Genes & Diseases (2015) 2, 186e196

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REVIEW ARTICLE

NKCC1 and NKCC2: The pathogenetic role ofcation-chloride cotransporters inhypertension

Sergei N. Orlov a,b,*, Svetlana V. Koltsova a,Leonid V. Kapilevich b,c, Svetlana V. Gusakova c,Nickolai O. Dulin d

a Faculty of Biology, M.V. Lomonosov Moscow State University, Russiab Tomsk State University, Russiac Siberian State Medical University, Russiad University of Chicago Department of Medicine, USA

Received 22 December 2014; accepted 16 February 2015Available online 25 February 2015

KEYWORDSHypertension;Intracellular chloride;Myogenic tone;Neuronal cell;NKCC cotransport;Smooth muscle

Abbreviations: CCC, sation-chloride cacid; GFR, glomerular filtration ratecotransport; NKCC, Naþ,Kþ,2Cl� cotracells; SNS, sympathetic nervous systemtubuloglomerular feedback; WNK, wit* Corresponding author. Laboratory

Russia.E-mail address: sergeinorlov@yandPeer review under responsibility o

http://dx.doi.org/10.1016/j.gendis.22352-3042/Copyright ª 2015, ChongqiCC BY-NC-ND license (http://creative

Abstract This review summarizes the data on the functional significance of ubiquitous(NKCC1) and renal-specific (NKCC2) isoforms of electroneutral sodium, potassium and chloridecotransporters. These carriers contribute to the pathogenesis of hypertension via regulation ofintracellular chloride concentration in vascular smooth muscle and neuronal cells and viasensing chloride concentration in the renal tubular fluid, respectively. Both NKCC1 and NKCC2are inhibited by furosemide and other high-ceiling diuretics widely used for attenuation ofextracellular fluid volume. However, the chronic usage of these compounds for the treatmentof hypertension and other volume-expanded disorders may have diverse side-effects due tosuppression of myogenic response in microcirculatory beds.Copyright ª 2015, Chongqing Medical University. Production and hosting by Elsevier B.V. This isan open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

otransporters; CNS, central nervous system; EFV, extracellular fluid volume; GABA, g-aminobutiric; JGA, juxtaglomerular apparatus; KCC, Kþ,Cl� cotransport; MD, macula densa; NCC, Naþ,Cl�

nsport; OSR1, oxidative stress response kinase; PVN, paraventricular nucleus; SMC, smooth muscle; SPAK, Ste20-related praline-alanine-rich kinase; TAHL, thick ascending limb of Henle’s loop; TGF,

h no K Z lysine kinase.of Biomemranes, Room 169, Faculty of Biology, MSU, Vorob’evy Gory 1, Build 12, Moscow 199899,

ex.ru (S.N. Orlov).f Chongqing Medical University.

015.02.007ng Medical University. Production and hosting by Elsevier B.V. This is an open access article under thecommons.org/licenses/by-nc-nd/4.0/).

NKCC1 and NKCC2 187

Naþ ,Kþ, 2Cl� cotransporters (NKCC) encoding by SLC12A2

Table 2 Effect of ouabain and bumetanide on the volumeof human lung fibroblasts.

Additions Cell volume, pl per cell

None (control) 0.23 � 0.04Ouabain 0.26 � 0.04Bumetanide 0.22 � 0.01Ouabain þ bumetanide 0.43 � 0.04*

Cells were incubated in the presence of 0.1 mM ouabain and/

(NKCC1) and SLC12A1 (NKCC2) belong to the subfamily ofcation-chloride cotransporters (CCCs) that provide elec-troneutral transport of sodium, potassium and chlorideacross the plasma membrane and are inhibited by bume-tanide, furosemide and several other structurally similarcompounds (Table 1). Because the major target of thesedrugs is inhibition of ion transport in thick ascending limb ofHenle’s loop (TAHL), they were termed as high-ceiling di-uretics. Gene structure, membrane architecture andpharmacology of SSSs were subjected to detailed analysisin several comprehensive reviews.1e3 This review focuseson physiological significance of NKCCs and their involve-ment in the pathogenesis of hypertension via regulation ofintracellular chloride concentration and sensing chlorideconcentration in the renal tubular fluid.

or 10 mM bumetanide for 24 h. Cell volume was measured as14C-urea available space.9 Means � S.E. obtained in experi-ments performed in quadruplicate are shown. *p < 0.005compared to control.

Functional implications of NKCCs

Cell volume regulation

Animal cells maintain their volume with an accuracy of1%e2% by means of systems providing inwardly andoutwardly directed fluxes of monovalent ions and organicosmolytes and termed as regulatory volume increase (RVI)and regulatory volume decrease (RVD), respectively.4,5 Inearly studies with mammalian erythrocytes, we and othershave shown that cell shrinkage and swelling result in acti-vation of NLCC and Kþ, Cl� cotransport (KCC),respectively.6e8 We also demonstrated that in vascularsmooth muscle cells (VSMC) subjected to hyperosmoticshrinkage, RVI is caused by activation of NLCC.9 It isnoteworsy that under the baseline isosmotic conditions,inhibition of NKCC by bumetanide does not significantlyaffect the volume of human lung fibroblasts (Table 2),suggesting that in the absence of external stimuli, NLCChas a minor impact on the generation of net osmolite fluxesin these cells. However, under inhibition of the Naþ, Kþ-ATPase by ouabain, treatment with bumetanide resulted inw2-fold elevation in the volume of human lung fibroblasts(Table 2). This observation suggests that dissipation oftransmembrane gradient of monovalent cations evoked by

Table 1 Major characteristics of Cl�-coupled cation cotranspor

Gene Human chromosome localization Protein Alterna

SLC12A2 5 NKCC1 NA

SLC12A1 15 NKCC2 Isoform

SLC12A3 16 NCC NASLC12A4 16 KCC1 NASLC12A5 20 KCC2 NA

SLC12A6 15 KCC3 Isoform

SLC12A7 3 KCC4 NA

NA, information not available.

Naþ, Kþ-ATPase inhibition results in generation of inwardly-directed ion flux mediated by NLCS1, the only isoform ofNLCS expressed in these cells. Functional consequences ofthe disturbances of cell volume regulatory machinery arediscussed elsewhere.4,10,11

NKCC as a regulator of intracellular ClL

concentration

In all types of cells studied so far, SSSs generate bothinwardly and outwardly-directed ion movements and thedirection of net flux depends on carrier’s stoichiometry andtransmembrane gradients of sations created by Naþ, Kþ-ATPase. Thus, the stoichiometry 1:1 predicts that thevalue of ion flux is in direct proportion to concentration ofco-transporting ions. Because [Naþ]o>>[Naþ]i,[Kþ]i>>[Kþ]p and [Sl�]o>[Cl�]i, net fluxes generated byNaþ, Cl�cotransport (NCC) and KCC exhibit inward andoutward directions, respectively. In the majority of cells[Sl�]o

2>>>[Cl�]i2, and net flux mediated by electroneutral

NKCC functioning with the stoichiometry 1Naþ:1Kþ:2Cl�

has inward direction.The above consideration suggests that SSSs play a key

role in the regulation of [Cl�]i whereas their contribution tothe adjustment of intracellular concentration of mono-valent cations is negligible because of the highly activeNaþ, Kþ-pump. Indeed, it was shown that inhibition of NCC

tes.1,3

tive spicing Tissue localization Inhibitors, IC50 (mM)

Ubiquitous Bumetanide, 0.05e0.60Furosemide, 10e50

s A, B and F Kidney Bumetanide, 0.10e0.50Furosemide 15e60

Kidney Polythiazide, 0.5Ubiquitous Bumetanide, 60Neurones Bumetanide, 55

Furosemide, 10s A and A Neurones Bumetanide, 40

Furosemide, 25Ubiquitous Bumetanide, 900

Furosemide, 900

188 S.N. Orlov et al.

and NLCC results in attenuation of [Cl�]i whereas sup-pression of LSS activity increases this parameter.12

Importantly, the alteration of CCC activity may lead toadjustment of [Cl�]i above or below the values corre-sponding to Nernst’s equilibrium potential. This means thatin cells abundant with anion channels, SSSs contribute tomaintenance of electrical potential thus having an impacton the whole spectrum of cellular functions controlled bypotential-sensitive proteins localized within the plasmamembrane. This conclusion is supported by data discussedin the next sections.

VSMC contraction

In skeletal and cardiac muscle, plasma membrane perme-ability for Lþ (PK) plays a major role in the maintenance ofelectrical resistance and resting potential (Em). In contrast,the values of PK and PCl in smooth muscles are about thesame,13 suggesting that in VSMC CCCs may be involved inregulation of the [Cl�]i/[Cl

�]o ratio and therefore of Em andexcitation-contraction coupling. Indeed, NKCC inhibition byfurosemide or bumetanide resulted in a decreased[Cl�]i

14,15 and led to hyperpolarization of rat VSMC.14 Thesedata suggest that decreased baseline tone seen in smoothmuscles treated with Henle’s loop diuretics,16e18 as well asattenuation by these compounds of contraction of smoothmuscle strips evoked by modest increment of [Kþ]o,

15 byelectrical stimulation,19 by histamine,20 angiotensin II,21

thrombaxane A2,22,23 oxitocyn,24,25 a-adrenergic15,26,27,28

or purinergic29 agonists is caused by Cl�i -dependent hyper-polarization and suppression of the activity of voltage-gated L-type Ca2þ channels.

Myogenic response

Myogenic tone (response) is a unique property of small(<100e200 mm) blood vessels to decrease rather than in-crease their inner diameter in response to elevated intra-luminal pressure. Both kinetic and the amplitude ofmyogenic response are different in blood vessels of differentorigins. Importantly, myogenic response in blood vesselsfrom the brain, skeletal muscle and renal afferent arterioleprovides constant blood supply of these tissues indepen-dently of the changes in the systemic blood pressure.30e32

It was reported that bumetanide decreased themyogenic tone of mesenteric arteries33 and completelyabolished myogenic response of renal afferent arteriole.23

We demonstrated that inhibitory action of bumetanide(but not of L-type Sa2þ channel blocker nicardipine) on themyogenic response as well as on contraction triggered by a-adrenergic stimulation is absent in mesenteric arteries fromNKCC1�/� mice.33 Because NKCC2 is not expressed in SMC,these data suffest that bumetanide and other loop’s di-uretics inhibit contraction and myogenic response ofvascular SMC via their interaction with NKCC1, i.e., anubiquitous isoform Naþ, Kþ, 2Cl� cotransporter.

Synaptic transmission

Neuroneneuron interactions are controlled by neurotrans-mitters via regulation of transduction of electrical signals.

Excitatory and inhibitory neurotransmitters lead to depo-larization and hyperpolarization of postsynaptic mem-brane, respectively. For example, ionotropic glutamatereceptor and acetylcholine receptors cause depolarizationof postsynaptic membrane via increment of ion currentmediated by ion channels permeable for Naþ and Ca2þ. Incontrast, hyperpolarization results from the increment ofthe permeability of Kþ channels triggered by activation ofmetabotropic acetylcholine receptors. Unlike above-listedneurotransmitters, gamma-aminobutiric acid (GABA) in-creases permeability for Cl� and other low molecularweight anions via its interaction with ionotropic GABAA re-ceptors. Direction of net flux mediated by these receptorsis determined by transmembrane chloride gradient andelectrical potential of postsynaptic membrane. In case RT/F � ln([Cl�]o/[Cl

�]i)<Em, the net chloride flux will bedirected into the cells which will lead to hyperpolarizationand attenuation of neuronal activity. An elevation of [Cl�]ichanges the direction of net chloride flux and in this caseactivation of GABAA receptors leads to elevation ofneuronal activity.

Keeping this in mind, one may assume that the ratio ofactivity of NKCC1 and LSS2, providing inwardly andoutwardly directed Cl� fluxes in neuronal cells, respec-tively, plays a key role in the function of GABAA receptors.Indeed, the attenuation of NKCC1 activity on the back-ground of elevated LSS2 is the major mechanism of thealteration of the functional properties of GABAA receptorsin central neuronal system (CNS) of mammals during onto-genesis: GABAA receptors function as activatory receptorsin prenatal stage but became inhibitory ones few days afterbirth (for review see34e36).

Salt reabsorption by NKCC2

A major impact of NKCC in salt reabsorption and regulationof extracellular fluid volume (EFV) is well-established bythe clinical use of furosemide and other inhibitors of thesecarriers as potent diuretics,37 as well as by salt-lastingcharacteristic of the loss-off-function mutations of NKCC2that underpin type I Barter syndrome.38 Importantly, inaddition to salt reabsorption in the TAHL, NKCC2 contrib-utes to the regulation of salt excretion via tubuloglomerularfeedback (TGF) in the juxtaglomerular apparatus (JGA)neighbouring to the distal segment of TAHL and consistingof epithelial cells of macula densa (MD), mesangial cellsand VSMC. Such location is unique along the nephron andplays a key role in the JGA function. Indeed, [NaCl] in renalfluid delivered to the MD is in the range of 20e60 mM. Thisis in sharp contrast to the proximal tubule, where concen-trations of most solutes deviate modestly from those inplasma (for more details, see 39e41). TGF is triggeredimmediately after elevation of salt concentration in thetubular fluid delivered to the JGA and results in thecontraction of VSMC of afferent arterioles, thus causingincreases in the exposure of proximal tubules to high-saltfluid via the attenuation of glomerular capillary pressureand the glomerular filtration rate (GFR). As a consequenceof this negative feedback loop, salt delivery to the distalnephron is kept within a narrow range. This process facili-tates the fine adjustment of salt handling in the distal

NKCC1 and NKCC2 189

tubules by corticosteroids and peptide hormones, such asaldosterone and arginine vasopressin (AVP).42,43

Similar to TAHL, the apical membrane of the MD cells isabundant in NKCC2, which provides up to 80% of apical NaClentry in these cells.44e46 Early studies of Wilcox and co-workers demonstrated that changes in the plasma [NaCl]affect renal blood flow in dogs mainly via modulation ofplasma chloride concentration.47,48 It was also shown thatregulation of renal blood flow is mediated by activation of[Cl�]o-sensitive, osmolality-independent TGF.49e51 Becauseof its stoichiometry (1Naþ:1Kþ:2Cl�), the [Cl�]o sensing byNKCC2 has an advantage compared to monovalent cations.Indeed, unlike MichaeleMenten’s pattern for Naþ and Kþ

dependencies, binding of Cl� to NKCC is a cooperativeprocess with a Hill coefficient of 2,52 providing high-efficiency regulation of carrier activity in the range of[Cl�]o existing in the JGA and close to the EC50 value of thetransporter.

Using isolated rabbit cortical TAHL with attachedglomeruli, Laamerti and co-workers determined that NKCCin MD cells was activated by Naþo and Cl�o with the EC50 of1.0 and 17 mM, respectively.45 Importantly, threealternatively-spliced NKCC2 isoforms (A, B and F) clonedfrom rabbit53 and mouse54 cDNA libraries. These splicevariants, are differently distributed along the nephron: theNKCC2B and the NKCC2A are co-expressed in the MD,whereas the NKCC2F is prevalent in the medullaryTAHL.55e57 In Xenopus oocytes, the EC50 values of mouseNKCC2B for Kþo , Naþo and Cl�o are 0.8, 3.0 and 12 mM,respectively.58 Very similar EC50 values for Naþ and Cl�

were obtained in a study of the rabbit NKCC2B.59 In Xen-opus oocytes injected with NKCC2A, the EC50 values for Na

þ

are very close to the those for NKCC2B, whereas the affinityfor Cl� is 2e5 folds lower.58,59 It was also shown that theNKCC2F-transfected oocytes are more energy-efficient inNaþ uptake thus indicating a key role of this splice-variantin overall salt reabsorption in TAHL.60 Indeed, experimentsperformed in gene knock-out animals demonstrated thatNKCC2B and NKCC2A contribute to salt absorption and MDfunction in the low and high NaCl concentration ranges,respectively.61,62 Importantly, the range of NKCC activationby Cl�o was consistent with the range of modulation of TGFand renin production by the apical NaCl and was similar tothe Cl� concentration in tubular fluid delivered to rat distaltubules.63 Downstream signaling events triggered by acutechanges in apical [Cl�] were discussed in several compre-hensive reviews.2,44,64

Salt secretion by NKCC1

Unlike apical location of NKCC2 in the absorptive epithelia,NKCC1 was found in the basolateral membrane of thesecretory epithelia.65 Several laboratories including ourteam reported that activity of NKCC is decreased by 2e3folds in erythrocytes from Blacks compared toCaucasians.66e70 Keeping in mind that NKCC1 is the onlyisoforms expressed in erythrocytes, we proposed thatattenuated activity of this carrier evokes salt retentionseen in African-Americans via its manifestation in thebasolateral membrane of sweat glands.71 Several observa-tions favour this hypothesis. First, both bumetanide-

sensitive 86Rb uptake and chloride fluxes across secretoryepithelium are suppressed in NKCC1�/� knock-out mice.72

Second, in humans, sweat glands contribute to up to 50%of total salt and water excretion during extensive exerciseand psychoemotional activity.73 Third, in a hot environ-ment, the function of sweat glands as a potent regulator ofEFV is suppressed in Blacks compared to Caucasians.74

Fourth, salt retention by sweat glands results inaugmented salt loading of renal distal tubule, which, inturn, leads to apical membrane depolarization andenhanced Kþ secretion.75 The latter is consistent with morethan a 5-fold prevalence of unprovoked hypokalemiadetected in African-Americans compared to Caucasians.76

In contrast to human, sweat glands have a negligibleimpact on EFV regulation in rodents and other small mam-mals.77 Because of this, NKCC1�/� knockout mice possess-ing slightly attenuated blood pressure than wild-typeanimals72 cannot be used to examine the sweat gland-mediated mechanism of NKCC1 involvement in salthandling and hypertension. Keeping this comment in mind,the comparative analysis of Naþ,Kþ,2Cl� cotransport andtranscellular ion transport in secretory epithelial cells fromBlacks and Whites is probably the best approach to examinethe relative impact of NKCC1 in blood pressure regulationvia its implication in the EFV adjustment.

Role of NKCC in the pathogenesis of hypertension

Elevation of systemic blood pressure has been documentedin 25% of adults and is a major risk factor for stroke, heartfailure, renal disease resulting in premature invalidizationand death.78 A priori, elevation of the systemic bloodpressure may be a consequence of the increase of periph-eral resistance of blood flow, heart rate and EFV. In turn,above-listed parameters are under the control of dozen ofhormones, neurotransmitters and sympathetic nerve sys-tem (SNS) affecting heart, blood vessels and renal func-tion.79 In several forms of systematic hypertension,servomechanisms underlying long-term elevation of bloodpressure are well-documented. This is the case of hyper-tension caused by adrenal tumors and renal insufficiency aswell as by a set of single gene mutations in monogenoushypertension and hypotension. These forms of diseasefound in less than 5% of patients with elevated bloodpressure are combined by common name of a secondaryhypertension. In the rest of patients with primary oressential hypertension, the mechanisms of blood pressureelevation remain poorly understood.

Monogenous forms of secondary hypertension andhypotension

Monogenous forms of hypertension and hypotension iden-tified so far are caused by mutations of genes involved inregulation of EFV by renal epithelial cells.80,81 This isconsistent with a key role of the kidney in long-termmaintenance of elevated blood pressure as described byArtur Guyton.82 Among monogenous forms of hypertensionand hypotension, three types of mutations result in alteredfunction of SSSs. The loss-off-function mutations ofNKCC2 and NCC detected in patients with Barter type I and

190 S.N. Orlov et al.

Gitelman syndrome, respectively, lead to attenuated saltreabsorption in thick ascending limb of Henle’s loop anddistal nephron, which, in turn, results in a decreased EFVand systemic blood pressure.38,83 In both diseases inheritedin accordance with classic Mendel’s genetics, hypotensionis accompanied by hypokalemia and alkaloidosis e universalmarkers of decreased reabsorption of salt in distal nephron.In contrast, in patients with pseudohypoaldosteronism typeII (ROAII) also known as Gordon’s syndrome, hypertensionis caused by mutations in with-no-K(lysine) kinases WNK1and WNK4 resulting in activation of NCC, increased sodiumreabsorption and hyperkalemia.84

Primary hypertension

Unlike monogenous forms of secondary hypertension,elevation of blood pressure in primary hypertension is aconsequence of a complex combination of inheriting traitsand several environmental factors including limited phys-ical activity, obesity, smoking, the augmented consumptionof salt and alcohol. Inherited traits are probably caused byaltered function of 4e5 genes whose combination may bedifferent even within the same human population,85

underlining mosaic origin of the pathogenesis of essentialhypertension as noted for the first time by Pickering.86

Up-to-now, there are no reports on mutations of NKCC2and other renal-specific CCCs in patients with essentialhypertension. Schiebl and co-workers found that in micedifferential splicing of NKCC2 pre-mRNA is modulated bydietary salt intake,87 which may be triggered by recentlydiscovered [Naþ]i/[K

þ]i-mediated excitation-transcriptioncoupling (for review, see88). Enhanced tubular reabsorp-tion of salt and osmotically-obliged water is importantdeterminant of obesity-related hypertension. Davies andco-workers reported that increased activity of NKCC2 inhigh-fat diet mice is caused by STE20/SPS1-related proline/alanine-rich kinase SPAK/OSR1-mediated phosphorylationof this carrier at serine-126.89 Additional studies should beperformed to examine the role of this signaling pathway inthe regulation of renal-specific CCCs in the pathogenesis ofessential hypertension.

In the early studies, it was shown that elevated perme-ability for monovalent cations of VSMC90 as well as eryth-rocytes from spontaneously hypertensive rats (SHR)91 andpatients with essential hypertension92 is at least partiallycaused by augmented activity of NKCC (for review,see.93e97). Because NKCC1 is the only isoform of Naþ,Kþ,2Cl� cotransporters identified in erythrocytes, thesedata indicate that at least in these experimental models ofhuman primary hypertension, augmented activity of thiscarrier contributes to activation of servomechanisms forlong-term elevation of systemic blood pressure. Thisconclusion can be supported by several observations. First,in erythrocytes of F1 hybrids obtained by crossing of Milanhypertensive strain (MHS) and Milan normotensive strain(MNS) rats and subjected to X-ray irradiation, NKCC activitywas increased after transplantation of bone marrow fromMHS but not from MNS.98 Second, in erythrocytes of F2MHS � MNS hybrids as well as F2 hybrids obtained bycrossing of SHR and normotensive Wistar-Kyoto (WKY) rats,NKCC activity positively correlated with blood

pressure.98,99 Third, several researchers demonstrateddecreased blood pressure in NKCC1�/� knock outmice.72,100,101 Fourth, administration of bumetanide, apotent inhibitor of Naþ, Kþ,2Cl� cotransport, decreasedblood pressure in wild-type but not in NKCC1�/� mice.27

These findings raise a question on the mechanisms forthe involvement of NKCC1 in the pathogenesis of hyper-tension. Data considered above suggest that these mecha-nisms may involve NKCC1-mediated regulation of [Cl�]iwhich, in turn, affects VSMC contraction and SNS activity.Indeed, it was shown that inhibitory action of bumetanideon the contraction of mesenteric arteries evoked by acti-vation of a-adrenergic receptor is increased in SHRcompared to normotensive controls.102,103 Due to themethodological problems, data on the activity of NKCC infreshly isolated VSMC in primary hypertension are limited tofew publications.94,95 It was shown, however, that thecontent of NKCC1 mRNA and immunoreactive protein isincreased in aorta and heart from SHR.102 Elevation ofsympathetic tone may lead to elevation of systemic bloodpressure via its impact on cardiovascular system and thekidney.104e107 This hypothesis is consistent with numerousreports on activation of SNS in patients with essential hy-pertension104 and SHR.108

The major role of CNS in activation of paraventricularnucleus (PVN) in hypothalamus possessing hyperactivity inprimary hypertension is well documented.109e111 It is alsoknown that presynaptic neurons in PVN are activated byexcitatory glutamatergic neurones and suppressed byinhibitory GABAergic neurons, respectively.112 Importantly,activity of GABAergic neurons is decreased in PVN ofSHR.112,113 As mentioned above, the relationship betweenthe inhibitory and stimulatory actions of GABAA receptors isdetermined by intracellular chloride concentration that isunder the control of the ratio of NKCC1 vs. KCC2 activities.Recent studies demonstrated that the value of the equi-librium potential for GABAA receptors (FGABA) is shifted topositive values in PVN of SHR by 15 mV, which correspondsto a 2-fold elevation of [Cl�]i compared to normotensivecontrol.114 This difference as well as decreased inhibitoryactivity of GABAergic neurons in SHR was abolished byaddition of low doses of bumetanide but not furosemide.These results allowed authors to propose that augmented[Cl�]i in SHR neurons is caused by activation of NKCC1rather than inhibition of LSS2. This conclusion is inagreement with elevated of NKCC1 mRNA and immunore-active protein levels with no changes in LSS2 content inPVN of SHR.114

Mechanisms of elevation of NKCC1 activity in primaryhypertension remain poorly investigated, which probablyreflects the polygenic and mosaic origin of this disease aswell as diverse mechanisms of regulation of the activity andexpression of CCCs. For example, elevation of [Ca2þ]i ac-tivates whereas production of cAMP inhibits this carrier inVSMC.26,115,116 Numerous investigations documentedabnormal activity of both signaling systems in primary hy-pertension.117,118 Recent studies demonstrated a key roleof WNK, SPAK and OSR1 kinases in regulation of severalCCCs including NKCC1 and NKCC2 (for reviews, see119e122).Bergaya and co-workers reported that both phosphorylationof NKCC1 and increment of blood pressure evoked by acti-vation of a-adrenergic receptors is decreased in Wnkþ/�

NKCC1 and NKCC2 191

mice.123 Bumetanide-sensitive component of vesselcontraction was also attenuated in SPAK knock-out mice.124

To further examine the role of this signalling pathway,Rafiqi and co-workers generated knock-in mice in whichSPAK cannot be activated by WNKs. These animals displayreduced salt-dependent increment of blood pressure aswell as decreased expression of NKCC2 and NCC proteinswith no changes in mRNA level.125 It is noteworthy, how-ever, that there is no evidence for mutations of genesencoding SSSs or WNK/SPAK/PSR1 regulatory pathway inprimary hypertension in contrast to monogenichypertension.

Epigenetic phenomena include DNA methylation, post-translation histone modification and expression of non-coding RNAs. Epigenetic mechanisms are under complexcontrol of diverse environmental factors, which make themcrucial for their involvement in the pathogenesis of hy-pertension and other complex and multifactorial disor-ders.126 Recent studies suggest that NKCC1-mediatedabnormalities of ion transport have epigenetic origin.127 Ithas been shown that the contents of NKCC1 mRNA andprotein are increased in aorta, heart and PVN neuronesfrom rats with spontaneous hypertension.102,114 In SHRaorta and heart, increased expression of NKCC1 is accom-panied by attenuated methylation of its promoter.102

Importantly, methylation of NLSS1 promoter exhibitedage-dependent increase in normotensive rats but not inSHR. It was also shown that the activity of DNA methyl-transferase 3C (DNTB3B) is 3-fold higher in normotensiverats as compared to age-matched SHR.103 These resultssuggest that in this experimental model of primary hyper-tension, decreased activity of DNTB3B results in hypo-methylation of NLSS1 promoter, which in turn leads toaugmented NKCC1 expression, increment of [Cl�]i, depo-larization and contraction of SMC, increased vascularresistance and elevation of blood pressure.

The role of epigenetic factors in augmented expressionof NKCC1 in neurones of PVN of SHR, controlling activity ofSNS, remains unknown. However, it was shown that in PVNfrom SHR, NKCC1 is highly glycosylated, which maycontribute to increased content of membrane-bound pro-tein, i.e., the fraction of the carrier providing Naþ, Kþ,2Cl�

cotransport.114 Thus, glycosylation of NKCC could representanother level of its regulation.

Complications caused by elevated blood pressure

The major cause of the premature death of patients withessential hypertension is a damage of the target organssuch as brain vessels and the kidney due to chronic eleva-tion of local blood pressure.128 Blood pressure elevation inthe brain microcirculation increases the probability ofnonreversible damage of the blood flow that results instroke, whereas in the kidney high blood pressure leads tostructural alterations in the nephron, changes of salt-waterhomeostasis and proteinuria.78

Keeping in mind that Rbf w 1/d4 (where Rbf is the bloodflow resistance and d is the inner vessel’s diameter),129

myogenic response plays a key role in the protection oftarget organs from elevation of systemic arterial pressure.It was shown that chronic suppression of myogenic

response in patients with essential hypertension as aconsequence of hypertrophy of vessel’s wall leads toattenuation of its sensitivity to the changes of intraluminalpressure. As a result of these changes, an increment ofsystemic blood pressure is transferred to microcirculationbeds incorporated into the brain, heart, retina, kidney,which causes irreversible changes in the structure andfunction of these and other target-organs ofhypertension.130,131

To investigate the role of myogenic response in thekidney function, Loutzenhiser and co-workers employedisolated perfused kidney, which allowed them to studyrenal microcirculation in the absence of its modulation byJGA.132 Using this model, they have shown that bumetanidecompletely suppresses myogenic response of afferentarteriole in the rat kidney.23 Together with the absence ofmyogenic response in Nkss1�/� mice33, these results maysuggest that augmented activity of NKCC1 in SHR, MHS ratsand in patients with essential hypertension protects kidneyfrom the damage by prolonged elevation of the systemicblood pressure whereas chronic administration of furose-mide and other NKCC inhibitors accelerates the renalinsufficiency and proteinuria.71,96,97 In other words, highactivity of NKCC1 in VSMC of the afferent arteriole main-tains the constant renal blood flow even after elevation ofsystemic blood pressure caused by activation of this carrierin mesenteric arteries and other vessels contributing toregulation of peripheral resistance (Fig. 1). This hypothesisis consistent with 4-fold increase of renal complications inAfrican-Americans with hypertension possessing up to 3-fold attenuation of NKCC activity in erythrocytes ascompared to age-matched hypertensive Caucasians.70,71

The relative contribution of Sa2þ channels and NKCC1 inthe myogenic response of coronary and SNS microcircula-tory beds remains unexplored.

Ischemia is the major consequence of even a briefdisturbance of blood circulation in the brain vessels thatleads to irreversible damage of the neuronal function. Itwas shown that attenuation of the oxygen partial pressureresults in astrocyte swelling, which in turn leads to arelease of glutamate and other neurotransmitters trig-gering massive Ca2þ influx in neurones and theirdeath.133,134 It was shown that bumetanide suppressesastrocyte swelling, neurotransmitter’s release135 and thedeath of neurones in the hippocampus subjected to hypoxiaand hypoglycemia.136 Moreover, both [Kþ]o-inducedswelling and release of neurotransmitters evokedby ischemia are sharply decreased in astrocytes fromNKCC1�/� mice,137 thus indicating that activation of NKCC1is a mechanism for astrocyte swelling. We have reportedthat elevation of [HCO3

e]o decreases NKCC activity in VSMCfrom rat aorta.138 Thus, activation of NKCC1 under hypoxicconditions might be caused by acidosis-mediated attenua-tion of [HCO3

�] in cerebrospinal fluid. Hypoxia is alsoaccompanied by sharp attenuation of intracellular ATPcontent and inhibition of the Naþ,Kþ-ATPase.139 As shown inTable 2, inhibition of the Naþ,Kþ-ATPase changes the di-rection of NKCC1-mediated net ion fluxes and evokes cellswelling. Additional experiments should be performed forevaluation of the relative impact of Naþ,Kþ-pump andNKCC1 in regulation of astrocyte volume in ischemicconditions.

Figure 1 The scheme showing implication of cation-chloride cotransporters in the pathogenesis of essential hypertension and itscardiovascular and renal complications. Augmented salt consumption leads to elevation of extracellular fluid volume (EFV) via saltreabsorption by NKCC2 in the thick ascending limb of Henle’s loop (TAHL), NCC in distal tubule (DT) and sensinig of Cl� concerationin tubular fluid delivered to juxtaglomerular apparatus (JGA) by NKCC2. Augmented activity of NKCC1 in vascular smooth musclecells (VSMC) resulted in elevation of peripheral resistance and systemic blood presure (SBP). Augmemted activity of NKCC1 andattenuated of KCC2 in neuronal cells of paraventricular nucleus (PVN) also contributes to SBP elevation via activation of sympa-thetic nervous system (SNS). On the other hand, augmented NKCC1 activity in VSMC of local microcirculatory bed (LMCB) resultedin suppression of myogenic tonus, elevation of local blood pressure (LBP) that, in turn, contributes to the pathogenesis of renalinsufficiency, heart failure and stroke.

192 S.N. Orlov et al.

Conclusion

The data discussed in this review leads to severalconclusions.

First, the transport of monovalent ions across the cellsas well as the regulation of intracellular chloride concen-tration and of cell volume are major functions of NKCCs. Inrenal epithelial cells, reabsorption of salt and osmotically-obliged water is provided by NKCC2, whereas NKCC1 has amajor impact on [Cl�]i, in VSMC and neuronal cells.

Second, in primary hypertension, NKCC1 activity isincreased in VSMC and neurons of PVN leading to elevationof peripheral resistance in systemic circulation and acti-vation of SNS, respectively. In both cases, these abnor-malities are mediated by elevation of [Cl�]i and plasmamembrane depolarization.

Third, furosemide and other loop’s diuretics decreasesystemic blood pressure via inhibition of NKCC2 in TAHL andof NKCC1 in smooth muscles of resistant vessels. However,the same compounds suppress the myogenic response inmicrocirculatory beds of the kidney and the brain thusincreasing the risk of renal and cerebral complications.

In spite of the progress in the understanding the role ofNKCC in the regulation of cellular functions at physiologicaland pathophysiological conditions, several questionsremain unanswered. In cultured VSMC as well as in isolatedblood vessels, NKCC1 is activated by phenylephrine,angiotensin II and other vasoconstrictors and is inhibited byvasodilators whose action is mediated by cAMP.26,115

Recently, Danielsson and co-workers have reported that

the combination of Cl-channel blockers and bumetanidepotentiates relaxation of airway smooth muscle rings byb-adrenergic agonists.140 Hence, the question is whetherNKCC1 contributes to the regulation of vascular tone bythese compounds. A key role of NKCC1 in regulation ofmyogenic response of renal afferent arteriole is well-documented. The question is what is the relative contri-bution of NKCC1 to the regulation of myogenic response ofmicrocirculatory beds of the brain and other target-organsof hypertension.

In vitro experiments have demonstrated that loop’s di-uretics might be used as a pharmacological tools aug-menting inhibitory function of GABAA receptors. It wasshown that at least furosemide fluently penetrates humanbloodebrain barrier.141 This finding together with the datadiscussed in the previous sections suggests that high-ceilingdiuretics may be used for the regulation of sympathetictone via inhibition of NKCC1 in neuronal cells. Indeed, itwas shown that orally administrated furosemide at dosescommonly used in clinic reduced spinal inhibitory inter-neuronal activity.142 It is important to point out thatcurrently used high-ceiling diuretics exhibit the same af-finity for NKCC1 and NKCC2. Because an apparent affinityfor furosemide and bumetanide is proportional to carrier’sactivity,143 inhibition of highly active NKCC2 and diureticaction of these compounds is much greater than their vas-odilatory and neuronal effects. It is also important tomention another side effect of these drugs, i.e., theirprolonged administration may result in the development ofdeafness due to inhibition of NKCC1 in epithelial cells of the

NKCC1 and NKCC2 193

inner ear.144,145 These issues become important for thedevelopment of novel antihypertensive drugs lacking side-effects caused by NKCC inhibition in epithelial cells andmyogenic tonus in microcirculatory beds.

Conflicts of interest

All authors have none to declare.

Acknowledgements

This work was supported by grants from the Canadian In-stitutes for Health Research (MOP-81392) (S.N.O.), RussianFoundation for Fundamental Research ##14-04-31705(S.V.K.), 15-04-00101 (S.N.O.), the Russian Scientific Foun-dation #14-15-00006 (S.N.O.) and the USA National In-stitutes of Health Award R01-GM85058 (N.O.D.).

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