Ginsenoside Rg1 protects mouse podocytes from aldosterone-induced injury in vitro
Abstract
Nan MAO1, #, Yuan CHENG2, #, Xin-li SHI3, Li WANG5, 6, Ji WEN1, Qiong ZHANG4, Qiong-dan HU4, Jun-ming FAN1, 4, 6, *
1Department of Nephrology, West China Hospital of Sichuan University, Chengdu 610041, China; 2Department of Nephrology, Shenzhen Second People’s Hospital, The First Affiliated Hospital of Shenzhen University, Shenzhen 518035, China; 3Department of Pathobiology and Immunology, Hebei University of Traditional Chinese Medicine, Shijiazhuang 050200, China; 4Department of Nephrology, The Affiliated Traditional Chinese Medicine Hospital of Luzhou Medical College, Luzhou 646000, China; 5The Research Center for Preclinical Medicine, Luzhou Medical College, Luzhou 646000, China; 6State Key Laboratory of Biotherapy of Human Disease, West China Hospital, Sichuan University, Chengdu 610041, China
Aim: Aldosterone is elevated in many diseases such as hypertension, diabetic nephropathy and chronic kidney disease, etc. The aim of this study was to investigate the effects of aldosterone on intracellular ROS production and autophagy in podocytes in vitro, and to explore the possibility of ginsenoside Rg1 (Rg1) being used for protecting podocytes from aldosterone-induced injury.
Methods: MPC5 mouse podocyte cells were tested. Autophagosome and autophagic vacuole formation were examined under confocal microscopy with MDC and acridine orange staining, respectively. ROS were detected with flow cytometry. Malondialdehyde content and superoxide dismutase (T-SOD) activity were measured using commercial kits. The expression of LC3-II, beclin-1, SOD2 and catalase was measured by Western blotting.
Results: Treatment with aldosterone (10 nmol/L) significantly increased ROS generation and the expression of SOD2 and catalase in MPC5 cells. Furthermore, treatment with aldosterone significantly increased the conversion of LC3-I to LC3-II, beclin-1 expression and autophagosome formation. Co-treatment with rapamycin (1 ng/mL) or chloroquine (10 μmol/L) further increased aldosterone-induced autophagosome formation. Co-treatment with Rg1 (80 ng/mL) effectively relieved oxidative stress and increased T-SOD activity at the early stage and subsequently decreased autophagy in aldosterone-treated podocytes. Co-treatment with 3-MA (4 mmol/L) or NAC (50 mmol/L) exerted similar effects against aldosterone-induced autophagy in podocytes.
Conclusion: Aldosterone enhances ROS generation and promotes autophagy in podocytes in vitro. Ginsenoside-Rg1 effectively relieves aldosterone-induced oxidative stress, thereby indirectly inhibiting aldosterone-induced podocyte autophagy.
Keywords: podocyte; aldosterone; autophagy; oxidative stress; ROS; ginsenoside; Rg1; rapamycin; chloroquine; 3-methyladenine; N-acetylcysteine; kidney
This work was supported by grants from the National Natural Science Foundation of China (81170667) and the Youth Science and Technology Support Program of Sichuan Province (2011JDT0014), China.
# These authors contributed equally to this work.
* To whom correspondence should be addressed.
E-mail junmingfan@163.com
Received 2013-08-01 Accepted 2013-12-08
Keywords:
1Department of Nephrology, West China Hospital of Sichuan University, Chengdu 610041, China; 2Department of Nephrology, Shenzhen Second People’s Hospital, The First Affiliated Hospital of Shenzhen University, Shenzhen 518035, China; 3Department of Pathobiology and Immunology, Hebei University of Traditional Chinese Medicine, Shijiazhuang 050200, China; 4Department of Nephrology, The Affiliated Traditional Chinese Medicine Hospital of Luzhou Medical College, Luzhou 646000, China; 5The Research Center for Preclinical Medicine, Luzhou Medical College, Luzhou 646000, China; 6State Key Laboratory of Biotherapy of Human Disease, West China Hospital, Sichuan University, Chengdu 610041, China
Aim: Aldosterone is elevated in many diseases such as hypertension, diabetic nephropathy and chronic kidney disease, etc. The aim of this study was to investigate the effects of aldosterone on intracellular ROS production and autophagy in podocytes in vitro, and to explore the possibility of ginsenoside Rg1 (Rg1) being used for protecting podocytes from aldosterone-induced injury.
Methods: MPC5 mouse podocyte cells were tested. Autophagosome and autophagic vacuole formation were examined under confocal microscopy with MDC and acridine orange staining, respectively. ROS were detected with flow cytometry. Malondialdehyde content and superoxide dismutase (T-SOD) activity were measured using commercial kits. The expression of LC3-II, beclin-1, SOD2 and catalase was measured by Western blotting.
Results: Treatment with aldosterone (10 nmol/L) significantly increased ROS generation and the expression of SOD2 and catalase in MPC5 cells. Furthermore, treatment with aldosterone significantly increased the conversion of LC3-I to LC3-II, beclin-1 expression and autophagosome formation. Co-treatment with rapamycin (1 ng/mL) or chloroquine (10 μmol/L) further increased aldosterone-induced autophagosome formation. Co-treatment with Rg1 (80 ng/mL) effectively relieved oxidative stress and increased T-SOD activity at the early stage and subsequently decreased autophagy in aldosterone-treated podocytes. Co-treatment with 3-MA (4 mmol/L) or NAC (50 mmol/L) exerted similar effects against aldosterone-induced autophagy in podocytes.
Conclusion: Aldosterone enhances ROS generation and promotes autophagy in podocytes in vitro. Ginsenoside-Rg1 effectively relieves aldosterone-induced oxidative stress, thereby indirectly inhibiting aldosterone-induced podocyte autophagy.
Keywords: podocyte; aldosterone; autophagy; oxidative stress; ROS; ginsenoside; Rg1; rapamycin; chloroquine; 3-methyladenine; N-acetylcysteine; kidney
This work was supported by grants from the National Natural Science Foundation of China (81170667) and the Youth Science and Technology Support Program of Sichuan Province (2011JDT0014), China.
# These authors contributed equally to this work.
* To whom correspondence should be addressed.
E-mail junmingfan@163.com
Received 2013-08-01 Accepted 2013-12-08