2016-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/645181摘要：流行病學顯示過去二十多年來急性腎損傷的發生率在美國增加約三倍多，但處理伴隨急性腎損傷而來的合併症所付出之醫療成本仍相對龐大。急性腎損傷是臨床常見的併發症，腎功能惡化對病人的長期預後會有不良的影響。游離鐵形成的自由基在急性腎損傷的致病機制中扮演重要的作用。目前已知體內鐵平衡可藉由調節鐵調素-骨形態發生蛋白-肝抑鐵素 (hemojuvelin-BMP-hepcidin axis)路徑控制。在缺血再灌流急性腎損傷時，鐵的吸收力受損以及巨噬細胞釋出的鐵無法回收利用會加重腎損傷。調節鐵調素(hemojuvelin, HJV)、弗林蛋白酶(furin protease)和第六型嵌膜絲胺酸蛋白酶(TMPRSS6)在調控肝抑鐵素(hepcidin)的表現及影響急性腎損傷的鐵沉積都有重要的作用。微核醣核酸(microRNA)已被證實可與鐵相關基因轉錄後調控有關，像是鐵的吸收、運送、儲存、利用以及調節全身性鐵平衡。然而，微核醣核酸在急性腎損傷調節鐵調素-肝抑鐵素訊號路徑的部分尚未有相關報導。本研究從急性腎損傷期間微核醣核酸表現的改變，探討直接及間接影響調節鐵調素表現及生理功能之關係。們已使用微核醣核酸微陣列晶片(miRNA array chip)分析人類急性腎損傷腎臟檢體，找出可能具調節HJV-BMP -hepcidin 路徑能力的微核醣核酸。並利用StarBase 資料庫分析、預測這些微核醣核酸可能作用於HJV (HFE2)、TMPRSS6和Furin 3’-UTR的序列位置。本計畫將探討調控HJV-BMP -hepcidin 路徑的微核醣核酸與急性腎損傷情況下鐵平衡失調機制間的相關性。第一年目標: 使用定量即時聚合酶鏈鎖反應(real-time PCR)確認急性腎損傷病人檢體的微核醣核酸表現，尤其是miR-187、miR-665、miR-137和miR-122。利用螢光原位雜合技術 (fluorescence in situ hybridization)及免疫組織染色(immunohistochemistry)技術，確認candidate microRNAs在腎臟上的分布。第二年目標: 探討與hemojuvelin-hepcidin 路徑相關的微核醣核酸在調控肝抑鐵素的表現以及於缺氧和鐵過量情況下細胞中鐵沉積的分子作用。第三年目標: 利用急性腎損傷動物模型探討hemojuvelin-hepcidin 路徑相關的微核醣核酸對於腎臟弗林蛋白酶、第六型嵌膜絲胺酸蛋白酶、肝抑鐵素以及膜蛋白調節鐵調素/水溶性調節鐵調素(mHJV/sHJV)比例的影響。並利用即時定量聚合酶連鎖反應微核醣)技術，分析NSARF/CAKS資料庫的臨床急性腎損傷尿液檢體，評估我們找到的candidate microRNAs做為診斷、預測急性腎損傷疾病和預後生物指標(biomarker)的臨床應用。我們的結果顯示，藉由調節HJV-BMP -hepcidin 訊號的鐵傳導可作為急性腎損傷診斷和進一步的治療策略。本研究將證實微核醣核酸在急性腎損傷情況下，可透過調控HJV-BMP -hepcidin 路徑而影響鐵平衡，並具有發展成新型急性腎損傷生物標記、及作為新治療標的可能性。我們的研究團隊業已建立NSARF/CAKS資料庫，大量保留了急性腎損傷患者的檢體，是本計畫成功的保證。<br> Abstract: Novel microRNAs modulates hemojuvelin-hepcidin pathway on iron homeostasis during acute kidney injuryThe incidence rate of acute kidney injury (AKI) in hospitalized patients is increasing, and the number of deaths associated with dialysis-requiring AKI has more than doubled. In hospitalized patients, AKI results in increased in-hospital and post-hospitalization resource utilization. Free iron plays an important role in the pathogenesis of acute kidney injury (AKI) via the formation of hydroxyl radicals. Systemic iron homeostasis is controlled by the hemojuvelin-BMP-hepcidin axis. Hepcidin excess results in impaired iron absorption and impaired ability to utilize iron that has been reclaimed by macrophages during I/R AKI. Hemojuvelin (HJV), furin and TMPRSS6 play an essential role in the regulation of hepcidin expression and affect the iron deposition during AKI. miRNAs have been demonstrated to post-transcriptionally regulate the expression of genes associated with iron acquisition, iron export, iron storage, iron utilization, and coordination of systemic iron homeostasis. However, the miRNAs modulating hemojuvelin-hepcidin signaling pathway during AKI has not been elucidated.Accordingly, we have identified the miRNAs, regulating hemojuvelin-BMP-hepcidin signaling pathway, from human kidney specimens at AKI by using miRNA array chip. The well-fitted conserved motif of 3’-UTR of HFE2 (HJV), TMPRSS6 and Furin as the target contains a matching with the ‘seed’ sequence of candidate microRNA are predicted by StarBase program analysisTherefore, the goals of this proposal will (1) To identify and validate the candidate miRNAs from humans specimen, especially miR-187, miR-665, and miR-137 and miR-122, by real-time quantitative reverse transcriptase polymerase chain reaction (PCR), fluorescence in situ hybridization (FISH) and immunohistochemistry. (2) To investigate the molecular role of hemojuvelin-hepcidin pathway-related miRNAs in the regulation of hepcidin expression and affects the iron deposition in cell model under hypoxia/iron overload. (3) To investigate the effect of HJV-BMP-hepcidin pathway-related miRNAs on furin protease, TMPRSS6, hepcidin, and the ratio of mHJV/sHJV in animal model and clinical using as a novel biomarker of AKI using the database from NSARF/CAKS.This study aimed to understand the mechanism of HJV-mediated iron sensing pathway during AKI, regulating by miRNAs through HJV/ Furin and TMPRSS6 signaling. We will show for the first time to our knowledge that systemic iron homeostasis, modulated by HJV is also controlled by miRNAs. The candidate miRNAs will be potential novel biomarker for diagnosis and prognosis of AKI. Our result will shed light for further therapeutic strategies to rescue through modulating iron sensing HJV-BMP -hepcidin signaling.流行病學顯示過去二十多年來急性腎損傷的發生率在美國增加約三倍多，但處理伴隨急性腎損傷而來的合併症所付出之醫療成本仍相對龐大。急性腎損傷是臨床常見的併發症，腎功能惡化對病人的長期預後會有不良的影響。游離鐵形成的自由基在急性腎損傷的致病機制中扮演重要的作用。目前已知體內鐵平衡可藉由調節鐵調素-骨形態發生蛋白-肝抑鐵素 (hemojuvelin-BMP-hepcidin axis)路徑控制。在缺血再灌流急性腎損傷時，鐵的吸收力受損以及巨噬細胞釋出的鐵無法回收利用會加重腎損傷。 調節鐵調素(hemojuvelin, HJV)、弗林蛋白酶(furin protease)和第六型嵌膜絲胺酸蛋白酶(TMPRSS6)在調控肝抑鐵素(hepcidin)的表現及影響急性腎損傷的鐵沉積都有重要的作用。微核醣核酸(microRNA)已被證實可與鐵相關基因轉錄後調控有關，像是鐵的吸收、運送、儲存、利用以及調節全身性鐵平衡。然而，微核醣核酸在急性腎損傷調節鐵調素-肝抑鐵素訊號路徑的部分尚未有相關報導。 本研究從急性腎損傷期間微核醣核酸表現的改變，探討直接及間接影響調節鐵調素表現及生理功能之關係。們已使用微核醣核酸微陣列晶片(miRNA array chip)分析人類急性腎損傷腎臟檢體，找出可能具調節HJV-BMP -hepcidin 路徑能力的微核醣核酸。並利用StarBase 資料庫分析、預測這些微核醣核酸可能作用於HJV (HFE2)、TMPRSS6和Furin 3’-UTR的序列位置。 本計畫將探討調控HJV-BMP -hepcidin 路徑的微核醣核酸與急性腎損傷情況下鐵平衡失調機制間的相關性。第一年目標: 使用定量即時聚合酶鏈鎖反應(real-time PCR)確認急性腎損傷病人檢體的微核醣核酸表現，尤其是miR-187、miR-665、miR-137和miR-122。利用螢光原位雜合技術 (fluorescence in situ hybridization)及免疫組織染色(immunohistochemistry)技術，確認candidate microRNAs在腎臟上的分布。第二年目標: 探討與hemojuvelin-hepcidin 路徑相關的微核醣核酸在調控肝抑鐵素的表現以及於缺氧和鐵過量情況下細胞中鐵沉積的分子作用。第三年目標: 利用急性腎損傷動物模型探討hemojuvelin-hepcidin 路徑相關的微核醣核酸對於腎臟弗林蛋白酶、第六型嵌膜絲胺酸蛋白酶、肝抑鐵素以及膜蛋白調節鐵調素/水溶性調節鐵調素(mHJV/sHJV)比例的影響。並利用即時定量聚合酶連鎖反應微核醣)技術，分析NSARF/CAKS資料庫的臨床急性腎損傷尿液檢體，評估我們找到的candidate microRNAs做為診斷、預測急性腎損傷疾病和預後生物指標(biomarker)的臨床應用。我們的結果顯示，藉由調節HJV-BMP -hepcidin 訊號的鐵傳導可作為急性腎損傷診斷和進一步的治療策略。 本研究將證實微核醣核酸在急性腎損傷情況下，可透過調控HJV-BMP -hepcidin 路徑而影響鐵平衡，並具有發展成新型急性腎損傷生物標記、及作為新治療標的可能性。我們的研究團隊業已建立NSARF/CAKS資料庫，大量保留了急性腎損傷患者的檢體，是本計畫成功的保證。Novel microRNAs modulates hemojuvelin-hepcidin pathway on iron homeostasis during acute kidney injury The incidence rate of acute kidney injury (AKI) in hospitalized patients is increasing, and the number of deaths associated with dialysis-requiring AKI has more than doubled. In hospitalized patients, AKI results in increased in-hospital and post-hospitalization resource utilization. Free iron plays an important role in the pathogenesis of acute kidney injury (AKI) via the formation of hydroxyl radicals. Systemic iron homeostasis is controlled by the hemojuvelin-BMP-hepcidin axis. Hepcidin excess results in impaired iron absorption and impaired ability to utilize iron that has been reclaimed by macrophages during I/R AKI. Hemojuvelin (HJV), furin and TMPRSS6 play an essential role in the regulation of hepcidin expression and affect the iron deposition during AKI. miRNAs have been demonstrated to post-transcriptionally regulate the expression of genes associated with iron acquisition, iron export, iron storage, iron utilization, and coordination of systemic iron homeostasis. However, the miRNAs modulating hemojuvelin-hepcidin signaling pathway during AKI has not been elucidated. Accordingly, we have identified the miRNAs, regulating hemojuvelin-BMP-hepcidin signaling pathway, from human kidney specimens at AKI by using miRNA array chip. The well-fitted conserved motif of 3’-UTR of HFE2 (HJV), TMPRSS6 and Furin as the target contains a matching with the ‘seed’ sequence of candidate microRNA are predicted by StarBase program analysis Therefore, the goals of this proposal will (1) To identify and validate the candidate miRNAs from humans specimen, especially miR-187, miR-665, and miR-137 and miR-122, by real-time quantitative reverse transcriptase polymerase chain reaction (PCR), fluorescence in situ hybridization (FISH) and immunohistochemistry. (2) To investigate the molecular role of hemojuvelin-hepcidin pathway-related miRNAs in the regulation of hepcidin expression and affects the iron deposition in cell model under hypoxia/iron overload. (3) To investigate the effect of HJV-BMP-hepcidin pathway-related miRNAs on furin protease, TMPRSS6, hepcidin, and the ratio of mHJV/sHJV in animal model and clinical using as a novel biomarker of AKI using the database from NSARF/CAKS. This study aimed to understand the mechanism of HJV-mediated iron sensing pathway during AKI, regulating by miRNAs through HJV/ Furin and TMPRSS6 signaling. We will show for the first time to our knowledge that systemic iron homeostasis, modulated by HJV is also controlled by miRNAs. The candidate miRNAs will be potential novel biomarker for diagnosis and prognosis of AKI. Our result will shed light for further therapeutic strategies to rescue through modulating iron sensing HJV-BMP -hepcidin signaling.調節鐵調素肝抑鐵素第六型嵌膜絲胺酸蛋白酶弗林蛋白酶微核醣核酸急性腎損傷HJV-BMP-hepcidinTMPRSS6FurinmicroRNAAKIkidney