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.

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.

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.

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.

Dose-response relationships for spironolactone in combination with a potassium-wasting diuretic.

The plasma and urine electrolyte responses to repeated doses of spironolactone, 25, 50, and 100 mg, in combination with metolazone, 2.5, 5.0, and 10 mg, were examined in 18 healthy subjects (six at each dose of metolazone). During the period of pharmacologic steady state, there were log linear spironolactone dose-response relationships for plasma potassium, sodium, and bicarbonate (P less than 0.001 in each case) with no contraindication to parallelism between the metolazone groups. In the absence of mineralocorticoid challenge, spironolactone dose-urine electrolyte responses could not be demonstrated. However, after fludrocortisone, spironolactone log dose-response trends were linear with respect to natriuresis (P = 0.027), antikaliuresis (P = 0.020), and log 10 Na/K (P = 0.001), which is usually considered the best single index of renal antimineralocorticoid activity, and exhibited parallelism between the metolazone doses. These observations suggest that a convenient bioassay for aldosterone antagonists in normal men may be provided by the electrolyte responses to repeated doses of such drugs in combination with potassium-wasting diuretics. In view of the limitations of other methods, this approach may have particular relevance to the evaluation of potassium-sparing properties.

Do diuretics have antihypertensive properties independent of natriuresis?

To ascertain whether diuretics have an antihypertensive effect independent of natriuresis, 12 stable patients on maintenance hemodialysis underwent a crossover evaluation with hydrochlorothiazide, 50 mg daily, metolazone, 5 mg daily, or placebo in 4-wk treatment periods for 6 mo. Compliance was assured by pill counts and serum drug concentrations. All patients had daily urine less than 100 ml. Pre- and postdialysis blood pressure, body weight, plasma volume, and plasma renin activity were monitored. Over the 6-mo study period there were no statistically significant changes in any parameter related to diuretic therapy. It is concluded that a functioning kidney with the ability to respond to diuretics with a natriuresis is necessary for the antihypertensive action of diuretics. Direct vascular effects of diuretics to lower peripheral resistance could not be demonstrated in this unique patient population.

Metolazone and spironolactone in cirrhosis and the nephrotic syndrome.

Eighteen patients with hepatic cirrhosis or nephrotic syndrome and having edema and/or ascites were treated during successive periods with metolazone 5 to 40 mg/day, spironolactone 100 mg/day, and with both diuretics concurrently. Metolazone alone produced a marked diuresis, natriuresis, and weight loss in 8 patients. Spironolactone alone had little effect, but the addition of metolazone renewed diuresis and natriuresis and resulted in additional substantial weight losses in all patients responsive to metolazone alone. Concurrent spironolactone and metolazone also induced moderate diuretic effects in some patients who failed to respond to either drug alone. The drugs were well tolerated; the administration of spironolactone with metolazone prevented decreases in serum potassium, which had occurred during treatment with metolazone alone.

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.

Antihypertensive and renin angiotensin effects of metolazone with and without propranolol.

Renin angiotensin system parameters and blood pressure (B.P.) were followed monthly in patients with essential hypertension on metolazone, 5 mg daily for three months and with added propranolol, 40 to 160 mg, for the subsequent three months. On metolazone alone at three months, sitting B.P. declined from 166/108 +/- 14/11 mm Hg to 145/98 +/- 14/9 mm Hg (P less than 0.005). Plasma renin activity (PRA) increased from 3.9 +/- ng/ml/hr to 10.4 +/- 8.6 ng/ml/hr (P less than 0.005); plasma angiotensinogen did not change. Venous blood angiotensin I and II levels (pg/ml) rose initially but returned toward control values. A significant decline in plasma renin substrate reactivity (PRSr) in index occurred. Propranolol addition caused further lowering of only systolic B.P. and predominantly in the standing position, more marked at one month (40 mg) than at three months (160 mg). No significant further changes were observed in any of the measured parameters of renin angiotensin system, except for a rise in PRSr index concomitant with B.P. elevation at three months. Metolazone-induced changes in B.P. showed significant correlations at three months with changes in PRSr index. It is concluded that during chronic metolazone administration, the overall activity of the renin angiotensin system was diminished or unchanged. Propranolol did not inhibit metolazone stimulated PRA but did cause further decline in B.P. in the first two months, unrelated to renin angiotensin system.

Long-term diuretic therapy with metolazone of renal failure and the nephrotic syndrome.

The effects of the new diuretic metolazone were studied in ten patients with chronic renal insufficiency and ten with nephrotic syndrome. Patients were maintained on metolazone for up to 44 months. Beneficial effects of treatment included loss of edema and improved control of blood pressure. The natriuretic effect of metolazone facilitated the use of sodium bicarbonate to treat acidosis in several patients. Concurrent administration of metolazone and furosemide produced a dramatic diuresis in one patient resistant to either diuretic alone. Adverse effects of metolazone therapy were those characteristic of other effective diuretics, Including serum electrolyte losses and hyperuricemia. Initial treatment produced small increases in serum creatinine among patients with renal insufficiency, suggesting that GFR was decreased secondary to diuresis-induced volume depletion. The study demonstrates that metolazone is both safe and effective over long periods of time.

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.

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.

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.

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.