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• W2903354687 abstract "Erythropoietin (Epo) is essential for erythropoiesis and is mainly produced by the fetal liver and the adult kidney following hypoxic stimulation. Epo regulation is commonly studied in hepatoma cell lines, but differences in Epo regulation between kidney and liver limit the understanding of Epo dysregulation in polycythaemia and anaemia. To overcome this limitation, we have generated a novel transgenic mouse model expressing Cre recombinase specifically in the active fraction of renal Epo-producing (REP) cells. Crossing with reporter mice confirmed the inducible and highly specific tagging of REP cells, located in the corticomedullary border region where there is a steep drop in oxygen bioavailability. A novel method was developed to selectively grow primary REP cells in culture and to generate immortalized clonal cell lines, called fibroblastoid atypical interstitial kidney (FAIK) cells. FAIK cells show very early hypoxia-inducible factor (HIF)-2α induction, which precedes Epo transcription. Epo induction in FAIK cells reverses rapidly despite ongoing hypoxia, suggesting a cell autonomous feedback mechanism. In contrast, HIF stabilizing drugs resulted in chronic Epo induction in FAIK cells. RNA sequencing of three FAIK cell lines derived from independent kidneys revealed a high degree of overlap and suggests that REP cells represent a unique cell type with properties of pericytes, fibroblasts, and neurons, known as telocytes. These novel cell lines may be helpful to investigate myofibroblast differentiation in chronic kidney disease and to elucidate the molecular mechanisms of HIF stabilizing drugs currently in phase III studies to treat anemia in end-stage kidney disease. Erythropoietin (Epo) is essential for erythropoiesis and is mainly produced by the fetal liver and the adult kidney following hypoxic stimulation. Epo regulation is commonly studied in hepatoma cell lines, but differences in Epo regulation between kidney and liver limit the understanding of Epo dysregulation in polycythaemia and anaemia. To overcome this limitation, we have generated a novel transgenic mouse model expressing Cre recombinase specifically in the active fraction of renal Epo-producing (REP) cells. Crossing with reporter mice confirmed the inducible and highly specific tagging of REP cells, located in the corticomedullary border region where there is a steep drop in oxygen bioavailability. A novel method was developed to selectively grow primary REP cells in culture and to generate immortalized clonal cell lines, called fibroblastoid atypical interstitial kidney (FAIK) cells. FAIK cells show very early hypoxia-inducible factor (HIF)-2α induction, which precedes Epo transcription. Epo induction in FAIK cells reverses rapidly despite ongoing hypoxia, suggesting a cell autonomous feedback mechanism. In contrast, HIF stabilizing drugs resulted in chronic Epo induction in FAIK cells. RNA sequencing of three FAIK cell lines derived from independent kidneys revealed a high degree of overlap and suggests that REP cells represent a unique cell type with properties of pericytes, fibroblasts, and neurons, known as telocytes. These novel cell lines may be helpful to investigate myofibroblast differentiation in chronic kidney disease and to elucidate the molecular mechanisms of HIF stabilizing drugs currently in phase III studies to treat anemia in end-stage kidney disease. Erythropoietin (Epo)-regulated red blood cell homeostasis is crucial for oxygen delivery in vertebrates, and Epo dysregulation causes anemia or polycythemia.1Semenza G.L. Involvement of oxygen-sensing pathways in physiologic and pathologic erythropoiesis.Blood. 2009; 114: 2015-2019Google Scholar, 2Wenger R.H. Kurtz A. Erythropoietin.Compr Physiol. 2011; 1: 1759-1794Google Scholar While the liver is the main site of Epo production in the embryo, the kidney produces about 90% of circulating Epo in the adult, and only the remaining 10% is of hepatic origin.3Schooley J.C. Mahlmann L.J. Erythropoietin production in the anephric rat. I. Relationship between nephrectomy, time of hypoxic exposure, and erythropoietin production.Blood. 1972; 39: 31-38Google Scholar, 4Fried W. The liver as a source of extrarenal erythropoietin production.Blood. 1972; 40: 671-677Crossref Google Scholar Local tissue oxygenation appears to be the major factor triggering Epo production. Intriguingly, Epo is almost exclusively regulated on the mRNA level by hypoxia-inducible factor (HIF)-dependent transcriptional activation.5Wenger R.H. Hoogewijs D. Regulated oxygen sensing by protein hydroxylation in renal erythropoietin-producing cells.Am J Physiol Renal Physiol. 2010; 298: F1287-F1296Google Scholar Whereas hepatic and neuronal cell lines are used to study Epo gene regulation,6Goldberg M.A. Glass G.A. Cunningham J.M. et al.The regulated expression of erythropoietin by two human hepatoma cell lines.Proc Natl Acad Sci USA. 1987; 84: 7972-7976Google Scholar, 7Stolze I. Berchner-Pfannschmidt U. Freitag P. et al.Hypoxia-inducible erythropoietin gene expression in human neuroblastoma cells.Blood. 2002; 100: 2623-2628Google Scholar a reliable cell culture model derived from renal Epo-producing (REP) cells is currently not available. Under normal conditions, REP cells are located in the peritubular interstitial space of the inner cortex.8Obara N. Suzuki N. Kim K. et al.Repression via the GATA box is essential for tissue-specific erythropoietin gene expression.Blood. 2008; 111: 5223-5232Google Scholar A number of attempts to generate REP cell models have been reported. Cell lines derived from renal cell carcinoma occasionally produce Epo, but oxygen-regulated Epo expression has not been found in these cancer cells.9Sherwood J.B. Shouval D. Continuous production of erythropoietin by an established human renal carcinoma cell line: development of the cell line.Proc Natl Acad Sci USA. 1986; 83: 165-169Google Scholar Transgenic mice bearing a SV40 large T antigen in the Epo locus principally allowed for the isolation and immortalization of REP cells, but cultured cells showed no inducible Epo or large T expression.10Maxwell P.H. Osmond M.K. Pugh C.W. et al.Identification of the renal erythropoietin-producing cells using transgenic mice.Kidney Int. 1993; 44: 1149-1162Google Scholar Kidney-derived mesenchymal progenitor cells were differentiated in vitro to produce cell lines capable of oxygen-regulated Epo expression.11Plotkin M.D. Goligorsky M.S. Mesenchymal cells from adult kidney support angiogenesis and differentiate into multiple interstitial cell types including erythropoietin-producing fibroblasts.Am J Physiol Renal Physiol. 2006; 291: F902-F912Google Scholar Transgenic mouse lines expressing fluorescent proteins under the control of regulatory elements derived from the Epo locus (knock-in allele in a background of severe neonatal anemia) or from the Col1a1 locus were used to isolate primary REP cells by flow cytometry.12Pan X. Suzuki N. Hirano I. et al.Isolation and characterization of renal erythropoietin-producing cells from genetically produced anemia mice.PLoS One. 2011; 6: e25839Google Scholar, 13Chang Y.T. Yang C.C. Pan S.Y. et al.DNA methyltransferase inhibition restores erythropoietin production in fibrotic murine kidneys.J Clin Invest. 2016; 126: 721-731Google Scholar Human mesenchymal-like CD133+/CD73+ progenitor cells isolated from the inner medulla showed increased Epo production following hypoxic stimulation.14Bussolati B. Lauritano C. Moggio A. et al.Renal CD133+/CD73+ progenitors produce erythropoietin under hypoxia and prolyl hydroxylase inhibition.J Am Soc Nephrol. 2013; 24: 1234-1241Google Scholar Finally, several conditional knockout mouse models with constitutive HIF (over-)expression resulted in ectopic renal Epo synthesis, including models that involve the promoters derived from the genes encoding for CD68, renin, connexin 40, PDGFRβ, and FOXD1 to drive Cre expression.15Franke K. Kalucka J. Mamlouk S. et al.HIF-1α is a protective factor in conditional PHD2-deficient mice suffering from severe HIF-2α-induced excessive erythropoiesis.Blood. 2013; 121: 1436-1445Google Scholar, 16Kurt B. Paliege A. Willam C. et al.Deletion of von Hippel-Lindau protein converts renin-producing cells into erythropoietin-producing cells.J Am Soc Nephrol. 2013; 24: 433-444Google Scholar, 17Kurt B. Gerl K. Karger C. et al.Chronic hypoxia-inducible transcription factor-2 activation stably transforms juxtaglomerular renin cells into fibroblast-like cells in vivo.J Am Soc Nephrol. 2015; 26: 587-596Google Scholar, 18Gerl K. Miquerol L. Todorov V.T. et al.Inducible glomerular erythropoietin production in the adult kidney.Kidney Int. 2015; 88: 1345-1355Google Scholar, 19Gerl K. Nolan K.A. Karger C. et al.Erythropoietin production by PDGFR-β+ cells.Eur J Physiol. 2016; 468: 1479-1487Google Scholar, 20Kobayashi H. Liu Q. Binns T.C. et al.Distinct subpopulations of FOXD1 stroma-derived cells regulate renal erythropoietin.J Clin Invest. 2016; 126: 1926-1938Google Scholar, 21Gerl K. Steppan D. Fuchs M. et al.Activation of Hypoxia Signaling in Stromal Progenitors Impairs Kidney Development.Am J Pathol. 2017; 187: 1496-1511Google Scholar In conclusion, none of these attempts resulted in a reliable renal cell culture with permanent capability of regulated Epo expression, maybe due to the transient nature of Epo locus activation by differentiation and oxygen-dependent signals. We hence reasoned that isolation of freshly isolated cells acutely tagged for an active Epo locus may enhance the chance of obtaining REP cells. Therefore, we generated novel transgenic mouse lines to conditionally tag REP cells and analyzed specific targeting of “on” REP cells by spatial comparison with oxygen bioavailability and hypoxic Epo mRNA expression. Using this mouse model, primary REP cultures as well as clonal REP cell lines can be generated, recapitulating hallmarks of Epo regulation. A CreERT2 expression vector was constructed using 220 kb of the mouse Epo locus as shown in Supplementary Figure S1. This construct was first tested in vitro to confirm hypoxia-inducible Cre expression and tamoxifen-dependent Cre activation (Figure 1a). Several transgenic founder lines were then generated and crossed with reporter mice, which allowed for the permanent tagging of REP cells by red fluorescent tdTomato expression. While under normoxic conditions no or only very few fluorescent cells could be observed in the absence or presence, respectively, of tamoxifen (data not shown), this number increased following exposure to hypoxic conditions (Figure 1b). Pixel area quantification demonstrated that hypoxia clearly activated more REP cells in founder lines 1, 240, and 241 than in 244 (Figure 1c). Epo-CreERT2 1 and 241 were chosen for subsequent analyses. As depicted in Figure 1d, tdTomato expression overlaps with immunoreactivity of CD73, an established marker of REP cells.22Bachmann S. Le Hir M. Eckardt K.U. Co-localization of erythropoietin mRNA and ecto-5'-nucleotidase immunoreactivity in peritubular cells of rat renal cortex indicates that fibroblasts produce erythropoietin.J Histochem Cytochem. 1993; 41: 335-341Google Scholar Furthermore, the CD73 surface marker revealed the long processes typical for REP cells.8Obara N. Suzuki N. Kim K. et al.Repression via the GATA box is essential for tissue-specific erythropoietin gene expression.Blood. 2008; 111: 5223-5232Google Scholar High-resolution microscopy showed in more detail the irregular shape and the long processes of REP cells, extending between multiple tubular cells (Figure 1e), which further confirms the reported phenotype.8Obara N. Suzuki N. Kim K. et al.Repression via the GATA box is essential for tissue-specific erythropoietin gene expression.Blood. 2008; 111: 5223-5232Google Scholar Whole-genome DNA sequencing revealed that both strains show single integration sites, but whereas the vector integrated in strain 1 into an intergenic region of chromosome 14 with the loss of only 330 bp, in strain 241 it integrated into chromosome 9 with the loss of 52.2 kb, including exons 4 to 13 of the Pou2f3 gene. The remaining Epo flanking regions in the integrated bacterial artificial chromosome vectors were 45.9 kb and 98.2 kb in 1, and 75.2 kb and 52 kb in 241, upstream and downstream, respectively, of the Epo-coding region (Supplementary Figure S2A). Both, the 5' and 3' boundaries of the integrated vectors were confirmed by polymerase chain reaction (examples shown in Supplementary Figure S2B). Although the gross phenotypes of both founder strains were indistinguishable from that of wild-type mice, and despite identical REP cell tagging by tdTomato expression, strain 1 was chosen for further analyses due to the gene-disrupting nature of the vector integration into strain 241, which potentially could have deleterious effects.23Matsumoto I. Ohmoto M. Narukawa M. et al.Skn-1a (Pou2f3) specifies taste receptor cell lineage.Nat Neurosci. 2011; 14: 685-687Google Scholar Renal Epo expression is exceptionally hypoxia-sensitive even though the HIF system and its hundreds of target genes are ubiquitously expressed.5Wenger R.H. Hoogewijs D. Regulated oxygen sensing by protein hydroxylation in renal erythropoietin-producing cells.Am J Physiol Renal Physiol. 2010; 298: F1287-F1296Google Scholar While the underlying molecular mechanisms remain to be elucidated, the unique oxygen distribution in the adult kidney is likely involved in REP cell activation. Indeed, mice ubiquitously expressing a fusion protein between the HIF-1α oxygen-dependent degradation (ODD) domain and the luciferase (Luc) reporter gene showed a strikingly kidney-specific bioluminescence, as reported previously.24Safran M. Kim W.Y. O'Connell F. et al.Mouse model for noninvasive imaging of HIF prolyl hydroxylase activity: assessment of an oral agent that stimulates erythropoietin production.Proc Natl Acad Sci USA. 2006; 103: 105-110Google Scholar Only distal extremities and the normally hypoxic testis but no other inner organs showed a comparable hypoxia-induced bioluminescence (Figure 2a). Because whole-body imaging cannot reveal the intra-organ ODD-Luc distribution, we analyzed fresh organ slices of the kidneys shown in Figure 2a. Despite the now uniform tissue thickness, bioluminescence intensity in the medulla was still much stronger than in the cortex. Both medulla and cortex displayed a quite homogenous bioluminescence distribution, with a steep O2 bioavailability gradient mainly located in the corticomedullary border region (Figure 2b). We next analyzed the localization of REP cells in Epo-CreERT2#1xtdTomato mice. In order to compare the delayed reporter protein accumulation with the short-lived Epo mRNA levels, we used 2 relatively strong hypoxic stimuli (each 4 hours of 0.1% CO inspiration) with 1 week recovery in between. While the first stimulus was used to permanently tag REP cells, the subsequent second stimulus was used to analyze acutely induced Epo mRNA. Both tdTomato (Figure 2c) as well as endogenous Epo mRNA (Figure 2d) localized to peritubular interstitial cells of the corticomedullary border region. Strikingly, a much higher number of REP cells was observed by histochemical Epo mRNA in situ hybridization than tdTomato immunofluorescence, most likely due to the single-molecule sensitivity of the former method, which detects Epo mRNA levels far below the corresponding Cre expression threshold required for recombination of the tdTomato reporter locus. Therefore, we analyzed Epo mRNA by the same technique following a more physiological hypoxic stimulus (3 hours of 8% O2 inspiration). As shown in Figure 2e, this treatment results in an REP cell pattern similar to the tdTomato-tagged REP cells shown in Figure 2c. While no red cells were detected in wild-type mice, only sporadic cells were seen in transgenic mice following tamoxifen treatment. This number did not further increase after 4 hours of 8% O2 inspiration (Figure 2f), suggesting that this stimulus is sufficient for Epo mRNA induction but borderline for functional Cre expression. However, there was a clear increase in REP cell numbers following 16 hours of 8% O2 inspiration, demonstrating that this mouse model faithfully recapitulates the spatio-temporal conditional regulation of the Epo gene. Because the results shown above revealed that only a very small fraction of all cells of the kidney are active “on” REP cells, we reasoned that selection against Epo-negative cells may be a more suitable strategy to isolate primary REP cells than selection for Epo-positive cells. Therefore, Epo-CreERT2#1xtdTomato reporter mice were crossed with the Terminator strain, which allowed for the killing of non–Cre-expressing cells using diphtheria toxin25Guo J.K. Shi H. Koraishy F. et al.The Terminator mouse is a diphtheria toxin-receptor knock-in mouse strain for rapid and efficient enrichment of desired cell lineages.Kidney Int. 2013; 84: 1041-1046Google Scholar (Supplementary Figure S1). To select the acutely Epo-expressing cells, freshly isolated renal cell suspensions were immediately exposed to hypoxia and tamoxifen to induce Cre-mediated recombination, followed by treatment with diphtheria toxin. While only few red cells were visible after 1.5 days of isolation, the abundance of tdTomato-positive REP cells increased after 7 days of normoxic cultivation (Figure 3a). Most but not all of the viable cells were tdTomato-positive after 10.5 days, suggesting that some cells may not have recombined the tdTomato allele. Epo mRNA was induced by hypoxia in primary REP cells after 6 days of isolation (Figure 3b), demonstrating that these proliferating primary REP cell cultures maintained the capacity to express and regulate the Epo gene. Because primary REP cells ceased to proliferate and lost inducible Epo expression after approximately 12 to 14 days of culture (data not shown), we generated permanent REP cell lines. Therefore, independent primary REP cell preparations were immortalized with SV40 large T antigen 3 or 6 days after isolation. Limited dilution cloning resulted in FAIK cell lines. Three independent clones were chosen for further analysis. FAIK cells maintained hypoxia-inducible Epo protein for at least 30 passages (Figure 3c and Supplementary Figure S3). Because the Epo signal intensity was rather weak, we reasoned that during propagation some of the cells may have lost Epo expression due to epigenetic silencing by DNA methylation of Epo-regulatory loci or the Epo locus itself. DNA methyltransferase inhibition may hence restore Epo expression in these cells as recently shown in fibrotic kidneys in vivo.13Chang Y.T. Yang C.C. Pan S.Y. et al.DNA methyltransferase inhibition restores erythropoietin production in fibrotic murine kidneys.J Clin Invest. 2016; 126: 721-731Google Scholar Indeed, 5-azacytidine could enhance both basal and hypoxia-inducible Epo protein levels in FAIK3-5 cells (Figure 3c). These results were confirmed by enzyme-linked immunosorbent assay using supernatants as well as cell lysates derived from the same cells (Figure 3d). A typical well-known feature of Epo gene expression is the early decrease after an initial strong induction, despite ongoing hypoxia and long before any change in hematocrit occurs.26Eckardt K.U. Dittmer J. Neumann R. et al.Decline of erythropoietin formation at continuous hypoxia is not due to feedback inhibition.Am J Physiol. 1990; 258: F1432-F1437Google Scholar, 27Tan C.C. Eckardt K.U. Firth J.D. et al.Feedback modulation of renal and hepatic erythropoietin mRNA in response to graded anemia and hypoxia.Am J Physiol. 1992; 263: F474-F481Crossref Google Scholar Interestingly, this feature could also be seen in FAIK cell lines as exemplified by Epo immunoblot detection in FAIK3-5 cells independent of whether 1 or 5 passages went by since the 5-azacytidine treatment (Figure 3e). Bisulfite sequencing of a short Epo promoter region revealed mostly methylation-free CpG dinucleotides proximal to the transcription start site, whereas the more 5' CpGs remained mostly methylated, independent of hypoxia or 5-azacytidine treatments (Figure 3f), suggesting that increased Epo expression following 5-azacytidine treatment is not due to direct action on the Epo promoter in FAIK cells. The mRNA levels in FAIK3-5 cells could efficiently be blocked using lentiviral transduction with short hairpin RNA constructs (Figure 3g and Supplementary Methods and corresponding Supplementary Figures). shHIF-2α but not shCtrl or shHIF-1α blunted Epo expression, confirming HIF-2–dependent Epo regulation. A well-established difference between HIF-1α and HIF-2α is the delayed (after 2 to 3 days) and prolonged hypoxic induction of HIF-2α protein often observed in cancer cell lines.28Stiehl D.P. Bordoli M.R. Abreu-Rodríguez I. et al.Non-canonical HIF-2α function drives autonomous breast cancer cell growth via an AREG-EGFR/ErbB4 autocrine loop.Oncogene. 2012; 31: 2283-2297Google Scholar However, such a delayed HIF-2α induction would not be consistent with the rapid induction of its target gene Epo in REP cells in vivo and FAIK cell lines in vitro. Intriguingly, while HIF-1α showed maximal protein levels after 12 hours, HIF-2α induction was even faster in REP cells, reaching maximal levels after 3 to 6 hours of hypoxia (Figure 4a). Because such rapid kinetics is dominated by the slow oxygen diffusion and exchange rates,29Wenger R.H. Kurtcuoglu V. Scholz C.C. et al.Frequently asked questions in hypoxia research.Hypoxia. 2015; 3: 35-43Google Scholar these experiments were repeated using cell culture media that had been pre-equilibrated with 0.2% O2. While HIF-2α was already induced at the earliest time point (1.5 hours), the phosphorylated high molecular weight form of HIF-1α maximally accumulated after 12 hours (Figure 4b, upper panel). Under these conditions, Epo protein followed the HIF-2α kinetics with a maximal level already after 1.5 hours followed by a gradual decrease, as shown by immunoblotting (Figure 4b, lower panel), enzyme-linked immunosorbent assay (Figure 4c), and mRNA quantification (Figure 4d). Of note, this rapid reversal of hypoxic Epo induction could not be observed using the prolyl-4-hydroxylase domain (PHD) inhibitor FG-4592 (roxadustat), which led to a permanent Epo expression until the end of the observation period after 48 hours, while the hypoxic levels were decreased after 24 hours (Figure 4e). As shown in Figure 4f, a single bolus of FG-4592 maintained Epo mRNA and protein levels for up to 72 hours, suggesting that chemical PHD inhibition blocks a potential negative feedback loop seen in prolonged hypoxia.30Stiehl D.P. Wirthner R. Köditz J. et al.Increased prolyl 4-hydroxylase domain proteins compensate for decreased oxygen levels. Evidence for an autoregulatory oxygen-sensing system.J Biol Chem. 2006; 281: 23482-23491Google Scholar The 3 independent FAIK cell lines were further phenotyped by RNA sequencing following exposure to hypoxia for 24 hours. Statistical evaluation confirmed the reproducibility of the REP cell isolation method (Supplementary Figure S4A). Nevertheless, unsupervised cluster analysis showed that FAIK2-5 were more distant from FAIK1-10 and FAIK3-5, with normoxic and hypoxic FAIK2-5 being more closely related than the distinct normoxic and hypoxic clusters formed by the 2 other cell lines (Supplementary Figure S4B). However, with a total of 16,357 expressed mRNA and lncRNA species, the majority (84.8%) was commonly expressed in all 3 cell lines. There was less overlap in the sequence read numbers of the hypoxically regulated (i.e., at least a 2-fold change) RNA species between the 3 cell lines, with 14.7% regulated, 21.1% induced, and 8.8% repressed RNA species, respectively (Supplementary Figure S4C). A rank order list of the 100 most strongly induced genes is shown in Supplementary Figure S5. In addition to the approximately 700 to 950 mRNA species also 120 to 200 lncRNA species accumulated under hypoxic conditions in the 3 FAIK cell lines (Supplementary Figure S4D), in line with our previous reports on hypoxic inducibility of lncRNAs.31Wollenick K. Hu J. Kristiansen G. et al.Synthetic transactivation screening reveals ETV4 as broad coactivator of hypoxia-inducible factor signaling.Nucl Acids Res. 2012; 40: 1928-1943Google Scholar, 32Lelli A. Nolan K.A. Santambrogio S. et al.Induction of long noncoding RNA MALAT1 in hypoxic mice.Hypoxia. 2015; 3: 45-52Google Scholar Gene ontology analysis revealed metabolic reprogramming toward HIF-1–dependent glycolysis under hypoxic conditions (Supplementary Table S1). Established HIF target genes,33Wenger R.H. Stiehl D.P. Camenisch G. Integration of oxygen signaling at the consensus HRE.Sci STKE. 2005; 2005: re12Crossref Scopus (815) Google Scholar including VEGFa, Glut1, DEC2, and NDRG1 showed a very robust hypoxia-inducible expression, while housekeeping ribosomal protein rpL28 was constitutively expressed (Figure 5a). However, CAIX, which is commonly induced in virtually all cancer cell lines, is almost completely absent even in hypoxic FAIK cells (data not shown), underlining the non-transformed nature of these cell lines. Of the mRNAs involved in the oxygen signaling cascade, PHD2 was most prominently expressed, in line with its predominant function in renal Epo regulation.34Takeda K. Aguila H.L. Parikh N.S. et al.Regulation of adult erythropoiesis by prolyl hydroxylase domain proteins.Blood. 2008; 111: 3229-3235Google Scholar PHD2 and PHD3, but not PHD1, were significantly induced by hypoxia, consistent with a role in an intrinsic negative feedback loop.30Stiehl D.P. Wirthner R. Köditz J. et al.Increased prolyl 4-hydroxylase domain proteins compensate for decreased oxygen levels. Evidence for an autoregulatory oxygen-sensing system.J Biol Chem. 2006; 281: 23482-23491Google Scholar Intriguingly in this context, also VHL (but not FIH) was induced by hypoxia, at least in the FAIK cell lines analyzed (Figure 5b). Epo mRNA did not reach the threshold of 10 normalized sequence reads. Considering (i) the basal levels close to zero, (ii) the nonoptimal time point for Epo mRNA induction, (iii) the small size of the Epo mRNA, and (iv) the low Epo versus housekeeping control ratio usually observed by reverse-transcription quantitative polymerase chain reaction, RNA sequencing was not sensitive enough for Epo detection under these experimental conditions. However, manual inspection revealed Epo mRNA sequencing reads below the threshold in all 3 FAIK cell lines under hypoxic conditions only (data not shown). To characterize the developmental origin of FAIK cell lines, a panel of cell-type–specific markers was analyzed. All FAIK cell lines were positive for the fibroblast markers PDGFRβ,19Gerl K. Nolan K.A. Karger C. et al.Erythropoietin production by PDGFR-β+ cells.Eur J Physiol. 2016; 468: 1479-1487Google Scholar CD73,22Bachmann S. Le Hir M. Eckardt K.U. Co-localization of erythropoietin mRNA and ecto-5'-nucleotidase immunoreactivity in peritubular cells of rat renal cortex indicates that fibroblasts produce erythropoietin.J Histochem Cytochem. 1993; 41: 335-341Google Scholar and Col1A113Chang Y.T. Yang C.C. Pan S.Y. et al.DNA methyltransferase inhibition restores erythropoietin production in fibrotic murine kidneys.J Clin Invest. 2016; 126: 721-731Google Scholar (Figure 5c). Also FSP135Higgins D.F. Kimura K. Bernhardt W.M. et al.Hypoxia promotes fibrogenesis in vivo via HIF-1 stimulation of epithelial-to-mesenchymal transition.J Clin Invest. 2007; 117: 3810-3820Google Scholar and the myofibroblast marker αSMA36Souma T. Nezu M. Nakano D. et al.Erythropoietin synthesis in renal myofibroblasts is restored by activation of hypoxia signaling.J Am Soc Nephrol. 2016; 27: 428-438Google Scholar were expressed, indicating that at least a subpopulation of these cells may have differentiated during cell culture. REP cells have recently been shown to express the neuronal markers MAP2 and NFL.8Obara N. Suzuki N. Kim K. et al.Repression via the GATA box is essential for tissue-specific erythropoietin gene expression.Blood. 2008; 111: 5223-5232Google Scholar While we could confirm the expression of MAP2, NFL was not found in FAIK cell lines, in line with a previous report.19Gerl K. Nolan K.A. Karger C. et al.Erythropoietin production by PDGFR-β+ cells.Eur J Physiol. 2016; 468: 1479-1487Google Scholar However, they were clearly positive for the neuronal markers NGF, BDNF, and nestin, as well as for the pericyte marker NG2/CSPG4 (Figure 5d) that has recently been used to trace brain pericytes as a major source of Epo in the mouse brain.37Urrutia A.A. Afzal A. Nelson J. et al.Prolyl-4-hydroxylase 2 and 3 coregulate murine erythropoietin in brain pericytes.Blood. 2016; 128: 2550-2560Google Scholar The neural crest markers P0 and Pax3, which have been used for the lineage tracing of REP cells,38Asada N. Takase M. Nakamura J. et al.Dysfunction of fibroblasts of extrarenal origin underlies renal fibrosis and renal anemia in mice.J Clin Invest. 2011; 121: 3981-3990Google Scholar, 39Suzuki N. Hirano I. Pan X. et al.Erythropoietin production in neuroepithelial and neural crest cells during primitive erythropoiesis.Nat Commun. 2013; 4: 2902Google Scholar could either not be detected in FAIK cells or only at low levels (data not shown). The transcription factors FoxD1 and WT1 are markers of the mesenchymal stroma precursor cells of the developing kidney that give rise to renal interstitial cells, including fibroblasts and pericytes. FoxD1 has previously been used to trace REP cells.20Kobayashi H. Liu Q. Binns T.C. et al.Distinct subpopulations of FOXD1 stroma-derived cells regulate renal erythropoietin.J Clin Invest. 2016; 126: 1926-1938Google Scholar Both markers are expressed in all FAIK cell lines, and WT1 was strongly induced by hypoxia. Because WT1 is known to regulate the Epo promoter,40Dame C. Kirschner K.M. Bartz K.V. et al.Wi" @default.
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• W2903354687 title "Generation of renal Epo-producing cell lines by conditional gene tagging reveals rapid HIF-2 driven Epo kinetics, cell autonomous feedback regulation, and a telocyte phenotype" @default.
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• W2903354687 doi "https://doi.org/10.1016/j.kint.2018.08.043" @default.
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