Dietary Electrolyte–Driven Responses in the Renal WNK Kinase Pathway In Vivo
Michelle O’Reilly*,
Elaine Marshall*,
Thomas MacGillivray,
Manish Mittal*,
Wei Xue*,
Chris J. Kenyon* and
Roger W. Brown*
* Endocrinology, Centre for Cardiovascular Science, Queen’s Medical Research Institute, and Wellcome Trust Clinical Research Facility, Western General Hospital, Edinburgh, United Kingdom
Address correspondence to: Dr. Roger W. Brown, Centre for Cardiovascular Science, Queen’s Medical Research Institute, 47 Little France Crescent, Edinburgh, UK EH16 4TJ. Phone: +44-1312426739/6777; Fax: +44-1312426779; E-mail: roger.brown{at}ed.ac.uk
Received for publication November 18, 2005.
Accepted for publication June 11, 2006.
WNK1 and WNK4 are unusual serine/threonine kinases with atypicalpositioning of the catalytic active-site lysine (WNK: With-No-K[lysine]).Mutations in these WNK kinase genes can cause familial hyperkalemichypertension (FHHt), an autosomal dominant, hypertensive, hyperkalemicdisorder, implicating this novel WNK pathway in normal regulationof BP and electrolyte balance. Full-length (WNK1-L) and short(WNK1-S) kinase-deficient WNK1 isoforms previously have beenidentified. Importantly, WNK1-S is overwhelmingly predominantin kidney. Recent Xenopus oocyte studies implicate WNK4 in inhibitionof both thiazide-sensitive co-transporter–mediated Na+reabsorption and K+ secretion via renal outer medullary K+ channeland now suggest that WNK4 is inhibited by WNK1-L, itself inhibitedby WNK1-S. This study examined WNK pathway gene expression inmouse kidney and its regulation in vivo. Expression of WNK1-Sand WNK4 is strongest in distal tubule, dropping sharply incollecting duct and with WNK4 also expressed in thick ascendinglimb and the macula densa. These nephron segments that expressWNK1-S and WNK4 mRNA have major influence on long-term NaClreabsorption, BP, K+, and acid-base balance, processes thatall are disrupted in FHHt. In vivo, this novel WNK pathway respondswith significant upregulation of WNK1-S and WNK4 with high K+intake and reduction in WNK1-S on chronic lowering of K+ orNa+ intake. A two-compartment distal nephron model explainsthese in vivo findings and the pathophysiology of FHHt well,with WNK and classic aldosterone pathways responding to driversfrom K+ balance, extracellular volume, and aldosterone and cross-talkthrough distal Na+ delivery regulating electrolyte balance andBP.
The role of WNK1 and WNK4 in control of electrolyte balanceand BP first became apparent with their mutation being associatedwith familial hyperkalemic hypertension (FHHt; also known asGordon syndrome and pseudohypoaldosteronism type 2), a humanautosomal dominant disorder that features hypertension, hyperkalemia,and acidosis that usually are hyperresponsive to thiazide diuretics(1,2). FHHt can be caused by intronic deletions in WNK1 or missensemutations in WNK4 (3). Mutations in either cause a broadly similarphenotype, suggesting that WNK1 and WNK4 function in a commonpathway. Unlike most monogenic disorders that affect BP, whichfeature reciprocal Na+ and K+ (and/or H+) imbalances and sharea relationship to the aldosterone pathway (4), FHHt featuresconcurrent NaCl and K+(and/or H+) retention (1,3,5). This unusualcharacteristic indicates the existence of a novel "WNK pathway"functioning in normal physiology, which may allow the "independentof aldosterone" regulation of K and Na balance (and extracellularvolume) by the kidney, ultimately also maintaining BP withinthe normal range. The BP-regulatory role of this WNK pathwayis conserved in evolution as WNK1+/– mice are hypotensive(6).
Previously, we and others demonstrated that a 5'-truncated kinase-deficientisoform (WNK1-S) predominates in kidney (7), this being conservedbetween human and mouse (7–9). Isoform-specific probesdistinguished ubiquitous low-level expression of full-lengthWNK1-long (WNK1-L) from abundant WNK1-S expression in distalnephron. Recent Xenopus oocyte studies implicate WNK4 in inhibitionof NaCl reabsorption by thiazide-sensitive Na+Cl– co-transporter(NCC) (10,11) and/or K+ transport via renal outer medullaryK+ (ROMK) (12), while WNK1-L may in turn inhibit WNK4 (11).Furthermore, very recent in vitro reports show that WNK1-S,the predominant isoform in kidney, may participate in regulationof electrolyte transport. In Xenopus oocyte studies, WNK1-Sacted as a dominant negative regulator of WNK1-L (13) (therebyWNK1-S would relieve repression of WNK4). In cultures of mousecortical collecting duct (CCD) cells, aldosterone induces WNK1-Sexpression, and this may mediate an increased epithelial Nachannel (ENaC) conductance (14).
This evidence suggests that this WNK pathway plays a functionalrole in normal physiology to control electrolyte homeostasisand BP. The full renal response that maintains normal BP orcompensates for altered electrolyte intake involves a substantialcomponent that takes many hours per day to develop fully andis accompanied by significant persistent changes in nephronultrastructure and gene expression. These current studies progressfrom valuable insights of Xenopus oocyte work to investigatethe physiologic role of the WNK pathway in vivo in mice, localizingit within the nephron and tracking WNK mRNA expression changesin response to chronic variations in dietary electrolyte intakeand aldosterone status. This leads to a working model of thedistal nephron, explaining the WNK pathway findings and thepathophysiology of FHHt.
Animal Treatment
All procedures were carried out under provisions of ethicallyapproved licenses and involved adult, 25- to 30-g, male C57BL/6mice (Charles River, Margate, UK). Modified electrolyte feedsfor mice were obtained from Special Diet Services (Witham, UK).
RNA Extraction
At conclusion of treatments of mice, both kidneys were removedunder terminal anesthetic, immediately frozen on dry ice, andstored at –80°C. Frozen kidneys were fragmented andimmediately homogenized in TRIzol Reagent (Invitrogen, Paisley,UK), and total RNA was extracted following the manufacturer’sguidelines.
Real-Time PCR
Assays used ABI PRISM 7900 relative quantification real-timemethods (Applied Biosystems, Foster City, CA). PCR was performedin 384-well plates (AB Gene) and used 10-µl reactionsthat contained 5.0 µl of TaqMan Master Mix (Applied Biosystems),200 nM of each primer, 5 nM of probe, and 4.5 µl of template(1:40 dilution of cDNA synthesized as described previously [7]).PCR conditions involved 95°C for 10 min, then 40 cyclesof 95°C for 15 s and 60°C for 60 s. Standard templatedilution curves enabled target gene quantification and normalizationto the endogenous control TATA-Box Binding Protein (TBP). Allgroup values were calibrated to their control groups.
Validation studies using mouse renal RNA established TBP asan excellent control for these studies, showing less variationthan 18S, actin, and several other reputed housekeeping genesand excellent reproducibility against an exogenous control gene.Previous literature (15) reinforces TBP as a particularly goodrenal endogenous control gene. Real-time PCR assays used theABI Assays-on-Demand (TBP: Mm00446973_m1) or Assays-by-Designservices (all other assays, see Table 1).
Table 1. Primer and probe sequences for assays by designa
In Situ Hybridization Analysis
RNA probes for in situ hybridization (ISH) were produced tospecific gene regions using nested PCR methods (7,16). For primersequences, see Table 2. ISH used renal cryostat sections (10µm) from each mouse mounted on silane-coated glass slides,then fixed, hybridized, RNase A treated, washed, and ethanol-dehydratedas described previously (7,16,17). Slides were exposed to Kodakx-ray film (BioMax MR-1; Sigma, Poole, UK), dipped in NTB-2photographic emulsion (Anachem Ltd., Bedfordshire, UK), exposed(within light-tight box) for up to 4 wk, and developed. Cresylviolet/eosin counterstaining with bright and dark-field illuminationwas used routinely to visualize and localize silver-grain distributionin emulsion-dipped slides. In counterstained slides, blue dark-fieldviews (dark-field view in blue-filtered light) also were usedto limit/eradicate counterstain dark-field artifacts. Serialsections allowed co-localizations and tracing of nephron structuresbeyond the plane of individual sections.
Table 2. Primer sequences for PCR construct probes used in ISHa
Image Analysis
Digital image analysis was performed using custom-written applicationswithin the Image Processing Toolkit MATLABR version 7 (The Mathworks,Inc., Natick, MA) environment on 16-bit grayscale TIFF images,having an intensity range from 0 (black) to 65535 (white). Thisallows semiquantitative image analysis of ISH autoradiographicfilms and dipped emulsion slides, measuring areas and grayscaleintensities (or silver grain densities) as an index of expressionlevel. Measured grayscale levels are converted to equivalentdpm of bound radioactive probe by calibrating from co-exposedradioactive microscales (Amersham, Little Chalfont, UK) thatare designed specifically for this purpose. Alignment of consecutivesections involved systematic rotation and linear displacementof one image relative to the next to calculate their normalizedcross-correlation and interatively proceeding to the optimalalignment, where this value is a maximum. The aligned imagescan be displayed as different red and green channels of a singleoverlaid merged image. Autoradiographic films were scanned ona high-resolution flatbed scanner. Any damaged or inadequatesections were excluded. The software greatly facilitated selectionof regions (typically complex shapes) showing expression thatwas highly significantly above background levels. Backgroundwas low, with sense-section backgrounds having a grayscale levelthat was not significantly different from zero.
Statistical Analyses
Data are expressed as mean ± SEM, P < 0.05 was consideredsignificant, and group comparisons were analyzed by one-wayANOVA and Neuman-Keuls post hoc testing, unless otherwise stated.Least significant difference post hoc test for planned comparisons(ANOVALSD) was used when Neuman-Keuls testing was borderlinenonsignificant. Routine changes are expressed as %change (mean± SEM) relative to control values. Larger scale changesare expressed as fold change (mean ± SEM), relative tocontrol values, and 95% confidence intervals are quoted.
Renal WNK1 and WNK4 Gene Expression
Renal WNK1 and WNK4 mRNA expression is illustrated in Figures 1 through 5,with WNK structure and probe positions shown in Figure 1, anoverview shown in Figure 2, the structures involved (and theirabbreviations) in superficial and deep distal nephrons in Figure 3,and key expression details in Figures 4 and 5. Both WNK1-S andWNK1-L are expressed in kidney (Figure 2, B, D, and E). WNK1-Lshows near background seemingly ubiquitous expression. The greatpreponderance of WNK1 expression is due to WNK1-S and limitedto renal cortex (Figure 2B). High levels of WNK1-S in distaltubules (Figure 2, D and E) fall off sharply distally from connectingtubule (CNT) to cortical collecting tubule (CCT) to CCD (Figure 5C;see Figure 3) (7) and proximally dropping 10-fold at the thickascending limb of the loop of Henle (TAL)–distal convolutedtubule (DCT) junction (Figure 5, A and B) with expression (includingthe macula densa) extinguishing in the cortical TAL. Hence,WNK1-S has only a limited weak extension into medullary rays.WNK4 expression levels are lower than WNK1 (more than threefoldlonger exposures for WNK4 than for WNK1[-S/-T] in Figures 2,4, and 5). WNK4 expression also is strongest in distal tubulestructures (DCT/CNT) but extends beyond distal tubule, at reducedlevels, more proximally into TAL, including macula densa andmedullary TAL (Figure 4, A, B, D, and G) and more distally atlow expression levels (compared with DCT) in collecting duct(CD; Figure 4F). Thus, substantial WNK4 expression extends intomedullary rays and outer (but not inner) medulla (Figures 2Aand 4G) involving much too high a proportion of tubules to bedue to outer medullary CD alone (ENaC -subunit versus WNK4;Figure 4G) rather resembling the density of medullary TAL tubules(shown by Na+K+2Cl– co-transporter type 2 [NKCC2]; Figure 4G).
Figure 1. Schematic representation of WNK cDNA structure showing 5'-exon composition and probe positions. The cDNA structures of the 5'-regions of kinase-intact full-length WNK1 (WNK1-L; containing exons 1 through 4), kinase-deficient WNK1 short (WNK1-S; containing exon 4a in place of exons 1 through 4), and WNK4 are illustrated with vertical bars representing exon:exon boundaries. Numbers within rectangles refer to exon numbers. Black boxes specify the cDNA region probed by in situ hybridization (ISH). Double-headed arrows indicate the exon:exon boundary crossed by probes used in real-time PCR studies. Horizontal dashed lines indicate further exons that were not included in this illustration.
Figure 2. WNK kinase gene expression in kidney. ISH for the genes indicated in mouse kidney. (A and B) Serial transverse sections, dark-field views. (C and D) Two-color dark-field views for paired serial sagittal sections, merging showing co-localization (yellow shift). (E) Two-color merge views of D (at higher magnification). WNK1-T expression (E) encompasses the striking distal nephron cortical expression of WNK1-S (B) and additional widespread expression near background levels of WNK1-L. Note: comparing (A)+(B), major coincidence of expression of WNK1-S and WNK4 in cortex, but WNK1-S proportionately very limited in medullary rays and absent in medulla; (E) strong WNK1-S cortical (labyrinth) expression is seen beyond thiazide-sensitive Na+Cl– co-transporter (NCC) overlap (distal convoluted tubule [DCT]), representing particularly extension into connecting tubule (CNT; NCC being DCT restricted).
Figure 3. Nephron structure. Several distal nephrons that drain into one cortical collecting duct (CCD) are shown. Distal nephron begins in the renal medulla in thick ascending limb of the loop of Henle (TAL; numbered), which ascends into cortical medullary rays and enters the cortical labyrinth, contacting its glomerulus (specialized macula densa cells at contact point) then shortly beyond this enters DCT, CNT, and finally initial/cortical collecting tubule (ICT/CCT), which re-enters medullary rays as CCD and re-descends into medulla. Nephrons of superficial glomeruli (e.g., TAL1) typically have a simple structure, with a short usually unbranched CNT. In contrast, in most species, nephrons of mid-cortical (e.g., TAL2) and some juxtamedullary glomeruli (TAL3 + 4) commonly drain through well-developed branched arcades of CNT. A considerably higher proportion of nephrons form arcades in mouse than in human. Arcades are arranged around vascular axes, with CNT being close to arcuate and especially intralobular vessels. The dashed boxes and lettering delimit regional views relevant to panels of Figure 4 (red letters) and Figure 5 (gray letters).
Figure 4. WNK4 gene expression in mouse kidney. ISH studies are shown in bright-field, dark-field, and blue dark-field (see In Situ Hybridization Analysis). Throughout a through g, similar regions of view are illustrated in Figure 3 (red labeled [A through G] boxes). (a) WNK4 expression in TAL(T) in medullary ray, crossing boundary (dashed line) to contact glomeruli (*) in cortical labyrinth. (b) Substantial expression in segment of macula densa (MD) and higher still in DCT (D), also seen in c. U, proximal tubule emerging from urinary pole of glomerulus. (d) WNK4 expression in TAL emerging from medulla, through MD segment, DCT, and CNT. DCT convolutions (dashed arrow) and a segment of TAL (red dashed outline) fall outside the plane of section. (e) Extensive WNK4 expression throughout a well-developed deep cortical arcade. Vascular axis (arcuate artery [AA] and vein [AV] and intralobular artery [IA]) and associated glomeruli are seen in left panel. Note extensive DCT loops adjacent to glomerulus (*) in left panel. Dashed arrow indicates connections of DCT convolutions (out of section plane). (f) Superficial cortex with loops of DCT and ICT/CCT passing near renal capsule. Although both segments have WNK4 expression, note that the level is strikingly higher in DCT. Dashed line indicates renal capsule. (g) Views of ISH studies for the genes indicated in inner stripe of renal outer medulla (having TAL and outer medullary CD [OMCD] as largest tubules). Note that the relative density of WNK4-positive tubules is greater than that for the subunit of epithelial Na channel (-ENaC; an OMCD marker) but similar to Na+K+2Cl– co-transporter type 2 (NKCC2; a TAL marker). Dashed red line indicates boundary of outer and inner medulla (I). Magnifications: x50 in g, top (dark-field view); x100 in g, bottom (blue dark-field view).
Figure 5. WNK1-S gene expression in mouse kidney. ISH studies: in bright-field view in a (top) and b, in blue dark-field view in A (bottom), and in dark-field view in c. Note that WNK1-S gene expression is present in late TAL (T) including at the macula densa (MD) but rises to much higher levels in DCT (D) and CNT (C), reducing somewhat by CCT. *Glomerulus; dashed line indicates position of renal capsule; U, proximal tubule emerging from urinary pole of glomerulus (see also Figure 3, gray-labeled boxes).
Effects of Chronic Variation in Dietary K+In Vivo Body Weight, Food Intake, and Fluid Balance.
Mice were given group treatments of varying dietary K+ intake(low K+ [LK], normal K+ [NK], and high K+ [HK]; see Table 3and Figure 6, A through C). The LK group showed borderline lowerweight that became significant versus HK (but not NK) at theend (25.6 ± 0.6 versus 27.3 ± 0.6 g, respectively;P = 0.03), but mice seemed healthy throughout. Both HK and LKgroups developed a higher fluid intake and urinary output comparedwith the NK group (HK versus NK significant at conclusion: >3.4-foldhigher intake [P < 0.05] and >4.9-fold higher output [P< 0.01]).
Figure 6. Effect of dietary K+ intake on metabolic measurements. The K+ diet study involved adult male C57BL/6 mice, housed in pairs in mouse metabolic cages (Techniplast). After 3 d of acclimation, groups (n = 6) of mice commenced specific diets: (1) Normal (0.33%) K+ feed (NK; control group), (2) low (0.006%) K+ feed (LK), and (3) high-KCl (3.3% K+, 4.4% Cl–) feed (HK). Batch analysis confirmed that diets were well matched for Na+ (0.28 ± 0.05%; all diets) and Cl– content (0.7 ± 0.12%; NK and LK). Mice took the specified diets for 10 d, and daily measurements of food intake (A), fluid intake (B), and urine output (C) were recorded. Urinary electrolyte excretion was measured daily, reflected changes in dietary electrolyte intake, and was expressed as a ratio of creatinine or Na+ excretion (Cl/Cre, K/Cre, K/Na [D through F]). A transient decrease in food intake was observed in the HK group on day 4 immediately after the introduction of treatment diet.
Urinary Electrolytes.
Mice were allowed a period of 3 d (days 1 through 3) to acclimateto metabolic cages. After this, during the initial 3 d of activetreatment with specific diets (days 4 through 6), K/Cre andCl/Cre rose 6.8-fold and 4.9-fold, respectively, with HK; remainedunchanged with NK; and K/Cre showed a dramatic 24.4-fold decreasewith LK. After days 5 to 6, group K/Cre ratios did not changesignificantly, indicating reestablishment of appropriate electrolytebalance. The HK and LK groups demonstrated a >17-fold increaseand a >14-fold decrease, respectively, in K/Na by day 6 (Figure 6,D through F).
Plasma Measurements.
As expected, HK induced a small but significant increase inplasma K+ within the normal range. Plasma aldosterone was elevatedwith HK but was unchanged with LK (see Table 3).
WNK Expression Responses to Dietary K+ and Na+ Challenges
For investigation of the effect of varied dietary K+ intakeon WNK expression, mice were fed diets with a specific K+ content(NK, LK, or HK) for 10 d. Specific real-time PCR assays indicatedthat renal WNK1-S was downregulated by 20 ± 9.3% withLK (P = 0.04) and upregulated by 30 ± 10.4% with HK (P= 0.01; a 50 ± 10.2% rise from LK to HK), as was totalWNK1 (WNK1-T: 24 ± 7% increase; P = 0.0009) comparedwith NK (Figure 7A). WNK4 was upregulated with HK (48 ±24.2%; P = 0.01) but unchanged with LK.
Figure 7. WNK expression in response to varied K+ intake. (A) Real-time PCR results from renal RNA from groups of mice (n = 6) with variations in dietary K+. WNK1-S expression is significantly downregulated with LK, whereas WNK1-S and WNK4 are upregulated with HK. No significant changes were observed in WNK1-L expression across the experimental groups. (B and C) ISH analysis for WNK1-S and WNK4 detected any major shifts in distribution and level of WNK expression with K+ intake at the regional level, undiluted by any widespread, invariant low-level expression. (i) Representative sections and (ii) densitometric analysis from ISH studies. WNK1-S (B) and WNK4 (C) have different expression profiles with WNK1-S expression restricted to the cortex, and WNK4 restricted to cortex and outer medulla. WNK1-S and WNK4 expression both are upregulated with HK. *P = 0.009; **P = 0.04; ***P = 0.01; #P = 0.003; ##P = 0.02.
In regions with clear WNK1-S expression (Figure 7B), ISH analysisshowed upregulation in cortex by HK (versus NK: 2.1 ±0.6-fold; 95% confidence interval 1 to 3.3; P = 0.003). WNK1-Sdistribution remained cortical without striking change. Overkidney regions with clear WNK4 expression, there was a 2.3 ±0.6-fold (1.1 to 3.5) upregulation in expression with HK (P= 0.02; Figure 7C); in contrast, the LK diet did not affectWNK4 mRNA significantly.
For testing whether varied dietary Na+ induces similar WNK expressionchanges, mice were fed diets with a specific Na+ content (normalNa+ [control], low Na+ [LNa], or high Na+ [HNa]) for 6 d. WNK1-Sshowed a borderline significant downregulation, by 39 ±16% of control levels, between HNa and LNa-diet (P = 0.049,ANOVALSD; Figure 8). Although WNK4 in particular showed a trendto similar changes, no other significant changes in WNK expressionwere observed across the treatment groups.
Figure 8. WNK expression in response to varied Na+ intake. The Na+ diet study involved adult male C57BL/6J mice, housed in pairs in mouse metabolic cages (Techniplast). Animals were allowed 1 wk to acclimate, after which they commenced specific dietary Na+ treatments that lasted 7 d: (1) low Na+ (0.03%) feed group (LNa), (2) normal/control Na+ (0.3%) feed group (NNa), or (3) high Na+ (3%) feed group (HNa). WNK1-S showed a marginally significant* downregulation (by ANOVALSD) between HNa and LNa diet. No other significant changes in WNK expression were observed across the treatment groups. *P = 0.049.
WNK Expression and Aldosterone Challenge
For examination of the effect of aldosterone levels on WNK expression,mice were given excess aldosterone via minipump (150 µgaldosterone/kg per d [18]) or adrenalectomized (supplementedwith 0.9% saline drinking water) to abolish aldosterone production.Real-time PCR showed that aldosterone treatment induced a 32± 6.7% (18.9 to 45.2) WNK1-S upregulation (P = 0.0002)without affecting WNK1-L or WNK4 expression (Figure 9). Adrenalectomyhad no significant effect on WNK expression, but WNK1-S rosesignificantly across the adrenalectomy-aldosterone excess range(43 ± 7.6%; P = 0.0002).
Figure 9. WNK expression in response to variations in aldosterone. The aldosterone study involved adult male C57BL/6 mice, housed in pairs in normal cages. Mice that had food and fluid intake and BP monitoring were given (n = 6) subcutaneous (Alzet) minipump (MP) treatments for 6 d: (1) Adrenalectomy (ADX): Bilateral adrenalectomy, 0.3% Na+ feed, 0.9% saline drinking water, saline-only MP; (2) control (CTRL): Normal 0.3% Na+ feed, saline-only MP; and (3) aldosterone excess (ALDO 150): 150 µg aldosterone/kg per d (18) by MP, 0.3% Na+ feed. Plasma renin activity and aldosterone measurements allowed confirmation of adequacy of treatments. WNK1-S expression was upregulated by chronic aldosterone treatment (150 µg/kg per d, 6 d) but was unchanged in the absence of aldosterone after adrenalectomy. No changes in WNK1-L or WNK4 were observed across the experimental groups. *P = 0.0002; **P = 0.02.
In beginning to understand the WNK pathway, Xenopus oocyte studiesprovided invaluable evidence of WNK pathway regulation of keymediators of distal nephron electrolyte transport. This studyis one of the first to investigate this pathway in vivo in amuch more physiologically relevant system, the mouse, reportingdetailed nephron segment localization and WNK expression responsesto dietary electrolyte challenges. The relevant aspects of humanphysiology and their disorders are very well modeled in mice,particularly mechanisms of electrolyte handling and associatedeffects on long-term BP control (19,20). Very few tools thatreliably differentiate WNK isoforms are currently availableto quantify expression changes and examine distribution simultaneously.Moreover, this study allows examination of changes within kidneyregions that are not easily accessible to micropuncture techniquesand have no good, well-validated, cell-line models (e.g., outermedullary CD, deep distal nephron arcades/CNT) and avoids dangersof unequal RNA degradation, a concern that is associated withmicrodissection.
Here we report strongest WNK1 and WNK4 expression in the distaltubule (DCT, CNT) with WNK1-S dropping to much lower levelsby CD, whereas WNK4 extends somewhat diminished into TAL andCD. WNK1-L has widespread, low-level, near-background expression.Figure 10 puts the distribution of WNK pathway expression incontext.
Figure 10. Distribution of gene expression of key genes involved in Na+ and K+ balance in distal nephron. Structure illustrated in Figure 3. mTAL, cTAL, medullary and cortical TAL; CNT/ARC, connecting tubule/arcade; CCD/OMCD/IMCD, cortical/outer medullary/inner medullary CD. ENaC, subunits of ENaC; ROMK, renal outer medullary K channel; WNK1-S/-L/-T, WNK1 short (kinase deficient), long (kinase intact), and total; sgk1, serum and glucocorticoid kinase 1. For ENaC/sgk expression, arrows represent regulated expression by rising aldosterone (A).
WNK4 expression in TAL and macula densa (discussed further below)has not been recognized before and may indicate different WNK4isoforms or posttranslational modifications in TAL (3). Theseare common in transport pathways (e.g., NKCC2 in TAL), whereasadditional bands on WNK4 immunoblots suggest varied posttranslationalmodification (3). A study of transgenic mice that express aFHHt-WNK4 cDNA in TAL-CNT (and intercalated cells) reportedthat mutant-WNK4 protein was absent from tight junctions andapically localized in TAL (21). The lack of FHHt phenotype inthese FHHt-WNK4 mice leaves some uncertainty. This may relateto transgenic WNK4 expression being driven from a cDNA and solacking normal in vivo regulation and potential for transcriptionaldiversity of the WNK4 genomic locus.
Our studies challenged mice with varied dietary K+ and Na+ intakeand aldosterone. All three classes of treatment showed notableWNK pathway gene expression responses in vivo to these physiologicdeterminants of electrolyte balance and BP. Changes with varieddietary K+ intake were particularly clear. WNK1-S expressionrose on HK and fell on LK diet, correlating significantly withK+ intake, while ISH findings revealed the importance of upregulationof this isoform with HK diet, in strongly expressing segments(DCT-CNT). HK intake also increased WNK4 expression. These coordinatedWNK expression changes seem functionally significant as merelyheterozygous changes in WNK1 or WNK4 cause substantial BP andelectrolyte abnormalities (1,3,5,6).
WNK1-S was significantly upregulated with chronic aldosteroneexcess. There were no significant changes in WNK1-L or WNK4across the aldosterone-adrenalectomy range. With variationsin Na+ intake, a fall in WNK1-S expression just reached significancecomparing HNa and LNa groups. It is intriguing that this couldrepresent a WNK pathway response to reductions in extracellularfluid volume as Na+ intake falls.
Thus, K+ intake, plasma aldosterone, and dietary Na+ intake/extracellularvolume seem to be possible in vivo regulators of the WNK pathway.K+ intake seems to be a relatively robust regulator, whereasin some circumstances, the role of aldosterone may be counterbalancedor secondary to another regulator. Thus, similar WNK1-S expressionaccompanies very different aldosterone elevations (2.3-fold[HK] and 11.2-fold [aldosterone]) or when aldosterone fell fromnormal to adrenalectomized levels (Figure 9). Aldosterone-independentregulation of WNK1-S expression clearly is present across LK-HKdietary groups, with individual WNK1-S expression correlatingwith K+ intake (P < 0.001) but not significantly with aldosterone.Moreover, reduced WNK1-S with LNa intake (lowering volume, stimulatingsecondary hyperaldosteronism) and increased WNK1-S with aldosterone(raising volume, primary hyperaldosteronism) implies that WNK1-Sresponds more to extracellular volume than aldosterone and suggestsfalling WNK1-S as a potential response to conserve volume. Furtherinvestigation is required to define fully the in vivo regulatoryroles of K+ intake, aldosterone, and volume acting on the WNKpathway, but integrating these findings into a more functionalcontext is of interest. One attempt to do so is outlined inFigure 11 and discussed further next.
Figure 11. Potential WNK pathway and two-compartment model of distal nephron Na+/K+ handling. (A) The WNK pathway is suggested to regulate the extent of NaCl reabsorption in (early) DCT in the manner of the alleged molecular switch proposed in explaining familial hyperkalemic hypertension (FHHt). A high K+ intake switches to lower Na+ reabsorption via NCC in DCT, so distal Na+ delivery rises, allowing K+ to be cleared. (B) Two-compartment model. (i) Normally, Na reabsorption is partitioned between an upper co-transport compartment (under WNK pathway repression), driving NaCl reabsorption and a lower electrogenic compartment (stimulated by aldosterone and Na+ delivery), where Na+ reabsorption via ENaC generates a lumen-negative charge facilitating K+ secretion. (ii) In hypovolemia, both compartments upregulate Na reabsorption, minimizing urinary NaCl loss and facilitating K+ secretion. (iii) In high K intake, the mechanism above delivers increased Na and aldosterone, which strongly drive Na+ reabsorption and K+ secretion. (iv) In FHHt, there is constitutive overreabsorption of NaCl, causing hypervolemia and hypertension with low renin, which limits the distal delivery to the electrogenic compartment without renin suppression and blunted aldosterone, promoting limited K secretion and hyperkalemia. WNK pathway regulation of NCC and the extent of Na+ delivery to the distal electrogenic compartment and inappropriate restriction of this in FHHt fits well with reported features of FHHt, including thiazide-sensitivity and NaCl dependence.
How do these results expand understanding of this novel WNKpathway? The FHHt phenotype and usual hyperresponsiveness tothiazide diuretics highlight the importance of DCT in electrolytebalance and BP control (1,5,22). The work above provides someof the first clues as to the role in vivo of WNK1-S, the predominantWNK1 isoform in kidney, and intriguingly shows that strongestWNK1-S and WNK4 expression co-localizes in DCT-CNT, where theymay contribute to a mechanism that regulates K+ homeostasisand BP. Xenopus oocyte work suggests that WNK4 may inhibit NCCin DCT, and WNK1-L prevents this inhibition (10,11). In a preliminarypresentation of this work (see Acknowledgments), we proposedthe hypothesis that both WNK1-S and WNK4 can limit NCC transport(Figure 11A). Xenopus oocyte work reported since supports asimilar mechanism (13). We propose, especially in DCT-CNT, thatWNK1-S could bind and counterbalance WNK1-L effects, so shieldingWNK4 from inhibition. Although WNK1-S is kinase deficient, itretains domains (coiled-coils) that likely facilitate multimeric/tetramericWNK1 assembly (23,24). Alternatively, WNK1-S could interactdirectly with a WNK-binding site on WNK4 or another regulatorykinase to repress NCC.
Figure 11 incorporates this WNK pathway in a two-compartmentmodel. In the upper co-transport compartment, increased WNK1-Sor WNK4 (as in our in vivo studies) downregulates NCC-mediatedNaCl reabsorption, increasing Na+ delivery to the lower electrogeniccompartment (from late DCT distally), where ENaC reabsorbs Na+,facilitating K+ secretion (19) (itself augmented by high distalflow-mediated Maxi-K channel activation). The two compartmentswill normally overlap in late DCT. LNa diet/hypovolemia andHK diet both will stimulate aldosterone, activating the lowercompartment. The in vivo studies above indicate that LNa diet/hypovolemiaalso will stimulate the upper compartment (Figure 11B[ii]),producing appropriate Na+ retention, whereas HK diet will repressit, producing appropriate K+ secretion (Figure 11B[iii]). Theupper co-transport compartment thus interconverts the same aldosteroneresponse between Na+ retention and K+ excretion. We proposethat the molecular switch that is alleged to explain FHHt (12)is based on distal delivery of Na+. This hypothesis is supportedby careful ultrastructural studies showing that HK diet (25),NCC–/– Gitelman mice, and high-dosage thiazide diuretics(19) all predispose to extensive hypertrophy and increased Na+/K+-ATPase(25) in early CNT (indicating higher Na+ delivery) and predisposeto greater K+ clearance and reduced BP (19). In WNK1+/–mice, global WNK1 reduction would repress NaCl reabsorption(in DCT, unopposed WNK4) and promote lower extracellular volume,in keeping with their lower BP.
FHHt jams the switch in the opposite direction (Figure 11B[iv]),inappropriately engaging a response (reducing distal Na+ deliveryand K+ secretion) that these studies suggest is normal whenbody K and/or Na/extracellular volume fall and require conservation.Although aldosterone level is low to normal, its contributionto FHHt pathophysiology and hypertension should not be underestimated(26). Both aldosterone-dependent and -independent mechanismscontribute to K+ secretion (19,27); blunting of both seems likelyin FHHt. Thus, distal Na+ delivery is blunted and hypervolemicsuppression of renin will restrain circulating aldosterone,blunting the aldosterone response to levels that are inadequateto restore normokalemia despite hyperkalemic drive (28). Thus,FHHt causes hypertension and hyperkalemia.
In vitro evidence suggests the WNK pathway also may directly(i) reduce surface ROMK (12), (ii) promote ENaC conductance(14), and (iii) increase paracellular Cl– flux (29,30)(depleting electrogenic lumen-negative charge). Although thepathway in Figure 11 seems not to require these effects, theseprocesses could impair K+ secretion, promote Na+ reabsorption,or both if they contributed significantly. Effect (ii) dependson WNK1-S and was demonstrated in CCD cells; it is unresolvedif it extends to late DCT-CNT, where the expression of WNK1-Sand ENaC and the potential physiologic influence all are stronger.The roles and significance in vivo, and in FHHt, of effects(i) through (iii) are not yet clear.
It is intriguing that we have found previously unexpected geneexpression of WNK4 in TAL and macula densa. WNK4 potentiallycould influence TAL NaCl transport via regulation of ROMK, NKCC2,CLC-Kb, or Barttin, because inactivation of any causes severeNaCl wasting in Bartter’s syndromes. ROMK surface localizationwas unaffected in transgenic mice that expressed FHHt-mutantWNK4 protein, which was apically distributed in TAL (21). Invitro, WNK4 can interact directly or with other kinases (e.g.,OSR1, SPAK, other WNK), to regulate proteins of key importancein TAL-DCT transport, including NCC (11,12), ROMK (12), and,it seems, NKCC2 (31,32). Hence, WNK4 might influence NKCC2 co-transportand expand the co-transport compartment (Figure 11) to morepowerful proportions. Overactivity of TAL-DCT NaCl reabsorptionseems compatible with FHHt. Certainly, increased BP is reportedwith activating mutation of CLC-Kb (T481S) (33). Moreover, consideringphenotypes of Gitelman plus Bartter syndromes (DCT+TAL salt-wastinghypokalemic alkalosis), it seems that the inverse of these mayencompass hyperkalemia, acidosis, and low-renin hypertension,all FHHt features. Other WNK4-FHHt features (e.g., degree ofthiazide sensitivity, hypercalcuria [34]) might depend on thespectrum of overactivation within DCT-TAL. Clearly, this makesWNK4 expression in TAL of interest, but much needs to be clarifiedbefore a role in physiology or FHHt pathophysiology could beattributed.
Acknowledgments
We acknowledge The Wellcome Trust (grant 065616 PhD Studentshipto M.D.), British Heart Foundation (grant PG2 0/01075), andScottish Hospitals Endowment Research Trust (grant 77/00) forsupport. The Wellcome Trust Clinical Research Facility enabledreal-time PCR and image analysis studies.
Portions of this work were presented at the 24th British HypertensionSociety Meeting, September 13 through 15, 2004, Cambridge, UK;the 37th annual meeting of American Society of Nephrology, October29 through November 1, 2004, St. Louis, MO; and the 24th BritishEndocrine Societies’ Meeting, April 4 through 6, 2005,Harrogate, UK; and appear in abstract form (Endocr Abstr 9:138, 2005).
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Top
Abstract
Introduction
-subunit versus WNK4; Figure 4G) rather resembling the density of medullary TAL tubules (shown by Na+K+2Cl– co-transporter type 2 [NKCC2]; Figure 4G). 
