Background and Purpose: For 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, CFTR_inh‐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 Approach: Using patch‐clamp technique, we tested the effects of CFTR_inh‐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 Ca ²⁺‐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 Results: We 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 CFTR_inh‐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 Implications: Both inhibitors affected Cl⁻ conductances apart from CFTR. Our results provided insights into their use in mouse models.

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 Drosophila 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 by N1-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.

Inhibitors of calcineurin phosphatase activity (CNIs) such as cyclosporin A (CsA) are widely used to treat tissue transplant rejection and acute graft-versus-host disease (aGVHD), for which inhibition of gene expression dependent on nuclear factor of activated T cells (NFAT) is the mechanistic paradigm. We recently reported that CNIs inhibit TCR-proximal signaling by preventing calcineurin-mediated dephosphorylation of Lck^S59, an inhibitory modification, raising the possibility of another mechanism by which CNIs suppress immune responses. Here we used T cells from mice that express Lck^S59A, which cannot accept a phosphate at residue 59, to initiate aGVHD. Although CsA inhibited NFAT-dependent gene upregulation in allo-aggressive T cells expressing either Lck^WT or Lck^S59A, it was ineffective in treating disease when the T cells expressed Lck^S59A. Two important NFAT-independent T cell functions were found to be CsA-resistant in Lck^S59A T cells: upregulation of the cytolytic protein perforin in tissue-infiltrating CD8⁺ T cells and antigen-specific T/DC adhesion and clustering in lymph nodes. These results demonstrate that effective treatment of aGVHD by CsA requires NFAT-independent inhibition of TCR signaling. Given that NFATs are widely expressed and off-target effects are a major limitation in CNI use, it is possible that targeting TCR-associated calcineurin directly may provide effective therapies with less toxicity.

The clinical use of the antineoplastic drug cisplatin is limited by its deleterious nephrotoxic side effect. Cisplatin-induced nephrotoxicity is associated with an increase in oxidative stress, leading ultimately to renal cell death and irreversible kidney dysfunction. Oxidative stress could be modified by the cystic fibrosis transmembrane conductance regulator protein (CFTR), a Cl⁻ channel not only involved in chloride secretion but as well in glutathione (GSH) transport. Thus, we tested whether the inhibition of CFTR could protect against cisplatin-induced nephrotoxicity. Using a renal proximal cell line, we show that the specific inhibitor of CFTR, CFTR_inh-172, prevents cisplatin-induced cell death and apoptosis by modulating the intracellular reactive oxygen species balance and the intracellular GSH concentration. This CFTR_inh-172-mediated protective effect occurs without affecting cellular cisplatin uptake or the formation of platinum-DNA adducts. The protective effect of CFTR_inh-172 in cisplatin-induced nephrotoxicity was also investigated in a rat model. Five days after receiving a single cisplatin injection (5 mg/kg), rats exhibited renal failure, as evidenced by the alteration of biochemical and functional parameters. Pretreatment of rats with CFTR_inh-172 (1 mg/kg) prior to cisplatin injection significantly prevented these deleterious cisplatin-induced nephrotoxic effects. Finally, we demonstrate that CFTR_inh-172 does not impair cisplatin-induced cell death in the cisplatin-sensitive A549 cancer cell line. In conclusion, the use of a specific inhibitor of CFTR may represent a novel therapeutic approach in the prevention of nephrotoxic side effects during cisplatin treatment without affecting its antitumor efficacy.