INTERSTITIAL FIBROSIS IN CHRONIC RENAL ALLOGRAFT DYSFUNCTION: A CONSEQUENCE OF TRANSDIFFERENTIATION OF TUBULAR EPITHELIUM TO A MYOFIBROBLAST PHENOTYPE?
Helen Robertson and John A Kirby
Applied Immunobiology Group, University of Newcastle, Newcastle upon Tyne NE2 4HH, U.K.
Background: The fibro-proliferative process responsible for graft failure is initiated by acute rejection during the first weeks after transplantation. However, the link between acute and chronic injury is not understood. We have reported the development of a population of CD103+ T cells in renal tubules during episodes of acute rejection that is maintained within the graft by locally produced IL-15. The primary lesion responsible for chronic graft dysfunction is tubulointerstital fibrosis associated with the loss of nephrons. Proliferating interstitial fibroblasts constitute the major effector cell-type responsible for fibrosis. In the mouse, the majority of these cells are generated from damaged and cytokine-stimulated tubular epithelial cells (TEC) by epithelial to mesenchymal transdifferentiation (EMT). Demonstration of the induction of fibroblast specific protein 1 (FSP1) within damaged tubules in vivo allows definitive visualisation of EMT. Importantly, expression of the human homologue of FSP1, S100A4, has not been investigated in the context of chronic renal allograft dysfunction. TGFb is thought to play a crucial role in the induction of EMT within the kidney. We have shown that the activity of this growth factor is increased within the kidney during both acute rejection and chronic graft dysfunction. The current study was designed to investigate the contribution of intratubular T cells to post transplant EMT.
Methods: Renal transplant biopsies were immunohistochemically labelled to detect S100A4, CD8, aSMA and MIB-1 and analysed using a Leica imaging system with QWin software. Primary renal cortical epithelial cells (RCC) were cultured with TGFb or co-cultured with the CD103+ T cell line MOLT 16; immunofluorescence labelling to detect S100A4, cytokeratin, E-cadherin and aSMA was analysed using scanning laser confocal microscopy.
Results: S100A4 positive tubular epithelial cells (TEC) were detected at an early stage after transplantation during episodes of graft dysfunction (acute rejection) and when the graft was in the recovery phase (post-acute rejection). S100A4 was differentially expressed in individual TEC which showed evidence of loss of orientation and migration into the surrounding interstitium. S100A4 was highly expressed in tubules within inflammatory foci infiltrated by CD8+ T cells. aSMA was rarely detected in tubules but identified activated myofibroblasts in the interstitium. The majority of TEC were strongly S100A4 positive in developing interstitial lesions and showed proliferative activity in the tubules. At the periphery of established fibrotic lesions, remnant tubules expressed S100A4 as did fibroblast-like cells within the lesion itself; CD8+ T cells were prominent within remnant tubules. RCC acquired a bi-polar morphology and expressed S100A4 but not aSMA following culture with TGFb for 72 hours. MOLT 16 T cells adhered to RCC, which also became elongated and expressed S100A4 within 72 hours; cytokeratin and E-cadherin were down-regulated. RCC cultured in medium alone did not express S100A4.
Conclusions: S100A4 expression is an important early indicator of TEC transition to a mesenchymal phenotype. After transplantation, long-lived allospecific CD103+ T cells within the tubules generate a microenvironment which stimulates TEC to transdifferentiate to proliferative S100A4-expressing fibroblasts and to migrate into the interstitium. Clearance of intratubular T cells after any episode of acute rejection could prevent EMT and progression to chronic tubulointerstitial fibrosis.
INTERSTITIAL FIBROSIS IN CHRONIC RENAL ALLOGRAFT DYSFUNCTION :
A CONSEQUENCE OF TRANSDIFFERENTIATION OF TUBULAR EPITHELIUM TO A
Helen Robertson and John A Kirby
Professor John A. Kirby
School of Surgical and Reproductive Sciences, Surgery
The Medical School
University of Newcastle
Newcastle upon Tyne
Telephone: +44 191 222 7158
Fax: +44 191 222 8514