Figure 1.

A: Relative PHD1-3 mRNA levels in murine collecting duct (M-1) cells and murine proximal tubular cells (mPT) under normoxic conditions. Results were obtained in triplicates from three different cell preparations and normalized to hypoxanthine-guanine phosphoribosyltransferase (HPRT) expression. Values are means ± SD; *P < 0.05 as compared with PHD1 expression in M-1 and mPT cells, respectively. B: Regulation of PHD expression by hypoxia in mPT and M-1 cells. mRNA was isolated after exposure of the cells to 1% O₂ or normoxia for 16 hours. Real-time PCR was performed in triplicates from three different cell preparations. Results were normalized to the HPRT signal and related to results from normoxic cells. Values are means ± SD; *P < 0.05. C: Hypoxic regulation of PHD2 and 3 protein expression. Western blot analyses revealed hypoxic induction of PHD2 and 3 in proximal tubular cells, whereas no induction was seen in M-1 cells. PHD1 protein levels were not altered by hypoxia in both cell types. D: Protein stability of the different PHDs in mPT cells determined by treatment of the cells with cycloheximide for the indicated times. PHD1 had the longest half life in mPT cells. E: In both cell types, hypoxia for 16 hours leads to HIF-1α accumulation. No significant HIF-2α induction was seen.