2012-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/645595摘要：足細胞是腎絲球過濾組織的重要結構，任何的傷害或遺傳缺陷都會造成蛋白尿。最 近研究發現：transient receptor potential cation channel canonical (TRPC) 6的突變，會造 成足細胞内鈣離子濃度增加，產生遺傳性腎絲球硬化。細胞内鈣離子的增加會活化下 游calcineurin-nuclear factor of activated T cells (NFAT)訊息傳導途徑。報導指出活化 calcineurin會使synaptopodin去場酸化，因而傷害足細胞，產生蛋白尿。但這樣的影響 是立即的，無法解釋在TRPC6突變家族蛋白尿發生於成人階段現象；因此，NFAT活 化激發的基因表現是讓足細胞表現型改變及腎絲球硬化的可能原因。在非遺傳性蛋白 尿中，許多刺激（如angiotensin II)會增加細胞I弓離子的濃度，活化calcineurin- (NFAT) 訊息傳導途徑，因此，NFAT引發的基因表現有可能在非遺傳性蛋白尿疾病及腎絲球 硬化扮演某種角色。活化NFAT引發的基因表現與心臟肥大及肺臟平滑肌重塑有關，我們的初步細胞研 究顯示：過度表現活性NFAT會改變足細胞的型性及肥大變化。在糖尿病腎病變及其 他蛋白尿疾病，足細胞的型性改變及肥大與後來發生的足細胞脫離、腎絲球硬化有相 當大的關連。因此，我們假設：在一些蛋白尿疾病，刺激引起的足細胞内I弓離子增加 會活化NFAT引發的基因表現，改變足細胞的型性，產生足細胞脫離，而有腎絲球硬 化。在本實驗，我們將在糖尿病及超過遽的動物模式中，檢視NFAT訊息傳導途徑的活性 及其與腎絲球病變的關連。細胞實驗將檢視過度表現活性NFAT對足細胞型性的影 響，並建立可激發足細胞表現活性NFAT基因轉植鼠，檢視是否會對足細胞型性、蛋 白尿及腎絲球硬化產生影響。預期：在蛋白尿疾病NFAT訊息傳導途徑會被活化，此 一傳導途徑的活化一些基因表現，改變足細胞型性及腎絲球硬化。此研究將有助於蛋 白尿及腎絲球病變產生機制的了解，提供治療研發的方向。<br> Abstract: Podocyte is one of the important elements of glomerular filtration barrier. Any damage orhereditary defect of podocyte will result in proteinuria. Recently, a mutation of transientreceptor potential cation channel canonical 6 (TRPC6) was found to cause increase ofcalcium influx in podocyte and familiar hereditary focal segmental sclerosis. But theunderlying mechanism leading to glomerulosclerosis is unknown. Increase of calcium influxwill result in downstream calcineurin-NFAT signaling pathway activation. There was areport revealed that activated calcineurin could dephosphorylate synaptopodin thereforedamage to podocyte structure and subsequent proteinuria. But this immediate effect onproteinuria can’t explain the late onset of proteinuria and segmental sclerosis in TRPC6mutation family members. Therefore, NFAT signaling pathway is still the most probableresponsible pathway leading to this podocyte phenotype change and hereditary nephropathy.In acquired proteinuric disease many damages or stimuli, such as angiotensin II, couldincrease intracellular calcium which probably also activate calcineurin-NFAT pathway.NFAT dependent gene expression may participate in the development of podocyte phenotypechange and subsequent glomerulosclerosis.In heart, activation of NFAT by calcineurin is associated hypertrophic change of stressinduced cardiomyopathy. NFAT pathway also mediates pulmonary smooth muscleremodeling. Our in vitro preliminary study revealed that over-expression of constitutiveactive NFATc3 results in podocyte phenotype change, including hypertrophic change.Podocyte phenotype change precedes podocyte depletion, and later glomerulosclerosis inDM nephropathy and other proteinuric renal disease. Therefore, we hypothesis that in somekind of kidney disease, the stimulus causes increase of calcium-calcineurin activity leadingto the activation of NFAT dependent gene transcription, therefore podocyte phenotypechange or hypertrophy, detachment and subsequent glomerulosclerosis.In this study, we will first examine the activity of NFAT signaling pathway in DMnephropathy or hyperfiltration animal model. The degree of NFAT dependent geneexpression will be correlated to podocyte phenotype change and glomerulosclerosis. In vitrostudy, we will activate the NFAT dependent gene expression by over-expression ofconstitutive active NFATc3 in podocyte through adenovirus infection. The podocytephenotype change, including cytoskeletal change, hypertrophy, cell-cell contract and motility,will be examined. In vivo, we will observe the podocyte hypertrophy, phenotype change,glomerular hypertrophy and glomerulosclerosis in podocyte specific inducibleover-expression of constitutive active NFAT mice.We expect that NFAT signaling pathway will be activated in podocyte in DM andhyperfiltration animal model. Activation of NFAT pathway in vitro and in vivo will result inpodocyte hypertrophy, phenotype change and glomerulosclerosis. This study will help usunderstand the underlying mechanism of the development of glomerulosclerosis and providethe rationale of blocking calcineurin-NFAT signaling pathway in proteinuric glomerularnephropathy.活化Ｔ細胞核因子足細胞蛋白尿腎絲球nuclear factor of activated T cellpodocyteproteinuriaglomerulus