糖化終產物引起腎膈細胞傷害之機制：內質網壓力、自噬、凋亡之角色探討

Abstract

糖尿病發病約20-25年之後，約有25-40%的病人會併發糖尿病性腎病。糖尿病性腎病是造成末期腎病變的主因，並會導致糖尿病患者殘障或有較高之死亡率。過去報導指出，糖化終產物在有末期腎病變的糖尿病患者組織中，累積量約為無末期腎病變之糖尿病患者的兩倍。然而，糖化終產物對腎膈細胞的影響尚未明瞭。因此，本研究將探討內質網壓力(ER stress)、凋亡(apoptosis)以及自噬(autophagy)作用在糖化終產物累積於腎膈細胞(mesangial cells)所扮演的角色，以及相互間之作用機制。給予腎膈細胞不同濃度之糖化終產物(10, 20, 40, 80 and 160 μg/ml)後，細胞存活率顯著地隨劑量增加而下降，顯示糖化終產物對腎膈細胞具有毒性。而beclin-1, Atg5, LC3-II, CHOP, p-eIF2α以及cleaved caspase-3之蛋白表現顯著增加，顯示糖化終產物會誘發自噬、內質網壓力以及凋亡作用。另外，處理糖化終產物後，凋亡細胞之比例顯著上升，進一步指出大量累積之糖化終產物會導致腎膈細胞凋亡。共同給予4-苯基丁酸(4PBA)抑制內質網壓力，發現LC3-II及cleaved caspase-3蛋白表現顯著下降，同時，凋亡細胞之比例顯著下降，顯示抑制內質網壓力可以進一步抑制自噬與凋亡作用。另外，細胞轉染(transfection) siAtg5抑制自噬作用，發現cleaved caspase-3蛋白表現顯著增加，並且細胞凋亡比例也顯著增加，指出在糖化終產物導致之細胞凋亡中，自噬作用扮演保護細胞之角色。綜合以上研究結果，糖化終產物可經由誘發內質網壓力而導致腎膈細胞凋亡，以造成腎膈細胞存活率下降；同時，糖化終產物可經由誘發內質網壓力而活化自噬作用，以避免腎膈細胞凋亡而達到保護之作用。

25-40 % of diabetic patients develop diabetic nephropathy within 20-25 years after the onset of diabetes. Diabetic nephropathy could lead to disability and high mortality rate in diabetic patients. Also, diabetic nephropathy is the most common cause of end-stage renal disease. It has been reported that the amount of advanced glycation end products (AGEs) in tissue of diabetic patients with end-stage renal disease is twice as much as diabetic patients without end-stage renal disease. Still, the influence of AGEs on mesangial cells remains unclear. Therefore, we investigated the effects of ER stress, apoptosis and autophagy responses in mesangial cells cultured with AGEs. Cells were cultured with BSA (160 μg/ml) and different concentrations of AGEs (10, 20, 40, 80 and 160 μg/ml), and the cell viability decreased by AGEs in a dose-dependent manner, suggesting that AGEs are cytotoxic to mesangial cells. Then, the induction of beclin-1, Atg5, LC3-II, CHOP, p-eIF2α and cleaved caspase-3 as well as the elevated apoptoic cell ratio indicated that AGEs could induce autophagy, ER stress and apoptosis response in mesangial cells. Further, mesangial cells were treated with siAtg5 and 4-phenylbutyric acid (4PBA) to study the role of autophagy and ER stress in AGEs-induced apoptosis. 4PBA, a chemical chaperone, significantly reduced the protein expression of LC3-II and cleaved caspase-3. Also, the ratio of apoptotic cell was significantly decreased with 4PBA. These data suggested that AGEs might induce autophagy and apoptosis through ER stress. On the other hand, transfection of siAtg5 significantly aggravated expression of cleaved caspase-3 and exacerbated apoptotic cell ratio, suggesting that autophagy played a protective role in AGEs-induced mesangial cells apoptosis. In conclusion, AGEs could induce apoptosis and autophagy through ER stress in mesangial cells. And, autophagy played a protective role in AGEs-induced mesangial cell apoptosis.