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PLoS By Category | Recent
PLoS Articles
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Biochemistry - Biotechnology - Diabetes and Endocrinology - Hematology - Molecular Biology - Neuroscience - Physiology
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Isolation and Characterization of Renal Erythropoietin-Producing Cells from Genetically Produced Anemia Mice
Published:
Tuesday, October 11, 2011
Author:
Xiaoqing Pan et al.
by Xiaoqing Pan, Norio Suzuki, Ikuo Hirano, Shun Yamazaki, Naoko Minegishi, Masayuki Yamamoto
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 (Epo?3'E). Mice harboring the mutant EpoGFP/?3'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 Hif2a and Hif3a 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 HIF2a and negative regulation by HIF3a might be necessary for correct renal Epo induction. (282 words)
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