The emerging role of VEGF in diabetic kidney disease

Mogher Khamaisi1,2, Bieke F. Schrijvers3, An S. De Vriese3, Itamar Raz2 and Allan Flyvbjerg1

1 Medical Department M (Diabetes and Endocrinology), Medical Research Laboratories, Institute of Experimental Clinical Research, Aarhus University Hospital, Aarhus, Denmark and 2 Departments of Internal Medicine and the Diabetes Center, Hadassah University Hospital, Jerusalem, Israel and 3 Renal Unit, Department of Internal Medicine, Ghent University Hospital, Ghent, Belgium

Correspondence and offprint requests to: Allan Flyvbjerg, MD, DMSc, Medical Department M/Medical Research Laboratories, Institute of Experimental Clinical Research, Aarhus University Hospital, Nørrebrogade 44, DK-8000 Aarhus C, Denmark. Email: allan.flyvbjerg{at}dadlnet.dk

Keywords: advanced glycation endproduct; angio-tensin converting enzyme; antibody; experimental diabetes; type 1 and type 2 diabetes

Introduction

Micro- and macrovascular complications are the most important cause of increased morbidity and mortality in type 1 and type 2 diabetic patients. Early renal changes in diabetes are characterized by kidney enlargement, glomerular hyperfiltration and increased synthesis of extracellular matrix. Twenty-five to 40% of diabetic patients develop microalbuminuria with a high risk of progression to overt diabetic nephropathy. Several classes of growth factors and cytokines have been suggested to be involved in mediating the adverse vascular effects of hyperglycaemia, most importantly growth hormone, insulin-like growth factors, transforming growth factors ß and vascular endothelial growth factor (VEGF) [13]. Most recently the latter has been implicated in the pathogenesis of both diabetic nephropathy and retinopathy. Overall, VEGF appears to play a central role in mediating diabetic endothelial dysfunction and vasculopathy [24]. Increasing understanding of the molecular mechanisms underlying the role of VEGF in these processes has stimulated the development of potential therapeutic approaches. Herein we review the extant evidence that VEGF is involved in the pathogenesis of diabetic kidney disease, identify some potential therapies and discuss their implications for the future management of diabetic renal vascular complications.

The VEGF system

The VEGF family consists of more than five different isoforms of highly conserved homodimeric glycoproteins, with heparin-binding properties [57]. VEGF was first recognized in 1983 and named vasopermeability factor due to its potent permeability-inducing properties. VEGF also exert potent mitogenic actions in endothelial cells [6] and has been shown to play an important role in pathological angiogenesis [8]. The most potent stimulus for VEGF is hypoxia and VEGF has been claimed to be a ‘survival factor’ during tissue ischaemia [5] (Table 1). Further, VEGF is an important factor for normal nephrogenesis [5]. The two best-described VEGF receptors (VEGFR-1 and VEGFR-2), also known as the fms-like tyrosine kinase (Flt-1) and fetal liver kinase 1 (Flk-1), are high-affinity transmembrane tyrosine kinase receptors [5] (Figure 1).


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Table 1. Stimuli of VEGF synthesis and action

 


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Fig. 1. Schematic depiction of the interaction between VEGF and its tyrosine-kinase receptor. This model is based primarily on data from Neufeld et al. [5].

 
In the human kidney, VEGF has been demonstrated in the podocytes, distal tubular cells and to a lesser extent in proximal tubular cells, while absent in mesangial and endothelial cells [8]. The VEGFRs are expressed predominantly in endothelium of preglomerular vessels, glomeruli and postglomerular vessels, but have also been identified in renal mesangial cells and tubules of normal kidney [8]. The specific mechanism by which VEGF may influence the glomerular fenestration is so far unknown.

Evidence for a role of VEGF in diabetic kidney disease

In vitro, mesangial cells, glomerular endothelial cells, vascular smooth muscle cells (VSMCs), proximal and distal tubular cells are capable of producing VEGF. Angiotensin II stimulates VEGF production in mesangial cells [9], and high glucose-induced VEGF production in VSMCs seems to be protein kinase C (PKC)-modulated and/or -dependent [10]. VEGF upregulates the expression of endothelial nitric oxide (NO) synthase (NOS3) in endothelial cells and increases the production of NO, thus indicating that NO may act as a downstream mediator for VEGF [11]. In vivo, increased renal VEGF gene expression, glomerular immunoreactivity and VEGFR binding has been described both in animal models of type 1 [i.e. streptozotocin (STZ)-diabetic animals] and type 2 diabetes [i.e. Otsuka-Long-Evans-Tokushima-Fatty (OLETF) rats and Zucker Diabetic Fatty (ZDF-rats)] [1214]. Data on serum VEGF levels obtained in type 1 diabetic subjects have been controversial. One study reported comparable serum VEGF levels in diabetic and non-diabetic children and adolescents [15], whereas two other studies found increased serum [16] and plasma VEGF levels [17] in diabetic individuals. Serum VEGF levels were reported to be influenced by glycaemic control, and to be markedly increased in young patients with microvascular complications [16]. Further, a study in adult type 1 diabetic men reported elevated plasma VEGF in the early course of diabetic nephropathy [18] while another study showed that plasma VEGF levels in adult type 1 diabetic patients were neither correlated with the extent of diabetic microvascular complications nor with other key risk factors [19]. In type 2 diabetic patients, plasma VEGF concentration tended to increase with increasing urinary albumin excretion (UAE) [20]. Recently, urinary and plasma VEGF levels were studied in 73 type 2 diabetic patients, and the relationship between these values and the severity of diabetic nephropathy was also searched [21]. The plasma VEGF concentration was significantly higher in type 2 diabetic patients with overt proteinuria than in patients with normo- or microalbuminuria. The VEGF excretion increased according to the degree of proteinuria in diabetic subjects and further a weak but significant correlation was found between urinary VEGF excretion and the levels of serum creatinine, creatinine clearance, microalbuminuria and proteinuria [21].

Treatments targeting VEGF

Substantial research has been focused on the development of VEGF/VEGFR antagonists to block the VEGF-mediated signalling pathway (Table 1). These strategies include monoclonal antibodies directed against VEGF (VEGF-ab), VEGFR tyrosine kinase inhibitors (VEGFR-TKi), angiotensin converting enzyme inhibitors (ACEi), PKC-inhibitors (PKCi), advanced glycation endproduct (AGE) inhibitors, VEGF aptamers and gene therapy by a soluble VEGFR.

In a series of recent studies the crucial role of VEGF in glomerular enlargement has been elucidated in non-diabetic models of glomerular hypertrophy. Administration of VEGF-ab to uninephrectomized mice blunted the compensatory renal enlargement and abolished the glomerular hypertrophy [22]. Likewise, administration of VEGF-ab in mice fed a high protein diet abolished the glomerular hypertrophy seen in placebo-treated animals on an identical diet, without affecting kidney enlargement or body weight [23]. Recently, two studies have been performed on the effect of VEGF-ab in diabetic animal models. Administration of VEGF-ab to a model of type 1 diabetes (i.e. STZ-diabetic rats) for 6 weeks abolished the diabetes-associated hyperfiltration and upregulation in NOS3 and partly the rise in glomerular enlargement, renal growth and the increase in UAE [3]. These effects were seen without any impact on metabolic control in diabetic animals and no renal effects of treatment were seen in non-diabetic controls [3]. Further, VEGF-ab adminstration in an obese mouse model of type 2 diabetes (i.e. db/db mice) showed amelioration of diabetic renal changes by attenuation of the diabetes-associated increases in kidney weight, glomerular volume and UAE, while the increase in basement membrane thickness and creatinine clearance was abolished. Finally, VEGF-ab administration tended to reduce total mesangial volume expansion [24] (Figure 2). VEGFR-TKi are rather newly developed substances designed primarily to block the angiogenic effect of VEGF in oncology, and accordingly no preclinical or clinical diabetes studies have yet been published. Studies on the renal effects of VEGFR-TKi are currently being performed in animal models of type 1 and type 2 diabetes.



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Fig. 2. The effect of 2 months administration of a neutralizing VEGF-ab on basement membrane thickness (BMT), urinary albumin excretion (UAE) and total mesangial volume in an animal model of type 2 diabetes (i.e. the db/db mice). Non-diabetic controls (open bars), placebo-treated db/db mice (black bars) and VEGF-ab-treated db/db mice (grey bars). *P < 0.05 vs non-diabetic controls and VEGF-ab-treated db/db mice; {Delta}P < 0.05 vs non-diabetic controls and placebo-treated db/db mice. Adapted from Flyvbjerg et al. [24].

 
Administration of ACEi in a model of type 1 diabetes (i.e. STZ rats) has been shown to reduce the diabetes-associated elevation in retinal VEGF gene expression and vascular permeability to albumin [25]. Further, clinical studies in type 1 and type 2 diabetic patients with proliferative retinopathy have showed a strong negative correlation between vitreous VEGF levels and the daily enalapril dose used [26]. However, the EURODIAB controlled trial of lisinopril in insulin-dependent diabetes mellitus (EUCLID) demonstrated a weak association between treatment with lisinopril and circulating plasma VEGF levels, despite definite effects of these agents on retinopathy and nephropathy, suggesting that these effects may not predominantly be mediated through circulating VEGF [27].

Hyperglycaemia-induced activation of PKC and diacyl-glycerol has been suggested to be an important mechanism in endothelial dysfunction and microvascular changes in experimental animal studies. Long-term administration of a specific PKCß inhibitor to both type 1 and type 2 diabetic animal models exerts beneficial renal effects. Interestingly, VEGF production in high glucose media is PKC dependent, therefore PKC inhibition might be a useful therapeutic strategy for the treatment of diabetic nephropathy [10]. Only a few studies have been published on the possible interrelationship between PKC and VEGF in vivo, and no studies with a renal focus have yet appeared. Of interest, however, it has been shown that a partially selective PKC inhibitor, that blocks phosphorylation by VEGF of several PKC isoforms, inhibits retinal neovascularization [28,29]. In addition, neutralizing PKC isoforms (i.e. {zeta} and {delta}) suppresses the aberrant VEGF overexpression in renal cell carcinoma cells and the resultant angiogenesis [30]. Administration of specific PKC isozyme inhibitors may be a noval approach in blocking the VEGF-mediated renal effects in diabetes.

In one study, using a type 2 diabetic rat model [12], it is shown that long-term treatment with OPB-9195, a novel inhibitor of AGE formation, abolished the enhanced renal VEGF mRNA and protein overexpression along with renoprotection, by restoring diabetes-induced renal collagen IV accumulation to normal and reducing the rise in UAE [12].

Finally, VEGF aptamers [31] or treatment by gene therapy may be new promising tools to blunt pathophysiological effects of VEGF as indicated in a recent study, where retinal neovascularization in a non-diabetic rat model, was reduced by local application of soluble VEGFR producing vector [32].

Conclusions

Despite intensified metabolic control and antihypertensive treatment of diabetic patients, the development of diabetic nephropathy remains a serious clinical problem. There is increasing evidence for a multifactorial pathogenesis of diabetic kidney disease, including various growth factors and cytokines as active players. Recently published data, mainly derived from in vitro and animal studies, support the hypothesis that a hyperglycaemia-induced rise in renal VEGF levels may be responsible for some of the renal changes leading to the development of diabetic nephropathy. Further, a series of new potential renoprotective agents may mediate their beneficial effects through down-regulation of a pathophysiologically enhanced renal VEGF-system. Further studies are warranted to elucidate the perspectives in targeting VEGF-mediated diabetic renal changes.

Acknowledgments

A.F. is supported by the Danish Medical Research Council (Grant # 9700592), the Eva and Henry Frænkels Memorial Foundation, the Danish Diabetes Association, the Novo Foundation, the Nordic Insulin Foundation, the Institute of Experimental Clinical Research, University of Aarhus, Denmark. M.K. is supported by the Israeli Diabetes Association, and S.De V. by the Fund for Scientific Research Flanders and B.F. Schrijvers by the Institute for the Promotion of Innovation by Science and Technology in Flanders.

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