The eukaryotic initiation factor 5A (eIF5A), which is highly conserved throughout evolution, has the unique characteristic of post-translational activation through hypusination. This modification is catalyzed by two enzymatic steps involving deoxyhypusine synthase (DHPS) and deoxyhypusine hydroxylase (DOHH). Notably, eIF5A may be involved in regulating the lifespan of during long-term hypoxia. Therefore, we investigated the possibility of a link between eIF5A hypusination and cellular resistance to hypoxia/anoxia. Pharmacologic targeting of DHPS by1-guanyl-1,7-diaminoheptane (GC7) or RNA interference-mediated inhibition of DHPS or DOHH induced tolerance to anoxia in immortalized mouse renal proximal cells. Furthermore, GC7 treatment of cells reversibly induced a metabolic shift toward glycolysis as well as mitochondrial remodeling and led to downregulated expression and activity of respiratory chain complexes, features characteristic of mitochondrial silencing. GC7 treatment also attenuated anoxia-induced generation of reactive oxygen species in these cells and in normoxic conditions, decreased the mitochondrial oxygen consumption rate of cultured cells and mice. In rats, intraperitoneal injection of GC7 substantially reduced renal levels of hypusinated eIF5A and protected against ischemia-reperfusion-induced renal injury. Finally, in the preclinical pig kidney transplant model, intravenous injection of GC7 before kidney removal significantly improved graft function recovery and late graft function and reduced interstitial fibrosis after transplant. This unconventional signaling pathway offers an innovative therapeutic target for treating hypoxic-ischemic human diseases and organ transplantation.

Background and PurposeFor decades, inhibitors of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel have been used as tools to investigate the role and function of CFTR conductance in cystic fibrosis research. In the early 2000s, two new and potent inhibitors of CFTR, CFTRinh-172 and GlyH-101, were described and are now widely used to inhibit specifically CFTR. However, despite some evidence, the effects of both drugs on other types of Cl−-conductance have been overlooked. In this context, we explore the specificity and the cellular toxicity of both inhibitors in CFTR-expressing and non–CFTR-expressing cells.Experimental ApproachUsing patch-clamp technique, we tested the effects of CFTRinh-172 and GlyH-101 inhibitors on three distinct types of Cl− currents: the CFTR-like conductance, the volume-sensitive outwardly rectifying Cl− conductance (VSORC) and finally the Ca2+-dependent Cl− conductance (CaCC). We also explored the effect of both inhibitors on cell viability using live/dead and cell proliferation assays in two different cell lines.Key ResultsWe confirmed that these two compounds were potent inhibitors of the CFTR-mediated Cl− conductance. However,GlyH-101 also inhibited the VSORC conductance and the CaCC at concentrations used to inhibit CFTR. The CFTRinh-172 did not affect the CaCC but did inhibit the VSORC, at concentrations higher than 5 µM. Neither inhibitor (20 µM; 24 h exposure) affected cell viability, but both were cytotoxic at higher concentrations.Conclusions and ImplicationsBoth inhibitors affected Cl− conductances apart from CFTR. Our results provided insights into their use in mouse models.

Adaptation to hypoxia is an essential physiological response to decrease in tissue oxygenation. This process is primarily under the control of transcriptional activator hypoxia-inducible factor (HIF1). A better understanding of the intracellular HIF1 stabilization pathway would help in management of various diseases characterized by anemia. Among human pathologies, cystic fibrosis disease is characterized by a chronic anemia that is inadequately compensated by the classical erythroid response mediated by the HIF pathway. Because the kidney expresses CFTR and is a master organ involved in the adaptation to hypoxia, we used renal cells to explore the relationship between CFTR and the HIF1-mediated pathway. To monitor the adaptive response to hypoxia, we engineered a hypoxia-induced fluorescent reporter system to determine whether CFTR modulates hypoxia-induced HIF1 stabilization. We show that CFTR is a regulator of HIF stabilization by controlling the intracellular reactive oxygen species (ROS) level through its ability to transport glutathione (a ROS scavenger) out of the cell. Moreover, we demonstrated in a mouse model that both the pharmacological inhibition and the ΔF508 mutation of CFTR lead to an impairment of the adaptive erythroid response to oxygen deprivation. We conclude that CFTR controls HIF stabilization through control of the level of intracellular ROS that act as signaling agents in the HIF-1 pathway.