Browsing by Subject "Transport"

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    A robust fluorescence-based assay for human erythrocyte Ca++ efflux suitable for high-throughput inhibitor screens
    (Springer, 2023) Sims, Jeremiah N.; Yun, EJun; Chu, Jonathan; Siddiqui, Mansoor A.; Desai, Sanjay A.; Surgery, School of Medicine
    Intracellular calcium is maintained at very low concentrations through the action of PMCA Ca++ extrusion pumps. Although much of our knowledge about these Ca++ extrusion pumps derives from studies with human erythrocytes, kinetic studies of Ca++ transport for these cells are limited to radioisotope flux measurements. Here, we developed a robust, microplate-based assay for erythrocyte Ca++ efflux using extracellular fluorescent Ca++ indicators. We optimized Ca++ loading with the A23187 ionophore, established conditions for removal of the ionophore, and adjusted fluorescent dye sensitivity by addition of extracellular EGTA to allow continuous tracking of Ca++ efflux. Efflux kinetics were accelerated by glucose and inhibited in a dose-dependent manner by the nonspecific inhibitor vanadate, revealing that Ca++ pump activity can be tracked in a 384-well microplate format. These studies enable radioisotope-free kinetic measurements of the Ca++ pump and should facilitate screens for specific inhibitors of this essential transport activity.
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    Claudin-2 deficiency associates with hypercalciuria in mice and human kidney stone disease
    (American Society for Clinical Investigation, 2020-03-09) Curry, Joshua N.; Saurette, Matthew; Askari, Masomeh; Pei, Lei; Filla, Michael B.; Beggs, Megan R.; Rowe, Peter S. N.; Fields, Timothy; Sommer, Andre J.; Tanikawa, Chizu; Kamatani, Yoichiro; Evan, Andrew P.; Totonchi, Mehdi; Alexander, R. Todd; Matsuda, Koichi; Yu, Alan S. L.; Anatomy and Cell Biology, School of Medicine
    The major risk factor for kidney stone disease is idiopathic hypercalciuria. Recent evidence implicates a role for defective calcium reabsorption in the renal proximal tubule. We hypothesized that claudin-2, a paracellular cation channel protein, mediates proximal tubule calcium reabsorption. We found that claudin-2–null mice have hypercalciuria due to a primary defect in renal tubule calcium transport and papillary nephrocalcinosis that resembles the intratubular plugs in kidney stone formers. Our findings suggest that a proximal tubule defect in calcium reabsorption predisposes to papillary calcification, providing support for the vas washdown hypothesis. Claudin-2–null mice were also found to have increased net intestinal calcium absorption, but reduced paracellular calcium permeability in the colon, suggesting that this was due to reduced intestinal calcium secretion. Common genetic variants in the claudin-2 gene were associated with decreased tissue expression of claudin-2 and increased risk of kidney stones in 2 large population-based studies. Finally, we describe a family in which males with a rare missense variant in claudin-2 have marked hypercalciuria and kidney stone disease. Our findings indicate that claudin-2 is a key regulator of calcium excretion and a potential target for therapies to prevent kidney stones.