Abstract
The sodium (Na+)-chloride cotransporter (NCC) expressed in the distal convoluted tubule (DCT) is a key molecule regulating urinary Na+ and potassium (K+) excretion. We previously reported that high-K+ load rapidly dephosphorylated NCC and promoted urinary K+ excretion in mouse kidneys. This effect was inhibited by calcineurin (CaN) and calmodulin inhibitors. However, the detailed mechanism through which high-K+ signal results in CaN activation remains unknown. We used Flp-In NCC HEK293 cells and mice to evaluate NCC phosphorylation. We analyzed intracellular Ca2+ concentration ([Ca2+]in) using live cell Ca2+ imaging in HEK293 cells. We confirmed that high-K+-induced NCC dephosphorylation was not observed without CaN using Flp-In NCC HEK29 cells. Extracellular Ca2+ reduction with a Ca2+ chelator inhibited high-K+-induced increase in [Ca2+]in and NCC dephosphorylation. We focused on Na+/Ca2+ exchanger (NCX) 1, a bidirectional regulator of cytosolic Ca2+ expressed in DCT. We identified that NCX1 suppression with a specific inhibitor (SEA0400) or siRNA knockdown inhibited K+-induced increase in [Ca2+]in and NCC dephosphorylation. In a mouse study, SEA0400 treatment inhibited K+-induced NCC dephosphorylation. SEA0400 reduced urinary K+ excretion and induced hyperkalemia. Here, we identified NCX1 as a key molecule in urinary K+ excretion promoted by CaN activation and NCC dephosphorylation in response to K+ load.
Highlights
Several epidemiological studies have reported that potassium (K+) intake is inversely related to blood pressure [1, 2], the risk of cardiovascular disease, and mortality [3,4,5]
Unlike in the in vivo setting [17], this renders the evaluation of NCC dephosphorylation in HEK293 cells difficult
Following transient CaN-B overexpression in Flp-In NCC HEK293 cells, we observed K+-induced NCC dephosphorylation, which did not occur in CaN-B untransfected cells (Fig 1B)
Summary
Several epidemiological studies have reported that potassium (K+) intake is inversely related to blood pressure [1, 2], the risk of cardiovascular disease, and mortality [3,4,5]. Evidence accumulated over the recent years has been drawing considerable attention to the importance of K+.
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