Heart and Soul Description
The Heart and Soul Study is an observational study originally designed to investigate the influence of psychosocial factors on the progression of CAD. Methods have been described previously. Briefly, participants were recruited from outpatient clinics in the San Francisco Bay area if they met one of the following inclusion criteria: history of myocardial infarction, angiographic evidence of >50% stenosis in ≥1 coronary vessels, evidence of exercise-induced ischemia by treadmill or nuclear testing, history of coronary revascularization, or documented diagnosis of CAD by an internist or cardiologist. Participants were excluded if they were not able to walk 1 block, had experienced myocardial infarction within the past 6 months, or were likely to move out of the area within 3 years. The study protocol was approved by the Institutional Review Boards of participating institutions, and all participants provided written informed consent. Between September 2000 and December 2002, 1024 participants enrolled and underwent a day-long baseline study appointment that included a medical history, physical examination, and comprehensive health status questionnaire. Of these, 982 participants provided DNA samples, and 879 of these provided 24-hour timed urine collections. After 5 years of follow-up, all surviving participants were invited to return for a repeat examination. Of the original 1024 enrollees, 195 had died before the 5-year examination. Between September 2005 and December 2007, 667 (80%) of the surviving 829 participants completed the 5-year follow-up examination.
The initial step of this study was to genotype the relevant UMOD sNP (rs12917707) identified from the CHARGE analysis across all Heart and Soul participants with available stored DNA and 24-hour urine samples (N = 879). In study 1, we evaluated the cross-sectional association of genotype with baseline creatinine clearance among all persons with available urine samples. In study 2, we evaluated whether the genotype at sNP (rs12917707) was associated with urine concentrations of uromodulin, as previously described in FHS and ARIC. In this analysis, we measured uromodulin concentrations among all participants who were homozygous for the minor allele (T/T, n = 24) and among 48 participants each who were heterozygous (G/T) or homozygous for the dominant allele (G/G). In study 3, we used a nested case-control design to determine whether urine uromodulin levels were associated with incidence of CKD. We identified cases defined by an initial measured creatinine clearance (CrCl) of > 70 ml/min and CrCl < 60 ml/min after 5 years of follow-up. For each case participant, a control was selected who was matched on age, sex, and race. We chose not to match on urine albumin excretion, so that we could compare it with urine uromodulin as a predictor of incident CKD. Urine uromodulin levels were then measured from baseline stored specimens with the technician blinded to case and control status. Stored urine specimens were available from 102 of the cases and 94 controls.
Of the 1024 participants, 982 individuals provided DNA for analysis. The SNP marker for rs12917707 was genotyped using TaqMan® SNP Genotyping Assays (Applied Biosystems, Foster City, CA, http://www.appliedbiosystems.com) functionally tested by Applied Biosystems and available on demand. TaqMan® PCR reactions were done with Universal Master Mix Amperase® UNG, 0.083 uL Taqman 40× probe mix and 1.417 uL of water, 1 ul of DNA normalized to 10 ng/ul, for a 5 uL total volume. The PCR conditions for the TaqMan® SNP Genotype Assays were: one enzyme activation step at 95.0°C for ten minutes, and 50 alternating cycles of denaturation at 95.0°C for 15 seconds and reannealing and extension at 60.0°C for one minute. All PCR reactions and allelic discrimination reactions were performed on an ABI 7900HT Real-Time PCR System (Applied Biosystems, Foster City, CA) and analyzed using SDS 2.3 software (Applied Biosystems, Foster City, CA).
Kidney Function Methods
Creatinine clearance (CrCl) was measured by a 24-hour urine collection in all participants. At the intake appointment, participants were provided with a 3-L collection jug and instructed to save all urine between the end of their intake appointment and the time when a researcher recovered the urine. Participants were instructed to keep the urine collections refrigerated at all times. Research personnel arrived at the participants' home 24 h after their inception appointments to ensure accurately timed specimens. At that time, participants were asked about the time of their first and last voids. When more than 1 h had passed since their last void, participants were instructed to void at that time to complete the collection. All participants were asked whether they were able to collect all urine or whether some fraction had been inadvertently discarded. When the sample was reported to be incomplete, participants were asked to repeat the collection, and research personnel returned 24 h later to re-collect the urine. When the 24-h urine volume was <1 L, participants were asked to repeat the collection to ensure an adequately collected specimen. Similarly, when the 3-L collection jug was completely full, participants were given two new jugs and asked to repeat the collection to ensure that no urine was inadvertently discarded. When participants were unable to collect all urine for any reason or had urinary incontinence, their samples were deemed inadequate and no data were recorded for these participants. CrCl was calculated using the following formula: urine creatinine (mg/dl) * 24-h urine volume (dl)/serum creatinine (mg/dl) * 1440 (min/d). This procedure was repeated at the 5-year follow-up visit. Baseline kidney function was also estimated by GFR equations using either creatinine or cystatin C.
Uromodulin Measurement Methods
Uromodulin was measured by the method of Lau et al with slight modifications. 96-well microtiter plates were coated with 100 μl of 10 μg/ml lectin WGA overnight at 4°C. Plates were washed and blocked with 200 μl blocking buffer (3% BSA in PBS) for 2 hours at room temperature, then washed again and allowed to dry at 37°C for 3 hours. After cooling to room temperature, plates were sealed and stored at 4°C. Urine samples and standards (Biomedical Techologies, Stoughton, MA) were diluted in TEA buffer (0.5% triton X-100, 20 mM EDTA, pH 7.5) and added to wells in duplicate. After one hour incubation at 37°C, wells are washed and anti-uromodulin antibody (Biomedical Techologies, Stoughton, MA) was added. After one hour incubation at 37°C, wells were washed and goat anti-rabbit IgG horseradish perxiodase (Bio-Rad, Hercules, CA) was added. After 1 hour incubation at 37°C, wells are washed and color was developed by the addition of TMB substrate solution and incubation at room temperature for 15 minutes. The reaction is stopped by adding 2N H2SO4, followed by reading immediately at OD450 and OD620. Urinary uromodulin concentration was determined by referring to standard curve.
The analysis began with Study 1, the comparison of UMOD genotype with baseline characteristics among the 879 participants with available DNA. These comparisons were stratified by race- Whites and non-Whites. Characteristics included demographics (age, sex, race), body mass index, prevalent hypertension and diabetes, and the urine albumin to creatinine ratio (ACR). Unadjusted comparisons were made by the Kruskall-Wallis test, Chi-square, or Fisher's exact test. We next compared baseline kidney function across genotype categories, using measured CrCl and estimated GFR by cystatin C and creatinine, separately. Because the T/T genotype group appeared to differ compared with the G/G and G/T groups, we also compared T/T versus all others using non-parametric statistics.
Our next step, Study 2, was to compare urine uromodulin concentrations by genotype using unadjusted and adjusted linear regression with log-transformed urine uromodulin as the outcome (N = 120). These analyses were repeated with restriction to Whites only. For Study 3, we compared uromodulin levels between incident CKD cases and controls (N = 196). The median uromodulin concentration, uromodulin-to-creatinine ratio (UCR), and total daily uromodulin were compared by descriptive statistics. We categorized both uromodulin and UCR into quartiles defined using cutpoints from the control group; the distribution of cases and controls across quartiles was evaluated using the Chi-square statistic. Then, we evaluated uromodulin, UCR levels, and total uromodulin as continuous variable predictors (log-transformed per SD) of case-control status using multivariate conditional logistic regression, adjusted for demographic characteristics, body mass index, hypertension, and diabetes.