Aldoctk 2

The presence of potassium, sodium, urea, and creatinine in urine samples was determined with a MEGA clinical chemistry analyzer (Merck). Potassium, and sodium were determined by indirect potentiometry, and urea was determined by a photometric test with the urease-GIDH method. Estimated glomerular filtration rate (eGFR) was calculated based on the combined creatinine and cystatin C Chronic Kidney Disease-Epidemiology Collaboration equation (12 ). Serum and urinary creatinine, plasma total cholesterol, and triglycerides were determined from fasting blood samples by using Kodak Ektachem dry chemistry (Eastman Kodak). Plasma HDL cholesterol was measured with a homogeneous method (direct HDL, Aeroset TM System, Abbott Laboratories). Plasma renin concentration was measured using an automated sandwich immunochemiluminescent assay (LIAISON®, Diasorin). Serum aldosterone concentration was measured using a competition-based radioimmunoassay (ALDOCTK-2, Diasorin). Serum cystatin C concentrations were measured by a Gentian Cystatin C Immunoassay (Gentian AS) on a modular analyzer (Roche Diagnostics). Urinary albumin concentration was determined by nephelometry (Dade Behring Diagnostic). Urinary albumin-to-creatinine ratio (ACR) was calculated by dividing the urinary albumin concentration (mg/L) by the urinary creatinine concentration (mmol/L).

After guillotine, blood was collected quickly in prechilled EDTA-coated or heparinized tubes. Plasma samples were separated and kept at −70°C until used. Plasma and tissue levels of renin activity and plasma ANP were measured by radioimmunoassay as reported previously [22 (link), 29 (link)]. Changes in renal renin contents were evaluated by angiotensin I (Ang I) generated with enough renin substrate from nephrectomized male rats and a small amount of whole kidney extract. Plasma renin activity was evaluated by Ang I generated with 1 ml of plasma. Radioimmunoassay for ANP was performed as reported previously [29 (link)]. The radioimmunoassay was performed in Tris-acetate buffer containing 0.2% neomycin, 1 mM EDTA, 50 BAEE-U/ml soybean-trypsin inhibitor, 0.02% sodium azide, 0.0004% phenylmethylsulfonyl fluoride, and 1% bovine serum albumin. Standards or samples were incubated following the addition of 100 μl anti-ANP-antibody and 100 μl [¹²⁵I]-ANP for 24 h at 4°C. The separation of free tracer from antibody-bound tracer was conducted by adding 1 ml of dextran charcoal suspension. Plasma levels of aldosterone (ALDOCTK-2, DiaSorin Inc., Stillwater, MN, USA) were measured by radioimmunoassay as recommended by manufacturers.