Nitric oxide reduces Cl⁻ absorption in the mouse cortical collecting duct through an ENaC-dependent mechanism

Abstract

Since nitric oxide (NO) participates in the renal regulation of blood pressure, in part, by modulating transport of Na⁺ and Cl⁻ in the kidney, we asked whether NO regulates net Cl⁻ flux (J_Cl) in the cortical collecting duct (CCD) and determined the transporter(s) that mediate NO-sensitive Cl⁻ absorption. Cl⁻ absorption was measured in CCDs perfused in vitro that were taken from aldosterone-treated mice. Administration of an NO donor (10 μM MAHMA NONOate) reduced J_Cl and transepithelial voltage (V_T) both in the presence or absence of angiotensin II. However, reducing endogenous NO production by inhibiting NO synthase (100 μM N^G-nitro-l-arginine methyl ester) increased J_Cl only in the presence of angiotensin II, suggesting that angiotensin II stimulates NO synthase activity. To determine the transport process that mediates NO-sensitive changes in J_Cl, we examined the effect of NO on J_Cl following either genetic ablation or chemical inhibition of transporters in the CCD. Since the application of hydrochlorothiazide (100 μM) or bafilomycin (5 nM) to the perfusate or ablation of the gene encoding pendrin did not alter NO-sensitive J_Cl, NO modulates J_Cl independent of the Na⁺-dependent Cl⁻/HCO₃⁻ exchanger (NDCBE, Slc4a8), the A cell apical plasma membrane H⁺-ATPase and pendrin. In contrast, both total and NO-sensitive J_Cl and V_T were abolished with application of an epithelial Na⁺ channel (ENaC) inhibitor (3 μM benzamil) to the perfusate. We conclude that NO reduces Cl⁻ absorption in the CCD through a mechanism that is ENaC-dependent.

aldosterone and angiotensin II increase NaCl absorption in the cortical collecting duct (CCD) by stimulating transporters such as epithelial Na⁺ channel (ENaC) and pendrin (12, 21, 24, 28, 39, 40), which increases NaCl absorption, thereby contributing to the hypertension expected following treatment with these hormones (41, 44).

In vascular and in renal tissue, many of the effects of angiotensin II and aldosterone are mediated by changes in O₂^·−/H₂O₂ or nitric oxide (NO) (1, 11, 19, 23, 48, 49). In kidney cells, aldosterone stimulates ENaC activity, in part, by increasing O₂^·− production and by reducing NO bioavailability (52). With increased NO bioavailability, a natriuresis and diuresis are observed (20) due to inhibition of renal Na⁺ transporters, such as ENaC (33). However, NO may also reduce Cl⁻ absorption by inhibiting pendrin since NO reduces HCO₃⁻ secretion in rabbit CCD (37) and since HCO₃⁻ secretion occurs largely through pendrin-dependent transport (31). Thus, the purpose of this study was to determine whether NO reduces Cl⁻ absorption in the mouse CCD and to explore the transport mechanism(s) by which this occurs.

METHODS

Animals.

All experiments were performed on male and female Slc26a4 ^−/− (or Pds^−/−) mice developed by Everett et al. (4) and in wild-type mice from the same strain (129S6/SvEv Tac; Taconic Farms), which were bred in parallel. Every third generation wild-type and pendrin-null mice were interbred to produce heterozygotes. These heterozygotes were interbred to generate new wild-type and homozygous pendrin-null breeders (25).

We generated mice that were both homozygous pendrin-null and homozygous for the Liddle's mutation (29) (Pds^−/−& L/L), which constitutively upregulates the ENaC. To do so, mice homozygous for the Liddle's mutation (29) on a C57Bl6 background were bred with homozygous pendrin-null mice (4) on a 129S6SvEvTac background. The progeny were backcrossed onto a 129 S6SvEvTac background over at least 10 generations. Mouse genotype was determined from tail biopsies using real-time PCR with specific probes designed for each gene (Transnetyx, Cordova, TN).

For 5–7 days before death, mice ate a balanced diet (53881300; Zeigler Brothers) prepared as a gel (0.6% agar, 74.6% water, and 24.8% mouse chow) supplemented with NaCl (∼0.8 meq NaCl/day) (12) and were treated with aldosterone by minipump (250 μg·kg body wt⁻¹·day⁻¹). The Institutional Animal Care and Use Commmittee at Emory University approved all treatment protocols.

In vitro perfusion of isolated CCDs.

CCDs were dissected from medullary rays and perfused at flow rates of 2–3 nl/min in the presence of symmetric, physiological solutions containing (in mmol/l) 125 NaCl, 2.5 K₂HPO₄, 24 NaHCO₃/5% CO₂, 2 CaCl₂, 1.2 MgSO₄, and 5.5 glucose bubbled with 95% air-5% CO₂ (31). Unless otherwise stated, 10 nM angiotensin II was present in the bath solution (24). Tubules were equilibrated at 37°C for 30 min before the collections were started. Stock solutions of benzamil hydrochloride (3 × 10⁻³ M) were prepared in deionized water. Hydrochlorothiazide stock solutions (10⁻¹ M) were prepared in DMSO, whereas stock solutions of bafilomycin (10⁻⁵ M) were prepared in absolute ethanol. All chemicals were purchased from Sigma (St. Louis, MO). MAHMA NONOate was purchased from Axxora (San Diego, CA). Stock solutions of 20 mM MAHMA were prepared in 0.01 M NaOH and used within 24 h. Uric acid was prepared in 1 M NaOH, diluted 1:1,000, and then titrated to pH 7.4 with 1 M HCl. Stock solutions of tempol and N^G-nitro-l-arginine methyl ester (l-NAME) were made in deionized water and diluted 1:1,000.

Measurement of net transepithelial Cl⁻ flux.

Cl⁻ concentration was measured in perfusate and collected samples using a continuous-flow fluorimeter and the Cl⁻-sensitive fluorophore 6 methoxy-N-(3-sulfopropyl) quinolinium (Molecular Probes, Eugene, OR), as described previously (5, 43, 44). Transepithelial Cl⁻ flux (J_Cl) was calculated according to the equation: J_Cl = (C_o − C_L)Q/L, where C_o and C_L are perfusate and collected fluid Cl⁻ concentrations, respectively. Q is flow rate in nanoliters per minute. L is tubule length. Net fluid transport was taken to be 0 since net fluid flux was observed in CCDs when perfused in vitro in the presence of symmetric solutions and in the absence of vasopressin (13, 14). J_Cl was expressed in picomoles per millimeter per minute.

Transepithelial voltage (V_T) was measured in the perfusion pipette connected to a high-impedance electrometer through an agar bridge saturated with 0.16 M NaCl and a calomel cell as described previously (42). The reference was an agar bridge from the bath to a calomel cell.

Statistics.

All data are presented as means ± SE. Data displayed usually represented a single measurement. Occasionally, data from two collections were averaged to obtain a single value. Each “n” used in the statistical analysis represents data from separate mice. To test for statistical significance between two groups, a paired or an unpaired Student's t-test was used, as appropriate. The criterion for statistical significance was P < 0.05.

RESULTS

NO reduces Cl⁻ absorption.

We examined the effect of NO donors on Cl⁻ absorption in CCDs from aldosterone-treated mice that were perfused in vitro with angiotensin II (10 nM) in the bath solution. Aldosterone was applied in vivo and angiotensin II was applied in vitro to upregulate the ENaC (21, 28), the H⁺-ATPase (27, 50), and pendrin (24, 39). As shown, while 1 μM MAHMA NONOate did not alter J_Cl or V_T (Fig. 1, A and B), application of 10 μM MAHMA NONOate reduced J_Cl and V_T by ∼50% (Fig. 1, C and D). This fall in J_Cl and V_T was not due to time-dependent changes (Fig. 1, E and F). To determine whether MAHMA reduces Cl⁻ absorption through a breakdown product of MAHMA, Cl⁻ absorption was measured before and after the addition of MAHMA that had been allowed to decay (Fig. 1, G and H). As shown, decayed MAHMA did not change J_Cl or V_T. We conclude that MAHMA reduces Cl⁻ absorption through a direct effect of NO.

Fig. 1.

Nitric oxide (NO) donors reduce Cl⁻ absorption (J_Cl) and transepithelial voltage (V_T). J_Cl and V_T were measured in cortical collecting ducts (CCDs) from aldosterone-treated mice before and after the application of the NO donor {MAHMA NONOate, (Z)-1-N-methyl-N-[6-(N-methylammoniohexyl)amino]-diazen-1-ium-1,2-diolate} at concentrations of 1 μM (A and B) or 10 μM (C and D). Angiotensin II (10 nM) was present in the bath solution. E and F: display time control experiments in the absence of MAHMA NONOate. G and H: display J_Cl and V_T when measured before and after application of 10 μM MAHMA NONOate that had been allowed to decay. To do so, MAHMA NONOate was incubated at 22°C for 60 min before use. Solid lines show individual tubules. Dashed lines show means ± SE. NS, not significant.

Further experiments asked whether reducing endogenous production of NO alters Cl⁻ absorption. Thus, Cl⁻ absorption was measured in the presence and absence of the NO synthase (NOS) inhibitor l-NAME (100 μM; Fig. 2) with angiotensin II in the bath solution. As shown, Cl⁻ absorption increased ∼30% with l-NAME application, although V_T did not change significantly.

Fig. 2.

Inhibiting endogenous NO synthases increases J_Cl. J_Cl (A) and V_T (B) were measured in CCDs from aldosterone-treated mice before and after application of the NO synthase inhibitor l-NAME (100 μM) to the bath. Angiotensin II (10 nM) was present in the bath solution throughout the experiment. Solid lines show individual tubules. Open squares show means ± SE.

Angiotensin II increases NO production in vascular tissue by stimulating NOS activity (7, 10, 30, 32) and stimulates NADPH oxidase activity, thereby increasing the production of O₂^·− (17, 34). Since impaired O₂^·−/NO balance in the CCD may alter the response to NO donors or NOS inhibitors, we examined the effect of NO donors and NOS inhibitors on Cl⁻ absorption in the absence of angiotensin II. As shown (Fig. 3), following NO donor (10 μM MAHMA) application, Cl⁻ absorption fell 64% (n = 3, P < 0.05), while V_T fell 47% (n = 3, P < 0.05) similar to the effects observed in the presence of angiotensin II. Therefore, NO donors modulate J_Cl both in the presence and in the absence of angiotensin II. In contrast, while NOS inhibition increased Cl⁻ absorption in the presence of angiotensin II (Fig. 2), in the absence of angiotensin II, NOS inhibition (100 μM l-NAME; Fig. 4) did not change Cl⁻ absorption. We conclude that Cl⁻ flux is much more sensitive to NOS inhibition following angiotensin II application in vitro, likely because angiotensin II increases NO production by stimulating NOS activity.

Fig. 3.

Removing angiotensin II from the bath does not alter the effect of NO donors on J_Cl. J_Cl (A) and V_T (B) were measured in CCDs from aldosterone-treated mice before and after the application of an NO donor (10 μM MAHMA NONOate) when angiotensin II was not present in the bath. In the absence of angiotensin II, application of MAHMA reduced J_Cl from 62.2 ± 5.8 to 22.2 ± 3.8 pmol·mm⁻¹·min⁻¹, and reduced V_T from −28.4 ± 2.6 to −14.9 ± 2.9 mV, respectively.

Fig. 4.

NO synthase inhibition does not increase J_Cl when angiotensin II is removed from the bath. J_Cl (A) and V_T (B) were measured in CCDs from aldosterone-treated mice before and after the application of the NO synthase inhibitor (100 μM l-NAME) without angiotensin II in the bath. In the absence of angiotensin II, application of l-NAME did not alter J_Cl or V_T. Solid lines show individual tubules. Open squares show means ± SE.

Since superoxide (O₂^·−) is involved in the breakdown of endothelium-derived vascular relaxing factor (NO) (6), we explored the effect of reducing O₂^·− through the application of a superoxide dismutase mimetic (50 μM tempol) to the bath. As shown (Fig. 5), tempol did not reduce Cl⁻ absorption in CCDs from aldosterone-treated mice, when perfused either in the presence or in the absence of angiotensin II in the bath solution. To exclude the possibility that tempol failed to reduce Cl⁻ absorption because of limited intracellular accumulation, we examined the effect of tempol in an unpaired experiment, in which CCDs were exposed to tempol for a longer period of time (i.e., at least 30 min) before each collection was started. Tubules were perfused in the presence of angiotensin II to stimulate O₂^·− production. Although Cl⁻ absorption was numerically lower with tempol present in the bath, the difference did not reach statistical significance (Fig. 5). We conclude that O₂^·− plays a limited role in regulating Cl⁻ absorption in CCDs from aldosterone-treated mice.¹

Fig. 5.

Superoxide dismutase mimetics do not reduce J_Cl. CCDs from aldosterone-treated mice were perfused in vitro in the presence (A and C) or in the absence (B) of angiotensin II (10 nM) in the bath. A and B: J_Cl was measured in individual tubules before and then after the application of the superoxide mimetic tempol (4-hydroxy 2,2,6,6,-tetramethyl piperidinoxyl; 50 μM) to the bath. Solid lines show individual tubules. Dashed lines show experiments conducted in reverse order (i.e., tempol was applied in the first period and then removed in the second). Open squares show means ± SE. C: J_Cl was measured in separate tubules after at least 30-min incubation with tempol (50 μM) or vehicle present in both the bath and luminal solution. The bar graph shows means ± SE.

Since angiotensin II application in vitro increases the sensitivity of Cl⁻ transport to NOS inhibition, further experiments were performed with angiotensin II present in the bath solution.

NO does not reduce Cl⁻ absorption through the action of ONOO⁻.

NO reacts with O₂^·− (superoxide) to form ONOO⁻, which is a highly oxidative and nitrosylating compound (49). We hypothesized that if NO reduces Cl⁻ absorption through ONOO⁻, ONOO⁻ donors should mimic the effect of NO on J_Cl. Therefore, we examined the effect of an ONOO⁻ donor (SIN-1) (15) on J_Cl and V_T. As shown (Fig. 6, A and B), Cl⁻ absorption and V_T were unchanged with SIN-1 application. These data suggest that the fall in Cl⁻ absorption observed with the NO donor MAHMA does not occur through the formation of ONOO⁻.

Fig. 6.

NO does not reduce J_Cl in the CCD through the action of ONOO⁻. A and B: J_Cl and V_T were measured before and after the application of the ONOO⁻ donor SIN-1 [5-amino-3-(4-morpholinyl)-1,2,3,-oxadiazolium chloride; 1 mM] to the bath solution. C and D: CCDs were perfused in vitro with uric acid (100 μM) present in the perfusate and the bath solution. J_Cl and V_T were measured before and after the application of MAHMA (10 μM). Angiotensin II (10 nM) was present in the bath in all experiments. Solid lines show individual tubules. Dashed lines show experiments conducted in reverse order (i.e., MAHMA was applied in the first period and then removed in the second). Open squares show means ± SE.

We further hypothesized that if NO reduces Cl⁻ absorption through the action of ONOO⁻, then ONOO⁻ scavengers should blunt the effect of NO on Cl⁻ absorption. To test this hypothesis, we examined the effect of MAHMA on J_Cl in CCDs that were perfused in vitro in the presence of uric acid, which is an effective ONOO⁻ scavenger (15). As shown, uric acid application did not prevent the fall in Cl⁻ absorption or V_T observed with NO donor (MAHMA) application (Fig. 6, C and D) (15). We conclude that NO does not reduce J_Cl through the action of ONOO⁻.

NO reduces Cl⁻ absorption through a benzamil-sensitive mechanism that does not involve the H⁺-ATPase or the Na⁺-dependent Cl⁻/HCO₃⁻ exchanger (Slc4a8).

Further experiments explored the transport pathways mediating NO-sensitive Cl⁻ absorption. Three transport inhibitors modulate Cl⁻ absorption in the CCD. Cl⁻ absorption falls in the CCD 1) when the basolateral plasma membrane H⁺-ATPase is inhibited with bafilomycin (24), 2) when the apical Na⁺-dependent Cl⁻/HCO₃⁻ exchanger encoded by Slc4a8 is inhibited with hydrochlorothiazide (18), and 3) when the apical plasma membrane ENaC is blocked with amiloride analogs (26).

The apical plasma membrane of type A intercalated cells mediates H⁺ secretion by way of the H⁺-ATPase, which may increase the driving force for apical Cl⁻/HCO₃⁻ exchange by reducing luminal HCO₃⁻ concentration. Since NO reduces H⁺-ATPase activity (36), we hypothesized that NO reduces Cl⁻ absorption by inhibiting the H⁺-ATPase. If so, the fall in Cl⁻ absorption observed with NO donor application should be eliminated with H⁺-ATPase blockade. To test this hypothesis, we examined the effect of NO on J_Cl and V_T with an H⁺-ATPase inhibitor (bafilomycin) present in the perfusate (Fig. 7, A and B). As shown, luminal bafilomycin did not eliminate MAHMA-induced changes in J_Cl and V_T. We conclude that MAHMA does not reduce J_Cl by inhibiting the apical plasma membrane H⁺-ATPase.

Fig. 7.

NO donors do not reduce J_Cl through an effect on the H⁺-ATPase. J_Cl and V_T were measured in CCDs from aldosterone-treated wild-type mice with angiotensin II (10 nM) in the bath solution. A and B: J_Cl and V_T measured before and after MAHMA application to the bath (10 μM) when a H⁺-ATPase inhibitor [5 nM bafilomycin (Baf)] was present in the perfusate in both periods. C and D: effect of bafilomycin (5 nM) on J_Cl and V_T when applied to the perfusate. Solid lines show individual tubules. Dashed lines show individual experiments conducted in reverse order. Open squares show means ± SE. Veh, vehicle.

Bafilomycin might not modify the effect of NO donors on J_Cl either because the H⁺-ATPase is not sensitive to this NO donor or because the H⁺-ATPase does not play a significant role in Cl⁻ absorption in this treatment model. To resolve this question, we measured Cl⁻ absorption in the CCD, in the presence and absence of the H⁺-ATPase inhibitor bafilomycin (5 nM) in the perfusate. As shown (Fig. 7, C and D), bafilomycin did not change J_Cl or V_T. We therefore conclude that inhibiting the apical H⁺-ATPase does not significantly alter Cl⁻ absorption in CCDs from aldosterone-treated mice.

In the CCD of NaCl-restricted mice, Cl⁻ absorption occurs through a thiazide-sensitive Na⁺-dependent Cl⁻/HCO₃⁻ exchanger encoded by Slc4a8 (18). Therefore, we asked whether thiazides attenuate the NO-sensitive component of Cl⁻ absorption in CCDs from aldosterone-treated mice. To test this hypothesis, we examined the effect of MAHMA on Cl⁻ absorption with hydrochlorothiazide (100 μM) in the perfusate. Figure 8 demonstrates that thiazides do not block the fall in Cl⁻ absorption and V_T observed with the application of NO donors. Because thiazides do not eliminate NO-sensitive changes in J_Cl, Slc4a8 is not likely a significant target of NO in CCDs from aldosterone-treated mice. These observations are in agreement with a recent study demonstrating that the thiazide-sensitive component of Cl⁻ absorption is very low in CCDs taken from mice following aldosterone treatment (26).

Fig. 8.

NO donors reduce J_Cl through a thiazide-insensitive pathway. CCDs from aldosterone-treated wild-type mice were perfused in vitro. Throughout the experiments, hydrochlorothiazide (HCTZ; 100 μM) was present in the perfusate and angiotensin II (10 nM) was present in the bath. A and B: J_Cl and V_T were measured before and after MAHMA application to the bath (10 μM). Solid lines show individual tubules. Dashed lines show individual experiments conducted in reverse order. Open squares show means ± SE.

To determine whether NO targets ENaC-dependent Cl⁻ absorption, we tested whether NO donors modulate the benzamil-sensitive component of Cl⁻ absorption. We observed that with an ENaC inhibitor (benzamil) in the perfusate, Cl⁻ absorption and V_T are low and unchanged with MAHMA application (Fig. 9, A and B). Moreover, in the presence of an ENaC inhibitor (benzamil), NOS inhibitors (l-NAME) did not stimulate Cl⁻ absorption or V_T (Fig. 9, C and D). We conclude that NO reduces Cl⁻ absorption through a benzamil-sensitive pathway that depends on the epithelial Na⁺ channel.

Fig. 9.

NO targets the benzamil-sensitive component of J_Cl. CCDs from aldosterone-treated wild-type mice were perfused in vitro with benzamil (3 μM) in the perfusate. A and B: J_Cl and V_T were measured in CCDs before and after MAHMA (10 μM) application to the bath. C and D: J_Cl and V_T were measured before and after the application of l-NAME (100 μM) to the bath. Solid lines show individual tubules. Dashed lines show individual experiments conducted in the reverse order. Open squares show means ± SE. Angiotensin II (10 nM) was present in the bath solution in all experiments.

NO does not reduce Cl⁻ absorption through a mechanism dependent on the Cl⁻/HCO₃⁻ exchanger pendrin.

Since pendrin mediates HCO₃⁻ secretion (31) and since HCO₃⁻ secretion is reduced with NO (38), we asked whether pendrin gene ablation alters NO-sensitive Cl⁻ absorption. To answer this question, we tested the effect of pendrin gene ablation (Slc26a4) on NO-sensitive Cl⁻ absorption. As shown, pendrin gene ablation did not eliminate the increase in Cl⁻ absorption observed with NOS inhibitor (100 μM l-NAME; Fig. 10) application. These data suggest that NO regulates J_Cl independent of pendrin. In contrast, pendrin gene ablation eliminated the fall in Cl⁻ absorption observed with NO donor administration (Fig. 11, A and B). Why Cl⁻ absorption changed with the application of a NOS inhibitor but not with an NO donor could be explained by two alternative hypotheses. If NO directly targets pendrin-mediated Cl⁻ absorption, NO donors might not reduce Cl⁻ absorption because the targeted transporter (pendrin) is absent in these mutant mice. However, if NO directly targets benzamil-sensitive Cl⁻ absorption, rather than pendrin-mediated transport, NO donors would not reduce Cl⁻ absorption because ENaC is already markedly downregulated in kidneys from pendrin-null mice (12), and therefore could not be downregulated much farther. To resolve this issue, we examined the effect of MAHMA on J_Cl in CCDs from mice that are homozygous for both the pendrin-null and the Liddle's mutation (Pds^−/−& L/L; Fig. 11, C and D). With the introduction of the Liddle's mutation, ENaC is constitutively upregulated in the pendrin-null mice (3). As shown, in CCDs from mice that are homozygous for both the pendrin-null and the Liddle's mutations (Pds^−/− & L/L), MAHMA reduced J_Cl and V_T. Thus, pendrin gene ablation does not eliminate NO-sensitive J_Cl and V_T. We conclude that NO does not inhibit J_Cl by directly targeting pendrin-mediated Cl⁻ absorption.

Fig. 10.

Pendrin gene ablation does not eliminate the increase in J_Cl observed with NO synthase inhibition. J_Cl was measured in CCDs from aldosterone-treated pendrin-null mice before and after the application of a NO synthase inhibitor (100 μM l-NAME) to the bath. Angiotensin II (10 nM) was present in the bath solution in all experiments. Solid lines show individual tubules. Open squares show means ± SE.

Fig. 11.

Pendrin gene ablation does not eliminate the fall in J_Cl observed with NO donor application. A and B: J_Cl and V_T were measured in CCDs from aldosterone-treated pendrin-null mice before and after the application of MAHMA (10 μM) to the bath. C and D: J_Cl and V_T were measured in CCDs from aldosterone-treated pendrin-null/Liddle's mice (Pds^−/−; L/L) before and after the application of MAHMA (10 μM) to the bath. Angiotensin II (10 nM) was present in the bath solution in all experiments. Solid lines show individual tubules. Dashed lines show individual experiments conducted in reverse order. Open squares show means ± SE.

DISCUSSION

NO is an important blood pressure regulator. With NO depletion, hypertension is observed in both rodents (16) and people (49). The present and previous studies showed that NO modulates blood pressure, in part, by targeting ENaC in the kidney. NO is produced through the action of NOS, which catalyze the oxidation of l-arginine to NO and l-citrulline (47, 51). Three NOS isoenzymes have been identified: neuronal NOS (nNOS, NOS 1), inducible NOS (iNOS, NOS II), and endothelial NOS (eNOS, NOS III) (47). eNOS (NOS III) expression has not been observed in the CCD. Within that segment, principal cells express nNOS (or NOS1) and ENaC (45), whereas type A and type B intercalated cells express iNOS, but do not express ENaC (2, 36). While the present study demonstrated that benzamil-sensitive Cl⁻ absorption is reduced by endogenous NO production, which NOS are responsible for this NO production remains to be determined. The ENaC might be targeted by NO produced in principal cells. However, NO produced in other cells might act on principal cells through a paracrine effect.

NO is a hydrophobic compound that partitions from the aqueous phase into biological membranes (22). By virtue of its biophysical properties, NO freely diffuses across artificial bilayers at a rate inversely proportional to the cholesterol content of the membrane (22). Since NO can be transported by water channels, such as aquaporin-1 (AQP1) (8, 9) and AQP4 (46), we cannot exclude the possibility that NO traverses cell membranes in the mouse CCD through a mechanism other than diffusion.

While NO might reduce Cl⁻ absorption in the CCD through NO·, NO⁺, ONOO⁻, or NO₂⁻, it is unlikely that MAHMA reduces Cl⁻ absorption through the action of NO₂⁻ since decayed MAHMA, which yields NO₂⁻ and NO₃⁻, had no effect on Cl⁻ absorption. Moreover, the present study shows that ONOO⁻ donors do not mimic the effect of NO on Cl⁻ absorption in the CCD. Instead, NO donors, such as MAHMA NONOate, most likely reduce Cl⁻ absorption through the action of NO· and/or NO⁺.

Our laboratory observed previously that in aldosterone-treated mice, Cl⁻ absorption and V_T are reduced by more than 50% with chemical inhibitors of the ENaC (26). ENaC-dependent changes in J_Cl do not occur exclusively through changes in paracellular Cl⁻ transport. Instead, ENaC blockade reduces Cl⁻ absorption, at least in part, by stimulating a Cl⁻ secretory pathway that is dependent on NKCC1 (26).

The present study demonstrates that in aldosterone-treated mice, benzamil-sensitive Cl⁻ transport is a major target of NO. Therefore, NO reduces amiloride (benzamil)-sensitive Na⁺ (33) and Cl⁻ absorption in the CCD. These data raise the possibility that the NO depletion that follows aldosterone administration contributes to the increase in benzamil-sensitive transport observed during this treatment model.

While NO does not modulate pendrin-dependent Cl⁻ absorption in vitro, NO modulates pendrin total protein abundance in vivo and in vitro (35, 40). We observed that in NaCl-restricted mice, the angiotensin type 1 receptor modulates pendrin protein abundance through a mechanism that is dependent on NO (40). The present study shows that while NO reduces Cl⁻ absorption in the CCD, it does so independent of pendrin. Thus, NO changes the number of pendrin transporters, and possibly pendrin-mediated Cl⁻ transport, in a span of hours to days, whereas NO changes pendrin-independent, ENaC-dependent Cl⁻ absorption within 10 min.

In summary, NO reduces benzamil-sensitive Cl⁻ absorption in the mouse CCD. The downstream signaling cascade mediating NO-induced ENaC inhibition remains to be determined.

GRANTS

This work was supported by Grant DK 46493 (to S. M. Wall) and by the American Society of Nephrology Career Development Award no. 145596 (to V. Pech).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

Author contributions: V.P., S.I.D., D.G.H., and S.M.W. conception and design of research; V.P. and M.T. performed experiments; V.P. and S.M.W. analyzed data; V.P., S.I.D., D.G.H., and S.M.W. interpreted results of experiments; V.P. and S.M.W. prepared figures; V.P. and S.M.W. drafted manuscript; V.P. and S.M.W. edited and revised manuscript; V.P., M.T., S.I.D., E.H., B.C.R., D.G.H., and S.M.W. approved final version of manuscript.