2015-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/645390摘要：慢性腎臟病(chronic kidney disease, CKD)已衍生成為全球公共衛生上重要議題且台灣末期腎病（end-stage renal disease, ESRD）盛行率亦高居全球第一。臨床上，無論是急性腎臟損傷、急性腎臟損傷至慢性腎臟病連續進程或是慢性腎臟病皆會進展至末期腎病。病患腎臟病理切片證據顯示：慢性腎病惡化的機轉中，慢性腎臟間質缺氧為共通的病理路徑，最終都是引起腎臟的纖維化，而慢性缺氧將激發細胞壓力反應，包括：內質網壓力（endoplasmic reticulum stress, ER stress）、氧化壓力及粒線體壓力。同時期，由於脫離常軌的氧氣代謝途徑使得細胞能量不足，進而減損內質網及粒線體功能。內質網在細胞內負責許多功能的執行，近三分之一的細胞蛋白質經內質網酵素及伴護蛋白（chaperone）修飾完成三級結構，成為完整功能蛋白質。因此，內質網蛋白質恆定（protein homeostasis或proteostasis）可以維持蛋白質品質並決定細胞命運。內質網蛋白質恆定不足量被認為在許多conformational疾病扮演病理及生理角色，如：Parkinson及Alzheimer病。近來，腎臟病惡化因子，如：蛋白尿、尿毒素及代謝失序（ie.糖尿病）將引發腎臟細胞功能失常，進而破壞蛋白質恆定。因此，使內質網蛋白質恆定正常化將可能成為「新興且有效」的腎臟病治療契機。Spliced X-Box binding protein-1 (sXBP-1) 在內質網蛋白質恆定反應中佔有重要角色，它是經由非傳統催化系統切割信息RNA而產生，進而影響許多細胞功能，如：(a) Metabolic and redox, (b)Apoptosis,(c) Autophagy, (d) ER-associated degradation (ERAD), (e) chaperones and foldases and(f)vesicle trafficking。不管腎臟傷害原因為何，慢性腎病表現出隨時間進展而腎功能持續惡化腎絲球傷害誘發腎膈細胞增生、發炎細胞浸潤及細胞外間質累積，導致腎絲球閉塞及有效腎元數目減少；為了維持有效廓清率，殘餘腎元肥大及超過率的調適作用，使的腎絲球過濾障壁破壞，進一步造成腎小管凋亡及細胞間質傷害，最終引發腎臟的纖維化。其中，慢性發炎及糖尿病引發內質網蛋白質恆定改變，可能對腎臟纖維化扮演著關鍵角色。吾人近期研究指出： 在缺血/再灌流急性腎臟傷害及單側輸尿管阻塞慢性腎臟纖維化研究中，都可以發現內質網蛋白質恆定被破壞及減損。基於上述發現，我們深信經由選擇性活化sXBP-1將可以逆轉腎臟所受到的損傷。經由Tet-on splicing XBP1基因轉殖技術，吾人將探討此搶救性機轉在急性腎臟傷害及腎臟纖維化的角色。此外，我們亦將利用此三年計劃，經由代謝體學(metabolomics)及轉譯體學(transcriptomics)切入研究，以找出新的內質網蛋白質恆定調控標的。本研究將分成三年執行：第一年，吾人將建立Tet-on splicing XBP1 基因轉殖系統，並利用其來分析內質網蛋白質恆定對急性及慢性腎臟損傷之細胞層級研究。第二年，吾人將建立腎臟專一Tet-onsplicing XBP1 基因轉殖鼠，並分析跨越傳統splicing XBP1 調控下的內質網蛋白質恆定，對急、慢性腎病及代謝疾病的影響。第三年，吾人將利用代謝體學及轉譯體學之工具，分析尋找splicing XBP1對“內質網蛋白質恆定“調控之新作用標的，借此找到全新且跳脫傳統對腎臟病治療的思維。希望藉由上述策略找出之標的，能對國家生技創藥計劃（NRPB）付出心力。<br> Abstract: Chronic kidney disease has emerged as a global public health burden. The prevalence of end-stage renaldisease (ESRD) in Taiwan stands at the highest ranking in the world. Acute kidney injury (AKI), AKI tochronic kidney disease (CKD) continuum and CKD all will lead to ESRD as a common ending of kidneydiseases. However, pathological analysis of biopsy samples from patients with ESRD commonly revealsimpaired glomeruli and tubulointerstitial fibrosis. Accumulating evidence has emphasized the important roleof chronic hypoxia in the tubulointerstitium in the final common pathway that leads to development of ESRD.Chronic hypoxia triggers cellular stresses, including endoplasmic reticulum (ER) stress, oxidative stress andmitochondrial stress. In parallel, energy loss owing to aberrant oxygen metabolism decreases ER andmitochondrial function. The ER is a key cellular organelle for protein maturation, and approximatelyone-third of all cellular proteins (including secretory, lumenal, and membrane proteins) are translocated intothe lumen of the ER, where they are modified to form their proper tertiary structure by ER-resident enzymesand chaperones. Protein homeostasis, or proteostasis, is conducted via sophisticated networks of mechanismsthat act to maintain the quality of proteins and the evolutionary diversity of the biological functions ofproteins. Therefore, proteostasis exerts substantial influence on cell homeostasis or, in other words, on cellfate. Insufficient proteostasis has been suggested to have a pathophysiological role in various diseases, suchas Parkinson disease and Alzheimer disease. Recently, renal pathogenic factors that include proteinuria,uraemic toxins and metabolic derangement act in association with renal cell dysfunction to disruptproteostasis. Thus, normalization of ER proteostasis might, therefore, be effective and novel for cellmaintenance, and protect against kidney pathologies.Splicing X-Box binding protein-1 (sXBP-1) catalyzes the unconventional processing of the mRNA,plays the central roles to activate the ER proteostasis. sXBP1 involved in different processes including (a)Metabolic and redox, (b) Apoptosis, (c) Autophagy, (d) ER-associated degradation (ERAD), (e) chaperonesand foldases and (f) vesicle trafficking. Regardless of etiology, all patients with chronic renal disease show aprogressive decline in renal function with time. Chronic inflammation and diabetes are associated with renalfibrosis, is a key factor of this pathophysiologic changes. Our recent publication demonstrated thedown-regulation of ER proteostasis in the ischemia/ reperfusion AKI and chronic unilateral ureteralobstruction CKD model. We believe that selective activation of splicing XBP1 pathway might play a role inovercoming the kidney insult. By using Tet-on splicing XBP1 transgene in vitro and in vivo, we can explorethe salvage mechanisms in AKI and chronic renal fibrosis. We will further explore the potential targets viametabolomics and transcriptomics within the 3 years’ project.In the first year, we will study the rescue ability of Tet-on splicing XBP1 gene in cellular models ofacute and chronic kidney injury. In the second year, we will generate kidney-specific Tet-on splicing XBP1transgenic mice, and study the consequences of the splicing XBP1 overexpression by passing the traditionaladaptive ER proteostasis in acute and chronic kidney and metabolic stress mice model. In the third year, wewill investigate the metabolomics and transcriptomics profiles of Tet-on splicing XBP-1 systems in vitro andin vivo. Using these available tools will help us to develop a novel approach in ER proteostasis, which mightbe a new convincing treatment approach in kidney diseases.慢性腎臟病(chronic kidney disease, CKD)已衍生成為全球公共衛生上重要議題且台灣末期腎病 （end-stage renal disease, ESRD）盛行率亦高居全球第一。臨床上，無論是急性腎臟損傷、急性腎臟 損傷至慢性腎臟病連續進程或是慢性腎臟病皆會進展至末期腎病。病患腎臟病理切片證據顯示：慢性 腎病惡化的機轉中，慢性腎臟間質缺氧為共通的病理路徑，最終都是引起腎臟的纖維化，而慢性缺氧 將激發細胞壓力反應，包括：內質網壓力（endoplasmic reticulum stress, ER stress）、氧化壓力及粒 線體壓力。同時期，由於脫離常軌的氧氣代謝途徑使得細胞能量不足，進而減損內質網及粒線體功能。 內質網在細胞內負責許多功能的執行，近三分之一的細胞蛋白質經內質網酵素及伴護蛋白 （chaperone）修飾完成三級結構，成為完整功能蛋白質。因此，內質網蛋白質恆定（protein homeostasis 或proteostasis）可以維持蛋白質品質並決定細胞命運。內質網蛋白質恆定不足量被認為在許多 conformational疾病扮演病理及生理角色，如：Parkinson及Alzheimer病。近來，腎臟病惡化因子， 如：蛋白尿、尿毒素及代謝失序（ie.糖尿病）將引發腎臟細胞功能失常，進而破壞蛋白質恆定。因此， 使內質網蛋白質恆定正常化將可能成為「新興且有效」的腎臟病治療契機。 Spliced X-Box binding protein-1 (sXBP-1) 在內質網蛋白質恆定反應中佔有重要角色，它是經由非 傳統催化系統切割信息RNA而產生，進而影響許多細胞功能，如：(a) Metabolic and redox, (b) Apoptosis,(c) Autophagy, (d) ER-associated degradation (ERAD), (e) chaperones and foldases and (f)vesicle trafficking。不管腎臟傷害原因為何，慢性腎病表現出隨時間進展而腎功能持續惡化腎絲球 傷害誘發腎膈細胞增生、發炎細胞浸潤及細胞外間質累積，導致腎絲球閉塞及有效腎元數目減少；為 了維持有效廓清率，殘餘腎元肥大及超過率的調適作用，使的腎絲球過濾障壁破壞，進一步造成腎小 管凋亡及細胞間質傷害，最終引發腎臟的纖維化。其中，慢性發炎及糖尿病引發內質網蛋白質恆定改 變，可能對腎臟纖維化扮演著關鍵角色。吾人近期研究指出： 在缺血/再灌流急性腎臟傷害及單側輸 尿管阻塞慢性腎臟纖維化研究中，都可以發現內質網蛋白質恆定被破壞及減損。基於上述發現，我們 深信經由選擇性活化sXBP-1將可以逆轉腎臟所受到的損傷。經由Tet-on splicing XBP1基因轉殖技術， 吾人將探討此搶救性機轉在急性腎臟傷害及腎臟纖維化的角色。此外，我們亦將利用此三年計劃，經 由代謝體學(metabolomics)及轉譯體學(transcriptomics)切入研究，以找出新的內質網蛋白質恆定調控標 的。 本研究將分成三年執行：第一年，吾人將建立Tet-on splicing XBP1 基因轉殖系統，並利用其來 分析內質網蛋白質恆定對急性及慢性腎臟損傷之細胞層級研究。第二年，吾人將建立腎臟專一Tet-on splicing XBP1 基因轉殖鼠，並分析跨越傳統splicing XBP1 調控下的內質網蛋白質恆定，對急、慢性 腎病及代謝疾病的影響。第三年，吾人將利用代謝體學及轉譯體學之工具，分析尋找splicing XBP1 對“內質網蛋白質恆定“調控之新作用標的，借此找到全新且跳脫傳統對腎臟病治療的思維。希望藉 由上述策略找出之標的，能對國家生技創藥計劃（NRPB）付出心力。Chronic kidney disease has emerged as a global public health burden. The prevalence of end-stage renal disease (ESRD) in Taiwan stands at the highest ranking in the world. Acute kidney injury (AKI), AKI to chronic kidney disease (CKD) continuum and CKD all will lead to ESRD as a common ending of kidney diseases. However, pathological analysis of biopsy samples from patients with ESRD commonly reveals impaired glomeruli and tubulointerstitial fibrosis. Accumulating evidence has emphasized the important role of chronic hypoxia in the tubulointerstitium in the final common pathway that leads to development of ESRD. Chronic hypoxia triggers cellular stresses, including endoplasmic reticulum (ER) stress, oxidative stress and mitochondrial stress. In parallel, energy loss owing to aberrant oxygen metabolism decreases ER and mitochondrial function. The ER is a key cellular organelle for protein maturation, and approximately one-third of all cellular proteins (including secretory, lumenal, and membrane proteins) are translocated into the lumen of the ER, where they are modified to form their proper tertiary structure by ER-resident enzymes and chaperones. Protein homeostasis, or proteostasis, is conducted via sophisticated networks of mechanisms that act to maintain the quality of proteins and the evolutionary diversity of the biological functions of proteins. Therefore, proteostasis exerts substantial influence on cell homeostasis or, in other words, on cell fate. Insufficient proteostasis has been suggested to have a pathophysiological role in various diseases, such as Parkinson disease and Alzheimer disease. Recently, renal pathogenic factors that include proteinuria, uraemic toxins and metabolic derangement act in association with renal cell dysfunction to disrupt proteostasis. Thus, normalization of ER proteostasis might, therefore, be effective and novel for cell maintenance, and protect against kidney pathologies. Splicing X-Box binding protein-1 (sXBP-1) catalyzes the unconventional processing of the mRNA, plays the central roles to activate the ER proteostasis. sXBP1 involved in different processes including (a) Metabolic and redox, (b) Apoptosis, (c) Autophagy, (d) ER-associated degradation (ERAD), (e) chaperones and foldases and (f) vesicle trafficking. Regardless of etiology, all patients with chronic renal disease show a progressive decline in renal function with time. Chronic inflammation and diabetes are associated with renal fibrosis, is a key factor of this pathophysiologic changes. Our recent publication demonstrated the down-regulation of ER proteostasis in the ischemia/ reperfusion AKI and chronic unilateral ureteral obstruction CKD model. We believe that selective activation of splicing XBP1 pathway might play a role in overcoming the kidney insult. By using Tet-on splicing XBP1 transgene in vitro and in vivo, we can explore the salvage mechanisms in AKI and chronic renal fibrosis. We will further explore the potential targets via metabolomics and transcriptomics within the 3 years’ project. In the first year, we will study the rescue ability of Tet-on splicing XBP1 gene in cellular models of acute and chronic kidney injury. In the second year, we will generate kidney-specific Tet-on splicing XBP1 transgenic mice, and study the consequences of the splicing XBP1 overexpression by passing the traditional adaptive ER proteostasis in acute and chronic kidney and metabolic stress mice model. In the third year, we will investigate the metabolomics and transcriptomics profiles of Tet-on splicing XBP-1 systems in vitro and in vivo. Using these available tools will help us to develop a novel approach in ER proteostasis, which might be a new convincing treatment approach in kidney diseases.