Urea assay kit

Urease (from Canavalia ensiformis), urea, glutaraldehyde, disodium hydrogen phosphate, potassium dihydrogen phosphate, fluorescein, fluorescein isothiocyanate (FITC), and ammonium carbamate were obtained from Merck (Darmstadt, Germany). The urea assay kit was purchased from BioAssay Systems (Hayward, CA, USA). Phosphate buffered saline (PBS) was obtained from LPS solution (Daejeon, Korea) and used as the supporting electrolyte for the electrochemical measurements. Phosphate buffer (PB) was prepared by mixing 20 mM disodium hydrogen phosphate and 20 mM potassium dihydrogen phosphate (pH = 7.4), and it was used as the immobilization buffer. Porous polytetrafluoroethylene (PTFE) membranes with a pore size of 1 μm were purchased from Advantec MFS (Dublin, CA, USA). The waste peritoneal dialysate of a patient with chronic renal failure was obtained from the Seoul National University Hospital (Seoul, Korea) with written consent, and they agreed to participate according to the Declaration of Helsinki. The study was approved by the institutional review boards of Seoul National University Hospital (protocol number: SNUH IRB No. 1610-016-797).

After exposure of the cells to 2 mM ammonium chloride (Sigma-Aldrich) for 24 h, urea productions in the culture media of hepatocyte-like cells were measured using Urea Assay Kit (BioAssay Systems). Fresh culture medium supplemented with 2 mM ammonium chloride was used as a negative control. Operation was performed according to the manufacturer’s recommendation.

Urea production by the differentiated cells was determined by using the Urea Assay Kit (BioAssay System) according to the manufacturer's instructions. Briefly, 25 µl of culture medium was added into 96-well plates and incubated with 100 µl of Assay Buffer for 50 minutes at room temperature while protected from light. The amount of urea present was measured at OD₄₃₀ using a micro plate reader (Promega). Urea concentration was stated as relative amount/10⁵ cells/ml.

The bioactive substances present in the culture supernatant were analyzed using the TGF-β1 ELISA kit (#KE00002, Protein Tech, Rosemont, IL, USA), IL-8 ELISA kit (#KE00006, Protein Tech), and Sonic hedgehog (SHH) ELISA kit (#ab100639, Abcam) according to the manufacturer's protocol. The samples were analyzed on day 3 or day 4 when ballooning was first observed (Fig. S2). On day 11, PHH/HSC sheets were homogenized, and protein lysates were prepared. Lysates were analyzed using the p62 ELISA kit (#ADI-900-212, ENZO New York, NY, USA), and protein carbonyl ELISA kit (#STA-310, CELL BIOLABS, San Diego, CA, USA) according to the manufacturer's protocol. The BCA protein assay kit (#T9300A, Takara Bio) was used to determine the protein concentration in the lysates. The Human Albumin ELISA kit (#E88-129, Bethyl Laboratories, Inc., Waltham, MA, USA) and Urea assay kit (#DIUR-100, BioAssay Systems, Hayward, CA, USA) were used to assess hepatocyte function. The samples were analyzed on days 0, 1, 5, and 10.