TY - JOUR AU - Wolf,, Gunter AB - Abstract Transforming growth factor-β (TGF-β) is a profibrotic cytokine found in chronic renal diseases, which initiates and modulates a variety of pathophysiological processes. It is synthesized by many renal cell types and exerts its biological functions through a variety of signalling pathways, including the Smad and MAPK pathways. In renal diseases, TGF-β is upregulated and induces renal cells to produce extracellular matrix proteins leading to glomerulosclerosis as well as tubulointerstitial fibrosis. Different types of renal cells undergo different pathophysiological changes induced by TGF-β, leading to apoptosis, hypertrophy and abnormalities of podocyte foot processes, which ultimately result in renal dysfunction. In this review, we describe the effects of TGF-β on different renal cell types and the means by which TGF-β participates in the pathomechanisms of glomerular and tubulointerstitial diseases. diabetic nephropathy, EMT, fibroblasts, progression of renal diseases, renal fibrosis TRANSFORMING GROWTH FACTOR BETA AND ITS PATHWAYS Transforming growth factor-beta (TGF-β) is a multifunctional regulator that modulates cell proliferation, differentiation, apoptosis, adhesion and migration of various cell types and induces the production of extracellular matrix proteins (ECM) [1]. Most cell types, including immature haematopoietic cells, activated T and B cells, macrophages, neutrophils and dendritic cells, produce TGF-β and/or are sensitive to its effects [1]. The TGF-β superfamily, characterized by 6 conserved cysteine residues, is encoded by 42 open reading frames in humans and consists of >30 related members in mammals, including 3 TGF-βs, 4 activins and over 20 bone morphogenetic proteins (BMPs) [1–3]. Although the diverse TGF-β ligands elicit very different cellular responses, they all share a set of common sequence and structural features [2]. The three mammalian isoforms of the TGF-β subfamily (TGF-β1, TGF-β2, TGF-β3) share 70–82% amino acid homology and have qualitatively similar activities in different systems [4]. The active form of a TGF-β cytokine is a dimer stabilized by hydrophobic interactions, which are further strengthened by an intersubunit disulphide bridge in most cases [2]. TGF-β initiates intracellular signalling by binding to receptor complexes that contain two distantly related transmembrane serine/threonine kinases called receptors type I and type II (TβR-I and TβR-II) (Figure 1) [5]. Both of these receptors have an N-glycosylated extracellular domain that is rich in cysteine residues, one transmembrane domain and an intracellular serine/threonine kinase domain [1]. The type II receptor kinase is a constitutively active kinase, whereas the type I receptor kinase needs to be activated by the type II receptor kinase [1]. On most cell types, TGF-β binds first and directly to TβR-II [1]. The bound TGF-β is then recognized by TβR-I, which is recruited into the complex and becomes phosphorylated by receptor II (Figure 1) [5]. The type III receptor (e.g. betaglycan and endoglin), a receptor-associated protein without any intrinsic enzymatic activity, acts as a co-receptor for binding/presenting TGF and as a regulator of TGF-β signalling [1, 6]. The intracellular mediators of TGF-β signalling are known as Smads, which act downstream of the TβR-I (Figure 1) [7]. Three classes of Smads have been identified. The receptor-regulated Smads (R-Smads) (e.g. Smad1, Smad2, Smad3, Smad5 and Smad8), which are directly phosphorylated and activated by TβR-I, form hetero-oligomeric complexes with a second class of Smad, the common mediator Smads (Co-Smads) (e.g. Smad4). These Smad complexes translocate into the nucleus, where they are recruited into DNA primarily by site-specific DNA-binding transcription factors, and participate in regulating the transcription of target genes (Figure 1) [7]. Smad2 and Smad3 respond to signalling by the TGF-β subfamily and Smads 1, 5 and 8 primarily by the BMP subfamily [2, 7]. Inhibitory Smads (e.g. Smad6 and Smad7) are the third identified class, which antagonize the activity of the receptor-regulated Smads by physical interaction with the activated TβR-I and subsequently prevent the docking and phosphorylation of R-Smads (Figure 1) [6–8]. In addition to Smad-mediated transcription, TGF-β can directly activate other signal transduction cascades, including MAPK pathways, such as Ras, Raf, Erk, JNK and p38 (Figure 1) [6, 8]. TGF-β can also activate the phosphatidylinositol-3 kinase (PI3K) cascade by phosphorylation of its effector Akt, as well as the Rho-like GTPases, including RhoA, Rac and Cdc42 (Figure 1) [6, 8]. FIGURE 1: Open in new tabDownload slide TGF-β binds to the primary TGF-β receptor type II (TβR-II), which is a constitutively active serine/threonine kinase that recruits receptor type I (TβR-I) to the complex. Subsequent transphosphorylation and activation of TβR-I by TβR-II allows the TβR-I kinase to phosphorylate selected Smads, which are inhibited by the so-called inhibitory Smads (Smad6 or Smad7). These receptor-activated Smads (R-Smads, e.g. Smad1/2/3/5/8) then form active complexes with the common Smad4, which translocate into the nucleus, where they regulate transcription of certain target genes. In addition, the activated receptor complex activates non-Smad signalling pathways, such as Ras, Erk, JNK, p38, RhoA and PI3K. (Figure modified after [1, 5, 6, 8]). FIGURE 1: Open in new tabDownload slide TGF-β binds to the primary TGF-β receptor type II (TβR-II), which is a constitutively active serine/threonine kinase that recruits receptor type I (TβR-I) to the complex. Subsequent transphosphorylation and activation of TβR-I by TβR-II allows the TβR-I kinase to phosphorylate selected Smads, which are inhibited by the so-called inhibitory Smads (Smad6 or Smad7). These receptor-activated Smads (R-Smads, e.g. Smad1/2/3/5/8) then form active complexes with the common Smad4, which translocate into the nucleus, where they regulate transcription of certain target genes. In addition, the activated receptor complex activates non-Smad signalling pathways, such as Ras, Erk, JNK, p38, RhoA and PI3K. (Figure modified after [1, 5, 6, 8]). THE ROLE OF TGF-β IN KIDNEY DISEASES There is a body of literature that has described the Janus-head-like activity of TGF-β. Physiological levels of TGF-β are thought to be essential for normal development, tissue repair and maintenance of organ functions. Further beneficial aspects of TGF-β include its anti-inflammatory actions via the inhibition of mitogenesis and cytokine responses of glomerular cells, and the suppression of accumulation and function of infiltrating cells [9]. TGF-β1 knockout mice showed multiorgan inflammation, including the kidney [9]. Yet, overexpression of TGF-β has also been closely linked with pathological alterations characteristic of various kidney diseases, which reflects the ‘dark side’ of this cytokine [9]. In human glomerular disease, such as in focal and segmental glomerulosclerosis (FSGS), IgA nephropathy, crescentic glomerulonephritis, lupus nephritis and diabetic nephropathy (DN), TGF-β has been regarded as a pivotal molecule that contributes to glomerulosclerosis [9]. In these diseases, which are mainly characterized by excessive ECM accumulation, a significantly increased expression of all three TGF-β isoforms as well as TGF-β receptors in the glomeruli and the tubulointerstitium has been demonstrated [6, 10, 11]. Although it has been proposed that the effects of TGF-β in human glomerular diseases may be the result of an interplay between individual TGF-β isoforms, primarily TGF-β1 shows a close relationship with matrix accumulation, whereas the roles of TGF-β2 and TGF-β3 are less clear [10, 12]. Furthermore, urinary levels of TGF-β are elevated in patients with various renal diseases, while the grade of interstitial fibrosis and mesangial matrix increases [13]. For example, urinary TGF-β1 excretion is increased in patients with proteinuria due to glomerular dysfunction, compared with healthy subjects and patients with glomerular disease without proteinuria. In addition, remission of proteinuria with immunosuppressive treatment is followed by decreased urinary TGF-β1 excretion [14]. It is well known that upregulated TGF-β stimulates the production of matrix proteins, decreases the activity of ECM-degrading proteinases and upregulates the synthesis of proteinase inhibitors, leading to excessive matrix deposition [9]. In addition, in progressive podocyte diseases, such as human FSGS, IgA nephropathy and DN, a strong TGF-β1 expression in the podocytes has been observed, indicating a role of TGF-β in podocyte injury [15]. In the pathophysiology of diabetic kidney diseases, the role of TGF-β as the key factor has been well characterized by numerous studies using experimental models of diabetic kidney disease as well as in patients with DN [16]. Patients with both type 1 and type 2 exhibit enhanced tubular and glomerular TGF-β expression in early and late stages of the disease and it has been shown that the expression is closely correlated with the degree of glycaemic control in these patients [16]. Whereas in the early stages of diabetic kidney disease TGF-β1 is stimulated by hyperglycaemia and glomerular stretch, in later stages persistent production of TGF-β may be due to stimulation by glycated proteins (e.g. AGEs), the influence of growth factors [angiotensin II (Ang II) and platelet-derived growth factor (PDGF)] and TGF-β auto-induction [16]. Ang II has been shown to stimulate TGF-β expression as well as its receptors [17, 18]. In contrast to chronic kidney diseases, the role of TGF-β in modulating the kidney's response to acute kidney injury is not well understood [19]. Although it was once thought that TGF-β1 could be a signal of potential recovery in acute renal failure, there has been no substantive evidence suggesting that it plays a critical role in the renal tubular repair response [19, 20]. It has been suggested that TGF-β initiates a variety of pathophysiological processes at the beginning of kidney injury, including tubular epithelial cell apoptosis, intrinsic cell dedifferentiation and ECM deposition, and it is correlated closely with an acute deterioration in renal function and the development of renal fibrosis [21]. Moreover, it has recently been shown that selective deletion of TβR-II in the proximal tubules of mice attenuates acute proximal tubule injury [19]. This interruption of the receptor-attenuated renal impairment and reduced tubular apoptosis suggests that TGF-β signalling in the proximal tubule has a detrimental effect on the response to acute kidney injury as a result of its proapoptotic effects [19]. TGF-β-MEDIATED RENAL PATHOMECHANISMS TGF-β significantly contributes to a number of key pathological events leading to reduced glomerular filtration and impaired renal function. At glomerular level, TGF-β mainly contributes to glomerular filtration barrier alteration, fibrosis and sclerosis, which reduce the filtration surface and finally cause glomerular collapse at the tubular level, and TGF-β has been shown to participate both directly and indirectly in tubule degeneration (Figure 2) [22]. FIGURE 2: Open in new tabDownload slide Role of TGF-β leading to glomerulosclerosis and tubulointerstitial fibrosis in renal diseases. TGF-β induces basement membrane thickening via stimulation of renal cells to produce more ECM. The induction of pathophysiological changes of renal cells, such as hypertrophy, apoptosis and podocyte abnormalities, leads to reduced glomerular filtration and loss of glomerular and interstitial capillaries, tubulointerstitial fibrosis and tubular atrophy, resulting in permanent renal dysfunction. EMT, epithelial-to-mesenchymal transition; EndMT, endothelial-to-mesenchymal transition. (Figure modified after [22]). FIGURE 2: Open in new tabDownload slide Role of TGF-β leading to glomerulosclerosis and tubulointerstitial fibrosis in renal diseases. TGF-β induces basement membrane thickening via stimulation of renal cells to produce more ECM. The induction of pathophysiological changes of renal cells, such as hypertrophy, apoptosis and podocyte abnormalities, leads to reduced glomerular filtration and loss of glomerular and interstitial capillaries, tubulointerstitial fibrosis and tubular atrophy, resulting in permanent renal dysfunction. EMT, epithelial-to-mesenchymal transition; EndMT, endothelial-to-mesenchymal transition. (Figure modified after [22]). TGF-β and mesangial cells Mesangial cells (MC), which are located in the intercapillary space, are crucial for glomerular function under both physiological and pathological conditions and share many characteristics with vascular smooth muscle cells. They regulate the glomerular filtration rate by providing structural support for the glomerular capillary loops and modulating glomerular capillary flow and the ultrafiltration surface [22]. The mesangium contributes to the pathophysiology of a variety of glomerular diseases; MCs are activated in response to numerous vasoactive substances as well as to growth factors/cytokines and mediate interactions with other glomerular cells (podocytes, endothelial cell and inflammatory cells) [22]. The increase in the mesangial compartment size, which is a result of mesangial matrix deposition as well as MC proliferation and hypertrophy, is the cornerstone of glomerulosclerosis (Figure 2) [22]. TGF-β is a major contributor to glomerular ECM accumulation by stimulating MCs to produce type I, III and IV collagen, laminin, fibronectin and heparan sulphate proteoglycans as well as by inhibiting matrix degradation [22, 23]. In MCs, it has been shown that TGF-β stimulates the synthesis and accumulation of ECM via different signalling pathways (e.g. Smad, p38, Erk1/2). In addition, TGF-β plays a role in MC hypertrophy associated with diabetes and other glomerulopathies [22]. In a high-glucose milieu, for example in patients with diabetes mellitus, MCs undergo proliferation, followed by cell hypertrophy, which is mediated, at least in part, by TGF-β (Figure 2) [23]. Cellular hypertrophy can be characterized by cell cycle arrest in the G1 phase. The molecular mechanism arresting MCs in the G1 phase of the cell cycle after prolonged exposure to high glucose is the induction of cyclin-dependent kinase (CdK) inhibitors such as p27Kip1 [24, 25]. The findings that deletion of the p27Kip1 gene in MCs attenuates the high-glucose-induced cell hypertrophy and that stimulation of TGF-β expression by high glucose is essential for stimulated expression of p27Kip1 as well as that TGF-β1 blockade prevents glucose-induced MC hypertrophy provided clear evidence that this effect is mediated by TGF-β1 [23, 24]. TGF-β and podocyte pathology Podocytes are highly differentiated polarized epithelial cells with a complex cellular morphology. The main cell body bulges into the urinary space and gives rise to long primary cytoplasmatic processes that divide into individual foot processes, which adhere to the outer surface of the glomerular basement membrane (GBM). The foot processes of neighbouring podocytes interdigitate, leaving between them filtration slits that are bridged by the slit diaphragm [15, 23]. Proteins that anchor foot processes to the GBM [e.g. α3β1-integrin and integrin-linked kinase (ILK)] and those associated with the slit diaphragm (e.g. nephrin and podocin) are crucial for a normal functioning of the filtration barrier [26]. Podocytes sustain the structural integrity of the GBM and synthesize most, if not all, components of the GBM, such as collagen type IV, laminin, fibronectin, agrin and heparan sulphate proteoglycan [23]. In cultured mouse podocytes, it has been shown that TGF-β is the key mediator of ECM production (e.g. fibronectin and collagen type IV α3 chain) by the podocyte, contributing to GBM thickening (Figure 2) [23, 27]. Furthermore, podocytes not only produce GBM components but also secrete matrix metalloproteinases (e.g. MMP-9), important enzymes for matrix remodelling [15]. Glomerulosclerosis in human and animal models is characterized in part by the depletion of podocytes [28]. Mechanical detachment of podocytes from GBM and loss in urinary space, due to altered cell adhesion, has been proposed as a potential mechanism (Figure 2) [28]. The loss of cell anchorage to the GBM may result from downregulation of α3β1-integrin, which is observed in the podocytes of patients with primary FSGS and diabetes [15, 29]. In nephrotic rats as well as in cultured podocytes, it has been shown that TGF-β1 significantly suppresses α3β1-integrin expression [15]. In the adult kidney, it is thought that podocytes are unable to undergo regenerative proliferation and to replace those that have been lost in most forms of glomerular injury, including DN [23, 28]. In DN, structural abnormalities of podocyte foot processes, such as foot process widening, effacement and narrower filtration slits, have been described (Figure 2) [23, 29]. The broadening of the foot process widths, which would be a compensatory response of the remaining podocytes in the attempt to cover areas of bare GBM, is caused by a TGF-β-stimulated decrease of nephrin protein production [23, 27, 29]. However, there is a reduced number of podocytes in podocyte-associated glomerular diseases, but it is not clear whether there is an absolute reduction in podocyte number or a relative reduction due to increased GBM area caused by both glomerular hypertrophy and enhanced glomerular volume expansion [15, 23]. Another mechanism that could explain the podocyte loss in glomerulosclerosis may relate to podocyte apoptosis. In podocytes, enhanced TGF-β1 expression and TGF-β1 activity induce podocyte apoptosis (Figure 2) [23, 29, 30]. In TGF-β1 transgenic mice with progressive renal disease induced by elevated circulating TGF-β1, podocytes undergo apoptosis at early stages in the course of glomerulosclerosis [30]. It has been shown that in the damaged podocytes of the TGF-β1 transgenic mice, as well as in cultured murine podocytes challenged with TGF-β, Smad7 expression is strongly induced [30]. Furthermore, whereas the TGF-β-mediated apoptosis of podocytes depends on p38 MAPK and caspase-3, the increased Smad7 expression independently causes apoptosis via blocking the transcriptional activation of the cell survival factor NF-κB, suggesting a novel functional role for Smad7 as amplifier of TGF-β-induced apoptosis in podocytes [30]. It should be noted that podocyte depletion by detachment and/or apoptosis often takes place in the advanced stages of chronic renal disease where proteinuria is already prominent, arguing against a causative role of early podocyte loss in the genesis of microalbuminuria [31]. Recent experimental evidence has supported the hypothesis that podocytes may undergo epithelial-to-mesenchymal transition (EMT) after injury, leading to podocyte dysfunction that ultimately leads to defective glomerular filtration (Figures 2 and 3) [31]. EMT is a process of reverse embryogenesis that occurs under pathological conditions in many organs; e.g. in diseased kidneys. Although it has been suggested that tubular cells may undergo EMT after chronic injury [32, 33], an increasing number of studies raise some doubts about the existence of this process in vivo [34]. The central issue is that to detect EMT in vivo, one would like to view epithelial cells transitioning into fibroblasts and migrating to the interstitium in real time. Unfortunately, this is not feasible with current technology. Instead, one is left with compelling snapshots of the transitional process through histochemical analysis of fibrogenic tissue [35]. To avoid the most controversial aspect of EMT, we use a weaker term such as ‘EMT-like changes’. However, in primary FSGS and idiopathic collapsing glomerulopathy, EMT-like changes have also been found in podocytes [36]. In both experimental and human DN, it has been reported that glomerular podocytes show decreased expression of specific markers, such as nephrin and ZO-1 (zonula occludens 1), as well as increased desmin and FSP-1 (fibroblast specific protein 1) expressions, characteristic of mesenchymal cells [31, 36]. Furthermore, in vitro experiments on cultured podocytes have shown that TGF-β1, which is known as a potent EMT inducer, suppresses the slit diaphragm-associated protein P-cadherin, ZO-1 and nephrin via induction of Snail as well as enhances the expression of the mesenchymal marker desmin [31]. FIGURE 3: Open in new tabDownload slide Hypothesis of progression of fibrosis by EMT and EndMT in the interstitium (A) or in the glomerulus (B). Of note, this is a hypothesis that is mainly derived from in vitro analysis. Whether the same is true in vivo is debated heatedly at present. Initiated by TGF-β1, epithelial (tubular cells, podocytes) or endothelial cells lose their cell–cell contacts and begin to express mesenchymal markers; they undergo EMT followed by EndMT. In the interstitium, these cells disengage themselves from the cell connective and transdifferentiate to interstitial myofibroblasts, which are responsible for the increased synthesis of ECM leading to tubulointerstitial fibrosis. In the glomerulus, the TGF-β1-induced biochemical changes in the cells contribute to excessive ECM deposition and podocyte loss, characteristics of glomerulosclerosis. (Figure modified after [49]). FIGURE 3: Open in new tabDownload slide Hypothesis of progression of fibrosis by EMT and EndMT in the interstitium (A) or in the glomerulus (B). Of note, this is a hypothesis that is mainly derived from in vitro analysis. Whether the same is true in vivo is debated heatedly at present. Initiated by TGF-β1, epithelial (tubular cells, podocytes) or endothelial cells lose their cell–cell contacts and begin to express mesenchymal markers; they undergo EMT followed by EndMT. In the interstitium, these cells disengage themselves from the cell connective and transdifferentiate to interstitial myofibroblasts, which are responsible for the increased synthesis of ECM leading to tubulointerstitial fibrosis. In the glomerulus, the TGF-β1-induced biochemical changes in the cells contribute to excessive ECM deposition and podocyte loss, characteristics of glomerulosclerosis. (Figure modified after [49]). TGF-β and endothelial cells Progressive renal disease is characterized in part by progressive loss of the glomerular and peritubular capillaries, associated with increased apoptosis of endothelial cells, and it correlates with the development of glomerulosclerosis and tubulointerstitial fibrosis (Figure 2) [28]. TGF-β regulates many endothelial functions including cell proliferation (e.g. in early DN) resulting in glomerular hypertrophy, but also the induction of apoptosis in microvascular endothelial cells (Figure 2) [22, 28]. Furthermore, it has been demonstrated that endothelial cells additionally contribute to the emergence of fibroblasts during kidney fibrosis via a mechanism called endothelial-to-mesenchymal transition (EndMT) in three different mouse models of chronic kidney disease: unilateral ureteral obstructive nephropathy, Alport renal disease and DN (Figure 2) [37]. EndMT is, similar to EMT, a process that is involved in fibrosis by ultimately increasing the number of glomerular myofibroblasts (Figure 3) [22]. It has been demonstrated that ∼30–50% of fibroblasts, detected in the three mouse models of renal fibrosis, coexpressed the endothelial marker CD31 and markers of fibroblasts/myofibroblasts such as FSP-1 and α-smooth muscle actin (α-SMA) [37]. The findings that TGF-β1 induced de novo expression of α-SMA and loss of expression of VE-cadherin and CD31 in primary cultures of renal endothelial cells and that a specific inhibitor for Smad3 abrogates TGF-β-induced EndMT demonstrate that TGF-β1 is a central inducer of EndMT via a Smad3-dependent pathway [38]. Tubular effects of TGF-β There are several consequential tubular pathological events that are mediated by TGF-β1 during the progression of chronic kidney diseases, such as interstitial fibrosis, EMT-like changes and epithelial cell apoptosis and proliferation (Figure 2) [22]. In various experimental models and human forms of progressive renal disease (e.g. DN), the expression of TGF-β1 has been associated with apoptotic tubular epithelial cells, causing tubular atrophy (Figure 2) [28]. In contrast, experiments using anti-TGF-β1 antibodies to inhibit TGF-β have shown reduced tubular apoptosis, and a reduced extent of tubular atrophy was seen in the tubular compartment in models of chronic kidney disease [22, 28]. In addition, tubuloepithelial hypertrophy, characterized by cell cycle arrest in the G1 phase, is a precursor of the later irreversible changes in the tubulointerstitial architecture leading to tubular atrophy and interstitial fibrosis (Figure 2). Part of this association between growth and fibrogenesis may be due to the fact that similar networks of cytokines and growth factors that induce cellular hypertrophy can also stimulate ECM synthesis and deposition [24]. For example, tubular hypertrophy is an early renal structural feature of DN and it has been shown that tubular epithelial cells obtained from TGF-β1 knockout mice failed to undergo hypertrophy and collagen synthesis when cultured in high-glucose medium [24, 39]. An interesting hypothesis is that early hypertrophy of tubular cells may facilitate apoptosis by arresting cells in the G1 phase of the cell cycle and exposing them to longer periods of apoptotic conditions without the opportunity of cell renewal through the progression of the cell cycle [24]. Tubular atrophy/loss as well as interstitial fibrosis can also occur by EMT in which proximal tubular epithelial cells, similar to podocytes and endothelial cells, transdifferentiate to acquire (myo)fibroblast phenotypes (Figures 2 and 3) [22, 28]. TGF-β in renal fibrosis and EMT Progressive renal damage is evidenced by glomerulosclerosis, tubulointerstitial fibrosis, infiltration of inflammatory mediators and the activation of α-SMA-positive myofibroblasts [40]. Of these fibrotic changes, progressive tubulointerstitial fibrosis and tubular atrophy represent the final common pathway characteristic of all kidney diseases leading to chronic renal failure [40, 41]. The most striking feature of tubulointerstitial fibrosis is the excessive deposition of ECM, in particular collagenous fibres. The widened interstitial space in fibrotic kidneys is filled with fibrillar material consisting predominantly of collagens type I and III and fibronectin [41]. The pathophysiology of tubulointerstitial fibrosis is divided into four arbitrary phases [42]. First in the cellular activation and injury phase, tubular, perivascular and mononuclear cells are activated and begin to populate the interstitium and release proinflammatory and injurious molecules. The second phase (fibrogenic signalling phase) is characterized by the production of fibrosis-promoting factors, such as TGF-β1, connective tissue growth factor (CTGF), Ang II and PDGF. In the third phase, ECM production increases and matrix degradation decreases, which results in the fourth phase (destruction phase), in which the number of intact nephrons progressively declines resulting in a continuous reduction in renal function [42]. Proximal tubular cells are the predominant cell type in the normal renal interstitium, in which there are normally relatively few resident interstitial fibroblasts. These resident interstitial fibroblasts lie in close apposition to the proximal TBM and accumulate in damaged tissues [39, 41]. The matrix-producing fibroblasts in the renal interstitium are the major source of the increased ECM, but their origin remains controversial [41–44]. Strutz et al. [32] hypothesized and Iwano et al. [33] confirmed that up to 36% of all interstitial fibroblasts may originate from tubular cells damaged by EMT (Figure 3). EMT is a highly regulated process, which is defined by four key events: (i) loss of epithelial cell adhesion molecules such as E-cadherin and ZO-1, (ii) de novo α-SMA expression and actin reorganization, (iii) disruption of TBM and (iv) enhanced cell migration and invasion of the interstitium (Figure 3) [40, 45, 46]. Evidence for EMT-like changes of tubular epithelial cells has been shown in human renal biopsies obtained from various renal diseases, including DN, membranous nephropathy, IgA glomerulopathy, primary FSGS and lupus nephritis [47]. In this study, tubular phenotype alterations were strictly associated with the degree of renal functional impairment independent of histological diagnosis, confirming the hypothesis that from a certain point tubulointerstitial events become independent of the original cause of renal disease and lead to progression of damage [47]. However, TGF-β1 is widely recognized as a strong inducer of fibrosis in renal structures during chronic kidney disease [22] and affects all four mentioned key events that occur in tubular EMT: (i) TGF-β1 downregulates the expression of E-cadherin and ZO-1 and induces the proteolytic shedding of E-cadherin by matrix metalloproteinases (MMPs) in vitro; (ii) in response to TGF-β1 cultured proximal tubular epithelial cells undergo dramatic reorganization accompanied by the de novo expression of α-SMA and (iii) TGF-β1 induces proteases such as MMP2 and MMP9 that disrupt intact tubular basement membranes, (iv) allowing the cells undergoing EMT to disengage themselves from cell connectivity and invade the interstitium [48, 49]. Of note, these are results obtained from in vitro analysis—whether the same is true of in vivo situation is currently not clear. THE TGF-β AXIS AS A POTENTIAL THERAPEUTICAL TARGET The complex role of TGF-β in renal diseases potentially offers a number of molecular targets for treatment approaches. As TGF-β is upregulated in almost all progressive renal disorders, TGF-β-suppressing treatments appear promising [16]. Treatment with antibodies to block TGF-β directly or to inhibit TβR-II dramatically decreases the excessive deposition of ECM and ameliorates renal fibrosis in chronic kidney disease as well as attenuates acute proximal tubule injury [19, 50–54]. In models of acute and chronic renal injury, increased levels of TGF-β are paralleled by a reduction in the expression of BMP-7 (bone morphogenic protein 7), a homodimeric protein and a member of the TGF-β superfamily [40]. In a mouse model of chronic renal injury, systemic administration of recombinant human BMP-7 reverses TGF-β1-induced de novo EMT and leads to the repair of severely damaged renal tubular epithelial cells, in association with a reversal of chronic renal injury [55]. An interesting study has shown that treatment with an angiotensin-converting enzyme (ACE) inhibitor lowers serum TGF-β1 levels in patients with DN. These treated patients have tended to better maintain renal function, suggesting that ACE inhibitor therapy may protect the kidney partly by lowering TGF-β1 production [6]. In addition, there are other strategies with proven efficacy for treatment of kidney diseases and tissue fibrosis, which exert some of their effects by interfering with components of the TGF-β pathways, but only a small fraction of these approaches have been studied in humans and even fewer have been successfully translated into clinical use for patients with kidney disease. CONCLUSION The cytokine TGF-β plays a central role in the glomerular and tubulointerstitial pathobiology of renal disease by contributing to pathological alterations which induce alterations of glomerular filtration barrier, glomerulosclerosis and fibrosis and the degeneration of tubules leading to permanent renal dysfunction. While the TGF-β axis can be a powerful potential target of therapeutic strategies, it should also be noted that a chronic inhibition of the actions of TGF-β could have grave side effects. Overall, a better understanding of the specific role of TGF-β and its crosstalk pathways, especially in modulating the response to acute kidney injury, could improve the development of effective therapeutic approaches to kidney disease. For example, a promising human trial with fresolimumab, a human monoclonal antibody that neutralizes all active isoforms of TGF-β, in treatment-resistant primary FSGS has just started. The results of the phase I clinical trial indicate that single-dose fresolimumab is safe and well tolerated in patients with treatment-resistant primary FSGS. Therefore, additional evaluation with larger randomized clinical trials to assess the efficacy of this agent will follow [56]. CONFLICT OF INTEREST STATEMENT None declared. REFERENCES 1 Dennler S , Goumans MJ , ten Dijke P . Transforming growth factor beta signal transduction , J Leukoc Biol , 2002 , vol. 71 (pg. 731 - 740 ) Google Scholar PubMed OpenURL Placeholder Text WorldCat 2 Shi Y , Massagué J . Mechanisms of TGF-beta signaling from cell membrane to the nucleus , Cell , 2003 , vol. 113 (pg. 685 - 700 ) Google Scholar Crossref Search ADS PubMed WorldCat 3 Massagué J . TGF-beta signal transduction , Annu Rev Biochem , 1998 , vol. 67 (pg. 753 - 791 ) Google Scholar Crossref Search ADS PubMed WorldCat 4 Kingsley DM . 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