 |
||||
| 2007 JASN IMPACT FACTOR 7.111 | HOME AUTHOR INFO EDITORIAL BOARD SUBSCRIBE FEEDBACK ALERTS HELP | |||
| CURRENT ISSUE | ARCHIVES | JASN Express | ONLINE SUBMISSION | |
|
||||
| 2007 JASN IMPACT FACTOR 7.111 | HOME AUTHOR INFO EDITORIAL BOARD SUBSCRIBE FEEDBACK ALERTS HELP | |||
| CURRENT ISSUE | ARCHIVES | JASN Express | ONLINE SUBMISSION | |
BASIC SCIENCE |
*Third Department of Internal Medicine, Gunma University School of Medicine, Maebashi, Japan; and Institute for Molecular and Cellular Regulation, Gunma University, Maebashi, Japan
Correspondence to Dr. Akito Maeshima, Third Department of Internal Medicine, Gunma University School of Medicine, 3-39-15 Showa, Maebashi 371-8511, Japan. Phone: +81-272-220-8166; Fax: +81-272-220-8173;
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-smooth muscle actin in a rat kidney fibroblast cell line (NRK-49F cells) as well as in primary cultured renal interstitial fibroblasts. In contrast, activin A did not affect the expressions of -smooth muscle actin and type I collagen in renal epithelial tubular cell lines LLC-PK1, and MDCK. Follistatin, an antagonist of activin A, significantly inhibited cell proliferation in NRK-49F cells. Blockade of activin signaling by overexpression of truncated type II activin receptor, which lacked the intracellular kinase domain, decreased cell proliferation and reduced the expression level of type I collagen mRNA in NRK-49F cells. The expression of activin A was induced by TGF-
1 or activin A itself. Induction of type I collagen expression by TGF-1 was reduced by follistatin or by overexpression of truncated type II activin receptor. These results suggest that activin A produced by tubular cells acts as a paracrine factor that activates renal interstitial fibroblasts during the fibrotic processes of the kidney. E-mail: amaeshima@ucsd.edu |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
is known to be a key mediator of renal fibrosis (8–12). Upregulated expression of TGF- has been observed in human and experimental models (13). Connective tissue growth factor, a novel profibrogenic factor, may be an important downstream mediator of TGF- profibrotic activities (14,15). Basic fibroblast growth factor (FGF), a mitogenic factor for various types of renal cells, is upregulated in fibrotic kidneys (16) and mediates TGF--induced cell proliferation in renal interstitial fibroblasts (17). Basic FGF is also implicated in tubular epithelial-mesenchymal transdifferentiation (TEMT) (18). Conversely, bone morphogenetic protein-7 (BMP-7), an antifibrogenic factor, ameliorates glomerular and interstitial fibrosis (19,20). Recently, BMP-7 was proved to counteract TGF-–induced TEMT and reverse chronic renal injury (21). PDGF (22), hepatocyte growth factor (23), and angiotensin II (24,25) were shown to be involved in fibrotic processes of the kidney. However, the precise mechanism of fibroblast activation in renal interstitial fibrosis remains unknown.
Activin A, a member of the TGF- superfamily, is a dimeric protein composed of two A subunits (26,27) and is expressed in various organs (28). Activin A regulates cell growth and differentiation in various types of cells (26,27). An important regulatory factor that modulates activin A action is follistatin (29). Follistatin stoichiometrically binds to activin A with high affinity and blocks its action (29,30). Follistatin is synthesized in the target cells of activin A and remains in the extracellular matrix (31). Activin A is trapped by follistatin, internalized by endocytosis, and subsequently degraded by proteolysis (32). Activin A is expressed in the embryonic kidney but not in the adult kidney (33). Activin A was shown to inhibit branching morphogenesis of the ureteric bud in an organ culture system (34) as well as in an in vitro tubulogenesis model (35). The number of glomeruli is increased in the kidney of transgenic mice expressing dominantly negative activin type II receptor (36). Collectively, activin A is a negative regulator of tubulogenesis. Recently, we demonstrated the involvement of the activin-follistatin system in tubular regeneration (37). The expression of activin A, which was not detected in normal kidney, was upregulated in tubular cells after ischemia/reperfusion injury (37). In contrast, follistatin, which is abundantly expressed in tubular cells (38), was decreased in ischemic kidneys. Blockade of activin action by intravenously administered follistatin improved renal dysfunction and histologic changes after renal ischemia (37), supporting the idea that activin A acts as an autocrine inhibitor of tubular cell growth (39). We then asked whether endogenous activin A in the damaged kidney is involved in renal interstitial fibrosis. To address this issue, we examined the expression of activin A in fibrosis model kidneys induced by unilateral ureteral obstruction (UUO) and studied the effect of activin A on TEMT in renal epithelial tubular cells as well as on fibroblast activation in renal interstitial fibroblasts.
The present study showed that activin A was upregulated in tubular cells of fibrotic kidneys. Activin A did not induce TEMT in MDCK cells and LLC-PK1 cells. In contrast, activin A promoted cell proliferation, induced differentiation into myofibroblasts, and enhanced the expression of type I collagen mRNA in NRK-49F cells as well as in primary cultured renal interstitial fibroblasts. The expression of activin A was induced by TGF-1. TGF-1 enhanced the expression level of type I collagen in NRK-49F cells, but its effect was canceled by follistatin or overexpression of activin mutant receptor. These results suggest that activin A acts as a profibrotic factor in the injured kidney and could be a new target for the treatment of chronic renal fibrosis.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1 was purchased from Wako (Osaka, Japan). Monoclonal anti-mouse -smooth muscle actin (-SMA) antibody was obtained from PROGEN Biotechnik (Heidelberg, German). Polyclonal goat anti-human Smad-2/3 antibody and polyclonal goat anti-human activin type II receptor antibody were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Polyclonal goat anti-human activin A antibody was purchased from R&D Systems (Minneapolis, MN). [3H]Thymidine and [-32P] dCTP were purchased from Dupont New England Nuclear (Boston, MA).
UUO
UUO was performed in male Wistar rats that weighed 200 g (NIHON SLC, Hamamatsu, Japan) as described previously (40). Briefly, after induction of general anesthesia by intraperitoneal injection of pentobarbital (50 mg/kg body wt), the abdominal cavity was exposed via a midline incision and the left ureter was ligated at two points with 4-0 silk. At the indicated times after UUO operation, rats were killed and the kidneys were removed for RNA extraction or histologic examination. UUO was confirmed by observation of dilation of the pelvis and proximal ureter and collapse of the distal ureter. Contralateral kidneys were used as controls. The experimental protocol was approved by the Ethics Review Committee for Animal Experimentation of Gunma University.
Reverse Transcription–PCR
Total RNA was extracted from whole kidney or cells with the TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. First-stranded cDNA was prepared using the Superscript Preamplification System (Invitrogen). Reverse transcription–PCR (RT-PCR) was performed with the primers as indicated in Table 1. Reactions included 5 µl of 10 x PCR buffer, 4 µl of MgCl2 (25 mM), 5 µl of dNTP mixture (2 mM), 1 µl of 3' primer, 1 µl of 5' primer, 0.5 µl of Taq polymerase (Roche, Branching, NJ), and 1 µl of cDNA. Samples were incubated at 94°C for 5 min, followed by the 30 cycles of 30 s at 94°C, 30 s at 55°C, and 45 s at 72°C, with final extension at 72°C for 7 min in a GeneAmp PCR system 9700 (Perkin Elmer, Shelton, CT). Reactions without cDNA were used as a negative control. Reactions were repeated at least twice.
|
Cell Culture
LLC-PK1 cells, MDCK cells, and NRK-49F cells (ATCC CRL-1392) were obtained from American Type Culture Collection (Rockville, MD) and cultured in DMEM (Sigma-Aldrich, St. Louis, MO) containing 5% FBS, penicillin, streptomycin, and amphotericin B in an atmosphere of 5% CO2-95% air at 37°C. For obtaining quiescent cells, cells were incubated in a serum-free medium for 24 h.
Rat renal interstitial fibroblasts were isolated from male Wistar rat kidneys as described previously (41). Renal cortex was minced and digested in a solution of 0.2% type I collagenase. For removing contaminating glomeruli and tubules, the digests were passed through a 36-µm mesh. Renal fibroblasts were cultured in DMEM containing 10% FBS in an atmosphere of 5% CO2-95% air at 37°C. Isolated cells were identified as interstitial fibroblasts by fibroblastic morphology and immunocytochemical staining (positive for a mesenchymal marker, vimentin, and negative for an epithelial marker, E-cadherin, and an endothelial marker, platelet-endothelial cell adhesion molecule [PECAM]). Primary cultured fibroblasts were used from three to five passages in this study.
Western Blot Analysis
Cells were washed three times with PBS and suspended in lysis buffer. After centrifugation, supernatant was collected, and the protein concentration was determined with the BCA protein assay kit (Pierce, Rockford, IL). Twenty micrograms of protein from each sample was separated by SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA). To reduce nonspecific antibody binding, the membrane was blocked with Block Ace (Yukijirushi, Osaka, Japan), incubated with primary antibody for 2 h, and washed with Tris-buffered saline (20 mM Tris-HCl, 150 mM NaCl, and 0.1% Tween 20). After incubation with peroxidase-labeled secondary antibody for 2 h, the membrane was washed with Tris-buffered saline and analyzed by exposure to X-ray film using Supersignal West Dura Extended Duration Substrate (Pierce).
Measurement of DNA Synthesis and Cell Proliferation
DNA synthesis was examined by measuring [3H]thymidine incorporation. Serum-starved cells were incubated in complete medium with the indicated concentrations of activin A for 20 h and pulse-labeled with 1 µCi/ml [3H]thymidine for an additional 4 h. [3H]Thymidine incorporation into TCA precipitable materials was performed as described previously (42). Cell proliferation was examined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay as described previously (39).
Immunocytochemical Analysis
Cells cultured on coverslips were washed with PBS, fixed with 4% paraformaldehyde, permeabilized with 0.1% (vol/vol) Triton X-100 in PBS, and then incubated overnight in PBS containing 3% BSA. Cells were then treated with primary antibodies at room temperature for 1 h. After washing with PBS, cells were incubated with a mixture of FITC-labeled rabbit anti-mouse IgG antibody or Cy3-labeled rabbit anti-goat IgG antibody and DAPI (4', 6-diamino-2'-phenylindole dihydrochloride; Boehringer Mannheim, Mannheim, Germany) for 1 h at room temperature. Immunofluorescence images were recorded as described previously (39).
RNA Extraction and Northern Blot Analysis
Total RNA was extracted from cells using TRIzol reagent according to the manufacturer’s instructions. Total RNA (20 to 25 µg) was electrophoresed on a 1.0% agarose gel containing 2.2 mol/L formaldehyde and blotted onto a Hybond-N+ membrane (Amersham Bioscience, NJ) by capillary blotting in 20x SSC. The membrane was prehybridized for 2 h at 42°C in a hybridization buffer comprising 50% (vol/vol) formamide, 5x standard saline phosphate-EDTA, 5x Denhart’s solution, 100 mg/ml denatured salmon sperm DNA, and 0.5% SDS, and then hybridized for 12 h at 42°C with the following probes: human A subunit cDNA (provided by Dr. Eto), human glyceraldehyde-3-phosphate dehydrogenase (Clontech Laboratories, Palo Alto, CA), rat type I collagen, rat matrix metalloproteinase-2 (MMP-2), rat tissue inhibitor of metalloproteinase-1 (TIMP-1), and rat plasminogen activator inhibitor-1 (PAI-1). These probes were labeled with [-32P] dCTP using the Ready-To-Go DNA labeling beads priming kit (Pharmacia, Uppsala, Sweden). The membrane was washed five times in 0.1x SSC-0.1% SDS for 5 min at room temperature and then at 55°C for 30 min. The autoradiograph was taken and analyzed with Fujix BAS 2000 (Fuji Photo Film, Tokyo, Japan) and photographed with Fujix Pictography 3000 (Fuji Photo Film). Probes of rat type I collagen, MMP-2, PAI-1, and TIMP-1 were generated by RT-PCR. RT-PCR was performed as described above using the primers shown in Table 1. PCR products were purified with SUPREC-01 (Takara, Shiga, Japan) and Microcom Centrifugal Filter Devices (Millipore).
Transfection and Establishment of Stable Cell Line
NRK-49F cells were transfected using Lipofectamine PLUS (Invitrogen) with PCXN2 plasmid containing truncated activin type II receptor (tARII) (35), and transfectants were selected by continuous growth in G418 (800 µg/ml geneticin; Invitrogen). After five passages, 10 individual clones were isolated and screened for tARII expression by Western blot analysis. Three clones expressing high levels of the receptor message were identified and used in this study. We observed no significant differences among the three clones in the experiments performed.
Statistical Analyses
Differences between the means was compared by t test with P < 0.05 considered significant.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
A subunit in rat fibrotic kidneys with UUO by RT-PCR. As shown in Figure 1A, the expression of activin A was not detected in normal and contralateral control kidneys. In contrast, the expression level of mRNA for A subunit was significantly increased at 3 d after UUO operation and thereafter. To examine the localization of activin A in the kidneys with UUO, we performed immunohistochemistry using anti-human activin A antibody. As shown in Figure 1B, d and g, progressive fibrosis occurred at 3 d after operation and thereafter in this UUO model. Immunoreactive activin A was not observed in normal (Figure 1B, a) or contralateral kidneys (Figure 1B, b and c). In contrast, consistent with results obtained by RT-PCR (Figure 1A), activin A was detected in the kidneys at 3 d after UUO operation. Activin A was localized in tubular cells in the cortex as well as in the outer medulla (Figure 1B, e and f, respectively) but not in collecting ducts in the inner medulla (data not shown) of the kidneys with UUO. At 7 d after operation, activin A was abundantly detected in tubular cells in the cortex and the outer medulla of the kidneys with UUO (Figure 1B, h and i). The expression of activin A was restricted to tubular cells and was not observed in glomeruli or interstitium of the kidneys with UUO throughout the experiments (data not shown).
|
-SMA and Type I Collagen in Renal Epithelial Tubular Cells-SMA, a phenotypic marker for myofibroblasts, in renal epithelial tubular cell lines LLC-PK1 and MDCK by Western blot analysis. The expression of -SMA was detected in both LLC-PK1 cells and MDCK cells. However, activin A did not affect the expression level of -SMA in these cells (Figure 2A).
|
Expressions of Activin A and Activin Type II Receptor in Renal Fibroblasts
We next examined the effect of activin A on renal fibroblasts. We used two kinds of fibroblasts, NRK-49F cells and primary cultured rat renal interstitial fibroblasts. First, we examined the expression of activin A and activin type II receptor in NRK-49F cells. As shown in Figure 3A, a, A subunit for activin A was detected in NRK-49F cells. Immunoreactive activin A was localized in the cytoplasm of NRK-49F cells (data not shown). Activin type II receptor was also expressed in NRK-49F cells (Figure 3A, b). These results suggest that activin A acts as an autocrine or paracrine factor in NRK-49F cells.
|
Effect of Activin A on the Expression of -SMA in Renal Fibroblasts
We then analyzed the effect of activin A on the expression of -SMA in renal fibroblasts. The expression of -SMA was minimally detected in NRK-49F cells (Figure 4A, a). In contrast, its expression was abundantly detected in the cytoplasmic region of activin-treated NRK-49F cells (Figure 4A, b). In primary cultured renal interstitial fibroblasts, activin A also enhanced the expression of -SMA (Figure 4C). Quantitative analysis showed that activin A significantly increased the number of -SMA–positive cells (Figure 4, B and D).
|
|
-SMA expression by activin A was abrogated in the presence of follistatin (data not shown), confirming the inactivation of activin action by follistatin. We then examined the effect of follistatin on cell proliferation in NRK-49F cells. As shown in Figure 6C, follistatin inhibited cell proliferation in NRK-49F cells in a dose-dependent manner. Proliferation rate of NRK-49F cells treated with follistatin was significantly slower than that of untreated cells (Figure 6D).
|
-SMA expression (Figure 7E) were not observed in NRK-49F–tARII cells, suggesting that overexpression of this mutant receptor completely blocked the activin signaling in a dominant-negative manner.
|
|
1 and Activin A in Renal Fibroblasts1, a potent inducer of renal fibrosis, on the expression of activin A in NRK-49F cells. As shown in Figure 9A, the expression level of activin A was significantly increased after TGF-1 treatment. Western blot analysis confirmed that the production of activin A protein was significantly enhanced by TGF-1 in NRK-49F cells (Figure 9B). We also examined the effect of activin A on the expression of activin A itself. The expression of activin A was significantly increased at 18 h after treatment of activin A (Figure 9C). In addition, the expression of activin A was increased after growth stimulation with FBS (data not shown).
|
1 Action by Blockade of Activin Signaling Pathway1 in renal fibroblasts, we examined the effect of TGF-1 on the expression of type I collagen mRNA in the presence of follistatin. TGF-1 significantly increased the mRNA expression of type I collagen in NRK-49F cells, but its effect was not observed in the presence of follistatin (Figure 10A). We also examined the effect of TGF-1 on the expression of type I collagen in NRK-49F–tARII cells. As shown in Figure 10B, the expression level of type I collagen was not increased by TGF-1 in NRK-49F–tARII cells.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In the present study, we showed that the expression of activin A was upregulated in tubular cells in the kidneys with UUO (Figure 1). Activin A has an inhibitory effect on cell proliferation in LLC-PK1 cells (39). Conversely, blockade of activin signaling by overexpressing activin mutant receptor reduced the expression of an epithelial marker, E-cadherin, and eventually induced an immature phenotype in LLC-PK1 cells (39,50). We observed here that activin A did not affect the expressions of -SMA and type I collagen in both LLC-PK1 cells and MDCK cells (Figure 2). Collectively, it is possible that activin A is one of the essential molecules for regulating cell proliferation and maintaining epithelial cell phenotype but is not involved in TEMT or ECM synthesis in renal epithelial tubular cells.
Differentiation from quiescent fibroblasts into myofibroblasts, which is defined by phenotypic changes such as the expression of -SMA, has been termed fibroblast activation. The presence of interstitial myofibroblasts correlates with the extent of tubulointerstitial scarring and functional outcome in clinical glomerulonephritis (51,52). Therefore, specific inhibition of fibroblast activation may lead to new approaches in the treatment of progressive renal disease. In this UUO model, the localization of activin A was restricted to tubules (Figure 1B). However, renal fibroblasts expressed activin type II receptor (Figure 3), suggesting the possibility that activin A produced by tubular cells plays a role on interstitial fibroblasts in a paracrine manner in the kidneys with UUO. In the present study, we demonstrated that activin A promoted cell proliferation in both NRK-49F cells and primary cultured renal interstitial fibroblasts (Figure 3). Activin A induced the expression of -SMA in these cells (Figure 4). Furthermore, activin A increased the expression level of type I collagen (Figure 5), suggesting that activin A acts as a potent activator of fibroblasts. Two protease cascades, the metalloproteinases and the plasminogen activator/plasmin family of serine proteases, are implicated in the turnover of interstitial matrix proteins (53). Given that activin A did not affect the expression of MMP-2, PAI-I, and TIMP-1 (data not shown), it is possible that activin A is involved in ECM synthesis such as type I collagen but not in matrix degradation in renal fibroblasts. The expression of activin A was detected in tubular cells at 3 d after operation, and its upregulated expression continued for 10 d after operation. Given that activation of interstitial fibroblasts generally occurs at the early phase in the UUO model (54), activin A may maintain interstitial fibroblasts in the activated state rather than initiate fibroblasts activation.
To examine the role of endogenous activin A in fibroblast activation, we used follistatin, an antagonist of activin A. Follistatin significantly inhibited cell growth in NRK-49F cells (Figure 6). This inhibitory effect of follistatin was observed when cells were cultured in complete medium containing 5% FBS but not in a serum-free medium. Given that the expression level of activin A was very low in serum-starved cells but was markedly increased after growth stimulation with FBS (data not shown), these results suggest that follistatin inhibited cell growth by blocking the action of endogenous activin A. This notion was supported by the following result: Proliferation rate of cells expressing truncated type II activin receptor was significantly reduced compared with that of wild-type or vector-transfected cells (Figure 8A). Activin A induced the expression of activin A itself in NRK-49F cells (Figure 9C). Given that activin expression might be accelerated by an autocrine loop, it is likely that blocking the action of activin A brings results larger than expected.
There is a difference in the effective dose of activin A between NRK-49F cells and primary cultured fibroblasts (Figure 3). In primary cultured fibroblasts, significant effect of activin A was observed at a lower concentration than in NRK-49F cells. This may be caused by the difference in the expression level of activin receptors between them. NRK-49F cells produce several growth factors endogenously and can divide several times even in a serum-free medium. Thus, basal proliferation rate in NRK-49F cells is obviously higher than that in primary cultured fibroblasts, which might blunt the effect of activin A on NRK-49F cells as compared with primary cultured fibroblasts.
TGF- is a key mediator of renal fibrosis (8–12), and its expression is known to be upregulated in fibrotic kidneys (13). However, the downstream mechanism of TGF- action in renal fibrosis is not totally elucidated. We demonstrated here that TGF-1 induced the expression of activin A in NRK-49F cells (Figure 9). TGF-1 also enhanced the expression of type I collagen in NRK-49F cells, but its effect was canceled by follistatin or overexpression of tARII (Figure 10). Although the precise mechanism of activin A in the action of TGF- is unknown at present, it is possible that the action of TGF-1 in fibroblast activation is at least partly mediated by activin A. Given that induction of type I collagen expression by TGF-1 (Figure 10) was preceded by the increase of activin production (Figure 9), TGF- may exert its action by activation of the machinery of activin secretion rather than the promotion of transcriptional activity. Further study will be needed to clarify this issue. With regard to the differentiation of fibroblasts into myofibroblast, induction of -SMA expression by TGF-1 was not blocked in NRK-49F–tARII cells (data not shown). Therefore, type I collagen synthesis is likely to be mediated by activin among various actions of TGF- in fibroblast activation.
Basic FGF mediates a part of TGF- actions in renal fibroblasts (17). We showed here that activin A promotes cell proliferation and, furthermore, mediates TGF-–induced collagen synthesis in NRK-49F cells. Given that activin A does not induce the expression of basic FGF in NRK-49F cells (Yamashita et al., unpublished observation), the action of activin A may be independent of basic FGF in fibroblast activation, although it is possible that there is a cross-talk in the intracellular signaling between them. Conversely, BMP-7 is known to be an antifibrogenic factor and ameliorate renal fibrosis (19,20). Recently, BMP-7 was proved to be capable of antagonizing TGF-–induced TEMT in vivo and in vitro (21). Given that BMP-7 exerts its antifibrotic effect via counteracting TGF-–induced TEMT (21), BMP-7 actions in the kidney may be independent of activin. In other words, combination of BMP-7 and follistatin may give rise to a synergistic therapeutic effect for the treatment of renal fibrosis. At all events, the present findings suggest that activin A is a new therapeutic target for the development of an antifibrotic agent. Follistatin may be an attractive candidate.
In summary, we demonstrated that the expression of activin A was upregulated in fibrotic kidneys with UUO. Activin A promoted cell proliferation, induced the expression of -SMA, and enhanced the expression of type I collagen mRNA in renal fibroblasts. Follistatin or overexpression of activin mutant receptor reduced the proliferation rate and suppressed the expression of type I collagen. TGF- induced the expression of activin A. In contrast, blockade of activin signaling reduced the TGF- action such as type I collagen synthesis. These results suggest that activin A is a potent inducer of fibroblast activation in the kidney.
|
Acknowledgments |
|---|
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
or platelet-derived growth factor gene into the rat kidney. J Clin Invest 92: 2597–2601, 1993
1. Nature 346: 371–374, 1990[CrossRef][Medline]
1 mRNA in the obstructed kidney of rats with unilateral ligation. Kidney Int 44: 313–321, 1993[Medline]
, A and B subunits in various tissues predicts diverse functions. Proc Natl Acad Sci U S A 85: 247–251, 1988A and B subunits and follistatin mRNA suggests multiple sites of action of the activin-follistatin system during human development. J Clin Endocrinol Metab 78: 1521–1524, 1994[Abstract]
This article has been cited by other articles:
 |
|
 |
|
  K. Fu, M. J. Corbley, L. Sun, J. E. Friedman, F. Shan, J. L. Papadatos, D. Costa, F. Lutterodt, H. Sweigard, S. Bowes, et al. SM16, an Orally Active TGF-{beta} Type I Receptor Inhibitor Prevents Myofibroblast Induction and Vascular Fibrosis in the Rat Carotid Injury Model Arterioscler. Thromb. Vasc. Biol., April 1, 2008; 28(4): 665 - 671. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. N Artaza, R. Singh, M. G Ferrini, M. Braga, J. Tsao, and N. F Gonzalez-Cadavid Myostatin promotes a fibrotic phenotypic switch in multipotent C3H 10T1/2 cells without affecting their differentiation into myofibroblasts J. Endocrinol., February 1, 2008; 196(2): 235 - 249. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Y Arai and T. Nishiyama Developmental Changes in Extracellular Matrix Messenger RNAs in the Mouse Placenta During the Second Half of Pregnancy: Possible Factors Involved in the Regulation of Placental Extracellular Matrix Expression Biol Reprod, December 1, 2007; 77(6): 923 - 933. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Mukhopadhyay, S. Y. Chan, I. J. Lim, D. J. Phillips, and T. T. Phan The role of the activin system in keloid pathogenesis Am J Physiol Cell Physiol, April 1, 2007; 292(4): C1331 - C1338. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. K. Sicklick, S. S. Choi, M. Bustamante, S. J. McCall, E. H. Perez, J. Huang, Y.-X. Li, M. Rojkind, and A. M. Diehl Evidence for epithelial-mesenchymal transitions in adult liver cells Am J Physiol Gastrointest Liver Physiol, October 1, 2006; 291(4): G575 - G583. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Aoki, M. Kurabayashi, Y. Hasegawa, and I. Kojima Attenuation of Bleomycin-induced Pulmonary Fibrosis by Follistatin Am. J. Respir. Crit. Care Med., September 15, 2005; 172(6): 713 - 720. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Yamashita, A. Maeshima, and Y. Nojima Involvement of Renal Progenitor Tubular Cells in Epithelial-to-Mesenchymal Transition in Fibrotic Rat Kidneys J. Am. Soc. Nephrol., July 1, 2005; 16(7): 2044 - 2051. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Gaedeke, T. Boehler, K. Budde, H.-H. Neumayer, and H. Peters Glomerular activin A overexpression is linked to fibrosis in anti-Thy1 glomerulonephritis Nephrol. Dial. Transplant., February 1, 2005; 20(2): 319 - 328. [Abstract] [Full Text] [PDF]
|
|
|
|
) or without (•) 10 nM follistatin for the indicated periods. Cell proliferation was determined by MTT assay. Results are presented as the means ± SE of three independent experiments. **P < 0.01 versus control.
