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. 2016 Oct 26;6:35986. doi: 10.1038/srep35986

Figure 5. Inhibition of WNK3-SPAK signaling prevents acute cell swelling in response to osmotic stress by stimulating KCC3 activity.

Figure 5

(A) Left panel: Relative change in cell water volume during hypotonic stress in WNK3 WT and WNK3-KD (kinase-dead) cells. Cells were sequentially exposed to isotonic HEPES-MEM (310 mOsm/kg H2O), followed by hypotonic HEPES-MEM (150 mOsm/kg H2O) to promote cell swelling for 20 min, and then isotonic HEPES-MEM for 5 min. Middle panel: Summary data of cell volume increase. Right panel: Rate constants from the slopes (blue and red lines in left panel) were calculated by fitting a linear regression to the cell water data (relative change) during the initial swelling response (5–7 min). WNK3-KD cells exhibited significantly less swelling in response to hypotonic stress. Data are mean ± SEM, n = 5–6 experiments. *p < 0.05 vs. WT. (B) Relative change in cell water after hypotonic swelling in WNK3-WT cells in the presence and absence of STOCK1S-50699. Cells were pre-incubated with 10 μM STOCK1S-50699 for 30 min at 37 degrees C and exposed to osmotic stress as described above in the presence of drug. STOCK1S-50699 prevented cells from swelling in response to acute hypotonic stress. Data are means ± SEM, n = 5–6 experiments. *p < 0.05 vs. WT. (C) Relative change in cell water after hypotonic swelling in WNK3-KD cells in the presence and absence of STOCK1S-50699, as described above. Data are means ± SEM, n = 4–5 experiments. No significant difference in either peak change of cell water content or the initial rate of cell swelling was noted with STOCK1S-50699 in WNK3-KD cells. (D) Relative change in cell volume in WNK3-WT and WNK3-KD cells in the presence or absence of furosemide. Cells were pre-incubated with 2 mM furosemide for 15 min prior to hypotonic stimulation as described above in the presence of drug. Right panel: Summary data of cell volume increase. The effect of WNK3-KD on decreasing cell swelling is reversed by 2 mM furosemide, revealing a dependence on KCC3 activity. Data are mean ± SEM, n = 3–4 experiments. *p < 0.05.