Am J Physiol Renal Physiol 305(7): F931-F942

Novel diuretic targets

ROMK (Kir1.1)

The renal outer medullary K^{(ROMK) channel, or Kir1.1, plays indispensable roles in the regulation of electrolyte and extracellular fluid-volume homeostasis by the kidney (}45, 148, 152). ROMK is the founding member of the inward rectifier potassium (Kir) channel family (58, 160) and is encoded by KCNJ1, 1 of 16 Kir channel genes in mammals (57). Three biophysically indistinguishable ROMK splice variants are differentially expressed in several nephron segments, including the thick ascending limb of Henle (TALH), distal convoluted tubule (DCT), and collecting duct (CD) (56). In the TALH (Fig. 1C), ROMK forms a ∼35-pS K^{-secretory pathway that is functionally coupled to the Na}^-K^-2Cl^{cotransporter and loop diuretic target NKCC2, which catalyzes the first step in the transepithelial NaCl reabsorption in this nephron segment (}49). Approximately 30% of the total filtered NaCl is reabsorbed in the TALH, and this contributes to a hypertonic medullary interstitium that promotes distal osmotic water reabsorption and generation of concentrated urine (9, 15). NKCC2 mediates the electroneutral symport of Na^{, K}^{, and 2 Cl}^{ions across the luminal membrane (}Fig. 1), after which Na^{and Cl}^{are transported across the basolateral membrane via the Na}^-K^{-ATPase and ClC-Kb, respectively. Since the concentration of K}^{in the tubular fluid is much lower than that of Na}^{or Cl}^{, the continual replenishment of tubular fluid K}^{by ROMK is necessary to maintain NKCC2 activity and NaCl reabsorption. A 70-pS K}^{channel is also expressed in the TALH and can account for up to 70% of the luminal K}^{conductance (}149). The molecular identity of the 70-pS channel has not yet been determined, but the 30- and 70-pS channels are both absent in the TALH of KCNJ1-null mice. This observation has led to the proposal that ROMK is a necessary subunit of the 70-pS channel (90). Inhibition of luminal K^{conductance by removal of K}^{or with the nonspecific blocker barium inhibits transepithelial NaCl reabsorption in isolated TALH segments (}50, 55).

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Fig. 1.

Putative diuretic targets. A: schematic of the nephron and vasa recta spanning the cortex, outer medulla, and inner medulla. AQP, aquaporin. B: thin ascending limb. CLC-Ka mediates Cl^{flux across both the apical and basolateral membranes. This Cl}^{flux helps to generate the steep solute gradient in the inner medulla that drives water reabsorption from the tubular fluid to the blood.}C: thick ascending limb. Na^-K^-2Cl^{cotransporter (NKCC2) mediates entry of Na}^{, K}^{, and Cl}^{across the apical membrane. Since luminal K}^{concentration is low compared with Na}^{and Cl}^{concentrations, K}^{must recycle across the apical membrane through the renal outer medullary potassium channel (ROMK) to maintain NKCC2 activity and NaCl reabsorption. Kinases such as STE20/SPS1-related proline/alanine-rich kinase (SPAK) and OSR1 independently regulate NKCC2 phosphorylation and activity. ClC-Kb mediates basolateral Cl}^efflux.D: distal convoluted tubule. NCC mediates NaCl entry across the apical membrane. SPAK stimulates NCC phosphorylation and activity. ClC-Kb mediates Cl^{exit across the basolateral membrane. Since extracellular K}^{concentration is low compared with Na}^{concentration, K}^{must recycle across the basolateral membrane through Kir4.1/5.1 to maintain Na}^-K^{-ATPase activity.}E, top: principal cell of the collecting duct. The epithelial sodium channel (ENaC) mediates apical Na^{entry. The resulting lumen negative potential drives K}^{secretion via ROMK.}E, middle: intercalated cell of the collecting duct. Pendrin mediates Cl^/HCO₃^{exchange across the apical membrane of type B and non-A, non-B intercalated cells. ClC-Kb mediates Cl}^{exit across the basolateral membrane. Alkalinization of the tubular fluid in the collecting duct lumen also stimulates apical Na}^{entry via ENaC across principal cells.}E, bottom: principal cell of the collecting duct. Arginine vasopressin (AVP) activates vasopressin-2 receptors in the basolateral membrane and stimulates apical insertion of AQP2 water channels to initiate water transport across the collecting duct. AVP also stimulates apical insertion of UTA-1/3 urea transporters to initiate rapid urea transport into the inner medullary interstitium. UT-B urea transporters in the vasa recta operate to recycle, trap, and maintain a high level of urea concentration in the inner medulla to drive water absorption in the collecting duct.

ROMK constitutes a major pathway for regulated K^{excretion in the mineralocorticoid-sensitive distal nephron (}45, 148, 152). Transepithelial K^{secretion in principal cells of the CD is a two-step process, whereby K}^{is initially pumped across the basolateral membrane by the Na}^-K^{-ATPase and subsequently conducted across the luminal membrane through ROMK (}Fig. 1E). K^{secretion shifts the luminal membrane potential toward the Nernst potential for K}^{, thus increasing the electrochemical driving force favoring Na}^{influx through ENaC, the rate-limiting step in Na}^{reabsorption in the CD (}Fig. 1E). ROMK inhibition with barium depolarizes the luminal membrane potential difference and reduces the driving force for ENaC-mediated Na^{reabsorption (}71).

The discovery of autosomal recessive loss-of-function (LOF) mutations in KCNJ1 in patients with type II Bartter syndrome (129) and studies in KCNJ1-deletion mice (87, 91) confirmed the importance of ROMK in renal function. The clinical presentation of type II Bartter's syndrome includes hypernatremia, hypokalemic alkalosis, polyuria, and low blood pressure (8). More recently, Lifton and colleagues (66) found that heterozygous carriers of inactivating KCNJ1 mutations have lower blood pressure and are protected from development of hypertension. These observations provide strong genetic validation for ROMK as a diuretic target and support the notion that pharmacological inhibitors of ROMK may lower blood volume and pressure without causing derangements in serum electrolytes. Specifically, by inhibiting Na^{reabsorption in the TALH and CD and blocking K}^{secretion in the distal nephron, ROMK inhibitors may induce natriuresis and limit the urinary loss of K}^{observed with conventional loop and thiazide diuretics. Furthermore, considering that ROMK participates in NaCl reabsorption in multiple nephron segments, it is conceivable that ROMK inhibitors may induce natriuresis and diuresis more effectively than conventional loop, thiazide, or K}^{-sparing diuretics, which target transport pathways present in only one nephron segment.}

Clark and colleagues (22) showed that administration of the sulfonylurea K_ATP channel inhibitor glyburide to rats led to a rapid and dose-dependent increase in renal Na^{excretion without affecting K}^{excretion. Wang et al. (}146, 147) later showed in patch-clamp experiments and isolated, perfused renal tubules that glyburide, as well as a nonsulfonylurea K_ATP channel antagonist, inhibit the ∼35-pS channel ROMK channel and reduce transepithelial Na^{and K}^{transport in the TALH and CD. Although these observations are consistent with ROMK-mediated effects, the high doses used and potential for off-target effects complicate interpretation of the data. Antagonists with higher affinity and greater selectivity for ROMK over other channels expressed in the nephron are required to verify ROMK as a diuretic target. With few exceptions, however, the molecular pharmacology of ROMK and most other Kir channels has remained largely undeveloped (}11), consequently stalling efforts to assess ROMK's therapeutic potential. However, the wait may soon be over, as targeted drug-discovery campaigns are leading to the development of promising pharmacological tools.

Denton and colleagues (78) employed high-throughput screening (HTS) to interrogate a small-molecule library of ∼225,000 compounds from the National Institutes of Health (NIH) Molecular Libraries Small-Molecule Repository (MLSMR) for pharmacological modulators of ROMK. This screen led to the discovery of VU590 (Fig. 2A), which inhibits ROMK currents by 50% at a concentration (IC₅₀) of ∼290 nM and exhibits at least 20-fold selectivity over Kir7.1 (IC₅₀ ∼8 μM), Kir2.1 (IC₅₀ >>10 μM), and Kir4.1 (IC₅₀ >>10 μM). The chemical structure of VU590 is shown in Fig. 2A. Lead optimization efforts to improve its potency were unsuccessful but revealed that the two nitro groups flanking the cryptan linker region of the compound are indispensable for VU590 potency. VU590 inhibits K^{secretion in isolated, perfused rat collecting ducts (Liu W and Satlin L, unpublished observations), consistent with inhibition of ROMK. However, given the limited selectivity of VU590 for ROMK over Kir7.1, both of which are expressed in the collecting duct (}102), these effects could be mediated by inhibition ROMK, Kir7.1, or both channels.

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Fig. 2.

Development of small-molecule ROMK inhibitors. A: chemical structure of VU590, a small-molecule inhibitor of ROMK and Kir7.1 discovered in an HTS at Vanderbilt University. Concentrations that inhibit channel activity by 50% (IC₅₀) are shown. B: BNBI, a weak but selective ROMK inhibitor identified in the HTS. C: VU591, an improved BNBI derivative that exhibits ∼30-fold higher potency toward ROMK. The asterisk (*) indicates VU591 is selective for ROMK over other Kir channels and >65 other ion channels and receptors (see Ref. 11 for details). The central linkers of VU590, BNBI, and VU591 are shown in bold to emphasize the common nitrogen- and oxygen-containing structures, which drove the medicinal chemistry efforts to optimize VU591 (see text for details). D: chemical structure of compound 5, a ROMK inhibitor discovered from an HTS at Merck. E: compound 30, a derivative of compound 5 with improved pharmacokinetic properties to enable in vivo pharmacology.

In an effort to develop a more selective ROMK antagonist, the investigators turned to another inhibitor identified in the screen, termed BNBI (Fig. 2B) (10). Unlike VU590, BNBI is a relatively weak (i.e., IC₅₀ ∼8 μM) ROMK channel inhibitor. Importantly, however, BNBI has no effect on Kir7.1 at doses up to 100 μM. As shown in Fig. 2, A and B, both VU590 and BNBI contain nitro groups flanking a variable seven-atom linker region containing two nitrogen atoms. A notable difference between the two compounds is the absence of a central oxygen atom in the linker of BNBI (Fig. 2B). Remarkably, addition of the oxygen increased the potency of the BNBI-derivative by 30-fold (IC₅₀ = 240 nM). The new compound was termed VU591 (Fig. 2C). VU591 is highly selective for ROMK over other members of the Kir channel family and >65 other ion channels and receptors. As predicted, VU591 inhibits K^{excretion in the isolated collecting duct (}10). Thus VU591 may afford the potency and selectivity to probe ROMK as a diuretic target in vivo.

Merck Research Laboratories recently disclosed their program to develop ROMK-directed diuretics with the publication of several nanomolar-affinity small-molecule ROMK antagonists (135). A high-throughput screen of ∼1.5 million compounds revealed a hit containing a 4-nitrophenyl group identical to that found in VU590 (Fig. 2A). Although it was only moderately potent (IC₅₀ ∼5 μM), the compound was highly selective for ROMK over Kir2.1 and Kir2.3. Purification of the molecule by HPLC led to a complete loss of ROMK inhibitory activity, suggesting the active component was a minor contaminant in the sample. Indeed, synthesis of a suspected impurity yielded a potent ROMK inhibitor (IC₅₀ ∼50 nM) that maintained selectivity over Kir2.1 and Kir2.3 (IC₅₀ >100 μM). As shown in Fig. 2D, the compound, termed “5,” consists of a piperazine ring flanked by two nitro-phenyl groups. Similar to VU590 (Fig. 2A), their initial lead optimization efforts revealed that the nitro groups are important for potency toward ROMK. Unfortunately, they also found that 5 is a potent inhibitor of the cardiac potassium channel hERG, which is predictive of cardiac toxicity and a major concern in drug development. After several rounds of additional chemistry, a compound termed “30” (Fig. 2E) was found to be potent (IC₅₀ ∼52 nM) and highly selective for ROMK over Kir2.1, Kir2.3, Kir4.1, and Kir7.1. Compound 30 also exhibited moderate (i.e., ∼3-fold) selectivity over hERG and satisfactory preclinical pharmacokinetic properties.

Foster et al. (41a) recently proposed that ROMK is a component of the mitochondrial K_ATP (MitoK_ATP) channel complex, which is involved in cardiomyocyte and neuronal ischemic preconditioning. A proteomic analysis of mitochondrial inner membrane protein fractions and RT-PCR analysis led to the identification of several ROMK splice variants in the heart, brain, and liver. The ROMK2 splice variant, which lacks 19 N-terminal amino acids present in the ROMK1 variant (160), is targeted to mitochondria of Chinese hamster ovary cells and H9C2 heart-derived immortalized cells by a mitochondrial localization sequence in the N terminus of the channel. The ROMK peptide inhibitor tertiapin-Q dose dependently inhibited flux of the K^{surrogate thallium in rat heart mitochondria, albeit with an IC}₅₀ three orders of magnitude lower than that observed for heterologously expressed ROMK. Consistent with a role in mitoK_ATP function, ROMK2 overexpression is cytoprotective against oxidative stress, whereas knockdown of ROMK enhances cell death. While this study points to a potentially critical and previously unappreciated role of ROMK in ischemic preconditioning, it also raises concerns that ROMK antagonists developed for use as diuretics may have untoward consequences on mitochondrial function. The development and use of small-molecule ROMK inhibitors should address this important and timely question.

Kir4.1/5.1

Recent genetic evidence suggests that heteromeric Kir4.1/Kir5.1 channels could represent novel drug targets for hypertension. Inactivating mutations in the gene encoding Kir4.1, KCNJ10, give rise to SeSAME (or EAST) syndrome, a complex disorder presenting with seizures, sensorineural deafness, ataxia, intellectual disability, and electrolyte imbalance (13, 122). The neurological deficits are presumably due in part to loss of extracellular “K^{buffering” mediated by homo- and heteromeric Kir4.1 channels expressed in astrocytes and glial cells (}27). The renal consequences, which include polyuria, hypokalemia, metabolic alkalosis, and elevated renin and aldosterone, are consistent with impaired NaCl reabsorption in the DCT. In the DCT, apical NaCl absorption is mediated by NCC, the thiazide-sensitive NaCl cotransporter (Fig. 1D). Na^{is pumped out of the cell, across the basolateral membrane, in exchange for K}^{by the Na-K-ATPase. Cl}^{moves down its electrochemical gradient and out of the cell through ClC-Kb. Kir4.1/5.1 channels are expressed in the basolateral membrane of the DCT (}Fig. 1D), where they perform at least two important functions. First, they recycle K^{back out across the basolateral membrane to help maintain the activity of the Na}^-K^{-ATPase. Second, Kir4.1/5.1 hyperpolarizes the basolateral and luminal membrane potentials and thus facilitates the electrogenic exit of Cl}^{ions and entry of Na}^{ions, respectively.}

Deletion of KCNJ10 in mice recapitulates the salt-wasting phenotype of subjects with SeSAME/EAST syndrome (13), whereas inactivation of the Kir5.1-encoding gene KCNJ16 paradoxically increases renal NaCl reabsorption (103). Heteromeric Kir4.1/5.1 channels are critically regulated by intracellular pH (pH_i), and unlike homomeric Kir4.1, Kir4.1/5.1 is partially inhibited at physiological pH_i. Patch-clamp analysis revealed that as a consequence of KCNJ16 deletion, and loss of this negative pH_i-dependent regulation, there is an increase in the basolateral homomeric Kir4.1 activity. The ensuing increased K^{conductance is proposed to stimulate transepithelial Na}^{and Cl}^{reabsorption in the DCT by increasing the electrochemical driving forces for these ions (}Fig. 1D) (122).

Kir4.1/5.1 antagonists would be expected to mimic the actions of thiazide diuretics, by indirectly inhibiting NCC-mediated NaCl reabsorption in the DCT. Importantly, however, inhibitors of Kir4.1/5.1 may offer some advantages over conventional diuretics due to the localization of the channel on the basolateral membrane (60, 76, 89). Loop and thiazide diuretics are first secreted into the renal tubular fluid by organic anion transporters and multidrug resistance proteins present in renal proximal tubule cells before reaching their sites of action on the luminal membrane (53, 54, 141, 142; for a review see Ref. 155). Competitive interactions between diuretics and other substrates, including β-lactam antibiotics, NSAIDs, antivirals, and organic acid in the setting of renal failure, can limit their secretion and natriuretic effects. Kir4.1/5.1 inhibitors acting directly on the basolateral membrane would presumably avoid this limitation of secreted diuretics.

Currently, the molecular pharmacology of Kir4.1 is limited to neurological drugs exhibiting weak off-target effects on the channel. These include the selective serotonin reuptake inhibitors fluoxetine and sertraline, as well as the tricyclic antidepressants amitriptyline, desipramine, imipramine, nortriptyline (42, 101, 134; for a review, see Ref. 11). There are no known inhibitors that discriminate between homomeric and heteromeric Kir4.1-containing channels. Identifying subtype-selective modulators will be important moving forward, because of the broad tissue distribution of Kir4.1 and potential for pleiotropic effects of general Kir4.1 inhibitors. Kir4.1 is broadly expressed in the periphery (i.e., kidney, stomach) and central nervous system (brain, spinal cord, retina, cochlea). In the kidney, Kir4.1 appears to exist predominantly as a heterotetramer with Kir5.1 (60, 76, 89). Developing small-molecule inhibitors of heteromeric Kir4.1/5.1 channels that are excluded from the central nervous system by the blood-brain barrier may enable the selective inhibition of the channel in renal DCT cells.

ClC-Ka/b

The chloride channel (CLC) family, with nine human homologs, is an eclectic group of anion-selective channels and transporters. Members share a similar molecular scaffold and selectivity for anions (20, 28, 29, 64, 94, 162), but vary significantly in their physiological distribution, function, and fundamental mechanisms. About half of all CLCs are secondary active transporters, catalyzing the stoichiometric exchange of two Cl^{ions for one proton, while the other half are passive channels, catalyzing rapid downhill movement of Cl}⁽1, 109, 120). Both types play important roles in kidney tubular function (23, 26, 30, 86, 88, 110, 151). In the channel branch of the family, the two kidney-specific homologs, ClC-Ka and ClC-Kb, have central roles in tubular Cl^{reabsorption that make them important targets for novel therapeutic interventions (}36, 41, 61, 63, 65, 75).

ClC-Kb is localized to the basolateral membrane in the TALH (Fig. 1C), DCT (Fig. 1D), and CD (Fig. 1E) (139). Mutations in ClC-Kb cause Bartter's syndrome type III, which is characterized by hypokalemic alkalosis with salt wasting and low blood pressure (127). The low blood pressure associated with defective ClC-Kb function suggests ClC-Kb is a target for antihypertensive drugs (41, 81). This suggestion is further supported by the observation of an association between a common ClC-Kb polymorphism that increases channel activity and a predisposition to hypertension (7, 37, 62, 73, 126). However, it should be noted that such a predisposition has not been detected in all studies (17, 37, 73, 132). It has been suggested that salt-loading conditions as well as sex and ethic segregation need to be considered, and that larger studies are necessary to resolve the issue (73, 126, 150).

ClC-Ka is expressed in both the apical and basolateral membranes of the thin ascending limb (92, 140) (Fig. 1B). It catalyzes the Cl^{flux necessary for maintaining the steep solute gradient in the kidney medulla and provides the driving force for water absorption from the urine to the blood (}72). In mice, genetic deletion of ClC-K1 (the mouse ortholog of human ClC-Ka) dissipates this solute gradient in the inner medulla and leads to an increase in urine water excretion (92). Importantly, deletion of ClC-K1 does not increase urine NaCl excretion or decrease extracellular fluid volume because renal NaCl transport in the more distal segments of the kidney tubule remain intact (2). This finding suggests ClC-Ka inhibitors may be useful as therapeutics to treat hyponatremia which develops in diseases that impair free water excretion, such as in decompensated heart failure, decompensated cirrhosis, renal failure, and syndrome of inappropriate anti-diuretic hormone secretion (41).

Traditional diuretics are not ideal therapies for hyponatremia because they can actually exacerbate hyponatremia by inducing excretion of Na^{in excess of free water. Thus a new class of pharmacological agents called aquaretics that selectively enhance renal water excretion has been developed. To date, antagonists that inhibit vasopressin-2 receptors (V2Rs) are the only class of aquaretics that have been approved by the FDA for the treatment of hyponatremia (}24). Although these inhibitors are effective for treatment of hyponatremia (74), some drawbacks of their clinical use have emerged. For example, not all patients respond optimally to V2R inhibitors (52), and their use with CYP3A inhibitors (which includes numerous and diverse drugs) is contraindicated (4). Response failure to V2R inhibitors has been attributed to increased thirst (52), increased responsiveness of the receptor to its ligand (163), and the presence of activating mutations in the receptor gene in the general population (25). In addition, the FDA recently issued a safety announcement that tolvaptan should not be used for longer than 30 days and is contraindicated in patients with liver disease. Risk of liver failure was observed in an open-label extension study of a recent clinical trial using tolvaptan to treat autosomal dominant polycystic kidney disease (138). This safety announcement seriously limits the utility of tolvaptan, and possibly the entire class of V2R antagonists. Thus, if prevention or treatment of hyponatremia is clinically desirable, aquaretics with different mechanisms of action will need to be developed. Inhibiting Cl^{transport in the thin ascending limb with a CLC-Ka inhibitor would dissipate the medullary concentration gradient, and hence the driving force for water movement; this would represent a novel mechanism for enhancing water excretion by the kidney.}

In addition to occurring in the diseases mentioned above, hyponatremia can be a serious side effect of thiazide diuretics. Thiazide-induced hyponatremia can lead to cognitive impairment, unsteady gait, falls, and hip fractures (6), which are particularly problematic in the elderly patient population (124, 133), as well as lethargy, vomiting, confusion, and even seizures (21). A combined regimen of a ClC-Ka inhibitor and thiazide diuretics would result in an increase in urine NaCl and water excretion so that hypertension can be controlled without causing hyponatremia. This strategy could lead to wider use of thiazide diuretics as a first-line therapy for hypertension.

ClC-Kb and ClC-Ka both are localized almost exclusively to the kidney and inner ear (125). In the inner ear, they are expressed in the basolateral membrane of the stria vascularis, where they are required for maintaining the K^{secretion performed by these cells (by allowing the parallel movement of Cl}^{) (}33). The redundant role of the ClC-K channels in the inner ear is indicated by the fact that loss of either ClC-Ka or ClC-Kb function has no effect on hearing, whereas loss of both results in deafness; simultaneous loss of both ClC-Ka and ClC-Kb function can occur either via direct effects on both ClC-Ka and ClC-Kb (100, 121), or via loss of function of the required accessory subunit Barttin (12, 33). In either case, the resulting disease (Bartter's syndrome type IV) involves severe renal salt wasting and bilateral sensorineural deafness. To preclude effects on hearing, it is crucial to develop drugs that are specific to ClC-Kb over ClC-Ka or vice versa.

In general, the pharmacology of CLC proteins is poorly developed. The so-called classic “chloride-channel inhibitors” have no selectivity among completely unrelated chloride channels, and are even known to inhibit cation channels (3, 18, 34, 67, 79). Nevertheless, recent progress demonstrates the potential for development of novel ClC-specific compounds. Compounds specific to the ClC-2 channel and to the ClC-ec1 antiporter have recently been described (59, 137). Of relevance to the kidney, Pusch and colleagues (48) have systematically characterized and developed small-molecule inhibitors and activators of the ClC-K channels. In a series of elegant studies, they evolved the low-affinity (mM) ClC-1 inhibitor p-chlorophenoxy-propionic acid (CPP) (5, 112) into a low-micromolar inhibitor of ClC-Ka, MT-189 (80, 83, 84, 108) (Fig. 3; see below). Administration of this drug to rats induced water excretion, as expected for a ClC-Ka inhibitor (81). The lack of decrease in solute accumulation in the inner medulla in this study suggests that ClC-Kb (discussed above) or other targets may be additional mediators of the effects. The excellent bioavailability of MT-189 motivates further development of this scaffold to improve specificity. The in vitro threefold selectivity of MT-189 for ClC-Ka over ClC-Kb (which is 90% identical to ClC-Ka) (83) arouses hope that it may be feasible to engineer more stringent specificity through molecular docking and rational design. Toward this end, a residue near the extracellular vestibule of the channel has been identified as a major determinant of inhibitor selectivity (83, 93).

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Fig. 3.

Evolution of a ClC-K channel inhibitor. Evolution of CLC-K channel inhibitors is shown, as described in the text. CPP (a) was tested against a chimera of ClC-Kb and mouse ClC-Ka (ClC-K1) (79).

In parallel studies, Pusch and colleagues (82, 83, 107) also characterized the interactions of another class of compounds, the fenamates, with the ClC-K channels. The fenamates are NSAID that are also known to inhibit and/or activate a wide variety of ion channels (14, 19, 44, 77, 117, 153). A ClC-K channel activator could be of use in amplifying residual ClC-Kb activity in patients with Bartter's syndrome (48, 99, 136). Extensive mutagenesis studies of ClC-Ka and ClC-Kb identified a set of three residues important for the activating effect (161). These residues form a cluster in three-dimensional space (as mapped onto a ClC-K homology model; see Ref. 47) in a region distinct from the site implicated in inhibition by MT-189. While it is not yet known whether these three residues constitute a binding site, these results will facilitate studies to design more specific drugs and thus further develop CLC pharmacology.

Pendrin

Pendrin is encoded by the Slc26a4 gene and mediates Na^{-independent Cl}^/HCO₃^{exchange. Inactivating mutations of}Slc26a4 results in Pendred's syndrome (123), which is characterized by hearing loss and goiter (106). In the kidney, pendrin is expressed in type B and non-A, non-B intercalated cells of the aldosterone-sensitive distal nephron (68, 116, 144), defined as the late DCT, connecting segment, and cortical CD. Pendred's syndrome is not associated with electrolyte abnormalities in either humans or mouse models (35, 116); however, under dietary Cl^{restriction, LOF mice reveal a requirement for pendrin in maintaining acid-base balance, extracellular fluid volume, and blood pressure (}145). Interestingly, although pendrin mediates Na^{-independent ion exchange, deletion of pendrin leads to urinary Na}^{wasting, indicating that pendrin interacts with Na}^{transport pathways (}69). Indeed, expression of the epithelial sodium channel (ENaC) is reduced in the kidneys of pendrin knockout mice under conditions of NaCl restriction or mineralocorticoid administration (69). In a series of elegant experiments, Wall and colleagues (105) demonstrated that raising the luminal HCO₃^{concentration in kidney tubules reversed the decrease in ENaC expression and function in the cortical collecting ducts of pendrin knockout mice. These findings suggest that pendrin, by setting the HCO}₃^{concentration or pH of the tubular fluid in the collecting duct, regulates ENaC activity and urine Na}^excretion.

Currently, no inhibitors against pendrin have been developed, but a very recent study suggests that dual inhibition of pendrin and the NCC could represent a potent K^{-sparing diuretic regimen. Soleimani and colleagues (}131) have demonstrated that pendrin/NCC double knockout mice exhibit severe urinary NaCl wasting, profound extracellular fluid volume depletion, and renal failure. In addition, these mice develop metabolic alkalosis and nephrogenic diabetes insipidus, but they do not develop hypokalemia. The absence of hypokalemia in pendrin/NCC double knockout mice indicates that dual inhibition of pendrin and NCC inhibits electroneutral NaCl reabsorption without stimulating K^{secretion. Thus a potential advantage for developing pendrin inhibitors is that they could be used in combination with thiazide diuretics (inhibitors of NCC) to induce a more potent diuresis without causing hypokalemia.}

Urea Transporters

Urea plays a critical role in the urine concentrating process because it contributes to the steep solute concentration gradient in the medullary interstitium, which drives water reabsorption. Studies in gene knockout mice demonstrate that UT-A1/3 and UT-B urea transporters are critical for urea accumulation in the inner medulla and water conservation. UT-A1/3 urea transporters are expressed in the apical membrane of the inner medullary collecting duct (40, 98) and respond to arginine vasopressin (AVP), which enhances urea permeability (70, 119) and enables urea to diffuse down a favorable concentration gradient from the collecting duct to the medullary interstitium (Fig. 1A). In UT-A1/3 double-knockout mice, isolated inner medulla CD lack urea permeability, and the urea concentration in the inner medulla of knockout mice is one-third of that in wild-type mice (39). UT-A1/3 knockout mice also demonstrate polyuria regardless of whether they are given free access to water (39). When urea excretion exceeds the ability of the inner medullary CD to reabsorb urea, urea becomes an osmotic diuretic in affected mice (38).

UT-B knockout mice also demonstrate defects in urine concentration. UT-B urea transporters are expressed in the vasa recta (Fig. 1A) and operate to ensure that urea is recycled, trapped, and maintained at a high level in the inner medulla (156). UT-B knockout mice with free access to water exhibit a higher plasma urea concentration and lower urine urea concentration than those in wild-type mice, suggesting that deletion of UT-B in vasa recta leads to washout of urea from the inner medulla. Indeed, the urea concentration of the inner medulla of knockout mice is more than two times lower compared with that in wild-type mice. Moreover, UT-B knockout mice with free access to water consume and excrete ∼50% more fluid compared with their wild-type counterparts; in response to water deprivation, knockout mice cannot appropriately increase urine osmolality to conserve water (157). Interestingly, humans lacking UT-B urea transporters also exhibit modest defects in urine concentration (118).

Since urea transporters play an integral role in urine concentration, small-molecule inhibitors directed against urea transporters have the potential to disrupt renal NaCl and water handling. Using an HTS approach, Verkman and colleagues (159) identified a novel class of triazolothieneopyrimidine UT-B inhibitors that selectively and reversibly inhibit urea transport with nanomolar potency. Docking studies between one compound called UTB(inh)-14 and a UT-B homology model suggest that this inhibitor binds to a site in a centrally located groove of the transporter. Intraperitoneal administration of UTB(inh)-14 in mice with free access to water increases urine output and decreases urine osmolality. Additionally, administration of UTB(inh)-14 blunts DDAVP-stimulated increases in urine osmolality (159). The diuretic effect of UTB(inh)-14 is not observed in UT-B knockout mice, suggesting that effects of UTB(inh)-14 on urine concentration are specific to UT-B (159). These data illustrate that urea transport inhibitors represent a novel class of diuretics that have a different mechanism of action from conventional diuretics or aquaretics. Inhibition of urea accumulation in the medullary interstitium, either by inhibiting urea transport in the inner medullary collecting duct or urea recycling in the vasa recta, renders urea itself an osmotic diuretic.

STE20/SPS1-Related Proline/Alanine-Rich Kinase/OSR1

In 2001, Lifton and colleagues (154) identified mutations in WNK1 and WNK4 that cause familial hyperkalemic hypertension (also referred to as pseudohypoaldosteronism type II or Gordon syndrome), a Mendelian form of hypertension that is also characterized by hyperkalemia and metabolic acidosis (46, 104). Mutations in WNK1 and WNK4 increase the activity of NCC in the DCT, but neither kinase directly phosphorylates the transporter. Instead, WNK1 and WNK4 signal through the STE20 kinases SPAK and OSR1. In cell-based studies, SPAK and OSR1 respond to similar signaling pathways (43) and phosphorylate and activate similar substrates such as NCC (97, 114, 143) and NKCC2 (111, 115). Moreover, both kinases are in the position to regulate NCC and NKCC2 in vivo (Fig. 1): SPAK is expressed specifically in the TALH (Fig. 1C) and DCT (Fig. 1D) (113); OSR1 is expressed in all nephron segments (96), including the TALH and DCT (158).

Transgenic knockout models, however, paint a more nuanced picture of how SPAK and OSR1 regulate NCC and NKCC2 in vivo. Kidney-specific deletion of OSR1 in mice leads to hypokalemia and hypercalciuria (85). When fed a low-Na^{diet, these knockout mice develop hypotension due to impaired renal conservation of sodium. This phenotype is reminiscent of Bartter's syndrome type I, which arises from defects in NKCC2 (}128). Indeed, expression of the activated (phosphorylated) form of NKCC2 is decreased in these knockout mice, indicating that OSR1 is a key kinase responsible for activating NKCC2 (85). On the other hand, expression of activated NCC is increased in kidney-specific OSR1 knockout mice, suggesting that NCC is not an in vivo target of OSR1 regulation but instead compensates for loss of NKCC2 function when OSR1 expression is disrupted. Consistent with this notion, the natriuretic and chloruretic effect of furosemide (an NKCC2 inhibitor), but not hydrochlorothiazide (an NCC inhibitor), is blunted in these knockout mice (85).

In contrast to the Barrter's-like phenotype of the kidney-specific OSR1 knockout mouse model, SPAK LOF mouse models exhibit phenotypes resembling Gitelman's syndrome, an autosomal recessive disease caused by LOF mutations in NCC (130). Similar to individuals with Gitelman's syndrome, SPAK LOF mice have low blood pressure accompanied by hypokalemia(158), hypomagnesemia, and hypocalciuria (113, 158). Furthermore, expression of total and activated (phosphorylated) forms of NCC is decreased in SPAK LOF mouse models (113, 158), a finding that is consistent with a defect in NCC activity. Finally, the natriuretic and chloruretic effect of hydrochlorothiazide, but not furosemide, is blunted in one SPAK LOF mouse model (158), confirming that NCC is indeed an in vivo target of SPAK.

Although it is clear that SPAK regulates NCC in vivo, it is not yet clear whether NKCC2 is also an in vivo target because the mouse models differ with respect to the status of NKCC2 phosphorylation, which represents a biochemical surrogate for activity. These discrepancies may relate to how SPAK function was disrupted in the different mouse models. SPAK knockin mice, in which SPAK catalytic activity is inactivated, exhibit a decrease in NKCC2 expression and phosphorylation (113), whereas SPAK-deletion mice exhibit an increase in NKCC2 expression and phosphorylation (158). Recent studies have indicated that deletion of SPAK eliminates expression of not only full-length SPAK but also other inhibitory SPAK isoforms in the TALH, which allows other kinases such as OSR1 or AMPK to compensate and increase NKCC2 phosphorylation and activity (51, 95). Inactivation of SPAK catalytic activity, on the other hand, specifically eliminates SPAK activity, but it does not alter the inhibitory activity of other SPAK isoforms in the TALH; as a result, NKCC2 phosphorylation remains suppressed (95).

If OSR1 and SPAK LOF mouse models accurately recapitulate human renal physiology, small-molecule inhibitors of SPAK or OSR1 would represent a novel, alternative strategy for inhibiting NaCl reabsorption in the TALH (Fig. 1C) and DCT (Fig. 1D). An OSR1-specific inhibitor would preferentially inhibit NKCC2 in the TALH, whereas a SPAK-specific inhibitor would inhibit NKCC2 in the TALH and NCC in the DCT. Inhibiting NaCl reabsorption in two successive nephron segments with a SPAK inhibitor would have a similar effect to that of combination therapy with loop and thiazide diuretics, a strategy that is currently used in clinical practice to induce a potent diuresis in individuals with diuretic resistance (31, 32). Another potential advantage for developing an OSR1 or SPAK inhibitor is that the side effect profile of either inhibitor may be distinct from that of loop and thiazide diuretics. Examples of side effects of thiazides include hyponatremia, hyperglycemia, hyperlipidemia, and hyperuricemia, whereas side effects of loop diuretics include severe hypokalemia and otoxicity. An OSR1 or SPAK inhibitor without these side effects may lead to wider use of diuretic therapy for the treatment of hypertension as well as diseases associated with elevated extracellular fluid volume.

Concluding Remarks

The molecular revolution that occurred in the late 20th century and continues today has led to an explosion in our understanding of disease pathways and potential therapeutic targets. With more than a century of elegant physiological investigations behind us, one of the grand challenges facing renal physiologists in the coming decades will be the translation of these discoveries into novel therapies to improve human health. Approximately one in three American adults have hypertension, and only about half of these are managing their blood pressure correctly due in part to inadequate therapy. A review of the physiological, genetic, and pharmacological data pointing to specific ion channels (i.e., Kir1.1, Kir4.1/5.1, ClC-Ka, ClC-Kb), transporters (i.e., UTA/B, pendrin), and regulatory proteins (i.e., SPAK) participating in the regulation of fluid-volume homeostasis by the kidney highlights the tremendous opportunities renal pharmacologists have to develop more diverse and efficacious antihypertensive drugs. Without exception, the molecular pharmacology of the putative diuretic targets described in this review is limited at best. The continued development of small-molecule ligands will provide critically needed tools to explore the integrative physiology and druggability of these pathways and accelerate efforts to bridge the bench and bedside.

GRANTS

This work was supported by National Institute of Digestive and Diabetes and Kidney Disease Grant 1R01DK082884 (J. S. Denton), National Institute of Neurological Disorders and Stroke Grant 1R21NS073097-01 (J. S. Denton), Foundation for the National Institutes Grant (J. S. Denton), American Heart Association Grant 10GRNT3890045 (M. Maduke), The Mathers Foundation (M. Maduke), The Rosalyn and Mervin Morris Fund (A. C. Pao), and a Stanford University SPARK award (A. C. Pao and M. Maduke).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

Author contributions: J.S.D., A.C.P., and M.M. prepared figures; J.S.D., A.C.P., and M.M. drafted manuscript; J.S.D., A.C.P., and M.M. edited and revised manuscript; J.S.D., A.C.P., and M.M. approved final version of manuscript.

^{Department of Anesthesiology, Vanderbilt University School of Medicine, Nashville, Tennessee;}

^{Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee;}

^{Institute of Chemical Biology, Vanderbilt University School of Medicine, Nashville, Tennessee;}

^{Institute for Global Health, Vanderbilt University School of Medicine, Nashville, Tennessee;}

^{Department of Medicine, Stanford University, Stanford, California;}

^{Department of Molecular and Cellular Physiology, Stanford University, Stanford, California; and}

^{Veterans Affairs Palo Alto Health Care System, Palo Alto, California}

^{Corresponding author.}

Address for reprint requests and other correspondence: J. S. Denton, T4208 Medical Center North, 1161 21st Ave. South, Nashville, TN 37232 (e-mail: ude.tlibrednaV@notneD.S.doreJ).

Received 2013 Apr 24; Accepted 2013 Jul 12.

Abstract

As the molecular revolution continues to inform a deeper understanding of disease mechanisms and pathways, there exist unprecedented opportunities for translating discoveries at the bench into novel therapies for improving human health. Despite the availability of several different classes of antihypertensive medications, only about half of the 67 million Americans with hypertension manage their blood pressure appropriately. A broader selection of structurally diverse antihypertensive drugs acting through different mechanisms would provide clinicians with greater flexibility in developing effective treatment regimens for an increasingly diverse and aging patient population. An emerging body of physiological, genetic, and pharmacological evidence has implicated several renal ion-transport proteins, or regulators thereof, as novel, yet clinically unexploited, diuretic targets. These include the renal outer medullary potassium channel, ROMK (Kir1.1), Kir4.1/5.1 potassium channels, ClC-Ka/b chloride channels, UTA/B urea transporters, the chloride/bicarbonate exchanger pendrin, and the STE20/SPS1-related proline/alanine-rich kinase (SPAK). The molecular pharmacology of these putative targets is poorly developed or lacking altogether; however, recent efforts by a few academic and pharmaceutical laboratories have begun to lessen this critical barrier. Here, we review the evidence in support of the aforementioned proteins as novel diuretic targets and highlight examples where progress toward developing small-molecule pharmacology has been made.

Keywords: ClC-K, Kir4.1/5.1, pendrin, ROMK, urea transporter

Abstract

approximately 67 million americans have hypertension, a chronic disease of excessive vascular tone and fluid volume that increases the risk of stroke, myocardial infarction, and chronic kidney disease. Hypertension costs the United States roughly $50 billion annually in direct medical expenses and lost productivity, and the incidence of hypertension is projected to rise considerably as rates of obesity and life expectancy increase. Diuretics continue to be a mainstay of antihypertensive therapy and work by reducing sodium chloride (NaCl) and osmotically obliged water reabsorption in the nephron, thereby lowering blood volume and pressure. Combination therapy, in which more than one drug is prescribed to reach a target blood pressure of <140/90, is required in ∼75% of hypertensive patients. Resistant hypertension, defined as blood pressure above 140/90, despite adherence to at least three antihypertensive drugs, is observed in about 20–30% of the population (16). Rotation between diuretics of different classes (e.g., loop, thiazide, potassium-sparing) or even within a class can overcome refractoriness in some diuretic-resistant patients; however, differences in drug bioavailability, metabolism, compatibility with other medications, as well as existing comorbidities like chronic kidney disease, can complicate establishing the correct combination to achieve the blood pressure goal. The development of new classes of drugs targeting diverse mechanisms would provide clinicians with greater flexibility in establishing effective antihypertensive regimens for their patients. An emerging body of physiological, genetic, and pharmacological evidence has recently implicated several ion-transport mechanisms in the nephron that may represent novel, but currently unexploited, molecular targets for diuretics. Here, we discuss this evidence and highlight, where possible, recent efforts to develop the molecular pharmacology for these emerging diuretic targets.

ROMK (Kir1.1)

Fig. 1.

Fig. 2.

Kir4.1/5.1

ClC-Ka/b

Fig. 3.

Evolution of a ClC-K channel inhibitor. Evolution of CLC-K channel inhibitors is shown, as described in the text. CPP (a) was tested against a chimera of ClC-Kb and mouse ClC-Ka (ClC-K1) (79).

Pendrin

Urea Transporters

STE20/SPS1-Related Proline/Alanine-Rich Kinase/OSR1

Concluding Remarks

ACKNOWLEDGMENTS

We gratefully acknowledge Rene Raphemot (Vanderbilt University School of Medicine) for generating the chemical structures used in Figs. 2 and and33 and Philip Change and Rhiannon Thomas (Stanford University) for critically reading the manuscript.

ACKNOWLEDGMENTS

REFERENCES

References

1. Accardi A, Miller C. Secondary active transport mediated by a prokaryotic homologue of ClC Cl- channels. Nature427: 803–807, 2004 [[PubMed]
2. Akizuki N, Uchida S, Sasaki S, Marumo F. Impaired solute accumulation in inner medulla of Clcnk1^{mice kidney}. Am J Physiol Renal Physiol280: F79–F87, 2001 [[PubMed]
3. Alper SL. Molecular physiology and genetics of Na^{-independent SLC4 anion exchangers}. J Exp Biol212: 1672–1683, 2009
4. Aperis G, Alivanis P. Tolvaptan: a new therapeutic agent. Rev Recent Clin Trials6: 177–188, 2011 [[PubMed]
5. Aromataris EC, Astill DS, Rychkov GY, Bryant SH, Bretag AH, Roberts ML. Modulation of the gating of CIC-1 by S-(-) 2-(4-chlorophenoxy) propionic acid. Br J Pharmacol126: 1375–1382, 1999
6. Ayus JC, Moritz ML. Bone disease as a new complication of hyponatremia: moving beyond brain injury. Clin J Am Soc Nephrol5: 167–168, 2010 [[PubMed]
7. Barlassina C, Dal Fiume C, Lanzani C, Manunta P, Guffanti G, Ruello A, Bianchi G, Del Vecchio L, Macciardi F, Cusi D. Common genetic variants and haplotypes in renal CLCNKA gene are associated to salt-sensitive hypertension. Hum Mol Genet16: 1630–1638, 2007 [[PubMed]
8. Bartter FC, Pronove P, Gill JR, Jr, Maccardle RC. Hyperplasia of the juxtaglomerular complex with hyperaldosteronism and hypokalemic alkalosis. A new syndrome. Am J Med33: 811–828, 1962 [[PubMed]
9. Bennett CM, Brenner BM, Berliner RW. Micropuncture study of nephron function in the rhesus monkey. J Clin Invest47: 203–216, 1968
10. Bhave G, Chauder BA, Liu W, Dawson ES, Kadakia R, Nguyen TT, Lewis LM, Meiler J, Weaver CD, Satlin LM, Lindsley CW, Denton JS. Development of a selective small-molecule inhibitor of Kir1.1, the renal outer medullary potassium channel. Mol Pharmacol79: 42–50, 2011
11. Bhave G, Lonergan D, Chauder BA, Denton JS. Small-molecule modulators of inward rectifier K^{channels: recent advances and future possibilities}. Future Med Chem2: 757–774, 2010
12. Birkenhager R, Otto E, Schurmann MJ, Vollmer M, Ruf EM, Maier-Lutz I, Beekmann F, Fekete A, Omran H, Feldmann D, Milford DV, Jeck N, Konrad M, Landau D, Knoers NV, Antignac C, Sudbrak R, Kispert A, Hildebrandt F. Mutation of BSND causes Bartter syndrome with sensorineural deafness and kidney failure. Nat Genet29: 310–314, 2001 [[PubMed]
13. Bockenhauer D, Feather S, Stanescu HC, Bandulik S, Zdebik AA, Reichold M, Tobin J, Lieberer E, Sterner C, Landoure G, Arora R, Sirimanna T, Thompson D, Cross JH, van't Hoff W, Al Masri O, Tullus K, Yeung S, Anikster Y, Klootwijk E, Hubank M, Dillon MJ, Heitzmann D, Arcos-Burgos M, Knepper MA, Dobbie A, Gahl WA, Warth R, Sheridan E, Kleta R. Epilepsy, ataxia, sensorineural deafness, tubulopathy, and KCNJ10 mutations. N Engl J Med360: 1960–1970, 2009
14. Bodendiek SB, Raman G. Connexin modulators and their potential targets under the magnifying glass. Curr Med Chem17: 4191–4230, 2010 [[PubMed]
15. Burg MB. Tubular chloride transport and the mode of action of some diuretics. Kidney Int9: 189–197, 1976 [[PubMed]
16. Calhoun DA, Jones D, Textor S, Goff DC, Murphy TP, Toto RD, White A, Cushman WC, White W, Sica D, Ferdinand K, Giles TD, Falkner B, Carey RM. Resistant hypertension: evaluation, treatment, diagnosis. A scientific statement from the American Heart Association Professional Education Committee of the Council for High Blood Pressure Research. Hypertension51: 1403–1419, 2008 [[PubMed]
17. Chang PY, Zhang XG, Su XL. Lack of association of variants of the renal salt reabsorption-related genes SLC12A3 and ClC-Kb and hypertension in Mongolian and Han populations in Inner Mongolia. Genet Mol Res10: 948–954, 2011 [[PubMed]
18. Chen B, Nicol G, Cho WK. Role of calcium in volume-activated chloride currents in a mouse cholangiocyte cell line. J Membr Biol215: 1–13, 2007 [[PubMed]
19. Chen GL, Zeng B, Eastmond S, Elsenussi SE, Boa AN, Xu SZ. Pharmacological comparison of novel synthetic fenamate analogues with econazole and 2-APB on the inhibition of TRPM2 channels. Br J Pharmacol167: 1232–1243, 2012
20. Chen TY, Hwang TC. CLC-0 and CFTR: chloride channels evolved from transporters. Physiol Rev88: 351–387, 2008 [[PubMed]
21. Chow KM, Kwan BC, Szeto CC. Clinical studies of thiazide-induced hyponatremia. J Natl Med Assoc96: 1305–1308, 2004
22. Clark MA, Humphrey SJ, Smith MP, Ludens JH. Unique natriuretic properties of the ATP-sensitive K^{-channel blocker glyburide in conscious rats}. J Pharmacol Exp Ther265: 933–937, 1993 [[PubMed]
23. Claverie-Martin F, Ramos-Trujillo E, Garcia-Nieto V. Dent's disease: clinical features and molecular basis. Pediatr Nephrol26: 693–704, 2011 [[PubMed]
24. Decaux G, Soupart A, Vassart G. Non-peptide arginine-vasopressin antagonists: the vaptans. Lancet371: 1624–1632, 2008 [[PubMed]
25. Decaux G, Vandergheynst F, Bouko Y, Parma J, Vassart G, Vilain C. Nephrogenic syndrome of inappropriate antidiuresis in adults: high phenotypic variability in men and women from a large pedigree. J Am Soc Nephrol18: 606–612, 2007 [[PubMed]
26. Devuyst O, Thakker RV. Dent's disease. Orphanet J Rare Dis5: 28, 2010
27. Djukic B, Casper KB, Philpot BD, Chin LS, McCarthy KD. Conditional knock-out of Kir4.1 leads to glial membrane depolarization, inhibition of potassium and glutamate uptake, and enhanced short-term synaptic potentiation. J Neurosci27: 11354–11365, 2007
28. Duran C, Thompson CH, Xiao Q, Hartzell HC. Chloride channels: often enigmatic, rarely predictable. Annu Rev Physiol72: 95–121, 2010
29. Dutzler R. A structural perspective on ClC channel and transporter function. FEBS Lett581: 2839–2844, 2007 [[PubMed]
30. Edvardsson VO, Goldfarb DS, Lieske JC, Beara-Lasic L, Anglani F, Milliner DS, Palsson R. Hereditary causes of kidney stones and chronic kidney disease. Pediatr Nephrol [Epub ahead of print]
31. Ellison DH. The physiologic basis of diuretic synergism: its role in treating diuretic resistance. Ann Intern Med114: 886–894, 1991 [[PubMed]
32. Epstein M, Lepp BA, Hoffman DS, Levinson R. Potentiation of furosemide by metolazone in refractory edema. Curr Ther Res21: 656–667, 1977 [PubMed]
33. Estevez R, Boettger T, Stein V, Birkenhager R, Otto E, Hildebrandt F, Jentsch TJ. Barttin is a Cl- channel beta-subunit crucial for renal Cl- reabsorption and inner ear K^secretion. Nature414: 558–561, 2001 [[PubMed]
34. Evanko DS, Zhang Q, Zorec R, Haydon PG. Defining pathways of loss and secretion of chemical messengers from astrocytes. Glia47: 233–240, 2004 [[PubMed]
35. Everett LA, Belyantseva IA, Noben-Trauth K, Cantos R, Chen A, Thakkar SI, Hoogstraten-Miller SL, Kachar B, Wu DK, Green ED. Targeted disruption of mouse Pds provides insight about the inner-ear defects encountered in Pendred syndrome. Hum Mol Genet10: 153–161, 2001 [[PubMed]
36. Fahlke C, Fischer M. Physiology and pathophysiology of ClC-K/barttin channels. Front Physiol1: 155, 2010
37. Fava C, Montagnana M, Almgren P, Rosberg L, Guidi GC, Berglund G, Melander O. The functional variant of the CLC-Kb channel T481S is not associated with blood pressure or hypertension in Swedes. J Hypertens25: 111–116, 2007 [[PubMed]
38. Fenton RA, Chou CL, Sowersby H, Smith CP, Knepper MA. Gamble's “economy of water” revisited: studies in urea transporter knockout mice. Am J Physiol Renal Physiol291: F148–F154, 2006 [[PubMed]
39. Fenton RA, Chou CL, Stewart GS, Smith CP, Knepper MA. Urinary concentrating defect in mice with selective deletion of phloretin-sensitive urea transporters in the renal collecting duct. Proc Natl Acad Sci USA101: 7469–7474, 2004
40. Fenton RA, Stewart GS, Carpenter B, Howorth A, Potter EA, Cooper GJ, Smith CP. Characterization of mouse urea transporters UT-A1 and UT-A2. Am J Physiol Renal Physiol283: F817–F825, 2002 [[PubMed]
41. Fong P. CLC-K channels: if the drug fits, use it. EMBO Rep5: 565–566, 2004
42. Foster DB, Ho AS, Rucker J, Garlid AO, Chen L, Sidor A, Garlid KD, O'Rourke B. Mitochondrial ROMK channel is a molecular component of mitoK(ATP). Circ Res111: 446–454, 2012
43. Furutani K, Ohno Y, Inanobe A, Hibino H, Kurachi Y. Mutational and in silico analyses for antidepressant block of astroglial inward-rectifier Kir4.1 channel. Mol Pharmacol75: 1287–1295, 2009 [[PubMed]
44. Gagnon KB, Delpire E. Molecular physiology of SPAK and OSR1: two Ste20-related protein kinases regulating ion transport. Physiol Rev92: 1577–1617, 2012
45. Garg P, Sanguinetti MC. Structure-activity relationship of fenamates as Slo2.1 channel activators. Mol Pharmacol82: 795–802, 2012
46. Giebisch G. Renal potassium channels: an overview. Kidney Int48: 1004–1009, 1995 [[PubMed]
47. Gordon RD, Geddes RA, Pawsey CG, O'Halloran MW. Hypertension and severe hyperkalaemia associated with suppression of renin and aldosterone and completely reversed by dietary sodium restriction. Australas Ann Med19: 287–294, 1970 [[PubMed]
48. Gradogna A, Fenollar-Ferrer C, Forrest LR, Pusch M. Dissecting a regulatory calcium-binding site of CLC-K kidney chloride channels. J Gen Physiol140: 681–696, 2012
49. Gradogna A, Pusch M. Molecular pharmacology of kidney and inner ear CLC-K chloride channels. Front Pharmacol1: 130, 2010
50. Greger R. Ion transport mechanisms in thick ascending limb of Henle's loop of mammalian nephron. Physiol Rev65: 760–797, 1985 [[PubMed]
51. Greger R, Schlatter E. Presence of luminal K^{, a prerequisite for active NaCl transport in the cortical thick ascending limb of Henle's loop of rabbit kidney}. Pflügers Arch392: 92–94, 1981 [[PubMed]
52. Grimm PR, Taneja TK, Liu J, Coleman R, Chen YY, Delpire E, Wade JB, Welling PA. SPAK isoforms and OSR1 regulate sodium-chloride co-transporters in a nephron-specific manner. J Biol Chem287: 37673–37690, 2012
53. Gross P, Marczewski T, Herbrig K. The vaptans ante portas: a status report. Nephrol Dial Transplant24: 1371–1373, 2009 [[PubMed]
54. Hasannejad H, Takeda M, Taki K, Shin HJ, Babu E, Jutabha P, Khamdang S, Aleboyeh M, Onozato ML, Tojo A, Enomoto A, Anzai N, Narikawa S, Huang XL, Niwa T, Endou H. Interactions of human organic anion transporters with diuretics. J Pharmacol Exp Ther308: 1021–1029, 2004 [[PubMed]
55. Hasegawa M, Kusuhara H, Adachi M, Schuetz JD, Takeuchi K, Sugiyama Y. Multidrug resistance-associated protein 4 is involved in the urinary excretion of hydrochlorothiazide and furosemide. J Am Soc Nephrol18: 37–45, 2007 [[PubMed]
56. Hebert SC, Andreoli TE. Control of NaCl transport in the thick ascending limb. Am J Physiol Renal Fluid Electrolyte Physiol246: F745–F756, 1984 [[PubMed]
57. Hebert SC, Desir G, Giebisch G, Wang W. Molecular diversity and regulation of renal potassium channels. Physiol Rev85: 319–371, 2005
58. Hibino H, Inanobe A, Furutani K, Murakami S, Findlay I, Kurachi Y. Inwardly rectifying potassium channels: their structure, function, and physiological roles. Physiol Rev90: 291–366, 2010 [[PubMed]
59. Ho K, Nichols CG, Lederer WJ, Lytton J, Vassilev PM, Kanazirska MV, Hebert SC. Cloning and expression of an inwardly rectifying ATP-regulated potassium channel. Nature362: 31–38, 1993 [[PubMed]
60. Howery AE, Elvington S, Abraham SJ, Choi KH, Dworschak-Simpson S, Phillips S, Ryan CM, Sanford RL, Almqvist J, Tran K, Chew TA, Zachariae U, Andersen OS, Whitelegge J, Matulef K, Du Bois J, Maduke MC. A designed inhibitor of a CLC antiporter blocks function through a unique binding mode. Chem Biol19: 1460–1470, 2012
61. Huang C, Sindic A, Hill CE, Hujer KM, Chan KW, Sassen M, Wu Z, Kurachi Y, Nielsen S, Romero MF, Miller RT. Interaction of the Ca^{-sensing receptor with the inwardly rectifying potassium channels Kir4.1 and Kir42 results in inhibition of channel function}. Am J Physiol Renal Physiol292: F1073–F1081, 2007 [[PubMed]
62. Jeck N, Seyberth HW. Loop disorders: insights derived from defined genotypes. Nephron Physiol118: p7–p14, 2011 [[PubMed]
63. Jeck N, Waldegger S, Lampert A, Boehmer C, Waldegger P, Lang PA, Wissinger B, Friedrich B, Risler T, Moehle R, Lang UE, Zill P, Bondy B, Schaeffeler E, Asante-Poku S, Seyberth H, Schwab M, Lang F. Activating mutation of the renal epithelial chloride channel ClC-Kb predisposing to hypertension. Hypertension43: 1175–1181, 2004 [[PubMed]
64. Jentsch TJ. Chloride transport in the kidney: lessons from human disease and knockout mice. J Am Soc Nephrol16: 1549–1561, 2005 [[PubMed]
65. Jentsch TJ. CLC chloride channels and transporters: from genes to protein structure, pathology and physiology. Crit Rev Biochem Mol Biol43: 3–36, 2008 [[PubMed]
66. Jentsch TJ, Maritzen T, Zdebik AA. Chloride channel diseases resulting from impaired transepithelial transport or vesicular function. J Clin Invest115: 2039–2046, 2005
67. Ji W, Foo JN, O'Roak BJ, Zhao H, Larson MG, Simon DB, Newton-Cheh C, State MW, Levy D, Lifton RP. Rare independent mutations in renal salt handling genes contribute to blood pressure variation. Nat Genet40: 592–599, 2008
68. Kasai M, Kawasaki T, Yamaguchi N. Regulation of the ryanodine receptor calcium release channel: a molecular complex system. Biophys Chem82: 173–181, 1999 [[PubMed]
69. Kim YH, Kwon TH, Frische S, Kim J, Tisher CC, Madsen KM, Nielsen S. Immunocytochemical localization of pendrin in intercalated cell subtypes in rat and mouse kidney. Am J Physiol Renal Physiol283: F744–F754, 2002 [[PubMed]
70. Kim YH, Pech V, Spencer KB, Beierwaltes WH, Everett LA, Green ED, Shin W, Verlander JW, Sutliff RL, Wall SM. Reduced ENaC protein abundance contributes to the lower blood pressure observed in pendrin-null mice. Am J Physiol Renal Physiol293: F1314–F1324, 2007 [[PubMed]
71. Knepper MA, Star RA. The vasopressin-regulated urea transporter in renal inner medullary collecting duct. Am J Physiol Renal Fluid Electrolyte Physiol259: F393–F401, 1990 [[PubMed]
72. Koeppen BM, Biagi BA, Giebisch GH. Intracellular microelectrode characterization of the rabbit cortical collecting duct. Am J Physiol Renal Fluid Electrolyte Physiol244: F35–F47, 1983 [[PubMed]
73. Kokko JP, Rector FC., Jr Countercurrent multiplication system without active transport in inner medulla. Kidney Int2: 214–223, 1972 [[PubMed]
74. Kokubo Y, Iwai N, Tago N, Inamoto N, Okayama A, Yamawaki H, Naraba H, Tomoike H. Association analysis between hypertension and CYBA, CLCNKB, and KCNMB1 functional polymorphisms in the Japanese population—the Suita Study. Circ J69: 138–142, 2005 [[PubMed]
75. Konstam MA, Gheorghiade M, Burnett JC, Jr, Grinfeld L, Maggioni AP, Swedberg K, Udelson JE, Zannad F, Cook T, Ouyang J, Zimmer C, Orlandi C. Effects of oral tolvaptan in patients hospitalized for worsening heart failure: the EVEREST Outcome Trial. JAMA297: 1319–1331, 2007 [[PubMed]
76. Kramer BK, Bergler T, Stoelcker B, Waldegger S. Mechanisms of disease: the kidney-specific chloride channels ClCKA and ClCKB, the Barttin subunit, and their clinical relevance. Nat Clin Pract Nephrol4: 38–46, 2008 [[PubMed]
77. Lachheb S, Cluzeaud F, Bens M, Genete M, Hibino H, Lourdel S, Kurachi Y, Vandewalle A, Teulon J, Paulais M. Kir4.1/Kir5.1 channel forms the major K^{channel in the basolateral membrane of mouse renal collecting duct principal cells}. Am J Physiol Renal Physiol294: F1398–F1407, 2008 [[PubMed]
78. Lawson K. Potassium channel openers as potential therapeutic weapons in ion channel disease. Kidney Int57: 838–845, 2000 [[PubMed]
79. Lewis LM, Bhave G, Chauder BA, Banerjee S, Lornsen KA, Redha R, Fallen K, Lindsley CW, Weaver CD, Denton JS. High-throughput screening reveals a small-molecule inhibitor of the renal outer medullary potassium channel and Kir7.1. Mol Pharmacol76: 1094–1103, 2009
80. Li L, Ma KT, Zhao L, Si JQ, Zhang ZS, Zhu H, Li J. Niflumic acid hyperpolarizes smooth muscle cells via calcium-activated potassium channel in spiral modiolar artery of guinea pigs. Acta Pharmacol Sin29: 789–799, 2008 [[PubMed]
81. Liantonio A, Accardi A, Carbonara G, Fracchiolla G, Loiodice F, Tortorella P, Traverso S, Guida P, Pierno S, De Luca A, Camerino DC, Pusch M. Molecular requisites for drug binding to muscle CLC-1 and renal CLC-K channel revealed by the use of phenoxy-alkyl derivatives of 2-(p-chlorophenoxy)propionic acid. Mol Pharmacol62: 265–271, 2002 [[PubMed]
82. Liantonio A, Gramegna G, Camerino GM, Dinardo MM, Scaramuzzi A, Potenza MA, Montagnani M, Procino G, Lasorsa DR, Mastrofrancesco L, Laghezza A, Fracchiolla G, Loiodice F, Perrone MG, Lopedota A, Conte S, Penza R, Valenti G, Svelto M, Camerino DC. In-vivo administration of CLC-K kidney chloride channels inhibitors increases water diuresis in rats: a new drug target for hypertension?J Hypertens30: 153–167, 2012 [[PubMed]
83. Liantonio A, Picollo A, Babini E, Carbonara G, Fracchiolla G, Loiodice F, Tortorella V, Pusch M, Camerino DC. Activation and inhibition of kidney CLC-K chloride channels by fenamates. Mol Pharmacol69: 165–173, 2006 [[PubMed]
84. Liantonio A, Picollo A, Carbonara G, Fracchiolla G, Tortorella P, Loiodice F, Laghezza A, Babini E, Zifarelli G, Pusch M, Camerino DC. Molecular switch for CLC-K Cl- channel block/activation: optimal pharmacophoric requirements towards high-affinity ligands. Proc Natl Acad Sci USA105: 1369–1373, 2008
85. Liantonio A, Pusch M, Picollo A, Guida P, De Luca A, Pierno S, Fracchiolla G, Loiodice F, Tortorella P, Conte Camerino D. Investigations of pharmacologic properties of the renal CLC-K1 chloride channel co-expressed with barttin by the use of 2-(p-chlorophenoxy)propionic acid derivatives and other structurally unrelated chloride channels blockers. J Am Soc Nephrol15: 13–20, 2004 [[PubMed]
86. Lin SH, Yu IS, Jiang ST, Lin SW, Chu P, Chen A, Sytwu HK, Sohara E, Uchida S, Sasaki S, Yang SS. Impaired phosphorylation of Na^-K^-2Cl^{cotransporter by oxidative stress-responsive kinase-1 deficiency manifests hypotension and Bartter-like syndrome}. Proc Natl Acad Sci USA108: 17538–17543, 2011
87. Lippiat JD, Smith AJ. The CLC-5 2Cl^/H^{exchange transporter in endosomal function and Dent's disease}. Front Physiol3: 449, 2012
88. Lorenz JN, Baird NR, Judd LM, Noonan WT, Andringa A, Doetschman T, Manning PA, Liu LH, Miller ML, Shull GE. Impaired renal NaCl absorption in mice lacking the ROMK potassium channel, a model for type II Bartter's syndrome. J Biol Chem277: 37871–37880, 2002 [[PubMed]
89. Lourdel S, Grand T, Burgos J, Gonzalez W, Sepulveda FV, Teulon J. ClC-5 mutations associated with Dent's disease: a major role of the dimer interface. Pflügers Arch463: 247–256, 2012 [[PubMed]
90. Lourdel S, Paulais M, Cluzeaud F, Bens M, Tanemoto M, Kurachi Y, Vandewalle A, Teulon J. An inward rectifier K^{channel at the basolateral membrane of the mouse distal convoluted tubule: similarities with Kir4-Kir5.1 heteromeric channels}. J Physiol538: 391–404, 2002
91. Lu M, Wang T, Yan Q, Wang W, Giebisch G, Hebert SC. ROMK is required for expression of the 70-pS K channel in the thick ascending limb. Am J Physiol Renal Physiol286: F490–F495, 2004 [[PubMed]
92. Lu M, Wang T, Yan Q, Yang X, Dong K, Knepper MA, Wang W, Giebisch G, Shull GE, Hebert SC. Absence of small conductance K^{channel (SK) activity in apical membranes of thick ascending limb and cortical collecting duct in ROMK (Bartter's) knockout mice}. J Biol Chem277: 37881–37887, 2002
93. Matsumura Y, Uchida S, Kondo Y, Miyazaki H, Ko SB, Hayama A, Morimoto T, Liu W, Arisawa M, Sasaki S, Marumo F. Overt nephrogenic diabetes insipidus in mice lacking the CLC-K1 chloride channel. Nat Genet21: 95–98, 1999 [[PubMed]
94. Matulef K, Howery AE, Tan L, Kobertz WR, Du Bois J, Maduke M. Discovery of potent CLC chloride channel inhibitors. ACS Chem Biol3: 419–428, 2008
95. Matulef K, Maduke M. The CLC ‘chloride channel’ family: revelations from prokaryotes. Mol Membr Biol24: 342–350, 2007 [[PubMed]
96. McCormick JA, Mutig K, Nelson JH, Saritas T, Hoorn EJ, Yang CL, Rogers S, Curry J, Delpire E, Bachmann S, Ellison DH. A SPAK isoform switch modulates renal salt transport and blood pressure. Cell Metab14: 352–364, 2011
97. Mercier-Zuber A, O'Shaughnessy KM. Role of SPAK and OSR1 signaling in the regulation of NaCl cotransporters. Curr Opin Nephrol Hypertens20: 534–540, 2011 [[PubMed]
98. Moriguchi T, Urushiyama S, Hisamoto N, Iemura S, Uchida S, Natsume T, Matsumoto K, Shibuya H. WNK1 regulates phosphorylation of cation-chloride-coupled cotransporters via the STE20-related kinases, SPAK and OSR1. J Biol Chem280: 42685–42693, 2005 [[PubMed]
99. Nielsen S, Terris J, Smith CP, Hediger MA, Ecelbarger CA, Knepper MA. Cellular and subcellular localization of the vasopressin- regulated urea transporter in rat kidney. Proc Natl Acad Sci USA93: 5495–5500, 1996
100. Nomura N, Tajima M, Sugawara N, Morimoto T, Kondo Y, Ohno M, Uchida K, Mutig K, Bachmann S, Soleimani M, Ohta E, Ohta A, Sohara E, Okado T, Rai T, Jentsch TJ, Sasaki S, Uchida S. Generation and analyses of R8L barttin knockin mouse. Am J Physiol Renal Physiol301: F297–F307, 2011 [[PubMed]
101. Nozu K, Inagaki T, Fu XJ, Nozu Y, Kaito H, Kanda K, Sekine T, Igarashi T, Nakanishi K, Yoshikawa N, Iijima K, Matsuo M. Molecular analysis of digenic inheritance in Bartter syndrome with sensorineural deafness. J Med Genet45: 182–186, 2008 [[PubMed]
102. Ohno Y, Hibino H, Lossin C, Inanobe A, Kurachi Y. Inhibition of astroglial Kir4.1 channels by selective serotonin reuptake inhibitors. Brain Res1178: 44–51, 2007 [[PubMed]
103. Ookata K, Tojo A, Suzuki Y, Nakamura N, Kimura K, Wilcox CS, Hirose S. Localization of inward rectifier potassium channel Kir7.1 in the basolateral membrane of distal nephron and collecting duct. J Am Soc Nephrol11: 1987–1994, 2000 [[PubMed]
104. Paulais M, Bloch-Faure M, Picard N, Jacques T, Ramakrishnan SK, Keck M, Sohet F, Eladari D, Houillier P, Lourdel S, Teulon J, Tucker SJ. Renal phenotype in mice lacking the Kir5.1 (Kcnj16) K^{channel subunit contrasts with that observed in SeSAME/EAST syndrome}. Proc Natl Acad Sci USA108: 10361–10366, 2011
105. Paver WK, Pauline GJ. Hypertension and hyperpotassaemia without renal disease in a young male. Med J Aust2: 305–306, 1964 [[PubMed]
106. Pech V, Pham TD, Hong S, Weinstein AM, Spencer KB, Duke BJ, Walp E, Kim YH, Sutliff RL, Bao HF, Eaton DC, Wall SM. Pendrin modulates ENaC function by changing luminal HCO₃. J Am Soc Nephrol21: 1928–1941, 2010
107. Pendred V. Deaf-mutism and goitre. Lancet ii, 1896 [PubMed]
108. Picollo A, Liantonio A, Babini E, Camerino DC, Pusch M. Mechanism of interaction of niflumic acid with heterologously expressed kidney CLC-K chloride channels. J Membr Biol216: 73–82, 2007 [[PubMed]
109. Picollo A, Liantonio A, Didonna MP, Elia L, Camerino DC, Pusch M. Molecular determinants of differential pore blocking of kidney CLC-K chloride channels. EMBO Rep5: 584–589, 2004
110. Picollo A, Pusch M. Chloride/proton antiporter activity of mammalian CLC proteins ClC-4 and ClC-5. Nature436: 420–423, 2005 [[PubMed]
111. Plans V, Rickheit G, Jentsch TJ. Physiological roles of CLC Cl^/H^{exchangers in renal proximal tubules}. Pflügers Arch458: 23–37, 2009 [[PubMed]
112. Ponce-Coria J, San-Cristobal P, Kahle KT, Vazquez N, Pacheco-Alvarez D, de Los Heros P, Juarez P, Munoz E, Michel G, Bobadilla NA, Gimenez I, Lifton RP, Hebert SC, Gamba G. Regulation of NKCC2 by a chloride-sensing mechanism involving the WNK3 and SPAK kinases. Proc Natl Acad Sci USA105: 8458–8463, 2008
113. Pusch M, Liantonio A, Bertorello L, Accardi A, De LA, Pierno S, Tortorella V, Camerino DC. Pharmacological characterization of chloride channels belonging to the ClC family by the use of chiral clofibric acid derivatives. Mol Pharmacol58: 498–507, 2000 [[PubMed]
114. Rafiqi FH, Zuber AM, Glover M, Richardson C, Fleming S, Jovanovic S, Jovanovic A, O'Shaughnessy KM, Alessi DR. Role of the WNK-activated SPAK kinase in regulating blood pressure. EMBO Mol Med2: 63–75, 2010
115. Richardson C, Rafiqi FH, Karlsson HK, Moleleki N, Vandewalle A, Campbell DG, Morrice NA, Alessi DR. Activation of the thiazide-sensitive Na^-Cl^{cotransporter by the WNK-regulated kinases SPAK and OSR1}. J Cell Sci121: 675–684, 2008 [[PubMed]
116. Richardson C, Sakamoto K, de los Heros P, Deak M, Campbell DG, Prescott AR, Alessi DR. Regulation of the NKCC2 ion cotransporter by SPAK-OSR1-dependent and -independent pathways. J Cell Sci124: 789–800, 2011
117. Royaux IE, Wall SM, Karniski LP, Everett LA, Suzuki K, Knepper MA, Green ED. Pendrin, encoded by the Pendred syndrome gene, resides in the apical region of renal intercalated cells and mediates bicarbonate secretion. Proc Natl Acad Sci USA98: 4221–4226, 2001
118. Sanders KM, Zhu MH, Britton F, Koh SD, Ward SM. Anoctamins and gastrointestinal smooth muscle excitability. Exp Physiol97: 200–206, 2012
119. Sands JM, Gargus JJ, Frohlich O, Gunn RB, Kokko JP. Urinary concentrating ability in patients with Jk(a-b-) blood type who lack carrier-mediated urea transport. J Am Soc Nephrol2: 1689–1696, 1992 [[PubMed]
120. Sands JM, Nonoguchi H, Knepper MA. Vasopressin effects on urea and H₂O transport in inner medullary collecting duct subsegments. Am J Physiol Renal Fluid Electrolyte Physiol253: F823–F832, 1987 [[PubMed]
121. Scheel O, Zdebik AA, Lourdel S, Jentsch TJ. Voltage-dependent electrogenic chloride/proton exchange by endosomal CLC proteins. Nature436: 424–427, 2005 [[PubMed]
122. Schlingmann KP, Konrad M, Jeck N, Waldegger P, Reinalter SC, Holder M, Seyberth HW, Waldegger S. Salt wasting and deafness resulting from mutations in two chloride channels. N Engl J Med350: 1314–1319, 2004 [[PubMed]
123. Scholl UI, Choi M, Liu T, Ramaekers VT, Hausler MG, Grimmer J, Tobe SW, Farhi A, Nelson-Williams C, Lifton RP. Seizures, sensorineural deafness, ataxia, mental retardation, and electrolyte imbalance (SeSAME syndrome) caused by mutations in KCNJ10. Proc Natl Acad Sci USA106: 5842–5847, 2009
124. Scott DA, Wang R, Kreman TM, Sheffield VC, Karniski LP. The Pendred syndrome gene encodes a chloride-iodide transport protein. Nat Genet21: 440–443, 1999 [[PubMed]
125. Sharabi Y, Illan R, Kamari Y, Cohen H, Nadler M, Messerli FH, Grossman E. Diuretic induced hyponatraemia in elderly hypertensive women. J Hum Hypertens16: 631–635, 2002 [[PubMed]
126. Sile S, Vanoye CG, George AL., Jr Molecular physiology of renal ClC chloride channels/transporters. Curr Opin Nephrol Hypertens15: 511–516, 2006 [[PubMed]
127. Sile S, Velez DR, Gillani NB, Narsia T, Moore JH, George AL, Jr, Vanoye CG, Williams SM. CLCNKB-T481S and essential hypertension in a Ghanaian population. J Hypertens27: 298–304, 2009
128. Simon DB, Bindra RS, Mansfield TA, Nelson-Williams C, Mendonca E, Stone R, Schurman S, Nayir A, Alpay H, Bakkaloglu A, Rodriguez-Soriano J, Morales JM, Sanjad SA, Taylor CM, Pilz D, Brem A, Trachtman H, Griswold W, Richard GA, John E, Lifton RP. Mutations in the chloride channel gene, CLCNKB, cause Bartter's syndrome type III. Nat Genet17: 171–178, 1997 [[PubMed]
129. Simon DB, Karet FE, Hamdan JM, DiPietro A, Sanjad SA, Lifton RP. Bartter's syndrome, hypokalaemic alkalosis with hypercalciuria, is caused by mutations in the Na-K-2Cl cotransporter NKCC2. Nat Genet13: 183–188, 1996 [[PubMed]
130. Simon DB, Karet FE, Rodriguez-Soriano J, Hamdan JH, DiPietro A, Trachtman H, Sanjad SA, Lifton RP. Genetic heterogeneity of Bartter's syndrome revealed by mutations in the K^{channel, ROMK}. Nat Genet14: 152–156, 1996 [[PubMed]
131. Simon DB, Nelson-Williams C, Bia MJ, Ellison D, Karet FE, Molina AM, Vaara I, Iwata F, Cushner HM, Koolen M, Gainza FJ, Gitleman HJ, Lifton RP. Gitelman's variant of Bartter's syndrome, inherited hypokalaemic alkalosis, is caused by mutations in the thiazide-sensitive Na-Cl cotransporter. Nat Genet12: 24–30, 1996 [[PubMed]
132. Soleimani M, Barone S, Xu J, Shull GE, Siddiqui F, Zahedi K, Amlal H. Double knockout of pendrin and Na-Cl cotransporter (NCC) causes severe salt wasting, volume depletion, and renal failure. Proc Natl Acad Sci USA109: 13368–13373, 2012
133. Speirs HJ, Wang WY, Benjafield AV, Morris BJ. No association with hypertension of CLCNKB and TNFRSF1B polymorphisms at a hypertension locus on chromosome 1p36. J Hypertens23: 1491–1496, 2005 [[PubMed]
134. Spital A. Diuretic-induced hyponatremia. Am J Nephrol19: 447–452, 1999 [[PubMed]
135. Su S, Ohno Y, Lossin C, Hibino H, Inanobe A, Kurachi Y. Inhibition of astroglial inwardly rectifying Kir4.1 channels by a tricyclic antidepressant, nortriptyline. J Pharmacol Exp Ther320: 573–580, 2007 [[PubMed]
136. Tang H, Walsh SP, Yan Y, de Jesus RK, Shahripour A, Teumelsan N, Zhu Y, Ha S, Owens KA, Thomas-Fowlkes BS, Felix JP, Liu J, Kohler M, Priest BT, Bailey T, Brochu R, Alonso-Galicia M, Kaczorowski GJ, Roy S, Yang L, Mills SG, Garcia ML, Pasternak A. Discovery of selective small molecule ROMK inhibitors as potential new mechanism diuretics. ACS Med Chem Lett3: 367–372, 2012
137. Teulon J, Eladari D. A new mouse model for Bartter's syndrome. Am J Physiol Renal Physiol301: F295–F296, 2011 [[PubMed]
138. Thompson CH, Olivetti PR, Fuller MD, Freeman CS, McMaster D, French RJ, Pohl J, Kubanek J, McCarty NA. Isolation and characterization of a high affinity peptide inhibitor of ClC-2 chloride channels. J Biol Chem284: 26051–26062, 2009
139. Torres VE, Chapman AB, Devuyst O, Gansevoort RT, Grantham JJ, Higashihara E, Perrone RD, Krasa HB, Ouyang J, Czerwiec FS. Tolvaptan in patients with autosomal dominant polycystic kidney disease. N Engl J Med367: 2407–2418, 2012
140. Uchida S. In vivo role of CLC chloride channels in the kidney. Am J Physiol Renal Physiol279: F802–F808, 2000 [[PubMed]
141. Uchida S, Sasaki S, Nitta K, Uchida K, Horita S, Nihei H, Marumo F. Localization and functional characterization of rat kidney-specific chloride channel, ClC-K1. J Clin Invest95: 104–113, 1995
142. Uwai Y, Saito H, Hashimoto Y, Inui KI. Interaction and transport of thiazide diuretics, loop diuretics, and acetazolamide via rat renal organic anion transporter rOAT1. J Pharmacol Exp Ther295: 261–265, 2000 [[PubMed]
143. Vallon V, Rieg T, Ahn SY, Wu W, Eraly SA, Nigam SK. Overlapping in vitro and in vivo specificities of the organic anion transporters OAT1 and OAT3 for loop and thiazide diuretics. Am J Physiol Renal Physiol294: F867–F873, 2008 [[PubMed]
144. Vitari AC, Deak M, Morrice NA, Alessi DR. The WNK1 and WNK4 protein kinases that are mutated in Gordon's hypertension syndrome phosphorylate and activate SPAK and OSR1 protein kinases. Biochem J391: 17–24, 2005
145. Wall SM, Hassell KA, Royaux IE, Green ED, Chang JY, Shipley GL, Verlander JW. Localization of pendrin in mouse kidney. Am J Physiol Renal Physiol284: F229–F241, 2003 [[PubMed]
146. Wall SM, Kim YH, Stanley L, Glapion DM, Everett LA, Green ED, Verlander JW. NaCl restriction upregulates renal Slc26a4 through subcellular redistribution: role in Cl^conservation. Hypertension44: 982–987, 2004 [[PubMed]
147. Wang T, Wang WH, Klein-Robbenhaar G, Giebisch G. Effects of a novel K_ATP channel blocker on renal tubule function and K^{channel activity}. J Pharmacol Exp Ther273: 1382–1389, 1995 [[PubMed]
148. Wang T, Wang WH, Klein-Robbenhaar G, Giebisch G. Effects of glyburide on renal tubule transport and potassium-channel activity. Ren Physiol Biochem18: 169–182, 1995 [[PubMed]
149. Wang W, Hebert SC, Giebisch G. Renal K^{channels: structure and function}. Annu Rev Physiol59: 413–436, 1997 [[PubMed]
150. Wang W, Lu M. Effect of arachidonic acid on activity of the apical K^{channel in the thick ascending limb of the rat kidney}. J Gen Physiol106: 727–743, 1995
151. Wang XF, Lin RY, Wang SZ, Zhang LP, Qian J, Lu DR, Wen H, Jin L. Association study of variants in two ion-channel genes (TSC and CLCNKB) and hypertension in two ethnic groups in Northwest China. Clin Chim Acta388: 95–98, 2008 [[PubMed]
152. Wellhauser L, D'Antonio C, Bear CE. ClC transporters: discoveries and challenges in defining the mechanisms underlying function and regulation of ClC-5. Pflügers Arch460: 543–557, 2010 [[PubMed]
153. Welling PA, Ho K. A comprehensive guide to the ROMK potassium channel: form and function in health and disease. Am J Physiol Renal Physiol297: F849–F863, 2009
154. Wiemuth D, Grunder S. The pharmacological profile of brain liver intestine Na^{channel: inhibition by diarylamidines and activation by fenamates}. Mol Pharmacol80: 911–919, 2011 [[PubMed]
155. Wilson FH, Disse-Nicodeme S, Choate KA, Ishikawa K, Nelson-Williams C, Desitter I, Gunel M, Milford DV, Lipkin GW, Achard JM, Feely MP, Dussol B, Berland Y, Unwin RJ, Mayan H, Simon DB, Farfel Z, Jeunemaitre X, Lifton RP. Human hypertension caused by mutations in WNK kinases. Science293: 1107–1112, 2001 [[PubMed]
156. Wright SH, Dantzler WH. Molecular and cellular physiology of renal organic cation and anion transport. Physiol Rev84: 987–1049, 2004 [[PubMed]
157. Yang B, Bankir L. Urea and urine concentrating ability: new insights from studies in mice. Am J Physiol Renal Physiol288: F881–F896, 2005 [[PubMed]
158. Yang B, Bankir L, Gillespie A, Epstein CJ, Verkman AS. Urea-selective concentrating defect in transgenic mice lacking urea transporter UT-B. J Biol Chem277: 10633–10637, 2002 [[PubMed]
159. Yang SS, Lo YF, Wu CC, Lin SW, Yeh CJ, Chu P, Sytwu HK, Uchida S, Sasaki S, Lin SH. SPAK-knockout mice manifest Gitelman syndrome and impaired vasoconstriction. J Am Soc Nephrol21: 1868–1877, 2010
160. Yao C, Anderson MO, Zhang J, Yang B, Phuan PW, Verkman AS. Triazolothienopyrimidine inhibitors of urea transporter UT-B reduce urine concentration. J Am Soc Nephrol23: 1210–1220, 2012
161. Zhou H, Tate SS, Palmer LG. Primary structure and functional properties of an epithelial K^channel. Am J Physiol Cell Physiol266: C809–C824, 1994 [[PubMed]
162. Zifarelli G, Liantonio A, Gradogna A, Picollo A, Gramegna G, De Bellis M, Murgia AR, Babini E, Camerino DC, Pusch M. Identification of sites responsible for the potentiating effect of niflumic acid on ClC-Ka kidney chloride channels. Br J Pharmacol160: 1652–1661, 2010
163. Zifarelli G, Pusch M. CLC chloride channels and transporters: a biophysical and physiological perspective. Rev Physiol Biochem Pharmacol158: 23–76, 2007 [[PubMed]
164. Zuber AM, Singer D, Penninger JM, Rossier BC, Firsov D. Increased renal responsiveness to vasopressin and enhanced V2 receptor signaling in RGS2^mice. J Am Soc Nephrol18: 1672–1678, 2007 [[PubMed]