Astragalus mongholicus ameliorates renal fibrosis by modulating HGF and TGF-β in rats with unilateral ureteral obstruction

Astragalus mongholicus ameliorates renal fibrosis by modulating HGF and TGF-β in rats with unilateral ureteral obstruction first-author †E-mail:zcsiren@163.comfanjunming2008@163.com †E-mail:zcsiren@163.comfanjunming2008@163.com zcsiren@163.com corresp ‡Corresponding Author ‡Corresponding Author †E-mail:zcsiren@163.comfanjunming2008@163.com †E-mail:zcsiren@163.comfanjunming2008@163.com zcsiren@163.com Department of Nephrology, West China Hospital of Sichuan University, Chengdu 610041, China Department of Nephrology, West China Hospital of Sichuan University, Chengdu 610041, China Astragalus mongholicus (AM) derived from the dry root of Astragalus membranaceus Bge. var. mongolicus (Bge.) Hsiao is a widely used traditional Chinese medicine. The present study investigated the potential role of AM on renal fibrosis on a rat model of unilateral ureteral obstruction (UUO). We divided 48 Sprague-Dawley rats randomly into 4 groups: sham-operated group (Sham), untreated UUO group, AM-treated (10 g/(kg·d)) UUO group, and losartan-treated (20 mg/(kg·d)) UUO group as positive control. Haematoxylin & eosin (HE) and Masson staining were used to study the dynamic histological changes of the kidneys 7 and 14 d after operation. The expressions of fibronectin (FN), type I collagen (colI), hepatocyte growth factor (HGF), transforming growth factor-β1 (TGF-β1), and α-smooth muscle actin (α-SMA) were analyzed by real-time polymerase chain reaction (PCR), immunohistochemistry staining, and Western blot. Results show that, similar to losartan, AM alleviated the renal damage and decreased the deposition of FN and colI from UUO by reducing the expressions of TGF-β1 and α-SMA ( P < 0.05), whereas HGF increased greatly with AM treatment ( P < 0.05). Our findings reveal that AM could retard the progression of renal fibrosis. The renoprotective effect of AM might be related to inhibition of myofibroblast activation, inducing of HGF and reducing of TGF-β1 expression. Astragalus mongholicus (AM) Myofibroblast Transforming growth factor-β1 (TGF-β1) Renal interstitial fibrosis Hepatocyte growth factor (HGF) INTRODUCTION 1 Renal interstitial fibrosis (RIF) is a common histopathological change and the major determinant in the progression of chronic kidney diseases that lead to end-stage renal failure. It is characterized by the accumulation of extracellular matrix (ECM) proteins in the renal tubulointerstitium (Eddy, 1996 1996 ; Remuzzi and Bertani, 1998 1998 ). The key event in the progression of RIF is the activation of α-smooth muscle actin (α-SMA)-positive myofibroblast cells, which is identified to be the main source of excessive ECM deposition and the best prognostic indicator of renal diseases both in human and experimental animal models (Roberts et al., 1997 1997 ; Essawy et al., 1997 1997 ). However, the origin of myofibroblast cells is still undefined. Emerging evidence indicates that a large proportion of interstitial fibroblasts actually originate from tubular epithelial cells via epithelial to myofibroblast transition (EMT) in diseased kidney (Liu, 2004a 2004a ). Previous studies from our laboratory showed that transforming growth factor-β1 (TGF-β1), a multifunctional cytokine with profibrogenic properties, was a central mediator that regulated transdifferentiation of tubular epithelial cells into α-SMA-positive myofibroblasts (Fan et al., 1999 1999 ). Furthermore, we demonstrated that hepatocyte growth factor (HGF) exhibited a remarkable ability to block this phenotypic transition both in vitro and in vivo (Zhong et al., 2005 2005 ; Xue et al., 2005 2005 ). These observations suggested that upregulation of endogenous HGF expression and reducing of TGF-β1 expression could be essential for maintaining the tubular epithelial cell phenotype by blocking EMT. This could provide a potential strategy for the treatment of renal fibrosis. During the recent decades, Chinese medicinal herbs and their extracts have drawn great attention to the prevention of renal fibrosis. Astragalus mongholicus (AM) derived from the dry root of Astragalus membranaceus Bge. var. mongolicus (Bge.) Hsiao, which belongs to leguminous plant of the Astragalus family, is one of the most popular traditional Chinese medicines. AM is composed of glycoside, astragalus polysaccharides, multi-amino acids, astragalus total saponin, caritinoid, astragalus total flavonoids, and microelement. It has been widely used in China and East Asia area for many years to treat myocardial ischemia, liver fibrosis, chronic nephritis, diabetes, etc. (Han et al., 2000 2000 ). Although the herb is commonly used in treating various diseases in Chinese herbal formula, scientific reports on its alleviation of tubulointerstitial fibrosis are limited. The mechanism of its renoprotection is lacking. In recent reports, AM has been shown to possess renoprotective activity by attenuating glomerular sclerotic injury in experimental diabetic nephropathy (Yang and Zhu, 2005 2005 ; Mou et al., 2002 2002 ). In the present study, we sought to investigate whether the anti-fibrotic action of AM goes beyond the glomeruli by using a unilateral ureteral obstruction (UUO) animal model. We provide evidence to support that AM may attenuate renal fibrosis likely by derivation of HGF and suppression of TGF-β1. MATERIALS AND METHODS 1 Drugs 2 Astragalus mongholicus injections, which were made of the extract of Astragalus mongholicus , were purchased from Chiatai Qingchunbao Pharmaceutical Co., Ltd. (Hangzhou, China) with batch No. 0508081 containing crude drug 2 g/ml. Animals and experimental design 2 A total of 48 male Sprague-Dawley (SD) rats (6 weeks old, weighing 180~200 g) were obtained from the Laboratory Animal Center of Sichuan University (Chengdu, China), and were treated strictly according to the Health Guide for the Care and Use of Laboratory Animals of the National Institutes. UUO was induced by left ureteral ligation with an established procedure as previously described (Chevalier et al., 2000 2000 ). Sham-operated rats had their ureters manipulated but not ligated. The animals that underwent surgery were randomly divided into four groups: UUO, Sham, AM, and losartan (an angiotensin type-1 receptor blocker, ARB) groups, with 12 rats each group. Treatment started within 1 h after the surgical procedure. Rats in the AM group were intraperitoneally injected with AM at 10 g/(kg·d) (this dose of AM has been shown to be effective in attenuating renal fibrosis without obviously adverse effect in UUO model in the pilot study from our laboratory), while rats in the Sham, UUO, and ARB groups were intraperitoneally injected with identical voluminal normal saline. Rats in the ARB group were intragastricly administrated with losartan (Merck Sharp & Dohme, USA) 20 mg/(kg·d), while those of the Sham, UUO, and AM groups were intragastricly administrated with identical volume of normal saline. Days 7 and 14 after surgery, 6 rats selected randomly from each group were sacrificed, and the left kidneys were harvested and kept at −80 °C. Some kidney tissue samples were immersed with 10% (w/v) neutral-buffered formalin for histopathological and immunohistochemical evaluations. Histopathological examination 2 Fixed tissues were dehydrated in graded alcohol and embedded in paraffin. Sections were stained with hematoxylin & eosin (HE) for general histology. Injury score was evaluated with a scale of 0~4 as previously reported (Taal et al., 2000 2000 ). Masson-Trichrome staining was performed in accordance with the standard protocol using a reagent kit (Sigma, USA). Ten separate fields of each section were scanned at 400× magnification and put into digital form using imaging software (Photoshop, Adobe, San Jose, California). The number of points covering the blue collagen staining was counted and the extent of collagen deposition was evaluated (Mizuguchi et al., 2004 2004 ; Xie et al., 2008 2008 ). Immunohistochemical staining analysis 2 Paraffin-embedded sections (5 μm) were deparaffinized, washed with phosphate-buffered saline (PBS), treated with 3% (v/v) H 2 O 2 for 5 min, blocked with 10% (w/v) normal goat serum for 1 h, and then incubated with primary antibodies, including rabbit antibodies to α-SMA (ABCAM, UK), rabbit antibodies to HGF (Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit antibodies to TGF-β1 (Santa Cruz Biotechnology), rabbit antibodies to collagen I (colI), and rabbit antibodies to fibronectin (FN) (Boshide Biological Technology Co., Ltd., Wuhan, China), at 4 °C overnight as described previously (Xie et al., 2008 2008 ). Slides then were incubated with appropriate biotin-conjugated secondary immunoglobulin G, and treated with reagents from a Vecta-Elite streptavidin-peroxidase kit (Vector Laboratories, Burlingame, CA, USA). The scores were determined as previously reported (Fu et al., 2006 2006 ). Briefly, the percentage of positive stained area in the examined tubulointerstitium was measured and scored in a blind manner on slides. Quantitative real-time polymerase chain reaction (PCR) analysis 2 Total RNA was extracted by RNeasy Mini RNA extracting kit (QIAGEN, USA) following the manufacturer’s protocol. The cDNA was synthesized and real-time PCR was run as described previously (Xie et al., 2008 2008 ). Primers and probes of α-SMA, TGF-β1, HGF, fibronectin, collagen I, and the internal calibrator glyceraldehyde-3-phosphate dehydrogenase (GAPDH, synthesized by Sangon, Shanghai, China) were listed in Table 1 1 . Primers Probes  FN Forward: 5′-CCATTGCAAATCGCTGCCAT-3′ TaqMan: 5′-CTCATGTGGCCTCCTCCAC-3′ Reverse: 5′-AACATTTCTCAGCTATTGGCTT-3′  ColI Forward: 5′-TGGCGCTTCAGGTCCAAT-3′ TaqMan: 5′-CCAGCTTCCCCATCATCTCC-3′ Reverse: 5′-TGTTCCAGGCAATCCACGAG-3′  α-SMA Forward: 5′-GTTCGAAACCTTCAATGTTCCT-3′ TaqMan: 5′-ATCTACGAGGGCTATGCACTG-3′ Reverse: 5′-CCATTCAAGCTGTGCTCTCGCT-3′  HGF Forward: 5′-GACCTTGTGAGGGAGATTAT-3′ TaqMan: 5′-CTTGGTGTCATTGTTCCTGGTC-3′ Reverse: 5′-ATGTGCCATCCCAAATCGTCC-3′  TGF-β1 Forward: 5′-CCAACTATTGCTTCAGCTCCA-3′ TaqMan: 5′-CCAAGGGCTACCATGCCAAC-3′ Reverse: 5′-GTGTCCAGGCTCCAAATGT-3′  GAPDH Forward: 5′-TGGGTGTGAACCAGGAGAA-3′ TaqMan: 5′-ACTGCACCACCAACTGCTTAGC-3′ Reverse: 5′-GGCATGGACTGTGGTCATGA-3′ A 30 μl of PCR reaction solution was amplified in Roche Lightcycler (Roche Diagnostics, USA). The thermocycles of PCR reaction were performed as reported previously (Xie et al., 2008 2008 ). PCR products then went through agarose-gel electrophoretic analysis, and fluorescence signal were collected to calculate Ct (cycles of threshold). Serial dilutions of sample RNA (500 ng), including α-SMA, TGF-β1, HGF, fibronectin, collagen I (10× dilution), GAPDH (10× dilution), were performed to establish the standard curves for determining the efficiency of real-time PCR. The method of Pfaffl et al.( 2002 2002 ) was used to analyze the real-time PCR results. Western blot analysis 2 Protein from kidney tissues was extracted and electrophoresed on SDS-polyacrylamide gel for Western blot analysis as previously reported (Fu et al., 2006 2006 ). In brief, samples were heated at 99 °C for 5 min and then transferred to a polyvinylidene difluoride membrane. Nonspecific binding to the membrane was blocked for 1 h at room temperature with 5% (w/v) bovine serum albumin (BSA) in Tris-buffered saline buffer (20 mmol/L Tris-HCl, 150 mmol/L NaCl, and 0.1% (v/v) Tween 20). The membranes were then incubated overnight at 4 °C with primary antibodies, including rabbit antibodies to α-SMA, rabbit antibodies to HGF, rabbit antibodies to TGF-β1, and GAPDH (Chemicon, Temecula, CA). After washing, the membranes were incubated with appropriate secondary immunoglobulin G-peroxidase conjugates (1:10 000 dilution; Kirkegaard Perry Laboratories, Gaithersburg, MD, USA), and developed using the enhanced chemiluminescence method. Band intensities were analyzed using a software named Quantity one. For quantification, the densities of the signals were normalized to that for GAPDH. Statistical analyses 2 All data were expressed as mean± SD . One-way analysis of variance (ANOVA) was used to perform comparisons between the different groups, and a P value < 0.05 was considered as statistically significant. All analyses were performed with the SPSS statistical software package (SPSS, Chicago, IL, USA). RESULTS 1 Effect of AM on the histological changes 2 On Day 7, the kidneys of rats subjected to ureteral obstruction developed a conspicuous tubulointerstitial injury characterized by tubular dilatation and atrophy, interstitial inflammation, and a marked interstitial fibrosis. The damage was more prominent on Day 14. Renal injury was ameliorated after treatments with AM and losartan as indicated by the lower tubulointerstitial injury scores and interstitial fibrosis expansion of these groups, with P < 0.05 for all comparisons (Table 2 2 and Fig. 1 1 in page 385). There was no significant difference between AM and ARB groups ( P > 0.05). Compared with the Sham group, P < 0.05; Compared with the UUO group, P < 0.05 2 Group 2 TIS 2 ICD (%) Day 7 Day 14 Day 7 Day 14 Sham 0.01±0.02 0.01±0.01 2.09±0.20 2.09±0.25 UUO 4.83±0.72 * * 6.85±1.12 * * 21.78±2.95 * * 39.11±3.87 * * AM 2.65±1.11 * * # # 4.00±0.65 * * # # 12.39±2.50 * * # # 28.00±3.20 * * # # ARB 2.61±0.82 * * # # 3.76±0.45 * * # # 11.99±3.05 * * # # 26.74±3.19 * * # # Effect of AM on the expressions of fibronectin and collagen I 2 A marked increase in tubulointerstitial fibronectin and collagen I accumulation was observed on Days 7 and 14 after UUO compared with the sham-operated animals. On Days 7 and 14, the mRNA and protein levels of collagen I and fibronectin reduced greatly in the AM and ARB groups as compared with the UUO group (Table 3 3 and Fig. 2 2 ), with P < 0.05 for all comparisons. Again, there was no significant difference between AM and ARB groups ( P > 0.05). Compared with the Sham group, P < 0.05; Compared with the UUO group, P < 0.05 3 Group 6 Protein expression (%) 2 α-SMA 2 FN 2 ColI Day 7 Day 14 Day 7 Day 14 Day 7 Day 14  Sham 0.37±0.12 0.43±0.12 3.05±0.73 3.06±0.73 3.04±0.71 2.93±0.50  UUO 8.69±2.21 * * 18.80±3.31 * * 17.65±3.29 * * 23.43±4.69 * * 19.31±3.25 * * 25.77±3.77 * *  AM 4.67±1.46 * * # # 13.45±2.13 * * # # 11.50±3.14 * * # # 15.27±4.01 * * # # 13.12±3.12 * * # # 18.91±3.37 * * # #  ARB 4.20±1.37 * * # # 11.12±3.12 * * # # 10.94±3.49 * * # # 13.84±4.77 * * # # 12.36±3.54 * * # # 17.95±3.19 * * # # Compared with the UUO group, AM and ARB groups had a greatly reduced deposition of FN and colI; Data are expressed as mean± SD ( n =6); * P < 0.05 vs the Sham group, # P < 0.05 vs the UUO group Effect of AM on α-SMA expression 2 The obstructed kidneys displayed a marked activation of myofibroblasts on Day 7 after surgery, characterized by a dramatic increase of α-SMA. This change was more prominent on Day 14. AM and losartan largely reduced renal α-SMA mRNA expression in the kidneys of UUO rats. Furthermore, Western blot and immunohistochemical findings proved that the protein level of α-SMA decreased in the UUO kidneys treated with AM or losartan compared with UUO rats without treatment (Table 3 3 and Fig. 3 3 ) ( P < 0.05 for all comparisons). Again, there was no significant difference between AM and ARB groups ( P > 0.05). Our results demonstrate that AM retarded renal myofibroblast activation in vivo. Effect of AM on TGF-β1 expression 2 A drastic increase of TGF-β1 is a key feature of the UUO kidney. We found that the levels of TGF-β1 mRNA in obstructed kidneys were increased by 5.657 and 14.254 times than those in the Sham group on Days 7 and 14, respectively, after ureteral ligation. Treatments with AM and losartan significantly decreased the expression of TGF-β1 mRNA, compared with the UUO control kidneys on Days 7 and 14. Immunohistochemical staining revealed that the expressions of TGF-β1 in tubular and interstitial cells were increased in the UUO control kidneys on Days 7 and 14 as compared with the sham-operated kidneys, which showed only the background with antibody against TGF-β1. AM and losartan treatments reduced the expression of TGF-β1 in tubulointerstitium on Days 7 and 14, in together with the reduced tubulointerstitial fibrosis. These results were consistent with the findings of Western blot analysis of TGF-β1 (Table 4 4 and Fig. 4 4 ) ( P < 0.05 for all comparisons). There was no significant difference between the AM and ARB groups ( P > 0.05). Compared with the Sham group, P < 0.05; Compared with the UUO group, P < 0.05 3 Group 4 Protein expression (%) 2 HGF 2 TGF-β1 Day 7 Day 14 Day 7 Day 14 Sham 1.08±0.21 1.09±0.15 3.18±0.65 3.07±0.78 UUO 6.01±0.54 * * 2.41±0.49 * * 12.90±2.51 * * 20.30±2.55 * * AM 7.54±0.57 * * # # 3.94±0.55 * * # # 7.73±2.39 * * # # 12.45±3.80 * * # # ARB 8.26±0.64 * * # # 4.33±0.37 * * # # 6.96±1.90 * * # # 11.11±3.85 * * # # Effect of AM on HGF expression 2 We next investigated the effect of AM on the expression of HGF, which has been identified as an antifibrotic molecular and an antagonist of TGF-β1. We found that the HGF mRNA and protein levels in UUO animals increased to peak on Day 7 after operation and decreased dramatically thereafter. HGF mRNA in the obstructed kidneys increased by 9.849 and 1.741 times than that in the Sham group on Days 7 and 14, respectively. But there was no significant difference between the UUO and Sham groups on Day 14. Both AM and losartan treatments evidently increased HGF mRNA levels to 1.741, 2.144 and 2.828, 3.249 times over those of the UUO control kidneys on Days 7 and 14, respectively. Furthermore, Western blot analysis of HGF in the UUO rats was also significantly induced after the treatments with AM and losartan, and this was in agreement with immunohistochemical evidence (Table 4 4 and Fig. 5 5 ). There was no significant difference between the AM and ARB groups ( P > 0.05). DISCUSSION 1 The progression of chronic kidney diseases is considered irreversible and eventually leads to end-stage renal failure. Renal interstitial fibrosis is the common final outcome and the remarkably histologic hallmark of chronic renal disease irrespective of diverse etiology. It is the result of destruction in architecture of the kidney and an imbalance of fibrogenesis and antifibrogenesis (Iwano and Neilson, 2004 2004 ). Angiotensin II (Ang II) was identified to be essential in the pathogenesis of renal fibrosis (Taal and Brenner, 2000 2000 ). Ang II inhibitors may suppress the expression of TGF-β1, block activation of myofibroblast cells via EMT, and ameliorate ECM accumulation (Nakao et al., 2003 2003 ; Gross et al., 2004 2004 ). Blocking Ang II has become a standard therapy in treating renal fibrosis. Nevertheless, it does not appear that they will entirely halt fibrosis (Peters et al., 1998 1998 ). It is thus worthwhile to seek other anti-fibrotic therapies, including herbal medicine. We report here that AM exhibited antifibrotic effect on UUO rats. This effect is comparable with losartan, an angiotensin type-1 receptor blocker (ARB) agent. UUO is an experimental model widely used to study the pathogenesis of tubulointerstitial fibrosis, because it is highly reproducible and induces pathogenetic events similar to those observed in human renal fibrosis (Chevalier, 1999 1999 ; Moon et al., 2006 2006 ; Wen et al., 1999 1999 ). In agreement with previous studies (Chevalier, 1999 1999 ; Klahr and Morrissey, 2002 2002 ; Moon et al., 2006 2006 ), the obstructed kidneys on Days 7 and 14 after UUO in the present study showed typical features of obstructive nephropathy including the presence of myofibroblasts in the interstitium, infiltration of inflammatory cells, tubular atrophy, and interstitial extracellular matrix deposition. Our study shows that both AM and losartan evidently suppressed the expressions of collagen and fibronectin, the specific biochemical markers for fibrosis, and attenuated the morphological changes of tubulointerstitial fibrosis in the UUO kidneys, although neither of them bestowed a complete protection from renal fibrosis in the UUO kidneys. Activation of α-SMA positive myofibroblasts plays a key role in the progression of chronic renal fibrosis. Therefore, the inhibition of α-SMA positive myofibroblasts becomes a potential antifibrotic strategy. On account of their absence in the renal interstitium, the exact origins and activation process of interstitial myofibroblasts under pathologic conditions remain largely unclear. Possible sources of renal myofibroblasts include interstitial fibroblasts, transdifferentiated tubular epithelial cells, and circulating mesenchymal precursor cells (Iwano et al., 2002 2002 ; Yang and Liu, 2002 2002 ; Yang et al., 2002 2002 ). Whether only one or all of these potential sources contribute to the myofibroblast pool in a given disease condition remains undefined. Yang et al.( 2002 2002 ; 2005 2005 )’s study proposed that myofibroblast activation in the obstructive kidney is a dynamic process, in which myofibroblasts are derived from local activation of residential fibroblasts at the earlier stage of obstructive nephropathy (3 d after obstruction) and are mainly derived from tubular epithelial cells via EMT at the later stage (7 d). Previous evidence indicated that more than 30% interstitial fibroblasts were actually from tubular epithelial cells via EMT in diseased kidney (Strutz et al., 2002 2002 ). In the process of EMT, tubular epithelial cells acquired mesenchymal features such as α-SMA expression and produced interstitial matrix components such as fibronectin and collagen I (Liu, 2004a 2004a ). In the present study, the obstructed kidneys displayed a marked activation of myofibroblasts on Day 7 after ureteral ligation, characterized by a dramatic increase in α-SMA abundance (Yang and Liu, 2002 2002 ). This change was more prominent on Day 14. AM largely reduced both renal α-SMA mRNA and protein. Given the data and previous reports, the results from our study demonstrate that AM prevented renal myofibroblast activation at least in part through the prevention of EMT in obstructive nephropathy, as did losartan. Our previous report showed that TGF-β1 was a key mediator in regulating transdifferentiation of tubular epithelial cells into α-SMA positive myofibroblasts (Fan et al., 1999 1999 ). A drastic increase of TGF-β1 was observed in obstructed kidneys in the present study. Administration of AM considerably inhibited the expression of TGF-β1 in the level of mRNA as well as protein, indicating that AM decreased the level of TGF-β1 in vivo. HGF is a pleiotropic factor that plays an imperative role in tubular repair and regeneration after acute renal injury (Yang and Liu, 2002 2002 ). Growing evidence indicates that HGF is a regenerative, cytoprotective molecule as well as a endogenous antifibrotic factor that shows an impressive efficacy in ameliorating tissue fibrosis in a wide variety of animal models, regulating motility, mitogenesis, and morphogenesis in a cell type-dependent fashion (Liu, 2004b 2004b ; Liu and Yang, 2006 2006 ). HGF and TGF-β1 regulate diversified cellular functions and often act antagonistically against each other. It has been reported that the reciprocal imbalance between TGF-β1 and HGF is closely involved in the progression of tissue fibrosis (Mizuno et al., 2000 2000 ). Previous studies indicated that TGF-β1 and HGF are counteracting in their biological activities (Mizuno et al., 2001 2001 ). TGF-β1 strongly stimulates the synthesis of ECM proteins and inhibits the degradation of ECM (Esposito et al., 2005 2005 ; Taipale et al., 1996 1996 ). In contrast, HGF accelerates the degradation of ECM by modulating proteinases involved in the breakdown of ECM proteins. These proteinases include matrix metalloproteinases/tissue inhibitors of metalloproteinases, and plasminogen activators/plasminogen activator inhibitors (Gong et al., 2003 2003 ; Mizuno et al., 2000 2000 ). TGF-β1 induces growth arrest and apoptosis in renal tubular cells and endothelial cells (Nowak and Schnellmann, 1996 1996 ; Taipale and Keski-Oja, 1996 1996 ), while HGF exhibits mitogenic, motogenic, morphogenic, and anti-apoptotic activities in these cell types (Bowes et al., 1999 1999 ; Dai et al., 2004 2004 ; Liu, 1999 1999 ). Furthermore, the epithelial to myofibroblast transition can be induced by TGF-β1 and reversed by HGF (Blobe et al., 2000 2000 ; Fan et al., 1999 1999 ; Yang and Liu, 2002 2002 ). Yang et al.( 2005 2005 ) reported a novel mode of interaction between the signals activated by HGF receptor tyrosine kinase and TGF-β1 receptor serine/threonine kinases. They found that HGF blocks EMT by antagonizing TGF-β1’s action via upregulating Smad transcriptional corepressor SnoN expression. In view of the fact that HGF expression increased at early stage and then decreased greatly later in a manner reciprocal to the increase in expression of TGF-β1 in obstructed kidney, our results likely provide novel insights into the mechanisms of impairment in the balance of profibrogenic and antifibrogenic cytokines in promoting renal fibrosis. In the AM-treated group, the enhancement of HGF expression and the reduction of TGF-β1 in kidney cells of rats might have contributed to the reduced fibrosis in the kidneys of these rats. However, the definitive mechanisms how AM suppresses TGF-β1 expression and augments HGF expression remain obscure. 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Blockage of tubular epithelial to myofibroblast transition by hepatocyte growth factor prevents renal interstitial fibrosis J Yang , RW Shultz , WM Mars , EW Rodney , Y Li , C Dai , N Kari , Y Liu . Disruption of tissue-type plasminogen activator gene in mice reduces renal interstitial fibrosis in obstructive nephropathy . J Clin Invest , 2002 . 110 ( 10 ): 1525 - 1538 . 12438450 DOI: 10.1172/JCI16219 http://doi.org/10.1172/JCI16219 . Disruption of tissue-type plasminogen activator gene in mice reduces renal interstitial fibrosis in obstructive nephropathy J Yang , C Dai , Y Liu . A novel mechanism by which hepatocyte growth factor blocks tubular epithelial to mesenchymal transition . J Am Soc Nephrol , 2005 . 16 ( 1 ): 68 - 78 . 15537870 DOI: 10.1681/ASN.2003090795 http://doi.org/10.1681/ASN.2003090795 . A novel mechanism by which hepatocyte growth factor blocks tubular epithelial to mesenchymal transition H Zhong , JM Fan , Z Li , F Liu , LC Yang , L Ji , ZH Sha , XY Ma . Effects of hepatocyte growth factor on TGF-beta1 triggered tubular epithelial-myofibroblast transdifferentiation by CTGF in vitro . Sichuan Da Xue Xue Bao Yi Xue Ban , 2005 . 36 ( 5 ): 653 - 656 . 653 (in Chinese) 16235529 . Effects of hepatocyte growth factor on TGF-beta1 triggered tubular epithelial-myofibroblast transdifferentiation by CTGF in vitro The authors are grateful to Prof. Qing-jie Xia (Laboratory of Molecular Genetics, West China Hospital of Sichuan University, China) for technical advice. 10.1631/jzus.B0820230 Project (No. 30170437) supported by the National Natural Science Foundation of China 2008-07-28 2009-04-08 2008-10-22 2022-01-05 Copyright © 2009, Journal of Zhejiang University Science 2009 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 05 10