Bumetanide Hyperpolarizes Madin–Darby Canine Kidney Cells and Enhances Cellular Gentamicin Uptake by Elevating Cytosolic Ca2+ Thus Facilitating Intermediate Conductance Ca2+-Activated Potassium Channels

Abstract

Loop diuretics such as bumetanide and furosemide enhance aminoglycoside ototoxicity when co-administered to patients and animal models. The underlying mechanism(s) is poorly understood. We investigated the effect of these diuretics on cellular uptake of aminoglycosides, using Texas Red-tagged gentamicin (GTTR), and intracellular/whole-cell recordings of Madin-Darby Canine kidney (MDCK) cells. We found that bumetanide and furosemide concentration-dependently enhanced cytoplasmic GTTR fluorescence by ~60%. This enhancement was suppressed by La3+, a non-selective cation channel (NSCC) blocker, and by K+ channel blockers Ba2+ and clotrimazole, but not by tetraethylammonium (TEA), 4-aminopyridine (4-AP) or glipizide, nor by Cl− channel blockers diphenylamine-2-carboxylic acid (DPC), niflumic acid (NFA), and CFTRinh-172. Bumetanide and furosemide hyperpolarized MDCK cells by ~14 mV, increased whole-cell I/V slope conductance; the bumetanide-induced net current I/V showed a reversal potential (Vr) ~−80 mV. Bumetanide-induced hyperpolarization and I/V change was suppressed by Ba2+ or clotrimazole, and absent in elevated [Ca2+]i, but not affected by apamin, 4-AP, TEA, glipizide, DPC, NFA or CFTRinh-172. Bumetanide and furosemide stimulated a surge of Fluo-4-indicated cytosolic Ca2+. Ba2+ and clotrimazole alone depolarized cells by ~18 mV and reduced I/V slope with a net current Vr near −85 mV, and reduced GTTR uptake by ~20%. La3+ alone hyperpolarized the cells by ~−14 mV, reduced the I/V slope with a net current Vr near −10 mV, and inhibited GTTR uptake by ~50%. In the presence of La3+, bumetanide caused negligible potential or I/V change. We conclude that NSCCs constitute a major cell entry pathway for cationic aminoglycosides; bumetanide enhances aminoglycoside uptake by hyperpolarizing cells that increases cation influx driving force; and bumetanide-induced hyperpolarization is caused by elevating the intracellular Ca2+ and thus a facilitation of the intermediate conductance Ca2+-activated K+ channels.

Sodium-Glucose Transporter-2 (SGLT2; SLC5A2) Enhances Cellular Uptake of Aminoglycosides

“…Heterologous expression of SGLT2 in distal tubule-derived KDT3 cells significantly enhanced GTTR uptake, and this enhanced uptake can be abolished by phlorizin. The residual uptake of GTTR in these cells after phlorizin treatment likely represents GTTR uptake via previously-identified gentamicin-permeant cation channels, as demonstrated previously [14] , [15] , [69] . Furthermore, siRNA knockdown of SGLT2 expression in KPT2 cells reduced cellular susceptibility to gentamicin-induced cytotoxicity.…”

A Mechanism to Explain Ototoxicity in Neonates Exposed to Bumetamide: Lessons to Help Improve Future Product Development in Neonates

“…Besides endocytosis, there are also endocytosis-independent pathways using cation channels [3]. Interestingly, in vitro models have documented that loop diuretics like furosemide or bumetanide enhance aminoglycoside uptake through hyperpolarization of the cell, increasing cation influx cation force [4]. It is therefore reasonable to hypothesize that in the NEMO trial the pharmacodynamic effect of bumetanide stimulated a cation influx force that resulted in intracellular aminoglycoside accumulation, subsequent toxicity, and unavoidable cell death [4].…”

“…Interestingly, in vitro models have documented that loop diuretics like furosemide or bumetanide enhance aminoglycoside uptake through hyperpolarization of the cell, increasing cation influx cation force [4]. It is therefore reasonable to hypothesize that in the NEMO trial the pharmacodynamic effect of bumetanide stimulated a cation influx force that resulted in intracellular aminoglycoside accumulation, subsequent toxicity, and unavoidable cell death [4]. In addition, based on animal studies, the mechanism of megalin-independent ion channel driven accumulation of aminoglycosides has also been suggested to explain the synergism between exposure to noise and aminoglycoside toxicity [5].…”

Bumetanide for neonatal seizures—back from the cotside

“…8 None of the infants included in our study had either high trough or high peak aminoglycoside levels, or hypoglycaemia. It is plausible that aminoglycosides and loop diuretics (such as bumetanide) act synergistically to produce ototoxicity, 9 and so extreme caution is required before co-administering these types of drug. We are also surprised at the authors' statement that "we do not know whether [hypothermia] is also beneficial in humans" in terms of reducing seizure burden.…”