Isolation and characterization of renal erythropoietin-producing cells from genetically produced anemia mice.
Isolation and characterization of renal erythropoietin-producing cells from genetically produced anemia mice.
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DOI：
10.1371/journal.pone.0025839
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发表时间：
2011
期刊：
影响因子：
3.7
通讯作者：
Yamamoto M
中科院分区：
文献类型：
--
作者：
Pan X;Suzuki N;Hirano I;Yamazaki S;Minegishi N;Yamamoto M
了解肾促红细胞生成素产生细胞（REPs）的性质仍然是阐明成年哺乳动物缺氧和/或贫血诱导的促红细胞生成素（Epo）产生机制的核心挑战。以往的研究表明，REPs是肾小管周细胞，但缺乏进一步的细节。在这里，我们描述了一种分离和表征REP的方法。我们将携带Epo基因等位基因的小鼠与携带缺乏3′增强子的Epo基因等位基因的小鼠（EpoΔ3′E）进行了交配，其中Epo基因等位基因敲入了绿色荧光蛋白（GFP）报告基因cDNA（EpoGFP）。携带突变型EpoGFP/Δ3′E基因的小鼠表现出贫血（出生后4至6天的平均红细胞压积为18%），这种围产期贫血使我们能够基于来自肾脏的GFP表达来鉴定和纯化REP。光镜和共聚焦显微镜显示，GFP免疫染色仅限于纤维母细胞，居住在肾小管周围的间隙，证实了我们以前的观察在Epo-GFP转基因报告基因检测。流式细胞术分析显示，GFP部分占整个肾细胞的约0.2%，并且63%的GFP阳性细胞共表达CD 73（肾中皮质成纤维细胞和Epo表达细胞的标志物）。定量RT-PCR分析证实，与未分选的细胞或CD 73阳性部分相比，纯化的REP群体中Epo表达增加了约100倍。基因表达分析表明，富集的HIF 2 α和HIF 3 α mRNA的纯化的REPs的人口。这里描述的遗传方法提供了一种手段，以分离一个纯的REPs的人口，允许分析的基因表达的一个定义的人口的细胞在肾脏中的Epo生产所必需的。这为HIF 2 α的正调节和HIF 3 α的负调节可能是正确的肾Epo诱导所必需的。(282（文字）
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)
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DOI：
10.1007/s004290050126
发表时间：
1998-02-01
期刊：
ANATOMY AND EMBRYOLOGY
影响因子：
--
作者：
Marxer-Meier, A;Hegyi, I;Kaissling, B
通讯作者：
Kaissling, B
博士丧失后小鼠中肝EPO合成的重生。
DOI：
10.1126/science.1192811
发表时间：
2010-07-23
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Minamishima YA;Kaelin WG Jr
通讯作者：
Kaelin WG Jr
影响因子：
64.8
作者：
Makino, Y;Cao, RH;Poellinger, L
通讯作者：
Poellinger, L
影响因子：
15.9
作者：
KOURY, MJ;BONDURANT, MC;SAWYER, ST
通讯作者：
SAWYER, ST
影响因子：
19.6
作者：
Maxwell, PH;Ferguson, DJP;Ratcliffe, PJ
通讯作者：
Ratcliffe, PJ