Patients diagnosed with type 2 diabetes mellitus (T2DM) based on the criteria of the American Diabetes Association (ADA) were collected from Shaanxi Provincial People’s Hospital (Xi’an, Shaanxi, China) between September 2020 and December 2021. Patients with type 1 diabetes, secondary diabetes, urinary tract infection, urolithiasis, pregnancy, superimposed systemic diseases and other glomerular diseases were excluded. 88 diabetic patients were enrolled in this study in which 22 were T2DM patients (patients with normalbuminuria, urine albumin creatinine ratio (UACR) < 30 mg/g) and 66 were DKD patients. DKD patients were divided into three groups according to UACR and serum creatinine: 1) Patients with microalbuminuria (UACR 30–300 mg/g), n = 23; 2) Patients with macroalbuminuria (UACR > 300 mg/g), n = 22; and 3) Patients with increased serum creatinine (renal dysfunction) (serum creatinine > 120µmol/L), n = 21. Meanwhile, 20 non-diabetic healthy volunteers were enrolled as control group. The present study was approved by the ethical committee for human investigation of Shaanxi Provincial People’s Hospital and was conducted according to the Declaration of Helsinki. Informed consent was obtained from all participants.
Clinical Data Collection
General data were collected as follow: gender, age, body height, body weight, systolic blood pressure (SBP), diastolic blood pressure (DBP) and duration of diabetes on admission. Body mass index (BMI) was calculated using the formula: BMI = body weight/body height2 (kg/m2). The following laboratory parameters were obtained from each patient: glycated hemoglobin A1c (HbA1c), UACR, urine β2-microglobulin (β2-MG), urine α1-microglobulin (α1-MG), serum creatinine (Scr), blood urea nitrogen (BUN), Cystatin C (CysC), neutrophil gelatinase-associated lipocalin(NGAL), uric acid (UA), white blood cell (WBC), hemoglobin (HGB), albumin (ALB), triglyceride (TG), total cholesterol (TC), high density lipoprotein (HDL) and low density lipoprotein (LDL) at the time of enrollment. UACR was calculated using the formula: ACR = urine albumin/creatinine. The eGFR was calculated according to the modified MDRD formula: eGFR = 186 × Scr− 1.154 × Age− 0.203 × gender (1 if male, 0.742 if female).
Peripheral Blood Samples Collection
Peripheral blood was collected by venipuncture with an ethylenediaminetetraacetic acid (EDTA) anticoagulant vacutainer from all patients and stored at − 80°C until analysis. Total RNA was extracted as soon as possible.
Quantitative Real-time Polymerase Chain Reaction
1mL peripheral blood was centrifuged at 3000rpm for 5min and the supernatants were removed. Then 3mL red cell lysis buffer added and mixed before being centrifuged at 3000rpm for 5min. The supernatant was then discarded and the extracted leukocytes were collected. The total RNA of leukocytes was extracted using Trizol reagent (Servicebio, Wuhan, China), and then dissolved in RNase-free water. The concentration of RNA was determined using NanoDrop 2000 (Thermo scientific, Waltham, MA, USA). Extracted RNA was reversibly transcribed into complementary DNA (cDNA) using Servicebio®RT First Strand cDNA Synthesis Kit (Servicebio, Wuhan, China). Quantitative real-time polymerase chain reaction was performed using SYBR Green qPCR Master Mix (Servicebio, Wuhan, China) on a CFX RT-PCR system (Bio-Rad). PCR reaction system was as follows: pre-degenerated at 95°C for 10min, followed by 40 cycles of 95°C for 15s and 60°C for 30s. The expression level of lncRNA ANRIL was normalized to the expression level of GAPDH as a housekeeping gene. The relative quantitative value was expressed by the 2−ΔΔCt method. ANRIL primer sequences were shown as follows: upstream: 5'-AGGGTTCAAGCATCACTGTTAGG-3'; downstream: 5'-GAAACCCCGTCTCTACTGTTACCT-3'.
SPSS software, version 18.0 (SPSS, Inc., Chicago, IL, USA) was used for statistical analysis. Normally distributed data were presented as mean ± S.D. and non-normally distributed data were expressed as median range. Differences between two groups for quantitative data and qualitative data were compared using a t test and chi-square test, respectively. Comparisons of normally distributed data in 3 or more groups were analyzed using one-way ANOVA, while non-normally distributed data were analyzed using nonparametric counterpart Kruskal-Wallis test. Correlations were examined using Pearson’s correlation analysis. Binary regression analysis was used to determine the influence factors of the presence of DKD. The diagnostic value of ANRIL was evaluated by ROC curve analysis. Area under the ROC curve (AUC) was calculated. When AUC = 0.5, diagnostic value was denied. The cut-off value and corresponding sensitivity and specificity were determined according to ROC curve analysis. P < 0.05 was considered to represent significant differences between groups.