Crep2 assay

Plasma DPP4 activity was measured by detecting p-nitroaniline (pNa) resolved from GP-pNa (Bachem L1880, Swiss), a DPP4 substrate^{43 (link)}. In brief, 5 µl blood samples were added to 150 µl of 50 mM tris-HCL containing 1 mM GP-pNa, and then the spectrophotometer at 405 nm was read immediately and 1 hour later. The activity is expressed as U/L, indicating that 1 mmol pNa is produced per minute at 37 °C. Biochemical measurements, including those of plasma glucose (GLUC3 Assay, Roche, Germany), cholesterol (CREP2/CHOL2/TRIGL/LDL_C/HDLC3 Assay, Roche, Germany), the MB isoenzyme of creatine kinase (CK-MB) (Elecsys CK-MB Assay, Roche, Germany), cardiac troponin T (cTNT) (Elecsys Troponin T hs Assay, Roche, Germany), NT-proBNP (Elecsys proBNP II Assay, Roche, Germany) and creatinine (CREP2 Assay, Roche, Germany) were obtained using standard clinical analytical methods (Cobas Roche, Germany). For plasma glucose, creatinine and lipid profile measurements we used the Cobas c701 platform (Roche, Swiss). For myocardial injury markers (CK-MB, cTnT and NT-proBNP) measurements we used the Cobas e602 platform (Roche, Swiss). The normal range of the NT-proBNP test was <150 pg/mL.

Rats were single-housed in metabolic cages (Techniplast) with free access to powdered chow and water. The excreted urine was collected for 16 h. Urine creatinine was quantified using a CREP2 assay (Roche Diagnostics) on the Cobas C-501 autoanalyzer as per manufacturer’s instructions. Urine albumin was measured using a rat albumin ELISA (Bethyl Laboratories). Urine neutrophil gelatinase-associated lipocalin (NGAL), soluble tumor necrosis factor receptors (sTNFR) I and II were measured using ELISA assays (R&D Systems), while urine nephrin and podocalyxin were quantified using ELISA assays from LSBio and CusaBio, respectively. All ELISA assays were run as per manufacturers’ instructions and levels of analytes were reported as the analyte-to-creatinine ratio (ACR) in urine. Urine ACR values were log10-transformed before statistical analyses.