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GROWTH, DIFFERENTIATION, AND APOPTOSIS

Cadmium-induced ceramide formation triggers calpain-dependent apoptosis in cultured kidney proximal tubule cells

Department of Physiology and Pathophysiology, University of Witten/Herdecke, Witten, Germany

Submitted 16 May 2007 ; accepted in final form 26 June 2007

	ABSTRACT

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A major target of cadmium (Cd²⁺) toxicity is the kidney proximal tubule (PT) cell. Cd²⁺-induced apoptosis of PT cells is mediated by sequential activation of calpains at 3–6 h and caspases-9 and -3 after 24-h exposure. Calpains also partly contribute to caspase activation, which emphasizes the importance of calpains for PT apoptosis by Cd²⁺. Upstream processes underlying Cd²⁺-induced calpain activation remain unclear. We describe for the first time that 10–50 µM Cd²⁺ causes a significant increase in ceramide formation by 22% (3 h) and 72% (24 h), as measured by diacylglycerol kinase assay. Inhibition of ceramide synthase with fumonisin B₁ (3 µM) prevents ceramide formation at 3 h and abolishes calpain activation at 6 h, which is associated with significant attenuation of apoptosis at 3–6 h with Hoechst 33342 nuclear staining and/or 3(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) death assays. This indicates that Cd²⁺ enhances de novo ceramide synthesis and that calpains are a downstream target of ceramides in apoptosis execution. Moreover, addition of C₆-ceramide to PT cells increases cytosolic Ca²⁺ and activates calpains. Apoptosis mediated by C₆-ceramide at 24 h is significantly reduced by caspase-3 inhibition, which supports cross talk between calpain- and caspase-dependent apoptotic pathways. We conclude that Cd²⁺-induced apoptosis of PT cells entails endogenous ceramide elevation and subsequent Ca²⁺-dependent calpain activation, which propagates kidney damage by Cd²⁺.

nephrotoxicity; cell signaling; cell biology and structure

The kidney is one of the primary organs adversely affected in humans after chronic oral or inhalational exposure to Cd²⁺. The majority of Cd²⁺ found in the kidney is localized in the epithelial cells lining the proximal tubule (PT), particularly in the S1 segment (15, 16, 61, 71). This occurs because any circulating Cd²⁺ in either bound or unbound form is ultrafiltrated by the kidney glomeruli and reabsorbed by the S1 segment of the PT, which possesses transport pathways and receptors for free Cd²⁺ as well as for the various forms of complexed Cd²⁺ (10, 61, 67). Hence, Cd²⁺-induced nephrotoxicity may result in a general transport defect of the PT with proteinuria, aminoaciduria, glucosuria, and phosphaturia (for review, see Ref. 66).

The cellular processes underlying Cd²⁺ nephrotoxicity usually culminate in the triggering of cell death by either apoptosis or necrosis (5, 57, 61, 72). PT apoptosis induced by Cd²⁺ has been described in vivo and in vitro (39, 59, 63) and involves increased generation of reactive oxygen species (ROS) (62). We (32) and others (37, 45) recently demonstrated that apoptosis is associated with the activation of calpain-dependent apoptotic pathways 3–6 h after exposure to 10–50 µM Cd²⁺. At later time points the mitochondria-dependent pathway predominates (51, 61), which entails the release of death-promoting factors such as cytochrome c and subsequent activation of executioner caspase-dependent apoptotic pathways (32, 33).

Ceramides are important regulators of cell proliferation, differentiation, inflammation, and apoptosis (14, 46, 52). They are either synthesized from serine and palmitate in a de novo pathway involving ceramide synthase or generated from the hydrolysis of the plasma membrane sphingolipid sphingomyelin (SM) by sphingomyelinases (SMases). There are several types of SMases; the most important in ceramide generation are the neutral and acidic forms (2, 31, 35). The generation of ceramides is triggered by a variety of stimuli such as cytokines, heat, UV radiation, hypoxia-reperfusion, or various cytotoxic agents and may involve oxidative stress (1, 25, 38). Thus the activity of neutral SMase is thought to be regulated by glutathione, because high levels of this reducing agent inactivate SMase. When high levels of ROS overwhelm the cell's antioxidant systems, SMase is activated and subsequently hydrolyzes SM to generate ceramides that go on to act as downstream signaling molecules (38).

Ceramides have long been known to be intimately involved in apoptotic signaling pathways (1, 24, 30, 31) and may act at a number of levels in the apoptotic program. There is substantial evidence for ceramides acting at the mitochondrial level to induce cytochrome c release leading to activation of executioner caspases without the requirement of initiator caspases (17, 18, 29, 56, 60). The study by Kim et al. (29) showed that C₆-ceramide induced cytochrome c release, which was mediated by a Bax-dependent and an initiator caspase-independent mechanism. Furthermore, Siskind et al. (56) provided evidence for a ceramide-induced increased outer membrane permeability in isolated mitochondria via ceramide channel formation. One mechanism by which ceramides could trigger apoptosis independently of initiator caspases may be the activation of calpains. Two reports so far associate ceramides with calpain-dependent cell death. These studies were performed in neuronal cells and showed that calpain activation required a mitochondrial step (49, 53), although cell death could also occur without activation of executioner caspases (49).

Despite the fact that a few studies in cultured PT cells have implicated ceramides as an "acute renal stress reactant" that increases in response to diverse ischemic or toxic insults (4, 28, 70), until now there have been no studies investigating the role of ceramides in Cd²⁺-induced PT cell death. Here we report for the first time that exposure to 10–50 µM Cd²⁺ for 3–6 h induces an increased formation of cellular ceramides, which trigger apoptosis of cultured rat kidney PT cells via a calpain-dependent pathway.

	MATERIALS AND METHODS

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Materials

Dulbecco's modified Eagle's medium-nutrient mixture F-12 (1:1), fetal bovine serum, and penicillin and streptomycin were all purchased from GIBCO (Carlsbad, CA). Apo-transferrin, dexamethasone, epidermal growth factor, ethidium bromide (EtBr), insulin, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), diethylenetriaminepentaacetic acid, octyl -D-glucopyranoside, adenosine 5'-triphosphate disodium salt (ATP), probenecid, and EDTA were all purchased from Sigma (St. Louis, MO). CdCl₂ and Silica-60 thin-layer chromatography (TLC) plates were from Merck (Darmstadt, Germany). Chloroform, acetic acid, and acetone were from AppliChem (Darmstadt, Germany). Hoechst 33342 (H-33342), fumonisin B₁, sn-1,2-diacylglycerol kinase (recombinant from Escherichia coli), and the caspase-3 inhibitor II benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val- Asp(OMe)-fluoromethyl ketone (z-DEVD-fmk) were from Calbiochem (San Diego, CA). Dithiothreitol (DTT) was from BDH Laboratory Supplies. Calpain activity fluorometric assay was from Biovision Research Products (Mountain View, CA). [-³²P]ATP (specific activity 4,500 Ci/mmol) was from MP Biomedicals (Eschwege, Germany). Heart bovine cardiolipin, N-hexanoyl-D-erythro-sphingosine (C₆-ceramide), and porcine brain ceramides were from Avanti Polar Lipids (Alabaster, AL). Fura-2 acetoxymethyl ester (AM) was purchased from Biotium (Hayward, CA). Inhibitors and drugs were dissolved in either water, ethanol, or dimethyl sulfoxide (DMSO), and in control experiments solvents were added to WKPT-0293 C1.2 cells at a concentration not exceeding 0.2% (vol/vol).

Methods

Cell culture. An immortalized S1 segment PT cell line from normotensive Wistar-Kyoto rats (WKPT-0293 C1.2) (69) was cultured as described previously (32, 33, 63). Briefly, cells were cultured in Dulbecco's modified Eagle's medium-F-12 (1:1) supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 µg/ml streptomycin, 1.2 mg/ml NaHCO₃, 5 µg/ml insulin, 4 µg/ml dexamethasone, 0.01 µg/ml epidermal growth factor, and 5 µg/ml apo-transferrin and passaged twice a week on reaching confluence. Experiments with Cd²⁺ (CdCl₂) were conducted with cells with a passage number <50 in serum-free medium.

Detection of apoptosis and necrosis with H-33422 and EtBr by fluorescence imaging. Staining experiments were conducted as previously described (32, 33). Briefly, 4 x 10⁴ cells were seeded per 35-mm dish (Corning, Corning, NY) in standard culture medium. After 48 h, the cells were treated with Cd²⁺ in serum-free medium. Control and Cd²⁺-treated cells were stained with 2 µg/ml H-33342 followed by 5 µg/ml EtBr. After being washed with 0.2 M HEPES, pH 7.4, cells were visualized with H-33342 and EtBr filters and a Visichrome High Speed Monochromator system (Visitron Systems, Puchheim, Germany) that was connected to a Zeiss Axiovert 200M microscope (Carl Zeiss, Jena, Germany) equipped with a x20 objective. Cells from five random microscopic fields of view at x200 magnification were counted per dish, and the average percentages of apoptotic and necrotic cells were calculated. Images were captured with a digital CoolSPAN ES charge-coupled device (CCD) camera (Roper Scientific, Tucson, AZ) and acquired, processed, and analyzed with MetaMorph software (Universal Imaging, Downingtown, PA).

MTT assay to determine mitochondrial function as a test for cell viability. The MTT assay determines the activity of mitochondrial succinate dehydrogenase and is therefore able to detect alterations of mitochondrial function. This is used as a measurement of cell viability, and hence an indicator of cell death. But this test does not distinguish between apoptosis and necrosis as well as the inhibition of cell growth (43). Assays were conducted according to Denizot and Lang (12), as previously described (32, 33). Reference wavelength values were subtracted from test wavelength values, and the differences obtained in controls were set to 100%, which was equivalent to 0% cell death.

Measurement of calpain activity. Calpain activity was determined according to manufacturer's instructions by cleavage of a calpain-specific substrate conjugated to the fluorophore 7-amino-4-trifluoromethylcoumarin (AFC), as described previously (32). Cells (9 x 10⁵) were grown for 48 h in 75-cm² flasks before treatment with or without Cd²⁺ or C₆-ceramide for 6 h in serum-free medium. Cells (0.3 mg protein) were lysed, and cytosolic extracts were mixed with Ac-LLY-AFC, incubated for 1 h at 37°C, and measured at excitation/emission wavelengths (_ex/_em) of 385/510 nm with a Berthold Mithras LB940 Multilabel Reader (Bad-Wildbad, Germany). Protein concentrations of cells and cytosolic extracts were determined by the methods of Bradford (9) and Lowry et al. (41), respectively.

Live Ca²⁺ imaging with fura-2 AM. WKPT-0239 Cl.2 cells (3 x 10⁵) were plated onto round glass coverslips with a diameter of 30 mm. The next day, medium was replaced with culture medium without phenol red indicator. Two days after plating, cells were washed once with probenecid-containing Ringer solution and incubated with 5 µM fura-2 AM in probenecid-Ringer solution for 1 h in the dark at room temperature. Probenecid was added to block extrusion of the fluorescent dye by anion transporters expressed in PT cells. After one wash in probenecid-Ringer solution for 20 min at 37°C, the coverslip was mounted onto a 1-ml chamber and gently perfused with probenecid-Ringer until a stable fluorescence signal was reached. Ratiometric Ca²⁺ measurements were performed by detecting fura-2 fluorescence at _ex of 340 and 380 nm and _em of 510 nm (22) with a Visichrome High Speed Monochromator system (Visitron Systems), which was connected to a Zeiss Axiovert 200M microscope equipped with a x20 Fluar objective (Carl Zeiss) and a Digital CoolSPAN ES CCD camera (Roper Scientific). Fluorescence values were obtained by selecting regions of interest, which were monitored with MetaFluor software (Universal Imaging) and background subtracted before being displayed. Emission ratios were measured every 2–5 s for up to a total of 50 min per experiment. When C₆-ceramide or DMSO was added to the chamber, the perfusion system was halted and fluorescence was continuously monitored.

Determination of cellular ceramides. Cellular ceramides was measured with the diacylglycerol (DAG) kinase assay as described elsewhere (8). WKPT-0293 Cl.2 cells (6 x 10⁵–1 x 10⁶) were seeded into 185-cm² flasks (Nunc, Nalge Nunc International, Rochester, NY) and grown in standard culture medium for 72 h before treatment with 50 µM Cd²⁺ for 3 or 24 h in serum-free medium. After treatment, cells were harvested into 10 ml of ice-cold buffered saline solution (BSS; in mM: 135 NaCl, 1.5 CaCl₂, 0.5 MgCl₂, 5.6 glucose, 10 HEPES pH 7.2) with a rubber policeman and centrifuged for 5 min at 400 g and 4°C. The cells were washed twice with ice-cold BSS and collected by centrifugation at 15,800 g for 1 min. The cell pellet was resuspended in 350 µl of BSS and sonicated twice for 10 s with a constant duty cycle and output control set at 3 on a Branson Sonifier 250 (Branson Ultrasonics, Danbury, CT). Unbroken cells were pelleted by centrifugation for 5 min at 400 g and 4°C and discarded. Protein concentration was determined by the method of Bradford (9).

Lipids were extracted from 0.4 mg of protein according to Bligh and Dyer (7) with 1 ml of 100:100:1 (vol/vol/vol) CHCl₃:MeOH:HCl at 4°C for 20 min in an Eppendorf Thermomixer (Eppendorf, Hamburg, Germany). The organic phase was dried under a constant stream of N₂. Dried lipids were digested with 500 µl of 100 mM KOH (dissolved in methanol) for 1 h at 37°C, followed by lipid extraction with 500 µl of chloroform, 270 µl of BSS, and 30 µl of 100 mM EDTA at 25°C for 20 min. Extracted lipids and ceramide standards (dissolved in chloroform to 0.5, 1.0, and 2.5 µM) were dried under a constant flow of N₂ and subsequently incubated for 30 min at room temperature with 150 µl of reaction mixture [150 µg cardiolipin dispersed in 20 µl of 1 mM diethylenetriaminepentaacetic acid by sonication in a water bath sonicator, 6.2 µl of 825 mM octyl -D-glucopyranoside, 50 µl of 2x reaction buffer (mM: 100 NaCl, 100 imidazole, 2 EDTA, 25 MgCl₂ pH 6.5), 7.2 µl of 10 mM imidazole pH 6.5, 0.8 µl of 1 mM diethylenetriaminepentaacetic acid, 2 µl of 100 mM DTT, 1 µl of 100 mM Na₂ATP pH 6.5, 7 µl of 2 U/mg protein DAG kinase, 50 µCi of [-³²P]ATP, and H₂O added up to 150 µl]. The DAG kinase reaction resulted in ³²P ceramide phosphorylation at position C₁. The reaction product was reextracted with 1 ml of 100:100:1 (vol/vol/vol) CHCl₃:MeOH:HCl additionally containing 120 µl of BSS and 30 µl of 100 mM EDTA (pH 7) for 20 min at room temperature. Lipids were then dried under a constant stream of N₂ and resolved for 135 min on a Silica-60 TLC plate with 50:20:15:10:5 (vol/vol/vol/vol/vol) CHCl₃:CH₃COCH₃:MeOH:CH₃COOH:H₂O as a mobile phase (6). The dried TLC plate was then exposed for 15 min to a FX Imaging-Screen-K (Bio-Rad Laboratories, Hercules, CA), and ³²P-ceramide radioactivity in standards and samples was quantified by scanning with a FX Personal Molecular Imager Scanner (Bio-Rad Laboratories).

Statistical analyses. Experiments were repeated at least three times, and representative data or means ± SE are shown. The dose-response curve of the effect of C₆-ceramide on cell death was fitted with the Sigma Plot 8.0 spreadsheet program assuming a sigmoidal function and using the Hill equation. Statistical analysis using unpaired Student's t-test was carried out with Sigma Plot 8.0. For more than two groups, statistical differences were compared with a one-way ANOVA assuming equality of variance with Levene's test and Tukey post hoc test for pairwise comparison. Statistical analysis was carried out with the SPSS 12.0 program. Results with P 0.05 were considered to be statistically significant.

	RESULTS

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Cd²⁺ Induces Cell Death of WKPT-0293 Cl.2 Cells

The ability of Cd²⁺ to induce cell death of PT cells is well documented in vivo (40, 59) as well as in vitro (62, 63). In this study, we further characterized the occurrence of apoptosis and necrosis in response to low micromolar Cd²⁺ at early time points in early-passage WKPT-0293 Cl.2 cells. Both types of cell death, determined by H-33342/EtBr nuclear staining, were measured after 10–50 µM Cd²⁺ incubation for 3 h (Fig. 1) and 6 h (shown previously in Ref. 32). Comparable rates of apoptosis (3–5% above control at both time points) were observed for all Cd²⁺ concentrations tested irrespective of time (3 or 6 h), and no significant increases in necrosis were detected. These results are in accordance with previous observations (62, 63). However, it must be stated that during the staining technique we could not avoid loss of dead cells that had already detached from neighboring cells. Thus it is possible that we are underestimating both apoptosis and necrosis.

View larger version (37K): [in this window] [in a new window]   Fig. 1. Cd2+-induced apoptotic cell death in proximal tubule (PT) cells. A: PT cells were grown as described in Methods and treated with varying Cd2+ concentrations for 3 h. Cells were stained with Hoechst 33342 (H-33342) and ethidium bromide (EtBr) to determine apoptosis (indicated by arrows) and necrosis. Bars = 50 µm. Nuclei are shown at higher magnification at top right. B: 5 microscopic fields were counted at x200 magnification. Values are means ± SE from 4 or 5 experiments. Statistical analysis using one-way ANOVA with Tukey post hoc test compared Cd2+-treated cells to controls.

Ceramide, which is considered to serve as a second messenger, is either generated by hydrolysis of SM through the action of SMase or by de novo synthesis and is regarded as an important cellular signal for inducing apoptosis (2, 47). To determine whether endogenous ceramides are involved in the cell death signaling pathways induced by Cd²⁺, intracellular ceramide levels of PT cells were measured with the DAG kinase assay (8). Ceramides were labeled with ³²P and then resolved by TLC. We measured ceramide levels after 10–50 µM Cd²⁺ exposure and found that 50 µM Cd²⁺ had the most reproducible and robust results (data not shown). Since the occurrence of apoptotic cell death is similar at these Cd²⁺ concentrations (Fig. 1B), 50 µM Cd²⁺ was selected for future experiments. Figure 2A shows a typical scan of radioactive-labeled ceramides. A standard curve was generated by loading differing concentrations of natural ceramides derived from brain, which consist of C₁₈-ceramides only. The concentration of natural ceramides extracted from PT cells was determined from the standard curve. The two spots observed on the TLC plate correspond to the hydroxy and nonhydroxy fatty acid derivatives of ceramide; therefore, both signals were included for densitometry analysis. Untreated PT cells had a basal level of ceramides (1.45 ± 0.09 µmol/mg protein; n = 9), but on treatment with 50 µM Cd²⁺ for 24 h the amount of ceramides was significantly augmented (Fig. 2A). Figure 2B shows a time course of ceramide levels induced by 50 µM Cd²⁺. Already after 3 h, a significant increase in ceramide formation above control values to 1.76 ± 0.23 µmol/mg protein was induced by 50 µM Cd²⁺, and ceramide formation almost doubled to 2.48 ± 0.26 µmol/mg protein after 24 h of Cd²⁺ incubation (Fig. 2B), when 40% cell death is induced by Cd²⁺ (32).

View larger version (26K): [in this window] [in a new window]   Fig. 2. Cd2+ increases ceramide formation in PT cells. A: representative scan of 32P-ceramide on a thin-layer chromatography (TLC) plate. Ceramides were extracted from PT cells treated with 50 µM Cd2+ for 24 h in serum-free medium and assayed with the diacylglycerol (DAG) kinase assay (see Methods). Natural ceramide standards were assayed in parallel. Ceramides were resolved by TLC. B: PT cells were treated with 50 µM Cd2+ for 3 or 24 h, followed by determination of cellular ceramides. 32P-ceramides were quantified by densitometry analysis. Values are means ± SE from 4–9 measurements. Basal ceramide levels in control cells were 1.45 ± 0.09 µmol/mg protein (n = 9). Control values were subtracted from the respective values in Cd2+-treated cells. Statistical analysis using one-way ANOVA with Tukey post hoc test compared ceramide formation in Cd2+-treated cells to controls.

To investigate whether Cd²⁺-induced apoptosis of PT cells and Cd²⁺-mediated generation of endogenous ceramides are mere coincidental phenomena or are linked in a causal relationship, WKPT-0293 Cl.2 cells were first depleted of sphingolipids. The Fusarium mycotoxin fumonisin B₁ potently inhibits ceramide synthase activity (IC₅₀ = 0.1 µM) and thereby blocks de novo sphingolipid biosynthesis (42, 65). PT cells were preincubated for 2 h with fumonisin B₁ and, in its continuing presence, subsequently treated with 50 µM Cd²⁺ for 3 h. Concentrations of fumonisin B₁ not exceeding 3 µM were chosen since fumonisin B₁ itself has apoptosis-inducing capacity at higher concentrations (13, 20, 54). After 3 h, 50 µM Cd²⁺ induced 2.6 ± 0.2% apoptosis compared with 0.5 ± 0.1% in controls as measured with the H-33342/EtBr staining method to determine levels of apoptosis and necrosis (P < 0.01; n = 3). When PT cells were incubated with 3 µM fumonisin B₁, Cd²⁺-induced apoptosis was abolished (0.9 ± 0.1%; n = 3; P < 0.01) (Fig. 3A). These data were confirmed with the MTT assay, which measures mitochondrial succinate dehydrogenase activity (data not shown). Similar effects of fumonisin B₁ (1–3 µM) on apoptosis were obtained when PT cells were exposed to 10 µM Cd²⁺ for 3 or 6 h, as measured with the MTT assay (data not shown) or the H-33342/EtBr staining method (Fig. 3B).

View larger version (14K): [in this window] [in a new window]   Fig. 3. Fumonisin B1 prevents Cd2+-induced ceramide formation and cell death of PT cells at 3 h. PT cells were treated with Cd2+ in serum-free medium for 3 h in the absence or presence of the ceramide synthase inhibitor fumonisin B1 (FB1, 3 µM), which was preincubated for 2 h before Cd2+ treatment. Apoptosis and necrosis induced by either 50 µM (A) or 10 µM (B) Cd2+ in the absence or presence of FB1 were ascertained by H-33342 and EtBr nucleic staining. Values are means ± SE from 3 experiments. C: PT cells were incubated with or without 50 µM Cd2+ and with or without 3 µM FB1. Ceramides were then extracted from PT cells. In control cells, 1.36 ± 0.13 µmol/mg protein of ceramides were measured (n = 6). Values are means ± SE from 4–6 experiments. Statistical analyses using one-way ANOVA with Tukey post hoc test compared ceramide formation or cell death of Cd2+- or FB1-treated cells to controls, FB1 to FB1+Cd2+, or FB1+Cd2+ to Cd2+-only cells; n.s., not significant.

Cd²⁺-Induced Activation of Calpains is Elicited by Ceramides in WKPT-0293 Cl.2 Cells

We recently demonstrated (32) that Cd²⁺-induced apoptosis of PT at 3- and 6-h exposure times is mediated by activation of calpains without the involvement of caspases. As a consequence, we investigated whether Cd²⁺-induced ceramide formation is part of the signaling pathway involving calpain activation and resulting in apoptosis. After exposure of PT to 10 µM Cd²⁺, calpain activity peaks at 6 h (32). Moreover, 10–50 µM Cd²⁺ cause similar increases in calpain activity after 6-h incubation (data not shown), which further supports the observations of Cd²⁺ concentration-independent apoptosis at early time points. Therefore we studied the relationship between ceramides and calpain activity at this time point. To prevent ceramide formation induced by Cd²⁺ we used fumonisin B₁. As shown in Fig. 4A, incubation of PT cells with 3 µM fumonisin B₁ marginally increased calpain activity [12.3 ± 2.2% above controls; n = 4; not significant (n.s.)]. Calpain activity was increased about twofold by 10 µM Cd²⁺ for 6 h (60.0 ± 17.1% above controls; n = 6; P < 0.01), which is the time point at which maximal calpain activation was detected (32). In the presence of 3 µM fumonisin B₁, Cd²⁺-induced calpain activity was abolished (7.9 ± 9.5% above controls; n = 5; n.s.) (Fig. 4A). We could further verify that ceramides were involved in mediating calpain activation since application of 3 µM C₆-ceramide, a synthetic short-chain ceramide that can easily diffuse across cellular membranes as opposed to natural long-chain ceramides, increased calpain activity after 6 h (60.1 ± 7.0% above controls, n = 3; P < 0.01) (Fig. 4B) to a similar extent as 10 µM Cd²⁺ (Fig. 4A). The data indicate that ceramide formation is part of the signaling cascade contributing to Cd²⁺-induced apoptosis at 3–6 h and that ceramides are generated upstream of calpain activation.

View larger version (12K): [in this window] [in a new window]   Fig. 4. Cd2+-induced activation of calpains is mediated by ceramide increase and Ca2+ release in PT cells at 6 h. A: PT cells were incubated with 3 µM FB1 for 2 h before addition of 10 µM Cd2+ for 6 h in serum-free medium. Calpain activity was determined in cytosolic extracts as described in Methods. Controls = 6,171.7 ± 2,297.4 fluorescence units/µg protein (n = 10). Values are means ± SE from 4–6 experiments. Statistical analyses using one-way ANOVA with Tukey post hoc test compared cell death of Cd2+- and/or FB1-treated cells to controls as well as FB1+Cd2+ to Cd2+-only cells. B: calpain activity was determined after 6-h incubation with or without 3 µM C6-ceramide (n = 3). Controls = 1,732.9 ± 137.4 fluorescence units/µg protein (n = 3). Statistical analysis using unpaired Student's t-test compared C6-ceramide-treated to control cells. C: cytosolic Ca2+ levels were monitored at excitation wavelengths of 340 and 380 nm and emission wavelength of 510 nm in fura-2-loaded cells on treatment with 3 µM C6-ceramide. A representative trace from 3 experiments is shown.

Cell Death of WKPT-0293 Cl.2 cells Induced by C₆-Ceramide is Partly Caspase-3 Dependent

Since long-chain ceramides (C₁₆–C₂₄) are cell impermeant, we investigated the death-inducing property of exogenously applied C₆-ceramide, which mimics the apoptosis-inducing effects of natural ceramides with acyl chain lengths of 16–24 carbon atoms (27, 44). By MTT assay, C₆-ceramide (1–50 µM) induced PT cell death in a dose-dependent manner with an EC₅₀ of 2.5–3.5 µM at all exposure times tested and reached saturation at 10 µM C₆-ceramide (Fig. 5A). The differences in EC₅₀ did not reach statistical significance. However, the slopes of the curves did differ: whereas the Hill coefficients of the curves at 3 and 6 h were similar (0.35 and 0.33, respectively), the Hill coefficient of the curve at 24 h had a value of 8.5, which suggests that a different mode of action of C₆-ceramide is responsible for cell death at 24 h. Furthermore, at concentrations of C₆-ceramide giving a maximal response, the magnitude of cell death (E_max) varied depending on the exposure time. Maximum cell death after 3- and 6-h exposure with 10–50 µM C₆-ceramide was 60%, but this increased significantly at the same concentrations to 90% cell death after 24-h exposure (Fig. 5A).

View larger version (18K): [in this window] [in a new window]   Fig. 5. C6-ceramide-induced calpain activation and cell death in PT cells. A: PT cells were incubated with increasing concentrations of C6-ceramide for 3, 6, or 24 h in serum-free medium. Cell death was determined with the MTT assay. EC50 was determined after sigmoidal fitting of each experiment. Values are means ± SE from 3 or 4 experiments. Statistical analysis using one-way ANOVA with Tukey post hoc test compared cells treated with C6 ceramide for 24 h to 3 and 6 h. B: caspase-3-dependent cell death (MTT) induced by C6 ceramide. Cells (5 x 103) were seeded in 24-well plates and grown for 48 h before incubation with caspase-3 inhibitor II z-DEVD-fmk (5 µg/ml) for 1 h and then with C6-ceramide (3 µM) or DMSO (0.1% vol/vol) for 24 h. Values are means ± SE from 11 experiments. Respective controls were deduced from C6-ceramide-exposed cells, where nontreated = 0.0 ± 0.0% and z-DEVD-fmk = 9.1 ± 4.3%. Statistical analysis using unpaired Student's t-test compared C6-ceramide-treated cells to C6-ceramide treated cells in the presence of z-DEVD-fmk.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
The aim of the present study was to determine whether ceramides play any role in Cd²⁺-induced apoptosis. Here, for the first time, we describe the generation of ceramides induced by exposure of kidney PT cells to low micromolar Cd²⁺ concentrations (Fig. 2). With the use of fumonisin B₁, an inhibitor of ceramide synthase, Cd²⁺-induced ceramide formation after 3 h (Fig. 3B) and apoptosis at 3–6 h (Fig. 3, A and B) are abolished. Because apoptosis at these time points is caspase independent and instead involves calpain activation (32), we investigated whether ceramide formation and calpain activation are linked processes. Indeed, using fumonisin B₁, we demonstrated ceramide formation to be a prerequisite for calpain activation and for subsequent apoptosis to occur (Fig. 4).

What is the Mechanism for Cd²⁺-Induced Formation of Ceramides in WKPT-0293 Cl.2 Cells?

The role of ceramide as a second messenger of apoptosis is indicated by numerous studies showing rapid increase (2–30 min) in ceramide levels, which were sustained over longer time periods, before the onset of apoptosis and the activation of caspases (36, 47, 58, 64). Increasing evidence indicates that ceramide formation and induction of oxidative stress are intimately connected in the process of apoptosis (1). For example, ceramide can stimulate ROS production (17, 50). On the other hand, increased ROS formation has been shown to induce increased ceramide generation by direct and/or indirect modulation of SMase (recently reviewed in Ref. 68). Our previous studies (62, 63) have shown that Cd²⁺ induces formation of ROS, which is involved in apoptosis of PT cells within 4–8 h and can be prevented by oxygen radical scavengers. It is therefore likely that ROS formation contributes to ceramide generation induced by Cd²⁺, although this remains to be demonstrated.

The data of the present study do not support a role for SMase in ceramide generation induced by Cd²⁺. We showed that fumonisin B₁ abolished ceramide formation, indicating that the de novo pathway (possibly ceramide synthase) was implicated in increased ceramide generation. In this context, it must be stated that 1–3 µM fumonisin B₁ alone did not induce apoptosis of PT cells, whereas 10–20 µM fumonisin B₁ caused significant cell death (data not shown). Further evidence indicating that the de novo pathway rather than SMase is involved in ceramide formation is the observation that Cd²⁺ induced a rapid drop of ceramides within 1 min of addition that lasted 30 min. This effect was balanced by a fumonisin B₁-sensitive increase of ceramide levels that rose above control levels within 1 h of Cd²⁺ addition and increased further up to 24 h (Fig. 2B; Torchalski and Thévenod, unpublished observations). Interestingly, another recent study demonstrated activation of ceramide synthase induced by a toxic hypoxia-reoxygenation stimulus in rat kidney tubular epithelial cells (4).

Possible Links Between Cd²⁺-Induced Formation of Ceramides in WKPT-0293 Cl.2 Cells and Calpain Activation

Cell death effects induced by Cd²⁺ observed at the early time points of 3–6 h appear to be independent of concentration (Fig. 1B and Ref. 63). In contrast, Cd²⁺ concentration plays a significant role at longer exposures (24 h), when the kinetics of Cd²⁺ accumulation into the cell is likely to differ between concentrations, which is exemplified by differences in caspase activation by 10 and 50 µM Cd²⁺ (32). Work performed in our laboratory (32) indicated that the mechanisms operating in early-onset Cd²⁺ apoptosis of PT cells (3–6 h) may involve the Ca²⁺-dependent proteases calpains, rather than caspases, as the executing enzymes. Calpains are a family of Ca²⁺-dependent intracellular cysteine proteases, including the ubiquitously expressed µ- and m-calpains as well as a number of distinct tissue-specific calpains (for review, see Ref. 19). µ- and m-calpains are both heterodimers consisting of a distinct large 80-kDa catalytic subunit encoded by the genes capn1 and capn2, respectively, and a common small 28-kDa regulatory subunit encoded by capn4. Interestingly, in vitro m-calpain activity requires calcium at the millimolar range, whereas micromolar concentrations activate µ-calpain. These calcium levels are unlikely to be achieved in vivo; however, highly localized concentrations of calcium might occur transiently in close proximity to ion channels or the endoplasmic reticulum during stress. Calpains are also negatively regulated by the endogenously expressed peptide inhibitor calpastatin. We previously speculated (32) that Cd²⁺ might activate calpains by a direct effect of the metal mimicking Ca²⁺. From the finding of the present study, calpain activation instead seems to depend on ceramide-induced increase of cytosolic Ca²⁺ (Fig. 4, A and C). Pinton et al. (48) reported that ceramide can directly cause Ca²⁺ release from endoplasmic reticulum, which leads to an increase in cytosolic Ca²⁺ levels. In this manner, pathological elevations in cytosolic Ca²⁺ could then induce calpain activation and subsequent apoptosis. However, we cannot exclude that Cd²⁺ itself might also induce Ca²⁺ release from intracellular stores (34, 37). Investigations of the effect and mechanisms of Cd²⁺ and ceramides on cytosolic Ca²⁺ in PT cells are the goals of future studies.

Consequences of Ceramide-Dependent Calpain Activation for Mitochondria- and Caspase-Dependent Cd²⁺ Apoptosis of WKPT-0293 Cl.2 Cells

If Cd²⁺-induced ceramide formation triggers activation of calpains (Fig. 4), then this should result in the activation of executioner caspases at later time points because we have previously observed calpain-dependent caspase activation (32). In other words, ceramide-induced cell death at 24 h should be partially caspase dependent. Indeed, the caspase-3 inhibitor z-DEVD-fmk (5 µg/ml) reduced ceramide-induced cell death (Fig. 5B). This observation, as well as differences in the magnitude and cooperativity of the cell death effect induced by C₆-ceramide at 3 and 6 h versus 24 h (Fig. 5A), further supports cross talk between early-onset apoptotic signaling pathways induced by Cd²⁺, i.e., ceramide formation inducing calpain activation, and late-onset apoptosis, involving release of proapoptotic mitochondrial components and activation of executioner caspases. Other studies have proposed that ceramide formation may contribute to activation of executioner caspases-9 and -3 by affecting mitochondrial function and/or stability: ceramides could affect mitochondrial stability through an indirect mechanism mediated by increased concentrations of cytosolic Ca²⁺ (see above; Refs. 21, 26, 48), or ceramide could be interacting either directly with the mitochondria by inhibiting the respiratory chain (11, 23) or by increasing the permeability of the mitochondrial outer membrane via ceramide channel formation (56). Our previous data (32) clearly indicated that calpains directly activate executioner caspases, because inhibition of calpains by PD-150606 reduced caspase-3 activity and Cd²⁺ apoptosis at 24 h by 50%. However, we cannot rule out that damaging effects of ceramides and/or Ca²⁺ on mitochondria may also contribute to the release of proapoptotic factors and activation of executioner caspases following incubation of PT cells with Cd²⁺ for 24 h.

In summary, these data further indicate that caspases represent a downstream target of calpain activity, as previously shown (32), and emphasize the central role of calpains in Cd²⁺-induced apoptosis of PT cells. A role of ceramide formation in the activation of mitochondria-dependent cell death pathways remains to be investigated.

	GRANTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Work in this laboratory is supported by the Deutsche Forschungsgemeinschaft (TH 345/8-1 and TH 345/10-1).

	ACKNOWLEDGMENTS

 
We thank Dr. U. Hopfer (Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, OH) for providing the WKPT-0293 Cl.2 rat proximal tubule cell line.

	FOOTNOTES

 

Address for reprint requests and other correspondence: F. Thévenod, Dept. of Physiology & Pathophysiology, Univ. of Witten/Herdecke, Faculty of Medicine, D-58448 Witten, Germany (e-mail: frank.thevenod{at}uni-wh.de)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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