Cell culture

Fibroblasts were isolated from WT and PFKFB2KI mouse kidneys using a modified method¹⁸.

Briefly, kidneys were removed from euthanized mice, and chopped into small pieces using a scalpel blade. Kidneys were then placed in 10 ml collagenase-dispase medium (ThermoFisher Scientific) and incubated at 37 °C for 45 min with constant shaking. Fibroblast medium (DMEM/F12; 15% FBS; 12.5 mM Hepes, Pen/Strep) was added to the digest to inactivate the enzymes and the samples centrifuged at 500×g for 5 min. The supernatant was removed and 20 ml fresh fibroblast medium added and the sample centrifuged as described above. This was repeated two more times and then the pellet was resuspended in 20 ml fibroblast medium and transferred to two 10 cm tissue culture dishes. The plates were checked daily and the media changed as required. Fibroblasts usually exit tissue fragments within 2–5 days. When there were sufficient patches of confluent cells, the cells were trypsinized and plated into 6-well plates. Once confluent, cell lysates were prepared.

Primary cultures of renal tubular epithelial cells (TEC) were prepared by sieving whole mouse kidneys, as we have recently reported⁴. These cultures are likely to contain cells of a variety of tubular lineages.