Big Mac Uncovered: Macrophage Induced Epithelial-Mesenchymal Transformation (EMT) Contributes to Late Kidney Allograft Failure
Résumé
Tubular atrophy/interstitial fibrosis (TA/IF) remains a major cause of late kidney allograft loss. EMT is now appreciated as a key feature in this process. While T cells have been implicated in EMT, the role of macrophages is not appreciated. We hypothesize that macrophages play a direct role in the development of TA/IF by stimulating EMT within the allograft. To test this hypothesis, mouse kidneys from H-2b mice were transplanted into MHC incompatible H-2 d recipients (allografts) or H-2 b littermates (isografts). Macrophage invasion in allografts was detected by immunostaining as early as 2 weeks post transplant prior to TA/IF changes and remained through week 6, when TA/IF is fully manifest. Coupled with this histologic invasion was increased gene expression in allografts compared to isografts for F4/80 (18±10-fold, p<0.05), a marker of macrophageinvasion, IL-13 (34±13-fold; p<0.01), a signal for alternative macrophage activation, and S100A4 (22±3-fold; p<0.001) and vimentin (24±4-fold; p<0.001), genes associated with EMT. To begin to understand this relationship, we evaluated gene expression by real-time PCR in mouse proximal tubule epithelium cells (PTECs) cultured with or without macrophages, using culture inserts to prevent direct cell-cell contact. After 6 hours of exposure to macrophages, PTECs showed significant upregulation of EMT markers S100A4 (8±1-fold, p<0.001), vimentin (7±1-fold, p<0.001), and growth factor TGFβ (10±5-fold, p<0.05), with down-regulation of E-cadherin (0.5±0.2 fold, p<0.01) and the negative regulator BMP7 (0.2±0.1-fold, p<0.001), compared to cells cultured alone. Additionally, there was substantial induction of arginase-1 (91±31-fold, p<0.001), IGF-1 (147±22-fold, p<0.001), and MMP9 (22±2-fold, p<0.001), markers of fibrosis and alternative macrophage activation. Furthermore, adding IL-13 accelerated EMT gene transcription by 3h of exposure, and boosted transcription of fibrosis markers arginase-1 (305±69-fold, p<0.01), IGF-1 (789±138-fold, p<0.001) and MMP9 (47±11- fold, p<0.05). These results indicate that macrophage co-culture with PTECs promotes EMT, even in the absence of physical contact. Moreover, the addition of IL-13, found in mouse allografts destined to develop TA/IF, promotes alternative macrophage activation and enhances EMT. Thus, in addition to their typical immune role, macrophages support a milieu within allografts that promotes TA/IF. Understanding this pathway should lead to new targets to ameliorate chronic graft injury.