Circulating angiopoietin-2 is a marker for early cardiovascular disease in children on chronic dialysis

Abstract

Cardiovascular disease (CVD) is increasingly recognised as a complication of childhood chronic kidney disease (CKD) even in the absence of diabetes and hypertension. We hypothesized that an alteration in angiopoietin-1 and -2, growth factors which regulate endothelial and vascular function could be involved. We report that the endothelial survival factor, angiopoietin-1 is low in children with pre-dialysis CKD whereas the pro-inflammatory angiopoietin-2 is elevated in children on dialysis. In dialysis patients, angiopoietin-2 positively correlated with time on dialysis, systolic blood pressure, and carotid artery intima media thickness. Elevated angiopoietin-2 levels in dialysis versus pre-dialysis CKD patients were also associated with an anti-angiogenic (high soluble VEGFR-1 and low VEGF-A) and pro-inflammatory (high urate, E-selectin, P-selectin and VCAM-1) milieu. Ang-2 was immunodetected in arterial biopsy samples whilst the expression of VEGF-A was significantly downregulated in dialysis patients. Serum urate correlated with angiopoietin-2 levels in dialysis patients and addition of uric acid was able to induce rapid release of angiopoietin-2 from cultured endothelial cells. Thus, angiopoietin-2 is a marker for cardiovascular disease in children on chronic dialysis and may act as an anti-angiogenic and pro-inflammatory effector in this context. The possibility that the release of angiopoietin-2 from endothelia is mediated by urate should be explored further.

Figure 1. Circulating Ang levels in pre-dialysis CKD and dialysis patients.

Serum Ang-1 levels (A) were significantly lower in pre-dialysis CKD patients compared with healthy controls. In dialysis patients Ang-1 levels were similar to values found in healthy controls. Similar levels of both circulating Ang-2 (B) and Ang-2/Ang-1 (C) were found in healthy children and those with pre-dialysis CKD, but these were significantly increased in the dialysis group.

Figure 2. Correlation of Ang-2 levels with clinical and vascular parameters.

Ang-2 levels in dialysis individuals correlated positively with time on dialysis (A), serum urate levels (B), systolic blood pressure SDS (C) and cIMT (D). Independent variables are shown on the x-axis. Regression lines account for dialysis patients only. Dotted line in D indicates the value for cIMT in healthy age-matched controls. There was no correlation between Ang-2 and any clinical and vascular measures in pre-dialysis CKD patients.

Figure 3. Circulating levels of VEGF-A and sFlt-1 in pre-dialysis CKD and dialysis patients.

VEGF-A levels were significantly lower in individuals on dialysis compared with pre-dialysis CKD patients (A). In contrast, sFlt-1 were significantly higher in the dialysis patients (B)

Figure 4. Circulating levels of soluble cell adhesion molecules.

Compared with pre-dialysis CKD individuals, patients treated with dialysis had significantly elevated levels of soluble E-selectin (A), P-selectin (B) and VCAM-1 (C); there was no difference in ICAM-1 levels (D).

Figure 5. Immunolocalisation of vascular growth factors in arteries.

Ang-1 was detected in the media of vessels from both pre-dialysis CKD (A) and dialysis patients (B); no differences in staining intensity were observed between the two groups (C). Ang-2 was immunodetected in both the media and endothelia (arrows) in pre-dialysis CKD (D) and dialysis (E) vessels with similar intensity (F). The endothelial later was also positive for von Willebrand factor (arrows, G and H). VEGF-A immunostaining was prominent in the media of pre-dialysis CKD vessels (I), but was significantly decreased in dialysis patients (J and K). All fields taken with ×40 objective.

Figure 6. Effect of uric acid on Ang-2 secretion in HUVECs.

A) Both non-stimulated and uric acid stimulated HUVECs expressed the mRNA for the transporter Urat1 but not Oat1-4; they were also positive for Ang-1, Ang-2, Tie-1, Tie-2 and Tlr4. Sizes were determined using a 100 bp marker (m), positive (+ive) controls consisted of total kidney cDNA and negative controls were without cDNA addition. (B) Uric acid stimulation for 15 minutes, but not 72 hours (C) led to elevated Ang-2 secretion in the conditioned media of HUVEC cells. Within the cells, uric acid stimulation led to a decreased abundance of Ang-2 mRNA after 6 hours of stimulation (D). a = p<0.05 compared with controls, b = p<0.01 compared with controls, c = p<0.001 compared with controls, d = p<0.01 compared with HUVEC stimulated with 3 mg/dl uric acid, e = p<0.01 compared with HUVEC stimulated with 6 mg/dl uric acid, f = p<0.05 compared with HUVEC stimulated with 9 mg/dl uric acid.