Epithelial-Mesenchymal Transition In Renal Interstitial Fibrosis And The Role Of Snail1 In EMT

Chronic kidney disease (CDS) is a worldwide threat to public health, with increasing prevalence, high costs, and poor prognosis. Progressive tubulointerstitial fibrosis is the terminal pathway for all CDS leading to chronic renal failure (CRF). There have been many studies stressing that tubulointerstitial fibrosis, characterized by loss of renal tubules and interstitial capillaries and the accumulation of extracellular matrix proteins, is responsible for renal dysfunction rather than glomerular changes. Fibroblasts and myofibroblasts are considered to be the key effector cells in renal tubulointerstitial fibrosis responsible for the synthesis and deposition of extracellular matrix components. In 1990, Gown claimed to have the mysteries of the myofibroblast (partially at least) unmasked. But 20 years later, the origin of activated ?broblasts and myo?broblasts still remains controversial. There are several potential sources for myofibroblasts in renal tubulointerstitial fibrosis than tissue-resident fibroblasts, involving circulating cells of bone marrow origin (fibrocytes), vascular pericytes, endothelial cells, and tubular epithelial cells. Since 1995, the transition of tubular epithelial cells to a mesenchymal phenotype (epithelial-mesenchymal transition, EMT) in the renal cortex has been firstly reported by Strutz et al, however, 15 years later, researchers are still continue to study the phenomenon and mechanism of EMT. In response to injury and inflammatory stimuli, it is proposed that epithelial cells undergo morphological changes, losing epithelial characteristics such as polarity and the expression of junctional markers, while inducing fibroblast markers such as FSP-1 (fibroblast specific protein 1), vimentin andα-SMA (α-smooth muscle actin). The cells begin to express stress fibres containingα-SMA and migrate through the basement membrane to become myofibroblasts in the interstitium. However, it is still controversial whether myofibroblasts were derived from tubular epithelial cells via EMT in renal tubulointerstitial fibrosis. In our study, we also tried to observe the phenomenon of EMT in vivo using mice unilateral ureteral obstruction (UUO) model and in vitro culturing immortalized human proximal tubular epithelial HK-2 cells.In in vitro study, we first observed the morphological change of HK-2 cells induced by TGF-β1 (5ng/ml) or EGF (10ng/ml) alone, and the combinating administration of TGF-β1 (5ng/ml) and EGF (10ng/ml). It is showed that the combinating use of EGF and TGF-β1 could more effectively stimulate the morphologic alteration of HK-2 cells from epithelial to mesenchymal phenotype when compared to TGF-β1 or EGF alone. Further, a follow-up study chose HK-2 cells induced by the combinating use of EGF and TGF-β1 as a model of renal tubular epithelial cells EMT in vitro. Using scratch test and matrigel invasion assays, we found that the cell migration velocity of HK-2 cells significantly increased when induced by the combinating use of EGF and TGF-β1. We also observed that the epithelial cell markers CK18 and E-cadherin was downregulated while mesenchymal cells marker Vimentin was upregulated using immunofluorescence. In conclusion, HK-2 cells have undertaken EMT when induced by the combinating use of EGF and TGF-β1.In order to explore the molecular mechanism of EMT, we then detected the EMT-related molecules'expressions via RT-PCR and immunofluorescence. We found that the transcriptive and expressing level of transcription factor Snail1 increased in HK-2 cells during EMT induced by the combinating use of EGF and TGF-β1. Western blot analysis also confirmed that ERK (p44, ERK1 and p42, ERK2) phosphorylation were more active and Snail1 expression level were higher when compared HK-2 cells that undertook EMT induced by the combinating use of EGF and TGF-β1 to those administrated with TGF-β1 or EGF alone. All the above suggested that ERK pathway mediates the increase of transcription factor Snail1 which may contribute to HK-2 cells EMT process.To further confirm the effect of ERK Pathway on Snail1 expression, MEK inhibitor U0126 was used to examine its impact on HK-2 cells EMT induced by the combinating use of EGF and TGF-β1. The results showed that MEK inhibitor U0126 could effectively block ERK1/2 phosphorylation, inhibited the expression of Snail1 while simutaneously inhibited the morphologic alteration and movement of HK-2 cells induced by the combinating use of EGF and TGF-β1. However, the level of Snail1 transcription is not inhibited in HK-2 cells using MEK inhibitor U0126. It is suggested that U0126 blocked ERK pathway as well as Snail1 protein expression, and subsequently inhibit TGF-β1/EGF-induced HK-2 cells EMT process.For in vivo studies, we used mice UUO-induced renal interstitial fibrosis model to observe the pathological changes of kidneys within 14 days after UUO establishment. The model mice suffered from renal interstitial fibrosis were identified by renal histological examinations. By further demonstration given by immunofluorescence and Western blottings, we observed that epithelial cell markers E-cadherin expression decrease in contrast to myofibroblast markerα-SMA increase. The increase ofα-SMA-positive cells were mainly located around small blood vessels at early stage on Day 4 after UUO establishment. On Day 14, we observed a few renal tubular epithelial cells begin to expressα-SMA, which demonstrated that EMT phenomenon indeed exist during renal interstitial fibrosis in mice. Gelatin zymographic analysis showed the expression of MMP2 and MMP9 were increased temporarily in early stage of renal interstitial fibrosis, suggesting that gelatinase may be involved in the degradation and balance of fibers in renal interstitium during this period, and help promoting renal tubular cells migration through the basement to interstitium. The number of PAX2-positive cells was increased among renal tubular epithelial cells detected by immunofluorescence, suggesting that renal tubular epithelial cells may first transited to metanephric mesenchymal-like cells. Also by immunofluorescence, we found a amount of BMP7-positive cells began to appear in renal cortical interstitium, we attrbuted this observation to the possible infiltration of mononuclear cells and thus may as a result take renal protective effects, which undoubtedly require further experiments to confirm. The transcriptive and protein level of EMT-related transcription factor Snail1 gradually increased as the time prolonging during this period, indicating that the proportion of the expression of Snail1 cells gradually increased.In additon, we also constructed a adenovirus vector containing human Snail1 and successful packaging of the adenovirus to study the phenotype impact of transient expression of transcription factors Snail1 in HK-2 cells. It was observed that phenotype changed in HK-2 cells infected with adenoviruses, showing a fibroblast-like shape. By RT-PCR, Western blot and immunofluorescence, we found that, compared with the control adenovirus, the expression of the epithelial cells marker E-cadherin reduced, E-cadherin-associatedβ-catenin translocation from the cell membrane to the cytoplasm, myofibroblasts markerα-SMA increased, and gelatinases (MMP2 and MMP9) transcription level were increased. All these demonstrated HK-2 cells infected with adenoviruses containing human Snail1 could lead to EMT.Through this study, the conclusion we could made were listed as follows①the combinating use of EGF and TGF-β1 can lead human renal proximal tubular epithelial cells line HK-2 cells to undertake EMT. The synergy of TGF-β1/EGF could enhance ERK1 / 2 (p44/42) phosphorylation and then increase Snail1. MEK inhibitor U0126 can block ERK1 / 2 (p44/42 ) phosphorylation and Snail1 expression at the protein level, and subsequently inhibit EMT, suggesting ERK pathway regulate the expression of Snail1 at the protein level in HK-2 cells during EMT.②In mice UUO model, myofibroblasts, which appeared firstly around the vessels, derived from perivascular cells or endothelial cells during early stage of renal interstitial fibrosis. The renal tubular epithelial cells transited into metanephric mesenchymal-like cells firstly, then could expressα-SMA and transited into myofibroblasts on Day 14, prompted myofibroblasts increase by EMT during later stage of renal interstitial fibrosis. At the same time, a large number of cortical interstitial mononuclear cells expressed BMP7, suggesting the potential renal protective effects, which require further experiments to confirm.③Transient expression of transcription factors Snail1 in human renal proximal tubular epithelial cell line HK-2 cells could lead to phenotype changes, cell migration, and EMT.