Kidney Week

Abstract: SA-PO1026

Molecular Mechanism of High-Potassium Induced Na-Cl Cotransporter Dephosphorylation

Session Information

• Fluid and Electrolytes: Basic - II
  October 27, 2018 | Location: Exhibit Hall, San Diego Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: Fluid and Electrolytes

• 901 Fluid and Electrolytes: Basic

Authors

• Shoda, Wakana, Tokyo Medical and Dental University, Tokyo, TOKYO, Japan

Wakana Shoda,

• Nomura, Naohiro, Tokyo Medical and Dental University, Tokyo, TOKYO, Japan

Naohiro Nomura,

• Ando, Fumiaki, Tokyo Medical and Dental University, Tokyo, TOKYO, Japan

Fumiaki Ando,

• Isobe, Kiyoshi, Tokyo Medical and Dental University, Tokyo, TOKYO, Japan

Kiyoshi Isobe,

• Mori, Takayasu, Tokyo Medical and Dental University, Tokyo, TOKYO, Japan

Takayasu Mori,

• Sohara, Eisei, Tokyo Medical and Dental University, Tokyo, TOKYO, Japan

Eisei Sohara,

• Rai, Tatemitsu, Tokyo Medical and Dental University, Tokyo, TOKYO, Japan

Tatemitsu Rai,

• Uchida, Shinichi, Tokyo Medical and Dental University, Tokyo, TOKYO, Japan

Shinichi Uchida,

Group or Team Name

• Tokyo Medical and Dental University Department of Nephrology

Background

Sodium-chloride cotransporter (NCC), expressed in distal convoluted tubules (DCT), is a key molecule in the regulation of urinary potassium (K⁺) excretion. We previously reported that tacrolimus (a calcineurin inhibitor) and W7 (a calmodulin inhibitor) inhibited the K⁺-induced NCC dephosphorylation and the increase in urinary K⁺ excretion in mouse kidneys. These results suggest that calmodulin and calcineurin (CaN) were activated by calcium (Ca²⁺) signal in response to the increase in extracellular K⁺ concentration ([K⁺]_ex) in DCT. However, the precise mechanism of K⁺-induced CaN activation in DCT cells remains unknown.

Methods

Flp-in NCC HEK293 cells were transfected with constitutively active CaN-A (CA-CaN-A), CaN-A, and -B plasmids to determine the role of CaN in NCC dephosphorylation. Intracellular Ca²⁺ concentration ([Ca²⁺]_i) were analyzed by live cell imaging using fluo-4 AM. SEA0400 (an NCX reverse-mode inhibitor) and ω-agatoxin (a P/Q type Ca²⁺ channel inhibitor) were injected to C57BL/6 mice 1 hour before K⁺ oral gavage.

Results

We confirmed that CA-CaN-A over-expression clearly dephosphorylated NCC in HEK293 cells. In the following experiments, we used HEK293 cells in which both CaN-A and CaN-B were transiently overexpressed. In these cells, K⁺-induced NCC dephosphorylation was inhibited by tacrolimus and EGTA treatment. The rapid increase in [Ca²⁺]_i after high K⁺ stimulation was not observed with EGTA treatment, suggesting that the increase in [Ca²⁺]_i after K⁺ stimulation was caused by Ca²⁺ influx. To determine the influx pathway, we treated the cells with several Ca²⁺ transporter inhibitors, and identified that SEA0400 and ω-agatoxin inhibited the K⁺-induced [Ca²⁺]_i increase and NCC dephosphorylation. Furthermore, we found that both SEA0400 and ω-agatoxin inhibited K⁺-induced NCC dephosphorylation in mouse kidneys.

Conclusion

This study showed that the K⁺-induced NCC dephosphorylation by CaN was mediated by high-[K⁺]_ex^-induced influx of Ca²⁺ through reverse-mode NCX and/or P/Q type Ca²⁺ channel.