The RIACE Italian Multicentre Study is an observational, prospective, cohort study on the impact of estimated glomerular filtration rate (eGFR) on morbidity and mortality in individuals with type 2 diabetes [21, 27].
The study population included 15,773 Caucasian patients (after excluding 160 individuals with missing or implausible values), consecutively attending 19 hospital-based, tertiary referral Diabetes Clinics of the National Health Service throughout Italy in the years 2006-2008. Exclusion criteria were dialysis or renal transplantation.
The vital status of study participants on 31 October 2015 was verified by interrogating the Italian Health Card database (http://sistemats1.sanita.finanze.it/wps/portal/), which provides updated and reliable information on all current Italian residents .
Baseline data were collected using a standardized protocol across participating centres [21, 27].
Participants underwent a structured interview in order to collect the following information: age at the time of the interview, smoking status, known diabetes duration, co-morbidities, and current glucose-, lipid-, and blood pressure (BP)-lowering treatments.
Body mass index (BMI) was calculated from weight and height, whereas waist circumference was estimated from log-transformed BMI values; BP was measured with a sphygmomanometer with the patients seated with the arm at the heart level.
Haemoglobin A1c (HbA1c) was measured by HPLC using DCCT-aligned methods; triglycerides and total and HDL cholesterol were determined in fasting blood samples by colorimetric enzymatic methods. The triglyceride:HDL cholesterol ratio (TG:HDL) was then calculated by dividing triglyceride for HDL cholesterol levels (both in mg/dl) and LDL cholesterol concentration was estimated using the Friedewald formula.
The presence of diabetic kidney disease (DKD) was assessed by measuring albuminuria and serum creatinine, as previously detailed [23, 29]. Albumin excretion rate was obtained from 24-hour urine collections or calculated from albumin-to-creatinine ratio in early-morning, first-voided urine samples, using a conversion formula developed in patients with type 1 diabetes and preliminarily validated in a subgroup of RIACE participants. Albuminuria was measured in fresh urine samples by immunonephelometry or immunoturbidimetry, in the absence of interfering clinical conditions. One-to-three measurements for each patient were obtained; in cases of multiple measurements, the geometric mean of 2-3 values was used for analysis. In individuals with multiple measurements, the concordance rate between the first value and the geometric mean was >90% for all albuminuria categories . Serum (and urine) creatinine was measured by the modified Jaffe method, traceable to IDMS, and eGFR was calculated using the Chronic Kidney Disease Epidemiology Collaboration equation . Patients were then classified into Kidney Disease: Improving Global Outcomes categories of albuminuria (A1 to A3) and eGFR (G1 to G5) and assigned to one of the following DKD phenotypes: no DKD (i.e., A1G1-A1G2), albuminuria alone (albuminuric DKD with preserved eGFR, i.e., A2G1-A2G2-A3G1-A3G2), reduced eGFR alone (non-albuminuric DKD, i.e., A1G3-A1G4-A1G5), or both albuminuria and reduced eGFR (albuminuric DKD with reduced eGFR, i.e., A2G3-A2G4-A2G5-A3G3-A3G4-A3G5), as previously reported .
In each centre, the presence of diabetic retinopathy (DR) was assessed by an expert ophthalmologist by dilated fundoscopy . Patients with mild or moderate non-proliferative DR were classified as having non-advanced DR, whereas those with severe non-proliferative DR, proliferative DR, or maculopathy were grouped into the advanced, sight threatening DR category. DR grade was assigned based on the worse eye.
Previous major acute CVD events, including myocardial infarction; stroke; foot ulcer/gangrene/amputation; and coronary, carotid, and lower limb revascularization, were adjudicated based on hospital discharge records by an ad hoc committee in each centre .
For the purpose of the current analysis, the whole RIACE cohort and men and women separately were divided into quartiles of triglycerides, HDL cholesterol, and TG:HDL.
Data are expressed as mean±SD or median (interquartile range) for continuous variables, and number of cases and percentage for categorical variables. Comparisons among quartiles were performed by one-way ANOVA or Kruskal–Wallis test, according to the parametric or non-parametric distribution of continuous variables, followed by Bonferroni correction or Mann-Whitney test, respectively, for post-hoc comparisons, and by Pearson’s χ2 test for categorical variables.
Crude mortality rates were described as events per 1,000 patient-years, with 95% exact Poisson confidence intervals (CIs) and adjusted for age and gender by a Poisson regression model. Kaplan-Meier survival probabilities for all-cause mortality were estimated according to the above categorizations and differences were analysed using the log-rank statistic. The hazard ratios (HRs) and their 95% CIs were estimated by Cox proportional hazards regression, using the lowest triglyceride, the highest HDL cholesterol or the lowest triglyceride TG:HDL quartile as reference. These analyses were adjusted for age and gender (model 1), plus CVD risk factors, i.e., smoking, diabetes duration, HbA1c, BMI, total cholesterol (or, in alternative, LDL or non-HDL cholesterol), and systolic and diastolic BP, and treatments, i.e., anti-hyperglycaemic, lipid-lowering, and anti-hypertensive therapy (model 2), plus presence of complications, i.e., DKD phenotypes, DR grade and any CVD, and/or severe comorbidity(ies), i.e., chronic obstructive pulmonary disease, chronic liver disease and/or cancer (model 3), and, for triglycerides and HDL cholesterol quartiles only, plus HDL cholesterol and triglyceride levels, respectively, as continuous variables (model 4). Covariates were selected a priori, as all of them potentially affect mortality. Appropriate tests were applied for assessing the interaction between gender and quartiles of triglycerides, HDL cholesterol, and TG:HDL and the analyses were then replicated separately for men and women. Finally, all the above analyses were rerun after substituting LDL cholesterol for total cholesterol and the association between HDL cholesterol and mortality was evaluated also among individuals falling in the lowest quartile of LDL cholesterol.
All p values were two-sided, and a p<0.05 was considered statistically significant. Statistical analyses were performed using SPSS version 13.0 (SPSS Inc., Chicago, IL, USA).