Ifn γ

Conditionally immortalized murine proximal tubular epithelial cells (IM-pTECs) were generated as previously described [31 (link)]. IM-pTECs were cultured in DMEM/F12 medium with 10% FCS, 5 ug/mL insulin and transferrin, 5 ng/mL sodium selenite (all from ThermoFisherScientific), 20 mg/mL triiodo-thyrionine (Sigma-Aldrich), 50 ng/mL hydrocortisone (Sigma-Aldrich), and 5 ng/mL prostaglandin E1 (Sigma-Aldrich) with 2 mM L-glutamine and 100 U/mL Penicillin/Streptomycin (both from ThermoFisherScientific). IM-PTECs were maintained at 33 °C in the presence of 10 ng/mL IFNγ (ProSpec) and differentiated at 37 °C without IFNγ for one week, resulting in loss of SV40 expression [31 (link)]. After one week of differentiation, cells were used for experimentation. Supernatant from damaged IM-pTECs was obtained via repeated freezing and thawing cycles, as described earlier by Sauter [32 (link)]. Total protein concentration in the supernatant was determined by BCA (Sigma-Aldrich). The supernatant was filtered through 0.22 µm filter and used to stimulate M0 bone marrow-derived macrophages at 0.15 mg/mL.

Cells were cultured in HK-2 medium (DMEM/F12 medium, supplemented with 10% FBS, 5 μg/ml insulin and transferrin, 5 ng/ml sodium selenite, 20 ng/ml triiodo-thyrionine, 50 ng/ml hydrocortisone, and 5 ng/ml prostaglandin E1 with l-glutamine and antibiotics). IM-TECs were cultured at 33°C in the presence of 10 ng/ml IFN-γ (ProSpec) and kept at 37°C without IFN-γ for another week before the start of the experiment (20 (link)). Primary TECs were isolated and cultured as described above (21 (link)).
Hypoxia/reoxygenation experiments were performed as follows. Cells (IM-TECs or primary TECs) were cultured until confluency, incubated in complete medium in the HypOxystation H35 (Don witley scientific) and cultured for 2 days in 1% Oxygen, 5% CO2 at 37°C, in order to mimic the hypoxic condition. Cell viability was assessed with the MTT staining (data not shown). Normoxic cells were kept in culture under standard conditions. After 2 days of hypoxia, both hypoxic and normoxic cells received fresh medium and were cultured for an additional 24 h under standard conditions (re-oxygenation). MitoTEMPO (100μM) or TREM-1 agonist (mTREM-1: 10μg/ml) were added in the culture media during the re-oxygenation time (24 h).

hpDPSCs and MDPSCs were stimulated with IFN-γ (PROSPEC, East Brunswick NJ, USA) at a concentration of 20 ng/ml in DMEM without serum for 24 h according to the previous studies [27 (link), 28 (link)] with slight modification. Non-stimulated cells were used as a control. RNA was extracted using Trizol, and the CM was collected and concentrated. The mRNA expression of immunosuppressive markers IDO, TGF-β1, PTGE, and IL-6 as our previous study [29 (link)] was examined by RT-PCR. The concentration of nitric oxide (NO) was examined by measuring its stable end product, nitrite, in the CM using a Griess reagent (Promega Corporation, Madison, WI, USA) according to manufacturer’s protocol. Absorbance at 540 nm was measured by microplate reader (SpectraMax Gemini XPS/EM, Molecular Devices, San Jose, CA, USA), and nitrite concentrations were calculated using a standard nitrite curve.