress protein expression,

in part, via activation of cPLA2. ER stress protein induction is a novel mechanism of protection from

complement attack.

by guest on March 27, 2018

http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

3

INTRODUCTION

Activation of the complement cascade near a cell surface leads to 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 (1,2). Assembly of C5b-9 results in formation

of transmembrane channels or rearrangement of membrane lipids with loss of membrane integrity.

Nucleated cells require multiple C5b-9 lesions for lysis, but at lower doses, C5b-9 induces sublethal

(sublytic) injury, and various metabolic effects (1-8). An example of sublytic C5b-9-mediated cell

injury in vivo is passive Heymann nephritis (PHN)1 in the rat, a widely-accepted model of human

membranous nephropathy (9). In PHN, antibody binds to visceral glomerular epithelial cell (GEC)

antigens, and leads to the in situ formation of subepithelial immune complexes (9,10). C5b-9

assembles in GEC plasma membranes, “activates” GEC, and leads to proteinuria and sublytic GEC

injury (9-14). Based on studies in GEC culture and in vivo, C5b-9 assembly induces transactivation

of receptor tyrosine kinases (15), an increase in cytosolic free Ca2+ concentration ([Ca2+]i), and

activation of protein kinase C, as well as cytosolic phospholipase A2-α (cPLA2)(16-19). cPLA2 is an

important mediator of C5b-9-dependent GEC injury. First, arachidonic acid (AA) released by cPLA2

is metabolized in GEC via cyclooxygenases-1 and -2 to prostaglandin E2 and thromboxane A2 (20),

and inhibition of prostanoid production reduces proteinuria in PHN (21-25) and in human

membranous nephropathy (26). Second, cPLA2 may mediate GEC injury more directly (16).

cPLA2 (group IV PLA2) is regulated by [Ca2+]i and phosphorylation (27,28). Stimuli that

raise [Ca2+]i in the submicromolar range may induce translocation of cPLA2 from cytosol to an

intracellular membrane, where cPLA2 would bind via its N-terminal Ca2+-dependent lipid binding

or C2 domain, gaining access to phospholipid substrate. We have demonstrated that cPLA2 is the

major PLA2 in GEC, and that complement enhances cPLA2 phosphorylation and catalytic activity


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

4

(16). Moreover, C5b-9 increases free AA in GEC, and release of AA is amplified by overexpression

of cPLA2 (16,17). Recently, we demonstrated that in GEC, cPLA2 localizes and hydrolyzes

phospholipids at the plasma membrane, the membrane of the endoplasmic reticulum (ER), and the

nuclear envelope, but not at mitochondria nor Golgi (29). Thus, the activation of cPLA2 and release

of AA are compartmentalized to specific organelles, but it is presently unknown if hydrolysis of

membrane phospholipids by cPLA2 leads to injury of these organelles in GEC.

Based on studies in cell culture, complement attack may injure cells, but may also activate

pathways that restrict injury or facilitate recovery. One mechanism of protection from complement

attack is “ectocytosis” (shedding) of C5b-9 complexes from cell membranes (1,2). There are

undoubtedly other recovery mechanisms, which require delineation. For example, exposure of cells

to environmental stress increases expression of stress proteins in cellular compartments, such as the

ER. There are several types of ER stress responses, including the “unfolded protein response” (30-

32). ER stress proteins are believed to be induced by accumulation of abnormal proteins or depletion

of ER Ca2+ stores, and include the glucose-related proteins (grp), grp94, bip (grp78) and erp72.

Tunicamycin, a nucleoside antibiotic that blocks N-linked glycosylation and is believed to cause an

accumulation of unfolded proteins in the ER, and the Ca2+ ionophore, ionomycin, which can deplete

Ca2+ from intracellular stores, can induce ER stress proteins (30-32). Under normal conditions, ER

stress proteins might serve as protein chaperones for exocytosis from ER, and they might 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 (31,32). Generally, regulation of ER

stress proteins appears to be controlled at the transcriptional level, and requires several hours and

de novo protein synthesis. Moreover, exposure of cells to mild stress, which induces ER stress

proteins, may be protective to additional insults (33,34), although prolonged or more substantial ER


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

5

stress may lead to cell death by apoptosis (35).

The aim of the present study was to determine if C5b-9-mediated activation of cPLA2 and

GEC injury are associated with induction of the ER unfolded protein response. We demonstrate that

C5b-9 may damage the ER and enhance expression of ER stress proteins via activation of cPLA2.

Induction of ER stress proteins limits complement-dependent GEC injury in culture and in PHN.

EXPERIMENTAL PROCEDURES

Materials

Tissue culture media, Trizol reagent, and Random Primer DNA Labelling System were

obtained from Life Technologies (Burlington, Ontario). Complement-deficient sera and purified C8

were purchased from Quidel (San Diego, CA). [3H]AA (100 Ci/mmol) and [α-32P]dCTP (3000

Ci/mmol) were purchased from New England Nuclear (Boston, MA). Adriamycin, puromycin

aminonucleoside, tunicamycin, ionomycin were from Sigma Chemical Co. (St. Louis, MO). Methyl

arachidonyl fluorophosphonate (MAFP) was from Biomol (Plymouth Meeting, PA). Antibodies to

bip, grp94 and erp72 were purchased from Stressgen Biotechnologies Corp. (Victoria, BC).

Fluorescein-conjugated antibodies were from Cedarlane Laboratoires Ltd. (Hornby, Ontario).

Electrophoresis and immunoblotting reagents were from Biorad Laboratories (Mississauga, Ontario).

Bip cDNA was kindly provided by Dr. M. Gething (University of Melbourne) and grp94 and erp72

cDNAs by Dr. M. Green (St. Louis University). Bip antisense cDNA constructed in pcDNA3 was

kindly provided by Dr. J. Stevens (University of Vermont) (33).

Cell culture and transfection

Rat GEC culture and characterization has been detailed previously (36). GEC were cultured


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

6

in K1 medium on plastic substratum. The method for stable transfection of GEC (e.g. to produce

GEC that stably overexpress cPLA2) was described previously (16,17), and the same method was

used to produce GEC that stably express bip antisense cDNA. GEC that express only the neomycin-

resistance gene (neo) were used as control. Briefly, GEC were co-transfected with the expression

vector of interest, and pRc/RSV (molar ratio 12.5:1), using a CaPO4 technique. Colonies of GEC

resistant to G418 were isolated, and expanded. Transfectants were selected for a high level of cPLA2

activity and protein expression, or in the case of bip antisense cDNA, inhibition of stimulated bip

protein expression (determined by immunoblotting). Compared with parental or neo GEC, PLA2

activity was increased ~5-fold in GEC that overexpress cPLA2, but was nevertheless within a

physiological range, and comparable to the level in Madin Darby canine kidney cells (16,17).

Incubation of GEC with antibody and complement

To activate complement, GEC in monolayer culture were incubated with rabbit anti-GEC

antiserum (5% vol/vol) 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 22oC (16,17). GEC were then incubated with normal human serum (diluted in Krebs-

Henseleit buffer in acute incubations, or K1 medium in chronic incubations), or with heat-inactivated

(decomplemented) human serum (56oC) in controls, at 37oC. In some experiments, antibody-

sensitized GEC were incubated with C8-deficient human serum, or C8-deficient serum supplemented

with purified C8 (80 µg/ml undiluted serum). We have generally used heterologous complement in

order to facilitate studies with complement-deficient sera, and to minimize possible signaling via

complement-regulatory proteins, however, in earlier studies, results of several experiments were

confirmed with homologous (rat) complement. There was some variability in sublytic and lytic


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

7

concentrations of complement among batches of sera. In GEC, complement is not activated in the

absence of antibody, and antibody does not independently affect free [3H]AA (16-18).

Induction and characterization of PHN

Sheep anti-rat Fx1A was prepared as described previously (37). Male Sprague-Dawley rats

(150 g; Charles River, St. Constant, Quebec) were injected with 350 µl of sheep anti-Fx1A

antiserum. This batch of antiserum caused little proteinuria in the heterologous phase (day 5 or

earlier) but induced significant proteinuria in the autologous phase (days 7-14). Rats were sacrificed

at various intervals, and glomeruli were isolated by differential sieving (37).

Immunofluorescence microscopy for sheep IgG, rat IgG and rat C3 was performed as

described previously (11). Briefly, 4 µm cryostat kidney sections were stained with fluorescein-

conjugated IgG fractions of monospecific antisera. The immunofluorescence signals from whole

glomeruli were evaluated using a Leitz immunofluorescence microscope with visual output

connected to a Nikon UFX-II photomultiplier and camera, similar to a method described earlier (38).

Densitometry readings were done under immersion oil, and the biopsy material was magnified 400

times, such that the glomerular cross-section filled the majority of the densitometry field. The time

required to collect an image from a glomerulus is inversely proportional to immunofluorescence

intensity. Times required to collect images from three representative glomeruli in each tissue section

were recorded and averaged.

Serum creatinine measurements were performed in the clinical biochemistry laboratory of

the Royal Victoria Hospital (Montreal, Quebec).

Measurement of free [3H]AA


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

8

GEC phospholipids were labelled to isotopic equilibrium with [3H]AA for 48-72 h, as

detailed previously (16-20). Lipids were extracted from ~1 × 106 cells and cell supernatants.

Methods for extraction and separation of radiolabelled lipids by thin layer chromatography are

published (16-20).

Northern hybridization and immunoblotting

Northern hybridization was performed as described previously (20). Briefly, total RNA was

extracted from GEC using the Trizol reagent. RNA was separated by gel electrophoresis (1% agarose

containing 1.9% formaldehyde), and was transferred to a nylon membrane. Coding regions of rat

bip, grp94 and erp72 cDNAs were radiolabelled with [α-32P]dCTP using the Random Primer DNA

Labelling System. Membranes were hybridized in buffer containing 1% BSA, 7% SDS, 0.5 M

sodium phosphate, pH 6.8, 1 mM EDTA, and 1-2 × 106 cpm/ml of radiolabelled probe for 16 h at

42oC. Membranes were washed in buffer containing 0.5% BSA, 5% SDS, 40 mM sodium phosphate,

pH 6.8, 1 mM EDTA twice for 20 min at 65oC, and then buffer containing 1% SDS, 40 mM sodium

phosphate, pH 6.8, 1 mM EDTA four times for 20 min at 65oC. Membranes were exposed to X-ray

film with an intensifying screen at -70oC for 48-72 h.

For immunoblotting, GEC or glomerular lysates were mixed with Laemmli sample buffer,

and were subjected to SDS-PAGE. Proteins were transferred to nitrocellulose paper, blocked with

5% fat-free dry milk in 20 mM Tris, 50 mM NaCl, pH 7.5, with 0.05% Tween 20 (15-17). Blots

were incubated with primary antibodies. After washing with Tris-buffered saline-Tween 20 solution,

blots were incubated with secondary antibody and developed using the enhanced chemiluminescence

technique.


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

9

Quantification of Northern blots and immunoblots was performed by densitometry. Blots

were scanned, specific bands of interest were selected, and the density of the bands was measured

using NIH Image software. Results are expressed in arbitrary units. Preliminary studies demonstrated

that there was a linear relationship between densitometric measurements and the amounts of protein

loaded onto gels.

Measurement of complement-dependent cytotoxicity

Complement-mediated cell lysis was determined by measuring release of lactate

dehydrogenase (LDH), as described previously (15,36). Specific release of LDH was calculated as

[NS-HIS]/[100-HIS], where NS represents the percent of total LDH released into cell supernatants

in incubations with normal serum, and HIS is the percent of total LDH released into cell supernatants

in incubations with heat-inactivated serum (36).

Statistics

Data are presented as mean±SEM. The t statistic was used to determine significant

differences between two groups. For more than two groups, one-way analysis of variance (ANOVA)

was used to determine significant differences among groups, and 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

Complement activates cPLA2 and induces GEC injury


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

10

Incubation of GEC with antibody and complement increases free AA (16-19). The increase

is evident within 40 min, and persists for at least 24 h (217±46% control at 24 h). Stable

overexpression of cPLA2 in GEC amplifies the complement-induced increase in free AA (465±183%

control at 24 h) (19). Table I shows levels of free [3H]AA in complement-treated GEC that stably

overexpress cPLA2. Incubation of antibody-sensitized GEC with normal serum as the complement

source increased [3H]AA more than 3-fold, as compared with heat-inactivated (decomplemented)

serum. C8 is the key component of the C5b-9 membrane attack complex. Incubation of GEC with

C8-deficient serum reconstituted with purified C8 also increased free [3H]AA more than 3-fold, as

compared with unreconstituted C8-deficient serum or with heat-inactivated serum. Therefore, PLA2-

mediated release of AA is due to assembly of C5b-9, while C5b-7 has no significant effect.

Complement-induced activation of cPLA2 leads to phospholipid hydrolysis at the membrane

of the ER (29). We assessed whether hydrolysis of ER membrane phospholipids may induce injury

to the ER compartment. Resting GEC express the ER stress proteins, bip and grp94, and these

proteins are localized mainly in the ER (Fig. 1). The integrity of the ER membrane was monitored

by the leakage of constitutively-expressed bip and grp94, from the ER compartment into the cytosol.

Brief incubation of GEC with complement (2.5% normal serum; 40 min) induced significant

increases in the amounts of bip or grp94 in the cytosol of the GEC that overexpress cPLA2 (a

representative immunoblot is shown in Fig. 1A, and densitometric quantification in Table IIA). The

amount of bip or grp94 in the cytosol represented only a minor proportion of the total (<15%), and

brief incubation with complement did not induce significant increases in total ER stress protein

expression (Fig. 1). At the concentration of complement that induced increases in bip and grp94 in

the cytosol of GEC that overexpress cPLA2, there were no significant increases in bip or grp94 in

the cytosol of neo GEC (Table IIA). However, increases in bip and grp94 could be detected in the


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

11

cytosol of neo GEC when the concentration of complement was increased (4.0% normal serum; Fig.

1B, Table IIB). These results suggest that activation of cPLA2 by complement perturbed the ER

membrane sufficiently to allow a small portion of bip or grp94 to leak into the cytosol. Sublytic

complement did not cause bip to leak out of cells, i.e. through the plasma membrane (not shown).

Overexpression of cPLA2 in GEC enhanced complement-mediated cytotoxicity (16). To

assess the role of endogenous cPLA2 in complement-mediated injury, antibody-sensitized neo GEC

were incubated with serially-increasing doses of complement in the presence or absence of the

cPLA2 inhibitor, MAFP (39). GEC injury was quantified by monitoring release of LDH, a sensitive

measure of cell viability. Cytolysis was lower in the presence of MAFP (Table III), suggesting that

inhibition of cPLA2 activity reduces complement-mediated injury.

Complement enhances expression of ER stress proteins in GEC

Assembly of C5b-9 may result in cell injury, and may in parallel activate mechanisms that

restrict or limit the amount of injury. Since C5b-9-induced activation of cPLA2 appeared to disrupt

the integrity of the ER membrane, as a consequence, the function of the ER may have been altered.

Thus, we proceeded to assess whether exposure of GEC to complement attack would lead to

increased expression of ER stress proteins. Incubation of antibody-sensitized GEC that overexpress

cPLA2 with sublytic complement for 4 h resulted in increased mRNA levels of bip, grp94 and erp72

(Fig. 2A). The signals on Northern blots were quantified by densitometric analysis. Bip, grp94 and

erp72 mRNAs increased 1.7±0.5-fold (N=4, p<0.02), 1.4±0.2-fold (N=6, p=0.01), and 1.3±0.2-fold

(N=4, p<0.04), respectively, as compared with control. Complement had no effect on the mRNA

level of the cytosolic stress protein, Hsp70 (not shown). Incubation of GEC with C5b-9 induces an

increase in [Ca2+]i and activates protein kinases (17,18). To determine the effect of increased [Ca2+]i


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

12

on bip, grp94 and erp72 mRNAs, GEC were incubated with the Ca2+ ionophore, ionomycin, for 24

h. Ionomycin increased bip, grp94 and erp72 mRNAs (Fig. 2B), 1.7-fold, 2.3-fold, and 1.4-fold,

respectively, as compared with control (2 experiments). The effect of tunicamycin, one of the most

potent inducers of ER stress proteins, is shown for comparison (Fig. 2B). Tunicamycin increased bip,

grp94 and erp72 mRNAs 5.5-fold, 5.4-fold, and 3.1-fold, respectively, as compared with control (2

experiments).

The next series of experiments addressed changes in protein levels of bip, grp94, and erp72

in GEC, and the role of cPLA2. Incubation with ionomycin for 24 h increased ER stress protein

expression (Fig. 3). The effect of ionomycin was more prominent in the GEC that overexpress cPLA2

(1.6-2.7-fold increases), as compared with neo (1.3-1.7-fold increases). Tunicamycin (shown for

comparison as a positive control) was generally a more potent inducer of ER stress proteins, and its

effect was similar in neo GEC and GEC that overexpress cPLA2 (1.7-2.9-fold increases). These

results indicate that the effect of ionomycin is, at least in part, mediated via activation of cPLA2,

while changes secondary to tunicamycin are cPLA2-independent. Chronic incubation of antibody-

sensitized GEC with complement (4-24 h) induced increases in bip and grp94 protein expression

(Fig. 4A-C). In these experiments, the increases in protein levels (30-50% above control at 24 h)

were evident mainly in the GEC that overexpress cPLA2. An upward trend was present in neo GEC,

but the change did not reach statistical significance. It should be noted that complement-induced

increases in [Ca2+]i are similar in magnitude in neo GEC and in GEC that overexpress cPLA2 (17).

Thus, the effect of complement on bip and grp94 expression is, at least in part, mediated via

activation of cPLA2. To address the involvement of endogenous cPLA2, antibody-sensitized neo

GEC were incubated with a higher concentration of complement, with or without the cPLA2

inhibitor, MAFP. At the higher dose, significant complement-induced increases in bip and grp94


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

13

protein levels were evident in neo GEC, and the increases were inhibited by MAFP (Fig. 4D). The

complement-induced increases in bip and grp94 were functionally important, as demonstrated in the

studies of complement-induced injury (discussed below). We did not study the effect of complement

on erp72, because preliminary studies demonstrated that complement-induced changes in erp72

protein levels were not readily detectable.

Increases in the expression of ER stress proteins could be due to cPLA2-mediated

phospholipid hydrolysis and membrane injury, or secondary to signals triggered by cPLA2-generated

lipid products or their metabolites (prostanoids). GEC that overexpress cPLA2 were incubated with

ionomycin, or ionomycin plus the cyclooxygenase inhibitor, indomethacin (10 µM), at a

concentration known to inhibit cyclooxygenase activity (20). Ionomycin increased bip and grp94

expression (as in Fig. 3), and this effect was not inhibited by indomethacin, suggesting that

prostanoid production was not involved in ER stress protein upregulation (Fig. 5). Products of PLA2-

induced phospholipid hydrolysis include AA and lysophospholipid. Addition of AA (10 µM) did not

enhance bip or grp94 expression (Fig. 5), although a similar concentration of AA can induce

prostanoid generation in GEC (20). Lysophosphatidylcholine was also ineffective in enhancing ER

stress protein expression (Fig. 5). Thus, increases in bip and grp94 are most likely triggered by

cPLA2-mediated phospholipid hydrolysis and membrane perturbation.

Bip-antisense mRNA expression and effects on GEC injury

To determine if complement-mediated induction of ER stress proteins is functionally

important, GEC were stably transfected with bip antisense cDNA. Three clones that demonstrated

a reduction in tunicamycin-stimulated bip protein expression, as compared with neo GEC were

selected for further studies (Fig. 6A). In these bip antisense clones, tunicamycin generally stimulated


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

14

increases in grp94 and erp72 protein expression similar to neo GEC. The increases tended to be

blunted in one of the clones (Fig. 6A), however, previous studies have reported that bip antisense

mRNA expression may also decrease induction of other ER stress proteins (33,40). When the GEC

clones that express bip antisense mRNA were incubated with 1 µM ionomycin for 24 h, there was

greater cytolysis (release of LDH) in these clones, as compared with neo GEC (Table IV). Thus,

inhibition of the ionomycin-induced increase in bip expression by bip antisense mRNA results in

enhanced GEC injury. There was also a tendency toward greater cytolysis with 24 h tunicamycin

incubation, but the difference was not statistically significant (Table IV). By light microscopy, bip

antisense clones had normal morphology, and proliferated at rates similar to neo GEC under standard

culture conditions.

The bip antisense clones and neo GEC were sensitized with antibody, and incubated with

serially-increasing doses of complement that induced minimal to moderate cell lysis at 18 h. This

protocol allows for C5b-9 to increase ER stress protein expression (Fig. 4), but with increasing

incubation time and complement dose, a portion of the cells will undergo lysis. After 18 h of

incubation, lysis (LDH release) was consistently greater in the GEC clones that express bip antisense

mRNA (Fig. 6B). Thus, attenuation of the C5b-9-induced increase in bip expression by bip antisense

mRNA results in enhanced cytolysis, indicating that induction of bip plays a functionally important

role in limiting the amount of complement-dependent injury. In other experiments, neo GEC and the

GEC that express bip antisense mRNA were incubated with antibody and serially-increasing doses

of complement for only 40 min. During brief incubation, there is insufficient time for C5b-9 to

increase ER stress protein expression. After 40 min, complement lysis was not enhanced in the bip

antisense clones, and actually tended to be lower in these clones, as compared with neo (Table V).

Thus, basal expression of ER stress proteins did not modulate complement cytotoxicity. Moreover,


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

15

these experiments indicate that expression of bip antisense mRNA in GEC does not exacerbate

complement-induced lysis non-specifically.

In the next series of experiments, we assessed whether other stimuli that induce ER stress

protein expression would affect complement-mediated GEC injury. We observed that some stimuli

provided protective effects, while others enhanced complement lysis. GEC in culture are particularly

sensitive to the cytotoxic effect of puromycin aminonucleoside (36), and injection of this compound

into rats may induce GEC injury and proteinuria in vivo (41). Incubation of cultured GEC with

puromycin aminonucleoside increased expression of bip at 2 and 4 h, however, expression returned

to basal levels at 24 h (Fig. 7A). In GEC that were preincubated with puromycin aminonucleoside

for 3 h, complement-dependent cytolysis was reduced significantly (Fig. 7B). We then examined the

effects of pretreatment with tunicamycin and ionomycin (Fig. 3). Unlike puromycin

aminonucleoside, 24 h preincubation with tunicamycin (a potent inducer of ER stress proteins)

enhanced complement-mediated lysis (Table VI). Preincubation with tunicamycin for only 6 h did

not affect complement-mediated GEC injury significantly, but this shorter preincubation time did

not increase expression of ER stress proteins consistently (data not shown). Ionomycin pretreatment

tended to enhance complement-mediated injury, although the change was not statistically significant

(Table VI). We also evaluated if exposure of GEC to an in vitro model of ischemia-reperfusion

injury (chemical anoxia) would increase expression of ER stress proteins, and protect from

subsequent complement attack. Incubation of GEC with 2-deoxyglucose + antimycin A for 90 min

(to inhibit glycolytic and/or oxidative metabolism), followed by reexposure to glucose-replete culture

medium for 24 h resulted in increased expression of bip and grp94 (Fig. 8). This protocol did not

protect, but rather enhanced complement-mediated injury significantly (Table VI).


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

16

Expression of ER stress proteins is enhanced in GEC injury in vivo

The above experiments demonstrated that C5b-9 increases bip and grp94 protein expression

in cultured GEC, but it is important to determine if analogous changes occur in C5b-9-mediated

GEC injury in vivo. To address this question, we assessed levels of bip and grp94 in PHN, where

GEC injury is due to C5b-9 assembly, and is associated with cPLA2 activation and production of

prostanoids. In our model of PHN, proteinuria begins to appear at approximately day 7, and is well-

established at day 13-14 (see below). On day 14, expression of glomerular bip and grp94 was

increased in rats with PHN, as compared with control (Fig. 9A and B). Increases in levels of bip and

grp94 proteins were not detected consistently on days 3 and 7 (data not shown). By analogy to

puromycin aminonucleoside, GEC in vivo are sensitive to the cytotoxic effects of adriamycin, and

injection of rats with adriamycin may lead to GEC injury, in association with proteinuria (41). A

second group of rats was injected with a subnephritogenic dose of adriamycin, i.e. a dose that did

not induce proteinuria for up to 14 days. Glomeruli of these rats showed a significant increase in

grp94 expression, while bip expression showed an upward trend (Fig. 9B). Thus, levels of ER stress

proteins also increased when GEC were injured in vivo by a toxin-mediated mechanism. Moreover,

the results with adriamycin suggest that development of proteinuria is not essential for enhanced ER

stress protein expression in GEC. Finally, injection of rats with tunicamycin enhanced glomerular

expression of bip and grp94 (Fig. 9C), without inducing proteinuria.

The experiments presented in Figs. 6 and 7 show that ER stress proteins restrict complement-

mediated cytolysis in cultured GEC. In the next series of experiments, we assessed whether stimuli

shown to induce ER stress protein expression would limit C5b-9-mediated GEC injury (i.e.

proteinuria) in vivo. Rats were treated with a subnephritogenic dose of adriamycin, or tunicamycin,

prior to the induction of PHN. Proteinuria developed in untreated rats with PHN (on days 7, 9, and


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

17

13), whereas the amount of proteinuria was significantly lower in the rats with PHN that had been

pretreated with adriamycin or tunicamycin (Fig. 10). Thus, increased ER stress protein expression

can reduce C5b-9-mediated GEC injury in vivo. By immunofluorescence microscopy and/or

immunoblotting, there were no apparent differences in the amounts of glomerular sheep antibody

IgG, rat IgG, and rat C3 among the three groups of rats (Fig. 11, Table VII), indicating that

adriamycin and tunicamycin did not decrease proteinuria by reducing the amount of glomerular

antibody deposition. Serum creatinine was not significantly different among the three groups of rats,

indicating that most likely there were no significant differences in renal function (Table VII). Urine

volumes ranged from 5.6-22.5 ml/24 h, and there was no consistent pattern among groups (data not

shown). In the PHN rats pretreated with adriamycin or tunicamycin, we did not detect further

increases in glomerular levels of bip and grp94 (day 14), as compared with the PHN-untreated group

(data not shown).

DISCUSSION

In this study, we show that cPLA2 activation in GEC was associated with leakage of lumenal

ER stress proteins (bip, grp94) into the cytosol (Fig. 1), suggesting that cPLA2-induced phospholipid

hydrolysis resulted in impairment of ER membrane integrity. These results are in keeping with our

previous studies, which demonstrated that overexpression of cPLA2 in GEC exacerbates

complement-mediated cytotoxicity (16), and that complement-induced activation of cPLA2 leads to

phospholipid hydrolysis at the membrane of the ER (29). Furthermore, we show that inhibition of

endogenous cPLA2 can attenuate complement-mediated cytotoxicity in GEC (Table III). The

mechanism of bip and grp94 leakage from the ER may have involved dysregulation of ER protein

pores (42), but it will require further delineation. Another consideration is that complement-induced


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

18

cPLA2 activation was associated with increased retrograde translocation of abnormal proteins from

the ER (the abnormal proteins being coupled with bip or grp94)(42), although it is less likely that

retrograde translocation could have occurred in an acute time frame. Impairment of ER membrane

integrity could potentially be injurious by causing leakage of Ca2+ and other ER lumenal

components, as well as impairment in ER Ca2+ uptake. While association of cPLA2 with membranes

of cell organelles has been established in other cell types (27), there is little information on potential

damage to organelle membranes due to phospholipid hydrolysis. In addition to the present study,

overexpression of cPLA2 in LLCPK1 kidney epithelial cells was associated with disruption of the

Golgi (43).

Chronic incubation of GEC with complement (4-24 h) enhanced expression of bip and grp94

mRNAs and proteins (Figs. 2 and 4), although there was no detectable increase in the cytosolic stress

protein, Hsp70. The increases in ER stress proteins were dependent on C5b-9 assembly (Fig. 4C).

At lower doses, complement-induced increases in bip and grp94 expression were seen mainly in the

GEC that overexpress cPLA2, but at a higher dose, expression was also induced in neo GEC, and the

increases were blocked with MAFP (Fig. 4). Thus, increases in bip and grp94 were, at least in part,

dependent on the activation of cPLA2. Further support for the role of cPLA2 in ER stress protein

induction was provided by experiments in which GEC were incubated chronically with the Ca2+

ionophore, ionomycin. Bip and grp94 protein expression was enhanced by ionomycin, and the

changes were significantly greater in the GEC that overexpress cPLA2 (Fig. 3).

Increases in ER stress proteins were not limited to cultured GEC, but also occurred in vivo.

Using the PHN model of C5b-9-induced GEC injury, it was demonstrated that glomerular bip and

grp94 proteins were upregulated in proteinuric rats with PHN (day 14), as compared with normal

controls (Fig. 9). Rats with PHN show increased glomerular activity of cPLA2 and AA metabolites


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

19

(19,20). At present, there are no specific inhibitors of cPLA2 that can be used in experimental

animals (44). Thus, while bip and grp94 upregulation in PHN is associated with the activation of

cPLA2, proof for the functional importance of cPLA2 in vivo will require development of suitable

cPLA2 inhibitors. Moreover, we have not been successful in establishing a proteinuric model of PHN

in mice, and consequently, cPLA2-null mice (45) could not be used to address the functional role of

cPLA2.

The ER serves as a site for folding, assembly and degradation of proteins (30-32). Membrane

and secreted proteins are translocated into the lumen of the ER shortly after initiation of synthesis,

and resident ER lumenal proteins, including bip and grp94, mediate protein folding. Moreover, bip

and grp94 are believed to bind to misfolded or abnormal proteins and prevent their aggregation,

either by rescuing such proteins from irreversible damage, or by increasing their susceptibility to

proteolytic attack. Other ER proteins, including erp72, may participate in disulfide isomerization.

Perturbation of the ER by stimuli, including accumulation of mutant proteins or Ca2+ depletion, may

increase expression of ER stress proteins. In yeast, misfolded proteins in the ER lead to activation

of Ire1p endonuclease (31,32). Ire1p splices HAC1 (homologous to ATF and CREB) mRNA in the

nucleus, which is then religated, exits the nucleus, and is translated into Hac1p transcription factor.

Hac1p translocates to the nucleus, binds to the unfolded response element and induces expression

of ER stress proteins. The mammalian unfolded protein response, while analogous to yeast, is more

complex, and appears to involve additional pathways and effectors (46).

C5b-9-induced sublethal cell injury may lead to a decline in cellular ATP, mitochondrial

lipid perturbation, or loss of mitochondrial membrane potential (47-49), whereas at high doses, C5b-

9 can induce mitochondrial damage and cell necrosis (50). Based on biochemical and morphologic

observations (36,49), it is likely that during complement-dependent GEC injury, integral membrane


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

20

and secretory proteins are altered. Such proteins may include integrins, transporters, and/or cell

junctional proteins, and these alterations may contribute to the permselectivity defect of the

glomerular capillary wall in PHN. Besides inducing cell injury, C5b-9 assembly leads to activation

of mechanisms that restrict injury or facilitate recovery of cells from complement attack. One such

mechanism of protection is “ectocytosis” (shedding) of C5b-9 complexes from cell membranes (1,2).

The present study identifies another mechanism that limits complement attack (Figs. 6, 8 and 10).

Expression of bip-antisense mRNA in GEC reduced stimulated bip protein expression, and when

these GEC were incubated with complement for 18 h, cytolysis was enhanced, as compared with neo

GEC (Fig. 6). Thus, induction of ER stress proteins is an important novel mechanism of protection

from sustained complement attack. The capability of the GEC to recover or limit the severity of

complement attack may depend on its capacity to resynthesize or reassemble integral membrane

proteins, which may require the presence of bip, or grp94. Induction of these ER stress proteins

during complement attack may also limit accumulation of abnormal proteins and help sustain

physiological functions and viability (31,32).

Exposure of cells to mild stress, sufficient to induce upregulation of ER stress proteins, may

be protective to additional insults (33,34), although progression to cell death may occur if the stress

is more intense or prolonged (35). The present study demonstrates that preincubation of cultured

GEC with a sublethal dose of puromycin aminonucleoside increased bip expression and protected

GEC from complement attack (Fig. 7). On the other hand, prior exposure of cultured GEC to

ischemia-reperfusion injury (chemical anoxia), despite increasing ER stress proteins (Fig. 8),

exacerbated complement-mediated cytotoxicity (Table VI). However, cellular effects of ischemia

and reperfusion are complex, and are manifested by a variety of changes that include a decline in

cellular ATP levels, alterations of cellular redox state and perturbations in intracellular Ca2+


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

21

homeostasis (34). In earlier studies, pretreatment with tunicamycin protected renal tubular epithelial

cells from chemical anoxia or toxin-induced injury (33,34). In contrast, ionomycin and tunicamycin,

despite increasing ER stress proteins, either had no effect or exacerbated complement-mediated

cytotoxicity in cultured GEC (Table VI). These compounds may have induced multiple alterations

in GEC leading to substantial injury, and with subsequent exposure to complement, the cells could

not withstand the additional stress. However, it is not clear why the effect of tunicamycin was

cytotoxic in culture, but protective in PHN. This question will require further study.

The functional role of ER stress proteins in cultured GEC was applicable to GEC injury in

vivo. Induction of ER stress proteins by pretreatment with a subnephritogenic dose of adriamycin

or tunicamycin significantly reduced proteinuria in PHN (Fig. 10), independently of changes in

glomerular immunoglobulin or complement deposition (Fig. 11, Table VII). It should be noted that

while tunicamycin was reported to induce a renal lesion resembling acute tubular necrosis in mice

(51), the PHN rats that had been pretreated with tunicamycin had no significant alterations in renal

function (Table VII). Our observations provide a rationale for developing non-toxic methods to

induce expression of ER stress protein in vivo, which may eventually have applications to therapy

of glomerular disease. The importance of these results extends beyond complement-induced GEC

injury. For example, preinduction of ER stress proteins prior to xenotransplantation may potentially

be a means of reducing hyperacute xenograft rejection, which is complement-dependent (52).


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

22

REFERENCES

1. Morgan, B.P. (1992) Curr. Topics. Microbiol. Immunol. 178, 115-140

2. Nicholson-Weller, A., and Halperin, J.A. (1993) Immunol. Res. 12, 244-257

3. Niculescu, F., Rus, H., and Shin, M.L. 1994. J. Biol. Chem. 269, 4417-4423

4. Niculescu, F., Rus, H., van Biesen, T., and Shin, M.L. (1997) J. Immunol. 158, 4405-4412

5. Rus, H.G., Niculescu, F., and Shin, M.L. (1996) J. Immunol. 156, 4892-4900

6. Halperin, J.A., Taratuska, A., and Nicholson-Weller, A. (1993) J. Clin. Invest. 91, 1974-1978

7. Shankland, S.J., Pippin, J.W., and Couser, W.G. (1999) Kidney Int. 56, 538-548

8. Kilgore, K.S., Schmid, E., Shanley, T.P., Flory, C.M., Maheswari, V., Tramontini, N.L.,

Cohen, H., Ward, P.A., Friedl, H.P., and Warren, J.S. (1997) Am. J. Pathol. 150, 2019-2031

9. Tischer, C.G., and Couser, W.G. (2000) J. Am. Soc. Nephrol. 10, 183-188

10. Cybulsky, A.V., Foster, M.H., Quigg, R.J., and Salant, D.J. (2000). The Kidney: Physiology

and Pathophysiology, 3rd edition. Seldin, D.W., and Giebisch, G., editors. Lippincott-Raven

Publishers, Philadelphia. 2645-2697

11. Cybulsky, A.V., Rennke, H.G., Feintzeig, I.D., and Salant, D.J. (1986) J. Clin. Invest. 77,

1096-1107

12. Cybulsky, A.V., Quigg, R.J., and Salant, D.J. (1986) J. Immunol. 137, 1511-1516

13. Kerjaschki, D., Schulze, M., Binder, S., Kain, R., Ojha, P.P., Susani, M., Horvat, R., Baker,

P.J., and Couser, W.G. (1989) J. Immunol. 143, 546-552

14. Baker, P.J., Ochi, R.F., Schulze, M., Johnson, R.J., Campbell, C., and Couser, W.G. (1989)

Am. J. Pathol. 135, 185-194

15. Cybulsky, A.V., Takano T., Papillon, J., and McTavish, A.J. (1999) Am. J. Pathol. 155,


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

23

1701-1711

16. Cybulsky, A.V., Monge, J.C., Papillon, J., and McTavish, A.J. (1995) Am. J. Physiol. 269,

F739-F749

17. Panesar, M., Papillon, J., McTavish, A.J., and Cybulsky, A.V. (1997) J. Immunol. 159, 3584-

3594

18. Cybulsky, A.V., Papillon, J., and McTavish, A.J. (1998) Kidney Int. 54, 360-372

19. Cybulsky, A.V., Takano, T., Papillon J., and McTavish, A.J. (2000) Kidney Int. 57, 1052-

1062

20. Takano, T., and Cybulsky, A.V. (2000) Am. J. Pathol. 156, 2091-2101

21. Zoja, C., Benigni A., Verroust, P., Ronco, P., Bertani, T., and Remuzzi, G. (1987) Kidney

Int. 31, 1335-1343

22. Kirschenbaum, M.A., Liebross, B.A., and Serros, E.R. (1985) Prostaglandins 30, 295-303.

23. Cybulsky, A.V., Lieberthal, W., Quigg, R.J., Rennke, H.G., and Salant, D.J. (1987) Am. J.

Pathol. 128, 45-51

24. Nagao, T., Nagamatsu, T., and Suzuki, Y. (1996) Eur. J. Pharmacol. 316, 73-80

25. Weise, W.J., Natori, Y., Levine, J.S., O'Meara, Y.M., Minto A.W., Manning, E.C, Goldstein,

D.J., Abrahamson, D.R., and Salant, D.J. (1993) Kidney Int. 43, 359-368

26. Golbetz, H., Black, V., Shemesh, O., and Myers, B.D. (1989) Am. J. Physiol. 256, F44-F51

27. Hirabayashi, T., and Shimizu, T. (2000) Biochim. Biophys. Acta. 1488, 124-138

28. Dessen, A. (2000) Biochim. Biophys. Acta. 1488, 40-47

29. Liu, J., Takano, T., Papillon, J., Khadir, A., and Cybulsky, A.V. (2001) Biochem. J. 353, 79-

90


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

24

30. Lee, A. (1992) Curr. Opin. Cell Biol. 4, 267-273

31. Pahl, H.L. (1999) Physiol. Rev. 79, 683-701

32. Kaufman, R.J. (1999) Genes. Dev. 13, 1211-1233

33. Liu, H., Bowes, R.C., van de Water, B., Sillence, C., Nagelkerke, J.F., and Stevens, J.L.

(1997) J. Biol. Chem. 272, 21751-21759

34. Bush, K.T., George, S.K., Zhang, P.L., and Nigam, S.K. (1999) Am. J. Physiol. 277, F211-

F218

35. Rao, R.V., Hermel, E., Castro-Obregon, S., del Rio, G., Ellerby, L.M., Ellerby, H.M, and

Bredesen, D.E. (2001) J. Biol. Chem. 276:33869-33874.

36. Quigg, R.J., Cybulsky, A.V., Jacobs, J.B., and Salant, D.J. (1998) Kidney Int. 34, 43-52

37. Salant, D.J., and Cybulsky, A.V. (1988) Methods in Enzymology, vol. 162. DiSabato, G.,

editor. Academic Press, New York, 421-461

38. Pruchno, C.J., Burns, M.M., Schultze, M., Johnson, R.J., Baker, P.J., Alpers, C.E., and

Couser, W.G. (1991) Am. J. Pathol. 138, 203-211

39. Balsinde, J., and Dennis, E.A. (1996) J. Biol. Chem. 271, 6758-6765

40. Li, L.J., Li, X., Ferrario, A., Rucker, N., Liu, E.S., Wong, S., Gomer, C.J., and Lee, A.S.

(1992) J. Cell. Physiol. 153, 575-582

41. Grond, J.G., Weening, J.J., and Elema, J.D. (1984) Lab. Invest. 51, 277-285

42. Kopito, R.R. (1997) Cell 88, 427-430

43. Choukroun, G.J., Marshansky V., Gustafson C.E., McKee, M., Hajjar, R.J., Rosenzweig, A.,

Brown, D., and Bonventre, J.V. (2000) J. Clin. Invest. 106, 983-993

44. Yedgar, S., Lichtenberg, D., and Schnitzer, E. (2000) Biochim. Biophys. Acta. 1488, 182-187


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

25

45. Bonventre, J.V., Huang, Z., Taheri, M.R., O'Leary, E., Li, E., Moskowitz, M.A., and

Sapirstein, A. (1997) Nature 390, 622-625

46. Ma, Y., and Hendershot, L.M. (2001) Cell 107, 827-830

47. Papadimitriou, J.C., Ramm, L., Drachenberg, C.B., Trump, B.F., and Shin, M.L. (1991) J.

Immunol. 147, 212-217

48. Papadimitriou, J.C., Phelps, P.C., Shin, M.L., Smith, M.W., and Trump, B.F. (1994) Cell

Calcium 15, 217-227

49. Topham, P.S., Haydar, S.A., Kuphal, R., Lightfoot, J.D., and Salant, D.J. (1999) Kidney Int.

55, 1763-1775

50. Papadimitriou, J.C., Drachenberg, C.B., Shin, M.L., and Trump, B.F. (1994) Virchows

Archiv. 424, 677-685

51. 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

52. Samstein, B., and Platt, J.L. (2001) J. Am. Soc. Nephrol. 12, 182-193


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

26

FOOTNOTES

* This work was supported by Research Grants from the Canadian Institutes of Health

Research and the Kidney Foundation of Canada. A.V. Cybulsky holds a Scholarship from

the Fonds de la Recherche en Santé du Québec. T. Takano holds a Scholarship and J. Liu

was the recipient of a Studentship from the Canadian Institutes of Health Research. H. Peng

was the recipient of a Fellowship from the McGill University Health Centre Research

Institute.

1. Abbreviations: AA, arachidonic acid; [Ca2+]i, cytosolic free Ca2+ concentration; cPLA2,

cytosolic phospholipase A2; GEC, glomerular epithelial cell; LDH, lactate dehydrogenase;

MAFP, methyl arachidonyl fluorophosphonate; PHN, passive Heymann nephritis.


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

27

FIGURE LEGENDS

Figure 1. Complement-induced activation of cPLA2 affects the integrity of the membrane of the ER.

A) Neo GEC (which express a low endogenous level of cPLA2) or GEC that overexpress cPLA2

were incubated in duplicate with anti-GEC antibody and 2.5% normal serum (NS; to form sublytic

C5b-9), or heat-inactivated serum (HIS) in controls, for 40 min. After collection of supernatants,

GEC plasma membranes were briefly permeabilized with digitonin (37 µg/ml) to recover the cytosol.

Then, membranes and contents of the ER were solubilized by the addition of 1% Triton X-100.

Digitonin and Triton fractions were immunoblotted with antibodies to bip or grp94 (in duplicate).

An increase in bip and grp94 is present in the digitonin fraction of complement-treated GEC that

overexpress cPLA2 (seventh and eighth lanes from the left). B) Antibody-sensitized neo GEC were

incubated with 4.0% normal serum, or heat-inactivated serum in controls, for 40 min. Bip and grp94

are increased in the digitonin fraction of complement-treated GEC.

Figure 2. Expression of ER stress protein mRNAs in GEC. GEC that overexpress cPLA2 were

incubated with anti-GEC antibody and 2.5% normal serum (NS; to form sublytic C5b-9), or heat-

inactivated serum (HIS) in controls, for 4 h (A). Total RNA was extracted and subjected to Northern

blotting. The control incubations demonstrate that there is some basal expression of bip, grp94 and

erp72 mRNAs in GEC. Complement increased expression of bip, grp94 and erp72 mRNAs. B) GEC

that overexpress cPLA2 were incubated with ionomycin (Iono, 1 µM), or buffer (Ctrl) for 24 h.

Ionomycin increased bip, grp94 and erp72 mRNAs. Tunicamycin (Tunic, 10 µg/ml, 24 h) was

employed as a positive control in these experiments.

Figure 3. Effect of ionomycin on expression of ER stress proteins in GEC. GEC that overexpress

cPLA2, or neo GEC were incubated with ionomycin (Iono, 1 µM), or buffer alone (control; Ctrl) for

24 h. Tunicamycin (Tunic, 10 µg/ml, 24 h) was employed as a positive control. Cell lysates were


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

28

immunoblotted with antibodies to bip, grp94, or erp72. A) Representative immunoblot. B)

Densitometric quantification of immunoblots. The control incubations demonstrate that there is some

basal expression of bip, grp94 and erp72 proteins in GEC. Ionomycin stimulated increases in grp94,

bip and erp72, and the effect of ionomycin was more prominent in the GEC that overexpress cPLA2.

+p<0.04 Iono vs Ctrl, *p<0.001 Iono vs Ctrl and p<0.004 cPLA2 vs neo (Iono-treated GEC), Xp=0.06

Iono vs Ctrl, ++p< 0.001 Iono vs Ctrl and p<0.03 cPLA2 vs neo (Iono-treated GEC), **p<0.01 Iono

vs Ctrl (5 experiments). Tunicamycin (positive control) increased ER stress proteins in both neo

GEC and GEC that overexpress cPLA2.

Figure 4. Effect of complement and cPLA2 on expression of ER stress proteins in GEC. Antibody-

sensitized GEC that overexpress cPLA2 or neo GEC were incubated with 2.5% normal serum (NS;

to form sublytic C5b-9), or heat-inactivated serum (HIS) in controls, for 4, 6 or 24 h. Cell lysates

were immunoblotted with antibodies to bip or grp94. A) Representative immunoblot. (On this gel,

the grp94 band appeared to run as a doublet.) B) Densitometric quantification of immunoblots.

Complement increased expression of bip and grp94 significantly in GEC that overexpress cPLA2 (9

experiments), while upward trends in bip and grp94 expression occurred in neo GEC (6

experiments). Bip: p<0.002 NS vs HIS and p=0.05 cPLA2 vs neo (at all time points); grp94: p<0.001

NS vs HIS and p<0.035 cPLA2 vs neo (at all time points). The effect of tunicamycin (Tunic) is

shown for comparison (positive control). C) Assembly of C5b-9 is required for increased expression

of ER stress proteins. Antibody-sensitized GEC that overexpress cPLA2 were incubated with 2.5%

C8-deficient serum (C8DS) or 2.5% C8-deficient serum reconstituted with purified C8 (C8DS+C8)

for 24 h. Cell lysates were immunoblotted with antibodies to bip or grp94. A representative

immunoblot and densitometric quantification of immunoblots are presented. C8DS+C8 increased

expression of bip and grp94 significantly, as compared with C8DS. Bip: p<0.0001 C8DS+C8 vs


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

29

C8DS; grp94: p<0.005 C8DS+C8 (6 incubations). D) Antibody-sensitized neo GEC were incubated

with 4.0% normal serum (NS; to form sublytic C5b-9), 4.0% normal serum plus MAFP (25 µM),

or heat-inactivated serum (HIS) in controls, for 24 h. A representative immunoblot and densitometric

quantification of immunoblots are presented. In control GEC (C), complement increased bip and

grp94 expression significantly (p<0.01), whereas MAFP (M) inhibited the complement-induced

increases (5 experiments).

Figure 5. Products of cPLA2-induced phospholipid hydrolysis do not increase expression of ER

stress proteins. A) Representative immunoblot. B) Densitometric quantification of immunoblots.

GEC that overexpress cPLA2 were incubated with ionomycin (Iono, 1 µM), ionomycin +

indomethacin (Indo, 10 µM), AA (10 µM), lysophosphatidylcholine (LPC, 10 µM), tunicamycin

(Tunic, 10 µg/ml; positive control), or buffer alone (Ctrl) for 24 h. Ionomycin increased bip and

grp94 expression, and the effect was not inhibited by indomethacin (*p<0.0005 vs control,

+p<0.0001 vs control). Bip and grp94 expression was not enhanced by exogenously-added AA, nor

lysophosphatidylcholine (4 experiments).

Figure 6. Effects of bip-antisense mRNA on GEC injury. A) Effect of bip antisense mRNA on ER

stress protein expression. GEC were stably transfected with bip antisense cDNA. The effect of bip

antisense mRNA expression on bip protein production was monitored by immunoblotting of lysates

from untreated (U) and tunicamycin (T)-treated GEC (10 µg/ml for 18 h). The figure shows three

clones of GEC in which bip protein induction by tunicamycin is reduced, as compared with neo

(bipAS-1-3). B) Complement-mediated cytotoxicity in GEC that express bip antisense mRNA. Neo

GEC and three clones of GEC that express bip antisense mRNA were incubated with anti-GEC

antibody (40 min), and normal serum (heat-inactivated serum in controls) for 18 h. Complement-

mediated cytotoxicity was determined by measuring release of LDH into cell supernatants.


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

30

Complement induced greater cytotoxicity in the bipAS-1 and bipAS-3 GEC (p<0.015 bipAS-1 vs

neo, p<0.003 bipAS-3 vs neo), while an upward trend in cytotoxicity was evident in bipAS-2

(p=0.078 bipAS-2 vs neo). Values represent 5 experiments. There were no significant differences

in background LDH release, i.e. in incubations with heat-inactivated serum (not shown).

Figure 7. Effects of puromycin aminonucleoside on bip expression (A) and complement-mediated

GEC injury (B). A) GEC that overexpress cPLA2 were incubated with puromycin aminonucleoside

(PA; 50 µg/ml) for 2, 4, or 24 h. An increase in bip expression is evident at 2 and 4 h (representative

immunoblot). The effect of tunicamycin is shown for comparison. B) GEC were preincubated

without (Ctrl) and with puromycin aminonucleoside for 3 h (to induce bip expression), and were then

incubated with antibody and normal serum (heat-inactivated serum in controls). LDH release

(reflecting cell lysis) was determined after 40 min. Pretreatment with puromycin aminonucleoside

reduced complement-dependent lysis significantly (p<0.002 PA vs Ctrl, 5 experiments).

Figure 8. Effect of chemical anoxia on ER stress protein expression (representative immunoblot).

GEC were incubated with glucose-free measurement buffer for 40 min at 37oC. Then, GEC were

incubated with 10 mM 2-deoxyglucose (DG), 10 mM 2-deoxyglucose + 10 µM antimycin A, or

buffer alone (Ctrl). Supernatants were removed after 90 min at 37oC, and GEC were placed into

culture medium for 24 h. Lysates were immunoblotted with antibodies to bip or grp94.

Deoxyglucose with or without antimycin A induced an increase in bip. There was a smaller increase

in grp94 with deoxyglucose + antimycin A. The effect of tunicamycin is shown for comparison.

Figure 9. Expression of ER stress proteins in vivo. Glomeruli were isolated from normal rats

(control) and from rats with PHN on day 14. Glomerular lysates were immunoblotted with antibodies

to bip or grp94 (A, representative immunoblots; B, densitometric quantification). Bip and grp94

expression was increased in glomeruli isolated from rats with PHN, as compared with control


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

31

(*p<0.002, +p<0.005 PHN vs control; 7-9 rats per group). Panel B also shows the densitometric

quantification of grp94 and bip expression in rats injected with subnephritogenic adriamycin, 6

mg/kg intravenously (ADR, day 14, **p<0.001 adriamycin vs control; 9-12 rats per group), as well

as in rats injected with tunicamycin, 1 mg/kg intraperitoneally (Tun, 24 h, the dots represent the

values of two individual rats in each group).

Figure 10. Pretreatment of rats with a subnephritogenic dose of adriamycin or tunicamycin reduces

proteinuria in PHN. Rats were untreated, or were injected with adriamycin, 6 mg/kg intravenously

(ADR), or tunicamycin, 1 mg/kg intraperitoneally (Tun) to upregulate ER stress proteins (Fig. 9).

Four days later, rats were injected with anti-Fx1A to induce PHN (day 0). Urine protein was

measured on days 0, 7, 9 and 13. Compared with the PHN-untreated group (4 rats), proteinuria was

significantly lower in the adriamycin (p<0.005, 3 rats) and tunicamycin groups (p<0.025, 3 rats).

Figure 11. Glomerular deposition of sheep (Sh) IgG, rat IgG and rat C3 in rats with PHN that were

pretreated with adriamycin or tunicamycin, or were untreated. Kidney sections were stained with

fluorescein-conjugated antibodies, and visualized using immunofluorescence microscopy

(magnification × 500).


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

32

Table I. C5b-9-induced release of [3H]AA.

A) [3H]AA-labelled GEC that overexpress cPLA2 were incubated with anti-GEC antibody and

normal serum (NS) at a sublytic concentration (2.5% vol/vol), or heat-inactivated serum (HIS) in

controls, for 40 min at 37oC. B) GEC were incubated with antibody and 2.5% C8-deficient serum

reconstituted with purified C8 (C8DS+C8), C8-deficient serum alone (C8DS) or heat-inactivated

serum for 40 min at 37oC. Lipids were extracted and [3H]AA measured by thin layer

chromatography. Free [3H]AA is presented as percent of total radioactivity. ap<0.01 vs HIS,

bp<0.005 vs C8DS and HIS; 3 experiments performed in duplicate.

Free [3H]AA Free [3H]AA

A) NS 2.96±0.66a B) C8DS+C8 5.47±0.47b

HIS 0.86±0.16 C8DS 1.57±0.42

HIS 1.25±0.20


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

33

Table II. Translocation of ER stress proteins from ER to cytosol.

A) Antibody-sensitized neo GEC and GEC that overexpress cPLA2 were incubated with 2.5%

normal serum, or heat-inactivated serum in controls, for 40 min (a representative experiment is

shown in Fig. 1A). B) Antibody-sensitized neo GEC were incubated with 4.0% normal serum, or

heat-inactivated serum in controls (a representative experiment is shown in Fig. 1B). The preparation

of cytosolic fractions is described in the legend to Fig. 1. Densitometry of bip and grp94 in cytosol

is presented as normal serum/heat-inactivated serum (fold control). ap<0.04, bp<0.0025, NS vs HIS

(A and B: 5 experiments performed in duplicate).

GEC NS(%) Densitometry of cytosolic fractions

bip grp94

A) neo 2.5 0.94±0.18 1.10±0.14

cPLA2 2.5 1.33±0.20a 1.52±0.23b

B) neo 4.0 1.36±0.28a 1.56±0.43a


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

34

Table III. Effect of the cPLA2 inhibitor, MAFP, on complement-mediated cytolysis.

Neo GEC were incubated with anti-GEC antibody (40 min), and normal serum (or heat-inactivated

serum in controls), in the presence or absence of MAFP (25 µM) for 18 h. Complement-mediated

cytolysis was determined by measuring release of LDH into cell supernatants. MAFP decreased the

amount of complement-induced cytotoxicity (p<0.05 MAFP vs control). Values represent 5

experiments.

Specific LDH Release (%)

Normal Serum: 10% 7% 4% (vol/vol)

Control 4.8±1.5 5.7±2.1 3.4±2.0

MAFP 2.6±1.5 2.1±0.9 1.4±0.7


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

35

Table IV. Effect of ionomycin and tunicamycin on LDH release in GEC that stably express bip

antisense mRNA.

Neo and bip antisense (bipAS) GEC were incubated with ionomycin, 1 µM (4 experiments), or

tunicamycin, 10 µg/ml (6 experiments). LDH release (reflecting cell lysis) was determined after 24

h. ap<0.025, bp<0.005 bipAS vs neo.

GEC Specific LDH Release (%)

Ionomycin Tunicamycin

bipAS-1 6.1±2.0a 4.6±2.9

bipAS-2 8.0±1.4b 3.1±1.6

bipAS-3 3.4±2.0 4.5±1.5

neo 0.1±0.08 0


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

36

Table V. Complement-mediated cytolysis (acute incubation) in GEC that express bip antisense

mRNA.

Neo GEC and three clones of GEC that express bip antisense mRNA were incubated with anti-GEC

antibody (40 min), and normal serum (or heat-inactivated serum in controls) for 40 min.

Complement-mediated cytolysis was determined by measuring release of LDH into cell supernatants.

There were no significant increases in cytolysis in the bip antisense clones, as compared with neo.

Values represent 6-10 experiments.



Neo 70±4 54±8 23±6

bipAS-1 40±7 44±8 34±8

bipAS-2 40±5 44±5 31±7

bipAS-3 40±6 31±7 21±5


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

37

Table VI. Effects of tunicamycin, ionomycin and chemical anoxia on complement-mediated GEC

injury.

GEC that overexpress cPLA2 were preincubated with tunicamycin (10 µg/ml, 24 h), ionomycin (1

µM, 24 h), or deoxyglucose (DG) + antimycin A (as in Fig. 8) to induce expression of ER stress

proteins. Then, GEC were incubated with antibody, followed by 5, 10 or 15% normal serum (or heat-

inactivated serum in controls). LDH release (reflecting cell lysis) was determined after 40 min.

Pretreatment with tunicamycin or prior exposure to chemical anoxia enhanced complement-

dependent lysis significantly (p<0.01 tunicamycin vs untreated, 4 experiments; p<0.04

DG+Antimycin A vs untreated, 3 experiments). A small upward trend in complement-dependent

lysis was evident with ionomycin pretreatment (5 experiments).



Untreated 60±2 52±5 28±8

Tunicamycin 66±4 66±7 52±11

Untreated 31±3 26±2 15±4

Ionomycin 38±4 31±3 17±5

Untreated 61±10 45±8 15±7

DG+Antimycin A 65±10 64±9 53±12


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

38

Table VII. Glomerular antibody and C3 deposition, and serum creatinine measurements in PHN.

Rats with PHN were untreated (4 rats) or were pretreated with adriamycin (ADR; 3 rats) or

tunicamycin (Tun; 3 rats). All measurements were performed on day 14 (see Fig. 10). Glomeruli

were isolated and the amounts of sheep IgG and rat IgG were assessed by immunoblotting, and were

quantified by densitometry. Values represent densitometry in arbitrary units. Kidney sections were

stained with fluorescein-conjugated specific antibodies, and the amounts of glomerular sheep IgG,

rat IgG and rat C3 were assessed by immunofluorescence microscopy (IF), and were quantified

(Methods). Values represent times required to collect images by the photomultiplier and camera

(seconds), and are inversely proportional to fluorescence intensity. For all parameters, there are no

significant differences among groups.

Densitometry Densitometry IF IF IF Creatinine

Sheep IgG Rat IgG Sheep IgG Rat IgG Rat C3 (µM)

PHN-ADR 0.58±0.08 0.83±0.13 1.14±0.08 1.91±0.13 1.95±0.09 28±3

PHN-Tun 0.81±0.03 0.76±0.05 1.37±0.18 1.87±0.12 1.76±0.11 25±2

PHN 0.82±0.07 0.64±0.06 1.15±0.11 1.78±0.12 2.07±0.29 25±2


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

Figure 1

neo cPLA2 neo cPLA2

HIS NS HIS NS HIS NS HIS NS

Digitonin Triton X-100

bip -

grp94 -

bip -HIS NS HIS NS

Digitonin Triton

grp94 -HIS NS HIS NS

Digitonin Triton

A

B


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

NS

HIS

Ctrl

Tuni

cIo

no

bip -

grp94 -

erp72 -

bip -

grp94 -

erp72 -

A B

Figure 2


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

A

B

cPLA2 neo

bip -

grp94 -C

trl

Iono

Tuni

c

Ctrl

Iono

Tuni

c

erp72 -

Tuni

cIo

no Ctrl

Tuni

cIo

no Ctrl

Tuni

cIo

no Ctrl

Tuni

cIo

no Ctrl

Tuni

cIo

no Ctrl

Tuni

cIo

no Ctrl

0.2

0.4

0.6

0.8

1.0

neo cPLA2 neo neocPLA2 cPLA2

grp94 bip erp72

Den

sito

met

ry (a

rbitr

ary

units

)

*

+

**++

x

Figure 3


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

Figure 4

bip -

grp94 -

NS,

4h

HIS

, 4h

Unt

r

Tuni

c

NS,

6h

HIS

, 6h

NS,

24h

HIS

, 24h

NS,

4h

HIS

, 4h

Unt

rTu

nic

NS,

6h

HIS

, 6h

NS,

24h

HIS

, 24h

neo cPLA2A

B

Fold

incr

ease

neo cPLA2

4h 6h 24h

Tuni

c 4h 6h 24h

Tuni

c1.01.21.41.61.82.02.22.42.6 bip

neo cPLA2

4h 6h 24h

Tuni

c 4h 6h 24h

Tuni

c1.01.21.41.61.82.02.2 grp94

C

C8D

S

C8D

S+C

8

bip -

grp94 -

Fold

incr

ease

bip

grp9

41.0

1.4

1.8

bip -

grp94 -

NS

HIS

NS+

MAF

P

1.01.21.41.61.8

1.01.21.41.61.8

Fold

incr

ease

grp94

bip

C M

D


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

Figure 5

bip -

grp94 -

Iono Iono AA LPC Tun Ctrl+ Indo

B

A

Iono

Iono

+Ind

oAA LP

CTu

nC

trl

Iono

Iono

+Ind

oAA LP

CTu

nC

trl0.2

0.4

0.6

0.8

1.0 * * * + + +

Den

sito

met

ry (a

rbitr

ary

units

) bip grp94


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

A

B

2 4 6 8 100

10

20

30

40

Normal Serum (%)

LDH

Rel

ease

(%)

bipAS-1bipAS-2

bipAS-3

neo

neo bipAS-1 bipAS-2 bipAS-3 U T U T U T U T

bip -

grp94 -

erp72 -

Figure 6


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

2h 4h 24h Tun CtrlPA

bip -A

B

Normal Serum (%)

LDH

Rel

ease

(%)

0.0 0.5 1.0 1.5 2.00

10

20

30

40Ctrl

PA

Figure 7


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

Tunic Ctrl DG DG+AnA

bip -

grp94 -

Figure 8


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

PHN d14 Control

bip -

grp94 -

A

B

Den

sito

met

ry(a

rbitr

ary

units

)

Ctrl

PHN

Ctrl

PHN

Ctrl

ADR

Ctrl

ADR

Ctrl

Tun

Ctrl

Tun0.4

0.6

0.8

1.0+

***

grp94 bip grp94 bip grp94 bip

h

hhh

hh

hh

Figure 9


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

Day 0 Day 7 Day 9 Day 130

500

1000

1500Pr

otei

nuria

(mg/

24 h

)PHN-ADRPHN-TunPHN

Figure 10


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

PHN-Adriamycin PHN-Tunicamycin PHN

Sh IgG

Rat IgG

Rat C3

Figure 11


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

Hongwei PengAndrey V. Cybulsky, Tomoko Takano, Joan Papillon, Abdelkrim Khadir, Jianhong Liu and

reticulum stress proteins in glomerular epithelial cellsComplement C5b-9 membrane attack complex increases expression of endoplasmic

published online August 20, 2002J. Biol. Chem.

10.1074/jbc.M204694200Access the most updated version of this article at doi:

Alerts:

When a correction for this article is posted•

When this article is cited•

to choose from all of JBC's e-mail alertsClick here


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/lookup/doi/10.1074/jbc.M204694200

http://www.jbc.org/cgi/alerts?alertType=citedby&addAlert=cited_by&cited_by_criteria_resid=jbc;M204694200v1&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/early/2002/08/20/jbc.M204694200.citation

http://www.jbc.org/cgi/alerts?alertType=correction&addAlert=correction&correction_criteria_value=early/2002/08/20/jbc&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/early/2002/08/20/jbc.M204694200.citation

http://www.jbc.org/cgi/alerts/etoc

http://www.jbc.org/

m2:04694 (revised) complement c5b-9 membrane attack

Documents