2013-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/645378摘要：紅血球生成素(EPO)是刺激紅血球生成的生長因子，在出生後生成EPO的細胞就由肝臟轉移到了腎臟細胞。利用原位雜交的技術，之前的研究就已推論出生成EPO的腎臟細胞是位於腎小管周邊的纖維母細胞。然而因為缺乏可靠且可以生成EPO的腎臟細胞株，調控EPO生成的確實分子機轉並不清楚。過去利用人類肝細胞癌細胞株的實驗雖已證實1α型低氧誘發因子(HIF1α)能經由拮合至EPO基因對氧氣敏感的促進區段以增加EPO基因轉錄，但最近的研究卻提出HIF2α才是促進EPO基因表現的重要轉錄因子。而且在罹患家族性紅血球過多症的人類基因研究中只發現基因突變位於HIF2α，而非HIF1α。雖然在腎臟病初期貧血通常表現輕微，但不管是病患罹患急性或慢性腎臟衰竭，貧血最後通常都會發生。根據過去的研究，當腎功能逐漸惡化時，血比容會持續降低，甚至於低到15至20%。隨著腎功能惡化，EPO生成的障礙會越來越明顯，而且血清EPO濃度顯得相當的低，不足以反應其貧血的程度。研究雖然顯示慢性腎病患者因貧血或低血氧誘發的EPO製造能力下降，但低血氧造成EPO製造、血色素升高的回饋機制仍然是存在的。一項在末期腎病需要血液透析治療的病患所進行的臨床試驗發現HIF的口服穩定劑可以經由增加HIF的表現量來增加血清EPO的濃度，而且EPO增加的幅度比正常的自願受試者還要高，隱約指出在纖維化的腎臟中仍存在、甚至於有更多的製造EPO的細胞。最近我們證實在腎臟中有一種微血管周邊細胞(pericytes)在正常狀況下會經由分泌血管內皮生長因子以穩定微血管循環，但在腎臟受到傷害時會增生及分化成造成腎臟纖維化的肌肉纖維母細胞。腎臟的血管構造相當特別，除了前後連接入、出小動脈的初級腎小球微血管外，其後緊接著腎小管周邊次級微血管網絡以供應包括近端腎小管在內的其他腎元構造。此腎小管周邊的次級微血管網絡處於低氧氣壓力下，而腎小管本身，尤其是近端腎小管與亨氏管粗上升支又是耗費能量的構造。因此正常的腎臟血管周邊細胞不僅可穩定微血管，更可能是感應氧氣分壓與貧血程度以製造EPO的腎臟細胞。但是當腎臟發生可以造成纖維化的傷害時，血管周邊細胞就會增生並分化成可以產生腎臟纖維化的肌肉纖維母細胞。然而目前對於為何血管周邊細胞分化成肌肉纖維母細胞會造成其EPO製造的減低仍不清楚，但目前已發現許多與纖維母細胞活化的基因會發生表基因的變化，尤其是過度甲基化。因此本研究將探討在正常腎臟與纖維化腎臟中製造EPO的細胞其EPO表現的分子調控機轉。我們將首先證實正常腎臟pericytes是製造EPO的主要細胞，而在纖維化腎臟中pericytes轉分化而來的肌肉纖維母細胞仍具有EPO製造能力，並探討其分子調控機轉，主要專注於來自HIF的調控。接著我們將研究DNA甲基轉移脢(尤其是第一型)是否造成纖維化腎臟肌肉纖維母細胞EPO基因促進子的高度甲基化並降低肌肉纖維母細胞表現EPO的能力。最後我們將探討纖維化腎臟中造成腎臟肌肉纖維母細胞EPO基因促進子甲基化的分子機轉，尤其是β1轉化生長因子(TGFβ1)的角色。我們相信本研究將釐清在正常腎臟與纖維化腎臟中表現EPO的分子機轉，尤其對於慢性腎病為何會降低其EPO表現的分子機轉有更新的認識。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: Erythropoietin (EPO) is the erythroid growth factor mainly produced by the renal cells inresponse to anemia and hypoxia after birth. Previous studies using in situ hybridization haveshown that EPO-producing renal cells are peritubular fibroblasts. However the molecularmechanisms responsible for the EPO synthesis in renal cells have not yet been clear till nowbecause of lacking established EPO-producing renal cell lines in vitro. Although hypoxiainducible factor (HIF) 1α has ever been suggested to promote the EPO transcription throughbinding to the oxygen-sensitive enhancer of the EPO gene in hepatoma cell lines, recent studiessupport that expression of EPO gene is largely dependent on the activation of the transcriptionfactor complex HIF2α. In genetic studies of patients with familial erythrocytosis only mutationsin HIF-2α gene, not in HIF-1α, have been found. Anemia, initially mild and inconsequential, isvirtually a constant feature of acute or chronic renal failure. However, as renal functionprogressively deteriorates, the hematocrit continues to decline and may reach concentrations aslow as 15% to 20%. The impairment of EPO production appears to parallel the progressivereduction of nephron mass, and the plasma EPO concentration becomes disproportionately lowfor the degree of reduction of hemoglobin concentration. The oxygen-EPO-hemoglobin feedbackloop is still operating, however the response of EPO production to anemia and hypoxia seem tobe low-set. A recent clinical trial studying an oral stabilizer of HIF in hemodialysis patients andhealthy volunteers demonstrated that pharmacologic increase of the HIF can stimulate productionof much more EPO in patients than in healthy volunteers, further suggesting no loss of, or evenmore, EPO-producing renal cells in fibrotic kidney. We recently proved a central role for kidneypericytes as progenitors of scar forming myofibroblasts that appear after injury, whereas normalkidney pericytes stabilize capillary networks in normal kidneys. The kidney has unusualvasculature which comprises a specialized primary capillary network in the glomeruli followeddirectly by secondary capillary plexus which supplies the remainder of the kidney. By design,this secondary capillary plexus (peritubular capillaries) is in low oxygen tension, with low flow.In addition to their capillary stabilizing properties by secreting angiotrophic growth factor, thenormal kidney pericytes are the good candidates to produce EPO in response to hypoxemia oranemia because they are enwrapping the peritubular capillaries which is in low oxygen tension.Once fibrogenic kidney injury happens the pericytes proliferate and differentiate intomyofibroblasts who will produce pathogenical extracellular collagenous matrix and lead tokidney fibrosis and function failure. However the molecular mechanisms involved in the EPOdownregulation when pericytes transit to myofibroblasts are still obscure. Therefore this studywill aim to identify the molecular mechanisms regulating the EPO production in renal cells ofmice with normal or fibrotic kidneys. We will first identify the expression of EPO in normalkidney pericytes and fibrotic kidney myofibroblasts and the molecular mechanisms regulatingtheir EPO expression. Secondly we will identify the role of epigenetic change by DNAmethylation responsible for the insufficient EPO production to anemia in fibrotic kidneys usingin vitro pericyte culture and in vivo knock out of DNA methyltransferase 1 in pericytes. Finallywe will study the role of increased transforming growth factor β1 in EPO promoter methylationthrough upregulation of DNA methyltransferase 1 in pericytes thereby downregulating the EPOresponse to anemia in fibrotic kidney. This study will advance our knowledge in the molecularmechanisms responsible for the EPO regulation in normal kidney pericytes and fibrotic kidneymyofibroblasts and discover novel therapeutic targets to improve the renal anemia.慢性腎病第一型DNA甲基轉移脢表基因紅血球生成素低氧誘發因子血 管周邊細胞Chronic kidney diseaseDNA methyltransferase 1epigeneticserythropoietinhypoxia inducible factorpericyte