The rats were individually placed in metabolic cages, and urine samples were collected for 24 h on the last week of the study to quantify UAE levels. Albumin concentrations were measured using an ELISA kit (Nephrat II; Exocell, Philadelphia, PA, USA). Protein concentrations were measured using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Kanagawa, Japan). Urinary and plasma creatinine concentrations were measured with an enzymatic Creatinine Plus version 2 reagent kit, using an automatic analyzer (COBAS INTEGRA 400 plus; Roche, Basel, Switzerland). Urinary corticosterone and aldosterone levels were measured using an enzyme immunoassay system (Cayman Chemical, Ann Arbor, MI, USA). UAE, UPE, urinary corticosterone and aldosterone levels were normalized to the urinary creatinine concentration.
Enzyme immunoassay system
Manufactured by Cayman Chemical
Sourced in United States
The Enzyme Immunoassay (EIA) System is a laboratory instrument used for the detection and quantification of specific analytes in a sample. It utilizes the principle of immunoassay, where an enzyme-labeled antibody or antigen is used to detect the presence and concentration of a target molecule. The system provides a reliable and sensitive method for the analysis of a wide range of substances, including proteins, hormones, drugs, and other molecules of interest.
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3 protocols using enzyme immunoassay system
1
Urinary Biomarker Quantification in Rats
2
Quantifying Urinary Albumin and Aldosterone
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Rats were individually placed in metabolic cages, and urine samples were collected for 24 hours in order to determine UAE levels. Albumin concentrations were measured with a commercially available ELISA kit (Nephrat II; Exocell Inc., Philadelphia, PA, USA). Aldosterone levels were measured using an enzyme immunoassay system (Cayman Chemical Company, Ann Arbor, MI, USA).
3
Cardiac and Metabolic Assessment in Mice
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Echocardiography and catheterization were performed as described previously5 (link), 17 (link). Echocardiography was performed using Nemio-XG echocardiography (Toshiba) with 14-MHz transducer under anesthesia with isoflurane (induction: 3–4%). LV hemodynamic parameters of mice 6-week after TAC or 4-week STZ treatment were assessed using a micronanometer catheter (Millar 1.4F, SPR 671, Millar Instruments) under anesthesia. Cardiac parameters of WT animals have been already reported17 (link).
Measurement of biochemical parameters in plasma glucose (Glucose Pilot, Technicon), total cholesterol, and high density lipoprotein cholesterol, and urinary protein (Pierce BCA protein assay kit, Thermo Fisher Scientific), urinary aldosterone and corticosterone (Enzyme immunoassay system, Cayman Chemical) were performed as described previously41 (link). Blood samples collected from cardiac apex at the time of euthanasia were centrifuged (3,000 × g) for 15 min to prepare plasma samples. Plasma total cholesterol (TCHO) and HDL cholesterol (HDLC) levels were measured using DRI-CHEM 7000Z (Fuji Film) with slides of TCHO-PIII or HDL-C-PIIID (Fuji Film) according to manufacturer’s instruction. Mice were individually placed in metabolic cages (Natsume), and urine samples were collected for 24 hours.
Measurement of biochemical parameters in plasma glucose (Glucose Pilot, Technicon), total cholesterol, and high density lipoprotein cholesterol, and urinary protein (Pierce BCA protein assay kit, Thermo Fisher Scientific), urinary aldosterone and corticosterone (Enzyme immunoassay system, Cayman Chemical) were performed as described previously41 (link). Blood samples collected from cardiac apex at the time of euthanasia were centrifuged (3,000 × g) for 15 min to prepare plasma samples. Plasma total cholesterol (TCHO) and HDL cholesterol (HDLC) levels were measured using DRI-CHEM 7000Z (Fuji Film) with slides of TCHO-PIII or HDL-C-PIIID (Fuji Film) according to manufacturer’s instruction. Mice were individually placed in metabolic cages (Natsume), and urine samples were collected for 24 hours.
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