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NKCC2 activity is inhibited by the Bartter's syndrome type 5 gain‐of‐function CaR‐A843E mutant in renal cells
Author(s) -
Carmosino Monica,
Gerbino Andrea,
Hendy Geoffrey N.,
Torretta Silvia,
Rizzo Federica,
Debellis Lucantonio,
Procino Giuseppe,
Svelto Maria
Publication year - 2015
Publication title -
biology of the cell
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.543
H-Index - 85
eISSN - 1768-322X
pISSN - 0248-4900
DOI - 10.1111/boc.201400069
Subject(s) - reabsorption , transfection , biology , mutant , hek 293 cells , intracellular , wild type , loop of henle , endocrinology , medicine , microbiology and biotechnology , kidney , bartter syndrome , mutation , receptor , biochemistry , gene , hypokalemia
Background information The gain‐of‐function A843E mutation of the calcium sensing receptor (CaR) causes Bartter syndrome type 5. Patients carrying this CaR variant show a remarkably reduced renal NaCl reabsorption in the thick ascending limb (TAL) of Henle's loop resulting in renal loss of NaCl in the absence of mutations in renal Na + and Cl − ion transporters. The molecular mechanisms underlying this clinical phenotype are incompletely understood. We investigated, in human embryonic kidney 293 (HEK 293) cells and porcine kidney epithelial (LLC‐PK1) cells, the functional cross‐talk of CaR‐A843E with the Na + :K + :2Cl – co‐transporter, NKCC2, which provides NaCl reabsorption in the TAL. Results The expression of the CaR mutant did not alter the apical localisation of NKCC2 in LLC‐PK1 cells. However, the steady‐state NKCC2 phosphorylation and activity were decreased in cells transfected with CaR‐A843E compared with the control wild‐type CaR (CaR WT)‐transfected cells. Of note, low‐Cl − ‐dependent NKCC2 activation was also strongly inhibited upon the expression of CaR‐A843E mutant. The use of either P450 ω‐hydroxylase (CYP4)‐ or phospholipase A2 (PLA2)‐blockers suggests that this effect is likely mediated by arachidonic acid (AA) metabolites. Conclusions The data suggested that the activated CaR affects intracellular pathways modulating NKCC2 activity rather than NKCC2 intracellular trafficking in renal cells, and throw further light on the pathological role played by active CaR mutants in Bartter syndrome type 5.