Urea nitrogen bun colorimetric detection kit

In part of the WT vs. SBP-1 KO experiments, mice were anesthetized with 3% isoflurane (Iso-Vet, Dechra, Nortwhich, United Kingdom) before retro-orbital puncture was performed with a heparinized (LEO Pharma, Lier, Belgium) glass capillary tube (Hirschmann-Laborgeräte, Eberstadt, Germany). The collected blood was injected in a cartridge in the Epoc Blood Analysis System (Siemens, Munich, Germany) for biochemical analysis. After the blood collection, mice were euthanized by performing heart puncture under anaesthesia with 3% isoflurane. After a transcardial perfusion with 20 ml PBS the left kidney was removed, laterally cut into two equal pieces and fixed in 4% formaldehyde (Klinipath, Duiven, The Netherlands) for 48 h at 4°C.
In the ART + CQ treatment experiments and part of the WT vs. SBP-1 KO experiments, mice were euthanized with Dolethal (Vétoquinol, Aartselaar, Belgium; 200 mg/mL, i.p. injection of 100 µL) followed by heart puncture at indicated time points. After a transcardial perfusion with 20 ml PBS the left kidney was removed and laterally cut into two equal pieces and fixed in 4% formaldehyde for 48 h at 4°C. In the plasma samples obtained from heart puncture, BUN levels were measured with the Urea Nitrogen (BUN) Colorimetric Detection Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA) for the WT vs. SBP-1 KO experiments.

Urinary NGAL was quantified in 50 μL of a 1:5000 dilution of urine using the Mouse Lipocalin-2/NGAL DuoSet ELISA kit from R&D Systems (Minneapolis, MN, USA) following the manufacturer’s protocol. Serum Cystatin-C was quantified in 50 μL of 1:1000 dilutions of serum, using the Mouse Cystatin C DuoSet ELISA kit from R&D Systems (Minneapolis, MN, USA) following the manufacturer’s protocol. All samples were measured in duplicate. BUN was quantified in 50 μL of a 1:10 dilution of serum using the Urea Nitrogen (BUN) Colorimetric Detection Kit from Invitrogen (Frederick, MD, USA) following the manufacturer’s protocol. Serum creatinine was quantified in 15 μL of serum using the Creatinine (serum) Colorimetric Assay Kit from Cayman Chemicals (Ann Arbor, MI, USA) following the manufacturer’s protocol. Urinary albumin was quantified in 20–40 μL of urine; Exton solution (Cole-Parmer, Vernon Hills, IL, USA) was added to urine at a 1:1 ratio and samples were incubated for 10 min in the dark. Absorbance was read at 620 nm and values were multiplied by total volume of urine (mL) to determine albumin in mg/day.

To assess indices of tissue injury, blood was collected from mice 24 h following sham or CLP procedure via cardiac puncture using a heparin-coated syringe. Blood sample was centrifuged at 10,000 rpm, and supernatant (plasma) was collected and stored at -80°C. For tissue injury assays, plasma was analyzed using the following kits according to the manufacturer’s protocol: Urea Nitrogen (BUN) Colorimetric Detection Kit (Invitrogen, Carlsbad, CA, USA, cat# EIABUN), Creatine Kinase Activity Assay Kit (Sigma-Aldrich, St. Louis, MO, USA: cat# MAK116), Alanine Aminotransferase (ALT) Activity Assay Kit (Sigma-Aldrich: cat# MAK052), Aspartate Aminotransferase (AST) Activity Assay Kit (Sigma-Aldrich: cat# MAK055), Amylase Assay Kit (Colorimetric) (Abcam, Cambridge, MA, USA: cat# ab102523), and Bilirubin Assay Kit (Direct Colorimetric) (Abcam: cat# ab235627).

Urine was sampled (every 2–4 weeks) throughout the study and stored at − 80 °C. Albumin concentrations in urine were analyzed using a mouse albumin simple-step ELISA kit (Abcam, Cambridge, UK) according to manufacturer instructions. The QuantiChrom™ Creatinine Assay Kit assay was performed according to the manufacturer’s instructions (DICT-500, BioAssay Systems, Hayward, CA, USA) to establish the albumin/creatinine ratio. Serum was sampled 2–3 weeks from vena saphena (Microvette® CB 300 Serum, Sarstedt, Germany) after each cycle and before sacrifice (6 weeks after third cycle) and blood urea nitrogen (BUN) was analyzed with the Urea Nitrogen (BUN) Colorimetric Detection Kit (Invitrogen, Waltham, Massachusetts, USA) according to the manufacturer’s instructions.