單側輸尿管阻塞誘發氧化壓力對大鼠腎小管細胞之凋亡與自噬表現之影響
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2008
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單側阻塞性尿路疾病是指尿液無法經由腎臟或輸尿管流入膀胱，尿液迴流造成腎水腫。其發生原因如單側輸尿管結石、狹窄、腫瘤、受傷等，但仍以腎結石最常見，而單側輸尿管阻塞則是一種適合模擬阻塞性尿路腎臟漸進式纖維化發展過程的病理模型。本研究主要在探討單側輸尿管阻塞造成腎臟損傷過中，活性氧、細胞凋亡與細胞自噬的角色以及細胞凋亡與細胞自噬的關係。本實驗利用大白屬於其右側輸尿管靠近腎臟三分之ㄧ處用兩條繩子分別打結，接著縫合腹腔。之後將老鼠隨機分成八組，第一組無進行結紮手術，其餘分別於術後4hr、8hr、12hr、1天、3天、5天、7天進行生理實驗並抽取500μl腎靜脈血進行自由基測定，另外動物犧牲後取阻塞腎臟，利用西方墨點法測定蛋白表現量；使用免疫組織染色觀察腎臟組織型態。結果發現腎靜脈血液H2O2/OH.-類的自由基於阻塞後四至十二小時期間有上升趨勢，而Catalase和MnSOD的蛋白表現從阻塞四小時開始就隨時間下降；腎臟組織Bax/Bcl-2比值及Caspase 3蛋白表現表現則增加；自噬作用的起始蛋白Beclin1及中期蛋白Atg5-Atg12和後期蛋白LC3表現則在阻塞3天後開始逐漸增加，從切片染色結果也發現自噬作用發生在腎臟近端與遠端腎小管處。我們的結果發現自噬作用蛋白和凋亡作用蛋白表現時間一致，表示自噬作用在單側輸尿管阻塞動物模式中有協同細胞死亡的功能。
Unilateral obstructive uropathy refers to the structural changes, like ureteral cot, stenosis, tumor and damage, that impede the normal flow of urine and the most common cause is renal calculus. Unilateral ureteral obstruction (UUO) is an animal model of mimic progressive fibrosis renal disease that can reproducibly demonstrate this pathological progress. The aim of the present study was to investigate the role of reactive oxygen species , apoptosis, autophagy and the relation between apoptosis and autophagy in UUO model. In this research, female wistar rats were performed ligation of the right ureter at the ureteropelvic junction using double silk suture. Rats were divided into 8 groups and the first group had no UUO operation and others were sacrificed after operating UUO for 4hrs, 8hrs, 12hrs, 1 day, 3 days, 5 days, 7 days. After then, rats were proceeded with physiological observation, 500μl volume of renal venous blood for free radical detection, harvested obstruction kidney for protein expression with western blot assay and immunohistochemistry stain for morphology changes. Our results found that, renal venous blood had increased hydrogen peroxide/hydroxyl radical production after UUO 4hrs to 12hrs. Catalase and MnSOD protein expression was decreased with a time-dependent manner from obstruction 4hrs. Bax and Bcl-2 ratio and Caspase 3 protein expression of UUO kidney was increased. UUO enhanced autophagy step Ⅰ protein, Beclin-1； step Ⅱ protein, Atg5-Atg12 complex； step Ⅲ protein, LC3 Ⅱ in a time-dependent after 3 days of UUO. We observed an incremental autophagy and apoptosis protein expression in renal proximal and distal tubules by immunohistochemistry. These results suggest that autophagy and apoptosis have identical tendency and represents that autophagy may play a role with apoptotic cell death in UUO animal models.
Unilateral obstructive uropathy refers to the structural changes, like ureteral cot, stenosis, tumor and damage, that impede the normal flow of urine and the most common cause is renal calculus. Unilateral ureteral obstruction (UUO) is an animal model of mimic progressive fibrosis renal disease that can reproducibly demonstrate this pathological progress. The aim of the present study was to investigate the role of reactive oxygen species , apoptosis, autophagy and the relation between apoptosis and autophagy in UUO model. In this research, female wistar rats were performed ligation of the right ureter at the ureteropelvic junction using double silk suture. Rats were divided into 8 groups and the first group had no UUO operation and others were sacrificed after operating UUO for 4hrs, 8hrs, 12hrs, 1 day, 3 days, 5 days, 7 days. After then, rats were proceeded with physiological observation, 500μl volume of renal venous blood for free radical detection, harvested obstruction kidney for protein expression with western blot assay and immunohistochemistry stain for morphology changes. Our results found that, renal venous blood had increased hydrogen peroxide/hydroxyl radical production after UUO 4hrs to 12hrs. Catalase and MnSOD protein expression was decreased with a time-dependent manner from obstruction 4hrs. Bax and Bcl-2 ratio and Caspase 3 protein expression of UUO kidney was increased. UUO enhanced autophagy step Ⅰ protein, Beclin-1； step Ⅱ protein, Atg5-Atg12 complex； step Ⅲ protein, LC3 Ⅱ in a time-dependent after 3 days of UUO. We observed an incremental autophagy and apoptosis protein expression in renal proximal and distal tubules by immunohistochemistry. These results suggest that autophagy and apoptosis have identical tendency and represents that autophagy may play a role with apoptotic cell death in UUO animal models.
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單側輸尿管阻塞, 自由基, 細胞凋亡, 自噬作用, 腎臟, unilateral ureteral obstruction, free radical, apoptosis, autophagy, kidney