induced cell damage [13–17]. Recent in vitro studies have shown that high levels of glucose can activate the p38 MAPK signaling pathway in renal cells and induce the phosphorylation of p38 MAPK, which promotes the production of fibronectin by the mesangial cells [18–21]. In addition, there is enough evidence that TGF-b signals through MAPKs [22], and the activation of p38 MAPK is required in TGF-b-induced EMT in mammary epithelial cells [23]. Therefore, we decided to examine whether the p38 MAPK signaling pathway contributes to the EMT that is induced by high glucose in human proximal tubular epithelial cells. AP-1, which is a transcription factor, is a heterodimeric protein that is composed of proteins of the c-Fos, c-Jun, ATF and JDP families. AP-1 regulates gene expression in response to a variety of stimuli, including cytokines, growth factors, stress, and bacterial and viral infections [24–28]. It has been shown that a tubular overactivation of AP-1 and a simultaneous up-regulation of certain proinflammatory and profibrogenic genes are markers of progres- sive renal disease in humans [29]. The activation of AP-1 through dimerization that is mediated by leucine zippers is known to be promoted by a variety of upstream protein kinases [30]. Upstream signaling pathways, which include mitogen-activated protein kinases (MAPKs), regulate the transcriptional activity and the half-life of certain proteins and produce AP-1 dimers of different transcriptional specificity [31]. Because MAPKs have been shown to be involved in a crucial step in the up-regulation of AP-1 activation in various types of cultured cells via multiple mechanisms, we attempted to examine the possible involvement of p38 MAPK, the major upstream kinase, in AP-1 activation in HK2 cells. The purpose of this study was to determine the role of p38 MAPK in high glucose-induced AP-1 activation and in the EMT in the HK-2 cells. A vector for small interfering RNA that targets p38 MAPK was constructed; the cells were then either transfected with p38 siRNA or pretreated with a chemical inhibitor of AP-1 and incubated with low glucose (5.5 mM glucose) plus TGF-b1 or high glucose (30 mM glucose) for 48 h. Cells that were not transfected or pretreated and were exposed to low glucose (5.5 mM glucose) with or without TGF-b1 or high glucose (30 mM glucose) for 48 h were considered to be the controls. In this study, it was found that the activation of p38 MAPK under high glucose conditions led to EMT in HK-2 cells. In addition, p38 siRNA partially suppressed a-SMA and vimentin expression and inhibited fibronectin synthesis. The activation of AP-1 in the high glucose-induced EMT and its partial abrogation by p38 siRNA were also shown. Furthermore, p38/AP-1 promoted the EMT via suppression of E-cadherin through up-regulated Snail expression. Results SiRNA silenced p38 MAPK in HK-2 cells cultured with high glucose To confirm whether high glucose could induce the epithelial- mesenchymal transition in the HK-2 cells, the cells were treated with low glucose (5.5 mM glucose) plus TGF-b1 or high glucose (30 mM glucose). We found that an increase in TGF-b1 of the HK-2 cells was induced by high glucose (Figure 1). The cells that were cultured in 5.5 mM glucose showed a typical epithelial cuboidal shape with the characteristic cobblestone morphology and abundant microvilli and mitochondria; however, the cells that were exposed to high glucose displayed distinct morphological changes (Figure 2 and 3). The expression of the epithelial phenotypic markers, E-cadherin and cytokeratin, as well as the mesenchymal phenotypic markers, a-SMA and vimentin, were p38 MAPK in Epithelial-Mesenchymal Transition determined (Figures 4 and 5). Under low glucose conditions, p38 MAPK gene silencing significantly inhibited the expression and phosphorylation of p38 MAPK (P,0.05) (Figure 6). To investigate the role of p38 MAPK in the high glucose-induced EMT, p38 MAPK and phosphorylated p38 were determined. The expres- sions of p38 MAPK under high glucose conditions were of no significant difference compared with that cultured in low glucose. But high glucose increased the phosphorylation of p38 MAPK in HK-2 cells. Knockdown of p38 gene lead to decrease of both expression and phosphorylation of p38 MAPK, the latter of which was induced by high glucose (P,0.05) (Figure 7). The role of p38 MAPK in the expression of E-cadherin, cytokeratin, vimentin, and a-SMA under high glucose conditions E-cadherin is a Ca2+-dependent cell adhesion molecule that plays a significant role in the maintenance of renal epithelial polarity. Cytokeratins are proteins of keratin-containing interme- diate filaments that are found in the intracytoplasmic cytoskeleton of the cells of epithelial tissue. Loss of E-cadherin and CK expression is considered to be the early changes in TGF-b-induced EMT [32]. High glucose or low glucose with TGF-b1 decreased the expression of E-cadherin and CK, but the expression of those proteins increased after pre-treatment with p38 siRNA (Figures 4, 5, 8). In addition, CK mRNA was down-regulated under high glucose conditions but was significantly up-regulated after pre- treatment with p38 siRNA (P,0.05) (Figure 5). a-SMA and vimentin are cytoskeletal proteins found in fibroblasts but not in TECs. In cells cultured with 5.5 mM glucose, a-SMA and vimentin were not detected. However, high glucose or low glucose plus TGF-b1 resulted in a significant increase in a-SMA and vimentin expression, which was blocked by pre-treatment with p38 siRNA (P,0.05) (Figures 4, 5, 8). It was further confirmed that the vimentin mRNA levels were signifi- cantly increased when the cells were treated with high glucose, but these levels were reduced after pre-treatment with p38 siRNA (P,0.05) (Figure 5). Figure 1. The expression of TGF-b1 in HK-2 cells by Western blot. TGF-b1 was measured by Western blot of the HK-2 cells cultured in 5.5 mM glucose (lane 1), 30 mM glucose for 24 h (lane 2), 30 mM glucose for 48 h (lane 3). aP,0.05 vs. 5.5 mM glucose. doi:10.1371/journal.pone.0022806.g001 PLoS ONE | www.plosone.org 2 July 2011 | Volume 6 | Issue 7 | e22806