E test 2

The serum levels of SP-D and KL-6 were measured using in vitro diagnostic kits, SP-D kit YAMASA EIA II (Yamasa, Chiba, Japan) and the E test TOSOH II (Tosoh, Tokyo), respectively. Serum kallistatin (KAL) was measured using DuoSet ELISA, and plasma C-C motif chemokine 18 (PARC) and plasma interleukin-1 receptor antagonist (IL-1Ra) were using Quantikine ELISA kits from R&D Systems (Minneapolis, MN, USA). Secreted phospholipase A2 (sPLA2) and apolipoprotein A-I (apo A-I) in plasma were measured using ELISA kits from Cayman Chemical (Ann Arbor, MI, USA) and Abcam (Cambridge, UK), respectively. All ELISA kits were used in accordance with the manufacturers’ instructions. The K18 and ccK18 ELISA kits (VLV bio, Nacka, Sweden) were used for detecting apoptosis in vitro experiments.

Blood samples were taken from the antecubital vein in the morning. We measured PG, hemoglobin A1c, insulin, and C-peptide levels. Enzymatic methods were used to assess PG (Glucose Assay Kit; Wako Pure Chemical Industries, Osaka, Japan). Serum insulin and HbA1c were measured by automated enzyme-linked immunosorbent assays (E-test TOSOH II; Tosoh, Tokyo, Japan) and high-performance liquid chromatography (HA-8180; Arkray, Tokyo, Japan), respectively. Serum C-peptide was measured using a chemiluminescent enzyme immunoassay (LUMIPULSE; Fujirebio, Tokyo, Japan). We calculated eGFR using the revised equation adjusted for the Japanese population^{52 (link)}. UACR, a marker for diabetic nephropathy, was also measured. The urinary albumin levels were measured using an immunoturbidimetric assay (SRL, Tokyo, Japan), and UACR was calculated by dividing the urinary albumin levels by the urinary creatinine concentration.

After a 12 h overnight fast, blood samples were taken from the antecubital vein and collected into tubes. FPG was measured by an enzymatic method (Glucose Assay Kit; Wako Pure Chemical Industries, Osaka, Japan). Serum insulin and HbA1c were measured by automated enzyme-linked immunosorbent assays (E-test TOSOH II; TOSOH, Tokyo, Japan) and high-performance liquid chromatography (HA-8180; Arkray, Tokyo, Japan), respectively. Total cholesterol, triglycerides, HDL cholesterol, and low-density lipoprotein (LDL) cholesterol were measured enzymatically using the following commercially available kits: Tcho-l, TG-LH (both from Wako Pure Chemical Industries), Cholestest N HDL, and Cholestest LDL (both from Daiichi Pure Chemicals, Tokyo, Japan), respectively. We calculated homeostasis model of assessment-insulin resistance (HOMA-IR) as the marker for insulin resistance by using FPG and insulin concentrations and the following equation: HOMA-IR = FPG (mg/dL) × fasting serum insulin (μU/mL)/405 [20 (link)].