Renal explanting provides a useful ex vivo model of epithelial-mesenchymal transition
Recognised by their de novo expression of alpha-smooth muscle actin (SMA), recruitment of myofibroblasts (MFs) is a key event in the pathogenesis of tubulointerstitial fibrosis. Increasingly we realise that epithelial-mesenchymal transition (EMT) is an important source of MFs in chronic kidney disease.
In this study we describe the establishment and validation of a novel model of renal EMT. Rat renal explants were finely diced on gelatin coated petri dishes and cultured in DMEM+20%FCS. Cell outgrowths were fixed at various time points between 3 and 17 days post-explanting. In each case, morphology and immunocytochemistry were used to identify de-differentiated (vimentin+), mesenchymal (SMA+, desmin+), epithelial (cytokeratin+) and endothelial (RECA+) cells.
Cell outgrowths from normal cortical tissue were almost exclusively epithelial cells (cuboidal in shape, cytokeratin+/SMA-) at day 3. By day 7, 66±20% (mean±SD) of cells were vimentin+, 21±18% SMA+, 26±18 desmin+, 79±12 cytokeratin+. None were RECA+. This indicated progressive EMT, as 50±12% of cytokeratin+ cells co-stained for SMA at day 10. Lectin staining established that the majority of cells had a proximal tubular origin. By day 17, cultures consisted only of MFs (SMA+/cytokeratin-). Cultures stimulated with an additional 10ng/ml TGFbeta1 had 35±18% more MFs at day 7, whilst lovastatin reduced cell number and the proportion of MFs by 20±8% (both p<0.05 vs control).
In conclusion, explanting of normal tissue is a reproducible ex vivo model of EMT. The ability to modify this transition provides a useful tool to study the regulation of tubulointerstitial fibrosis.