Amicon 10kda ultrafiltration units

Fasting blood and urine samples were collected at the baseline study visit. Blood samples were stored at room temperature for 30 minutes, then centrifuged for 10 minutes at 1,500× g. Serum was aliquoted and stored at -80°C. Spot urine samples were aliquoted and stored at -80°C. Angiotensinogen, gremlin-1 and MMP-7 were measured using commercial immunoassays, as previously described.^{22 (link)} Urine samples were either used directly for immunoassays (MMP-7, angiotensinogen) or concentrated 5-fold (gremlin-1) using Amicon 10kDa ultrafiltration units (Millipore, Billerica, MA). Serum samples were used after being thawed. Angiotensinogen was measured using a quantitative sandwich Enzyme-Linked-Immunosorbent Assay (ELISA) (IBL-America, Minneapolis, MN), with a minimum detection limit of 30 pg/mL, and intra- and inter-assay coefficients of variations (CV's) of 8 and 14%, respectively.^{22 (link)} Gremlin-1 was measured using a quantitative ELISA (Uscnk Life Sciences, Wuhan, China), with a minimum detection limit of 60 pg/mL, and intra- and inter-assay CV's of 13 and 20%.^{22 (link)} MMP-7 was measured using a quantitative ELISA (R & D Systems, Minneapolis, MN), with a minimum detection limit of 20 pg/mL, and intra- and inter-assay CV's of 5 and 9%.^{22 (link)}