A role for HO-1 in the epithelial-to-mesenchymal-transdifferentiation of renal tubular cells

Abstract

Chronic allograft nephropathy (CAN) is the major barrier to long-term graft survival. Kidney graft function is intimal related to the degree of the tubulointerstitial fibrosis (TIF). Recent studies suggest that some interstitial myofibroblasts might be derived from kidney tubular epithelial cells that have undergone epithelial-to-mesenchymal transdifferentiation (EMT). The additional effect of the immunosuppressive drugs in delaying or accelerating the EMT in kidney transplant receptors is still not clear. Heme oxygenase-1 (HO-1) gene expression, with anti-proliferative effects, can potentially suppress the fibrotic process and limit CAN development in vitro and in vivo. Sub-project 1: Effect of HO-1 expression on tubular proximal epithelial renal cells in EMT in vitro. Specific aim: Assess the role of HO-1 expression upon EMT in proximal tubular epithelial cells. Study design: Human immortalized cells from kidney proximal tubular epithelia will be treated with inductors or inhibitors of HO-1, or will be transduced with HO-1 gene, prior EMT induction. The potential of BMP-7 will be also investigated as a natural regulator of EMT. Sub-project 2: Modulation of HO-1 expression by immunosuppressive drugs on proximal tubular epithelial cells submitted to EMT. Specific aim: To verify the capacity of immunosuppressive drugs in accelerate or in delaying EMT process in vitro, and it possible effects on HO-1 expression. Study design: Human immortalized cells from kidney proximal tubular epithelia will be treated with inductors and inhibitors of HO-1 drugs, or will be transduced with HO-1 gene, prior EMT induction. At the different time points, immunosuppressive (rapamycin, mycophenolate mofetil and cyclosporine) will administered in the culture. Methods: EMT will be induced by TGF-b and by hypoxia; EMT will be assessed by confocal microscopy, immunohistochemistry and by western blot; Enzymatic activity assay for HO-1 and evaluation of the transcripts expression of HO-1, TGF-b1, Smads 2/3 e and 7 by Real time PCR will be also performed; Protein expression by western blot will be done to complement those results; HO-1 will be cloned and transduced; Cell motility assay for fibroblasts will be carried out. Expected results: That the overexpression or the induction of HO-1 would be associated with dedifferentiation of the myofibroblasts into epithelial cells. (AU)