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. 2010 Apr 12;120(5):1627–1635. doi: 10.1172/JCI40145

Figure 7. Effects of HCTZ (10–4 M) on NDCBE or PDS activity in isolated collecting ducts or on recombinant NDCBE or PDS expressed in Xenopus oocytes.

Figure 7

(A) Effects of HCTZ and Ndcbe disruption on Na+-dependent pHi changes measured in intercalated cells of CCD isolated from Na-depleted Ndcbe–/– or Ndcbe+/+ mice fed a low-Na+ diet. Traces are the average of pHi changes recorded when luminal Na+ was removed and then readded, in the presence of extracellular Cl– (122 mM) and HCO3– (25 mM). Intracellular Na+-dependent acidification was detected in Ndcbe+/+ mice but absent in Ndcbe–/– mice or when HCTZ 10–4 M was present in the perfusate. In these 3 different experimental conditions, mean starting pHi values were 7.10 ± 0.02, 6.93 ± 0.11, and 7.01 ± 0.04, respectively. (B) Effects of HCTZ on apical Cl–/HCO3– exchange activity in intercalated cells of CCDs isolated from Na-depleted animals. Traces are the average of pHi changes recorded when luminal Cl– was removed and then readded, in the presence of extracellular HCO3– (25 mM) and in Na+-free solutions. Intracellular Cl–-dependent alkalinization, reflecting apical Cl–/HCO3– exchange, was completely abolished when 10–4 M HCTZ was present in the perfusate. Mean starting pHi values (immediately before Cl– removal) were 6.91 ± 0.03 and 6.89 ± 0.08, in the absence and presence of HCTZ, respectively. (C) Effects of HCTZ on mNdcbe-mediated HCO3– influx. Oocytes had been injected with mNdcbe cRNA or H2O and incubated with HCTZ (0.25 mM). As a control, NDCBE-expressing and H2O-injected oocytes were incubated with vehicle (methanol). Values are mean ± SEM with 6–9 oocytes per group. **P < 0.01, ***P < 0.001 versus H2O-injected oocyte. HCO3– flux was unaffected by the application of HCTZ (0.25 mM) compared with vehicle alone (P = 0.279). (D) Effects of HCTZ on Pds-mediated 36Cl– uptake. Pendrin-expressing oocytes (mPds) were incubated in ND96 containing 0.1 or 1 mM HCTZ during the uptake period (16 minutes). As a control, pendrin-expressing and H2O-injected oocytes were incubated with vehicle (methanol). Values are mean ± SEM, with 6–16 oocytes per group. *P < 0.001 versus H2O; †P < 0.001 versus mPds, HCTZ 0.1 mM.