Cell membrane water permeability is an important determinant of epithelial fluid secretion, tissue swelling, angiogenesis, tumor spread and other biological processes. Cellular water channels, aquaporins, are important drug targets. Water permeability is generally measured from the kinetics of cell volume change in response to an osmotic gradient. Here, we developed a microfluidic platform in which cells expressing a cytoplasmic, volume-sensing fluorescent dye are rapidly subjected to an osmotic gradient by solution mixing inside a ~0.1 nL droplet surrounded by oil. The solution mixing time was <10 ms. Osmotic water permeability was deduced from a single, time-integrated fluorescence image of an observation area in which the time after mixing was determined through spatial position. Water permeability was accurately measured in aquaporin-expressing erythrocytes with half-times for osmotic equilibration down to <50 ms. Compared with conventional water permeability measurements using costly stopped-flow instrumentation, the microfluidic platform here utilizes sub-microliter blood sample volume, does not suffer from mixing artifacts, and replaces challenging kinetic measurements by single image capture using a standard laboratory fluorescence microscope.

Urea transporter (UT) proteins, including UT-A in kidney tubule epithelia and UT-B in vasa recta microvessels, facilitate urinary concentrating function. A screen for UT-A inhibitors was developed in MDCK cells expressing UT-A1, water channel aquaporin-1, and YFP-H148Q/V163S. An inwardly directed urea gradient produces cell shrinking followed by UT-A1-dependent swelling, which was monitored by YFP-H148Q/V163S fluorescence. Screening of ~90,000 synthetic small molecules yielded four classes of UT-A1 inhibitors with low micromolar half-maximal inhibitory concentration that fully and reversibly inhibited urea transport by a noncompetitive mechanism. Structure-activity analysis of >400 analogs revealed UT-A1-selective and UT-A1/UT-B nonselective inhibitors. Docking computations based on homology models of UT-A1 suggested inhibitor binding sites. UT-A inhibitors may be useful as diuretics ("urearetics") with a mechanism of action that may be effective in fluid-retaining conditions in which conventional salt transport-blocking diuretics have limited efficacy.

Conventional diuretics such as furosemide and thiazides target salt transporters in kidney tubules, but urea transporters (UTs) have emerged as alternative targets. UTs are a family of transmembrane channels expressed in a variety of mammalian tissues, in particular the kidney. UT knockout mice and humans with UT mutations exhibit reduced maximal urinary osmolality, demonstrating that UTs are necessary for the concentration of urine. Small-molecule screening has identified potent and selective inhibitors of UT-A, the UT protein expressed in renal tubule epithelial cells, and UT-B, the UT protein expressed in vasa recta endothelial cells. Data from UT knockout mice and from rodents administered UT inhibitors support the diuretic action of UT inhibition. The kidney-specific expression of UT-A1, together with high selectivity of the small-molecule inhibitors, means that off-target effects of such small-molecule drugs should be minimal. This Review summarizes the structure, expression and function of UTs, and looks at the evidence supporting the validity of UTs as targets for the development of salt-sparing diuretics with a unique mechanism of action. UT-targeted inhibitors may be useful alone or in combination with conventional diuretics for therapy of various oedemas and hyponatraemias, potentially including those refractory to treatment with current diuretics.

Small-molecule inhibitors of urea transporter (UT) proteins in kidney have potential application as novel salt-sparing diuretics. The urea analog dimethylthiourea (DMTU) was recently found to inhibit the UT isoforms UT-A1 (expressed in kidney tubule epithelium) and UT-B (expressed in kidney vasa recta endothelium) with IC50 of 2-3 mM, and was shown to have diuretic action when administered to rats. Here, we measured UT-A1 and UT-B inhibition activity of 36 thiourea analogs, with the goal of identifying more potent and isoform-selective inhibitors, and establishing structure-activity relationships. The analog set systematically explored modifications of substituents on the thiourea including alkyl, heterocycles and phenyl rings, with different steric and electronic features. The analogs had a wide range of inhibition activities and selectivities. The most potent inhibitor, 3-nitrophenyl-thiourea, had an IC50 of ~0.2 mM for inhibition of both UT-A1 and UT-B. Some analogs such as 4-nitrophenyl-thiourea were relatively UT-A1 selective (IC50 1.3 vs. 10 mM), and others such as thioisonicotinamide were UT-B selective (IC50>15 vs. 2.8 mM).

The aquaporin-1 (AQP1) water channel is a potentially important drug target, as AQP1 inhibition is predicted to have therapeutic action in edema, tumor growth, glaucoma, and other conditions. Here, we measured the AQP1 inhibition efficacy of 12 putative small-molecule AQP1 inhibitors reported in six recent studies, and one AQP1 activator. Osmotic water permeability was measured by stopped-flow light scattering in human and rat erythrocytes that natively express AQP1, in hemoglobin-free membrane vesicles from rat and human erythrocytes, and in plasma membrane vesicles isolated from AQP1-transfected Chinese hamster ovary cell cultures. As a positive control, 0.3 mM HgCl2 inhibited AQP1 water permeability by >95%. We found that none of the tested compounds at 50 µM significantly inhibited or increased AQP1 water permeability in these assays. Identification of AQP1 inhibitors remains an important priority.

Kidney urea transporters are targets for development of small-molecule inhibitors with action as salt-sparing diuretics. A cell-based, functional high-throughput screen identified 2,7-bisacetamido fluorenone 3 as a novel inhibitor of urea transporters UT-A1 and UT-B. Here, we synthesized twenty-two 2,7-disubstituted fluorenone analogs by acylation. Structure-activity relationship analysis revealed: (a) the carbonyl moiety at C9 is required for UT inhibition; (b) steric limitation on C2, 7-substituents; and (c) the importance of a crescent-shape structure. The most potent fluorenones inhibited UT-A1 and UT-B urea transport with IC₅₀ ~ 1 μM. Analysis of in vitro metabolic stability in hepatic microsomes indicated metabolism of 2,7-disubstituted fluorenones by reductase and subsequent elimination. Computational docking to a homology model of UT-A1 suggested UT inhibitor binding to the UT cytoplasmic domain at a site that does not overlap with the putative urea binding site.

Urea transporter (UT) proteins, which include isoforms of UT-A in kidney tubule epithelia and UT-B in vasa recta endothelia and erythrocytes, facilitate urinary concentrating function. Inhibitors of urea transporter function have potential clinical applications as sodium-sparing diuretics, or 'urearetics,' in edema from different etiologies, such as congestive heart failure and cirrhosis, as well as in syndrome of inappropriate antidiuretic hormone (SIADH). High-throughput screening of drug-like small molecules has identified UT-A and UT-B inhibitors with nanomolar potency. Inhibitors have been identified with different UT-A versus UT-B selectivity profiles and putative binding sites on UT proteins. Studies in rodent models support the utility of UT inhibitors in reducing urinary concentration, though testing in clinically relevant animal models of edema has not yet been done.

Background and aims

Induced pluripotent stem cells (iPSCs) provide an important tool for the generation of patient-derived cells, including hepatocyte-like cells, by developmental cues through an endoderm intermediate. However, most iPSC lines fail to differentiate into endoderm, with induction resulting in apoptosis.

Approach and results

To address this issue, we built upon published methods to develop an improved protocol. We discovered that doxycycline dramatically enhances the efficiency of iPSCs to endoderm differentiation by inhibiting apoptosis and promoting proliferation through the protein kinase B pathway. We tested this protocol in >70 iPSC lines, 90% of which consistently formed complete sheets of endoderm. Endoderm generated by our method achieves similar transcriptomic profiles, expression of endoderm protein markers, and the ability to be further differentiated to downstream lineages.

Conclusions

Furthermore, this method achieves a 4-fold increase in endoderm cell number and will accelerate studies of human diseases in vitro and facilitate the expansion of iPSC-derived cells for transplantation studies.