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NKCC2 activity is inhibited by the Bartter's syndrome type 5 gain-of-function CaR-A843E mutant in renal cells
Monica Carmosino1, Andrea Gerbino, Geoffrey N Hendy
1Department of Biosciences, Biotechnologies, and Biopharmaceutics, University of Bari, 70125 Bari, Italy; Department of Sciences, University of Basilicata, 85100 Potenza, Italy.
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.
Insights
The gain-of-function calcium sensing receptor (CaR) A843E mutation impairs renal NaCl reabsorption by reducing NKCC2 transporter activity. This study reveals CaR-A843E affects intracellular pathways, not NKCC2 trafficking, in Bartter syndrome type 5.
Area of Science:
- Nephrology
- Molecular Biology
- Cell Biology
Background:
- Bartter syndrome type 5 is caused by a gain-of-function mutation (A843E) in the calcium sensing receptor (CaR).
- This mutation leads to reduced renal NaCl reabsorption in the thick ascending limb (TAL), causing salt loss.
- The underlying molecular mechanisms linking CaR-A843E to impaired NaCl transport are not fully understood.
Purpose of the Study:
- To investigate the functional cross-talk between the CaR-A843E mutant and the Na+:K+:2Cl- co-transporter (NKCC2).
- To elucidate the molecular mechanisms by which CaR-A843E affects NKCC2 activity in renal cells.
Main Methods:
- Experiments were conducted in human embryonic kidney 293 (HEK 293) and porcine kidney epithelial (LLC-PK1) cells.
- Functional assays assessed NKCC2 localization, phosphorylation, and activity in cells expressing CaR-A843E versus wild-type CaR (CaR WT).
- Inhibitors of P450 ω-hydroxylase (CYP4) and phospholipase A2 (PLA2) were used to explore the role of arachidonic acid (AA) metabolites.
Main Results:
- CaR-A843E expression did not affect NKCC2's apical localization.
- Steady-state NKCC2 phosphorylation and activity were significantly decreased in cells expressing CaR-A843E compared to CaR WT.
- Low-Cl(-)-dependent NKCC2 activation was inhibited by CaR-A843E, suggesting involvement of arachidonic acid metabolites.
Conclusions:
- The activated CaR-A843E mutant modulates NKCC2 activity through intracellular pathways, rather than altering NKCC2 trafficking.
- These findings provide further insight into the pathological role of active CaR mutants in Bartter syndrome type 5.
- The study highlights the complex interplay between CaR signaling and renal ion transport.
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