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Secreted Products of Macrophages Exposed to Calcium Oxalate Crystals Induce Epithelial Mesenchymal Transition of Renal Tubular Cells via RhoA-Dependent TGF-β1 Pathway

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Abstract

Kidney stone disease is associated with renal fibrosis by the unclear mechanisms. We hypothesized that calcium oxalate (CaOx), a major crystalline component of kidney stones, could induce secretion of fibrotic factors from macrophages leading to “epithelial mesenchymal transition/transdifferentiation” (EMT) of renal tubular cells. Western blot analysis revealed an increased level of vimentin (mesenchymal marker) but decreased levels of E-cadherin and cytokeratin (epithelial markers) in MDCK cells treated with “secreted products from CaOx-exposed macrophages” (CaOx-M-Sup). Immunofluorescence study confirmed the increased level of vimentin and decreased level of cytokeratin, and also revealed the increased level of fibronectin (another mesenchymal marker). The data also showed decreased levels and disorganization of F-actin (cytoskeletal marker) and zonula occludens-1 (ZO-1) (tight junction marker) induced by CaOx-M-Sup. ELISA demonstrated the increased level of transforming growth factor-β1 (TGF-β1), the well-defined EMT inducer, in CaOx-M-Sup. Downstream signaling of TGF-β1 was involved as demonstrated by the decreased level of RhoA. Interestingly, pretreatment with a proteasome inhibitor (MG132) could restore RhoA to its basal level, most likely through ubiquitin-proteasome pathway (UPP). Moreover, MG132 successfully sustained cytoskeletal assembly and tight junction, and could prevent the cells from EMT. Altogether, these data demonstrate for the first time that CaOx-M-Sup could induce EMT in renal tubular cells by TGF-β1 signaling cascade via RhoA and UPP. This may be, at least in part, the underlying mechanism for renal fibrosis in kidney stone disease.

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Acknowledgments

We thank Ms. Paleerath Peerapen for her technical assistance. This study was supported by The Thailand Research Fund (RTA5380005 and TRG5480005), Office of the Higher Education Commission and Mahidol University under the National Research Universities Initiative, and Faculty of Medicine Siriraj Hospital. KS is supported by the Royal Golden Jubilee PhD Program, whereas VT is also supported by the “Chalermphrakiat” Grant, Faculty of Medicine Siriraj Hospital.

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  1. Medical Proteomics Unit, Office for Research and Development, Faculty of Medicine Siriraj Hospital, and Center for Research in Complex Systems Science, Mahidol University, 6th Floor, SiMR Building, 2 Prannok Road, Bangkoknoi, Bangkok, 10700, Thailand

    Rattiyaporn Kanlaya, Kitisak Sintiprungrat & Visith Thongboonkerd

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  1. Rattiyaporn Kanlaya
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  2. Kitisak Sintiprungrat
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Correspondence to Visith Thongboonkerd.

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Kanlaya, R., Sintiprungrat, K. & Thongboonkerd, V. Secreted Products of Macrophages Exposed to Calcium Oxalate Crystals Induce Epithelial Mesenchymal Transition of Renal Tubular Cells via RhoA-Dependent TGF-β1 Pathway. Cell Biochem Biophys 67, 1207–1215 (2013). https://doi.org/10.1007/s12013-013-9639-z

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