The glomerular circadian clock temporally regulates basement membrane dynamics and glucocorticoid response. Cultured podocyte cells circadian clock
The glomerular circadian clock temporally regulates basement membrane dynamics and glucocorticoid response. Cultured podocyte cells circadian clock
The care and use of all mice in this study was carried out in accordance with the UK Home Office regulations, UK Animals (Scientific Procedures) Act of 1986 under the Home Office Project Licences: P1AE9A736, PP4262564 and PP7202624. Mice were maintained at 20°C to 22°C, humidity of 40-50%, with ad libitum access to water and standard rodent chow. Wild type C57/BL6J mice housed in 12-h light/12-h dark cycle (LD) were used for time-series cell harvesting.  Podocyte cells were cultured from glomeruli from 3 mice independently (n=3). For glomerular isolation, kidneys were cut into 1-2mm3 pieces and passed through a 100 μm cell strainer (Endecotts, UK). Flow-through was passed through a 70 μm cell strainer (Endecotts, UK) and retained glomeruli were seeded onto cell culture dishes and cultured for 14 days. During this period, cells underwent transformation from undifferentiated cobblestone-like cells into terminally differentiated arborised cells resembling podocytes in vivo. Cells were cultured in complete media at 37oC, in 5% CO2; RPMI-1640 medium containing L-glutamine, supplemented with 10% foetal bovine serum (FBS) (Thermo Fisher Scientific, UK), 5% insulin, transferrin and selenium (ITS), 50µg/ml each of penicillin and streptomycin (Invitrogen, UK). To synchronise the circadian clocks of a population of cells, cells were incubated with either 100nm Dexamethasone (Merck) for 1h, 10 M forskolin for 1h, or 50% horse serum (v/v) (Thermo Fisher Scientific) for 2h, in complete media at 37oC, in 5% CO2. Podocyte cells were collected at 4-hourly intervals for 48 hours starting 15 hours post synchronisation with dexamethasone. Podocytes were harvested in BL buffer containing 0.1% 1-Thioglycerol (Promega) and immediately snap-frozen at 4-hourly intervals for 48 hours starting at 15-hours post-synchronisation with dexamethasone. The purpose of delaying cell harvest to 15-hours post-synchronisation was to exclude immediate dexamethasone-responsive genes which would skew rhythmic gene analysis.  RNA was isolated and purified using a ReliaPrep RNA Cell Miniprep System (Promega) as per manufacturers protocol. In brief, podocytes were washed in ice-cold sterile PBS and resuspended in BL buffer containing 0.1% 1-Thioglycerol. The lysate was either frozen at -80oC for later use or used immediately for RNA isolation. Isopropanol was added to the lysate which was centrifuged prior to a series of washes and subsequent DNase treatment. Following further washes, RNA was eluted in nuclease-free water and concentration measured using a NanoDrop 2000 (Thermo Fisher Scientific). The quality of resuspended mRNA was then measured using a QubitTM Fluorometer (Thermo Fisher Scientific) and RNA integrity measured using a 2200 TapeStation (Agilent Technologies) to ensure good quality for downstream analysis.  Illumina stranded mRNA library