TOXICOLOGICAL SCIENCES 126(1), 101–113 (2012) doi:10.1093/toxsci/kfr330 Advance Access publication December 6, 2011 Cyclosporine A–Induced Oxidative Stress in Human Renal Mesangial Cells: A Role for ERK 1/2 MAPK Signaling Séin O’Connell,1 Niamh Tuite,1 Craig Slattery, Michael P. Ryan, and Tara McMorrow2 UCD School of Biomolecular and Biomedical Research, UCD Conway Institute, University College Dublin, Dublin 4, Ireland Received July 29, 2011; accepted November 28, 2011 Cyclosporine A (CsA) is a potent immunosuppressant used to prevent organ transplant rejection and in the treatment of autoimmune diseases. However, chronic CsA nephropathy is the major limiting factor to its widespread use. The exact mechanisms of CsA-induced renal damage remain to be fully elucidated. The objective of the current research was to examine whether CsA treatment induced any glomerular mesangial cell alterations. In this research goal, human mesangial cells (HMCs) were treated with CsA for various time points. CsA caused an increase in the production of reactive oxygen species (ROS). Microarray analysis of mesangial cells treated with CsA also indicated 282 dysregulated genes. Bioinformatic analysis of these 282 genes indicated enriched apoptotic oxidative stress, mitogen-activated protein kinase (MAPK), and transforming growth factor-b signaling in response to CsA treatment. The focus of this study was directed on oxidative stress and MAPK signaling as potential novel mechanisms of CsA nephrotoxicity. One key contributor to oxidative stress, thioredoxin interacting protein, was significantly upregulated following CsA treatment. Inhibition of the MAPK pathway resulted in attenuation of the CsA-induced mesangial cell alterations. These findings suggest a major role for ROS, oxidative stress, and MAPK signaling in promoting CsA-induced glomerular dysfunction and subsequent nephrotoxicity. Key Words: CsA; glomerulus; MAPK; oxidative stress; thioredoxin interacting protein. Cyclosporine A (CsA) has improved allograft survival and the quality of life for solid-organ transplant recipients. Its effectiveness in transplantation by suppression of the immune system has led to its use in treating autoimmune diseases (Ruperto et al., 2006). CsA inhibits immune system cell proliferation by binding to cytoplasmic cyclophilin. This complex then inhibits calcineurin, which in turn inhibits the translocation of the nuclear factor of activated T cells and gene transcription of cytokines such as interleukin-2 (Flanagan et al., 1991). The most significant side effect caused by CsA administration is nephrotoxicity (Burdmann et al., 2003). This comprises both acute and chronic nephrotoxic effects. Acute CsA nephrotoxicity involves renal vasoconstriction with accompanying increased serum creatinine. These acute effects are largely reversible with reduction of dose or cessation of CsA therapy (Remuzzi and Perico, 1995; Shihab, 1996). In contrast, chronic CsA nephrotoxicity is characterized by irreversible progressive afferent arteriolopathy, tubular atrophy, striped tubulointerstitial fibrosis, and glomerulosclerosis (Cattaneo et al., 2004; Myers et al., 1984; Waiser et al., 2006). Several key factors have been studied for their involvement in chronic CsA nephrotoxicity, but the mechanisms are still not fully elucidated. Although tubulointerstitial fibrosis is widely regarded as the final common pathway in chronic CsA toxicity, glomerulosclerosis is also a major histopathological hallmark (Waiser et al., 2006). The mesangial cells of the glomerulus play an important role in regulating glomerular structure and function of the kidney as a whole. Mesangial cells establish and maintain the structural integrity and organization of both the individual tufts and the glomerulus itself. The mesangium provides mechanical strength for maintaining normal glomerular basement membrane structure (Kwoh et al., 2006). Disruption of the normal mesangium contributes to the development of proteinuria and deterioration of renal function (Wolf et al., 2005). Reactive oxygen species (ROS) production and oxidative stress signaling has been implicated in a variety of renal diseases such as IgA nephropathy and chronic kidney disease (Coppo et al., 2010; Rodrı́guez-Iturbe and Garcı́a Garcı́a, 2010). ROS are generated as by-products of normal cellular metabolism. Oxidative stress occurs when there is an imbalance between the production of ROS and the capacity of a cell’s antioxidant scavenging mechanisms (Halliwell and Whiteman, 2004). The thioredoxin system and the glutathione system are the two most important systems for maintaining the cell interior in a reduced state (Droge et al., 1994; Holmgren, 1985). Impairment of these systems or an increased production of ROS can lead to excess ROS levels within the cell. Thioredoxin-interacting protein (TXNIP), also known as vitamin D3 upregulated protein 1 (VDUP1), was first identified Ó The Author 2011. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please email: journals.permissions@oup.com Downloaded from https://academic.oup.com/toxsci/article/126/1/101/1712314 by guest on 06 June 2022 1 These authors contributed equally to this work. To whom correspondence should be addressed at Renal Disease Research Group, School of Biomolecular and Biomedical Science, UCD Conway Institute, University College Dublin, Dublin 4, Ireland. Fax: þ353-1-7166456. E-mail: tara.mcmorrow@ucd.ie. 2 102 O’CONNELL ET AL. MATERIALS AND METHODS Cell culture and treatment. The HMC was used during the course of this work (Sraer et al., 1996). The HMCs were maintained in RPMI 1640 containing 5% fetal calf serum, penicillin, streptomycin, and L-glutamine and maintained at 37°C in a humidified atmosphere containing 95% air and 5% CO2. CsA was obtained from Sigma-Aldrich (Cat no. C1832). Confluent HMCs were treated with 4.2lM CsA. Cells used in the inhibitor studies were pretreated for 1 h with U0126, prior to incubation with CsA treatment. Cell morphology and viability. Mesangial cell morphology was assessed using phase contrast microscopy. Mesangial cell viability was assessed using the resazurin (Sigma-Aldrich, Cat no. 7017) cell viability assay. This assay was conducted according to the manufacturer’s protocol. The viability of the cells was expressed as a percentage of the absorbance recorded for control cells. BrdU cell proliferation assay. BrdU is a synthetic thymidine analog, which is incorporated into the DNA of actively replicating cells. The BrdU assay was performed using the commercially available Calbiochem kit, according to the manufacturer’s protocol (Cat no. QIA58). This colorimetric assay detects BrdU incorporation into proliferating cells, and proliferation was expressed as a percentage of control incorporation. Western blot assay. Total protein was isolated from mesangial cells using the RIPA buffer method (Sigma-Aldrich, R0278) according to the manufacturer’s protocol. The SDS-polyacrylamide gel electrophoresis procedure used was that of Laemmli (Laemmli, 1970). Expression levels of renal proteins following CsA treatment was determined by Western blot and has been described previously (Feighery et al., 2008; McMorrow et al., 2005; Slattery et al., 2005). Proteins of interest were detected using the following antibodies according to the manufacturer’s protocol (rabbit anti-ERK 1/2, Cell Signaling Technology, 9211S and 9211). Time-matched controls were used in the phosphorylation studies. Mesangial cell adhesion assay. Following CsA treatment, cells were trypsinized to form a single cell suspension using a balanced salt solution containing 0.5% (wt/vol) trypsin and 0.2% (wt/vol) EDTA. Trypsin activity was inhibited by addition of serum and then cells were harvested by centrifugation. Cells were resuspended in growth medium and counted. An equal number of cells were applied to wells of a 96-well plate. Cells were permitted to adhere to the plate for 30 min at 37°C. Nonadherent cells were removed by aspiration, and the remaining adherent cells were fixed with 3.7% formaldehyde. Cells were stained for 30 min with crystal violet solution (0.5% (wt/vol) crystal violet and 20% (vol/vol) MeOH). Cells were lysed and absorbance was read at 590 nm on a spectrophotometer. Plates were also coated with 10 ng/ml collagen I, 10 ng/ml collagen IV, or 5 ng/ml fibronectin in a sterile environment overnight under UV light. Detection of ROS production using fluorescence measurement of the oxidant sensitive probe CM-H2DCFDA. Commercially available CMH2DCFDA (5-(and-6)-chloromethyl 2#,7# dichloro-dihydro fluorescein diacetate, acetyl ester) was prepared as a stock solution according to the manufacturer’s instructions (Molecular Probes, Cat no. C6827). This assay was performed according to the manufacturer’s protocol. Briefly, cells were given 10lM CM-H2DCFDA for 30 min at 37°C. Cells were then with 4.2lM CsA and fluorescence measured on a Wallac Victor2 1420 Multilabel HTS counter plate reader (485 nm excitation and 535 nm emission). RNA isolation. Total RNA was isolated using TRIzol reagent (Invitrogen) and quantitated by absorbance at 260 nm. RNA integrity was controlled by electrophoretic analysis on 1.2% agarose gels. RNA was purified for microarray analysis using Qiagen Mini-Spin clean up columns. Microarray analysis. This process has been described previously (Slattery et al., 2005). Briefly, human mesangial cells were incubated in the presence of 4.2lM CsA. RNA was isolated at 0, 12, and 48 h posttreatment. Complementary DNA was synthesized from the total RNA using SuperScript Choice kit (Invitrogen) with a T7-(dT)24 primer. Complementary RNA (cRNA) was prepared and biotin labeled by in vitro transcription (Enzo Biochemical). Labeled cRNA was fragmented and 15 mg of fragmented cRNA from each time point was hybridized for 16 h at 45°C to an HG-U133A array (Affymetrix, Santa Clara, CA). After hybridization, each gene chip was automatically washed and stained with streptavidin-phycoerythrin. Finally, probe arrays were scanned at 3mM resolution using GeneChip System confocal scanner made for Affymetrix by Agilent. Affymetrix Microarray suite 5.0 was used to analyze the relative abundance of each gene. Data was analyzed with the GeneSpring (Silicon Genetics, San Carlos, CA) to generate lists of genes with differential expression. Additional data and statistical analyses were carried out using Microarray Suite version 4.0.1 (Affymetrix). To identify differentially expressed genes, we directly compared gene expression profiles of CsA-treated HMCs with time-matched controls at Downloaded from https://academic.oup.com/toxsci/article/126/1/101/1712314 by guest on 06 June 2022 as a gene upregulated in HL-60 cells following stimulation with vitamin D3 (Chen and DeLuca, 1994). Subsequently, VDUP1 was identified as a thioredoxin-binding protein, which can inhibit the function of thioredoxin and decrease thioredoxin expression (Nishiyama et al., 1999; Yamanaka et al., 2000). Increased TXNIP results in the inhibition of thioredoxinmediated scavenging of ROS leading to enhanced sensitivity of cells to oxidative stress. Recently in murine fibroblast cells, a variety of extracellular stimuli have been shown to induce a transient increase in the intracellular concentration of ROS, and specific inhibition of the ROS generation resulted in a complete blockage of this stimulant-dependent signaling (Gulati et al., 2001). Important observations on the role of ROS as physiological regulators of intracellular signaling cascades activated by growth factors through their tyrosine kinase receptors have shed new light on the possible mechanisms underlying the growth regulatory activity of oxygen species (Chiarugi et al., 2003). However, there is still a debate whether oxidative stress is a cause or a result of these diseases, largely due to a lack of our understanding of the mechanisms by which ROS function in both normal physiological and disease states. Just this year, the potential interaction between ROS and the various mitogenactivated protein kinase (MAPK) signaling pathways has been reviewed and the evidence for the interaction examined (Son et al., 2011). The MAPK pathway is an enzymatic cascade pathway of evolutionary conserved enzymes that transduce signals from cell-surface receptors to the nucleus in response to a variety of extracellular stimuli. The mammalian MAPKs are well-characterized and include the extracellular signal–regulated kinases (ERKs), the c-jun-terminal kinases, and the p38 MAPKs (Roux and Blenis, 2004). MAPKs activity is regulated through three-tier cascades composed of a MAPK, a MAPK kinase (MAPKK or MEK), and a MAPKK kinase (MAPKKK or MEKK) (English et al., 1999). Evidence of MAPK activation has been observed in both acute and chronic renal diseases (Islam et al., 2011; Masaki et al., 2003). However, the role of ROS and MAPK signaling in CsA-induced toxicity in renal mesangial cells has yet to be defined. The aim of this study was to investigate the effects of CsA on human mesangial cell lines (HMCs) and identify novel mechanisms involved in CsA-induced nephrotoxicity. CsA-INDUCED MESANGIAL CELL DYSFUNCTION 12 and 48 h using GeneSpring version 4.2. Genes had to exhibit a fold change of greater than two with a p-value of less than 0.05 to be regarded as differentially expressed. The data discussed in this publication have been deposited in NCBI’s Gene Expression Omnibus and are accessible through GEO Series accession number GSE30952. The data can be viewed at the following location: http:// www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token¼pjedzsamgqwqshi&acc¼ GSE30952. Statistical analysis. Statistical analyses were performed using GraphPad Prism 4.0. Data was analyzed by one-way ANOVA and multiple comparisons between control and treatment groups were made using the Bonferroni posttest. A Student’s t-test was used for assessing statistical differences between two groups. A probability of 0.05 or less was deemed statistically significant. Results were expressed as the mean ± SEM. The following scheme was used throughout the work. (*p < 0.05, **p < 0.01 and ***p < 0.001). RESULTS also evident. CsA at 42lM caused much more pronounced alterations in HMC morphology and accompanying cell death was indicated by floating cells observed in the well. HMC viability following treatment with the same range of concentrations of CsA was assessed using a resazurin conversion assay (Fig. 1B). Treatment with CsA at concentrations up to and including 4.2lM resulted in no significant effect on cell viability. There was a trend toward decreased HMC viability at 4.2lM. CsA at 42lM resulted in a significant reduction in HMC viability to 85 ± 6% of control. Based on these results, the subcytotoxic concentration of 4.2lM CsA was chosen for all further experiments. Although this concentration is outside the range of plasma values observed in patients receiving CsA, which typically peak at 1lM (approximately 1000 ng/ml) (Kovarik et al., 1994), Lensmeyer et al. (1991) reported significant renal concentration of CsA compared with blood, of up to eightfold. This suggests that 4.2lM CsA is an achievable renal CsA concentration in patients and is therefore clinically relevant. HMC proliferation was analyzed in the presence of CsA using the BrdU incorporation assay (Fig. 1C). Treatment with CsA for 24 h had no significant effect on BrdU incorporation. However, a significant decrease in BrdU incorporation was observed after exposure to CsA for 48 h (100 ± 7.4 vs. 78 ± 4.7; *p < 0.05). CsA Exposure Resulted in Dose-Dependent Toxicity in HMCs The effect of a range of concentrations of CsA on the morphology of HMCs for 48 h was investigated (Fig. 1A). There was no significant alteration in HMC morphology at concentrations below 4.2lM. However, 4.2lM caused an elongation of HMCs, and gaps in the HMC monolayer were CsA Significantly Altered the Gene Expression Profile of HMCs Microarray analysis of gene expression changes induced in HMCs by CsA (4.2lM) over a 48-h period is shown in Tables 1 and 2. A minimum twofold change was used as a cutoff for FIG. 1. CsA caused dose-dependent toxicity in human mesangial cells. Confluent HMCs were treated with increasing concentrations of CsA (0.042–42lM) for 48 h. (A) HMC morphology was assessed by phase contrast microscopy (Magnification 3200) or (B) HMC viability was assessed by the resazurin conversion assay. (C) HMC proliferation was assessed using the BrdU uptake assay after 24- and 48-h CsA treatment. *indicates statistically significant difference to control (*p < 0.05 and **p < 0.01). Downloaded from https://academic.oup.com/toxsci/article/126/1/101/1712314 by guest on 06 June 2022 Bioinformatic pathway and biological process analysis. Gene lists generated from GeneSpring of differentially expressed genes following CsA treatment were uploaded onto the publicly available PANTHER website (www.pantherdb.org) or the commercially available Ingenuity Pathway Analysis (IPA) (Ingenuity Systems, Redwood City, CA) program. Gene lists were categorized into pathways and biological processes. Following software analysis significantly enriched pathways and processes were identified. 103 104 O’CONNELL ET AL. TABLE 1 Upregulated Genes in Mesangial Cells Following CsA Treatment as Detected by Microarray Analysis Downloaded from https://academic.oup.com/toxsci/article/126/1/101/1712314 by guest on 06 June 2022 Notes. Confluent HMCs were treated with 4.2lM CsA for 0 (control), 12, or 48 h. Total RNA was extracted, purified, and complementary DNA synthesized. cRNA was then prepared before labeling, washing, and hybridization to a HG-U133A array. Affymetrix Microarray suite 5.0 was used to analyze the relative abundance of each gene. Data were analyzed with the GeneSpring (Silicon Genetics, San Carlos, CA) to generate lists of genes with differential expression. Listed here are the downregulated genes following 12- or 48-h CsA treatment compared with control. CsA-INDUCED MESANGIAL CELL DYSFUNCTION 105 TABLE 2 Downregulated Genes in Mesangial Cells Following CsA Treatment as Detected by Microarray Analysis Downloaded from https://academic.oup.com/toxsci/article/126/1/101/1712314 by guest on 06 June 2022 Notes. Confluent HMCs were treated with 4.2lM CsA for 0 (control), 12, or 48 h. Total RNA was extracted, purified, and complementary DNA synthesized. cRNA was then prepared before labeling, washing, and hybridization to a HG-U133A array. Affymetrix Microarray suite 5.0 was used to analyze the relative abundance of each gene. Data were analyzed with the GeneSpring (Silicon Genetics, San Carlos, CA) to generate lists of genes with differential expression. Listed here are the downregulated genes following 12- or 48-h CsA treatment compared with control. 106 O’CONNELL ET AL. Pathway and Biological Process Alterations Following CsA Treatment Computational approaches were employed to identify biological pathways among the list of differentially expressed genes that respond to CsA (Fig. 2). Major cellular processes dysregulated by CsA treatment were identified through analysis of the 282 dysregulated genes using the publicly available PANTHER software (www.pantherdb.org) and IPA (Ingenuity Systems, Redwood City, CA). These programs sort genes into canonical pathways based on the scientific literature and identify significantly overrepresented pathways in a gene expression dataset. PANTHER identified a number of canonical signaling pathways, including a number of pathways relevant to apoptosis, oxidative stress, and MAPK signaling (Fig. 2A). IPA analysis was performed using the toxicology pathway analysis module. The 10 most significantly enriched toxicological pathways identified are shown in Figure 2B. These included renal cell death, mitochondrial dysfunction, oxidative stress, and transforming growth factor (TGF)-b signaling. CsA-Induced Alterations in TXNIP RNA and Protein Levels in HMCs Since microarray and pathway analysis experiments highlighted the potential role of oxidative stress in this model, mechanisms regulating this pathway were further examined. One potentially significant member of the oxidative stress pathway, TXNIP, was significantly increased in HMCs treated with CsA. This finding was confirmed by RT-PCR (Fig. 3A). This increased expression was also apparent in TXNIP protein levels following CsA treatment in HMCs, with a significant increase observed following 48-h treatment (Fig. 3A, *p < 0.05). CsA also increased ROS production in HMCs. ROS production was measured using the oxidant sensitive probe CM-H2DCFDA (Bass et al., 1983; LeBel et al., 1992). This dye is oxidized to a fluorescent substance in the presence of ROS; the level of fluorescence is proportional to the amount of ROS present. HMCs were treated with CsA (4.2lM) for 6 or 24 h in the presence of CM-H2DCFDA, and an increase in fluorescence, indicating increased ROS production in CsA-treated cells, was observed following 24-h treatment (Fig. 3B). HMC adhesion to the surrounding matrix is important for a number of reasons, including maintenance of normal glomerular structure and function as well as signal transduction and secretion of a variety of signaling molecules. The effect of treatment with CsA at 4.2lM for 24 and 48 h on HMC adhesion to tissue culture plastic (TCP) and to a range of extracellular matrix (ECM) proteins was examined. HMC adhesion following 24-h treatment with CsA is shown in Figure 3C. Adhesion of HMCs to TCP (p < 0.05), collagen I (p < 0.05), collagen IV (p < 0.05), and fibronectin (p < 0.01) was significantly reduced following treatment with CsA, 4.2lM for 24 h. Both control and treated cells displayed the highest affinity for fibronectin-coated plastic. Cells were less adherent to collagens I and IV, and HMCs adhered to TCP with the least affinity. Similar results were obtained following 48-h HMC treatment (Fig. 4). Since the potential involvement of MAPK signaling was also suggested by pathway analysis (Ras signaling, epidermal growth factor signaling, and platelet-derived growth factor and TGF-b signaling), we investigated the role of MAPK in CsA-induced effects using the MEK1 inhibitor, U0126. The levels of ERK 1/2 activation following treatment with CsA in the presence and absence of the MEK inhibitor U0126 are shown in Figure 5A. ERK 1/2 phosphorylation was significantly increased following treatment with CsA at early time points. At later time points, ERK signaling had returned to control levels. Pretreatment with U0126 abolished both the CsA-induced increase in ERK 1/2 activity and basal ERK 1/2 activity. Time-matched controls remained constant under all conditions as represented by the total ERK 1/2 blot. Inhibition of ERK 1/2 signaling also blocked the CsA-induced increase in TXNIP protein expression at 48 h (Fig. 5B). Inhibition of ERK 1/2 MAPK Attenuated CsA-Induced ROS Production in HMCs The morphology of HMCs treated with CsA in the presence or absence of U0126 is demonstrated in Figure 6A. Inhibition of ERK 1/2 signaling prevented the CsA-induced alterations in HMC morphology. HMC shape was similar to control with only a few elongated cells observed. However, some gaps in the monolayer were still evident indicating only partial protection by ERK inhibition. No alteration in HMC morphology was observed in the presence of U0126 alone. Inhibition of ERK signaling also did not result in any decrease in HMC viability (Fig. 6A). Inhibition of ERK 1/2 activity also attenuated the CsA-induced increase in ROS production in HMCs (Fig. 6B). At 6 and 24 h in the presence of U0126, there was no longer a significant increase in ROS production with CsA treatment. Basal levels of ROS were also reduced in the presence of U0126. Downloaded from https://academic.oup.com/toxsci/article/126/1/101/1712314 by guest on 06 June 2022 statistical analysis. Following CsA treatment, a total of 136 genes were upregulated. Thirty-five genes were increased after 12-h CsA treatment, and 114 genes were increased at the 48 h time point. Only 13 genes were common to both time points (Table 1). Following CsA treatment, 146 genes in total were downregulated. Twenty-six genes were decreased by 12 h, and 125 genes were decreased at 48 h. Five genes were downregulated at both time points (Table 2). In a list of all upregulated and downregulated genes, the fold change and their associated p-values are presented in Tables 1 and 2. A number of these gene expression changes were independently confirmed by realtime reverse transcription polymerase chain reaction (RT-PCR) (data not shown). CsA-INDUCED MESANGIAL CELL DYSFUNCTION 107 Downloaded from https://academic.oup.com/toxsci/article/126/1/101/1712314 by guest on 06 June 2022 FIG. 2. Bioinformatic analysis of enriched biological pathways and processes following CsA treatment in mesangial cells. Gene lists identified from the microarray analysis were bioinformatically analyzed using Panther and IPA software applications. (A) The top 10 significantly enriched pathways using Panther pathway analysis. The total number of genes in the pathway and the number of genes from our dataset in that pathway and the p value are shown here. (B) The top 10 significantly enriched pathways and their logarithmic p values using IPA is shown here. DISCUSSION In this study, the mechanisms of CsA-induced HMC dysfunction were investigated, and the hypothesis that CsA treatment increased ROS generation, which contributed to HMC dysfunction was tested. Exposure to CsA resulted in increased ROS generation, which promoted HMC dysfunction. These detrimental alterations were then attenuated upon inhibition of MAPK signaling. The results of this study suggest novel mechanisms of CsA-induced mesangial cell toxicity, which could potentially contribute to the development of progressive glomerulosclerosis, which can lead to end-stage renal disease (ESRD). Meta-analysis of signaling pathways and biological processes following a particular stimulus is a relatively new 108 O’CONNELL ET AL. phenomenon but is becoming widely accepted as a means for qualitatively observing cellular changes on a global scale. Several bioinformatic programs are available for such analysis. In the current study, two programs, Panther and IPA, were employed. Using these programs, dysregulated pathways following drug treatment can be analyzed and their importance examined (D’Alessandro et al., 2010; Szabo et al., 2010). In this current study, the strategy of combining experimental data with high-throughput gene expression analysis and pathway analysis was extremely useful in highlighting important signaling pathways and identifying which gene changes were particularly relevant to the biological alterations observed. Components of the oxidative stress pathway have been recently implicated in the proposed molecular mechanisms of the response to cadmium and other nephrotoxin exposure in renal proximal tubular epithelial cells (Wilmes et al., 2011). In the current study, oxidative stress was one of the major pathways enriched upon CsA treatment indicating an enhanced signaling through this pathway. The TGF-b pathway was also significantly enriched following CsA treatment which was to be FIG. 4. Altered mesangial cell adhesion following CsA treatment. HMC adhesion to TCP or different ECM proteins was examined following 4.2-lM CsA treatment for 24 or 48 h. Graphical results are represented the mean ± SEM of three independent experiments. *indicates statistically significant difference to control (*p < 0.05). Downloaded from https://academic.oup.com/toxsci/article/126/1/101/1712314 by guest on 06 June 2022 FIG. 3. Increased TXNIP and ROS production following CsA treatment in human mesangial cells. (A) Confirmation of microarray results showing increased TXNIP gene expression following 48-h CsA treatment by RT-PCR and densitometric analysis. Western blot and densitometric analysis of TXNIP protein following 24-, 48-, or 72-h CsA treatment. (B) Hydrogen peroxide production was examined following 6- and 24-h CsA treatment in HMCs. This was conducted using the oxidant sensitive probe CM-H2DCFDA. Graphical results are represented the mean ± SEM of three independent experiments. *indicates statistically significant difference to control (*p < 0.05). CsA-INDUCED MESANGIAL CELL DYSFUNCTION 109 expected as it is widely accepted that TGF-b is one of the major profibrotic mediators of CsA nephrotoxicity (Martin-Martin et al., 2010; McMorrow et al., 2005; O’Connell et al., 2011; Slattery et al., 2005). The MAPK signaling pathway was also identified as being significantly active following CsA treatment. Again, it has been proposed that MAPK signaling has an important role promoting CsA-induced renal dysfunction (Feldman et al., 2007; Kiely et al., 2003). These detrimental effects observed following CsA treatment in the human mesangial cells were correlated with enhanced ROS production. Increased ROS levels have been implicated in a number of in vitro and in vivo nephrotoxicity models (Parra et al., 1998; Pérez de Lema et al., 1997). In the current study, microarray analysis also demonstrated that TXNIP, an inhibitor of thioredoxin, which is an important ROS scavenger, was significantly increased following CsA treatment (Choksi et al., 2011). Increased TXNIP also known as VDUP1 and increased ROS production have been observed in mesangial cells exposed to high glucose. Exposing cells to high glucose is widely accepted as a model of diabetic nephropathy (DN) Downloaded from https://academic.oup.com/toxsci/article/126/1/101/1712314 by guest on 06 June 2022 FIG. 5. The effect of MAPK inhibition on the CsA-induced alterations in TXNIP protein. (A) Western blot and densitometric analysis of ERK 1/2 activity. ERK 1/2 activity was assessed following 30 min, 6-, 24-, and 48-h CsA treatment in the presence and absence of the MAPK inhibitor U0126. Whole cell ERK activity was used as a control. Densitometric analysis results are expressed as the ratio of phosphorylated ERK to whole cell ERK. (B) Western blot and densitometric analysis of TXNIP following CsA treatment in the presence or absence of U0126 for 24 or 48 h. Graphical results are represented the mean ± SEM of three independent experiments. *indicates statistically significant difference to control (*p < 0.05, **p < 0.01 and ***p < 0.001). 110 O’CONNELL ET AL. Downloaded from https://academic.oup.com/toxsci/article/126/1/101/1712314 by guest on 06 June 2022 FIG. 6. Inhibition of ERK 1/2 MAPK in the attenuated the CsA-induced increase in ROS production in human mesangial cells. HMCs were treated with CsA for 24 or 48 h in the presence or absence of U0126. (A) HMC morphology and viability was assessed by phase contrast microscopy (Magnification 3200) or a resazurin conversion assay, respectively. (B) Hydrogen peroxide production was examined following 6- or 24-h CsA treatment in the presence or absence of U0126 in HMCs. This was conducted using the oxidant sensitive probe CM-H2DCFDA. Graphical results are represented the mean ± SEM of three independent experiments. *indicates statistically significant difference to control (*p < 0.05 and **p < 0.01). 111 CsA-INDUCED MESANGIAL CELL DYSFUNCTION attenuated the CsA-induced alterations in renal epithelial cell barrier function (Feldman et al., 2007). In the current study, inhibition of the ERK 1/2 MAPK pathway resulted in decreased ROS and improved HMC viability by reducing one potential contributor to this oxidative stress, TXNIP. Clinical studies have also demonstrated oxidative stress in kidney transplant patients treated with CsA (Calo et al., 2002). Therefore, this study provides a novel mechanism of CsAinduced nephrotoxicity involving enhanced ROS production as a result of increased TXNIP expression, mediated by ERK 1/2—MAPK signaling. In vivo, this would contribute to prolonged oxidative stress and promote mesangial cell dysfunction, which can lead to glomerulosclerosis, tubulointerstitial fibrosis, and ultimately ESRD. FUNDING This work was supported by grants from the Health Research Board, the Hadwen Trust, and Science Foundation of Ireland. This project was also funded by the EU 7th Framework grant ‘‘SysKid,’’ HEALTH–F2–2009–241544 and the Conway Institute of Biomolecular and Biomedical Research and the Dublin Molecular Medicine Center, under the Programme for Research in Third Level Institutions administered by the Higher Education Authority. C.S. is funded by a Government of Ireland Research Fellowship from the Irish Research Council for Science, Engineering, and Technology. ACKNOWLEDGMENTS Thanks to the Transcriptomics Core Technology of Conway Institute. REFERENCES Bass, D. A., Parce, J. W., Dechatelet, L. R., Szejda, P., Seeds, M. C., and Thomas, M. (1983). Flow cytometric studies of oxidative product formation by neutrophils: A graded response to membrane stimulation. J. Immunol. 130, 1910–1917. Billiet, L., and Rouis, M. (2008). Thioredoxin-1 is a novel and attractive therapeutic approach for various diseases including cardiovascular disorders. Cardiovasc. Hematol. Disord. Drug Targets 8, 293–296. Burdmann, E. A., Andoh, T. F., Yu, L., and Bennett, W. M. (2003). Cyclosporine nephrotoxicity. Semin. Nephrol. 23, 465–476. Calo, L. A., Davis, P. A., Giacon, B., Pagnin, E., Sartori, M., Riegler, P., Antonello, A., Huber, W., and Semplicini, A. (2002). Oxidative stress in kidney transplant patients with calcineurin inhibitor-induced hypertension: Effect of ramipril. J. Cardiovasc. Pharmacol. 40, 625–631. Cattaneo, D., Perico, N., Gaspari, F., and Remuzzi, G. (2004). Nephrotoxic aspects of cyclosporine. Transplant. Proc. 36, 234S–239S. Chen, K. S., and DeLuca, H. F. (1994). Isolation and characterization of a novel cDNA from HL-60 cells treated with 1,25-dihydroxyvitamin D-3. Biochim. Biophys. Acta 1219, 26–32. Downloaded from https://academic.oup.com/toxsci/article/126/1/101/1712314 by guest on 06 June 2022 (Kobayashi et al., 2003). TXNIP modulates thioredoxin, which can then alter cellular redox balance promoting oxidative stress. Controlling the redox system has recently been proposed as a novel and attractive approach in treating a variety of pathologies (Billiet and Rouis, 2008). In the current study, increased TXNIP was observed at later time points than the observed increase in ROS, so it is possible that we are observing both increased ROS generation and reduced ROS scavenging. In renal proximal tubular epithelial cells, TXNIP was upregulated in response to high glucose suggesting it had a role in the pathogenesis of DN (Qi et al., 2007). More recently, it has been demonstrated in rat mesangial cells that p38 MAPK inhibition attenuated increased TXNIP expression in response to high glucose (Fang et al., 2011). However, it has not been previously shown that TXNIP and its attenuation by ERK 1/2 inhibition has a role in CsA-mediated mesangial cell dysfunction. Increased ROS has also been proposed as a trigger of various signaling pathways and one of these included the focal adhesion kinase (FAK). Chiarugi et al. (2003) observed two major conclusions: (1) ROS have a major role in the signaling cascade triggered by integrins during cell–ECM interaction and (2) modulation of integrin signaling and cell adhesion through FA formation by ROS is mediated, at least in part, by an upregulation of FAK in mouse embryonic fibroblasts. Increased ROS in the current study may have resulted in the decreased mesangial cell adhesion observed following CsA treatment. In the kidney, adhesion of mesangial cells to the glomerular ECM plays a central role in regulating cell proliferation, contraction, and survival (Rupprecht et al., 1996). Any change in the adhesive properties of mesangial cells is likely to have far-reaching implications for cell function and for the integrity of the kidney as a whole. Mesangial cells provide vital structural support for the glomerulus and are essential for maintaining the balance of ECM production and breakdown within the glomerulus, appropriate adhesion of mesangial cells is essential for preserving these and other functions. A change in the adhesive properties may also result in an altered capacity for cell migration as coordinated adhesion and deadhesion is essential for cell migration (Slattery et al., 2005; Webb et al., 2002). Increased mesangial cell migration may have a role to play in the mesangial cell response to injury. Impaired adhesion of mesangial cells exposed to CsA may also affect the integrity of the glomerular barrier. We observed decreased adhesion of mesangial cells to TCP and decreased adhesion to the ECM components, collagen I and IV, and fibronectin following treatment with CsA. Although ROS-induced signal transduction is becoming clearer, to our knowledge, this is the first report that inhibition of the ERK 1/2-MAPK pathway attenuated CsA-induced increases in ROS generation in human mesangial cells. It has been shown previously that proinflammatory mediators caused mesangial cell dysfunction and that this was mediated by ERK 1/2 and p38 MAPK (Nee et al., 2007). ERK 1/2 inhibition also 112 O’CONNELL ET AL. Chiarugi, P., Pani, G., Giannoni, E., Taddei, L., Colavitti, R., Raugei, G., Symons, M., Borrello, S., Galeotti, T., and Ramponi, G. (2003). 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