Vascular Endothelial Growth Factor-A165b Is Protective and Restores Endothelial Glycocalyx in Diabetic Nephropathy

Diabetic nephropathy is the leading cause of ESRD in high-income countries and a growing problem across the world. Vascular endothelial growth factor-A (VEGF-A) is thought to be a critical mediator of vascular dysfunction in diabetic nephropathy, yet VEGF-A knockout and overexpression of angiogenic VEGF-A isoforms each worsen diabetic nephropathy. We examined the vasculoprotective effects of the VEGF-A isoform VEGF-A₁₆₅b in diabetic nephropathy. Renal expression of VEGF-A₁₆₅b mRNA was upregulated in diabetic individuals with well preserved kidney function, but not in those with progressive disease. Reproducing this VEGF-A₁₆₅b upregulation in mouse podocytes in vivo prevented functional and histologic abnormalities in diabetic nephropathy. Biweekly systemic injections of recombinant human VEGF-A₁₆₅b reduced features of diabetic nephropathy when initiated during early or advanced nephropathy in a model of type 1 diabetes and when initiated during early nephropathy in a model of type 2 diabetes. VEGF-A₁₆₅b normalized glomerular permeability through phosphorylation of VEGF receptor 2 in glomerular endothelial cells, and reversed diabetes-induced damage to the glomerular endothelial glycocalyx. VEGF-A₁₆₅b also improved the permeability function of isolated diabetic human glomeruli. These results show that VEGF-A₁₆₅b acts via the endothelium to protect blood vessels and ameliorate diabetic nephropathy.     

Vascular endothelial growth factor and its soluble receptor, Flt-1, are not correlated to erythropoietin in diabetics with normal or reduced renal function

BACKGROUND AND AIMS: Recombinant erythropoietin upregulates the expression of the vascular endothelial growth factor (VEGF) receptors, Flt-1 (VEGFR-1) and KDR/Flk-1 (VEGFR-2), in endothelial cells. The integrity of the VEGF system seems to be crucial for the regulation of endothelial permeability and thus for the avoidance of renal protein leakage. As albuminuria/proteinuria is a hallmark of diabetic nephropathy, we examined cross-sectionally in 35 type 1 and 37 type 2 diabetic patients with various degrees of renal dysfunction and albuminuria whether there was an interrelationship between intrinsic erythropoietin (EPO) and VEGF/Flt-1. METHODS AND RESULTS: In patients with plasma creatinine values < or =1.5 (n = 53) or >1.5 mg/dL (n = 19), the mean serum EPO was 5.6 +/- 4.4 and 10.2 +/- 7.0 mU/mL (P = 0.02), respectively. In the two groups, urinary and serum VEGF(165) concentrations were similarly distributed (mean 94.3 +/- 91.8 vs 108 +/- 72.2 ng/L and 91.7 +/- 76.8 vs 91.9 +/- 74.9 ng/L, respectively; both P = NS). The mean urinary Flt-1 for the two groups amounted to 0.14 +/- 0.35 and 0.51 +/- 0.93 ng/mL (P = 0.045), respectively. No correlation between VEGF or Flt-1 and EPO was apparent. CONCLUSION: Our data suggest that in vivo EPO does not affect the functionality and/or production of components of the VEGF/Flt-1 system in diabetics with normal or reduced renal function.     

Local VEGF activity but not VEGF expression is tightly regulated during diabetic nephropathy in man

Several studies have implicated the angiogenic cytokine vascular endothelial growth factor (VEGF) in the development of diabetic nephropathy, but no data are available about its local activity during human disease. Glomeruli from 52 archival biopsies from type II diabetics were evaluated and compared to 10 renal biopsies without kidney disease (controls). Glomerulosclerosis, capillary rarefaction, glomerular and endothelial cell proliferation, apoptosis, VEGF expression, as well as receptor-bound VEGF indicating local VEGF activity, and phosphorylation of the signal transduction molecule Akt were investigated. Owing to substantial heterogeneity of glomerular lesions in individual biopsies, these parameters were correlated with the degree of injury in individual glomeruli rather than biopsies. Severe glomerular capillary rarefaction was linked to the degree of glomerulosclerosis. While cellular apoptosis was detected independent of the stage of injury, endothelial cell proliferation indicating capillary repair was markedly increased only in mildly/moderately injured glomeruli. In controls, VEGF was predominantly expressed in podocytes, whereas receptor-bound VEGF was confined to the glomerular endothelium. VEGF expression was increased in all diabetic glomeruli by many different cell types. In contrast, VEGF receptor activation was increased predominantly in the endothelium of only mildly injured glomeruli, but significantly decreased in more severely injured glomeruli. Diabetic nephropathy is associated with glomerular capillary rarefaction. Despite overall increased glomerular VEGF, the decreased receptor-bound VEGF on the endothelium may be an indicator of an insufficient capillary repair reaction.     

Increased expression of vascular endothelial growth factor in kidney leads to progressive impairment of glomerular functions

Vascular endothelial growth factor (VEGF) is an important mediator in maintaining normal kidney functions. In addition, several lines of evidence suggest that upregulation of VEGF in glomeruli may be associated with or cause renal dysfunction such as diabetic nephropathy. For elucidation of the pathologic consequences of high levels of VEGF in glomeruli, transgenic (Tg) rabbits that express human VEGF(165) isoform in both kidney and liver under the control of the human alpha-1-antitrypsin promoter were generated and characterized. With the use of heterozygous Tg rabbits and their littermates aged 8 to 55 wk, renal functions and structures were investigated. Compared with control rabbits, Tg rabbits exhibited progressive proteinuria with increased GFR at the early stage and decreased GFR at the later stage. Histologic examinations revealed that Tg rabbit kidneys were characterized by considerable glomerular hypertrophy as a result of increased proliferation of both glomerular capillaries and mesangial cells accompanied by prominent podocyte hypertrophy. With increasing age starting from 20 wk, Tg rabbit kidneys showed prominent formation of microaneurysms and capillary proliferation at the vascular pole area. At a later stage (55 wk), many glomeruli showed sclerosis and tuft collapse with the formation of glomerular cysts on a background of tubular atrophy and interstitial fibrosis. This study provides the first evidence that increased expression of VEGF in glomeruli directly causes the glomerular hypertrophy that is associated with proteinuria, suggesting that VEGF exerts multiple effects on the glomerular pathophysiologic processes.     

Gene expression of VEGF and its receptors Flk-1/KDR and Flt-1 in cultured and transplanted rat islets

BACKGROUND: Vascular endothelial growth factor (VEGF) and its two receptor tyrosine kinases, Flk-1/KDR and Flt-1, may play an important role in mediating the revascularization of transplanted pancreatic islets. METHODS: Using semiquantitative multiplex reverse-transcribed polymerase chain reaction we determined the gene expression of VEGF and its receptors in cultured and transplanted rat islets. RESULTS: After exposure of islet cells to hypoxia in vitro, increases were found in the gene expression of the VEGF120 and VEGF164 isoforms, with simultaneous increases in VE-cadherin, Flk-1/KDR, and Flt-1. In vivo studies consisted of analysis of islet grafts transplanted into both normal and diabetic recipients. Expression of both VEGF120 and VEGF164 in grafts was up-regulated for the first 2-3 days after transplantation, with the response being more prolonged in the diabetic rats. These increases were followed by reduced expression of VEGF on days 5, 7, and 9. Increases in the expression of VE-cadherin in islet grafts in normal and diabetic recipients tended to parallel VEGF expression, with the increases in both probably being caused by hypoxia. The early increases of VEGF expression were followed by a rise in the expression of VEGF receptors, which probably represents the early stages of angiogenesis. Graft expression of Flk-1/KDR and Flt-1 was enhanced at 3 and 5 days in the normoglycemic recipients, while in the diabetic recipients increases were found later on days 5, 7, and 14. CONCLUSIONS: The delayed expression of VEGF receptors in the diabetic recipients could reflect impaired angiogenesis caused by the diabetic milieu; this delay could contribute to the less outcomes of grafts transplanted into a hyperglycemic environment.     

补阳还五汤对早期糖尿病肾病患者血管内皮生长因子及其受体Flt-1的影响

目的：探讨补阳还五汤对早期糖尿病肾病患者血管内皮生长因子（VEGF）及其受体（Flt-1）的影响。方法：将60例早期糖尿病肾病患者随机分为治疗组和对照组,两组均用西药常规治疗,治疗组加服补阳还五汤。采用酶联免疫法检测各组血清VEGF及其受体Flt-1水平。结果：治疗组血清VEGF及其受体Flt-1水平较治疗前明显下降（P＜0．05）,24h尿蛋白量较治疗前明显下降（P＜0．05）,且治疗组优于对照组（P＜0．05）。VEGF、Flt—1与尿蛋白呈正相关。结论：VEGF及其受体Flt-1在早期糖尿病肾病发病机制中起重要作用,补阳还五汤可降低早期糖尿病肾病患者血清VEGF及其受体Flt—1蛋白表达,是其保护肾功能的可能作用机制之一。     

A neutralizing VEGF antibody prevents glomerular hypertrophy in a model of obese type 2 diabetes, the Zucker diabetic fatty rat.

BACKGROUND: Antagonism of vascular endothelial growth factor (VEGF) has improved the outcome in experimental nephropathies of various origins, including diabetic nephropathy in a type 1 diabetic rat model and a type 2 diabetic mouse model. Neutralizing VEGF antibodies prevented glomerular hypertrophy in these models. We examined the renal effects of VEGF blockade in an obese rat model of type 2 diabetic nephropathy and investigated the mechanism underlying the inhibition of glomerular hypertrophy. METHODS: Twenty female Zucker diabetic fatty (ZDF) rats, fed a high-fat diet and aged 10 weeks, were treated with VEGF antibodies or an irrelevant isotype-matched IgG. Ten heterozygous (fa/+) littermates served as additional non-diabetic, lean controls. Urinary albumin excretion (UAE) and creatinine clearance (CrCl) were assessed at baseline, and at 3 and 5 weeks. Kidney weight and glomerular volume were determined at the end of the study. Glomerular apoptosis was examined with anti-active caspase-3 immunohistochemistry. RESULTS: All obese animals had established diabetes, hyperlipidaemia and normal blood pressure, which were not influenced by VEGF antibody treatment. ZDF control rats had increased UAE, CrCl, kidney weights and glomerular volumes compared with non-diabetic, lean control rats. VEGF antibody treatment prevented the glomerular hypertrophy, but did not affect UAE, CrCl and kidney weight. Glomerular anti-active caspase-3 immunostaining was not different between the groups. CONCLUSIONS: Inhibition of VEGF prevented early glomerular hypertrophy in ZDF rats with established diabetes. Increased apoptosis of glomerular endothelial cells does not appear to underly the inhibition of glomerular growth.     

Apoptosis in the kidneys of patients with type II diabetic nephropathy

The occurrence and extent of apoptosis in the kidneys of patients with diabetic nephropathy is largely unknown. We evaluated apoptosis in renal biopsies obtained from patients with early or advanced type II diabetic nephropathy. Apoptosis was about 6- and 3-fold higher, respectively, in glomeruli and tubules in kidneys of patients with early nephropathy than in the normal kidney and this was not further increased in advanced diabetic nephropathy. Glomerular apoptosis was related directly to hemoglobin A1(c) and systolic blood pressure, whereas tubular cell apoptosis correlated to diabetes duration and low-density lipoprotein-cholesterol. Fas, Fas ligand, and p38 mitogen-activated protein kinase expressions were enhanced in glomeruli and tubules; however, this did not correlate with apoptosis. In patients with proteinuria, apoptosis was associated with the subsequent loss of kidney function. When these parameters were subjected to multivariate analysis, only glomerular apoptosis retained a significant independent predictive value. Our findings suggest that apoptosis might be a clinically relevant mechanism of glomerular and tubular cell loss in proteinuric type II diabetic patients.     

A Mildly Altered Vascular Homeostasis in Early Stage of CKD

Background. A continuous increase in number of CKD patients entering ESRD is a growing public health threat, which reflects the present therapeutic failure usually initiating at the late stage of CKD. Objective. To study the mechanism of vascular repair in CKD patients associated with mildly impaired renal function, which included angiogenic factors such as VEFG, angiopoietin-1, and flt-1 (VEGFR1); and antiangiogenic factors such as angiopoietin-2 and KDR (VEGFR2). Results. A mild defect in angiogenic factor—namely, angiopoietin-1—was observed, whereas VEGF and flt-1 (VEGFR1) were within normal limit. Also, antiangiogenic factor—namely, angiopoietin-2—was mildly elevated, whereas KDR (VEGFR2) remained within normal limit. Conclusion. The mechanism of vascular repair appears to be adequately functional in the early stage of CKD. Therapeutic intervention at this stage can improve renal perfusion and restore renal function as indicated in normoalbuminuric, type 2 diabetic nephropathy. The authors encourage changing the conceptual view of treatment under common treatment at late stage of CKD to treatment at early stage of CKD under an environment favorable for renal regeneration.     

Impaired glomerular maturation and lack of VEGF165b in Denys-Drash syndrome

Individuals with Denys-Drash syndrome (DDS) develop diffuse mesangial sclerosis, ultimately leading to renal failure. The disease is caused by mutations that affect the zinc finger structure of the Wilms' tumor protein (WT1), but the mechanisms whereby these mutations result in glomerulosclerosis remain largely obscure. How WT1 regulates genes is likely to be complex, because it has multiple splice forms, binds both DNA and RNA, and associates with spliceosomes. Herein is described that in DDS podocytes, the ratio of both WT1 +KTS isoforms C to D differs considerably from that of normal child and adult control podocytes and more closely resembles fetal profiles. Aside from the delay in podocyte maturation, DDS glomeruli show swollen endothelial cells, reminiscent of endotheliosis, together with incompletely fused capillary basement membranes; a dramatic decrease in collagen alpha4(IV) and laminin beta2 chains; and the presence of immature or activated mesangial cells that express alpha-smooth muscle actin. Because appropriate vascular endothelial growth factor A (VEGF-A) expression is known to be essential for the development and maintenance of glomerular architecture and function, this article addresses the question of whether VEGF-A expression is deregulated in DDS. The data presented here show that DDS podocytes express high levels of the proangiogenic isoform VEGF165, but completely lack the inhibitory isoform VEGF165b. The VEGF165/VEGF165b ratio in DDS resembles that of fetal S-shaped bodies, rather than that of normal child or adult control subjects. The alteration in VEGF-A expression presented here may provide a mechanistic insight into the pathogenesis of DDS.     

Heparan sulfate proteoglycans are lost in patients with diabetic nephropathy.

The pathogenesis of diabetic nephropathy relative to the changes in the glomerular extracellular matrices was investigated. Renal tissues from 10 diabetic patients were immunostained with antibodies directed against heparan sulfate proteoglycans (HS-PGs), laminin, type IV collagen and fibronectin. Seven patients were nephrotic and had advanced glomerulosclerosis with nodular lesion, while the other 3 had no renal manifestations or minor glomerular tissue alterations. Controls included kidneys removed from patients with renal tumors and specimens obtained by renal biopsy from patients with IgA nephropathy. Relationships among proteinuria, intensity of fluorescence and glomerular changes were studied. In diabetes 3 patients with minor glomerular lesions were found to have no changes in various components of extracellular matrices. A marked reduction in the intensity of staining with anti-HS-PG antibodies was observed in renal specimens from patients with nodular glomerulosclerosis and proteinuria, while a mild decrease in the intensity of fluorescence was observed in tissues stained with antilaminin antibodies. An increase compared to normal control sample findings in type IV collagen and fibronectin was observed in the mesangium of sclerosing glomeruli. No loss of HS-PG was observed in patients with IgA nephropathy. These results indicate that glomerular extracellular matrix HS-PG is lost in association with diabetic nephropathy; this loss results in alteration of the charge-selective properties of glomerular capillaries. This alteration may, in part, be the cause of the proteinuria associated with diabetic nephropathy.     

The role of vascular endothelial growth factor (VEGF) in renal pathophysiology

Vascular endothelial growth factor (VEGF) is an endothelial-specific growth factor that promotes endothelial cell proliferation, differentiation and survival, mediates endothelium-dependent vasodilatation, induces microvascular hyperpermeability and participates in interstitial matrix remodeling. In the kidney, VEGF expression is most prominent in glomerular podocytes and in tubular epithelial cells, while VEGF receptors are mainly found on preglomerular, glomerular, and peritubular endothelial cells. The role of VEGF in normal renal physiology is essentially unknown. The absence of prominent effects of VEGF blockade in normal experimental animals suggests a limited function during homeostasis, although a role in the formation and maintenance of glomerular capillary endothelial fenestrations has been suggested. VEGF and its receptors are up-regulated in experimental animals and humans with type 1 and type 2 diabetes. Inhibition of VEGF has beneficial effects on diabetes-induced functional and structural alterations, suggesting a deleterious role for VEGF in the pathophysiology of diabetic nephropathy. VEGF is required for glomerular and tubular hypertrophy and proliferation in response to nephron reduction, and loss of VEGF is associated with the development of glomerulosclerosis and tubulointerstitial fibrosis in the remnant kidney. No firm conclusions on the role of VEGF in minimal change or membranous glomerulonephritis can be drawn. VEGF may be an essential mediator of glomerular recovery in proliferative glomerulonephritis. Glomerular and tubulointerstitial repair in thrombotic microangiopathy and cyclosporin nephrotoxicity may also be VEGF-dependent. In conclusion, VEGF is required for growth and proliferation of glomerular and peritubular endothelial cells. While deleterious in some, it may contribute to recovery in other forms of renal diseases.     

Altered Vascular Homeostasis in Type 2 Diabetic Nephropathy

Type 2 diabetic nephropathy is a primary cause of ESRD worldwide. Therapeutic strategy in patients with microalbuminuric or macroalbuminuric type 2 diabetic nephropathy usually fails to restore renal function but merely slows the renal disease progression. In contrast, a recent study implies that the restoration of renal function as well as renal perfusion can be accomplished in early stage of type 2 diabetic nephropathy (normoalbuminuria) by correcting the hemodynamic maladjustment in renal microcirculation with vasodilators. Therefore, we intend to study the mechanism of vascular homeostasis to explain why treatment in the late stage of diabetic nephropathy during microalbuminuria or macroalbuminuria fails to enhance renal perfusion or restore renal function. The results indicate that such therapeutic failure in late-stage type 2 diabetic nephropathy likely relates to multiple defects in vascular repair, namely deficiencies in angiogenic factors such as endothelial progenitor cell, angiopoietin-1, flt-1 receptor, as well as elevated levels of antiangiogenic factors such as angiopoietin-2 and KDR.     

Detection of glomerular sialic acids in patients with diabetic nephropathy

A study on immunofluorescence of sialic acids in glomeruli from patients with diabetic nephropathy is described. Measurement of sialic acid in sera from 25 patients with diabetes mellitus was also performed. Renal biopsy specimens from 12 patients with diabetic nephropathy were stained with FITC-labeled antihuman IgG antiserum and rhodamine-labeled Triticum vulgaris (WGA) or Limulus polyphemus (LPA). These specimens were also stained with such reagents after treatment with neuraminidase, trypsin or citrate buffer. Both deposition of IgG and binding of WGA in the glomerular capillary walls were observed in all patients with diabetic nephropathy. The binding of WGA in the glomerular capillary walls in diabetic nephropathy was significantly increased compared with that in four normal renal tissues. However, the binding of LPA was hardly observed in the glomerular capillary walls of patients with diabetic nephropathy. The binding of WGA in the glomeruli was markedly decreased after treatment with neuraminidase although it was hardly decreased after treatment with trypsin or citrate buffer. The levels of sialic acid in sera from patients with diabetic nephropathy were markedly increased. It is suggested that accumulated substances in the glomerular capillary walls with an affinity for WGA are mainly composed of N-acetyl glucosamine and/or N-acetyl neuraminic acid in patients with diabetic nephropathy.     

Vascular endothelial growth factor receptors in human mesangium in vitro and in glomerular disease

Mesangial cell proliferation and growth factor over-expression are characteristic features of several glomerular diseases. Vascular endothelial growth factor (VEGF), a potent mitogen, is expressed in podocytes in the glomerulus, and VEGF receptors (flt-1, KDR, and neuropilin-1) are present on endothelial cells and other cell types. This study examined whether human mesangial cells (HMC) express VEGF receptors in vitro and ex vivo and evaluated the effect of VEGF on HMC proliferation. All receptor types were detected in HMC in vitro by immunofluorescence and Western blotting. VEGF(165) induced a dose-responsive increase in (3)H-thymidine incorporation (25 ng/ml VEGF(165) : 2.3-fold increase; 50 ng/ml : 3.8-fold; 100 ng/ml : 4. 8-fold; 200 ng/ml : 3.4-fold; P = 0.016) and in cell number (50 ng/ml VEGF(165) : 1.2-fold increase; 100 ng/ml : 1.6-fold; 200 ng/ml : 1.4-fold; P = 0.005), effects prevented by an anti-VEGF(165) polyclonal neutralizing antibody (100 microg/ml). The proliferative effect was confirmed by a tetrazolium dye-based assay (100 ng/ml VEGF(165) : 1.4-fold increase). In ex vivo experiments, VEGF receptors in biopsy material from normal and diseased kidneys were detected by immunohistochemistry. No mesangial flt-1 receptor staining was seen in normal renal cortical tissue samples, and only weak mesangial KDR staining was detected. In contrast, mesangial flt-1 and KDR receptor staining were both clearly seen in biopsy samples from proliferative renal diseases. In conclusion, flt-1, KDR, and neuropilin-1 are present on cultured HMC, and VEGF(165) induces HMC proliferation. In addition, the flt-1 and KDR receptors are expressed in the mesangium in mesangioproliferative disease.     

Evidence linking glycated albumin to altered glomerular nephrin and VEGF expression, proteinuria, and diabetic nephropathy

BACKGROUND: Albumin modified by Amadori-glucose adducts has been linked to the development of diabetic nephropathy through its ability, independent of hyperglycemia, to activate protein kinase C-beta (PKC-beta), up-regulate the transforming growth factor-beta (TGF-beta) system, and stimulate expression of extracellular matrix proteins in glomerular cells, and by the demonstration that reducing the burden of glycated albumin ameliorates renal structural and functional abnormalities in the db/db mouse. METHODS: To probe whether the salutary effects consequent to lowering glycated albumin, which include reduction of albuminuria, relate to an influence of the Amadori-modified protein on nephrin, the podocyte protein critical to regulation of protein excretion, and on the angiogenic vascular endothelial growth factor (VEGF), which induces microvascular permeability, diabetic db/db mice were treated with a small molecule that inhibits the nonenzymatic glycation of albumin. RESULTS: Compared to nondiabetic db/m mice, diabetic controls exhibited increased urinary excretion of albumin and type IV collagen, elevated renal TGF-beta1 protein levels, reduced glomerular nephrin immunofluorescence and nephrin protein by immunoblotting, and increased glomerular VEGF immunostaining and renal VEGF protein content. Diabetic animals receiving test compound showed significant lowering of proteinuria, normalization of renal TGF-beta1 protein, and significant restoration of altered glomerular nephrin and VEGF expression. CONCLUSION: The findings causally implicate the increased glycated albumin associated with the diabetic state in the abnormal renal nephrin and VEGF expression found in diabetes, thereby promoting proteinuria and glomerulosclerosis.