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Title: Autophagy attenuates renal fibrosis in obstructive nephropathy through inhibiting epithelial-to-mesenchymal transition.
Author: Zhang B, Ru F, Chen X, Chen Z.
Journal: Zhong Nan Da Xue Xue Bao Yi Xue Ban; 2021 Jun 28; 46(6):601-608. PubMed ID: 34275928.
Abstract:
OBJECTIVES: To explore the relationship between autophagy and epithelial-to-mesenchymal transition (EMT), and to evaluate whether autophagy can affect the progression of renal fibrosis in obstructive nephropathy by regulating the EMT process. METHODS: Unilateral ureteral obstruction (UUO) renal fibrosis model of rat was constructed, and the animals were divided into a sham group, an UUO group, an UUO+low-dose rapamycin group, and an UUO+high-dose rapamycin group. HE staining was used to observe the structure of the kidney, and Masson staining was used to observe renal interstitial collagen deposition. The expressions of E-cadherin, alpha-smooth muscle actin (α-SMA), Snail 1, and microtubule-associated protein-1 light chain 3II (LC3II) were detected by Western blotting, reflecting cellular EMT and autophagy. Transforming growth factor β1 (TGF-β1) induced-NRK52E cells model was constructed, and the cells were divided into a control group, a TGF-β1 group, and a TGF-β1+ Snail 1 siRNA group. To explore the effect of autophagy on EMT, the cells were also divided into a control group, a rapamycin group, and a Beclin 1 siRNA group. Western blotting was used to detect the expressions of E-cadherin, α-SMA, Snail 1, LC3II, collagen I, and fibronectin. RESULTS: Compared with the sham group, the kidney damage in the UUO group was significantly worse; compared with the sham group, the collagen deposition in the kidney tissues in the UUO group was significantly increased, which were significantly reduced in the UUO+high-dose rapamycin group and the UUO+low-dose rapamycin group compared with the UUO group; compared with the sham group, the E-cadherin level in the kidney of the UUO group was significantly reduced, and the expression levels of α-SMA and LC3II were significantly increased (all P<0.05). Compared with the UUO group, the expression levels of E-cadherin and LC3II in the UUO+high-dose rapamycin group and the UUO+low-dose rapamycin group were significantly increased (P<0.01 and P<0.05, respectively), and the expression level of α-SMA was significantly decreased (P<0.01 and P<0.05, respectively). The expression levels of Snail 1, α-SMA, collagen I and fibronectin were significantly higher, and the E-cadherin level was significantly lower in the TGF-β1 group than those in the control group (all P<0.05). Compared with the TGF-β1 group, the expression of E-cadherin was increased significantly, and the expressions of α-SMA, collagen I and fibronectin were decreased significantly in the TGF-β1+Snail 1 siRNA group (all P<0.05). Compared with the control group, the expression levels of LC3II and E-cadherin were significantly elevated, and the expression levels of α-SMA and Snail 1 in the rapamycin group were significantly reduced (all P<0.05); the expression levels of LC3II and E-cadherin were significantly reduced, and the expression levels of α-SMA and Snail 1 were significantly increased in the Beclin 1 siRNA group (all P<0.05). CONCLUSIONS: Autophagy plays an essential role in the regulation of EMT in obstructive nephropathy fibrosis. Autophagy can alleviate renal fibrosis by inhibiting EMT. 目的: 探讨细胞自噬与上皮间质转化(epithelial-to-mesenchymal transition，EMT)之间的关系，以及自噬是否可以通过调节EMT进程影响梗阻性肾病肾纤维化进展。方法: 构建大鼠单侧输尿管梗阻(unilateral ureteral obstruction，UUO)肾纤维化模型，并将实验大鼠随机分为假手术组、UUO组、UUO+低剂量雷帕霉素组、UUO+高剂量雷帕霉素组。采用HE染色观察肾结构，Masson染色观察肾间质胶原沉积；采用蛋白质印迹法检测钙黏附蛋白E(E-cadherin)、α平滑肌肌动蛋白(alpha-smooth muscle actin，α-SMA)、锌指转录因子1(Snail 1)表达以反映细胞EMT，检测微管相关蛋白1轻链3II(microtubule-associated protein-1 light chain 3II，LC3II)表达以反映细胞自噬。构建转化生长因子-β1(transforming growth factor-β1，TGF-β1)处理的大鼠肾小管上皮细胞NRK52E模型，并将其分为对照组、TGF-β1组、TGF-β1+Snail 1 siRNA组；为探讨自噬对EMT的影响，再将细胞分为对照组、雷帕霉素组、Beclin 1 siRNA组。采用蛋白质印迹法检测E-cadherin、α-SMA、Snail 1、LC3II、I型胶原(collagen I)和纤维黏连蛋白(fibronectin)的表达。结果: HE染色结果显示：与假手术组比较，UUO组的肾损伤明显加重；Masson染色结果显示：与假手术组比较，UUO组的肾组织中胶原沉积明显增加；与UUO组比较，UUO+高剂量雷帕霉素组和UUO+低剂量雷帕霉素组胶原沉积明显减轻。蛋白质印迹法结果显示：与假手术组比较，UUO组肾组织中E-cadherin水平明显降低，α-SMA及LC3II表达水平明显升高(均P<0.05)；与UUO组比较，UUO+高剂量雷帕霉素组和UUO+低剂量雷帕霉素组E-cadherin和LC3II表达水平均明显升高(分别P<0.01和P<0.05)，α-SMA表达水平明显降低(分别P<0.01和P<0.05)。TGF-β1组中Snail 1、α-SMA、collagen I和fibronectin表达水平均显著高于对照组，E-cadherin水平明显低于对照组(均P<0.05)。与TGF-β1组比较，TGF-β1+Snail 1 siRNA组E-cadherin的表达显著增加，α-SMA、collagen I和fibronectin表达均显著减少(均P<0.05)。与对照组相比，雷帕霉素组LC3II及E-cadherin的表达水平均显著升高，α-SMA及Snail 1表达水平显著降低(均P<0.05)；Beclin 1 siRNA组LC3II及E-cadherin表达水平显著降低，α-SMA及Snail 1表达水平均显著升高(均P<0.05)。结论: 自噬在梗阻性肾病肾纤维化的EMT调控中发挥了重要的作用。激活自噬可以通过抑制EMT而减轻肾纤维化的进程。. OBJECTIVE: To explore the relationship between autophagy and epithelial-to-mesenchymal transition (EMT), and to evaluate whether autophagy can affect the progression of renal fibrosis in obstructive nephropathy by regulating the EMT process. METHODS: Unilateral ureteral obstruction (UUO) renal fibrosis model of rat was constructed, and the animals were divided into a sham group, an UUO group, an UUO+low-dose rapamycin group, and an UUO+high-dose rapamycin group. HE staining was used to observe the structure of the kidney, and Masson staining was used to observe renal interstitial collagen deposition. The expressions of E-cadherin, alpha-smooth muscle actin (α-SMA), Snail 1, and microtubule-associated protein-1 light chain 3II (LC3II) were detected by Western blotting, reflecting cellular EMT and autophagy. Transforming growth factor β1 (TGF-β1) induced-NRK52E cells model was constructed, and the cells were divided into a control group, a TGF-β1 group, and a TGF-β1+ Snail 1 siRNA group. To explore the effect of autophagy on EMT, the cells were also divided into a control group, a rapamycin group, and a Beclin 1 siRNA group. Western blotting was used to detect the expressions of E-cadherin, α-SMA, Snail 1, LC3II, collagen I, and fibronectin. RESULTS: Compared with the sham group, the kidney damage in the UUO group was significantly worse; compared with the sham group, the collagen deposition in the kidney tissues in the UUO group was significantly increased, which were significantly reduced in the UUO+high-dose rapamycin group and the UUO+low-dose rapamycin group compared with the UUO group; compared with the sham group, the E-cadherin level in the kidney of the UUO group was significantly reduced, and the expression levels of α-SMA and LC3II were significantly increased (all P<0.05). Compared with the UUO group, the expression levels of E-cadherin and LC3II in the UUO+high-dose rapamycin group and the UUO+low-dose rapamycin group were significantly increased (P<0.01 and P<0.05, respectively), and the expression level of α-SMA was significantly decreased (P<0.01 and P<0.05, respectively). The expression levels of Snail 1, α-SMA, collagen I and fibronectin were significantly higher, and the E-cadherin level was significantly lower in the TGF-β1 group than those in the control group (all P<0.05). Compared with the TGF-β1 group, the expression of E-cadherin was increased significantly, and the expressions of α-SMA, collagen I and fibronectin were decreased significantly in the TGF-β1+Snail 1 siRNA group (all P<0.05). Compared with the control group, the expression levels of LC3II and E-cadherin were significantly elevated, and the expression levels of α-SMA and Snail 1 in the rapamycin group were significantly reduced (all P<0.05); the expression levels of LC3II and E-cadherin were significantly reduced, and the expression levels of α-SMA and Snail 1 were significantly increased in the Beclin 1 siRNA group (all P<0.05). CONCLUSION: Autophagy plays an essential role in the regulation of EMT in obstructive nephropathy fibrosis. Autophagy can alleviate renal fibrosis by inhibiting EMT.

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