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Differential Regulation of Collecting Duct Na⁺,K⁺-ATPase and K⁺ Excretion by Furosemide and Piretanide: Role of Bradykinin

Bénédicte Buffin-Meyer^*,, Mauricio Younes-Ibrahim^*,, Ghazi El Mernissi^*,, Lydie Cheval^*, Sophie Marsy^*, Michèle Grima^¶, Jean-Pierre Girolami and Alain Doucet^*

*Laboratoire de Physiologie et Génomique des Cellules Rénales (UMR 7134 CNRS/Université Paris 6), Institut des Cordeliers, Paris, France; Laboratoire de Pharmacologie Moléculaire et Physiopathologie Rénale, (INSERM U388), Institut Louis Bugnard, Toulouse, France; Laboratorio de Fisiopatologia Renal, Faculdade de Ciencias Médicas, Universidade do Estado do Rio de Janeiro, Rio de Janeiro, Brazil; Laboratoire des Biomembranes, Faculté des Sciences, Université Caddi Ayyad, Marrakech, Marocco; ^¶Institut de Pharmacologie et de Médecine Expérimentale, Université Louis Pasteur, Faculté de Médecine, Strasbourg, France.

Correspondence to: Dr. Bénédicte Buffin-Meyer, INSERM U388, IFR31, CHU Rangueil, 1, avenue Jean Poulhès, 31403 Toulouse Cedex 4, France. Phone: 33-5-61-32-30-90; Fax: 33-5-62-17-25-54; E-mail: benedicte.buffin-meyer{at}toulouse.inserm.fr

	Abstract

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ABSTRACT. In response to chronic treatment with furosemide, collecting ducts adapt their function to the initial loss of Na⁺ to prevent further Na⁺ loss and extracellular volume decrease. This adaptation, which includes the overexpression of Na⁺,K⁺-ATPase, is thought to account for most of the kaliuretic effect of furosemide. Because piretanide is reported to be less kaliuretic than equidiuretic doses of furosemide, the authors compared the effects of 1-wk treatment with the two loop diuretics on urinary potassium excretion and on Na⁺,K⁺-ATPase activity in the collecting duct. At equidiuretic and equinatriuretic doses, furosemide increased urinary potassium excretion as well as collecting duct Na⁺,K⁺-ATPase activity, whereas piretanide had no effect on either parameter. These effects of furosemide were curtailed by concomitant administration of the angiotensin-converting enzyme inhibitor enalapril, but they were not altered either by clamping changes in plasma aldosterone or by blocking type I angiotensin receptors. Treatment with the antagonist of bradykinin B2 receptors Hoe140 mimicked the two effects of furosemide. In addition, the effects of Hoe140 and furosemide were not additive. Finally, piretanide increased urinary bradykinin excretion, whereas furosemide did not. These results suggest that induction of collecting duct Na⁺,K⁺-ATPase (a) accounts for the kaliuretic effect of furosemide, (b) is independent of the renin/angiotensin/aldosterone system, (c) results from increased Na⁺ delivery to the collecting duct and enhanced intracellular Na⁺ concentration, and (d) is prevented in piretanide treated rats by increased bradykinin production that may limit apical Na⁺ entry in collecting duct principal cells.

	Introduction

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Loop diuretics are commonly prescribed for the treatment of hypertension and edema. In kidneys, they inhibit the apical Na⁺-K⁺-2Cl^- cotransporter in thick ascending limbs of Henle’s loop (TAL) (1). In the short term, this results in the inhibition of NaCl reabsorption in TAL (2), which increases natriuresis, and a decrease in the corticopapillary gradient of osmotic pressure, which increases diuresis through inhibition of water reabsorption along collecting ducts. Within a few days of continuous treatment, the kidneys, particularly the distal tubules and collecting ducts, adapt their function to the initial loss of Na⁺ by activating antinatriuretic mechanisms that prevent further Na⁺ loss and extracellular volume decrease (3).

Urinary loss of potassium and the resulting risk of hypokalemia are the main side effects of long-term therapy with loop diuretics. The loss of K⁺ induced by loop diuretics results from the combined inhibition of its reabsorption along the TAL (4) and stimulation of its secretion along the collecting duct (5,6). Loop diuretic-induced secretion of K⁺ along the collecting ducts likely results from increased delivery of Na⁺ to these segments, since K⁺ secretion through the apical membrane of collecting duct principal cells increases with luminal Na⁺ concentration (7).

Furosemide and piretanide are two loop diuretics that share the Na⁺-K⁺-2Cl^- cotransporter of the TAL (2) as primary target. Accordingly, the natriuretic and diuretic effects of furosemide and piretanide are similar. Curiously, however, several authors reported a weaker kaliuretic effect of piretanide as compared with equinatriuretic doses of furosemide (8–10). This suggests that kidneys may adapt their function differentially in response to long-term treatment with furosemide and piretanide. This study was therefore designed to confirm the different kaliuretic effects of chronic treatments with furosemide and piretanide and to elucidate the mechanism responsible for such a difference.

It was previously reported that chronic treatment of rats with furosemide induces cellular hypertrophy and upregulation of Na⁺,K⁺-ATPase specifically in collecting ducts (11,12). Because Na⁺,K⁺-ATPase is the motor for K⁺ secretion along the collecting duct, we evaluated the mechanism of upregulation of Na⁺,K⁺-ATPase in furosemide-treated rats and its relationship with kaliuresis, and we compared the effects of furosemide and piretanide treatments on Na⁺,K⁺-ATPase in the collecting duct.

	Materials and Methods

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Animals
Experiments were performed on Male Sprague Dawley rats (190 to 210 g) housed in individual cages with free access to usual food and tap water. The day before the experiment, some animals were individually placed in metabolic cages, and 24-h urine was collected.

In the first experimental series, we evaluated the effects of furosemide and piretanide on urinary K⁺ excretion and collecting duct Na⁺,K⁺-ATPase. Furosemide and piretanide were administered by constant infusion (1 µl · h^-1) using osmotic minipumps (Alzet) implanted intraperitonealy under light ether anesthesia. The dose of furosemide used in this study (150 µg · h^-1) was intermediate between those previously used in similar protocols (11,12), and the dose of piretanide (75 µg · h^-1) was adjusted to induce similar natriuretic and diuretic effects in the long term. Furosemide and piretanide (Hoechst, Paris) were dissolved in isotonic NaCl at alkaline pH (9.0). Control rats received the same volume of diluent via osmotic minipumps. Animals were sacrificed 6 d after the onset of the diuretic treatment.

The second experimental series evaluated the effect of inhibiting angiotensin-converting enzyme (ACE) on furosemide action on collecting duct Na⁺,K⁺-ATPase. For this purpose, the ACE inhibitor enalapril (Merck, Sharp, Dohme & Chibret) was given to furosemide-treated rats (at the same dose as in series one) by daily oral gavage in the morning at the dose of 600 µg · d^-1 in 10% glucose solution (1 ml/animal). Rats in control groups (untreated rats) received the glucose solution. Animals were studied 6 d after starting furosemide and enalapril treatments.

In the third series, we evaluated the involvement of corticosteroids on furosemide action. Rats were bilaterally adrenalectomized under light ether anesthesia and supplemented with 1 µg/100 g body wt aldosterone per day and 1.4 µg/100 g body wt dexamethasone per day through subcutaneous osmotic pump (13). Animals were treated with either furosemide or the diluent as above starting on the day of surgery, and they were studied 6 d later.

In a fourth series, we evaluated the effect on furosemide action of blocking type I angiotensin II (AngII) receptor with irbesartan. In this series, animals were fed a gelified diet (25 g/100 g body wt per d) consisting of 30% powdered diet, 69.6% water, and 0.4% agar-agar with or without irbesartan (2 mg/100 g body wt per d). Treatment with furosemide (as above) was started at the same time as irbesartan, and animals were studied 6 d later.

In the fifth series of experiments, the effect of the bradykinin B₂ receptor antagonist Hoe140 (Hoechst Marion Roussel, Frankfurt am Main, Germany) was compared with that of furosemide. Hoe140 was administered to either furosemide-untreated or furosemide-treated rats (at the same dose as in series one) at the dose of 4 µg · h^-1 by constant infusions (1 µl · h^-1) using osmotic minipumps implanted subcutaneously under light ether anesthesia. Hoe140 was dissolved in isosmotic NaCl. Control rats received the same volume of diluent via osmotic minipumps. Animals were sacrificed 6 d after the onset of the diuretic treatment.

Microdissection of Collecting Ducts
Cortical and outer medullary collecting ducts (CCD and OMCD, respectively) were dissected from collagenase-treated kidneys. Briefly, after anesthesia (pentobarbital sodium, 50 mg/kg body wt intraperitoneally), the left kidney was infused via the abdominal aorta with 4 ml of microdissection solution (see below) containing 0.24% (wt/vol) collagenase (from Clostridium histolyticum, 0.32 U/mg; Boehringer Mannheim) and 0.2% (wt/vol) bovine serum albumin (BSA). The kidney was excised, cut into small corticopapillary pieces that were incubated at 30°C for 20 min in microdissection solution containing 0.15% collagenase and 0.2% BSA, rinsed, and stored in the cold until use. The microdissection solution contained (in mM): 137 NaCl, 5 KCl, 0.8 MgSO₄, 1 CaCl₂, 0.33 Na₂HPO₄, 1 MgCl₂, and 10 tris(hydroxymethyl)amino-methane hydrochloride (Tris.HCl), pH 7.4.

Cortical and outer medullary collecting ducts were dissected from the cortex below the last branching and in the inner stripe of the outer medulla, respectively. The length of each piece of nephron, which served as reference for ATPase activities, was determined by automatic image analysis.

Na⁺,K⁺-ATPase Assay
ATPase activity was determined as described previously (11). Briefly, tubular samples were rinsed in ice-cold hypotonic solution (Tris.HCl 10 mM, pH 7.4) and frozen in 0.2 µl of hypotonic solution. After thawing and addition of 1 µl of assay medium (see below), samples were incubated for 15 min at 37°C. Incubation was stopped by cooling and addition of 5 µl of ice-cold trichloracetic acid (5% wt/vol). Each sample was then transferred into 2 ml of an ice-cold suspension of 10% (wt/vol) activated charcoal; after mixing and centrifugation, the radioactivity of a 0.5 ml aliquot of the supernatant containing P_i was determined by liquid scintillation.

ATPase assay solution contained (in mM): 50 NaCl, 5 KCl, 10 MgCl₂, 1 EDTA, 100 Tris-HCl, 10 Na₂ATP, and tracer amounts of [-³²P]ATP (10 Ci/mmol; Dupont de Nemours, Boston MA) for the measurement of total ATPase activity. For the measurement of basal Mg²⁺-ATPase activity, NaCl and KCl were omitted, Tris-HCl was 150 mM, and ouabain 1 mM was added. The pH of both solutions was adjusted at 7.40. Total and Mg²⁺-ATPase activities were each determined on 4 to 6 replicates. Na⁺,K⁺-ATPase activity was taken as the difference between the mean total and the mean Mg-ATPase, and it was expressed in pmol · min^-1 · h^-1. Data are means ± SEM from several animals.

Dosage of Plasma Renin and Angiotensin
Renin and AngI were determined in the plasma of control, furosemide-treated, and piretanide-treated rats. Nonanesthetized animals were decapitated. For the plasma AngI determination, 5 ml of blood were collected in a tube containing 250 µl of renin inhibitors (50 mM orthophenantroline, 62.5 mM Na₂EDTA, 0.2% neomycin) and 5 µl of 1.2 mM pepstatin. For the renin dosage, 1.5 ml of blood was collected in a Vacutainer EDTA K3 tube. Each tube was immediately centrifuged for 10 min at 3000 rpm at 4°C. Plasma were transferred in tubes devoided of inhibitors and were frozen in liquid nitrogen. Dosage of plasma renin was determined by radioimmunoassay (RIA) of AngI produced during a 30 min of incubation with 8-hydroxyquinoleine. The dosage of AngI was determined by RIA, as described previously (14).

Urinary Excretion of Bradykinin
Urinary excretion of bradykinin was measured in control rats and rats treated with furosemide or with piretanide. Animals were anesthetized with Inactin (100 mg/kg body wt intraperitoneally; Byk Gulden, Konstanz, Germany), a tracheotomy was performed, and catheters (PE50) were introduced into a jugular vein for solution infusion and into the bladder. Animals were perfused with 0.9% saline solution at a rate of 40 µl · min^-1. After a 60-min equilibration period, urine was collected for 90 min on refrigerated tubes. After measurement of urinary rate, samples were frozen until assay. Bradykinin concentration was measured by RIA as described previously (15).

Statistical Analyses
Comparison between groups was performed by t test or, when necessary, by variance analysis according to ANOVA. P values less than 0.05 were considered as significant.

	Results

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Comparative Effects of Furosemide and Piretanide on Urine Excretion of K⁺ and Na⁺,K⁺-ATPase Activity in the Collecting Duct
As already mentioned, the chosen doses of furosemide (150 µg · h^-1) and piretanide (75 µg · h^-1) induced similar effects on urinary excretion of water (+50%) and of sodium (+24%). They also reduced the osmotic pressure of the urine by a same factor (-30%). However, furosemide increased urinary K⁺ excretion by 21%, whereas piretanide did not (Table 1). Furosemide treatment also increased Na⁺,K⁺-ATPase activity in the CCD (+82%) and OMCD (+60%), as described previously (11), whereas piretanide had no effect on Na⁺,K⁺-ATPase activity in either CCD or OMCD (Figure 1). Neither furosemide nor piretanide altered Na⁺,K⁺-ATPase activity in the medullary thick ascending limb of Henle’s loop (data not shown). These results confirm the absence of kaliuretic effect of piretanide as compared with equidiuretic doses of furosemide and suggest that the kaliuretic effect of furosemide might be related to increased collecting duct Na⁺,K⁺-ATPase activity.

View larger version (49K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 1. Effects of loop diuretics on Na+,K+-ATPase activity in the collecting duct. Na+,K+-ATPase activity was determined in cortical and outer medullary collecting duct (CCD and OMCD, respectively) from normal rats (open bars) and rats treated with either 150 µg · h-1 furosemide (hatched bars) or 75 µg · h-1 piretanide (gray bars) for 6 d. Results are means ± SE from n animals. Statistical significance between groups was determined by variance analysis. ***, P < 0.001 versus controls.

View larger version (52K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 2. Effects of angiotensin-converting enzyme (ACE) inhibitor enalapril on Na+,K+-ATPase stimulation induced by furosemide in the collecting duct. Na+,K+-ATPase activity was determined in CCD and OMCD from normal rats (open bars) and rats treated with either 150 µg · h-1 furosemide alone (hatched bars) or 600 µg · h-1 enalapril plus furosemide (dotted bars) for 6 d. Results are means ± SE from n animals. Statistical significance between groups was determined by variance analysis; *P < 0.05 versus controls; P < 0.05 versus furosemide; P < 0.01 versus furosemide.

Involvement of aldosterone in furosemide-induced activation of Na⁺,K⁺-ATPase might be considered because aldosterone induces Na⁺,K⁺-ATPase synthesis in rat collecting ducts (19–21). However, clamping aldosterone level did not prevent furosemide-induced stimulation of Na⁺,K⁺-ATPase in CCD (in pmol · mm^-1 · h^-1 ± SE: Clamp-Aldo, 752 ± 50, n = 6; Clamp-Aldo + Furosemide, 1220 ± 144, n = 6; P < 0.025) or natriuresis (in mEq/100 g body wt per day ± SE: Clamp-Aldo, 0.65 ± 0.01, n = 5; Clamp-Aldo + Furosemide, 0.85 ± 0.04, n = 6; P < 0.001) or kaliuresis (in mEq/100 g body wt per day ± SE: Clamp-Aldo, 1.08 ± 0.04, n = 5; Clamp-Aldo + Furosemide, 1.55 ± 0.03, n = 6; P < 0.001). These findings, which confirm previous results showing that spironolactone did not prevent stimulation of Na⁺,K⁺-ATPase by furosemide (11), rule out an involvement of aldosterone.

Although AngII may increase renal Na⁺,K⁺-ATPase activity (22), its involvement in furosemide action was ruled out also on the following two bases. (1) Furosemide treatment did not alter plasma renin activity (in ng · ml^-1 · h^-1 ± SE: Control, 14.7 ± 1.2, n = 8; Furosemide, 15.8 ± 0.9, n = 10, NS) or plasma AngI (in pM ± SE: Control, 280 ± 37, n = 9; Furosemide, 238 ± 18, n = 14, NS). (2) Treatment with irbesartan did not prevent the stimulatory effects of furosemide on Na⁺,K⁺-ATPase in CCD (in pmol · mm^-1 · h^-1 ± SE: Irbesartan, 782 ± 41, n = 6; Irbesartan + Furosemide, 1221 ± 42, n = 6; P < 0.001) or on natriuresis (in mEq/100g body wt per day ± SE: Irbesartan, 0.85 ± 0.03, n = 6; Irbesartan + Furosemide, 0.97 ± 0.02, n = 5; P < 0.01) or on kaliuresis (in mEq/100g body wt per day ± SE: Irbesartan, 1.16 ± 0.04, n = 6; Irbesartan + Furosemide, 1.40 ± 0.02, n = 5; P < 0.005). In conclusion, furosemide effects on collecting duct Na⁺,K⁺-ATPase and urinary Na⁺ and K⁺ excretion are independent of the renin/angiotensin/aldosterone system.

Role of Bradykinin
Enalapril increases bradykinin concentration in the kidney (23), and bradykinin inhibits Na⁺ reabsorption in rat CCD (24); therefore, chronic increase in bradykinin level might be responsible for the antagonizing effect of enalapril on furosemide-induced regulation of Na⁺,K⁺-ATPase. Because most effects of bradykinin are mediated through its B₂ receptors, we evaluated whether blocking bradykinin B₂ receptors with Hoe140 (25) would mimic furosemide action.

Chronic treatment with Hoe140 induced a stimulation of Na⁺,K⁺-ATPase in CCD similar to that induced by furosemide (Figure 3). This suggests the presence in control rats of a basal tonic inhibition of collecting duct Na⁺,K⁺-ATPase mediated by bradykinin and prevented by Hoe140. From a metabolic point of view (Table 3), Hoe140 had no effect on urine volume, it reduced urinary Na⁺ excretion, and it increased urinary K⁺ excretion, although to a lesser extent than furosemide. The effect of bradykinin antagonist on Na⁺ and K⁺ renal losses might be explained by the activation of collecting duct Na⁺,K⁺-ATPase. When Hoe140 and furosemide were administered together, their effects on Na⁺,K⁺-ATPase activity were not additive (Figure 3). Furthermore, the stimulatory effect of Hoe140 on K⁺ excretion was not additive with that of furosemide, whereas the opposite effects of the two drugs on Na⁺ excretion were additive (Table 3).

View larger version (58K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 3. Effects of bradykinin B2 receptor antagonist Hoe140 on Na+,K+-ATPase stimulation induced by furosemide in the collecting duct. Na+,K+-ATPase activity was determined in cortical collecting duct (CCD) from normal rats (open bars) and rats treated with either 150 µg · h-1 furosemide alone (hatched bars) or 4 µg · h-1 Hoe140 alone (dotted bars) or Hoe140 plus furosemide (gray bars) for 6 d. Results are means ± SE from n animals. Statistical significance between groups was determined by variance analysis; ***P < 0.001 versus controls.

	Discussion

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Furosemide and Piretanide Induce Different Effects on Kaliuresis
Present results show that chronic treatment with piretanide did not modify urinary excretion of K⁺, whereas chronic treatment with furosemide increased it (Table 1). Previous data from the literature suggest that the effect of piretanide on K⁺ excretion might depend of the duration of treatment. As summarized in Table 4 (27–47), all reports in humans indicate that a single administration of piretanide increases urinary excretion of K⁺, whereas sustained administration for 5 d to 6 mo alters neither the kaliuresis nor the kalemia. Acute administration of piretanide to rats also increases urinary excretion of K⁺ (48), whereas chronic administration for 1 to 8 wk does not (49). These findings in humans and rats are confirmed by present results in rats treated for 1 wk with piretanide.

Because urinary loss of potassium and the resulting risk of hypokalemia are the main side effects of long-term therapy with loop diuretics, piretanide might be more suitable than furosemide to treat hypertension and edema.

Differential Adaptation of Collecting Duct Na⁺,K⁺-ATPase in Response to Furosemide and Piretanide
In collecting duct principal cells, adaptation to chronic furosemide treatment includes the overexpression of basolateral Na⁺,K⁺-ATPase (11) and of apical amiloride-sensitive sodium channels (ENaC) (50,51). In these cells, Na⁺ reabsorption is stoichiometrically coupled to K⁺ secretion because K⁺ accumulated within the cells by Na⁺,K⁺-ATPase preferentially leaks out the cells by apical K⁺ channels. We therefore hypothesized that the different kaliuretic effect of furosemide and piretanide might result from a differential adaptation of the collecting duct Na⁺ transport. As a matter of fact, results show that chronic treatment with piretanide did not modify Na⁺,K⁺-ATPase activity in collecting duct, conversely to furosemide which increased it (Figure 1). Furthermore, a linear relationship was found between Na⁺,K⁺-ATPase activity in collecting duct and urinary excretion of K⁺ (figure 4). This suggests that indeed differential adaptation of the collecting duct Na⁺,K⁺-ATPase in piretanide- and furosemide-treated animals likely accounts in part for the different kaliuretic action of the two loop diuretics. This means that in piretanide-treated rats, TAL are the only sites of K⁺ loss; in furosemide-treated rats, loss of K⁺ originates in both TAL and collecting ducts.

View larger version (16K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 4. Correlation between Na+,K+-ATPase activity in CCD and urinary K+ excretion. Mean Na+,K+-ATPase activities measured in CCD from control rats and rats treated with Furosemide, Piretanide, Furosemide + Enalapril, Hoe140, Hoe140 + Furosemide, Clamp-Aldo, Clamp-Aldo + Furosemide, Irbesartan or Irbesartan + Furosemide are plotted against urinary K+ excretion in the same groups of animals. Values for Na+,K+-ATPase activity and for urinary K+ excretion are from the different series presented in Figures 1 through 3, in Tables 1 through 3, and in the text.

Mechanism of Differential Regulation of Collecting Duct Na+,K+-ATPase by Furosemide and Piretanide
Adaptation of Na⁺,K⁺-ATPase in collecting ducts of furosemide-treated rats is thought to be brought about by the increased delivery of Na⁺ to the collecting duct, and to the resulting increase in intracellular Na⁺ concentration ([Na⁺]_i). In turn, increasing [Na⁺]_i induces the rapid recruitment of a latent pool of Na⁺,K⁺-ATPase (53) and the delayed synthesis of new Na⁺,K⁺-ATPase units (54). Because furosemide and piretanide inhibit Na⁺ reabsorption in TAL (55,56), one would expect them to induce Na⁺,K⁺-ATPase in the collecting duct. It could be argued that piretanide did not induce Na⁺,K⁺-ATPase in the collecting duct because it promoted a weaker natriuretic effect in the TAL (see above). However, this is unlikely because treatment with a much lower dose of furosemide (4 µg · h^-1 instead of 150 µg · h^-1), which expectedly had a weaker effect in the TAL than the dose of piretanide used in the present study, induced collecting duct Na⁺,K⁺-ATPase to a similar extent (11) as in the present study.

Alternatively, it can be proposed that piretanide promotes two opposite actions on Na⁺,K⁺-ATPase. First, it would trigger its upregulation by increasing Na⁺ delivery to the collecting duct; second, it would trigger a mechanism that counterbalances this inductory mechanism either by blocking the increase in [Na⁺]_i or by downregulating Na⁺,K⁺-ATPase. Bradykinin might be responsible for the antagonizing mechanism observed in piretanide-treated rats because (1) bradykinin inhibits Na⁺ reabsorption by in vitro microperfused CCD (24), (2) chronic piretanide increased the urinary excretion of bradykinin, whereas furosemide did not, (3) enalapril, which curtailed furosemide effects on collecting duct Na⁺,K⁺-ATPase activity, also increases renal bradykinin concentration (7), and (4) blocking bradykinin B₂ receptors with Hoe140 mimicked furosemide action on Na⁺,K⁺-ATPase. Neither the mechanism underlying the differential regulation of renal bradykinin by the two loop diuretics nor the pathway through which bradykinin might prevent the increase in [Na⁺]_I and the upregulation of Na⁺,K⁺-ATPase by increased distal Na⁺ delivery are known.

Finally, one cannot exclude that the differential effects of furosemide and piretanide on Na⁺,K⁺-ATPase and potassium handling might be related to an effect of either drug on a molecular target other than the Na⁺-K⁺-2Cl^- cotransporter. For example, furosemide has been reported to antagonize some GABA receptor subtypes (57) and to inhibit the anion transporter pendrin (58) and 11-hydroxysteroid dehydrogenase (59), and it is not known whether piretanide also shares these properties. It should be noted, however, that these side effects of furosemide are unlikely to participate to the adaptation of the collecting duct because (1) GABA receptors are not expressed in the kidney, (2) pendrin is specifically expressed in intercalated cells in collecting duct (60) and therefore is unlikely to interfere with Na⁺,K⁺-ATPase in principal cells, and (3) the effect of furosemide is independent of corticosteroids.

In summary, results of this study confirm that in the long-term piretanide is less kaliuretic than furosemide and that furosemide-induced kaliuresis is associated with upregulation of collecting duct Na⁺,K⁺-ATPase. The lack of adaptation of the collecting duct to increased Na⁺ delivery in piretanide-treated rats may be accounted in part by increased renal bradykinin concentration, which controls Na⁺ entry in collecting duct principal cells. The mechanisms of differential regulation of bradykinin by piretanide and furosemide and of bradykinin action in collecting duct remain to be determined.

	Acknowledgments

 
This work was supported in part by grants from Laboratoires Hoechst, Puteaux, France. Authors are grateful to Dr K. Wirth, Hoechst Marion Roussel, Frankfurt am Main, Germany, for the gift of Hoe140.

	References

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