A Defective Angiogenesis in Chronic Kidney Disease

Abstract

Background. A progressive reduction in peritubular capillary flow is observed in chronic kidney disease (CKD) patients as the disease severity progresses. This suggests an altered vascular homeostasis in CKD patients, but such a defective mechanism needs to be verified. Methods. To study the vascular injury as reflected by circulating endothelial cell (CEC), the balance between angiogenic factor, vascular endothelial growth factor (VEGF), and antiangiogenic factor, endostatin. Results. A deficient VEGF was observed, whereas the value of endostatin and CEC were abnormally elevated in CKD patients. Discussion. Enhanced CEC reflects an increased activity of vascular injury. A deficient VEGF in the presence of enhanced antiangiogenesis (endostatin) implies a defective angiogenesis. This may explain the progressive nature of renal microvascular disease observed in late stage of CKD patients.

Keywords:

• VEGF
• endostatin
• angiogenesis
• chronic kidney disease
• circulating endothelial cell (CEC)

INTRODUCTION

The National Kidney Foundation / Kidney Disease Outcome Quality Initiative clinical practice guidelines define chronic kidney disease (CKD) patient as having creatinine clearance value of less than 60 mL/min/1.73m², which is equivalent to late stage of CKD (stages 3–5).[1],[2] Such a definition depends on the insensitiveness of present diagnostic markers such as serum creatinine determination or microalbuminuria, which is able to screen only the late stage of CKD, but is unable to screen early stage of CKD (stages 1 and 2). It has been a general consensus that therapeutic strategy at present for this late stage of CKD patients is usually ineffective in preventing them from entering end-stage renal disease.[3],[4] In addition, accumulative evidence renders support that renal microvascular disease, which is the crucial determinant of renal disease progression, has become progressive as the disease severity progresses.[5] The progressive nature of renal microvascular disease implies that the mechanism relevant to the balance of vascular homeostasis between vascular injury and vascular repair is likely to be altered. Therefore, it is the purpose of this study to determine circulating endothelial cell, vascular endothelial growth factor, and endostatin, which reflects vascular injury, angiogenic factor, and antiangiogenic factor, respectively.

MATERIALS AND METHOD

Sixty chronic kidney disease patients associated with moderately impaired renal function and 15 normal controls were subject to the following studies.

Enumeration of Circulating Endothelial Cells (CEC)

The number of CEC was examined using the buffy coat smear technique as described by Solovey.[6] Diluted blood was layered on a Ficoll-Hypaque density gradient (Histopaque-1077, Sigma, St. Louis, Missouri, USA) and sedimented for 30 min at 250 g. The pooled supernatant and interface were centrifuged for 5 min at 1200 g, and a smear was made of buffy-coat pellet. After being air-dried, the smear was fixed with 4% paraformaldehyde for 10 min and stained with FITC-conjugated sheep antihuman von Willebrand factor (Seroter, Oxford, UK). The number of CEC/mL of whole blood was examined using fluorescence microscopy. Negative controls were provided by the white cells on the smears and by parallel slides prepared with control negative antibody.

ELISA for Vascular Endothelial Growth Factor (VEGF)

This assay employs the quantitative sandwich enzyme immunoassay technique. Standards and samples are pipetted into the wells, and any VEGF present is bound by the immobilized antibody. After washing away any unbound substances, an enzyme-linked polyclonal antibody specific for VEGF is added to the wells. Following a wash to remove any unbound antibody enzyme reagent, a substance solution is added to the wells, and color develops in proportion to the amount of VEGF bound in the initial step. The color development is then stopped, and the intensity of the color is measured.

Endostatin

This assay employs the quantitative sandwich enzyme immunoassay technique in a similar manner as described above.

RESULTS

As illustrated in Table 1, a deficient VEGF and enhanced levels of endostatin and circulating endothelial cells were substantiated in CKD patients.

Table 1 The altered vascular homeostasis observed in late stage of CKD

	Normal	CKD	p value
VEGF	355 ± 167	36 ± 35	< 0.001
Endostatin pg/mL	122 ± 21	356 ± 126	< 0.001
Circulating endothelial cells/mL	112 ± 185	1893 ± 1325	0.001

DISCUSSION

Recently, various circulating toxins such as oxidative stress, immunocirculatory disturbance, lipid disorder, sugar, vasoconstrictors, stress, and aging were found to induce endothelial cell injury in CKD patients .[7–9] This is substantiated by enhanced numbers of endothelial cell loss into the circulation. Injury to the glomerular endothelium is also reflected by intrarenal hemodynamic study, which reveals a unique pattern of hemodynamic maladjustment characterized by a preferential constriction of the efferent arteriole.[10] Such constriction induces a sustained and progressive reduction in peritubular capillary flow, a chronic ischemic injury to the tubulointerstitium, and (eventually) tubulointerstitial fibrosis. The progressive reduction in peritubular capillary flow reflects a progressive nature of renal microvascular disease.[11] The result of our study has supported this view. An enhanced circulating endothelial cell loss has continuously been observed in our CKD patients, by which it indicates a continuous process of renal microvascular injury. Impaired angiogenesis is supported by the deficient state of VEGF. In addition, the enhanced activity of antiangiogenesis is reflected by an elevated level of endostatin. Endostatin is an active metabolite of platelets. The activation of platelets resulting in the formation of local intravascular coagulation in the renal microcirculation has previously been reported, and is reflected by the increased consumption of platelet and shortened platelet half life in CKD patients.[12] Endostatin released from platelet activation is accumulated, and appears to be directly correlated with the serum creatinine level. Increased levels of endostatin may participate in the antiangiogenic activity observed in late stage of CKD patients, in which a defective vascular repair has recently been demonstrated.[5] Thus, the late stage of CKD patients appears to be an unfortunate environment in favor of renal regression. This study proposes that the preventive therapeutic strategy should be initiated in the early stage of CKD (stages 1 or 2), in which the environment associated with an adequate renal functional reserve, is favorable towards renal regeneration.[13]

ACKNOWLEDGMENT

This study is supported by the Thailand Research Fund and the Thailand Research Council Fund.