mRNA levels were calculated and compared after normalization to beta-actin. The primers for the RT-PCR reaction were purchased from Sangon Biotech (China). The sequences were as follows: Snail (human), sense 59-CATTC CACGCCCAGC- TACCC-39 and antisense 59-CGCCCAGGCTC ACA- TATTCC-39; CK mRNA, sense 59-CCCAGAGCCTTGAGA- TAGAA C-39 and antisense 59-CACGACCTTGCCATCCAC- 39; vimentin mRNA, sense 59-CGCTTCGCCAACTACAT C-39 and antisense 59-GGTCAGGCTT GGAAACATC-39; and beta- actin, sense 59-CACCAACTGGGACG ACAT-39 and antisense 59-AC AGCCTGGATAGCAACG-39. Transmission electron microscopy For transmission electron microscope analysis, HK-2 cells were harvested, pre-fixed with 2% glutaraldehyde for 2 h at 4uC, washed twice with PBS, and then post-fixed with 1% osmic acid for 2 hours at 4uC. After an additional two washes in PBS, the samples were dehydrated with an ethanol gradient, washed twice with propylene oxide, soaked in ethoxyline resin overnight, and mounted at 60uC for 48 hours. Thin sections were cut with an ultramicrotome and then viewed under a transmission electron microscope (Hitachi, Japan). ELISA (enzyme-linked immunosorbent assay) Fibronectin release was determined using a Fibronectin ELISA kit (Jingmei BioTech, China). After exposure to the defined experimental conditions, culture supernatants were harvested. Different concentrations of supernatants were added to 96-well plates for 2 h at 37uC, followed by three washes with PBST buffer. Next, 100 ml of peroxidase-conjugated IgG (diluted with PBS to a concentration of 1:5000) was added to each well, and the plates were incubated at 25uC for 2 h. After the plates were washed three times with PBST, 100 ml of substrate solution (3, 39, 5, 59- tetramethylbenzidine, TMB) was added to each well using a multichannel pipet. The reaction was stopped by adding 50 ml of 2.0 M sulfuric acid to each well. After sufficient color development, absorbance values were immediately determined with an ELISA reader at a wavelength of 450 nm. Immunocytochemistry Cells under different conditions were plated onto different six- well plates at a density of 16105 cells/well; each well contained a polylysine-coated slide. Cells were fixed in 4% PFA for 15 min at 4uC and individually stained with monoclonal antibodies to E- cadherin, a-SMA, vimentin and CK using immunocytochemistry according to the instructions in the immunocytochemical detection References 1. Zeisberg M, Kalluri R (2004) The role of epithelial-to-mesenchymal transition in renal fibrosis. J Mol Med 82: 175–181. 2. Masayuki I, David P, Theodore MD, Chengsen X, Hirokazu O, et al. (2002) Evidence that fibroblasts derive from epithelium during tissue fibrosis. J Clin Invest 110: 341–350. 3. Zeisberg M, Strutz F, Muller GA (2000) Role of fibroblast activation in inducing interstitial fibrosis. J Nephrol 13: 111–120. 4. Yang J, Liu Y (2001) Dissection of key events in tubular epithelial to myofibroblast transition and is implications in renal interstitial fibrosis. Am J Pathol 159: 1465–1475. 5. Massague J (1998) TGF-b Signal Tranduction. Annu Rev Biochem 67: 753–791. 6. Paivi JM, Reinhard E, Alfredo RL, Rik D (1994) TGF-b Induced Transdifferentiation of Mammary Epithelial Cells to Mesenchymal Cells: Involvement of Type I Receptors. J Cell Biol 127: 2021–2036. 7. Ester P, Aristidis M, Akira K, Carl-Henrik H, Peter TD (1999) TGF-b type I receptor/ALK-5 and Smad proteins mediate epithelial to mesenchymal transdifferentiation in NMuMG breast epithelial cells J Cell Sci 112: 4557–4568. p38 MAPK in Epithelial-Mesenchymal Transition kit (Zhongshan Goldbridge Biotech, China). Briefly, HK-2 cells were rinsed in phosphate-buffered saline, fixed in 2% paraformal- dehyde, preincubated with 10% fetal calf serum to block nonspecific binding, and then incubated with antibodies for 60 min. After washing with phosphate-buffered saline, endogenous peroxidase was inactivated by incubation with 0.3% H2O2 in methanol for 15 min; this was followed by incubation with peroxidase-conjugated IgG and peroxidase-conjugated anti-peroxidase complexes and color development with diaminobenzidine to produce a brown color. All procedures were performed at room temperature. Data from five experiments were expressed as the mean percentage. EMSA The nuclear extracts were prepared from the cells under the different conditions. The EMSA was performed using the LightShift chemiluminescent EMSA kit (Pierce, Rockford, USA) according to the manufacturer’s instructions. Briefly, the biotin- labeled probes were incubated with the nuclear extracts for 30 min at 37uC, and the reaction samples were electrophoresed on a 4% non-denaturing acrylamide gel in a 0.5X Tris-borate-EDTA buffer. After transfer to nylon membranes, the biotin-labeled probes were detected. The following AP-1 probe was used for the EMSA: AP-1 response element (AP1, 59CGC TTG ATG AGT CAG CCG GAA39) Data analysis All experiments were repeated at least three times. Values were reported as mean 6 SD. Data were analyzed using SPSS 13.0 software. Statistical significance was assessed using ANOVA and LSD-t test, and P values of less than 0.05 were considered to be statistically significant. Supporting Information Figure S1 HK-2 cells apoptosis were measured by flow cytometry. (A) shows the rate of apoptosis in HK-2 cells under 5.5 mM glucose conditions for 48 h (lane 1), (B) shows the rate of apoptosis in HK-2 cells under 30 mM glucose conditions for 48 h (lane2). (TIF) Author Contributions Conceived and designed the experiments: Z-ML RW. Performed the experiments: Z-ML. Analyzed the data: Z-ML Q. Wang Q. Wan M-SH Y- XL. Wrote the paper: Z-ML. Finished the intellectual input and review of the manuscript: RW J-GL. 8. Erwin PB, Markus B (2002) TGF-b Signaling in Renal Disease. J Am Soc Nephrol 13: 2600–2610. 9. Koh O, Jiahuai H (2000) The p38 signal transduction pathway Activation and function. Cell Signal 12: 1–13. 10. John MK, Joseph A (1996) Sounding the alarm: protein kinase cascades activated by stress and inflammation. J Biol Chem 271: 24313–24316. 11. Cano E, Mahadevan LC (1995) Parallel signal processing among mammalian MAPKs. Trends Biochem Sci 20: 117–122. 12. Tomlinson DR (2003) Mitogen activated protein kinase as glucose transducers for diabetic complications. Diabetologia 46: 1271–1281. 13. Tanaka T, Kanai H, Sekiguchi K, Aihara Y, Yokoyama T, et al. (2000) Induction of VEGF gene transcription by IL-1 beta is mediated throughstress-activated MAP kinases and Sp1 sites in cardiac myocytes. J Mol Cell Cardiol 32: 1955–1967. 14. Jung YD, Liu W, Reinmuth N, Ahmad SA, Fan F, et al. (2001) Vascular endothelial growth factor is upregulated by interleukin-1 beta in human vascular smooth muscle cells via the p38 mitogen-activated protein kinase pathway. Angiogenesis 49: 155–162. 15. Duyndam MC, Hulscher ST, van der Wall E, Pinedo HM, Boven E (2003) Evidence for a role of p38 kinase in hypoxia-inducible factor-1-independent PLoS ONE | www.plosone.org 11 July 2011 | Volume 6 | Issue 7 | e22806