Immunostaining

For histological analysis sections of formalin-fixed, paraffin-embedded (FFPE) mouse renal tissue (4 μm) were deparaffinized using xylene followed by graded ethanols for rehydration and stained with hematoxylin and eosin, Periodic-acid schiff (PAS) or Masson’s trichrome according to standard protocol (HistoLab Products AB, Göteborg, Sweden). For IHC boiling in 10 mmol/L citrate buffer at pH 6 was performed as antigen retrieval. Staining was detected using the EnVision system and DAKO Techmate 500 staining equipment according to the instructions of the manufacturer (DAKO, Carpenteria, CA). Paraffin sections were incubated with the following primary antibodies GFP (A290, Abcam, Cambridge, UK), CaIX (AF2344, R&D Systems, Minneapolis, MN), Glut1 (07–1401, Merek Millipore, Darmstadt, Germany), Ki67 (SP6 RM-9601-S, Thermo Scientific, Waltham, MA) and Podocalyxin (AF1556, R&D Systems, Minneapolis, MN). For Oil red O (ORO) stain kidneys were fixed in formalin over night followed by cryo preservation in 30% sucrose over night followed by embedding in optimal cutting temperature compound (O.C.T.) and sectioned (10 μm) on a cryostat.