Formulation, Characterization, and the Diuretic Effects of a New Intravenous Metolazone Emulsion.

OBJECTIVE: Acute decompensated heart failure is often treated with a combination of loop and thiazide-like diuretics. Of these thiazide-like diuretics, two common choices are intravenous chlorothiazide or oral metolazone. Metolazone is more potent and has a longer duration of action, but since it is an oral formulation, it has a longer on-set time as compared to chlorothiazide. In addition, metolazone is poorly water-soluble, thereby rendering intravenous formulation more challenging. To address these issues, we proposed the formulation of a solvent-free metolazone emulsion for intravenous administration. METHODS: An oil-in-water emulsion containing 1 mg/mL of metolazone was formulated by homogenizing soybean oil and l-lecithin in water in the presence of optimized concentrations of glycerin with tween 80 or poloxamer 188 as surfactant. The emulsion was characterized on the basis of particle size, zeta potential, morphology and metolazone release kinetics. The diuretic effect of the metolazone emulsion was evaluated in rats. RESULTS: The 1 mg/mL metolazone emulsion prepared with 5% tween 80 displayed the best physical stability. The emulsion exhibited a hydrodynamic diameter of 157.13±1.52 nm. About 93% of metolazone was released from the formulation within 2 h. The 2 mg/kg and 4 mg/kg dose of the metolazone emulsion increased urine output in the rats by 68.9 and 134%, respectively, as compared to control rats. Furthermore, the 4 mg/kg dose exhibited a 168.8%, 25.8%, and 150.9% increase in sodium, potassium, and chloride, respectively. CONCLUSION: This metolazone emulsion was capable of increasing urine volume output and demonstrated both natriuretic and kaliuretic properties.

Association of QT-Prolonging Medication Use in CKD with Electrocardiographic Manifestations.

BACKGROUND AND OBJECTIVES: Several drugs used in CKD can prolong electrocardiographic conduction. We examined the use of electrocardiogram QT-prolonging medications in predialysis CKD and their association with QT duration. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: In total, 3252 Chronic Renal Insufficiency Cohort participants with at least one study electrocardiogram between 2003 and 2011 were included. QT-prolonging medications used in 100 or more visits (n=16,451 visits) along with diuretics and proton pump inhibitors, given their potential for electrolyte disturbances, were examined for QT interval prolongation. RESULTS: Mean QT interval corrected for heart rate was at 414±21 (±SD) milliseconds and prolonged (≥450 milliseconds) in 4.6% of electrocardiograms. QT interval corrected for heart rate was inversely related to serum potassium and calcium. Medications classified as QT prolonging were taken at 76% of visits, with two or more of these taken at 33% of visits. Of 30 medications examined, eight were associated with statistically significant QT interval corrected for heart rate prolongation after adjustment for comorbidities, potassium, and calcium, including amiodarone (+10±2 milliseconds), metolazone (+7±2 milliseconds), fluoxetine (+4±1 milliseconds), citalopram (+4±1 milliseconds), hydroxyzine (+4±1 milliseconds), escitalopram (+3±2 milliseconds), venlafaxine (+3±1 milliseconds), and furosemide (+3±0 milliseconds). Potassium-depleting diuretics were associated with minimal decrements in potassium (between 0.1 and 0.3 mEq/L) and smaller changes in calcium. Diuretics associated with a change in QT interval corrected for heart rate before adjustment for potassium and calcium were metolazone (+8±3 milliseconds), furosemide (+4±1 milliseconds), and spironolactone (-3±3 milliseconds). Most of the QT prolongation associated with metolazone and furosemide, but not spironolactone, remained after adjustment for potassium and calcium. Proton pump inhibitors were not associated with QT prolongation. CONCLUSIONS: Use of medications associated with QT prolongation is common in CKD; the safety implications of these findings should be considered in these high-risk patients. PODCAST: This article contains a podcast at https://www.asn-online.org/media/podcast/CJASN/2017_08_09_CJASNPodcast_17_09_b.mp3.

Thiazide-like diuretic drug metolazone activates human pregnane X receptor to induce cytochrome 3A4 and multidrug-resistance protein 1.

Human pregnane X receptor (hPXR) regulates the expression of drug-metabolizing enzyme cytochrome P450 3A4 (CYP3A4) and drug transporters such as multidrug-resistance protein 1 (MDR1). PXR can be modulated by small molecules, including Federal Drug Administration (FDA)-approved drugs, thus altering drug metabolism and causing drug-drug interactions. To determine the role of FDA-approved drugs in PXR-mediated regulation of drug metabolism and clearance, we screened 1481 FDA-approved small-molecule drugs by using a luciferase reporter assay in HEK293T cells and identified the diuretic drug metolazone as an activator of hPXR. Our data showed that metolazone activated hPXR-mediated expression of CYP3A4 and MDR1 in human hepatocytes and intestine cells and increased CYP3A4 promoter activity in various cell lines. Mammalian two-hybrid assays showed that hPXR recruits its co-activator SRC-1 upon metolazone binding in HepG2 cells, explaining the mechanism of hPXR activation. To understand the role of other commonly-used diuretics in hPXR activation and the structure-activity relationship of metolazone, thiazide and non-thiazide diuretics drugs were also tested but only metolazone activates hPXR. To understand the molecular mechanism, docking studies and mutational analysis were carried out and showed that metolazone binds in the ligand-binding pocket and interacts with mostly hydrophobic amino acid residues. This is the first report showing that metolazone activates hPXR. Because activation of hPXR might cause drug-drug interactions, metolazone should be used with caution for drug treatment in patients undergoing combination therapy.

Comparative characterization of Na+ transport in Cyprinodon variegatus variegatus and Cyprinodon variegatus hubbsi: a model species complex for studying teleost invasion of freshwater.

The euryhaline fish Cyprinodon variegatus variegatus is capable of tolerating ambient salinities ranging from 0.3 to 160 PSU, but is incapable of long-term survival in freshwater (<2 mmol l(-1) Na(+)). A population isolated in several freshwater (0.4-1 mmol l(-1) Na(+)) lakes in central Florida is now designated as a subspecies (Cyprinodon variegatus hubbsi). We conducted a comparative study of Na(+) transport kinetics in these two populations when acclimated to different ambient Na(+) concentrations. Results reveal that the two subspecies have qualitatively similar low affinity Na(+) uptake kinetics (K(m)=7000-38,000 µmol l(-1)) when acclimated to 2 or 7 mmol l(-1) Na(+), but C. v. hubbsi switches to a high affinity system (K(m)=100-140 µmol l(-1)) in low-Na(+) freshwater (≤1 mmol l(-1) Na(+)). Inhibitor experiments indicate that Na(+) uptake in both subspecies is EIPA-sensitive, but sensitivity decreases with increasing external Na(+). EIPA induced a 95% inhibition of Na(+) influx in C. v. hubbsi acclimated to 0.1 mmol l(-1) Na(+), suggesting that this subspecies is utilizing a Na(+)/H(+) exchanger to take up Na(+) in low-Na(+) environments despite theoretical thermodynamic constraints. Na(+) uptake in C. v. hubbsi acclimated to 0.1 mmol l(-1) Na(+) is phenamil-sensitive but not bafilomycin-sensitive, leading to uncertainty about whether this subspecies also utilizes Na(+) channels for Na(+) uptake. Experiments with both subspecies acclimated to 7 mmol l(-1) Na(+) also indicate that a Cl(-)-dependent Na(+) uptake pathway is present. This pathway is not metolazone-sensitive (NCC inhibitor) in either species but is bumetanide-sensitive in C. v. variegatus but not C. v. hubbsi. This suggests that an apical NKCC is increasingly involved with Na(+) uptake for this subspecies as external Na(+) increases. Finally, characterization of mitochondria-rich cell (MRC) size and density in fish acclimated to different ambient Na(+) concentrations revealed significant increases in the number and size of emergent MRCs with decreasing ambient Na(+). A linear relationship between the fractional area of emergent MRCs and Na(+) uptake rate was observed for both subspecies. However, C. v. variegatus have lower Na(+) uptake rates at a given MRC fractional area compared with C. v. hubbsi, indicating that the enhanced Na(+) uptake by C. v. hubbsi at low ambient Na(+) concentrations is not strictly a result of increased MRC fractional area, and other variables, such as differential expression of proteins involved in Na(+) uptake, must provide C. v. hubbsi with the ability to osmoregulate in dilute freshwater.

Role of SLC12A10.2, a Na-Cl cotransporter-like protein, in a Cl uptake mechanism in zebrafish (Danio rerio).

The thiazide-sensitive Na(+)-Cl(-) cotransporter (NCC), a member of the SLC12 family, is mainly expressed in the apical membrane of the mammalian distal convoluted tubule (DCT) cells, is responsible for cotransporting Na(+) and Cl(-) from the lumen into DCT cells and plays a major role in the mammalian renal NaCl reabsorption. The NCC has also been reported in fish, but the functional role in fish ion regulation is yet unclear. The present study used zebrafish as an in vivo model to test the hypothesis of whether the NCC plays a role in Na(+) and/or Cl(-) uptake mechanisms. Four NCCs were cloned, and only one of them, zebrafish (z) slc12a10.2 was found to predominately and specifically be expressed in gills. Double in situ hybridization/immunocytochemistry in zebrafish skin/gills demonstrated that the specific expression of zslc12a10.2 mRNA in a novel group of ionocytes differed from those of the previously-reported H(+)-ATPase-rich (HR) cells and Na(+)-K(+)-ATPase-rich (NaR) cells. Gill mRNA expression of zslc12a10.2 was induced by a low-Cl environment that stimulated fish Cl(-) influx, while a low-Na environment suppressed this expression. Incubation with metolazone, a specific inhibitor of the NCC, impaired both Na(+) and Cl(-) influx in 5-day postfertilization (dpf) zebrafish embryos. Translational knockdown of zslc12a10.2 with a specific morpholino caused significant decreases in both Cl(-) influx and Cl(-) content of 5-dpf zebrafish embryos, suggesting that the operation of zNCC-like 2 results in a net uptake of Cl(-) in zebrafish. On the contrary, zslc12a10.2 morphants showed increased Na(+) influx and content that resulted from upregulation of mRNA expressions of Na(+)-H(+) exchanger 3b and carbonic anhydrase 15a in HR cells. These results for the first time provide in vivo molecular physiological evidence for the possible role of the NCC in the Cl(-) uptake mechanism in zebrafish skin/gills.

The evaluation of the diuretic action of parenteral formulations of metolazone.

BACKGROUND AND OBJECTIVES: This study was design to compare the diuretic and natriuretic effects of the 2 parenteral formulations of metolazone and the combination of these 2 formulations of metolazone with the parenteral administration of furosemide. Metolazone is an anthracrene acid derivate and manifests a dual diuretic effect on the proximal and distal tubule with a minimal kaluretic effect. It is currently only marketed in an orally administrable formulation, and this has limited its utility in critically ill patients. Metolazone given orally and furosemide given orally or parenterally are frequently administrated together when furosemide alone is clinically inadequate at producing a desired diuresis. METHODS: Sprague Dawley male rats (400 to 450 g) were divided into groups to receive a parenteral formulation of metolazone or furosemide administrated separately intraperitoneally (IP) or administrated IP in combination with one another. Tris buffer-administered IP was used as a control vehicle comparator. The urine volume voided over the following 24 hours was collected, measured and analyzed for sodium content. RESULTS: Vehicle (Tris buffer) caused 9 +/- 1 mL/d output of urine with a sodium [Na+] concentration of 194 +/- 41 micromol/L (n=6 per group). Metolazone 2 mg/kg resulted in 16 +/- 3 mL/d urine output and sodium [Na+] of 278 +/- 76 micromol/L (n=6 per group). Furosemide 2, 4, and 6 mg/kg resulted in a volume of urine 9 +/- 1, 14 +/- 2 and 17 +/- 2 mL/d and [Na+] micromol/L of 194 +/- 41, 206 +/- 108, and 229 +/- 91, respectively. Metolazone 4 mg/kg combined with furosemide 4 mg/kg resulted in a urine volume of 21 +/- 1 mL/d and [Na+] of 326 +/- 108 micromol/L. CONCLUSION: Combining metolazone and furosemide can cause an increase in urine volume and sodium excretion. Metolazone administrated parenterally in combination with the parenteral administration of furosemide appears to have an important clinical potential.

Functional expression of mutations in the human NaCl cotransporter: evidence for impaired routing mechanisms in Gitelman's syndrome.

Gitelman's syndrome is an autosomal recessive renal tubular disorder characterized by hypokalemic metabolic alkalosis, hypomagnesemia, and hypocalciuria. This disorder results from mutations in the thiazide-sensitive NaCl cotransporter (NCC). To elucidate the functional implications of mutations associated with this disorder, metolazone-sensitive (22)Na(+) uptake, subcellular localization, and glycosidase-sensitive glycosylation of human NCC (hNCC) were determined in Xenopus laevis oocytes expressing FLAG-tagged wild-type or mutant hNCC. Injection of 10 ng of FLAG-tagged hNCC cRNA resulted in metolazone-sensitive (22)Na(+) uptake of 3.4 +/- 0.2 nmol Na(+)/oocyte per 2 h. Immunocytochemical analysis revealed sharp immunopositive staining at the plasma membrane. In agreement with this finding, a broad endoglycosidase H-insensitive band of 130 to 140 kD was present in Western blots of total membranes. The plasma membrane localization of this complex-glycosylated protein was confirmed by immunoblotting of purified plasma membranes. The mutants could be divided into two distinct classes. Class I mutants (G439S, T649R, and G741R) exhibited no significant metolazone-sensitive (22)Na(+) uptake. Immunopositive staining was present in a diffuse band just below the plasma membrane. This endoplasmic reticulum and/or pre-Golgi complex localization was further suggested by the complete absence of the endoglycosidase H-insensitive band. Class II mutants (L215P, F536L, R955Q, G980R, and C985Y) demonstrated significant metolazone-sensitive (22)Na(+) uptake, although uptake was significantly lower than that obtained with wild-type hNCC. The latter mutants could be detected at and below the oocyte plasma membrane, and immunoblotting revealed the characteristic complex-glycosylated bands. In conclusion, this study substantiates NCC processing defects as the underlying pathogenic mechanism in Gitelman's syndrome.

Characterization of the thiazide-sensitive Na(+)-Cl(-) cotransporter: a new model for ions and diuretics interaction.

The thiazide-sensitive Na(+)-Cl(-) cotransporter (TSC) is the major pathway for salt reabsorption in the apical membrane of the mammalian distal convoluted tubule. When expressed in Xenopus laevis oocytes, rat TSC exhibits high affinity for both cotransported ions, with the Michaelis-Menten constant (K(m)) for Na(+) of 7.6 +/- 1.6 mM and for Cl(-) of 6.3 +/- 1.1 mM, and Hill coefficients for Na(+) and Cl(-) consistent with electroneutrality. The affinities of both Na(+) and Cl(-) were increased by increasing concentration of the counterion. The IC(50) values for thiazides were affected by both extracellular Na(+) and Cl(-). The higher the Na(+) or Cl(-) concentration, the lower the inhibitory effect of thiazides. Finally, rTSC function is affected by extracellular osmolarity. We propose a transport model featuring a random order of binding in which the binding of each ion facilitates the binding of the counterion. Both ion binding sites alter thiazide-mediated inhibition of transport, indicating that the thiazide-binding site is either shared or modified by both Na(+) and Cl(-).

The effect of nonsteroidal agents (NSAIDs) on the pharmacokinetics and pharmacodynamics of metolazone.

NSAIDs attenuate the natriuretic response to loop diuretics. Their effect on the action of distal tubular diuretics is poorly explored. Accordingly, the pharmacokinetics and pharmacodynamics of metolazone [M], a distal tubular diuretic, with and without indomethacin [M+I] or sulindac [M+S] were examined in six healthy volunteers. Urine samples were obtained over 36 hours post-metolazone dosing for the determination of sodium, potassium and metolazone concentration. Though cumulative M excretion 750 +/- 247 [mucg/36 h] [M]; 749 +/- 239 [M+I]; 848 +/- 443 [M+S] was comparable between treatment groups, total sodium [Na+] excretion was significantly depressed in the presence of S or I, 685 +/- 114 [mEq/36 h] [M]; 454 +/- 90 [M+I] (p < or = 0.05); 553 +/- 123 [M+S] (p < or = 0.05). Peak Na+ excretion [muEq/min] was significantly decreased by I only and time to peak Na+ excretion did not differ amongst treatment groups. Total potassium [K+] excretion 160 +/- 39 [mEq/36 h] [M]; 111 +/- 53 [M+I] (p < or = 0.05); 135 +/- 31 [M+S] significantly decreased with I. This phenomenon was most evident between 12 and 36 hours. The administration of I or S with M significantly blunted sodium excretion on a purely pharmacodynamic basis while the decline in urinary potassium excretion upon addition of I to M related probably to an attenuation of braking phenomenon induced kaliuresis. These findings likely reflect NSAID-induced sodium reabsorption at loci prior to the site of action of metolazone.

Metolazone pharmacokinetics and pharmacodynamics in renal transplantation.

Metolazone pharmacokinetics and pharmacodynamics were investigated in five renal transplant patients and five creatinine clearance matched controls. Whereas the time to peak metolazone excretion was similar in both groups (with the exception of one renal transplant patient who subsequently was found to have chronic rejection), the percent of the administered dose excreted over 48 h was significantly less in renal transplant patients (8% in renal transplant patients vs 24% in controls). Diminished bioavailability of metolazone or tubular secretion likely accounted for this disparity in metolazone excretion. Both groups developed diuretic tolerance as indicated by hysteresis in the dose response relationship. Cumulative sodium excretion over three successive 12-h time intervals did not differ between groups. Despite this comparable natriuresis, potassium excretion was significantly less in the renal transplant patients during the first day of the study. Accordingly, metolazone administration may provide a means to "unmask" subclinical, tubular secretory dysfunction in the transplanted kidney, as exemplified by defects in metolazone secretion and potassium excretion.

Interactions of diuretics with the peripheral-type benzodiazepine receptor in rat kidney.

The peripheral-type benzodiazepine receptor (PBR) has been autoradiographically localized to the thick ascending limb and early distal tubule. To elucidate further the role of this receptor in kidney function, we have examined the effects of all classes of diuretics on the binding of labeled PBR-specific ligands (R05-4864, PK 11195) to rat kidney membranes (13,000 X g X 10 min). Drugs capable of inhibiting R05-4864 binding by 50% at less than 200 microM included: metolazone (IC50 = 1 microM), indacrinone (IC50 = 42 microM), indapamide (IC50 = 58 microM), hydrochlorothiazide (HCTZ; IC50 = 117 microM) and trichloromethiazide (IC50 = 175 microM). Conversely, diuretics of the loop (e.g., furosemide), K+-sparing (e.g., triamterene), and carbonic anhydrase inhibitor (e.g., acetazolamide) classes exerted no significant effects on R05-4864 binding (IC50S greater than or equal to 1 mM). Inhibition by indacrinone was stereoselective. Thiazide-like compounds inhibited R05-4864 binding with a rank-order of potencies similar to that for their enhancement of in vivo natriuresis (metolazone greater than HCTZ approximately equal to trichlormethiazide greater than chlorothiazide). Scatchard analysis revealed that metolazone, indacrinone, indapamide and HCTZ inhibited R05-4864 binding by reducing Kd, with no effect on maximum binding. The apparent Kd of metolazone for the renal PBR was 3.8 X 10(-7) M. IC50 values of 14 metolazone derivatives for inhibition of R05-4864 binding correlated well (r = .71, P less than .01) with their natriuretic efficacies. PK 11195 binding to digitonin (1.2 mg/mg of protein)-solubilized membranes displayed the same rank-order of, but was twice as sensitive to inhibition by metolazone, indacrinone, indapamide and HCTZ.(ABSTRACT TRUNCATED AT 250 WORDS)

Some effects of metolazone on electrolyte transport.

Metolazone action was studied 1) in vitro on isolated operculum of Fundulus heteroclitus (active chloride transport) using an Ussing chamber (metolazone conc 500 microM) and in vivo 2) using the modified Sperber technique in the hen (metolazone infusion rate 0.75-1.2 micrograms/kg/min) and 3) in healthy volunteers using clearance techniques (metolazone infusion rate 10 mg/h). Metolazone reduced (p less than 0.05) short circuit current potential differences with 20% from average control values (p less than 0.05), while direct current resistance was unchanged. This is comparable to thiazide but much lower than loop diuretic effects. True tubular excretion fraction of metolazone before and after novobiocin (2.7 mumol/kg/min coinfusion averaged 14.1 and 4.5%, resp. (p less than 0.01; n = 8). Thus metolazone is partly eliminated by renal tubular secretion. However, the diuretic effect (sodium, chloride and potassium excretion)--and clearances of Cr51-EDTA and I125-Na-o-iodohippurate--were symmetrical, i.e. independent of metolazone urinary excretion rate, as previously shown for thiazides. Renal clearance of metolazone in healthy volunteers. (HPLC-method) averaged 173 +/- 20 ml/min (n = 8). Probenecid (1 g iv.) significantly reduced the renal clearance of metolazone to 33 +/- 7 ml/min and potassium excretion with maximum 30%, while diuretic and saluretic effects were significantly increased with maximum 30%. Thus, also in humans the diuretic effect of metolazone is not coupled to the urinary excretion rate of the drug, but suggests that its diuretic effect is elicited primarily from the peritubular side of the nephron. Probenecid apparently dissociates sodium from potassium excretion effects of metolazone. This implies a luminal, sodium-independent kaliuretic effect of the drug.

Diuretic effects on renal brush border membrane transport and metabolism.

Previous studies have indicated that the thiazide diuretics exert effects on proximal electrolyte transport. To determine whether the locus of these effects is at the brush border membrane (BBM) and if renal metabolism is affected, adult female Sprague-Dawley rats were acutely treated with either 1 mg/kg metolazone, 20 mg/kg chlorothiazide followed by a 20 mg/kg/hr maintenance infusion, 10 mg/kg acetazolamide followed by a 10 mg/kg/hr maintenance infusion, or the vehicles only. Administration of these agents resulted in an approximately tenfold increase in sodium excretion. Neither urinary phosphate nor inulin excretion changed significantly in any group. Sodium dependent BBM vesicle phosphate transport was examined at 0.15, 0.5, and 1 and 120 minute incubation periods in the diuretic treated groups and their respective control groups. Decreased uptake was seen in all pre-equilibrium time points in rats treated with metolazone: 0.15 minutes: 221 +/- 24 pmoles/mg protein (pmol/mg prot) in control rats versus (vs) 185 +/- 23 pmoles/mg prot in metolazone-treated animals (P less than .05) ; 0.5 minutes: 463 +/- 54 vs 369 +/- 49 pmol/mg prot (P less than .005); 1 minute: 549 +/- 74 vs 460 +/- 61 pmol/mg prot (P less than .05); no significant difference in phosphate transport was noted at the two hour equilibrium time point. No significant differences in sodium dependent phosphate transport existed between chlorothiazide or acetazolamide treated rats and control animals. Substrate-stimulated renal gluconeogenesis did not differ between metolazone treated and control animals. We therefore conclude that metolazone inhibits phosphate transport through an effect on the BBM and does not affect renal gluconeogenesis in the rat.

Pharmacokinetic and pharmacodynamic interaction between furosemide and metolazone in man.

In various clinical situations a poor diuretic response to furosemide may be improved by the addition of metolazone. The mechanism of this additive effect is unclear. The purpose of the present investigation was to establish whether metolazone changes the pharmacokinetics of furosemide and by this mechanism enhances the diuretic effect. Eight volunteers were given an intravenous infusion of 4 mg h-1 of furosemide for 12 h. After 6 h 2.5 mg metolazone were administered orally. The addition of metolazone increased diuresis, urinary excretion of sodium and chloride (P less than 0.01), but decreased urinary excretion of calcium (P less than 0.01), while furosemide excretion remained unchanged. Total body clearance and renal clearance values of furosemide were similar before and after administration of metolazone. Our data confirm the additive diuretic effect of the combination treatment metolazone-furosemide and show for the first time a distinct hypocalciuric action of metolazone, similar to thiazides. Moreover metolazone does not affect the pharmacokinetics of furosemide.

Combined effect of bumetanide and metolazone in normal volunteers.

Bumetanide 1 to 2 mg or metolazone 2.5 mg were administered by mouth separately and then in combination to eight normal men and women in order to determine whether a sequential blockade of sodium reabsorption with diuretic agents that act at different sites within the nephron leads to a supra-additive diuretic effect. All three treatment regimens resulted in a significant weight loss and increased urine volume and the excretion of sodium, potassium, and chloride. A prolonged diuretic effect lasting up to 48 hours after administration occurred with metolazone alone. Although absolute and fractional chloride and potassium excretion and urine volume were higher after combined therapy (P less than .05 or better) than after either drug alone, absolute sodium excretion after combination therapy was higher than excretion after bumetanide (P less than .05) but not after metolazone. The percent of fractional sodium excretion after both drugs was greater than after either drug alone (P less than .05). However, excretion of chloride, sodium, potassium, and fluid on the combined therapy day was less than the sum of excretion on each single drug therapy day. Thus, the combination of bumetanide and metolazone did not have a supra-additive effect in normal subjects.

Mechanisms of the synergistic combination of metolazone and bumetanide.

We assessed the effects of metolazone and of bumetanide alone and in combination in normal volunteers when both sodium replete and when sodium deprived. In both conditions a supra-additive natriuretic (P less than .025) and chloruretic (P less than .05) effect of the combination was observed, the magnitude of which was somewhat greater in the sodium-deprived state. Neither phosphate excretion nor an index of delivery to the diluting segment was supra-additive, indicating that the synergistic effect most likely did not emanate from the proximal tubule. In contrast, in the sodium-deprived subjects, free water clearance relative to the delivery term was diminished 72 +/- 2% with the combination compared to 64 +/- 2% for the two diuretics individually (P = .003). These data indicate that the supra-additive response is due to a synergistic effect of the combination at the diluting segment.

Sodium chloride absorption by the urinary bladder of the winter flounder. A thiazide-sensitive, electrically neutral transport system.

The urinary bladder of the winter flounder absorbs NaCl by a process independent of the transepithelial voltage. In contrast to most other epithelia which have a neutral NaCl-absorptive system, the flounder bladder has a high transepithelial resistance. This feature simplifies analysis of the cellular transport system because the rate of ion transfer through the paracellular pathway is rather low. Experiments were designed to distinguish among three possible mechanisms of neutral NaCl absorption: (a) Na/K/2Cl cotransport; (b) parallel Na/H and Cl/OH exchange; (c) and simple NaCl cotransport. A clear interdependency of Na and Cl for net absorption was demonstrated. NaCl absorption was not dependent on mucosal K and was minimally sensitive to loop diuretics. Thus a Na/K/2Cl transport system was unlikely. The mechanism was not parallel exchange as evidenced by insensitivity to amiloride and to 4,4'-diisothiocyano-2,2'-disulfonic stilbene, an inhibitor of anion exchange. In addition, inhibitors of carbonic anhydrase had no effect. Net absorption was almost completely abolished by hydrochlorothiazide (0.1 mM). Its action was rapid, reversible, and effective only from the mucosal surface. Metolazone, a structurally dissimilar diuretic in the benzothiadiazide class had qualitatively similar actions. The mechanism of NaCl absorption in this tissue appears to be a simple interdependent process. Its inhibition by thiazide diuretics appears to be a unique feature. The flounder bladder may be a model for NaCl absorption in the distal renal tubule.

Use of a bioassay in healthy men to evaluate diuretic-spironolactone combinations.

The plasma potassium responses to 1 week's treatment with metolazone 0.625 mg, 1.25 mg and 2.5 mg in combination with spironolactone 50 mg, and metolazone 2.5 alone were examined in a double-blind, crossover study in twelve healthy subjects. Spironolactone attenuated the hypokalaemia induced by metolazone--addition of spironolactone 50 mg to metolazone 2.5 mg raised plasma potassium by 0.18 mmol/l (P less than 0.025). In the presence of spironolactone, a linear log metolazone dose-plasma potassium response relationship (P less than 0.01) was demonstrated. Spironolactone was unable to compensate fully for metolazone's hypokalaemic effect although in combination with metolazone 0.625 mg and 1.25 mg, plasma potassium concentration was maintained close to pretreatment levels. The human bioassay employed provided conveniently quantitative information which allows the rational development of a fixed dose diuretic-spironolactone combination tablet.