p38 MAPK in Epithelial-Mesenchymal Transition Figure 6. Effect of knockdown of p38MAPK by p38 MAPK siRNA on p38 MAPK expression by Western blot and RT-PCR analysis. (A)P38 MAPK expression were determined by RT-PCR of the HK-2 cells cultured for 72 h in 5.5 mM glucose (lane 1), 5.5 mM glucose+Cont siRNA (lane 2), 5.5 mM glucose+p38 siRNA (lane 3). (B)P38 MAPK expression were determined by Western blot of the HK-2 cells cultured for 72 h in 5.5 mM glucose (lane 1), 5.5 mM glucose+Cont siRNA (lane 2), 5.5 mM glucose+p38 siRNA (lane 3).Values represent the mean 6 SD, aP,0.05 vs. 5.5 mM glucose. doi:10.1371/journal.pone.0022806.g006 Discussion TIF has been recognized as a common terminal pathway for almost all forms of chronic kidney disease. TECs are pivotal in the Figure 7. The expression of p38 MAPK and phosphorylated P38 MAPK by Western blot analysis. p38 MAPK and phosphory- lated p38 MAPK were measured by Western blot of the HK-2 cells cultured in 5.5 mM glucose (lane 1), 30 mM glucose (lane 2), 30 mM glucose+Cont siRNA(lane 3), 30 mM glucose+p38 siRNA for 24 h(lane 4), 30 mM glucose+p38 siRNA for 48 h(lane 5). Values represent the mean 6 SD, aP,0.05 vs. 5.5 mM glucose, bP,0.05 vs. 30 mM glucose. doi:10.1371/journal.pone.0022806.g007 processes of TIF through EMT because TECs are considered to be an essential source of myofibroblasts, which promote ECM synthesis [38,39]. Increasing evidence indicates that TECs, which are the major constituents of the renal parenchyma, are not innocent bystanders but play a decisive role in the evolution of renal fibrosis. Recent clinical studies have also demonstrated the existence of EMT in human renal biopsies [40]. p38 MAPK activation is a key modulator in the progression of renal diseases and is thought to occur in various kinds of cells, including mesangial cells [41] and vascular endothelial cells [42]; however, little is known about the activation of p38 MAPK in renal tubular epithelial cells. Currently, there is a wealth of data that supports the central role of the p38 MAPK signaling pathway in high glucose-induced cell damage [43–46]. Therefore, We focused on the p38 MAPK signaling pathway to determine whether it mediates high glucose-induced EMT. Becasuse the activation of TGF-b signaling is sufficient to induce EMT in cultured epithelial cells and MAPK signaling pathways have been implicated inTGF-b-induced EMT [47], expression of TGF-b1 under high glucose conditions were detected to further confirm whether EMT could be induced by high glucose. The results showed that high glucose could induce EMT in human renal epithelial cells, which became more elongated and less adhered and lost their apical-to-basal polarity. During this process, cells lost E-cadherin and CK, showed increased de novo expression of a- SMA and vimentin and synthesized extracellular matrix mole- cules, such as fibronectin, which are features that are consistent with a more fibroblast-like phentoype. The activation of myofibroblasts plays a critical role in the processes of cell adhesion, actin re-organization and enhanced cellular progression of chronic renal disease. The role of p38 MAPK in tubular EMT is further supported by the p38 siRNA findings. In this study, p38 siRNA was shown to reverse the loss in E-cadherin and CK, suppress the expression of a-SMA and vimentin and inhibit fibronectin synthesis: these effects limited the development of the high glucose-induced epithelial mesenchymal transition. More PLoS ONE | www.plosone.org 6 July 2011 | Volume 6 | Issue 7 | e22806