Like cyclosporine, FK506 is a calcineurin inhibitors (CNI) and binds to a corresponding protein FK506-binding protein 12 (FKBP12). These tacrolimus–immunophilin complexes inhibit the activity of calcineurin, which will impede calcium-dependent transduction and inactivate transcription factors, particularly the NFAT. NFAT is believed to initiate gene transcription for the formation of lymphokines such as interleukin-2 and interferon-c . Thus, FK506 reduce acute rejection rates and improve patient and allograft survivals by interfering with T-cell activation. Nevertheless, CNI are toxic, and in particular, they are nephrotoxic. When strategies of reducing dose or withdrawal of FK506 with addition of MMF are implemented, there is a trend toward better kidney function . Mycophenolatemofetil (MMF) is the first pharmaceutical prodrug of mycophenolic acid (MPA) and is used for preventing acute rejection. MPA inhibits the activity of inosine 5’-monophosphate dehydrogenase (IMPDH), the rate-limiting enzyme in the de novo pathway of guanosine nucleotides synthesis [17–19]. The pharmacological inhibition of IMPDH is considered to cause the depletion of guanosine nucleotides, leading to the suppression of cell proliferation in activated lymphocytes and a decrease of the adhesionmolecules expression [19, 20]. This study aimed to determine its related mechanism through comparing the effect of FK506 and MMF on CAN as a single factor after transplantation. And we observed that MMF could inhibit renal fibrosis in an experimental animal CAN model by down-regulating the expression of TGF-β1 and Smad2 in grafts . EMT is an important event in fibrogenesis of renal grafts, in which CTGF has been used potentially as a biomarker of EMT . In the present study, we evaluated the expression of CTGF and fibrosis-associated genes in CAN rats treated with FK-506 or MMF to determine whether there are differences between these compounds. The pathological profile demonstrated that CAN lesions were milder in the MMF group than those in the vehicle and FK506 groups, and MMF could effectively prevent fibrosis in kidney allografts, possibly by reducing the expression of CTGF, α-SMA and collagen. In addition, although there was obvious histopathological damage in the MMF group at 12 week, HE and Masson’s trichrome staining results showed some parts of glomeruli remain functional. Since Scr levels significantly increased only when glomerular filtration rate was reduced to 1/3 of normal levels, it maintains a significant low level in MMF group compared with other groups at 12 week.
Renal fibrosis correlated with renal TEC lose their polarized phenotype and acquire new characteristic features of myofibroblasts, the major effector cells responsible for the excess deposition of interstitial extracellular matrixc (ECM) under pathological conditions . Alpha-SMA is a specific molecular marker of activated myofibroblasts, and a high expression level of α-SMA is associated with the differentiation from epithelial cells to myofibroblasts [22, 23]. E-cadherin is an adhesive junction protein expressed in differentiated and polarized epithelial cells . Under pathological conditions, TEC lose E-cadherin. The disappearance of E-cadherin in epithelial cells indicates that the cell undergoes an EMT process . Both α-SMA and E-cadherin are extracellular matrix proteins correlated with the renal fibrosis process [26, 27]. Once secreted by ECM producing cells, collagen accumulates in the basement or interstitial space. Disordered synthesis and secretion and unsatisfactory degradation leads to the excessive deposition of ECM components and destruction of tissue architecture. In this study, we evaluated fibrosis-associated genes to analyze whether FK506 and MMF modulate the renal fibrosis process in a different manner. Our results showed that FK506 significantly up-regulated the expression of α-SMA and collagen, and down-regulated the expression of E-cadherin in rat kidney grafts. MMF reduced these effects, suggesting that MMF could effectively attenuate the fibrosis process in renal allografts by decreasing the expression of fibrosis-associated genes.
In this study, we also noticed that there was no significant difference in the level of CAN and renal fibrosis between FK506 and vehicle groups. In this study, we used an enhanced ischemic model which we established previously, it could successfully accelerate the development and progression of CAN in the early stage. We used CsA to prevent AR after kidney transplantation. And after that, vehicle group did not use immunosuppressive agents and therefore, the rejection may accelerate the development of CAN and renal fibrosis. To some extent, the use of FK506 could suppress immune rejection, but its renal toxicity had also accelerated the development of CAN and renal fibrosis. Although there was difference between these two groups in the progression of CAN and renal fibrosis, however, with no statistical significant difference. In addition, the combination of MMF and FK506 is often used in clinical kidney transplantations, further studies are needed to clarify the combined effects.
CTGF is one of the key cytokines in renal fibrosis, and it plays an important role in EMT of TEC. Our study demonstrated that MMF, but not FK506, down-regulated the expression of CTGF in rat kidney allografts. A correlation between MMF and EMT might provide an explanation of the mechanism of action of MMF to ameliorate transplant fibrosis in experimental models of CAN. The above results suggested that fibrosis-associated genes, especially CTGF, are involved in the compatibility of MMF to ameliorate the process of renal fibrosis within an experimental model of CAN. Furthermore, our previous results also showed that the change in CTGF expression was earlier than the pathological changes (data not shown). Therefore, CTGF has the potential to be a biomarker of CAN, as well as a therapeutic target in the management of graft fibrosis .