Figure 7: Effects of SPAK or ClC-K knockdown on NCC and intracellular chloride.

(a) SPAK knockdown using siRNA (siSPAK) prevented the up-regulation of NCC & p-NCC in TK-treated mDCTs. Total lysate protein loading was normalized by GAPDH. n=4–6 in each group. Data are means±s.e. *P<0.01 versus Control; #P<0.01 versus sham+TK (ANOVA). (b) Membrane expression of ClC-K was detected by western blot. The binding of membrane ClC-K to its subunit barttin was detected by immunoprecipitation (IP) of barttin and immunoblot (IB) of ClC-K. Membrane protein loading for both western blot and IP/IB were normalized to Na-K-ATPase (see Methods). n=4–6 in each group. Data are means±s.e. *P<0.01 versus Control (t-test). (c) ClC-K knockdown using siRNAs (siClC-K) prevented the reduction of intracellular chloride in TK-treated mDCTs. Intracellular chloride measured with MQAE. The difference between PBS and HCTZ+Bume of each line reflects NCC/NKCC compensated chloride efflux in each group of mDCTs. TK-induced chloride efflux (in presence of HCTZ+Bume) was compared between the groups without siClC-K (Sham-si, Dashed lines) and with siClC-K (solid lines). Data are means±s.e. n=6–10 in each group. P<0.01 (t-test). (d) NCC-mediated sodium uptake in control or TK-treated mDCTs with or without ClC-K knockdown was measured using the same method as in Fig. 5. Data are representative of nine (sham-si) to ten (siClC-K) experiments. (e) TK-induced change (Δ) in the proportion of cells with elevated sodium. n=9–10 groups. Data are means±s.e. P<0.01 (t-test).