Isolation and Characterization of Renal Erythropoietin-Producing Cells: Contribution of HIF3α In Vivo.

Abstract 1541

Understanding the nature of renal erythropoietin-producing cells (REPs) remains a central challenge for understanding the mechanisms involved in hypoxia/anemia-induced erythropoietin (Epo) production in adult mammals. Previous studies have shown that REPs are renal peritubular cells, but further details are lacking. Here, we describe our approach used to isolate and characterize REPs. We bred mice bearing Epo/GFP combined with Epo-3’-enhancer deletion. These mice exhibit perinatal anemia (average Hct = 18% at P4–6). This enabled identification and purification of REPs based on GFP expression in the kidney. Light and confocal microscopy revealed that GFP immunostaining was confined to fibroblasts residing in the peritubular interstitium, which is consistent with our previous observation from the 180-Kb Epo/GFP reporter assay. Flow cytometry revealed that the GFP fraction constituted approximately 0.2% of the whole kidney cells; with most (63%) cells co-expressing CD73, a well-known marker for Epo-expressing cells. qRT-PCR confirmed that Epo expression was increased by approximately 100-fold in the purified REP population compared with that of the unsorted cells or CD73 fraction. Gene expression profiling showed Hif2α and Hif3α enrichment in the purified GFP/REP population. Moreover, in mouse hypoxia experiments, Epo expression was approximately 2.5-fold higher in Hif3α^–/– kidney cells than in wild-type cells. The molecular strategy described here provides a means to isolate a pure population of REPs, allowing the analysis of gene expression profile of a defined population of cells essential for Epo production in the kidney. Our findings have led to a novel hypothesis that positive regulation by HIF2α and negative regulation by HIF3α might be necessary for correct renal Epo induction.