近期上市的治療第二型糖尿病的新藥當中，第二型鈉-葡萄糖共同轉運通道（Sodium-glucose cotransporter-2, SGLT2）抑制劑，主要透過抑制近端腎小管細胞此一轉運通道對葡萄糖的再吸收，讓過多的葡萄糖由尿液中排出體外，藉此降低血液中葡萄糖的濃度。然而最新的研究顯示SGLT-2抑制劑除了降低血糖之外，更具有保護腎臟的作用，其中的機轉仍有待釐清。目前已有充分的研究證據指出，糖尿病會誘發腎小管細胞粒線體氧化壓力增加，造成其粒線體功能的損害；SGLT-2抑制劑會調控腸道細胞內ROS的製造，來減緩細胞發炎與死亡。因此我們假設SGLT-2抑制劑會透過粒線體對於近端腎小管細胞帶來正面的影響，從而達成其腎臟保護的作用。 本研究中，我們發現高糖高脂飲食誘發的糖尿病動物，其腎小管細胞粒線體融合蛋白減少，裂解蛋白增加。在電子顯微鏡觀察到葡萄糖會造成人類近端腎小管細胞 (HK2 cells) 粒線體呈現皺縮斷裂的型態。SGLT-2抑制劑empagliflozin能保護粒線體，減緩其走向皺縮斷裂。接著利用西方墨點分析粒線體動態平衡相關蛋白，發現empagliflozin使得受葡萄糖刺激之HK2細胞Mitofusion1 (MFN1)與Mitofusion2 (MFN2)增加，裂解蛋白Dynamin-related protein (DRP1)與Fission1 (FIS1)減少。在細胞凋亡的實驗發現葡萄糖造成TUNEL螢光表現增強，empagliflozin會減緩細胞凋亡；進一步測量細胞凋亡相關蛋白發現葡萄糖導致Bcl-2減少，Bid/t-bid，Bax, cytochrome C增加，並活化caspase-8, 9, 3的連鎖效應，而empagliflozin能降低這些相關蛋白表現。流式細胞儀的分析結果顯示empagliflozin可以讓細胞ROS及粒線體膜電位回復，呈現較正常的狀態。並且在高葡萄糖狀態下，empagliflozin可促使HK2細胞粒線體DNA拷貝數(copy number)增加，顯示它加強粒線體的有氧代謝，促進氧化磷酸化作用使得ATP產量增加。根據以上結果，我們認為empagliflozin藉由強化HK2細胞粒線體功能，減少粒線體斷裂來減緩高葡萄糖對HK-2細胞造成的傷害。

Sodium-glucose cotransporter 2 (SGLT2) inhibitors are newly marketed oral hypoglycemic agents used to treat diabetic patients. They block reabsorption of filtered glucose by inhibiting the primary glucose transporter in the proximal tubular cells (PTC), leading to glycosuria and lowering of serum glucose. A growing body of evidence from diabetic animal studies shows that SGLT2 inhibitors offer kidney benefits in better function, less hyperfiltration, less albuminuria and ameliorating pathology. However, little is known about the underlying mechanisms of these phenomenon. Glucose is known to produce oxidative stress in glomerulus and renal tubules and eventually leads to its mitochondrial damage. SGLT2 inhibitors is reported to attenuated inflammation and reduce cellular apoptosis in the intestinal cells by regulating its reactive oxygen species (ROS) production. Therefore, we hypothesize that SGLT2 inhibitor offers renal protection via mitochondrial-dependent pathways. In the present study, it was found that mitochondrial fusion proteins were downregulated, whereas fission proteins were upregulated in renal proximal tubules of mice fed with high fat/high sucrose diet. By using transmission electron microscopy, it was shown that glucose induced a shrinkage appearance of mitochondria. Human proximal tubules HK2 cells were susceptible to glucose-induced mitochondria fragmentation, which was rescued by SGLT2 inhibitor empagliflozin. Western blots analysis showed that high glucose exposure reduced the expression of Mitofusion 1 (MFN1) and mitofusion 2 (MFN2), but increased the expression of Dynamin-related protein 1 in HK2 cells. In addition, empagliflozin application attenuated the glucose-induced apoptosis of HK2 cells Mechanistic study revealed that high glucose elicited down-regulation of Bcl-2 and up-regulation of t-Bid/Bid, Bax and cytochrome C in HK2 cells. Moreover, excessive glucose also triggered the activation of caspase-8, 9, 3. Application of empagliflozin rescued HK2 cells from apoptosis by down-regulating the above pro-apoptotic genes. Furthermore, empagliflozin supply protected the mitochondria from fragmentation by reducing ROS production and restoring mitochondrial membrane potential. Besides, empagliflozin improved the mitochondrial functions by increasing oxidative metabolism, ATP production, and mitochondrial DNA contents in high glucose treated HK2 cells. In conclusion, the present study suggested that SGLT2 inhibitor protected renal proximal tubules cells from glucose-mediated injuries through mitochondria-dependent mechanism.

中文摘要…………………………………………………………. i
英文摘要………………………………………..………………... ii
第 一 章 前言….……………………………………………… 1
第一節 糖尿病…………………………………………… 1
第二節 粒線體…………………………………………….. 3
第三節 第二型鈉葡萄糖轉運通道抑制劑- Empagliflozin. 5
第四節 Empagliflozin與粒線體…………………………… 7
第 二 章 材料與方法……………………………………………. 10
第 二 章 結果…………………………………………………... 18
第 三 章 討論…………………………………………………. 25
第 四 章 圖表…………………………………………………. 31
參考文獻…………………………… ……….…… …………….. 49
附錄………………………………………………………………. 52

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