1,721,091 research outputs found
High-Throughput NKCC Functional Assay in Adherent Epithelial Cells
No full textBackground: The kidney-specific isoform of the Na-K-2Cl cotransporter NKCC2 is involved in the Na+ reabsorption in the TAL cells and in the regulation of body fluid volume. In contrast, the isoform NKCC1 represents the major pathway for Cl- entry in endothelial cells, playing a crucial role in cell volume regulation and vascular tone. Indeed both NKCC isoforms are involved in the regulation of blood pressure and represent important potential drug targets for hypertension treatment. Accordingly a high-throughput screening for NKCC inhibitors is extremely useful in the development and characterization of new anti-hypertensive drugs. So far the high-throughput screening of NKCC transporters activity has been done by 86Rb+ influx assays.
Methods: We developed a Tallium (Tl+) based fluorescent influx assay that can accurately and rapidly measure NKCC transport activity in adherent epithelial cells in the high-throughput Flex station device (FLEXA). We assessed the feasibility of this assay in the renal epithelial LLC-PK1 cells stably transfected with a previously characterized chimeric NKCC2 construct (c-NKCC2).
Results: In the absence of Cl- in the assay buffer, Tl+ addition did not induce any increase in fluorescence. However a robust Tl+ influx was observed after Cl-addition in c-NKCC2 transfected cells but not in mock-transfected or in parental LLC-PK1 cells suggesting that the Tl+ influx is actually mediated by the c-NKCC2 cotransporter. The c-NKCC2-driven FLEXA signal displays a rapid linear increased phase within the first 20 s after Cl- addition followed by a slower increase and a plateau phase. The initial rate of Tl+-dependent Cl- influx observed in c-NKCC2 transfected cells is about 3-fold over the background signal in mock-transfected LLC-PK1 cells. The preincubation with furosemide prevented the Cl-dependent Tl+ influx confirming the specificity of the NKCC-mediated Tl+ influx.
Conclusions: We demonstrated that this assay is highly reproducible, offers high temporal resolution of NKCC-mediated ion flux profiles and, importantly, as a continuous assay, it offers improved sensitivity over endpoint NKCC functional assay.

Histamine-induced AQP4 internalization in gastric cells is paralleled to an increase in AQP4 phosphorylation
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The cultured human gastric cells HGT-1 express the principal transporters involved in acid secretion.
No full textI-IGT-I is a human cell line sharing a number of physiological features with gastric parietal cells. HGT-1 cell monolayers were able to secrete H+ when stimulated with histamine (calculated external pH variation, Delta pH(e) 0.46+/-0.05) as assessed using the impermeant, pH-sensitive: fluorescence dye 8-hydroxypyrene-1,3,6-trisulphonic acid, trisodium salt (HPTS). Treatment with 100 muM omeprazole inhibited the histamine-induced apical acidification by about 60%. Intracellular pH (pH(i)) measurements using the fluorescent pH-sensitive dye 2',7'-bis-carboxyethyl-5(6)-carboxyfluorescein (BCECF) demonstrated the expression of a functional, omeprazole-sensitive H+/K+-pump. A monoclonal antibody directed against the alpha subunit of the H+/K+-ATPase immunoprecipitated a 95-kDa protein From HGT-1 cells and human stomach which corresponds to the expected molecular size of the native protein. HGT-1 cells were also positive for the anion exchanger AE2 that is expressed in gastric parietal cells. In addition, we identified a histamine- and pH(i)-sensitive Na+/H+ exchanger in HCT-I cells, which might correspond to the functional expression of the NHE4 isoform that has been detected in gastric epithelial cells as well as in primary cultured parietal cells. HGT-1 cells therefore display the principal features of parietal cells and might represent an interesting cell culture model for studying the regulatory mechanisms involved in acid secretion
Stimulation of AQP2 translocation and water transport by Okadaic Acid in AQP2-transfected renal epithelial cells
No full textAdipocytes support cAMP-dependent translocation of Aquaporin 2 (AQP2) from intracellular sites distinct from the insulin-responsive GLUT4 storage compartment
No full textAquaporin-2 (AQP2), when expressed in fully differentiated 3T3-L1 adipocytes, displays cAMP-dependent plasma membrane translocation in a manner similar to its behavior in renal epithelial cells. The translocation of AQP2 required phosphorylation at serine 256, as the expression of AQP2/S256D was constitutively plasma membrane localized, whereas AQP2/S256A was refractory to forskolin stimulation. Unlike GLUT4, this property is not inhibited by depolymerization of cortical actin. In addition, coexpression with the dominant negative form of TC10 (TC10/T31N) or inhibition of phosphatidylinositol 3-kinase did not abrogate the cAMP-mediated response. Under basal conditions, AQP2 is localized in both the perinuclear region and in punctate vesicles scattered within the periphery of the cell. Two- and three-dimensional confocal immunofluorescence microscopy demonstrated that the adipocyte AQP2 cAMP-responsive compartment was distinct from the GLUT4 insulin-responsive compartment. Consistent with this conclusion, insulin was an effective stimulator of GLUT4 translocation but had no effect on AQP2. Conversely, forskolin induced AQP2 translocation but not GLUT4. Colocalization studies with the early endosomal marker EEA1 and transferrin receptor suggested that the AQP2 compartment is mostly distinct from endosomal vesicles. Interestingly, however, the peripheral AQP2 vesicles significantly overlapped vesicle-associated membrane protein-2, underscoring the role of the latter in hormone-regulated exocytosis. To acquire insulin responsiveness following biosynthesis, GLUT4 undergoes a slow sorting step that requires 6 - 9 h. In contrast, AQP2 rapidly acquires forskolin responsiveness ( 3 h following biosynthesis) and directly enters the cAMP-regulated compartment without transiting the plasma membrane. Together, these data demonstrate that adipocytes display two different intracellular sorting mechanisms that direct distinct hormone-sensitive partitioning of GLUT4 and AQP2
Cell culture models and animal models for studying the patho-physiological role of renal aquaporins
Full text linkAquaporins (AQPs) are key players regulating urinary-concentrating ability. To date, eight aquaporins have been characterized and localized along the nephron, namely, AQP1 located in the proximal tubule, thin descending limb of Henle, and vasa recta; AQP2, AQP3 and AQP4 in collecting duct principal cells; AQP5 in intercalated cell type B; AQP6 in intercalated cells type A in the papilla; AQP7, AQP8 and AQP11 in the proximal tubule. AQP2, whose expression and cellular distribution is dependent on vasopressin stimulation, is involved in hereditary and acquired diseases affecting urine-concentrating mechanisms. Due to the lack of selective aquaporin inhibitors, the patho-physiological role of renal aquaporins has not yet been completely clarified, and despite extensive studies, several questions remain unanswered. Until the recent and large-scale development of genetic manipulation technology, which has led to the generation of transgenic mice models, our knowledge on renal aquaporin regulation was mainly based on in vitro studies with suitable renal cell models. Transgenic and knockout technology approaches are providing pivotal information on the role of aquaporins in health and disease. The main goal of this review is to update and summarize what we can learn from cell and animal models that will shed more light on our understanding of aquaporin-dependent renal water regulation
β3 adrenergic receptor in the kidney may be a new player in sympathetic regulation of renal function
No full textTo date, the study of the sympathetic regulation of renal function has been restricted to the important contribution of β1- and β2-adrenergic receptors (ARs). Here we investigate the expression and the possible physiologic role of β3-adrenergic receptor (β3-AR) in mouse kidney. The β3-AR is expressed in most of the nephron segments that also express the type 2 vasopressin receptor (AVPR2), including the thick ascending limb and the cortical and outer medullary collecting duct. Ex vivo experiments in mouse kidney tubules showed that β3-AR stimulation with the selective agonist BRL37344 increased intracellular cAMP levels and promoted 2 key processes in the urine concentrating mechanism. These are accumulation of the water channel aquaporin 2 at the apical plasma membrane in the collecting duct and activation of the Na-K-2Cl symporter in the thick ascending limb. Both effects were prevented by the β3-AR antagonist L748,337 or by the protein kinase A inhibitor H89. Interestingly, genetic inactivation of β3-AR in mice was associated with significantly increased urine excretion of water, sodium, potassium, and chloride. Stimulation of β3-AR significantly reduced urine excretion of water and the same electrolytes. Moreover, BRL37344 promoted a potent antidiuretic effect in AVPR2-null mice. Thus, our findings are of potential physiologic importance as they uncover the antidiuretic effect of β3-AR stimulation in the kidney. Hence, β3-AR agonism might be useful to bypass AVPR2-inactivating mutations.To date, the study of the sympathetic regulation of renal function has been restricted to the important contribution of β1- and β2-adrenergic receptors (ARs). Here we investigate the expression and the possible physiologic role of β3-adrenergic receptor (β3-AR) in mouse kidney. The β3-AR is expressed in most of the nephron segments that also express the type 2 vasopressin receptor (AVPR2), including the thick ascending limb and the cortical and outer medullary collecting duct. Ex vivo experiments in mouse kidney tubules showed that β3-AR stimulation with the selective agonist BRL37344 increased intracellular cAMP levels and promoted 2 key processes in the urine concentrating mechanism. These are accumulation of the water channel aquaporin 2 at the apical plasma membrane in the collecting duct and activation of the Na-K-2Cl symporter in the thick ascending limb. Both effects were prevented by the β3-AR antagonist L748,337 or by the protein kinase A inhibitor H89. Interestingly, genetic inactivation of β3-AR in mice was associated with significantly increased urine excretion of water, sodium, potassium, and chloride. Stimulation of β3-AR significantly reduced urine excretion of water and the same electrolytes. Moreover, BRL37344 promoted a potent antidiuretic effect in AVPR2-null mice. Thus, our findings are of potential physiologic importance as they uncover the antidiuretic effect of β3-AR stimulation in the kidney. Hence, β3-AR agonism might be useful to bypass AVPR2-inactivating mutations
Method of treatment of nephrogenic diabetes insipidus
No full textNephrogenic diabetes insipidus is treated with statins
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