2011-08-012024-05-14https://scholars.lib.ntu.edu.tw/handle/123456789/658232摘要：足細胞是腎臟過濾組織中重要的結構，任何的先天缺陷或後天傷害都會造成足細 胞足突融合或細胞脫離，而導致蛋白尿及腎絲球硬化。最近發現：一個鈣離子通道(transient receptor potential canonical 6)的突變，會增加足細胞内I弓離子的濃度， 並與遺傳性腎絲球硬化有關。細胞内转離子的增加會活化calcineurin訊息傳導途徑， 使得一些蛋白去磚酸化，促進基因的表現。活化的calcineurin會使synaptopodin去磚 酸化，使其被分解，導致細胞骨幹（cytoskeleton)改變及足突融合；以cyclosporin 抑制calcineurin可以預防synaptopodin被分解及減缓蛋白尿；因此，calcineurin的過度 活化極可能在一些蛋白尿疾病扮演重要角色。Calcineurin抑制劑普遍被使用於治療 蛋白尿，但往往會引起慢性的腎臟間質傷害，臨床使用有其限制。Myocyte-enriched calcineurin-interacting protein (MCIP)1 會與calcineurin結合而抑制其活性；過度表現 MCIP1可以經由抑制calcineurin訊息傳導途徑，而防止心臟肥大。我們推測：過度表 現MCIP1可以抑制calcineurin的活性，減少足細胞傷害及蛋白尿。在本實驗，我們將在細胞實驗以LPS或puromycin aminoglycoside引發足細胞傷害， 檢視calcineurin的活性及MCIP1的基因表現；以腺病毒將MCIP1基因帶入足細胞内， 分析MCIP1的過度表現是否會減缓足細胞傷害，包括：細胞骨幹的改變、結構蛋白 的分解、細胞與細胞的銜接及細胞活動力的改變。在動物實驗，我們將配對 podocin-rtTA瓦pTRELacZ/MCIP】轉殖良，繁殖出可藉doxycyllin刺激，在足細胞過度 表現MCIP1的基因轉殖鼠，以lipopolysaccharide (LPS)引發蛋白尿，檢視MCIP1過 度表現是否可以抑制calcineurin活性，減少足細胞傷害及降低蛋白尿。我們預期：一些蛋白尿疾病會有足細胞calcineurin的過度活化及MCIP1的表現改 變，過度表現MCIP1可以抑制calcineurin的活性，減少足細胞的傷害及蛋白尿。這個 實驗將有助於愛清calcineurin訊息傳遞途徑在蛋白尿疾病扮演的角色，並提供以 MCIP1為治療模式的理論基礎。<br> Abstract: Podocyte injury is an important filtration structure of glomerular barrier. Any hereditarydefect or injury of podocyte will result in foot process enfacement and cell detachmentleading to proteinuria and glomerulosclerosis. Recently, a mutation of calcium channel(transient receptor potential canonical 6) was found to result in calcium influx in podocyteand hereditary familiar segmental glomerulosclerosis. Influx of calcium into cell will lead tothe activation of calcineurin signaling pathway, subsequent protein dephosphorylation andgene expression. Activated calcineurin was known to dephosphorylate synaptopodin leadingto degradation of synaptopodin and foot process enfacement. Inhibiting calcineurin withcyclosporin could prevent synaptopodin degradation and proteinuria. Calcineurin inhibitor isalso used in human proteinuric renal disease. However, long-term use of calcineurininhibitor is associated with kidney injury, which limit its use in clinical practice.Myocyte-enriched calcineurin-interacting protein (MCIP) 1 (or regulator of calcineurin 1,RCAN1) is expressed primarily in cardiac and skeletal muscles in mice and humans. MCIP1binds to catalytic subunit of calcineurin (CnA) and inhibit its activating effect.Over-expression of MCIP1 could prevent cardiac hypertrophy through inhibiting calcineurinsignaling pathway. It is possible over-expressing MCIP1 could also inhibit calcineurinhyperactivity and related injury in podocyte, therefore could attenuate proteinuria.In this study, we are going to demonstrate the calcineurin hyperactivity in podocyte injuryin culture system and proteinuric animal model. We will induce podocyte injury withlipopolysaccharide (LPS) or puromycin aminoglycoside (PAN) in vitro. The LPS proteinuricmice are used for animal model. Calcineurin activity is measured with commercial kit andMCIP1 expression is examined with quantitative PCR and Western blotting. Theover-expression of MCIP1 is achieved by transfection of MCIP1 cDNA through adenovirusinfection in differentiated podocyte cell culture system. To see if there is any protectiveeffect of over-expression MCIP1 on podocyte injury, we will examine the cytoskeletalchange, structure protein degradation (such as synaptopodin), cell-cell contact and motility.We will also generate inducible podocyte specific MCIP1 over-expression transgenic miceby breeding podocin-rtTA (will be bought from Jackson Laboratory) and pTRE LacZ/MCIP1(will be generated by ourselves). The over-expression MCIP1 is induced by administrationof doxycyllin. We will see its effect on proteinuria and podocyte damage (foot processenfacement).We expect there will be calcineurin hyperactivity and change of MCIP1 expression inpodocyte injury disease. Over-expression of MCIP1 could inhibit the calcineurinhyperactivity therefore attenuate podocyte injury and proteinuria. This study will furtherclarify the role of calcineurin signaling pathway in proteinuric renal disease and provideanother probable treatment modality by targeting calcineurin through MCIP1over-expression.足細胞鈣依賴磷酸?肌細胞鈣依賴磷酸?作用蛋白蛋白尿podocytecalcineurinmyocyte enriched calcineurin inhibitor 1proteinuria