IN-1130, a novel transforming growth factor-β type I receptor kinase (ALK5) inhibitor, suppresses renal fibrosis in obstructive nephropathy
2006; Elsevier BV; Volume: 70; Issue: 7 Linguagem: Inglês
10.1038/sj.ki.5001775
ISSN1523-1755
AutoresJ.-A. Moon, Hongduk Kim, I.-S. Cho, Yhun Yhong Sheen, D.-K. Kim,
Tópico(s)Connective Tissue Growth Factor Research
ResumoThe transforming growth factor-β (TGF-β) plays a central role in the progression of renal fibrosis. TGF-β transduces its signal through the activin receptor-like kinase (ALK)5. IN-1130, a novel small molecule ALK5 inhibitor, inhibited the purified kinase domain of ALK5-mediated Smad3 phosphorylation with an IC50 value of 5.3 nM. IN-1130 proved to be highly selective in a panel of 27 serine/threonine and tyrosine kinases including p38α mitogen-activated protein kinase. We evaluated the efficacy of IN-1130 to block renal fibrogenesis induced by unilateral ureteral obstruction (UUO) in rats. Either vehicle (saline) or IN-1130 (10 and 20 mg/kg/day) was intraperitoneally administered to UUO rats for 7 and 14 days. Phosphorylated Smad2 (pSmad2) and markers of fibrosis were analyzed in kidney tissues. In UUO control kidneys, interstitial fibrosis including tubular atrophy, loss and dilation, inflammatory cell infiltration, and fibroblast cell proliferation was prominent. These morphological changes were notably reduced by IN-1130 treatment. IN-1130 decreased levels of TGF-β1 messenger RNA (mRNA), type I collagen mRNA, and pSmad2, compared to UUO control rats. As determined by measuring the hydroxyproline content, total kidney collagen amount was increased in UUO control kidneys, but significantly reduced by IN-1130 treatment, which was comparable to results of histochemical staining for collagen. IN-1130 also suppressed the expression of α-smooth muscle actin (α-SMA) and fibronectin in UUO kidneys. Our results show that IN-1130 suppressed the fibrogenic process of UUO, further underscoring the potential clinical benefits of IN-1130 in the treatment of renal fibrosis. The transforming growth factor-β (TGF-β) plays a central role in the progression of renal fibrosis. TGF-β transduces its signal through the activin receptor-like kinase (ALK)5. IN-1130, a novel small molecule ALK5 inhibitor, inhibited the purified kinase domain of ALK5-mediated Smad3 phosphorylation with an IC50 value of 5.3 nM. IN-1130 proved to be highly selective in a panel of 27 serine/threonine and tyrosine kinases including p38α mitogen-activated protein kinase. We evaluated the efficacy of IN-1130 to block renal fibrogenesis induced by unilateral ureteral obstruction (UUO) in rats. Either vehicle (saline) or IN-1130 (10 and 20 mg/kg/day) was intraperitoneally administered to UUO rats for 7 and 14 days. Phosphorylated Smad2 (pSmad2) and markers of fibrosis were analyzed in kidney tissues. In UUO control kidneys, interstitial fibrosis including tubular atrophy, loss and dilation, inflammatory cell infiltration, and fibroblast cell proliferation was prominent. These morphological changes were notably reduced by IN-1130 treatment. IN-1130 decreased levels of TGF-β1 messenger RNA (mRNA), type I collagen mRNA, and pSmad2, compared to UUO control rats. As determined by measuring the hydroxyproline content, total kidney collagen amount was increased in UUO control kidneys, but significantly reduced by IN-1130 treatment, which was comparable to results of histochemical staining for collagen. IN-1130 also suppressed the expression of α-smooth muscle actin (α-SMA) and fibronectin in UUO kidneys. Our results show that IN-1130 suppressed the fibrogenic process of UUO, further underscoring the potential clinical benefits of IN-1130 in the treatment of renal fibrosis. Transforming growth factor (TGF)-β family has a pivotal role in the regulation of a variety of physiological processes. Three TGF-β isoforms (TGF-β1, TGF-β2, and TGF-β3) are expressed in mammals, and each is encoded by a unique gene and expressed in a tissue-specific manner. TGF-β signals through a transmembrane receptor serine/threonine complex that comprises the type I and type II receptor kinases. Once activated, TGF-β binds to the constitutively active type II receptor, and the type I receptor kinase activin receptor-like kinase (ALK)5 is subsequently recruited into the complex and is activated by TGF-β type II receptor-mediated phosphorylation. Phosphorylation of serine/threonine residues in the ALK5 subsequently phosphorylates the major downstream signaling molecules Smad2 and 3 proteins. Phosphorylated Smad2 (pSmad2) and 3 form a complex with Smad4. This complex translocates into nucleus and regulates the transcription of specific genes involved in cell growth, differentiation, development, and immune response.1.Heldin C.H. Miyazono K. ten Dijke P. TGF-beta signalling from cell membrane to nucleus through SMAD proteins.Nature. 1997; 390: 465-471Crossref PubMed Scopus (3206) Google Scholar, 2.Massague J. TGF-beta signal transduction.Annu Rev Biochem. 1998; 67: 753-791Crossref PubMed Scopus (3848) Google Scholar, 3.Leask A. Abraham D.J. 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Hollinger M.A. Effect of antibody to transforming growth factor beta on bleomycin induced accumulation of lung collagen in mice.Thorax. 1993; 48: 959-966Crossref PubMed Scopus (389) Google Scholar The extensive knowledge regarding TGF-β-mediated ALK5-dependent signaling pathway as an initiating point at the receptor level has highlighted the therapeutic potential of TGF-β signaling antagonist. Recent studies have shown that several small molecule adenosine triphosphate (ATP)-competitive ALK5 inhibitors inhibit or retard progressive fibrosis in kidney, lung, and liver.23.Grygielko E.T. Martin W.M. Tweed C. et al.Inhibition of gene markers of fibrosis with a novel inhibitor of transforming growth factor-beta type I receptor kinase in puromycin-induced nephritis.J Pharmacol Exp Ther. 2005; 313: 943-951Crossref PubMed Scopus (147) Google Scholar, 24.Bonniaud P. Margetts P.J. Kolb M. et al.Progressive transforming growth factor {beta}1-induced lung fibrosis is blocked by an orally active ALK5 kinase inhibitor.Am J Respir Crit Care Med. 2005; 171: 889-898Crossref PubMed Scopus (209) Google Scholar, 25.de Gouville A.C. Boullay V. Krysa G. et al.Inhibition of TGF-beta signaling by an ALK5 inhibitor protects rats from dimethylnitrosamine-induced liver fibrosis.Br J Pharmacol. 2005; 145: 166-177Crossref PubMed Scopus (142) Google Scholar We have synthesized and developed a series of ALK5 inhibitors that possess an imidazole-based molecular scaffold, acting as a competitive inhibitor of the ATP binding site of ALK5. We previously showed that IN-1130, a representative molecule of our compounds, inhibited transcription of reporter genes induced by TGF-β, but did not show significant activity against p38α mitogen-activated protein kinase.26.Kim DK, Bang YJ, Kim HT et al. 2-pyridyl substituted imidazoles as ALK5 and/or ALK4 inhibitors. WO 2005/103028 A1; US 2005/0261299 A1; EP 1620426 A1Google Scholar In addition, IN-1130 effectively prevented hepatic fibrosis in the bile duct ligated rat model (submitted). Progressive renal disease is associated with interstitial fibrosis that is characterized by monocyte infiltration, fibroblast proliferation/differentiation, and extracellular matrix (ECM) protein accumulation. Interstitial fibrosis is a common sequel to renal injury and is the final common pathway in several renal disorders leading to end-stage renal failure. Various inflammatory mediators such as cytokines, nitric oxide, and growth factors influence the progression of renal fibrosis.3.Leask A. Abraham D.J. TGF-beta signaling and the fibrotic response.FASEB J. 2004; 17: 816-827Crossref Scopus (1837) Google Scholar, 4.Wang W. Koka V. Lan H.Y. Transforming growth factor-beta and Smad signalling in kidney diseases.Nephrology (Carlton). 2005; 10: 48-56Crossref PubMed Scopus (299) Google Scholar, 27.Eddy A.A. 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Wrana J.L. Sodek J. Independent regulation of collagenase, 72-kDa progelatinase, and metalloendoproteinase inhibitor expression in human fibroblasts by transforming growth factor-beta.J Biol Chem. 1989; 264: 1860-1869Abstract Full Text PDF PubMed Google Scholar These points have provided the therapeutic potential of antagonizing the TGF-β pathway in renal fibrotic disorders. In this study, we evaluated the efficacy of IN-1130 for the treatment of renal fibrosis in a rat unilateral ureteral obstruction (UUO) model and demonstrated for the first time that IN-1130 is effective in the prevention and treatment of renal fibrosis in a rat UUO model. IN-1130 inhibited ALK5 phosphorylation of Smad3 with an IC50 value of 5.3 nM (Figure 1). The selectivity of IN-1130 for ALK5 versus other kinases was evaluated using a panel of various kinases (Table 1). IN-1130 inhibited ALK5 phosphorylation of casein with an IC50 values of 36 nM and inhibited p38α mitogen-activated protein kinase with an IC50 value of 4.3 μM, demonstrating that IN-1130 is approximately 100-fold more selective for ALK5 than p38α. IN-1130 exhibited IC50 values greater than 5 μM for other kinases.Table 1IC50 values for IN-1130 against various protein kinasesaAll protein kinases were expressed as human recombinant glutathione-S transferase-fusion proteins or His-tagged proteins in Sf9 insect cells by using a baculovirus expression system. Kinases were purified by affinity chromatography using either GSH-agarose or Ni-NTA-agarose. ProQinase GmBH (Freiburg, Germany) performed radioisotopic protein kinase assays for measuring kinase activities for all enzymes except for p38α. In vitro kinase inhibition assay of IN-1130 against p38α was performed in MDS Pharma services. IC50 values were measured by testing 12 concentrations of IN-1130 in each kinase assay. The IC50 values for IN-1130 against ALK5 were determined from an experiment run in triplicate.Protein kinaseIC50 (μM)Protein kinaseIC50 (μM)ALK50.036bCasein was used as a substrate. The IC50 values for IN-1130 were determined from an experiment run in triplicate.JNK330ABL126MAPKAPK5>100ACV-R18.5MST4>100ARK5>100NEK2>100Aurora-A>100NLK29B-Raf12p38α4.3CK2a1>100PAK1>100COT>100PDGFR-a7.8CSK32PIM1>100DAPK1>100PRK1>100FGF-R113S6K>100FLT3>100SGK1>100IGF1-R>100SRC>100IRAK4>100ZAP70>100a All protein kinases were expressed as human recombinant glutathione-S transferase-fusion proteins or His-tagged proteins in Sf9 insect cells by using a baculovirus expression system. Kinases were purified by affinity chromatography using either GSH-agarose or Ni-NTA-agarose. ProQinase GmBH (Freiburg, Germany) performed radioisotopic protein kinase assays for measuring kinase activities for all enzymes except for p38α. In vitro kinase inhibition assay of IN-1130 against p38α was performed in MDS Pharma services. IC50 values were measured by testing 12 concentrations of IN-1130 in each kinase assay. The IC50 values for IN-1130 against ALK5 were determined from an experiment run in triplicate.b Casein was used as a substrate. The IC50 values for IN-1130 were determined from an experiment run in triplicate. Open table in a new tab Histological examination showed that, in UUO control kidneys, architecture was drastically altered with acute and chronic tubular damages including tubular atrophy, loss and dilation, infiltration of inflammatory cells, and development of interstitial fibrosis (Figure 2). Most prominent damage was observed in collecting ducts and loops of Henle in the medulla area, which is characterized by expansion of interstitial space with the accumulation of fibroblasts and inflammatory cells at day 7. Tubulointerstitial damage was more prominent in both cortex and medulla at day 14. These changes were notably reduced or absent in UUO animals treated with IN-1130 (Figure 2). A drastic increase in TGF-β is a key feature of UUO kidney. Level of TGF-β1 messenger RNA (mRNA) in UUO control kidneys was increased 4.4- and 4.1-fold at days 7 and 14 after ureteral ligation, respectively, compared to sham-operated animals (Figure 3; P<0.01). IN-1130 dose-dependently decreased levels of TGF-β1 mRNA. We determined that increase in TGF-β in UUO kidneys is accompanied by the increase in pSmad2. Western blotting analysis showed that level of pSmad2 in UUO control kidneys at day 14 was increased 40-fold compared to sham-operated kidneys. IN-1130 significantly decreased levels of pSmad2 in a dose-dependent manner (Figure 4). Levels of pSmad2 in sham-operated kindeys and UUO kidneys treated with IN-1130 were too low to quantitatively measure at day 7 (data not shown). Next, we performed pSmad2 immunohistochemistry on kidney sections. pSmad2 immunoreactivity was weak or absent in sham-operated kidneys. Strong pSmad2 immunoreactivity was present in the nuclei of tubular and interstitial cells in UUO kidneys, whereas weak staining was observed in UUO kidneys treated with IN-1130 (Figure 5).Figure 5Representative photomicrographs of pSmad2 immunohistochemistry on kidney sections from sham-operated rats, UUO control rats, and UUO rats treated with IN-1130. (a and d) In sham-operated animals, no significant nuclear pSmad2 staining was observed at (a) day 7 and (d) day 14. (b and e) Nuclear localization of pSmad2 in tubular and interstitial cells was increased in UUO control kidneys at (b) day 7 and (e) day 14 (brown, arrowheads) compared to sham-operated kidneys. (c and f) IN-1130 (20 mg/kg) treatment reduced nuclear staining intensity (brown, arrowheads) in interstitial and tubular areas of kidneys at (c) day 7 and (f) day 14, which are associated with the decrease in the extent of interstitial nephritis and fibrosis compared to UUO control kidneys. Bar=80 μm.View Large Image Figure ViewerDownload (PPT) The appearance of α-smooth muscle actin (α-SMA)-positive myofibroblasts is considered a key event in the progression of chronic renal diseases. Western blotting analysis for α-SMA demonstrated that the level of α-SMA in UUO kidneys increased approximately 35- and 90-fold at days 7 and 14, respectively, compared to sham-operated animals (Figure 6; P<0.01). IN-1130 decreased α-SMA expression in a dose-dependent manner. Immunohistochemical staining for α-SMA was performed to localize the expression of α-SMA on kidney sections (Figure 7). In UUO control kidneys at day 7, elongated myofibroblasts stained positively for α-SMA were observed in interstitial areas, and some α-SMA-positive tubular epithelial cells were present. The abundance and location of α-SMA expression in UUO kidneys treated with IN-1130 at a dose of 20 mg/kg/day was similar to that of sham-operated animals. In kidneys with UUO for 14 days, strong α-SMA immunoreactivity was extensively present in tubular and interstitial cells, but reduced by IN-1130 treatment. Levels of type I collagen mRNA in UUO control kidneys at days 7 and 14 were increased 3.5- and 9.7-fold, respectively, compared to sham-operated animals (Figure 8; P<0.01). IN-1130 treatment dose dependently decreased levels of type I collagen mRNA both at days 7 and 14. Total kidney collagen content was determined by measuring the amount of hydroxyproline, a quantitative biochemical assay. As shown in Figure 9, hydroxyproline contents in the UUO control kidneys increased to 151 and 245% at days 7 and 14, respectively, compared to that (12.8±0.7 μg/mg protein) of normal rats at the starting point (day 0). IN-1130 significantly reduced hydroxyproline contents in UUO kidneys. We further examined the expression of collagen on Masson's trichrome stained kidney sections. The collagen accumulation were prominent in the interstitial space of UUO control kidneys, compared to sham-operated animals (Figure 10). IN-1130 markedly reduced the collagen accumulation in UUO control kidneys (Figure 10).Figure 10Representative photomicrographs of Masson's trichrome staining on kidney sections from sham-operated rats, UUO control rats, and UUO rats treated with IN-1130. (a, d, and g) In sham-operated animals, collagen expression is limited to blood vessels (blue, arrows). (blue in b and e) Collagen expression is prominent in interstitial and tubular areas of UUO control kidneys at day 7 after UUO (b and e). (blue in h) Collagen expression is present in a widespread pattern at day 14 after UUO. Once-daily treatment with IN-1130 (20 mg/kg) for 7 (c and f) and 14 days (i) reduced staining intensity in interstitial and tubular areas of kidneys, which is associated with the decrease in the extent of interstitial nephritis and fibrosis. Collagen is present only around blood vessels (arrows in c) in UUO kidneys once daily treated with IN-1130 (20 mg/kg) for 7 days. Bar=30 μm.View Large Image Figure ViewerDownload (PPT) We also investigated the effect of IN-1130 on the expression of fibronectin, a major interstitial matrix component, in UUO kidneys (Figure 11). Compared to sham-operated animals, UUO kidneys exhibited 17- and 53-fold increases in the expression of fibronectin at days 7 and 14, respectively. IN-1130 suppressed the levels of fibronectin in UUO kidneys in a dose-dependent manner. We previously identified IN-1130 as a potent small molecule inhibitor of TGF-β signaling pathway in cell-based assays. The present study clearly shows that IN-1130 effectively inhibits the in vitro ALK5 activity without affecting other serine/threonine and tyrosine kinases examined. Recently, several ALK5 inhibitors have been developed as a potential therapy in fibrosis diseases. Among them, SB-525334 is a small molecule ALK5 inhibitor and has a similar molecular scaffold with IN-1130.23.Grygielko E.T. Martin W.M. Tweed C. et al.Inhibition of gene markers of fibrosis with a novel inhibitor of transforming growth factor-beta type I receptor kinase in puromycin-induced nephritis.J Pharmacol Exp Ther. 2005; 313: 943-951Crossref PubMed Scopus (147) Google Scholar We compared the potency of IN-1130 with SB-525334 and found out that IN-1130 is more potent than SB-525334 in both enzyme inhibition and in vitro cell-based assays (data not shown). The obstructed kidneys at 7 days after ureteral ligation in the present study showed typical features of obstructive nephropathy such as the presence of myofibroblasts and inflammatory cells in the interstitium, tubular degeneration and atrophy, and interstitial fibrosis. Tubulointerstitial damages progressed in UUO kidneys at day 14. These morphological changes are accompanied by increases in the amounts of α-SMA, hydroxyproline, collagen, and fibronectin that are specific biochemical markers for fibrosis. Our study shows that IN-1130 almost completely suppressed the expression of α-SMA, collagen, and fibronectin in UUO kidneys at an early stage (day 7), which is accompanied by the attenuation of tubulointerstitial fibrosis. Although IN-1130 did not confer complete protection from renal fibrosis in UUO kidneys at a later stage (day 14), IN-1130 significantly inhibited fibrogensis in UUO kidneys. TGF-β is believed to be the prime fibrogenic molecule in UUO model. A drastic increase in TGF-β1 was observed in UUO kidneys. Interestingly, IN-1130 treatment decreased the amount of TGF-β1 mRNA in UUO kidneys being accompanied by the attenuation of all fibrogenic features examined. Decreased TGF-β1 mRNA expression by IN-1130 may be the indirect consequence of reduced fibrogenic cellular components including fibroblasts and myofibroblasts that produce TGF-β. Also, this reduction of TGF-β1 mRNA by IN-1130 may contribute to less numbers of inflammatory cells such as macrophages and fibroblasts in the interstitium as TGF-β is a potent chemoattractant for those cell types.32.Eddy A.A. Progression in chronic kidney disease.Adv Chronic Kidney Dis. 2005; 12: 353-365Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar Indeed, we found that IN-1130 decreased the number of infiltrating macrophages in UUO kidneys which were identified by immuohistochemistry using a monoclonal ED1 antibody (data not shown). In the present study, we did not identify the cellular source of TGF-β in UUO kidneys, but several reports have indicated that the major cell component that produces TGF-β in UUO model is interstitial cells. However, a contribution of tubular cells to the increased TGF-β levels cannot be excluded as tubular cells are known to upregulate TGF-β.33.Sutaria P.M. Ohebshalom M. McCaffrey T.A. et al.Transforming growth factor-beta receptor types I and II are expressed in renal tubules and are increased after chronic unilateral ureteral obstruction.Life Sci. 1998; 62: 1965-1972Crossref PubMed Scopus (39) Google Scholar,34.Kaneto H. Morrissey J. Klahr S. Increased expression of TGF-beta 1 mRNA in the obstructed kidney of rats with unilateral ureteral ligation.Kidney Int. 1993; 44: 313-321Abstract Full Text PDF PubMed Scopus (298) Google Scholar In the present study, levels of TGF-β1 mRNA in UUO control kidneys were similar between at days 7 and 14 after unilateral ligation, which is consistent with the previous reports in which, unlike other fibrogenic markers, level of TGF-β was not prominently elevated from early to late stages.35.Fukasawa H. Yamamoto T. Togawa A. et al.Down-regulation of Smad7 expression by ubiquitin-dependent degradation contributes to renal fibrosis in obstructive nephropathy in mice.Proc Natl Acad Sci USA. 2004; 101: 8687-8692Crossref PubMed Scopus (169) Google Scholar,36.Sommer M. Eismann U. Deuther-Conrad W. et al.Time course of cytokine mRNA expression in kidneys of rats with unilateral ureteral obstruction.Nephron. 2000; 84: 49-57Crossref PubMed Scopus (17) Google Scholar This discrepancy may be explained by the observation that, in the late stage (day 14), more than 60–70% of renal tubular parenchyma which may produce TGF-β was replaced by fibrotic components. IN-1130 suppressed phosphorylation of Smad2, a marker of TGF-β/ALK5 signaling activation. This reduction of pSmad2 is well correlated with the reductions seen for other fibrogenic markers, indicating that pSmad2 is a major downstream signaling molecule in the fibrogenesis of UUO kidneys. Taken together, our study demonstrates that the inhibition of ALK5 signaling pathway by IN-1130 could interfere with the autocrine/paracrine effect of TGF-β in fibrogenic and inflammatory cells, leading to the blocking of the vicious fibrosis cycle in UUO model. It should be mentioned here that this reduction of the number of fibrogenic cells is not related to the direct effect of IN-1130 on the cell cycle-related events because IN-1130 did not show an antiproliferative effect on several cell lines in vitro at the concentration of up to 100 μM (data not shown). Myofibroblasts are not present in normal kidney. The appearance of myofibroblast cells expressing α-SMA protein is one of the early changes leading to fibrosis in the UUO kidney. α-SMA is considered specific for epithelial myofibroblast transdifferentiation. Elongated myofibroblasts expressing α-SMA were found in moderate numbers in fibrotic interstitium of UUO kidneys, and some tubular epithelial cells were stained with α-SMA. These findings indicate that there is a continuous transition between α-SMA-positive epithelial cells and positively staining cells with myofibroblast morphology. This event is probably induced by TGF-β that is increased during the pathogenesis of interstitial nephritis and fibrosis. Myofibroblasts have been known to produce interstitial ECM components such as type I collagen and fibronectin.37.Liu Y. Epithelial to mesenchymal transition in renal fibrogenesis: pathologic significance, molecular mechanism, and therapeutic intervention.J Am Soc Nephrol. 2004; 15: 1-12Crossref PubMed Scopus (915) Google Scholar As shown in Figures 7 and 10, in UUO control kidneys, type I collagen was colocalized on the α-SMA-positive cells in the interstitium. IN-1130 treatment significantly reduced the expression of α-SMA-positive cells, type I collagen, and fibronectin. This finding suggests that blocking of TGF-β signaling pathway is sufficient to prevent the generation of ECM components. A similar result was previously obtained when IN-1130 was used for the treatment of hepatic fibrosis induced by bile duct ligation in rats in which IN-1130 successfully decreased levels of α-SMA protein and type I collagen (submitted). Our study clearly demonstrates that the inhibition of fibrogenesis in UUO kidneys by interfering TGF-β signaling is sufficient to prevent the fibrogenesis, which is consistent with the previous reports in which another TGF-β signaling antagonists including anti-TGF-β antibody could prevent the fibrogenic process in UUO kidneys.15.Giri S.N. Hyde D.M. Braun R.K. et al.Antifibrotic effect of decorin in a bleomycin hamster model of lung
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