Abstract
Understanding the nature of renal erythropoietin-producing cells (REPs) remains a central challenge for elucidating the mechanisms involved in hypoxia and/or 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 an approach to isolate and characterize REPs. We bred mice bearing an Epo gene allele to which green fluorescent protein (GFP) reporter cDNA was knocked-in (EpoGFP) with mice bearing an Epo gene allele lacking the 3′ enhancer (EpoΔ3′E). Mice harboring the mutant EpoGFP/Δ3′E gene exhibited anemia (average Hematocrit 18% at 4 to 6 days after birth), and this perinatal anemia enabled us to identify and purify REPs based on GFP expression from the kidney. Light and confocal microscopy revealed that GFP immunostaining was confined to fibroblastic cells that reside in the peritubular interstitial space, confirming our previous observation in Epo-GFP transgenic reporter assays. Flow cytometry analyses revealed that the GFP fraction constitutes approximately 0.2% of the whole kidney cells and 63% of GFP-positive cells co-express CD73 (a marker for cortical fibroblasts and Epo-expressing cells in the kidney). Quantitative RT-PCR analyses confirmed that Epo expression was increased by approximately 100-fold in the purified population of REPs compared with that of the unsorted cells or CD73-positive fraction. Gene expression analyses showed enrichment of Hif2α and Hif3α mRNA in the purified population of REPs. The genetic approach described here provides a means to isolate a pure population of REPs, allowing the analysis of gene expression of a defined population of cells essential for Epo production in the kidney. This has provided evidence that positive regulation by HIF2α and negative regulation by HIF3α might be necessary for correct renal Epo induction. (282 words)
Highlights
As we previously reported in EpoD39E/D39E mice, deletion of the 39 enhancer provokes transient anemia at late embryonic and neonatal stages due to defect in hepatic Epo production and erythropoiesis
Increased Epo mRNA could be detected in the kidney of the EpoGFP/D39E newborns by quantitative (q) RT-polymerase chain reaction (PCR)
By generating EpoGFP/D39E mice, we isolated a specific type of renal cells namely renal Epo-producing cells (REPs), which are responsible for Epo production after birth
Summary
Epo is a glycoprotein hormone mainly produced in the kidney and liver in response to changes in tissue oxygen tension. Epo production is considered to be controlled primarily at the level of gene transcription and Epo gene expression is strictly regulated in a tissue/cell-specific and hypoxia/anemiainduced manner [3,4,5,6,7]. Several tissues have been reported to express the Epo gene; but the ability to produce substantial amounts of Epo during hypoxia/ anemia is restricted to the fetal liver and adult kidney [4,5,6,7,8]. Difficulties in identification and purification of the renal Epo-producing cells (REPs) have limited the understanding of the mechanism for controlling Epo production in kidney. Further details remain to be elucidated [5,7,13]
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