Creatinine companion

The animal care protocols were approved in advance by the NIDDK Animal Care and Use Committee (approval No. K097-KDB-08) and the Institutional Animal Care and Use Committee, which is convened at the Graduate School of Veterinary Medicine, Hokkaido University (approval No. 13-0032). We followed the NIH Guide for the Care and Use of Laboratory Animals and the Guide for the Care and Use of Laboratory Animals of Hokkaido University, Graduate School of Veterinary Medicine. For short-term exposure, indoxyl sulfate (Sigma-Aldrich, St. Louis, MO) dissolved in phosphate-buffered saline (PBS) was injected into C57BL/6 mice (800 mg/kg, i.p. given once). For chronic exposure, indoxyl sulfate in 4% dimethyl sulfoxide (DMSO)/PBS was injected daily into FVB/N mice, having a susceptibility to glomerular sclerosis compared to C57BL/6 mice [27] (link), [28] (link), for receiving 600 mg/kg, i.p. for 8 w. Kidneys, serum, and urine were collected, and glomeruli were isolated following a bead perfusion method [29] . Kidney tissue was either frozen at −80°C for RNA analysis and immunoblotting or fixed with 4% paraformaldehyde (PFA) or 2.5% glutaraldehyde for histopathological analysis. Urinary albumin/creatinine ratio (uACR) was measured using Albuwell and the Creatinine Companion (Exocell, Philadelphia, PA).

To measure albuminuria, 24 hours urine was collected using metabolic cages. Urinary albumin (Exocell NephratII; Exocell Inc., Philadelphia, PA, USA) and creatinine (The Creatinine Companion; Exocell Inc.) levels were measured using ELISA kit.

Urinary albumin (Exocell Nephrat II; Exocell Inc., Philadelphia, PA, USA) and creatinine (The Creatinine Companion; Exocell Inc.) levels in urine collected over 24 hours were measured according to the manufacturer’s instructions.

Mice were placed in individual mouse metabolic cages (Tecniplast, Gazzada, Italy) with access to water and food for 24 h. Urine collection was done every four weeks and data from month 18 and 24 was used in this experiment. Albuminuria (Albuwell M, Exocell, Philadelphia, PA, USA) and urine creatinine concentration (The Creatinine Companion, Exocell, Philadelphia, PA, USA) were measured using ELISA kits. Serum creatinine concentrations and Blood urea nitrogen were measured using i-STAT system Cartridges (CHEM8+, Abbott Point of Care, Abbott Park, IL, USA). Creatinine clearance was calculated using a standard formula (urine creatinine (mg/dL) × urine volume (mL/24 h)/serum creatinine (mg/dL) × 1440 (min/24 h)).

Each mouse was placed in an individual mouse metabolic cage (Tecniplast, Gazzada, Italy) and 24-h urine was collected every 4 weeks. Urine collected at 18 and 24 months was used in the experiment. In aging urine, ELISA kits were used to measure albumin concentration (Albuwell M, Exocell, Philadelphia, PA, USA) and urine creatinine concentration (The Creatinine Companion, Exocell). Blood, serum creatinine, BUN, and HbA1c concentrations of aged mice were measured using i-STAT system cartridges (CHEM8+, Abbott Point of Care Inc., Abbott Park, IL, USA). The fasting blood glucose was measured using an Accu-Chek meter (Roche Diagnostics, St Louis, MO, USA). Creatinine clearance was calculated using the standard formula: [urine creatinine (mg/dL) × urine volume (mL/24 h)] / [serum creatinine (mg/dL) × 1440 (min/24 h)].

Renal function was investigated at the initiation and end of the study. The mice were placed in individual mouse metabolic cages (Tecniplast, Gazzada, Italy) to collect urine and provided with water and food for 24 h. Enzyme-linked immunosorbent assay (ELISA) kits were used to measure albuminuria in 24-h urine (Albuwell M, Exocell, Philadelphia, PA, USA) and creatinine concentration (The Creatinine Companion, Exocell) in urine. Measurement of the serum creatinine level was assessed by i-STAT system cartridges (CHEM8+, Abbott Point of Care Inc., Abbott Park, IL, USA). Creatinine clearance was calculated using the standard formula: (urine creatinine (mg/dL) × urine volume (mL/24 h))/(serum creatinine (mg/dL) × 1440 (min/24 h)). The sandwich enzyme immunoassay technique (CUSABIO Biotech, Wuhan, China) was used to measure the serum cystatin-C concentrations in mice from each group according to the manufacturer’s protocols.

Urine was collected on day 0 prior to injection of saline (control) or DT and then on days 5, 8, 15, 23, 30, 42, and 56 after DT administration and stored at − 80 °C. Urinary albumin concentration was calculated using the Albuwell M Albumin ELISA (1011; Exocell, Philadelphia, PA), and urinary creatinine concentration was calculated using The Creatinine Companion (1012; Exocell). An ACR ratio was then generated.