Since TGF-b 1 is up-regulated in the fibrotic kidney and is the main inducer of EMT in RTECs [17, 18] , we determined TGFb 1 concentration in the co-culture media of HK-2 cells and PBMCs with non-direct contacted culture with HK-2 cells pre-stimulated by TGF-b 1 using transwell plate to investigate whether prestimulation of HK-2 cells by TGF-b 1 contributes to EMT on RTECs or just induces the CXCL12 that activates LFA-1 on PBMCs. TGF-b 1 concentrations of the co-culture media of HK-2 cells and PBMCs were about threefold up-regulated compared with those of HK-2 cells without PBMCs. These results suggested that the pre-stimulation of HK-2 cells by TGF-b 1 contributes to increase not only chemokine production but also up-regulation of TGF-b 1 that induced EMT in RTECs upon the interaction of RTECs and PBMCs. We conducted real-time RT-PCR to detect the change of the TGF-b 1 mRNA in the HK-2 cells that underwent EMT and PBMCs in the lower plate separately at several time points after co-culture; however, we could not detect up-regulation of TGF-b 1 mRNA in both these HK-2 cells and PBMCs (data not shown). Although further study will be needed to investigate the regulation of TGF-b 1 on the interaction of LFA-1 on PBMCs and ICAM-1 on RTECs, the up-regulation of TGF-b 1 concentrations of the co-culture media of HK-2 cells and PBMCs also suggested In the EMT of RTECs, smad2 and smad3 were mainly mediated by TGF-b 1 [33] . In the present study, the phosphorylation of smad2 and smad3 was not detected in HK-2 cells by the stimulation of TGF-b 1 for 24 hrs before and after co-culture with PBMCs. This lack of phosphorylation of smad2 and smad3 is considered to be due to the low concentration of TGF-b 1 (0.1 ng/ ml) because phosphorylation of smad2 and smad3 on HK-2 cells was detected after stimulation with higher concentrations of TGFb 1 (10.0 ng/ml) for 24 hrs (data not shown). Xie et al. reported that the activation of ERK pathway was required for TGF-b 1induced EMT of RTECs [34] . Schramek et al. reported that longterm ERK1/2 activation was an important mechanism involved in the EMT of HK-2 cells [35, 36] . In addition to the role of ICAM-1 as an adhesion molecule, ICAM-1 also has roles as a signal HK-2 cells co-cultured with PBMCs for 24 hrs following TGF-b 1 (0.1 ng/ml) stimulation (D). HK-2 cells co-cultured with PBMCs with pre-incubated anti-LFA-1 mAb following TGF-b 1 (0.1 ng/ml) stimulation for 24 hrs (E). HK-2 cells pre-incubated with anti-ICAM-1 mAb co-cultured with PBMCs following TGF-b 1 (0.1 ng/ml) stimulation for 24 hrs (F). HK-2 cells pre-incubated with isotype control mAb co-cultured with PBMCs following TGF-b 1 (0.1 ng/ml) stimulation for 24 hrs (G). Magnification 1006. HK cells were co-cultured with PBMCs for 24 hrs following TGF-b 1 (0.1 ng/ml) stimulation for 24 hrs. Then, the changes of the epithelial markers (E-cadherin and occludin) and the mesenchymal markers (vimentin and fibronectin 1) on HK-2 cells (HK-2-TGF-b 1 (0.1)-PBMCs) were assessed by western blotting analysis (H). For blocking the interaction of LFA-1 and ICAM-1, before co-culture, PBMCs were pre-incubated with anti-LFA-1 mAb for 20 minutes (Anti-LFA-1 mAb ) or HK-2 cells were pre-incubated with anti-ICAM-1 for 20 minutes (Anti-ICAM-1 mAb) or isotype control mAb for 20 minutes (Isotype mAb). HK-2 cells without co-culture with PBMCs following TGF-b 1 (0.1 ng/ml) stimulation for 24 hrs (TGF-b 1 (0.1)) and HK-2 cells without stimulation of TGF-b 1 co-cultured with PBMCs (PBMCs) were investigated as control. One representative immunoblot is presented at the top of the graph. Values are expressed as mean 6 SEM of at least three experiments. *P,0.05. doi:10.1371/journal.pone.0023267.g005 transduction molecule [37, 38, 39] . With the interaction of LFA-1 with ICAM-1 and the formation of ICAM-1 multimers within the cell membrane, signal transduction is initiated [40, 41] . ICAM-1 with associated proteins such as Grb2, SOS and Shc can lead to downstream activation of the mitogen-activated protein (MAP) kinase pathway, including ERK1/2 [38, 42] . The ERK1/2 proteins were most commonly activated through ICAM-1 ligation [38, 40] . In the present study, phosphorylation of ERK1/2 was detected on HK-2 cells after stimulation of TGF-b 1 co-cultured with PBMCs. This phosphorylation of ERK1/2 was blocked by anti-LFA-1 mAb or anti-ICAM-1 mAb. These results suggested that ERK 1/2 may be activated through ICAM-1 on HK-2 cells by the interaction of ICAM-1 on HK-2 cells and LFA-1 on PBMCs. LFA-1 also acts as a signal transduction molecule when it binds ligands [7] . Although ICAM-1 expression still showed a high level in HK-2 cells by TGF-b 1 stimulation, its suppression by TGF-b 1 might be an inhibition factor for EMT of HK-2 cells by the interaction of ICAM-1 of HK-2 cells and LFA-1 of PBMCs. Further study will be needed to investigate the mechanism and significance of the regulation of ICAM-1 expression, the signaling pathway that affects EMT of HK-2 cells on PBMCs by the interaction of LFA-1 on PBMCs and ICAM-1 on HK-2 cells.