Soluble αKlotho protein containing the ectodomain of mouse αKlotho (amino acid number 31–982) with C-terminal V5 and 6×His tags was generated and purified in our laboratory in mammalian cells as described previously [11 (link), 22 (link), 23 (link)]. For the experiment of αKlotho administration in CP-treated mice, CP nephrotoxicity was induced with low dose (10 mg/kg) or vehicle [24 (link)] and high phosphate (2%) diet was given 2 weeks after CP injection [8 (link)–10 (link)]. At 4 weeks after injection, recombinant αKlotho protein (0.3 mg/kg body weight/month) or vehicle (normal saline) was given for 16 weeks via ALZET® 1004 osmotic minipumps (DURECT Corporation, Cupertino, CA). The effective lifespan for this type of minipump is 4 weeks and a new one was implanted to replace every 4 weeks resulting in 4 minipumps per animal [11 (link)]. There were 10 mice (equal male and female) at the beginning and ~8 mice at 20 weeks after CP injection.