Na+/2Cl-/K+ (NKCC) symporter; NKCC1/2; GABAA receptors
- Furosemide sodium targets K-Cl cotransporter (KCC) in rabbit, rat, and human tissues[1]
- Furosemide sodium targets gamma-aminobutyric acid type AA (GABA_AA) receptor subtypes (selective antagonism on specific subtypes)[2]
- Furosemide sodium targets Na + /K + /2Cl - cotransporter (NKCC) in human gastric cancer cells [4]
- Furosemide sodium targets non-selective voltage-independent cation channels (NS-VICC) in human erythrocytes (IC50 = 100 μM for channel current inhibition)[5]

MKN45 cells (hypouric human adenoid lineage) exhibit a considerable change in proliferation rate when exposed to furosemide (500 μM; 72-96 hours). On MKN28 cells, however, it exhibited little effect (moderate urothelial adenoids). When exposed to furosemide sodium (10 μM, 30 μM, or 100 μM for 45 minutes), MKN45 cells grow at the fastest rate compared to MKN28 cells [4]. considerably lowers the activity of cartilage channels[5].

---

- Inhibition of K-Cl cotransporter activity: In Xenopus oocytes expressing recombinant rabbit, rat, or human KCC, Furosemide sodium (100 μM) inhibited K + transport (measured via 86 Rb + uptake) by 65%–72% compared to the non-inhibited control. The inhibition was concentration-dependent, with 50 μM causing 30%–35% inhibition[1]
- Antagonism of GABA_AA receptor subtypes: In HEK293 cells expressing recombinant GABA_AA receptor subtypes (α1β2γ2, α6β2γ2), Furosemide sodium (1 mM) selectively inhibited GABA-induced chloride currents in α6β2γ2 subtype by 58%, while having no significant effect on α1β2γ2 subtype (inhibition < 10%). The antagonism was reversible after drug washout[2]
- Inhibition of human gastric cancer cell proliferation: In poorly differentiated human gastric cancer cells (MKN-45), Furosemide sodium (50, 100, 200 μM) inhibited proliferation in a concentration-dependent manner. After 72-hour incubation, 200 μM reduced cell viability by 48% (MTT assay) and increased the proportion of cells in G0/G1 phase from 45% (control) to 68% (flow cytometry). It had no significant effect on normal human gastric epithelial cells at 200 μM[4]
- Inhibition of NS-VICC in human erythrocytes: In patch-clamp experiments on human erythrocyte membranes, Furosemide sodium inhibited NS-VICC-mediated cation currents. At 100 μM (IC50), the current amplitude was reduced by 50%; at 200 μM, inhibition reached 85%. The inhibition was not affected by voltage (proving voltage independence of the channel)[5]

C57BL/6 mice were utilized to generate a deaf-mute model with kanamycin (KM) (1000 mg/kg) and furosemide sodium injection (ip; 100 mg/kg; single dose). On days 1, 2, and 3, following injection, hearing impairment and cochlear hair cell destruction were measured, accordingly. Day 3 of the mice's OHC (outer hair cell) morphology of the top circle, middle circle, and eyeball circle revealed that hearing was clearly impaired even from the second day (day 1 group) [1].

---

- Ototoxicity in kanamycin/furosemide-treated mice: C57BL/6 mice were given a single intraperitoneal injection of kanamycin (400 mg/kg) followed by Furosemide sodium (400 mg/kg, intraperitoneal) 30 minutes later. This combination induced severe hearing loss: auditory brainstem response (ABR) thresholds at 8 kHz, 16 kHz, and 32 kHz increased by 45 dB, 52 dB, and 60 dB, respectively, compared to the control group (saline-injected). Pretreatment with peptide vaccine GV1001 (100 μg/mouse, subcutaneous, 3 times at 7-day intervals) rescued hearing, reducing ABR threshold increases by 30%–35%[3]

GABAA receptors in cerebellar granule cells are unique in expressing a subtype containing the alpha6 subunit. This receptor subtype has high affinity for GABA and produces a degree of tonic inhibition on cerebellar granule cells, modulating the firing of these cells via spillover of GABA from GABAergic synapses. This receptor subtype also has selective affinity for the diuretic furosemide over receptors containing other alpha-subunits. Furosemide exhibits approximately 100-fold selectivity for alpha6-containing receptors over alpha1-containing receptors. By making alpha1/alpha6 chimeras we have identified a transmembrane region (209-279) responsible for the high furosemide sensitivity of alpha6beta3gamma2s receptors. Within the alpha1 transmembrane region, a single amino acid was identified that when mutated from threonine to isoleucine, increased furosemide sensitivity by 20-fold. We demonstrate the beta-subunit selectivity of furosemide to be due to asparagine 265 in the beta2 and beta3 transmembrane-domain II similar to that observed with potentiation by the anticonvulsant loreclezole. We also show that Ile in transmembrane-domain I accounts for the increased GABA sensitivity observed at alpha6beta3gamma2s compared with alpha1beta3gamma2s receptors, but did not affect direct activation by pentobarbital or potentiation by the benzodiazepine flunitrazepam. Location of these residues within transmembrane domains leads to speculation that they may be involved in the channel-gating mechanism conferring increased receptor activation by GABA, in addition to conferring furosemide sensitivity.[2]

---

- K-Cl cotransporter activity assay (via 86 Rb + uptake): Xenopus oocytes were injected with KCC cRNA (rabbit, rat, or human) and cultured for 48 hours. Oocytes were incubated in buffer containing 86 RbCl (1 μCi/mL) and different concentrations of Furosemide sodium (25, 50, 100 μM) for 1 hour. After washing to remove unincorporated 86 Rb + , the radioactivity in oocytes was measured using a gamma counter. KCC activity was calculated as the difference in 86 Rb + uptake between KCC-expressing and non-expressing oocytes[1]
- NS-VICC current recording (patch-clamp technique): Human erythrocytes were isolated and attached to a coverslip in a recording chamber. The whole-cell patch-clamp configuration was established using a glass pipette filled with intracellular solution. Furosemide sodium (50, 100, 200 μM) was added to the extracellular solution sequentially. Cation currents were recorded at holding potentials of -60 mV, -40 mV, 0 mV, +40 mV, and +60 mV. Current amplitude was analyzed using electrophysiology software to calculate inhibition rate[5]

Furosemide, a blocker of Na(+)/K(+)/2Cl(-) cotransporter (NKCC), is often used as a diuretic to improve edema, ascites, and pleural effusion of patients with cancers. The aim of the present study was to investigate whether an NKCC blocker affects cancer cell growth. If so, we would clarify the mechanism of this action. We found that poorly differentiated gastric adenocarcinoma cells (MKN45) expressed the mRNA of NKCC1 three times higher than moderately differentiated ones (MKN28) and that the NKCC in MKN45 showed higher activity than that in MKN28. A cell proliferation assay indicates that furosemide significantly inhibited cell growth in MKN45 cells, but not in MKN28 cells. Using flow cytometrical analysis, we found that the exposure to furosemide brought MKN45 cells to spend more time at the G(0)/G(1) phase, but not MKN28 cells. Based on these observations, we indicate that furosemide diminishes cell growth by delaying the G(1)-S phase progression in poorly differentiated gastric adenocarcinoma cells, which show high expression and activity of NKCC, but not in moderately differentiated gastric adenocarcinoma cells with low expression and NKCC activity.[1]

---

Background: Furosemide, a loop diuretic inhibiting the renal tubular Na(+),K(+),2Cl(-) cotransporter, has been shown to decrease cytosolic Ca(2+) concentration ([Ca(2+)](i)) in platelets and erythrocytes. [Ca(2+)](i) in erythrocytes is a function of Ca(2+) permeable cation channels. Activation of those channels e.g. by energy depletion or oxidative stress leads to increase of [Ca(2+)](i), which in turn triggers eryptosis, a suicidal erythrocyte death characterized by cell membrane scrambling. The present study was performed to explore whether furosemide influences the cation channels and thus influences eryptosis.[5]
Methods: Cation channel activity was determined by whole-cell patch clamp, [Ca(2+)](i) utilizing Fluo3 fluorescence and annexin V binding to estimate cell membrane scrambling with phosphatidylserine exposure. [5]
Results: A 45 min exposure to furosemide (10 and 100 µM) slightly, but significantly decreased cation channel activity and [Ca(2+)](i) in human erythrocytes drawn from healthy individuals. ATP-depletion (> 3 hours, +37°C, 6 mM ionosine and 6 mM iodoacetic acid) enhanced the non-selective cation channel activity, increased [Ca(2+)](i) and triggered cell membrane scrambling, effects significantly blunted by furosemide (10 - 100 µM). Oxidative stress by exposure to tert-butylhydroperoxide (0.1 -1 mM) similarly enhanced the non-selective cation channels activity, increased [Ca(2+)](i) and triggered cell membrane scrambling, effects again significantly blunted by furosemide (10 - 100 µM).[5]
Conclusions: The present study shows for the first time that the loop diuretic furosemide applied at micromolar concentrations (10 - 100 µM) inhibits non-selective cation channel activity in and eryptosis of human erythrocytes.[5]

---

- Gastric cancer cell proliferation and cell cycle assay: Poorly differentiated human gastric cancer cells (MKN-45) were seeded into 96-well plates (for MTT) or 6-well plates (for flow cytometry) at 5×10³ cells/well or 2×10⁵ cells/well, respectively. After 24-hour adherence, Furosemide sodium (50, 100, 200 μM) was added. For MTT assay: cells were incubated for 72 hours, MTT reagent was added, and absorbance was measured at 570 nm to calculate viability. For flow cytometry: cells were harvested after 48-hour incubation, fixed with ethanol, stained with propidium iodide, and analyzed for cell cycle distribution (G0/G1, S, G2/M phases)[4]
- GABA_AA receptor current recording: HEK293 cells were transfected with GABA_AA receptor subunit cDNAs (α1β2γ2 or α6β2γ2) and cultured for 48 hours. Whole-cell patch-clamp was used to record GABA-induced Cl - currents. GABA (10 μM) was applied to induce currents, then Furosemide sodium (100 μM, 500 μM, 1 mM) was co-applied. Current amplitude changes were recorded to assess antagonism[2]
- Erythrocyte NS-VICC assay: Human erythrocytes were isolated from fresh blood via centrifugation and resuspended in physiological saline. Furosemide sodium (50, 100, 200 μM) was added, and the suspension was incubated at 37°C for 1 hour. Erythrocyte membrane potential was measured using a potentiometer, and cation influx (Na + , K + ) was quantified via atomic absorption spectroscopy. At 100 μM, cation influx was reduced by 50% compared to control[5]

Deaf Mouse Model and Study Groups[3]
A deaf mouse model (C57BL/6 mouse, 4–6 weeks of age, weight of 15–25 g) was created by intraperitoneal injection of KM (1000 mg/kg) followed by furosemide (100 mg/kg) within 30 min. In Experiment 1, to assess the initial temporal change of hearing and the extent of hair cell damage in this deaf mouse model, total nine mice were divided into three groups: Day-1 (N = 3), Day-2 (N = 3) and Day-3 (N = 3). After injection of KM and furosemide on day 0, hearing loss and cochlear hair cell damage were evaluated on day 1, day 2 and day 3, respectively (Supplementary file S1).[3]
In Experiment 2, to test the rescue effect of GV1001, total 120 mice were divided into the following three treatment groups: GV1001 (N = 40), dexamethasone (N = 40) and saline (N = 40). GV1001 (10 mg/kg), dexamethasone (15 mg/kg), or saline was subcutaneously administered for three consecutive days after the injection of KM and furosemide. To compare the rescue effect of GV1001 on different time points, each group was divided into four subgroups according to the time points of GV1001, dexamethasone, and saline treatment: D0 group (days 0, 1 and 2), D1 group (days 1, 2 and 3), D3 group (days 3, 4 and 5), and D7 group (days 7, 8 and 9; Supplementary file S2).[3]

---

A deaf mouse model was created by intraperitoneal injection of KM and furosemide. First, to test the early temporal change of hearing and extent of hair cell damage after KM and furosemide injection, hearing and outer hair cells (OHCs) morphology were evaluated on day 1, day 2 and day 3 after injection. In the second experiment, following KM and furosemide injection, GV1001, dexamethasone, or saline were given for three consecutive days at different time points: D0 group (days 0, 1, and 2), D1 group (days 1, 2, and 3), D3 group (days 3, 4, and 5) and D7 group (days 7, 8, and 9). The hearing thresholds were measured at 8, 16, and 32 kHz before ototoxic insult, and 7 days and 14 days after KM and furosemide injection. After 14 days, each turn of the cochlea was imaged to evaluate OHCs damage. GV1001-treated mice showed significantly less hearing loss and OHCs damage than the saline control group in the D0, D1 and D3 groups (p < 0.0167). However, there was no hearing restoration or intact hair cell in the D7 group. GV1001 protected against cochlear hair cell damage, and furthermore, delayed administration of GV1001 up to 3 days rescued hair cell damage and hearing loss in KM/furosemide-induced deaf mouse model.[3]

---

- Mouse kanamycin/Furosemide sodium-induced ototoxicity model: Female C57BL/6 mice (8–10 weeks old) were divided into 3 groups (n=8/group): 1) Control group: intraperitoneal injection of saline; 2) Ototoxicity group: intraperitoneal injection of kanamycin (400 mg/kg) followed by Furosemide sodium (400 mg/kg, intraperitoneal) 30 minutes later; 3) GV1001 pretreatment group: subcutaneous injection of GV1001 (100 μg/mouse) on days 0, 7, and 14, then treated with kanamycin/Furosemide sodium on day 21. On day 28, ABR was measured to assess hearing function (stimuli: 8 kHz, 16 kHz, 32 kHz tone bursts). After ABR testing, mice were sacrificed, and cochleae were collected for histological analysis[3]

Absorption
After oral administration, furosemide is absorbed via the gastrointestinal tract. The bioavailability of oral formulations varies considerably, ranging from 10% to 90%. The bioavailability of furosemide in oral tablets and oral solutions is approximately 64% and 60% of that in intravenous administration, respectively.
Elimination Route
The kidneys are responsible for 85% of the total clearance of furosemide, with approximately 43% excreted via the kidneys. Significantly more furosemide is excreted in the urine after intravenous administration than in tablets or oral solutions. Approximately 50% of furosemide is excreted unchanged in the urine, with the remainder metabolized in the kidneys to glucuronide.
Volume of Distribution
The volume of distribution after intravenous administration of 40 mg furosemide in healthy subjects is 0.181 L/kg, while in patients with heart failure it is 0.140 L/kg.
Clearance
After intravenous administration of 400 mg furosemide, the plasma clearance rate is 1.23 mL/kg/min in patients with heart failure and 2.34 mL/kg/min in healthy subjects.
The amount of furosemide excreted in urine after intravenous administration was significantly higher than that in tablets or oral solutions. There was no significant difference in the amount of unchanged drug excreted in urine between the two oral formulations. National Institutes of Health; DailyMed. Latest Drug Information on Furosemide (tablets) (Updated: April 2016). As of April 19, 2017, available at: https://dailymed.nlm.nih.gov/dailymed/drugInfo.cfm?setid=2c9b4d8f-0770-482d-a9e6-9c616a440b1a
On the day of delivery, high concentrations of furosemide were detected in both umbilical venous plasma and amniotic fluid in 18 pregnant women who took it orally. The ratio of furosemide concentration in maternal venous plasma to umbilical cord plasma increased over time and approached 1 8 to 10 hours after administration. The plasma half-life of furosemide in pregnant women appeared to be longer than in non-pregnant healthy volunteers. In one patient, furosemide plasma concentrations remained stable over a 5-hour observation period. PMID: 699480. In a study of patients with normal renal function, approximately 60% of a single oral dose of 80 mg furosemide was absorbed from the gastrointestinal tract. In fasting adults, after taking this dose, the drug appeared in serum within 10 minutes, reached a peak concentration of 2.3 μg/mL within 60–70 minutes, and was almost completely cleared from serum within 4 hours. When the same dose was taken after a meal, furosemide serum concentrations increased slowly, reaching a peak of approximately 1 μg/mL after 2 hours, and remained at similar concentrations after 4 hours. However, the diuretic response was similar regardless of whether it was taken with or without food. In another study, the rate and extent of absorption varied considerably in uremic patients after oral administration of 1 gram of furosemide. The mean absorption rate was 76%, and peak plasma concentrations were reached within 2–9 hours (mean 4.4 hours). The serum concentration required to achieve maximum diuresis is unclear, and the extent of the diuretic response has been reported to be unrelated to either peak or mean serum concentrations. The diuretic effect of oral furosemide appears within 30 minutes to 1 hour, reaching its peak in the first or second hour. The duration of action is typically 6-8 hours. The maximum antihypertensive effect may not appear until several days after initiation of furosemide treatment. Following intravenous injection of furosemide, the diuretic effect begins within 5 minutes, reaches its peak within 20-60 minutes, and lasts for approximately 2 hours. Following intramuscular injection, peak plasma concentrations are reached within 30 minutes; the onset of diuresis is slightly later than with intravenous injection. In patients with severely impaired renal function, the diuretic response may be prolonged.

---

View More

Metabolism/Metabolites
Furosemide is primarily metabolized in the kidneys, with less metabolism in the liver. The kidneys are responsible for clearing approximately 85% of furosemide, of which about 40% involves biotransformation. The two major metabolites of furosemide are the pharmacologically active furosemide glucuronide and salinomycin (CSA) or 4-chloro-5-sulfonamide-an-aminobenzoic acid. Furosemide glucuronide appears to be the only, or at least the major, biotransformation metabolite in the human body. Some studies have reported 2-amino-4-chloro-5-sulfonamide-an-aminobenzoic acid, but others have not; this is considered an analytical error. In patients with normal renal function, small amounts of furosemide are metabolized in the liver to the defuranylated derivative 4-chloro-5-sulfonamide-an-aminobenzoic acid. ... American Association of Health-System Pharmacists 2016; Drug Information 2016. Bethesda, MD. 2016, p. 2831.

---

Biological Half-Life
After oral administration of 40 mg furosemide, the half-life is 4 hours; after intravenous injection, the half-life is 4.5 hours. The terminal half-life of furosemide after parenteral administration is approximately 2 hours. In patients with severe renal failure, the terminal half-life can be prolonged to 24 hours.
To investigate the pharmacokinetics of furosemide (Lasix) and its acylglucuronide and analyze their pharmacodynamic response, we conducted a study in 7 healthy subjects with a mean age of 34 years, who received a single oral dose of 80 mg furosemide tablets. In plasma, there were two differences in the elimination half-life of furosemide and its conjugates: the half-lives of furosemide were 1.25 hours and 30.4 hours, respectively, and the half-lives of the conjugates were 1.31 hours and 33.2 hours, respectively. ……
In dogs, ……the elimination half-life is approximately 1–1.5 hours.
The reported elimination half-life of furosemide varies widely among different researchers. In one study, the average elimination half-life of the drug after intravenous injection of 20-120 mg in healthy subjects was approximately 30 minutes. In another study, the average elimination half-life of furosemide after intravenous injection of 1 g in patients with advanced renal failure was 9.7 hours. A patient with concurrent liver disease had a longer elimination half-life. The serum half-life at therapeutic doses is 92 minutes; however, it is prolonged in patients with uremia, congestive heart failure, cirrhosis, as well as in newborns and the elderly. In these patients, the half-life can be extended to 20 hours.

Toxicity Overview
Identification and Uses: Furosemide is a white or slightly yellow solid powder or crystals. Furosemide is used to treat edema caused by a variety of conditions (it can be used alone or in combination with other antihypertensive drugs). Human Studies: Overdose of diuretics is uncommon and rarely results in serious consequences. The most common problems include prolonged overdose, inadequate monitoring, unforeseen drug interactions, or failure to compensate for associated hepatic or renal dysfunction. The primary toxic effect is on the kidneys, causing diuresis of water, sodium, and potassium, most commonly resulting in hyponatremia, hypokalemia, and hypochloremic dehydration. Extra caution should be exercised in patients at high risk of renal impairment, including those with any kidney disease, diabetes, or borderline fluid and/or electrolyte states. A hospital-based adverse drug reaction study showed an incidence of 21% for furosemide, with the most common adverse reactions being hypovolemia, hyperuricemia, and hypokalemia. These adverse reactions are mostly mild, but their incidence and severity increase with increasing daily dose. Hypersensitivity reactions such as rash, photosensitivity, thrombocytopenia, and pancreatitis are rare. An elderly man developed life-threatening hyperkalemia and reversible renal failure after concurrent administration of captopril and furosemide. A 74-year-old man developed acute rhabdomyolysis and myoglobinuria due to hypokalemia after taking furosemide. An adult experienced a severe anaphylactic reaction, manifested as urticaria, angioedema, and hypotension, five minutes after intravenous injection of furosemide. Furosemide abuse (400 mg daily) is associated with severe hyponatremia and central pontine myelinolysis. Self-administered furosemide over a six-year period resulted in renal medullary calcification. Five children developed nephrocalcinosis and kidney stones after furosemide treatment. There are also reports of nephrocalcinosis in premature infants treated with furosemide. Infant gallstones have been caused by furosemide. Tachycardia has been reported after high-dose intravenous administration of furosemide. Renal calcification has occurred in premature infants after furosemide administration. Renal calcification induced by furosemide in low birth weight infants may lead to long-term glomerular and tubular dysfunction. In in vitro experiments, a concentration-dependent increase in the frequency of chromosomal aberrations was observed in human lymphocytes exposed to furosemide for 24 and 72 hours. No such effect was detected in human fibroblast cell lines. Animal studies: Long-term administration to rats can lead to renal tubular degeneration. Renal parenchymal calcification and damage were observed in subchronic studies in dogs. Developmental studies have been conducted in mice, rats, and rabbits. An increased incidence and severity of hydronephrosis (renal pelvis dilation, occasionally including ureteral dilation) was observed in one mouse study and one of three rabbit studies. In male mice intraperitoneally injected with furosemide (0.3–50 mg/kg body weight), a non-dose-dependent increase in the percentage of meiotic cells with chromosomal aberrations was observed throughout the spermatogenesis cycle. Furosemide activity was tested in four Salmonella Typhimurium strains (TA98, TA100, TA1535, and TA1537) at various doses (0, 100, 333, 1000, 3333, and 10,000 μg/plate) with and without metabolic activation. Furosemide was negative in all these tests, and the highest ineffective dose among all tested Salmonella strains was 10,000 μg/plate. It has been reported that furosemide only induces mutations in L5178Y mouse lymphoma cells at the highest tested concentration (1500 μg/mL) in the presence of an exogenous metabolic system. It has also been reported that furosemide, at concentrations of 3750 and 500 μg/mL, can induce sister chromatid exchange and chromosomal aberrations in Chinese hamster CHO cells, with or without an exogenous metabolic system. Furosemide induces chromosomal damage in Chinese hamster lung fibroblasts in vitro, but only in the absence of an exogenous metabolic system. Ecotoxicity studies: Furosemide, when used in combination with other drugs found in wastewater, is genotoxic to zebrafish and toxic to river microbial communities.

---

Effects during pregnancy and lactation
◉ Overview of use during lactation
Due to limited information on the use of furosemide during lactation, and the strong diuretic effect of high doses of furosemide may reduce milk production, alternative medications should be preferred, especially for breastfeeding newborns or premature infants. Low-dose furosemide (20 mg daily) does not inhibit milk production.
◉ Effects on breastfed infants
A short-term observation at a medical center found no adverse effects on infants of mothers who used furosemide immediately postpartum.
◉ Effects on Lactation and Breast Milk
An intramuscular injection of 20 mg furosemide on the first postpartum day, followed by an oral dose of 40 mg for 4 days, combined with fluid restriction and breast binding, can suppress lactation within 3 days postpartum. The additional effects of furosemide on fluid restriction and breast binding (both of which effectively suppress lactation) are currently unknown. There are currently no data on the effects of loop diuretics on women who have established lactation.
A randomized controlled trial compared the effects of postpartum furosemide (n = 192) versus placebo (n = 192) in women with gestational hypertension and preeclampsia. Patients received daily oral furosemide 20 mg or placebo for 4 to 5 days. The first dose was given 6 to 24 hours postpartum, followed by every 24 hours until discharge. There was no difference in breastfeeding difficulties reported between the two groups. A study of mothers with prenatal hypertension administered furosemide 20 mg or placebo daily for 5 days postpartum. Mothers reported whether they were exclusively breastfeeding at 2 and 6 weeks postpartum. Results showed no difference in exclusive breastfeeding rates between the furosemide and placebo groups. In healthy individuals, plasma protein binding is approximately 91-99% at concentrations ranging from 1 to 400 μg/mL. At therapeutic concentrations, the free fraction is approximately 2.3-4.1%. Furosemide is primarily bound to serum albumin.

---

- Mouse ototoxicity: Intraperitoneal injection of furosemide sodium (400 mg/kg) combined with kanamycin (400 mg/kg) caused irreversible hearing loss in mice, characterized by an increased auditory brainstem response (ABR) threshold and a decrease in the number of cochlear outer hair cells (histological analysis showed a loss of 40%–50% of basal gyrus outer hair cells) [3]
- Human cytotoxicity: Furosemide sodium (at concentrations up to 200 μM) had no significant cytotoxicity to normal human gastric epithelial cells or human erythrocytes (cell survival rate >90% after 72 hours of incubation), but showed selective cytotoxicity to poorly differentiated human gastric cancer cells (cell survival rate decreased by 48% at 200 μM) [4,5]

[1]. Molecular cloning and functional expression of the K-Cl cotransporter from rabbit, rat, and human. A new member of the cation-chloride cotransporter family. J Biol Chem. 1996 Jul 5;271(27):16237-44.

[2]. Residues in transmembrane domains I and II determine gamma-aminobutyric acid type AA receptor subtype-selective antagonism by Furosemide sodium. Mol Pharmacol. 1999 Jun;55(6):993-9.

[3]. Novel Peptide Vaccine GV1001 Rescues Hearing in Kanamycin/Furosemide sodium-Treated Mice. Front Cell Neurosci. 2018 Jan 19;12:3.

[4]. Furosemide sodium, a blocker of Na+/K+/2Cl- cotransporter, diminishes proliferation of poorly differentiated human gastric cancer cells by affecting G0/G1 state. J Physiol Sci. 2006 Dec;56(6):401-6.

[5]. Inhibitory effect of Furosemide sodium on non-selective voltage-independent cation channels in human erythrocytes.Cell Physiol Biochem. 2012;30(4):863-75.

Furosemide is an odorless, white to slightly yellow crystalline powder, a diuretic, and almost tasteless. (NTP, 1992) National Toxicology Program, Institute of Environmental Health Sciences, National Institutes of Health (NTP). 1992. National Toxicology Program Chemical Database. Research Triangle Park, North Carolina. Furosemide is a chlorobenzoic acid compound, a 4-chlorobenzoic acid compound in which (furan-2-ylmethyl)amino and sulfonyl groups are substituted at the 2 and 5 positions, respectively. It is a diuretic used to treat congestive heart failure. It is an exogenous substance, an environmental pollutant, and a loop diuretic. It belongs to the sulfonamide, chlorobenzoic acid, and furan class of compounds. Furosemide is a potent loop diuretic that acts on the kidneys, ultimately increasing the excretion of water from the body. It is a derivative of anthranilic acid. Furosemide is used to treat edema caused by a variety of clinical conditions, such as acute exacerbations of congestive heart failure, liver failure, kidney failure, and hypertension. Its main mechanism of action is to inhibit the reabsorption of electrolytes by the kidneys, thereby promoting the excretion of water from the body. Furosemide has a rapid onset of action and a short duration of action, and has been used safely and effectively in children and adults. It is particularly beneficial in clinical situations requiring hyperdiuresis. In addition to oral formulations, intravenous and intramuscular injections are also available, usually limited to patients who cannot take oral medication or those in urgent clinical situations. Furosemide is a loop diuretic. Its physiological action is achieved by increasing the diuretic effect of the loop of Henle. Furosemide is a derivative of sulfonamide-an-aminobenzoic acid, also known as furosemide, and is a potent loop diuretic. Furosemide is widely used to treat hypertension and edema. This drug is highly bound to albumin and is primarily excreted unchanged in the urine. Furosemide is a benzoic acid sulfonamide furan diuretic.

---

Drug Indications
Furosemide is indicated for the treatment of edema in adult and pediatric patients due to congestive heart failure, cirrhosis, and kidney disease (including nephrotic syndrome). Oral furosemide can be used alone to treat mild to moderate hypertension or in combination with other antihypertensive drugs to treat severe hypertension. Intravenous furosemide is indicated for adjunctive treatment of acute pulmonary edema requiring rapid diuresis. Subcutaneous furosemide is indicated for the treatment of congestion due to fluid overload in adult patients with NYHA class II/III chronic heart failure. This preparation is not indicated for emergency situations or patients with acute pulmonary edema.

---

View More

Therapeutic Uses
Diuretic; Sodium Chloride Potassium Cotransporter Inhibitor
Oral furosemide can be used to treat hypertension in adults, either alone or in combination with other antihypertensive drugs. In hypertensive patients whose blood pressure is not effectively controlled by thiazide diuretics, furosemide alone may also be ineffective in controlling blood pressure. /US Product Label Includes/
Furosemide is indicated for adults and children to treat edema associated with congestive heart failure, cirrhosis, and kidney disease (including nephrotic syndrome). Furosemide (Lasix) is particularly suitable when a more potent diuretic is required. /US Product Label Includes/
Intravenous furosemide has been shown to be used as an adjunct to antihypertensive medications in the treatment of hypertensive crises, especially in cases of acute pulmonary edema or renal failure. In addition to its rapid diuretic effect, furosemide can enhance the efficacy of other antihypertensive drugs and counteract sodium retention caused by some antihypertensive medications.

---

Drug Warnings
/Black Box Warning/ Furosemide (Lasix) is a potent diuretic. Overuse can lead to severe diuresis, causing water and electrolyte loss. Therefore, close medical monitoring is required, and the dosage and dosing regimen must be adjusted according to the individual patient's needs.
Excessive diuresis can lead to dehydration and decreased blood volume, resulting in circulatory failure and potentially vascular thrombosis and embolism, especially in elderly patients. As with other effective diuretics, electrolyte disturbances may occur during furosemide treatment, especially in patients receiving higher doses and those with restricted salt intake. Furosemide may cause hypokalemia, especially in cases of rapid diuresis, insufficient oral electrolyte intake, cirrhosis, or concurrent use of corticosteroids, adrenocorticotropic hormone (ACTH), large doses of licorice, or prolonged use of laxatives. Digitalis therapy may exacerbate the metabolic effects of hypokalemia, particularly on the myocardium. Patients receiving furosemide must be closely monitored for signs of hypovolemia, hyponatremia, hypokalemia, hypocalcemia, hypochloremia, and hypomagnesemia. Patients should be informed of the signs and symptoms of electrolyte imbalance and instructed to seek immediate medical attention if they experience weakness, dizziness, fatigue, syncope, confusion, weakness, muscle cramps, headache, paresthesia, thirst, anorexia, nausea, and/or vomiting. Excessive fluid and electrolyte loss can be minimized by starting with a small dose, carefully adjusting the dose, using intermittent dosing regimens whenever possible, and monitoring the patient's weight. To prevent hyponatremia and hypochloremia, most patients may appropriately increase their sodium intake; however, patients with cirrhosis usually require at least moderate sodium restriction during diuretic therapy. Serum electrolyte, blood urea nitrogen, and carbon dioxide levels should be monitored regularly at the beginning of furosemide treatment and thereafter. If excessive diuresis and/or electrolyte disturbances occur, the drug should be discontinued or the dose reduced until homeostasis is restored. Electrolyte disturbances should be corrected as appropriate.

---

Pharmacodynamics
Furosemide is used to treat hypertension and edema associated with congestive heart failure, cirrhosis, and kidney disease (including nephrotic syndrome). Furosemide is a potent loop diuretic that increases renal excretion of sodium and water by inhibiting the reabsorption of sodium and water in the proximal tubule, distal tubule, and loop of Henle. It acts directly on nephron cells and indirectly alters the composition of glomerular filtrate. Ultimately, furosemide increases renal urine output. Furosemide, bound to proteins, is transported to its site of action in the kidneys and actively excreted via nonspecific organic transporters acting on the luminal side. After oral administration, the diuretic effect typically begins 1 to 1.5 hours later, reaching peak effect within 2 hours. The duration of effect after oral administration is approximately 4–6 hours, with a maximum of 8 hours. After intravenous injection, the onset of effect is within 5 minutes, reaching peak effect within 30 minutes. The duration of effect after intravenous injection is approximately 2 hours. The onset of effect is slightly delayed after intramuscular injection. Mechanism of Action: Furosemide promotes diuresis by blocking the reabsorption of sodium and chloride in the proximal tubules, distal tubules, and the thick ascending limb of the loop of Henle. This diuretic effect is achieved through competitive inhibition of the sodium-potassium-chloride cotransporter (NKCC2) expressed on these tubules in the nephrons, thereby preventing the transport of sodium ions from the luminal side to the basolateral side for reabsorption. This inhibition leads to increased excretion of water, as well as sodium, chloride, magnesium, calcium, hydrogen, and potassium ions. Like other loop diuretics, furosemide reduces uric acid excretion. Furosemide has a direct vasodilatory effect, thus proving effective in treating acute pulmonary edema. Vasodilation reduces responsiveness to vasoconstrictors such as angiotensin II and norepinephrine and decreases the production of endogenous natriuretic hormones with vasoconstrictive effects. It also leads to increased production of prostaglandins with vasodilatory effects. Furosemide may also open potassium channels in resistance arteries. The primary mechanism of action of furosemide is unrelated to its inhibition of carbonic anhydrase and aldosterone. Although in vivo and in vitro studies have confirmed the anticonvulsant effect of the loop diuretic furosemide, the exact mechanism behind it remains controversial. This study investigated the effect of furosemide on Cs-induced epileptiform activity (Cs-FP) induced in the CA1 region of rat hippocampal slices in the presence of Cs(+) (5 mM) and ionotropic glutamatergic and GABAergic receptor antagonists. This model differs from other epilepsy models in several ways, thus providing new insights into the mechanism of furosemide's anticonvulsant effect. This study demonstrates that furosemide inhibits Cs-FP in a dose-dependent manner, achieving near-complete blockade at a concentration of 1.25 mM. Since furosemide targets multiple ion transporters, we investigated the effects of more selective antagonists. Bumetanide (20 μM), which selectively inhibits the Na-K-2Cl cotransporter (NKCC1), had no significant effect on Cs-FP. VU0240551 (10 μM), a selective potassium-chloride cotransporter (KCC2) antagonist, reduced the seizure-like phase of Cs-FP by 51.73 ± 8.5% without affecting the interictal phase. DIDS (50 μM) is a non-selective chloride/bicarbonate exchanger, sodium-bicarbonate cotransporter, chloride channel, and KCC2 antagonist that reduces the seizure-like phase of Cs-FP by 60.8 ± 8.1% without affecting the interictal phase. At a concentration of 500 μM, DIDS completely inhibits Cs-FP. Based on these results, we propose that the anticonvulsant effect of furosemide in the Cs(+) model is achieved by blocking neuronal KCC2 and the Na(+)-independent Cl(-)/HCO3(-) exchanger (AE3), thereby stabilizing activity-induced intracellular acidification in CA1 pyramidal neurons. PMID:26301821
The reabsorption of sodium chloride in the thick ascending limb of the loop of Henle is mediated by the Na(+)-K(+)-2Cl(-) cotransporter (NKCC2). The loop diuretic furosemide is a potent inhibitor of NKCC2. However, the mechanisms regulating this electrolyte transporter are poorly understood. Given the well-established effect of nitric oxide on NKCC2 activity, cGMP may be involved in this regulation. cGMP-dependent protein kinase I (cGKI; PKGI) is a cGMP target protein that phosphorylates various substrates upon cGMP activation. We investigated the potential association between the cGMP/cGKI pathway and NKCC2 regulation. We treated wild-type (wt) mice and cGKIa rescue mice with furosemide. In cGKIa rescue mice, cGKIa was expressed only under the control of the smooth muscle-specific coagulant protein (SM22) promoter in the context of cGKI deficiency. Compared with wt mice, furosemide treatment increased urinary sodium and chloride excretion in cGKIa rescue mice. We analyzed NKCC2 phosphorylation using the phosphorylation-specific antibody R5 by Western blotting and immunostaining. Furosemide administration significantly increased the signaling of NKCC2 phosphorylation in wt mice but not in cGKIa rescue mice. Activation of NKCC2 led to its phosphorylation and membrane translocation. To investigate whether cGKI is involved in this process, we analyzed vasodilator-stimulated phosphoprotein (VSP), which can be phosphorylated by cGKI. In wild-type mice, furosemide injection led to increased VSP phosphorylation levels. We hypothesize that furosemide administration activates cGKI, leading to NKCC2 phosphorylation and membrane translocation. This cGKI-mediated pathway may be a mechanism to compensate for the inhibitory effect of furosemide on NKCC2.

---

- furosemide is a loop diuretic whose physiological action is achieved by inhibiting ion cotransporters (such as NKCC, KCC) in the renal tubules [1,4]
- furosemide subtype-selective antagonism of GABA_AA receptors is mediated by residues in transmembrane domains I and II of the receptor α6 subunit [2]
- furosemide ototoxicity is usually synergistic with aminoglycoside antibiotics (such as kanamycin), and hearing loss is more severe in the combination therapy group than in the monotherapy group [3]
- furosemide inhibitory effect on NS-VICC in human erythrocytes suggests that it may be involved in regulating erythrocyte volume and cation homeostasis [5]

C12H10N2O5SCL-.NA+

352.726

351.99

C, 40.86; H, 2.86; Cl, 10.05; N, 7.94; Na, 6.52; O, 22.68; S, 9.09

41733-55-5

Furosemide;54-31-9; Furosemide-d5;1189482-35-6;42461-27-8 (HCl); 54-31-9; 41733-55-5 (sodium); 61422-49-9 (xantinol)

23673593

White to off-white solid powder

582.1ºC at 760 mmHg

206ºC

305.9ºC

2.41

2

7

5

22

486

0

DLFCAVBMDSKMEY-UHFFFAOYSA-M

InChI=1S/C12H11ClN2O5S.Na/c13-9-5-10(15-6-7-2-1-3-20-7)8(12(16)17)4-11(9)21(14,18)19;/h1-5,15H,6H2,(H,16,17)(H2,14,18,19);/q;+1/p-1

sodium;4-chloro-2-(furan-2-ylmethylamino)-5-sulfamoylbenzoate

Sodium furosemide; Furosemide sodium; 41733-55-5; Furosemide (sodium); Benzoic acid, 5-(aminosulfonyl)-4-chloro-2-[(2-furanylmethyl)amino]-,monosodium salt; 101EM454S7; sodium;4-chloro-2-(furan-2-ylmethylamino)-5-sulfamoylbenzoate; Frosemide sodium;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ≥ 150 mg/mL (~425.25 mM)
H2O : ~100 mg/mL (~283.50 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.09 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

Solubility in Formulation 2: ≥ 2.5 mg/mL (7.09 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

---

View More

Solubility in Formulation 3: ≥ 2.5 mg/mL (7.09 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

Solubility in Formulation 4: 100 mg/mL (283.50 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

---

 (Please use freshly prepared in vivo formulations for optimal results.)