2014-01-012024-05-14https://scholars.lib.ntu.edu.tw/handle/123456789/660759摘要：蛋白尿是末期腎病變重要的原因。足細胞、基底膜及內皮細胞構成了腎絲球的過濾屏障，任何的傷害會導致蛋白尿產生。Angiopoietin 1（Ang 1）、angiopoietin 2（Ang 2）及 tyrosine kinase with Ig and EGF homology domains 2（Tie 2）為血管生成及成熟的重要因子。Ang 1與Tie 2結合，會促進血管成熟及維持完整，Ang 2則會拮抗Ang 1。過去的研究顯示：Ang 1/Ang 2比值的減少與daunorubicin 引起的腎絲球硬化有關；糖尿病腎病變中，足細胞及內皮細胞Ang 2的表現會增加；拮抗內皮細胞生長因子在糖尿病腎病變早期可以減少腎絲球的肥大。因此，angiopoietin可能會影響足細胞及腎絲球內皮細胞的生理，不平衡的Ang 1/Ang 2訊息傳導有可能會影響腎絲球病理變化。我們過去的研究顯示：單側腎臟切除後，腎絲球的Ang 1及Ang 2表現會增加，投與Ang 1、Ang 2或二者的拮抗劑可以減緩腎絲球肥大。在糖尿病，Ang 2表現則會增加。在腎絲球，Ang 1主要表現在足細胞，而Tie 2受體則在內皮細胞。因此，我們推測：在正常腎臟或腎絲球疾病，足細胞可以透過Ang 1的表現來影響腎絲球內皮細胞生理，從而改變腎絲球生理或產生病變。我們將利用基因轉植或踢除技術來測試我們的假說。我們將以podocin引子帶動Cre recombinase表現的基因轉植鼠（NPHS2-CreErt2）與Ang 1兩邊有loxP DNA序列的基因踢除鼠交配，以產生可引發性足細胞Ang 1踢除鼠，以此引發性基因踢除鼠來檢視踢除足細胞Ang 1後，是否可以預防單側腎臟切除引起的腎絲球肥大，也將檢視足細胞Ang 1踢除後，對個別腎絲球內皮細胞及足細胞本身有關生長肥大基因（如PDGF、IGF-1及 transforming growth factor ）表現的影響。 我們將拮抗或補充這些由內皮細胞分泌因Ang 1踢除被改變的因子，檢視是否能逆轉足細胞踢除Ang 1的效果。NPHS2-CreErt2鼠也將與 兩邊有loxP DNA序列stop codon其後接有Ang 1基因過度表現鼠交配，以產生以產生可引發性足細胞Ang 1過度表現基因轉植鼠，該鼠在recombinase切除lopxP後，stop codon被移除，Ang 1即被過度表現。以此基因轉植鼠來檢視足細胞過度表現Ang 1是否可以減輕糖尿病腎病變；同樣的，我們也將分析個別腎絲球內皮細胞及足細胞本身有關基因在Ang 1過度表現後的變化， 我們將拮抗或補充這些由內皮細胞分泌因Ang 1細胞過度被改變的因子，檢視是否能逆轉足細胞過度表現Ang 1的效果。我們預期：足細胞踢除Ang 1將減緩因單側腎臟切除引起的腎絲球肥大，並改變腎絲球內皮細胞有關生長肥大基因表現，這些內皮細胞產生的因子也在腎絲球肥大扮演重要角色。在糖尿病腎病變，過度表現足細胞Ang 1可以減緩腎絲球病變，增加內皮細胞保護腎絲球的因子。這些研究將有助於瞭解腎絲球疾病的致病機轉，並提供蛋白尿疾病治療的方向。<br> Abstract: Proteinuric renal disease is an important cause of end stage renal disease. Podocyte, basement membrane and glomerular endothelium form the barrier of glomerulus. Damage to these component results in proteinuria. Angiopoietin 1 (Ang1), angiopoietin 2 (Ang 2) and tyrosine kinase with Ig and EGF homology domains 2 (Tie 2) are involved in the process of vascular generation and maturation. Ang 1 binds to Tie2 to promote vascular maturation and integrity, whereas Ang 2 acts as a naturally occurring antagonist of Ang 1. Previous studies revealed that: A decreasing ratio of Ang-1/Ang-2 expression ratio correlated with glomerulosclerosis in daunorubicin-induced glomerular disease. Ang-2 is prominent in podocyte and glomerular endothelium in animal DM nephropathy model. The over-expression of podocyte-specific angiopoietin-2 causes proteinuria and apoptosis of glomerular endothelium. Anti-angiogenic ameliorates renal change and glomerular hypertrophy in early stage of DM nephropathy. Thus, angiopoietins affect podocyte as well as glomerular endothelial biology, and imbalanced angiopoietin signaling contributes to glomerular pathobiology. Our previous studies revealed that there was up-regulation of glomerular Ang 1 and Angpt 2 in uninephrectomy, a model of glomerular hypertrophy. Systemic administration of antagonist of Ang 1, Ang 2 or both attenuates glomerular hypertrophy. In DM model, only Ang 2 was up-regulated in glomerulus 2 months after induction of hyperglycemia. In glomerulus, podocyte is the major source of Ang 1while Tie 2 located mainly in endothelium. Therefore, we hypothesize that podocyte may exert its influence on endothelium through the change of secreting angiopoietin into glomerulus in the process of glomerular development and disease status.We will use knock out and transgenic mice to test this hypothesis. A podocin promoter driven Cre recombinase transgenic mice strain (NPHS2-CreErt2) will mate with loxP flanked Ang 1 knock out mice to generate inducible podocyte specific Ang 1 knock out mice. We will see whether podocyte Ang 1 knock out would prevent glomerular hypertrophy in uninephrectomy animal model. The effect of podocyte Ang 1 knock out on gene (hypertrophy related growth factors and cytokines, such as PDGF, IGF-1 and transforming growth factor) expression change of podocyte and endothelium respectively will be analyzed. We will antagonize or supply the endothelium derived factors which are changed by podocyte Ang 1 knock out in the uninephrectomized mice to rescue Ang 1 knock out effect.NPHS2-CreErt2 mice will also mate will loxP flanked stop codon sequence preceding Ang 1 transgenic mice to generate inducible podocyte specific Ang 1 over expression transgenic mice. Ang1 will be over expressed after loxP excised by recombinase. We will see whether podocyte Ang 1 over expression would attenuate DM nephropathy or not. Similarly, the effect of podocyte Ang 1 over expression on gene expression change of podocyte and endothelium respectively will be analyzed. We will antagonize or supply the endothelium derived factors which is changed by podocyte Ang 1 over expression in the DM mice to reverse Ang 1 over expression effect.We expect podocyte Ang 1 knock out would attenuate glomerular hypertrophy and change endothelium gene expression in uninephrectomy model. The endothelium derived gene products also play a role in hypertrophy. In DM nephropathy model the over expression of podocyte Ang 1 would attenuate glomerular damage and change endothelium gene expression that is beneficial to glomerulus. These results will support that there is an interaction between podocyte and glomerular endothelium through angiopoietin 1. The study will help us understand the pathophysiology of glomerular disease and provide another treatment strategy in proteinuric renal disease.足細胞蛋白尿血管生成因子1podocyteproteinuriaangiopoietin 1