VEGF IN RENAL PHYSIOLOGY

This section elaborates on the expression and the potential role of VEGF, angiopoietins, and their receptors in the normal adult kidney. A comprehensive discussion of the role of the VEGF system in renal development is beyond the scope and space limitations of this review and has been published elsewhere¹⁵. Cultured rat and human mesangial cells express both mRNA of VEGF₁₂₁, VEGF₁₆₅, and VEGF₁₈₉, and VEGF protein^16,17. In rodent and human kidneys, VEGF mRNA and/or protein were detected predominantly in glomerular podocytes, distal tubules, and collecting ducts, and to a lesser extent in some proximal tubules^{3,16,18,19,20,21,22,23}. The expression of the different VEGF isoforms in normal human glomeruli was complex and variable with substantial inter- and intraindividual variation³. Ang-1, but not Ang-2, was identified in adult human glomeruli, particularly in podocytes²⁴. VEGFR-1 and VEGFR-2 were detected in cultured rat and human mesangial cells^25,26,27,28 and in cultured rat renal tubular epithelial cells²⁹, but not in cultured primary human podocytes³⁰. In contrast, conditionally immortalized human podocytes expressed VEGFR-1, VEGFR-3 and Neuropilin-1 but not VEGFR-2³¹. Cultured mouse glomerular endothelial cells and transformed tubular epithelial cells expressed Neuropilin-1 and Neuropilin-2³². Neuropilin-1 was also detected in cultured human mesangial cells^25,27 and in cultured primary human glomerular podocytes³⁰. The expression of VEGFR-1, VEGFR-2, sVEGFR-1, and Neuropilin-1 in isolated human glomeruli was also heterogenous^3,30. In human kidney, VEGFR-1 and VEGFR-2 were predominantly expressed on preglomerular, glomerular, and peritubular endothelial cells^20,23,27,33. In rat kidney, VEGFR-2 expression was detected in glomerular and peritubular endothelial cells, in distal convoluted tubules and collecting ducts, and in cortical interstitial fibroblast and medullary interstitial cells, whereas VEGFR-1 was expressed more diffuse in proximal and distal tubules^19,29. In human kidney, Neuropilin-1 was detected in glomerular podocytes³⁰. Neuropilin-1 and Neuropilin-2 were localized in peritubular capillary endothelial cells in adult mouse and rat kidney³². Tie2 was demonstrated in glomerular capillary endothelial cells of human and rat glomeruli and in cultured human microvascular endothelial cells²⁴. In summary, in vivo, capillary endothelial cells express VEGFR-1, VEGFR-2, and Tie2, glomerular podocytes express Neuropilin-1 and produce VEGF and Ang-1.

Although the functions of constitutively expressed VEGF and VEGF receptors in the normal kidney are largely unknown, some hypotheses may be derived from the peculiar distribution of the molecule and its receptors in the kidney. VEGF is strongly expressed by visceral epithelial cells while its binding sites are localized on glomerular endothelial cells. If one assumes the existence of a paracrine loop in the glomerulus, VEGF must move in the opposite direction of the glomerular filtrate in order to bind to its receptors. The presence of such complex mechanism suggests that the strategic localization of podocytes is required for the correct sensing and interpretation of the stimulus for VEGF release. VEGF may be involved in the induction and maintenance of the fenestrae in glomerular capillary endothelial cells³⁷. Given the role of VEGF in promoting microvascular permeability, it has been speculated that VEGF may regulate glomerular permeability, although it is generally acknowledged that the capillary fenestrations do not represent the ultimate barrier to filtration. Recently, in vitro evidence indicated that VEGF may act as an autocrine factor on calcium homeostasis and cell survival in human podocytes³¹. In contrast to the prominent expression of the VEGF system in the adult kidney, the administration or inhibition of VEGF in normal adult animals appears to have only minimal effects. In the isolated perfused rat kidney, administration of VEGF increased the renal blood flow but did not influence the glomerular filtration rate or the permselectivity of the glomerular barrier wall³⁴. In vivo infusions of VEGF into the renal artery of rats did not influence protein excretion rate³⁴. The administration of neutralizing monoclonal anti-VEGF-antibodies to normal rats had no effect on glomerular filtration rate or glomerular volume³⁵. Injection of a VEGF₁₆₅ aptamer, an oligonucleotide-based VEGF₁₆₅ antagonist, in normal rats had no effect on kidney and glomerular morphology, did not induce proteinuria and did not affect glomerular cell proliferation and the number of endothelial fenestrations³⁶. In contrast, podocyte-specific heterozygous and homozygous deletions of VEGF in mice resulted in proteinuria and endotheliosis by 2½ weeks of age, and in perinatal lethality, respectively, with loss of endothelial fenestrations or failure to form fenestrations³⁷. Conversely, podocyte-specific overexpression of VEGF₁₆₅ led to a collapsing glomerulopathy³⁷.

VEGF IN RENAL PATHOPHYSIOLOGY

Diabetic nephropathy

Type 1 diabetes

VEGF mRNA and protein expression were increased at the onset of diabetes in genetically diabetic BioBreeding rats³⁸, in glomerular podocytes, distal tubules and collecting ducts after 3 weeks and 32 weeks of streptozotocin (STZ)-diabetes in rats¹⁹ and in renal tubular and vascular compartments in STZ-diabetic rats with superimposed hypertension³⁹. More specifically, the VEGF₁₆₄ and VEGF₁₈₈ isoforms increased after STZ-diabetes induction which was reversed by insulin treatment⁴⁰. Glomerular VEGFR-1 and VEGFR-2 mRNA expression were higher after 6 weeks of STZ-diabetes⁴⁰. Similarly, VEGFR-2 mRNA was increased in glomerular and peritubular endothelial cells and interstitial cells after 3 weeks of STZ-diabetes but not after 32 weeks¹⁹. To assess the role of VEGF in the pathophysiology of early renal dysfunction in diabetes, type 1 diabetic rats were treated with monoclonal neutralizing anti-VEGF-antibodies for 6 weeks. Inhibition of VEGF abolished the diabetes-associated glomerular hyperfiltration, glomerular hypertrophy, and urinary albumin excretion (UAE) without an effect on metabolic control³⁵. In addition, the diabetes-associated up-regulation of NOS3 expression was prevented, further supporting the evidence that nitric oxide acts as a downstream mediator of VEGF³⁵.

Both increased [abstract; Abdel Aziz MY, Nephrol Dial Transplant 12:1538a, 1997]^41,42 and unaltered^43,44 serum or plasma VEGF levels were observed in type 1 diabetic patients versus control subjects. Various studies examined a correlation between circulating VEGF levels and parameters reflecting the severity of diabetic nephropathy. VEGF levels were higher in macroalbuminuric type 1 diabetics than in patients without complications^41,45 and microalbuminuric patients [abstract; Abdel Aziz MY, Nephrol Dial Transplant 12:1538a, 1997]. Other studies reported no differences in VEGF levels between type 1 diabetic patients with and without (micro)albuminuria^43,44,46. Correlations between VEGF levels and glycemic control, severity of diabetic nephropathy, and degree of UAE were observed in some [abstract; Abdel Aziz MY, Nephrol Dial Transplant 12:1538a, 1997]⁴¹ but not in all studies^43,44,46,47. A positive correlation was found between serum VEGF levels and nailfold capillary permeability in type 1 diabetics⁴⁷. Urinary VEGF excretion was unchanged in patients with diabetic nephropathy compared with control subjects⁴⁸. Renal biopsies from patients with diabetic nephropathy showed fewer glomerular VEGF mRNA positive cells than biopsies from controls¹⁸. Glomerular VEGF expression was highest in the patients with mildest sclerotic changes and was particularly strong in viable glomerular podocytes²⁰, but reduced or absent in sclerotic glomeruli^20,49. Atrophic tubules were negative for VEGF mRNA, except for a weak signal in distal tubules, whereas interstitial cells often had pronounced VEGF mRNA and protein positivity²⁰. In patients with type 1 diabetes and a minimum diabetes duration of 10 years, the frequency of the +405 CC genotype, which is associated with lower VEGF protein production in peripheral blood mononuclear cells (PBMC)⁹, was increased compared with control subjects [abstract; Summers AM, J Am Soc Nephrol 13:248A, 2002]. An increase in the -460 T allele was found in patients with microalbuminuria or proteinuria compared to patients with normoalbuminuria [abstract; Summers AM, J Am Soc Nephrol 13:248A, 2002]. The D allele and DD genotype of the VEGF D/I polymorphism at -2549 of the promotor region were associated with susceptibility to diabetic nephropathy in type 1 diabetics and the presence of the D allele was linked to increased transcriptional activity¹².

Type 2 diabetes

Renal and/or glomerular VEGF mRNA were increased in diverse experimental models of type 2 diabetes [i.e., modestly in insulin-resistant Zucker fatty/fatty rats and more pronounced in Zucker diabetic fatty (ZDF) rats⁴⁰, in db/db mice⁵⁰, and in Otsuka Long-Evans Tokushima fatty (OLETF) rats]⁵¹. In OLETF rats, VEGF mRNA expression was increased only in the early period of diabetic nephropathy⁵¹. In ZDF rats, VEGF mRNA levels rose early in the course of diabetes and remained elevated up to 7 months⁵². At 9 months, when glomerulosclerosis was most pronounced, renal VEGF mRNA levels were reduced⁵². VEGFR-1 and VEGFR-2 mRNA were also elevated in ZDF rats⁴⁰. Treatment with anti-VEGF-antibodies in db/db mice attenuated the diabetes-associated increases in kidney weight, glomerular volume and UAE, and abolished the increase in basement membrane thickness and creatinine clearance⁵³. In addition, the antibodies tended to reduce the mesangial matrix expansion⁵³. In contrast, in Goto-Kakizaki rats, an experimental model of lean type 2 diabetes, anti-VEGF-antibodies only tended to reduce glomerular hypertrophy and had no effect on UAE or creatinine clearance [abstract; Schrijvers BF, J Am Soc Nephrol 13:764A, 2002]. Treatment of OLETF rats with temocapril or CS-866, an angiotensin II type 1 receptor (AT1) antagonist, improved glomerulosclerosis, reduced urinary protein excretion and VEGF staining, suggesting that AT1 may be involved in the overproduction of VEGF in diabetic nephropathy⁵⁴.

Plasma VEGF levels were higher in type 2 diabetics than in controls⁵⁵. In another study, plasma VEGF levels were elevated only in type 2 diabetic patients with characteristics of atherosclerosis⁵⁶. Plasma VEGF tended to rise with increasing UAE⁵⁵, however, other studies reported no such correlation^47,57. In type 2 diabetic patients, VEGF did not correlate with serum creatinine⁵⁷ or capillary permeability⁴⁷. There were no differences in sVEGFR-1 plasma levels between diabetic patients with or without atherosclerosis and control subjects⁵⁶. Urinary VEGF excretion increased with the progression of diabetic nephropathy and correlated weakly with the levels of serum creatinine, creatinine clearance, microalbuminuria, and proteinuria¹⁶. In biopsies with mild changes of diabetic nephropathy, VEGF was up-regulated in glomerular podocytes and distal tubular cells. In biopsies with advanced changes, VEGF staining was decreased or negative in sclerotic glomeruli but remained intense in tubules¹⁶. More recently, in patients with type 2 diabetic nephropathy, glomerular VEGF mRNA expression, observed predominantly in podocytes, was higher than in normal kidneys, but in contrast to the previous study, VEGF expression was higher in patients with advanced renal morphologic changes than in patients with mild-moderate mesangial expansion and tubulointerstitial changes [abstract; Kanesaki Y, J Am Soc Nephrol 13:8A, 2002]. In glomeruli of type 2 diabetic patients, VEGF mRNA level was inversely related to albumin excretion rate [abstract; Bortoloso E, Diabetologia 42:273A, 1999]. Tubulointerstitial VEGF and VEGFR-1 mRNA were lower in patients with severe diabetic nephropathy⁵⁸. While the ratio between VEGF₁₂₁ and VEGF₁₆₅ correlated with mesangial matrix expansion in one study [abstract; Bortoloso E, Diabetologia 42:273A, 1999], no relation with the severity of the histologic changes was found in another⁵⁸. As VEGF₁₂₁ is freely diffusable and VEGF₁₆₅ is mostly bound to extracellular matrix, the pathophysiologic relevance of the ratio between both isoforms remains to be determined.

Conclusion

Data regarding circulating VEGF levels in patients with diabetes are highly discrepant. As VEGF is a paracrine mediator, systemic levels may not adequately reflect changes in the local VEGF system⁴³. In addition, the assay methods vary substantially between the studies (i.e., VEGF isoform specificity, detection of total or free VEGF and interference with VEGF-binding molecules such as ₂-macroglobulin)⁵⁹. Finally, determinations of serum and plasma VEGF levels may differ, owing to release from platelets and leukocytes after sampling^60,61. Renal expression of VEGF and its receptors is more consistently up-regulated in experimental animals and patients with type 1 and type 2 diabetes, especially early in the course of diabetes Table 1. Inhibition of VEGF resulted in beneficial effects on the diabetes-associated renal changes, underlining a deleterious role for VEGF in the pathophysiology of diabetic nephropathy. Although the cause of the up-regulation of the VEGF system in diabetes remains unknown, several factors relevant to the pathogenesis of diabetic nephropathy have been shown to promote VEGF expression in different cell types, including hyperglycemia, AGEs, PKC, angiotensin II, various cytokines, and aldose reductase.

Table 1 - Summary of the expression and intervention data per disease type in experimental models and patient populations.

Full table

High protein–induced nephropathy

One study examined the potential role of VEGF in high protein–induced renal and glomerular enlargement Table 1⁶². Mice fed a diet containing 45% protein exhibited early glomerular hypertrophy and kidney enlargement compared with mice on a 20% protein containing diet. Treatment with a monoclonal neutralizing VEGF-antibody abolished the high protein–induced glomerular hypertrophy without affecting kidney or body weight. Kidney IGF-I was up-regulated in high protein–fed mice and not affected by treatment with the VEGF-antibody. Consequently, as IGF-I has been implicated in high protein–induced renal growth, VEGF could be a downstream mediator of IGF-I. This study illustrates that VEGF plays a major role in high protein-induced glomerular growth⁶².

Nephron reduction

Uninephrectomized mice are characterized by an increased glomerular volume and kidney weight in the remnant kidney and an early transient increase in kidney IGF-I concentration⁶³. To explore a role for VEGF in compensatory renal changes after uninephrectomy, these animals were treated with anti-VEGF-antibodies, which abolished the glomerular enlargement and partially blocked the renal growth without affecting renal IGF-I levels⁶³. After a 75% surgical nephron reduction, the remnant kidneys of C57Bl6xDBA2/F1 mice displayed compensatory glomerular and tubular hypertrophy with prominent cortical peritubular capillary growth, whereas another strain, FVB/N mice, additionally developed severe tubulointerstitial lesions and glomerulosclerosis⁶⁴. While total kidney VEGF protein levels were increased, VEGF immunostaining revealed a decrease in proximal tubules, a marked increase in some distal tubules and no change in the glomeruli. In mice with severe tubulointerstitial injury, the changes in the peritubular microcirculation and VEGF expression were more prominent⁶⁴. In the rat, experimental nephron reduction resulted in early peritubular and glomerular endothelial cell proliferation followed by a progressive loss of peritubular and glomerular capillaries, the latter associated with a decreased VEGF staining in tubules and glomerular podocytes^65,66,67,68. In another study, VEGF mRNA and protein expression was increased 2 weeks, but decreased 4 weeks after 5/6 nephrectomy [abstract; Horike H, J Am Soc Nephrol 13:164A, 2002]. Administration of VEGF₁₂₁ after experimental nephron reduction increased glomerular and peritubular endothelial cell proliferation and reduced tubulointerstitial fibrosis⁶⁶. In addition, VEGF₁₂₁ increased renal NOS3 staining and 24-hour urinary nitrite and nitrate excretion⁶⁶, which is consistent with the observation that VEGF-induced angiogenesis is mediated by nitric oxide. NOS blockade with L-N(G)-nitro-arginine-methyl-ester (L-NAME) in animals with remnant kidneys accelerated the development of proteinuria, glomerulosclerosis, and tubulointerstial fibrosis, along with more glomerular and peritubular endothelial cell loss and impaired endothelial proliferation⁶⁷. VEGF immunostaining in glomerular podocytes and tubules was not different between the groups⁶⁷. In rats with subtotal nephrectomy, treatment with perindopril improved glomerular filtration rate and endothelial cell density, reduced UAE and glomerulosclerosis and restored VEGF expression⁶⁸. Taken together, the experimental studies Table 1 suggest that VEGF is required for the glomerular and tubular hypertrophy and endothelial cell proliferation in response to nephron reduction. Down-regulation of VEGF may result in the development of glomerulosclerosis and tubulointerstitial fibrosis.

Human data regarding uremia and VEGF expression are rather scarce. VEGF plasma levels were higher in uremic patients than in control subjects^55,69. The reasons for higher VEGF levels in uremia are unknown, but excess production, tissue hypoxia, or reduced clearance of VEGF have been suggested^55,68. The negative correlation between plasma VEGF levels and residual renal function observed in peritoneal dialysis patients suggests that renal clearance contributes to the elimination of VEGF. Conversely, VEGF may have a negative impact on residual renal function⁷⁰.

Glomerulonephritis

Minimal change nephropathy

Minimal change nephropathy (MCN) has been speculated to result from the release of soluble circulating factors by mononuclear cells that alter glomerular permeability. As VEGF may be a regulator of glomerular permeability, the release of VEGF by PBMC has been scrutinized. In cultured PBMC from patients with MCN, the effects of various cytokines on the secretion of VPF in the culture supernatant were investigated^{71,72,73,74,75,76,77,78,79,80}. VPF is an older term for VEGF and is determined by quantifying the intradermal permeability in guinea pigs. Cultured PBMC, activated by concanavalin-A or lipopolysaccharide, secreted VPF^{71,72,73,74,75,76,77,78,79,80,81,82} and the VPF levels in the culture supernatant were higher in PBMC from nephrotic MCN patients than from patients in remission or control subjects^{71,72,73,74,75,76,77,78,79,80}. IL-2, IL-12, IL-15, and IL-18 stimulated and TGF-1, IL-4, IL-10, and IL-13 inhibited VPF release by activated PBMC from patients with MCN^{71,72,73,74,75,76,77,78,79}. Patients treated with steroids had lower VPF levels in PBMC supernatant than untreated patients^{71,72,73,74,75,76,77,78}. In contrast, VEGF mRNA expression in unstimulated PBMC was not different between children with steroid-sensitive nephrotic syndrome (SSNS) in relapse and children with SSNS in remission⁸³ or controls⁸⁴. The potential role of VEGF in the development of proteinuria was also investigated in experimental MCN^85,86. In puromycin aminonucleoside nephrosis (PAN) rats, an experimental model of human MCN, the renal mRNA expression of VEGF and its receptors VEGFR-2 and VEGFR-1 was down-regulated⁸⁵. In contrast, in hyperalbuminemia-induced rat nephrosis, another model of human MCN, VEGF and its receptors were up-regulated and correlated with the severity of proteinuria⁸⁶. In PAN rats, treatment with a VEGF₁₆₅ aptamer for 6 days did not affect proteinuria, podocyte swelling, foot-process fusion, or glomerular endothelial fenestrations³⁶, suggesting that VEGF₁₆₅ is not involved in the disease process, although a pathogenic role for other isoforms is not excluded. Plasma VEGF concentrations (free VEGF₁₂₁ and VEGF₁₆₅) and urine VEGF/creatinine ratios were not elevated during relapses of childhood SSNS when compared with remission and normal controls⁸⁴. Likewise, plasma or urinary VEGF levels were not different between patients with MCN in the nephrotic state and those in remission⁸³ or healthy controls^87,88. In contrast, urinary VEGF levels were increased in patients with MCN with nephrotic syndrome when compared with patients without nephrotic syndrome and healthy controls, and correlated with the degree of proteinuria⁸⁹. Urinary VEGF levels decreased rapidly after steroid therapy⁸⁹. Patients with MCN in the nephrotic state and in remission displayed a similar VEGF protein and mRNA expression mainly localized in glomerular podocytes⁹⁰. In contrast, in situ hybridization revealed that VEGF mRNA expression was up-regulated in podocytes in MCN and correlated with the urinary protein excretion¹⁸. In two patients with systemic lupus erythematosus (SLE) and minimal light microscopic changes VEGF expression was normal⁴⁹. The allele frequencies of the dinucleotide repeat polymorphisms within the VEGFR-1 gene were not different between patients with MCN and normal controls⁹¹. Similarly, polymorphisms in the VEGF gene promotor were not associated with the development of proteinuria in MCN [abstract; Watson CJ, J Am Soc Nephrol 9:A2497, 1998]. Further, the genotype frequencies of the VEGF gene polymorphisms -460 C/T, -141 A/C, and +405 G/C were not different in children with SSNS versus controls⁹².

The lack of consistent alterations in the VEGF system in experimental animals or patients with MCN Table 1, does not allow firm conclusions to be drawn about the role of VEGF in the pathophysiology of MCN. Methodologic difficulties may interfere with the correct interpretation of the results. PBMC in culture may be in anaerobic metabolism, which may affect the expression of VEGF. Pitfalls in the determination of circulating VEGF levels have been described above. Further, high urinary VEGF levels may merely reflect podocyte loss and urinary podocyte excretion⁹³ rather than an active involvement of VEGF in the disease process. It has even been suggested that urinary VEGF may be derived from the circulation and as such may be nothing more than an assay for proteinuria⁸⁹.

Membranous glomerulonephritis

Cultured lymphocytes from nephrotic membranous glomerulonephritis (MGN) subjects did not release VPF in their supernatants⁸². In rats with passive Heymann nephritis, an experimental model of MGN, proteinuria, podocyte foot-process fusion and subepithelial immune deposits were not affected by treatment with a VEGF₁₆₅ aptamer for 7 days³⁶. Urinary VEGF excretion was decreased in patients with idiopathic MGN compared with control subjects, but did not correlate with serum VEGF, renal function or proteinuria⁴⁸. In the follow-up of some patients, however, a reduction of proteinuria was associated with increasing urinary VEGF excretion and the change in proteinuria over one year correlated inversely with the change in urinary VEGF⁴⁸. As mentioned above, decreased VEGF excretion in MGN may be explained by podocyte injury and increased VEGF excretion during clinical improvement by partial recovery of podocytes. In 10 nephrotic MGN patients with already a significant degree of glomerulosclerosis, there was no difference in renal VEGF mRNA expression compared with normal kidneys and no correlation between VEGF expression and the degree of proteinuria¹⁸. Another study, including two patients with membranous nephropathy, one idiopathic and one lupus nephritis class IV, reported kidney VEGF protein and mRNA expression to be similar as in controls and localized mainly in podocytes⁹⁰. In three other cases of MGN, VEGF mRNA and protein expression was strong in relatively preserved glomeruli but decreased or absent in sclerotic areas⁴⁹. A recent study demonstrated reduction of VEGF immunostaining in the tubular epithelium of MGN patients versus controls⁹⁴. The C allele of the VEGF -460 C/T polymorphism was associated with progression toward end-stage renal failure in patients with idiopathic membranous nephropathy [abstract; Summers AM, J Am Soc Nephrol 13:260A, 2002].

No consistent pattern of changes in the VEGF system emerges from these studies Table 1. Difficulties with the interpretation of urinary VEGF levels have been described above. The discrepant results of VEGF expression in renal biopsies of patients with MGN are likely due to differences in the degree of glomerulosclerosis, as loss of podocytes implicates loss of VEGF. It thus remains unclear whether VEGF plays a role in the pathophysiology of MGN.

Membranoproliferative glomerulonephritis

The POEMS syndrome is a multisystemic syndrome characterized by polyneuropathy, organomegaly, endocrinopathy, M protein, and skin changes. Renal involvement can occur and has been described as membranoproliferative glomerulonephritis-like lesions⁹⁵. Circulating VEGF levels were elevated in POEMS syndrome compared with normal controls^96,97 or with patients with primary membranoproliferative glomerulonephritis⁹⁸. There was no difference in serum VEGF levels between POEMS patients with and without renal involvement⁹⁸. Further, serum VEGF levels in POEMS patients did not correlate with glomerular alterations⁹⁵.

Mesangioproliferative glomerulonephritis (MPGN)

PBMC of patients with IgA nephropathy (IgAN) responded similarly to cytokines as those of MCN patients, with stimulation of VPF release by IL-2, IL-12, IL-15, and IL-18 and inhibition by TGF-1, IL-4, IL-10, and IL-13^{71,72,73,74,75,76,77,78}. In contrast, VEGF mRNA levels in unstimulated PBMC were not different between IgAN patients and healthy volunteers and did not correlate with clinical or pathologic parameters⁹⁹. Incubation of human mesangial cells with aberrantly glycosylated IgA resulted in a down-regulation of VEGF mRNA expression and decreased VEGF release in the culture medium¹⁰⁰. During recovery of anti-Thy 1.1 nephritis, an experimental model of MPGN, up-regulation of VEGF mRNA in podocytes and the mesangial region, and of VEGFR-2 mRNA in the glomerular capillary walls was associated with endothelial cell proliferation Table 1^27,101. In accordance, administration of a VEGF₁₆₅ antagonizing aptamer reduced glomerular endothelial cell proliferation and inhibited glomerular capillary repair resulting in glomerular sclerosis³⁶. Glucocortocoids decreased VEGF release and aggravated proteinuria in anti-Thy 1.1 nephritic rats possibly due to impairment of glomerular endothelial repair^102,103. Rats with anti-Thy 1.1 nephritis combined with uninephrectomy¹⁰³ or Habu-snake venom injection¹⁰⁴ developed progressive glomerulosclerosis and renal insufficiency. In the former model, regeneration of glomerular endothelial cells and VEGF mRNA expression were decreased¹⁰³. In the latter model, the administration of recombinant human VEGF₁₆₅ enhanced endothelial cell proliferation and glomerular capillary repair¹⁰⁴. Serum VEGF levels were not different in patients with IgAN compared with normal controls^87,88. In contrast, urinary VEGF levels were increased in patients with IgAN with nephrotic syndrome when compared with patients without nephrotic syndrome and healthy controls, and correlated with the degree of proteinuria⁸⁹. In patients with MPGN due to IgAN, Henoch-Schönlein nephritis, non-IgA proliferative glomerulonephritis and lupus nephritis, VEGF protein and mRNA were expressed by glomerular podocytes and mesangial cells but were faint or absent in areas of glomerulosclerosis Table 1^49,90. In patients with early primary MPGN⁹⁰ or IgAN, expression of VEGF and its receptors was up-regulated, but in IgAN patients with more severe interstitial damage, VEGF staining was decreased [abstract; Horike H, J Am Soc Nephrol 13:164A, 2002]²⁷. In patients with MPGN, including IgAN, the presence of the VEGF -460 CC genotype or C allele was associated with progression toward end-stage renal disease [abstract; Summers AM, J Am Soc Nephrol 13:260A, 2002].

In conclusion, VEGF may be essential for glomerular repair in MPGN. Depressed VEGF synthesis resulting from podocyte injury may contribute to endothelial cell loss and favor the development of glomerulosclerosis.

Crescentic glomerulonephritis

In mice with antiglomerular basement membrane glomerulonephritis, an experimental model of crescentic glomerulonephritis, loss of glomerular capillaries was temporally associated with decreased VEGF, VEGFR-2, Ang-1, and Tie2 immunostaining and up-regulation of Ang-2, especially in glomeruli with crescents or sclerosis Table 1¹⁰⁵. In antiglomerular basement membrane glomerulonephritis rats, treatment with VEGF₁₆₅ resulted in recovery of the necrotizing and crescentic lesions, proliferation of endothelial cells and capillary repair, and improved renal function and proteinuria [abstract; Shimizu A, J Am Soc Nephrol 13:36A, 2002]. Serum VEGF levels were higher in patients with crescentic glomerulonephritis, including pauci-immune rapidly progressive glomerulonephritis, Henoch-Schönlein purpura nephritis and IgAN, than in normal control subjects or in patients with MCN, IgAN, and focal segmental glomerular sclerosis (FSGS)^49,87,88. Serum VEGF levels correlated with crescent frequency, tubulointerstitial injury, and glomerular monocyte infiltration, but not with urinary protein excretion or serum creatinine levels^49,87,88. Serum VEGF levels decreased after corticosteroid therapy^49,88. VEGF expression was normal in glomeruli with preserved architecture but decreased or absent in glomeruli compressed by crescents and in sclerotic areas Table 1^49,88. Taken together, the experimental data suggest that VEGF accelerates glomerular recovery in crescentic glomerulonephritis. Further, for the first time, a role for angiopoietins in glomerular disease is suggested. Up-regulation of Ang-2 may be an appropriate adaptive response to promote new vessel formation, but in the presence of low VEGF levels it may contribute to endothelial apoptosis and vessel regression¹⁴.

FSGS

VEGF mRNA expression in unstimulated PBMC was higher in children with FSGS than in those with MCN but the difference was not significant⁸³. Rats developed proteinuria after the injection of the supernatant of cultured PBMC from FSGS patients suggesting that a "glomerular permeability factor" released from PBMC may change glomerular permeability and result in proteinuria in FSGS⁸¹. In a murine FSGS model, adriamycin nephrosis, areas of interstitial expansion and tubular atrophy were associated with increased staining of hypoxia inducable factor-1 (HIF-1) in tubular and interstitial cells, reduced VEGF staining and loss of cortical microvasculature [abstract; Kairaitis LK, J Am Soc Nephrol 13:336A, 2002). Plasma VEGF levels were higher in proteinuric patients with FSGS than in those with MCNS but the difference was not significant⁸³. Serum VEGF levels in FSGS patients were not different from those in control subjects^48,87,88. Urinary VEGF excretion, on the other hand, was higher than normal in patients with FSGS, but did not correlate with serum VEGF, renal function, or proteinuria⁴⁸. In patients with FSGS, VEGF protein and mRNA expression were normal in preserved glomeruli but absent or very low in glomeruli with segmental or global sclerosis Table 1^49,90. Rare polymorphisms in the VEGF gene promotor were not associated with the development of proteinuria in FSGS [abstract; Watson CJ, J Am Soc Nephrol 9:A2497, 1998]. In contrast, the CC genotype and C allele of the more common -460 C/T polymorphism were associated with progression to end-stage renal disease in patients with FSGS [abstract; Summers AM, J Am Soc Nephrol 13:260A, 2002].

The low renal VEGF expression and increased urinary VEGF excretion in FSGS may be secondary to podocyte injury and loss in the urine. No solid conclusions on the role of VEGF in the pathophysiology of FSGS can thus be drawn.

Thrombotic microangiopathy (TMA)/hemolytic uremic syndrome (HUS)

A rat model of renal TMA with histologic features similar to human HUS was induced by antiglomerular endothelial cell antibodies which produced loss of glomerular and peritubular capillary endothelium¹⁰⁶. An early transient increase in VEGF immunostaining was observed in glomerular podocytes and cortical tubules, but despite proliferation of glomerular and peritubular endothelial cells, progressive glomerular and tubulointerstitial damage, and renal failure developed¹⁰⁶. The hypothesis that angiogenic growth factors may accelerate recovery of renal microvascular injury was studied in experimental TMA Table 1^107,108. Subcutanous administration of VEGF₁₂₁ resulted in greater recovery of the renal microvasculature together with a better renal function and less tubulointerstitial fibrosis¹⁰⁷. VEGF₁₂₁-treated rats had higher urinary nitrites and nitrates excretion, suggesting that the angiogenic effects of VEGF were mediated by nitric oxide¹⁰⁷. In a more severe form of renal TMA in rats with acute massive renal infarction, VEGF administration resulted in reduced glomerular endothelial cell apoptosis, preserved microvascular endothelium and less cortical and medullary necrosis¹⁰⁸. Elevated serum and plasma VEGF levels were found in HUS patients compared with controls and correlated with the severity of the disease [abstract; te Loo DM, J Am Soc Nephrol 13:251A, 2002]. In three patients with HUS, immunohistochemistry of renal biopsy material showed increased VEGFR-1 and VEGFR-2 in the glomeruli, and absent VEGF staining in tubuli compared to controls [abstract; te Loo DM, J Am Soc Nephrol 13:251A, 2002). In conclusion, VEGF may be required for glomerular and tubulointerstitial repair in TMA.

Renal transplantation

Acute rejection

To study the possible relationships between VEGF gene polymorphisms and the risk of acute renal allograft rejection, single nucleotide substitutions in the VEGF gene were identified in 173 white renal allograft recipients¹⁰⁹. The correlation between VEGF gene polymorphisms and VEGF production was investigated in vitro in PBMC from healthy individuals. Homozygotes with -1154G/G genotype and -2578C/C genotype showed the greatest risk of rejection and had the highest production of VEGF by stimulated PBMC from healthy volunteers, as compared with -1154A/A and -2578A/A genotypes, respectively. Heterozygotes with -1154G/A and -2578C/A genotypes demonstrated an intermediate risk. In biopsies of patients with acute rejection or temporary allograft dysfunction, VEGF immunostaining appeared to be quite similar to that of normal human kidney, but biopsies from normal kidneys were not included in this study Table 1¹¹⁰.

Chronic rejection

In the Fischer (F344) donor to Lewis recipient rat renal allograft, a well-established experimental model of chronic allograft rejection, no differences in VEGF mRNA expression were observed between allografts and isografts at any time. VEGF expression did not correlate with the extent of macrophage or myofibroblast infiltration¹¹¹. In kidneys from four patients with chronic vascular rejection, VEGF mRNA and protein expression was increased compared with normal kidneys, most notably in proximal and distal tubular cells and interstitial cells and to a lesser extent in vascular smooth muscle cells and the mononuclear inflammatory infiltrate²⁰. Viable glomerular podocytes displayed marked VEGF mRNA expression, but VEGF mRNA labeling was reduced or absent in sclerosed glomeruli²⁰. Similarly, increased renal VEGF protein expression was observed in glomerular podocytes and mesangial cells, vascular smooth muscle cells, and some endothelial cells and tubulointerstitium of kidneys in 17 patients with chronic renal allograft rejection, particularly in interstitial cells likely to be macrophages¹¹⁰. Renal VEGF immunostaining was increased in patients with chronic rejection when compared with patients with acute rejection or temporary allograft dysfunction¹¹⁰. The increased VEGF expression in glomeruli and particularly interstitium of human kidneys with chronic allograft rejection may be induced by hypoxia, as a consequence of reduced blood flow²⁰. Other hypotheses for the up-regulation of VEGF include production of VEGF by macrophages or proliferating glomerular mesangial cells¹¹⁰. Alternatively, VEGF may contribute to the recruitment of macrophages into the interstitium.

Cyclosporine nephrotoxicity

In a rat model of chronic cyclosporine nephrotoxicity, VEGF mRNA and protein expression as well as VEGFR-1 and VEGFR-2 mRNA expression were up-regulated as early as 7 days after exposure Table 1^112,113,114. The increased VEGF and VEGFR-1 expression continued until day 28, whereas VEGFR-2 expression declined but remained higher than in control rats^112,113. Immunostaining for VEGF was particularly strong in proximal and distal tubular cells and occasionally in glomerular podocytes¹¹³. Treatment with enalapril or losartan improved the tubulointerstitial fibrosis and afferent arteriolopathy, and decreased VEGF mRNA and protein expression and VEGFR-2 mRNA expression¹¹³. L-NAME worsened both glomerular filtration rate and cyclosporine-induced interstitial fibrosis and arteriolopathy, and further increased VEGF mRNA and protein expression while L-arginine had the opposite effect, suggesting that nitric oxide down-regulates VEGF expression in cyclosporine nephrotoxicity¹¹⁴. In a mouse model of acute cyclosporine toxicity, specific blockade of VEGF by a monoclonal antibody increased the deleterious effects of cyclosporine on the kidney¹¹⁵. Mice treated with the VEGF-antibody showed enhanced tubular toxicity, more apoptotic nuclei, increased blood urea nitrogen and hematuria compared with mice treated with cyclosporine alone or with cyclosporine and an irrelevant IgG¹¹⁵. However, urinary protein excretion was higher in cyclosporine-treated mice than in cyclosporine and VEGF-antibody–treated mice¹¹⁵. In vitro, the cyclosporine-induced toxicity in cultured murine proximal tubular epithelial cells also increased in the presence of a VEGF antibody¹¹⁵. In a rat model of chronic cyclosporine nephrotoxicity, administration of exogenous VEGF₁₂₁ resulted in renoprotective effects (i.e., osteopontin expression, macrophage infiltration, collagen III deposition were decreased versus control rats and afferent arteriolopathy was dramatically improved)¹¹⁶. Treatment with VEGF₁₂₁ also lowered blood pressure, which was suggested to be mediated by the accelerated recovery from tubulointerstitial and microvascular injury in VEGF-treated rats¹¹⁶. The role of VEGF in human cyclosporine nephrotoxocity has not been evaluated.

In summary, the expression of the VEGF system is increased in experimental cyclosporine nephrotoxicity. VEGF-blockade aggravated and VEGF-administration ameliorated the cyclosporine-induced injury, suggesting a role for VEGF in the repair process induced by cyclosporine nephrotoxicity.

Other kidney diseases

Cystic kidney diseases

One experimental study investigated the expressions of VEGF, HIF-1 and HIF-3 in polycystic kidney lesions that occurred spontaneously in two rats¹¹⁷. Compared with the kidneys of control rats, VEGF protein expression was increased in the inner stripe of the outer medulla where cystic alterations were prominent. HIF-1 and HIF-3 protein expressions were increased in proximal tubuli and in the thin loop of Henle, respectively. In kidneys from 14 patients with autosomal-dominant polycystic kidney disease (ADPKD), increased angiogenesis was identified, especially in the cyst walls and around the cysts¹¹⁸. In addition, VEGF protein was expressed in the epithelial cysts cells, VEGFR-2 protein in some capillaries surrounding the cysts and some glomeruli, whereas VEGFR-1 was expressed irregularly in some cyst cells and remnant tubular cells. Furthermore, VEGF₁₆₅ was demonstrated in cultured ADPKD cells and in their supernatant, and VEGF secretion was increased during hypoxia¹¹⁸. In patients with acquired cystic kidney disease, IL-6, IL-8, and VEGF concentrations were higher in the cyst fluid than in the blood, and higher than in patients with other cystic nephropathies (i.e., ADPKD or renal cell carcinoma). Taken together, these findings suggest an angiogenic role for VEGF in the pathogenesis of cystic kidney diseases, likely triggered by local hypoxia¹¹⁷ and involving the development of a pericystic circulation which may be necessary for cyst cells to grow¹¹⁸.

Ischemia/reperfusion injury

Cultured rat kidney tubular epithelial cells subjected to hypoxia showed increased VEGF staining in the periphery of the cells¹¹⁹. In a rat model of renal ischemia/reperfusion injury, whole kidney VEGF mRNA and protein expression was not increased following ischemia or ischemia and reperfusion, although preexisting VEGF in tubular epithelial cells redistributed from the cytoplasm to the basolateral surface¹¹⁹. Further, VEGFR-1 mRNA expression was not changed, whereas VEGFR-2 mRNA expression was up-regulated most prominent in glomerular endothelial cells and peritubular capillaries, but also in some tubular epithelial cells¹²⁰. It was speculated that the increased VEGFR-2 expression in peritubular capillaries during ischemia/reperfusion injury may direct the effects of VEGF released by ischemic tubular epithelial cells to adjacent endothelial cells in order to preserve the capillary blood supply and to promote tubular cell survival and recovery¹²⁰.

Congenital nephrotic syndrome of the Finnish type

In kidney samples of infants with congenital nephrotic syndrome of the Finnish type, expression and localization of VEGF and VEGFR-2 mRNA was similar to normal adult human kidney¹²¹. VEGF protein expression was also not different from normal kidneys except for an intense juxtaglomerular staining¹²¹.

Lupus nephritis

VEGF mRNA in PBMC of patients with SLE did not differ from those of controls¹²². VEGF plasma levels were higher in SLE patients than in controls, and SLE patients with renal failure had higher levels than those with normal renal function¹²³. VEGF protein expression was increased in distal tubules, collecting ducts and some podocytes in SLE patients with moderate renal failure¹²³. In two cases of SLE with diffuse endocapillary proliferative glomerulonephritis, renal VEGF expression was reduced⁴⁹.

Wegener's granulomatosis

Serum VEGF levels were higher in patients with Wegener's granulomatosis than in normal controls¹²⁴. Serum VEGF levels were markedly elevated in patients with major disease versus minor disease activity, suggesting that VEGF may be a marker of disease activity¹²⁴.

Renal cell carcinoma

VEGF promotes the growth of renal cell carcinoma, by virtue of its angiogenesis-inducing potential¹²⁵. A detailed description of the role of VEGF in renal cell carcinoma is, however, beyond the scope and space limitations of this review.

INTERFERENCE WITH THE VEGF AXIS

Several strategies exist to target VEGF and its receptors, including VEGF neutralizing antibodies, VEGF antagonizing aptamers, VEGF receptor-blocking antibodies, VEGF receptor antagonists, and angiopoietins¹²⁶. So far, the experience with these treatments in renal disease is limited to animal models. A humanized anti-VEGF monoclonal antibody (bevacizumab) and an anti-VEGF pegylated aptamer (EYE001) are currently evaluated in clinical trials to treat various types of cancer, as well as macular degeneration and diabetic retinopathy^127,128. VEGF receptor antagonists are also available. SU5416 and ZM323881 are specific for VEGFR-2, whereas PTK787/ZK222584 inhibits both VEGFR-1 and VEGFR-2¹²⁶. Other strategies to reduce VEGF-mediated effects, including placenta growth factor inhibition and drugs that interfere with signaling molecules in the VEGF signal transduction pathway such as Src/Fyn kinase inhibitors, are in development or in early stages of testing¹²⁶. Targeting specific VEGF signaling molecules might be a way to distinctively inhibit specific actions of VEGF.

Interference with the renin-angiotensin system may be an indirect means to affect the VEGF axis, but the interaction between both systems is complex. In cultured mesangial cells angiotensin II induced VEGF expression¹²⁹, whereas in tubular epithelial cells angiotensin II diminished VEGF expression⁶⁵. In agreement, divergent effects have been reported with in vivo blockade of the renin-angiotensin system. Angiotensin-converting enzyme (ACE) inhibition or AT1 antagonism reduced VEGF expression in diabetic nephropathy⁵⁴ and in cyclosporine nephrotoxicity¹¹³, suggesting that AT1 may be involved in the VEGF overexpression observed in these pathologies. In contrast, ACE inhibition was associated with an increased VEGF expression in the remnant kidney⁶⁸.

CONCLUSION

The strategic localization of VEGF in the vicinity of the filtration barrier and its known effects on microvascular permeability have engendered the hypothesis that VEGF controls glomerular permeability in the normal adult kidney and induces proteinuria in pathologic conditions. Although a large number of studies have been designed to examine this hypothesis, none has been able to firmly support it. Correlations of plasma or urinary VEGF levels with proteinuria in diverse glomerular pathologies have been inconsistent. Positive correlations between urinary VEGF levels and proteinuria may relate to urinary podocyte loss rather than to a causative link between renal up-regulation of VEGF and development of proteinuria. The inhibition of VEGF in experimental glomerulonephritis did not affect proteinuria. The laudable effect of VEGF-blockade on proteinuria in experimental diabetes may be indirect through inhibition of the disease process. Finally, podocyte-specific overexpression of VEGF caused a collapsing glomerulopathy rather than proteinuria.

The paramount task of VEGF in the adult glomerulus appears to be the stimulation of capillary endothelial cell growth and proliferation. This may be inappropriate in diabetic nephropathy where it contributes to glomerular hypertrophy and hyperfiltration, but may be an essential repair mechanism in glomerulonephritis and TMA. Similarly, tubular cells may respond to hypoxia or injury with the production of VEGF that stimulates proliferation of peritubular capillaries in order to overcome the tubular damage.

As the VEGF system is affected in a wide variety of kidney diseases, interventions to manipulate VEGF may be promising therapeutic tools. Several strategies to either inhibit or enhance the VEGF axis have shown promising results in animal models of renal disease, but no data in humans are presently available.

The VEGF gene is highly polymorphic and certain polymorphisms may be associated with alterations in the expression of VEGF. Although the diagnostic importance of genotyping renal patients remains to be established, some VEGF polymorphisms may develop into useful markers of disease susceptibility and/or progression.

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Acknowledgments

B. Schrijvers is supported by the Institute for the Promotion of Innovation by Science and Technology in Flanders (IWT), A. Flyvbjerg is supported by the Danish Diabetes Association, the Eva and Henry Frænkels Memorial Foundation, and the Danish Medical Research Council.