Transdifferentiation of Pericytes into Myofibroblasts is Responsible for the Inadequate EPO Production in Fibrotic Kidneys

幾乎所有的慢性腎臟病患都會產生貧血的現象。當健康的成人處於貧血的情況，血清中的紅血球生成素濃度會增加100到1000多倍以刺激造血；然而慢性腎衰竭病人血中的紅血球生成素卻不足以矯正貧血現象。我們在先前的研究證實，在腎臟纖維化的過程中，肌肉纖維母細胞主要的前驅細胞是血管周邊細胞。正常腎臟中的血管周邊細胞與血管內皮細胞共同擁有一基底膜，並具有合成基底膜、維持血管內皮細胞完整，與穩定血管的功能。利用Col1a1-GFPTg報導小鼠，我們發現血管周邊細胞與前人研究中所描述在腎臟中製造紅血球生成素的細胞具有類似的特徵，並表現許多與之相同的標幟蛋白。而在給予放血或缺氧刺激之後，血管周邊細胞能表現紅血球生成素。同時，我們也發現將血管周邊細胞的Hif2a剔除，會導致腎臟中的紅血球生成素表現降低。利用單側輸尿管結紮的動物疾病模式，我們發現在放血刺激前與刺激後，纖維化的腎臟比起對側控制組腎臟，其紅血球生成素表現有下降的趨勢。除此之外，當纖維化腎臟中的肌纖維母細胞受到放血刺激時，其製造的紅血球生成素，比起對側控制組腎臟當中的血管周邊細胞所製造的紅血球生成素，明顯較低。為了釐清紅血球生成素在血管周邊細胞轉分化為肌肉纖維母細胞時，其紅血球生成素表現降低的機制，我們分析了紅血球生成素基因的去氧核醣核酸甲基化修飾情形。我們確認了肌肉纖維母細胞的紅血球生成素基因，其啟動子與五端非轉譯區發生去氧核醣核酸過度甲基化的現象。我們也發現當給予5’-Azactiding以去除甲基化修飾後，腎臟中紅血球生成素的基因表現提升。因此我們的實驗結果支持：血管周邊細胞是腎臟臟中製造紅血球生成素的細胞；以及當血管周邊細胞轉分化為肌肉纖維母細胞，其紅血球生成素基因的啟動子與五端非轉譯區的過度甲基化，會導致在慢性腎臟病時，腎臟的紅血球生成素製造不足。Anemia is present in almost all of the chronic kidney disease (CKD) patients. Serum erythropoietin (EPO) concentrations in healthy adults increase 100- to 1000-fold under anemia conditions to stimulate erythropoiesis, whereas EPO in CKD patients fail to response adequately to correct the anemia. Our previous studies shown that pericytes are the major progenitors of myofibroblasts during renal fibrosis. Using Col1a1-GFPTg reporter mice, we found that pericytes displayed similar characteristics and markers to renal EPO-producing cells (REPCs) described in literature, and produced EPO after phlebotomy or hypoxic stimulation. Also, we found that knockout of hypoxia-inducible factor 2 in pericytes led to decreased Epo expression in kidney. In animal model of unilateral ureteral obstruction (UUO), Epo expression was decreased in fibrotic UUO kidneys before and after phlebotomy when compared with contra-lateral kidneys (CLK). Besides, EPO induction in response to phlebotomy was inferior in UUO kidney myofibroblasts than that in CLK pericytes. To get an insight into the mechanisms responsible for the decreased Epo expression during pericyte-myofibroblast transition, we analyzed the DNA methylation modification of Epo gene. Hypermethylation of Epo promoter and 5’-UTR was confirmed in myofibroblasts. Demethylation by administration of 5’-azacytidine led to increased Epo expression in fibrotic kidneys. Hence our data support pericytes are the REPC and hypermethylation of Epo promoter and 5’-UTR during pericyte-myofibroblast transition is responsible for the inadequate EPO production in CKD.口試委員會審定書 i 摘要 ii Abstract iii Table of Contents iv List of Figures viii List of Tables ix Abbreviations x Chapter 1 Introduction 1 1.1 Erythropoietin 1 1.1.1 The function of erythropoietin 1 1.1.2 The character of erythropoietin 1 1.1.3 The Regulation of erythropoietin 2 1.2 Renal Anemia 4 1.2.1 The causes and effects of renal anemia 5 1.2.2 Causes of inadequate erythropoietin production 6 1.2.3 Treatment of renal anemia 6 1.3 Pericyte in kidneys 7 1.3.1 Characteristics of pericytes 7 1.3.2 Pericytes and erythropoietin producing cells 8 1.4 Renal fibrosis 9 1.4.1 Chronic kidney disease and renal fibrosis 9 1.4.2 Pericytes transdifferentiate into myofibroblasts 10 1.4.3 Renal fibrosis and inadequate EPO production 11 1.5 DNA Methylation 11 1.5.1 Overview of DNA methylation 11 1.5.2 Mechanisms of DNA methylation 12 1.5.3 The relation between DNA Methylation, Epo gene and renal anemia 14 1.6 Purpose of Study 15 Chapter 2 Materials and Methods 17 2.1 Materials 17 2.1.1 Animals 17 2.1.2 Chemicals and Reagent 17 2.1.3 Buffer 23 2.1.4 Antibodies 25 2.1.5 Kits 27 2.1.6 Instruments 28 2.2 Methods 29 2.2.1 Animal model 29 2.2.2 Animal sample collection 30 2.2.3 Animal sample preparation 30 2.2.4 Immunofluorescence staining 31 2.2.5 Isolation of cells by fluorescence-activated cell sorting (FACS) 32 2.2.6 EPO inductions in isolated pericytes 34 2.2.7 ELISA for plasma EPO levels quantification 35 2.2.8 Kidney genomic DNA extraction 36 2.2.9 General PCR for confirming Hif2a knock out in kidney genomic DNA 36 2.2.10 Quantitative polymerase chain reaction (QPCR) 37 2.2.11 Methylation analysis 38 2.2.12 Statistical analyses 41 Chapter 3 Results 43 3.1 Pericytes are EPO-producing cells in kidneys. 43 3.2 EPO production was decreased during pericyte-myofibroblast transition. 44 3.3 Dnmt1 was up-regulated in myofibroblasts in fibrotic kidneys. 46 3.4 Epo gene is hypermethylated in fibrotic kidney myofibroblasts. 47 3.5 Administration of demethylating agent attenuated Epo methylation in fibrotic kidney myofibroblasts and increased EPO production in fibrotic kidney. 48 Chapter 4 Discussion 51 4.1 A more specific way to trace EPO producing cells is required. 51 4.2 Dnmt1 might be induced by TGFβ1 during pericyte-myofibroblast transition. 51 4.3 Epo expression was increased in fibrotic kidneys with 5’-Azacytidine treatment, but still minor compared to that of control kidneys. 52 4.4 5’-azacytidine did not specifically demethylate genes in pericytes and myofibroblasts in kidneys. 53 4.5 5’-Azacytidine as a treatment of renal anemia 55 4.6 Involvement of hypoxia-responsive element in inadequate EPO production during renal fibrosis 55 4.7 There might be models better than unilateral ureteral obstruction model for studying renal anemia in chronic kidney disease. 56 Chapter 5 Conclusion and Future prospect 58 Chapter 6 References 76 Appendix 9