Homocysteine induces mesangial cell apoptosis via activation of p38-mitogen-activated protein kinase

original article

http://www.kidney-international.org & 2007 International Society of Nephrology

Homocysteine induces mesangial cell apoptosis via activation of p38-mitogen-activated protein kinase S Shastry1, AJ Ingram2, JW Scholey3 and LR James1 1

Department of Medicine, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas, USA, 2Department of Medicine, McMaster University, Hamilton, Ontario, Canada and 3Department of Medicine, University of Toronto, Ontario, Canada

Hyperhomocysteinemia is prevalent among patients with chronic kidney disease (CKD) and has been linked to progressive kidney and vascular diseases. Increased glomerular mesangial cell (MC) turnover, including proliferation and apoptosis, is a hallmark of CKD. Activation of p38-mitogen-activated protein kinase (p38-MAPK) has been linked to apoptosis in many cell lines. Accordingly, we studied the effect of homocysteine (Hcy) on MC p38-MAPK signalling and apoptosis. Hcy (50 lM/24 h) increased MC apoptosis as determined by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick end labelling (TUNEL) and single-stranded DNA (ssDNA) analysis. In addition to increases in pro-caspase-3 protein and caspase-3 activity, cells exposed to Hcy manifested enhanced reactive oxygen species content. Hcy increased p38-MAPK activity (fivefold), with maximal effect at 50 lM and 20 min; p38-MAPK activation was attenuated by N-acetylcysteine (Nac) and catalase (Cat), further indicating that the effect was via oxidative stress. Confocal microscopy revealed activation and nuclear translocation of p38-MAPK that was attenuated by Cat. In addition, Hcy-induced apoptosis as determined by TUNEL and ssDNA assay was abrogated by Nac, Cat, and SB203580 (p38-MAPK inhibitor). We conclude that in MC, Hcy (i) activates p38-MAPK and increases p38MAPK nuclear translocation via an oxidative stress dependent mechanism and (ii) induces DNA damage and apoptosis that is dependent on oxidative stress and p38-MAPK activation. Kidney International (2007) 71, 304–311. doi:10.1038/sj.ki.5002031; published online 6 December 2006 KEYWORDS: mesangial cells; p38-MAPK; oxidative stress; signalling; apoptosis

The mechanism(s) that lead to reduction in glomerular cell numbers and progressive glomerulosclerosis are unresolved. Podocyte loss is an early event in proteinuric kidney disease and glomerulosclerosis.1 On the other hand, mesangial cell (MC) proliferation and hypertrophy and mesangial matrix expansion are key events in progression of some forms of kidney disease.2,3 Importantly, MC apoptosis or programmed cell death plays a key role in both clinical and experimental kidney diseases.4,5 Although apoptosis is a necessary mechanism for tissue development and remodelling,6 in certain circumstances it may contribute to organ dysfunction and disease progression.4,7 Homocysteine (Hcy) or its metabolite S-adenosylhomocysteine cause apoptosis of neuronal cells,8 myeloid leukemia cell line HL60,9 and vascular endothelial cell.10 Hyperhomocysteinemia characterizes all forms of chronic kidney diseases (CKDs), but its role in the development and progression of renal disease is not well defined.11,12 Recently, p38-mitogenactivated protein kinase (p38-MAPK) has been implicated in apoptosis of diverse cells, including cardiac myocytes13 and vascular smooth muscle cells (VSMCs).14 The generation of reactive oxygen species (ROS) activates p38-MAPK and p38MAPK-dependent signalling in various cells and tissue.15,16 MC apoptosis is also induced by ROS,17,18 but the role of p38-MAPK in this ROS-dependent MC apoptosis is not defined. Given the observations that (i) ROS activate p38-MAPK,15,16 (ii) Hcy increases oxidant stress,19,20 and (iii) both hyperhomocysteinemia and p38-MAPK activation have been associated with apoptosis13,14 we hypothesize that Hcy induces oxidative stress which leads to MC apoptosis via a p38-MAPK-dependent mechanism. RESULTS Hcy induced apoptosis in MC

Correspondence: LR James, Department of Medicine/Nephrology Division, University of Texas Southwestern Medical Center at Dallas, Room H5.102, 5323 Harry Hines Blvd, Dallas, TX 75287, USA. E-mail: [email protected] Received 23 December 2005; revised 26 September 2006; accepted 10 October 2006; published online 6 December 2006 304

These experiments examined the impact of Hcy on MC apoptosis. Incubation of MC with Hcy (50 mM; 24 h) led to apoptosis as determined by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick end labelling (TUNEL) (Figure 1a and b) and DNA fragmentation (Figure 1c). In addition, MC pretreated with Hcy (15–100 mM; 371C/24 h) manifested a dose-dependent Kidney International (2007) 71, 304–311

original article

S Shastry et al.: Homocysteine-induced mesangial cell apoptosis

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Figure 1 | MC undergo apoptosis following exposure to Hcy. (a and b) MC were incubated with Hcy (50 mM/24 h), fixed (3.7% formaldehyde), permeabilized (triton X-100), then labelled (DMEM/ 0.5% FBS; 24 h) by TUNEL. Cells (b, green) were mounted using antifade with 40 ,6-diamidino-2-phenylindole, dihydrochloride (blue nuclei; a) as detailed in ‘Materials and Methods’; original magnification  40. Nuclear condensation and fragmentation is noted in (a) (arrows). (b) Depicts TUNEL staining of cells from 1A identifying apoptotic nuclei (fluorescein isothiocyanate (FITC)-green fluorescence; arrowhead); non-apoptotic cells do not reveal FITC staining. (c) DNA fragmentation: Lane 1 – molecular weight marker; Lane 2 – Control; Lane 3 – L-cysteine (100 mm); Lanes 4 to 6 – Hcy 50, 100, and 250 mm, respectively. (d) ssDNA increases in MC (70–80% confluent) exposed to Hcy (50–250 mM) whereas L-cysteine (100 mM) is ineffective. *Po0.05, n ¼ 5; #Po0.01, n ¼ 5.

increase in ssDNA levels, with a 1.5 to twofold increase occuring at 50 mM and increasing to threefold with 100 mM Hcy when compared with control and L-cysteine-treated cells (Figure 1d). These results suggest induction of MC injury and death by high Hcy concentrations.

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Figure 2 | Hcy increases procaspase-3 protein and caspase-3 activity. (a) Procaspase-3 protein and (b) caspase-3 activity are increased by Hcy (a). Cells were exposed to L-cysteine (100 mM) or Hcy (15–100 mM; 24 h) as detailed in ‘Materials and Methods’. Caspase-3 was detected with rabbit polyclonal anti-caspase-3 primary. Protein loading was assessed by b-actin immunoblots. *Po0.05, n ¼ 4. (b) Caspase-3 activity was determined using a Caspase-3 cellular activity assay kit (product No. 235419; Calbiochem). Assays were performed on cell lysate using 200 mM DEVD-pNA caspase-3 substrate (3 h/371C); recombinant caspase-3 was used as a positive control. DEVD-pNA substrate cleavage (pmol) was estimated by the product optical density (OD405nm) of cleaved species (pNA)  pmol pNA/ OD405nm (determined with supplied pNA). Caspase-3 activity (pmol/ min/mg) was determined from the quotient of the rate (pmol/min) of DEVD-pNA cleavage and cell extract protein. *Po0.05, n ¼ 5.

Induction of caspase-3 by Hcy in MCs

Caspase activation is a characteristic and stereotyped feature of cell undergoing apoptosis.21,22 To further support our observation of MC apoptosis in response to pathophysiologic Hcy levels, caspase-3 activity assay, and procaspase-3 immunoblotting were performed on total cell lysates. MC exposed to Hcy (50 mM) manifested increases in procaspase-3 protein and caspase-3 activity (Figure 2; Po0.05, n ¼ 4) supporting our observation of Hcy-induced MC apoptosis, and indicating the activation an execution pathway involving caspase-3. p38-MAPK activation and translocation in MC in response to Hcy

Subsequently, the effect of Hcy on MC p38-MAPK signalling was characterized. Incubation of MC with Hcy (5 mM to 1 mM; 1 h) led to an increase in p38-MAPK activity that was maximal at 50 mM (Figure 3a). Accordingly, we chose to study 50 mM, as this plasma level is commonly seen in patients with CKD.12 Time course studies with Hcy (50 mM) revealed maximal p38-MAPK activity after 20 min, with decay thereafter (Figure 3b). Consequently, all subsequent experiments Kidney International (2007) 71, 304–311

were performed at this time point. Activation of p38-MAPK was specific to Hcy, as L-cysteine (50 mM for 20 min) was without effect (data not shown). Subsequently, we observed that Hcy-induced caspase-3 enzymatic activity was abrogated by the p38-MAPK inhibitor SB203580 (Figure 3c). p38-MAPK nuclear translocation occurs subsequent to its activation.23 Using confocal microscopy, we observed that p38-MAPK activation was accompanied by translocation of phospho-(activated) p38-MAPK into the nucleus (Figure 3e and f) that was abolished by preincubation with catalase (Cat) (Figure 3g and h). Increased intracellular ROS in MC exposed to Hcy

Given the ability of Cat to prevent Hcy-induced p38-MAPK translocation, ROS generation was assessed using a cellpermeant fluoroprobe (carboxy 5-(and 6)-chloromethyl20 ,70 -dichlorodihydrofluorescein diacetate (CM-H2DCFDA)), after cells were exposed to Hcy with and without Nac and Cat. In accord with our earlier findings,24 we observed that MC exposed to Hcy exhibited increased fluorescence 305

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S Shastry et al.: Homocysteine-induced mesangial cell apoptosis

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Figure 3 | Hcy activates p38-MAPK in a concentration- and time-dependent manner, increases caspase-3 activity through p38-MAPK and induces p38-MAPK nuclear translocation. (a and b) p38-MAPK was measured in MC lysates following immunoprecipitation and subsequent detection of phosphorylation of an ATF-2 fusion protein. *Po0.05, n ¼ 3. (c) Demonstrates p38-MAPK-dependent Hcy-induced caspase-3 activity in MC lysates that was measured as detailed in the ‘Materials and Methods’. (d–h) *Po0.05, #Po0.01; n ¼ 4. Assess nuclear translocation of phospho-p38-MAPK in Hcy-challenged MC without or with inhibitors. (d) Negative control (secondary antibody only); (e) Background cytoplasmic and nuclear staining in control MC; (f) Hcy (50 mM) increased nuclear translocation of p38 MAPK; (g) preincubation with Cat (300 U/ml) attenuated the nuclear phospho-p38 MAPK. (h) Quantification of nuclear p38-MAPK was estimated using Image J (NIH, USA); 40 nuclei were analyzed/condition. Relative intensity represents the ratio pixel density/nucleus from (d) control to that from MC exposed to (e) Hcy or (f) Hcy and Cat. n ¼ 3, *Po0.05.

(Figure 4c and f; Po0.001, n ¼ 4) that was diminished by the Nac and Cat (Figure 4d–f). These observations are consistent with an interpretation of Hcy-induced intracellular ROS. Role of oxidative stress and p38MAPK in MC exposed to Hcy

To further link ROS to p38-MAPK activation, hydrogen peroxide (H2O2) was employed as an ROS.25,26 MC exposed 306

to H2O2 (50 mM; 20 min) manifested a significant (6-fold) elevation in p38-MAPK activity (Figure 5a), suggesting that in MC, ROS activate p38-MAPK. Oxidative stress may lead to DNA damage and apoptosis17 and, in some instances, p38-MAPK activation may mediate apoptosis.13 To determine whether Hcy-induced MC apoptosis was mediated by oxidative stress and p38-MAPK, MCs Kidney International (2007) 71, 304–311

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S Shastry et al.: Homocysteine-induced mesangial cell apoptosis

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Figure 4 | Assessment of Hcy-induced oxidative stress. Generation of intracellular ROS was determined with the cell-permeant fluoroprobe CM-H2DCFDA. (a) Control cells (no Hcy), (b) L-cysteine (100 mM), (c) Hcy with either (d) Cator (e) Nac. Analyses were performed as detailed in ‘Materials and Methods’. Representative images from four such experiments performed in triplicates are depicted; original magnification  40. (f) Relative intensity represents the ratio pixel density/cell (estimated using Image J (NIH, USA)) from (a) control to that from (b) MC exposed to L-cysteine, (c) Hcy, (d) Hcy and Cat or (e) Hcy and Nac. Forty cells were analyzed from each condition. *Po0.001, n ¼ 4; #Po0.01, n ¼ 4.

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Figure 5 | Hcy-induced p38-MAPK activation in MC is ROS-dependent. (a) H2O2 activates p38 MAPK in glomerular MC. Representative blot for three experiments performed in duplicates are shown. Lower panel depicts densitometry. (b) p38-MAPK activation by Hcy (50 mM) is inhibited by Nac, (1 mM), Cat. (300 U/ml) and SB203580 (10 mM). Activated p38-MAPK was measured in immunoprecipitates from lysates by detection of phosphorylation of an ATF-2 fusion protein by Western blotting; experiments were performed three times in duplicates. *Po0.01, n ¼ 3; #Po0.02, n ¼ 3.

were incubated with Hcy (50 mM) in the presence of known inhibitors of oxidative stress27,28 (Cat (300 U/ml) or Nac (1 mM)) or a p38MAPK inhibitor (SB203580 (10 mM)). Our analysis revealed that Hcy-mediated p38-MAPK activation was abolished by the SB203580, as well as by Cat and Nac (Figure 5b). Finally, to determine whether oxidative stress and p38MAPK activation by Hcy, were important for apoptosis, MC were preincubated (10 min) with Cat (300 U/ml) or SB203580 before Hcy exposure. TUNEL and ssDNA analyses Kidney International (2007) 71, 304–311

demonstrated that Hcy mediated MC apoptosis was inhibited by Cat and SB203580 (Figures 6a and b), further implicating oxidative stress and p38-MAPK activation as mediators of Hcy-induced MC apoptosis.

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CKD is characterized by a progressive increase in serum Hcy levels11,29 that may contribute to cardiovascular disease,30 and some pathologic effects of Hcy have been attributed to oxidative stress.19,20 VSMCs proliferate in the presence of Hcy31,32 with upregulation of cyclin A33 and the antioxidant Nac inhibits VSMC proliferation in response to Hcy.34 However, apoptosis has also been observed when VSMC are exposed to Hcy.35 In contrast, vascular endothelial cells respond to Hcy challenge with growth inhibition and apoptosis.36–38 Recently, we have demonstrated that in MC Hcy increased DNA synthesis, proliferation and endoplasmic reticulum stress in association with activation of p42/44 MAPK (extracellular signal-regulated kinase).24 In addition, whereas ROS increased in MC exposed to Hcy, ROS generation was unrelated to extracellular signal-regulated kinase activation. The present study further examined the influence of Hcy on MC turnover in vitro; specifically, we assessed Hcy’s role in MC DNA damage and apoptosis. These studies are particularly relevant given (a) our prior observations of Hcy’s influence on MC growth, (b) Hcy’s effect on growth, proliferation and death of the closely related VSMC, and (c) data derived from epidemiologic studies demonstrating that Hcy levels predict CKD, independent of other known risk factors.39 307

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Figure 6 | Hcy-induced MC death is mediated by oxidative stress and p38-MAPK activation. (a) Hcy-induced MC apoptosis is abrogated by Cat, Nac, and SB203580. TUNEL analyses were performed as described in text. *Po0.05, n ¼ 5; #Po0.02, n ¼ 5. (b) Hcy-induced immunoreactive ssDNA is inhibited by Cat. and SB203580. *Po0.5, n ¼ 4.

Our initial observation was that in MC challenged with Hcy (50 mM/24 h), there was increased DNA damage and apoptosis (Figure 1). ssDNA assay confirmed that Hcy dose dependently increased MC DNA damage in the concentration range from15 mM to100 mM (Figure 1). In unchallenged MC and rat glomeruli, apoptotic cells represent about 2%40,41 and 0.1%,40 respectively; thus moderate changes could contribute significantly to disease progression or resolution.42,43 Increases in apoptosis (from 0.05% at baseline to 0.5% at 120 days) has been observed in experimental CKD using subtotal nephrectomized rats.44 Though numerically small, these changes in glomerular apoptotic cells represent significant increases in total apoptotic population. Accordingly, our observed changes in apoptotic MC are of a magnitude consistent with these other observations. In concert with increased MC apoptosis following exposure to Hcy, the activity of caspase-3 was increased in Hcy-challenged MC. Caspases are cysteine proteases that are activated by apoptotic stimuli and caspase-3 is a key enzyme involved in execution of caspase-dependent apoptosis. Others have observed caspase activation in MC undergoing apoptosis.45,46 Our current observations are consistent with these reports. 308

The second major observations that we made were that (i) Hcy activates p38-MAPK in a time- and dose-dependent manner that is linked to caspase-3 enzymatic activity (Figure 3) and (ii) Hcy-induced p38-MAPK activation was curtailed by inhibitors of oxidative stress (Figure 5). H2O2 may serve as an important surrogate and positive control for demonstrating ROS involvement in physiologic and pathophysiologic processes.25,26 In addition, p38-MAPK activation may be mediated by ROS generation47,48 and/or p38-MAPK activation may mediate some adverse consequences of ROS generation.49 In our investigation of the effect of H2O2 on p38-MAPK in glomerular MC, we observed that H2O2 manifested marked activation of p38-MAPK (Figure 5). On the basis of these results we infer that ROS generation may play a role in Hcy-induced p38-MAPK activity in glomerular MC. Following MAPK activation, the active species are translocated to the nucleus where they exert influences on various processes.23,50 We next determined whether p38MAPK is translocated to MC nucleus in response to Hcy and whether ROS were important in this regard. Indeed, in MC exposed to Hcy (50 mM) there is enhanced nuclear phosphop38-MAPK above that observed in the basal state (Figure 3). Furthermore, this latter event is attenuated by Cat suggesting a role for H2O2 in enhanced nuclear localization of p38MAPK following exposure to Hcy. The final observation in the current study is the finding that the effect of Hcy on MC apoptosis could be attenuated by Nac, Cat, and p38-MAPK inhibition (Figure 6). This implicates an ROS-p38-MAPK pathway as a possible mediator in Hcy-induced MC injury and death and is consistent with previous observations in MC-related VSMC.35,51 In the case of glomerular MC, although there are no reports linking Hcy to cellular injury and death, we and other have previously observed enhanced ROS generation when MC were exposed to Hcy.24,52 ROS generation following Hcy exposure may be secondary to either enhanced NADH oxidase activity,53,52 auto-oxidation54 or increased intracellular H2O2.9 In support of a role for Hcy in intracellular ROS generation and in keeping with our previous findings,24 we observed that MC exposed to Hcy manifested increased ROS levels that were attenuated by antioxidants Cat and Nac (Figure 4). Therefore, we infer that Hcy causes MC injury and apoptosis through oxidant stressdependent mechanism(s), and our data indicate that p38MAPK activation downstream of ROS is the major producer of the apoptotic effect. Hcy causes renal injury in rats.42,55 Albumin excretion rate was found be directly related to serum Hcy,55 whereas creatinine clearance was inversely related to serum Hcy levels.42 Hcy is also known to induce apoptosis and proliferation in VSMC,35 which are phenotypically similar to MC and respond similarly to injurious stress, proliferation and matrix production. Hcy (10–20 mM) dose dependently increased VSMC proliferation and induced VSMC apoptosis.35 Similarly, MC apoptosis have been correlated with Kidney International (2007) 71, 304–311

S Shastry et al.: Homocysteine-induced mesangial cell apoptosis

mitotic activity.40 Accordingly, our earlier observations that Hcy caused cell proliferation via extracellular signal-regulated kinase signaling24 and the current findings that Hcy caused apoptosis via p38-MAPK suggest a dual effect of Hcy on cellular behaviour as have been demonstrated for other agents, like angiotensin II.56 In summary, the findings of these series of experiments identify Hcy as a cause of MC apoptosis, an event that is linked to ROS generation and is mediated by p38-MAPK activation (Figure 6). Our recent report documenting calcium-dependent, extracellular signal-regulated kinasemediated MC proliferation in response to Hcy,24 together the current findings suggest a role for Hcy in MC turnover and may have implications for progression of glomerular disease. Above all, these observations demonstrate a central role for MAPK in cellular responses to Hcy. High plasma Hcy levels are commonly seen are seen in CKD and hyperhomocysteinemia levels have been associated with CKD risk.11,12 Given that apoptosis of glomerular MC have been observed in various forms of CKD, these findings may have important implication for development and progression of CKD. Future studies aimed at testing this hypothesis in in vivo models are necessary. MATERIALS AND METHODS Cell culture Sprague–Dawley rat MC were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 20% fetal bovine serum (FBS) (Invitrogen, CA, USA), streptomycin (100 g/ml), penicillin (100 U/ml), and 2 mM glutamine at 371C in 95% air/5% CO2 as described previously.57 Cells from passage 5–10 were used throughout these studies. All chemicals were obtained from SigmaAldrich (St Louis, MO, USA) unless otherwise indicated. Apoptosis detection TUNEL assay. MC apoptosis was quantified with a commercially available in situ cell death detection (Roche Canada, Laval, Quebec, Canada). MCs were seeded onto glass cover slips in 12-well plastic plates (1.5  105 cells/well) and maintained in DMEM/20% FBS for 24 h. Culture media was changed to DMEM/0.5% FBS and Hcy (50 mM) with and without Cat (300 U/ml), Nac (1 mM), or SB203580 (10 mM) for 24 h. Cells were fixed (3.7% formaldehyde in phosphate-buffered saline (PBS); 20 min; room temperature), washed (  3, PBS), then permeabilized (Triton X-100 (0.1%; 2 min, 41C). Fixed cells were labelled by TUNEL (371C 1.0 h), washed (PBS) and mounted onto glass slides using antifade mount media with 40 ,6-diamidino-2-phenylindole, dihydrochloride (Vector Laboratories Inc. CA, USA). Subsequently, confocal (Bio-Rad MRC600 confocal microscope, Bio-Rad, Mississauga, ON, USA) and fluorescence microscopic (Nikon Instruments Inc., Lewisville, TX, USA) analyses were performed. ssDNA assay. This method is based on selective denaturation of apoptotic cells by formamide and detection of denatured DNA by monoclonal antibody.58 MC were seeded on to 96-well plates (11  103 cells/well) and maintained in DMEM/10%FBS until cells were 70–80% confluent. Culture media was changed to DMEM/ 0.5% FBS for 24 h. Cells were then pretreated with L-cysteine (100 mM) or Hcy (15 mM, 50 mM,100 mM and 250 mM) with or without

Kidney International (2007) 71, 304–311

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Nac (1 mM), Cat (300 U/ml), or SB203580(10 mM) at 371C for 24 h and analyzed for ssDNA apoptosis by enzyme-linked immunosorbent assay as per the manufacturer’s protocol (Chemicon International Inc, Temecula, CA, USA). DNA fragmentation analysis. Cells (106) were washed with PBS (  1) and dislodged from culture dishes with a rubber policeman. DNA fragmentation analysis was performed with a commercially available kit according to the manufacturer’s specification (R&D Systems, MN, USA). Caspase-3 protein detection by Western blotting Cultures were serum-starved overnight (DMEM/0.5% FBS for 24 h) before the addition of L-cysteine (100 mM) or Hcy (15 mM, 50 mM and 100 mM). Subsequently, washed cells (PBS, 41C) were harvested under non-denaturing conditions (41C/5 min) with lysis buffer (20 mM Tris, pH 7.4; 150 mM NaCl; 1 mM ethylenediaminetetraacetic acid; 1 mM ethylene glycol-bis(aˆ-aminoethyl ether)-N,N,N’,N’,tetraacetic acid; 1% Triton X-100; 1 mM glycerolphosphate, 1 mM sodium orthovanadate; 1 mg/ml leupeptin; 1 mM phenyl methylsulphonyl fluoride). Protein was separated (10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel), electroblotted to a nitrocellulose membrane (Schleicher and Schuell, Keene, NH, USA). Procaspase-3 was detected with rabbit polyclonal anti-caspase-3 primary antibody (Biovision, Mountain View, CA, USA; antibody diluted 1:2000 in 1  tris-buffered saline, 0.1% Tween-20 trisbuffered saline) and an horseradish peroxidase-conjugated antirabbit secondary antibody (Cell Signalling Technology, Danvers, MA, USA; antibody diluted 1:4000 with Tween-20 tris-buffered saline). Appropriate bands were identified with ECL Chemiluminescence Reagent (Amersham Biosciences, Little Chalfont, Buckinghamshire, England) and followed by exposure to X-ray film (X-OMAT, Kodak, Rochester NY, USA). Subsequently, immune complexes were removed from the membrane (stripping buffer (100 mM 2-mercaptoethanol, 2%sodium dodecyl sulfate, 62.5 mM Tris-HCl (pH 6.7); 501C; 30 min) and protein loading was assessed by re-blotting with anti-actin antibody (1:2000, Sigma, St Louis, MO, USA) and an horseradish peroxidase-conjugated anti-rabbit secondary antibody (1:4000) (Cell Signalling Technology). Caspase-3 activity assay Caspase-3 activity was determined using a Caspase-3 cellular activity assay kit (product 235419; Calbiochem, San Diego, CA, USA). MCs were pre-incubated with L-cysteine (100 mM), Hcy (15 mM, 50 mM and 100 mM), and Hcy (50 mM) plus SB203580 (10 mM). Cells were washed (PBS; 41C), lysed and cell debris was cleared (centrifugation 10 000 g/ 10 min at 41C). Cell lysates were transferred to 96-well microplate containing 200 mM DEVD-pNA caspase-3 substrate and incubated (3 h/371C); recombinant caspase-3 was used as a positive control. DEVD-pNA substrate cleavage (pmol) was estimated by the product: -optical density (OD405nm) of cleaved species (pNA)  pmol pNA/ OD405nm (determined with supplied pNA). Caspase-3 activity (pmol/ min/mg) was determined from the quotient of the rate (pmol/min) of DEVD-pNA cleavage and protein content of cell extract. p38-MAPK protein and activity Protein isolation and Western blotting. These procedures were performed using standard methodologies as we have previously described.23 Initially, the time course and concentration dependence of p38-MAPK activation in response to Hcy was studied and subsequent experiments performed at 50 mM Hcy at 20 min. Serum-

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starved MCs (DMEM/0.5% FBS; 18 h) were exposed to Hcy and/or inhibitors, and cell lysate was assayed for p38-MAPK protein and activity as we have previously described.23 For Western blot analysis, 40 mg cell protein was separated on a 12% sodium dodecyl sulfatepolyacrylamide gel electrophoresis gel and electroblotted to a nitrocellulose membrane. p38-MAPK protein was identified with p38-MAPK polyclonal primary antibody (1:1000) (New England Biolabs, Beverly, MA, USA), horseradish peroxidase-conjugated anti-rabbit secondary antibody (1:2000 New England Biolabs) and LumiGlo chemiluminescent reagent (KPL Inc., Gaithersburg, MD, USA). Activity assays. After protein isolation from total cell lysate as above, total protein (200 mg) was used for determination of p38MAPK activity as we have described.23 In brief, cell lysate phosphop38-MAPK was captured with phospho-p38-MAPK monoclonal antibody (Thr180/Tyr 182) immobilized on protein A beads (1:100) (New England Biolabs). P38-MAPK activity was determined with ATP (200 mM) and activating transcription factor 2 (ATF-2) fusion protein (2 mg) as substrates. Following electrophoresis (10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and electroblotting to nitrocellulose membranes, phosphorylated ATF-2 was identified with a phospho-specific ATF-2 (Thr 71) primary antibody (1:1000) and an horseradish peroxidase-conjugated anti-rabbit secondary antibody (1:2000).23

S Shastry et al.: Homocysteine-induced mesangial cell apoptosis

between the means of two groups, whereas the Tukey and Bonferroni tests were used for comparison of differences between multiple means. Differences were considered significant at Po0.05. ACKNOWLEDGMENTS

We wish to thank Hao Ly, Kerry Thai, Michael Mellon, and Maile Princena for technical assistance on this project. This work was supported by grants from the NIH Grant DK062799 (to LRJ) and Canadian Institute of Health Research (to JWS). REFERENCES 1.

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5.

6. 7.

Immunofluorescent microscopy for p38-MAPK nuclear translocation This procedure was performed as described previously.23 After incubation with Hcy with or without inhibitors, washing (thrice with 1  PBS), fixation (3.7% formaldehyde, 10 min, ambient temperature) and permeabilization (0.1% Triton X-100, 41C for 2 min), cells were incubated (30 min; room temperature) with antiphospho-p38-MAPK (1:25 dilution; Thr180/Tyr182; New England Biolabs). Subsequently, cells were incubated (30 min; room temperature, light protected) with an Alexa 488 goat anti-rabbit IgG (H þ L) conjugate (1:50 in PBS; Molecular Probes, Eugene, OR, USA). Laser scanning confocal microscopy (Bio-Rad MRC-600 confocal microscope; Bio-Rad) was performed anti-fade mounted specimen. Pixel density per nucleus was estimated using Image J (NIH, USA); 40 nuclei were analyzed per condition.

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Detection of ROS The generation of intracellular ROS was determined with the cellpermeant fluoroprobe carboxy 5–(and 6)-chloromethyl-20 ,70 -dichlorodihydrofluorescein diacetate (CM-H2DCFDA; Molecular Probes) in cells exposed to Hcy, with and without NAC or Cat. Analyses were performed as have been previously detailed.24 Washed cells were loaded with CM-H2DCFDA (10 mMol/l; 20 min at 371C), excess CM-H2DCFDA was removed, cells were washed (PBS) and allowed to recover (45 min/371C/5% CO2). Digital images of fluorescent cells (excitation 488 nm, emission 520 nm) were captured a using Nikon Fluorescence Microscope (Nikon Instruments Inc, Lewisville, TX, USA) and Olympus C-4000 Digital Camera (Olympus America Inc, Melville, NY, USA). As above, pixel density per cell was estimated using Image J (NIH, USA); 40 cells were analyzed per condition. Statistical analyses Instat statistical package (Graphpad Inc) was used for statistical analyses. Unpaired Student’s t-test was used to evaluate differences 310

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