Canagliflozin Impairs Blood Reperfusion of the Ischemic Lower Limb by Inhibiting the Retention and Paracrine Function of Mesenchymal Stem Cells

Background: Canagliflozin (CANA) administration increases amputation risk of the lower limb in the clinic. The present study aimed to investigate whether and how CANA interferer with intracellular physiological processes of mesenchymal stem cells (MSCs), and its contribution to ischemic lower limb. Methods: The in vivo blood flow recovery of ischemic lower limb following CANA treatment was evaluated. The cellular function of MSCs after CANA treatment were also assessed in vitro. In silico docking analysis and mutant substitution assay were conducted to confirm the interaction of CANA and glutamate dehydrogenase 1 (GDH1). Findings: Following CANA treatment, attenuated angiogenesis and hampered recovery of blood flow were observed in ischemic region, and the proliferation inhibition, migration inhibition, and apoptosis of MSCs was evidenced. CANA was involved in MSC mitochondrial respiratory malfunction, ATP production inhibition, cytochrome c release and vessel endothelial growth factor A (VEGFA) secretion inhibition, which contribute to reduced tissue repair capacity of MSCs, resulting in compromised angiogenesis and blood reperfusion in ischemic region. The detrimental effects of CANA on MSCs result from the inhibition of GDH1 by CANA (evidenced by in silico docking analysis and H199A-GDH1/ N392A-GDH1 mutant substitution). Interpretation: Our work highlights that GDH1 activity inhibition by CANA interferes with metabolic activity of mitochondria, which deteriorates retention and VEGFA secretion of MSCs, hampering angiogenesis and the recovery of blood flow following lower limb ischemia. Funding Statement: This study was financially supported by National Natural Science Foundation of China (grant # 81800314 for J. N.), Natural Science Foundation of Zhejiang Province (grant # LQ18H090007 for Z. L.) and Wenzhou Science and Technology Bureau Foundation (grant # Y20190163 for Y. L.). Declaration of Interests: The authors declare no competing financial interests. Ethics Approval Statement: All experimental protocols were approved by the Animal Care and Use Committee of Wenzhou Medical University.