- Email: [email protected]
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ALK5 inhibition in renal disease Nicholas J Laping Recent advances have identified novel small molecule inhibitors of the transforming growth factor-b (TGF-b) type I receptor kinase as a potential therapy in organ remodeling diseases, such as chronic renal disease. Because TGF-b is central to the progression of fibrosis, selective inhibition of this signaling pathway could provide a novel treatment in many fibrotic diseases. The rationale for inhibition of TGF-b signaling in renal disease includes prevention of fibrosis, tubular dedifferentiation and vascular effects.
against TGF-b1 can prevent the accumulation of extracellular matrix in nephritic rats and diabetic rodents [8,9]. This review addresses, firstly, recent advances in the understanding of the TGF-b signaling pathways; secondly, its role in apoptosis, epithelial–mesenchymal transformation (EMT), fibrosis and glomerular vascular effects in renal disease; and thirdly, recent advances in the development of inhibitors of TGF-b type I receptor (ALK5) for the treatment of renal disease.
Addresses GlaxoSmithKline Pharmaceuticals, 709 Swedeland Road, PO 1539 King of Prussia, PA 19406, USA e-mail: [email protected]
Signaling pathways
Current Opinion in Pharmacology 2003, 3:204–208 This review comes from a themed issue on Cardiovascular and renal Edited by Anthony P Davenport and Colin H Macphee 1471-4892/03/$ – see front matter ß 2003 Elsevier Science Ltd. All rights reserved. DOI 10.1016/S1471-4892(03)00002-X
Abbreviations ALK5 TGF-b type I receptor CTGF connective tissue growth factor EMT epithelial–mesenchymal transformation TGF-b transforming growth factor-b
Introduction The prototypic member of the transforming growth factorb (TGF-b) superfamily was discovered over 20 years ago in normal rat kidney fibroblasts [1]. Since then, the family has grown to include many factors that are structurally and functionally related to TGF-b, which plays critical roles during development, including regulation of phenotype, cell division and deposition of extracellular matrix. The inappropriate expression and activity of TGF-b can be linked to a variety of chronic organ remodeling diseases, such as renal fibrosis. Renal fibrosis disturbs the complex cytoarchitecture of the kidney, with a concomitant loss in filtration function. A causal relationship between TGF-b1 and renal fibrosis has been established. Renal cells can be induced to both produce extracellular matrix protein and inhibit protease activity by exogenous TGF-b1 in vitro [2–4]. Similarly, rapid development of glomerulosclerosis and a concomitant elevation of matrix markers was seen in both TGF-b1 transgenic mice and normal rats transfected in vivo with the TGF-b1 gene [5–7]. Finally, neutralizing antibodies Current Opinion in Pharmacology 2003, 3:204–208
Several studies have shown that TGF-b signaling requires both ALK5 and the TGF-b type II receptor (reviewed in [10]); however, the regulation of proliferation by TGF-b might not require ALK5 [11,12]. A novel type I receptor, ALK8, which was shown to bind TGF-b, was found in zebra fish [13]. A mammalian orthologue is expressed in murine mesangial cells, as determined by ALK8 antibodies and northern blot techniques using the zebra fish ALK8 probe [13]. Unlike other ALKs, ALK8 appears to be preferentially expressed in the heart, kidney and liver, and might play a unique role in cardiovascular and renal function. Another type I receptor relevant to the cardiovascular system is ALK1, which can mediate the actions of TGF-b specifically in vascular endothelial cells [14,15]. Thus, whereas TGF-b signaling is primarily mediated by ALK5, ALK1 and ALK8 could be responsible for specific cellular responses to TGF-b. The cytoplasmic mediators of TGF-b receptor activation are the pathway-selective Smad1, Smad2, Smad3 and Smad5, which bind to the common Smad4 [10,16]. ALK1 phosphorylates Smads 1 and 5, whereas ALK5 phosphorylates Smads 2 and 3. Using a novel ALK5 kinase inhibitor (SB-431542), recent studies have further delineated that nuclear accumulation of Smad2 and Smad3 is dependent on ALK5 kinase activity, and that the maintained nuclear presence of these Smads is entirely dependent on continued ALK5 activity [17–19]. The ALK5 activity, and subsequent nuclear presence of Smads 2 and 3, is responsible for activation of genes that participate in the deposition of extracellular matrix proteins in tubular dedifferentiation and vascular endothelial cell changes that can lead to progressive renal disease.
Renal fibrosis A central hypothesis of TGF-b-induced renal disease is that TGF-b activation causes accumulation of extracellular matrix components, leading to fibrosis. This was most directly demonstrated in transgenic mice and transfected rats overexpressing TGF-b [5–7]. The literature is replete with reports and reviews extolling the influence www.current-opinion.com
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that TGF-b has over extracellular matrix production. Thus, TGF-b continues to be associated with renal fibrosis [20,21] and the regulation of expression of many components of the extracellular matrix [22]. Now, the pathways through which TGF-b regulates these genes are being elaborated [19,23]. Connective tissue growth factor (CTGF) is an important factor that mediates some of the effects of TGF-b, which include hypertrophy of glomerular mesangial cells [24] and production of extracellular matrix [25]. The direct mechanism of TGF-b regulation was explored in mesangial cells by examining the promoter, mRNA and protein for CTGF. TGF-b-induced CTGF transcription in rat mesangial cells was inhibited by mutation of the Smad response-element in the CTGF promoter, dominant– negative Ras transfection and MEK2 inhibition with U0-126 [26]. This illustrates that TGF-b regulation of extracellular matrix components utilizes the direct ALK5–Smad pathway, as well as additional kinase pathways and growth factors. While several recent reports continue to support the concept that Jun kinase (JNK) and extracellular signalregulated kinase (ERK) pathways are activated in parallel to the Smad signaling pathway [26,27], new inhibitors of ALK5 and p38 have been used to identify which extracellular matrix components use the p38 pathway in addition to Smad [18]. In renal epithelial cells, TGF-b increased the mRNA for collagen Ia1, which was inhibited by a selective ALK5 inhibitor but not by a selective p38 inhibitor. Conversely, fibronectin mRNA was also increased by TGF-b but was inhibited by both the ALK5 and p38 inhibitors [19]. Consistent with this finding, TGF-b can increase fibronectin in fibroblasts derived from Smad3-deficient mice [28]. The prolonged exposure of TGF-b needed to produce changes in fibronectin RNA, and the requirement of p38 activity, suggest an indirect mechanism for the regulation of this gene. However, it has been shown recently that overexpression of Smad3 can increase fibronectin mRNA [29], presumably through Smad response-elements in the fibronectin promoter. This suggests that fibronectin may respond to both Smad and p38 activation. Thus, certain components of the extracellular matrix are primarily regulated through the ALK5–Smad pathway, whereas others utilize additional kinases, including p38, although it is likely that this is cell- and tissue-specific.
Renal tubular dedifferentiation Tubular atrophy is a feature of most renal diseases and has been previously reviewed in detail [30]. Recent advances have been described in the transgenic Ren-2 rat, which displays tubular epithelial apoptosis when made diabetic with streptozotocin. This degeneration of renal tubules was associated with increased TGF-b mRNA levels [31]. Further indirect evidence for a role for TGF-b in interwww.current-opinion.com
stitial apoptosis of tubular epithelial cells was seen in decorin knockout mice [32]. Decorin is a natural binding protein that can sequester and limit the actions of TGF-b [33]. Thus, its absence in the knockout mice resulted in more severe renal damage following ureteral obstruction [32]. Recent evidence supporting a direct role for TGF-b in renal apoptosis includes the release of TGF-b in glomerular visceral epithelial cells treated with either TGF-b or angiotensin II. These factors induce apoptosis, and Fas and Bax expression, in glomerular visceral epithelial cells, which can be prevented by antibodies to TGF-b [34]. Thus, angiotensin II acting through TGF-b can contribute to glomerulosclerosis by increasing the rate of apoptosis. The dedifferentiation of the tubular epithelium comprises not only apoptosis, but also transformation of the epithelial cells to a mesenchymal phenotype such as fibroblasts. A role for TGF-b in EMT has been elaborated in many systems, and markers of EMT correlate with the severity of renal disease in patients [35]. In proteinuric patients, abnormal uptake of ultra-filtered proteins by proximal tubular cells occurs, and is associated with increased presence of TGF-b1 mRNA in the tubular epithelium [36]. In response to TGF-b, CTGF might also participate in the induction of EMT [25]. In human proximal tubule epithelial cells, CTGF increases the expression of tenascin-c, a marker of EMT [25]. However, CTGF was not as potent as TGF-b, suggesting that CTGF is a secondary pathway for TGF-b in EMT. The signaling mechanism by which TGF-b effects EMT involves the stress-activated kinase p38. A kinase inhibitor of p38 (SB-202190), and dominant–negative expression of the MAP kinase-3 that normally activates p38, both prevented TGF-b-induced EMT [37,38]. However, a p38independent mechanism also exists that involves the expression of collagen I. TGF-b increased collagen Ia1 mRNA in renal epithelial cells independent of p38 [19]. Whereas collagen IV suppresses EMT, collagen I promotes it [39]. Thus, TGF-b can induce EMT directly through p38, as well as indirectly through managing increased levels of collagen components that favour EMT.
Vascular mechanisms Several studies suggest an association between cardiovascular dysfunction, renal disease and TGF-b. These studies have shown actions of TGF-b on vascular endothelial cells, activation of angiogenic factors in glomerular podocytes [40] and, most recently, an effect of TGF-b on the myogenic mechanism that regulates glomerular filtration rate through changes in afferent arteriolar resistance [41]. The mechanism by which TGF-b regulates vascular endothelial cell function includes an interaction between ALK5 and ALK1 [15]. Transfection studies have demonstrated that endothelial cell proliferation is inhibited by Current Opinion in Pharmacology 2003, 3:204–208
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the TGF-b/ALK5 pathway, whereas migration and proliferation is induced by the TGF-b/ALK1 pathway [15]. Thus, endothelial cells can determine their angiogenic response to TGF-b by the relative levels of ALK5 versus ALK1 expression. In association with its modulation of angiogenesis, TGF-b has been shown to induce the expression of the angiogenic vascular-endothelial-growth-factor in glomerular podocytes [40]. Whether this mechanism contributes to renal disease or is a protective mechanism remains to be determined. However, because high glucose also increases the expression of collagen IV and vascular-endothelial-growthfactor in podocytes, it is possible that this mechanism contributes to the glomerular expansion seen in diabetic nephropathy [40]. A novel vascular mechanism by which TGF-b might contribute to renal disease has been identified using the blood-perfused juxtamedullary nephron technique. As described in a recent abstract [41], TGF-b completely blocks the autoregulatory vasoconstriction of the afferent arteriole to increased perfusion pressure. These data suggest that, in addition to the profibrotic mechanism, TGF-b may increase intra-glomerular pressure leading to further glomerular damage. The functional consequence of reduced glomerular function is invariably associated with hypertension. Thus, protecting the kidney from TGF-b-induced fibrosis and impaired regulation of renal vasculature should also slow the development of hypertension. This was demon-
strated nicely in Dahl salt-sensitive rats. Renal deposition of extracellular matrix was dramatically reduced with preservation of renal histology and consequently protection from hypertension by treating the Dahl salt-sensitive rats with neutralizing antibodies to TGF-b [42]. These data clearly demonstrate that there continues to be a strong rationale for inhibition of the TGF-b signaling pathway to treat renal disease.
Pharmacological intervention Given the strength of the rationale for inhibiting TGF-b to treat renal disease, there continues to be many attempts to interrupt TGF-b signaling in models of renal disease. Indirect inhibition of TGF-b signaling has been accomplished with angiotensin II receptor inhibitors, resulting in lowering of urinary TGF-b in patients, and decreased renal matrix markers in rodents [43,44]. Similarly, inhibition of the formation of glycated proteins, which also activate TGF-b, can also reduce matrix markers in diabetic kidneys [45]. Consequently, TGF-b antibody treatment continues to prove to be effective in reducing kidney weight, glomerular hypertrophy and renal extracellular matrix mRNA, as exemplified in the diabetic mouse [46]. Whether antibodies can be delivered chronically in patients for the treatment of renal disease remains to be established. Because ALK5 mediates most actions of TGF-b, including the pro-fibrotic actions, this appears to be an ideal target for pharmacological intervention (Figure 1). Recently, compounds capable of inhibiting the kinase activity of ALK5 have been described. These pyridinylimidazoles interact with the ATP-binding site of ALK5
Figure 1
Diabetes
↑ Glomerular permeability
Elevated glucose
Albuminuria Urinary collagen, TGFβ1
AGEs
↑ TGF-β1 glomerular
ALK5 TGFβ type I receptor
↑ TGF-β1 interstitial
Extracellular matrix
↑ Renal fibronectin, collagen, PAI-1
Glomerulosclerosis
Interstitial fibrosis
Impaired renal function Current Opinion in Pharmacology
Diagram showing the mechanisms of diabetic nephropathy, including the contribution of TGF-b to renal disease. AGEs, advanced glycation endproducts, PAI-1; plasminogen activator inhibitor-1. Current Opinion in Pharmacology 2003, 3:204–208
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ALK5 inhibition in renal disease Laping 207
[47]. Because certain similarities exist in the ATP-binding pocket between ALK5 and the serine/threonine kinase p38, it was shown that the p38 inhibitor SB203580 inhibited in vitro ALK5 phosphorylation of Smad3 with an IC50 value between 0.7 mM and 3 mM [19,48,49]. However, a more selective ALK5 inhibitor, SB-431542, was developed to inhibit ALK5 kinase activity and TGFb-induced extracellular matrix, with IC50 values under 100 nM [19,47]. Although this inhibitor is useful in preventing many actions of TGF-b in vitro [17–19], compounds with adequate in vivo half-life need to be developed to evaluate the utility of this approach in treating progressive renal disease. At a recent meeting, an example of an ALK5 inhibitor with in vivo activity, SB525334, was presented [50]. This inhibitor not only blocked TGF-b-induced signaling and extracellular matrix mRNA in cultured renal cells, but also inhibited renal expression of collagen Ia1 mRNA and fibronectin protein after renal injury caused by puromycin aminonucleoside [50]. In addition, the ALK5 inhibitor also reduced the injury-induced proteinuria, which is consistent with the vascular effects of TGF-b on glomerular function described above. Therefore, the application of ALK5 inhibitors may also be useful in reducing renal fibrosis in chronic renal disease such as diabetic nephropathy.
Conclusions It is understood that TGF-b plays a central role in the progression of renal disease, as concluded in many reviews. With the advent of specific inhibitors of the TGF-b signaling pathway, it is becoming clear that ALK5 mediates many of the actions of TGF-b, including extracellular matrix production and proliferation. However, the contribution of ALK5 to tubular dedifferentiation and intra-glomerular pressure regulation still needs further investigation. Therefore, it appears that ALK5 is well positioned as a target for intervention of TGF-bmediated diseases, and is supported by preliminary studies using an ALK5 inhibitor in models of renal disease. We must bear in mind, however, that TGF-b is a pleiotropic factor with actions that also modulate cell growth, angiogenesis and immune functions with both a pro- and anti-inflammatory nature, which are likely to involve other type I receptors. The challenge in using therapeutics that block ALK5 activity will lie not only in designing specific kinase inhibitors, but also in striking a balance between disease-relevant fibrotic actions and the immunemodulatory actions of TGF-b.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1.
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36. Abbate M, Zoja C, Rottoli D, Corna D, Tomasoni S, Remuzzi G: Proximal tubular cells promote fibrogenesis by TGF-beta 1-mediated induction of peritubular myofibroblasts. Kidney Int 2002, 61:2066-2077. 37. Bakin AV, Rinehart C, Tomlinson AK, Arteaga CL: p38 mitogenactivated protein kinase is required for TGF beta-mediated fibroblastic transdifferentiation and cell migration. J Cell Sci 2002, 115:3193-3206. 38. Yu L, Hebert MC, Zhang YE: TGF-beta receptor-activated p38 MAP kinase mediates smad-independent TGF-beta responses. EMBO J 2002, 21:3749-3759. Using a mutant of ALK5 that is unable to activate Smads but still retains kinase activity, the authors demonstrate that TGF-b can activate p38 and inhibit proliferation of NmuMG cells. However, EMT is prevented with this mutant. 39. Zeisberg M, Bonner G, Maeshima Y, Colorado P, Muller GA, Strutz F, Kalluri R: Renal fibrosis — collagen composition and assembly regulates epithelial–mesenchymal transdifferentiation. Am J Pathol 2001, 159:1313-1321. 40. Iglesias-de la Cruz MC, Ziyadeh FN, Isono M, Kouahou M, Han DC, Kalluri R, Mundel P, Chen S: Effects of high glucose and TGF-beta 1 on the expression of collagen IV and vascular endothelial growth factor in mouse podocytes. Kidney Int 2002, 62:901-913. 41. Sharma K, Cook AK, Insho EW: TGF-beta inhibits pressureinduced afferent arteriolar autoregulatory behavior [abstract]. J Am Soc Nephrol Abstracts 2002, 13:FC094. 42. Dahly AJ, Hoagland KM, Flasch AK, Jha S, Ledbetter SR, Roman RJ: Antihypertensive effects of chronic anti-TGF-beta antibody therapy in Dahl S rats. Am J Physiol Regul Integr Comp Physiol 2002, 283:R757-R767. This is the first demonstration that TGF-b inhibition can lower blood pressure, albeit secondarily through preservation of renal function. 43. Agarwal R, Siva S, Dunn SR, Sharma K: Add-on angiotensin II receptor blockade lowers urinary transforming growth factorbeta levels. Am J Kidney Dis 2002, 39:486-492.
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44. Abrahamsen CT, Barone FC, Campbell WG, Nelson AH, Contino LC, Pullen MA, Grygielko ET, Edwards RM, Laping NJ, Brooks DP: The angiotensin type 1 receptor antagonist, eprosartan, attenuates the progression of renal disease in spontaneously hypertensive stroke-prone rats with accelerated hypertension. J Pharmacol Exp Ther 2002, 301:21-28.
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47. Callahan JF, Burgess JL, Fornwald JA, Gaster LM, Harling JD, Harrington FP, Heer J, Kwon C, Lehr R, Mathur A et al.: Identification of novel inhibitors of the transforming growth factor-beta type I receptor (ALK5). J Med Chem 2002, 45:999-1001. First disclosure of an inhibitor (with a low nanomolar IC50) of ALK5 kinase activity. 48. Eyers PA, Craxton M, Morrice N, Cohen P, Goedert M: Conversion of SB 203580-insensitive MAP kinase family members to drugsensitive forms by a single amino-acid substitution. Chem Biol 1998, 5:321-328.
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ALK5 inhibition in renal disease Nicholas J Laping Recent advances have identified novel small molecule inhibitors of the transforming growth factor-b (TGF-b) type I receptor kinase as a potential therapy in organ remodeling diseases, such as chronic renal disease. Because TGF-b is central to the progression of fibrosis, selective inhibition of this signaling pathway could provide a novel treatment in many fibrotic diseases. The rationale for inhibition of TGF-b signaling in renal disease includes prevention of fibrosis, tubular dedifferentiation and vascular effects.
against TGF-b1 can prevent the accumulation of extracellular matrix in nephritic rats and diabetic rodents [8,9]. This review addresses, firstly, recent advances in the understanding of the TGF-b signaling pathways; secondly, its role in apoptosis, epithelial–mesenchymal transformation (EMT), fibrosis and glomerular vascular effects in renal disease; and thirdly, recent advances in the development of inhibitors of TGF-b type I receptor (ALK5) for the treatment of renal disease.
Addresses GlaxoSmithKline Pharmaceuticals, 709 Swedeland Road, PO 1539 King of Prussia, PA 19406, USA e-mail: [email protected]
Signaling pathways
Current Opinion in Pharmacology 2003, 3:204–208 This review comes from a themed issue on Cardiovascular and renal Edited by Anthony P Davenport and Colin H Macphee 1471-4892/03/$ – see front matter ß 2003 Elsevier Science Ltd. All rights reserved. DOI 10.1016/S1471-4892(03)00002-X
Abbreviations ALK5 TGF-b type I receptor CTGF connective tissue growth factor EMT epithelial–mesenchymal transformation TGF-b transforming growth factor-b
Introduction The prototypic member of the transforming growth factorb (TGF-b) superfamily was discovered over 20 years ago in normal rat kidney fibroblasts [1]. Since then, the family has grown to include many factors that are structurally and functionally related to TGF-b, which plays critical roles during development, including regulation of phenotype, cell division and deposition of extracellular matrix. The inappropriate expression and activity of TGF-b can be linked to a variety of chronic organ remodeling diseases, such as renal fibrosis. Renal fibrosis disturbs the complex cytoarchitecture of the kidney, with a concomitant loss in filtration function. A causal relationship between TGF-b1 and renal fibrosis has been established. Renal cells can be induced to both produce extracellular matrix protein and inhibit protease activity by exogenous TGF-b1 in vitro [2–4]. Similarly, rapid development of glomerulosclerosis and a concomitant elevation of matrix markers was seen in both TGF-b1 transgenic mice and normal rats transfected in vivo with the TGF-b1 gene [5–7]. Finally, neutralizing antibodies Current Opinion in Pharmacology 2003, 3:204–208
Several studies have shown that TGF-b signaling requires both ALK5 and the TGF-b type II receptor (reviewed in [10]); however, the regulation of proliferation by TGF-b might not require ALK5 [11,12]. A novel type I receptor, ALK8, which was shown to bind TGF-b, was found in zebra fish [13]. A mammalian orthologue is expressed in murine mesangial cells, as determined by ALK8 antibodies and northern blot techniques using the zebra fish ALK8 probe [13]. Unlike other ALKs, ALK8 appears to be preferentially expressed in the heart, kidney and liver, and might play a unique role in cardiovascular and renal function. Another type I receptor relevant to the cardiovascular system is ALK1, which can mediate the actions of TGF-b specifically in vascular endothelial cells [14,15]. Thus, whereas TGF-b signaling is primarily mediated by ALK5, ALK1 and ALK8 could be responsible for specific cellular responses to TGF-b. The cytoplasmic mediators of TGF-b receptor activation are the pathway-selective Smad1, Smad2, Smad3 and Smad5, which bind to the common Smad4 [10,16]. ALK1 phosphorylates Smads 1 and 5, whereas ALK5 phosphorylates Smads 2 and 3. Using a novel ALK5 kinase inhibitor (SB-431542), recent studies have further delineated that nuclear accumulation of Smad2 and Smad3 is dependent on ALK5 kinase activity, and that the maintained nuclear presence of these Smads is entirely dependent on continued ALK5 activity [17–19]. The ALK5 activity, and subsequent nuclear presence of Smads 2 and 3, is responsible for activation of genes that participate in the deposition of extracellular matrix proteins in tubular dedifferentiation and vascular endothelial cell changes that can lead to progressive renal disease.
Renal fibrosis A central hypothesis of TGF-b-induced renal disease is that TGF-b activation causes accumulation of extracellular matrix components, leading to fibrosis. This was most directly demonstrated in transgenic mice and transfected rats overexpressing TGF-b [5–7]. The literature is replete with reports and reviews extolling the influence www.current-opinion.com
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that TGF-b has over extracellular matrix production. Thus, TGF-b continues to be associated with renal fibrosis [20,21] and the regulation of expression of many components of the extracellular matrix [22]. Now, the pathways through which TGF-b regulates these genes are being elaborated [19,23]. Connective tissue growth factor (CTGF) is an important factor that mediates some of the effects of TGF-b, which include hypertrophy of glomerular mesangial cells [24] and production of extracellular matrix [25]. The direct mechanism of TGF-b regulation was explored in mesangial cells by examining the promoter, mRNA and protein for CTGF. TGF-b-induced CTGF transcription in rat mesangial cells was inhibited by mutation of the Smad response-element in the CTGF promoter, dominant– negative Ras transfection and MEK2 inhibition with U0-126 [26]. This illustrates that TGF-b regulation of extracellular matrix components utilizes the direct ALK5–Smad pathway, as well as additional kinase pathways and growth factors. While several recent reports continue to support the concept that Jun kinase (JNK) and extracellular signalregulated kinase (ERK) pathways are activated in parallel to the Smad signaling pathway [26,27], new inhibitors of ALK5 and p38 have been used to identify which extracellular matrix components use the p38 pathway in addition to Smad [18]. In renal epithelial cells, TGF-b increased the mRNA for collagen Ia1, which was inhibited by a selective ALK5 inhibitor but not by a selective p38 inhibitor. Conversely, fibronectin mRNA was also increased by TGF-b but was inhibited by both the ALK5 and p38 inhibitors [19]. Consistent with this finding, TGF-b can increase fibronectin in fibroblasts derived from Smad3-deficient mice [28]. The prolonged exposure of TGF-b needed to produce changes in fibronectin RNA, and the requirement of p38 activity, suggest an indirect mechanism for the regulation of this gene. However, it has been shown recently that overexpression of Smad3 can increase fibronectin mRNA [29], presumably through Smad response-elements in the fibronectin promoter. This suggests that fibronectin may respond to both Smad and p38 activation. Thus, certain components of the extracellular matrix are primarily regulated through the ALK5–Smad pathway, whereas others utilize additional kinases, including p38, although it is likely that this is cell- and tissue-specific.
Renal tubular dedifferentiation Tubular atrophy is a feature of most renal diseases and has been previously reviewed in detail [30]. Recent advances have been described in the transgenic Ren-2 rat, which displays tubular epithelial apoptosis when made diabetic with streptozotocin. This degeneration of renal tubules was associated with increased TGF-b mRNA levels [31]. Further indirect evidence for a role for TGF-b in interwww.current-opinion.com
stitial apoptosis of tubular epithelial cells was seen in decorin knockout mice [32]. Decorin is a natural binding protein that can sequester and limit the actions of TGF-b [33]. Thus, its absence in the knockout mice resulted in more severe renal damage following ureteral obstruction [32]. Recent evidence supporting a direct role for TGF-b in renal apoptosis includes the release of TGF-b in glomerular visceral epithelial cells treated with either TGF-b or angiotensin II. These factors induce apoptosis, and Fas and Bax expression, in glomerular visceral epithelial cells, which can be prevented by antibodies to TGF-b [34]. Thus, angiotensin II acting through TGF-b can contribute to glomerulosclerosis by increasing the rate of apoptosis. The dedifferentiation of the tubular epithelium comprises not only apoptosis, but also transformation of the epithelial cells to a mesenchymal phenotype such as fibroblasts. A role for TGF-b in EMT has been elaborated in many systems, and markers of EMT correlate with the severity of renal disease in patients [35]. In proteinuric patients, abnormal uptake of ultra-filtered proteins by proximal tubular cells occurs, and is associated with increased presence of TGF-b1 mRNA in the tubular epithelium [36]. In response to TGF-b, CTGF might also participate in the induction of EMT [25]. In human proximal tubule epithelial cells, CTGF increases the expression of tenascin-c, a marker of EMT [25]. However, CTGF was not as potent as TGF-b, suggesting that CTGF is a secondary pathway for TGF-b in EMT. The signaling mechanism by which TGF-b effects EMT involves the stress-activated kinase p38. A kinase inhibitor of p38 (SB-202190), and dominant–negative expression of the MAP kinase-3 that normally activates p38, both prevented TGF-b-induced EMT [37,38]. However, a p38independent mechanism also exists that involves the expression of collagen I. TGF-b increased collagen Ia1 mRNA in renal epithelial cells independent of p38 [19]. Whereas collagen IV suppresses EMT, collagen I promotes it [39]. Thus, TGF-b can induce EMT directly through p38, as well as indirectly through managing increased levels of collagen components that favour EMT.
Vascular mechanisms Several studies suggest an association between cardiovascular dysfunction, renal disease and TGF-b. These studies have shown actions of TGF-b on vascular endothelial cells, activation of angiogenic factors in glomerular podocytes [40] and, most recently, an effect of TGF-b on the myogenic mechanism that regulates glomerular filtration rate through changes in afferent arteriolar resistance [41]. The mechanism by which TGF-b regulates vascular endothelial cell function includes an interaction between ALK5 and ALK1 [15]. Transfection studies have demonstrated that endothelial cell proliferation is inhibited by Current Opinion in Pharmacology 2003, 3:204–208
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the TGF-b/ALK5 pathway, whereas migration and proliferation is induced by the TGF-b/ALK1 pathway [15]. Thus, endothelial cells can determine their angiogenic response to TGF-b by the relative levels of ALK5 versus ALK1 expression. In association with its modulation of angiogenesis, TGF-b has been shown to induce the expression of the angiogenic vascular-endothelial-growth-factor in glomerular podocytes [40]. Whether this mechanism contributes to renal disease or is a protective mechanism remains to be determined. However, because high glucose also increases the expression of collagen IV and vascular-endothelial-growthfactor in podocytes, it is possible that this mechanism contributes to the glomerular expansion seen in diabetic nephropathy [40]. A novel vascular mechanism by which TGF-b might contribute to renal disease has been identified using the blood-perfused juxtamedullary nephron technique. As described in a recent abstract [41], TGF-b completely blocks the autoregulatory vasoconstriction of the afferent arteriole to increased perfusion pressure. These data suggest that, in addition to the profibrotic mechanism, TGF-b may increase intra-glomerular pressure leading to further glomerular damage. The functional consequence of reduced glomerular function is invariably associated with hypertension. Thus, protecting the kidney from TGF-b-induced fibrosis and impaired regulation of renal vasculature should also slow the development of hypertension. This was demon-
strated nicely in Dahl salt-sensitive rats. Renal deposition of extracellular matrix was dramatically reduced with preservation of renal histology and consequently protection from hypertension by treating the Dahl salt-sensitive rats with neutralizing antibodies to TGF-b [42]. These data clearly demonstrate that there continues to be a strong rationale for inhibition of the TGF-b signaling pathway to treat renal disease.
Pharmacological intervention Given the strength of the rationale for inhibiting TGF-b to treat renal disease, there continues to be many attempts to interrupt TGF-b signaling in models of renal disease. Indirect inhibition of TGF-b signaling has been accomplished with angiotensin II receptor inhibitors, resulting in lowering of urinary TGF-b in patients, and decreased renal matrix markers in rodents [43,44]. Similarly, inhibition of the formation of glycated proteins, which also activate TGF-b, can also reduce matrix markers in diabetic kidneys [45]. Consequently, TGF-b antibody treatment continues to prove to be effective in reducing kidney weight, glomerular hypertrophy and renal extracellular matrix mRNA, as exemplified in the diabetic mouse [46]. Whether antibodies can be delivered chronically in patients for the treatment of renal disease remains to be established. Because ALK5 mediates most actions of TGF-b, including the pro-fibrotic actions, this appears to be an ideal target for pharmacological intervention (Figure 1). Recently, compounds capable of inhibiting the kinase activity of ALK5 have been described. These pyridinylimidazoles interact with the ATP-binding site of ALK5
Figure 1
Diabetes
↑ Glomerular permeability
Elevated glucose
Albuminuria Urinary collagen, TGFβ1
AGEs
↑ TGF-β1 glomerular
ALK5 TGFβ type I receptor
↑ TGF-β1 interstitial
Extracellular matrix
↑ Renal fibronectin, collagen, PAI-1
Glomerulosclerosis
Interstitial fibrosis
Impaired renal function Current Opinion in Pharmacology
Diagram showing the mechanisms of diabetic nephropathy, including the contribution of TGF-b to renal disease. AGEs, advanced glycation endproducts, PAI-1; plasminogen activator inhibitor-1. Current Opinion in Pharmacology 2003, 3:204–208
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[47]. Because certain similarities exist in the ATP-binding pocket between ALK5 and the serine/threonine kinase p38, it was shown that the p38 inhibitor SB203580 inhibited in vitro ALK5 phosphorylation of Smad3 with an IC50 value between 0.7 mM and 3 mM [19,48,49]. However, a more selective ALK5 inhibitor, SB-431542, was developed to inhibit ALK5 kinase activity and TGFb-induced extracellular matrix, with IC50 values under 100 nM [19,47]. Although this inhibitor is useful in preventing many actions of TGF-b in vitro [17–19], compounds with adequate in vivo half-life need to be developed to evaluate the utility of this approach in treating progressive renal disease. At a recent meeting, an example of an ALK5 inhibitor with in vivo activity, SB525334, was presented [50]. This inhibitor not only blocked TGF-b-induced signaling and extracellular matrix mRNA in cultured renal cells, but also inhibited renal expression of collagen Ia1 mRNA and fibronectin protein after renal injury caused by puromycin aminonucleoside [50]. In addition, the ALK5 inhibitor also reduced the injury-induced proteinuria, which is consistent with the vascular effects of TGF-b on glomerular function described above. Therefore, the application of ALK5 inhibitors may also be useful in reducing renal fibrosis in chronic renal disease such as diabetic nephropathy.
Conclusions It is understood that TGF-b plays a central role in the progression of renal disease, as concluded in many reviews. With the advent of specific inhibitors of the TGF-b signaling pathway, it is becoming clear that ALK5 mediates many of the actions of TGF-b, including extracellular matrix production and proliferation. However, the contribution of ALK5 to tubular dedifferentiation and intra-glomerular pressure regulation still needs further investigation. Therefore, it appears that ALK5 is well positioned as a target for intervention of TGF-bmediated diseases, and is supported by preliminary studies using an ALK5 inhibitor in models of renal disease. We must bear in mind, however, that TGF-b is a pleiotropic factor with actions that also modulate cell growth, angiogenesis and immune functions with both a pro- and anti-inflammatory nature, which are likely to involve other type I receptors. The challenge in using therapeutics that block ALK5 activity will lie not only in designing specific kinase inhibitors, but also in striking a balance between disease-relevant fibrotic actions and the immunemodulatory actions of TGF-b.
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