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標題: | 藉由飲食誘導肥胖豬隻探討粒線體功能在慢性腎臟
疾病扮演的角色 The role of mitochondrial function in chronic kidney disease in diet induced obese minipigs |
作者: | Miao-Ju Chien 簡妙如 |
指導教授: | 陳靜宜(Ching-Yi Chen) |
關鍵字: | 李宋豬,高脂飲食,腎損傷,粒線體功能, Lee Sung minipig,High fat diet,Renal damage,Mitochondria function., |
出版年 : | 2019 |
學位: | 碩士 |
摘要: | 肥胖是近年來急遽增加的健康問題,且被認為是慢性腎臟疾病 (Chronic kidney disease, CKD) 的重要危險因素。腎臟是人體中高能量需求的器官之一,其粒線體含量和耗氧量僅次於心臟。近期研究指出,腎臟粒線體功能異常,可能加速其相關病程的進展。故本研究以高脂飲食誘導肥胖之李宋小型豬隻為模式探討粒線體功能與CKD之間的關係。
本研究共分三部分,第一部分使用5月齡之李宋豬 (10頭閹公豬和10頭母豬),起始體重為22.6±5.9公斤,並隨機分為兩組:對照飼糧 (Control, C) 組和高脂飼糧 (High fat diet, HFD) 組。餵飼六個月後,收集腎臟皮質進行分析。結果顯示,相較於C組,HFD組具有較重之體重和較厚的背部脂肪,且於腹部及頸部具有較多的油脂堆積。此外,血液生化值也指出HFD組別有較高的血糖、三酸甘油酯、總膽固醇以及游離脂肪酸和葡萄糖不耐之情況。HFD組中具有較重之腎臟重量以及較低之腎臟/體重比。HFD組之血液和尿液的肌酸酐、尿素及尿蛋白濃度亦高於C組。HFD組之腎絲球結構混亂、環間膜基質擴張、腎小管基底膜增厚和腎絲球纖維化的比例增加。此外, HFD豬隻腎臟皮質中發現三酸甘油酯累積、脂質過氧化增加以及腎臟抗氧化能力下降,推測其氧化壓力的發生。據此判定長期餵飼HFD確實可誘導李宋豬之腎皮質損傷。 為了觀察肥胖導致腎損傷之豬隻是否造成粒線體失常,所以第二部分為檢測肥胖豬隻其腎臟皮質粒線體功能表現,結果發現HFD組之ATP產量低於C組。於mRNA基因表現方面,HFD組之粒線體抗氧化能力指標 (UCP2) 表現量增加;粒線體生合成基因 (SIRT1和PGC-1α) 表現量下降;粒線體動態平衡上,HFD抑制粒線體分裂基因 (FIS1) 的表現量;蛋白質表現量則發現,HFD組別中分裂之DRP1、FIS1和融合之MFN2顯著增加,此結果顯示HFD可能造成腎臟粒線體功能異常的現象。 第三部分進一步透過體外實驗探討HFD誘發腎損傷的相關機制。利用棕櫚酸酯處理大鼠腎臟近端小管上皮細胞NRK-52E細胞株誘發脂毒性。結果發現,當棕櫚酸酯濃度增高,細胞存活率則會隨之降低、細胞凋亡現象也更為嚴重,且細胞的ATP產量也伴隨棕櫚酸酯濃度增加而下降,而在蛋白質表現量中也發現FIS1隨濃度增加而降低。 綜上所述,李宋小型豬隻經長期HFD餵飼會導致肥胖、血脂異常和腎臟損傷,同時伴隨著腎臟粒線體功能異常之現象,顯示出肥胖與腎臟疾病之關聯性。體外實驗發現,經棕櫚酸酯處理後即會造成細胞ATP產量降低,同時也顯示其對粒線體功能相關蛋白質有一定影響。因此,本研究推測粒線體功能對於肥胖導致腎損傷中扮演著重要角色。 Obesity is a common and complex health problem which has dramatically increased in the recent decades. It is also considered as an independent risk factor for chronic kidney disease. Kidney is one of the most energy-demanding organs in the body, which exhibits a high mitochondrial content and oxygen consumption only next to the heart. Recent studies suggested that mitochondrial dysfunctions in kidney would accelerate the progression of renal diseases. The purpose of this study was to investigate the nexus between mitochondrial function and kidney injury using diet-induced obesity minipigs model. This study was divided into three parts. The first part: five-month-old Lee-Sung minipigs (10 castrated male and 10 female pigs) with an initial weight of 22.6 ± 5.9 kg were randomly assigned into two groups; fed either a control diet (C) or a high-fat diet (HFD) for six months. The kidney cortex was collected for analysis. Compared with C group, HFD group had a heavier body weight, thicker back fat, higher plasma levels of glucose, triacylglycerol, total cholesterol, free fatty acid and glucose intolerance. A heavier kidney was observed in HFD pigs; whereas a smaller ratio of kidney weight to body weight was found in HFD group. Furthermore, the biomarkers of kidney injury, including blood creatinine, urine creatinine, urine urea, and urine protein were higher in HFD group. In histologic section of kidney cortex, glomerular disarranged structure, mesangial matrix dilatation, tubular basement membrane thickening and more glomerular fibrosis were observed in the HFD pigs. Moreover, greater triacylglycerol accumulation, increased lipid peroxidation, and decreased antioxidant capacity were found in the kidney cortex of HFD pigs. The results showed that long-term HFD feeding induced kidney injury in Lee-Sung minipigs. In order to observe whether HFD caused the renal injury through mitochondrial mechanisms. We next analyzed the function of the mitochondrial of renal cortex in obese pigs. The result showed that HFD pigs displayed a lower ATP production in the kidney than C group did. The expression of mitochondrial antioxidant capacity index (UCP2) was increased in the HFD group, whereas mitochondrial biogenesis genes (SIRT1 and PGC-1α) was downregulated by HFD, and mitochondrial dynamics gene (FIS1) was suppressed by HFD. The protein expressions of DRP1, FIS1, and MFN2 was upregulated in the HFD group, suggesting a mitochondrial dysfunction in the kidney. Finally, we explored the mechanism of HFD-induced renal injury through the in vitro model. Lipotoxicity was induced by palmitate treatment in NRK-52E cells (rat kidney proximal tubule epithelial cells). Palmitate treatment resulted in cell death in a dose dependent manner. In addition, ATP production was decreased with increasing palmitate concentration, and the protein expression of FIS1 was also decreased with increasing concentration. In conclusion, long-term feeding of HFD in Lee-Sung minipigs induced obesity, dyslipidemia and renal injury, in accompany with abnormal mitochondrial function in the kidney, suggesting an interrelationship with renal disease progression. Meanwhile, the in vitro study showed that palmitate treatment impaired ATP production, and it also showed an effect on mitochondrial function-related protein. Therefore, it is speculated that mitochondrial function plays an important role in obesity-induced kidney injury. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/72495 |
DOI: | 10.6342/NTU201902206 |
全文授權: | 有償授權 |
顯示於系所單位: | 動物科學技術學系 |
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