Novel Aspects of Transforming Growth Factor-Beta in Diabetic Kidney Disease

Over the past 10 years, it has been established that the multifunctional cytokine, transforming growth factor-beta (TGF-β) plays a critical role in the development of diabetic kidney disease. Among the myriad of growth factors and cytokines described, TGF-β was recognized early on as a likely candidate to mediate the renal manifestations of diabetes [1, 2].Diabetic kidney disease is characterized in its early stages by hyperfiltration, glomerular hypertrophy, and tubular hypertrophy. In the chronic stage, there is glomerular basement membrane (GBM) thickening, microalbuminuria, and diffuse mesangial matrix expansion [3, 4]. In the progressive stage of diabetic nephropathy as clinically identified by a decline in the glomerular filtration rate (GFR) and increase in albuminuria, there is a further increase in mesangial matrix expansion, hyalinization of the arterioles, tubulointerstitial fibrosis, and tubular atrophy [3, 4]. Recently, it has been recognized that a reduction in podocyte number, but not mesangial or glomerular endothelial cell numbers, is noted with progressive diabetic nephropathy [5, 6].It is striking that very similar effects of TGF-β are found in cell culture and animal models of kidney disease. TGF-β induces cell hypertrophy by inhibiting cell proliferation and increasing cellular protein synthesis in mesangial cells and proximal tubular epithelial cells [7, 8, 9]. TGF-β stimulates production of type IV collagen by glomerular epithelial cells [10]and stimulates production of type IV collagen, type I collagen, fibronectin (FN), and plasminogen activator inhibitor-1 (PAI-1) in mesangial cells and thus may contribute to GBM thickening and mesangial matrix expansion [11, 12, 13]. The recent description that TGF-β induces apoptosis of podocytes [14]may have relevance to loss of podocytes in patients with progressive diabetic nephropathy [5, 6]. TGF-β also stimulates type I and type III collagen production in fibroblasts [15, 16]and regulates apoptosis in tubular epithelial cells [17]. Therefore, TGF-β may contribute to tubulointerstitial fibrosis and tubular cell atrophy.In the present review, we will briefly highlight the experimental and clinical data implicating TGF-β in diabetic kidney disease. We will then focus on three areas that are closely related to TGF-β and the pathogenesis of diabetic kidney disease. We will discuss (1) the mechanisms underlying high glucose-induced stimulation of TGF-β; (2) the signaling pathways underlying TGF-β-induced matrix production, and (3) the novel role of TGF-β in mediating diabetic vascular dysfunction.Several studies in experimental animal models of diabetic kidney disease provide evidence that TGF-β production is stimulated at both early and late time periods of diabetes. In two animal models of spontaneous diabetes (BB rat and NOD mouse), a short duration of hyperglycemia (3–7 days) was sufficient to stimulate renal TGF-β1 mRNA expression [18]. TGF-β1 mRNA and protein levels are also significantly increased in the kidney cortex of streptozotocin (STZ)-induced type 1 diabetic rat [19, 20, 21], and mouse [22]. Glomerular expression of TGF-β1 mRNA is increased as early as 2 days after onset of hyperglycemia [21, 23]and progressively increases up to 16–24 weeks of diabetes [20, 24]. In addition, the TGF-β2 isoform and TGF-β type II receptor are also increased in different rat and mouse models of type 1 and type 2 diabetes [22, 24, 25, 26]. With the use of neutralizing monoclonal pan-selective anti-TGF-β antibody [22]or TGF-β2 specific anti-TGF-β antibody [26], it is clear that TGF-β plays a critical role in the early changes of glomerular hypertrophy and increased matrix mRNA as noted in the STZ-induced mouse and STZ-induced rat model of type 1 diabetes. In addition, chronic inhibition of TGF-β with a neutralizing monoclonal pan-selective anti-TGF-β antibody in db/db mice prevents the glomerulosclerosis and renal insufficiency resulting from type 2 diabetes [24].Several studies have also implicated an important role for TGF-β in the pathogenesis of human diabetic nephropathy [2]. Patients with diabetic nephropathy demonstrate upregulation of TGF-β1 mRNA and protein in the glomerulus [28, 29]and tubulointerstitium [28]. We previously demonstrated that nondiabetic patients exhibit net renal extraction of circulating TGF-β1 whereas diabetic patients exhibit net renal production of TGF-β1, even before established kidney disease [30]. Recently, we found that captopril treatment was associated with reduction in circulating levels of TGF-β1 and that the reduction of TGF-β1 levels correlated with preservation of renal function [31]. These findings implicate a critical role of TGF-β in the pathogenesis of both human and experimental diabetic nephropathy.The initial description that high glucose concentration is a sufficient stimulus for TGF-β1 stimulation was originally described by Ziyadeh and colleagues [7, 8]. Using transformed murine mesangial cells and murine proximal tubular cells, it was found that high glucose (450 mg/dl) stimulated the steady state mRNA levels and protein of TGF-β1 after a 48- to 72-hour incubation period. Based on these results, it is clear that high glucose for a relatively short period of time is a potent stimulus of TGF-β production independent of the formation of early or advanced glycation products and is independent of hemodynamic stress. Subsequently, it has been recognized that modification of proteins by glycation, by both the Amadori products [32]as well as advanced glycated end products (AGEs) [27, 33, 34]are also able to stimulate TGF-β1 production independent of high glucose. Additionally, a variety of other stressors that are recognized to play a role in progressive diabetic nephropathy have been identified to stimulate TGF-β1. These stressors, among others, include cell stretch and oxidative stress (fig. 1).There are several pathways by which high glucose may stimulate TGF-β1 production in mesangial cells. Primarily, the mechanisms underlying high glucose-induced TGF-β1 stimulation appears to be at the transcriptional level, as the mRNA half-life was not affected by high glucose and nuclear run-off studies demonstrated increased TGF-β1 gene transcription by high glucose [35]. These studies were performed in murine mesangial cells, and although similar processes may be occurring in human cells, definitive studies have not been performed as yet.The initial necessary step is glucose entry into the cells. In mesangial cells, this occurs via the glucose transporter-1 (GLUT1) [36]. Overexpression of GLUT1 in mesangial cells leads to an increase in intracellular glucose levels in normal glucose conditions and an increase in TGF-β1 expression [37]. It appears that only certain cell types, such as mesangial and vascular smooth muscle cells, upregulate their glucose transporter in the presence of high glucose whereas other cell types do not [38]. TGF-β1 itself stimulates glucose uptake by enhancing the expression of GLUT1 in mesangial cells [39].The major intracellular pathway underlying the high glucose stimulation appears to involve the PKC-mitogen-activated protein kinase (MAPK) pathway. Several studies have found that PKC-inhibitors block the high glucose effect and PKC-activators stimulate TGF-β1 production [35, 40]. However, these studies are limited by the non-specific nature of the inhibitors and the potent effect of the phorbol ester to stimulate PKC isoforms, which may exceed the capacity of high glucose to stimulate PKC isoforms. With the recognition of multiple PKC isoforms, several groups have attempted to identify which PKC isoform is critical to this process. The majority of the studies have found that the PKC-β isoform is preferentially up-regulated by high glucose in mesangial cells and in the glomeruli of diabetic rats [42, 42]. The most convincing evidence to date is the use of the selective PKC-β inhibitor, LY333531, to block PKC-β activation in diabetic glomeruli and block glomerular TGF-β1 production in the diabetic glomeruli [43, 44]. However, it should be noted that other PKC isoforms have been found to be stimulated by high glucose as well, including PKC-ζ, PKC-δ, PKC-ε, etc. [45, 46, 47]. Further studies with specific inhibitors or the use of genetic manipulation to selectively block discrete isoforms is required to ascertain the relative importance of the PKC isoforms in this context.The role of the MAPK pathway has recently been recognized to be important in mediating diabetic glomerular pathology. The initial study by Haneda et al. [48]described MAPK activation in glomeruli of diabetic rats. In murine mesangial cells, high glucose-induced activation of extracellular signal-regulated kinases (ERK) is blocked by both MAPK inhibitors as well as PKC inhibitors [49]. Furthermore, MAPK inhibitors blocked high glucose-induced stimulation of TGF-β1 mRNA and the TGF-β1 promoter [49]. One theory put forward is that high glucose initially stimulates PKC, which then stimulates MAPK to regulate transcription factors that regulate TGF-β1 gene transcription [49, 50].The identification of the various transcriptional factors regulating high glucose-induced TGF-β1 gene transcription remains unclear. Several groups have implicated the activated protein-1 (AP-1) complex as being critical for this effect. High glucose does stimulate c-fos formation and increased AP-1 binding by gel-shift assays [51]. Inhibitors of PKC can block formation of AP-1 complex and decrease its binding. Furthermore, AP-1 and Sp1 has been implicated in the stimulation of gene transcription of the TGF-β1 promoter [51]. An inhibitor of AP-1 (curcumin) was able to inhibit high glucose-induced TGF-β1 promoter activity whereas an inhibitor of Sp1 (mithramycin) failed to do so [51].There was a small increase in AP-1 binding from cells grown in high glucose and both JunD and c-Fos were present in the complex [51].Although it is likely that the AP-1 complex is important in mediating stimulation of TGF-β1 by high glucose, there appears to be alternative pathways how this may occur. The high glucose-induced transcriptional stimulation of TGF-β1 has been studied in depth in the murine system [35]. The murine TGF-β1 promoter does not have conserved AP-1 binding sites and AP-1 binding has not been demonstrated with sites in the murine promoter [52]. Our recent studies have identified a region in the murine promoter that exhibits a strong high glucose-induced binding of several transcription factors [35]. Ongoing studies are in process to identify the nature of these transcription factors. Our group has recently identified an E-box element as important for mediating high glucose stimulation of the murine TGF-β1 promoter. High glucose is also able to stimulate binding of nuclear proteins that recognize the E-box element specifically. Interestingly, the E-box has been recognized to be an important site mediating carbohydrate-induced gene transcription of a number of carbohydrate-induced genes in the liver [53, 54, 55]. These genes, including L-type pyruvate kinase and S14, contain two E-box-like elements separated by 5 bases [54]. The upstream stimulatory factor (USF) family of transcription factors has been implicated in carbohydrate-induced regulation of these promoters [56]. Interestingly, in preliminary studies we found that high glucose markedly stimulates USF-1 binding and USF-1 protein levels in murine mesangial cells [57]. Ongoing studies are evaluating the significance of the USF family in mediating high glucose-induced effects on the TGF-β1 promoter.Sp1 has also been implicated in mediating glucose-induced TGF-β1 stimulation. In an elegant series of studies, Brownlee’s group found that high glucose stimulates Sp1 O-glycosylation [58]. This modification of Sp1 appears to be capable of driving TGF-β1 gene transcription. O-glycosylation of Sp1 may be via the hexosamine pathway and driven by oxidant radical formation [58, 59]. Thus a unified theory would glucose-induced radical PKC and MAPK and hexosamine by Sp1 and AP-1 from TGF-β1 gene it is clear that there is increased TGF-β1 as well [35]. TGF-β1 is initially in a that be activated in for the to to its Recently, it has been recognized that the protein is able to TGF-β1 from its to its The site in for TGF-β activation has been to a specific Several groups have demonstrated that is stimulated in mesangial cells by high glucose as well as in various models of progressive kidney In a cell culture to block are able to block high glucose-induced TGF-β activation this pathway may also involve PKC activation from high glucose and other factors in diabetic kidney such as II cell stretch are potent of TGF-β The II is by the II type 1 receptor whereas the cell stretch effect may be by the MAPK pathway a variety of stressors are important in TGF-β in the of diabetic kidney disease. from the initial stressors, it is well that TGF-β has a potent pathway can its and of these pathways to stimulate TGF-β would be an important to in progressive diabetic TGF-β is it can then potent matrix effects in the kidney TGF-β has been to stimulate the production of including type III and IV collagen and production both in glomerular mesangial and epithelial cells [11, In addition, TGF-β the of by inhibiting the synthesis of and matrix and the synthesis of and inhibitor of Recently, growth factor expression has been to be by and also to stimulate protein synthesis may be in the pathogenesis of fibrosis, as a of These mechanisms contribute to and to glomerulosclerosis in diabetic evidence that TGF-β contribute to in of TGF-β1 gene into the rat kidney has been to glomerulosclerosis In mouse models TGF-β1 in the glomerulus of the promoter in the liver of the murine promoter there was renal of collagen type IV and mice a human TGF-β1 of the rat tubulointerstitial fibrosis, with collagen and increased fibronectin and of TGF-β that cells and that TGF-β is activated or from protein complex by or before binding its and that intracellular signaling is to the The intracellular pathways to matrix production include protein kinase and other pathways identification of the family has to the of mechanisms by which TGF-β from the cell to genes in TGF-β its effects by binding to two types of with kinase The to the type II which the type I receptor by The activated type I receptor then such as and that with The complex into the it transcription of genes two and have been identified in only has been genes are by binding of to specific promoter which are elements or by of with other transcriptional such as AP-1 and such as have been identified in the promoters of TGF-β genes, including type I collagen, type collagen, and the region and which are related to the other as by the activity with activated type I and the of Interestingly, inhibition of TGF-β signaling by and may be increased binding of to type I receptor TGF-β also stimulates itself to activation also MAPK pathway are three major of MAPK the the protein kinase and of have been to be activated by TGF-β in several cell types including mesangial cells TGF-β activation of the MAPK pathway and pathways to of element binding protein with kinase 1 be a potent activator of both the and pathways TGF-β receptor family of small have also been in the TGF-β and activation MAPK has been to on by several groups of by or can to of the of resulting in inhibition of nuclear of and of have previously that TGF-β stimulated the signaling pathway by in kinase assays as well as by of the of from to We also demonstrated that activation to TGF-β stimulation of and gene expression in murine mesangial cells The effects of high glucose on synthesis in mesangial cell have been also to be by the hexosamine pathway via regulation of and signaling pathway recent also demonstrated PKC PKC of leads to of the growth effect of TGF-β the role of PKC in TGF-β-induced matrix production has not been are on the of the signaling of production by TGF-β in the kidney and in other previously TGF-β stimulates a variety of matrix and matrix It is clear that matrix gene has its signaling pathways and transcriptional factors that are in regulation of that gene by this we will discuss the signaling pathways that are to TGF-β of type I collagen, and I collagen is of two and which are but by are several that TGF-β stimulates the type I collagen gene the pathway by binding of a complex to the in the promoter Recently, several groups that Sp1 and proteins and their plays an important role in mediating TGF-β-induced expression the have recently found that the of type I collagen expression is also on at the and after stimulation with TGF-β These that pathway is a necessary and critical factor for both and gene In to these studies, et al. that the by TGF-β is on the MAPK pathway in rat mesangial cells a et al. that both and of inhibit TGF-β-induced expression and promoter In this and MAPK pathway may also function as a in the signaling of production by TGF-β1. inhibition of type I collagen synthesis by TGF-β by of by cytokines and growth factors may be of of the signaling pathway is likely to TGF-β-induced type I collagen production as both the and of type I collagen are on or and However, enhancing may be as has recently been to apoptosis of podocytes In addition, identification of the transcription factors that with and are in type I collagen gene stimulation may provide for the use of transcription to the of type I collagen by TGF-β via there are several that the is for TGF-β-induced This has been in cell such as and cell well as fibroblasts fibroblasts In these cell the TGF-β-induced expression was the in cells as to cells. have been studies TGF-β-induced stimulation in mesangial cells with to We recently demonstrated that TGF-β-induced stimulation was not in mesangial cell from the other pathways have been implicated in TGF-β-induced stimulation. et al. that TGF-β-induced expression was on the pathway in human cell we have also demonstrated that TGF-β stimulates the signaling pathway and that activation may contribute to TGF-β stimulation of and gene expression in murine mesangial cells et al. that TGF-β increases activity in rat mesangial cells and the inhibitor, TGF-β-induced expression was on the activation of MAPK pathway. TGF-β can increase the activity of AP-1 of by or the activity of of These findings the that TGF-β upregulate AP-1 or genes such as in a It is so which TGF-β signaling pathway is most critical for production in kidney has been implicated in as well as renal of diabetes It has been recognized that TGF-β is a potent of gene transcription et al. that the TGF-β element of the gene three binding of to was found to be critical for of expression by TGF-β However, other studies have that the expression of with TGF-β stimulation may not be on et al. that there were and elements in the promoter that of a TGF-β-induced complex on of two in the human promoter not but on binding site et al. that the promoter is by TGF-β by two The promoter is activated by TGF-β-induced binding of a nuclear complex to the The proximal promoter and on the other two Sp1 sites and of independent of binding. these mechanisms may be required for of the although are very important in the of TGF-β in production, it is also likely that both pathway and other pathways PKC are important for the production with stimulation by These pathways may contribute to TGF-β production and the signaling pathway be critical for the of of specific of matrix proteins at stages of disease on an of TGF-β signaling would likely to kidney disease is not only characterized by mesangial matrix but also by hemodynamic The and of diabetic nephropathy is related to the vascular of diabetes and as in the early stages of diabetic kidney disease. is closely associated with glomerular hypertrophy in diabetic kidney disease. The that anti-TGF-β prevents early diabetic glomerular hypertrophy that TGF-β is in the of early diabetes. there are vascular in early appears to be related to and increased glomerular This increase in glomerular may be as a of to the process in which is with in In the normal an increase in in to the increase in glomerular and at a The of via in the of increased is important to glomerular from in may the development of in early diabetic kidney disease is via a as well as In the increased is as increased to the has been to in membrane in both the appears to be via stretch or activated to there is entry into cells via is blocked by by extracellular from the The intracellular of the in to an and is has also been to resulting in increased levels of and It is role this it may contribute to membrane or cell The may be stimulated via this pathway and membrane by inhibiting occurs via the in increased This increase in tubular is by the as an increased concentration the of a of thus been as both entry and from intracellular of is from the in the of increased mediate and mediate via and is on normal of smooth muscle cells in the renal An increase in is a critical step of smooth muscle the complex to kinase the of at can then to the and resulting in in diabetes would an to be to the glomerular to increased and progressive glomerular The for in diabetes may be to of vascular smooth muscle cells to regulate intracellular are several of evidence that hyperglycemia the increase in intracellular in to or to of that is critical for the process to occur. uptake and was in vascular smooth muscle cells in high glucose as by conditions to high glucose in a decrease in In further the pathway was by membrane and stimulation of the by and increased uptake but failed to an increase of the uptake in the of high glucose. also uptake in to II and et al. the in to the in uptake with in STZ-induced diabetic rats. was significantly in the diabetic and in in in whereas only the these from diabetic The the the role entry via These studies implicate a in regulation to a of the in the as a of in diabetes It was that this may be by of the or of cells in the glomerulus increased intracellular glucose leads to increased synthesis of in increasing PKC et al. increased of and PKC activity in glomeruli of STZ-induced diabetic rats. with a inhibitor the PKC activity in these in the diabetic the and filtration to normal and significantly the Haneda et al. that inhibiting PKC production with which the increase in and in STZ-induced diabetic rats. These effects were independent of changes in glucose. also found that treatment the and extracellular matrix protein in the diabetic inhibition of PKC or MAPK also high glucose-induced TGF-β from intracellular the of the glomerular may be affected by PKC activity has been to mesangial cell is for mesangial cell and vascular smooth muscle cell and to et al. and increased PKC activation in mesangial cells in high glucose. These were in the PKC was by the inhibitor or for with the phorbol ester It likely that upregulation of the PKC pathway has a role in the glomerular of diabetes. this occurs via regulation of and the or is by such as is PKC activation can to increased TGF-β production and it is that TGF-β of the effects of high glucose and PKC to inhibit vascular smooth muscle cell function via regulation and has been to be an important in the development of diabetic expression of TGF-β occurs early in the of experimental with renal hypertrophy and treatment with anti-TGF-β glomerular hypertrophy, which is the of increased [22]. In addition, high levels of TGF-β and the TGF-β type II receptor are present in glomerular a role for TGF-β in the of in diabetes is an and an of has been to intracellular in mesangial cells well as the increase in intracellular in vascular smooth muscle cells The in increase may be to the effects of TGF-β on the receptor which regulates from intracellular of mesangial cells to TGF-β has been to the type I receptor at it TGF-β type I receptor mRNA protein type III receptor protein in mesangial cells. I and type III are also in renal vascular smooth muscle cells In STZ-induced diabetic mice and rats have type I receptor mRNA and protein levels as well as for this receptor in renal smooth muscle cells and glomeruli in with increased levels of TGF-β by a in TGF-β may cellular to has also been to increase production in early diabetes. weeks after rats were to have increased production demonstrated by increased levels of the products and as well as increased mRNA and protein in endothelial cells for the role of TGF-β from the that of human endothelial cells to TGF-β also increased mRNA This effect of TGF-β may be by increased the as increases in mRNA and protein with the increases in on several in studies in patients and the vascular effects of TGF-β are only to be of TGF-β2 to a decrease in in patients with multiple studies that of TGF-β2 in the Interestingly, the effect of TGF-β to be in the but not in the In a model of anti-TGF-β the development of is likely that TGF-β effects on specific vascular in the kidney disease We that TGF-β has potent effects on the glomerular not only in of cell growth and matrix formation but also in of vascular The to TGF-β may in glomeruli glomeruli to the in these The even as there are multiple TGF-β isoforms and TGF-β that are in the normal kidney and is in the diabetic kidney [22, studies discrete vascular of the renal with regulation of the TGF-β system is required to into this of TGF-β on the