Q33120

Plasma glucose levels were measured with a hexokinase glucose-6-phosphate dehydrogenase method by biochemical analyzer (BS-380; Mindray Medical International, Shenzhen, China). Serum total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-c), and low-density lipoprotein cholesterol (LDL-c) were measured enzymatically on an automatic analyzer (Model 7080; Hitachi, Tokyo, Japan) with reagents purchased from Leadman Biochemistry Co. (Beijing, China). Serum creatinine and cystatin C were measured with the use of an automatic biochemical analyzer (Modular DDP, Roche).
A commercially available kit (Q33120, USA, Thermo), which was described in a previous study,5 (link) was used to determine the concentrations of serum cfDNA. All experimental procedures were carried out according to the manufacturer’s instructions. Renal function was measured as eGFR calculated by the Modification of Diet in Renal Disease. We tested two indices of change in eGFR as done in previous studies.6 (link) Progression of DKD was defined as two-continuous decreases in eGFR and changes of UACR from less than 300 mg/g at baseline to higher than 300 mg/g at follow-up.

Faecal samples were extracted on the QIAcube HT system using the DNeasy 96 PowerSoil Pro QIAcube HT Kit (Qiagen 47021, Hilden, Germany), according to the manufacturer’s instructions, with a modified initial processing step (Qiagen 9001793). Mechanical lysis was performed using PowerBead Pro beads (Qiagen 19311). The resulting DNA was quantified using QuantIT, a high-sensitivity dsDNA fluorometric assay (Thermo Fisher, Q33120, Waltham, MA, USA). Samples were required to reach a minimum of 0.2 ng/μL to pass quality control requirements.