Bumetanide As An Inhibitor Of The Nkcc Cotransporter Biology Essay

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Hypertension is present in 20-30% of the adult population and is particularly prevalent in the developed world. An increased arterial pressure can lead to a variety of diseases including ischaemic heart disease, peripheral vascular disease and cerebrovascular events. All of these are associated with a marked increased risk of mortality and morbidity. Most patients (80-90%) suffer from primary hypertension with unknown cause, while secondary hypertension can have a wide variety of causes including: renal disease, endocrine disorders, congenital vascular diseases, drug use or pregnancy. Alterations in the architecture of resistance vessels, left ventricular hypertrophy and changes in the renal vasculature characterize a state of chronic hypertension. Without treatment, hypertension can result in cerebrovascular disease and coronary artery disease. This frequently leads to death, while patients suffering from hypertension additionally have an increased risk of renal failure and stroke (Kumar and Clark, 2005).

The human body employs various mechanisms to control and regulate blood pressure. The central nervous system regulates blood pressure by controlling heart rate and vessel diameter. The kidneys are the main organs influencing blood pressure as they can modulate blood volume. Low blood pressure activates the Renin-Angiotensin-Aldosterone System (RAAS), which eventually decreases salt and water secretion and increases blood pressure (Silverthorn, 2008). Mechanisms to decrease elevated blood pressure also target the total volume of extracellular fluid, as well as decreasing vascular resistance.

First line treatment of non-severe hypertension mainly comprises therapy without pharmacological intervention. Patients are advised to reduce their weight and alcohol consumption, to follow a low fat and low sodium diet, exercise regularly and in addition, to consume fruits and vegetables. Drug therapy is usually initiated after a six-month period of continuous assessment of blood pressure. The most frequently used drugs are ACE inhibitors or angiotensin receptor antagonists, beta-blockers, calcium channel blockers or diuretics. Loop diuretics, acting on the loop of Henle to increase dieresis, can be used to treat hypertension and renal impairment. Usually however, thiazides, which act on distal convoluted tubules to decrease sodium and water retention, are the first choice (Rang and Dale, 2007).

Bumetanide was developed in the seventies for treatment of hypertensive conditions(SOURCE), but is currently indicated for treatment of edema secondary to heart failure, hepatic or renal disease (Wargo and Banta). Bumetanide belongs to the group of loop diuretics and exerts its action mainly on the Na-K-2Cl cotransporter influencing ion transport. In the kidney Bumetanide stimulates the production of large quantities of urine, in the central nervous system however, the NKCC1 receptor is inhibited by the drug, exerting a possible anti-epileptic effect.

Recently new areas of treatment have been considered for the application of Bumetanide. Thus, in the past decade much research has been conducted investigating the effect of treatment with Bumetanide on neonatal seizures and autism.

Haven't used bumetanide at all in introduction!

I. Physiology

1. The Loop of Henle

The functional unit of the kidney is the nephron. The nephron consists of the proximal tubule, the descending and ascending loops of Henle, the distal tubule and the collecting duct. In the nephron, water and small molecules are partly reabsorbed out of the filtered blood plasma. In the proximal tubule, bulk reabsorption of iso-osmotic fluid takes place. The main function of the loop of Henle is to create a concentrated filtrate. In the distal tubule and the collecting duct, fine regulation of salt and water balance occurs under hormonal influence (Silverthorn, 2008). The loop op Henle consists of a descending limb and an ascending one. Filtrate initially moves through the thin descending limb of Henle, which is permeable to water but not to ions and urea. The thin ascending limb, on the other hand, is impermeable to water but permeable to ions. Filtrate then flows through the thick ascending limb where sodium, potassium, chloride and other cations are actively transported to the blood and urine (Kumar and Clark, 2005). The thick ascending limb is thus responsible for the hypertonic interstitium of the renal medulla.

2. Effect of bumetanide on a physiological level

Bumetanide was developed in the seventies as a treatment for hypertensive conditions (Asbury et al., 1972). Currently, however, it is most commonly indicated for treatment of edema secondary to heart failure, hepatic or renal disease (Wargo and Banta, 2009). Bumetanide belongs to the group of loop diuretics, which exert their action on the thick ascending loop of Henle. These drugs enhance diuresis and decrease blood pressure: they increase the amount of urinary fluid secreted and to a decreased volume of fluid reabsorbed in the thick ascending loop of Henle (TALH). The Na+-K+-2Cl- cotransporter (NKCC) is one of the ion transporters found in this part of the nephron (fig. 1). Bumetanide (and other loop diuretics) binds to, and inhibit the function of this transporter. When the NKCC is inhibited, the volume of urine secreted is enhanced and the fluid in the tubule becomes isosmotic with plasma (reviewed by Russell, 2000).

Inhibition of NKCC2 leads to inhibition of transcellular Na+, K+ and Cl- transport. Mouse and rabbit models have shown that in the presence of vasopressin or hypertonicity, there is a switch of transport mode from the Na+-Cl- transporter to NKCC2 (reviewed by Gamba, 1999). Inhibition of the NKCC2 receptor prevents the re-absorption of Na+, K+ and Cl- in the epithelial cells. The Na+/K+ ATPase on the basolateral side can be seen as the energy source for NKCC2 in normal circumstances, and is less active because of decreased [Na+] in the cell. This means that sodium is not reabsorbed; rather, it is excreted in the urinary fluid. Potassium concentration in the blood, on the other hand, is increased. Moreover, although the TAL is impermeable to water, bumetanide has an effect on the volume of excreted urine. The increased ion concentration in the lumen increases the osmolarity of the urine, and less water is reabsorbed from the collecting duct into the blood, decreasing venous capacitance (and more generally, extra-cellular fluid) (fig. 1)./ As the ion concentration in the lumen increases, so does the osmolarity of the urine, which means that less water is reabsorbed from the collecting duct into the blood. This decreases venous capacitance, and more generally, the volume of extra-cellular fluid (Fig.1).

Re-absorbed or reabsorbed?

Figure 1: Physiology of sodium re-absorption in the thick ascending limb. 50% of the potassium transported via NKCC2 is recycled and

returns to the lumen via the rectifying K+ channel (ROMK). Potassium gradients play a role in the transport of Cl- via the K/Cl transporter, and the Na+/K+ ATPase on the basolateral side can be seen as the energy source for NKCC2 (Gamba, 1999).

II. Physicochemical properties of bumetanide

Bumetanide (3-n-buytilamino-4.phenoxy-5-sulfamoyl-benzoic acid) is a derivative of metanilamide. Its chemical structure (displayed in fig. 2) differs from furosemide, which is another loop diuretic with a similar mechanism of action. Furosemide is derived from suphanilamide and does not have the phenoxy group that is present in bumetanide (Karlander et al., 1973 and Asbury et al., 1972). In addition, bumetanide has a sylfamyl- group and a carboxyl-group at the position of a benzene ring in furosemide (Henning and Lundval, 1973). These chemical groups might indicate the binding site of bumetanide to the NKCC co-transporter, as explained in further details in part (Hofstetter, 1973)

Figure 2: Chemical structure of bumetanide (Hofstetter, 1973F).

Bumetanide comes in the form of a white powder and has a molecular weight of 364.42 (u). Its molecular formula is C17H20N2O5S (Hofstetter, 1974). Bumetanide is only slightly soluble in water because it is a highly lipophilic molecule, but it readily dissolves in acidic or basic environments. Since the pKa values of the functional groups of bumetanide differ (pKa of the carboxylic group is 3.6 and the pKa of the secondary amino group is 7.7), the drug exists in variable forms depending on the solution. Bumetanide is photoliable and consequently needs to be protected from light when being stored. A recent study by Fiori and colleagues (2003) has determined that the drug does not have any phototoxic effects (Fiori et al, 2003).

Many studies that have observed the role of bumetanide in inhibition of the NKCC co-transporter have used [3H]bumetanide, a labeled derivative of bumetanide. Displacement studies (with e.g. furosemide) have shown that [3H]bumetanide binds with the same 'rank order' as bumetanide (Forbrush et al, 1983). Since the derivative binds with the same properties as bumetanide itself, results obtained from these studies are considered to be representative for investigating bumetanide.

What is rank order?

III. Pharmacodynamics

1. The NKCC Cotransporter

NKCC is a transmembrane protein belonging to a larger ion family that includes many cation-chloride cotransporters, such as the NaCl and KCl co-transporters. The NKCC transporter is found in a wide variety of tissues. This protein transports 2 cations and 2 anions (stoichiometry 1Na:1K:2Cl-) from the extracellular fluid into the cell. There is no net movement of charge and hence the transport is both passive and electroneutral. Two isoforms of the NKCC cotransporter have been identified. The NKCC1 transporter is mainly expressed on the apical membranes of secretory epithelia (e.g. glands) to maintain proper levels of electrolytes. In absorptive epithelia, such as formed in the thick ascending loop of Henle (TALH), NKCC2 can be found on the basolateral membrane, and is responsible for electrolyte re-absorption. NKCC2's amino acid sequence is 60% identical to that of NKCC1.

The NKCCs have large hydrophilic amino (NH) and carboxyl (COOH) termini. Both NH and COOH can be phosphorylated from intracellular sites (fig. 3). The 500 residue containing central domain crosses the membrane 12 times (12TM). All these sites (NH, central domain and COOH) are highly conserved among species. Differences in primary structure consequently lead to differences in inhibition, ion selectivity and affinity (Xu et al., 1983). NKCC binds ions in the following order: Na+-Cl-K+-Cl- (fig. 4). The affinity of one ion binding to NKCC influences the affinity of the binding of other ions. This is especially true for the subsequent binding ion.

Thus two principles, the ordered and the cooperative ion-binding hypothesis, apply to the NKCC (Miyamoto et al., 1986). Co-transport of the ions is enhanced when the extracellular fluid increases in hypertonicity, (WHY) but also when intracellular chloride concentrations drop. Activation is established by phosphorylation of intracellular domains. The activity of the NKCC is furthermore influenced by hormones and by changes in cell volume (reviewed by Russel, 2000).

Figure 3: Model of the shark rectal gland NKCC1 (adapted from Payne et al., 1995).

Figure 4: Ordered binding of ions to the NKCC cotransporter (adapted from Russell, 2000).maybe order differently?!

2. Molecular mechanisms of drug action

Bumetanide inhibits NKCC with a high affinity: ~1*10-7M (reviewed by Russel, 2000). Dose response curves can be found in fig. 5 and fig. 6. It must be noted that the affinity of bumetanide is different for the two different isoforms of NKCC. In shark rectal gland, the dissociation constants (Kd) for NKCC1 is higher than 1*10-6M; whereas this constant for NKCC2 (found in TALH and GI-tract in humans?) is significantly lower than 1*10-6M (Lytle et al., 1992). These affinity constants were compared by Isenring et al. (1998) to affinity of bumetanide for NKCC in HEK203 cell (human embryonic kidney cell line). This study found dissociation constants of 0.28*10-6M for NKCC1 and 0.08*10-6M for NKCC2. Thus the Kd was significantly lower for both isoforms in human cells, as compared to cells from shark rectal gland.

Figure 5: Inhibition of the rat NKCC2 by bumetanide.

Inhibitory action measured by Rb+(K+) flux (adapted from Hannert et al., 2002).

Figure 6: Same figure as Fig. 3,?? however IC50 (~10-6.5) indicated.

Both figures show that bumetanide is fully inhibitory at ~10-4M.

The bumetanide-sensitive NKCC2 co-transporter (BSC1) itself has been found in three isoforms. In cells from the rat kidney, these isoforms show different binding properties (Plata et al., 2002), as illustrated by figure 7.

Figure 7: IC50 curves representing inhibition by bumetanide of BSC1 isforms A (circles), B (squares) and F (triangles). (Plata et al, 2002).

One molecule of bumetanide binds to one NKCC co-transporter and fully inhibits the transporter (Forbrush et al, 1983). Bumetanide is specific for NKCC, but no other chloride-cation cotransporters, as long as no more than 10µM is administrated. Higher concentrations of bumetanide do not only lead to inhibition of NKCC channels, but also inhibit Cl-/HCO3- transporter, Cl- channels and KCC transporter. Hence, at these concentrations, bumetanide can be responsible for observations found other than those related to NKCC action (reviewed by Russell, 2000). Not only the concentration of the drug itself, but also the concentration of the co-transported ions influences bumetanide specificity.

The NKCC co-transporter is only fully active when all three co-transported ions (K+, Na+, and Cl-) are present in the extracellular medium (fig. 8). Consequently, bumetanide can only exert its inhibitory effect fully in the presence of extracellular potassium, sodium and chloride. The potency of the drug is inversely related to the concentration of [Cl-] and positively correlated to [K+] and [Na+] (Palfrey et al, 1980). For instance, in turkey RBC(red blood cells?) the dose response curve of bumetanide shifts to the right when [Na+] and [K+] were reduced in the extracellular fluid medium. This indicates a reduced affinity of bumetanide for the co-transporter when potassium and sodium concentrations are lowered. The opposite is true for chloride: the affinity of bumetanide for NKCC is reduced when [Cl-] is increased in the ECF(extracellular fluid). In the absence of any of the three co-transported ions, the binding of bumetanide is reduced to 10-20% of the maximum binding level calculated when all three ions are present (Forbrush et al, 1983). Similar results have been found by Hedge et al, (1992), who studied the role and presence of different ions on bumetanide binding to NKCC in avian erythrocytes.

Figure 8: Experiments measuring the influx of the cotransported ions, controlling for 1 ion (either Na+, K+ or Cl-). Intracellular ion concentrations were kept constant; bumetanide was added after some time to each experiment (adapted from Russell, 2000).

3. Bumetanide binding to the NKCC co-transporter: affinity

As higher concentrations of chloride lower the specific binding of bumetanide to NKCC, various studies have found indications that bumetanide, like furosemide, competes for the second chloride binding site. This hypothesis is based on various findings: bumetanide is negatively charged at physiological pH and might therefore be able to replace the anion Cl-. Moreover, furosemide inhibits NKCC at the second binding site for chloride; and raising the extracellular concentration of chloride in controlled experiments reduces the specific binding of bumetanide to the co-transporter (reviewed by Russell, 2000). Nevertheless, Hedge et al. (1992) in a study on duck erythrocytes proved that bumetanide does not bind to the second chloride binding side. Increasing the extracellular concentration of chloride indeed negatively influenced the binding of bumetanide to the NKCC cotransporter. Hedge et al also found a reduction in Bmax . However, after analysis of the data, no change in affinity was found. These results indicate that although bumetanide inhibits the co-transporter in a non-competitive way, it does not do so by binding to the second chloride-binding site.

Isenring and colleagues (1998) used chimeras to study the critical binding regions of the human and shark NKCC. BRIEFLY EXPLAIN TMs Especially TMs 2, 4 and 7 are different between the shark NKCC1 and the human isoform. This might explain the different kinetics for ion transportation these isoforms have. These TMs in particular are likely to be the helices involved in the translocation pore of Na+, K+ and Cl- (fig. 9). Changes in TM11 and TM12 seem to affect bumetanide binding mostly. No single chimera showed the same affinity as the unchanged NKCC, suggesting either that multiple TMs are involved in bumetanide binding, or that extracellular or intracellular loops are involved in binding. However, it cannot be excluded that some residues of NKCC are involved in both chloride transportation and bumetanide binding. There is no evidence that bumetanide exclusively binds to the EC part of the co-transporter, which is the case for other diuretics. Consequently, this drug might also have its inhibitory site of action intracellularly. This hypothesis is supported by the fact that bumetanide is largely lipophilic; implying that the drug can easily pass the cell membrane (reviewed by Russell, 2000).

Figure 9: Isenring et al. (1998) showed that mainly TM2 is responsible for cation binding, because when this region is altered the affinity of the NKCC for positively charged ions is changed (graphs for Na and Rb). Especially TM4 and TM7 are involved in anion binding (see graph Cl). Moreover, this study showed that bumetanide binding does not happen at the same site as chloride binding (adapted from Isenring et al.,


4. Binding to NKCC: insights from molecular modeling

As mentioned in the section on physicochemical properties, bumetanide has several chemical groups: a phenoxy-group, a sylfamyl-group (X2) and a carboxyl-group (X1). It might be the case that X1 of bumetanide bind to TM4/TM7, as this group of bumetanide is anionic and these TMs are involved in anion binding. The cationic group X3 might bind to TM2 (cation binding) and X2/X4 to TM11/TM12 (fig. 9).

Figure 10: Bumetanide with its chemical groups; the nature of the chemical groups might determine the binding sites of the drug to the different transmembrane parts of the NKCC (adapted from Hannaert, 2000).

One study that visualized molecular modelling of the NKCC2 co-transporter was carried out by Fraser and colleagues (Fraser et al., 2007). The N-terminus of the co-transporter is a potential site for phosphorylation, as mentioned earlier. Moreover, the threonine residues: Thr104, Thr99 and Thr117 are also prone to phosphorylation. This does not explain the entire mechanism of the co-transporter however, since after mutation of the genes expressing these residues NKCC2 retains 50% of its normal activity. One extra site of phosphorylation proposed by Fraser and colleagues is the serine residue 126 (Ser126). Phosphorylation of NKCC2 by an AMP-activated kinase (AMPK) was demonstrated in vitro, in cells of a rat kidney (fig. 11). This newly discovered site could be linked to the action of bumetanide.

Shall we add this part to 'NKCC cotransporter'? In this part we discuss phosphorylation of NKCC as well. Awesome picturesJ Maybe it's fine here because it is linked to the previous part??

Figure 11: NKCC2 associates with AMPK. Laser scanning confocal microscopy was used to co-localize NKCC2 and AMPK in paraformaldehyde-fixed rat kidney. NKCC2 was detected using a T4 monoclonal antibody (A; green fluorescence), with active AMPK detected using an anti-AMPK phospho-Thr172 (B; red fluorescence). Areas of co-localization of AMPK and NKCC2 result in a accumulation of the emission, which appears yellow (Fraser et al, 2007).

Animal Models

- Dog kidney outer medulla cells are used in Forbush et al (1983) to demonstrate binding of [3H]bumetanide to the isolated membranes.

- Duck erythrocytes provide a model for the NKCC receptors (Hegde et al, 1992). Particularly, there seem to be two binding sites for chloride. Indeed, low [Cl-] stimulates binding of bumetanide, whereas high [Cl] inhibits binding. The two sites are recognized in these cells.

- Shark and human NKCCs show 74% identity in amino acid sequence, but also marked differences in binding affinities for ions and bumetanide. (Susanne's part), Isenring et al, 1998.

- In rat astrocytes, bumetanide was shown to be a more potent inhibitor of NKCC2 than Furosemide (Su et al., 2000).

- Turkey RBC

- Rat model (Hannaert et al, 2002): no selectivity of bumetanide for NKCC1 and NKCC2 in activated state. Selectivity for NKCC2 when inactive.

Shall we leave this out? à Maybe we can make a table of it?

Furosemide vs. Bumetanide

The family of loop diuretics has many members, and bumetanide is part of these types of diuretics. All of these different drugs exert their effects on the loop of Henle and increase diuresis. Their properties, however, such as binding affinities and potencies are different. The relative binding affinities of loop-diuretics is as follows: benzmetanide > bumetanide > piretanide > furosemide. Bumetanide is 40 times more potent than furosemide. More specifically, in rat astrocytes bumetanide was shown to be a more potent inhibitor of NKCC2 than Furosemide (Su et al., 2000). It has been proven that the inhibitory site for furosemide is the binding site for the second chloride ion. The second chloride binding site has a lower affinity for chloride and larger affinity for other negatively charged molecules, like sulfate, acetate, gluconate and loop diuretics. However, as described previously, the binding of bumetanide to the NKCC is not as simple and as one-to-one as is the case with furosemide (Kinne et al, 1985; Turner et al, 1988; Palfrey et al, 1980). Therefore, results obtained from studies with furosemide cannot be translated to the action of bumetanide on a molecular level.

Bumetanide's chemical structure (displayed in fig. 2) differs from the structure of furosomide (derived from suphanilamide) in the presence of a phenoxyl group (Karlander et al, 1973; Asbury et al, 1972). Besides the aromatic ring, bumetanide has four active groups, of which X1, X2 and X3 are also present in furosemide (see fig. 9). The X4 (phenoxy) group has high lipophilycity, which makes bumetanide more lipophilic than furosemide. In addition, bumetanide has a sylfamyl- group and a carboxyl-group at the position of a benzene ring in furosomide (Henning and Lundval, 1973).

I think the previous part needs to be integrated elsewhere. à Maybe after discussing chemical structure bumetanide or after 3. Bumetanide binding to the NKCC co-transporter: affinity?

IV. Side Effects

As mentioned before, bumetanide increases the amount of urinary fluid that is secreted by acting on NKCC2 in the loop of Henle. Frequently observed side effects result from a change in water-electrolyte balance. High doses and frequent administration of bumetanide could lead to profound H2O loss, dehydration and electrolyte depletion (BUMEX).

1. Electrolyte Depletion

The observed alternations in electrolyte balance typically reflect the pharmacological activity of Bumetanide. Inhibition of NKCC2 results in decreased serum concentrations of electrolytes Na+, K+ and Ca2+, Cl- and Mg2+.' Bumetanide increases Na+ excretion, which could lead to hypoatremia (Sica, 2004). Hypokalemia (serum [K+] ≤ 4.5 mmol/L) is caused by an increase in the excretion of K+, leading to a decrease in serum K+ levels. The risk of developing hypokalaemia with the use of loop diuretics increases with age. (Zuccalà, et al., 2002). Arrhythmias have been suggested to have a relation with K+ loss; the exact connection still requires more investigation (Sica, 2004). Hyponatremia and hypokalemia often coexist with hypomagnesaemia. Low serum [Mg2+] has been associated with both neurological changes such as mental status changes, muscular irritability, muscle twitching and tetany (very rare). On an ECG hypomagnesaemia may present as prolongation of Q-T and P-R intervals, depression in T waves and ST segment. Furthermore, hypomagnesaemia may also contribute to tachyarrhythmias (Sica, 2004).

2. Binding to NKCC1

The selectivity of bumetanide (and other loop diuretics) for the NKCC1/NKCC2 transporter was studied in rats (Hannaert et al., 2002). Cells from the thick ascending loop (TAL) of Henle, rat thymocytes and rat ertythrocytes were used to determine a difference in selectivity. Both isoforms were found to bind to bumetanide with equal potency (bumetanide pIC50 = 6.48 (TAL NKCC2), 6.48 (thymocyte NKCC1) and 6.47 (erythrocyte NKCC1)). Both cells with the NKCC1 are inhibited with similar potency. When comparing activated NKCC1 and NKCC2, no selectivity of bumetanide for either cotransporter could be found. However, when dephosphorylated and inactive, a slight selectivity can be found for NKCC2, indicating that the renal and gastrointestinal isoform is modestly favored. NKCC1 is expressed in the basolateral membrane of many secretory epithelia, where it regulates Cl- and H2O secretion. In non-epithelial cells this isoform regulates the cell's volume and [Cl]i (Carmosino, et al., 2008). For example, it has been found in the cells of the inner ear, the heart, skeletal muscle and neurons (Wright, 2009; Haas & Forbush, 1998). NKCC1 is also found on vascular smooth muscle, where it has been suggested to effect systemic blood pressure. In rats, the relation between the NKCC1 co-transporter and blood pressure where studied by administering bumetanide. Inhibition of NKCC1 on vascular smooth muscle led to vasodilation of resistance vessels, and these rats showed a decrease in blood pressure (Garg, et al. 2007). These findings suggest that bumetanide may have a hypotensive effect via both NKCC isoforms. Not many adverse side effects resulting from the action of bumetanide on NKCC1 co-transporters have been mentioned in the literature. Further applications of the inhibition of this isoform are discussed in the next section.

V. Secondary Applications of Bumetanide

Recently, new areas of treatment have been considered for the application of bumetanide.

1. Neonatal Seizures

Neonatal seizures are difficult to control and respond poorly to anticonvulsive medication. This unresponsiveness to therapy constitutes a major challenge, as ongoing seizure activity often results in developmental problems regarding the brain. In the developing nervous system GABA, one of the major neurotransmitters of the CNS, activates the GABAa-Rs receptor resulting in Cl- export from neurons via the NKCC1 receptor. The resulting depolarization contrasts with the situation in adults, where GABAa-Rs activation results in transport of Cl- into the cell (via KCC2) and thus leads to hyperpolarization and a low amount of action potential. Dzhala and colleagues (2005) found that the inhibition of NKCC1 by bumetanide produces superior anticonvulsive effects to Phenobarbital in neonates. This indicates that bumetanide could possibly be a future treatment of neonatal seizures.

2. Osteoporosis

A study by Lim et al. (2008) suggested a relation between osteoporosis and hypocalcemia in elderly patients (aged 65 years and older), who used loop diuretics for a long period of time. This longitudinal research included elderly who continuously, regularly or never used loop diuretics. Their bone mineral density (BMD) was measured after an extended period of time. The results showed that the subjects who were continuous loop diuretic users had the greatest average rate of bone loss, about 2.5 times as much as subjects who did not use diuretic treatment. This suggests that there might be a relation between prolonged use of loop diuretics, subsequent increase in urinary calcium excretion and osteoporosis. The biological mechanisms behind this observation are not yet understood and still need more research.

3. Autism

A recent study by Lemonnier & Ben-Ari (2010) has suggested that bumetanide could be used to ameliorate the symptoms associated with infantile autistic syndromes (IAS). Patients with IAS have been associated with a different GABA signaling pattern in comparison to healthy controls. It has been suggested that this difference in signaling results from the accumulation of chloride during brain maturation. The mechanism that is thought to take place of is the same as the mechanism that was described in targeting neonatal seizures, namely reducing chloride concentration by inhibiting NKCC1. It was found that treatment with bumetanide improved behavioral aspects of IAS, most likely by means of reducing [Cl-].