Background. Glomerular cellular crescents consist of epithelial cells and macrophages, which can undergo an irreversible process of fibrous organization. However, the origin of the fibroblast-type cells that mediate this fibrous organization is unclear. Methods. This study examined glomerular epithelial myofibroblast transdifferentiation (GEMT) in the formation and evolution of glomerular crescents in two distinct rat models of glomerulonephritis: 5/6 nephrectomy and antiglomerular basement membrane (GBM) disease. Results. Early in the course of both disease models, and prior to crescent formation, immunohistochem-istry staining and in-situ hybridization demonstrated de novo expression of alpha-smooth-muscle actin (α-SMA), a marker of smooth muscle cells and myofib-roblasts, by glomerular parietal epithelial cells (GPEC). The expression of α-SMA by GPEC was accompanied by a loss of E-cadherin staining, a marker of epithelial cells. At this early stage of GEMT, ultra-structural studies identified the presence of character-istic actin microfilaments and dense bodies within GPEC which retained a normal epithelial morphology with apical-basal polarity and microvilli. A late stage of transdifferentiation was seen in fibrocellular cres-cents. In this case, GPEC attached to intact segments of the capsular basement membrane contained large bundles of actin microfilaments throughout the cell, and this was accompanied by a loss of polarity, micro-villi, and tight junctions. There was a significant cor-relation between the presence of α-SMA+ GPEC and glomerular crescent formation. Cellular crescents con-tained small numbers of α-SMA+ myofibroblasts. These cells become the dominant population in fibro-cellular crescents, which was associated with marked local proliferation. Relatively few α-SMA+ myofibro-blasts remained in fibrotic/organizing crescents. Most cells within cellular and fibrocellular crescents expressed transforming growth factor-β(TGF-β) and basic fibroblast growth factor (FGF-2), suggesting that these growth factors may regulate this GEMT process during the evolution of glomerular crescents. Conclusions. This study provides the first phenotypic and morphological evidence that glomerular epithelial-myofibroblast transdifferentiation participates in the formation and evolution of glomerular crescents.

Transforming growth factor-β regulates tubular epithelial-myofibroblast transdifferentiation in vitro. Background. We recently found evidence of tubular epithe-lial-myofibroblast transdifferentiation (TEMT) during the de-velopment of tubulointerstitial fibrosis in the rat remnant kid-ney. This study investigated the mechanisms that induce TEMT in vitro. Methods. The normal rat kidney tubular epithelial cell line (NRK52E) was cultured for six days on plastic or collagen type I-coated plates in the presence or absence of recombinant trans-forming growth factor-j31 (TGF-β1). Transdifferentiation of tubular cells into myofibroblasts was assessed by electron mi-croscopy and by expression of α-smooth muscle actin (α-SMA) and E-cadherin. Results. NRK52E cells cultured on plastic or collagen-coated plates showed a classic cobblestone morphology. Culture in 1 ng/ml TGF-β caused only very minor changes in morphology, but culture in 10 or 50ng/ml TGF-β1 caused profound changes. This involved hypertrophy, a loss of apical-basal polarity and microvilli, with cells becoming elongated and invasive, the for-mation of a new front-end back-end polarity, and the appear-ance of actin microfilaments and dense bodies. These morpho-logjcal changes were accompanied by phenotypic changes. Double immunohistochemistry staining showed that the addi-tion of TGF-β1 to confluent cell cultures caused a loss of the epithelial marker E-cadherin and de novo expression of α-SMA. An intermediate stage in transdifferentiation could be seen with hypertrophic cells expressing both E-cadherin and α-SMA. De novo α-SMA expression was confirmed by Northern blotting, Western blotting, and flow cytometry. In particular, cells with a transformed morphology showed strong α-SMA immunostaining of characteristic microfilament struc-tures along the cell axis. There was a dose-dependent increase in the percentage of cells expressing α-SMA with increasing concentrations of TGF-β1, which was completely inhibited by the addition of a neutrafizing ant-TGF-β1 antibody. Corn