ENaC inhibition stimulates Cl- secretion in the mouse cortical collecting duct through an NKCC1-dependent mechanism

Abstract

In cortical collecting ducts (CCDs) perfused in vitro, inhibiting the epithelial Na(+) channel (ENaC) reduces Cl(-) absorption. Since ENaC does not transport Cl(-), the purpose of this study was to determine how ENaC modulates Cl(-) absorption. Thus, Cl(-) absorption was measured in CCDs perfused in vitro that were taken from mice given aldosterone for 7 days. In wild-type mice, we observed no effect of luminal hydrochlorothiazide on either Cl(-) absorption or transepithelial voltage (V(T)). However, application of an ENaC inhibitor [benzamil (3 μM)] to the luminal fluid or application of a Na(+)-K(+)-ATPase inhibitor to the bath reduced Cl(-) absorption by ∼66-75% and nearly obliterated lumen-negative V(T). In contrast, ENaC inhibition had no effect in CCDs from collecting duct-specific ENaC-null mice (Hoxb7:CRE, Scnn1a(loxlox)). Whereas benzamil-sensitive Cl(-) absorption did not depend on CFTR, application of a Na(+)-K(+)-2Cl(-) cotransport inhibitor (bumetanide) to the bath or ablation of the gene encoding Na(+)-K(+)-2Cl(-) cotransporter 1 (NKCC1) blunted benzamil-sensitive Cl(-) absorption, although the benzamil-sensitive component of V(T) was unaffected. In conclusion, first, in CCDs from aldosterone-treated mice, most Cl(-) absorption is benzamil sensitive, whereas thiazide-sensitive Cl(-) absorption is undetectable. Second, benzamil-sensitive Cl(-) absorption occurs by inhibition of ENaC, possibly due to elimination of lumen-negative V(T). Finally, benzamil-sensitive Cl(-) flux occurs, at least in part, through transcellular transport through a pathway that depends on NKCC1.

Fig. 1.

Hydrochlorothiazide (HCTZ) does not reduce Cl⁻ absorption in the cortical collecting duct (CCD) of aldosterone (Aldo)-treated mice. Transepithelial Cl⁻ flux (J_Cl; left) and transepithelial voltage (V_T; right) were measured simultaneously in CCDs from Aldo-treated mice (treatment 1) in either the presence or absence of 100 μM HCTZ in the luminal fluid. ANG II (10⁻⁸ M) was present in the bath solution. Each solid line displays data from a single experiment. Open squares show means ± SE of all experiments. NS, not significant.

Fig. 2.

Benzamil reduces Cl⁻ absorption in the CCD of Aldo-treated mice. J_Cl (left) and V_T (right) were measured simultaneously in CCDs from Aldo-treated mice (treatment 1) in either the presence or absence of 3 μM benzamil in the luminal fluid. ANG II (10⁻⁸ M) was present in the bath. Solid lines indicate results from experiments where the vehicle was applied in the first period and benzamil was applied in the second period. Dashed lines indicate experiments where the order was reversed (i.e., benzamil was applied in the initial period and then removed in the second period). Open squares show means ± SE under each condition.

Fig. 3.

Na⁺-K⁺-ATPase inhibition reduces J_Cl and V_T. CCDs from Aldo-treated mice (treatment 1) were perfused with ANG II (10⁻⁸ M) in the bath. J_Cl (left) and V_T (right) were measured in the presence of vehicle. In separate tubules, J_Cl and V_T were measured when the Na⁺-K⁺-ATPase inhibitor ouabain (2 mM) was added to the bath. *P < 0.05.

Fig. 4.

Ablation of the gene encoding the α-subunit of the epithelial Na⁺ channel (ENaC) eliminates benzamil-sensitive J_Cl and V_T in the CCD. The effect of benzamil (3 μM) on J_Cl and V_T was examined in CCDs taken from Aldo-treated collecting duct-specific ENaC-null mice (B; Hoxb7:CRE, Scnn1a^loxlox) and wild-type, CRE-negative littermates (A) (treatment 1). C: time control experiments performed in collecting duct-specific ENaC-null mice. In all experiments, vehicle was applied in the first period and benzamil (or vehicle) was applied in the second period. ANG II was present in the bath in all experiments. The solid lines display data from a single experiment. Open squares show means ± SE of all experiments.

Fig. 5.

Benzamil increases Cl⁻ secretion. CCDs taken from mice that received a high-NaCl [high-salt (HS)] diet (treatment 3) were perfused in vitro in the absence of ANG II. Under basal conditions (vehicle), J_Cl was very low. With the application of benzamil to the perfusate, Cl⁻ secretion was observed. Solid lines display results from experiments in which the vehicle was applied in the first period and benzamil was applied in the second period. Dashed lines indicate experiments where the order was reversed (i.e., benzamil was applied in the initial period and then removed in the second period). Open squares show means ± SE under each condition.

Fig. 6.

Na⁺-K⁺-2Cl⁻ cotransporter 1 (NKCC1) localizes to the basolateral regions of intercalated cells in CCDs from Aldo-treated mice but not from vehicle-treated mice. Shown are micrographs of cortical sections labeled for NKCC1 that were taken from mice given Aldo or vehicle (treatments 1 or 2). No NKCC1 labeling was noted in CCDs taken from vehicle-treated mice (A and C). However, in mice given Aldo, NKCC1 label was observed in the basolateral regions of a minority subtype within the CCD (B and D, arrows). C and D are higher-magnifications of the boxed regions in A and B, respectively.

Fig. 7.

NKCC1 localizes to the basolateral regions of intercalated cells in the outer medullary collecting duct (OMCD) in Aldo-treated mice but not in vehicle-treated mice. Shown are sections of the outer medulla from mice given Aldo or vehicle (treatments 1 or 2) that were labeled for NKCC1. Areas in the boxes of A and B are shown at higher power in C and D. No NKCC1 labeling was noted in OMCDs taken from vehicle-treated mice (A and C). However, in mice given Aldo, NKCC1 label was observed in the basolateral regions of a minority cell type within the OMCD (B and D, arrows).

Fig. 8.

NKCC1 localizes to the basolateral regions of type A intercalated cells in the mouse CCD and OMCD. A and B: sections of the cortex (A) and outer medulla (B) from Aldo-treated mice (treatment 1) were labeled for NKCC1 (brown) and both pendrin (Pds) and aquaporin 2 (AQP2; blue-gray). NKCC1 labeling (arrows) was observed in the basolateral regions of cells within the CCD and OMCD that did not label for either pendrin or APQ2. C: NKCC1 labeling (brown) and H⁺-ATPase labeling (blue-gray) in sections of the outer medulla from Aldo-treated mice. NKCC1 labeling was observed in cells that express H⁺-ATPase (arrows). Thus, NKCC1 localizes to type A intercalated cells in the CCD and OMCD of Aldo-treated mice.

Fig. 9.

Possible role of NKCC1 in Cl⁻ transport in the mouse CCD. The transporters present in the CCD are shown (4, 11, 14, 46). In the CCD of Aldo-treated mice, Cl⁻ secretion may be through NKCC1-mediated Cl⁻ uptake across the basolateral membrane of type A intercalated cells in tandem with an apical Cl⁻ conductive pathway. This apical conductive pathway should be stimulated when the lumen-negative voltage is abolished with amiloride analogs, such as benzamil.

Fig. 10.

When ENaC is inhibited, Na⁺-K⁺-2Cl⁻ cotransport blockade increases Cl⁻ absorption. CCDs taken from Aldo-treated mice (treatment 1) were perfused with ANG II (10⁻⁸ M) in the bath. A and B: J_Cl and V_T were measured in the absence (A) or presence (B) of benzamil (3 μM) in the luminal fluid. Solid lines display results from experiments where the vehicle was applied in the first period and benzamil was applied in the second period. Dashed lines indicate experiments where the order was reversed (i.e., benzamil was applied in the initial period and then removed in the second period). Open squares show means ± SE of data under each condition.

Fig. 11.

Effect of bumetanide on benzamil-sensitive J_Cl and V_T. The effects of benzamil (3 μM) on J_Cl (left) and V_T (right) were measured in CCDs from Aldo-treated mice (treatment 1) that were perfused with bumetanide (100 μM) with ANG II (10⁻⁸ M) present in the bath solution. Solid lines display results from experiments where the vehicle was applied in the first period and benzamil was applied in the second period. Dashed lines indicate experiments where the order was reversed (i.e., benzamil was applied in the initial period and then removed in the second period). Open squares show means ± SE of data under each condition.

Fig. 12.

Effect of bumetanide (Bumet) on benzamil-sensitive J_Cl and V_T. A and B: benzamil-sensitive changes in J_Cl (A) and V_T (B) taken from Aldo-treated mice (treatment 1) are shown. C: ratio of benzamil-sensitive J_Cl to benzamil-sensitive V_T (ΔJ_Cl/ΔV_T). Data from Figs. 2 (n = 10), 4A (n = 3), and 9 (n = 9) were included.

Fig. 13.

Benzamil-sensitive J_Cl is blunted in NKCC1-null mice. J_Cl (left) and V_T (right) were measured in CCDs from Aldo-treated NKCC1-null mice (treatment 1) before and after the application of benzamil (3 μM) to the luminal fluid. ANG II (10⁻⁸ M) was present in the bath. Solid lines display results from experiments where the vehicle was applied in the first period and benzamil was applied in the second period. The dashed line indicates an experiment where the order was reversed (i.e., benzamil was applied in the initial period and then removed in the second period). Open squares show means ± SE of data under each condition.

Fig. 14.

CFTR inhibition does not significantly alter J_Cl. CCDs from Aldo-treated mice (treatment 1) were perfused with benzamil (3 μM) present in the perfusate with ANG II (10⁻⁸ M) present in the bath. J_Cl (left) and V_T (right) were measured before and after the application of CFTR inhibitor [CTFR_ihn 172 (5 μM)] to the perfusate. The dashed lines show means ± SE.