title: Secreted products of macrophages exposed to calcium oxalate crystals induce epithelial mesenchymal transition of renal tubular cells via RhoA-dependent TGF-β1 pathway.
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.
authors.affiliation: 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.
authors.forename: Rattiyaporn
authors.initials: R
authors.lastname: Kanlaya
authors.forename: Kitisak
authors.initials: K
authors.lastname: Sintiprungrat
authors.forename: Visith
authors.initials: V
authors.lastname: Thongboonkerd
mesh_terms: D000818:Animals; D015820:Cadherins / Q000378:metabolism; D002129:Calcium Oxalate / Q000494:pharmacology*; D002460:Cell Line; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004285:Dogs; D058750:Epithelial-Mesenchymal Transition / Q000187:drug effects*; D006801:Humans; D007633:Keratins / Q000378:metabolism; D007684:Kidney Tubules / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D007976:Leupeptins / Q000494:pharmacology; D008264:Macrophages / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D061985:Madin Darby Canine Kidney Cells; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D053773:Transforming Growth Factor beta1 / Q000378:metabolism*; D025801:Ubiquitin / Q000737:chemistry / Q000378:metabolism; D014746:Vimentin / Q000378:metabolism; D062826:Zonula Occludens-1 Protein / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*
chemical_list: D015820:Cadherins; D015853:Cysteine Proteinase Inhibitors; D007976:Leupeptins; C569042:TJP1 protein, human; D053773:Transforming Growth Factor beta1; D025801:Ubiquitin; D014746:Vimentin; D062826:Zonula Occludens-1 Protein; D002129:Calcium Oxalate; D007633:Keratins; D046988:Proteasome Endopeptidase Complex; D020742:rhoA GTP-Binding Protein; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde