Abstract: SA-PO125
ATG5-Mediated G2/M Arrest Through ATR-Chk1 Signaling Contributes to Renal Fibrosis After Injury Induced by Aristolochic Acid I
Session Information
- AKI: Mechanisms - AKI-CKD Transition
November 09, 2019 | Location: Exhibit Hall, Walter E. Washington Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Acute Kidney Injury
- 103 AKI: Mechanisms
Authors
- Wu, Meiju, The First Affiliated Hospital, Sun Yat-sen University, Guang Zhou, China
- Huang, Xuan, The First Affiliated Hospital, Sun Yat-sen University, Guang Zhou, China
- Peng, Yuan, The First Affiliated Hospital, Sun Yat-sen University, Guang Zhou, China
- Xiao, Xi, The First Affiliated Hospital, Sun Yat-sen University, Guang Zhou, China
- Yu, Xueqing, Department of Nephrology, Guangdong Provincial People's Hospital, Guangzhou, China
- Yang, Xiao, The First Affiliated Hospital, Sun Yat-sen University,, Guangzhou, China
Background
Recent studies have shown that autophagy is involved in the regulation of G2/M arrest and plays a renoprotective role after kidney injury. However, its role in aristolochic acid (AA)-induced AKI transition CKD remains largely unclarified.
Methods
Here, we investigated whether autophagy-related gene 5 (ATG5) modulation of Chk1 phosphate expression during AKI contributes to the progression to CKD. We tested this hypothesis by administration of aristolochic acid-I (AAI) in vitro.
Results
(1) CCK-8 showed that the viability of HK-2 cells treated with different concentrations of AAI (0, 2.5, 5, 10, 20, 40 μM) for 48 hours decrease in a concentration-dependent manner. The cell viability decreased in a time-dependent manner when HK-2 were stimulated with 5 μM AAI for different durations (0, 3, 6, 12, 24, 48 hours). (2) Western blot showed that the expression of LC3-II/I, DNA double-stranded damage marker γH2AX and FN in concentration-dependent, time-dependent increase after AAI stimulation. Phosphorylation level of Chk1(s345) and its upstream regulatory protein ATR(t1989) showed parabolic trends at 48 h after AAI stimulation. Chk1(345) and ATR(t1989) phosphorylation level increased in a time-dependent manner when AAI stimulation concentration was 5 μM. (3) Flow cytometry: After AAI treat of HK-2 cells for 48 h, the cell cycle G2/M arrest rate increased most at AA-I concentrations of 5 μM and 10 μM and the proportion of G2/M arrest was 39.12±9.1% and 40.88±10.1% respectively; the difference was statistically significant compared with 0μM (8.09±1.5%) (p<0.05, 0.05). While stimulation with AAI concentration of 5 μM for different times. The proportion of G2/M arrest gradually increased with time, and it was 41.57±16.6%at 48h; the difference was statistically significant compared with 0h (6.94±0.7%) (p<0.05).(3) ATG5 deletion led to enhanced G2/M cell-cycle arrest and increased expression of the Chk1 and of profibrotic factors (Fibronectin(FN), Connective Tissue Growth Factor (CTGF)). Autophagy activator (rapamycin) down-regulated phosphorylation level of chk1 and aggravated fibrosis (CTGF). Treatment with a Chk1 inhibitor reduced mRNA expression of fibrosis gene (FN).
Conclusion
Taken together，these findings indicated that ATG5 mediated G2/M arrest through Chk1 contributes to renal fibrosis after injury induced by AAI.