Wei-Cheng Tseng1,2,3,4,5, Pei-Ying Lee6, Ming-Tsun Tsai1,2,3,5, Fu-Pang Chang2,3,5,7, Nien-Jung Chen5,8, Chiang-Ting Chien9, Shih-Chieh Hung10,11,12,13, Der-Cherng Tarng14,15,16,17,18,19,20. 1. Division of Nephrology, Department of Medicine, Taipei Veterans General Hospital, 201, Section 2, Shih-Pai Road, Taipei, 11217, Taiwan. 2. Faculty of Medicine, School of Medicine, National Yang-Ming University, Taipei, Taiwan. 3. Institute of Clinical Medicine, School of Medicine, National Yang-Ming University, Taipei, Taiwan. 4. Center for Intelligent Drug Systems and Smart Bio-devices (IDS2B), National Chiao-Tung University, Hsinchu, Taiwan. 5. Institute of Clinical Medicine, School of Medicine, National Yang Ming Chiao Tung University, Taipei, Taiwan. 6. Holistic Education Center, Tzu Chi University of Science and Technology, Hualien, Taiwan. 7. Department of Pathology and Laboratory Medicine, Taipei Veterans General Hospital, Taipei, Taiwan. 8. Institute of Microbiology and Immunology, School of Medicine, National Yang-Ming University, Taipei, Taiwan. 9. Department of Life Science, School of Life Science, National Taiwan Normal University, Taipei, Taiwan. 10. Institute of Clinical Medicine, School of Medicine, National Yang-Ming University, Taipei, Taiwan. hung3340@gmail.com. 11. Institute of Clinical Medicine, School of Medicine, National Yang Ming Chiao Tung University, Taipei, Taiwan. hung3340@gmail.com. 12. Integrative Stem Cell Center, Department of Orthopedics, and Institute of New Drug Development, New Drug Development Center, China Medical University, Taichung, Taiwan. hung3340@gmail.com. 13. Institute of Biomedical Sciences, Academia Sinica, 128, Section 2, Academia Road, Taipei, 11529, Taiwan. hung3340@gmail.com. 14. Division of Nephrology, Department of Medicine, Taipei Veterans General Hospital, 201, Section 2, Shih-Pai Road, Taipei, 11217, Taiwan. dctarng@vghtpe.gov.tw. 15. Faculty of Medicine, School of Medicine, National Yang-Ming University, Taipei, Taiwan. dctarng@vghtpe.gov.tw. 16. Institute of Clinical Medicine, School of Medicine, National Yang-Ming University, Taipei, Taiwan. dctarng@vghtpe.gov.tw. 17. Center for Intelligent Drug Systems and Smart Bio-devices (IDS2B), National Chiao-Tung University, Hsinchu, Taiwan. dctarng@vghtpe.gov.tw. 18. Institute of Clinical Medicine, School of Medicine, National Yang Ming Chiao Tung University, Taipei, Taiwan. dctarng@vghtpe.gov.tw. 19. Department and Institute of Physiology, School of Medicine, National Yang-Ming University, Taipei, Taiwan. dctarng@vghtpe.gov.tw. 20. Department of Biological Science and Technology, College of Biological Science and Technology, National Chiao-Tung University, Hsinchu, Taiwan. dctarng@vghtpe.gov.tw.
Abstract
BACKGROUND: Acute kidney injury (AKI) is an emerging global healthcare issue without effective therapy yet. Autophagy recycles damaged organelles and helps maintain tissue homeostasis in acute renal ischemia-reperfusion (I/R) injury. Hypoxic mesenchymal stem cells (HMSCs) represent an innovative cell-based therapy in AKI. Moreover, the conditioned medium of HMSCs (HMSC-CM) rich in beneficial trophic factors may serve as a cell-free alternative therapy. Nonetheless, whether HMSCs or HMSC-CM mitigate renal I/R injury via modulating tubular autophagy remains unclear. METHODS: Renal I/R injury was induced by clamping of the left renal artery with right nephrectomy in male Sprague-Dawley rats. The rats were injected with either PBS, HMSCs, or HMSC-CM immediately after the surgery and sacrificed 48 h later. Renal tubular NRK-52E cells subjected to hypoxia-reoxygenation (H/R) injury were co-cultured with HMSCs or treated with HMSC-CM to assess the regulatory effects of HSMCs on tubular autophagy and apoptosis. The association of tubular autophagy gene expression and renal recovery was also investigated in patients with ischemic AKI. RESULT: HMSCs had a superior anti-oxidative effect in I/R-injured rat kidneys as compared to normoxia-cultured mesenchymal stem cells. HMSCs further attenuated renal macrophage infiltration and inflammation, reduced tubular apoptosis, enhanced tubular proliferation, and improved kidney function decline in rats with renal I/R injury. Moreover, HMSCs suppressed superoxide formation, reduced DNA damage and lipid peroxidation, and increased anti-oxidants expression in renal tubular epithelial cells during I/R injury. Co-culture of HMSCs with H/R-injured NRK-52E cells also lessened tubular cell death. Mechanistically, HMSCs downregulated the expression of pro-inflammatory interleukin-1β, proapoptotic Bax, and caspase 3. Notably, HMSCs also upregulated the expression of autophagy-related LC3B, Atg5 and Beclin 1 in renal tubular cells both in vivo and in vitro. Addition of 3-methyladenine suppressed the activity of autophagy and abrogated the renoprotective effects of HMSCs. The renoprotective effect of tubular autophagy was further validated in patients with ischemic AKI. AKI patients with higher renal LC3B expression were associated with better renal recovery. CONCLUSION: The present study describes that the enhancing effect of MSCs, and especially of HMCSs, on tissue autophagy can be applied to suppress renal tubular apoptosis and attenuate renal impairment during renal I/R injury in the rat. Our findings provide further mechanistic support to HMSCs therapy and its investigation in clinical trials of ischemic AKI.
BACKGROUND:Acute kidney injury (AKI) is an emerging global healthcare issue without effective therapy yet. Autophagy recycles damaged organelles and helps maintain tissue homeostasis in acute renal ischemia-reperfusion (I/R) injury. Hypoxic mesenchymal stem cells (HMSCs) represent an innovative cell-based therapy in AKI. Moreover, the conditioned medium of HMSCs (HMSC-CM) rich in beneficial trophic factors may serve as a cell-free alternative therapy. Nonetheless, whether HMSCs or HMSC-CM mitigate renal I/R injury via modulating tubular autophagy remains unclear. METHODS: Renal I/R injury was induced by clamping of the left renal artery with right nephrectomy in male Sprague-Dawley rats. The rats were injected with either PBS, HMSCs, or HMSC-CM immediately after the surgery and sacrificed 48 h later. Renal tubularNRK-52E cells subjected to hypoxia-reoxygenation (H/R) injury were co-cultured with HMSCs or treated with HMSC-CM to assess the regulatory effects of HSMCs on tubular autophagy and apoptosis. The association of tubular autophagy gene expression and renal recovery was also investigated in patients with ischemic AKI. RESULT: HMSCs had a superior anti-oxidative effect in I/R-injured rat kidneys as compared to normoxia-cultured mesenchymal stem cells. HMSCs further attenuated renal macrophage infiltration and inflammation, reduced tubular apoptosis, enhanced tubular proliferation, and improved kidney function decline in rats with renal I/R injury. Moreover, HMSCs suppressed superoxide formation, reduced DNA damage and lipid peroxidation, and increased anti-oxidants expression in renal tubular epithelial cells during I/R injury. Co-culture of HMSCs with H/R-injured NRK-52E cells also lessened tubular cell death. Mechanistically, HMSCs downregulated the expression of pro-inflammatory interleukin-1β, proapoptotic Bax, and caspase 3. Notably, HMSCs also upregulated the expression of autophagy-related LC3B, Atg5 and Beclin 1 in renal tubular cells both in vivo and in vitro. Addition of 3-methyladenine suppressed the activity of autophagy and abrogated the renoprotective effects of HMSCs. The renoprotective effect of tubular autophagy was further validated in patients with ischemic AKI. AKI patients with higher renal LC3B expression were associated with better renal recovery. CONCLUSION: The present study describes that the enhancing effect of MSCs, and especially of HMCSs, on tissue autophagy can be applied to suppress renal tubular apoptosis and attenuate renal impairment during renal I/R injury in the rat. Our findings provide further mechanistic support to HMSCs therapy and its investigation in clinical trials of ischemic AKI.