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The aquaglyceroporins are a subfamily of aquaporins that conduct both water and glycerol. Aquaporin-3 (AQP3) has an important physiological function in renal water reabsorption, and AQP3- mediated hydrogen peroxide (H 2O 2) permeability can enhance cytokine signaling in several cell types. The related aquaglyceroporinAQP7is required for dendritic cell chemokine responses and antigen uptake. Selective small-molecule inhibitors are desirable tools for investigating the biological and pathological roles of these and other AQP isoforms. Here, using a calcein fluorescence quenching assay,wescreened a library of 7360 drug-like small molecules for inhibition of mouse AQP3 water permeability. Hit confirmation and expansion with commercially available substances identified the ortho-chloride-containing compound DFP00173, which inhibitedmouseandhumanAQP3with an IC 50 of ∼0.1-0.4 μM but had low efficacy toward mouse AQP7 and AQP9. Surprisingly, inhibitor specificity testing revealed that the methylurealinked compound Z433927330, a partial AQP3 inhibitor (IC50, ∼0.7-0.9μM), is a potent and efficacious inhibitor of mouseAQP7 water permeability (IC 50, ∼0.2 μM). Stopped-flow light scattering measurements confirmed that DFP00173 and Z433927330 inhibit AQP3 glycerol permeability in human erythrocytes. Moreover, DFP00173, Z433927330, and the previously identified AQP9 inhibitor RF03176 blocked aquaglyceroporin H2O2 permeability. Molecular docking to AQP3, AQP7, and AQP9 homology models suggested interactions between these inhibitors and aquaglyceroporins at similar binding sites. DFP00173 and Z433927330 constitute selective and potent AQP3 and AQP7 inhibitors, respectively, and contribute to a set of isoform-specific aquaglyceroporin inhibitors that will facilitate the evaluation of these AQP isoforms as drug targets.

Aquaporin-4 (AQP4), a water channel protein, is expressed mainly in the perivascular end-feet of astrocytes in the brain and spinal cord. Dysregulation of AQP4 is critically associated with abnormal water transport in the astrocytes. We aimed to examine whether peripheral nerve injury (PNI) could induce the changes of AQP4 expression and astrocytic morphology in the spinal cord. Two different PNI models [partial sciatic nerve transection (PST) and chronic constriction injury (CCI)] were established on the left sciatic nerve in Sprague-Dawley rats, which decreased the pain withdrawal threshold in the ipsilateral hind paws. Both PNI models were associated with a persistent up-regulation of AQP4 in the ipsilateral dorsal horn at the lower lumbar region over 3 weeks, despite an absence of direct injury to the spinal cord. Three-dimensional reconstruction of astrocytes was made and morphometric analysis was done. Up-regulation of AQP4 was accompanied by a significant increase in the length and volume of astrocytic processes and the number of branch points. The most prominent changes were present in the distal processes of the astrocytes and the changes were maintained throughout the whole experimental period. Extravasation of systemically administered tracers Evans Blue and sodium fluorescein was not seen in both models. Taken together, PNI was associated with a long-lasting AQP4 up-regulation and enlargement of astrocytic processes in the spinal cord in rats, both of which were not related to the disruption of blood-spinal cord barrier. The findings could provide novel insights on the understanding of pathophysiology of spinal cords after PNI.