第二型血管生成素與慢性腎病的白蛋白尿及動脈硬化之關聯

Abstract

台灣末期腎病患者的發生率與盛行率皆居世界首位，慢性腎病不僅是個體健康問題，相關併發症治療的花費更是社會經濟的巨大負擔。心血管疾病是造成慢性腎病患者死亡的主要原因，多年來的研究已證實慢性腎病本身即等同於心血管疾病的危險因子，而在慢性腎病患者，即使在使用藥物矯正傳統心血管疾病危險因子，如糖尿病、高血壓、高血脂等，患者的死亡率仍較一般族群高，意味著存在一些與慢性腎病相關的特殊危險因子，對患者的預後有極為重要的影響，其中最為注目的就是鈣磷不平衡所造成的血管鈣化。臨床研究已證實慢性腎病患者的血管壁中層鈣化所造成的血管硬化，與病人的心血管疾病預後有明確相關。一些研究認為致病機轉可能與血管平滑肌細胞之形態轉變有相關，而血管壁細胞包括血管平滑肌細胞與血管內皮細胞等細胞間之相互作用可能也與血管鈣化的發生有關聯。此外，發炎反應與內皮失衡也是重要的非傳統心血管疾病危險因子。臨床研究顯示發炎反應與患者心血管疾病之發生及死亡率有明確相關，而在臨床上白蛋白尿不僅代表內皮受損，臨床研究亦證實其與慢性腎病患者之心血管疾病預後有明確關聯。 在眾多致病因子及媒介物中，臨床研究發現第二型血管生成素(angiopoietin-2)與透析患者之冠狀動脈及周邊動脈疾病相關，同時血中第二型血管生成素濃度與白蛋白尿程度皆與慢性患者之心血管疾病發生及死亡有明顯關聯。在動物實驗中，第二型血管生成素除了在血管新生有其角色之外，與發炎反應的誘發更是密切相關。第二型血管生成素會經由影響巨噬細胞浸潤及吸引血管平滑肌細胞而促進動脈血管生成(arteriogenesis)，我們認為在慢性腎病中，第二型血管生成素可能透過活化血管內皮細胞及血管平滑肌細胞，影響血管塑造及發炎反應之過程。因此在我們的研究計畫中，第一部分為探討慢性腎病患者血漿中第二型血管生成素濃度與發炎因子及白蛋白尿之關聯；第二部分則是探討第二型血管生成素與脈波速度(pulse wave velocity)所代表的動脈硬化程度，兩者之間的關聯性；在第三部分我們利用慢性腎病的動物模式及細胞實驗，來觀察第二型血管生成素與動脈硬化的發生之關聯及其可能之病理生成機轉。 我們首先分析慢性腎病第3期至第5期共416位患者，發現患者血漿中第二型血管生成素濃度隨慢性腎病惡化而升高、與患者血中之高敏感度C-反應蛋白呈正相關。隨著患者白蛋白尿程度愈厲害，血漿中之第二型血管生成素濃度亦愈高。統計分析更顯示血漿中之第二型血管生成素濃度與用脈波速度測量的動脈硬化程度呈正相關。 在動物實驗中，我們利用六分之五腎臟切除及單側輸尿管阻塞之模式，發現小鼠血漿中之第二型血管生成素濃度增加，同時發現在纖維化的腎臟中，受傷害的腎小管細胞明顯表現較高的第二型血管生成素。而在六分之五腎臟切除模式中，除了腎臟之外，其他器官包括主動脈及肺臟，第二型血管生成素的表現都較控制組老鼠之表現為低。此外，在六分之五腎臟切除模式中，主動脈的膠原蛋白基因、促纖維化生長因子基因、及促發炎反應因子基因，表現都明顯較控制組為增加。我們另外還利用腺病毒在小鼠的循環表現人類第二型血管生成素，發現表現人類第二型血管生成素，會刺激在主動脈血管平滑肌細胞的膠原蛋白基因、促纖維化生長因子基因之表現，同時發現在此實驗中，主動脈的巨噬細胞次族群之表現會受到影響，Ly6C低表現之巨噬細胞會增加，而這群細胞會產生促纖維化生長因子，β型轉型生長因子(TGF-β1)。在細胞實驗中，我們發現主動脈血管內皮細胞會受到第二型血管生成素的刺激，表現趨化因子(chemokines)、黏附分子(adhesion molecules)及β型轉型生長因子，促進發炎細胞的趨附反應。最後我們於小鼠接受六分之五腎臟切除的動物模式中，利用重組拮抗劑蛋白L1-10來阻斷活體內第二型血管生成素之作用，發現主動脈中增加的膠原蛋白基因、促纖維化生長因子基因、及促發炎反應因子基因表現都受到明顯的抑制。 我們的研究從臨床到基礎實驗，首先證實在臨床慢性腎病患者，血漿中第二型血管生成素濃度與發炎因子、白尿白尿、及脈波速度有正相關。在動物及細胞實驗中，我們證實了第二型血管生成素在纖維化腎臟及動脈硬化之間可能扮演的角色：第二型血管生成素在纖維化腎臟本身及循環中上升、直接作用在血管內皮細胞及巨噬細胞產生不同的生長因子及分子物質、間接刺激主動脈平滑肌細胞膠原蛋白之產生，而造成慢性腎病之血管硬化。因此針對第二型血管生成素，致力於減少發炎反應及血管硬化之產生，可能是治療慢性腎病患者之心血管疾病的新契機。

Cardiovascular disease (CVD) is the major cause of morbidity and mortality in patients with chronic kidney disease (CKD). Even though attempting to correct the traditional risk factors, the mortality rates still exceed the expected. Among all the CKD-related risk factors, much attention has been paid to the mineral metabolism and vascular calcification. Medial calcification, leading to arterial stiffness clinically, is recognized detrimental to cardiovascular outcome. The arterial stiffness in CKD patients is characterized by arterial intima-media hypertrophy resulting from alterations of the intrinsic properties of arterial wall materials. Arterial stiffness is accelerated in CKD patients in comparison with age-, sex-, and pressure-matched controls, suggesting that there are unique CKD-related factors leading to such acceleration. Treatment with phosphate binders to correct mineral disturbance, however, has not shown improvement in cardiovascular outcome. Phenotype changes of vascular smooth muscle cells (VSMCs), cross-talks between endothelial cells, VSMCs and other cells through humoral and mechanical mechanisms are possible involving pathogenesis. Besides, inflammation and endothelial dysfunction also play important roles in the CKD-related risk factors. Clinically, albuminuria is indicative of endothelial damage and vascular disease in CKD population. Albuminuria is also an independent predictor of cardiovascular events in patients with CKD. Systemic microinflammation as well infers the increased cardiovascular morbidity and mortality. Although albuminuria and microinflammation explain the complex interplay in CKD, the effectors mediating the cross talk between CKD and CVD are still not confirmative. Among all these humoral factors, increased levels of circulating angiopoietin-2 (Angpt2) are correlated with scores of coronary and peripheral artery diseases in dialysis patients. Elevated albuminuria and circulating Angpt2 both predict CVD and mortality in CKD. Angiopoietin-1 (Angpt1) and Angpt2 are ligands of the Tie-2 receptor, a second class of vascular specific receptor tyrosine kinases. The Angpt/Tie-2 system tightly controls the endothelial phenotype during angiogenesis in a unique and nonredundant fashion. In addition to angiogenesis, Angpt2 is an important regulator in numerous diseases including inflammation. Angpt1-mediated Tie-2 activation is required to maintain the quiescent endothelium. By contrast Angpt2 destabilizes the quiescent endothelium and primes it to respond to exogenous stimuli, thereby modulate the activities of inflammatory (tumor necrosis factor α and interleukin-1) and angiogenic (vascular endothelial growth factor, VEGF) cytokine. In the mice undergoing femoral artery ligation, Angpt2 is shown to induce expression of intercellular and vascular adhesion molecules, infiltration of macrophage and recruitment of VSMCs, thereby promoting arteriogenesis and blood flow recovery. Hence it is reasonable to speculate that dysregulation of Angpt/Tie-2 system in favor of Angpt2 may affect vascular remodeling through activation of endothelial cells and VSMCs. First of all, we aim to define the relationship between plasma levels of Angpt2 with albuminuria and markers of systemic microinflammation in patients with CKD stage 3 to 5. Secondly, we delineate the relationship between plasma levels of Angpt2 and arterial stiffness, measured by pulse wave velocity (PWV), in the clinical cohort of patients with CKD stage 3 to 5. Finally, we attempt to design experimental arterial stiffness in mice to study whether the elevated systemic Angpt2 contribute to arterial stiffness in CKD animal model. We further use in vivo and in vitro cell experiment to investigate the underlying mechanisms through which elevated circulating Angpt2 leads to aortic remodeling in CKD animal model. A total of 416 patients with CKD stages 3 to 5 are stratified by urine albumin-creatinine ratio (ACR) as normoalbuminuria (<30 mg/g), microalbuminuria (30–300 mg/g), or macroalbuminuria (>300 mg/g). The levels of plasma Angpt2 and VEGF increase, and soluble Tie-2 receptor (sTie-2) decrease in the subgroups of albuminuria; whereas Angpt1 does not change. Linear regression analysis shows a positive correlation between urine ACR and plasma Angpt2 (correlation coefficient r=0.301, 95% confidence interval 0.211–0.386, P<0.0001), but not between ACR and VEGF or sTie-2. Multivariate linear regression analysis confirms that plasma Angpt2 is independently associated with ACR (P=0.025). Furthermore, plasma Angpt2 is positively correlated with levels of high sensitive C-reactive protein (hsCRP) (r=0.114, 95% confidence interval 0.018–0.208, P=0.020). Among the cohort of 416 patients with CKD, plasma levels of Angpt2 are independently correlated with PWV by multivariate linear regression analysis (P=0.023). Using murine CKD models induced by 5/6 subtotal nephrectomy or unilateral ureteral obstruction, we demonstrate the increase of plasma Angpt2 independent of plasma creatinine. We demonstrate that Angpt2 expression markedly increases in tubular epithelial cells of fibrotic kidney by immunofluorescence and in situ hybridization. With quantitative polymerase chain reaction, we find the decrease of Angpt2 transcript in other tissues including aorta and lung of mice in the model of 5/6 subtotal nephrectomy. Quantitative polymerase chain reaction also reveals increased collagen transcripts, profibrotic and proinflammatory genes in aorta of mice after 5/6 subtotal nephrectomy. Expression of collagen and profibrotic genes in aortic VSMCs is increased in mice producing human Angpt2. In vivo study of mice producing human Angpt2 demonstrates that Angpt2 induces discrete subpopulation of macrophages defined by Ly6C in aorta. Angpt2 increases the population of Ly6Clow macrophages in aorta, producing profibrotic cytokine transforming growth factor β1 (TGF-β1). In vitro cell experiment shows that Angpt2 stimulates endothelial expression of chemokines and adhesion molecules for monocytes. Angpt2 also induces profibrotic cytokine TGF-β1. In the blocking experiments, we prove that Angpt2 blockade with recombinant antagonist L1-10 attenuates expression of monocyte chemokines, profibrotic cytokines, and collagen expression in aorta of mice after 5/6 subtotal nephrectomy. In the first part of the study, we could only demonstrate the association between Angpt2 with symbolized markers of microinflammation (hsCRP) and vascular injury (ACR). Likewise, the cross-sectional study demonstrating the association between Angpt2 and PWV cannot provide the causal relationship. In the animal experiment, the subjective tool to evaluate arterial stiffness in mice is still lacking. We also need to design the delicate animal model to investigate the origin of increasing systemic Angpt2, the exact receptor and mechanism through which Angpt2 induces arterial stiffness in CKD. In conclusion, plasma Angpt2 is associated with albuminuria and markers of systemic microinflammation in patients with CKD. In addition, plasma Angpt2 is associated with worsening of arterial stiffness measured by PWV in patients with CKD. In the experimental study, we identify a link between fibrotic kidney and arterial stiffness through Angpt2, which is increased in kidney and circulation, induces pro-arteriosclerotic signaling to endothelia and macrophages thereby promoting collagen production of aortic VSMCs. Targeting Angpt2 to attenuate inflammation and collagen expression may provide a novel therapy for CVD in CKD.