View.50 the Small ﬁgure Shows the R2 Between the Two Snps Highlighted in Red

META-ANALYSIS www.jasn.org

Common Variants in UMOD Associate with Urinary Uromodulin Levels: A Meta-Analysis

† ‡ | †† Matthias Olden,* Tanguy Corre, § Caroline Hayward, Daniela Toniolo,¶** Sheila Ulivi, ††‡‡ ‡ Paolo Gasparini, Giorgio Pistis,¶ Shih-Jen Hwang,* Sven Bergmann, § Harry Campbell,§§ ††‡‡ ††‡‡ || Massimiliano Cocca,¶ Ilaria Gandin, Giorgia Girotto, Bob Glaudemans, | ††† Nicholas D. Hastie, Johannes Loffing,¶¶ Ozren Polasek,*** Luca Rampoldi,§§ Igor Rudan, ‡‡‡ ††‡‡ Cinzia Sala,¶ Michela Traglia,¶ Peter Vollenweider, Dragana Vuckovic, || || | ‡ ||| ||| Sonia Youhanna, §§§ Julien Weber, §§§ Alan F. Wright, Zoltán Kutalik, § Murielle Bochud, || Caroline S. Fox,*¶¶¶ and Olivier Devuyst §§§ *National Heart, Lung, and Blood Institute’s Framingham Heart Study, Framingham, Massachusetts; †Department of Genetic Epidemiology, Institute of Epidemiology and Preventive Medicine, University of Regensburg, Regensburg, Germany; ‡Department of Medical Genetics, University of Lausanne, Lausanne, Switzerland; §Swiss Institute of Bioinformatics, Lausanne, Switzerland; |Institute of Genetics and Molecular Medicine, Western General Hospital, and ‡‡‡Centre for Population Health Sciences, University of Edinburgh, Edinburgh, Scotland, United Kingdom; ¶Division of Genetics and Cell Biology, San Raffaele Scientific Institute, Milano, Italy; **Institute of Molecular Genetics, National Research Council, Pavia, Italy; ††Institute for Maternal and Child Health, Burlo Garofolo Pediatric Institute Trieste, Italy; ‡‡Department of Medical Sciences, University of Trieste, Trieste, Italy; §§Dulbecco Telethon Institute, San Raffaele Scientific Institute, Milan, Italy; ||Institute of Physiology, Zurich Center for Integrative Human Physiology, and ¶¶Institute of Anatomy, University of Zurich, Zurich, Switzerland; §§§Division of Nephrology, Catholic University of Louvain Medical School, Brussels, Belgium; ***Department of Public Health, Faculty of Medicine, University of Split, Croatia; ‡‡‡Department of Internal Medicine, Lausanne University Hospital, and |||Institute of Social and Preventive Medicine, Lausanne University Hospital, Lausanne, Switzerland; and ¶¶¶Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts

ABSTRACT Uromodulin is expressed exclusively in the thick ascending limb and is the most abundant protein excreted in normal urine. Variants in UMOD, which encodes uromodulin, are associated with renal function, and urinary uromodulin levels may be a biomarker for kidney disease. However, the genetic factors regulating uromodulin excretion are unknown. We conducted a meta-analysis of urinary uromodulin levels to identify associated common genetic variants in the general population. We included 10,884 individuals of European descent from three genetic isolates and three urban cohorts. Each study measured uromodulin indexed to creatinine and conducted linear regression analysis of approximately 2.5 million single nucleotide polymorphisms using an additive model. We also tested whether variants in genes expressed in the thick ascending limb associate with uromodulin levels. rs12917707, located near UMOD and previously associated with renal function and CKD, had the strongest association with urinary uromodulin levels (P,0.001). In all cohorts, carriers of a G allele of this variant had higher uromodulin levels than noncarriers did (geometric means 10.24, 14.05, and 17.67 mg/g creatinine for zero, one, or two copies of the G allele). rs12446492 in the adjacent gene PDILT (protein disulfide isomerase-like, testis expressed) also reached genome-wide significance (P,0.001). Regarding genes expressed in the thick ascending limb, variants in KCNJ1, SORL1, and CAB39 associated with urinary uromodulin levels. These data indicate that commonvariantsintheUMOD promoter region may influence urinary uromodulin levels. They also provide insights into uromodulin biology and the association of UMOD variants with renal function.

J Am Soc Nephrol 25: 1869–1882, 2014. doi: 10.1681/ASN.2013070781

Received July 25, 2013. Accepted January 13, 2014. Correspondence: Dr. Olivier Devuyst, Institute of Physiology, Zurich Center for Integrative Human Physiology, University of M.O., T.C., and C.H. contributed equally to this work, and Z.K., Zurich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland. M.B., C.S.F., and O.D. jointly oversaw this work. Email: [email protected] META-ANALYSIS

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CKD is a major public health problem affecting 5%–10% of INGI-Val Borbera) with urinary uromodulin levels. Detailed adults worldwide,1,2 and is associated with increased risks of information on samples, assays, genotyping, and imputation cardiovascular morbidity and mortality.3,4 Although hyper- platforms are included in Supplemental Tables 1 and 2. tension and diabetes are established risk factors for developing Heritability measures performed in the Framingham Heart CKD,5,6 they do not fully identify individuals at risk for this Study showed that uromodulin levels are heritable at 17% condition.7 Therefore, further exploration of novel biomark- (P=0.003), whereas the Croatia-Korcula study showed herita- ers for early detection and development of CKD beyond the bility of 28%. current measures of kidney function and damage is necessary.8 Uromodulin (Tamm–Horsfall protein), the most abundant Variants Identified through Meta-Analysis of GWAS protein excreted in the urine, is increasingly considered as a Figure 1 displays P values from the meta-analysis of GWAS of potential biomarker for CKD.9–11 Uromodulin is exclusively urinary uromodulin indexed to urinary creatinine on a produced in the epithelial cells lining the thick ascending limb 2log10 scale versus their genomic position. A genome-wide (TAL) of the loop of Henle, where it is sorted to the apical significant signal (P,1E-72) was present on chromosome 16 plasma membrane and released into the tubular lumen by in and near the UMOD gene, followed by two loci, which did proteolytic cleavage. Uromodulin is an abundant transcript in not reach genome-wide significance. In addition, an excess of the TAL cells, and its rate of secretion in the urine ranges from low P values was present in the quantile-quantile plot of the 20 to 100 mg/d in physiologic conditions.12 Recent genome- –log10 observed versus expected P values (Figure 2), which wide association studies (GWAS)13–15 and sequencing efforts16 was still present after removing a 1 Mb–long region around the have identified common single-nucleotide polymorphism UMOD gene on chromosome 16 (Figure 2, inset). The re- (SNP) variants in the UMOD gene in association with the gional association plot of UMOD (Figure 3) shows that the eGFR and the risk of developing CKD.14 On the other hand, urinary uromodulin association signal spreads over several mutations in UMOD cause dominantly inherited forms of linkage disequilibrium (LD) blocks. tubulointerstitial kidney disease collectively referred to as Table 2 presents results from the inverse-variance meta- uromodulin-associated kidney disease (UAKD).17 These disor- analysis for urinary uromodulin indexed to urinary creatinine. ders include defective urinary concentrating ability, hyperuri- The variant with the lowest P value (7.85E-73) was rs12917707 cemia, gout, and progression to ESRD between the second and located in the 59 promoter region of the UMOD gene. Each copy fourth decades of life. Analyses of renal biopsies and in a trans- of the G allele was associated with high levels of uromodulin in genic mouse model of UAKD revealed that UAKD is coupled our analyses (P=6.6E-71) and lower levels of eGFR in CKDGen to a significant reduction of uromodulin excretion in the urine. Consortium participants (P=1.2E-20). This SNP was previ- Studies in UMOD knockout mice showed that uromodulin ously reported in association with eGFR and CKD.13 A second may play a role in the transport processes operating in the variant (rs12446492, within the PDILT gene located near the TAL18,19 and protects against urinary tract infection20 and for- UMOD gene) showed similar associations with uromodulin mation of kidney stones.21 (P=6.6E-26) and eGFR (P=6.9E-07). This variant has low R2 Despite these data, obtained essentially in mouse or cellular but high D9 values (R2=0.17, D9=0.83) with the lead GWAS models, the biologic function of uromodulin isstillundefined and SNP,rs12917707. The next two independent variants identified the genetic contribution to common variation in uromodulin through meta-analysis (rs4533720 on chromosome 4 near the production and release remains to be determined. Thus, this MARCH1 gene and rs6988636 on chromosome 8 near the study aimed to investigate the genetic association of urinary FAM83A gene) did not reach genome-wide significance uromodulin levels in the general population. We performed a (P=2.41E-07 and P=7.97E-07, respectively) and were not as- meta-analysis of GWAS for urinary uromodulin concentration sociated with eGFR in 67,093 individuals in the CKDGen Con- in six studies including three urban cohorts and three genetic sortium (P=0.47 and P=0.58, respectively).15,22 A sensitivity isolates amounting to 10,884 participants of European ancestry. meta-analysis of raw unindexed uromodulin (results in Sup- Wealso used a candidate gene approach to seek whether urinary plemental Table 3) yielded similar values to the ones indexed to uromodulin concentrations are associated with variants in creatinine. The association of eGFR with the four variants re- genes regulating transport processes in the TAL. stricted to the six cohorts included in this study is not significant. In the CoLaus cohort (N=5077), stratified analyses for the RESULTS association of urinary uromodulin indexed to urinary creatinine with rs127917707 by hypertension status (–0.284960.0418 Table 1 shows study sample characteristics for 10,884 partic- [P=9.64E-12] in individuals with hypertension versus ipants from all six cohorts (Cohort Lausannoise [CoLaus], 20.312360.0318 [P=9.37E-23] in normotensive people; P for Croatia-Korcula, Croatia-Split, Framingham Heart Study, difference between strata=0.60) or by age groups (20.3160.04 Network Italiano Isolati Genetici (INGI)-Carlantino, and [P=1.58E-17] in people aged#53 years versus 20.3060.04

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[P=6.88E-17] in younger people; P for between strata difference=0.79) led to similar results. Adding hypertension as a 30.37) 20.74) 33) 88.68) 26.85) – – – – – 73.7) covariate in the model barely affected this same association –

g/ml) (20.296060.0354 [P=6.49E-17] including hypertension m ( versus 20.301860.0254 [P=1.22E-32] not including hyper-

UMOD Unindexed tension).

Association of rs12917707 Genotypes with Uromodulin Levels 34.54) 6.75 (1.52 29.43) 5.09 (0.97 48.82) 28.6 (5.05 27.98) 6.9 (2.28 – – – – 27) 7.42 (0.30 46.7) 25.8 (4.9 – – Table 3 presents the association of rs12917707 genotypes with urinary uromodulin levels. The G allele of rs12917707 was consistently associated with higher urinary uromodulin UCr (mg/g Cr)

UMOD Indexed to levels in an additive manner. The 2-fold increase in participants harboring the GG genotype was observed in each of the six cohorts investigated, with a meta P value of 2.41E-58.

Joint Analyses Table 4 shows the results from a joint analysis conditioned on the rs12917707 SNP. This analysis revealed another nearby HTN DM variant in the PDILT gene, rs4494548, in low LD with the conditioned SNP (R2=0.02, D9=0.16), which is associated P ) with uromodulin (meta-analysis, =3.9E-15; joint condi- 2 19.5 36.0 (1830) 4.5 (229) 18.4 (3.6 22.4 41.4 (593) 6.6 (95) 8 (1.54 25.7 40.4 (122) 10.0 (29) 6.7 (1.44 25 40.0 (1055) 9.4 (249) 9.5 (0.3 23.8 35.2 (172) 3.2 (16) 20.6 (3.54 29 55.9 (496) 14.2 (126) 6.4 (1.37 tional analysis, P=2.3E-08). This variant was not signiﬁcantly 6 6 6 6 6 6 associated with eGFR in CKDGen Consortium participants eGFR Cr 1.73 m 13,22 (ml/min per (P=4.3E-04, N=67,093). Variance analysis using genotype data from the CoLaus study showed that the lead ) 2 GWAS hit rs12917707 explains 1.2% of the variance of uri- 4.5 88.9 5.9 94.8 5.1 87.5 4.5 90.1 4.2 95.6 4.1 91.3 6 6 6 6 6 6 nary uromodulin levels, with an additional 0.8% variance explained by the second SNP, rs4494548. These results suggest that at least two independent signals for urinary uromodulin exist at the UMOD locus. 18.3 25.9 20.4 26.7 9.6 27.9 10.7 25.7 14.7 26.9 13.9 28.0 6 6 6 6 6 6 Gene-Based Association Analyses Results from the Versatile Gene-based Association Study (VEGAS) analysis are included in Supplemental Table 4. This gene-based association analysis identiﬁed the region of chromosome 16 containing UMOD, PDILT, GP2,andACSM5 as ) for categorical variables, or median (5th percentile, 95th percentile). BMI, body mass index; Cr, creatinine; HTN, hypertension; DM, diabetes

Women Age (yr) BMI (kg/m ﬁ n being the only region with a statistically signi cant result 2 (P,2.8310 6) and identiﬁed the top SNP from the meta-

) P n analysis (rs12917707, =7.9E-73) as the main contributor to the finding. No other genes were identified. Size ( Sample Candidate Gene Analyses Results Uromodulin is exclusively synthesized in the epithelial cells lining the TAL (Figure 4). These cells reabsorb approximately fi Type 5% of the ltered NaCl and generate a lumen-positive SD for continuous variables, % ( 6

Population transepithelial voltage. Previous studies suggested that uri-

Nonisolate urban 2640 53.2 (1404) 58.4 nary uromodulin concentration reﬂects the transport activ- ity of the TAL.9,12 The potential association of 24 candidate genes expressed in the TAL and involved in TAL transport Study sample size characteristics processes revealed that SNPs in KCNJ1(ROMK), CAB39, and SORL1 genes were associated (P value lower than the Study gene-speciﬁc threshold) with the level of uromodulin in- Heart Study INGI-Val Borbera Isolate 1432 55.7 (797) 54.1 INGI-Carlantino Isolate 360 56.9 (205) 48.1 mellitus; UCr, urine creatinine. Framingham Table 1. Data are presented as mean CoLaus Nonisolate urban 5077 53.5 (2716) 53.3 Croatia-Split Nonisolate urban 488 56.6 (276) 49 Croatia-Korcula Isolate 887 63.7 (565) 56.3 dexed to urinary creatinine (Table 5). Of the other 21

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Figure 1. The 2log10(P value) plot by genomic position (Manhattan plot) for results from the meta-analysis of GWAS of uromodulin indexed to creatinine in six population-based studies. The reported locus (P,1E-72) is highlighted in orange. SNPs with minor allele frequency ,5% are excluded. P values are double corrected for genomic control. investigated candidate genes, 5 genes were only nominally as- included in a LD block encompassing the UMOD promoter. We sociated with uromodulin and the rest did not show any asso- show that the G allele of the rs12917707 variant, associated with ciation. We calculated the effective number of independent higher risk of CKD in previous studies and with lower eGFR in tests of the 1739 SNPs examined in the 24 candidate genes to this study, is associated with up to 2-fold higher levels of be 129. When accounting for all candidate SNPs tested, declar- uromodulin in urine. This finding is consistent across all co- ing the four most significant SNPs (rs2855800, rs2438298, horts, in both genetic isolates and nonisolated populations. rs1532763, rs4239217) as true associations would result in rs4494548 is also associated with urinary uromodulin levels, a false discovery rate below 75% (Table 5). SNP rs28555800 even upon adjustment by rs12917707. rs4494548 is an intronic in KCNJ1 was also associated with eGFR (P=0.02; N=67,093) in SNP of the PDILT gene, which is known to be transiently ex- previously published CKDGen meta-analysis results.13,22 Ex- pressed during urinary tract development and is associated with pression studies performed on well characterized microdissected male infertility.23 Finally, we used a candidate gene approach to tubule segments showed that Kcnj1, Cab39,andSorl1 were show that SNPs in three genes (ROMK, CAB39,andSORL1) enriched with Umod in the TAL of mouse kidney (Ct values: known to mediate or regulate transport processes in TAL cells Kcnj1, 26.4360.41; Cab39, 30.3660.44; Sorl1, 28.3460.40; are associated with urinary uromodulin levels. Taken together, Umod, 19.7160.41; n=3 independent experiments performed our findings suggest that common SNP variants identified on tubular fractions pooled from two kidneys), where they are through GWAS and conditional analysis may account for vari- known to be involved in the regulation of NaCl transport ac- ation in urinary uromodulin and eGFR. tivity (Figure 4). Previous GWAS have shown that promoter variants in the UMOD gene in high LD with our top rs12917707 SNP are associated with eGFR and CKD. A first GWAS with nearly 20,000 DISCUSSION individuals from population-based studies in the Cohorts for Heart and Aging Research in Genomic Epidemiology consor- Our study provides the first meta-analysis of uromodulin levels tium identified rs12917707, whereby each copy of the minor in urine, including six population-based cohorts amounting to allele T was associated with higher levels of eGFR and a lower .10,000 individuals of European descent. Our analyses risk of CKD.24 On the basis of these findings, investigators then document a robust locus with two independent signals around measured urinary uromodulin in several nested case-control the UMOD gene (rs12917707 and rs4494548), with rs12917707 studies. Among 200 individuals in the Framingham Heart Study,

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Figure 2. Quantile-quantile plot of results from the meta-analysis of GWAS of uromodulin indexed to creatinine in six population-based studies. The inset graph shows the remaining signals after excluding SNPs in and 1 Mb around the UMOD gene. SNPs with minor allele frequency ,5% are excluded from the meta-analysis. P values are double corrected for genomic control.

the C allele at rs4293393 (in perfect LD with rs12917707, R2=1) around the UMOD gene. We confirm the strong and consistent was associated with lower urinary uromodulin levels, and these association of the promoter variant with an up to 2-fold in- levels predicted higher eGFR at 10 years.15 In light of these crease in urinary excretion of uromodulin. That association is intriguing case-control studies, we sought to measure urinary detected in each cohort analyzed, either from urban or genetic uromodulin in much larger sample sizes, and to apply genome- isolate origin, and (in CoLaus) is not altered by adjustment for wide association to uncover loci in association with urinary hypertension nor upon stratification for hypertension status uromodulin levels. Our study advances knowledge in this and for age. Of note, large differences in the median value of uri- area in several important ways. First, we evaluated the associ- nary uromodulin have been observed across cohorts even when ation between genetic variants and urinary uromodulin at a using the same sensitive assay, and they are not related to storage genome-wide level, evidencing two robust independent signals conditions. We do not think that this variation influenced our

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Figure 3. Regional association plot of UMOD gene for the analysis indexed to urinary creatinine. The lead GWAS hit (rs12917707) and GCTA hit (rs4494548) are highlighted. The plot is modified from LocusZoom49 and Haploview.50 The small figure shows the R2 between the two SNPs highlighted in red. LD information is based on HapMap CEU. meta-analysis results, because we first performed study-specific genomic regions suggested through gene expression and func- analyses that served to internally calibrate the data. Large varia- tional studies in order to better understand the link between tions in the daily physiologic excretion of uromodulin have been uromodulin and ion transport in the TAL segment. reported, probably reflecting the complex secretory pathway of The potential mechanisms of the association of SNPs the protein and its potential interactions with multiple urinary (including the top rs12917707) in the promoter of UMOD components.9 Second, we used a joint conditional analysis to include potential cis-acting effects that could modulate the extract other independent signals at the same locus that account transcription of uromodulin by the TAL cells, and thus its for a higher variation of the observed signal. Third, we leveraged excretion in the urine. The dose-dependent effect, which is our existing urinary uromodulin GWAS data set to interrogate consistently observed in all cohorts, supports the latter

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Table 2. Top loci with P values up to 1E-7 associated with uromodulin indexed to creatinine in the overall population eGFR Cr Effect Nearest Imputation Direction eGFR Cr R2 to Top SNP SNP ID Chr RA NRA P Valuea SEMa P Valuea RAF Size Geneb Qualityc Relative P Value rs12917707 to RA rs12917707 16 T G 20.32 7.85E-73 0.02 7.85E-73 0.18 UMOD 0.94 + 1.20E-20 Same SNP rs12446492 16 A T 20.15 5.60E-27 0.01 5.60E-27 0.45 PDILT 0.72 + 6.90E-07 0.16 rs4533720 4 A G 0.07 3.50E-7 0.01 3.50E-7 0.53 MARCH1 1 + 0.47 NA rs6988636 8 T C 20.13 9.47E-7 0.03 9.47E-7 0.92 FAM83A 0.99 2 0.58 NA Genes nearby were based on RefSeq genes (build 36). Chr, chromosome; RA, reference allele; NRA, nonreference allele; RAF, reference allele frequency; Cr, creatinine; NA, not available. aP value from inverse-variance meta-analysis, corrected for genomic control. Sample size weighted meta-analysis yielded similar results. bThe gene closest to the SNP is listed ﬁrst and is marked bold if the SNP is located within the gene. cMedian imputation quality across participating studies.

Table 3. Association of rs12917707genotypes with urinary uromodulin levels in the six cohorts UMOD Indexed to UCr (mg/g Cr) Sample Size (n) Total Analyzed P Value in Cohort a TT TG GG TT TG GG Sample Size (n) Each Cohort CoLaus 13.9068.92 18.74613.08 22.80617.27 195 1472 3410 5077 2.31E-26 Croatia-Korcula 4.8363.63 8.0768.16 9.97610.83 12 207 655 874 4.97E-03 Croatia-Split 12.8064.97 17.43610.03 26.09616.83 11 123 347 481 9.27E-10 Framingham Heart Study 5.27 (4.35) 8.9867.53 12.60611.88 103 851 1689 2643 7.37E-25 INGI-Carlantino 5.6766.25 9.0068.49 11.13615.2 13 108 220 341 6.08E-02 INGI- Val Borbera 6.2164.31 10.27610.86 12.43611.89 26 319 1028 1373 1.50E-04 Data are presented as mean6SD unless otherwise indicated. UCr, urine creatinine; Cr, creatinine. aThe meta P value for the six cohorts is 2.41E-58.

Table 4. Joint conditional analysis for loci associated with urinary uromodulin indexed to creatinine in the overall population Meta-Analysisa Joint Analysis R2 eGFR Cr Sample to Top SNP Variance P Chr SNP RA RAF Effect GAF b Value in SEM P ValueSize (n) Effect SEM P Value rs12917707 (%) Size CKDGen 16 rs12917707 T 0.18 20.32 0.02 7.9E-73 10,417 0.17 20.31 0.018 3.4E-64 1.0 1.2 1.2E-20 16 rs4494548 A 0.13 0.17 0.02 3.9E-15 10,417 0.10 0.12 0.022 2.3E-08 0.0 0.8 4.3E-04 Joint analysis of the meta-analysis results for unindexed urinary uromodulin did not yield any additional signal beyond the lead SNP rs12917707. Chr,chromosome; RA, reference allele; RAF, reference allele frequency; GAF, genotype allele frequency; Cr, creatinine. aValues from the inverse-variance meta-analysis. bLinear model using phenotype and genotype data from the CoLaus cohort. hypothesis, in line with the recent results of Trudu et al. show- Our candidate gene analysis revealed that variants in ing an effect of UMOD promoter variants on gene expres- KCNJ1/ROMK, SORL1,andCAB39 are associated with the sion.25 The fact that age does not modify the association of level of uromodulin in urine. In TAL cells, uromodulin is traf- urinary uromodulin with rs12917707 may suggest that ele- ficked to the apical plasma membrane and released into the vated urinary uromodulin could lead to progressive renal tubule lumen via proteolytic cleavage. This cleavage is neces- damage. Indeed, evidence obtained in a transgenic mouse sary for the polymerization of uromodulin in the urine.9,26 model suggest that such deleterious effects of uromodulin The cells lining the TAL reabsorb approximately 25% of the would take a long time and it could reflect multiple hits.25 filtered NaCl, a process that involves the apical, bumetanide- 2 Alternatively, increased urinary uromodulin levels may be sec- sensitive Na+/K+/2Cl cotransporter NKCC2, organized in ondary to renal damage caused by mechanisms unrelated to parallel with the renal outer medullary K+ (ROMK) channel, 2 uromodulin. This second hypothesis would be in line with a which is essential to recycle K+.Na+ and Cl are transported protective effect of urinary uromodulin.9 The mechanism of across the basolateral membrane via the Na+/K+-ATPase 2 the SNPs in PDILT is not predictable, although it is unlikely and the Cl channel ClC-Kb, respectively. These transport that the association is driven by LD with the UMOD promoter processes are highly regulated: the Ste20- and SPS1-related variants. proline and alanine-rich kinase (SPAK) and oxidative

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Figure 4. Candidate gene analysis and nephron segmentation. (A) Expression levels of UMOD, ROMK, SORL1, and CAB39 in microdissected mouse tubule segments (relative to TAL taken as 100%, white bars). Expression levels are relative to reference gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Data are means6SEM of at least three independent experiments obtained for pooled tubules from two mouse kidneys. (B) Model for the intersection of ROMK, SORL1, and CAB39 with uromodulin in cells lining the TAL.

stress-responsive kinase (OSR1) activate NKCC2 by N-terminal addition, we used common genetic variants (minor allele fre- phosphorylation.27 In turn, SPAK and OSR1 require regulatory quency .5%) and it is possible that there may be other low cofactors, such as sorting protein-related receptor with A-type frequency variants that could additionally explain this associ- repeats 1 (SORL1) and calcium-binding protein 39 (CAB39). ation. Future GWAS conducted in larger cohorts and using CAB39 is a scaffolding protein that interacts with SPAK and variants derived from exome sequencing could reveal other OSR1, increasing kinase activation.28 SORL1 was previously genetic associations with urinary uromodulin concentrations. shown to mediate SPAK membrane sorting, allowing the phos- In conclusion, a meta-analysis of urinary uromodulin phorylation of NKCC2 by SPAK.29 The fact that uromodulin concentrations revealed the association of common variants expression is limited to the TAL segment in mice and in the promoter region of the UMOD gene, together with a humans30–32 andtheenrichmentofROMK,SORL1,and second independent signal located within the flanking gene CAB39 in that very segment emphasize the potential link be- PDILT. Each copy of the G allele at rs12917707 (risk allele for tween transport processes and uromodulin secretion in the CKD) was associated with higher urinary uromodulin con- cells lining the TAL. Furthermore, a functional link between centrations. Variants in the KCNJ1, CAB39, and SORL1 genes ROMK and uromodulin has already been documented in vitro regulating transport processes in the TAL cells producing and in mouse models.18 Future studies will address the biologic uromodulin were also associated with its urinary concentra- mechanisms that underlie these genetic and functional associ- tion. These results advance our understanding of the biology ations. of uromodulin and substantiate the association of UMOD Our study combines the advantages of the first and large variants with renal function. GWAS on urinary uromodulin, performed using a robust assay on various types of well documented cohorts, with an in- terrogation of candidate genes operating in the very tubular CONCISE METHODS segment that is releasing uromodulin in the urine. Limitations of this study include the availability of data only for individuals Urinary uromodulin and creatinine levels were measured in 10,884 of European descent, restricting the generalizability of our individuals of European descent from six cohorts in both urban findings. The lack of external replication is balanced by the and isolate communities (CoLaus, Croatia-Korcula, Croatia-Split, genome-wide significance of the association of rs12917707 in Framingham Heart Study, INGI-Carlantino, and INGI-Val Borbera). the CoLaus cohort, with replication of this main hit in five All studies performed genome-wide associations for both urinary independent cohorts. Conversely, the subgenome-wide find- uromodulin indexed to urinary creatinine and raw urinary uromodulin ings around the FAM83A and MACH1 genes should be inter- only, which were then meta-analyzed. Participants provided written preted with caution given the absence of replication. In informed consent.

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82 04GntcDtriat fUiayUromodulin Urinary of Determinants Genetic 2014 1882, Table 5. Candidate gene analysis for uromodulin indexed to creatinine in the overall population LD Blocks SNPs in Gene with Lowest eGFR Cr SNPs Found Gene-Specific SNP ID with Lowest Gene Name Chr in Gene at P Value Below P Value FDR P Value Gene Category in Gene (n) Threshold P Value in Gene R2=0.2 (n) Threshold (n) in Gene in CKDGen KCNJ1(ROMK)a 11 40 5 0.010 1 rs2855800 0.001 0.1914 0.023 Ion transport CAB39a 2 43 6 0.008 1 rs2438298 0.002 0.2962 0.065 Regulator, ion transport SORL1a 11 120 9 0.006 3 rs1532763 0.004 0.4381 0.29 Regulator, ion transport HNF1B 17 36 9 0.006 0 rs4239217 0.01 0.7178 0.38 Transcription factor REN 1 21 6 0.008 0 rs11571093 0.02 .0.75 0.64 Regulator, ion transport STK39 2 362 15 0.003 0 rs2203703 0.02 .0.75 0.19 Regulator, ion transport CLDN14 21 165 12 0.004 0 rs4816538 0.03 .0.75 0.9 Paracellular, ion transport CLDN16 3 56 6 0.008 0 rs17445530 0.03 .0.75 0.69 Paracellular, ion transport CNNM2 10 137 6 0.008 0 rs12769080 0.06 .0.75 0.1 Ion transport SLC9A3 5 27 7 0.007 0 rs1035908 0.07 .0.75 0.94 Ion transport CLCNKB 1 27 5 0.010 0 rs2007471 0.07 .0.75 0.21 Ion transport SLC9A4 2 168 7 0.007 0 rs17027258 0.07 .0.75 0.45 Ion transport NFAT5 16 75 6 0.008 0 rs1064825 0.08 .0.75 0.017 Transcription factor SLC9A2 2 81 12 0.004 0 rs13011360 0.11 .0.75 0.49 Ion transport AGT 1 48 5 0.010 0 rs3789664 0.11 .0.75 0.52 Regulator, ion transport WNK1 12 104 8 0.006 0 rs7137188 0.13 .0.75 0.15 Regulator, ion transport CLCNKA 1 34 3 0.017 0 rs1889785 0.15 .0.75 0.34 Ion transport CASR 3 75 8 0.006 0 rs3804593 0.15 .0.75 0.65 Regulator, ion transport OXSR1 3 19 5 0.010 0 rs4955408 0.22 .0.75 0.75 Regulator, ion transport SLC12A1 15 44 1 0.050 0 rs1843144 0.22 .0.75 0.38 Ion transport CLDN19 1 8 3 0.017 0 rs7548008 0.31 .0.75 0.73 Paracellular, ion transport BSND 1 20 2 0.025 0 rs12134118 0.31 .0.75 0.55 Ion transport WNK4 17 1 1 0.050 0 rs873084 0.34 .0.75 0.2 Regulator, ion transport www.jasn.org HIF1A 14 28 2 0.025 0 rs7143164 0.43 .0.75 0.19 Transcription factor The table is sorted after lowest P value in the gene. Significance was defined as P value lower than a gene-specific threshold, calculated as 0.05 divided by the number of found LD blocks at R2=0.2 in that gene. Chr, chromosome; FDR, false discovery rate; Cr, creatinine. aThree loci with at least one SNP with a P value below the threshold in the gene. The FDR values ,0.75 are provided as an alternative threshold (see the Concise Methods). META-ANALYSIS 1877 META-ANALYSIS www.jasn.org

Study Samples determined at 3.28% and 5.46%, respectively. The assay had a de- The CoLaus study is a population-based study including .6000 peo- tection range between 3.9 and 500 ng/ml (Supplemental Table 1). ple aged 35–75 years from the city of Lausanne, Switzerland, as Uromodulin was indexed to creatinine to compensate for variations previously described.33 All participants were of European descent, in urine concentrations. Uromodulin levels below the detection defined as having both parents and grandparents born in a restricted threshold were set to the lower limit of the urinary uromodulin assay. list of countries. A baseline examination, including collection of morning spot urine, a fasting venous blood sample, as well as a de- Genotyping Platforms and Imputation tailed health questionnaire, was conducted by trained health care Genotyping and imputation were conducted as described in Supple- workers between 2003 and 2006. The study was approved by the mental Table 2. Genotyped SNPs that passed quality control proce- ethical committee of Lausanne University Hospital. dures were then imputed to approximately 2.5 million HapMap CEU The Croatia-Korcula study is a family-based, cross-sectional study in SNPs. the isolated island of Korcula, Croatia, that included 965 examinees aged – 18 95 years. Urine samples were collected in 2007, along with clinical Heritability and Proportion of Variance Explained and biochemical measures and lifestyle and health questionnaires. Heritability of urinary uromodulin levels was assessed in the The Croatia-Split study is a population-based, cross-sectional Framingham Heart Study using SOLAR software (version 1.4),36 study in the Dalmatian City of Split, Croatia, that included 1012 with age, sex, and age by sex as covariates, and in the Croatia-Korcula examinees aged 18–95 years. Urine samples were collected in 2009. study using an estimate of heritability derived from analysis of the The Framingham Heart Study is a community-based multigen- polygenic model in GenABEL software,37 with age and sex as eration family study, with three generations (1971, original cohort; covariates. The proportion of urinary uromodulin variance explained 1984, offspring cohort; 2002, third generation). The design of this by the reported SNPs was calculated on the basis of the meta-analysis study was previously described.34 The sample used here is a subset of results and the SD of uromodulin levels in the CoLaus cohort using 2640 participants from the offspring cohort at examination 6 (1995– GCTA software (version 1.04).38 1998), with urinary uromodulin and eGFR levels measured. Partic- ipants self-reported their European descent ancestry. Statistical Analyses The INGI-Carlantino study is a population-based, cross-sectional Each participating study performed a GWAS of approximately 2.5 study in avillage situated in the southeastern part of the Apennines in a million SNPs on both urinary uromodulin indexed to urinary creatinine hilly area of the Puglia region. We used a subset of 360 individuals and raw urinary uromodulin (unindexed) using linear regression and an (from a total sample size of 1478) who had genotyping information additive genetic model, adjusting for age and sex and principal and urine samples available. components where applicable. Data from the Framingham Heart Study, The INGI-Val Borbera project was initiated in 2005, involving Croatia-Korcula,Croatia-Split,INGI-ValBorbera,andINGI-Carlantino inhabitants of the geographically isolated Borbera Valley of Northwest cohorts were adjusted for family relatedness using the kinship matrices Italy, in Piedmont. Information on a large set of phenotypes and and for population stratification using the first three principal compo- biologic samples was obtained from 1803 inhabitants aged between 18 nents.Theseadjustmentsaccountforallknownandcrypticrelationships and 102 years. within these cohorts. The results were then meta-analyzed with METAL software (version 2011-03-05)39 using both sample size and inverse- Uromodulin and Creatinine Measurements variance weighted fixed-effects models. The statistical significance All spot urine samples were aliquoted immediately aftercollection and threshold was set to 5E-8 for genome-wide significance. P values were were frozen and stored at 280°C before analysis. In the Framingham corrected for genomic control both at the study level and after meta- Heart Study, the Rules-Based Medicine array (Rules-Based Medicine, analysis. The l value was around 1.0. SNPs with a minor allele frequency Inc., Austin, TX) measured urinary uromodulin via immunoassay ,5% or not present in at least 50% of the studies were dropped from the with a bead Luminex platform. This assay has coefficient of variations meta-analysis results. For each locus, independent SNPs with the lowest of 5.0% at a mean concentration of 37 mg/ml and 11.4% at a mean P value and in low linkage disequilibrium (R2,0.2) were reported. concentration of 9.4 mg/ml. The assay range is between 6.2 and 135 ng/ml. All other studies measured urinary uromodulin concen- Stratified Analyses for the Lead SNP tration by a well established ELISA35 based on a sheep anti-human UMOD variants have been associated with hypertension,40 and the uromodulin antibody (K90071C; Meridian Life Science, Memphis, association of renal function with UMOD variants was modified by TN) as the capture antibody, a mouse monoclonal anti-human age.14 We therefore conducted stratified analyses for the lead SNP Tamm–Horsfall protein antibody (CL 1032A; Cedarlane Laborato- (rs12917707) by hypertension status (defined as being on antihyper- ries, Burlington, NC) as the primary antibody, and a goat anti-mouse tensive treatment or having systolic/diastolic BP$140/90 mmHg) IgG (H+L) horseradish peroxidase–conjugated protein (172.1011; and by age group (below and above the median age of 53 years) in Bio-Rad Laboratories, Inc., Hercules, CA) as a secondary antibody. CoLaus, the largest cohort available in our study (n=5077), to Human uromodulin (AG 733, stock solution: 100 mg/ml; EMD explore a potential effect modification by hypertension or age. We Millipore, Temecula, CA) was used to establish the standard curve. comparedp effectffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi sizes across strata using a two-sample test ðb 1 b Þ= ðSE2 1 SE2Þ 41 The ELISA for human uromodulin showed a sensitivity of 2.8 ng/ml 1 2 1 2 . We also conducted sensitivity analyses and a linearity of 1.0. The interassay and intraassay variability were adding hypertension status as a covariate in the analysis.

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Associations with Renal Function SYBR Green Real-Time Quantitative PCR We tested the association with kidney function in the six population- The isolated fractions were characterized for their enrichment in based studies using eGFR, determined by the simplified Modification of mRNA expression of Nphs2 (podocin, glomerulus), Slc38a3 (SNAT3, Diet in Renal Disease study equation. We defined CKD as eGFR,60 proximal convoluted tubule), Slc5a2 (SGLT2, proximal straight tu- ml/min per 1.73 m2. We also tested the association of SNPs most sig- bule), Umod (uromodulin, TAL), Slc12a3 (NCC, distal convoluted nificantly associated with indexed urinary uromodulin with eGFR by tubule), and Aqp2 (AQP2, collecting ducts). Total RNA was extracted querying these in publicly available meta-analysis results of 67,093 in- from isolated segments using the RNAqueousR kit (Applied Biosystems, dividuals of European ancestry from the CKDGen Consortium.13 Inc., Foster City, CA), subjected to DNase treatment, and reverse transcribed by using the iScript cDNA Synthesis Kit (Bio-Rad, Gene-Based Association Analyses Munich, Germany). Quantitative RT-PCR analyses were performed The SNP association P values from the meta-analysis of urinary in duplo using 100 nM of both sense and anti-sense primers (Sup- uromodulin were analyzed using VEGAS,42 a program for performing plementary Table 5) using iQ SYBR Green Supermix (Bio-Rad) and gene-based tests for association using the summary statistics from ge- an iCycler IQ System (Bio-Rad). All amplicons showed expected netic association studies. The gene-based approach reduces the multiple- sizes and the dissociation curves showed one melting peak. Glycer- testing problem of GWAS by only considering statistical tests for aldehyde 3-phosphate dehydrogenase (Gapdh) was used routinely 17,787 genes giving a Bonferroni-corrected threshold of P,2.8E-6. as a reference gene, because preliminary experiments showed no differences with other reporter genes (Cyclophilin, Hprt1, Actb, Joint Analyses 36b4). The relative changes in target gene/GAPDH mRNA ratio 2DD Toexplore the presence of multiple independent signals at the UMOD were determined by the formula: 2 ct. locus, we ran a joint step-wise model selection (massoc-slct function) with GCTA38 using the meta-analysis association results for the uromodulin to creatinine ratio. The CoLaus cohort was used as ref- ACKNOWLEDGMENTS erence to estimate the LD between SNPs. The threshold of significance was set to the default parameters, 5E-8. The resulting estimates The authors acknowledge Viviane Beaujean and Sebastien Druart (UCL fi are equivalent to that of a multivariate linear model tting. The LD Medical School, Brussels, Belgium) for establishing the uromodulin 2 expressed in R between these SNPs, by definition ,1, is also indi- ELISA. The authors wish to specially thank the recruitment teams, cated in the results file. To quantify the level of variance explained by the administrative teams, and the study participants in the various 2 the different significant SNPs, the adjusted R was estimated in the cohorts. CoLaus population using the LM function in R software.43 This research was conducted in part using data and resources from the Framingham Heart Study of the National Institutes of Health Candidate Gene Analyses National Heart Lung and Blood Institute (NHLBI) and Boston Wecompiled a list of 24 candidate genes for urinary uromodulin levels University School of Medicine. This work was partially supported by on the basis of expression profiles showing enrichment in the TAL, and the NHLBI FraminghamHeart Study(ContractN01-HC-25195). The functional data supporting a role in transport processes operating in funders had no role in the study design, data collection and analysis, the cells lining the TAL.44–46 Each gene was queried in the meta- decision to publish, or preparation of the manuscript. The CoLaus analysis results, by extracting all variants within that gene (including study received financial contributions from GlaxoSmithKline, the the promoter and the 59 region around it). For each gene, we accounted Faculty of Biology and Medicine of Lausanne, and the Swiss National for region-specific multiple testing using a gene-specific threshold Science Foundation (33CSCO-122661, 3200BO-111361/2, 3100AO- (0.05 divided by the number of found LD blocks at R2=0.2). SNPs 116323/1, and 310000-112552). The computations for CoLaus impu- with a median imputation quality ,60% and minor allele frequency tationwere performedin part atthe Vital-IT center for high-performance ,5% were excluded before this analysis. Variants that had a P value computing of the Swiss Institute of Bioinformatics. M.B. is supported lower than gene-specific thresholds were declared significant. Toassess by the Swiss School of Public Health Plus. The Croatia-Korcula and the overall significance of SNPs in the 24 candidate genes, we esti- Croatia-Split studies were funded by grants from the United Kingdom mated the effective number of tests for the 1739 tested SNPs using the Medical Research Council, the European Commission Framework method by Gao et al.47 The SNP-by-SNP pairwise correlation matrix EUROSPAN 6 Project (Contract LSHG-CT-2006-018947), and the was calculated based on the CoLaus data. We then adjusted the Republic of Croatia Ministry of Science, Education, and Sports (Grant Benjamini–Hochberg step-down procedure to compute the false dis- 108-1080315-0302 to I.R.). The SNP genotyping for the Croatia- covery rate for the best SNP in each gene.48 Korcula cohort was performed by Helmholtz Zentrum München (Neuherberg, Germany). The SNP genotyping for the Croatia-Split Microdissection and Isolation of Tubular Segments cohort was performed by AROS Applied Biotechnology (Aarhus, Tubular segments and glomeruli from mouse kidney were micro- Denmark). The INGI-Carlantino study was funded by Regione FVG dissected using a previously described protocol.32 The collagenase- (L.26.2008). The INGI-Val Borbera study was supported by funds digested segments were sieved and selected on the basis of morphology from Compagnia di San Paolo (Torino, Italy), Fondazione Cariplo, characteristics. Pooled segments (from two kidneys for each sample) and the Ministry of Health (Ricerca Finalizzata 2008 to D.T.). Other were frozen in liquid nitrogen and kept at 280°C until RNA extraction. funding sources for this study include the European Commission

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Seventh Framework Programme (FP7/2007-2013 under Grant 246539 13. Köttgen A, Pattaro C, Böger CA, Fuchsberger C, Olden M, Glazer NL, of the Marie Curie Actions Programme and Grant 305608 of the Parsa A, Gao X, Yang Q, Smith AV, O’Connell JR, Li M, Schmidt H, EURenOmics project), Action de Recherche Concertée (ARC10/15- Tanaka T, Isaacs A, Ketkar S, Hwang SJ, Johnson AD, Dehghan A, Teumer A, Paré G, Atkinson EJ, Zeller T, Lohman K, Cornelis MC, 029, Communauté Française de Belgique), Fonds de la Recherche Probst-Hensch NM, Kronenberg F, Tönjes A, Hayward C, Aspelund T, fi fi Scienti que and Fonds de la Recherche Scienti que Medicale, Eiriksdottir G, Launer LJ, Harris TB, Rampersaud E, Mitchell BD, Arking Belgium Federal Government Inter-University Attraction Pole, DE, Boerwinkle E, Struchalin M, Cavalieri M, Singleton A, Giallauria F, Gebert Rüf Stiftung (Project GRS-038/12), and the Swiss National Metter J, de Boer IH, Haritunians T, Lumley T, Siscovick D, Psaty BM, Science Foundation (Grant 310030-146490 and the NCCR Kidney. Zillikens MC, Oostra BA, Feitosa M, Province M, de Andrade M, Turner ST, Schillert A, Ziegler A, Wild PS, Schnabel RB, Wilde S, Munzel TF, CH program). Leak TS, Illig T, Klopp N, Meisinger C, Wichmann HE, Koenig W, Zgaga L, Zemunik T, Kolcic I, Minelli C, Hu FB, Johansson A, Igl W, Zaboli G, DISCLOSURES Wild SH, Wright AF, Campbell H, Ellinghaus D, Schreiber S, Aulchenko YS, Felix JF, Rivadeneira F, Uitterlinden AG, Hofman A, Imboden M, None. Nitsch D, Brandstätter A, Kollerits B, Kedenko L, Mägi R, Stumvoll M, Kovacs P, Boban M, Campbell S, Endlich K, Völzke H, Kroemer HK, Nauck M, Völker U, Polasek O, Vitart V, Badola S, Parker AN, Ridker PM, REFERENCES Kardia SL, Blankenberg S, Liu Y, Curhan GC, Franke A, Rochat T, Paulweber B, Prokopenko I, Wang W, Gudnason V, Shuldiner AR, Coresh J, Schmidt R, Ferrucci L, Shlipak MG, van Duijn CM, Borecki I, 1. 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1882 Journal of the American Society of Nephrology J Am Soc Nephrol 25: 1869–1882, 2014 JASN-2013-07-0781.R2

Supplementary materials

Common Variants in UMOD are associated with Urinary Uromodulin Levels:

a Meta-Analysis of 10,884 Individuals from 6 cohorts

Matthias Olden* 1,2, Tanguy Corre* 3,4, Caroline Hayward* 5, Daniela Toniolo6,17, Sheila Ulivi7 , Paolo Gasparini8, Giorgio Pistis6, Shih-Jen Hwang1, Sven Bergmann3,4, Harry Campbell12, Massimiliano Cocca6, Ilaria Gandin8, Giorgia Girotto8, Bob Glaudemans9, Nicholas D Hastie5, Johannes Loffing10, Ozren Polasek11, Luca Rampoldi12, Igor Rudan13, Cinzia Sala6, Michela Traglia6, Peter Vollenweider14, Dragana Vuckovic8, Sonia Youhanna9, Julien Weber9, Alan F Wright5, Zoltán Kutalik#3,4,15, Murielle Bochud# 15, Caroline S. Fox# 1,16, Olivier Devuyst# 9.

* MO, TC and CH jointly contributed. # ZK, MB, CSF and OD jointly oversaw the work.

Supplementary Tables 1 to 5

Supplementary Table 1 . Mouse primers used in real-time RT-PCR analyses.

Gene (Protein) Forward primer (5’-3’) Reverse primer (5’-3’) PCR Efficiency

Product (bp) Slc38a3 (SNAT3) GTT ATC TTC GCC CCC AAC AT TGG GCA TGA TTC GGA AGT AG 109 0.97 ± 0.03

Slc5a2 (SGLT2) TTG GGC ATC ACC ATG ATT TA GCT CCC AGG TAT TTG TCG AA 164 1.02 ± 0.03

Nphs2 (Podocin) GTC TAG CCC ATG TGT CCA AA CCA CTT TGA TGC CCC AAA TA 162 1.03 ± 0.03

Slc12a3 (NCC) TTT TCT GGA CCA CCA TCT CC GTG AAG TTC CAG CCA TAG CC 148 1.01 ± 0.02

Aqp2 (AQP2) TGA GCC TCA AGA AGG GTC TC TCT CCA GAG CTC TCC GTC TC 142 0.98 ± 0.03

Umod (UMOD) TTG CGA AGA ATG CAG GGT AG TGG CAC TTT CTG AGG GAC AT 156 1.01 ± 0.02 Kcnj1 (ROMK) CCG TGT TCA TCA CAG CCT TCT T CCG TAA CCT ATG GTC ACT TGG G 190 1.03 ± 0.03

Sorl1 (SORL1) CAG CCT ATC CAG GTG TAT GGA CTA ATG CCA CGA TCA CGT TG 100 0.99 ± 0.02 Cab39 (CAB39) GTG AGC TGC TGT TGG ACA GA CCA CAA ACA CCT TGA ACA CG 146 1.01 ± 0.03

Gapdh (GAPDH) TGC ACC ACC AAC TGC TTA GC GGA TGC AGG GAT GGG GGA GA 176 1.04 ± 0.03

The primers were designed using Beacon Design 2.0 (Premier Biosoft International, Palo Alto, CA).

Supplementary Table 2: Uromodulin assay and urine samples processing.

No of No of Interval Lower unindexed Upper unindexed Temperature Mode of between Assay name, detection uromodulin detection uromodulin at which the Collection of No thawing Study urine collection specifications limit below lower limit above upper samples were samples before assay (N) collection and freezing (ng/mL) detection (ng/mL) detection stored (oC) (Minutes) limit limit (N)

In-house Morning UMOD 3.9 0 500 0 60-90 -80°C 2003 to 2006 None COLAUS urine sample ELISA

In-house Morning CROATIA- UMOD 3.9 0 500 0 ~30 -70C 2008 to 2010 None Korcula urine sample ELISA

In-house Morning CROATIA- UMOD 3.9 0 500 0 ~30 -70C 2008 to 2010 None Split urine sample ELISA

Rule Based Morning Medicine 6.2 61 135 3 120 -80°C 1995 to 1998 Once FHS urine sample multiplex array

In-house Morning INGI- UMOD 3.9 0 500 0 ~5 -20°C 2005 Twice Carlantino urine sample ELISA

In-house -20°C for 1-4 Morning INGI-Val UMOD 3.9 0 500 0 ~120 weeks; then - 2005 to 2008 None Borbera urine sample ELISA 80°C

Supplementary Table 3: Genotyping and imputation platforms.

Imputation # SNPs Backbone for Filtering of Data management Genotype QC filters for genotyped SNPs Study Array type used for Imputation phased CEU imputed and calling used for imputation imputation haplotypes genotypes statistical analysis (NCBI build)

HapMap release 21 COLAUS Affymetrix 500K BRLMM call rate <70%, MAF <1%, pHWE <1E-7 390631 IMPUTE v0.2.0 none Matlab (build 35)

phased CEU HWEp<1e-6, callrate<98%, Croatia-KORCULA Illumina 370CNV GenomeStudio 307625 MACH v1.0.16 haplotypes, HapMap none GenABEL, ProbABEL MAF<0.01 release 22 (build 36 phased CEU MACH Croatia-SPLIT Illumina 370CNV GenomeStudio HWEp<1e-6, callrate<98%, MAF<0.01 321456 haplotypes, HapMap none GenABEL, ProbABEL v1.0.16 release 22 (build 36 HWEp<1e-6, callrate<97%, Mishapp<1e-9, MAF<0.01, Affymetrix 500K mapping phased CEU Mendelian MACH R, SAS FHS array and Affymetrix 50K Affymetrix 378,163 haplotypes, HapMap none errors>100, SNPs not v1.0.15 supplemental array release 22 (build 36) in Hapmap or with strand issues when merging with Hapmap

phased CEU R INGI- Carlantino Illumina HH 370 CNV Bead Studio HWEp<1e-6, callrate<95%, MAF<0.01, Mach1 285,569 haplotypes, HapMap RSQ < 0.3 GenABEL/ProbABEL

release 22 (build 36)

Sample Call Rate >95%, SNP Call R, Illumina SNP array 370K - 324,319 MACH MAF < 0.01, INGI-Val Borbera BeadStudio Rate >90%, HWE P-value >10-4, release 22 (build 36) GenABEL/ProbABEL HumanCNV370-Quadv3 v1.0.15 r^2 < 0.3 MAF>0.01%,

Supplementary Table 4: Top loci with p-values up to 1e-7 associated with raw (unindexed to urinary creatinine) urinary uromodulin in the overall population.

Median Refe- Imputation Non- rence eGFRcrea direction Reference Quality eGFRcrea p- R2 to top SNP SNP ID Chr reference Effect size p-value * allele Nearest Gene relative to reference allele Across value rs12917707 allele freque allele Participating ncy Studies rs12917707 16 T G -0.29 1.7E-56 0.1786 UMOD 0.94 + 1.20E-20 same snp rs12446492 16 A T -0.12 7.0E-17 0.4487 PDILT 0.72 + 6.90E-07 0.16 rs4238595 16 T C -0.11 1.7E-09 0.2906 UMOD 0.90 + 0.15 0.05 rs11641045 16 A G -0.13 3.6E-10 0.8793 UMOD 0.99 + 0.00048 0.03

* P-value from inverse-variance meta-analysis, corrected for genomic control. Sample size weighted meta-analysis yielded similar results.

Note: Genes nearby were based on RefSeq genes (build 36). The gene closest to the SNP is listed first and is marked bold if the SNP is located within the gene. Effects and p-values from inverse-variance meta-analysis were consistent with the sample-size weighted results.

Supplementary Table 5: VEGAS analysis for loci associated with urinary uromodulin indexed to urinary creatinine in the overall population.

Chr Nearest Gene nSNPs Simulations Start position Stop position Test statistic VEGAS Pvalue SNP SNP-pvalue EGFR Pvalue 16 UMOD 120 1.00E+06 20251873 20271538 6827.690262 0 rs12917707 7.85E-73 1.2E-20 16 PDILT 127 1.00E+06 20277992 20323534 6852.990812 0 rs12917707 7.85E-73 1.2E-20 16 GP2 129 1.00E+06 20229311 20246336 5482.984445 0 rs12917707 7.85E-73 1.2E-20 16 ACSM5 110 1.00E+06 20328356 20359782 2827.046129 0 rs11864909 1.02E-41 1.0E-12 8 C8orf76 66 1.00E+06 124301411 124322798 558.5728862 8.00E-06 rs4377946 9.46E-07 0.58 1 TDRD5 212 1.00E+06 177827647 177927021 1138.352998 0.000118 rs12039153 1.54E-05 0.58 8 FAM83A 112 1.00E+06 124263932 124291499 713.532712 0.000165 rs4377946 9.46E-07 0.58 6 SESN1 135 1.00E+06 109414337 109521970 1138.513874 0.000171 rs3734649 8.63E-05 0.93 8 WDR67 203 1.00E+06 124154100 124233571 1007.030324 0.000178 rs4377946 9.46E-07 0.25 20 STK35 203 1.00E+06 2031518 2077198 928.9085244 0.00047 rs6046998 0.0001195 0.19 2 DUSP11 48 1.00E+06 73842836 73860730 460.5605177 0.000671 rs10203740 0.0004001 0.72 1 ATAD3C 6 1.00E+06 1374931 1395401 48.45550841 0.000687 rs6690515 0.0001865 0.00029 1 ATAD3B 15 1.00E+06 1397026 1421445 105.386289 0.000835 rs6690515 0.0001865 0.00029 2 FLJ43987 51 1.00E+06 73864823 73897782 276.6775208 0.000854 rs10203740 0.0004001 0.00029

The table is sorted by SNP and VEGAS P-value, with significant results highlighted in bold.

Results shown for sample size weighted meta-analysis results. Inverse-variance meta-analysis yielded similar results.