ToxSci Advance Access originally published online on August 18, 2006
Toxicological Sciences 2006 94(1):118-128; doi:10.1093/toxsci/kfl084
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Calpain-Induced Endoplasmic Reticulum Stress and Cell Death following Cytotoxic Damage to Renal Cells
Department of Biomedical Sciences, Atlantic Veterinary College, University of Prince Edward Island and the PEI Health Research Institute, Charlottetown, PEI, Canada C1A 4P3
1To whom correspondence should be addressed at Faculty of Veterinary Medicine, University of Calgary, 3330 Hospital Drive NW Calgary, AB, Canada T2N 4N1. Fax: (403) 220-7922. E-mail: acribb{at}ucalgary.ca.
Received April 19, 2006; accepted August 4, 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Calpains and endoplasmic reticulum (ER) stress have both been implicated in renal cell death following exposure to reactive chemical toxicants (RCTs). Therefore, we explored the link between ER stress, calpain, and cell death in renal cell injury due to model RCTs (iodoacetamide, menadione, tert-butyl hydroperoxide) and ER stress inducers (tunicamycin [TUN], thapsigargin [THAPS]). The calpain inhibitor, PD150606, significantly reduced the RCT and TUN-induced cell death in the renal cell line LLC-PK1, but not death induced by THAPS. ER stress was confirmed by the significant induction of GRP78 following exposure to RCTs and ER stress inducers. While GRP94 induction was observed following RCTs and TUN, it was not statistically significant because of variability. THAPS at 5µM significantly induced GRP94, while 20µM caused a calpain-dependent cleavage of GRP94. Caspase-12 and m-calpain were variably induced and/or cleaved following exposure to all toxicants, supporting activation of these signaling pathways. Inhibition of calpain blocked the induction of GRP78 following exposure to RCTs suggesting that calpain was contributing to the observed ER stress following RCTs. In contrast, calpain inhibition did not block ER stress protein induction following exposure to nontoxic concentrations of TUN or THAPS, indicating that calpain inhibition did not block the ER stress protein induction pathways directly. These studies demonstrate a previously unappreciated link between calpain activation and ER stress–associated cell death in renal cells. While further studies are required to clarify the molecular events involved, these results confirm that calpain activation and the ER are important related players in chemically induced renal cell damage.
Key Words: nephrotoxicity; ER stress; tunicamycin; thapsigargin; reactive chemical toxicants; m-calpain; caspase-12.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Nephrotoxicity remains a significant clinical problem for many therapeutically useful drugs. Renal cell damage may occur following exposure to directly toxic compounds or following the formation of reactive metabolites from nontoxic parent compounds (Lock, 1999
). Several different mechanisms have been invoked by which nephrotoxicants can damage renal tubular cells, including covalent binding to critical cellular proteins, oxidative stress, or disruption of lysosomal structures. To date, mechanisms for reducing the occurrence or severity of nephrotoxicity have been limited to reducing exposure, blocking formation or enhancing removal of reactive metabolites, or drug removal and symptomatic therapy following the onset of damage (Lock, 1999). If common cell death pathways could be identified, it may be possible to develop a general nephroprotective strategy that could be employed to reduce or protect against the toxicity of a number of nephrotoxic drugs and chemicals.
Two candidates for common links in renal cell death that have been identified are the calpain system and the endoplasmic reticulum (ER) (Harriman et al., 2002; Liu et al., 1997). The ER is the principal cellular organelle that regulates folding and trafficking of secretory proteins (Liu and Kaufman, 2003). The ER is also an important storage site for cellular calcium and is gaining recognition as an important cellular signaling organelle (Berridge, 2002). The ER is highly sensitive to any disturbance to its internal environment. Moderate disruption of ER function may lead to the accumulation of misfolded or unfolded protein, triggering the ER stress or unfolded protein response (UPR) (Harding et al., 2002). The UPR is a cytoprotective response, but if the ER stress is severe or prolonged, ER-associated cell death pathways can be activated. Cell death may also occur following loss of calcium homeostasis if ER calcium regulation is disrupted.
Studies in porcine kidney cells (LLC-PK1) have demonstrated that induction or overexpression of ER stress proteins protects renal cells from a variety of reactive chemical toxicants (RCTs), including iodoacetamide (IDAM), tert-butyl hydroperoxide (TBHP), S-(1,1,2,3-tetrafluoroethyl)-L-cysteine, and hydrogen peroxide, suggesting the involvement of the ER in renal cell death (Asmellash et al., 2005; Bedard et al., 2004; Liu et al., 1997, 1998; Ryan et al., 2005). While disruption of the ER can trigger the release of calcium leading to cell death (Liu et al., 1997, 1998; van De Water et al., 1999), calcium is not consistently involved (Ryan et al., 2005; van De Water et al., 1999), and several other signaling pathways have been implicated in modulating and effecting ER stress–induced renal cell death (Cribb et al., 2005; Rao et al., 2004). The major ER-related cell death–signaling pathways include CHOP/GADD153, calpain, c-jun N-terminal kinase, and caspase-12. Recently, activation of these pathways has been identified in renal cells following exposure to cisplatin, acetaminophen, oxidative stress, and ischemia-reperfusion injury (Cribb et al., 2005). Together, these results suggest that disruption of the ER may be a common event in many instances of renal cell damage from diverse sources (di Mari et al., 1999; Liu and Baliga, 2005; Lorz et al., 2004).
Several studies have demonstrated the involvement of calpains in cell death following renal ischemia/reperfusion injury, mitochondrial toxins, and RCTs (Chatterjee et al., 2005; Harriman et al., 2002; Liu et al., 2001). Calpains have been shown to trigger degradation of the plasma membrane and, subsequently, loss of cellular integrity in renal cells (Liu and Schnellmann, 2003). Therefore, calpains have been suggested as another common link in necrotic and apoptotic renal cell death following exposure to renal toxins.
In investigating the role of ER-associated cell damage and death in LLC-PK1 cells using IDAM and TBHP as model RCTs, we observed cleavage of GRP94 and caspase-12 (Ryan et al., 2005). Both GRP94 and caspase-12 can be cleaved by m-calpain (Nakagawa and Yuan, 2000; Reddy et al., 1999), suggesting that calpains may be involved in these ER-associated cell death events in renal cells (Ryan et al., 2005). Release of calcium from the ER has also been implicated in the activation of calpains (Harriman et al., 2002), suggesting that there may be a link between ER stress and calpain activation.
We therefore hypothesized that exposure of LLC-PK1 cells to model RCTs (IDAM, menadione [MEN], and TBHP) would cause ER damage, leading to activation of calpain-dependent cell death pathways. In order to determine if activation of calpains was subsequent to ER stress, we also explored whether the selective ER stress inducers tunicamycin (TUN), a protein N-glycosylation inhibitor, and thapsigargin (THAPS), an inhibitor of the ER Ca2+-ATPase, triggered activation of calpain-dependent cell death pathways in LLC-PK1 cells. The results of these studies demonstrated a link between calpain and ER stress, but the sequence of events was different from that initially proposed.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Materials.
The LLC-PK1 cell line was obtained from the American Type Culture Collection (Manassas, VA). T-75 tissue culture flasks and 24-well tissue culture plates were obtained from Costar (Cambridge, MA). Fetal bovine serum was from Invitrogen (Burlington, ON, Canada). Enhanced chemiluminescence (ECL) reagents were obtained from Amersham Biosciences U.K. Ltd (Little Chalfont Buckinghamshire, England). IDAM, MEN, TBHP, TUN, THAPS, dimethylsulfoxide (DMSO), L-glutamine, Dulbecco's modified Eagles medium, protease inhibitor cocktail, and calpain inhibitor II were obtained from Sigma (Oakville, ON, Canada). The calpain inhibitor, PD150606, was from Calbiochem. The colorimetric lactate dehydrogenase (LDH) assay kits were obtained from Promega Corporation (Madison, WI). Anti-GRP94 antibody from StressGen Biotechnologies (CATALOG# SPA850), anti-GRP78 antibody from BD transduction laboratories (CATALOG# 610978), anti-caspase-12 antibody from Sigma (CATALOG# C7611), anti-m-calpain antibody from Triple Point Biologics (CATALOG# RP1-Calpain 2), and anti-ß-actin antibody from Sigma (CATALOG# A5441) were used in the study. All antibodies selected were specific for the targets identified.
Cell culture.
LLC-PK1 cells were cultured in Dulbecco's modified Eagles medium supplemented with 5% fetal bovine serum and 1% L-glutamine. Cells were cultured in a humidified 5% CO2 incubator at a temperature of 37°C. Cells grown to 75% confluency were harvested and seeded out at a density of 1.5 x 104 cells/cm2 in T-75 tissue culture flasks for protein expression experiments or 24-well plates for cytotoxicity experiments. The cells were grown for a period of 48 h with a single media change prior to initiating experiments.
Drug treatments.
TUN, THAPS, RCTs (oxidative stress inducers [TBHP and MEN] and covalent binding compound [IDAM]) were used as model cytotoxins in the study. Cells were exposed to all toxins for 3 h in Hank's balanced salt solution (HBSS) at the concentrations specified in the figure legends. MEN and TBHP were soluble in HBSS. IDAM was initially dissolved in DMSO and further diluted in HBSS so that the final DMSO concentration was 0.001%. The calpain inhibitor (PD150606; 5µM dissolved in DMSO [final concentration 0.05%]) was added at the same time as the toxicants. After 3 h, all treatments were removed and the cells were returned to complete media. At specified periods as described in the figure legends, cells were assessed for toxicity or harvested for protein expression determination by immunoblotting. All results are from at least three independent experiments, as described in figure legends.
Cytotoxicity.
Cytotoxicity at 3 h was assessed by measuring release of LDH and relative survival by the LDH content of remaining cells at 24 h (Nuss et al., 1996). The CytoTOX96 cytotoxicity assay kit (Promega Corporation) was used for all the LDH measurements.
Immunoblotting.
Cells were harvested using a nonenzymatic cell stripper solution (Cellgro; Media Tech, Inc, Herndon, VA). After incubation with the cell stripper solution for 30 min, the cells were removed from the plates by gentle scraping. The cells were pelleted and extracted with lysis buffer (composition: HEPES, 10mM, pH 7.4, NaCl, 200mM, MgCl2, 2.5mM, CaCl2, 2mM, Triton X-100, 0.1%) containing the protease inhibitor cocktail (1:100). The nuclei and cellular debris were removed by centrifugation for 10 min at 9000 x g. The protein concentrations were determined with the Biorad D/C protein assay (Biorad, Hercules, CA). Equal amounts (50 µg) of total protein from the cell lysate supernatants were separated in 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and blotted onto nitrocellulose membranes. This amount of protein was used to ensure that blots were within the linear range. Immunostaining of the blots was performed using a rabbit anti-m-calpain (1:1000) or mouse anti-caspase-12 (1:1000), rat anti-GRP94 (1:1000), mouse anti-GRP78 (1:1000), and mouse anti-ß-actin (1:2000). The appropriate secondary antibodies conjugated with peroxidase enzyme were used at 1:10,000 dilution. The immunoreactive bands were detected by ECL (Amersham). Densitometric analysis was performed using the Kodak Image Station 440CF (IS440CF). The value for each protein was normalized to the amount of ß-actin.
Statistical analysis.
A minimum of three independent experiments were performed for each experimental procedure. The immunoblots presented are representative of at least three independent experiments. The results are presented as the mean ± SEM, relative to control cells. All statistical analyses were carried out using the statistical analysis software program GraphPad Prism Version 3.03 (GraphPad Software, San Diego, CA). Single exposure experiments were analyzed by one-way ANOVA and double-exposure experiments were analyzed by two-way ANOVA, followed by Bonferroni's post hoc analysis. A p-value < 0.05 was considered significant. Statistical results are reported as *p < 0.05, **p < 0.01, or ***p < 0.001.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Calpain Inhibitors Block Cytotoxicity
The five toxicants caused significant morphological changes in the cells as early as 3 h, with clear evidence of cell damage and loss of cells by 24 h (Fig. 1). IDAM, TBHP, and TUN tended to be associated with rounding of the cells and membrane blebbing, with evidence of chromatin condensation in the nuclei. MEN caused minimal morphological changes at 3 h, but led to marked cellular contraction and loss of cell number by 24 h. THAPS was associated with cellular contraction and the formation of smaller round cell body remnants by 24 h. Cells treated with HBSS as a control did not show any morphological changes.
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All toxicants except MEN and THAPS caused a significant, dose-dependent increase in LDH release by 3 h (Figs. 2 and 3). By 24 h, except for TUN, the maximum concentration of each toxicant resulted in approximately 95–100% loss of cell number, as assessed by total LDH content (Figs. 2 and 3). TUN, at close to the limits of solubility, caused a 50% loss of cell viability.
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The calpain inhibitor, PD150606, reduced the baseline LDH release in control cells and completely blocked the LDH release induced by IDAM, TBHP, and TUN at 3 h (Figs. 2 and 3). PD150606 had no direct effect itself on cell survival at 24 h. However, it significantly (p < 0.001) and markedly increased the cell survival at 24 h following exposure to all the three RCTs. The greatest protective effect was observed for TBHP. The decrease in cell number associated with TUN at 24 h was also reversed by PD150606, but there was no effect on the THAPS-induced cell death.
To confirm the effects observed with PD150606, the effect of another calpain inhibitor, calpain inhibitor II (5µM) was assessed against IDAM and TBHP-induced cytotoxicity. Although the effects were not as great as PD150606, it significantly inhibited cell death at 3 and 24 h in a similar manner (data not shown).
Activation of ER Stress–Associated Pathways
In order to confirm that the ER had been disrupted, we assessed the induction of the ER stress proteins GRP78 and GRP94 in response to a high (> 75% loss of cell viability at 24 h) and a low concentration (< 25% loss of viability) of the RCT at 3, 8, and 24 h. All three RCTs led to significant induction of GRP78, when normalized to actin expression (Fig. 4). There was little to no induction at 3 h, mild induction at 8 h, and marked induction by 24 h. TUN and THAPS (Fig. 5) caused time-dependent induction of GRP78 with maximal induction by 24 h, consistent with their known ability to induce ER stress. Induction of GRP94 by RCTs and TUN was apparent (as shown in Fig. 4), but overall the observed induction was not significant because of markedly greater induction in one experiment. THAPS at 5µM caused a significant induction of GRP94 by 24 h, but 20µM led to an apparent degradation of GRP94 at all time points.
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Caspase-12 showed neither a change in levels nor an appearance of active bands after 3 h of RCT exposure (not shown). By 8 h, pro-caspase-12 was markedly induced by all three RCT, although cleaved caspase-12 was not detected (Fig. 6). By 24 h, a consistent induction of pro-caspase-12 was no longer apparent, but cleaved caspase-12 bands were observed. Caspase-12 cleavage products were more apparent at the lower concentrations of RCT and particularly when pro-caspase-12 concentrations were also decreased.
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TUN and THAPS did not consistently alter pro-caspase-12 expression at 3 h (not shown). At 8 h, TUN caused a loss of pro-caspase-12, but THAPS had no effect. By 24 h, both TUN and THAPS induced a loss of pro-caspase-12, but cleavage products were not consistently observed (Fig. 6).
Induction and Cleavage of m-Calpain
In order to further confirm the involvement of m-calpain in ER stress–associated cell death, cell lysates at 3, 8, and 24 h after RCT exposure were immunoblotted for m-calpain (Fig. 7). At 3 h, there was an increase in the number of immunoreactive bands of smaller molecular weight. By 8 h after the exposure, there was a marked induction of m-calpain and the largest cleavage product. By 24 h, after the initial exposure, the lower concentration of the RCT showed induction of m-calpain and continued presence of cleavage products, while the higher concentrations led to a reduction of total calpain and cleavage products.
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TUN and THAPS caused little change in m-calpain expression at 3 h (Fig. 8). By 8 h, TUN led a reduction in m-calpain, but cleaved products were not apparent. TUN and THAPS caused a significant decrease in m-calpain expression at 24 h, but only small amounts of cleavage products were observed.
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Effect of Calpain Inhibition on ER Stress Protein Expression
The induction of GRP78 at 3, 8, and 24 h following RCT exposure was almost completely inhibited by PD150606 (Figs. 9A and 9B). In 24-h experiments where these was an induction of GRP94, its induction was also inhibited by PD150606. In order to determine if PD150606 was blocking the events causing the ER stress, or if it was blocking the pathways of ER stress protein induction, the effects of PD150606 on TUN- and THAPS-dependent GRP78 and GRP94 induction were assessed. The induction of the ER stress proteins was only partially blocked at 3 and 8 h following exposure to the highest toxic concentration of TUN (Fig. 10) and not at the lower, relatively nontoxic concentrations. PD150606 had no effect on induction of GRP78 at 24 h (Fig. 10B). For THAPS (Figs. 11A and 11B), there was no inhibition of GRP78 or GRP94 induction. However, the cleavage of GRP94 following THAPS (20µM) exposure was markedly reduced, so that induction of GRP94 was apparently enhanced by the calpain inhibitor (Fig. 11A).
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To confirm the effects observed with PD150606, the effect of another calpain inhibitor, calpain inhibitor II, on the induction of GRP78 and GRP94 by RCT and ER stressors was assessed. Similar inhibitory effects were observed on the RCT-induced expression of GRP78 and GRP94 (data not shown). Also, consistent with the effects of PD150606, induction by THAPS or TUN at 24 h was not inhibited by calpain inhibitor II (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In vitro and in vivo studies have implicated both calpain and the ER in cell death pathways following exposure to nephrotoxicants and ischemia/reperfusion injuries (Chatterjee et al., 2005
; Harriman et al., 2002; Liu and Baliga, 2005; Liu et al., 2001; Lorz et al., 2004; Ryan et al., 2005). Activation of markers of ER-associated cell death (caspase-12; CHOP/GADD153) and induction of ER stress proteins following exposure to nephrotoxicants have been reported, and induction of ER stress proteins protects renal cells from a variety of toxins (reviewed in Cribb et al., 2005). Calpains have also been implicated in renal cell death following exposure to a range of nephrotoxicants and renal ischemia/reperfusion injuries (Harriman et al., 2002; Schnellmann and Williams, 1998). Therefore, both the ER and the calpain system have been identified as potentially common pathways in renal cell death.
In recent work (Ryan et al., 2005), we observed that exposure of LLC-PK1 cells to RCTs (IDAM, TBHP, and the reactive metabolite of sulfamethoxazole) led to the cleavage of caspase-12 and GRP94, events that have been attributed to m-calpain activation (Nakagawa and Yuan, 2000; Reddy et al., 1999). Therefore, we proposed that m-calpain activation would be an important part of the downstream cell death–signaling pathways in response to RCT targeting the ER in LLC-PK1 cells. Harriman et al. (2002) have suggested that there is a link between the ER and calpain activation through the release of ER calcium. However, we did not observe an increase in cytosolic calcium following exposure of LLC-PK1 cells to low concentrations of the RCTs investigated (Ryan et al., 2005), suggesting that another pathway may be involved. Therefore, we wished to further explore the relationship between calpain activation, ER stress, and cell death in LLC-PK1 following exposure to RCTs.
We elected to use three model RCTs: IDAM, associated with extensive covalent binding but also causing oxidative damage; MEN, that causes oxidative stress but also covalently binds to proteins, and TBHP, primarily associated with oxidative stress. The different morphological changes observed following exposure to the RCTs (Fig. 1) are consistent with there being known differences between these three RCTs in terms of their cellular effects. We also compared the effects of the RCTs to the effects of two well-known ER stress inducers. TUN and THAPS are classical ER stress inducers that are considered to produce relatively selective ER dysfunction, albeit by different mechanisms. These compounds were used at high concentrations to tip the balance from inducing a protective ER stress response to inducing ER stress–induced cell death (Cribb et al., 2005).
Calpain inhibition by PD150606 virtually eliminated LDH release at 3 h and significantly reduced cell death at 24 h, following exposure to all the RCTs. It is important to note that this similarity in response contrasts with the known differences in mechanisms and morphological characteristics of cell death was observed for these compounds. Calpain inhibition also effectively blocked or reduced the toxicity associated with 20µM TUN, while it had no effect on the toxicity of THAPS. To confirm that the observed result was not a result of an unexpected effect of PD150606, we assessed the effects of a second calpain inhibitor. Calpain inhibitor II, a relatively nonselective calpain inhibitor, produced a similar inhibition of cell death as PD150606.
THAPS is an ER Ca2+-ATPase inhibitor and is believed to lead to cell death and ER dysfunction primarily through the disruption of ER and, subsequently, cellular calcium homeostasis. The failure of calpain inhibition to block THAPS-induced cell death while inhibiting RCT-induced cell death is consistent with our previous observations suggesting that disruption of calcium regulation is not the key trigger behind cell death following exposure to moderate concentrations of RCTs in LLC-PK1 cells. We have previously observed that prior ER stress induction by TUN protects LLC-PK1 cells against RCTs (Bedard et al., 2004), but not against THAPS (Cribb, unpublished data) consistent with a different cell death–signaling pathway for THAPS and RCT. These data, therefore, suggest that the disruption of ER produced by RCTs more closely resembles that produced by TUN.
To confirm that RCTs were indeed causing a disruption of the ER and ER stress, we assessed induction of GRP78 and GRP94, and the expression and cleavage of caspase-12. All three RCT led to significant induction of GRP78. We did not, however, observe the extensive cleavage of GRP94 previously seen (Ryan et al., 2005), but induction of GRP94 was variable and did not reach significance as observed with GRP78. Part of the variability could have been the result of a balance between induction and cleavage of GRP94. Caspase-12 induction and cleavage was observed in a time- and dose-dependent manner, consistent with significant ER stress and activation of related caspase-12 pathways. These data confirm that the RCTs led to ER stress and activation of ER-associated cell death pathways.
Both ER stressors led to the induction of ER stress proteins, although 20µM THAPS also induced cleavage of GRP94. TUN and THAPS exposure led to a time-dependent loss of pro-caspase-12, consistent with cleavage of the pro-caspase-12, although cleaved products were not consistently observed.
The involvement of the calpain system was further explored by immunoblotting for the expression of calpain. We observed significant alterations in the expression of calpain and formation of immunoreactive cleavage products following exposure to RCTs. Active calpain may be difficult to differentiate from pro-calpain because of their similar molecular weights (80 vs. 78 kDa) (Goll et al., 2003). While smaller cleavage products also exist (as observed in our blots), it has also been shown that full-length calpain can be active calpain without cleavage (Goll et al., 2003). Therefore, while it is not possible to clearly assign activity to a single band, changes in expression of full-length calpain (induction or loss) and appearance of cleaved products can all be consistent with activation of the calpain system. In our experiments, we observed marked changes in calpain and its cleavage products in a time- and concentration-dependent fashion for the RCTs and the ER stressors, confirming the involvement of the calpain system.
As with caspase-12, TUN and THAPS led to a dose- and time-dependent loss of full-length calpain, although cleaved products were not observed. These results suggest that calpain activation was occurring with both compounds, consistent with the loss of pro-caspase-12. The extensive cleavage of GRP94 following 20µM THAPS was blocked by the calpain inhibitor, PD150606, suggesting that it was calpain dependent. This result is consistent with the previous reports that THAPS leads to activation of calpain in rabbit renal proximal tubular cells (Harriman et al., 2002). The failure of calpain inhibitors to block THAPS-induced cell death suggests, however, that other significant noncalpain-dependent cell death pathways must be activated by the extensive disruption of cellular calcium homeostasis produced by THAPS.
To further explore the relationship between calpain activation and the ER stress induced by RCTs examined, we assessed ER stress protein expression in the presence of calpain inhibition. We expected to observe induction of ER stress proteins in the absence of cell death. Unexpectedly, the induction of ER stress proteins following exposure to RCTs was almost completely blocked by the calpain inhibitor. This suggested that, contrary to our expectations, calpain activation was an early event following exposure to RCTs and may precede, rather than follow, the disruption of the ER. It was also possible, however, that the calpain inhibitor interfered with the induction of GRP78 and GRP94. Therefore, we examined the effect of calpain inhibition on the induction of these proteins following exposure to TUN and THAPS.
Similar to the effects observed with RCTs, the induction of GRP78 and GRP94 following exposure to the highest toxic concentration of TUN was inhibited at 3 and 8 h by calpain inhibition. However, there was no inhibition of induction at 24 h. There was no significant inhibition of ER stress protein induction at low concentrations of TUN or at any concentration of THAPS. As mentioned previously, the cleavage of GRP94 following exposure to high concentrations of THAPS was inhibited by PD150606, confirming that this cleavage was calpain dependent. These results indicate that the calpain inhibitor does not directly interfere with the induction mechanism of the ER stress proteins. Overall, therefore, these results suggest that the RCTs are first activating calpain and that the activation of calpain leads to a disruption of the ER and subsequent ER stress.
The results from this study identify a connection between calpain activation and disruption of the ER following exposure of renal cells to RCTs. This may then provide a link between studies identifying the calpain as a critical and common player in nephrotoxicity and other studies demonstrating that induction of ER stress proteins is a common protective pathway. These results suggest that calpain may not only play a role in propagating ER stress–associated events (e.g., cleavage of caspase-12) but in fact be an executor of ER damage and contribute to subsequent development of ER stress. The observations in this study and with IDAM and TBHP in previous studies (Liu et al., 1997, 1998) demonstrate that multiple cell death pathways can be involved when the ER is an important cellular target.
Further investigations are required to understand how calpain activation contributes to ER damage after exposure to RCTs. Investigations of clinically relevant nephrotoxins are required to determine the role of calpain and the ER in nephrotoxicity. These studies are underway in our laboratory.
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ACKNOWLEDGMENTS |
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This work was supported by a grant from the Canadian Institutes of Health Research to A.E.C. S.M. was supported by a CIHR Regional Partnership Fellowship. A.E.C. holds a Canada Research Chair. The work was made possible by an infrastructure grant from the Canada Foundation for Innovation.
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REFERENCES |
|---|
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Asmellash S, Stevens JL, Ichimura T. (2005) Modulating the endoplasmic reticulum stress response with trans-4,5-dihydroxy-1,2-dithiane prevents chemically induced renal injury in vivo. Toxicol. Sci. 88:576–584.
Bedard K, MacDonald N, Collins J, Cribb AE. (2004) Cytoprotection following endoplasmic reticulum stress protein induction in continuous cell lines. Basic Clin. Pharmacol. Toxicol. 94:124–131.[Web of Science][Medline]
Berridge MJ. (2002) The endoplasmic reticulum: A multifunctional signaling organelle. Cell Calcium 32:235–249.[CrossRef][Web of Science][Medline]
Chatterjee PK, Todorovic Z, Sivarajah A, Mota-Filipe H, Brown PA, Stewart KN, Mazzon E, Cuzzocrea S, Thiemermann C. (2005) Inhibitors of calpain activation (PD150606 and E-64) and renal ischemia-reperfusion injury. Biochem. Pharmacol. 69:1121–1131.[CrossRef][Web of Science][Medline]
Cribb AE, Peyrou M, Muruganandan S, Schneider L. (2005) The endoplasmic reticulum in xenobiotic toxicity. Drug Metab. Rev. 37:405–442.[CrossRef][Web of Science][Medline]
di Mari JF, Davis R, Safirstein RL. (1999) MAPK activation determines renal epithelial cell survival during oxidative injury. Am. J. Physiol. 277:F195–F203.
Goll DE, Thompson VF, Li H, Wei W, Cong J. (2003) The calpain system. Physiol. Rev. 83:731–801.
Harding HP, Calfon M, Urano F, Novoa I, Ron D. (2002) Transcriptional and translational control in the Mammalian unfolded protein response. Annu. Rev. Cell Dev. Biol. 18:575–599.[CrossRef][Web of Science][Medline]
Harriman JF, Liu XL, Aleo MD, Machaca K, Schnellmann RG. (2002) Endoplasmic reticulum Ca(2+) signaling and calpains mediate renal cell death. Cell Death Differ. 9:734–741.[CrossRef][Web of Science][Medline]
Liu CY and Kaufman RJ. (2003) The unfolded protein response. J. Cell Sci. 116:1861–1862.
Liu H and Baliga R. (2005) Endoplasmic reticulum stress-associated caspase 12 mediates cisplatin-induced LLC-PK1 cell apoptosis. J. Am. Soc. Nephrol. 16:1985–1992.
Liu H, Bowes RC, Water VB, Sillence C, Nagelkerke JF, Stevens JL. (1997) Endoplasmic reticulum chaperones GRP78 and calreticulin prevent oxidative stress, Ca2+ disturbances, and cell death in renal epithelial cells. J. Biol. Chem. 272:21751–21759.
Liu H, Miller E, Water VB, Stevens JL. (1998) Endoplasmic reticulum stress proteins block oxidant-induced Ca2+ increases and cell death. J. Biol. Chem. 273:12858–12862.
Liu X, Rainey JJ, Harriman JF, Schnellmann RG. (2001) Calpains mediate acute renal cell death: Role of autolysis and translocation. Am. J. Physiol. Renal Physiol. 281:F728–F738.
Liu X and Schnellmann RG. (2003) Calpain mediates progressive plasma membrane permeability and proteolysis of cytoskeleton-associated paxillin, talin, and vinculin during renal cell death. J. Pharmacol. Exp. Ther. 304:63–70.
Lock EA. (1999) Responses of the kidney to toxic compounds. In Ballantyne B, Marrs TC, Syversen TLM (Eds.). General and Applied Toxicology 2nd ed (Grove's Dictionaries Inc, New York) pp. 675–700.
Lorz C, Justo P, Sanz A, Subira D, Egido J, Ortiz A. (2004) Paracetamol-induced renal tubular injury: A role for ER stress. J. Am. Soc. Nephrol. 15:380–389.
Nakagawa T and Yuan J. (2000) Cross-talk between two cysteine protease families. Activation of caspase-12 by calpain in apoptosis. J. Cell Biol. 150:887–894.
Nuss CE, Grant DM, Spielberg SP, Cribb AE. (1996) Further investigations of the role of acetylation in sulphonamide hypersensitivity reactions. Biomarkers 1:267–272.
Rao RV, Ellerby HM, Bredesen DE. (2004) Coupling endoplasmic reticulum stress to the cell death program. Cell Death Differ. 11:372–380.[CrossRef][Web of Science][Medline]
Reddy RK, Lu J, Lee AS. (1999) The endoplasmic reticulum chaperone glycoprotein GRP94 with Ca(2+)-binding and antiapoptotic properties is a novel proteolytic target of calpain during etoposide-induced apoptosis. J. Biol. Chem. 274:28476–28483.
Ryan PM, Bedard K, Breining T, Cribb AE. (2005) Disruption of the endoplasmic reticulum by cytotoxins in LLC-PK1 cells. Toxicol. Lett. 159:154–163.[CrossRef][Web of Science][Medline]
Schnellmann RG and Williams SW. (1998) Proteases in renal cell death: Calpains mediate cell death produced by diverse toxicants. Ren. Fail. 20:679–686.[Web of Science][Medline]
van De Water B, Wang Y, Asmellash S, Liu H, Zhan Y, Miller E, Stevens JL. (1999) Distinct endoplasmic reticulum signaling pathways regulate apoptotic and necrotic cell death following iodoacetamide treatment. Chem. Res. Toxicol. 12:943–951.[CrossRef][Web of Science][Medline]
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