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Serum Uromodulin and Mortality Risk in Patients Undergoing Coronary Angiography Graciela E. Delgado,* Marcus E. Kleber,*†‡ Hubert Scharnagl,§ Bernhard K. Krämer,* Winfried März,*§| and Jürgen E. Scherberich¶ *Vth Department of Medicine (Nephrology, Hypertensiology, Rheumatology, Endocrinology, Diabetology), Medical Faculty Mannheim, University of Heidelberg, Mannheim, Germany; †Competence Cluster of Nutrition and Cardiovascular Health, Halle-Jena-Leipzig, Leipzig, Germany; ‡Department of Nutritional Biochemistry and Physiology, Institute of Nutrition, Friedrich Schiller University Jena, Jena, Germany; §Clinical Institute of Medical and Chemical Laboratory Diagnostics, Medical University of Graz, Graz, Austria; |Synlab Academy, Synlab Holding Deutschland GmbH, Mannheim, Germany; and ¶Department of Nephrology and Clinical Immunology, Klinikum Muenchen-Harlaching, Teaching Hospital of the Ludwig-Maximilians University, Munich, Germany
ABSTRACT The mucoprotein uromodulin is the most abundant protein in mammalian urine and has important roles in ion transport, maintenance of water and electrolyte balance, and clearance of bacteria from the urinary tract. Low urinary uromodulin concentrations have been associated with increased mortality risk. However, measuring uromodulin in urine has several preanalytic drawbacks, and sensitive assays for the detection of uromodulin in blood have become available. In this study, we investigated the association of serum uromodulin concentration with cardiovascular biomarkers and mortality risk in a large cohort of patients referred for coronary angiography. Uromodulin concentrations were available in 3057 of 3316 participants of the Ludwigshafen Risk and Cardiovascular Health Study. Higher serum uromodulin concentration associated with a favorable metabolic profile, lower prevalence rates of comorbidities (arterial hypertension, diabetes mellitus, and heart failure), and a lower risk for 10-year mortality, with hazard ratios (95% confidence intervals) of 0.65 (0.54 to 0.78), 0.71 (0.58 to 0.88), and 0.57 (0.45 to 0.73) in the second, third, and fourth quartiles, respectively, compared with the first quartile. The association with reduced mortality was independent of other cardiovascular risk factors, including eGFR, and stronger after adjustment for the genotype of the rs12917707 polymorphism at the UMOD locus. Adding serum uromodulin concentration to established cardiovascular risk prediction scores improved risk prediction. Uromodulin may, therefore, be a useful marker for cardiovascular and renal health. J Am Soc Nephrol 28: 2201–2210, 2017. doi: https://doi.org/10.1681/ASN.2016111162
Uromodulin was isolated in 1950 as a mucoprotein from human urine that inhibits the agglutination of viruses1 and later, named according to its discoverers Tamm and Horsfall. Uromodulin is synthesized on the rough ER of epithelial cells of the thick ascending limb of the loop of Henle. After transport to the apical plasma membrane, it is cleaved and released into the tubular fluid, where it constitutes the most abundant protein present in the urine of humans. Extracellularly, it polymerizes into a high molecular weight polymer resembling a three-dimensional matrix with pores.2 The filamentous gel–like structures made up by uromodulin serve as a physical barrier to water permeation.3 It also has important roles in ion J Am Soc Nephrol 28: 2201–2210, 2017
transport, maintenance of water and electrolyte balance,4 and clearing bacteria from the urinary tract (e.g., by direct binding to type-1 fimbriated Escherichia coli).5 Received November 2, 2016. Accepted January 23, 2017. G.E.D. and M.E.K. contributed equally to this work. Published online ahead of print. Publication date available at www.jasn.org. Correspondence: Dr. Marcus E. Kleber, Vth Department of Medicine (Nephrology, Hypertensiology, Endocrinology, Diabetology, Rheumatology), Medical Faculty of Mannheim, University of Heidelberg, Theodor-Kutzer-Ufer 1-3, 68167 Mannheim, Germany. Email:
[email protected] Copyright © 2017 by the American Society of Nephrology
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Animal studies with uromodulin knockout mice showed an increased susceptibility to urinary tract infections.6 In addition, uromodulin seems to play a key role in innate immunity of the kidney. Uromodulin is able to activate myeloid dendritic cells via Toll-like receptor 4, and TLR4 knockout mice have been reported to be severely impaired in the uromodulin-specific humoral immune response.7 An interaction of the uromodulin protein with lymphotoxin-a has been inferred from interaction databases. Lymphotoxin-a mediates a large variety of inflammatory, immunostimulatory, and antiviral responses. Furthermore, lower urinary uromodulin has been linked to increased all-cause mortality in an observational study of risk factors for cardiovascular disease8 and increased cardiovascular mortality in patients with type 1 diabetes.9 Recently, genetic and genome-wide association studies linking polymorphisms in the UMOD gene located on 16p12.3 with eGFR10 and increased risk of CKD,10–12 hypertension,13 and diabetic nephropathy14 renewed the interest in uromodulin. Although most studies of uromodulin have been conducted using urinary uromodulin, new and more sensitive assays measuring uromodulin in blood have become available. Apart from the intracellular route of uromodulin to the apical cell pole, a second pathway to the basolateral cell site was recently identified, in which the protein is released from the endoplasmic reticulum via the Golgi apparatus and transferred by cytoplasmic vesicles to the contraluminal plasma membrane. From there, it is then shed into the renal interstitium and blood compartment.15 An association with low concentrations of serum or plasma uromodulin with increased risk of CKD has been reported previously.16,17 The aim of our study was to analyze the association of serum uromodulin with all-cause and cardiovascular mortality in a large and well characterized cohort of patients who had been referred for coronary angiography and investigate its potential predictive value in addition to established cardiovascular risk scores.
with eGFR. Mean serum uromodulin concentration was slightly higher in women compared with men (Supplemental Figure 1). Regarding prevalent disease, participants with higher uromodulin were less likely to suffer from coronary artery disease (CAD), diabetes mellitus, and hypertension. Accordingly, participants in higher quartiles of uromodulin were less likely to use antihypertensive medication. We also analyzed the association of uromodulin with different immune cell subtypes. Although there was no association with natural killer cells and monocytes, we found higher percentages of lymphocytes and lower percentages of neutrophils and eosinophils with increasing serum uromodulin concentration (Supplemental Table 1).
RESULTS
Serum Uromodulin and Mortality
Serum Uromodulin and Anthropometric Characteristics
Uromodulin was available in 3057 of 3316 participants of the Ludwigshafen Risk and Cardiovascular Health (LURIC) Study. We split our cohort into quartiles of uromodulin and investigated the association with anthropometric data and biomarkers by ANOVA and chi-squared test (Table 1). Study participants with higher serum uromodulin were younger, had a lower body mass index (BMI), and had lower blood pressure (BP) as well as had lower triglycerides, fasting glucose, hemoglobin A1c, highsensitivity C-reactive protein (hsCRP), parathyroid hormone, galectin-3, and N-terminal pro–B-type natriuretic peptide (NT-proBNP). LDL cholesterol, HDL cholesterol, 25OH vitamin D, and 1,25(OH)2 vitamin D were higher in patients with higher uromodulin. Uromodulin was inversely associated with serum creatinine, cystatin C, and b-trace protein and directly correlated 2202
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Correlation of Serum Uromodulin with Markers of Kidney Function
Uromodulin showed a strong direct correlation with eGFR at lower concentrations that flattens in the higher ranges (i.e., in people with normal kidney function) (Figure 1). Serum concentrations of uromodulin were directly correlated with LDL cholesterol and HDL cholesterol and inversely correlated with age, BMI, systolic BP, triglycerides, glucose, hemoglobin A1c, hsCRP, fibrinogen, galectin-3, uric acid, and NT-proBNP (Table 2). After adjustment for age, sex, and eGFR, the correlation coefficients were attenuated but still nominally significant. For urinary uromodulin, meta-analyses have identified a genetic variant in the UMOD promotor rs12917707, the minor allele of which is associated with lower uromodulin but higher eGFR.12 Using genotypic data of 2984 participants in the LURIC Study that had been imputed to the HapMap2 reference panel, we performed a genome-wide association study with serum uromodulin as phenotype, identified the same variant as significantly associated with the serum concentration (Supplemental Figures 2 and 3), and replicated the association of the T allele of rs12917707 with higher eGFR (Figure 1, Table 3). Genetic data were only available for 2826 study participants, limiting our sample size for all analyses involving rs12917707.
We examined the association of uromodulin with all-cause and cause-specific mortality by means of Cox regression. Modeling uromodulin as restricted cubic spline, we plotted the predicted hazard against the uromodulin concentration (Figure 2). The risk decreased strongly up to a uromodulin concentration of about 200 ng/dl, after which the slope of the curve flattened. Sex-stratified analysis revealed a more linear relationship in women (Supplemental Figure 4). Overall, the rs12917707 variant was not significantly associated with mortality, but we found evidence for a possible protective effect of the minor uromodulinlowering allele in study participants younger than 67 years old (now the regular retirement age in Germany) with hazard ratios (HRs) of 0.74 (95% confidence interval [95% CI], 0.58 to 0.94) and 0.71 (95% CI, 0.37 to 1.39) for study participants heterozygous or homozygous for the T allele, respectively. Because this stands in contrast to the association of low serum uromodulin J Am Soc Nephrol 28: 2201–2210, 2017
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Table 1. Anthropometric and biochemical characteristics of study participants shown as mean6SD or median (25th–75th percentile) according to quartiles of serum uromodulin Characteristic N Age, yr Sex, % men BMI, kg/m2 Systolic BP, mmHg Diastolic BP, mmHg LDL-C, mg/dl HDL-C, mg/dl TG, mg/dl Fasting glucose, mg/dl HbA1c, % HOMA-IR hsCRP, mg/dl Fibrinogen, mg/dl Galectin-3, mg/L NT-proBNP, pg/ml Renin, pg/ml Aldosterone, ng/L Uric acid, mg/dl PTH, pg/ml 25OH vitamin D , mg/L 1,25(OH)2 vitamin D, ng/L b-Trace protein, mg/L Creatinine, mg/dl Cystatin C, mg/L eGFR, ml/min per 1.73 m2 CAD, % Heart failure, % Diabetes mellitus, % Hypertension, % Smoking (active/ex/never), % ACE inhibitors, % Diuretics, % Calcium antagonists, % AT2 receptor blocker, % b-Blocker, % Lipid-lowering therapy, %
Uromodulin, ng/ml
