腺苷單磷酸活化蛋白質激酶誘發細胞自噬以保護缺血再灌流所引起之腎小管細胞凋亡

Abstract

腎臟缺血再灌流 (ischemia/reperfusion, I/R)為常見造成急性腎衰竭的原因，其造成的傷害常發生於腎臟移植、腎臟動脈血管狹窄或是其他原因所導致。過去文獻指出，腎臟I/R會造成內皮細胞功能異常、引發嚴重的發炎反應、活化細胞死亡相關的蛋白及造成氧化壓力增加等而導致腎臟傷害。腎臟I/R所產生大量的過氧化物 (reactive oxygen species, ROS)被認為是造成腎臟傷害的主要原因，這些ROS會經由蛋白質氧化、脂質過氧化、DNA的傷害而導致腎臟急性傷害以及細胞凋亡等。許多抗氧化劑像是N-乙醯半胱胺酸 (N-acetyl-L-cysteine)、超氧化物歧化酶 (superoxide dismutase)等能藉由抑制ROS生成而對急性I/R腎傷害具有保護的作用。近期研究指出I/R會誘發細胞產生自噬作用 (autophagy)，而細胞可能會透過此機制走向死亡或存活。然而，I/R引起腎臟細胞傷害的分子機制並不完全清楚，有進一步研究的必要。在本研究中，我們以模擬的I/R細胞模式探討腎臟近曲小管細胞受到傷害時所牽涉到的分子調控機制。我們將豬腎臟近曲小管上皮細胞株 (LLC-PK1)處理1.5個小時的抗黴素A (antimycin A)及二去氧2-去氧-D-葡萄糖 (2-deoxy-D-glucose)，並分別以干擾粒線體呼吸傳遞鏈及抑制糖解作用方式，模擬細胞化學性缺氧的情形，之後移除藥物模擬細胞再灌流情形。實驗結果顯示，細胞在缺氧時並不會發生嚴重損傷，但隨著再灌流時間延長會造成細胞凋亡。此外，由西方墨點法實驗結果發現，細胞處理化學性缺氧藥物時，會活化與能量調控相關的蛋白-腺苷單磷酸活化蛋白質激酶 (adenosine monophosphate-activated protein kinase, AMPK)磷酸化的表現，並且隨著再灌流時間延長，藉由monodansylcadaverine染色及轉染標記LC3綠螢光蛋白 (green fluorescent protein-labeled LC3)實驗都證實I/R會誘發腎臟細胞自噬作用的情形。在流式細胞儀實驗中，我們以細胞自噬作用專一性抑制劑-3-methylamphetamine抑制細胞自噬作用的表現，觀察到細胞於處理I/R後24小時凋亡情形明顯增加。接著，為了確認AMPK在I/R中扮演之角色，我們利用干擾性核醣核酸抑制細胞AMPK的表現，結果顯示細胞於I/R處理後mTOR蛋白磷酸化情形增加、細胞自噬作用表現受抑制，並令細胞凋亡情形更嚴重；而處理AMPK專一性抑制劑-compound C於細胞中也有相同結果。另外，利用mTOR專一性抑制劑-RAD001抑制細胞mTOR磷酸化表現，我們發現細胞在I/R處理後會明顯活化細胞自噬作用的表現，並且能保護細胞不走向凋亡。接著，我們探討抗氧化劑-槲黃素 (quercetin)是否能減緩腎臟細胞所受到I/R之傷害，並探討其中分子調控機制。我們將細胞處理於化學性缺氧的過程同時合併給予quercetin，觀察控制組與加藥組之間的不同。實驗結果發現，處理quercetin的細胞會造成AMPK蛋白磷酸化表現增加、mTOR蛋白磷酸化表現下降、細胞自噬作用活化，並減低I/R誘發的細胞凋亡；利用干擾性核醣核酸抑制細胞AMPK的表現，我們發現quercetin能恢復原先受抑制的細胞自噬作用。綜合以上實驗結果，顯示AMPK能透過負調控mTOR以誘發細胞自噬作用來保護I/R引起腎臟細胞凋亡之傷害，而quercetin可能經由此調控機制來保護腎臟細胞在I/R時所造成的傷害。希望藉由此研究確認細胞自噬作用在腎臟I/R所扮演的角色與其分子調控機制，未來可望開發為藥物治療之新方向。

Renal ischemia/reperfusion (I/R) injury is the most common cause of acute kidney injury. Renal I/R injury occurs in many clinical conditions such as hypovolemic shock, thromboembolism, and renal transplantation. Several mechanisms participate in renal I/R injury including deleterious inflammatory responses, endothelial dysfunction, nitric oxide dysregulation, oxidative stress, and caspase activation. Among of them, oxidative stress is considered a major event. Moreover, the increasing lines of evidence also suggest that autophagy may participate in I/R injury, and will lead cell to death or survival. However, the detail molecular mechanisms of I/R injury on renal tubular cells still remain to be clarified. Therefore, the aim of study is to investigate the molecular mechanisms of I/R injury on renal proximal tubule epithelial cells. To mimic renal I/R injury in vivo, LLC-PK1 cells were incubated with antimycin A and 2-deoxy-D-glucose for 1.5 h to induce ischemia injury, which could disturb mitochondrial respiratory chain and inhibit energy generation via blocking glycolysis. The reperfusion was achieved by replacing the ischemic medium by a glucose-replete complete growth medium. We demonstrated I/R induced tubular cell apoptosis in a reperfusion time-dependent manner at first. By using western blotting analysis, transfection green fluorescent protein (GFP)-labeled LC3, and stainings of monodansylcadaverine, I/R induced the LC3-II forms protein expression and autophagosome formations in LLC-PK1 cells, which were obvious after 6 h of reperfusion. Analysed by flow cytometry, it revealed that inhibit autophagy by 3-methyladenine significantly enhanced I/R-induced renal tubular cell apoptosis. In addition, the phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) was also increased by I/R treatment in LLC-PK1 cells. Inhibition of AMPK by shRNA for AMPKα1 or compound C increased the phosphorylation of mammalian target of rapamycin (mTOR) protein and decreased induction of autophagy, and then enhanced I/R-induced renal tubular cell apoptosis. Moreover, RAD001, a mTOR inhibitor, could increase the autophagy activation and attenuate I/R-induced renal tubular cell apoptosis. On the other hand, we investigated the protective effect of antioxidant quercetin on I/R injury in renal tubular cells. We observed that quercetin significantly up-regulated the AMPK phosphorylation, down-regulated the mTOR phosphorylation, activated the autophagy, and decreased the I/R-induced renal cell apoptosis. In I/R-treated renal tubular cells, quercetin could also reverse the shRNA of AMPKα1-reduced renal tubular cells autophagy. Taken together, these findings suggest that autophagy protects renal tubular cells from I/R injury through an AMPK-regulated mTOR pathway. Quercetin may reduce the I/R-induced renal tubular cell injury by AMPK-regulated autophagy induction. These findings suggested that induction of autophagy by AMPK phosphorylation and mTOR reduction in renal tubular cells as a potential target for intervention renal I/R injury. Besides, quercetin may be as a potential treatment for I/R-induced renal cell injury.