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Diuretics and Salt Transport Along the Nephron
Diuretics and Salt Transport Along the Nephron Paul L. Bernstein, MD,* and David H. Ellison, MD† Summary: The clinical use of diuretics almost uniformly predated the localization of their site of action. The consequence of diuretic specificity predicts clinical application and side effect, and the proximity of the sodium transporters, one to the next, often dictates potency or diuretic efficiency. All diuretics function by inhibiting the normal transport of sodium from the filtrate into the renal tubular cells. This movement of sodium into the renal epithelial cells on the apical side is facilitated by a series of transporters whose function is, in turn, dependent on the adenosine triphosphate (ATP)-dependent Na-K cotransporter on the basolateral side of the cell. Our growing understanding of the physiology of sodium transport has spawned new possibilities for diuretic development. Semin Nephrol 31:475-482 © 2011 Elsevier Inc. All rights reserved. Keywords: Diuretics, sodium transport, loop of Henle, proximal tubule, distal convoluted tubule, cortical collecting tubule
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ffective circulating volume is maintained by capturing sodium from the filtrate in an active process by various transporters along the length of the nephron. The molecular identification of these transporters over the past 20 years has resulted in their classification as part of the family of solute-carrier SLC genes. The identification of the complementary DNA consensus sequences has permitted confirmation of mutations originally deduced from clinical observation because disturbances in sodium transport are associated with loss of other electrolytes and acid-base homeostasis.1 The clinical use of diuretics almost uniformly predated the localization of their site of action. The consequence of diuretic specificity predicts clinical application and side effect, and the proximity of the sodium transporters, one to the next, often dictates potency or diuretic efficiency (urinary milliequivalents of sodium per milligram of diuretic). This article focuses on the interaction of diuretics and the sodium transporters they affect. Site-specific clinical relevance as a consequence of diuretic use or classified mutation also is discussed. It is important to remember that cells in many other organs will contain the identical sodium transporters found along the nephron, so that systemic effects may not be uniquely attributed to diuretic inhibition in the kidney. In 1959, for example, Wilson and Freis2 showed that although chlorothiazideinduced blood pressure reduction could be mitigated by *Division of Nephrology, Rochester General Hospital, University of Rochester School of Medicine and Dentistry, Rochester, NY. †Division of Nephrology and Hypertension, Oregon Health and Science University, Portland, OR. Address reprint requests to Paul L. Bernstein, MD, Rochester General Hospital, University of Rochester School of Medicine and Dentistry, 1425 Portland Ave, Rochester, NY 14621. E-mail: Paul.Bernstein@ rochestergeneral.org 0270-9295/ - see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.semnephrol.2011.09.002
Seminars in Nephrology, Vol 31, No 6, November 2011, pp 475-482
infusion of 6% dextran in isotonic saline in acute treatment with diuretic, the saliuretic effects plateau in approximately 48 hours. However, chronic blood pressure lowering from prolonged use of the same diuretic may not be volume-dependent because the hypotensive effect of the drug remained despite replenishment of plasma volume,2 implicating additional mechanisms other than salt loss through the kidney. At a cellular level it is known that isoforms of the electroneutral sodium transporters, which move a balanced combination of cations and anions across kidney epithelia, are involved in cellvolume regulation. These isoforms respond to osmolar stress that would swell or shrink the cell’s volume by shuttling ions, and the water that follows by obligation, to offset these effects. A Na⫹-K⫹-2Cl⫺ (NKCC1) isoform (also known as the bumetanide-sensitive cotransporter) will pump Na⫹, K⫹, and 2 Cl⫺ intracellularly when placed in an environment of high osmolarity to recapture water and maintain volume.3 These cation-chloride transporters are distributed ubiquitously and function independently of renal handling of sodium. Extrarenal actions may explain the beneficial effect of thiazide diuretics on bone mineral density. The thiazide-sensitive Na⫹-Cl⫺ cotransporter (NCC) is expressed in bone and the addition of thiazides to osteoblasts in culture increases the formation of mineralized nodules, an effect that is dependent on the presence of NCC.4 In the kidney, all diuretics function by inhibiting the normal transport of sodium from the filtrate into the renal tubular cells (Fig. 1). This movement of Na⫹ into the renal epithelial cells on the apical side is facilitated by a series of transporters whose function is, in turn, dependent on the adenosine triphosphate (ATP)-dependent Na⫹-K⫹ cotransporter on the basolateral side of the cell. This enzyme consumes energy while electrogenically moving three Na⫹ molecules from the inside of the cell, after internal phosphorylation, to the cell exterior. The phosphorylation causes a conformational change that permits binding to two K⫹ ions from the cell exterior and 475
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Figure 1. Schematic representation of the nephron illustrating sodium reabsorption in the proximal convoluted tubule (PCT), thick ascending limb (TAL), distal convoluted tubule (DCT), connecting tubule and collecting duct (CNT,CD). Other abbreviations: Na K-ATPase, sodium pump; NHE3, sodium-hydrogen exchanger isoform 3; NKCC2, sodium-potassium-chloride co-transporter; ROMK, renal outer medulla potassium channel; CLCNKB, chloride channel; NCCT, thiazide-sensitive sodium chloride co-transporter; WNK, “without lysine” kinase; ENaC, amiloridesensitive epithelial sodium channel; MR, mineralocorticoid receptor. Data from Glover.39
transports them into the cell. This transport, coupled with a resulting leak of potassium across the basolateral cell membrane, causes a voltage that is oriented with the cell interior negative, with respect to the cell exterior. This net negative intracellular charge promotes passive movement of Na⫹ facilitated by their transporters on the apical or luminal side of the renal epithelial cell. The Na⫹-K⫹ATPase protein is a structural dimer with several isotypes for the ␣ and  unit. The alpha-1 subtype predominates in the renal epithelial cells, but as with all of the isoforms there is no functional activity for either the ␣ or  unit alone.5 The Na⫹-K⫹ATPase, or Na⫹ pump, is itself regulated by some of the same influences that regulate the sodium transporters.6 Regulation of salt homeostasis represents the summative effect of filtered salt and the reabsorptive actions of the apical and basolateral transport. Alterations in sodium intake must be sensed as a perturbation and then salt transport adjusted, to restore balance. A sudden excess of sodium intake, for example, initiates a negative feedback response that promotes sodium excretion in a gradual manner and simultaneously guards against overshoot excretion.7
SODIUM TRANSPORTERS AND SITE-SPECIFIC DIURETICS Proximal Tubule There are multiple sodium transporters on the apical membrane of the proximal tubule where about two thirds of the filtered sodium is reclaimed. For example, the sodium-glucose cotransporter is part of a family of transporters that transfer glucose, amino acids, and vitamins. The sodium-glucose co-transporter 2 (SGLT-2) is the major apical glucose transport protein in the kidney, predominantly in the S1 portion of the proximal tubule. It is now the focus of a new family of medications (gliflazocin) to treat type 2 diabetes. This sodium transporter, when inhibited, obligates a diuresis from a combination of blocking sodium and glucose (osmotic diuresis) uptake. These medications work in a dose-dependent fashion and in early trials show antihypertensive as well as improved serum glucose control.8 Congenitally defective transport by SGLT-2 causes familial renal glucosuria, a heritable benign condition that causes clinically significant glucose losses and, rarely, clinically important salt losses as well. For example, low blood pressure and
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increased plasma renin and aldosterone despite daily excretion of 280 mmol of Na was reported in one patient with a deletion in the SLC5A2 gene coding for SGLT-2.9 More typically, under chronic conditions, sodium loss in the proximal tubule is reabsorbed, or compensated, by more distal transporters. This glucose wasting with a predominantly osmotic diuresis then serves as the first clinical model of the consequence of a diuretic, although this medication is intended for glucose control in diabetic patients. There are a number of different sodium transporters that move other ions such as phosphate or bicarbonate in the proximal tubule (PT), as well as several isoforms of the workhorse for sodium transport, the sodium-hydrogen exchangers (NHE). The predominant isoform expressed in the proximal tubule is NHE3. The proximal tubule has a rich anatomy of microvilli on the apical border used to increase surface area and facilitate bulk transfer of sodium. On the basolateral side, in turn, there is a rich supply of mitochondria to serve as the energy source for the Na⫹ pump. In this way the proximal tubule is well situated to transfer the bulk of filtered sodium, which amounts to approximately 60% of the filtered load. Increase of blood pressure in rats by arterial constriction causes natriuresis, and a redistribution of NHE3 and the sodium pump, thus limiting sodium uptake. After normalization of blood pressure, the transporters reassume their distribution and functionality within minutes, suggesting alterations in trafficking and sequestration.10 In the PT, if we excluded the SGLT-2 inhibitors, there are only two classes of effective diuretics, osmotic agents (eg, mannitol) and carbonic anhydrase inhibitors (eg, acetazolamide). The osmotic diuretics have limited use chronically but they often are used to treat acute cerebral edema and acute glaucoma, in which their effectiveness is caused primarily by local effects, promoting water egress from neuronal cells and from the aqueous humor. Mannitol and other osmotic diuretics, which are freely filtered at the glomerulus but are not absorbed, do not directly alter the activity of sodium transporters; rather, by retarding the passive absorption of water from the tubular lumen, they act indirectly, by increasing the concentration gradient against which these transports must work. Similar to the osmotic diuresis that accompanies severe hyperglycemia, mannitol-induced sodium and water losses may be great enough to cause clinical volumeinduced hypotension and hypernatremia along with hypokalemia and metabolic acidosis.11 Sodium is exchanged for H⫹ by NHE3 along the apical membrane of proximal tubule cells. The origin of the secreted H⫹ is the intracellular breakdown of carbonic acid, H2CO3, to HCO3⫺ and H⫹ catalyzed by carbonic anhydrase II. The HCO3 ions that are generated exit the cell with sodium by cotransport on the basolateral side and the H⫹ becomes available for exchange apically. The ultimate formation of CO2 and H2O is catalyzed similarly by a second pool of carbonic anhy-
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drase (isoform IV) located on the brush border after the apically secreted H⫹ recombines with filtered HCO3⫺ to form H2CO3. Both pools of carbonic anhydrase are inhibited by acetazolamide and other carbonic anhydrase inhibitors.12 Carbonic anhydrase inhibitors increase bicarbonate excretion by 25% to 30% with a minimal increase in sodium and chloride excretion because these ions are largely reabsorbed by more distal segments. Potassium excretion increases because the absorption of Na⫹ delivered downstream with HCO3⫺ is absorbed electrogenically by the epithelial sodium channel (ENaC), creating a negative intraluminal voltage that favors potassium secretion. Although the majority of Na⫹ transport occurs in the PT, inhibition of this pathway by chronic use of acetazolamide does not cause profound volume loss because of up-regulation of distal Na⫹ transport. The carbonic anhydrase inhibitor class is useful in glaucoma because of the presence of carbonic anhydrase in the eye, and is also used to correct metabolic alkalosis, which occurs during chronic loop diuretic use or after hypercapnea. Resultant hypokalemia should be anticipated in either of these uses, and there have been no clinical trials showing improved outcomes. Carbonic anhydrase inhibitors also have been used for high-altitude mountain climbing for mild diuretic effect as well as to combat the ensuing chronic respiratory alkalosis.11 They also are used commonly to treat pseudotumor cerebri. Ascending Limb and Distal Tubule: Sodium-Chloride Electroneutral Transporters The salt transporters in the thick ascending limb (TAL) and distal convoluted tubule are part of a family of electroneutral sodium transporters that move an equal number of cations and anions. The SLC12 gene family, which includes these transporters, is categorized according to ion specificity. The family includes K⫹Cl⫺ electroneutral transporters as well, but these are not included in this discussion because they are not targets of diuretic drugs.3 In the TAL, the sulfamoylbenzoic or bumetanidesensitive transporters predominate, and in the distal convoluted tubule (DCT), the benzothiadiazine or thiazidesensitive transporters are the major transporters. The electroneutral transporters were first isolated from the shark salt-secreting rectal gland and flounder bladder and their functionality was tested in the Xenopus laveis frog egg, a monstrous single cell. Once they were identified and sequenced, these transporters became useful as probes to discover the locations and variations of expression in the mammalian kidney and to understand their physiological role in both health and disease. In time it was discovered that two genes code for bumetanidesensitive transporters: SCL12A1 (coding for NKCC2, the predominant renal form, that is expressed on the apical side of the TAL) and SCL12A2 (coding for NKCC1, the transporter that is expressed on the basolateral side of epithelial cells, when present).3
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Figure 2. Glide symmetry model for consecutive binding of ions by NKCC2. Data from Lytle et al.13
NKCC2
Na⫹,
The basic structure of NKCC2, which cotransports K⫹, and 2 Cl⫺ ions, involves 12 membrane-spanning domains of 475 amino acid residues, with both the amino and carboxy termini as hydrophilic regions of approximately 165 and 450 residues, respectively.3 A model of NKCC2 has been proposed with four ion binding sites arrayed in a pocket in such a way that they can load only in a strictly ordered sequence (Fig. 2). Once the sites are fully occupied, the transporter can oscillate between two major conformations, alternately exposing it to the tubular lumen and then the cell. A major feature of the model is that net cotransport in either direction can occur only after the ions bind in a fixed sequence. This first-on, first-off behavior has been called glide symmetry.13 Splice variants of NKCC2 have been discovered based on variations in sequence in a specific transmembrane domain and the area immediately adjacent to it, with A, B, and F isoforms distributed in the cortex and medulla, uniquely cortex, and uniquely medulla, respectively.14 Experimental manipulation of sodium balance results in a physiologic-responsive distribution of the isoforms.15 In addition to splice variants, NKCC2 control is accomplished by regulation of phosphorylation sites. The NKCC2 protein expressed at the apical membrane of the TAL and macula densa cells lies in parallel with a K channel (renal outer medullary K channel [ROMK]) that permits potassium that has been transported from the lumen into the cell to return to the tubular lumen. The net effect of the transport processes in the TAL is the creation of a lumen-positive voltage that drives absorption of Na, K, Ca, and Mg through the paracellular pathway, accounting for 50% of the total transepithelial transport by this segment. By directly inhibiting transcellular Na transport, loop diuretics also inhibit paracellular trans-
port, enhancing their natriuretic effect and increasing excretion of potassium, calcium, and magnesium. Loop diuretics (furosemide, bumetamide, and torsemide) are organic anions that bind to NKCC2 from the luminal surface, inhibiting transport activity with a stoichiometry of one inhibitor molecule per transporter protein. Based on studies using [3H]bumetanide binding to renal outer medulla membranes it was concluded that bumetanide binds to the second Cl⫺ site on the NCC2 molecule.16 However, recent studies of the Na⫹K⫹-2Cl⫺ cotransporters using human-shark chimeras and point mutations have shown that altering the second transmembrane domain affects Na⫹, K⫹, and bumetanide kinetics, but not Cl⫺ kinetics. If bumetanide and Cl⫺ bind to the same site on the protein, the kinetics of both should be affected by the same alterations in amino acid sequence. Thus, bumetanide binding to the Na-K-2Cl cotransporter appears to be distinct from the Cl⫺ binding site.17 There are several glycosylation sites that impact NKCC2 activity, and two, N442 and N452, are conserved in the NCC as well, where they regulate transport activity. These appear in an extracellular loop, and in experiments of site-directed mutagenesis, activity of the NKCC2 was diminished 50% with a single phosphorylation site mutation, and 80% when both glycosylation sites were mutated. Simultaneous fluorescent microscopy revealed that the diminished activity was a result of down-regulated surface expression of the transporter, not damaged binding kinetics.18 NCC
Simon et al19 isolated the human thiazide-sensitive cotransporter and found that Gitelman’s syndrome of hypokalemic alkalosis is caused by loss-of-function mu-
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tations in NCC. The protein, 1021 amino acids in length, is similar to NKCC2 in that it has a central hydrophobic domain comprising 12 membrane-spanning domains, with a large cytoplasmic amino and carboxyl termini. The gene is named SLC12A3, and shows 50% homology with NKCC2.3 In 1990, Tran et al used [3H]-metolazone binding to rat kidney membranes as a surrogate for NCC activity. The investigators showed that Na⫹ ions promoted metolazone binding, but Cl⫺ ions inhibited it, implicating a competitive relationship between Cl⫺ and metolazone. These findings led to the conclusion that thiazide-type diuretics act as a surrogate for Cl⫺ and are bound to the NCC transporter in the presence of Na⫹ such that the bound diuretic blocks Cl⫺ transport and hence Na⫹ transport as well.20 More recent findings showing that Cl⫺ and thiazides bind to separate regions of NCC do not support the idea that thiazide diuretics and chloride compete for the same binding site. The creation of chimera in which segments of the NCC transporter were interchanged between the rat and the flounder (whose NCC molecules are functionally different in their affinities for sodium, chloride, and thiazides), revealed that the central hydrophobic domain of the molecule determines its affinity to Cl⫺ or to thiazides. The affinity of NCC for Cl⫺ was located on transmembrane segments 1 to 7, whereas affinity-defining residues for thiazide were located on transmembrane segments 8 to 12.4 It is interesting to note that although there is a good deal of homology between NKCC and NCC and a similarity in basic structure, the residues or motifs that convey diuretic specificity to the proteins remain undefined. Although NCC is the predominant apical Na transport pathway in the DCT, the DCT is now known to encompass a transitional segment in which NCC is expressed with the epithelial Na transporter ENaC.21 This segment is recognized increasingly as essential for the control of NaCl and K balance.22 Regulation of NKCC2 and NCC
More recently, NKCC2 and NCC have been discovered to be under the control of a novel regulatory network called “with no lysine” kinases (WNK), because lysine is present in more typical kinases, sterile 20/SPS1-related proline/alanine-rich kinase (SPAK) and oxidative stress response kinase (OSR1). WNKs interact with SPAK and OSR1 to modulate the activity of the NKCC1 and NCC.23 Mutations of WNK1 and WNK4 cause the human disease familial hyperkalemic hypertension (also called pseudohyperaldosteronism type 2 or Gordon’s syndrome). Yang et al24 showed, using the frog oocyte model system, that WNK4 inhibits NCC activity, owing to a decrease in NCC abundance at the plasma membrane. WNK1, in contrast, increases NCC activity, both by blocking WNK4 effects and by activating SPAK. Mutations that increase expression of WNK1 therefore
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activate NCC, as occurs in some patients with familial hyperkalemic hypertension.25 Lalioti et al26 made transgenic mice with a mutation in WNK4 that caused hypertension and hyperkalemia similar to Gordon’s syndrome in human beings. The effect of the engineered mutation to WNK4 could be circumvented when the mice were given thiazide diuretics. This study underscored the important role of these kinases, acting on NCC along the DCT, to generate human hypertension. WNK4 mutations keep the NCC in a “locked on phosphorylated state” so that it is functioning all the time. WNK4 may affect NCC activity both by altering its trafficking to the plasma membrane and by affecting its activation via phosphorylation.27 Both dietary NaCl intake and plasma aldosterone are known to modulate distal salt transport. To clarify whether effects of NaCl and aldosterone were mediated by the WNK network, mice with the pseudohyperaldosteronism type 2 (PHA-2) mutation and wild-type controls were given diets with low, normal, and high sodium content. In the wild-type mice, a high-salt diet decreased and a low-salt diet increased phosphorylation of the SPAK. In contrast, phosphorylation of SPAK was unaffected by diet in the mice carrying the PHA-2 WNK4 mutation. The enhanced phosphorylation in the wild-type mice could be blocked by spironolactone, which had no effect on the WNK4 mutated mice. This lack of regulation in the WNK4 mutated mice underscores that despite a volume-overloaded state achieved with a high-salt diet, the PHA-2 WNK4 mutation keeps NCC turned on, explaining the clinical findings in Gordon’s syndrome (PHA-2)28 (Fig. 3). Loss of function mutations
Families with loss-of-function mutations to the electroneutral transporters at the TAL and DCT present clinically as if treated with diuretics. Patients with Bartter’s syndrome, which simulates diuretic treatment with the TAL diuretics, were first described in 1962. Bartter’s syndrome has five subtypes, but all present with hypokalemia and metabolic alkalosis to varying degrees. Type I, an autosomal-recessive mutation, which affects SCL12A1 (NKCC2), typically presents with hypotension in newborns. Other types involve apical K⫹ channels (ROMK), basolateral Cl- channels, or subunits of the Clchannel; all of these proteins are highly expressed along the TAL. Type V involves a calcium-sensing receptor on the basolateral side of TAL cells that, when activated, inhibits NKCC2 activity. A mutation of this receptor constitutively decreases expression of NKCC2, thus mimicking a nonfunctioning transporter.3 Gitelman’s syndrome also presents with hypokalemia and metabolic alkalosis, but the phenotype is generally milder. It typically is diagnosed in the second or third decades of life and is inherited as an autosomal-recessive disease. These patients present clinically as if they are being treated with thiazide diuretics. In addition to pro-
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Figure 3. Regulation of thiazide-sensitive Na-Cl cotransporter. The thiazide-sensitive NCC is synthesized and then glycosylated (green forks) within the Golgi apparatus (not shown). NCC then moves to and into the apical membrane where it exists as a dimer. To be fully active, NCC undergoes phosphorylation along its amino-terminal cytoplasmic domain, mediated by SPAK, thereby permitting NaCl transport. Arginine vasopressin (AVP), aldosterone (Aldo), and angiotensin I (Ang II) all stimulate NCC activity. Trafficking may be a rapid effect, modulated predominantly by AVP and Ang II. Phosphorylation may occur within the membrane and is enhanced by all three factors. Reprinted with permission from Ellison.35
found hypokalemia and metabolic alkalosis, they also show hypocalciuria and hypomagnesemia. Patients with Gitelman’s syndrome almost invariably have mutations in the SCL12A3 gene (encoding NCC). There are more than 100 known Gitelman mutation sites, but the largest number fall into the category of mutations that result in defects in protein processing.3 Cortical Collecting Tubule: Electrogenic Sodium Transport Used predominantly to inhibit K⫹ secretion rather than Na⫹ absorption, drugs that inhibit sodium transport in the cortical collecting tubule are considered weaker diuretics because of the limited amount of Na⫹ reabsorbed in the distal nephron (approximately 3% of the filtered load). It is here that aldosterone alters sodium absorption and K⫹ excretion. These diuretics typically are divided into two classes: the aldosterone antagonists, spironolactone and eplerenone, and the Na channel blockers, including the pteridine, triamterene, and the pyrazinoylguanidine, amiloride. All inhibit transport in the aldosterone-sensitive distal nephron made up of the terminal portion of the distal convoluted tubule, the connecting tubule, and the cortical collecting duct.29 Along this segment, Na⫹ moves into the cells through the apically present ENaC, driven by the electrogenic gradient generated by the basolateral Na, K-ATPase. ENaC is made up of three subunits, ␣, , and ␥, which form a pore that can select for Na⫹ over K⫹ at a greater than 100:1
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ratio. The channel has two membrane-spanning domains, M1 and M2, linked by a large extracellular loop. Sitedirected mutagenesis on specific areas of the ␣, , and ␥ regions promote more rapid dissociation of amiloride, or triamterene from the pore, implicating critical areas that bind these diuretics. ENaC, expressed in the colon and the lung, is equally amiloride-sensitive, and the expression of ENaC is tightly regulated.30 ENaC action is enhanced by aldosterone, acting via serum-and-glucocorticoid-induced protein kinase (SGK1), which increases its abundance at the plasma membrane. Aldosterone also increases the apical abundance of ROMK, thereby increasing electrogenic Na⫹ absorption and K⫹ secretion. Aldosterone antagonists, therefore, inhibit these processes; thus, antagonism to aldosterone differs from the inhibition of cotransporters present early in the nephron by thiazides and loop diuretics.29 The effects of aldosterone are biphasic and can be divided into nongenomic and genomic actions. The nongenomic effects of the hormone on Na⫹ transport occur within 15 minutes and are not inhibitable by cycloheximide; the slower genomic effects occur at 120 minutes and are inhibited by drugs that inhibit DNA replication or protein synthesis. Both effects, elucidated with Na22 labeling, are inhibitable by spironolactone. The same nongenomic effects are present in colonic and vascular smooth cells, which express ENaC.31 Genomic effects of aldosterone involve hormone binding to a cytosolic mineralocorticoid receptor. The mineralocorticoid-receptor complex migrates to the nucleus where it up-regulates aldosterone-induced proteins. One of the important aldosterone-induced proteins is SGK1, which can interact with neural precursor cell expressed developmentally down-regulated protein (NEDD)4-2, as described later. Mineralocorticoid-receptor knock-out mice show relatively preserved levels of ENaC messenger RNA, suggesting that the effects of aldosterone on ENaC activity must be primarily post-transcriptional because the knockout mice die of salt wasting despite extraordinary levels (⬎10-fold of wild type) of renin, aldosterone, and angiotensin II.32 Liddle’s syndrome is a Mendelian-inherited form of hypertension involving a gain-of-function mutation in ENaC. The gain of function occurs in part because the mutation affects a ubiquitin-protein ligase named Nedd4 that targets ENaC for degradation. A failure of the targeted degradation allows for an increased expression of ENaC at the apical membrane. In addition, however, Nedd4 mutations can result in activation of existing ENaC, which then increases the proportion of channels that are open for conductance. Conversely, overexpression of Nedd4 diminishes active or cleaved ENaC and diminishes Na conductance.33 The overexpression of ENaC in Liddle’s syndrome is treated with a low-sodium diet and diuretics that block the channel such as amiloride or triamterene. Spironolactone has no effect, however, because sodium retention in the disease is the
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result of activated channels that do not require aldosterone. Although it previously was assumed that aldosterone’s actions were primarily to stimulate ENaC, it is now clear that aldosterone can stimulate NCC activity and abundance. Studies of adrenalectomized rats given angiotensin II or physiologic and supraphysiologic doses of aldosterone show that angiotensin II up-regulates NCC in a manner that is independent of aldosterone, partially inhibited by thiazide diuretics, and associated with SPAKmediated phosphorylation of NCC. Similarly, aldosterone increased the expression of NCC in a manner that is independent of angiotensin II.34 These differential effects of angiotensin II and aldosterone allow for differential regulation of NCC and ENaC, based on stimulus: volume depletion promotes both angiotensin II and aldosterone secretion and will increase Na⫹ absorption along the entire nephron resulting in Na⫹ absorption with little K⫹ secretion. Alternatively, when aldosterone is secreted without angiotensin II in response to hyperkalemia, K⫹ excretion is accomplished because this is predominantly an ENaC-regulated phenomenon.35 Spironolactone binds to the cytosolic mineralocorticoid receptor after entering the cell and inhibits further binding to the nucleus where aldosterone acts by uncapping DNA binding sites.29 Although typically thought of as a weaker diuretic, the cortical collecting tubule diuretics may be the preferred drug in high aldosterone states, such as hypertension driven by excess aldosterone or congestive heart failure. Clinically, they are used more often than loop or DCT diuretics in patients with hepatic cirrhosis because end-stage liver disease is associated with very high levels of aldosterone. In a study of a cirrhotic population, high doses of spironolactone evoked greater diuresis than did loop diuretics,36 but more commonly this group of diuretics is used in synergy with other diuretics to limit K⫹ loss.
FUTURE DIRECTIONS The story of diuretics and sodium transport has evolved simultaneously, each helping to define the other. Our growing understanding of the physiology of sodium transport has spawned new possibilities for diuretic development. For example, a specific inhibitor of the ROMK channel has been developed and could lead to a loop diuretic with minimal potassium wasting.37 SPAK and WNK kinases also have been targeted as new therapeutic agents.38 Because one of the primary roles of the kidney is the transport of sodium, disorders of this vital function will continue to drive diuretic use and help elucidate models of disease associated with abnormal transporters.
REFERENCES 1. Gitelman H, Grahm J, Walt L. A new familiar disorder characterized by hypokalemia, hypomagnesemia. Trans Assoc Am J Physiol. 1966;79:221-35.
481 2. Wilson I, Freis E. Relationship between plasma and extracellular fluid volume deprivation and the anti-hypertensive effect of chlorthiazide. Circulation. 1959;20:1028-36. 3. Gamba G. Molecular physiology and pathophysiology of electroneutral cation-chloride cotransporters. Physiol Rev. 2005;85: 423-93. 4. Gamba G. The thiazide-sensitive Na⫹Cl- cotransporter: molecular biology, functional properties, and regulation by WNKs. Am J Physiol Renal Physiol. 2009;297:F838-48. 5. Kaplan J. Biochemistry of Na, K-ATPase. Ann Rev Biochem. 2002;71:511-35. 6. McDonough A. Mechanism of proximal tubule sodium transport regulation that link extracellular fluid volume and blood pressure. Am J Physiol Regul Integr Compar Physiol. 2010;298:R851-61. 7. Singh P, Thomson S. Renal homeostasis and tubuloglomerular feedback. Curr Opin Nephrol Hypertens. 2010;19:59-64. 8. Komoroski B, Vacharajani N, Boulton D, Kornhauser D, Geraldes M, Li L. Dapagliflozin, a novel SGLT2 inhibitor, induces dose-dependent glucosuria in healthy subjects. Clin Pharmacol Ther. 2009;85:520-6. 9. Calado J, Loeffler J, Sakallioglu O, Gok F, Lhotta K, Barata J, et al. Familial renal glucosuria: SLC5A2 mutation analysis and evidence of salt-wasting. Kidney Int. 2006;69:852-5. 10. Zhang Y, Magyar C, Norian J, Holstein-Rathlou N, Mircheff A, McDonough A. Reversible effects of acute hypertension on proximal tubule sodium transporters. Am J Physiol Cell Physiol. 1998;274:1090-100. 11. Brater DC. Pharmacology of diuretics. Am J Med Sci. 2000;319: 38-50. 12. Sarafidis P, Georgianos P, Lasaridis A. Diuretics in clinical practice. Part I: mechanisms of action, pharmacological effects and clinical indications of diuretic compounds. Expert Opin Drug Saf. 2010;9:243-57. 13. Lytle C, McManus T, Haas M. A model of Na-K-2Cl cotransport based on ordered ion binding and glide symmetry. Am J Physiol. 1998;274:C299-309. 14. Payne J, Forbush B 3rd. Alternatively spliced isoforms of the putative renal Na-K-Cl cotransporter are differentially distributed within the rabbit kidney. Proc Natl Acad Sci U S A. 1994;91: 4544-8. 15. Brunet G, Gagnon E, Simard C, Daigle N, Caron L, Noel M, et al. Novel insights regarding the operational characteristics and teleological purpose of the renal Na-K-Cl2 cotransporter (NKCC2s) splice variants. J Gen Physiol. 2005;126:325-37. 16. Haas M, McManus T. Bumetanide inhibits Na⫹K⫹2Cl- cotransport at a chloride site. Am J Physiol Cell Physiol. 1983;245: C235-240. 17. Gagnon E. Molecular mechanisms of Cl transport by the renal Na-K-Cl cotransporter. J Biol Chem. 2004;279:5648-54. 18. Anahi-Paredes C, Plata C, Rivera M, et al. Activity of the renal Na-K-2Cl cotransporter is reduced by mutagenesis of N-glycosylation sites: role for protein surface charge in Cl transport. Am J Physiol Renal Physiol. 2006;290:F1094-102. 19. Simon D, Nelson-Williams C, Bia M, Ellison D, Karet F, Molina A, et al. Gitelman’s variant of Barter’s syndrome, inherited hypokalemic alkalosis, is caused by mutations in the thiazide-sensitive Na-Cl cotransporter. Nat Genet. 1996;12:24-30. 20. Tran J, Farrell M, Fanestil D. Effect of ions on binding of the thiazide-type diuretic metolazone to kidney membrane. Am J Physiol. 1990;258:F908-15. 21. Obermuller N, Kunchaparty S, Ellison D, Bachmann S. Expression of the Na-K-2Cl cotransporter by macula densa and thick ascending limb cells of rat and rabbit nephron. J Clin Invest. 1996;98:635-40. 22. Meneton P, Loffing J, Warnock D. Sodium and potassium handling by the aldosterone-sensitive distal nephron: the pivotal role of the distal and connecting tubule. Am J Physiol Renal Physiol. 2004;287:F593-601. 23. Gimenez I. Molecular mechanisms and regulation of furosemide-
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24.
25. 26.
27.
28.
29.
30.
31.
P.L. Bernstein and D.H. Ellison sensitive Na-K-Cl cotransporters. Curr Opin Nephrol Hypertens. 2006;15:517-23. Yang C-L, Angell J, Mitchell R, Ellison D. WNK kinases regulate thiazide-sensitive Na-Cl cotransport. J Clin Invest. 2003;111: 1039-45. Wilson F. Human Hypertension caused by mutations in WNKkinases. Science. 2001;293:1107-12. Lalioti M, Zhang J, Volkman H, Kahle K, Hoffmann K, Toka H, et al. WNK-4 controls blood pressure and potassium homeostasis via regulation of mass and activity of the distal convoluted tubule. Nat Genet. 2006;38:1124-32. Subramanya A, Ellison D. Sorting out lysosomal trafficking of the thiazide-sensitive Na-Cl Co-transporter. J Am Soc Nephrol. 2010; 21:7-9. Chiga M, Rai T, Yang S-S, Ohta A, Takizawa T, Sasaki S, et al. Dietary salt regulates the phosphorylation of OSR1/SPAK-kinases, and the sodium-chloride cotransporter through aldosterone. Kidney Int. 2008;74:1403-9. Okusa M, Ellison D. Physiology and pathophysiology of diuretic action. In: Alpern RJ, Hebert SC editors. Seldin and Giebisch’s the kidney. 4th ed. Philadelphia: Elsevier; 2008. p 1051-94. Kellenberger S. Mutations in the epithelial Na channel outer pore disrupt amiloride block by increasing its dissociation rate. Mol Pharmacol. 2003;64:848-56. Funder JW. The nongenomic effects of aldosterone. Endocr Rev. 2005;26:313-21.
32. Berger S, Bleich M, Schmid W, Cole T, Peters J, Watanabe H, et al. Mineralocorticoid receptor knockout mice: pathophysiology of Na metabolism. Proc Natl Acad Sci U S A. 1998;95: 9424-9. 33. Knight K, Olson D, Zhou R, Snyder P. Liddle’s syndrome mutations increase Na transport through dual effects on epithelial Na channel surface expression and proteolytic cleavage. Proc Natl Acad Sci U S A. 2006;103:2805-8. 34. Van der Lubbe N, Lim C, Fenton R, Meima M, Danser A, Zietse R, et al. Angiotensin II induces phosphorylation of thiazidesensitive sodium chloride cotransporter independent of aldosterone. Kidney Int. 2011;79:66-76. 35. Ellison D. Through a glass darkly: salt transport by the distal tubule. Kidney Int. 2011;79:5-8. 36. Laffi G, La Villa G, Carloni V, Foschi M, Bartoletti L, Quartini M, et al. Loop diuretic therapy in liver cirrhosis with ascites. J Cardiovasc Pharmacol. 1993;22 Suppl 3:S51-8. 37. Bhave G, Chauder B, Liu W, Dawson E, Kadakia R, Nguyen T, et al. Development of a selective small-molecule inhibitor of Kir1.1, the renal outer medullary potassium channel. Mol Pharmacol. 2011;79:42-50. 38. Glover M, O’Shaughnessy K. SPAK and WNK kinases: a new treatment for blood pressure treatment? Curr Opin Nephrol Hypertens. 2011;20:16-22. 39. Glover M, Zuber A, O’Shaugnessy K. Hypertension, salt intake, and the role of the thiazide-sensitive sodium chloride transporter NCCT. Cardiovascular therapeutics. 2011;29:68-76.
E
ffective circulating volume is maintained by capturing sodium from the filtrate in an active process by various transporters along the length of the nephron. The molecular identification of these transporters over the past 20 years has resulted in their classification as part of the family of solute-carrier SLC genes. The identification of the complementary DNA consensus sequences has permitted confirmation of mutations originally deduced from clinical observation because disturbances in sodium transport are associated with loss of other electrolytes and acid-base homeostasis.1 The clinical use of diuretics almost uniformly predated the localization of their site of action. The consequence of diuretic specificity predicts clinical application and side effect, and the proximity of the sodium transporters, one to the next, often dictates potency or diuretic efficiency (urinary milliequivalents of sodium per milligram of diuretic). This article focuses on the interaction of diuretics and the sodium transporters they affect. Site-specific clinical relevance as a consequence of diuretic use or classified mutation also is discussed. It is important to remember that cells in many other organs will contain the identical sodium transporters found along the nephron, so that systemic effects may not be uniquely attributed to diuretic inhibition in the kidney. In 1959, for example, Wilson and Freis2 showed that although chlorothiazideinduced blood pressure reduction could be mitigated by *Division of Nephrology, Rochester General Hospital, University of Rochester School of Medicine and Dentistry, Rochester, NY. †Division of Nephrology and Hypertension, Oregon Health and Science University, Portland, OR. Address reprint requests to Paul L. Bernstein, MD, Rochester General Hospital, University of Rochester School of Medicine and Dentistry, 1425 Portland Ave, Rochester, NY 14621. E-mail: Paul.Bernstein@ rochestergeneral.org 0270-9295/ - see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.semnephrol.2011.09.002
Seminars in Nephrology, Vol 31, No 6, November 2011, pp 475-482
infusion of 6% dextran in isotonic saline in acute treatment with diuretic, the saliuretic effects plateau in approximately 48 hours. However, chronic blood pressure lowering from prolonged use of the same diuretic may not be volume-dependent because the hypotensive effect of the drug remained despite replenishment of plasma volume,2 implicating additional mechanisms other than salt loss through the kidney. At a cellular level it is known that isoforms of the electroneutral sodium transporters, which move a balanced combination of cations and anions across kidney epithelia, are involved in cellvolume regulation. These isoforms respond to osmolar stress that would swell or shrink the cell’s volume by shuttling ions, and the water that follows by obligation, to offset these effects. A Na⫹-K⫹-2Cl⫺ (NKCC1) isoform (also known as the bumetanide-sensitive cotransporter) will pump Na⫹, K⫹, and 2 Cl⫺ intracellularly when placed in an environment of high osmolarity to recapture water and maintain volume.3 These cation-chloride transporters are distributed ubiquitously and function independently of renal handling of sodium. Extrarenal actions may explain the beneficial effect of thiazide diuretics on bone mineral density. The thiazide-sensitive Na⫹-Cl⫺ cotransporter (NCC) is expressed in bone and the addition of thiazides to osteoblasts in culture increases the formation of mineralized nodules, an effect that is dependent on the presence of NCC.4 In the kidney, all diuretics function by inhibiting the normal transport of sodium from the filtrate into the renal tubular cells (Fig. 1). This movement of Na⫹ into the renal epithelial cells on the apical side is facilitated by a series of transporters whose function is, in turn, dependent on the adenosine triphosphate (ATP)-dependent Na⫹-K⫹ cotransporter on the basolateral side of the cell. This enzyme consumes energy while electrogenically moving three Na⫹ molecules from the inside of the cell, after internal phosphorylation, to the cell exterior. The phosphorylation causes a conformational change that permits binding to two K⫹ ions from the cell exterior and 475
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Figure 1. Schematic representation of the nephron illustrating sodium reabsorption in the proximal convoluted tubule (PCT), thick ascending limb (TAL), distal convoluted tubule (DCT), connecting tubule and collecting duct (CNT,CD). Other abbreviations: Na K-ATPase, sodium pump; NHE3, sodium-hydrogen exchanger isoform 3; NKCC2, sodium-potassium-chloride co-transporter; ROMK, renal outer medulla potassium channel; CLCNKB, chloride channel; NCCT, thiazide-sensitive sodium chloride co-transporter; WNK, “without lysine” kinase; ENaC, amiloridesensitive epithelial sodium channel; MR, mineralocorticoid receptor. Data from Glover.39
transports them into the cell. This transport, coupled with a resulting leak of potassium across the basolateral cell membrane, causes a voltage that is oriented with the cell interior negative, with respect to the cell exterior. This net negative intracellular charge promotes passive movement of Na⫹ facilitated by their transporters on the apical or luminal side of the renal epithelial cell. The Na⫹-K⫹ATPase protein is a structural dimer with several isotypes for the ␣ and  unit. The alpha-1 subtype predominates in the renal epithelial cells, but as with all of the isoforms there is no functional activity for either the ␣ or  unit alone.5 The Na⫹-K⫹ATPase, or Na⫹ pump, is itself regulated by some of the same influences that regulate the sodium transporters.6 Regulation of salt homeostasis represents the summative effect of filtered salt and the reabsorptive actions of the apical and basolateral transport. Alterations in sodium intake must be sensed as a perturbation and then salt transport adjusted, to restore balance. A sudden excess of sodium intake, for example, initiates a negative feedback response that promotes sodium excretion in a gradual manner and simultaneously guards against overshoot excretion.7
SODIUM TRANSPORTERS AND SITE-SPECIFIC DIURETICS Proximal Tubule There are multiple sodium transporters on the apical membrane of the proximal tubule where about two thirds of the filtered sodium is reclaimed. For example, the sodium-glucose cotransporter is part of a family of transporters that transfer glucose, amino acids, and vitamins. The sodium-glucose co-transporter 2 (SGLT-2) is the major apical glucose transport protein in the kidney, predominantly in the S1 portion of the proximal tubule. It is now the focus of a new family of medications (gliflazocin) to treat type 2 diabetes. This sodium transporter, when inhibited, obligates a diuresis from a combination of blocking sodium and glucose (osmotic diuresis) uptake. These medications work in a dose-dependent fashion and in early trials show antihypertensive as well as improved serum glucose control.8 Congenitally defective transport by SGLT-2 causes familial renal glucosuria, a heritable benign condition that causes clinically significant glucose losses and, rarely, clinically important salt losses as well. For example, low blood pressure and
Diuretics and salt transport
increased plasma renin and aldosterone despite daily excretion of 280 mmol of Na was reported in one patient with a deletion in the SLC5A2 gene coding for SGLT-2.9 More typically, under chronic conditions, sodium loss in the proximal tubule is reabsorbed, or compensated, by more distal transporters. This glucose wasting with a predominantly osmotic diuresis then serves as the first clinical model of the consequence of a diuretic, although this medication is intended for glucose control in diabetic patients. There are a number of different sodium transporters that move other ions such as phosphate or bicarbonate in the proximal tubule (PT), as well as several isoforms of the workhorse for sodium transport, the sodium-hydrogen exchangers (NHE). The predominant isoform expressed in the proximal tubule is NHE3. The proximal tubule has a rich anatomy of microvilli on the apical border used to increase surface area and facilitate bulk transfer of sodium. On the basolateral side, in turn, there is a rich supply of mitochondria to serve as the energy source for the Na⫹ pump. In this way the proximal tubule is well situated to transfer the bulk of filtered sodium, which amounts to approximately 60% of the filtered load. Increase of blood pressure in rats by arterial constriction causes natriuresis, and a redistribution of NHE3 and the sodium pump, thus limiting sodium uptake. After normalization of blood pressure, the transporters reassume their distribution and functionality within minutes, suggesting alterations in trafficking and sequestration.10 In the PT, if we excluded the SGLT-2 inhibitors, there are only two classes of effective diuretics, osmotic agents (eg, mannitol) and carbonic anhydrase inhibitors (eg, acetazolamide). The osmotic diuretics have limited use chronically but they often are used to treat acute cerebral edema and acute glaucoma, in which their effectiveness is caused primarily by local effects, promoting water egress from neuronal cells and from the aqueous humor. Mannitol and other osmotic diuretics, which are freely filtered at the glomerulus but are not absorbed, do not directly alter the activity of sodium transporters; rather, by retarding the passive absorption of water from the tubular lumen, they act indirectly, by increasing the concentration gradient against which these transports must work. Similar to the osmotic diuresis that accompanies severe hyperglycemia, mannitol-induced sodium and water losses may be great enough to cause clinical volumeinduced hypotension and hypernatremia along with hypokalemia and metabolic acidosis.11 Sodium is exchanged for H⫹ by NHE3 along the apical membrane of proximal tubule cells. The origin of the secreted H⫹ is the intracellular breakdown of carbonic acid, H2CO3, to HCO3⫺ and H⫹ catalyzed by carbonic anhydrase II. The HCO3 ions that are generated exit the cell with sodium by cotransport on the basolateral side and the H⫹ becomes available for exchange apically. The ultimate formation of CO2 and H2O is catalyzed similarly by a second pool of carbonic anhy-
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drase (isoform IV) located on the brush border after the apically secreted H⫹ recombines with filtered HCO3⫺ to form H2CO3. Both pools of carbonic anhydrase are inhibited by acetazolamide and other carbonic anhydrase inhibitors.12 Carbonic anhydrase inhibitors increase bicarbonate excretion by 25% to 30% with a minimal increase in sodium and chloride excretion because these ions are largely reabsorbed by more distal segments. Potassium excretion increases because the absorption of Na⫹ delivered downstream with HCO3⫺ is absorbed electrogenically by the epithelial sodium channel (ENaC), creating a negative intraluminal voltage that favors potassium secretion. Although the majority of Na⫹ transport occurs in the PT, inhibition of this pathway by chronic use of acetazolamide does not cause profound volume loss because of up-regulation of distal Na⫹ transport. The carbonic anhydrase inhibitor class is useful in glaucoma because of the presence of carbonic anhydrase in the eye, and is also used to correct metabolic alkalosis, which occurs during chronic loop diuretic use or after hypercapnea. Resultant hypokalemia should be anticipated in either of these uses, and there have been no clinical trials showing improved outcomes. Carbonic anhydrase inhibitors also have been used for high-altitude mountain climbing for mild diuretic effect as well as to combat the ensuing chronic respiratory alkalosis.11 They also are used commonly to treat pseudotumor cerebri. Ascending Limb and Distal Tubule: Sodium-Chloride Electroneutral Transporters The salt transporters in the thick ascending limb (TAL) and distal convoluted tubule are part of a family of electroneutral sodium transporters that move an equal number of cations and anions. The SLC12 gene family, which includes these transporters, is categorized according to ion specificity. The family includes K⫹Cl⫺ electroneutral transporters as well, but these are not included in this discussion because they are not targets of diuretic drugs.3 In the TAL, the sulfamoylbenzoic or bumetanidesensitive transporters predominate, and in the distal convoluted tubule (DCT), the benzothiadiazine or thiazidesensitive transporters are the major transporters. The electroneutral transporters were first isolated from the shark salt-secreting rectal gland and flounder bladder and their functionality was tested in the Xenopus laveis frog egg, a monstrous single cell. Once they were identified and sequenced, these transporters became useful as probes to discover the locations and variations of expression in the mammalian kidney and to understand their physiological role in both health and disease. In time it was discovered that two genes code for bumetanidesensitive transporters: SCL12A1 (coding for NKCC2, the predominant renal form, that is expressed on the apical side of the TAL) and SCL12A2 (coding for NKCC1, the transporter that is expressed on the basolateral side of epithelial cells, when present).3
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Figure 2. Glide symmetry model for consecutive binding of ions by NKCC2. Data from Lytle et al.13
NKCC2
Na⫹,
The basic structure of NKCC2, which cotransports K⫹, and 2 Cl⫺ ions, involves 12 membrane-spanning domains of 475 amino acid residues, with both the amino and carboxy termini as hydrophilic regions of approximately 165 and 450 residues, respectively.3 A model of NKCC2 has been proposed with four ion binding sites arrayed in a pocket in such a way that they can load only in a strictly ordered sequence (Fig. 2). Once the sites are fully occupied, the transporter can oscillate between two major conformations, alternately exposing it to the tubular lumen and then the cell. A major feature of the model is that net cotransport in either direction can occur only after the ions bind in a fixed sequence. This first-on, first-off behavior has been called glide symmetry.13 Splice variants of NKCC2 have been discovered based on variations in sequence in a specific transmembrane domain and the area immediately adjacent to it, with A, B, and F isoforms distributed in the cortex and medulla, uniquely cortex, and uniquely medulla, respectively.14 Experimental manipulation of sodium balance results in a physiologic-responsive distribution of the isoforms.15 In addition to splice variants, NKCC2 control is accomplished by regulation of phosphorylation sites. The NKCC2 protein expressed at the apical membrane of the TAL and macula densa cells lies in parallel with a K channel (renal outer medullary K channel [ROMK]) that permits potassium that has been transported from the lumen into the cell to return to the tubular lumen. The net effect of the transport processes in the TAL is the creation of a lumen-positive voltage that drives absorption of Na, K, Ca, and Mg through the paracellular pathway, accounting for 50% of the total transepithelial transport by this segment. By directly inhibiting transcellular Na transport, loop diuretics also inhibit paracellular trans-
port, enhancing their natriuretic effect and increasing excretion of potassium, calcium, and magnesium. Loop diuretics (furosemide, bumetamide, and torsemide) are organic anions that bind to NKCC2 from the luminal surface, inhibiting transport activity with a stoichiometry of one inhibitor molecule per transporter protein. Based on studies using [3H]bumetanide binding to renal outer medulla membranes it was concluded that bumetanide binds to the second Cl⫺ site on the NCC2 molecule.16 However, recent studies of the Na⫹K⫹-2Cl⫺ cotransporters using human-shark chimeras and point mutations have shown that altering the second transmembrane domain affects Na⫹, K⫹, and bumetanide kinetics, but not Cl⫺ kinetics. If bumetanide and Cl⫺ bind to the same site on the protein, the kinetics of both should be affected by the same alterations in amino acid sequence. Thus, bumetanide binding to the Na-K-2Cl cotransporter appears to be distinct from the Cl⫺ binding site.17 There are several glycosylation sites that impact NKCC2 activity, and two, N442 and N452, are conserved in the NCC as well, where they regulate transport activity. These appear in an extracellular loop, and in experiments of site-directed mutagenesis, activity of the NKCC2 was diminished 50% with a single phosphorylation site mutation, and 80% when both glycosylation sites were mutated. Simultaneous fluorescent microscopy revealed that the diminished activity was a result of down-regulated surface expression of the transporter, not damaged binding kinetics.18 NCC
Simon et al19 isolated the human thiazide-sensitive cotransporter and found that Gitelman’s syndrome of hypokalemic alkalosis is caused by loss-of-function mu-
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tations in NCC. The protein, 1021 amino acids in length, is similar to NKCC2 in that it has a central hydrophobic domain comprising 12 membrane-spanning domains, with a large cytoplasmic amino and carboxyl termini. The gene is named SLC12A3, and shows 50% homology with NKCC2.3 In 1990, Tran et al used [3H]-metolazone binding to rat kidney membranes as a surrogate for NCC activity. The investigators showed that Na⫹ ions promoted metolazone binding, but Cl⫺ ions inhibited it, implicating a competitive relationship between Cl⫺ and metolazone. These findings led to the conclusion that thiazide-type diuretics act as a surrogate for Cl⫺ and are bound to the NCC transporter in the presence of Na⫹ such that the bound diuretic blocks Cl⫺ transport and hence Na⫹ transport as well.20 More recent findings showing that Cl⫺ and thiazides bind to separate regions of NCC do not support the idea that thiazide diuretics and chloride compete for the same binding site. The creation of chimera in which segments of the NCC transporter were interchanged between the rat and the flounder (whose NCC molecules are functionally different in their affinities for sodium, chloride, and thiazides), revealed that the central hydrophobic domain of the molecule determines its affinity to Cl⫺ or to thiazides. The affinity of NCC for Cl⫺ was located on transmembrane segments 1 to 7, whereas affinity-defining residues for thiazide were located on transmembrane segments 8 to 12.4 It is interesting to note that although there is a good deal of homology between NKCC and NCC and a similarity in basic structure, the residues or motifs that convey diuretic specificity to the proteins remain undefined. Although NCC is the predominant apical Na transport pathway in the DCT, the DCT is now known to encompass a transitional segment in which NCC is expressed with the epithelial Na transporter ENaC.21 This segment is recognized increasingly as essential for the control of NaCl and K balance.22 Regulation of NKCC2 and NCC
More recently, NKCC2 and NCC have been discovered to be under the control of a novel regulatory network called “with no lysine” kinases (WNK), because lysine is present in more typical kinases, sterile 20/SPS1-related proline/alanine-rich kinase (SPAK) and oxidative stress response kinase (OSR1). WNKs interact with SPAK and OSR1 to modulate the activity of the NKCC1 and NCC.23 Mutations of WNK1 and WNK4 cause the human disease familial hyperkalemic hypertension (also called pseudohyperaldosteronism type 2 or Gordon’s syndrome). Yang et al24 showed, using the frog oocyte model system, that WNK4 inhibits NCC activity, owing to a decrease in NCC abundance at the plasma membrane. WNK1, in contrast, increases NCC activity, both by blocking WNK4 effects and by activating SPAK. Mutations that increase expression of WNK1 therefore
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activate NCC, as occurs in some patients with familial hyperkalemic hypertension.25 Lalioti et al26 made transgenic mice with a mutation in WNK4 that caused hypertension and hyperkalemia similar to Gordon’s syndrome in human beings. The effect of the engineered mutation to WNK4 could be circumvented when the mice were given thiazide diuretics. This study underscored the important role of these kinases, acting on NCC along the DCT, to generate human hypertension. WNK4 mutations keep the NCC in a “locked on phosphorylated state” so that it is functioning all the time. WNK4 may affect NCC activity both by altering its trafficking to the plasma membrane and by affecting its activation via phosphorylation.27 Both dietary NaCl intake and plasma aldosterone are known to modulate distal salt transport. To clarify whether effects of NaCl and aldosterone were mediated by the WNK network, mice with the pseudohyperaldosteronism type 2 (PHA-2) mutation and wild-type controls were given diets with low, normal, and high sodium content. In the wild-type mice, a high-salt diet decreased and a low-salt diet increased phosphorylation of the SPAK. In contrast, phosphorylation of SPAK was unaffected by diet in the mice carrying the PHA-2 WNK4 mutation. The enhanced phosphorylation in the wild-type mice could be blocked by spironolactone, which had no effect on the WNK4 mutated mice. This lack of regulation in the WNK4 mutated mice underscores that despite a volume-overloaded state achieved with a high-salt diet, the PHA-2 WNK4 mutation keeps NCC turned on, explaining the clinical findings in Gordon’s syndrome (PHA-2)28 (Fig. 3). Loss of function mutations
Families with loss-of-function mutations to the electroneutral transporters at the TAL and DCT present clinically as if treated with diuretics. Patients with Bartter’s syndrome, which simulates diuretic treatment with the TAL diuretics, were first described in 1962. Bartter’s syndrome has five subtypes, but all present with hypokalemia and metabolic alkalosis to varying degrees. Type I, an autosomal-recessive mutation, which affects SCL12A1 (NKCC2), typically presents with hypotension in newborns. Other types involve apical K⫹ channels (ROMK), basolateral Cl- channels, or subunits of the Clchannel; all of these proteins are highly expressed along the TAL. Type V involves a calcium-sensing receptor on the basolateral side of TAL cells that, when activated, inhibits NKCC2 activity. A mutation of this receptor constitutively decreases expression of NKCC2, thus mimicking a nonfunctioning transporter.3 Gitelman’s syndrome also presents with hypokalemia and metabolic alkalosis, but the phenotype is generally milder. It typically is diagnosed in the second or third decades of life and is inherited as an autosomal-recessive disease. These patients present clinically as if they are being treated with thiazide diuretics. In addition to pro-
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Figure 3. Regulation of thiazide-sensitive Na-Cl cotransporter. The thiazide-sensitive NCC is synthesized and then glycosylated (green forks) within the Golgi apparatus (not shown). NCC then moves to and into the apical membrane where it exists as a dimer. To be fully active, NCC undergoes phosphorylation along its amino-terminal cytoplasmic domain, mediated by SPAK, thereby permitting NaCl transport. Arginine vasopressin (AVP), aldosterone (Aldo), and angiotensin I (Ang II) all stimulate NCC activity. Trafficking may be a rapid effect, modulated predominantly by AVP and Ang II. Phosphorylation may occur within the membrane and is enhanced by all three factors. Reprinted with permission from Ellison.35
found hypokalemia and metabolic alkalosis, they also show hypocalciuria and hypomagnesemia. Patients with Gitelman’s syndrome almost invariably have mutations in the SCL12A3 gene (encoding NCC). There are more than 100 known Gitelman mutation sites, but the largest number fall into the category of mutations that result in defects in protein processing.3 Cortical Collecting Tubule: Electrogenic Sodium Transport Used predominantly to inhibit K⫹ secretion rather than Na⫹ absorption, drugs that inhibit sodium transport in the cortical collecting tubule are considered weaker diuretics because of the limited amount of Na⫹ reabsorbed in the distal nephron (approximately 3% of the filtered load). It is here that aldosterone alters sodium absorption and K⫹ excretion. These diuretics typically are divided into two classes: the aldosterone antagonists, spironolactone and eplerenone, and the Na channel blockers, including the pteridine, triamterene, and the pyrazinoylguanidine, amiloride. All inhibit transport in the aldosterone-sensitive distal nephron made up of the terminal portion of the distal convoluted tubule, the connecting tubule, and the cortical collecting duct.29 Along this segment, Na⫹ moves into the cells through the apically present ENaC, driven by the electrogenic gradient generated by the basolateral Na, K-ATPase. ENaC is made up of three subunits, ␣, , and ␥, which form a pore that can select for Na⫹ over K⫹ at a greater than 100:1
P.L. Bernstein and D.H. Ellison
ratio. The channel has two membrane-spanning domains, M1 and M2, linked by a large extracellular loop. Sitedirected mutagenesis on specific areas of the ␣, , and ␥ regions promote more rapid dissociation of amiloride, or triamterene from the pore, implicating critical areas that bind these diuretics. ENaC, expressed in the colon and the lung, is equally amiloride-sensitive, and the expression of ENaC is tightly regulated.30 ENaC action is enhanced by aldosterone, acting via serum-and-glucocorticoid-induced protein kinase (SGK1), which increases its abundance at the plasma membrane. Aldosterone also increases the apical abundance of ROMK, thereby increasing electrogenic Na⫹ absorption and K⫹ secretion. Aldosterone antagonists, therefore, inhibit these processes; thus, antagonism to aldosterone differs from the inhibition of cotransporters present early in the nephron by thiazides and loop diuretics.29 The effects of aldosterone are biphasic and can be divided into nongenomic and genomic actions. The nongenomic effects of the hormone on Na⫹ transport occur within 15 minutes and are not inhibitable by cycloheximide; the slower genomic effects occur at 120 minutes and are inhibited by drugs that inhibit DNA replication or protein synthesis. Both effects, elucidated with Na22 labeling, are inhibitable by spironolactone. The same nongenomic effects are present in colonic and vascular smooth cells, which express ENaC.31 Genomic effects of aldosterone involve hormone binding to a cytosolic mineralocorticoid receptor. The mineralocorticoid-receptor complex migrates to the nucleus where it up-regulates aldosterone-induced proteins. One of the important aldosterone-induced proteins is SGK1, which can interact with neural precursor cell expressed developmentally down-regulated protein (NEDD)4-2, as described later. Mineralocorticoid-receptor knock-out mice show relatively preserved levels of ENaC messenger RNA, suggesting that the effects of aldosterone on ENaC activity must be primarily post-transcriptional because the knockout mice die of salt wasting despite extraordinary levels (⬎10-fold of wild type) of renin, aldosterone, and angiotensin II.32 Liddle’s syndrome is a Mendelian-inherited form of hypertension involving a gain-of-function mutation in ENaC. The gain of function occurs in part because the mutation affects a ubiquitin-protein ligase named Nedd4 that targets ENaC for degradation. A failure of the targeted degradation allows for an increased expression of ENaC at the apical membrane. In addition, however, Nedd4 mutations can result in activation of existing ENaC, which then increases the proportion of channels that are open for conductance. Conversely, overexpression of Nedd4 diminishes active or cleaved ENaC and diminishes Na conductance.33 The overexpression of ENaC in Liddle’s syndrome is treated with a low-sodium diet and diuretics that block the channel such as amiloride or triamterene. Spironolactone has no effect, however, because sodium retention in the disease is the
Diuretics and salt transport
result of activated channels that do not require aldosterone. Although it previously was assumed that aldosterone’s actions were primarily to stimulate ENaC, it is now clear that aldosterone can stimulate NCC activity and abundance. Studies of adrenalectomized rats given angiotensin II or physiologic and supraphysiologic doses of aldosterone show that angiotensin II up-regulates NCC in a manner that is independent of aldosterone, partially inhibited by thiazide diuretics, and associated with SPAKmediated phosphorylation of NCC. Similarly, aldosterone increased the expression of NCC in a manner that is independent of angiotensin II.34 These differential effects of angiotensin II and aldosterone allow for differential regulation of NCC and ENaC, based on stimulus: volume depletion promotes both angiotensin II and aldosterone secretion and will increase Na⫹ absorption along the entire nephron resulting in Na⫹ absorption with little K⫹ secretion. Alternatively, when aldosterone is secreted without angiotensin II in response to hyperkalemia, K⫹ excretion is accomplished because this is predominantly an ENaC-regulated phenomenon.35 Spironolactone binds to the cytosolic mineralocorticoid receptor after entering the cell and inhibits further binding to the nucleus where aldosterone acts by uncapping DNA binding sites.29 Although typically thought of as a weaker diuretic, the cortical collecting tubule diuretics may be the preferred drug in high aldosterone states, such as hypertension driven by excess aldosterone or congestive heart failure. Clinically, they are used more often than loop or DCT diuretics in patients with hepatic cirrhosis because end-stage liver disease is associated with very high levels of aldosterone. In a study of a cirrhotic population, high doses of spironolactone evoked greater diuresis than did loop diuretics,36 but more commonly this group of diuretics is used in synergy with other diuretics to limit K⫹ loss.
FUTURE DIRECTIONS The story of diuretics and sodium transport has evolved simultaneously, each helping to define the other. Our growing understanding of the physiology of sodium transport has spawned new possibilities for diuretic development. For example, a specific inhibitor of the ROMK channel has been developed and could lead to a loop diuretic with minimal potassium wasting.37 SPAK and WNK kinases also have been targeted as new therapeutic agents.38 Because one of the primary roles of the kidney is the transport of sodium, disorders of this vital function will continue to drive diuretic use and help elucidate models of disease associated with abnormal transporters.
REFERENCES 1. Gitelman H, Grahm J, Walt L. A new familiar disorder characterized by hypokalemia, hypomagnesemia. Trans Assoc Am J Physiol. 1966;79:221-35.
481 2. Wilson I, Freis E. Relationship between plasma and extracellular fluid volume deprivation and the anti-hypertensive effect of chlorthiazide. Circulation. 1959;20:1028-36. 3. Gamba G. Molecular physiology and pathophysiology of electroneutral cation-chloride cotransporters. Physiol Rev. 2005;85: 423-93. 4. Gamba G. The thiazide-sensitive Na⫹Cl- cotransporter: molecular biology, functional properties, and regulation by WNKs. Am J Physiol Renal Physiol. 2009;297:F838-48. 5. Kaplan J. Biochemistry of Na, K-ATPase. Ann Rev Biochem. 2002;71:511-35. 6. McDonough A. Mechanism of proximal tubule sodium transport regulation that link extracellular fluid volume and blood pressure. Am J Physiol Regul Integr Compar Physiol. 2010;298:R851-61. 7. Singh P, Thomson S. Renal homeostasis and tubuloglomerular feedback. Curr Opin Nephrol Hypertens. 2010;19:59-64. 8. Komoroski B, Vacharajani N, Boulton D, Kornhauser D, Geraldes M, Li L. Dapagliflozin, a novel SGLT2 inhibitor, induces dose-dependent glucosuria in healthy subjects. Clin Pharmacol Ther. 2009;85:520-6. 9. Calado J, Loeffler J, Sakallioglu O, Gok F, Lhotta K, Barata J, et al. Familial renal glucosuria: SLC5A2 mutation analysis and evidence of salt-wasting. Kidney Int. 2006;69:852-5. 10. Zhang Y, Magyar C, Norian J, Holstein-Rathlou N, Mircheff A, McDonough A. Reversible effects of acute hypertension on proximal tubule sodium transporters. Am J Physiol Cell Physiol. 1998;274:1090-100. 11. Brater DC. Pharmacology of diuretics. Am J Med Sci. 2000;319: 38-50. 12. Sarafidis P, Georgianos P, Lasaridis A. Diuretics in clinical practice. Part I: mechanisms of action, pharmacological effects and clinical indications of diuretic compounds. Expert Opin Drug Saf. 2010;9:243-57. 13. Lytle C, McManus T, Haas M. A model of Na-K-2Cl cotransport based on ordered ion binding and glide symmetry. Am J Physiol. 1998;274:C299-309. 14. Payne J, Forbush B 3rd. Alternatively spliced isoforms of the putative renal Na-K-Cl cotransporter are differentially distributed within the rabbit kidney. Proc Natl Acad Sci U S A. 1994;91: 4544-8. 15. Brunet G, Gagnon E, Simard C, Daigle N, Caron L, Noel M, et al. Novel insights regarding the operational characteristics and teleological purpose of the renal Na-K-Cl2 cotransporter (NKCC2s) splice variants. J Gen Physiol. 2005;126:325-37. 16. Haas M, McManus T. Bumetanide inhibits Na⫹K⫹2Cl- cotransport at a chloride site. Am J Physiol Cell Physiol. 1983;245: C235-240. 17. Gagnon E. Molecular mechanisms of Cl transport by the renal Na-K-Cl cotransporter. J Biol Chem. 2004;279:5648-54. 18. Anahi-Paredes C, Plata C, Rivera M, et al. Activity of the renal Na-K-2Cl cotransporter is reduced by mutagenesis of N-glycosylation sites: role for protein surface charge in Cl transport. Am J Physiol Renal Physiol. 2006;290:F1094-102. 19. Simon D, Nelson-Williams C, Bia M, Ellison D, Karet F, Molina A, et al. Gitelman’s variant of Barter’s syndrome, inherited hypokalemic alkalosis, is caused by mutations in the thiazide-sensitive Na-Cl cotransporter. Nat Genet. 1996;12:24-30. 20. Tran J, Farrell M, Fanestil D. Effect of ions on binding of the thiazide-type diuretic metolazone to kidney membrane. Am J Physiol. 1990;258:F908-15. 21. Obermuller N, Kunchaparty S, Ellison D, Bachmann S. Expression of the Na-K-2Cl cotransporter by macula densa and thick ascending limb cells of rat and rabbit nephron. J Clin Invest. 1996;98:635-40. 22. Meneton P, Loffing J, Warnock D. Sodium and potassium handling by the aldosterone-sensitive distal nephron: the pivotal role of the distal and connecting tubule. Am J Physiol Renal Physiol. 2004;287:F593-601. 23. Gimenez I. Molecular mechanisms and regulation of furosemide-
482
24.
25. 26.
27.
28.
29.
30.
31.
P.L. Bernstein and D.H. Ellison sensitive Na-K-Cl cotransporters. Curr Opin Nephrol Hypertens. 2006;15:517-23. Yang C-L, Angell J, Mitchell R, Ellison D. WNK kinases regulate thiazide-sensitive Na-Cl cotransport. J Clin Invest. 2003;111: 1039-45. Wilson F. Human Hypertension caused by mutations in WNKkinases. Science. 2001;293:1107-12. Lalioti M, Zhang J, Volkman H, Kahle K, Hoffmann K, Toka H, et al. WNK-4 controls blood pressure and potassium homeostasis via regulation of mass and activity of the distal convoluted tubule. Nat Genet. 2006;38:1124-32. Subramanya A, Ellison D. Sorting out lysosomal trafficking of the thiazide-sensitive Na-Cl Co-transporter. J Am Soc Nephrol. 2010; 21:7-9. Chiga M, Rai T, Yang S-S, Ohta A, Takizawa T, Sasaki S, et al. Dietary salt regulates the phosphorylation of OSR1/SPAK-kinases, and the sodium-chloride cotransporter through aldosterone. Kidney Int. 2008;74:1403-9. Okusa M, Ellison D. Physiology and pathophysiology of diuretic action. In: Alpern RJ, Hebert SC editors. Seldin and Giebisch’s the kidney. 4th ed. Philadelphia: Elsevier; 2008. p 1051-94. Kellenberger S. Mutations in the epithelial Na channel outer pore disrupt amiloride block by increasing its dissociation rate. Mol Pharmacol. 2003;64:848-56. Funder JW. The nongenomic effects of aldosterone. Endocr Rev. 2005;26:313-21.
32. Berger S, Bleich M, Schmid W, Cole T, Peters J, Watanabe H, et al. Mineralocorticoid receptor knockout mice: pathophysiology of Na metabolism. Proc Natl Acad Sci U S A. 1998;95: 9424-9. 33. Knight K, Olson D, Zhou R, Snyder P. Liddle’s syndrome mutations increase Na transport through dual effects on epithelial Na channel surface expression and proteolytic cleavage. Proc Natl Acad Sci U S A. 2006;103:2805-8. 34. Van der Lubbe N, Lim C, Fenton R, Meima M, Danser A, Zietse R, et al. Angiotensin II induces phosphorylation of thiazidesensitive sodium chloride cotransporter independent of aldosterone. Kidney Int. 2011;79:66-76. 35. Ellison D. Through a glass darkly: salt transport by the distal tubule. Kidney Int. 2011;79:5-8. 36. Laffi G, La Villa G, Carloni V, Foschi M, Bartoletti L, Quartini M, et al. Loop diuretic therapy in liver cirrhosis with ascites. J Cardiovasc Pharmacol. 1993;22 Suppl 3:S51-8. 37. Bhave G, Chauder B, Liu W, Dawson E, Kadakia R, Nguyen T, et al. Development of a selective small-molecule inhibitor of Kir1.1, the renal outer medullary potassium channel. Mol Pharmacol. 2011;79:42-50. 38. Glover M, O’Shaughnessy K. SPAK and WNK kinases: a new treatment for blood pressure treatment? Curr Opin Nephrol Hypertens. 2011;20:16-22. 39. Glover M, Zuber A, O’Shaugnessy K. Hypertension, salt intake, and the role of the thiazide-sensitive sodium chloride transporter NCCT. Cardiovascular therapeutics. 2011;29:68-76.