Growth Factors and the Development of Diabetic Nephropathy

Gunter Wolf, MD
Address University of Hamburg, University Hospital Eppendorf, Department of Medicine, Division of Nephrology, Rheumatology, and Osteology, Pavilion N26, Martinistrae 52, Hamburg D-20246, Germany. E-mail: Wolf@uke.uni-hamburg.de Current Diabetes Reports 2003, 3:485490 Current Science Inc. ISSN 1534-4827 Copyright 2003 by Current Science Inc.

Growth factors play an important role in the development of functional and structural changes associated with diabetic nephropathy. Although it has been known for years that these factors are important for early renal hypertrophy and subsequent development of glomerulosclerosis and tubulointerstitial fibrosis, the exact molecular mechanism of many of these factors has only recently been more elucidated. Furthermore, growth factors also link the metabolic theory of diabetic complications with renal hemodynamic changes in diabetic nephropathy because some growth factors could directly influence glomerular hemodynamics and tubular transport in diabetic nephropathy. The high glucose environment with stimulated cellular uptake of glucose and accelerated nonenzymatic reactions resulting in Amadorimodified proteins and the later-developing advanced glycation end products are the main stimulators for intrarenal induction of growth factors. Intracellular generation of reactive species is an important signal intermediate in these stimulated expressions of growth factors. Taking into consideration the pivotal role of growth factors in the development of diabetic nephropathy, a therapeutic strategy to antagonize growth factor effects appears to be straightforward. However, the pleiotropic function of many of these factors and their physiologic role in normal renal homeostasis may make this approach difficult.

Introduction
Diabetic nephropathy due to type 2 diabetes mellitus has become the single most common cause of end-stage renal disease (ESRD) in the entire Western world. Apart from the individual human suffering that cannot be expressed in numbers, patients with type 2 diabetes undergoing maintenance dialysis consume significantly more financial resources than those with nondiabetic ESRD. In addition, type 2 diabetic patients do poorly on dialysis and have an excessive mortality. Although functional and structural changes of the kidney start

directly with the onset of diabetes mellitus, it takes years for the development of dialysis-dependent renal insufficiency. Thus, there is an evolution and progression of diabetic nephropathy. Hemodynamic alterations such as glomerular hyperfiltration and structural changes of renal growth (eg, proliferation, hypertrophy) are the prevailing findings early in the course of diabetic nephropathy. Later, a progressive decrease in renal function, glomerulosclerosis, interstitial fibrosis, and tubular atrophy are found [1]. In parallel, the evolution of protein excretion in patients with type 1 and 2 diabetes from the microalbuminuric stage to one of overt proteinuria represents a continuum of progressively altered glomerular capillary wall function and structure. Meticulously performed morphometric studies on renal tissue from patients with type 1 diabetes provided convincing evidence that glomerular and tubular hypertrophy and basement membrane thickening are among the earliest pathologic alterations found in diabetic nephropathy. Moreover, similar observations have been made in patients with type 2 diabetes and in various experimental models of diabetic nephropathy. Various growth factors, cytokines, chemokines, and vasoactive agents have been implicated in these structural changes of diabetic nephropathy [15], although the classification of what substance is a growth factor has become somewhat arbitrary more recently because vasoactive substances such as angiotensin II and endothelins, or chemokines such as monocyte chemotactic protein-1, could all exert growth stimulatory actions on renal cells. Therefore, the present article focuses on insulin-like growth factor (IGF), transforming growth factor- (TGF-), connective tissue growth factor (CTGF), and vascular endothelial growth factor (VEGF). Due to space restrictions other classical peptide growth factors that could play a role in diabetic nephropathy (eg, epidermal growth factor or platelet-derived growth factor) could not be covered and the interested reader is referred to excellent recent reviews [6]. The potential renal functions of leptin, a hormone that has recently been discovered to exhibit growth stimulatory effects on renal cells and may also qualify as a growth factor, will also not be described [2].

Pathogenesis of Diabetic Nephropathy

For many years there was a hot debate on whether hemodynamic mechanisms or structural changes such as mesangial hypertrophy are more important in the development of diabetic nephropathy. However, more recently it became clear

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that hemodynamic changes and growth response are two sides of one medal and are intimately interwoven [1]. This is underscored by the observation that classical vasoactive substances such as angiotensin II exhibit renal growth stimulatory effects. Conversely, there is increasing evidence that growth factors such as IGFs or TGF- may modulate some of the renal hemodynamic alterations and stimulated transport processes associated with diabetic nephropathy [3]. Finally, mechanical stress can induce the release of growth factors from various cells, and this response is amplified in the presence of high-ambient glucose. Recent evidence suggests that increased superoxide formation following high glucose-induced throughput in the mitochondrial electron-transport chain generates reactive oxygen species, which are involved in the development of diabetic complications [4]. This increase in oxygen species requires an increased transport of glucose into the cell, indicating that high-ambient glucose is one of the major causes of diabetic organ complications. Furthermore, proteins modified by glucose or glucose-derived products such as methylglyoxal (ie, Amadori products), and advanced glycation end products (AGEs), are all important for the development of diabetic nephropathy. Increased mitochondrial oxidation of glucose activates protein kinase C (PKC) and subsequently mitogen-activated protein (MAP) kinases. Activation of PKC and MAP kinases are major signal transduction intermediates involved in the transcription, synthesis, and release of growth factors. Gene expression profile studies with mesangial cells exposed to high glucose revealed the induction of growth factors [5]. Induction of growth factors stimulates early glomerular and tubular hypertrophy in diabetes and also modulates extracellular matrix production, which ultimately leads to glomerulosclerosis and interstitial fibrosis [1]. Finally, growth factors could induce other cytokines, making them a pivotal step in a cascade of pathophysiologic alterations ultimately mediating diabetic nephropathy.

Insulin-like Growth Factors

Growth hormone (GH) and IGFs are among the most widely and earliest studied growth factors in diabetic nephropathy [6,7]. GH has been linked with the development of diabetes mellitus since the 1940s, when it was shown that anterior pituitary preparations could induce diabetes mellitus in dogs. GH and IGFs constitute a rather complex system with endocrine, paracrine, and autocrine functions involving IGFI and -II, two specific receptors for IGF, and several IGF-binding proteins (IGFBPs) [6]. The endocrine axis of this system involves GH that binds to specific receptors that have been demonstrated on collecting duct cells where GH stimulates IGF-I synthesis. GH may also directly influence transport processes in the proximal tubule. IGF-I itself binds to different renal cells along the nephron, including glomerular endothelial, mesangial, and proximal tubular cells where it exhibits growth stimulatory effects.

Cell culture studies have demonstrated that IGF-I induces proliferation and extracellular matrix production in various renal cell populations. In a more recent study, IGF-I induces hypertrophy of mesangial cells as determined by an increase in cell size and protein to DNA ratio [7]. Further studies into signal transduction pathways revealed that IGF-I activates Erk 1,2 MAP kinases and phosphatidylinositol 3-kinase (PI3K), but these pathways were not required for cell hypertrophy. Instead, IGF-I mediates hypertrophy of cultured rat mesangial cells via a calcineurin-dependent pathway that required nuclear translocalization NFAT (nuclear factor of activated T cell [7]). In addition, mesangial cells cultured from diabetic NOD mice synthesized increased amounts of IGF-I and exhibited decreased collagen turnover that was mediated by IGF-I [8]. Incubation of cultured human mesangial cells with IGF-I induces other growth factors, such as VEGF [9]. The nonreceptor tyrosine kinase Src plays an important role in this process [9]. Human diabetes mellitus type 1 and mouse models are characterized by hypersecretion of GH, whereas circulating IGF-I levels are reduced [10]. In a cross-sectional study, Cummings et al. [10] reported an increase in urinary IGF-I in children and adolescents with type 1 diabetes [11]. The findings in diabetic humans are in contrast to streptozotocin (STZ)induced diabetes in rats with reduced circulating GH [12]. There is an early and temporary increase in renal IGF-I protein after the onset of diabetes in various models. This increase is closely linked to hyperglycemia and is prevented by insulin application. Furthermore, a very early increase in IGFBP-1 and the IGF-II/mannose-6-phosphate receptor is found that precedes the development of diabetic hypertrophy. Renal expression studies of IGF-I mRNA have shown conflicting results [6,12], but it is clear that the increase in renal IGF-I protein content in diabetes is not paralleled by an increase in transcript expression as shown in the remnant kidney model. This dissociation may indicate a post-transcriptional mechanism of IGF-I synthesis, but a more convincing explanation is that the complex changes in renal IGFBPs expression during diabetes could lead to enhanced IGF-I trapping in the kidney and modification of bioactivity [6]. Interestingly, transgenic mice for IGFBP-1 developed a glomerulosclerosis without glomerular hypertrophy [13]. Although one would assume that an increase in IGFBP-1 would bind IGF-I resulting in an increase in GH because the negative feedback between IGF-I and GH is inhibited, the transgenic animals had no GH excess [13]. An interesting question is whether GH exerts direct renal effects in diabetes independently of IGF-I. However, such a hypothesis is not easy to test because many experimental manipulations to interfere with GH also directly or indirectly inhibit downstream IGF-I effects. Experiments in diabetic dwarf rats that have an isolated GH deficiency demonstrated a diminished renal hypertrophy. Renal enlargement was restored by GH infusions. However, diabetic dwarf rats also had a smaller rise in kidney IGF-I compared with diabetic control animals. Diabetic mice with a disrupted gene for the GH receptor did not develop diabetic nephropathy despite a

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similar degree of hyperglycemia as wild-type animals [14]. In diabetic mice treated with a long-acting GH antagonist, glomerular hypertrophy and renal IGF-I accumulation were abolished without affecting serum IGF-I [15]. Long-acting analogues of somatostatin, the endogenous GH releaseinhibiting hormone, have also been extensively used in various models of diabetic nephropathy. Chronic, but not acute treatment with octreotide prevented the renal hyperfiltration in STZ-induced diabetes of rats [16]. Octreotide treatment partially prevented renal hypertrophy and led to a decrease in renal IGF-I content of diabetic animals. Interestingly, this somatostatin analogue prevented the early increase of IGFBP1 mRNA and protein expression in diabetic kidneys [17]. These findings suggest that the protective effects of octreotide may be caused by less renal IGF-I binding or trapping. Mice transgenic for GH or IGF-I both had an increase in glomerular size, but only the mice with high circulating GH levels developed glomerulosclerosis [18]. Rats made diabetic with STZ showed more extensive glomerular matrix accumulation when injected with bovine GH. Taken together, there is good evidence that the GH/IGF-I system plays a role in the development of diabetic nephropathy. However, the exact contribution of the various components of this complex hormonal axis remain to be established.

The role of TGF- and its receptors type I and II in diabetic nephropathy begun with the seminal work of Ziyadeh et al. who demonstrated that high glucose medium increases TGF- mRNA and protein in renal tubular and mesangial cells. High glucose-induced TGF- expression is, at least to some extent, mediated by hexosamine pathway products such as glucosamine [19]. In addition, Amadori products as well as AGEs stimulate TGF- expression in various renal cells and upregulate TGF- receptors [19,20]. Glomerular hypertension is an additional potent stimulus for renal TGF- expression linking metabolic and hemodynamic effects [21]. TGF- and its type II receptor expression are increased in models of type 1 and 2 diabetes [22]. Finally, upregulation of the TGF- system and production of TGF- with release in the systemic circulation has been found in humans with diabetes [19]. A consensus nucleotide sequence termed the glucose response element has been characterized in the TGF- promoter [23,24]. Furthermore, activation of PKC and p38 MAP kinase plays a role through activating protein-1 (AP-1) binding sites in the transcriptional stimulation of the TGF- gene [24]. This high glucose-induced TGF-1 gene activation in mesangial cells is prevented by low molecular weight heparin that attenuates the enhanced binding of nuclear proteins to the regulatory AP-1 site. Finally, a posttranscriptional activation of latent to active TGF- is induced in mesangial cells through thrombospondin-1 [25]. TGF- stimulates the production of several extracellular matrix proteins including fibronectin, and types I, III, and IV collagens

Transforming Growth Factor-

[19]. However, some cell culture data show that high glucose-induced production of extracellular matrix can occur independently of TGF- [26]. Transforming growth factor- inhibits the degradation of extracellular matrix components through inhibition of matrix metalloproteinases. TGF-mediated upregulation of collagen type I expression in mesangial cells is blocked by activation of the peroxisome proliferator-activated receptor , suggesting a potential therapeutic pathway to interfere with extracellular matrix synthesis [27]. Conversely, TGF- has been shown in vitro to induce cell cycle arrest of mesangial and tubular cells with concomitant cellular hypertrophy [28]. This hypertrophic effect requires the presence of cell cycle inhibitors such as p21 and p27 [28]. In contrast, TGF- stimulates proliferation of tubulointerstitial fibroblasts. Recent evidence suggests a role of TGF- in the altered renal hemodynamics of early diabetic nephropathy. McGowan et al. [3] showed that TGF- phosphorylates the type 1 receptor for inositol 1,4,5-trisphophate (IP3), resulting in downregulation. Expression of this receptor is reduced in murine diabetes models. Because this receptor is important in IP3 mediated intracellular calcium release and vasoconstriction, a TGF-induced downregulation could explain the attenuated glomerular vascular contraction inducing hyperfiltration in early diabetic nephropathy [3]. Long-term treatment of mice with either type 1 or 2 diabetes and nephropathy with neutralizing antiTGF- antibodies prevented mesangial matrix expansion and attenuated the decrease in renal function [29]. Interestingly, the increased excretion of urinary albumin was not influenced [29]. Moreover, inhibition of renal TGF- activity with neutralizing antibodies partially reversed extracellular matrix expansion and glomerular basement thickening in a mouse model of already established diabetic nephropathy [30]. These findings suggest that inhibition of TGF- would be useful even if diabetic nephropathy has already developed. Although application of an anti TGF- antibody for prolonged time is not feasible in diabetic humans, alternative strategies to interfere with TGF- expression have emerged. Bone morphogenetic protein (BMP)-7, a member of the TGF- superfamily that plays a major role during embryonic development, can antagonize TGF-dependent fibrogenesis in renal cells [31]. Because BMP-7 expression is reduced in diabetic nephropathy, supplementation with BMP-7 could be a potential innovative concept to interfere with TGF-mediated changes of diabetic nephropathy.

Connective Tissue Growth Factor

Connective tissue growth factor is one of the more recently characterized growth factors and is a 36- to 38-kD cysteinerich peptide. CTGF may be the direct downstream mediator of some effects previously thought to be caused by TGF-. TGF- induces CTGF mRNA and protein expression in several renal cells [32]. Although TGF- and CTGF have overlapping effects

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on mesangial cells, it appears that CTGF exclusively mediates induction of a mesenchymal phenotype in proximal tubular cells [33]. Furthermore, CTGF regulates expression of adhesion molecules such as 51 integrin in mesangial cells [34]. This integrin is important in fibronectin matrix assembly and interaction of mesangial cells with extracellular matrix proteins. Exposing cultured mesangial cells to high glucose increased CTGF. This response is mediated by TGF-, because a TGF-neutralizing antibody blocked the stimulation [32]. CTGF expression is increased in glomeruli and tubules of diabetic mice [32]. Aminoguanidine prevents renal CTGF expression, suggesting AGEs as an important inducer [35]. Incubation of mesangial cells to recombinant CTGF or transfection of cells with a CTGF expression plasmid significantly increased fibronectin and collagen type I synthesis [36]. In contrast, CTGF-antisense constructs reduce the elevated synthesis of this extracellular matrix protein, and in high glucose culture CTGF induces cellular hypertrophy in mesangial cells by causing p21- and p27-mediated cell cycle arrest [37]. Interestingly, hepatocyte growth factor, a pleiotropic growth factor that is a ligand for the c-Met receptor tyrosine kinase, inhibits the expression of CTGF in tubular cells [38]. Because hepatocyte growth factor and its receptor are induced in the kidney of diabetic rats [39], this mechanism may represent an intrinsic response to counteract the profibrogenic effects transduced by CTGF [38]. Further studies such as interference with CTGF expression or receptor blockade are ultimately necessary to test its role in diabetic nephropathy.

Vascular Endothelial Growth Factor

The first two angiogenic growth factors, then thought to be highly specific for vascular cells, were isolated on the basis of their ability to mediate vascular leakage (vascular permeability factor) and proliferation of endothelial cells (VEGFs) in the late 1980s [40]. It turned out by sequencing that these proteins were identical and share approximately 20% of their amino acid sequence with platelet-derived growth factor. VEGF is an approximately 34- to 46-kD homodimeric glycoprotein that exists in at least five different isoforms that are produced by differential exon splicing [40]. In humans, VEGF165 is the most abundantly expressed and secreted isoform. VEGF binds on target cells to two high-affinity tyrosine kinase receptors called VEGFR-1 and VEGFR-2. In addition, a soluble VEGFR-2 protein (sFlt-1) has been found in the amniotic fluid. In the normal kidney, VEGFRs are expressed on endothelial and mesangial cells, interstitial fibroblasts, and renomedullary interstitial cells [41]. VEGF itself is expressed in podocytes, distal tubules, and collecting duct [42]. It has been speculated that heavier VEGF isoforms may be carried across the glomerular basement membrane exerting their effects on endothelial cells. An in vitro study demonstrated VEGF mRNA and protein expression in cultured human proximal tubules [43]. A pivotal role of VEGF in the neovascularization in proliferative diabetic retinopathy is well established. Because

VEGF was originally characterized as a vascular permeability factor, it has consequently been implicated in several proteinuric renal diseases including diabetic nephropathy. Circulating VEGF concentrations are significantly increased in type 1 and 2 diabetes [40]. Several studies indicate that VEGF mRNA and protein expression is enhanced in kidneys with early diabetic nephropathy [42]. In contrast, a decreased VEGF content, probably due to podocyte loss, has been described in more advanced diabetic nephropathy with glomerulosclerosis. High glucose stimulates VEGF protein expression in cultured mouse podocytes and this effect was mediated by TGF- [44]. Furthermore, AGEs bind to their specific receptor, named RAGE, on podocytes and contribute to the expression of VEGF [45]. Consequently, the synthetic thiazolidine derivate OPB-9196, a novel AGE inhibitor, suppressed overproduction of VEGF in OLETF rats, a model of type 2 diabetes [46]. A functional role of VEGF in diabetic nephropathy was demonstrated by the observation that monoclonal anti-VEGF antibodies administered to diabetic rats decreased hyperfiltration, albuminuria, and glomerular hypertrophy in these animals [47]. Because VEGF165 phosphorylates and activates endothelial nitric oxide synthase resulting in a local increase in nitric oxide, prevention of nitric oxide formation could therefore explain how anti-VEGF antibody attenuates hyperfiltration in diabetic nephropathy. A neutralizing anti-VEGF antibody also ameliorated long-term functional and structural changes in obese type 2 diabetic mice [48]. Exogenous VEGF leads to tyrosine phosphorylation of the VEGFR-2 in cultured mouse proximal tubular cells [49]. VEGF stimulates PI3K and also activated the downstream target of PI3K, Akt (protein kinase B). This treatment was associated with an increase in de novo protein synthesis, suggesting tubular hypertrophy. VEGF stimulated eukaryotic initiation factor 4E-binding protein phosphorylation in an Akt-dependent manner, indicating that early events in protein translation may explain the induced protein synthesis [49]. However, the situation may be much more complex in vivo. Neutralizing circulating VEGF caused proteinuria in normal mice that was associated with downregulation of nephrin expression [50]. In addition, exogenous VEGF 165 significantly enhanced capillary repair and convincingly improved renal function in rats with experimentally induced glomerulonephritis [42]. These data indicate that probably a certain amount of VEGF is necessary to maintain normal glomerular structure and function. Disturbance of this delicate balance by either under- or overexpression of VEGF may then lead to changes in glomerular function and structure of diabetes nephropathy.

Conclusions
In vitro and in vivo studies, as well as investigations in patients, have convincingly suggested that growth factors are important for the development of diabetic nephropa-

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thy. They mediate functional as well as structural renal changes. Several, but not all growth factors, fulfill Kochs postulate to qualify as a causative factor of diabetic nephropathy. The case of CTGF demonstrates that novel growth factors are still discovered that are involved in the kidney pathophysiology in diabetes. Targeting various growth factors at multiple points will be necessary to attenuate the development of diabetic nephropathy, but this approach must take into account that these factors are also involved in the regulatory systems of normal physiology.

Acknowledgments
The work in the authors laboratory is supported by the Deutsche Forschungsgemeinschaft.

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