Tnfr1

Urine was collected by metabolic caging to measure the urinary albumin concentration and to calculate the urinary albumin excretion rate (UAER). Mice were single-housed in metabolic cages for 18 hours on days 2-3, 6-7, 16-17, and 36-37. Urinary markers were measured by ELISA: albumin (Bethyl Laboratories, cat.no. E90-134), Cystatin C (R&D systems, Minneapolis, MN Cat. number MSCTCO) and TNFR1 (R&D systems, Minneapolis MN Cat. number MRT10). Urinary creatinine was measured by high-performance liquid chromatography (HPLC). Creatinine was measured in serum by acetonitrile deproteinization, followed by isocratic, cation exchange HPLC as previously described [20 (link)].
Blood plasma was prepared from blood samples collected on days 7, 14, 21, 28, and 42. Serum Amyloid P (SAP) was measured by ELISA (Genway, San Diego, CA). Cystatin C was measured using ELISA (R&D systems, Minneapolis, MN Cat. number MSCTCO).

The following cytokines/chemokines were measured by sandwich ELISA kits: IL-18 (R&D), MCP-3 (R&D), TNFR1 (R&D).

Blocking studies were performed with cell-free supernatants obtained from freshly isolated NK cells activated overnight in serum-free AIM-V^® medium and IL-2 (1.000 U/ml) supplemented with FMKp (10 μg/ml) or poly(I:C)HMW (50 μg/ml). The receptor-blocking was performed by pre-incubating iDC with blocking antibodies for 20 min before their addition into flat-bottom 96-well plates containing the cell-free NK cell supernatant supplemented with IL-4 (500 U/ml) and GM-CSF (500 U/ml). The following receptor blocking antibodies were used: IFNGR1 (20 μg/ml), TNFR1 (20 μg/ml), TNFR2 (20 μg/ml), or isotype control (all purchased from R&D systems). The blocking of the cytokines (IFN-γ and TNF-α) in the NK cell-derived supernatants was performed by pre-incubating the supernatants with anti-TNF-α (20 μg/ml; BD) or anti-IFN-γ (10 μg/ml; BD) before adding the iDC. As reference value, iDC were incubated with NK cell-derived supernatant in the absence of blocking agents. As a negative control, iDC were incubated with medium supplemented with FMKp or poly(I:C) and IL-2 (control ‘supernatant’). After 48 h of maturation, the supernatant was harvested to determine the DC cytokine and chemokine profiles.