2015-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/645387摘要：腎臟血管周邊細胞是源自間葉組織的微血管旁細胞。我們之前的研究已經證實腎臟血管周邊細胞 就是腎臟内製造促紅血球生成素的細胞。在健康的狀態下，低氧或貧血都會透過活化2a型低氧誘發因 子來促進腎臟血管周邊細胞製造促紅血球生成素。在小鼠接受單侧輸尿管阻塞手術(UUO)或食用腺嘌 吟〔adenine〕之後，血管周邊細胞會轉化成為肌肉纖維母細胞，腎臟會產生纖維化。在穩定狀態下或接 受急性放血之後，肌肉纖維母細胞製造促紅血球生成素的表現都大為降低下來。相較於促紅血球生成 素的表現降低，肌肉纖維母細胞反而表現較高的第一型DNA甲基轉移腾(Dnmtl〕以及在促紅血球生成 素基因〔郎0〕的促進子與5端非轉譯區發生高度甲基化。我們也發現pi轉化生長因子會增加第一型DNA 甲基轉移腾基因的表現與促紅血球生成素基因促進子與5端非轉譯區發生高度甲基化。利用去甲基化 藥物5-azacytidine來治療UUO手術後的小鼠，我們發現無論在穩定狀態下或接受急性放血之後的纖維 化腎臟都能部份增加促紅血球生成素的表現。因為我們無法在促紅血球生成素基因的促進子與5端非 轉譯區找出低氧反應元件(HRE〕以及我們使用的兩種腎臟纖維化小鼠模式(UUO與每日攝取adenine〕在 實驗觀察期間都不表現一般慢性腎病的腎性貧血，因此我們提出此一三年計畫，首先要研究小鼠促紅 血球生成素基因在遠5端的低氧反應元件是否在腎臟血管周邊細胞扮演角色，是否促紅血球生成素基 因在遠5端的低氧反應元件在腎臟血管周邊細胞轉化成肌肉纖維母細胞後會發生高度甲基化，以及 5-azacytidine是否能有效地將遠5端的低氧反應元件高度甲基化去除。然後我們將優化在試驗實驗中發 展的慢性腎病模式，利用間歇性餵食腺嘌呤以產生表現高氮血症與貧血的小鼠模式。之後我們將利用 此一腺嘌吟慢性腎病模式來研究5-azacytidine是否能夠在我們的促紅血球生成素報告鼠(EPO-reporter〕 有效地矯正貧血與提高腎臟肌肉纖維母細胞製造促紅血球生成素。為了更進一步了解腎臟肌肉纖維母 細胞促紅血球生成素表現受抑的分子機轉，我們將利用基因改造小鼠，專一性地剔除肌肉纖維母細胞 的Dnmd基因，以研究Dnmtl對於慢性腎病肌肉纖維母細胞促紅血球生成素表現受抑的影響。我們相 信本研究將釐清在慢性腎臟病的肌肉纖維母細胞為何促紅血球生成素表現受抑的分子機轉。本研究也 一定可以開發出新的治療標的來改善腎性貧血。<br> Abstract: Kidney pericytes are mesenchyme-derived perivascular cells attached to the abluminal surface of capillaries. Our previous study has shown that pericytes are the renal erythropoietin-producing cells. In health pericytes produce erythropoietin (Epo) through hypoxia inducible factor 2a under the condition of hypoxia or anemia. In the mouse model of kidney injury induced by surgery of unilateral ureteral obstruction (UUO) or administration of adenine daily, pericytes transit to myofibroblasts and the kidneys develop fibrosis. Decreased Epo expression in steady state as well as after acute phlebotomy is found in myofibroblasts. Upregulated DNA methyltransferase 1 (Dnmtl) and hypermethylation of Epo promoter and 5' untranslated region (5'-UTR) are found in myofibroblasts, not in pericytes. Transforming growth factor-pi (TGF-pi) can upregulate Dnmtl and lead to hypermethylation of Epo promoter and 5'-UTR. Demethylation with 5-azacytidine in mice after UUO is found to increase Epo expression in fibrotic kidney in steady state as well as after acute phlebotomy partially. Because hypoxia responsive element (HRE) is not found in Epo promoter and 5'-UTR and the mouse fibrotic kidney model after UUO surgery or daily adenine administration do not develop anemia in the period of experiment, we would like to propose this 3-year project to study the role of distal 5'-HRE in Epo regulation of kidney pericytes, whether Epo 5'-HRE is hypermethylated in fibrotic kidney myofibroblasts, and the demethylating effect of 5-azacytidine on Epo distal 5'-HRE in the first. Then we will fine-tune the chronic kidney disease (CKD) mouse model induced by intermittent administration of adenine, which has presented with chronic azotemia and anemia in preliminary experiments. This chronic adenine model will be used to study whether demethylation by 5-azacytidine can rescue anemia and EPO production by kidney myofibroblasts in our EPO-reporter mice. To further get insight into the molecular mechanisms repressing EPO production in fibrotic kidney myofibroblasts, myofibroblast-specific Dnmtl knockout will be used to study the role of Dnmtl in repressed EPO production in CKD. This project will advance our knowledge in the molecular mechanisms responsible for the EPO downregulation in fibrotic kidney myofibroblasts and discover novel therapeutic targets to improve the renal anemia.慢性腎病DNA甲基轉移腾促紅血球生成素高度甲基化低氧誘發因子肌肉纖維母細 胞血管周邊細胞Chronic kidney diseaseDNA methyltransferaseerythropoietinhypermethylationhypoxia inducible factormyofibroblastpericyte