血液透析病人器官併發症的分子探索

腎衰竭末期病人相較於一般正常族群有較高的死亡與併發症風險。末期腎臟疾病與系統性併發症的風險增加有關，例如心臟肥大，血管鈣化，礦物質骨疾病和腦損傷。儘管在一般人群中已知道血管疾病與失智症間的關係，但在腎衰竭末期病人仍需證明其相關。再者，血液透析患者的骨病變與腦損傷之間的相關研究很少，而蛋白質結合尿毒素(吲哚3乙酸[IAA]，硫酸吲哚酚[IS]，對甲酚硫酸酯[PCS])與心血管蛋白質組學間的關聯亦少有研究評估。因此，我們利用在新型蛋白質生物標誌物平台，分析血液透析患者血中尿毒素與併發症的關係來了解骨血管軸，尿毒素和腦損傷間的分子關聯。具體目的包括：(1)闡明踝臂指數(ABI)與臂踝脈搏波速度(baPWV)和認知障礙之間的聯繫；(2)評估骨轉換指標與認知功能的關係；(3)研究吲哚3乙酸與誘發有絲分裂原活化蛋白激酶(MAPK)信號反應的相關蛋白之關聯；(4)研究硫酸吲哚酚/對甲酚硫酸酯與181種心血管蛋白間的關係；(5)分析蛋白質結合尿毒素，骨轉化蛋白與臨床踝臂指數(ABI)和臂踝脈搏波速度(baPWV)間的關係。我們從兩個血液透析單位(高雄醫學大學醫院和高雄市立小港醫院)收案331位血液透析患者。利用Montreal Cognitive Assessment (MoCA)與Cognitive Abilities Screening Instrument (CASI)來檢測受試者的認知功能。利用質譜儀測定受試者血中結合蛋白質的尿毒素(IAA, IS, PCS)濃度，並使用螢光珠的技術(Luminex)透過免疫測定法來定量骨轉換蛋白。心血管相關蛋白(包括MAPK相關蛋白)則是透過鄰近延伸分析方法(Olink蛋白質組學)來進行分析。在研究1中，ABI與MoCA評分和CASI評分獨立呈正相關，發現baPWV與CASI之間存在負相關。在研究2中，核因子κB配體的受體激活劑(RANKL)是唯一發現與認知功能(MoCA和CASI)相關的骨轉換指標，控制潛在的干擾因子後，此相關依然顯著。在研究3中，IAA在多重因子校正後發現與幹細胞因子(SCF)呈正相關。在多變量回歸校正模型中，校正傳統心血管危險因子後，其相關性仍然顯著。在研究4中，利用多重校正模型與多變量校正模型來探討IS / PCS與心血管蛋白間的相關，結果發現IS與六種蛋白質(C-C motif chemokine 15, complement component C1q receptor, perlecan, bleomycin hydrolase, cluster of differentiation 166 antigen, and signaling lymphocytic activation molecule family member 5)呈負相關，與一種蛋白質(fibroblast growth factor 23)呈正相關。PCS與三種蛋白質(C-C motif chemokine 15, complement component C1q receptor, and interleukin-1 receptor-like 2)為負相關。在調整心血管危險因子後的多變量模型後亦得出相似的結果。在研究5中，我們發現蛋白質結合尿毒素與alkaline phosphatase和osteoprotegerin呈現正相關，特別是在高副甲狀腺功能的狀態下，其相關性更為顯著，而osteoprotegerin也同是與臂踝脈搏波速度呈現正相關。總結本研究之結果，我們發現血液透析患者的低ABI或高baPWV與較低的認知功能相關(研究1)。血液透析患者的血清RANKL濃度與較佳的認知功能有關(研究2)。至於蛋白結合的尿毒素，IAA與與細胞遷移/增殖相關，也與參與心臟修復的SCF有關(研究3)。IS和PCS與數種與心血管的蛋白相關，這些蛋白與細胞粘附，內皮屏障功能，炎症，補體系統和磷調節有關(研究4)。蛋白質結合尿毒素，骨轉化指標與臨床周邊血管疾病檢測之間都有相關性(研究5)。這些研究結果可以闡述骨血管軸，蛋白結合尿毒素和器官併發症的相關聯。透過新的分子技術平台檢測這些蛋白質可提供一個新面向，從臨床研究中聯繫血液透析患者的器官併發症及其複雜性。

關鍵字

血液透析；周邊血管疾病；認知功能障礙；骨代謝指標；蛋白質組學；蛋白質結合尿毒素

並列摘要

Patients with end-stage kidney disease (ESKD) increase the risk of mortality and morbidity compared to the general population. Patients receiving hemodialysis also have many organ complications, such as cardiac hypertrophy, vascular calcification, mineral bone disorder, and brain damage. Although the link between vascular disease and dementia is recognized in the general population, however, the association in patients with ESKD still needs to elucidate. Besides, the relationship between bone and brain damage is sparse in hemodialysis patients. Also, a limited study evaluates the association between protein-bound uremic toxins (indole-3 acetic acid [IAA], indoxyl sulfate [IS], or p-cresyl sulfate [PCS]) and cardiovascular proteomics. Therefore, we investigate the molecules association between bone-vascular axis, uremic toxins, and brain damage using a novel protein biomarkers platform in hemodialysis patients. The specific aim of this doctoral thesis included (1) elucidate the link between ankle-brachial index (ABI) and brachial-ankle pulse wave velocity (baPWV) and cognitive impairment; (2) evaluate the association of bone turnover markers and cognitive function; (3) investigate the relationship between IAA and part of cardiovascular proteins that related to mitogen-activated protein kinase (MAPK) signaling cascades; (4) investigate the association between IS/PCS and 181 cardiovascular proteins; (5) evaluate the association between protein-bound uremic toxins, bone turnover markers, and ABI/baPWV. We recruited 331 prevalent hemodialysis patients from 2 hemodialysis units (Kaohsiung Medical University Hospital and Kaohsiung Municipal Hsiao-Kang Hospital). Cognitive performance was measured using the Montreal Cognitive Assessment (MoCA) and Cognitive Abilities Screening Instrument (CASI) by trained psychiatrists. Protein-bound uremic toxins (IAA, IS, PCS) in patients’ serum were measured by mass spectrometry. Bone turnover proteins were quantified by sandwich immunoassay with the fluorescent-bead-based technology (Luminex). Cardiovascular-related proteins (including MAPK cascade associated proteins) were accessed by a proximity extension assay (Olink proteomics). In Study 1, ABI was independently positively associated with the MoCA score and CASI score. A negative association between baPWV and CASI was found. In Study 2, receptor activator of nuclear factor kappa-B ligand (RANKL) was the only bone turnover marker found associated with cognitive function (MoCA and CASI test). The association remained significant in MoCA and CASI after controlling potential confounders. In Study 3, IAA was positively associated with stem cell factor (SCF) in the discovery phase with multiple testing. The association remained significant after adjusting for traditional cardiovascular risk factors in the second phase with a multivariable-adjusted regression model. In Study 4, we investigated the associations between the IS/PCS and the cardiovascular proteins in the discovery phase with multiple testing and the second phase with multivariable-adjusted models. IS was negatively associated with six proteins (C-C motif chemokine 15, complement component C1q receptor, perlecan, bleomycin hydrolase, cluster of differentiation 166 antigen, and signaling lymphocytic activation molecule family member 5) and positively associated with one protein (fibroblast growth factor 23). PCS was negatively associated with three proteins (C-C motif chemokine 15, complement component C1q receptor, and interleukin-1 receptor-like 2). Adjusting for the classical cardiovascular risk factors in multivariable models yielded similar results. In Study 5, a positive correlation between protein-bound uremic toxins and alkaline phosphatase or osteoprotegerin, especially in hyperparathyroidism status. Besides, the circulating osteoprotegerin was also positively associated with baPWV. In summary of this doctoral thesis, we found a low ABI or high baPWV were associated with a lower cognitive function in hemodialysis patients (Study 1). Serum RANKL levels were potentially associated with better cognitive function in hemodialysis patients (Study 2). As for protein-bound uremic toxins, IAA was associated with SCF that linked to cell migration/proliferation and involved in cardiac repair (Study 3). IS and PCS were associated with several cardiovascular-related proteins involved in cell adhesion, endothelial barrier function, inflammation, complement system, and phosphate homeostasis (Study 4). There are links between protein-bound uremic toxins, bone turnover markers, and examination of peripheral vascular disease (Study 5). These associations interplay the bone-vascular axis, protein-bound uremic toxins, and organ complications. Detection of these proteins association by new molecules technological platform provides a novel aspect to link the complexity of organ complications in hemodialysis patients from a clinical research setting.

並列關鍵字

hemodialysis ； peripheral vascular disease ； cognitive impairment ； bone turnover markers ； cardiovascular proteomics ； protein-bound uremic toxins

參考文獻

1. Collaboration GBDCKD. Global, regional, and national burden of chronic kidney disease, 1990-2017: a systematic analysis for the Global Burden of Disease Study 2017. Lancet 2020;395(10225):709-733

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