Renal injury due to renin angiotensin aldosterone system activation of the transforming growth factor-b pathway

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1 review & 2006 International Society of Nephrology Renal injury due to renin angiotensin aldosterone system activation of the transforming growth factor-b pathway G Wolf 1 1 Klinik für Innere Medizin III, Klinikum der Friedrich-Schiller-Universität, Jena, Germany Glomerulosclerosis, interstitial fibrosis, and tubular atrophy occur with end-stage kidney failure, irrespective of the primary etiology. The transforming growth factor-b (TGF-b) is a key factor in these alterations either directly, by stimulating synthesis of extracellular matrix components and reducing collagenase production, or indirectly through other profibrogenic factors such as connective tissue growth factor (CTGF). TGF-b is important for the proliferation of intrarenal fibroblasts and the epithelial mesenchymal transition through which tubular cells become fibroblasts. Although several factors induce TGF-b expression in the kidney, one very interesting aspect is the link between the renin angiotensin aldosterone (Aldo) system (RAAS) and TGF-b. Angiotensin II (ANG II) stimulates TGF-b expression in the kidney by various mechanisms and upregulates receptors for TGF-b. ANG II can directly phosphorylate Smads without inducing TGF-b. Recent data provide compelling evidence that other components of the RAAS including ANG III, renin, and Aldo also activate the TGF-b system. As direct modulation of the TGF-b system is not yet feasible in humans, angiotensin-converting enzyme (ACE) inhibitors and angiotensin type 1 (AT 1 )-receptor blockers are currently the most potential drugs to interfere with this ANG II-mediated TGF-b expression. This review highlights some current aspects of the interaction between the RAAS and the TGF-b axis. Kidney International (2006) 70, doi: /sj.ki ; published online 20 September 2006 KEYWORDS: chronic renal insufficiency; aldosterone; ACE inhibitors; TGF-beta; angiotensin Correspondence: G Wolf, University of Jena, Department of Medicine, Erlanger Allee 101, D Jena, Germany. Gunter.Wolf@med.uni-jena.de Received 23 June 2006; revised 27 July 2006; accepted 1 August 2006; published online 20 September 2006 Many chronic renal diseases, irrespective of the primary etiology, progress to end-stage renal disease with an irreversible loss of renal tissue. 1 Glomerulosclerosis, fibrosis of the tubulointerstitial microenvironment, and tubular atrophy all constitute the major morphological correlates of such end-stage kidneys. Deposition of extracellular matrix proteins including fibronectin, collagen types I, III, and IV is an important component of the scarring observed during the evolution of glomerulosclerosis and tubulointerstitial fibrosis. 1 An increase in the synthesis as well as a decrease in turnover of these proteins is, as a simplification, responsible for the net accumulation of extracellular matrix. Transforming growth factor-b (TGF-b) serves as a paradigm for a profibrogenic cytokine. 2,3 Its function in the renal pathophysiological process has been investigated in landmark studies by Border et al. 2 TGF-b directly stimulates transcription of many extracellular matrix genes in renal cells including mesangial, endothelial, and tubular cells. 3 During renal fibrosis new fibroblasts are derived mainly through epithelial mesenchymal transition, a process principally driven by TGF-b. 4 On the other hand, TGF-b reduces collagenase production and simultaneously stimulates expression of tissue inhibitor of metalloproteinases, resulting in an overall inhibition of extracellular matrix turnover. 5 Members of the TGF-b family interact with specific receptors resulting in the activation of distinct signal-transduction pathways, mainly involving Smad proteins. 6,7 Some of the profibrotic effects of TGF-b are actually mediated by the connective tissue growth factor (CTGF), a member of the CCN (cyr61, ctgf, nor) family of early response genes. 8 For example, CTGF stimulates proliferation of renal fibroblasts and induces extracellular matrix synthesis. An early feature of chronic renal disease, before glomerulosclerosis and tubulointerstitial fibrosis have yet developed, is the recruitment of macrophages/monocytes from the circulation into the local tissue. 9 It has been reported that TGF-b functions as a chemoattractant for macrophages/monocytes and regulates the survival of lymphocytes, natural killer cells, and dendritic cells. 9 On the other hand, TGF-b can also exert anti-inflammatory properties as shown in atherosclerosis. 10 Compensatory growth processes, such as hypertrophy and/or hyperplasia, are characteristic findings of chronic kidney 1914 Kidney International (2006) 70,

2 G Wolf: RAAS and TGF-b review disease as nephrons surviving the injury may undergo adaptive growth to compensate for the loss of functional renal tissue. 11 Cell culture studies have revealed that TGF-b arrests renal cells in the G 1 phase of the cell cycle, and stimulates cellular hypertrophy by a variety of mechanisms including inhibition of cyclin-dependent kinases by induction of cyclin-dependent kinase inhibitors such as p27 Kip1 and p It has also been demonstrated that TGF-b may exert proliferative actions on renal cells under specific conditions. 13 TGF-b additionally exerts hemodynamic effects. It impairs renal autoregulation via the generation of reactive oxygen species. 14 Recent evidence shows that Emilin1 (an inhibitor of active TGF-b formation) knockout mice have increased blood pressure, increased peripheral vasculature resistance, and reduced vessel size indicating a potential role of TGF-b in hypertension. 15 Interestingly, TGF-b stimulates angiotensinogen gene expression, at least in proximal tubular cells, indicating a positive feedback loop that further enhances renal injury. 16 Thus, activation of the TGF-b axis explains many morphological alterations of chronically failing kidneys. The landmark studies by Anderson et al. 17 showing the superiority of angiotensin-converting enzyme (ACE) inhibitors in halting the progression of renal disease, suggest that angiotensin II (ANG II) plays a pivotal role in the pathophysiology of chronic renal disease. 18 There is now ample evidence that activation of the renin angiotensin aldosterone (Aldo) system (RAAS) is a key mediator in the progression of renal disease. There is a close interaction between the RAAS and the TGF-b systems In fact, many profibrotic effects of ANG II are mediated by stimulation of TGF-b. The current review describes more recent insights into the link between the RAAS and the TGF-b axis. ANG II-MEDIATED STIMULATION OF THE TGF-b AXIS TGF-b1 is the prototype of a family of more then 35 structurally related cytokines. This family also includes TGFb2, TGF-b3, activins, and bone morphogenetic proteins. Although these other members, particularly bone morphogenetic protein-7 as an antifibrotic agent, are of considerable interest, they will not be further discussed here and the reader is referred to excellent editorials 23,24 The synthesis of TGF-b is a complex process. The translated protein is a TGF-b precursor termed preprotgf-b, which contains a signal peptide. Pro-TGF-b is processed in the Golgi apparatus by a furin-like protease. A homodimer of this new protein, the latency-associated protein, is noncovalently bound to the homodimer of the mature TGF-b. The latency-associated protein/tgf-b complex can be secreted as such or can associate with the latent TGF-b-binding protein. In order to bind to its putative receptors, TGF-b must be liberated from latency-associated protein. Several mechanisms have been found to lead to the release of TGF-b. For example, thrombospondin, through the physical interaction with latency-associated protein, releases free TGF-b. Principally, components of the RAAS can upregulate TGF-b concentration by influencing several of the mechanisms described above. Early evidence that ANG II stimulates TGF-b messenger RNA (mrna) expression stems from observations of cultured vascular smooth muscle cells. 25 Moreover, in cultured vascular smooth muscle cells, ANG II not only stimulated transcription of TGF-b, but also promoted its conversion to the biologically active form. 26 Our group was the first to demonstrate that ANG II stimulates TGF-b synthesis in renal cells. 27 We had previously noticed that ANG II decreased proliferation, but induced cellular hypertrophy of cultured proximal tubular cells. 28 This enlargement of tubular cells in the presence of ANG II was associated with stimulated transcription and synthesis of collagen type IV, but not of type I. 6 Application of exogenous TGF-b mimicked the growth effects observed with ANG II in proximal tubular cells (inhibition of DNA synthesis and stimulation of hypertrophy). 27 Cells stimulated with ANG II expressed more TGF-b 1 mrna and produced bioactive TGFb. 27 These effects were transduced through angiotensin type 1 (AT 1 ) receptors. Moreover, neutralizing anti-tgf-b antibody, but not control immunoglobulin G, abolished the ANG IImediated inhibition of proliferation as well as the induction of cellular hypertrophy indicating that part of ANG IIinduced growth effects were in fact mediated by TGF-b. 27 In a subsequent study, we provided evidence that ANG II directly stimulates transcription of the TGF-b 1 gene. 29 The murine as well as the human TGF-b 1 promoter contain two putative transcriptional start sites, with several sequence specific transcription factor 1-binding and activator protein (AP)-2-like sequences and AP-1 binding motifs located in the 5 0 end. Transient transfection of different chimeric constructs into proximal tubular cells revealed that ANG II stimulates TGF-b 1 transcription from both start sites. 29 As ANG II induces c-fos and c-jun, and the protein products of these two immediate early genes form a leucine zipper which can bind to AP-1 sites, it is likely that the ANG II-stimulated transcription of TGF-b 1 is mediated through these AP-1 sites. Subsequent studies have confirmed that ANG II, through protein kinase C and p38 mitogen-activated protein kinase-dependent pathways, activate the binding of nuclear proteins to the AP-1-box B of the TGF-b1 promoter and stimulate transcriptional activity. 30 In addition to direct stimulation of TGF-b1 transcription, ANG II also enhances the concentration of the TGF-b1 protein by additional mechanisms. ANG II stimulates through the p38-mitogen-activated protein kinase and c-jun N-terminal kinase signaling thrombospondin-1 that, in turn, leads to an increased release of active TGF-b1 from the inactive latent complex. 31,32 Thus, ANG II-dependent transcriptional and post-transcriptional mechanisms enhance TGF-b expression. ANG II-mediated TGF-b induction in the kidney is not restricted to the proximal tubule. It has been described that ANG II treatment of rat mesangial cells in culture increases TGF-b as well as the matrix components biglycan, fibronectin, and collagen type I. 33 Kidney International (2006) 70,

3 review G Wolf: RAAS and TGF-b TGF-b mediates its biological functions through binding to type I and II receptors. Endoglin is a glycoprotein that functions as a type III receptor for TGF-b. Interestingly, TGFb2 is only able to signal in the presence of the type III receptor whereas TGF-b1 and TGF-b3 do not require endoglin as receptor for activation of downstream signaling. Endoglin cannot bind TGF-b in the absence of type II receptors, but may modulate signal transduction of type I and II heterodimers. The active form of TGF-b1 initially engages the TGF-b receptor type II. In a second step this brings the type II receptor in proximity and then activates the type I receptor, which phosphorylates intracellular Smad proteins. Eight Smad proteins have been identified and are grouped into three categories (receptor-associated Smads: 1, 2 3, 5, and 8); one common Smad (Smad 4), and two inhibitory Smads (6 and 7). Some Smads such as 1, 5, and 8 are exclusively involved in signaling of other ligands of the TGF-b superfamily. 34 The common Smad 4 forms heteromeric complexes with receptor-associated Smads. The complexes then translocate to the nucleus and modulate gene expression. 34 There is also evidence of Smad-independent TGF-b signaling pathways. 35 We have shown in proximal tubular cells that ANG II stimulated protein expression of TGF-b receptor type II, but not that of type I. 36 This stimulated receptor expression was reflected in an overall increase of specific binding of TGF-b1 to cells. 36 Moreover, ANG II increased mrna expression for TGF-b receptor type II. Reporter gene experiments indicated that AP-1 sites in the promoter for TGF-b receptor type II are a necessary prerequisite for ANG II-induced transcriptional activity. 36 In a culture system, albumin (as a factor mimicking proteinuria) leads to upregulation of type II TGF-b receptors via stimulation of ANG II. 37 This can amplify profibrogenic actions of TGF-b suggesting a mechanism for how proteinuria leads to tubulointerstitial fibrosis. ANG II increased the expression of endoglin mrna and protein in cardiac fibroblasts without effects on type I and II TGF-b receptors. 38 As endoglin is essential for the binding of TGF-b2, this effect would presumably stimulate TGF-b2 signaling. Rodriguez-Vita et al. 39 as well as Wang et al. 40 have shown new twists in how ANG II can stimulate the TGF-b axis. These investigators discovered that ANG II phosphorylates Smad 2 and 3 independently of TGF-b. The effects were transduced by AT1 receptors, and dependent on extracellular signal-regulated kinase (Erk) 1, 2, and p38 mitogen-activated protein kinase signal transduction. These fascinating data suggest that Smad pathways are not exclusively activated by the classic TGF-b-triggered mechanisms, and that profibrogenic pathways can be activated by ANG II without directly stimulating TGF-b. 39,40 ANG II also induces other profibrotic factors. CTGF, a downstream key mediator of TGF-b s profibrogenic effects, is directly and independently of TGF-b, induced by ANG II via a calcineurin-associated pathway In addition, Iwanciw et al. 44 found that ANG II-mediated CTGF expression requires Erk 1,2, and Rho signaling. In podocytes, ANG II stimulates the expression of the vascular endothelial growth factor. 45 Vascular endothelial growth factor, in turn, leads to an upregulation of the TGF-b receptor type II and increased Smad2 phosphorylation. 46 As a consequence, a3(iv) collagen protein synthesis is stimulated in podocytes, 45 an effect that presumably contributes to the thickening of the glomerular basement membrane in pathophysiological conditions such as diabetic nephropathy. ACTIVATION OF THE TGF-b AXIS BY OTHER RAAS COMPONENTS ANG III, through binding to AT 1 receptors, increases TGF-b1 mrna expression in mesangial cells and in renal interstitial fibroblasts. 47 This peptide also increases extracellular matrix synthesis in these cells in a TGF-b-dependent manner. 47 ANG IV does not directly stimulate TGF-b expression, but influences extracellular matrix turnover by increasing expression of the plasminogen activator inhibitor-1, a potent inhibitor of metalloproteinases that are involved in extracellular matrix degradation. 48 Although Aldo is profibrotic through induction of plasminogen activator inhibitor-1, 49 it has been found to additionally induce TGF-b protein by a post-transcriptional mechanism without influencing TGF-b transcripts. 50 Moreover, Aldo promotes ANG II-mediated signal-transduction effects such as Erk 1,2, and c-jun N-terminal kinase phosphorylation. 51 As TGF-b has been shown to activate Erk 1,2 through Smad-independent pathways, 35 Aldo and TGF-b may act synergistically in stimulating downstream signaling events. Fascinating recent evidence suggests that renin increases mesangial cell TGF-b expression and matrix proteins through a specific receptor, independent of ANG II. 52 Specific receptors for renin have been characterized on mesangial cells and vascular smooth muscle cells that provoke a rapid activation of Erk 1,2. 53 Remarkably, renin-stimulated TGF-b expression was independent of its proteolytic activity. 52 These findings suggest that renin can contribute to TGF-b expression and renal fibrosis through two different mechanisms: directly, by binding to specific receptors and indirectly, via the generation of ANG II and subsequent TGF-b induction. 54 CLINICAL EVIDENCE OF RAAS-MEDIATED STIMULATION OF TGF-b AXIS The role of ANG II and other RAAS components in the activation of the TGF-b axis has also been found in animal models ranging from reduction of renal mass, Marfan syndrome, renal artery stenosis to calcineurin-inhibitor toxicity One of the first models in which the link between the RAAS and TGF-b was comprehensively studied was unilateral ureteral ligation. 59 In this model, TGF-b 1 mrna levels are increased in the obstructed kidney 3 days after surgery, but do not change significantly in the contralateral kidney. 59 Treatment with the ACE inhibitor enalapril significantly blunted this increase in TGF-b 1 mrna. 59,60 Moreover, an AT 1 -receptor antagonist was 1916 Kidney International (2006) 70,

4 G Wolf: RAAS and TGF-b review equally effective in halting the increase of TGF-b 1 mrna in the obstructed kidney. 61 Naturally, the clinical evidence is more indirect and stems mainly from measuring urinary TGF-b excretion that is thought to be mainly derived from the kidney under RAAS blockade in various diseases Table 1 gives an overview of some of the published studies. The data indicate that ACE inhibitor as well as AT 1 -receptor antagonists reduce urinary TGF-b protein levels. Biopsy studies in patients with membranous nephropathy also reveal that renal in situ formation of ANG II could participate in TGF-b upregulation. 76 Table 1 Urinary TGF-b excretion, as a parameter of renal synthesis, is reduced by ACE inhibitors and/or AT 1 -receptor antagonists Disease entity Reference 62,63 Diabetes type 1 Diabetes type Mild hypertension 67 Hypertension with minor renal disease 68 Renal transplantation with calcineurin inhibitors 69,70 Membranous glomerulonephritis 71 IgA nephropathy ACE, angiotensin-converting enzyme; AT, angiotensin; IgA, immunoglobulin A; TGF-b, transforming growth factor-b. ANG II CTGF Fibrosis Smad3 ANG III Renin TGF-β TGF-β receptors Inflammation Aldo Figure 1 Overview of the interaction between the RAAS and the TGF-b1 axis. ANG II, ANG III, renin, and Aldo all upregulate TGF-b1 expression. In addition, ANG II also stimulates expression of TGF-b receptors leading to a further amplification of TGF-b s effect. Independently, ANG II also leads to activation of the Smad pathway via phosphorylation of Smad3. Downstream effects of TGF-b are activation of CTGF expression that is additionally directly stimulated by ANG II. Fibrosis and inflammation of renal tissue are the final mediators of this interaction between the RAAS and the TGF-b axis. CONCLUSION Convincing and accumulating evidence increasingly indicates that ANG II and other members of the RAAS activate the TGF-b axis in the kidney by both direct and indirect mechanisms (Figure 1). As renal TGF-b is a key player in the development of morphological changes such as glomerulosclerosis and tubulointerstitial fibrosis, strategies to hinder this profibrogenic cytokine are essential to prevent the loss of functioning renal tissue. Despite the fact that a wide array of new approaches to interfere with TGF-b expression has been tested including application of neutralizing antibodies, antisense oligonucleotides, overexpression of decorin, and tranilast, these treatments are not easily adaptable to humans. However, ACE inhibitors and AT 1 -receptor blocker are drugs that interfere with TGF-b expression and should be part of every regimen to halt progression of renal disease. As RAAS can activate the TGF-b axis by several mechanisms, a more complete blockade would presumably also reduce the deleterious effects of TGF-b on the kidney. 77 ACKNOWLEDGMENTS I apologize to all investigators whose work was not directly referenced owing to space constraints. I thank my friend and colleague Fuad N Ziyadeh, MD for the work together on the TGF-b system. Original studies in the author s laboratory are supported by the Deutsche Forschungsgemeinschaft (Wo 460/2-6, 2-7, 2-8). REFERENCES 1. Remuzzi G, Benigni A, Remuzzi A. 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