Study design and sample
The DHS cohort includes 1,220 self-described EA individuals from 475 families. Briefly, the DHS recruited siblings concordant for T2D without advanced renal insufficiency, manifesting as serum creatinine concentration >2 mg/dL, or end-stage renal disease. When possible, one non-T2D affected sibling was also recruited. T2D was clinically defined as diabetes developing after the age of 35 years and actively treated with insulin and/or oral agents, in the absence of historical evidence of ketoacidosis. Diagnoses were confirmed by baseline measurement of fasting blood glucose and glycosylated hemoglobin (HbA1C). Full ascertainment and recruitment criteria have been previously described in detail [19, 20].
Study protocols were approved by the Institutional Review Board at Wake Forest School of Medicine, and all participants provided written informed consent prior to participation. Participant examinations were conducted in the Clinical Research Unit of Wake Forest Baptist Medical Center, and included interviews for medical history (including self-reported history of prior CVD events or intervention) and health behaviors, anthropometric measures, resting blood pressure, electrocardiography, fasting blood sampling for laboratory analyses including fasting glucose, HbA1C, lipids, serum albumin and creatinine concentration. Estimated GFR was calculated using the 4-variable Modification of Diet in Renal Disease (MDRD) equation . A spot urine collection was obtained for determination of urine albumin: creatinine ratio (UACR).
Coronary artery calcified plaque (CAC) was measured using fast-gated helical CT scanners, with calcium scores calculated as previously described [22, 23]. CAC is widely accepted as reflecting the burden of subclinical CVD.
Vital status was determined for all participants from the National Social Security Death Index maintained by the United States Social Security Administration. For participants confirmed as deceased, length of follow-up was determined from the date of the initial study visit to date of death. For deceased participants, copies of death certificates were obtained from relevant county Vital Records Offices to confirm cause of death. For all other participants the length of follow-up was determined from the date of the initial study visit to the end of 2011. Cause of death was categorized based on information contained in death certificates as CVD-related (myocardial infarction, congestive heart failure, cardiac arrhythmia, sudden cardiac death, peripheral vascular disease, and stroke), cancer, infection, end-stage renal disease, accidental, or other (including obstructive pulmonary disease, pulmonary fibrosis, liver failure and Alzheimer’s dementia).
Summary statistics were calculated including means and standard deviations (SD), medians and ranges for continuous variables, and count and percentages for categorical variables. Continuous variables were transformed prior to analysis to approximate normality. To evaluate the association of serum albumin, serum creatinine, UACR and eGFR with all-cause mortality and CVD-mortality a survival analysis was used. Variables reflecting kidney disease and serum albumin were considered as both ordinal (Q1-4 derived from increasing quartile ranges) and continuous variables. In order to compare the relative importance, kidney disease (continuous) variables were standardized for analysis of associations with outcome.
Curves of cumulative incidence of both all-cause mortality and CVD-mortality for increasing quartiles of each of the kidney disease measures were plotted for exploratory analyses (Additional file 1: Figures S1-S4). Due to the inclusion of related individuals in the DHS, Cox proportional hazards models with sandwich-based variance estimation were used to examine the relationships between measures of kidney disease and both all-cause mortality and CVD-mortality. Initially an exploratory test for trend across increasing quartiles of the measures of kidney disease was performed to examine relationships with all-cause and CVD-mortality. Analyses of the simple univariate associations using continuous variables were then performed. Each of these associations was subsequently adjusted for (i) age, sex, T2D affection status and use of angiotensin-converting enzyme (ACE) inhibitor and angiotensin-receptor blocker (ARB) medications (partially adjusted) and (ii) age, sex, T2D affection status, ACE/ARB medication use, body mass index (BMI), current smoking, hypertension, dyslipidemia, and self-reported history of prior CVD (fully adjusted). To assess whether measures of kidney disease predicted mortality independently of subclinical CVD the fully adjusted models were further adjusted for CAC.
Receiver operating characteristic (ROC) curves were computed for models containing traditional CVD risk factors (as used in fully adjusted models above) and with addition of either a measure of kidney disease, CAC, or both kidney disease measures and CAC. The areas under the curves were used to assess the ability of measures of kidney disease to predict all-cause mortality or CVD-mortality after adjusting for traditional CVD risk-factors and CAC. The difference in area under the curve between two models was tested using Delong’s method .
All analyses were performed using SAS version 9.2 (SAS Institute Inc., Cary, NC) with the exception of the ROC analyses which were performed using Stata software, version 12.1 (StataCorp, College Station, TX). Statistical significance was accepted at p<0.05.