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Abstract: SA-PO1060

Low Chloride Increased NCC Expression through Modulating Both ERK1/2 and SPAK Signaling Pathways

Session Information

  • Na+, K+, Cl-
    November 04, 2017 | Location: Hall H, Morial Convention Center
    Abstract Time: 10:00 AM - 10:00 AM

Category: Fluid, Electrolytes, and Acid-Base

  • 703 Na+, K+, Cl- Basic

Authors

  • Xiao, Jia, Emory University School of Medicine, Atlanta, Georgia, United States
  • Feng, Xiuyan, Emory University School of Medicine, Atlanta, Georgia, United States
  • Chen, Xinxin, Emory University School of Medicine, Atlanta, Georgia, United States
  • Chen, Shan, Emory University School of Medicine, Atlanta, Georgia, United States
  • Cai, Hui, Emory University School of Medicine, Atlanta, Georgia, United States
Background

Previous studies have showed that low chloride concentration activates NCC activity via WNK-SPAK signaling pathway. We also found that ERK1/2 signal pathway plays an important role in regulating NCC. We previously showed that ERK 1/2 phosphorylation increased in SPAK KO mice, suggesting that SPAK signaling affects ERK 1/2 signaling pathway. Therefore, we investigated whether low chloride concentration affects NCC expression through modulating interaction between ERK1/2 and SPAK signaling pathways.

Methods

Cell culture, transfection, siRNA knock-down, and western blot analysis were used in this study.

Results

We first treated the Cos-7 Cells transfected with NCC with different low chloride concentrations (chloride concentrations decreased from 142 to 7 mEq/L) for 12 hours. The western blot analysis showed that NCC protein expression increased with the decrease in chloride ion concentration in a dose-dependent manner while ERK1/2 phosphorylation decreased and S373-SPAK phosphorylation increased. To further confirm the role of MAPK-EKR1/2 signaling pathway in modulating NCC in response to low chloride concentration, we knocked down the ERK1/2 expression in Cos-7 cells using ERK1/2 siRNA and treated the cells with the low chloride solution. The western blot analysis showed that the basal NCC expression increased in the ERK1/2 siRNA knocked down group compared with the control group transfected with the scramble siRNA as expected. However, NCC expressions further increased with lowering chloride concentration while ERK 1/2 phosphorylation was also further decreased. We also found that SPAK phosphorylation increased with the ERK1/2 siRNA knocked down. To investigated the interation role of SPAK signal pathway with ERK 1/2 signaling, we knocked down SPAK expression in Cos-7 cells using SPAK siRNA and treated the cells with some chloride solutions. We found that the increase in NCC expression was partially reversed by SPAK Knocked down while the ERK1/2 phosphorylation increased.

Conclusion

These data suggested that both MAPK-ERK1/2 and SPAK signaling pathways are involved in the regulation of NCC in response to low chloride stimulation.

Funding

  • Veterans Affairs Support