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標題: | 內質網壓力於急性腎損傷至慢性腎臟病轉換:專注於早期硫酸吲哚酚移除與XBP1s作用 Endoplasmic Reticulum Stress Implicated in AKI to CKD Transition: Focus on Early Elimination of Indoxyl Sulfate and Impact of XBP1s Signaling |
作者: | Jia-Huang Chen 陳佳煌 |
指導教授: | 姜至剛(Chih-Kang Chiang) |
關鍵字: | 急性腎損傷至慢性腎臟病轉換,未折疊蛋白反應,硫酸吲哚酚,AST-120,剪切型X-box 結合蛋白,腫瘤壞死因子受器相關蛋白1, AKI to CKD transition,unfolded protein response,indoxyl sulfate,AST-120,XBP1s,Trap1, |
出版年 : | 2022 |
學位: | 博士 |
摘要: | 慢性腎臟病(Chronic kidney disease, CKD)為多重器官衰竭之惡化因子,臨床上不僅引起健康危害,於醫療保健支出所造成的經濟負擔同樣為全球關注的議題。因此找尋潛在致病因子及治療策略仍然是臨床上的當務之急。急性腎損傷(Acute kidney injury, AKI)為慢性腎臟病進展的長期風險因子以及急性腎損傷後異常修復為造成慢性腎臟病轉變的觀念於近年來逐漸受到重視。然而,急性腎損傷後導致的異常修復的因子眾多且尚未研究透徹。硫酸吲哚酚(Indoxyl sulfate, IS)為一種親蛋白性尿毒素,過去研究指出其在CKD患者體內濃度與腎臟纖維化成正相關。此外,未折疊蛋白反應(unfolded protein response, UPR)活化與腎損傷進展有所關聯。團隊先前研究發現XBP1s (Spliced X box binding protein 1),一種適應性調控轉錄因子,蛋白質表現量伴隨腎臟纖維化進展而逐漸下降。然而,IS是否引起急性腎損傷後的異常修復以及XBP1s在AKI至CKD轉變過程中的角色尚未完全釐清。因此,本研究將探討IS於AKI後的作用機轉以及XBP1s參與在AKI至CKD轉變過程中的角色。 首先,我們以C57BL6小鼠利用單側腎損傷再灌流建立AKI to CKD模型,發現隨著腎纖維化進展,血清中IS濃度逐漸增加並且伴隨有機陽離子通道蛋白(organic anion transporter 1, OAT1)表現降低,然而小分子尿毒素則未有顯著上升情形。進一步探討IS於AKI to CKD過程中作用,小鼠於早期腎損傷後第五天餵食IS前驅物(Indole)及AST-120 (親蛋白性尿毒素吸附劑)並於損傷後第十天將對側腎臟切除以加速尿毒素累積,而第十五天進行犧牲。結果顯示餵食indole組別顯著增加小鼠血清中IS濃度並加劇腎臟纖維化、衰老相關旁泌生長因子基因表現及內質網壓力蛋白恆定失調,而合併給予AST-120組別則能緩解IS累積所誘導腎損傷惡化情形。進一步探討IS暴露加速腎損傷作用機轉,利用人類腎小管上皮細胞HK-2暴露於IS合併培養於厭氧袋中以模擬缺氧再灌流(Hypoxia/reperfusion, H/R)損傷。結果顯示IS處理能夠加劇H/R引起之細胞週期G2/M停滯、上皮間質轉換(Epithelial-mesenchymal transition, EMT)與內質網壓力;透過合併給予化學伴護因子(4-phenylbutyric acid, 4-PBA)減緩內質網壓力能夠改善IS加劇H/R所誘導之EMT及細胞週期G2/M停滯作用,說明IS累積於AKI後介導內質網壓力恆定失調導致CKD進展扮演重要角色。另一方面,為證實AKI 至CKD進展過程中XBP1蛋白表現降低為腎纖維化進展之致病因子,本研究建立近端腎小管上皮細胞XBP1條件性敲除轉殖鼠(XBP1 conditional knockout mice, XBP1cKO)並以UIRI誘導之。結果顯示XBP1cKO相較基因型對照組以UIRI誘導後能夠引起更顯著之腎臟纖維化、腎損傷、細胞週期G2/M停滯、DNA損傷及發炎相關mRNA表現,說明XBP1喪失為腎損傷後導致腎功能失衡之重要因素。進一步探討可能受XBP1s影響之下游標的,HK-2細胞以基因減量技術降低XBP1表現並進行蛋白質體學分析。結果顯示腫瘤壞死因子受體相關蛋白1 (TNF receptor-associated protein 1, Trap1)表現下降最為顯著,並且經由in vitro驗證XBP1s能夠轉錄調控Trap1表現,此外,本研究也以UIRI、單側輸尿管結紮(Unilateral ureteral obstruction, UUO)及腺嘌呤誘導腎病變模式證實Trap1伴隨XBP1s表現逐漸下降。最後我們以大量表現Trap1發現能夠回復腎小管細胞因XBP1缺失所引起之細胞週期G2/M停滯及促纖維生長因子分泌。 總結以上,本研究探討兩種造成AKI後加速進展至CKD之風險因子:IS累積與XBP1s缺失。AKI早期移除親蛋白性尿毒素IS能夠作為延緩患者進展至CKD之有效治療策略;以及XBP1s缺失於腎損傷後扮演促纖維化作用,透過活化腎小管細胞partial-EMT及旁泌與自泌作用加速腎纖維化進展,此外本研究也證實XBP1s-Trap1能夠作為恆定AKI後腎臟纖維化進展之新穎訊號途徑。未來也將持續探索XBP1s所調控之下游標的以及評估選擇性活化XBP1s作為治療AKI to CKD之潛力。 Chronic kidney disease (CKD) development is one of the deterioration factors for multiorgan failure which not only raises public health issues but also imposes socioeconomic burden worldwide. Exploring the potential pathogenetic factors and therapeutic strategies are still emerging unmet medical needs. Acute kidney injury (AKI) is a long-term risk for CKD development and the concept of maladaptive repair after AKI contributes to CKD transition has been extensively emphasized. Nevertheless, aberrant kidney recovery after AKI is multifactorial and still poorly understood. The accumulation of indoxyl sulfate (IS), a protein-bound uremic toxin, has been identified as a threat for the progression of renal fibrosis. Besides, the activation of the unfolded protein response (UPR) is also closely linked to the pathophysiology of renal injuries. Our previous study revealed that XBP1s, a crucial regulator of adaptive UPR, was downregulated and associated with renal fibrosis progression. However, the underlying mechanisms regarding IS caused aberrant kidney recovery after AKI and loss of XBP1s during AKI to CKD transition are still unknown. Therefore, we aim to investigate the effects of IS on tubular damage and the involvement of XBP1s in the pathophysiology of AKI to CKD transition in this study. First of all, we established AKI to CKD model in C57BL6 mice utilizing unilateral ischemia-reperfusion injury (UIRI), which revealed that IS accumulation without the synergetic small molecule uremic toxins increased and that renal fibrosis development is associated with serum IS accumulation. To evaluate the impact of IS on AKI to CKD transition, we administered indole or AST-120, a precursor of IS and an oral spherical carbonaceous adsorbent respectively, on the early stage of UIRI (day 5). Then, a contralateral nephrectomy (Nx) was performed on day 10 to accelerate uremic toxins accumulation and animals were sacrificed on day 15. Our results demonstrated IS potentiated renal fibrosis, senescence-associated secretory phenotype (SASP), and impaired endoplasmic reticulum (ER) homeostasis, which can be ameliorated by concomitant AST-120 administration. To further investigate the underlying mechanism, human proximal tubular epithelial cells were exposed to IS and an anaerobic bag to mimic hypoxia-reperfusion (H/R) insults. The results showed that IS exposure potentiated H/R induced cell cycle G2/M arrest, epithelial-mesenchymal transition (EMT), and aggravated ER stress activation. The ER chemical chaperon, 4-phenylbutyric acid (4-PBA), successfully reversed the IS potentiated maladaptive repair process, suggesting that IS accumulation plays an important role in proteostasis imbalance during AKI to CKD progression. On the other hand, the loss of XBP1s expression was found during AKI to CKD transition and negatively correlated with renal fibrosis development. Therefore, we further investigate the role of XBP1s in UIRI model using proximal tubular XBP1 conditional knockout (XBP1cKO) transgenic mice. XBP1cKO mice exhibited more severe renal fibrosis, cell cycle arrest in G2/M phase, and DNA damage in UIRI model than wide-type littermates. To figure out the potential downstream effector protein regulated by XBP1s, proteomic analysis was performed in HK-2 cells silenced of XBP1. Proteomic analysis identified that TNF receptor-associated protein 1 (Trap1) was a potential downstream target transcriptionally regulated by XBP1s. We also examined that the downregulation of XBP1s coincided with decreased Trap1 protein expression in UIRI, unilateral ureteral obstruction (UUO), adenine induced nephropathy model. Trap1 overexpression can alleviate silencing XBP1 induced profibrotic factor expressions and cell cycle arrest. In summary, we identified the potential risk factors that contribute to advanced CKD development after AKI insults including IS accumulation and the loss of XBP1s in tubular epithelial cells. Timely IS elimination in the early stage of AKI is likely to be an effective strategy in the prevention and treatment of the AKI to CKD transition. In addition, we also confirmed the loss of XBP1 in kidney injury was profibrotic, and the process was mediated by autocrine and paracrine regulations in combination. We also identified the XBP1-Trap1 axis as an instrumental mechanism responsible for post-AKI fibrosis, which is a novel regulatory pathway. The more potential regulatory effectors of XBP1s are still needed to be explored and the therapeutic approach through selective XBP1s activation during AKI to CKD to be determined in future studies. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/85286 |
DOI: | 10.6342/NTU202201837 |
全文授權: | 同意授權(限校園內公開) |
電子全文公開日期: | 2022-10-03 |
顯示於系所單位: | 毒理學研究所 |
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