2017-01-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/645935摘要：本計畫將持續探討我們的研究主題之一：如何藉由引導腎臟肌肉纖維母細胞(myofibroblasts)再分化回血管周細胞(pericytes)，以治療慢性腎臟病及回復肌肉纖維母細胞製造促紅血球生成素(erythropoietin)的功能。我們之前的研究已證實腎臟血管周細胞除了具有維持微血管穩定的功能，也是製造促紅血球生成素的細胞。腎臟發生疾病之後，血管周細胞卻受到許多生長因子與細胞激素的刺激而轉分化成製造疤痕組織的肌肉纖維母細胞，不僅微血管循環密度受破壞而降低，腎臟也發生纖維化走向功能喪失。在眾多的慢性腎臟病臨床表現中，因為腎臟促紅血球生成素生成的降低而造成漸進性的腎性貧血發生。我們之前的研究已經證實：當腎臟血管周細胞轉分化成肌肉纖維母細胞時，其促紅血球生成素(Epo)基因5’端的調節元件會發生高甲基化，因而降低了促紅血球生成素的基因表現與蛋白生成。我們的研究進一步證實：脫氧核糖核酸甲基移換酶抑制劑5-azacytidine可以回復小鼠慢性腎臟病模式的促紅血球生成素生成與改善貧血。同時我們也發現慢性腎臟病的肌肉纖維母細胞會再分化回血管周細胞。因為5-azacytidine會嵌入脫氧核糖核酸並導致細胞毒性，因此我們提出此計畫以研究是否可以經由誘導高甲基化的胞嘧啶產生內生性的羥甲基化(hydroxymethyaltion)以回復纖維化腎臟促紅血球生成素的生成與血管周細胞功能。因為涉及表觀基因調控與腎臟血管周細胞重分化，因此我們認為本計劃符合研究領域: 1-4 Epigenetic regulation of tumor metabolism in cancer progression, 或 6-2 Tissue Engineering & Regenerative Medicine。我們的研究利用5-azacytidine以抑制脫氧核糖核酸甲基移換酶雖然可以清楚證實甲基化表觀基因的變化是造成慢性腎臟病的肌肉纖維母細胞降低促紅血球生成素生成的原因，而降低高甲基化的藥物治療可以回復促紅血球生成素的生成，改善貧血與腎臟纖維化，但目前的臨床用藥5-azacytidine卻可能因為嵌入脫氧核糖核酸並導致細胞毒性而無法安全使用於慢性腎臟病患。因此我們期待經由本研究釐清活化內生性的去甲基化是否能夠產生相同的效果。為了活化內生性的去甲基化，我們將聚焦於10-11異位酶(ten-eleven translocation enzyme, Tet)，該異位酶可以將甲基化胞嘧啶氧化成羥甲基化以達到去甲基化的效果。為了調控Tet3表現與作用，我們將利用分子生物學的技術與基因改造小鼠來增加或剔除腎臟肌肉纖維母細胞的Tet3表現，以研究該細胞的Epo基因5’端調節元件的甲基化/羥甲基化變化與促紅血球生成素的表現，並研究慢性腎臟病小鼠的腎臟纖維化，促紅血球生成素的生成與貧血的變化等等。我們相信這些技術將證實在慢性腎臟病的肌肉纖維母細胞誘導Tet3表現可以達到回復促紅血球生成素生成與改善慢性腎臟病的效果。目前臨床有一些藥物，包括高血壓藥hydralazine，可以經由誘導Tet3表現以促進去甲基化。我們將利用非降血壓的低劑量與濃度來研究hydralazine是否可以應用於改善腎臟纖維化，回復促紅血球生成素生成與改善腎性貧血，並探討血管周細胞的微血管循環穩定功能是否回復。基於過去我們的研究證實高甲基化的表觀基因變化在腎臟纖維化，促紅血球生成素生成降低與貧血的發生所扮演的重要角色，我們預期本研究所探討的透過誘導Tet3表現以活化內生性去甲基化的效果，將可以很快的轉譯到臨床研究與應用，用以治療慢性腎臟病與改善腎性貧血。<br> Abstract: This proposed project is going to continue one of our research interests in how to restore erythropoietin (EPO) production in fibrotic kidney myofibroblasts and attenuate the progression of chronic kidney disease (CKD) by induction of myofibroblast re-differentiation back to pericytes. Our previous studies have shown that normal kidney pericytes are renal EPO-producing cells (REPCs) in addition to maintain microvascular stability. Upon injury to kidneys, pericytes transit to scar-producing myofibroblasts, leading microvascular rarefaction and kidney fibrosis. Anemia due to decreased EPO production by the fibrotic kidney is a common complication in CKD patients. Our previous studies demonstrate that hypermethylation of Epo 5’-regulatory elements happens during pericyte-myofibroblast transition and leads to decrease of EPO production. Our studies further show that DNA methyltransferase inhibition by 5-azacytidine restores EPO production and ameliorates anemia in murine fibrotic kidney models. Moreover, re-differentiation of myofibroblasts back to pericytes is shown. Because 5-azacytidine may incorporate into DNA and lead to cytotoxic effect, we propose this project to investigate whether activation of endogenous hydroxymethylation is able to restore EPO production in fibrotic kidneys and recover the normal pericyte function. Therefore we consider this proposed application fits in the research fields: 1-4 Epigenetic regulation of tumor metabolism in cancer progression, or 6-2 Tissue Engineering & Regenerative Medicine.Although DNA methyltransferase inhibition by 5-azacytidine provides robust evidence for the mechanistic role for epigenetic changes in the reduced function of REPCs in fibrosis and demonstrates that targeting these changes can restore EPO production and ameliorate anemia in mice with fibrotic kidneys, the inherent cytotoxicity due to incorporation of 5-azacytidine into DNA might hinder the clinical application in CKD patients. We therefore look forward to the potential effect of activating endogenous demethylation on the restoration of EPO production and attenuation of kidney fibrosis. To study the endogenous demethylation, we will focus on ten-eleven translocation (Tet) enzymes in fibrotic kidneys and myofibroblasts. Evidence has shown that Tet3 will specifically demethylate hypermethylated CpG through hydroxylation. To manipulate the function of Tet3, we will use molecular biology tools and genetically modified mice to induce or knock out the expression of Tet3 in kidney myofibroblasts in cell culture and fibrotic kidneys. Methylation and hydroxymethylation of Epo 5’-regulatory elements in kidney myofibroblasts will be studied. EPO production, hemoglobin concentration and kidney fibrosis in murine fibrotic kidney models will be evaluated. We believe these specific tools will prove the demethylating effect and EPO restoration by Tet3 induction in kidney myofibroblasts and murine fibrotic kidney models. Chemicals including hydralazine have shown demethylating effect through Tet3 induction. We will study whether non-antihypertensive low dose of hydralazine will produce demethylating effect on Epo 5’-regulatory elements through Tet3 induction and lead to restoration of EPO production in kidney myofibroblasts and fibrotic kidneys as well as the re-differentiation of myofibroblasts back to pericytes. Since epigenetic hypermethylation plays an important role in both kidney fibrosis and EPO repression, we expect the strategy to activate the endogenous demethylation through Tet3 will be translated into clinical therapy soon after this study.erythropoieitnchronic kidney disease