Figure 7.

NCC-mediated sodium uptake and NCC-phosphorylation are reduced with complex IV inhibition. (A and B) ²²Na⁺ uptake in HEK293 cells transfected with NCC or mock, with or without inhibition of OXPHOS complex IV with KCN. KCN 1 mmol/L or KCl 1 mmol/L (control) was added during both preincubation and the uptake period, as indicated; the same applies to hydrochlorothiazide (HCTZ) 100 µmol/L. Bars represent mean with SD. (A) HCTZ-sensitive ²²Na⁺ uptake of NCC-transfected cells over a period of 30 minutes. Data in (A) are on the basis of (B). Significance was assessed with an unpaired t test. (B) ²²Na⁺ uptake in 30 minutes after preincubation with hypotonic-low-chloride buffer or isotonic buffer. Cells were transfected with NCC or mock and treated with KCl or KCN (n=4 of triplicates in each experiment). (C) Representative immunoblots showing NCC and phosphorylated NCC after a 30-minute incubation in hypotonic-low-chloride or isotonic buffer, with either KCN or KCl treatment. The mock condition has been incubated in hypotonic-low-chloride buffer as well. (D–E) Densitometry analysis of pNCC band intensity, and pNCC/tNCC ratio (n=3 duplicates in each experiment). Significance was assessed with unpaired t tests and corrected for multiple testing. pNCC, NCC phosphorylated at Thr60. **P<0.005; ****P<0.00005.