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High-Mobility Group Box Protein 1: A Novel Mediator of Inflammatory-Induced Renal Epithelial-Mesenchymal Transition

Background: High-mobility group box protein 1 (HMGB-1) is a chromatin-binding protein that bends DNA, thereby facilitating gene transcription. HMGB-1 has also been observed as an extracellular secreted protein in serum of patients with sepsis and has putative intracellular signalling effects regulating the production of interleukin-1 and tumour necrosis factor in a number of inflammatory conditions. Methods: We established a model of immune-mediated epithelial-mesenchymal transition (EMT) in human proximal tubular epithelial cells (PTECs). PTECs were cultured with conditioned medium containing supernatant from activated peripheral blood mononuclear cells (aPBMCs). The model was characterized using phenotypic and transcriptomic approaches and suppression subtractive hybridisation was performed to identify differentially regulated genes. Results: Activation of PBMCs resulted in increased secretion of HMGB-1. In addition, treatment of PTECs with aPBMC-conditioned medium resulted in significant upregulation of HMGB-1 in PTECs. Direct treatment of PTECs with recombinant human HMGB-1 induced alterations in epithelial morphology consistent with EMT including reduced E-cadherin expression, increased α-smooth muscle actin expression and enhanced cell migration. HMGB-1 effects were mediated at least in part by the receptor for advanced glycation end products and through induction of transforming growth factor-β1 secretion from PTECs. Conclusions: These results suggest that HMGB-1 is a key mediator of immune-mediated EMT of PTECs and a potentially important signalling molecule in the development of renal fibrosis.


Introduction
Tubulointerstitial fibrosis is characterized by loss of renal tubules, an increased myofibroblast population, and accumulation of extracellular matrix proteins (ECM) such as collagen, fibronectin and laminin [1] . Myofibroblasts, identified by expression of mesenchymal marker ␣ -smooth muscle actin ( ␣ -SMA), are the primary source of ECM and are the best prognostic indicator of disease progression in both human and experimental glomerulonephritis [2] . These cells are virtually absent in the normal kidney, but have been shown to be critical in renal fibrosis progression. The origin of myofibroblasts within the diseased kidney remains a point of debate [3] .
Evidence suggests that one source of these myofibroblasts may be proximal tubular epithelial cells (PTECs) by a process termed epithelial-mesenchymal transition (EMT) [4][5][6] . While there is currently debate regarding the role of PTEC EMT in disease progression in vivo, EMT has been extensively observed in vitro in response to a variety of stimuli including transforming growth factor-␤ , epidermal growth factor and interleukin-1 (IL-1) [7] . Mononuclear cell infiltration, predominantly consisting of lymphocytes and macrophages [8] is another widely observed characteristic of tubulointerstitial fibrosis. It has been suggested that these infiltrating immune cells play a role in promoting myofibroblast activation/ accumulation through their ability to secrete cytokines [9] . The aim of this study was to further investigate the interaction of the infiltrating mononuclear cells with PTECs in the context of tubulointerstitial fibrosis development.
High-mobility group box protein-1 (HMGB-1), also known as amphoterin, is best known as an architectural transcription factor because of its ability to regulate gene activity through DNA bending [10] . However, additional roles for HMGB-1 have been identified including the ability to bind to and modulate NF-B [11] , and as a secreted factor capable of regulating the expression of proinflammatory cytokines tumour necrosis factor-␤ (TNF-␤ ) and interferon-␤ [12] . As an extracellular signalling molecule, HMGB-1 elicits its effects through the receptor for advanced glycation end products (RAGE) which has been shown to interact with HMGB-1 with high affinity [13] . A member of the immunoglobulin superfamily of cell surface molecules, RAGE is a multi-ligand receptor that also interacts with amyloid-␤ and advanced glycation end products [14,15] . RAGE is expressed in vascular smooth muscle cells, endothelial cells, mononuclear phagocytes, mesangial cells, neurons and lung alveolar epithelial cells [16] . Binding of HMGB-1 to RAGE has been shown to induce cell migration, cell invasion, tumour growth and metastasis [17] . The role of RAGE-HMGB-1 interactions in disease has been widely suggested [18] ; however, the context of the interaction and mechanisms underlying the involvement in disease remain poorly understood.
Previously, we reported an in vitro model of immunemediated PTEC damage using conditioned medium con-taining supernatant from activated peripheral blood mononuclear cells (aPBMC-CM) and human PTECs [19] . Using similar techniques, Nightingale et al. [20] suggested that aPBMC-CM induced EMT in primary renal epithelial cells. In the current study, we have used the PTEC line HK-2 to investigate the effects of aPBMC-CM on PTEC phenotype and identify key mediators of the effects observed.

Reagents
Anti-HMGB-1 and anti-E-cadherin antibodies were obtained from BD Transduction Laboratories (Lexington, Ky., USA). Anti-RAGE antibody was obtained from Chemicon Ltd. Anti-TGF-␤ 1 antibody was obtained from R&D systems (UK). Recombinant HMGB-1 protein was obtained from Sigma. FITC secondary antibodies were obtained from DAKO. Cell culture dishes and slides were purchased from Falcon (UK).

Cell Culture and Treatments
HK-2 PTECs were maintained as previously described [21] . For the indicated treatments: 10 g/ml of HMGB-1 (Sigma-Aldrich) was prepared according to manufacturer's instructions and added in the indicated concentrations. aPBMC-CM was prepared as previously described [19] . Primary human renal proximal tubular cells (RPTECs) were purchased from Clonetics and maintained as per the manufacturer's instructions.

PCR Analysis
Total RNA was extracted from cells using TRIzol reagent (Life Technologies-BRL) as per manufacturer's instructions. mRNA abundance was assessed by real-time PCR or by RT-PCR. Briefly, real-time PCR was performed on an ABI 7900HT real-time PCR system (Applied Biosystems, Foster City, Calif., USA). Relative gene expression levels of E-cadherin, ZO-1, vimentin genes were assayed using pre-optimized single tube primer/probe Taqman Gene Expression Assays (Applied Biosystems), Ribosomal 18S RNA levels were used as an endogenous normalising control throughout. Reaction cycling conditions were: 2 min at 50 ° C, 10 min at 95 ° C for enzyme activation and 40 cycles of 15 s at 95 ° C for denaturation followed by 1 min at 60 ° C for annealing and extension. A negative-RT control was run alongside all assays to control for genomic DNA contamination. Results were analysed using the Ct method. For RT-PCR, the number of PCR cycles used were determined to be within the linear range of the reactions. PCR primers for E-cadherin, HMGB-1 and ␣ -SMA were supplied by Sigma-Genosys (UK). The sequences of the primers were as previously described [21,22] . Primer sequences for human HMGB-1 were as follows, forward: 5 -GGAGAGATGTGGAATA-3 , reverse: 5 -GGGAGTGAGTTGTGTA-3 .

Immunofluorescence and Western Blot Analysis
Immunofluorescent staining was performed as previously described [21,22] . Cells were visualized using a Zeiss Axioplan 2 imaging fluorescent microscope. Western blot analysis was also performed as previously described [21,22] .

Migration Assay
The migratory behaviour of PTECs was analysed using an in vitro wound healing assay. Briefly, monolayers of confluent cultures were experimentally wounded and subsequently cultured under different conditions as indicated. Cultures were observed at 24-hour intervals for up to 72 h after wounding.

Suppression Subtractive Hybridisation
Briefly, mRNA was prepared from control HK-2 cells and cells treated with 30% (v/v) aPBMC-CM using a PolyATtract mRNA Isolation System III Kit (Promega; n = 3 for each condition). From this mRNA, cDNA was synthesized using a SMARTTM PCR cDNA synthesis kit (Clontech). A suppression subtractive hybridisation (SSH) was then performed using a PCR-SELECT cDNA subtraction kit (Clontech) according to manufacturer's instructions. The subtracted cDNAs were cloned into PCR2.1 vector using the original TA cloning kit (Invitrogen) and transformed into TOP10 One-Shot Chemically Competent Escherichia coli (Invitrogen). Subtracted expressed sequence tag clones were isolated and stored at -70 ° C as glycerol stocks for PCR amplification of the cDNA inserts and sequence analysis. Sequencing of cloned DNA was performed using an ABI prism dye terminator cycle sequencing ready reaction kit (Perkin-Elmer) and performed by GATA-Biotech, Ltd., Belgium, using Run-24 sequencing. Sequences were then analyzed with the BLAST programs provided by the National Center for Biotechnology Information (http:// www.ncbi.nlm.nih.gov/BLAST).

Statistical Analysis
Data were analysed by one-way analysis of variance (ANOVA), and multiple comparisons between treatment groups were made using the Student t test using GraphPad Prism 3.0 (GraphPad Software, Inc., San Diego, Calif., USA). Results were expressed as the mean 8 SEM. A probability of 0.05 or less was deemed statistically significant. The following scheme was used throughout * p ! 0.05, * * p ! 0.01, * * * p ! 0.001.

aPBMC-CM Induced EMT in PTECs
To model the interactions between immune cells and renal tubular epithelial cells, PTECs were exposed to 30% (v/v) of control or aPBMC conditioned medium (aPBMC-CM). Cell morphology was assessed after 48 h of treatment with aPBMC-CM by phase contrast microscopy ( fig. 1 a). Control cells exhibited typical epithelial, cuboidal morphology with extensive cell-cell contacts. Following treatment with aPBMC-CM, PTEC morphology was significantly affected. PTECs were more elongated, formed less cell-cell attachments and produced filopodia. Similar effects were observed in primary proximal tubular cells, whereby treatment with aPBMC-CM induced a more severe morphological change. The expression and distribution of key epithelial proteins was examined by immunofluorescent microscopy. The cytoplasmic F-actin network regulates cell shape and modulates many aspects of cell phenotype. In control PTECs, F-actin filaments were observed in concentrated peripheral bundles, which is indicative of polarized epithelial cells with established cell-cell junctions. aPBMC-CM treatment disrupted this distribution with F-actin filaments appearing as stress fibres which are associated with a more mesenchymal phenotype ( fig. 1 b). Junctional integrity is critical to epithelial monolayer function. We examined the distribution and expression of E-cadherin (adherens junctions) and ZO-1 (tight junctions). E-cadherin mRNA was significantly reduced after 48 h of aPBMC-CM treatment ( fig. 2 a). This reduction was also reflected at the protein level as assessed by immuno-fluorescent microscopy and by Western blot ( fig. 1 b, 2 b). ZO-1 mRNA was also significantly downregulated by aPBMC-CM treatment in our model ( fig. 2 a). Again, this was also reflected at the protein level ( fig. 1 b, 2 b). Taken together, these results suggest significant disruption of the adherens and tight junctions. The phenotypic changes associated with EMT include increased expression of myofibroblast-associated proteins. Therefore, effects of aPBMC-CM on ␣ -SMA and vimentin expression were assessed ( fig. 1  highly expressed in myofibroblasts significantly increased both at the transcript and whole cell protein levels. Taken together, the effects on cell morphology, epithelial junctions and myofibroblast-associated proteins suggest that PTECs exposed to aPBMC-CM undergo significant phenotypic changes consistent with those observed during EMT [5] .

HMGB-1 Is Upregulated in PTECs Exposed to aPBMC-CM and Is Secreted by Activated PBMCs
In the current study, a SSH was performed to identify genes that were differentially regulated in PTECs by exposure to aPBMC-CM. After amplification and sequencing, a number of potentially interesting genes were identified as being differentially regulated in PTECs after aPBMC-CM treatment. Upregulated genes included IL-8, matrix metalloprotease 1 (MMP-1), p27, interferon-␣inducible-protein-27 (IFI27), macrophage inflammatory protein 3 ␣ (MIP-3 ␣ ) and HMGB-1. Peptidase D was the most notable downregulated gene. HMGB-1 was of particular interest to us since HMGB-1 is a member of the high-mobility group (HMG) box of proteins. Other proteins containing a HMG box domain include Wnt tran- Upregulation of HMGB-1 was confirmed by RT-PCR demonstrating increased HMGB-1 mRNA in PTECs exposed to aPBMC-CM ( fig. 3 a). This effect was then examined at protein level. In whole cell lysates, equal amounts of HMGB-1 were observed in control and aPBMC-CM treated PTECs across all time points, suggesting that intracellular levels of HMGB-1 were unchanged ( fig. 3 b). In contrast, HMGB-1 protein levels in cell supernatants were significantly increased in response to aPBMC-CM treatment compared to controls, at all time points ( fig. 3 c). Also of note was a modest increase in HMGB-1 protein in the supernatant of PBMCs after activation compared to inactivated controls ( fig. 3 d).
Together, these results demonstrate that exposure to aPBMC-CM resulted in significant induction and secretion of HMGB-1 in PTECs.

Recombinant HMGB-1 Protein Induces EMT in PTECs
To investigate the potential role of secreted HMGB-1 in this model of immune-mediated PTEC damage, the effects of direct application of recombinant human HMGB-1 to PTECs were examined. Firstly, the effects of increasing concentrations of HMGB-1 on PTEC morphology were investigated ( fig. 4 a). PTECs were treated with 1, 5 and 10 g/ml recombinant HMGB-1 protein for 72 h. HMGB-1 induced dose-dependent alterations in PTEC morphology, inducing EMT-like effects as cells became scattered, elongated and formed filopodia. The effects of HMGB-1 on PTEC phenotype were further examined by RT-PCR assessment of E-cadherin and ␣ -SMA mRNA levels ( fig. 4 b, c). After 72 h, exposure to 5 g/ml HMGB-1 resulted in significant downregulation of E-cadherin mRNA in PTECs. After the same treatment, ␣ -SMA mRNA was significantly upregulated, and this effect was also observed at the protein level by indirect immunofluorescent microscopy ( fig. 4 d). Aberrant expression of HMGB-1 has been observed in some invasive cancers [23,24] . Since our studies demonstrated loss of cell-cell contacts following treatment with HMGB-1, we investigated the effect of HMGB-1 on cell motility using an in vitro wound healing assay. Mi-grating cells were quantified at 24, 48 and 72 h after wounding. At each time point, significantly higher numbers of PTECs cultured in the presence of HMGB-1 had entered the wound area compared to control cells ( fig. 4 e).

HMGB-1-Induced EMT in PTECs Is Mediated by RAGE and TGF-␤ 1
HMGB-1 binds to the RAGE receptor with high affinity, and ligand binding to RAGE has been shown to activate positive autoregulatory signals to cause RAGE upregulation in cells to enhance RAGE signalling [25] . This potentially important mechanism was investigated in this study. PTECs were treated with 5 g/ml HMGB-1 over 72 h. By 48 h, treatment with HMGB-1 had increased RAGE compared to time-matched controls ( fig. 5 a). By 72 h, levels of RAGE were strongly increased by HMGB-1 treatment. The role of RAGE in HMGB-1-induced EMT was examined using a RAGE-neutralising antibody (nAb). PTECs were pretreated with increasing doses of RAGE nAb for 1 h prior to treatment with 5 g/ml HMGB-1. RAGE nAb caused a dose-dependent protection against the alterations in epithelial morphology induced by HMGB-1 with the greatest degree of protection observed using 2 g/ml RAGE nAb. PTECs pretreated with RAGE nAb retained a more epithelial morphology compared to HMGB-1 alone ( fig. 5 b). This protective effect on PTEC morphology coincided with a significant inhibition of HMGB-1-induced ␣ -SMA mRNA upregulation ( fig. 5 c). A number of studies  HK-2 cells were grown on 60-mm tissue culture dishes and were cultured in the absence or presence of 5 g/ml HMGB-1, with and without a RAGE nAb (2 g/ml) for 24, 48 or 72 h. a Equal amounts of HK-2 whole cell lysates were analysed by Western blot using an anti-RAGE antibody. Images shown are representative of at least three independent experiments performed in duplicate. ␤ -Actin levels were used as a loading control. b Cell morphology was assessed by phase contrast microscopy. c RT-PCR was carried out using primers designed against ␣ -SMA and GAPDH as described in 'Materials and Methods'. Band intensity was quantified densitometrically. Results are expressed as mean relative optical density units 8 SEM of three independent experiments performed in duplicate. *** p ! 0.001.

Ctrl
HMGB-1 (5 μg/ml) have suggested links between RAGE activation and TGF-␤ 1 signalling. TGF-␤ 1 plays a significant role in renal fibrosis development through the regulation of ECM production and the induction of EMT in PTECs. We investigated the role for TGF-␤ 1 in mediating the effects of HMGB-1 in this model. In PTECs exposed to increasing concentrations of HMGB-1, dose and time-dependent increases in TGF-␤ 1 secretion were detected ( fig. 6 a). In PTECs pretreated with 100 g/ml of a TGF-␤ 1 nAb, significant inhibition of HMGB-1-induced ␣ -  Fig. 6. Effects of HMGB-1 are dependent on TGF-␤ 1 secretion in RPTECs. HK-2 cells were grown on 60-mm tissue culture dishes and were cultured in the absence or presence of increasing concentrations of HMGB-1, with and without a RAGE nAb (100 g/ml) for 24, 48 or 72 h. a Equal amounts of HK-2 whole cell lysates were analysed by Western blot using an anti-TGF-␤ 1 antibody. ␤ -Actin levels were used as a loading control. Images shown are representative of at least three independent experiments performed in duplicate. b RT-PCR was carried out using primers designed against ␣ -SMA and GAPDH as described in 'Materials and Methods'. Band intensity was quantified densitometrically. Results are expressed as mean relative optical density units 8 SEM of three independent experiments performed in duplicate. *** p ! 0.001. c HK-2 cells were grown on 60-mm tissue culture dishes and cultured in the presence of 30% (v/v) control or aPBMC-CM, with and without a TGF-␤ 1 nAb (100 g/ml) for 48 h. SMA mRNA upregulation was observed ( fig. 6 b). This finding would suggest that HMGB-1 is indeed inducing TGF-␤ 1 expression and that TGF-␤ 1 could be a key mediator in HMGB-1 induced EMT. To investigate the wider implications of this finding, we investigated the effects of the TGF-␤ 1 nAb on aPBMC-CM-induced EMT in this model ( fig. 6 c). As previously observed, exposure to 30% (v/v) aPBMC-CM resulted in significant alterations to PTEC morphology by 48 h. Co-treatment with the TGF-␤ 1 nAb appeared to provide some measure of protection from these effects; however, significant alterations in PTEC morphology were still evident as were changes to ␣ -SMA mRNA levels (data not shown). These results are in close agreement with the findings of Nightingale et al. [20] . Taken together, these results suggest the involvement of both RAGE and TGF-␤ 1 in mediating the profibrotic effects of HMGB-1 on PTEC phenotype in this model.

Discussion
Activated immune cells secrete significant quantities of cytokines and growth factors at the site of renal injury [26] . Many of these factors are capable of activating fibroblasts and have so been implicated in renal disease progression [27] . However, the role of the tubular epithelial cell and in particular the interaction between the immune cell and tubular epithelial cell in renal fibrosis development is less well studied. Tubular EMT is a process by which renal tubular epithelial cells lose features of their epithelial phenotype and acquire new characteristic features of myofibroblasts [5] . The process of EMT is essential for morphogenetic movements within the developing embryo. The capacity for this phenotypic conversion suggests an incredible plasticity of the differentiated tubular epithelial cell and has been associated with generation of the matrix-producing fibroblasts under pathological settings such as fibrosis and in tumour invasion [6] . A number of features associated with EMT have been characterized including loss of epithelial cell adhesion, F-actin reorganisation, de novo expression of fibroblast proteins (such as ␣ -SMA and vimentin), and altered cell migration [28] . While the importance of the later feature is currently under debate, the likely contribution of PTECs to renal fibrosis development is widely accepted. Despite advances in our understanding of the EMT process, mechanistic links between the in vitro observations and the in vivo disease environment are lacking. In the current work, we have utilized an ex-vivo preparation to model immune cell activation in a disease context and investigate the interaction of the tubular epithelium with the immune system.
Previously, we demonstrated significant effects on primary tubular epithelial cells following treatment with the secretome of aPBMCs [19] . The current study confirms and extends these observations using a well-established PTEC line. The current study investigated the effects of aPBMC-CM on PTEC phenotype, examining a number of the previously outlined features associated with EMT. aPBMC-CM caused significant morphological changes consistent with those reported previously in primary tubular epithelial cells [19] . This was likely underpinned by the observed alterations in F-actin distribution. aPBMC-CM also induced strong upregulation of fibroblast associated proteins ␣ -SMA and vimentin at mRNA and protein level. ␣ -SMA was distributed in a stretched and elongated pattern indicative of stress fibre formation, suggesting a dynamic alteration of PTEC cellular architecture likely contributing to the more mesenchymal appearance [29] . aPBMC-CM treatment also significantly impacted cell-cell adhesions in PTECs. Both E-cadherin and ZO-1 were significantly downregulated at mRNA and protein level following treatment with aPBMC-CM. Together, these results strongly suggest that exposure of PTECs to aPBMC-CM resulted in significant restriction of epithelial phenotype, suggesting an EMT-like effect.
EMT can be initiated by a number of extracellular signals [30] . ECM components such as collagen, as well as soluble factors including epidermal growth factor, hepatocyte growth factor/scatter factor, IL-1, TNF-␣ , members of the fibroblast growth factor and TGF-␤ 1 have all been shown to contribute to EMT induction in vitro and in vivo [29] . In an effort to identify novel factors of importance in this ex-vivo model of EMT, an SSH approach was employed. Sequence analysis of the cDNA library generated in our laboratory revealed the upregulation of a number of genes that had not been reported to be upregulated by aPBMC-CM. IL-8, MMP-1, MIP-3 ␣ , IFI27, p27 and HMGB-1 were all upregulated. Peptidase D was downregulated by aPBMC-CM treatment. IL-8 is a major chemoattractant for neutrophils, which have been suggested to contribute to scarring and fibrosis formation in other organs including the lungs [31] . Overproduction of MMP-1 is associated with reduced cell adhesion, and may promote enhanced epithelial cell motility [32] . MIP-3 ␣ is an NF-B-responsive chemokine that is proposed to play a role in immune-mediated diseases such as inflammatory bowel disease, rheumatoid arthritis and disorders of the airways involving pulmonary inflammation and tissue fibrosis [33] . IFI27 is upregulated in epithelial cancer and is a TGF-␤ 1 responsive gene [34] . p27 is also upregulated in the presence of TGF-␤ 1 and is a potent negative regulator of cell cycle progression [35] . Peptidase D regulates collagen I metabolism and its downregulation may promote collagen accumulation in the context of tissue fibrosis [36] . While a number of these genes were potentially relevant in the context of aPBMC-induced EMT, HMGB-1 was of particular interest to us as a number of other HMG box-containing proteins have roles in regulating cell differentiation, inflammatory disease states and metastatic cancers.
Upregulation of HMGB-1 by aPBMC-CM in PTECs was confirmed by RT-PCR and was also observed at protein level. Importantly, however, the increases in HMGB-1 production appeared to be restricted to the secreted protein fraction. As a result of this finding, we next investigated the effect of recombinant human HMGB-1 protein on PTEC morphology and phenotype. We observed alterations consistent with EMT demonstrating, for the first time, a possible link between HMGB-1 and EMT in PTECs.
Several characteristics of HMGB-1 suggest associations with mesenchymal phenotypes. HMGB-1 has been shown to localize at infiltrating filopodial edges, and HMGB-1 is associated with enhanced outgrowth and migration in embryonic neurons, lung cells and tumour cells [37] . HMGB-1 may associate with ␣ -SMA at filopodial membranes. Another member of the HMGB family, HMG-Y, has been shown to induce EMT in the MCF-7 breast epithelial cell line [38] . Our results suggest that the effects of HMGB-1 in PTECs are mediated, at least in part by the RAGE receptor and by TGF-␤ 1 . In addition to the effects observed on cell phenotype, HMGB-1 treatment also resulted in upregulation of RAGE expression. This is similar to the effects observed by Arumugam et al. [39] , demonstrating that ligand binding to RAGE upregulated RAGE expression, thereby amplifying the proinflammatory effects of RAGE and its ligands. In PTECs pre-treated with an nAb against RAGE, significant protection of epithelial phenotype was observed. It has also been observed that the downstream effects of RAGE activation can be mediated by TGF-␤ 1 [24,40] . Evidence suggests that positive crosstalk links RAGE activation and TGF-␤ 1 signaling leading to enhancement of both signals [41] . TGF-␤ 1 induced a time-dependent increase in de novo synthesis of RAGE protein in hepatic stellate cells and RAGE agonist AGE-BSA caused upregulated TGF-␤ 1 mRNA levels in tubular epithelial cells [42,43] . In addition, upregulation of TGF-␤ 1 in sub-mesothelial fibrosis was prevented using anti-RAGE antibodies [40] . In the current study, HMGB-1 induced TGF-␤ 1 secretion from PTECs in a dose-dependent manner. Inhibition of TGF-␤ 1 with an nAb prevented induction of ␣ -SMA in HMGB-1 treated cells, thus suggesting that it is also involved in the subsequent signalling from the HMGB-1/ RAGE interaction. TGF-␤ 1 has been shown to be a key signalling molecule in the induction of EMT in renal epithelial cells; therefore, this would suggest that it may also play an important role in HMGB-1 induced EMT [21,22] .
In summary, we have demonstrated that the supernatant from aPBMCs induced EMT in PTECs. Differential gene expression screening identified HMGB-1 as being significantly upregulated by aPBMC-CM treatment. HMGB-1 alone was capable of inducing EMT in PTECs. Inhibition of RAGE and TGF-␤ 1 conferred a protective effect against the alterations induced by HMGB-1, suggesting their involvement in mediating HMGB-1 actions. Our results strongly implicate HMGB-1 in the induction of immune-mediated EMT. A schematic representation of the key signalling events involved is outlined in figure 7 . These results contribute to our better understanding of the molecular mechanisms of EMT, indicating the importance of the immune-cell secretome in renal EMT induction and subsequent fibrosis development.