The possible role of VEGF in diabetic nephropathy has been investigated in several animal models of diabetes mellitus, initially in essentially descriptive studies but more recently in experiments in which the effects of VEGF have been blocked. Cooper et al. (21) demonstrated an early and sustained increase in VEGF gene and protein expression in the visceral epithelial cells of the glomerulus from rats with streptozotocin-induced diabetes. On the other hand, the expression of VEGFR-2, mainly in the glomeruli was increased at the earlier 3-wk time-point but not at 32 wk, suggesting a more important role for VEGF in the earlier stages of diabetic renal disease. A similar increased expression of VEGF mRNA and protein has been reported at early timepoints in the development of nephropathy in spontaneously diabetic rats. These investigators suggested that hypoxia and cAMP played a regulatory role in the expression of VEGF at the onset of diabetes (29). Changes in VEGF expression have been examined in a longitudinal manner in Otsuka-Long-Evans-Tokushima-Fatty rats, a model of type-2 diabetes mellitus. These rats had increased albuminuria throughout the study (30). Urinary VEGF levels were greater from 25 to 37 wk and then decreased but remained higher than observed in control rats. Urinary VEGF levels correlated with albuminuria. VEGF was found mainly in the podocytes, as had been reported in the type-1 models of diabetes. Interestingly, the increase in VEGF gene expression was observed in the early period of diabetic nephropathy and was associated with increased urinary albumin excretion. Other investigators have described similar findings in these Otsuka-Long-Evans-Tokushima-Fatty rats (31) as well as in Zucker Diabetic fatty rats, another model of type-2 diabetes (32).
The importance of this early increase in VEGF expression in the diabetic kidney has been further investigated using a variety of different interventions. Monoclonal antibodies against VEGF were administered intraperitoneally to rats with streptozo-tocin diabetes. The antibodies were shown to inhibit serum VEGF levels and caused a decrease in hyperfiltration, albuminuria, and glomerular hypertrophy (33). As experimental diabetes is associated with renal hypertrophy, Schrijvers et al. (34) used another model of glomerular hypertrophy induced by high protein intake in mice. The administration of a neutralizing antibody to VEGF prevented the glomerular hypertrophy in that model but had no effect on kidney and body weight, consistent with a specific action of VEGF on glomerular hypertrophy. Furthermore, these investigators demonstrated in a mouse model of obese type-2 diabetes the db/db mouse that the administration of a neutralizing VEGF antibody resulted in attenuation in the increases in kidney weight, glomerular volume, and basement membrane thickness, seen in untreated diabetic mice (35). In addition the increase in creatinine clearance, a relatively crude marker of hyperfiltration, was prevented. These findings are consistent with the view that direct inhibition of VEGF may prevent the renal damage seen in diabetes.
Of particular interest is whether other agents, which ameliorate diabetic nephropathy, work through VEGF-dependent mechanisms. Diabetic rats were treated with either an angiotensin converting enzyme inhibitor (ACE-I) (enalapril) or an angiotensin II receptor blocker (ARB) (candesartan) (36). Both treatment regimens reduced the urinary excretion of albumin, whereas only the ACE-I attenuated renal VEGF protein content, consistent with the antialbuminuric effect of ACE inhibition, which may be partly via reducing renal VEGF accumulation. Recently Satoh et al. (37) examined the effects of an ARB and a calcium channel blockade in hypertensive Wistar fatty rats, a model of type-2 diabetes. ARB administration reduced blood pressure and the increase in urinary albumin excretion, whereas the calcium channel blocker did not affect albuminuria despite reducing blood pressure to a similar extent. The ARB reduced renal expression of VEGF, whereas the calcium channel blocker had no effect on this growth factor. The authors concluded that in this model the concomitant systemic hypertension accelerates nephropathy through hemodynamic mechanisms involving angiotensin II's action on VEGF synthesis. Of interest is the report showing that the administration of a cyclo-oxygenase-2 inhibitor to a rat model of diabetes and hypertension resulted in the reduction of several putative mediators of renal injury including PAI-I, VEGF, and tumor growth factor (TGF)-Pj (38). This was associated with a decrease in proteinuria and mesangial sclerosis, suggesting that various mediators of renal disease in diabetes, such as VEGF may involve cyclo-oxygenase-2 dependent pathways. In a study to determine if HMG Coenzyme A reductase inhibitors influence the progression of diabetic nephropathy diabetic rats were treated with losartan (ARB), simvastatin or both (39). Injury to the kidney was attenuated by losartan but not by simvastatin. Both drugs individually and in combination resulted in a similar attenuation of the increase in not only glomerular expression of TGF-Pj but also VEGF. However, only the combination resulted in a reduction in the plasma concentrations of urea and creatinine.
In a study investigating the relationship between AGE products and the intracellular second messenger, protein kinase C (PKC) in diabetic nephropathy, diabetic rats were treated with pharmacological approaches to attenuate renal AGE accumulation, either the crosslink breaker, ALT-711 or the inhibitor of AGE formation, aminoguanidine (40). Both treatments reduced the glomerular deposition of AGEs and attenuated increased expression of certain PKC isoforms. However, the increased renal expression of VEGF was attenuated only by ALT-711 and not by aminoguanidine. Further studies indicated that ALT-711, in both in vitro and in vivo experiments, inhibited phosphorylation of the PKC-a isoform suggesting a link between certain PKC isoforms and VEGF expression. The importance of the PKC-a isoform has been further suggested in experiments in which induction of diabetes in PKC-a-/- mice was associated with reduced renal VEGF expression and interestingly less albuminuria (41).
In an attempt to correlate the effects of VEGF with other angiogenic growth factors, Rizkalla et al. (42) measured the expression not only of VEGF and the receptor VEGF-R2 but other angiogenesis related factors such as, angiopoietin (Ang)-1, Ang-2, and their cognate receptor, Tie2 in diabetic rat, kidneys. Furthermore, the investigators assessed the effects of blockade of the RAS with AT-1 and AT-2 receptor antagonists on these growth factors and their receptors. Diabetes was associated with increased gene and protein expression of VEGF, the VEGF receptor VEGFR-2, Ang-1, Ang-2, and Tie-2. Blockade of the AT-1 receptor prevented the increased expression of these cytokines and their respective receptors, whereas the AT-2 blocker had less widespread effects, reducing VEGF receptor and Ang-1 gene expression and decreasing VEGF, Ang-1, and Ang-2 protein levels.
Although not directly relevant to this chapter, further evidence linking the RAS to expression of VEGF, related proteins and receptors has been described in AII infused rodent kidneys (43) as well as in the diabetic retina (44) and in a nondiabetic model of retinal neovascularization (45).
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