Abstract
Human cytomegalovirus (HCMV) infection is associated epidemiologically with poor outcome of renal allografts due to mechanisms which remain largely undefined. Transforming growth factor-β1 (TGF-β1), a potent fibrogenic cytokine, is more abundant in rejecting renal allografts that are infected with either HCMV or rat CMV as compared to uninfected, rejecting grafts. TGF-β1 induces renal fibrosis via epithelial-to-mesenchymal transition (EMT) of renal epithelial cells, a process by which epithelial cells acquire mesenchymal characteristics and a migratory phenotype, and secrete molecules associated with extracellular matrix deposition and remodeling. We report that human renal tubular epithelial cells infected in vitro with HCMV and exposed to TGF-β1 underwent morphologic and transcriptional changes of EMT, similar to uninfected cells. HCMV infected cells after EMT also activated extracellular latent TGF-β1 via induction of MMP-2. Renal epithelial cells transiently transfected with only the HCMV IE1 or IE2 open reading frames and stimulated to undergo EMT also induced TGF-β1 activation associated with MMP-2 production, suggesting a role for these viral gene products in MMP-2 production. Consistent with the function of these immediate early gene products, the antiviral agents ganciclovir and foscarnet did not inhibit TGF-β1 production after EMT by HCMV infected cells. These results indicate that HCMV infected renal tubular epithelial cells can undergo EMT after exposure to TGF-β1, similar to uninfected renal epithelial cells, but that HCMV infection by inducing active TGF-β1 may potentiate renal fibrosis. Our findings provide in vitro evidence for a pathogenic mechanism that could explain the clinical association between HCMV infection, TGF-β1, and adverse renal allograft outcome.
Highlights
Human cytomegalovirus (HCMV) has been associated with poor renal allograft outcome in numerous seroepidemiologic studies [1,2,3,4]
This result differs from Transforming growth factor-b1 (TGF-b1) effects upon human foreskin fibroblasts (HFFs), where HCMV replication is induced by exposure to TGF-b1 [37]
These studies were performed with equivalent volume samples obtained from the same experimental cultures, confirming that the picomolar measurements differed between the two assays; significant induction of active TGF-b1 was measured in the HCMV infected, raTGF-b1 treated cells by both methods. These studies have not been performed in parallel by other investigators, and the differing results obtained from assays of the same samples suggests that the luciferase bioassay may detect the downstream function of a given quantity of protein more sensitively than the measurement of protein by antibody binding in the ELISA. These results suggested that HCMV infection of HK-2 cells, without epithelial-to-mesenchymal transition (EMT), did not induce de novo production of active TGF-b1, whereas the HCMV infected epithelial cells after EMT appeared to acquire the capacity to produce de novo active transforming growth factor-b1 (TGFb1), similar to the phenotype demonstrated by HCMV infected fibroblasts [25]
Summary
Human cytomegalovirus (HCMV) has been associated with poor renal allograft outcome in numerous seroepidemiologic studies [1,2,3,4]. In a rat renal allograft model, infection with rat CMV accelerates and intensifies rejection in infected allografts as compared to uninfected allografts [6,7,8] These studies support an association between HCMV and adverse renal allograft outcome, but the mechanisms by which HCMV contributes to renal allograft loss remain cryptic. During EMT, renal tubular cells demonstrate loss of epithelial characteristics and cellular adhesions, develop changes in the actin cytoskeleton, induce expression of fibrogenic molecules, and acquire a migratory phenotype [22]. These fibroblastoid renal tubular cells are key contributors to renal fibrosis, as inhibition of TGF-b1 mediated EMT prevents and reverses experimentally induced renal fibrosis in animal models [22,23,24]. The association between CMV and TGF-b1 in renal allografts raises the possibility that CMV might accelerate renal allograft loss via viral induction of TGF-b1 with resultant fibrosis within the allograft
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