source: https://doi.org/10.7892/boris.48779 | downloaded: 13.3.2017 © 2013 Nature America, Inc. All rights reserved. Correspondence should Correspondence be addressed to L.R. ( 12 Milan, Italy. 8 Ca’ Granda, Ospedale Maggiore Policlinico, Milan, Italy. of Nephrology and Dialysis, San Raffaele Scientific Institute, Milan, Italy. Human Physiology, University of Zurich, Zurich, Switzerland. 1 disorders rare inherited of causing mutations identification from the and stems CKD tension of of hyper architecture the complex genetic Current understanding lowering blood pressure and preserving renal function. These findings point to uromodulin as a therapeutic target for and uromodulin’s effect on salt reabsorption in the kidney. to hypertension and CKD to the level of uromodulin expression hypertensive patients. Our findings link genetic susceptibility homozygous for in lowering blood pressure in hypertensive patients who are inhibition pharmacological of NKCC2 was more effective the relevance of this mechanism in humans by showing that of the renal sodium NKCC2. cotransporter We demonstrated between uromodulin and hypertension is due to activation homozygous for renal lesions similar to those observed in elderly individuals sensitive hypertension and to the presence of age-dependent Uromodulin in overexpression transgenic mice led to salt- variants increased is not understood. Here, we demonstrate that the general population, the underlying biological mechanism between Despite compelling genetic evidence for the association cause independent to susceptibility CKD and hypertension. uromodulin, the major protein secreted in normal urine, that variants in the promoter of the genome-wide association studies have identified common traits representing major global health problems Hypertension and chronic kidney disease (CKD) are complex team Kamel Laghmani Lorena Citterio Matteo Trudu increasing uromodulin expression salt-sensitive hypertension and damagekidney by Common noncoding nature medicine nature Received 14 June; accepted 19 September; published online 3 November 2013; School School of Nephrology, University Vita-Salute San Raffaele, Milan, Italy. Dulbecco Telethon Institute, Division of Genetics and Cell Biology, San Raffaele Scientific Institute, Milan, Italy. Institute Institute of Anatomy, Zurich Center for Integrative Human Physiology, University of Zurich, Zurich, Switzerland. 11 , , Johannes Loffing UMOD 10 Department Department of Pathology, San Raffaele Scientific Institute, Milan, Italy. risk variants and disease in susceptibility 1 UMOD UMOD , , Sylvie Janas
4 UMOD , , Sylvie Demaretz advance online publication online advance 7 , , Giacomo Dell’Antonio 10 promoter risk variants than in other promoter risk variants. The link , 1 1 expression and of several susceptibility loci through through loci and susceptibility of several 12 UMOD , , Maria P Rastaldi 2 , 3 , Chiara , Lanzani Chiara [email protected] in in vitro gene 7 , , Francesco Trevisani 3– 6 and Fondazione Fondazione D’Amico per la Ricerca sulle Malattie Renali, Milan, Italy. 9 UMOD , , which encodes 3 1 10 UMOD Division Division of Nephrology, Université catholique de Louvain, Medical School, Brussels, Belgium.
, in in vivo 2 , , the Swiss Project Kidney on in Genes Hypertension (SKIPOGH) . . Multiple 5 ) ) or O.D. ( risk , 6 4 , , Paolo Manunta , , Huguette Debaix . 9 Department Department of Research, Cardiovascular Istituto di Ricerce Mario Farmacologiche Negri, 5 Renal Renal Research Laboratory, Fondazione Istituto di Ricovero e Cura a Carattere Scientifico [email protected] - doi:10.1038/nm.338 8 gene variants induce , , Giuseppe Ristagno biological mechanisms underlying these genetic associations has has associations genetic these underlying mechanisms biological studies association population-based dominantly inherited disorders causing kidney damage and CKD and damage kidney causing disorders inherited dominantly TAL the by segment reabsorption NaCl in involved transporters ion main two the (ROMK), channel potassium medullary outer renal the and (NKCC2) transporter of sodium-potassium-chloride the activity the transport lar infection tract knockout mice that revealed uromodulin may protect against urinary kidney the in Henle of loop the of (TAL) limb ascending thick the lining cells epithelial the pro by secreted and duced specifically is and protein urinary abundant most the is tein) uromodulin gene encoding the in hypertension and CKD function, for renal variants ity susceptibil of have European individuals identified ancestry 200,000 challenge. major a be to proven homozygous for either the risk or protective alleles at lead variants variants lead at alleles protective or risk the either for homozygous UMOD hypothesis this Wetested expression. gene on effect an with associated be could variants these that hypothesized the includes that block librium (SNPs) by tide polymorphisms identified GWAS in a disequi linkage function uromodulin affect they how and risk associated their of nature the understand to need pressing 20% increased risk for CKD and 15% for hypertension emphasizes the high frequency (about 0.8) in the general population and confer about that The variants observation susceptibility in the 11 Recent genome-wide association studies (GWAS) in more than than more in (GWAS) studies association genome-wide Recent Given the localization of the most significant (lead) single nucleo single (lead) significant most the of localization the Given Full Full list of members and affiliations appears at the end of the paper. 8 transcript levels in nephrectomy samples from individuals individuals from samples nephrectomy in levels transcript , Olivier , Devuyst Olivier 19 2 , , 2 3 0 ). , Céline , Schaeffer Céline . Mutations in in Mutations . 1 8 1 . Recent evidence suggests that uromodulin regulates regulates uromodulin that suggests evidence Recent . 4 6 and kidney stones kidney and 9 13 2 , , Bob Glaudemans Institute Institute of Physiology, Zurich Center for Integrative These These authors contributed equally to this work. 3– UMOD 9 2 . Uromodulin . (or Uromodulin Tamm-Horsfall pro , 3 UMOD , 13 1 & Luca Rampoldi 1 , , Masami Ikehata 7 have been associated with rare rare with associated been have 7 INSERM INSERM UMRS 872, Paris, France. and modulate electrolyte tubu electrolyte modulate and 12– 2 2 . gene promoter ( promoter gene 1 4 . . However,the deciphering in vivo in 2 1 , 5 UMOD
. Studies in in Studies .
s r e t t e l by measuring measuring by Fig. 1 Fig. 5 gene have a 1 , 6 , 13 , 4 Division Division UMOD Umod a ), we we ), 2 1 - - - - - .
© 2013 Nature America, Inc. All rights reserved. allele on the promoter response to glucocorticoids ( glucocorticoids to response promoter risk the on allele rs4293393 the of effect predicted the confirmed also We ings. ( tested cells of types the of each luciferase in twofold about the by allele of protective the to expression relative reporter the increased allele risk the Notably, high retain that cells primary TAL mouse tiated differen highly including cells, of renal types different ment in three (kb) 3.7-kilobase a employing assay reporter UMOD human the of activity transcriptional the affected SNP this at alleles ( element this disrupt would allele protective the that such element, response ( maps rs4293393 to a region of conserved highly the silico In ( (SKIPOGH) cohort population-based large a in levels lin uromodu urinary in increase dose-dependent similar a showing by of ers of the protective haplotype ( twofold higher the of Carriers promoter. the in localized both are which rs4293393, and rs12917707 the promoterless vector). Data are expressed as means fragment containing either the C or T allele was cloned upstream of the firefly luciferase ( cells. The data are from four independent experiments. The schematic shows the luciferase reporter constructs used, in which a 3.7-kb differentiated mouse primary TAL cells retaining uromodulin expression), immortalized MKTAL (mouse kidney TAL) and HEK293 (human embryonic kidney) SNP rs4293393 on the transcriptional activity of (C) allele would disrupt this predicted binding site. T, risk allele. ( the color, the higher the degree of nucleotide conservation). The rs4293393 SNP is predicted to lie within a glucocorticoid response element; the protectivemacaque (Mm), cow (Bt) and dog (Cf) urinary uromodulin-to-creatinine ratio in a mixed linear model. C, protective allele; T, risk allele. ( n (Mann-Whitney test). ( differing TAL content in the samples. Each dot represents an individual, and the distribution and mean values of expression levels are shown. *** promoter variants rs12917707 and rs4293393. All samples were genotyped for both variants. We normalized (quantitative RT-PCR (qRT-PCR)) in nephrectomy samples of individuals homozygous for either protective ( by the linkage disequilibrium plot ( SNPs in the Figure 1 s r e t t e l Fig. Supplementary Supplementary Fig. 1 d = 532 TT). Data are expressed as means Protective a
UMOD
2 Consensus Risk ). The finding that glucocorticoids induced transcription from from transcription induced glucocorticoids that finding The ). Mm –570 Gg –569 Hs –569 gene promoter. We performed a standard standard a Wepromoter.performed gene
Cf –597 Bt –578 Nl –570 analysis revealed that among the lead SNPs from GWAS, from SNPs lead the among that revealed analysis Effects of C A Fig. 1 Fig. promoter risk variants with higher uromodulin expression promoter uromodulin with risk variants higher rs12917707 G A UMOD rs12922822 C G A
UMOD rs13329952 d T ). We hence tested whether the risk and protective protective and risk the whether tested Wehence ).
promoter associated with hypertension and CKD are shown. All variants are within the same linkage disequilibrium block, as shown rs13333226 UMOD c C T ) Urinary uromodulin concentrations in individuals of the SKIPOGH cohort by genotype at rs4293393 ( 2 1 expression in kidney samples compared to carri rs4293393 ) ) and is predicted to lie within a glucocorticoid SNP variants on uromodulin expression. ( Fig. 1 Fig. Glucocorticoid responseelement UMOD rs4293393 Fig. 1 r T/C 2 e 83 values, data from HapMap CEU, release #28). Genotyped SNPs are shown in red. ( 4 3 ), in accord with the the with accord in ), G A 87 UMOD rs13335818 promoter risk variants showed showed variants risk promoter 83 96 b 96 88 ). We confirmed the association 96 88 ± s.e.m. The 88 promoter sequences. The intensity of the blue color shading corresponds to nucleotide conservation (the darker 84 6 5 UMOD UMOD ± P s.e.m. ** promoter as assessed by a luciferase reporter assay in three types of kidney cells: mTAL, (highly Umod in vitro in value reflects significant association of rs4293393 genotype with square-root–transformed daytime UMOD Supplementary Supplementary promoter frag promoter C TG rs9928936 in vivo in C expression. expression.
7 rs9928757 e luciferase luciferase UMOD promoter P ) Quantitative analysis of the relative effects of the protective (C) and risk (T) alleles of < 0.01; *** Fig. 1 Fig. UMOD a ) Schematic representation of human find (chr.16p12.3) c Basic ). ). - - - - - e
8 C T P 2 1 0 been been implicated in rare by disorders inherited characterized defective nephron ( the of TAL in segments exclusively expressed was protein transgenic for homozygous on expression in subjects rable uromodulin to effect the Tg amounts in higher Fig. 3a ( secretion and expression uromodulin in increase control mice). The presence of the transgene caused a dose-dependent indistinguishable from control nontransgenic mice (here referred to as Transgenic animals from both lines(Tg were transgene viable, apparentlythe for healthy homozygous and line a generated Tg as to referred here (Tg(Umod)416Lura, uromodulin wild-type (HA)-tagged hemagglutinin expressing line expression. uromodulin higher with associated is block, disequilibrium linkage same the within variants other with the complex hormonal regulation. these Overall, results demonstrate that data not shown) and suggest that expression uromodulin is to subject response glucocorticoid elements in the predicted additional of presence the with line in is constructs both < 0.001 (ANOVA followed by Bonferroni’s test). Relative luciferase As uromodulin is expressed in the is TAL,As expressed uromodulin that a has segment tubular To model this effect UMOD UMOD b activity
mTAL UMOD transcript level
– LUC ** (relative to NKCC2) c 0 1 2 3 4 5
). ). Notably, was uromodulin atexpressed approximately 80%
Supplementary Fig. 3d Fig. Supplementary
promoter risk variant rs4293393, probably acting together ** risk variants. Similarly to endogenous uromodulin, the the uromodulin, endogenous to Similarly variants. risk ) gene in the pGL3-Basic reporter vector (Basic corresponds to *** Protective d 3 advance online publication online advance ) Partial alignment of human (Hs), gorilla (Gg), gibbon (Nl), pGL3 –3,68 Basic Haplotype UMOD Umod in vivo 4 C *** T n 1 0 UMOD = 17) or risk ( Relative luciferase wt/wt UMOD , we took advantage of a transgenic mouse Risk HEK293 rs429339
activity
gene structure. The positions of lead mice relative to control mice, compa control mice, to relative mice C/T *** expression to + – 2 1 f ; see also ref. also ; see 3 b
c )
n LUC
UMOD *** Urinary uromodulin
= 27) alleles for 3 UMOD *** (mg per g creatinine,
n square-root–transformed) = 18 CC, 0 1 2 3 4 5 6 7 expression levels Umod NKCC2 Basic promoter (prediction
CC UMOD 2 nature medicine nature C wt T 3 ). 1 0 mice). We also also We mice). P n Supplementary Supplementary Umod Relative luciferase to account for rs4293393 <1 = 214 CT and UMOD promoter CT
×
MKTAL activity wt/wt 10 P *** –8 < 0.001
2 mice). mice).
TT *** *** 3
-
© 2013 Nature America, Inc. All rights reserved. return to a standard NaCl diet ( diet NaCl standard a to return by control levels upon a levels but to diet low-NaCl elevated returned genic mice was salt sensitive, as blood pressure could be normalized to ( state hypertensive chronic a with consistent hypertrophy, ventricular left and mass heart total the of pressure in Tg aging control mice, as described previously age. This age-dependent effect was partly due as to low blood pressure in early as mice with increased pressure control blood in difference the and to of age, 2 months relative higher significantly was mice post hoc ( fashion dependent to relative mice control markedly in mice a in transgenic higher indeed was pressure Blood reabsorption. NaCl on effect pressure by blood an having affect could overexpression uromodulin pressure blood low and reabsorption NaCl ( shown) are significance statistical reaching parameters (only analysis 100 bars, (PAS, scale rows) bottom and (top casts by filled mostly row) (top tubules dilated numerous means Tg and control in 14-month-old in Tg hypertrophy ventricular Tg and control 16-month-old in (heart/tibia) length tibia to relative weight heart of distribution and and control 2 Figure nature medicine nature exposure also caused the modifications in expected aldosterone levels *** Tg and control 16-month-old ( Havcr1 bar, 100 Scale material. (arrows) uromodulin-negative PAS-positive by or (arrowhead) uromodulin by mostly formed are and (uromodulin-negative) tubules distal more or TALs in (uromodulin-positive) present are mouse means as expressed are Data shown). are significance statistical reaching parameters (only ( row) (bottom casts tubular of presence and row) (top epithelium ( row) (bottom membrane basement tubular the of thickness increased with damage tubular focal mild and row) (top compartment interstitial normal shows variants protective the for homozygous Tissueindividuals from rs12917707. and rs4293393 of variants risk or protective the for homozygous subjects elderly from tissue renal h a d
) Representative immunoblot images and quantitative data ( data quantitative and images immunoblot ) Representative Systolic blood pressure P Capillary loop
< 0.001 determined by unpaired unpaired by determined < 0.001 (mmHg) 100 120 140 160
dilation 80 (encoding Kim-1) and chemokines chemokines and Kim-1) (encoding ± 0 1 2 0
s.e.m. ( s.e.m. Ctr test ( for trend) linear Uromodulin overexpression leads to hypertension and renal damage. ( damage. renal and hypertension to leads overexpression Uromodulin * Ctr Umod 6 2 * * Umod
d Tubular casts 0 1 2 3 Age (months) transgenic mice at the indicated ages ( ages indicated the at mice transgenic ) Top, representative renal histological images of 16-month-old control (Ctr) and Tg and (Ctr) control 16-month-old of images histological renal ) Top, representative Tg wt/wt
Umod * advance online publication online advance Tg * P mice was associated with a significant increase wt/w Umod 000, nlss f aine (ANOVA) variance of analysis 0.0001, < n
= 9), whereas tissue from subjects homozygous for the risk variants shows dilated tubules with detachment of the tubular tubular the of detachment with tubules dilated shows variants risk the for homozygous subjects from tissue whereas = 9), Tubular dilation t 0 1 2 3 Umod wt/w Umod *** Umod 14 t * Tg Ctr Fig. Fig. 2 wt/wt wt/wt Umod Fig. 2 Fig. wt/wt mice (right) (H&E 1× magnification). Each dot represents an individual mouse. ( mouse. Each an dot mice (H&E individual represents (right) 1× magnification). e mice normalized to the loading control, control, loading the to normalized mice wt/wt Fig. 2 Fig. a t mice on standard (1% NaCl) or low-sodium (0.01% NaCl) diet ( diet NaCl) (0.01% low-sodium or NaCl) (1% standard on mice -test ( -test ). Blood pressure in ). Tg Blood c Protective ). The changes in dietary salt salt dietary in changes The ). Ccl2 Systolic blood pressure b a Tubular casts (mmHg) 100 120 140 160 1 24 ). Hypertension in trans in Hypertension ). (left), (left), 0 1 2 and and 80 0 Protective UMOD , we first tested whether whether tested , first we 0 , 2 5 *** ( Age (months) Fig. Fig. 2 Ccl5 b haplotype , c 16 *** and and
in kidneys from 16-month-old mice ( mice 16-month-old from kidneys in Tubular dilation
n 0.5 1.0 1.5 ** Umod a = 6–10 mice per group). Bars represent minimum and maximum values. ( values. maximum and minimum represent Bars group). per mice = 6–10 0 ). ). High blood Risk h n Tg Tg Ctr Risk ), ANOVA followed by Bonferroni’s test ( test Bonferroni’s by followed ANOVA ), * = 13 mice per group) showing levels of Lcn2 and Kim-1 in kidney lysates from from lysates kidney in Kim-1 and Lcn2 of levels showing group) per mice = 13 Umod Umod Umod dosage– n = 15) (PAS, scale bars, 100 100 bars, (PAS, scale = 15) wt/wt wt wt/wt n µ =5 mice per group). Data are expressed as means means as expressed are Data group). per mice =5 m. ( m. b - –1
Heart/tibia (mg mm ) 0 5 6 7 8 g h ) Transcript levels (qRT-PCR) of renal damage markers markers damage (qRT-PCR) renal of levels ) Transcript β f always stained positive for uromodulin and localized to TALs and and TALs to localized and uromodulin for positive stained always Tg in tigation ( variants protective for individuals in increased was for lesions homozygous these of area the and age, of years 65 than older individuals from samples nephrectomy human ( mice segmen control to relative with area cast tubular segments, increased and distal dilation tal affecting and to localized mainly Tg aging from neys ( clearance FITC-sinistrin by measured as tion, months 16 to ( up age mice of control to relative parameters plasma and transgenic and ( animals control in similar were which handling, NaCl and Fig. upeetr Tbe 1 Table Supplementary -actin. Data are expressed as means means as expressed are Data -actin. a Umodwt/wt ) Box-and-whisker plots showing systolic blood pressure at baseline in baseline at pressure blood systolic showing plots ) Box-and-whisker 37 kDa 20 kDa Transgenic mice had a similar body weight and baseline urine urine baseline and weight body similar a had mice Transgenic Tg Ctr Umod
2 d Supplementary Table 1 Table Supplementary and Online Methods). We detected similar focal lesions in in lesions focal similar Wedetected Methods). Online and Tg * Supplementary Fig. 4 Fig. Supplementary Lcn2 Umod Ctr ± 0 1 2 s.d. ( s.d. wt/w Umod Tg n Umod Ctr t = 5 per group). Data are expressed as means means as expressed are Data group). = 5 per µ Tg UMOD m). Bottom, quantification of the histological analysis analysis histological the of quantification Bottom, m). * wt/wt Tg wt/wt Umod f Umod Ctr ) Tubular casts in renal tissue from a Tg from tissue renal in ) Tubular casts Umod Umod Umod wt/w µ mice revealed that tubular casts often but not not but often casts tubular that revealed mice a wt/wt wt/wt Fig. m). Bottom, quantification of the histological histological the of quantification Bottom, m). n (right) and and (right) t risk variants relative to those homozygous homozygous those to relative variants risk wt/wt = 7 mice per group). Data are expressed as expressed are Data group). per = 7 mice wt/wt c . oee, itlgcl nlss f kid of analysis histological However, ). mice (left) and heart histology showing left left showing histology heart and (left) mice β Lcn2 Systolic blood pressure
-actin 2 mice. Kidneys from Tg from Kidneys mice. g mice revealed signs of renal damage, damage, renal of signs revealed mice e (mmHg) 110 130 Time (d) and Online Methods). Further inves Further Methods). Online and 90 0 ). and data not shown). Renal func Renal shown). not data and 37 kDa 75 kDa Expression level Standard ± g c diet s.d. * s.d.
) or Mann-Whitney test ( test Mann-Whitney ) or (fold change relative to Ctr) ) ) Average blood pressure systolic 0 1 2 3 4 *** 0 Ctr Lcn2 ± P * s.d. ( s.d. ** 3 * Low-sodium < 0.05; ** < 0.05;
Kim-1 Ctr 0 1 2
Tg diet Umod Ctr Havcr1 9 6 Tg e 2 ) Top, representative ) Top, representative Tg 6 *
wt/wt Umod Lcn2 , was also normal normal also was , Umod Umod b ) Average value value ) Average wt s r e t t e l P wt/wt wt/wt < 0.01; < 0.01; and and Ccl2 12 Umod Standard * diet mice show show mice β Kim-1 d ± Tg -actin
and and Tg Ctr s.e.m. s.e.m.
wt/wt 15 ** Umod Umod Ccl5
* ***
wt/wt * e * 18 wt/wt ).
- - - - © 2013 Nature America, Inc. All rights reserved. Bars indicate means means indicate Bars ( means as expressed are and experiments independent eight from are Data in transgenic and control mice ( mice ( Nkcc2 In line with a post-translational mechanism for the regulation of Nkcc2, of TAL cells, which lacks Nkcc1, and by the 40-foldzation ofhigherthe phosphorylated globalprotein signal expressiononthe apical membrane of bodies used in this experiment, is supportedNkcc2, by rather thanthe on Nkcc1,almost which can alsoexclusive be recognized by the antilocali test for linear trend). The specificity of this effect for phosphorylationwas linearlyon correlated with tive to control mice ( Nkcc2 at activating sites (Thr96 and Thr101)hypothesis,we found asignificantly higher level of phosphorylation on of Nkcc2, the main sodium transporter in the TAL. Consistent with this tensionofthetransgenic micecould caused be byabnormal activation ( expression renin decreased slightly or normal showed which mice, above). (see salt by dietary in changes induced BP response chronic to ascribed ( be hypertension could that kidney the in remodeling cular and ( mice these in loops capillary glomerular of dilation nificant (data not microalbuminuria shown). There substantial was also a sig chemokines ( and damage tubule of established markers of expression renal increased included also mice genic ( segments distal more (means analysis Densitometric control. cells. transfected Ctr, mock uromodulin. UMOD) (Sol. soluble or UMOD) (WT wild-type either with transfected and Nkcc2 expressing stably cells HEK293 in (UMOD) uromodulin and Nkcc2 total and phosphorylated of levels showing data quantitative and images immunoblot ( Tg (SBP, 16-month-old in pressure right) blood systolic and (left) excretion sodium ( means as expressed are Data mice. 16-month-old ( data 3 Figure s r e t t e l type of 100 kDa 150 kDa 150 kDa 150 kDa 150 kDa d a n n b SupplementaryFig. 6
37 kDa 75 kDa = 4 mice per group) showing levels of phosphorylated and total Spak and Osr1 in kidney lysates from 16 month-old control and Tg and control month-old 16 from lysates kidney in Osr1 and Spak total and phosphorylated of levels showing group) per = 4 mice = 8 Ctr mice, mice, = 8 Ctr ) Nkcc2 To test the functional importance of Nkcc2 to the hypertensive pheno of function renal normal the of context the In Nkcc2 Supplementary Fig. 5a Fig. Supplementary Havcr1 n transcript levels were not different between transgenic and control Fig. 3 = 10–13 mice per group) showing levels of phosphorylated (Thr96 and Thr101) Nkcc2 (p-Nkcc2) and total Nkcc2 in kidney lysates from from lysates kidney in Nkcc2 total and (p-Nkcc2) Nkcc2 Thr101) and (Thr96 phosphorylated of levels showing group) per mice = 10–13 Umod
as compared to Increased activation of Nkcc2 co-transporter and Spak kinase in Tg in kinase Spak and co-transporter Nkcc2 of activation Increased mRNA (qRT-PCR) in total kidney extracts from 16-month-old mice ( mice 16-month-old from extracts kidney (qRT-PCR) total mRNA in Ctr b , encoding kidney injury molecule-1 (Kim-1)) and the the and (Kim-1)) molecule-1 injury kidney encoding , ). Romk expression and membrane localization were similar Ccl2 transgenic mice, we assessed the response of these mice to Ctr Supplementary Fig. 5b Fig. Supplementary
n WT UMOD = 6 Tg and and Fig. 3 ±
s.d. * s.d. Sol. UMOD Ccl5 ),wehypothesized that thesalt-sensitive hyper Umod Nkcc1 Fig. 2 Fig. β UMOD Nkcc2 p-Nkcc2 Tg a -actin ). The increase in phosphorylated Nkcc2 levels Umod Umod P ( wt/wt < 0.05; ** < 0.05; Fig. 2g Fig. ). There were no signs of interstitial vas interstitial of signs no were There ). in the kidney ( f wt ). Evidence of renal damage in trans in damage renal of Evidence ). Supplementary Fig. 8a Lcn2 mice (sodium excretion); excretion); (sodium mice gene dosage ( p-Nkcc2/Nkcc2 Tg 0 1 2 , , encoding lipocalin 2 (Lcn2), (Lcn2), 2 lipocalin encoding , h ± WT UMODCtr Umod ), and transgenic mice also had had also mice transgenic and ), s.d. of three independent experiments) is shown. ( shown. is experiments) independent three of s.d. P ), in line with the fairly rapid rapid fairly the with line in ), < 0.01 determined by ANOVA followed by Bonferroni’s test ( test Bonferroni’s by followed ANOVA by determined < 0.01
Sol. UMOD wt/w * 27 t Supplementary Fig. , * 2 8 P p-Nkcc2 Calnexin # Nkcc2 in Tg < 0.01, ANOVA ± s.d. of four independent experiments. #, unspecific signal. Calnexin was used as a loading control. control. a loading as used was Calnexin signal. unspecific #, experiments. independent four of s.d. e Umod
Umod Nkcc2 activity – (dpH dt–1) c i 0.05 0.10 0.15 wt/wt n p-Nkcc2/Nkcc2 ). 0 1 2 = 5 mice per group (SBP)). Data are expressed as means means as expressed are Data (SBP)). group per = 5 mice 0 transgenic Tg Ctr
mice rela WT UMODCtr Umod Tg Umod post hoc **
Fig. wt
Sol. UMOD *
7a wt/wt ,
* b 2 ). d ------±
s.e.m. ( s.e.m. p-Nkcc2/normalizer Umod 0 1 2 with with this effect, the transcript level of kidney-specific of this network regulatory by uromodulin overexpression. Consistent and Tg in Osr1 total to relative Thr185) (at Osr1 phosphorylated of level the in increase an towards trend a also was Tg in significantly increased was Spak total to relative Spak) active (i.e., Thr243 at stress response 1 kinase (OSR1) oxidative by and (SPAK) kinase proline-alanine–rich SPS1-related suggest that this effect is exerted by membrane-anchored uromodulin. of uromodulin on NKCC2 phosphorylation and activity findings confirm in a mammalian system the previously described effect was completely lost in cells transfected with soluble uromodulin. These its activity ( increasein Nkcc2 phosphorylation that correlated with an increase in site (S614X) isoform truncated at the glycosylphosphatidylinositol (GPI)-anchoring with either human wild-type uromodulin or with a soluble uromodulin tion in kidney cells stably expressing Nkcc2 and transiently transfected as well as similar levels of aldosterone ( lial sodium channel (ENaC) (collecting ducts)sodium-chloride ( symporter (Ncc) (distal convoluted tubules)ron segments, as control and and transgenic epithe mice had comparable levels of to adaptive downregulation of NaCl reabsorption in more distal neph Nkcc2 activity and increased sodium reabsorption increasedprobably tointreatment furosemidedueis to control miceto the TAL rather than pressure ( nificantly enhanced natriuretic effect and a significant of reductionTg of blood furosemide, a loop diuretic that specifically targets NKCC2. Treatment n 37 kDa 58 kDa 69 kDa 58 kDa 69 kDa f Ctr = 5 per group). Data are expressed as means means as expressed are Data group). = 5 per Tg
NKCC2 phosphorylation is, at least in part, mediated by STE20/ by mediated part, in least at is, phosphorylation NKCC2 We next investigated the direct effect of uromodulin on Nkcc2 activa Umod wt/wt Tg Umod
Supplementary Fig. 7c Fig. Supplementary Umod wt ** Umod f ) Representative immunoblot images and quantitative data data quantitative and images immunoblot ) Representative
mice. ( mice. wt/wt wt/wt Fig. 3 wt/wt Fig. 3d 2 and control mice 2 h after treatment with furosemide furosemide with treatment 2 h after mice control and b e 9 Ctr . Expression of wild-type uromodulin led to a significant mice with a single dose of ) Quantification of Nkcc2 activity as assessed by dpH by assessed as activity Nkcc2 of ) Quantification a Nkcc2 ) Representative immunoblot images and quantitative quantitative and images immunoblot ) Representative c advance online publication online advance Umod ). The enhanced response of transgenic mice compared transcript level 0.4 0.8 1.2 , e 0 ). The effect on Nkcc2 phosphorylation and activity
wt/wt Ctr Tg Umod a , mice compared to control mice, and there there and mice, control to compared mice b
Tg Tg wt Umod , d Umod and and ). These data demonstrate upregulation upregulation demonstrate data These ). wt/wt wt/wt 30 , e 3 ) or unpaired unpaired ) or 1 . . The level of Spak phosphorylated
+ c ± Supplementary Fig. 4b ∆Na
s.e.m. ( s.e.m. furosemide induced both a sig –1 β Osr1 Spak p-Osr1 p-Spak (µmol min ) -actin β 0.8 1.0 1.2 -actin was used as a loading a loading as used was -actin 0 Supplementary Fig. 8d ±
s.e.m. ( s.e.m. Tg Ctr
Umod Umod t d test ( test ) Representative ) Representative
p-Spak/Spak wt/wt * 0 2 4 6 8 nature medicine nature Tg wt/wt Umod Umod Ctr Spak c c
wt/wt * and and ) Change in ) Change 1 wt/wt mice ( mice SBP (mmHg) 9 ∆ –16 –12 and strongly –8 –4 , , an isoform 0 f mice. mice. ). p-Osr1/Osr1 Tg Ctr 0 1 2 3 4
). Umod
Fig. 3 Fig. Tg
Umod ** i Ctr wt/wt d
t wt/wt , −1 e ), . - - - - f
© 2013 Nature America, Inc. All rights reserved. transport in the TAL to hypertension. with hypertension and to elucidate the contribution of increasedprospective sodiumstudies will be necessary to confirm our findings in patients populationgeneral( the variantsin relevant to human hypertension, given the high frequency NaCl ofwith blood pressure regulation. This mechanism could be of widely transport renal links that paradigm the completes here described hypoaldosteronism type 2) (Liddle’ssegments distal morepseudo these andsyndrome affecting exclusivelylinked hypertension toleading been mutationstionhas to pseudohypoaldosteronism type 1). Thus far, increased sodium (Bartter’sreabsorp syndrome) or more distal segments (Gitelman’sTALthe affecting pressuremonogenic diseases in blood reducedandsyndrome and impairing sodium reabsorption have been associated with salt wasting in the kidney and blood pressure regulation. Loss-of-function mutationscurrent knowledge of the functional relationship disorders betweenmonogenic rare NaClof handling GWAS.multipleStudies in identified been had which hypertension, humans. in hypertension to contribute may uromodulin) of overexpression to linked activity NKCC2 (i.e., increased mice transgenic in hypertension causing mechanism the that retrospec suggest results these involved cohort, which small-sized relatively a study, of analysis tive this of limitations the Despite ( SBP similar a for and DBP of trend decrease the in difference significant a with ( values baseline diu over of natriuresis increase higher increased a significantly an response,with retic showed allele risk available. the also for were homozygous tests Patients furosemide from data subjects, these ( of allele protective the for or homozygous were heterozygous were that individuals to relative homozygous allele risk the individuals for hypertensive in higher significantly was tus ( stratified cohort) (MI_HPT subjects hypertensive (never-treated) naive of cohort characterized of a well- advantage took we humans, in pressure blood modulating ( mice transgenic in reduced was regulator of Nkcc2 negative phosphorylation a as acts which and TAL the in mainly expressed is which means ( DBP and SBP ( excretion sodium in Changes genotypes. rs4293393 indicated ( monitoring. pressure blood ambulatory 24-h by assessed as + CT), (CC heterozygous or genotype protective the for homozygous either individuals for and (TT) genotype risk rs4293393 the for homozygous individuals in pressure blood (DBP) diastolic and (SBP) systolic for values for homozygous patients hypertensive 4 Figure nature medicine nature establishes the importance of uromodulin in modulating NaCl handling in b a , Throughevidence obtained in mouse and cellular models, this study This work identifies a causal role for a major risk locus for CKD and Finally, to whether investigate
c BP ) Response to furosemide treatment in hypertensive patients with the the with patients hypertensive in treatment furosemide to ) Response
Supplementary TableSupplementary 2 (mmHg) 138 142 90 94 ± 0
s.e.m. ( s.e.m. Increased blood pressure and response to furosemide in furosemide to response and pressure blood Increased ( n CC+CT SBP =161) c ) 4 h after treatment are shown. Data are expressed as expressed are Data shown. are treatment ) 4 h after a and and Fig. 4 Fig. P * ( n 00) ( 0.06) = DBP
=310) TT advance online publication online advance c ) or means means ) or b ) and a more marked drop in blood pressure, pressure, ) in drop blood and a more marked b
10 ∆Na+ , ). Baseline ). mean Baseline diastolic pressureblood i. 4 Fig. a a posteriori
1 –1 3
4 (µmol min ) 3 300 400 500 600 700 . The new gain-of-function mechanism have contributed considerably to our to considerably contributed have 0 ± UMOD Supplementary Fig. 7d Fig. Supplementary ( CC+CT s.d. ( s.d. n =33) c UMOD Supplementary TableSupplementary 3 3 and and 2 , , but not that of full-length b * for their rs4293393 SNP sta rs4293393 for their ( ). * ). variants could have could a variants role in n risk variants. ( variants. risk =94) upeetr Tbe 2 Table Supplementary TT P < 0.05 (ANOVA). < 0.05 Fig. 4 Fig. c ∆BP (mmHg) –6 –4 –2 0 2 a
). For a subset subset a For ). ( CC+CT n SBP a =47) ) Average ) Average , UMOD e
b ). ). Further ). ) and ) and * ( n DBP =118) TT Spak risk
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in the general population. population. general the in traits complex in role causal a play may disorder monogenic rare a conditions comorbid additional with age groups older the in the association of that evidence recent by supported is hypothesis This kidney. the ing increases with age, would be triggered by additional conditions harm but could predispose to CKD. In this scenario, CKD, whose incidence failure renal to lead to unlikely is overexpression uromodulin kidneys aging in observed changes of reminiscent are expression, Kim-1 and Lcn2 of upregulation the as in other rodent models of hypertension be secondary to chronic hypertension, as no such lesions were detected Tg in observed damage kidney focal lesions, tubular dilation renal and casts,focal despiteof normalpresence kidneythe function. with The associated was expression modulin for homozygous pressure regulation and have been shown to act on TAL cells coids, which are known to have a centraldemonstrate role that in ion home lin are increased by high sodium intake in rats the TAL. Notably, both the expression and urinary excretion of uromodu Telethon Scientist. Foundation and is (33CM30-124087/1 L.R. 33CM30_140331). an Associate The (EURenOmics). projectSKIPOGH is funded by the Swiss National Science under (FP7/2007-2013) grant agreement 246539 (Marie Curie) and grant 305608 and310030_146490 the European Community’s Seventh Framework Programme Foundation),Science the Swiss National FoundationScience project grant the National Centre of Competence in Research Kidney. CH (Swiss National by Policy the Science Belgian Rüfthe Office, Gebert Stiftung (Project GRS-038/12), Research Action (10/15-029), an Interuniversity Attraction Pole program initiated Scientifique and Fonds pour la Recherche Scientifique Médicale, a Concerted per il Bambino Nefropatico, the Belgian Fonds National de la Recherche (TCR08006), the Italian of Ministry Health (grant RF-2010-2319394), Associazione reagents and technical assistance. This work was supported by Telethon-Italy Kusch and N. Gretz (University of Heidelberg) for clearanceFITC-sinistrin B. (University of forDundee) antibodies to SPAK and phospho-SPAK/OSR1, to to S. S. Youhanna for help, technical assistance anddiscussions. Wefruitful are grateful S. We thank M. Azizi, A. Blanchard, G. Capasso, M. Carrel, Y. Cnops, A. Creatore, onli Note: Any Supplementary Information and Source Data files are available in the the versi in available are references associated any and Methods Me function. renal preserving and pressure blood controlling for evant rel be may function or expression uromodulin targeting therapies same variants could now be exerting a Accordingly,deleterious effect. these intake, salt higher and conditions hygienic better expectancy, life increased with However, reabsorption. salt renal of stimulation and its infections tract in urinary due effect to its modulin protective UMOD infection but Americans to resistance increase APOL1 ease-associated variant is atypical but dis not a unprecedented; for for pressure example, Selective pressure. selective of action the gesting frequency in all ethnic groups studied ( A ck
Delli Delli Carpini, P. Houillier, X. Jeunemaitre, R. Latini, N. Morel, S. Terryn and Forbush (Yale University) for antibody to phospho-NKCC2 and to D. Schock- Our study shows that common variants in a gene associated with with associated gene a in variants common that shows study Our Tg aging in Both ne version of the pape
thod Bourgeois Bourgeois (University of Zurich) for providing MKTAL cells, to D. Alessi n on of the pape the of on o variants are strongly associated with kidney disease in African w variants leading to high expression and urinary levels of levels uro and to urinary expression high leading variants 4 l 0 s e . We speculate that selective pressure might have favored favored have might pressure selective that We . speculate dgm UMOD en UMOD UMOD r ts . r . Umod promoter activity is modulated by glucocorti promoter risk variants, upregulation of uro upregulation variants, promoter risk promoter risk variants with CKD is stronger UMOD wt/wt mice and in elderly individuals individuals elderly in and mice risk variants are present in a high high a in present are variants risk Umod Trypanosoma brucei rhodesiense 38 3 Supplementary TableSupplementary 3 7 , 3 . Rather, these lesions, as well 9 wt/wt . These results suggest that that suggest results These . 3 4 mice seems unlikely to unlikely seems mice and humans o s r e t t e l stasis and blood 3 3 6 5 . . Here, we 7,9 online online per se per ), ), sug .
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© 2013 Nature America, Inc. All rights reserved. Primary Primary Care Medicine, Department of Community Medicine and Primary Care and Emergency Medicine, Geneva University Hospitals, Geneva, Switzerland. 19 Pharmacology, Inselspital, University Hospital and University of Bern, Bern, Switzerland. Lausanne, Switzerland. 14 Idris Guessous Fred Paccaud Murielle Bochud The teamSKIPOGH 15. 14. 13. 12. 11. 10. 9. 8. 7. 6. 5. 4. 3. 2. 1. reprints/index.htm at online available is information permissions and Reprints The authors declare no competing interests.financial approved the manuscript. L.R., O.D. and M.T. wrote the manuscript. All authors critically reviewed and staining; L.R. and O.D. designed the study and supervised the experiments; assessment; M.T. and M.I. performed histological and immunohistochemistry work on mouse and human kidneys; G.D.A., M.P.R. and M.I. carried out histological activity that were performed by S.D.; G.D.A. and M.P.R. supervised the histology contributed in designing the L.C. performed DNA extraction and genotyping on human samples; K.L. P.M. and C.L. designed and performed the study on human hypertensive patients; to the hypertensive patient (MI_HPT) cohort patient recruitment and assessment; urinary uromodulin determination (H.D. and O.D.); P.M., C.L. and F.T. contributed The SKIPOGH investigators provided the population-based cohort used for on the L.R., H.D. and M.T. did bioinformatics analysis; H.D. carried out measurements; S.J. and H.D. carried out plasma and urine analyses on mice; on mouse and human kidneys; M.T., S.J. and G.R. performed blood pressure salt transporters; M.T. and C.S. performed RNA extraction and qRT-PCR analysis and immunoblot analyses on mouse tissue; J.L. carried out expression studies for M.T. and S.J. characterized the mouse model and carried out immunofluorescence s r e t t e l COMP AUTHOR CO
Cardiology, Department of Specialties of Internal Medicine, Geneva University Hospitals, Geneva, Switzerland. Institute of Social and Preventive Medicine, Lausanne University Hospital, Lausanne, Switzerland.
atr, C. Pattaro, Böger,C.A. D.F. Gudbjartsson, C. Pattaro, A. Köttgen, Köttgen, A. S. Padmanabhan, A.S. Levey, P.M. Kearney, loci for kidney function. kidney for loci ESRD. and CKD diseases. comorbid and (2010). age e1001039 of stones-role kidney and disease creatinine serum with GABRR2 and level. SYT1, COL22A1, of association an reveals disease. disease. kidney with hypertension. associated (2010). UMOD e1001177 near variant identifies Improving Disease Kidney Outcomes. Global from statement position initiatives–a and approaches Lancet Rampoldi, L., Scolari, F., Amoroso, A., Ghiggeri, G. & Devuyst, O. The rediscovery The O. Devuyst, & G. Ghiggeri, A., F.,Amoroso, Scolari, L., Rampoldi, blood of basis Genetic A.F. Dominiczak, & C. Newton-Cheh, S., Padmanabhan, kidney chronic of studies association Genome-wide C.S. Fox, & C.M. O’Seaghdha, K.U. Eckardt, human of mechanisms Molecular L.M. Boyden, D.S. Geller, & A.G. Gharavi, R.P., Lifton, f rmdln Tm-osal rti) fo tblitrtta nprpty to nephropathy tubulointerstitial from disease. kidney chronic protein): (Tamm-Horsfall uromodulin of hypertension. and pressure learned? we have what disease: burden. health global abnormalities. electrolyte hypertension. UMOD E TI BMC Med. Genet. Med. BMC
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© 2013 Nature America, Inc. All rights reserved. showing association of the risk allele T with hypertension GWAS with line in is finding This 15.3). expected versus 9 (observed C allele protective the for homozygous individuals of underrepresentation by driven was cohort MI_HPT the in deviation The (SKIPOGH). 0.34 and (MI_HPT) the SKIPOGH cohort. The cohort and MI_HPT of C in the minor was in 0.180 rs4293393 0.163 the allele and 4.5% in the SKIPOGH cohorts) showed 100% concordance. The frequency inter- and intraplate duplicate testing and clear separation of signal clusters. (KASPar v4.0) (call rate of 96.4%). Additional quality-control criteria included technique PCR Specific Allele Competitive the using KBioscience) (formerly performed on genomic DNA extracted from white blood cells at LGC Genomics 97.2%). of rate (call instructions manufacturer’s to according 31122302_10) ID (Assay C__ and rs12917707 ID (Assay C__27865986_10) SNPs rs4293393 marker for Biosystems) Applied (TaqMan, probes MGB allele-specific with 5 out carried and (Qiagen) Kit Mini DNAQIAamp the We used respectively. tissue, renal or samples blood from DNA extracted genomic on nephrectomy underwent who individuals in and Genotyping. ( years) for age and sex with at least one individual homozygous for the risk alleles ( alleles tive We matched individuals homozygous for the rs12917707 and rs4293393 protec not under antitumor treatment at the time of explant were eligible for this study. four-variable Modification of Diet in Renal Disease formula min ml 90 > eGFR (i.e.,function renalnormal with RNAlater (Qiagen) at −80 °C for molecular analysis. Only kidneys from patients 3- into cut sequently sub and paraffin in formaldehyde,bufferedembedded 4% in fixed dissected, were tissue normal of Samplescarcinoma. renal of because Hospital Raffaele San the nephrectomyat underwent who individuals from weresamples renal Hospital.Raffaele participantsAll provided informed written consent. Human Comitatobythe committeeEticoethicalSanRaffaele, Ospedalethe Sanofthe Human kidney samples. of uromodulin urinary levels was performed for all available samples. daytime, which had a median duration of 16 h (interquartilein Switzerland), as range,previously 2).described The analysis from December 2009 until April 2013 in threegenes centers in blood (Bern,pressure Geneva and andkidney functionLausanne regulation. We recruited participants consent. written informed provided participants All (Inselspital). of Bern Kantonale Ethikkommission Bern, the ethical committee of University Hospital centrale d’éthique, the ethical committee of Geneva University Hospitals and the nique, the ethical committee of Lausanne University Hospital, the Commission study (SKIPOGH) was approved by d’éthiqueCommission de la recherche cli cohort. SKIPOGH foods and compliancyverified by assessing sodium excretion.24-h urinary We gave patients dietary instructions including a list of suggested and forbidden estrogen or progestin therapy or with a history of addiction and/or arterial hypertension.ary alcoholWe abuse.also excluded subjects under antihypertensive or min ml <80 cardiomy clearance creatinine ischemic vasculopathy, cerebral for decompensation, cardiac opathy, history clinical pathologies, acute or chronic with patients study We Hg.the frommm excluded <160/110 andHg mm >140/90 kg/m <30 index mass Hospital Raffaele San at Clinic Outpatient the to referred patients hypertensive treated consent. written informed provided participants All Etico Ospedale San Raffaele, the ethical committee of the San Raffaele Hospital. cohort. MI_HPT ONLIN doi: n = 27; 33.3% females; age at the time of nephrectomy 67.9 A A reproducibility test on randomly selected duplicates (10% in the MI_HPT was cohort SKIPOGH the in individuals in rs4293393 SNP of Genotyping The SKIPOGH study is a family-based cross-sectional study exploring the role of In study,this we enrolled a cohort of and471 (391 males 80 never-females) 10.1038/nm.3384 4 E 1 M . Patients were between 20 and 65 years old and each had a body body a had each and old years 65 and 20 between were Patients . n −1 = 17; 41.2% females; age at the time of nephrectomyof 67.7 time the atage females; 41.2% 17; = We out carried genotyping in in individuals the cohort MI_HPT E , liver failure, diabetes, severe essential hypertension or second or hypertension essential severe diabetes, failure, liver , THOD The MI_HPT cohort study was approved by the Comitato Comitato the by approved was study cohort MI_HPT The The Swiss Kidney Project on Genes in Hypertension Hypertension in Genes on Project Kidney Swiss The µ 2 m sections for histology. Tissue samples were stored in stored were sampleshistology.Tissue for sections m S and systolic and diastolic blood pressure (SBP/DBP) (SBP/DBP) pressure blood diastolic and systolic and The study on adult human kidney samples was approved P values for Hardy-Weinberg equilibrium were 0.048 ′ 4 nuclease allelic discrimination assays assays discrimination allelic nuclease 2 . Participants collected urine during the −1 , calculated by using the usingby calculated , 3 4 . 3 ± ) and from patients 9.6 years). ± 12.7 - - - - -
Human urinary uromodulin measurements. uromodulin urinary Human (Sorin radioimmunoassay commercial by Laboratories). activity renin plasma and test age, sex, BMI or renal function. for the risk variant (TT, n pressure monitoring, we grouped patients carrying one (CT, blood ambulatory for As timepoints. the of each at minute every done ments treatment.arevalues after furosemide average the Reported of measurethree min 30 every and period equilibration the during min 60 every BP measured collected urine immediately before and 4 h after furosemide administration. We equilibration, we orally administered 25 mg of furosemide (Sanofi-Aventis). We renal function (see (TT, variant risk the for homozygous those with them compared and variant tive (CT,one carrying patients nighttime. during min 30 every and (daytime) weekly activity. Recordings were typical performed every 10 for min during waking chosen hours day a on Medical) Spacelab 90207; (Spacelab monitoring ( cohort MI_HPT the in Patient ambulatory blood pressure monitoring and furosemide test. (Beckman Coulter) and measured aldosterone using a validated Aldosterone Aldosterone a validated using aldosterone measured and Coulter) (Beckman analyzer CX5 Synchron a on acid uric and urea determination), (enzymatic creatinine electrolytes, plasma albumin, and creatinine electrolytes, urinary by killing of time the Wegroups. in all were at identical measured procedures Sampling decapitation. or puncture venous by blood obtained We mice. the of training appropriate after furosemide weight) body kg after per h mg 10 2 administration, and measurement baseline for overnight h (14 cages metabolic individual using urine collected We SSNIFF). (E15430-24, chow and chow standard AO3, (Diet GR 25/18 SAFE; Srl) Mucedola or low-sodium in a light- and temperature-controlled room with from age- and gender-matched transgenic and control mice. Mice were housed analysis. and collection urine and Plasma respectively. Committee, Ethics Animals/Animal for Guide and Care the Use of Council Laboratory Research National Belgian by and the Useand Care Committee Animal Institutional Raffaele San by the approved protocols to according Belgium, Brussels, Louvain, de Catholique Italy, Milan, Institute, and at Université Scientific at mice San Raffaele female mals in the same isogenic background. We carried ani out all animal nontransgenic procedureswere mice on Control background. FVB/N pure in are lines verified transgene homozygosity by Southern blot analysis. The two transgenic Tg We generated terminus. N 3 entire the and 11 to 2 exon from sequence coding the intron, first the exon, noncoding composed of construct a 2.9-kb fragment of the uromodulin transgenic the with strain mouse FVB/N the injected we Briefly, mice. Transgenic concentration. urine in variations for compensate to ratio) to-creatinine concentration to uromodulin malized concentration (uromodulin- creatinine Synchron Clinical System) levels using Beckman Coulter Synchron System Creatinine Assay (Unicell DxC creatinine Weurinary . 5.5% of measured variability intra-assay an and 3.3% ml ng 2.8 of sensitivity a had 100 antibody ondary as radish 1:2,000) antibody 1721011, peroxidase–conjugated (BioRad, the sec 1 CL1032A, (Cedarlane, body, THP antibody anti-human a monoclonal mouse 5 K90071C, Science, Life (Meridian antibody uromodulin anti-human sheep a on using ELISA by concentration modulin = 3) copies of the protective variant and compared them with homozygous with them compared and variant protective the of copies 3) = µ We measured urinary Na urinary Wemeasured A subgroup of 165 patients underwent a furosemide test, as follows. After 2 grouped we allele, protective rs4293393 the of frequency low the to Owing g ml g
µ n g ml g = 310). The two groups did not significantly differ by age, sex, BMI or BMI sex, age, by differ significantly not did groups two The 310). = −1 ) as the primary antibody and a goat anti-mouse IgG (H+L) horse (H+L) IgG anti-mouse goat a and antibody primary the as ) −1 ′ untranslated region. An HA tag was inserted at the uromodulin uromodulin the at inserted was HA tag An region. untranslated ) to establish the standard curve standard the establish to ) 4 4 We generated Tg generated We . We used human uromodulin (Millipore, stock solution solution stock (Millipore, uromodulin human used We . Supplementary Supplementary Table 2 n n = 118). The two groups did not significantly differ in = 471) underwent 24-h ambulatory blood pressure pressure blood ambulatory 24-h underwent = 471) 4 6 n following the manufacturer’s instructions. We nor + = 152) or two (CC, (CC, two or 152) = −1 by flame photometry, creatinine by picric acid acid picric by creatinine photometry, flame by Umod , a linearity of 1.0, an interassay variability of of variability interassay an 1.0, of linearity a , wt/wt Umod mice by breeding Tg breeding by mice wt ). mice as described previously described as mice 4 We obtained urine and plasma plasma and urine Weobtained 5 . The uromodulin ELISA assay assay ELISA uromodulin The . µ Umod e esrd rnr uro urinary measured We ad libitum g ml g n = 9) copies of the protec the of copies 9) = gene promoter, the first −1 ) as the capture anti capture the as ) nature medicine nature n access to tap water = 44) or two (CC, Umod wt mice and and mice Patients 2
3 h ------.
© 2013 Nature America, Inc. All rights reserved. Firefly luciferase activity was corrected for transfection efficiency using meas forwas corrected luciferase activity Firefly efficiency transfection (Promega) with 10 s of integration time (the duration of measurement per well). Luciferase AssayReporter System (Promega) using a GloMax 96 luminometer (Invitrogen) according to manufacturer’s instructions. a of orcarrying firefly luciferase reporter plasmid Promega),(pGL3-Basic, either promoterless cells, HEK293 shown). not data (qRT-PCR, and respectively MKTAL in detected was expression uromodulin no or weak state, differentiated fully their with consistent expression uromodulin mTAL high mTAL,in Whereas moter retain cells. cells MKTAL HEK293 and the of activity transcriptional the on alleles risk and protective UMOD previously described anchoringGPI- site the (S614X), lacks into which the expression UMOD), (Sol. vector isoform pcDNA3.1 soluble (+) uromodulin (Invitrogen), truncated as by sequencing. plasmids all We protocol. manufacturer’s confirmed the following Agilent) (Stratagene, Kit QuickChange Mutagenesis the Site-Directed using Lightning mutagenesis site-directed by C) −551 (nucleotide allele protective rs4293393 the containing construct promoter the generated MluI (reverse, 5 5 with SacI (forward, primers using PCR by −3684 to −1093 nucleotide from region promoter the 5 (reverse, XhoI 5 (forward, MluI with by primers +1 to using PCR −1092 nucleotide from region promoter the We steps. two obtained in (Promega) vector Reporter Luciferase pGL3-Basic the into T) −551 (nucleotide rs4293393 SNP the at allele risk the containing Constructs. CO 5% containing atmosphere humidified a expression uromodulin endogenous of level high TAL of the segment those to similar properties structural and functional morphological, by characterized are monolayers These ayers. monol confluent of well-polarized formation the for 14 d, allowing supports morphology their of basis the on according to by the described method Terryn kidneys mouse C57BL/6 collagenase-treated of the outer 5-week-old medulla of tubules microdissected (mTAL) from cultures primary TAL Weobtained gentamicin/amphotericin. and transferrin triiodothyronine, insulin, phrine, with the SingleQuots Kit (Lonza) containing hydrocortisone, hEGF, FBS, epine by Bourgeois acterized penicillin/ 1% and char and isolated (Invitrogen) TAL line immortalized an are MKTAL FBS cells streptomycin. 10% with supplemented DMEM in cultures. Cell analysis. immunofluorescence and immunohistochemical histological, for paraffin in embedding before paraformaldehyde 4% in tissue cardiac and renal Wefixed extraction. RNA or protein for organs homogenized diately We (Abbott). imme Sevoflurane with anesthesia after dislocation or cervical decapitation by killed were mice the testis), and aorta glands, adrenal brain, preparation. and collection Tissue mice. the of measurements. successive We six to four training averaged appropriate after animals conscious in days different two on Systems) Visitech BP-2000, or Narco (Physiograph method tail-cuff measurements. pressure blood Mouse osmometer Instruments). (Advanced Fiske a on instruc osmolality Synchron measured We Coulter Assay). manufacturer’s (Beckman Creatinine System the creatinine urinary to to normalizing according and tions Chemical) (Cayman kit EIA nature medicine nature of urement e vlae lcfrs atvt 4 h fe taseto wt te Dual- the with transfection after h 48 activity luciferase evaluated We We transiently mTAL, transfected MKTAL and with 2 cells HEK293 and UMOD) (WT uromodulin wild-type human HA-tagged cloned We Renilla promoter activity activity promoter luciferase vector (pRL-SV40, Promega) using Lipofectamine 2000 2000 Lipofectamine Promega)using (pRL-SV40, vector luciferase UMOD Renilla We cloned a 3.7-kb fragment of the human We maintained human embryonic kidney (HEK) 293 cells cells 293 (HEK) kidney embryonic human maintained We ′ -TTACGCGTCAGGTTTGTTACATATGTATAC-3 ′ -TCTCGAGTGGTCATGATGTGCCTCATAC-3 luciferase activity. We used the promoterless pGL3-Basic pGL3-Basic promoterless the Weused activity. luciferase promoter fragments (see constructs paragraph) and 10 ng 10 and paragraph) constructs promoter (see fragments ′ -TTACGCGTCACGTTGTGCACTTGTACCC-3 2 ′ 9 -TTGAGCTCATTCAGGACACGGTGTAAGG-3 . et et al. 4 7 n vitro in . . We MKTAL cultured (1:1) in cells DMEM:F12 4 9 and cultured them on permeable filter filter permeable on them cultured and To collect organs (kidney, heart, spleen, spleen, heart, (kidney, organs To collect . We tested the effect of rs4293393 rs4293393 of effect the tested We We measured systolic BP by the the by BP systolic measured We 2 et et al. . 4 4 9 8 . We kept cells at 37 °C in in °C 37 at We . cells kept . . We TALselected tubules UMOD in in vivo gene promoter UMOD , including a , including ′ ) tails, and ) tails, ′ ) tails. We ′ µ ) and and ) ′ g g of a ) ) and pro ------
activity activity is therefore expressed as dpH NH by (dpH recovery to induce cellular alkalinization and measured the initial rate of intracellular pH Hepes-Tris buffered medium. We NH added then on coverslips. We measured pH baseline tive dye BCECF to measured cytoplasmic pH (pH as previously described shown). not qRT-PCR (data by verified was isoforms uromodulin transfected of two the expression Equal instructions. manufacturer’s the to according (Invitrogen) Lipofectamine Reagent Plus and using vector empty or uromodulin truncated or full-length expressing plasmids of transfection transient out We clone. carried same the (G418, geneticin to 500 resistance for selected cells, the transfected we Nkcc2, expressing stably cells To HEK293 (Promega). generate vector expression get cDNA a into with for pTarthe Myc Nkcc2 to mouse its N-terminus tag fused activity cotransporter Nkcc2 and assayed in extracts duplicate. UMOD sone (Sigma) and/or 10 glucocorticoids, 24 h after transfection cells were treated with 1 negativecontrol. a To as vector the of response the assess at room temperature with primary antibody and for 1 h at room temperatureroom at h 1 for and antibody primary with temperatureroom at albumin or 10% donkey serum (1 h at room temperature) and incubated for 1 min. 2.5 for twice 6.0), pH M, (0.01 buffer citrate heated with incubation by obtained was retrieval water. tap Antigenic cold and ethanol 70% ethanol, hydrated in the following solutions (5 min each): 2× xylene, 100% ethanol, 80% thick) were first deparaffinized by heating for 60 min at 56 °C and subsequently Immunofluorescence and immunohistochemistry. value of five or six measurements per vessel) per organ ( (average vessels six least at in width media tunica determined quantitatively as glomerulosclerosis percentage of the total number of glomeruli. of the whole area). section We quantified the presence of segmental and global tubular casts and tubular dilation (0, absent; 3, 61–100% 2, 1, 31–60%; 1–30%; of the tuft area), as we did for interstitial inflammation and fibrosis, presence of and capillary loop dilation semiquantitatively (0, absent; 1, 1–50%; 2, 51–100% expansion Wemesangial Zeiss). assessed (Carl 4.3 AxioVision software using equipped with an AxioCam MRc5 digital video camera. Images were recorded type. Sections were viewed using a Zeiss Axioscope 40FL microscope (Carl andexamined unawarescored Zeiss)by two observers of mouse the or patient geno for the quantification of histological features. The sections were independently (Zeiss). camera digital Midi Mirax a with images digital capturing microscopy, light by tion evalua for H&E with sections heart We stained techniques. standard to ing Histology. the relative mRNA expression of genes of interest following the content in each renal tissue sample, and in mouse samplesexpression to in human samples to determinedamplification efficiency by dilution curves.We normalized (ref. forSYBR Assay (Eurogentec SA).We designed specificprimers using Primer 3 of target genes by qRT-PCR on LightCycler 480 (Roche) using the qPCR (BioRad)Coreaccording kit to themanufacturer’s instructions. We analyzed expression reagent (Invitrogen) and reverse-transcribed extracted RNA using the iScript kit protocol. manufacturer’s the ing mirVana the using follow Technologies), Life kit miRNA (Ambion, Isolation qRT-PCR. We measured Nkcc2 cotransport activity as bumetanide-sensitive NH In mouse renal sections, we scored vascular damage as present or absent and patient) or mouse per sections (three sections renal PAS-stained Weused We extracted total RNA from mouse whole kidney by homogenization in TRIzol For immunofluorescence (IF), slides were blocked in either 10% bovine serum µ 5 g ml g 1 4 ) (primer sequences and PCR conditions available upon request). Werequest). upon availableconditions PCR and sequences (primer ) allele and cell type, we carried out at least four independent transfections Cl addition to calculate the cell buffer capacity. Nkcc2 cotransporter cotransporter Nkcc2 capacity. buffer cell the calculate to addition Cl −1 We performed routine staining (PAS, Trichrome and H&E) accord We used 50–200 mg of human renal tissue for total RNA extraction ) and isolated single clones. We performed all experiments on on experiments all performed We clones. single isolated and ) i d t −1 ) over the first 20 s of s recording.20 over ) first We the dpH the used 5 µ 0 . Briefly, we used the intracellularly trapped pH-sensi M M RU-486 (Sigma) for 48 h. For each combination of NKCC2 in vitro in i d ( t . SLC12A1 −1 i We cloned a construct containing a containing construct a Wecloned in cells bathed at 37 °C in a CO . i ) in cells grown to confluence ), to account for TAL segment 4 Cl (20 mM) tomM) (20 medium Cl the Kidney sections (4–5 n = 5 per group). doi: UMOD Hprt1 10.1038/nm.3384 ∆ µ ∆ M dexametha . We calculated C promoter to T method i caused caused UMOD 4 influx 2 -free -free µ 5 m
2 h ------.
© 2013 Nature America, Inc. All rights reserved. Protease-Inhibitor Cocktail 1:1,000 (Sigma-Aldrich) (adapted from ref. ref. from (adapted (Sigma-Aldrich) 1:1,000 Cocktail Protease-Inhibitor SDS containing 1%, NaF 10 mM, Na in PBS and acetone with resuspended SDS, 0.1% precipitated against dialyzed at 11,000 homog were in enized TRIzol (Invitrogen) and proteins tissues after were centrifugation extracted mouse Alternatively, supernatant. the used we which 16,000 at centrifugation and sonication by followed buffer, lysis in tissue the of by solubilization mice 16-month-old from lysates protein We obtained 7.4). pH 20mM, Tris-HCl and (Sigma-Aldrich) 1:1,000 10 mM, Na mM, 150 (NaCl buffer lysis in °C 4 at animals Immunoblotting. equipped with an AxioCam MRc5 digital video camera (Carl Zeiss). (Invitrogen). Sections were analyzed using a Zeiss Axioscope 40FL microscope vidin (Invitrogen). Peroxidase activity was detected with 3,5-diaminobenzidine secondary antibody (Vector Laboratories, 1:300) and peroxidase-labeled strepta body, followed by incubation for 30 min at normalroom goattemperature serum and incubatedwith forthe 1 biotinylatedh at room temperature 10% with blocked activity. were then peroxidase endogenousquench Sections with primary anti Briefly, after antigen retrieval, sections were incubated for 15 min in 3% H acquisition parameters for the same antibody in different sections. kidney a 40× 1.4 Plan-Apochromat oil-immersion objective (Zeiss). We used identical Microsystems) or an LSM510Meta Confocal microscope (Zeiss) equipped with DFC Twain DFC480 camera, Leica (Leica scope Software, lens; 0.75 Leica 40× micro upright fluorescence 5000B DM a using viewed were slides All 1:500) Technologies, (Life antibody secondary AlexaFluor-labeled appropriate with doi: kidneys in ice-cold lysis buffer (200 mM mannitol, 80 mM HEPES, 41 mM mM 41 HEPES, mM 80 mannitol, mM (200 buffer lysis ice-cold in kidneys tions of 16-month-old mice as previously described We analyzed the expression of Ncc and ENaC from kidney membrane prepara goat polyclonal antibody against renin (Santa Cruz Biotechnology, sc-27318, sc-27318, Biotechnology, Cruz (Santa renin against antibody polyclonal goat against polyclonal rabbit or WB), for (1:2,000 pT58NCC against pT53NCC against polyclonal rabbit NCC total against polyclonal rabbit WB), for 1:500 M09, H00009943- (Abnova, (OXSR1) OSR1 against monoclonal mouse D. Alessi), (Thr185) pho-OSR1 phos and (Thr243) phospho-SPAK both against antibody polyclonal sheep and for WB) SPAK 1:1,000 against (S150C, antibody sheep B. from Forbush), (phosphorylated Thr96 and Thr101) phospho-NKCC2 against antibody polyclonal rabbit WB), for 1:1,000 and IF NKCC2 against AB3562P, (Merck bit antibody Millipore, polyclonal for 1:250 body against HA (Roche, 11 867 423 001, 1:500 for IF and 1:1,000 for WB), anti rab monoclonal rat uromodulin, against antibodies WB) for 1:1,000 55140, Biomedicals, (MP polyclonal goat and IHC) for and IF for 1:200 ab9029-1, Antibodies. software ( protocols. Quantification was performed using the gel analysis option of ImageJ brane (GE Healthcare). We performedmem nitrocellulosewestern onto transferredblotting and conditions(WB) experiments) followingother (all standard rated on 8–12% SDS-PAGE gel in nonreducing (total uromodulin) or in reducing sequently loaded onto gels with normalization to urinary creatinine levels. times in PBS (Invitrogen). three washed was protein immunocomplex purified using The (Dynal). purification beads G–agarose affinity by followed 1:200). 631206, (Clontech, antibody anti-Myc with immunoprecipitation following levels Nkcc2 ylated phosphor and total Weassessed Diagnostics)). Roche (Complete, inhibitors Hepes 10 mM, pH 7.9; glycerol 5% (v/v); Nonidet P-40 0.5% (v/v) and protease MgCl mM; EGTA0.5 M; 0.4 (NaCl buffer lysis in cells the bilizing (100,000 trifugation ULTRA and PhosSTOP, Roche), obtained membrane(Complete preparations cocktails by ultracen inhibitor phosphatase and protease with 7.5) pH KOH, Immunohistochemistry (IHC) was performed following standard protocols. Proteinlysates, immunoprecipitated proteins proteinsurinaryor weresepa Urinary proteins were precipitated with acetone, resuspended in PBS and sub bysolu We transfection after h 48 cells protein obtained HEK fromlysates 10.1038/nm.3384 g http://rsbweb.nih.gov/ij for 15 min at that of was then phase the organic 4 °C and collection 3 VO We used the following antibodies: sheep polyclonal (Abcam, (Abcam, polyclonal sheep antibodies: following the used We 4 1 mM, glycerophosphate 1 mM, Protease-Inhibitor Cocktail Cocktail 1 mM, Protease-Inhibitor 1 mM, glycerophosphate We homogenized mouse tissues from 6- to 24-week-old 24-week-old to 6- from tissues mouse homogenized We 5 4 g (1:5,000 for WB), rabbit polyclonal against against polyclonal rabbit WB), for (1:5,000 5 , 1 h, 4 °C) and solubilized in Laemmli buffer. Laemmli in solubilized and °C) 4 h, 1 , 6 (S204C, 1:200 for IF and 1:2,000 for WB) (provided by for (provided WB) for IF and 1:2,000 1:200 (S204C, / ). 5 3 5 VO 5 (R5, 1:100 for IF and 1:750 for WB) (gift 4 (1:5,000 for WB), rabbit polyclonal polyclonal rabbit WB), for (1:5,000 4 1 mM, 1 glycerophosphate mM and N γ -octylglucoside 60 mM, NaF mM, 60 -octylglucoside -ENaC 5 4 . Briefly, we homogenized g for 1 min at 4 °C, after after °C, 4 at min 1 for 5 7 5 (1:20,000 for WB), WB), for (1:20,000 4 (1:5,000 for WB), WB), for (1:5,000 α 2 1.5 mM, mM, 1.5 -ENaC 2 O 5 2 3 to 5 ). ). 4 ------
distribution of the residuals and to take familial correlations into account. account. into correlations familial take to and residuals the of distribution a approximate normal in to order ratio better uromodulin-to-creatinine nary the explore to model of linear association mixed a used We figures. the in indicated are comparisons pairwise significant statistically Only compared). were groups Bonferroni’s by ANOVAfollowed one-way or test Student’s unpaired tailed analyses. Statistical ( Jalview with them displayed sequences with Clustal Omega ( and Informatics Laboratory, University of Pennsylvania). We aligned promoter grams.htm cgi-b ( (TESS) Software Search Element Transcription silico In against antibody monoclonal mouse and WB) for 1:20,000 C4731, (Sigma, calnexin against antibody polyclonal rabbit (Novus WB), for 1:500 (TIM-1) NBP1-76701, KIM-1 Biologicals, against polyclonal rabbit Systems, WB), D & for (R 1:500 2/NGAL AF1857, Lipocalin against polyclonal goat IF), for 1:200 1:500 for WB), rabbit against antibody polyclonal 2 aquaporin A7310, (Sigma, sc-10692, Biotechnology, Cruz (Santa ROMK against 1:50 polyclonal goat IF), for AB5196, (Chemicon, ROMK against polyclonal rabbit WB), for 1:500 58. 57. 56. 55. 54. 53. 52. 51. 50. 49. 48. 47. 46. 45. 44. 43. 42. 41. mean as ments mean as measures continuous Weexpressed
aehue AM, rce, .. Mri, .. Cap M & atn GJ Jalview G.J. Barton, & M. Clamp, D.M., Martin, J.B., Procter, A.M., Waterhouse, Wagner, C.A. F.H. Rafiqi, Activation B. Forbush, & R.B. B.F.X.,Darman, Dowd, I., A.W.,Flemmer,Gimenez, M.V. Sorensen, inreal-time Hummon, A.B., Lim, S.R., Difilippantonio, M.J. quantification & Ried, forrelative T. Isolation and solubilization model mathematical Anew M.W. Pfaffl, biologist for and users general for WWW the on Primer3 Skaletsky,H. & S. Rozen, highly A K. Laghmani, & D. Mordasini, N., Defontaine, S., Demaretz, N., Zaarour, B. Glaudemans, Terryn, S. A primary culture of mouse proximal tubular cells, established on collagen- S. Bourgeois, D.B. Barr, K. Dahan, S. Youhanna, kidney chronic for guidelines practice clinical K/DOQI Foundation. Kidney National M. Pruijm, Manunta, P. eso 2a utpe eune lgmn eio ad nlss workbench. analysis and editor alignment sequence Bioinformatics multiple 2–a Version 294 proteins. water-transporting and sodium- renal of pressure. antibody. phospho-specific Chem. Biol. a J. with detected NKCC1 cotransporter Na-K-Cl the of (2013). cotransporter in response to oral potassium intake in mice. storage. prolonged following material patient from DNA and RNA of extraction TRIzol after proteins of RT-PCR. programmers. and NKCC2. exit of reticulum expression surface endoplasmic cell dictates terminus COOH the at motif processing. conserved uromodulin and function preserved do with cells membranes.coated oxide. nitric of effect isoform: NHE2 apical functional express mice transgenic SV40 from measurements. monitoring biologic Perspect. urinary for implications Nephrol. uromodulin. of expression abnormal with nephropathy hyperuricemic 2013). October processing. (3 and storage stratification. (2002). S1–S266 and classification, evaluation, disease: study. population family-based (2008). regulation. pressure blood sodium-related on pathways in/tess/tes i:10.1007/s00424-013 , F1373–F1380 (2008). F1373–F1380 , β analysis. -actin (Sigma, A2228, 1:20,000 for WB). for 1:20,000 A2228, (Sigma, -actin Pflugers Arch. Pflugers l
) and the associated Transfac database (Computational Biology Biology (Computational database Transfac associated the and ) Nucleic Acids Res. Acids Nucleic
14 EMBO Mol. Med. Mol. EMBO 113 et al. et et al. et UMOD , 2883–2893 (2003). 2883–2893 , et al. t al. et t al. et et al. s ± et al. et ) or PATCH( or ) 1.0 , 192–200 (2005). 192–200 ,
Methods Mol. Biol. Mol. Methods
t al. et s.e.m. We set the significance level to to level We significance the s.e.m. set 25 277 A cluster of mutations in the UMOD gene causes familial juvenile familial causes gene UMOD the in mutations of cluster A Heritability, determinants and reference values of renal length: a length: renal of values reference and Heritability,determinants t al. et t al. et Physiological interaction between We analyzed the the analyzed We , 1189–1191 (2009). 1189–1191 , Mouse model of type II Bartter’s syndrome. II. Altered expression oe f h WKatvtd PK iae n euaig blood regulating in kinase SPAK WNK-activated the of Role We comparisons groups performed between using a two- rnr cetnn cnetain i te .. population: U.S. the in concentrations creatinine Urinary Differentiated thick ascending limb (TAL)limb ascending thick Differentiated derived cells cultured Biotechniques , 37551–37558 (2002). 37551–37558 , Am. J. Physiol. Renal Physiol. Renal Physiol. J. Am. rs4293393 with square-root–transformed daytime uri daytime square-root–transformed with rs4293393 Determination of uromodulin in human urine: influence of influence urine: human in uromodulin of Determination
446 piay utr sse o mue hc acnig limb ascending thick mouse of system culture primary A ai dpopoyain f h rnl oim chloride sodium renal the of dephosphorylation Rapid , 672–683 (2003). 672–683 , t -1321- -test, two-tailed nonparametric Mann-Whitney Mann-Whitney nonparametric two-tailed -test,
eho. il Transplant. Dial. Nephrol. 2
29 http://www.jalview.org , 63–75 (2010). 63–75 , http://www.ebi.ac. Eur. Radiol. Eur. , e45 (2001). e45 , 1
http:// 42 132 (26 July 2013). July (26 J. Biol. Chem. Biol. J. , 467–470, 472 (2007). 472 467–470, , , 365–386 (2000). 365–386 , www.gene-regulation. UMOD
23 ± , 2899–2905 (2013). 2899–2905 , s.d. and averages of measure of averages and s.d.
promoter sequence using using sequence promoter
284 http://www. 293 uk/Tools/msa/clustal m J Pyil Rnl Physiol. Renal Physiol. J. Am. α -adducin and WNK1-NEDD4L post hoc post , 21752–21764 (2009). 21752–21764 , Hypertension / , F476–F485 (2007).F476–F485 , )
m J Kde Dis. Kidney J. Am. 5 Kidney Int. P 8 doi:10.1093/ndt/gft34 nature medicine nature . < 0.05. < test (when three three (when test cbil.upenn.edu/ nio. Health Environ. com/pub/pro Pflugers Arch. Pflugers
52 83 . m Soc. Am. J. , 366–372 , , 811–824 o / ) and
39 5 - - - ,